Egr-1 and serum response factor are involved in growth factors- and serum-mediated induction of E2-EPF UCP expression that regulates the VHL-HIF pathway.

PubMed

Lim, Jung Hwa; Jung, Cho-Rok; Lee, Chan-Hee; Im, Dong-Soo

2008-11-01

E2-EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel-Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia-inducible factor (HIF)-1alpha. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response-1 (Egr-1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12-myristate 13-acetate induced UCP expression following early induction of Egr-1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence-specific DNA-binding of Egr-1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr-1 or SRF increased UCP expression. RNA interference-mediated depletion of endogenous Egr-1 or SRF impaired EGF- or serum-mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr-1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF-1alpha protein level under non-hypoxic conditions, suggesting that the Egr-1/SRF-UCP-VHL pathway is in part responsible for the increased HIF-1alpha protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr-1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth.

Inhibition of hypoxia inducible factor-1alpha by dihydroxyphenylethanol, a product from olive oil, blocks microsomal prostaglandin-E synthase-1/vascular endothelial growth factor expression and reduces tumor angiogenesis.

PubMed

Terzuoli, Erika; Donnini, Sandra; Giachetti, Antonio; Iñiguez, Miguel A; Fresno, Manuel; Melillo, Giovanni; Ziche, Marina

2010-08-15

2-(3,4-dihydroxyphenil)-ethanol (DPE), a polyphenol present in olive oil, has been found to attenuate the growth of colon cancer cells, an effect presumably related to its anti-inflammatory activity. To further explore the effects of DPE on angiogenesis and tumor growth we investigated the in vivo efficacy of DPE in a HT-29 xenograft model and in vitro activities in colon cancer cells exposed to interleukin-1beta (IL-1beta) and prostaglandin E-2 (PGE-2). DPE (10 mg/kg/day for 14 days) inhibited tumor growth, reducing vessel lumina and blood perfusion to tumor, and diminished expression of hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), and microsomal prostaglandin-E synthase-1 (mPGEs-1). In vitro, DPE (100 mumol/L) neither affected cell proliferation nor induced apoptosis in HT-29 and WiDr cells. DPE prevented the IL-1beta-mediated increase of mPGEs-1 expression and PGE-2 generation, as it did the silencing of HIF-1alpha. Moreover, DPE blocked mPGEs-1-dependent expression of VEGF and inhibited endothelial sprouting induced by tumor cells in a coculture system. PGE-2 triggers a feed-forward loop involving HIF-1alpha, which impinges on mPGEs-1 and VEGF expression, events prevented by DPE via extracellular signal-related kinase 1/2. The reduction of PGE-2 and VEGF levels, caused by DPE, was invariably associated with a marked decrease in HIF-1alpha expression and activity, independent of proteasome activity, indicating that the DPE effects on tumor growth and angiogenesis are dependent on the inhibition of HIF-1alpha translation. We show that the in vivo DPE antitumor effect is associated with anti-inflammatory and antiangiogenic activities resulting from the downregulation of the HIF-1alpha/mPGEs-1/VEGF axis.

Tumor necrosis factor-alpha activates signal transduction in hypothalamus and modulates the expression of pro-inflammatory proteins and orexigenic/anorexigenic neurotransmitters.

PubMed

Amaral, Maria E; Barbuio, Raquel; Milanski, Marciane; Romanatto, Talita; Barbosa, Helena C; Nadruz, Wilson; Bertolo, Manoel B; Boschero, Antonio C; Saad, Mario J A; Franchini, Kleber G; Velloso, Licio A

2006-07-01

Tumor necrosis factor-alpha (TNF-alpha) is known to participate in the wastage syndrome that accompanies cancer and severe infectious diseases. More recently, a role for TNF-alpha in the pathogenesis of type 2 diabetes mellitus and obesity has been shown. Much of the regulatory action exerted by TNF-alpha upon the control of energy stores depends on its action on the hypothalamus. In this study, we show that TNF-alpha activates canonical pro-inflammatory signal transduction pathways in the hypothalamus of rats. These signaling events lead to the transcriptional activation of an early responsive gene and to the induction of expression of cytokines and a cytokine responsive protein such as interleukin-1beta, interleukin-6, interleukin-10 and suppressor of cytokine signalling-3, respectively. In addition, TNF-alpha induces the expression of neurotransmitters involved in the control of feeding and thermogenesis. Thus, TNF-alpha may act directly in the hypothalamus inducing a pro-inflammatory response and the modulation of expression of neurotransmitters involved in energy homeostasis.

TNF-alpha infusion impairs corpora cavernosa reactivity.

PubMed

Carneiro, Fernando S; Zemse, Saiprazad; Giachini, Fernanda R C; Carneiro, Zidonia N; Lima, Victor V; Webb, R Clinton; Tostes, Rita C

2009-03-01

Erectile dysfunction (ED), as well as cardiovascular diseases (CVDs), is associated with endothelial dysfunction and increased levels of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). We hypothesized that increased TNF-alpha levels impair cavernosal function. In vitro organ bath studies were used to measure cavernosal reactivity in mice infused with vehicle or TNF-alpha (220 ng/kg/min) for 14 days. Gene expression of nitric oxide synthase isoforms was evaluated by real-time polymerase chain reaction. Corpora cavernosa from TNF-alpha-infused mice exhibited decreased nitric oxide (NO)-dependent relaxation, which was associated with decreased endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) cavernosal expression. Cavernosal strips from the TNF-alpha-infused mice displayed decreased nonadrenergic-noncholinergic (NANC)-induced relaxation (59.4 +/- 6.2 vs. control: 76.2 +/- 4.7; 16 Hz) compared with the control animals. These responses were associated with decreased gene expression of eNOS and nNOS (P < 0.05). Sympathetic-mediated, as well as phenylephrine (PE)-induced, contractile responses (PE-induced contraction; 1.32 +/- 0.06 vs. control: 0.9 +/- 0.09, mN) were increased in cavernosal strips from TNF-alpha-infused mice. Additionally, infusion of TNF-alpha increased cavernosal responses to endothelin-1 and endothelin receptor A subtype (ET(A)) receptor expression (P < 0.05) and slightly decreased tumor necrosis factor-alpha receptor 1 (TNFR1) expression (P = 0.063). Corpora cavernosa from TNF-alpha-infused mice display increased contractile responses and decreased NANC nerve-mediated relaxation associated with decreased eNOS and nNOS gene expression. These changes may trigger ED and indicate that TNF-alpha plays a detrimental role in erectile function. Blockade of TNF-alpha actions may represent an alternative therapeutic approach for ED, especially in pathologic conditions associated with increased levels of this cytokine.

Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts.

PubMed

Rapisarda, Annamaria; Zalek, Jessica; Hollingshead, Melinda; Braunschweig, Till; Uranchimeg, Badarch; Bonomi, Carrie A; Borgel, Suzanne D; Carter, John P; Hewitt, Stephen M; Shoemaker, Robert H; Melillo, Giovanni

2004-10-01

We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1alpha protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1alpha protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1alpha inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

Hypoxia preconditioning protection of corneal stromal cells requires HIF1alpha but not VEGF.

PubMed

Xing, Dongmei; Bonanno, Joseph A

2009-05-18

Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1alpha (Hypoxia Inducible Factor) is responsible and whether this is promulgated by VEGF (Vascular Endothelial Growth Factor). Cultured bovine stromal cells were preconditioned with hypoxia in the presence of cadmium chloride, a chemical inhibitor of HIF-1alpha, and HIF-1alpha siRNA to test if HIF-1alpha activity is needed for hypoxia preconditioning protection from UV-irradiation induced cell death. TUNEL assay was used to detect cell apoptosis after UV-irradiation. RT-PCR and western blot were used to detect the presence of HIF-1alpha and VEGF in transcriptional and translational levels. During hypoxia (0.5% O2), 5 muM cadmium chloride completely inhibited HIF-1alpha expression and reversed the protection by hypoxia preconditioning. HIF-1alpha siRNA (15 nM) reduced HIF-1alpha expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning. Since VEGF is induced by hypoxia, can be HIF-1alpha dependent, and is often protective, we examined the changes in transcription of VEGF and its receptors after 4 h of hypoxia preconditioning. VEGF and its receptors Flt-1 and Flk-1 are up-regulated after hypoxia preconditioning. However, the transcription and translation of VEGF were paradoxically increased by siHIF-1alpha, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1alpha. These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1alpha, but that VEGF is not a component of the protection.

Enhanced actions of insulin-like growth factor-I and interferon-alpha co-administration in experimental cirrhosis.

PubMed

Tutau, Federico; Rodríguez-Ortigosa, Carlos; Puche, Juan Enrique; Juanarena, Nerea; Monreal, Iñigo; García Fernández, María; Clavijo, Encarna; Castilla, Alberto; Castilla-Cortázar, Inma

2009-01-01

Cirrhosis is a diffuse process of hepatic fibrosis and regenerative nodule formation. The liver is the major source of circulating insulin-like growth factor-I (IGF-I) whose plasma levels are diminished in cirrhosis. IGF-I supplementation has been shown to induce beneficial effects in cirrhosis, including antifibrogenic and hepatoprotective effects. On other hand, interferon-alpha (IFN-alpha) therapy seems to suppress the progression of hepatic fibrosis. The aim of this study was to investigate the effect of the co-administration of IGF-I+IFN-alpha to Wistar rats with CCl(4)-induced cirrhosis, exploring liver function tests, hepatic lipid peroxidation and histopathology. The mechanisms underlying the effects of these agents were studied by reverse transcription-polymerase chain reaction, determining the expression of some factors [hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-beta), alpha-smooth muscle actin, collagen, tissular inhibitor of metalloproteinases-1 and pregnane X receptor (PXR)] involved in fibrogenesis, fibrolysis and/or hepatoprotection. Both IGF-I and IFN-alpha exerted significant effects on fibrogenesis. IGF-I significantly increased serum albumin and HGF whereas IFN-alpha-therapy did not. The inhibition of TGF-beta expression was only observed by the effect of IFN-alpha-therapy. In addition, only the co-administration of IGF-I and IFN-alpha was able to increase the PXR. The combined therapy with both factors improved liver function tests, hepatic lipid peroxidation and reduced fibrosis, inducing a relevant histological improvement, reducing fibrosis and recovering hepatic architecture. The co-administration IGF-I+IFN enhanced all the beneficial effects observed with each factor separately, showing an additive action on histopathology and PXR expression, which is involved in the inhibition of fibrogenesis.

Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Dang; Fang, Liurong; Luo, Rui

2010-08-13

Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reductionmore » of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.« less

[Induction of hypoxia-inducible factor-1alpha in two kinds of rats asphyxiation death models].

PubMed

Zhang, Bei-lei; Yang, Zhi-hui; Ran, Peng; Liang, Wei-bo; Zhou, Bin; Zhang, Geng-qian; Lu, Mei-li; Zhang, Lin

2007-02-15

To investigate the expression of hypoxia-inducible factor 1-alpha (HIF1-alpha) in the heart, lung, liver and kidney in rats died of two typical models of asphyxia. Two asphyxia models were made and tissue samples of the dead rats were collected from different groups at various postmortem duration. The expression and the changes of HIF1-alpha in various tissues were examined by immunohistochemistry and image analysis techniques. Results Significant expression of HIF1-alpha was observed in the myocardial fibers, kidney cells, liver cells and lung cells in both asphyxia models, but not in the control group. The expression of HIF1-alpha in various tissues in the rat died of nitrogen gas breathing was found in the nuclei at 0 hour and the expression level decreased gradually thereafter. The HIF1-alpha expression level and duration in various tissues of the rat died of hanging were higher and longer than that of the former group, with a peak of the expression level observed 6 hours after death, and then started to decline in all tissues except the heart where the expression still showed an increase 24 hours after death. The control groups showed a steady expression in the cytoplasm but not in the nuclei. HIF1-alpha appears to be a valuable biomarker in the diagnosis of asphyxia within 24 hours after death.

Regulation of the human SLC25A20 expression by peroxisome proliferator-activated receptor alpha in human hepatoblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp; Takeuchi, Kentaro; Inada, Hirohiko

2009-11-20

Solute carrier family 25, member 20 (SLC25A20) is a key molecule that transfers acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane in the mitochondrial {beta}-oxidation. The peroxisome proliferator-activated receptor alpha (PPAR{alpha}) is a ligand-activated transcription factor that plays an important role in the regulation of {beta}-oxidation. We previously established tetracycline-regulated human cell line that can be induced to express PPAR{alpha} and found that PPAR{alpha} induces the SLC25A20 expression. In this study, we analyzed the promoter region of the human slc25a20 gene and showed that PPAR{alpha} regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element.

The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1{alpha} expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Wei; Department of cardiology, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin 150081; Guo, Ting

2011-05-01

Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPAR{gamma}) coactivator-1 alpha (PGC-1{alpha}) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1{alpha} in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1{alpha} expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1{alpha} mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1{alpha} expression while stimulating VSMC migration.more » Mechanistic study showed that suppression of PGC-1{alpha} by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1{alpha} had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1{alpha} decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPAR{gamma} activation by a PPAR{gamma} antagonist GW9662 abolished the suppressive effects of PGC-1{alpha} on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1{alpha} were enhanced by a PPAR{gamma} agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1{alpha} expression. PGC-1{alpha} suppresses PDGF-induced VSMC migration through PPAR{gamma} coactivation and, consequently, p38 MAPK inhibition.« less

Expression of the transforming growth factor alpha protooncogene in differentiating human promyelocytic leukemia (HL-60) cells.

PubMed

Walz, T M; Malm, C; Wasteson, A

1993-01-01

The process of myeloid differentiation in human promyelocytic leukemia cells (HL-60) is accompanied by the coordinate expression of numerous protooncogenes. To investigate the expression of transforming growth factor alpha (TGF-alpha) in myeloid differentiation, HL-60 cells were induced to differentiate into granulocytes with 1.25% dimethyl sulfoxide, 0.2 microM all-trans retinoic acid, or 500 microM N6,O2-dibutyryladenosine-3'5'-cyclic monophosphate or differentiated along the monocyte/macrophage pathway with 0.1 microM phorbol-12-myristate-13-acetate. Using Northern blot analyses, TGF-alpha transcripts were detected within 24 h of treatment in cells differentiating toward granulocytes; maximal levels of gene expression were reached after 3 days or later and remained essentially constant throughout the observation period. These cells released TGF-alpha protein, as demonstrated by analysis of the incubation medium. In contrast, no TGF-alpha RNA or protein was detectable in HL-60 cell cultures when induced with phorbol-12-myristate-13-acetate. Epidermal growth factor receptor transcripts could not be detected either in undifferentiated or in differentiated HL-60 cells; therefore it appears as if an autocrine loop involving TGF-alpha in HL-60 cells is unlikely. In conclusion, the results demonstrate, for the first time, the expression of TGF-alpha in human granulocyte precursor cells. Our findings may indicate novel regulatory pathways in hematopoiesis.

Modulation of Mycoplasma arthritidis-derived superantigen-induced cytokine gene expression by dexamethasone and interleukin-4.

PubMed

Mehindate, K; al-Daccak, R; Rink, L; Mecheri, S; Hébert, J; Mourad, W

1994-11-01

Activation of human monocytes or monocytic cell lines with all known stimuli coordinately induces the gene expression of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and the IL-1 receptor antagonist (IL-1Ra). In contrast, superantigens induce TNF-alpha and IL-1 beta but fail to affect IL-1Ra gene expression, suggesting that activation of monocytes via major histocompatibility complex class II is distinct from other signal transduction pathways. In the present study, we analyzed the regulation of the Mycoplasma arthritidis-derived superantigen (MAM)-induced IL-1 beta and TNF-alpha gene expression by studying the effects of two different anti-inflammatory agents: dexamethasone (DEX) and the T-cell-derived cytokine IL-4. Both agents contributed to the downregulation of MAM-induced IL-1 beta and TNF-alpha gene expression. They accelerated the normal decline of the gene expression of both MAM-induced cytokines by decreasing the stability of mRNAs via the induction or enhanced synthesis of one or more regulatory proteins. In addition, IL-4, but not DEX, induced a strong and rapid expression of IL-1Ra mRNA in MAM-stimulated and unstimulated THP-1 cells in a de novo protein synthesis-independent manner. The capacity of IL-4 to induce IL-1Ra gene expression reinforces its anti-inflammatory activity. This study illustrates some of the mechanisms by which MAM-induced proinflammatory monokine gene expression can be downregulated by IL-4 and DEX.

The proinflammatory cytokines IL-1beta and TNF-alpha induce the expression of Synoviolin, an E3 ubiquitin ligase, in mouse synovial fibroblasts via the Erk1/2-ETS1 pathway.

PubMed

Gao, Beixue; Calhoun, Karen; Fang, Deyu

2006-01-01

The overgrowth of synovial tissues is critical in the pathogenesis of rheumatoid arthritis (RA). The expression of Synoviolin (SYN), an E3 ubiquitin ligase, is upregulated in arthritic synovial fibroblasts and is involved in the overgrowth of synovial cells during RA. However, the molecular mechanisms involved in the elevated SYN expression are not known. Here, we found that SYN expression is elevated in the synovial fibroblasts from mice with collagen-induced arthritis (CIA). The proinflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) induce SYN expression in mouse synovial fibroblasts. Cultivation of mouse synovial fibroblasts with IL-1beta activates mitogen-activated protein kinases, including extra-cellular signal-regulated kinase (Erk), JNK (c-Jun N-terminal kinase), and p38, while only Erk-specific inhibitor blocks IL-1beta-induced SYN expression. Expression of transcription factor ETS1 further enhances IL-1beta-induced SYN expression. The dominant negative ETS1 mutant lacking the transcription activation domain inhibits SYN expression in a dose-dependent manner. The activation of both Erk1/2 and ETS1 is increased in the CIA synovial fibroblasts. Inhibition of Erk activation reduces ETS1 phosphorylation and SYN expression. Our data indicate that the proinflammatory cytokines IL-1beta and TNF-alpha induce the overgrowth of synovial cells by upregulating SYN expression via the Erk1/-ETS1 pathway. These molecules or pathways could therefore be potential targets for the treatment of RA.

Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Konnai, Satoru; Usui, Tatsufumi; Ikeda, Manabu

2005-09-01

Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-{alpha} and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-{alpha}-induced responses, in this study we examined the TNF-{alpha}-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-{alpha} (rTNF-{alpha}) was significantly higher than those from ALmore » cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5{sup +} or sIgM{sup +} cells and these cells showed resistance to TNF-{alpha}-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-{alpha}-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp; Kameshima, Satoshi; Usui, Tatsuya

Highlights: Black-Right-Pointing-Pointer Chemerin is a novel adipocytokine with almost unknown function in vasculature. Black-Right-Pointing-Pointer Chemerin activates Akt/eNOS/NO pathways in endothelial cells. Black-Right-Pointing-Pointer Chemerin inhibits TNF-{alpha}-induced monocyte adhesion to endothelial cells. Black-Right-Pointing-Pointer Chemerin inhibits TNF-induced VCAM-1 via suppressing NF-{kappa}B and p38 signal. Black-Right-Pointing-Pointer Chemerin is anti-inflammatory through producing NO in vascular endothelium. -- Abstract: Chemerin is a recently identified adipocytokine which plays a role on inflammation and adipocytes metabolism. However, its function in vasculature is largely unknown. We examined the effects of chemerin on vascular endothelial inflammatory states. Treatment of human umbilical vein endothelial cells with chemerin (300 ng/ml, 20 min)more » induced phosphorylation of Akt (Ser473) and endothelial nitric oxide (NO) synthase (eNOS) (Ser1177). Consistently, chemerin increased intracellular cyclic GMP content. Pretreatment with chemerin (1-300 ng/ml, 24 h) significantly inhibited phosphorylation of nuclear factor (NF)-{kappa}B p65 (Ser536) and p38 as well as vascular cell adhesion molecule (VCAM)-1 expression induced by tumor necrosis factor (TNF)-{alpha} (5 ng/ml, 20 min-6 h). Inhibitor of NF-{kappa}B or p38 significantly inhibited the TNF-{alpha}-induced VCAM-1 expression. Chemerin also inhibited TNF-{alpha}-induced VCAM-1 expression in rat isolated aorta. Moreover, chemerin significantly inhibited monocytes adhesion to TNF-{alpha}-stimulated endothelial cells. The inhibitory effect of chemerin on TNF-{alpha}-induced VCAM-1 was reversed by a NOS inhibitor. Conversely, an NO donor, sodium nitroprusside significantly inhibited TNF-{alpha}-induced VCAM-1. The present results for the first time demonstrate that chemerin plays anti-inflammatory roles by preventing TNF-{alpha}-induced VCAM-1 expression and monocytes adhesion in vascular endothelial cells. The effect is mediated via inhibiting activation of NF-{kappa}B and p38 through stimulation of Akt/eNOS signaling and NO production.« less

Withaferin A inhibits tumor necrosis factor-alpha-induced expression of cell adhesion molecules by inactivation of Akt and NF-kappaB in human pulmonary epithelial cells.

PubMed

Oh, Jung Hwa; Kwon, Taeg Kyu

2009-05-01

We here investigated the functional effect of withaferin A on airway inflammation and its action mechanism. Withaferin A inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human lung epithelial A549 cells stimulated with tumor necrosis factor-alpha (TNF-alpha), resulting in the suppression of leukocyte adhesion to lung epithelial A549 cells. In addition, withaferin A inhibited TNF-alpha-induced expression of adhesion molecules (ICAM-1 and VCAM-1) protein and mRNA in a dose-dependent manner. Withaferin A prevented DNA binding activity of nuclear factor-kappaB (NF-kappaB) and nuclear translocation of NF-kappaB. It also inhibited phosphorylation of Akt and extracellular signal-regulated kinase (ERK), which are upstream in the regulation of adhesion molecules by TNF-alpha. Furthermore, withaferin A inhibited U937 monocyte adhesion to A549 cells stimulated by TNF-alpha, suggesting that it may inhibit the binding of these cells by regulating the expression of critical adhesion molecules by TNF-alpha. Taken together, these results suggest that withaferin A inhibits cell adhesion through inhibition of ICAM-1 and VCAM-1 expression, at least in part, by blocking Akt and down-regulating NF-kappaB activity.

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

PubMed

Williams, C M; Coleman, J W

1995-10-01

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs.

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

PubMed Central

Williams, C M; Coleman, J W

1995-01-01

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs. Images Figure 1 Figure 2 Figure 3 PMID:7490125

Transforming growth factor-{beta} inhibits CCAAT/enhancer-binding protein expression and PPAR{gamma} activity in unloaded bone marrow stromal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahdjoudj, S.; Kaabeche, K.; Holy, X.

2005-02-01

The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-{beta}2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP){alpha} and C/EBP{beta} {alpha} at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}2) transcripts at 7 days. TGF-{beta}2more » administration in unloaded rats corrected the rise in C/EBP{alpha} and C/EBP{beta} transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPAR{gamma}2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBP{alpha} and C/EBP{beta} expression by TGF-{beta}2 was associated with increased PPAR{gamma} serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPAR{gamma} transactivating activity. The sequential inhibitory effect of TGF-{beta}2 on C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma}2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-{beta}2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma} expression and activity, which provides a sequential mechanism by which TGF-{beta}2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.« less

Akt mediates 17beta-estradiol and/or estrogen receptor-alpha inhibition of LPS-induced tumor necresis factor-alpha expression and myocardial cell apoptosis by suppressing the JNK1/2-NFkappaB pathway.

PubMed

Liu, Chung-Jung; Lo, Jeng-Fan; Kuo, Chia-Hua; Chu, Chun-Hsien; Chen, Li-Ming; Tsai, Fuu-Jen; Tsai, Chang-Hai; Tzang, Bor-Show; Kuo, Wei-Wen; Huang, Chih-Yang

2009-09-01

Evidence shows that women have lower tumour necrosis factor-alpha (TNF-alpha) levels and lower incidences of heart dysfunction and sepsis-related morbidity and mortality. To identify the cardioprotective effects and precise cellular/molecular mechanisms behind estrogen and estrogen receptors (ERs), we investigated the effects of 17beta-estradiol (E(2)) and estrogen receptor alpha (ERalpha) on LPS-induced apoptosis by analyzing the activation of survival and death signalling pathways in doxycycline (Dox)-inducible Tet-On/ERalpha H9c2 myocardial cells and ERalpha-transfected primary cardiomyocytes overexpressing ERalpha. We found that LPS challenge activated JNK1/2, and then induced IkappaB degradation, NFkappaB activation, TNF-alpha up-regulation and subsequent myocardial apoptotic responses. In addition, treatments involving E(2), membrane-impermeable BSA-E(2) and/or Dox, which induces ERalpha overexpression, significantly inhibited LPS-induced apoptosis by suppressing LPS-up-regulated JNK1/2 activity, IkappaB degradation, NFkappaB activation and pro-apoptotic proteins (e.g. TNF-alpha, active caspases-8, t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3) in myocardial cells. However, the cardioprotective properties of E(2), BSA-E(2) and ERalpha overexpression to inhibit LPS-induced apoptosis and promote cell survival were attenuated by applying LY294002 (PI3K inhibitor) and PI3K siRNA. These findings suggest that E(2), BSA-E(2) and ERalpha expression exert their cardioprotective effects by inhibiting JNK1/2-mediated LPS-induced TNF-alpha expression and cardiomyocyte apoptosis through activation of Akt.

Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1alpha variant for local induction of angiogenesis.

PubMed

Trentin, Diana; Hall, Heike; Wechsler, Sandra; Hubbell, Jeffrey A

2006-02-21

Hypoxia-inducible factor (HIF) constitutes a target in therapeutic angiogenesis. HIF-1alpha functions as a sensor of hypoxia and induces expression of vascular endothelial growth factor (VEGF), which then induces angiogenesis. To explore the potential of HIF-1alpha gene therapy in stimulating wound healing, we delivered a gene encoding a stabilized form of HIF-1alpha, lacking the oxygen-sensitive degradation domain, namely HIF-1alpha deltaODD, by using a previously characterized peptide-based gene delivery vector in fibrin as a surgical matrix. The peptide vector consisted of multiple domains: (i) A cysteine-flanked lysine hexamer provided DNA interactions that were stable extracellularly but destabilized intracellularly after reduction of the formed disulfide bonds. This DNA-binding domain was fused to either (ii) a fibrin-binding peptide for entrapment within the matrix or (iii) a nuclear localization sequence for efficient nuclear targeting. The HIF-1alpha deltaODD gene was expressed and translocated to the nucleus under normoxic conditions, leading to up-regulation of vascular endothelial growth factor (VEGF)-A165 mRNA and protein levels in vitro. When the peptide-DNA nanoparticles entrapped in fibrin matrices were applied to full-thickness dermal wounds in the mouse (10 microg per wound in 30 microl of fibrin), angiogenesis was increased comparably strongly to that induced by VEGF-A165 protein (1.25 microg per wound in 30 microl of fibrin). However, the maturity of the vessels induced by HIF-1alpha deltaODD was significantly higher than that induced by VEGF-A165 protein, as shown by stabilization of the neovessels with smooth muscle. Nonviral, local administration of this potent angiogenesis-inducing gene by using this peptide vector represents a powerful approach in tissue engineering and therapeutic angiogenesis.

DNA-binding activity of TNF-{alpha} inducing protein from Helicobacter pylori

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuzuhara, T.; Suganuma, M.; Oka, K.

2007-11-03

Tumor necrosis factor-{alpha} (TNF-{alpha}) inducing protein (Tip{alpha}) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-{alpha} and chemokine genes and activation of nuclear factor-{kappa}B. Since Tip{alpha} enters gastric cancer cells, the Tip{alpha} binding molecules in the cells should be investigated. The direct DNA-binding activity of Tip{alpha} was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tip{alpha} and DNA, revealed that the affinity of Tip{alpha} for (dGdC)10 is 2400 times stronger than that of del-Tip{alpha}, an inactive Tip{alpha}. This suggestsmore » a strong correlation between DNA-binding activity and carcinogenic activity of Tip{alpha}. And the DNA-binding activity of Tip{alpha} was first demonstrated with a molecule secreted from H. pylori.« less

Modulation of Mycoplasma arthritidis-derived superantigen-induced cytokine gene expression by dexamethasone and interleukin-4.

PubMed Central

Mehindate, K; al-Daccak, R; Rink, L; Mecheri, S; Hébert, J; Mourad, W

1994-01-01

Activation of human monocytes or monocytic cell lines with all known stimuli coordinately induces the gene expression of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and the IL-1 receptor antagonist (IL-1Ra). In contrast, superantigens induce TNF-alpha and IL-1 beta but fail to affect IL-1Ra gene expression, suggesting that activation of monocytes via major histocompatibility complex class II is distinct from other signal transduction pathways. In the present study, we analyzed the regulation of the Mycoplasma arthritidis-derived superantigen (MAM)-induced IL-1 beta and TNF-alpha gene expression by studying the effects of two different anti-inflammatory agents: dexamethasone (DEX) and the T-cell-derived cytokine IL-4. Both agents contributed to the downregulation of MAM-induced IL-1 beta and TNF-alpha gene expression. They accelerated the normal decline of the gene expression of both MAM-induced cytokines by decreasing the stability of mRNAs via the induction or enhanced synthesis of one or more regulatory proteins. In addition, IL-4, but not DEX, induced a strong and rapid expression of IL-1Ra mRNA in MAM-stimulated and unstimulated THP-1 cells in a de novo protein synthesis-independent manner. The capacity of IL-4 to induce IL-1Ra gene expression reinforces its anti-inflammatory activity. This study illustrates some of the mechanisms by which MAM-induced proinflammatory monokine gene expression can be downregulated by IL-4 and DEX. Images PMID:7927746

Inhibitory effects of clotrimazole on TNF-alpha-induced adhesion molecule expression and angiogenesis.

PubMed

Thapa, Dinesh; Lee, Jong Suk; Park, Min-A; Cho, Mi-Yeon; Park, Young-Joon; Choi, Han Gon; Jeong, Tae Cheon; Kim, Jung-Ae

2009-04-01

Cell adhesion molecules play a pivotal role in chronic inflammation and pathological angiogenesis. In the present study, we investigated the inhibitory effects of clotrimazole (CLT) on tumor necrosis factor (TNF)-alpha-induced changes in adhesion molecule expression. CLT dose-dependently inhibited monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expressions in TNF-alpha-stimulated HT29 colonic epithelial cells. This inhibitory action of CLT correlated with a significant reduction in TNF-alpha-induced adhesion of monocytes to HT29 cells, which was comparable to the inhibitory effects of anti-ICAM-1 and VCAM-1 monoclonal antibodies on monocyte-epithelial adhesion. These inhibitory actions of CLT were, at least in part, attributable to the inhibition of redox sensitive NF-kappaB activation, as CLT inhibited TNF-alpha-induced ROS generation as well as NF-kappaB nuclear translocation and activation in HT29 cells. Furthermore, the inhibition of TNF-alpha-induced monocyte adhesion was also mimicked by the specific NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). Inflammatory mediators including TNF-alpha have known to promote angiogenesis, which in turn further contributes to inflammatory pathology. Therefore, we additionally evaluated whether CLT modulates TNF-alpha-induced angiogenesis using in vivo chick chorioallantoic membrane (CAM) assay. The CAM assay showed that CLT dose-dependently attenuated TNF-alpha-induced angiogenesis, and the effect was correlated with decreased inflammation of the CAM tissue. In conclusion, our results suggest that CLT can inhibit TNF-alpha-triggered expression of adhesion molecules, ICAM-1 and VCAM-1, and angiogenesis during inflammation.

Granulocyte-macrophage and macrophage colony-stimulating factors differentially regulate alpha v integrin expression on cultured human macrophages.

PubMed

De Nichilo, M O; Burns, G F

1993-03-15

The colony-stimulating factors (CSFs) greatly influence mature macrophage function in vitro: macrophage (M)-CSF induces maturation of monocytes and enhances differentiated cell function; granulocyte-macrophage (GM)-CSF stimulates a variety of antimicrobial functions. In vivo M-CSF is thought to promote differentiation, and GM-CSF is thought to potentiate the inflammatory response. One mechanism by which these differential effects may be achieved is through the receptor-mediated interaction of macrophages with their extracellular matrix. Here we show that M-CSF induces specifically the expression of the alpha v beta 5 integrin receptor, whereas GM-CSF rapidly induces mRNA and surface expression of the alpha v beta 3 integrin. The M-CSF-treated cells acquire a flattened epitheloid phenotype, and on vitronectin the alpha v beta 5 is located in adhesion plaques. These cells do not bind collagen or laminin. In contrast, cells treated with GM-CSF adopt an elongated phenotype on a number of substrates, including collagen and laminin, and express alpha v beta 3 at the leading edge of cells on vitronectin. These results suggest that a primary means by which the CSFs exert their individual effects on mature cells may be through regulating integrin expression.

Immunohistochemical detection of osteopontin in advanced head-and-neck cancer: prognostic role and correlation with oxygen electrode measurements, hypoxia-inducible-factor-1alpha-related markers, and hemoglobin levels.

PubMed

Bache, Matthias; Reddemann, Rolf; Said, Harun M; Holzhausen, Hans-Jürgen; Taubert, Helge; Becker, Axel; Kuhnt, Thomas; Hänsgen, Gabriele; Dunst, Jürgen; Vordermark, Dirk

2006-12-01

The tumor-associated glycoprotein osteopontin (OPN) is discussed as a plasma marker of tumor hypoxia. However, the association of immunohistochemical OPN expression in tumor sections with tumor oxygenation parameters (HF5, median pO(2)), the hypoxia-related markers hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase IX (CAIX), or hemoglobin and systemic vascular endothelial growth factor (VEGF) levels has not been investigated. Tumor tissue sections of 34 patients with advanced head-and-neck cancer treated with radiotherapy were assessed by immunochemistry for the expression of OPN, HIF-1alpha, and CA IX. Relationship of OPN expression with tumor oxygenation parameters (HF5, median pO(2)), HIF-1alpha and CA IX expression, hemoglobin and serum VEGF level, and clinical parameters was studied. Bivariate analysis showed a significant correlation of positive OPN staining with low hemoglobin level (p = 0.02), high HIF-1alpha expression (p = 0.02), and high serum vascular endothelial growth factor level (p = 0.02) for advanced head-and-neck cancer. Furthermore, considering the 31 Stage IV patients, the median pO(2) correlated significantly with the OPN expression (p = 0.02). OPN expression alone had only a small impact on prognosis. However, in a univariate Cox proportional hazard regression model, the expression of either OPN or HIF-1alpha or CA IX was associated with a 4.1-fold increased risk of death (p = 0.02) compared with negativity of all three markers. Osteopontin expression detected immunohistochemically is associated with oxygenation parameters in advanced head-and-neck cancer. When the results of OPN, HIF-1alpha, and CA IX immunohistochemistry are combined into a hypoxic profile, a strong and statistically significant impact on overall survival is found.

Immunohistochemical detection of osteopontin in advanced head-and-neck cancer: Prognostic role and correlation with oxygen electrode measurements, hypoxia-inducible-factor-1{alpha}-related markers, and hemoglobin levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bache, Matthias; Reddemann, Rolf; Institute of Pathology, Martin-Luther-University Halle-Wittenberg, Halle

2006-12-01

Purpose: The tumor-associated glycoprotein osteopontin (OPN) is discussed as a plasma marker of tumor hypoxia. However, the association of immunohistochemical OPN expression in tumor sections with tumor oxygenation parameters (HF5, median pO{sub 2}), the hypoxia-related markers hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and carbonic anhydrase IX (CAIX), or hemoglobin and systemic vascular endothelial growth factor (VEGF) levels has not been investigated. Methods and Materials: Tumor tissue sections of 34 patients with advanced head-and-neck cancer treated with radiotherapy were assessed by immunochemistry for the expression of OPN, HIF-1{alpha}, and CA IX. Relationship of OPN expression with tumor oxygenation parameters (HF5, median pO{sub 2}), HIF-1{alpha}more » and CA IX expression, hemoglobin and serum VEGF level, and clinical parameters was studied. Results: Bivariate analysis showed a significant correlation of positive OPN staining with low hemoglobin level (p = 0.02), high HIF-1{alpha} expression (p = 0.02), and high serum vascular endothelial growth factor level (p = 0.02) for advanced head-and-neck cancer. Furthermore, considering the 31 Stage IV patients, the median pO{sub 2} correlated significantly with the OPN expression (p = 0.02). OPN expression alone had only a small impact on prognosis. However, in a univariate Cox proportional hazard regression model, the expression of either OPN or HIF-1{alpha} or CA IX was associated with a 4.1-fold increased risk of death (p = 0.02) compared with negativity of all three markers. Conclusion: Osteopontin expression detected immunohistochemically is associated with oxygenation parameters in advanced head-and-neck cancer. When the results of OPN, HIF-1{alpha}, and CA IX immunohistochemistry are combined into a hypoxic profile, a strong and statistically significant impact on overall survival is found.« less

N-acetylcysteine attenuates TNF-alpha-induced human vascular endothelial cell apoptosis and restores eNOS expression.

PubMed

Xia, Zhengyuan; Liu, Min; Wu, Yong; Sharma, Vijay; Luo, Tao; Ouyang, Jingping; McNeill, John H

2006-11-21

The circulatory inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in pathological conditions, such as diabetes, which initiate or exacerbate vascular endothelial injury. Both nitric oxide (NO) and reactive oxygen species may play a dual role (i.e., inhibiting or promoting) in TNF-alpha-induced endothelial cell apoptosis. We investigated the effects of the antioxidant N-acetylcysteine on TNF-alpha-induced apoptosis in human vascular endothelial cell (cell line ECV304) apoptosis, NO production and lipid peroxidation. Cultured vascular endothelial cell (ECV304) were either not treated (control), or treated with TNF-alpha (40 ng/ml) alone or TNF-alpha in the presence of N-acetylcysteine at 30 mmol/l or 1 mmol/l, respectively, for 24 h. Cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was assessed by flow cytometry. TNF-alpha-induced endothelial cell apoptosis was associated with increased inducible NO synthase but reduced endothelial NO synthase (eNOS) protein expression. NO production and the levels of the lipid peroxidation product malondialdehyde were concomitantly increased. Treatment with NAC at 30 mmol/l restored eNOS expression and further increased NO production as compared to TNF-alpha alone, resulting in improved cell viability and reduced apoptosis. This was accompanied by increased superoxide dismutase activity, increased glutathione peroxidase production and reduced malondialdehyde levels. N-acetylcysteine at 1 mmol/l, however, did not have significant effects on TNF-alpha-induced endothelial cell apoptosis and cell viability despite it slightly enhanced glutathione peroxidase production. N-acetylcysteine attenuation of TNF-alpha-induced human vascular endothelial cell apoptosis is associated with the restoration of eNOS expression.

Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF-alpha-induced angiogenesis.

PubMed

Pandey, A; Shao, H; Marks, R M; Polverini, P J; Dixit, V M

1995-04-28

B61, a cytokine-inducible endothelial gene product, is the ligand for the Eck receptor protein tyrosine kinase (RPTK). Expression of a B61-immunoglobulin chimera showed that B61 could act as an angiogenic factor in vivo and a chemoattractant for endothelial cells in vitro. The Eck RPTK was activated by tumor necrosis factor-alpha (TNF-alpha) through induction of B61, and an antibody to B61 attenuated angiogenesis induced by TNF-alpha but not by basic fibroblast growth factor. This finding suggests the existence of an autocrine or paracrine loop involving activation of the Eck RPTK by its inducible ligand B61 after an inflammatory stimulus, the net effect of which would be to promote angiogenesis, a hallmark of chronic inflammation.

Diesel exhaust particles induce the over expression of tumor necrosis factor-alpha (TNF-alpha) gene in alvelor machrophage and failed to induce apoptosis through activation of nuclear factor-kappaB (NF-kappaB)

EPA Science Inventory

Exposure to particulate matter (PM2.5-10), including diesel exhaust particles (DEP) has been reported to induce lung injury and exacerbation of asthma and chronic obstructive pulmonary disease. Alveolar macrophages play a major role in the lung's response to inhaled particles and...

Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogura, Hirotsugu; Tsukumo, Yoshinori; Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501

2008-04-01

The transcription factor nuclear factor {kappa}B (NF-{kappa}B) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-{alpha}-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in amore » dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-CHX profoundly decreased constitutive and inducible expression of c-FLIP, whereas these compounds potentiated TNF-{alpha}-induced caspase-8 activation only when metalloprotease inhibitors were present. Thus, our results indicate that ectodomain shedding of TNF-R1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8.« less

The involvement of hypoxia-inducible transcription factor-1-dependent pathway in nickel carcinogenesis.

PubMed

Salnikow, Konstantin; Davidson, Todd; Zhang, Qunwei; Chen, Lung Chi; Su, Weichen; Costa, Max

2003-07-01

Nickel is a potent environmental pollutant in industrial countries. Because nickel compounds are carcinogenic, exposure to nickel represents a serious hazard to human health. The understanding of how nickel exerts its toxic and carcinogenic effects at a molecular level may be important in risk assessment, as well as in the treatment and prevention of occupational diseases. Previously, using human and rodent cells in vitro, we showed that hypoxia-inducible signaling pathway was activated by carcinogenic nickel compounds. Acute exposure to nickel resulted in the accumulation of hypoxia-inducible transcription factor (HIF)-1, which strongly activated hypoxia-inducible genes, including the recently discovered tumor marker NDRG1 (Cap43). To further identify HIF-1-dependent nickel-inducible genes and to understand the role of the HIF-dependent signaling pathway in nickel-induced transformation, we used the Affymetrix GeneChip to compare the gene expression profiles in wild-type cells or in cells from HIF-1 alpha knockout mouse embryos exposed to nickel chloride. As expected, when we examined 12,000 genes for expression changes, we found that genes coding for glycolytic enzymes and glucose transporters, known to be regulated by HIF-1 transcription factor, were induced by nickel only in HIF-1 alpha-proficient cells. In addition, we found a number of other hypoxia-inducible genes up-regulated by nickel in a HIF-dependent manner including BCL-2-binding protein Nip3, EGLN1, hypoxia-inducible gene 1 (HIG1), and prolyl 4-hydroxylase. Additionally, we found a number of genes induced by nickel in a HIF-independent manner, suggesting that Ni activated other signaling pathways besides HIF-1. Finally, we found that in HIF-1 alpha knockout cells, nickel strongly induced the expression of the whole group of genes that were not expressed in the presence of HIF-1. Because the majority of modulated genes were induced or suppressed by nickel in a HIF-1-dependent manner, we elucidated the role of HIF-1 transcription factor in cell transformation. In HIF-1 alpha-proficient cells, nickel exposure increased soft agar growth, whereas it decreased soft agar growth in HIF-1 alpha-deficient cells. We hypothesize that the induction of HIF-1 transcription factor by nickel may be important during the nickel-induced carcinogenic process.

Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival.

PubMed

Schipani, E; Ryan, H E; Didrickson, S; Kobayashi, T; Knight, M; Johnson, R S

2001-11-01

Breakdown or absence of vascular oxygen delivery is a hallmark of many common human diseases, including cancer, myocardial infarction, and stroke. The chief mediator of hypoxic response in mammalian tissues is the transcription factor hypoxia-inducible factor 1 (HIF-1), and its oxygen-sensitive component HIF-1alpha. A key question surrounding HIF-1alpha and the hypoxic response is the role of this transcription factor in cells removed from a functional vascular bed; in this regard there is evidence indicating that it can act as either a survival factor or induce growth arrest and apoptosis. To study more closely how HIF-1alpha functions in hypoxia in vivo, we used tissue-specific targeting to delete HIF-1alpha in an avascular tissue: the cartilaginous growth plate of developing bone. We show here the first evidence that the developmental growth plate in mammals is hypoxic, and that this hypoxia occurs in its interior rather than at its periphery. As a result of this developmental hypoxia, cells that lack HIF-1alpha in the interior of the growth plate die. This is coupled to decreased expression of the CDK inhibitor p57, and increased levels of BrdU incorporation in HIF-1alpha null growth plates, indicating defects in HIF-1alpha-regulated growth arrest occurs in these animals. Furthermore, we find that VEGF expression in the growth plate is regulated through both HIF-1alpha-dependent and -independent mechanisms. In particular, we provide evidence that VEGF expression is up-regulated in a HIF-1alpha-independent manner in chondrocytes surrounding areas of cell death, and this in turn induces ectopic angiogenesis. Altogether, our findings have important implications for the role of hypoxic response and HIF-1alpha in development, and in cell survival in tissues challenged by interruption of vascular flow; they also illustrate the complexities of HIF-1alpha response in vivo, and they provide new insights into mechanisms of growth plate development.

Inhibition of HIF-1{alpha} activity by BP-1 ameliorates adjuvant induced arthritis in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shankar, J.; Thippegowda, P.B., E-mail: btprabha@uic.edu; Kanum, S.A.

Rheumatoid arthritis (RA) is a chronic inflammatory, angiogenic disease. Inflamed synovitis is a hallmark of RA which is hypoxic in nature. Vascular endothelial growth factor (VEGF), one of the key regulators of angiogenesis, is overexpressed in the pathogenesis of RA. VEGF expression is regulated by hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), a master regulator of homeostasis which plays a pivotal role in hypoxia-induced angiogenesis. In this study we show that synthetic benzophenone analogue, 2-benzoyl-phenoxy acetamide (BP-1) can act as a novel anti-arthritic agent in an experimental adjuvant induced arthritis (AIA) rat model by targeting VEGF and HIF-1{alpha}. BP-1 administered hypoxic endothelial cells andmore » arthritic animals clearly showed down regulation of VEGF expression. Further, BP-1 inhibits nuclear translocation of HIF-1{alpha}, which in turn suppresses transcription of the VEGF gene. These results suggest a further possible clinical application of the BP-1 derivative as an anti-arthritic agent in association with conventional chemotherapeutic agents.« less

The PPARdelta agonist GW501516 suppresses interleukin-6-mediated hepatocyte acute phase reaction via STAT3 inhibition.

PubMed

Kino, T; Rice, K C; Chrousos, G P

2007-05-01

Interleukin-6 and downstream liver effectors acute phase reactants are implicated in the systemic inflammatory reaction. Peroxisome proliferator-activated receptor delta (PPARdelta), which binds to and is activated by a variety of fatty acids, was recently shown to have anti-inflammatory actions. We examined the ability of the synthetic PPARdelta agonist GW501516 to suppress interleukin-6-induced expression of acute phase proteins in human hepatoma HepG2 cells and rat primary hepatocytes. Results GW501516 dose-dependently suppressed interleukin-6-induced mRNA expression of the acute phase protein alpha1-antichymotrypsin in HepG2 cells. The compound also suppressed interleukin-6-induced mRNA expression of alpha2-acid glycoprotein, beta-fibrinogen and alpha2-macroglobulin in and the secretion of C-reactive protein by rat primary hepatocytes. Depletion of the PPARdelta receptor, but not of PPARalpha or gamma, attenuated the suppressive effect of GW501516 on interleukin-6-induced alpha1-antichymotrypsin mRNA expression, indicating that PPARdelta specifically mediated this effect. Since interleukin-6 stimulates the transcriptional activity of the alpha1-antichymotrypsin promoter by activating the signal transducer and activator of transcription (STAT) 3, we examined functional interaction of this transcription factor and PPARdelta on this promoter. Overexpression of PPARdelta enhanced the suppressive effect of GW501516 on STAT3-activated transcriptional activity of the alpha1-antichymotrypsin promoter, while GW501516 suppressed interleukin-6-induced binding of this transcription factor to this promoter. These findings indicate that agonist-activated PPARdelta interferes with interleukin-6-induced acute phase reaction in the liver by inhibiting the transcriptional activity of STAT3. PPARdelta agonists might be useful for the suppression of systemic inflammatory reactions in which IL-6 plays a central role.

Zinc finger protein 267 is up-regulated in hepatocellular carcinoma and promotes tumor cell proliferation and migration.

PubMed

Schnabl, Bernd; Valletta, Daniela; Kirovski, Georgi; Hellerbrand, Claus

2011-12-01

Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulates diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 mRNA is up-regulated in liver cirrhosis, which is the main risk factor for hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in human HCC cells and tissue specimens and found a significant up-regulation compared to primary human hepatocytes and corresponding non-tumorous liver tissue. Over-expression of the transcription factor Ets-1 further enhanced ZNF267 expression, and reporter gene assays revealed that mutation of the Ets-1 binding site to the ZNF267 promotor markedly inhibited ZNF267 promotor activity. Hypoxic conditions induced Ets-1 in HCC cells via HIF1alpha activation, and hypoxia induced ZNF267 expression while HIF1alpha inhibition significantly reduced both hypoxia-induced as well as basal ZNF267 expression in HCC cells. It is known that hypoxic conditions in tumorous tissues induce the formation of reactive oxygen species (ROS), and ROS have been identified as important factor in the regulation of Ets-1 expression in tumor cells. Here, we found that ROS induction induced and ROS scavenging reduced ZNF267 expression in HCC cells, respectively. Loss and gain of function analysis applying siRNA directed against ZNF267 or transient transfection revealed that ZNF267 promotes proliferation and migration of HCC cells in vitro. These findings indicate Ets-1 and HIF1alpha as critical regulators of basal and hypoxia- or ROS-induced ZNF267 expression in HCC, and further suggest that the pro-tumorigenic effect of these factors is at least in part mediated via increased ZNF267 expression in HCC. Since ZNF267 is already elevated in cirrhosis, ZNF267 appears as promising target for both prevention as well as treatment of HCC in patients with chronic liver disease. Copyright © 2011 Elsevier Inc. All rights reserved.

Zinc finger protein 267 is up-regulated in hepatocellular carcinoma and promotes tumor cell proliferation and migration

PubMed Central

Schnabl, Bernd; Valletta, Daniela; Kirovski, Georgi; Hellerbrand, Claus

2012-01-01

Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulates diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 mRNA is up-regulated in liver cirrhosis, which is the main risk factor for hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in human HCC cells and tissue specimens and found a significant up-regulation compared to primary human hepatocytes and corresponding non-tumorous liver tissue. Over-expression of the transcription factor Ets-1 further enhanced ZNF267 expression, and reporter gene assays revealed that mutation of the Ets-1 binding site to the ZNF267 promotor markedly inhibited ZNF267 promotor activity. Hypoxic conditions induced Ets-1 in HCC cells via HIF1alpha activation, and hypoxia induced ZNF267 expression while HIF1alpha inhibition significantly reduced both hypoxia-induced as well as basal ZNF267 expression in HCC cells. It is known that hypoxic conditions in tumorous tissues induce the formation of reactive oxygen species (ROS), and ROS have been identified as important factor in the regulation of Ets-1 expression in tumor cells. Here, we found that ROS induction induced and ROS scavenging reduced ZNF267 expression in HCC cells, respectively. Loss and gain of function analysis applying siRNA directed against ZNF267 or transient transfection revealed that ZNF267 promotes proliferation and migration of HCC cells in vitro. These findings indicate Ets-1 and HIF1alpha as critical regulators of basal and hypoxia- or ROS-induced ZNF267 expression in HCC, and further suggest that the pro-tumorigenic effect of these factors is at least in part mediated via increased ZNF267 expression in HCC. Since ZNF267 is already elevated in cirrhosis, ZNF267 appears as promising target for both prevention as well as treatment of HCC in patients with chronic liver disease. PMID:21840307

Angiotensin II type 1 receptor blockers prevent tumor necrosis factor-alpha-mediated endothelial nitric oxide synthase reduction and superoxide production in human umbilical vein endothelial cells.

PubMed

Kataoka, Hiroki; Murakami, Ryuichiro; Numaguchi, Yasushi; Okumura, Kenji; Murohara, Toyoaki

2010-06-25

Decrease in endothelial nitric oxide synthase (eNOS) expression is one of the adverse outcomes of endothelial dysfunction. Tumor necrosis factor-alpha (TNF-alpha) is known to decrease eNOS expression and is an important mediator of endothelial dysfunction. We hypothesized that an angiotensin II type 1 (AT1) receptor blocker would improve endothelial function via not only inhibition of the angiotensin II signaling but also inhibition of the TNF-alpha-mediated signaling. Therefore we investigated whether an AT1 receptor blocker would restore the TNF-alpha-induced decrease in eNOS expression in cultured human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with an antioxidant (superoxide dismutase, alpha-tocopherol) or AT1 receptor blockers (olmesartan or candesartan) restored the TNF-alpha-dependent reduction of eNOS. The AT1 receptor blocker decreased the TNF-alpha-dependent increase of 8-isoprostane. The superoxide dismutase activities in HUVEC were stable during AT1 receptor blocker treatment, and the AT1 receptor blocker did not scavenge superoxide directly. The AT1 receptor blocker also decreased TNF-alpha-induced phosphorylation of I kappaB alpha and cell death. These results suggest that AT1 receptor blockers are able to ameliorate TNF-alpha-dependent eNOS reduction or cell injury by inhibiting superoxide production or nuclear factor-kappaB activation. (c) 2010 Elsevier B.V. All rights reserved.

Expression and function of CD8 alpha/beta chains on rat and human mast cells.

PubMed

Kim, Mi-Sun; Kim, Sung-Hoon; Lee, Hye-Jung; Kim, Hyung-Min

2004-03-01

The expression and functional role of CD8 glycoprotein, a marker of cytotoxic/suppressor T lymphocytes and NK cells, were not studied on freshly isolated connective tissue type rat peritoneal mast cells, a rat mucosal type mast cell line (RBL 2H3), or human mast cell line (HMC-1). We used the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, immunohistochemistry and enzyme-linked immunosorbent assay. RT-PCR and Western blot analysis identified the presence of CD8 alpha/beta chains on the mast cells, and immunohistochemistry confirmed CD8alpha expression on rat or human mast cells. Functional studies demonstrated that stimulation of CD8 alpha/beta chains on rat mast cells induced the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), which are regarded as important mediators during infection. However, co-stimulation with stem cell factor had no effect on CD8-induced mediator secretion. Our findings demonstrate novel biological roles of CD8 molecules in mast cells.

The coffee diterpene kahweol inhibits tumor necrosis factor-{alpha}-induced expression of cell adhesion molecules in human endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil

2006-12-15

Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNF{alpha}-induced monocytes to endothelial cells and suppressed the TNF{alpha}-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNF{alpha}-induced JAK2-PI3K/Akt-NF-{kappa}B activation pathway in these cells. Overall, kahweol hasmore » anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells.« less

Induction of hypoxia-inducible factor-1alpha and activation of caspase-3 in hypoxia-reoxygenated bone marrow stroma is negatively regulated by the delayed production of substance P.

PubMed

Qian, J; Ramroop, K; McLeod, A; Bandari, P; Livingston, D H; Harrison, J S; Rameshwar, P

2001-10-15

The bone marrow (BM), which is the major site of immune cell development in the adult, responds to different stimuli such as inflammation and hemorrhagic shock. Substance P (SP) is the major peptide encoded by the immune/hemopoietic modulator gene, preprotachykinin-1 (PPT-I). Differential gene expression using a microarray showed that SP reduced hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA levels in BM stroma. Because long-term hypoxia induced the expression of PPT-I in BM mononuclear cells, we used timeline studies to determine whether PPT-I is central to the biologic responses of BM stroma subjected to 30-min hypoxia (pO(2) = 35 mm Hg) followed by reoxygenation. HIF-1alpha mRNA and protein levels were increased up to 12 h. At this time, beta-PPT-I mRNA was detected with the release of SP at 16 h. SP release correlated with down-regulation of HIF-1alpha to baseline. A direct role for SP in HIF-1alpha expression was demonstrated as follows: 1) transient knockout of beta-PPT-I showed an increase in HIF-1alpha expression up to 48 h of reoxygenation; and 2) HIF-1alpha expression remained baseline during reoxygenation when stroma was subjected to hypoxia in the presence of SP. Reoxygenation activated the PPT-I promoter with concomitant nuclear translocation of HIF-1alpha that can bind to the respective consensus sequences within the PPT-I promoter. SP reversed active caspase-3, an indicator of apoptosis and erythropoiesis, to homeostasis level after reoxygenation of hypoxic stroma. The results show that during reoxgenation the PPT-I gene acts as a negative regulator on the expression of HIF-1alpha and active caspase-3 in BM stroma subjected to reoxygenation.

Equine colostral carbohydrates reduce lipopolysaccharide-induced inflammatory responses in equine peripheral blood mononuclear cells.

PubMed

Vendrig, J C; Coffeng, L E; Fink-Gremmels, J

2012-12-01

Increasing evidence suggests that reactions to lipopolysaccharide (LPS), particularly in the gut, can be partly or completely mitigated by colostrum- and milk-derived oligosaccharides. Confirmation of this hypothesis could lead to the development of new therapeutic concepts. To demonstrate the influence of equine colostral carbohydrates on the inflammatory response in an in vitro model with equine peripheral blood mononuclear cells (PBMCs). Carbohydrates were extracted from mare colostrum, and then evaluated for their influence on LPS-induced inflammatory responses in PBMCs isolated from the same mares, mRNA expression of tumour necrosis factor-alpha, interleukin-6 and interleukin-10 was measured as well as the protein levels of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10). Equine colostral carbohydrates significantly reduced LPS-induced TNF-alpha protein at both times measured and significantly reduced LPS-induced TNF-alpha, IL-6 and IL-10 mRNA expression by PBMCs. Moreover, cell viability significantly increased in the presence of high concentrations of colostral carbohydrates. Carbohydrates derived from equine colostrum reduce LPS-induced inflammatory responses of equine PBMCs. Colostrum and milk-derived carbohydrates are promising candidates for new concepts in preventive and regenerative medicine.

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis.

PubMed

Hardie, William D; Davidson, Cynthia; Ikegami, Machiko; Leikauf, George D; Le Cras, Timothy D; Prestridge, Adrienne; Whitsett, Jeffrey A; Korfhagen, Thomas R

2008-06-01

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.

Mechanism of alpha-lipoic acid in attenuating kanamycin-induced ototoxicity☆

PubMed Central

Wang, Aimei; Hou, Ning; Bao, Dongyan; Liu, Shuangyue; Xu, Tao

2012-01-01

In view of the theory that alpha-lipoic acid effectively prevents cochlear cells from injury caused by various factors such as cisplatin and noise, this study examined whether alpha-lipoic acid can prevent kanamycin-induced ototoxicity. To this end, healthy BALB/c mice were injected subcutaneously with alpha-lipoic acid and kanamycin for 14 days. Auditory brainstem response test showed that increased auditory brainstem response threshold shifts caused by kanamycin were significantly inhibited. Immunohistochemical staining and western blot analysis showed that the expression of phosphorylated p38 mitogen-activated protein kinase and phosphorylated c-Jun N-terminal kinase in mouse cochlea was significantly decreased. The experimental findings suggest that phosphorylated p38 and phosphorylated c-Jun N-terminal kinase mediated kanamycin-induced ototoxic injury in BALB/c mice. Alpha-lipoic acid effectively attenuated kanamycin ototoxicity by inhibiting the kanamycin-induced high expression of phosphorylated p38 and phosphorylated c-Jun N-terminal kinase. PMID:25317129

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Xiao-dong; Tobo, Masayuki; Mogi, Chihiro

Highlights: Black-Right-Pointing-Pointer Glucocorticoid (GC) induced the expression of proton-sensing TDAG8 in macrophages. Black-Right-Pointing-Pointer GC enhanced acidic pH-induced cAMP accumulation and inhibition of TNF-{alpha} production. Black-Right-Pointing-Pointer The enhancement of the GC-induced actions was lost by TDAG8 deficiency. Black-Right-Pointing-Pointer GC-induced anti-inflammatory actions are partly mediated by TDAG8 expression. -- Abstract: Dexamethasone (DEX), a potent glucocorticoid, increased the expression of T-cell death associated gene 8 (TDAG8), a proton-sensing G protein-coupled receptor, which is associated with the enhancement of acidic pH-induced cAMP accumulation, in peritoneal macrophages. We explored the role of increased TDAG8 expression in the anti-inflammatory actions of DEX. The treatment of macrophagesmore » with either DEX or acidic pH induced the cell death of macrophages; however, the cell death was not affected by TDAG8 deficiency. While DEX inhibited lipopolysaccharide-induced production of tumor necrosis factor-{alpha}, an inflammatory cytokine, which was independent of TDAG8, at neutral pH, the glucocorticoid enhanced the acidic pH-induced inhibition of tumor necrosis factor-{alpha} production in a manner dependent on TDAG8. In conclusion, the DEX-induced increase in TDAG8 expression is in part involved in the glucocorticoid-induced anti-inflammatory actions through the inhibition of inflammatory cytokine production under the acidic pH environment. On the other hand, the role of TDAG8 in the DEX-induced cell death is questionable.« less

Effect of transforming growth factor-alpha on enterocyte apoptosis is correlated with EGF receptor expression along the villus-crypt axis during methotrexate-induced intestinal mucositis in a rat.

PubMed

Sukhotnik, Igor; Shteinberg, Dan; Ben Lulu, Shani; Bashenko, Yulia; Mogilner, Jorge G; Ure, Benno M; Shaoul, Ron; Coran, Arnold G

2008-11-01

The purpose of the present study was to evaluate the effect of transforming-growth factor-alpha (TGF-alpha) on enterocyte apoptosis following methotrexate (MTX) induced intestinal mucositis in a rat and in Caco-2 cells. Non-pretreated and pretreated with MTX Caco-2 cells were incubated with increasing concentrations of TGF-alpha. Cell apoptosis was determined by FACS cytometry. Adult rats were divided into four groups: Control, Control-TGF-alpha, MTX, and MTX- TGF-alpha rats. Three days later rats were sacrificed. Enterocyte apoptosis were measured at sacrifice. RT-PCR and Western Blotting was used to determine the level of Bax and Bcl-2 mRNA and protein. Real time PCR was used to measure epidermal growth factor receptor (EGFr) expression along the villus-crypt axis. The in vitro experiment has shown that treatment with TGF-alpha of Caco-2 cells results in a significant inhibition of cell apoptosis in a dose-dependent manner. In vivo experiment, a decreased levels of apoptosis in MTX- TGF-alpha rats corresponded with the decrease in Bax and with the increase in Bcl-2 at both mRNA and protein levels. The inhibiting effect of TGF-alpha on enterocyte apoptosis was strongly correlated with EGFr expression along the villus-crypt axis. In conclusion, treatment with TGF-alpha inhibits enterocyte apoptosis following MTX- injury in the rat.

Early growth response protein 1 (EGR1) regulates pro-inflammatory gene expression in response to palmitate and TNF alpha in human placenta cells and is induced in obese placenta

USDA-ARS?s Scientific Manuscript database

Maternal obesity has been hypothesized to induce a pro-inflammatory response in the placenta. However, the specific factors contributing to this pro-infalmmatory response are yet to be determined. Our objective was to examine the effects of palmitic acid (PA), tumor necrosis factor alpha (TNF alph...

Immunohistochemical detection of HIF-1alpha and CAIX in advanced head-and-neck cancer. Prognostic role and correlation with tumor markers and tumor oxygenation parameters.

PubMed

Kappler, Matthias; Taubert, Helge; Holzhausen, Hans-Jürgen; Reddemann, Rolf; Rot, Swetlann; Becker, Axel; Kuhnt, Thomas; Dellas, Kathrin; Dunst, Jürgen; Vordermark, Dirk; Hänsgen, Gabriele; Bache, Matthias

2008-08-01

Tumor hypoxia has an impact on the outcome of cancer patients treated with radiotherapy. The validity of endogenous markers such as hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase isozyme IX (CAIX) to detect therapeutically relevant Levels of hypoxia within tumors is controversially discussed. Furthermore, the association of these hypoxia markers with tumor markers or tumor oxygenation parameters is of importance for understanding the relationship between the different factors. Tumortissue sections of 34 patients with advanced head-and-neck cancertreated with radio(chemo)therapy were assessed by immunohistochemistry for the expression of HIF-1alpha and CAIX. The relationships of both markers with tumor oxygenation parameters, molecular factors like P53, OPN, VEGF, VHL, survivin, and Ki67 levels, and clinical parameters were studied. Bivariate analysis showed a significant correlation of HIF-1alpha expression with high P53 and high OPN expression, high serum VEGF Levels, and low VHL and low Ki67 expression. The CAIX expression was inversely correlated with pH value and directly correlated with T-stage. However, no correlation was found between HIF-1alpha and CAIX expression. Neither in a univariate Cox proportional hazard regression nor in a Kaplan-Meier analysis did expression of HIF-1alpha or CAIX have a significant impact on clinical outcome. However, in a Kaplan-Meier analysis, the combination of both factors showed that patients with intratumoral overexpression of either HIF-1alpha or CAIX or both markers died on average 2 years earlier than patients whose tumors had low expression of both factors (p < 0.05). Expression of HIF-1alpha and CAIX was correlated with different tumor parameters. Only combined HIF-1alpha and CAIX expression was significantly predictive of patients' overall survival.

Synergistic regulation of the acute phase protein SIP24/24p3 by glucocorticoid and pro-inflammatory cytokines.

PubMed

Liu, Quan-Sheng; Nilsen-Hamilton, Marit; Xiong, Si-Dong

2003-10-25

SIP24/24p3 is a secreted murine acute phase protein which has been speculated to play an anti-inflammatory role in vivo. Recently SIP24/24p3 has been found to be able to specifically induce apoptosis in leukocytes. By using (35)S metabolic labeling method, we studied the regulation of SIP24/24p3 by glucocorticoid and pro-inflammatory cytokines IL-6 and TNF-alpha in cultured Balb/c 3T3 and BNL cells. The following results were observed: (1) dexamethasone induced the expression of SIP24/24p3 in both Balb/c 3T3 and BNL cells, the induction was more significant in BNL cells; (2) dexamethasone and IL-6 synergistically induced the expression of SIP24/24p3 in both Balb/c 3T3 and BNL cells; (3) in Balb/c 3T3 cells dexamethasone and TNF-alpha acted synergistically to induce the expression of SIP24/24p3, whereas in BNL cells dexamethasone and TNF-alpha induced the expression of SIP24/24p3 in an additive manner; (4) dexamethasone and IL-6/TNF-alpha acted synergistically in Balb/c 3T3 cells and additively in BNL cells to induce the expression of SIP24/24p3. The inducibility of SIP24/24p3 by multiple factors will help to explain its highly specific expression in vivo. The difference in the expression patterns of SIP24/24p3 in different cell types is also suggestive to its expression and regulation in hepatic and extrahepatic tissues. Finally, the fact that SIP24/24p3 protein can be induced by both pro-inflammatory as well as anti-inflammatory factors is indicative of the important role of SIP24/24p3 in the entire acute phase response process.

Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

PubMed Central

Gerritsen, M. E.; Carley, W. W.; Ranges, G. E.; Shen, C. P.; Phan, S. A.; Ligon, G. F.; Perry, C. A.

1995-01-01

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking. Images Figure 7 Figure 8 Figure 11 PMID:7543732

The protein level of hypoxia-inducible factor-1alpha is increased in the plateau pika (Ochotona curzoniae) inhabiting high altitudes.

PubMed

Li, Hong-Ge; Ren, Yong-Ming; Guo, Song-Chang; Cheng, Long; Wang, De-Peng; Yang, Jie; Chang, Zhi-Jie; Zhao, Xin-Quan

2009-02-01

The plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant species living only at 3,000-5,000 m above sea-level on the Qinghai-Tibetan plateau. Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates a variety of cellular and systemic adaptations to hypoxia. To investigate how the plateau pika adapts to a high-altitude hypoxic environment at the molecular level, we examined the expression pattern of the HIF-1alpha protein in the pika by Western blot and immunohistochemical analysis. We found that HIF-1alpha protein is expressed at a significantly high level in the pika, which is higher in most tissues (particularly in the lung, liver, spleen and kidney) of the plateau pika than that of mice living at sea-level. Importantly, we found that the protein levels of HIF-1alpha in the lung, liver, spleen and kidney of the pika were increased with increased habitat altitudes. We observed that the plateau pika HIF-1alpha localized to the nucleus of cells by an immunostaining analysis, and enhanced HRE-driven gene expression by luciferase reporter assays. Our study suggests that the HIF-1alpha protein levels are related to the adaptation of the plateau pika to the high-altitude hypoxic environment.

The potential role of Brachyury in inducing epithelial-to-mesenchymal transition (EMT) and HIF-1α expression in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Chao; Zhang, Jingjing, E-mail: jingjingzhangzs@163.com; Fu, Jianhua

One of transcription factors of the T-box family, Brachyury has been implicated in tumorigenesis of many types of cancers, regulating cancer cell proliferation, metastasis, invasion and epithelial-to-mesenchymal transition (EMT). However, the role of Brachyury in breast cancer cells has been scarcely reported. The present study aimed to investigate the expression and role of Brachyury in breast cancer. Brachyury expression was analyzed by qRT-PCR and Western blot. The correlations between Brachyury expression and clinicopathological factors of breast cancer were determined. Involvement of EMT stimulation and hypoxia-inducible factor-1α (HIF-1α) expression induction by Brachyury was also evaluated. Moreover, the effect of Brachyury onmore » tumor growth and metastasis in vivo was examined in a breast tumor xenograft model. Brachyury expression was enhanced in primary breast cancer tissues and Brachyury expression was correlated with tumor stage and lymph node metastasis. Hypoxia enhanced Brachyury expression, the silencing of which blocked the modulation effect of hypoxia on E-cadherin and vimentin expression. Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling as well as accelerated cell proliferation and migration in vitro. Additionally, Brachyury accelerated breast tumor xenograft growth and increased lung metastasis in nude mice. In summary, our data confirmed that Brachyury might contribute to hypoxia-induced EMT of breast cancer and trigger HIF-1alpha expression via PTEN/Akt signaling. - Highlights: • Brachyury expression was correlated with tumor stage and lymph node metastasis. • Hypoxia enhanced Brachyury expression, which contributes to hypoxia-induced EMT. • Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling. • Brachyury accelerated tumor xenograft growth and increased lung metastasis.« less

Leptin potentiates Prevotella intermedia lipopolysaccharide-induced production of TNF-alpha in monocyte-derived macrophages.

PubMed

Kim, Sung-Jo

2010-06-01

In addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production in differentiated THP-1 cells, a human monocytic cell line. LPS from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-alpha and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-alpha and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis. Leptin enhanced P. intermedia LPS-induced TNF-alpha production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-alpha expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-alpha production was not mediated by the leptin receptor. The ability of leptin to enhance P. intermedia LPS-induced TNF-alpha production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.

Reduction of isoprenaline-induced myocardial TGF-{beta}1 expression and fibrosis in osthole-treated mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Rong; The First Hospital Affiliated to Soochow University, Suzhou 215006, Jiangsu Province; Xue Jie

Peroxisome proliferator-activated receptor (PPAR) {alpha} and PPAR{gamma} ligands can attenuate myocardial fibrosis. Osthole, an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson, may be a dual PPAR{alpha}/{gamma} agonist, but there has been no report on its effect on myocardial fibrosis. In the present study, we investigated the inhibitory effect of osthole on myocardial fibrotic formation in mice and its possible mechanisms. A mouse model with myocardial fibrosis was induced by hypodermic injection of isoprenaline while the mice were simultaneously treated with 40 and 80 mg/kg osthole for 40 days. After the addition of osthole, the cardiac weightmore » index and hydroxyproline content in the myocardial tissues were decreased, the degree of collagen accumulation in the heart was improved, and the downregulation of myocardial PPAR{alpha}/{gamma} mRNA expression induced by isoprenaline was reversed. Moreover, the mRNA expression of transforming growth factor (TGF)-{beta}1 and the protein levels of nuclear factor (NF)-{kappa}B and TGF-{beta}1 in the myocardial tissues were decreased. These findings suggest that osthole can prevent isoprenaline-induced myocardial fibrosis in mice, and its mechanisms may be related to the reduction of TGF-{beta}1 expression via the activation of PPAR{alpha}/{gamma} and subsequent inhibition of NF-{kappa}B in myocardial tissues. - Highlights: > Osthole could inhibit the myocardial fibrosis induced by isoprenaline in mice. > The mechanism was related to reduction of TGF-{beta}1 expression in myocardial tissue. > The result of osthole was from the activation of PPAR{alpha}/{gamma} and inhibition of NF-{kappa}B.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arpiainen, Satu; Jaervenpaeae, Sanna-Mari; Manninen, Aki

The nutritional state of organisms and energy balance related diseases such as diabetes regulate the metabolism of xenobiotics such as drugs, toxins and carcinogens. However, the mechanisms behind this regulation are mostly unknown. The xenobiotic-metabolizing cytochrome P450 (CYP) 2A5 enzyme has been shown to be induced by fasting and by glucagon and cyclic AMP (cAMP), which mediate numerous fasting responses. Peroxisome proliferator-activated receptor {gamma} coactivator (PGC)-1{alpha} triggers many of the important hepatic fasting effects in response to elevated cAMP levels. In the present study, we were able to show that cAMP causes a coordinated induction of PGC-1{alpha} and CYP2A5 mRNAsmore » in murine primary hepatocytes. Furthermore, the elevation of the PGC-1{alpha} expression level by adenovirus mediated gene transfer increased CYP2A5 transcription. Co-transfection of Cyp2a5 5' promoter constructs with the PGC-1{alpha} expression vector demonstrated that PGC-1{alpha} is able to activate Cyp2a5 transcription through the hepatocyte nuclear factor (HNF)-4{alpha} response element in the proximal promoter of the Cyp2a5 gene. Chromatin immunoprecipitation assays showed that PGC-1{alpha} binds, together with HNF-4{alpha}, to the same region at the Cyp2a5 proximal promoter. In conclusion, PGC-1{alpha} mediates the expression of CYP2A5 induced by cAMP in mouse hepatocytes through coactivation of transcription factor HNF-4{alpha}. This strongly suggests that PGC-1{alpha} is the major factor mediating the fasting response of CYP2A5.« less

Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNF{alpha}-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ichikawa, Tomonaga; Li, Jinqing; Dong, Xiaoyu

2010-01-01

Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNF{alpha})-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNF{alpha}-induced activation of ERK andmore » DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNF{alpha} hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.« less

Dexamethasone impairs hypoxia-inducible factor-1 function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagner, A.E.; Huck, G.; Stiehl, D.P.

2008-07-25

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of {alpha}- and {beta}-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1{alpha} levels in the cytosol of HepG2 cells, while nuclear HIF-1{alpha} levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in amore » reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.« less

Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

PubMed

Martinez, Victor G; Ontoria-Oviedo, Imelda; Ricardo, Carolina P; Harding, Sian E; Sacedon, Rosa; Varas, Alberto; Zapata, Agustin; Sepulveda, Pilar; Vicente, Angeles

2017-09-29

Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK cell lysis and, hence, their potential in vivo lifespan. Our results further support the use of HIF-1 alpha-expressing dental MSCs for cell therapy in tissue injury and immune disorders.

Metformin inhibits inflammatory response via AMPK-PTEN pathway in vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sun Ae; Choi, Hyoung Chul, E-mail: hcchoi@med.yu.ac.kr

2012-09-07

Highlights: Black-Right-Pointing-Pointer PTEN was induced by metformin and inhibited by compound C and AMPK siRNA. Black-Right-Pointing-Pointer Metformin suppressed TNF-{alpha}-induced COX-2 and iNOS mRNA expression. Black-Right-Pointing-Pointer Compound C and bpv (pic) increased iNOS and COX-2 protein expression. Black-Right-Pointing-Pointer NF-{kappa}B activation was restored by inhibiting AMPK and PTEN. Black-Right-Pointing-Pointer AMPK and PTEN regulated TNF-{alpha}-induced ROS production in VSMCs. -- Abstract: Atherosclerosis is a chronic inflammation of the coronary arteries. Vascular smooth muscle cells (VSMCs) stimulated by cytokines and chemokines accelerate the inflammatory response and migrate to the injured endothelium during the progression of atherosclerosis. Activation of AMP activated protein kinase (AMPK), amore » key sensor maintaining metabolic homeostasis, suppresses the inflammatory response. However, how AMPK regulates the inflammatory response is poorly understood. To identify the mechanism of this response, we focused on phosphatase and tensin homolog (PTEN), which is a negative regulator of inflammation. We investigated that activation of AMPK-induced PTEN expression and suppression of the inflammatory response through the AMPK-PTEN pathway in VSMCs. We treated with the well-known AMPK activator metformin to induce PTEN expression. PTEN was induced by metformin (2 mM) and inhibited by compound C (10 {mu}M) and AMPK siRNA. Tumor necrosis factor-alpha (TNF-{alpha}) was used to induce inflammation. The inflammatory response was confirmed by cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) expression, and activation of nuclear factor (NF)-{kappa}B. Metformin suppressed COX-2 and iNOS mRNA and protein expression dose dependently. Treatment with compound C and bpv (pic) in the presence of metformin, iNOS and COX-2 protein expression increased. NF-{kappa}B activation decreased in response to metformin and was restored by inhibiting AMPK and PTEN. Inhibiting AMPK and PTEN restored ROS levels stimulated with TNF-{alpha}. Taken together, PTEN could be a possible downstream regulator of AMPK, and the AMPK-PTEN pathway might be important in the regulation of the inflammatory response in VSMCs.« less

A HIF-1alpha-related gene involved in cell protection from hypoxia by suppression of mitochondrial function.

PubMed

Kakinuma, Yoshihiko; Katare, Rajesh G; Arikawa, Mikihiko; Muramoto, Kazuyo; Yamasaki, Fumiyasu; Sato, Takayuki

2008-01-23

Recently, we reported that acetylcholine-induced hypoxia-inducible factor-1alpha protects cardiomyocytes from hypoxia; however, the downstream factors reducing hypoxic stress are unknown. We identified apoptosis inhibitor (AI) gene as being differentially expressed between von Hippel Lindau (VHL) protein-positive cells with high levels of GRP78 expression and VHL-negative cells with lower GRP levels, using cDNA subtraction. AI decreased GRP78 level, suppressed mitochondrial function, reduced oxygen consumption and, ultimately, suppressed hypoxia-induced apoptosis. By contrast, knockdown of the AI gene increased mitochondrial function. Hypoxic cardiomyocytes and ischemic myocardium showed increased AI mRNA expression. These findings suggest that AI is involved in suppressing mitochondrial function, thereby leading to cellular stress eradication and consequently to protection during hypoxia.

Bletilla striata polysaccharide stimulates inducible nitric oxide synthase and proinflammatory cytokine expression in macrophages.

PubMed

Diao, Huajia; Li, Xin; Chen, Jiangning; Luo, Yi; Chen, Xi; Dong, Lei; Wang, Chunming; Zhang, Chenyu; Zhang, Junfeng

2008-02-01

Bletilla striata, a traditional Chinese medicine, has been used for the treatment of alimentary canal mucosal damage, ulcers, bleeding, bruises and burns. B. striata polysaccharide (BSP) isolated from B. striata was found to enhance vascular endothelial cell (EC) proliferation and vascular endothelial growth factor (VEGF) expression. However, the wound healing mechanism of BSP is not well understood. In this study, the results show that treatment with BSP induces coordinate changes in inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1beta) mRNA levels and enhances the expression of these cytokines, but has no effect on interferon gamma (IFN-gamma) level. In this study, we partially elucidate the wound healing mechanism of BSP.

Ethylenediaminetetraacetic acid induces antioxidant and anti-inflammatory activities in experimental liver fibrosis.

PubMed

González-Cuevas, J; Navarro-Partida, J; Marquez-Aguirre, A L; Bueno-Topete, M R; Beas-Zarate, C; Armendáriz-Borunda, J

2011-01-01

Experimental liver fibrosis induced by carbon tetrachloride (CCl(4)) is associated with oxidative stress, lipid peroxidation, and inflammation. This work was focused on elucidating the anti-inflammatory and antioxidant effects of ethylenediaminetetraacetic acid (EDTA) in this model of hepatotoxicity. Wistar male rats were treated with CCl(4) and EDTA (60, 120, or 240 mg/kg). Morphometric analyses were carried out in Masson's stained liver sections to determine fibrosis index. Coagulation tests prothrombin time (PT) and partial thromboplastin time (PTT) were also determined. Gene expression for transforming growth factor beta (TGF-beta1), alpha1(I) procollagen gene (alpha1 Col I), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and superoxide dismutase (SOD) was monitored by real-time PCR. Antioxidant effect of EDTA was measured by its effects on lipid peroxidation; biological activity of ceruloplasmin (Cp), SOD, and catalase (Cat) were analyzed by zymography assays. Animals with CCl(4)-hepatic injury that received EDTA showed a decrement in fibrosis (20%) and lipid peroxidation (22%). The mRNA expression for TNF-alpha (55%), TGF-beta1 (50%), IL-6 (52%), and alpha1 Col I (60%) was also decreased. This group of animals showed increased Cp (62%) and SOD (25%) biological activities. Coagulation blood tests, Cat activity, and gene expression for SOD were not modified by EDTA treatment. This study demonstrates that EDTA treatment induces the activity of antioxidant enzymes, decreases lipid peroxidation, hepatic inflammation, and fibrosis in experimental liver fibrosis induced by CCl(4).

Lipopolysaccharide mitagates methamphetamine-induced striatal dopamine depletion via modulating local TNF-alpha and dopamine transporter expression.

PubMed

Lai, Yu-Ting; Tsai, Yen-Ping N; Cherng, Chianfang G; Ke, Jing-Jer; Ho, Ming-Che; Tsai, Chia-Wen; Yu, Lung

2009-04-01

Systemic lipopolysaccharide (LPS) treatment may affect methamphetamine (MA)-induced nigrostriatal dopamine (DA) depletion. This study was undertaken to determine the critical time window for the protective effects of LPS treatment and the underlying mechanisms. An LPS injection (1 mg/kg) 72 h before or 2 h after MA treatment [three consecutive, subcutaneous injections of MA (10 mg/kg each) at 2-h intervals] diminished the MA-induced DA depletion in mouse striatum. Such an LPS-associated effect was independent of MA-produced hyperthermia. TNF-alpha, IL-1beta, IL-6 expressions were all elevated in striatal tissues following a systemic injection with LPS, indicating that peripheral LPS treatment affected striatal pro-inflammatory cytokine expression. Striatal TNF-alpha expression was dramatically increased at 72 and 96 h after the MA treatment, while such TNF-alpha elevation was abolished by the LPS pretreatment protocol. Moreover, MA-produced activation of nuclear NFkappaB, a transcription factor following TNF-alpha activation, in striatum was abolished by the LPS (1 mg/kg) pretreatment. Furthermore, thalidomide, a TNF-alpha antagonist, treatment abolished the LPS pretreatment-associated protective effects. Pretreatment with mouse recombinant TNF-alpha in striatum diminished the MA-produced DA depletion. Finally, single LPS treatment caused a rapid down-regulation of dopamine transporter (DAT) in striatum. Taken together, we conclude that peripheral LPS treatment protects nigrostriatal DA neurons against MA-induced toxicity, in part, by reversing elevated TNF-alpha expression and subsequent signaling cascade and causing a rapid DAT down-regulation in striatum.

Alleviative effects of s-allyl cysteine and s-ethyl cysteine on MCD diet-induced hepatotoxicity in mice.

PubMed

Lin, Chun-che; Yin, Mei-chin; Liu, Wen-hu

2008-11-01

Alleviative effects of s-allyl cysteine (SAC) and s-ethyl cysteine (SEC) upon methionine and choline deficient (MCD) diet-induced hepatotoxicity in mice were examined. SAC or SEC at 1g/L was added into drinking water for 7 weeks with MCD diet. MCD feeding significantly increased hepatic triglyceride and cholesterol levels, and elevated the activity of glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme, fatty acid synthase (FAS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (P < 0.05). However, the intake of SAC or SEC significantly decreased hepatic triglyceride accumulation, and reduced G6PDH and FAS activities (P < 0.05). MCD feeding significantly lowered serum and hepatic glutathione (GSH) levels, increased malondialdehyde (MDA) and oxidized glutathione (GSSG) formation, and suppressed the activity and mRNA expression of glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (P < 0.05). The intake of SAC or SEC significantly increased serum and hepatic GSH levels, decreased MDA and GSSG formation, restored the activity and mRNA expression of GPX, SOD and catalase (P < 0.05). MCD feeding significantly enhanced the mRNA expression of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, matrix metalloproteinases-9 (MMP-9) and collagen-alpha1 (P < 0.05). The intake of SAC and SEC significantly blunted the mRNA expression of IL-1beta, IL-6, TNF-alpha, TGF-beta1 and collagen-alpha1 (P < 0.05). SEC was greater than SAC in suppressing IL-6 and TNF-alpha expression (P < 0.05), but SAC was greater than SEC in suppressing collagen-alpha1 and TGF-beta1 expression (P < 0.05). These data suggest that SAC and SEC are potent agents against MCD-induced hepatotoxicity.

IFN-{gamma} sensitizes MIN6N8 insulinoma cells to TNF-{alpha}-induced apoptosis by inhibiting NF-{kappa}B-mediated XIAP upregulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hun Sik; Kim, Sunshin; Lee, Myung-Shik

2005-10-28

Although X-linked inhibitor of apoptosis protein (XIAP) is an important intracellular suppressor of apoptosis in a variety of cell types, its role in cytokine-induced pancreatic {beta}-cell apoptosis remains unclear. Here, we found that: (i) XIAP level was inversely correlated with tumor necrosis factor (TNF)-{alpha}-induced apoptosis in MIN6N8 insulinoma cells; (ii) adenoviral XIAP overexpression abrogated the TNF-{alpha}-induced apoptosis through inhibition of caspase activity; (iii) downregulation of XIAP by antisense oligonucleotide or Smac peptide sensitized MIN6N8 cells to TNF-{alpha}-induced apoptosis; (iv) XIAP expression was induced by TNF-{alpha} through a nuclear factor-{kappa}B (NF-{kappa}B)-dependent pathway, and interferon (IFN)-{gamma} prevented such an induction in amore » manner independent of NF-{kappa}B, which presents a potential mechanism underlying cytotoxic IFN-{gamma}/TNF-{alpha} synergism. Taken together, our results suggest that XIAP is an important modulator of TNF-{alpha}-induced apoptosis of MIN6N8 cells, and XIAP regulation in pancreatic {beta}-cells might play an important role in pancreatic {beta}-cell apoptosis and in the pathogenesis of type 1 diabetes.« less

[Effect of Yiguan Decoction on differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells: an experimental research].

PubMed

Ping, Jian; Chen, Hong-Yun; Yang, Zhou; Yang, Cheng; Xu, Lie-Ming

2014-03-01

To observe the effect of Yiguan Decoction (YGD) on differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro. Rat BMSCs were isolated using whole bone marrow adherent method. The properties of BMSCs were identified by analyzing the expression of surface cytokines by flow cytometry. The third passage cells were differentiated into fat cells to identify their features. BMSCs were incubated with hepatocyte growth factor (HGF) plus fibroblast growth factor 4 (FGF4) or YGD containing serum YGD for 21 days. The mRNA expression of alpha-fetoprotein (alphaAFP), albumin (Alb), and hepatocyte nuclear factor 4alpha (HNF4alpha) were detected by real time PCR. Expression of AFP and cytokeratin 18 (CK18) protein was detected by cell immunofluorescence. Glycogen synthesis was observed using periodic acid-Schiff stain (PAS). CK18, Wnt 3alpha, and alphacatenin protein expressions were detected by Western blot. High expression of CD90, CD29, and CD44, and low expression of CD34 and CD11b were observed in BMSCs isolated by whole bone mar- row adherent method, and numerous lipid droplets were observed in BMSCs using oil red O staining. Both YGD containing serum and growth factor stimulated the expression levels of Alb, AFP, HNF4alpha mRNA and CK18 protein. The down-regulated expression of Wnt 3alpha and beta-catenin could be detected at 21 days after induction. The synthesized glycogen granule could be seen. Down-regulated Wnt 3alpha and beta-catenin expression could also be observed. YGD could induce the differentiation of rat BMSCs into hepatocyte-like cells, which was related to down-regulating Wnt/beta-catenin signal pathway.

Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor {alpha}/retinoid X receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belanger, Adam J.; Luo Zhengyu; Vincent, Karen A.

2007-12-21

In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid {beta}-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid {beta}-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1{alpha}/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferasemore » I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for {beta}-oxidation. Furthermore, adenovirus-mediated expression of HIF-1{alpha}/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})/retinoid X receptor (RXR), in the presence or absence of a PPAR{alpha} ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPAR{alpha}/RXR.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reed, S.A.; Senf, S.M.; Cornwell, E.W.

Research highlights: {yields} Independent inhibition of Foxo, IKK{alpha} and IKK{beta} activities does not alter muscle fiber size in weight bearing muscles. {yields} Inhibition of Foxo activity plus IKK{alpha} or IKK{beta} activities increases muscle fiber size. {yields} Independent inhibition of Foxo and IKK{beta} activities attenuates cast immobilization-induced muscle fiber atrophy. {yields} Disuse muscle fiber atrophy is abolished by inhibition of Foxo activity plus IKK{alpha} or IKK{beta} activities. -- Abstract: Two transcription factor families that are activated during multiple conditions of skeletal muscle wasting are nuclear factor {kappa}B (NF-{kappa}B) and forkhead box O (Foxo). There is clear evidence that both NF-{kappa}B andmore » Foxo activation are sufficient to cause muscle fiber atrophy and they are individually required for at least half of the fiber atrophy during muscle disuse, but there is no work determining the combined effect of inhibiting these factors during a physiological condition of muscle atrophy. Here, we determined whether inhibition of Foxo activation plus inhibition of NF-{kappa}B activation, the latter by blocking the upstream inhibitor of kappaB kinases (IKK{alpha} and IKK{beta}), would prevent muscle atrophy induced by 7 days of cast immobilization. Results were based on measurements of mean fiber cross-sectional area (CSA) from 72 muscles transfected with 5 different mutant expression plasmids or plasmid combinations. Immobilization caused a 47% decrease in fiber CSA in muscles injected with control plasmids. Fibers from immobilized muscles transfected with dominant negative (d.n.) IKK{alpha}-EGFP, d.n. IKK{beta}-EGFP or d.n. Foxo-DsRed showed a 22%, 57%, and 76% inhibition of atrophy, respectively. Co-expression of d.n. IKK{alpha}-EGFP and d.n. Foxo-DsRed significantly inhibited 89% of the immobilization-induced fiber atrophy. Similarly, co-expression of d.n. IKK{beta}-EGFP and d.n. Foxo-DsRed inhibited the immobilization-induced fiber atrophy by 95%. These findings demonstrate that the combined effects of inhibiting immobilization-induced NF-{kappa}B and Foxo transcriptional activity has an additive effect on preventing immobilization-induced atrophy, indicating that NF-{kappa}B and Foxo have a cumulative effect on atrophy signaling and/or atrophy gene expression.« less

Requirement for STAT1 in LPS-induced gene expression in macrophages.

PubMed

Ohmori, Y; Hamilton, T A

2001-04-01

This study examines the role of the signal transducer and activator of transcription 1 (STAT1) in induction of lipopolysaccharide (LPS)-stimulated gene expression both in vitro and in vivo. LPS-induced expression of an interferon (IFN)-inducible 10-kDa protein (IP-10), IFN regulatory factor-1 (IRF-1), and inducible nitric oxide synthase (iNOS) mRNAs was severely impaired in macrophages prepared from Stat1-/- mice, whereas levels of tumor necrosis factor alpha and KC (a C-X-C chemokine) mRNA in LPS-treated cell cultures were unaffected. A similar deficiency in LPS-induced gene expression was observed in livers and spleens from Stat1-/- mice. The reduced LPS-stimulated gene expression seen in Stat1-/- macrophages was not the result of reduced activation of nuclear factor kappaB. LPS stimulated the delayed activation of both IFN-stimulated response element and IFN-gamma-activated sequence binding activity in macrophages from wild-type mice. Activation of these STAT1-containing transcription factors was mediated by the intermediate induction of type I IFNs, since the LPS-induced IP-10, IRF-1, and iNOS mRNA expression was markedly reduced in macrophages from IFN-alpha/betaR-/- mice and blocked by cotreatment with antibodies against type I IFN. These results indicate that indirect activation of STAT1 by LPS-induced type I IFN participates in promoting optimal expression of LPS-inducible genes, and they suggest that STAT1 may play a critical role in innate immunity against gram-negative bacterial infection.

Distinct roles of NF-{kappa}B p50 in the regulation of acetaminophen-induced inflammatory mediator production and hepatotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dambach, Donna M.; Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, NJ 08543; Durham, Stephen K.

2006-03-01

Oxidative stress plays an important role in acetaminophen (APAP)-induced hepatotoxicity. In addition to inducing direct cellular damage, oxidants can activate transcription factors including NF-{kappa}B, which regulate the production of inflammatory mediators implicated in hepatotoxicity. Here, we investigated the role of APAP-induced oxidative stress and NF-{kappa}B in inflammatory mediator production. Treatment of mice with APAP (300 mg/kg, i.p.) resulted in centrilobular hepatic necrosis and increased serum aminotransferase levels. This was correlated with depletion of hepatic glutathione and CuZn superoxide dismutase (SOD). APAP administration also increased expression of the proinflammatory mediators, interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF{alpha}), macrophage chemotactic protein-1 (MCP-1), andmore » KC/gro, and the anti-inflammatory cytokine, interleukin-10 (IL-10). Pretreatment of mice with the antioxidant, N-acetylcysteine (NAC) prevented APAP-induced depletion of glutathione and CuZnSOD, as well as hepatotoxicity. NAC also abrogated APAP-induced increases in TNF{alpha}, KC/gro, and IL-10, but augmented expression of the anti-inflammatory cytokines interleukin-4 (IL-4) and transforming growth factor-{beta} (TGF{beta}). No effects were observed on IL-1{beta} or MCP-1 expression. To determine if NF-{kappa}B plays a role in regulating mediator production, we used transgenic mice with a targeted disruption of the gene for NF-{kappa}B p50. As observed with NAC pretreatment, the loss of NF-{kappa}B p50 was associated with decreased ability of APAP to upregulate TNF{alpha}, KC/gro, and IL-10 expression and increased expression of IL-4 and TGF{beta}. However, in contrast to NAC pretreatment, the loss of p50 had no effect on APAP-induced hepatotoxicity. These data demonstrate that APAP-induced cytokine expression in the liver is influenced by oxidative stress and that this is dependent, in part, on NF-{kappa}B. However, NF-{kappa}B p50-dependent responses do not appear to play a major role in the pathogenesis of toxicity in this model.« less

Carbachol induces TGF-alpha expression and colonic epithelial cell proliferation in sensory-desensitised rats.

PubMed

Bulut, Kerem; Felderbauer, Peter; Hoeck, Karoline; Schmidt, Wolfgang E; Hoffmann, Peter

2010-03-01

Signals for the expression of the peptide growth factors epidermal growth factor and transforming growth factor-alpha (TGFalpha) in the gastrointestinal mucosa are largely unknown. We have shown earlier that extrinsic afferents in the gastrointestinal tract induce TGFalpha expression in colonic mucosa via the deliberation of neurotransmitters substance P and calcitonin gene-related peptide. The aim of our present study was to determine the effects of carbachol on mucosal TGFalpha expression and epithelial cell proliferation in vivo. Rats were divided in three groups. Group 1 was treated with vehicle only, group 2 received one single subcutaneous injection of 250 microg/kg of carbachol and animals in group 3 were sensory-desensitised prior to the injection of 250 microg/kg carbachol. TGFalpha expression and epithelial cell proliferation was evaluated by polymerase chain reaction, Western blot analysis and bromodeoxyuridine staining. Carbachol induced a significant increase in mucosal epithelial cell proliferation and TGFalpha expression. Sensory desensitisation did neither abolish the increased TGFalpha expression nor the increase in epithelial cell proliferation. Parasympathetic pathways are involved in the control of TGFalpha expression in gastrointestinal mucosa as well as in epithelial cell proliferation.

Phenotypic differences between oral and skin fibroblasts in wound contraction and growth factor expression.

PubMed

Shannon, Diane B; McKeown, Scott T W; Lundy, Fionnuala T; Irwin, Chris R

2006-01-01

Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, alpha-smooth muscle actin expression (alpha-SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor -beta1 and -beta3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7-day period. alpha-SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme-linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of alpha-SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor-beta1 and -beta3, over the concentration range of 0.1-10 ng/mL, had similar effects on cell function, stimulating both gel contraction and alpha-SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.

Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55

PubMed Central

1993-01-01

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine triggering cell responses through two distinct membrane receptors. Stimulation of leukocyte adhesion to the endothelium is one of the many TNF-alpha activities and is explained by the upregulation of adhesion molecules on the endothelial cell surface. Human umbilical vein endothelial cells (HUVEC) were isolated, cultured, and demonstrated to express both TNF receptor types, TNF-R55 and TNF-R75. Cell adhesion to HUVEC was studied using the HL60, U937, and MOLT-4 cell lines. HUVEC were activated by either TNF-alpha, binding to both TNF-R55 and TNF- R75, and by receptor type-specific agonists, binding exclusively to TNF- R55 or to TNF-R75. The TNF-alpha-induced cell adhesion to HUVEC was found to be controlled almost exclusively by TNF-R55. This finding correlated with the exclusive activity of TNF-R55 in the TNF-alpha- dependent regulation of the expression of the intercellular adhesion molecule type 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule type 1 (VCAM-1). The CD44 adhesion molecule in HUVEC was also found to be upregulated through TNF-R55. However, both TNF-R55 and TNF- R75 upregulate alpha 2 integrin expression in HUVEC. The predominant role of TNF-R55 in TNF-alpha-induced adhesion in HUVEC may correlate with its specific control of NF-kappa B activation, since kappa B elements are known to be present in ICAM-1, E-selectin, and VCAM-1 gene regulatory sequences. PMID:8386742

TNF-alpha sensitizes HT-29 colonic epithelial cells to intestinal lactobacilli.

PubMed

McCracken, Vance J; Chun, Taehoon; Baldeón, Manuel E; Ahrné, Siv; Molin, Göran; Mackie, Roderick I; Gaskins, H Rex

2002-09-01

The ability of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) to influence epithelial interleukin (IL)-8 responses to the intestinal bacterium Lactobacillus plantarum 299v was analyzed in the human HT-29 colonic epithelial cell line. In the absence of TNF-alpha, IL-8 mRNA expression was not detectable by Northern blot analysis in HT-29 cells alone or in HT-29 cells co-cultured with L. plantarum 299v. However, TNF-alpha induced IL-8 mRNA expression, and co-culture of TNF-alpha-treated HT-29 cells with L. plantarum 299v significantly increased IL-8 mRNA expression above levels induced by TNF-alpha alone in an adhesion-dependent manner. The increase in IL-8 mRNA expression was not observed in TNF-alpha-treated HT-29/L. plantarum 299v co-cultures using heat-killed lactobacilli or when L. plantarum adhesion was prevented using mannoside or a trans-well membrane. Paradoxically, IL-8 secretion was decreased in TNF-alpha-treated HT-29 cells with L. plantarum 299v relative to cells treated with TNF-alpha alone. TNF-alpha-mediated responsiveness to L. plantarum 299v was further investigated by analyzing expression of a coreceptor for bacterial cell wall products CD14. HT-29 cells expressed CD14 mRNA and cell-surface CD14; however, TNF-alpha did not alter CD14 mRNA or cell-surface expression, and blockade of CD14 with monoclonal antibody MY4 did not alter the IL-8 response to L. plantarum 299v in TNF-alpha-treated HT-29 cells. These results indicate that although TNF-alpha sensitizes HT-29 epithelial cells to intestinal lactobacilli, the bacteria exert a protective effect by downregulating IL-8 secretion.

Low-level laser therapy (LLLT) attenuates RhoA mRNA expression in the rat bronchi smooth muscle exposed to tumor necrosis factor-alpha.

PubMed

de Lima, Flávia Mafra; Bjordal, Jan M; Albertini, Regiane; Santos, Fábio V; Aimbire, Flavio

2010-09-01

Low-level laser therapy (LLLT) has been found to produce anti-inflammatory effects in a variety of disorders. Bronchial smooth muscle (BSM) hyperreactivity is associated with increased Ca+2 sensitivity and increased RhoA mRNA expression. In the current study, we investigated if LLLT could reduce BSM contraction force and RhoA mRNA expression in tumor necrosis factor-alpha (TNF-alpha)-induced BSM hyperreactivity. In the study, 112 male Wistar rats were divided randomly into 16 groups, and BSM was harvested and suspended in TNF-alpha baths for 6 and 24 h, respectively. Irradiation with LLLT was performed with a wavelength of 660 nm for 42 s with a dose of 1.3 J/cm2. This LLLT dose was administered once in the 6-h group and twice in the 24-h group. LLLT significantly decreased contraction force in BSM at 6 h (TNF-alpha + LLLT: 11.65+/-1.10 g/100 mg of tissue) (F=3115) and at 24 h (TNF-alpha+ LLLT: 14.15+/-1.1 g/100 mg of tissue) (F=3245, p<0.05) after TNF-alpha, respectively, when compared to vehicle-bathed groups (control). LLLT also significantly decreased the expression of RhoA mRNA in BSM segments at 6 h (1.22+/-0.20) (F=2820, p<0.05) and 24 h (2.13+/-0.20) (F=3324, p<0.05) when compared to BSM segments incubated with TNF-alpha without LLLT irradiation. We conclude that LLLT administered with this protocol, reduces RhoA mRNA expression and BSM contraction force in TNF-alpha-induced BSM hyperreactivity.

Chondrogenic differentiation of growth factor-stimulated precursor cells in cartilage repair tissue is associated with increased HIF-1alpha activity.

PubMed

Gelse, K; Mühle, C; Knaup, K; Swoboda, B; Wiesener, M; Hennig, F; Olk, A; Schneider, H

2008-12-01

To investigate the chondrogenic potential of growth factor-stimulated periosteal cells with respect to the activity of Hypoxia-inducible Factor 1alpha (HIF-1alpha). Scaffold-bound autologous periosteal cells, which had been activated by Insulin-like Growth Factor 1 (IGF-1) or Bone Morphogenetic Protein 2 (BMP-2) gene transfer using both adeno-associated virus (AAV) and adenoviral (Ad) vectors, were applied to chondral lesions in the knee joints of miniature pigs. Six weeks after transplantation, the repair tissues were investigated for collagen type I and type II content as well as for HIF-1alpha expression. The functional role of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling on BMP-2/IGF-1-induced HIF-1alpha expression was assessed in vitro by employing specific inhibitors. Unstimulated periosteal cells formed a fibrous extracellular matrix in the superficial zone and a fibrocartilaginous matrix in deep zones of the repair tissue. This zonal difference was reflected by the absence of HIF-1alpha staining in superficial areas, but moderate HIF-1alpha expression in deep zones. In contrast, Ad/AAVBMP-2-stimulated periosteal cells, and to a lesser degree Ad/AAVIGF-1-infected cells, adopted a chondrocyte-like phenotype with strong intracellular HIF-1alpha staining throughout all zones of the repair tissue and formed a hyaline-like matrix. In vitro, BMP-2 and IGF-1 supplementation increased HIF-1alpha protein levels in periosteal cells, which was based on posttranscriptional mechanisms rather than de novo mRNA synthesis, involving predominantly the MEK/ERK pathway. This pilot experimental study on a relatively small number of animals indicated that chondrogenesis by precursor cells is facilitated in deeper hypoxic zones of cartilage repair tissue and is stimulated by growth factors which enhance HIF-1alpha activity.

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

PubMed

Khurana, Satish; Jaiswal, Amit K; Mukhopadhyay, Asok

2010-02-12

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

Effects of tumor necrosis factor-alpha and interferon-gamma on expressions of matrix metalloproteinase-2 and -9 in human bladder cancer cells.

PubMed

Shin, K Y; Moon, H S; Park, H Y; Lee, T Y; Woo, Y N; Kim, H J; Lee, S J; Kong, G

2000-10-31

We have investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon (INF-gamma), the potent Bacillus Calmette-Guerin (BCG)-induced cytokines on the production of MMP-2, MMP-9, TIMP-1, TIMP-2 and MT1-MMP in high grade human bladder cancer cell lines, T-24, J-82 and HT-1376 cell lines. MMP-2 expression and activity were decreased in T-24 cells treated with both cytokines in a dose dependent manner. However, J-82 cells treated with TNF-alpha and INF-gamma revealed dose dependent increases of MMP-9 expression and activity with similar baseline expression and activity of MMP-2. HT-1376 cells after exposure to TNF-alpha only enhanced the expression and activity of MMP-9. These results indicate that TNF-alpha and INF-gamma could regulate the production of MMP-2 or MMP-9 on bladder cancer cells and their patterns of regulation are cell specific. Furthermore, this diverse response of bladder cancer cells to TNF-alpha and INF-gamma suggests that BCG immunotherapy may enhance the invasiveness of bladder cancer in certain conditions with induction of MMPs.

Tumor necrosis factor alpha induces gamma-glutamyltransferase expression via nuclear factor-kappaB in cooperation with Sp1.

PubMed

Reuter, Simone; Schnekenburger, Michael; Cristofanon, Silvia; Buck, Isabelle; Teiten, Marie-Hélène; Daubeuf, Sandrine; Eifes, Serge; Dicato, Mario; Aggarwal, Bharat B; Visvikis, Athanase; Diederich, Marc

2009-02-01

Gamma-glutamyltransferase (GGT) cleaves the gamma-glutamyl moiety of glutathione (GSH), an endogenous antioxidant, and is involved in mercapturic acid metabolism and in cancer drug resistance when overexpressed. Moreover, GGT converts leukotriene (LT) C4 into LTD4 implicated in various inflammatory pathologies. So far the effect of inflammatory stimuli on regulation of GGT expression and activity remained to be addressed. We found that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) induced GGT promoter transactivation, mRNA and protein synthesis, as well as enzymatic activity. Remicade, a clinically used anti-TNFalpha antibody, small interfering RNA (siRNA) against p50 and p65 nuclear factor-kappaB (NF-kappaB) isoforms, curcumin, a well characterized natural NF-kappaB inhibitor, as well as a dominant negative inhibitor of kappaB alpha (IkappaBalpha), prevented GGT activation at various levels, illustrating the involvement of this signaling pathway in TNFalpha-induced stimulation. Over-expression of receptor of TNFalpha-1 (TNFR1), TNFR-associated factor-2 (TRAF2), TNFR-1 associated death domain (TRADD), dominant negative (DN) IkappaBalpha or NF-kappaB p65 further confirmed GGT promoter activation via NF-kappaB. Linker insertion mutagenesis of 536 bp of the proximal GGT promoter revealed NF-kappaB and Sp1 binding sites at -110 and -78 relative to the transcription start site, responsible for basal GGT transcription. Mutation of the NF-kappaB site located at -110 additionally inhibited TNFalpha-induced promoter induction. Chromatin immunoprecipitation (ChIP) assays confirmed mutagenesis results and further demonstrated that TNFalpha treatment induced in vivo binding of both NF-kappaB and Sp1, explaining increased GGT expression, and led to RNA polymerase II recruitment under inflammatory conditions.

Ketamine inhibits tumor necrosis factor-{alpha} and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, G.-J.; Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

2008-04-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 {mu}M ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 {mu}M of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-{alpha}more » and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-{alpha} and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 {mu}M) significantly inhibited LPS-induced TNF-{alpha} and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-{alpha} and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-{alpha} and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated sequential activations of c-Jun N-terminal kinase and activator protein-1.« less

Ketamine inhibits tumor necrosis factor-alpha and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation.

PubMed

Wu, Gone-Jhe; Chen, Ta-Liang; Ueng, Yune-Fang; Chen, Ruei-Ming

2008-04-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 microM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 microM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-alpha and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-alpha and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 microM) significantly inhibited LPS-induced TNF-alpha and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-alpha and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-alpha and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated sequential activations of c-Jun N-terminal kinase and activator protein-1.

Gonadotropin-releasing hormone (GnRH) agonist triptorelin inhibits estradiol-induced serum response element (SRE) activation and c-fos expression in human endometrial, ovarian and breast cancer cells.

PubMed

Gründker, Carsten; Günthert, Andreas R; Hellriegel, Martin; Emons, Günter

2004-11-01

The majority of human endometrial (>80%), ovarian (>80%) and breast (>50%) cancers express GnRH receptors. Their spontaneous and epidermal growth-factor-induced proliferation is dose- and time-dependently reduced by treatment with GnRH and its agonists. In this study, we demonstrate that the GnRH agonist triptorelin inhibits estradiol (E2)-induced cancer cell proliferation. The proliferation of quiescent estrogen receptor alpha (ER alpha)-/ER beta-positive, but not of ER alpha-negative/ER beta-positive endometrial, ovarian and breast cancer cell lines, was significantly stimulated (P<0.001) (ANOVA) after treatment with E2 (10(-8) M). This effect was time- and dose-dependently antagonized by simultaneous treatment with triptorelin. The inhibitory effect was maximal at 10(-5) M concentration of triptorelin (P<0.001). In addition, we could show that, in ER alpha-/ER beta-positive cell lines, E2 induces activation of serum response element (SRE) and expression of the immediate early-response gene c-fos. These effects were blocked by triptorelin (P<0.001). E2-induced activation of estrogen-response element (ERE) was not affected by triptorelin. The transcriptional activation of SRE by E2 is due to ER alpha activation of the mitogen-activated protein kinase (MAPK) pathway. This pathway is impeded by GnRH, resulting in a reduction of E2-induced SRE activation and, in consequence, a reduction of E2-induced c-fos expression. This causes downregulation of E2-induced cancer cell proliferation.

alpha1B-Adrenergic receptor phosphorylation and desensitization induced by transforming growth factor-beta.

PubMed Central

Romero-Avila, M Teresa; Flores-Jasso, C Fabián; García-Sáinz, J Adolfo

2002-01-01

Transforming growth factor-beta (TGF-beta) induced alpha(1B)-adrenergic receptor phosphorylation in Rat-1 fibroblasts stably expressing these adrenoceptors. This effect of TGF-beta was rapid, reaching a maximum within 30 min and decreasing thereafter, and concentration-dependent (EC(50) 0.3 pM). The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, and the protein kinase C inhibitors staurosporine, Ro 318220 and bisindolylmaleimide, blocked the effect of this growth factor. alpha(1B)-Adrenergic receptor phosphorylation was associated with desensitization, as indicated by a reduction in the adrenergic-mediated production of [(3)H]inositol phosphates. Phosphorylation of alpha(1B)-adrenergic receptors by TGF-beta was also observed in Cos-1 cells transfected with the receptor. Co-transfection of the dominant-negative mutant of the regulatory subunit of phosphoinositide 3-kinase (Deltap85) inhibited the phosphorylation of alpha(1B)-adrenergic receptors induced by TGF-beta. Our results indicate that activation of TGF-beta receptors induces alpha(1B)-adrenergic receptor phosphorylation and desensitization. The data suggest that phosphoinositide 3-kinase and protein kinase C play key roles in this effect of TGF-beta. PMID:12234252

alpha1B-Adrenergic receptor phosphorylation and desensitization induced by transforming growth factor-beta.

PubMed

Romero-Avila, M Teresa; Flores-Jasso, C Fabián; García-Sáinz, J Adolfo

2002-12-01

Transforming growth factor-beta (TGF-beta) induced alpha(1B)-adrenergic receptor phosphorylation in Rat-1 fibroblasts stably expressing these adrenoceptors. This effect of TGF-beta was rapid, reaching a maximum within 30 min and decreasing thereafter, and concentration-dependent (EC(50) 0.3 pM). The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, and the protein kinase C inhibitors staurosporine, Ro 318220 and bisindolylmaleimide, blocked the effect of this growth factor. alpha(1B)-Adrenergic receptor phosphorylation was associated with desensitization, as indicated by a reduction in the adrenergic-mediated production of [(3)H]inositol phosphates. Phosphorylation of alpha(1B)-adrenergic receptors by TGF-beta was also observed in Cos-1 cells transfected with the receptor. Co-transfection of the dominant-negative mutant of the regulatory subunit of phosphoinositide 3-kinase (Deltap85) inhibited the phosphorylation of alpha(1B)-adrenergic receptors induced by TGF-beta. Our results indicate that activation of TGF-beta receptors induces alpha(1B)-adrenergic receptor phosphorylation and desensitization. The data suggest that phosphoinositide 3-kinase and protein kinase C play key roles in this effect of TGF-beta.

Methanol extract of Xanthium strumarium L. possesses anti-inflammatory and anti-nociceptive activities.

PubMed

Kim, In-Tae; Park, Young-Mi; Won, Jong-Heon; Jung, Hyun-Ju; Park, Hee-Juhn; Choi, Jong-Won; Lee, Kyung-Tae

2005-01-01

As an attempt to identify bioactive natural products with anti-inflammatory activity, we evaluated the effects of the methanol extract of the semen of Xanthium strumarium L. (MEXS) on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) production in RAW 264.7 cells. Our data indicate that MEXS is a potent inhibitor of NO, PGE2 and TNF-alpha production. Consistent with these findings, the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and iNOS, COX-2 and TNF-alpha mRNA were down-regulated in a concentration-dependent manner. Furthermore, MEXS inhibited nuclear factor kappa B (NF-kappaB) DNA binding activity and the translocation of NF-kappaB to the nucleus by blocking the degradation of inhibitor of kappa B-alpha (IkappaB-alpha). We further evaluated the anti-inflammatory and anti-nociceptive activities of MEXS in vivo. MEXS (100, 200 mg/kg/d, p.o.) reduced acute paw edema induced by carrageenin in rats, and showed analgesic activities in an acetic acid-induced abdominal constriction test and a hot plate test in mice. Thus, our study suggests that the inhibitions of iNOS, COX-2 expression, and TNF-alpha release by the methanol extract of the semen of Xanthium strumarium L. are achieved by blocking NF-kappaB activation, and that this is also responsible for its anti-inflammatory effects.

TNF-alpha-induced apoptosis is prevented by erythropoietin treatment on SH-SY5Y cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pregi, Nicolas; Wenker, Shirley; Vittori, Daniela

2009-02-01

The growth factor erythropoietin (Epo) has shown neuronal protective action in addition to its well known proerythroid activity. Furthermore, Epo has dealt with cellular inflammation by inhibiting the expression of several proinflammatory cytokines, such as IL-1 and TNF-{alpha}. The action of TNF can have both apoptotic and antiapoptotic consequences due to altered balance between different cell signalling pathways. This work has focused on the apoptotic effects of this cytokine and the potential protective action of Epo. The model we used was neuroblastoma SH-SY5Y cells cultured in the presence of 25 ng/ml TNF-{alpha} or pretreated with 25 U/ml Epo for 12more » h before the addition of TNF-{alpha}. Apoptosis was evaluated by differential cell count after Hoechst staining, analysis of DNA ladder pattern, and measurement of caspase activity. Despite its ability to induce NF-{kappa}B nuclear translocation, TNF-{alpha} induced cell death, which was found to be associated to upregulation of TNF Receptor 1 expression. On the other hand, cells activated by Epo became resistant to cell death. Prevention of death receptor upregulation and caspase activation may explain this antiapoptotic effect of Epo, which may be also favoured by the induction of a higher expression of protective factors, such as Bcl-2 and NF-{kappa}B, through mechanisms involving Jak/STAT and PI3K signalling pathways.« less

Apoptosis induced by tumor necrosis factor-alpha in rat hepatocyte cell lines expressing hepatitis B virus.

PubMed Central

Guilhot, S.; Miller, T.; Cornman, G.; Isom, H. C.

1996-01-01

Three well differentiated SV40-immortalized rat hepatocyte cell lines, CWSV1, CWSV2, and CWSV14, and Hepatitis B Virus (HBV)-producing cell lines derived from them were examined for sensitivity to tumor necrosis factor (TNF)-alpha. CWSV1, CWSV2, and CWSV14 cells were co-transfected with a DNA construct containing a dimer of the HBV genome and the neo gene and selected in G418 to generate stable cell lines. Characterization of these cell lines indicated that they contain integrated HBV DNA, contain low molecular weight HBV DNA compatible with the presence of HBV replication intermediates, express HBV transcripts, and produce HBV proteins. The viability of CWSV1, CWSV2, and CWSV2 cells was not significantly altered when they were treated with TNF-alpha at concentrations as high as 20,000 U/ml. The HBV-expressing CWSV1 cell line, SV1di36, and the HBV-expressing CWSV14 cell line, SV14di208, were also not killed when treated with TNF-alpha. However, the HBV-expressing CWSV2 cell line, SV2di366, was extensively killed when treated with TNF-alpha at concentrations ranging from 200 to 20,000 U/ml. Analysis of several different HBV-producing CWSV2 cell lines indicated that TNF-alpha killing depended upon the level of HBV expression. The TNF-alpha-induced cell killing in high HBV-producing CWSV2 cell lines was accompanied by the presence of an oligonucleosomal DNA ladder characteristic of apoptosis. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 9 Figure 10 Figure 11 PMID:8774135

Gigantol isolated from the whole plants of Cymbidium goeringii inhibits the LPS-induced iNOS and COX-2 expression via NF-kappaB inactivation in RAW 264.7 macrophages cells.

PubMed

Won, Jong-Heon; Kim, Ji-Yeon; Yun, Kyung-Jin; Lee, Jin-Hee; Back, Nam-In; Chung, Hae-Gon; Chung, Sun A; Jeong, Tae-Sook; Choi, Myung-Sook; Lee, Kyung-Tae

2006-10-01

During our efforts to find bioactive natural products with anti-inflammatory activity, we isolated gigantol from the whole plants of Cymbidium goeringii (Orchidaceae) by activity-guided chromatographic fractionation. Gigantol was found to have potent inhibitory effects on LPS-induced nitric oxide (NO) and prostaglandin E (2) (PGE (2)) production in RAW 264.7 cells. Consistent with these findings, gigantol suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in RAW 264.7 cells in a concentration-dependent manner. Our data also indicate that gigantol is a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) release and influenced the mRNA expression levels of these cytokines in a dose-dependent manner. Furthermore, a reporter gene assay for nuclear factor kappa B (NF-kappaB) and an electromobility shift assay (EMSA) demonstrated that gigantol effectively inhibited the activation of NF-kappaB, which is necessary for the expression of iNOS, COX-2, TNF-alpha, IL-1beta and IL-6. Thus, our studies suggest that gigantol inhibits LPS-induced iNOS and COX-2 expression by blocking NF- kappaB activation.

Apolipoprotein CIII-induced THP-1 cell adhesion to endothelial cells involves pertussis toxin-sensitive G protein- and protein kinase C alpha-mediated nuclear factor-kappaB activation.

PubMed

Kawakami, Akio; Aikawa, Masanori; Nitta, Noriko; Yoshida, Masayuki; Libby, Peter; Sacks, Frank M

2007-01-01

Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC alpha as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC alpha in human monocytic THP-1 cells, leading to NF-kappaB activation. Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC alpha activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC alpha activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC alpha activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-kappaB (NF-kappaB) through PKC alpha in THP-1 cells and augmented beta1-integrin expression. The NF-kappaB inhibitor peptide SN50 partially inhibited apoCIII-induced beta1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. PTX-sensitive G protein pathway participates critically in PKC alpha stimulation in THP-1 cells exposed to apoCIII, activating NF-kappaB, and increasing beta1-integrin. This action causes monocytic cells to adhere to endothelial cells. Furthermore, because leukocyte NF-kappaB activation contributes to inflammatory aspects of atherogenesis, apoCIII may stimulate diverse inflammatory responses through monocyte activation.

Novel function of STAT1beta in B cells: induction of cell death by a mechanism different from that of STAT1alpha.

PubMed

Najjar, Imen; Schischmanoff, Pierre Olivier; Baran-Marszak, Fanny; Deglesne, Pierre-Antoine; Youlyouz-Marfak, Ibtissam; Pampin, Mathieu; Feuillard, Jean; Bornkamm, Georg W; Chelbi-Alix, Mounira K; Fagard, Remi

2008-12-01

Alternate splicing of STAT1 produces two isoforms: alpha, known as the active form, and beta, previously shown to act as a dominant-negative factor. Most studies have dealt with STAT1alpha, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1-deficient human B cell line was transfected to express STAT1alpha or STAT1beta. STAT1alpha, expressed alone, enhanced cell death, potentiated the fludarabine-induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1beta, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1beta-expressing B cells, p53 was strictly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1beta in programmed cell death, which is independent of p53.

Down-regulation of Jab1, HIF-1alpha, and VEGF by Moloney murine leukemia virus-ts1 infection: a possible cause of neurodegeneration.

PubMed

Lungu, Gina F; Stoica, George; Wong, Paul K Y

2008-05-01

Moloney murine leukemia virus-temperature sensitive (MoMuLV-ts1)-mediated neuronal death is a result of both loss of glial support and release of cytokines and neurotoxins from ts1-infected glial cells. Here the authors propose vascular endothelial growth factor (VEGF) down-regulation as another contributory factor in neuronal degeneration induced by ts1 infection. To determine how ts1 affects VEGF expression in ts1-infected brain, the authors examined the expression of several proteins that are important in regulating the expression of VEGF. The authors found significant decreases in Jun-activating domain-binding protein 1 (Jab1), hypoxia-inducible factor (HIF)-1alpha, and VEGF levels and increases in p53 protein levels in ts1-infected brains compared to noninfected control brains. The authors suggest that a decrease Jab1 expression in ts1 infection leads to accumulation of p53, which binds to HIF-1alpha to accelerate its degradation. A rapid degradation of HIF-1alpha leads to decreased VEGF production and secretion. Considering that endothelial cells are the most conspicuous in virus replication and production in ts1 infection, but are not killed by the infection, the authors examined the expression of these proteins using infected and noninfected mouse cerebrovascular endothelial (CVE) cells. The ts1- infected CVE cells showed decreased Jab1, HIF-1alpha, and VEGF mRNA and protein levels and increased p53 protein levels compared with noninfected cells, consistent with the results found in vivo. These results confirm that ts1 infection results in insufficient secretion of VEGF from endothelial cells and may result in decreased neuroprotection. This study suggested that ts1-mediated neuropathology in mice may result from changes in expression and activity of Jab1, p53, and HIF-1alpha, with a final target on VEGF expression and neuronal degeneration.

HNF4alpha dysfunction as a molecular rational for cyclosporine induced hypertension.

PubMed

Niehof, Monika; Borlak, Jürgen

2011-01-27

Induction of tolerance against grafted organs is achieved by the immunosuppressive agent cyclosporine, a prominent member of the calcineurin inhibitors. Unfortunately, its lifetime use is associated with hypertension and nephrotoxicity. Several mechanism for cyclosporine induced hypertension have been proposed, i.e. activation of the sympathetic nervous system, endothelin-mediated systemic vasoconstriction, impaired vasodilatation secondary to reduction in prostaglandin and nitric oxide, altered cytosolic calcium translocation, and activation of the renin-angiotensin system (RAS). In this regard the molecular basis for undue RAS activation and an increased signaling of the vasoactive oligopeptide angiotensin II (AngII) remain elusive. Notably, angiotensinogen (AGT) is the precursor of AngII and transcriptional regulation of AGT is controlled by the hepatic nuclear factor HNF4alpha. To better understand the molecular events associated with cyclosporine induced hypertension, we investigated the effect of cyclosporine on HNF4alpha expression and activity and searched for novel HNF4alpha target genes among members of the RAS cascade. Using bioinformatic algorithm and EMSA bandshift assays we identified angiotensin II receptor type 1 (AGTR1), angiotensin I converting enzyme (ACE), and angiotensin I converting enzyme 2 (ACE2) as genes targeted by HNF4alpha. Notably, cyclosporine represses HNF4alpha gene and protein expression and its DNA-binding activity at consensus sequences to AGT, AGTR1, ACE, and ACE2. Consequently, the gene expression of AGT, AGTR1, and ACE2 was significantly reduced as evidenced by quantitative real-time RT-PCR. While RAS is composed of a sophisticated interplay between multiple factors we propose a decrease of ACE2 to enforce AngII signaling via AGTR1 to ultimately result in vasoconstriction and hypertension. Taken collectively we demonstrate cyclosporine to repress HNF4alpha activity through calcineurin inhibitor mediated inhibition of nuclear factor of activation of T-cells (NFAT) which in turn represses HNF4alpha that leads to a disturbed balance of RAS.

Fanconi anemia protein, FANCG, is a phosphoprotein and is upregulated with FANCA after TNF-alpha treatment.

PubMed

Futaki, M; Watanabe, S; Kajigaya, S; Liu, J M

2001-02-23

Fanconi anemia (FA) is a genetic syndrome characterized by bone marrow failure, birth defects, and a predisposition to malignancy. At this time, six FA genes have been identified, and several gene products have been found to interact in a protein complex. FA cells appear to overexpress the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). We therefore examined the effects of TNF-alpha on the regulation of FA complementation group proteins, FANCG and FANCA. We found that treatment with TNF-alpha induced FANCG protein expression. FANCA was induced concurrently with FANCG, and the FANCA/FANCG complex was increased in the nucleus following TNF-alpha treatment. Inactivation of inhibitory kappa B kinase-2 modulated the expression of FANCG. We also found that both nuclear and cytoplasmic FANCG fractions were phosphorylated. These results show that FANCG is a phosphoprotein and suggest that the cellular accumulation of FA proteins is subject to regulation by TNF-alpha signaling.

Tumor Necrosis Factor alpha (TNF{alpha}) regulates CD40 expression through SMAR1 phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Kamini; Sinha, Surajit; Malonia, Sunil Kumar

2010-01-08

CD40 plays an important role in mediating inflammatory response and is mainly induced by JAK/STAT phosphorylation cascade. TNF{alpha} is the key cytokine that activates CD40 during inflammation and tumorigenesis. We have earlier shown that SMAR1 can repress the transcription of Cyclin D1 promoter by forming a HDAC1 dependent repressor complex. In this study, we show that SMAR1 regulates the transcription of NF-{kappa}B target gene CD40. SMAR1 recruits HDAC1 and forms a repressor complex on CD40 promoter and keeps its basal transcription in check. Further, we show that TNF{alpha} stimulation induces SMAR1 phosphorylation at Ser-347 and promotes its cytoplasmic translocation, thusmore » releasing its negative effect. Concomitantly, TNF{alpha} induced phosphorylation of STAT1 at Tyr-701 by JAK1 facilitates its nuclear translocation and activation of CD40 through p300 recruitment and core Histone-3 acetylation. Thus, TNF{alpha} mediated regulation of CD40 expression occurs by dual phosphorylation of SMAR1 and STAT1.« less

Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, MiRan; Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr

Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the {alpha} subunit of Gq protein (G{alpha}q) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active G{alpha}q (G{alpha}qQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of G{alpha}q with shRNA in HaCaT human keratinocytes. G{alpha}q was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipasemore » C (PLC), protein kinase C{delta} (PKC{delta}), and matrix metaloprotease-2 (MMP-2). Moreover, G{alpha}qQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that G{alpha}q mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKC{delta} and MMP-2 in HaCaT cells.« less

Hsp27 and its expression pattern in diffusely infiltrating astrocytomas.

PubMed

Mäkelä, Katri S; Haapasalo, Joonas A; Ilvesaro, Joanna M; Parkkila, Seppo; Paavonen, Timo; Haapasalo, Hannu K

2014-09-01

Heat shock protein 27 (Hsp27) is induced by cell stress conditions. In the presence of oxidative stress it functions as an antioxidant. To study the putative expression patterns and clinical significance of Hsp27, we assessed the associations between Hsp27, R132H mutation of Isocitrate dehydrogenase1 (IDH1-R132H), Hypoxia-inducible factor subunit alpha (HIF-1 alpha), Carbonic anhydrase IX (CA IX), and patient prognosis in astrocytic gliomas. Tissue micro-array samples of 295 grade II-IV astrocytomas were stained immunohistochemically for Hsp27, IDH1-R132H, HIF-1 alpha, and CA IX. We tested their relationship with clinicopathological features and patient survival. There was a significant correlation between Hsp27 expression and increasing WHO grade (p<0.001). Hsp27 expression correlated significantly with IDH1 mutation when studied within the entire cohort (p<0.001) as well as separately in WHO grade II and III tumors (p=0.006 and 0.002, respectively). IDH1 mutation and HIF-1 alpha positive staining were detected simultaneously (p<0.001). In IDH1 mutated tumors, positive HIF-1 alpha staining correlated with CA IX expression (p=0.027), whereas no such correlation was found in IDH1 non-mutated tumors. IDH1 mutation was associated with a low cell proliferation index (p=0.001) and HIF-1 alpha with increasing proliferation (p = 0.003). Hsp27 expression was associated with a shorter rate of patient survival in univariate survival analysis (p=0.001). In multivariate survival analysis, patient age, IDH1 mutation and HIF-1 alpha appeared as independent prognostic factors (p<0.000, <0.000 and 0.011 respectively) Hsp27 expression is associated with increasing WHO grade and patient prognosis in astrocytic gliomas. The results suggest that IDH1 mutation may have an effect on the expression pathways of Hsp27 and CA IX.

Involvement of Mst1 in tumor necrosis factor-{alpha}-induced apoptosis of endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohtsubo, Hideki; Ichiki, Toshihiro; Imayama, Ikuyo

2008-03-07

Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-{alpha}-induced apoptosis of ECs. Western blot analysis revealed that TNF-{alpha} induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-{alpha}-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting ofmore » propidium iodide-stained cells showed that TNF-{alpha} induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-{alpha}-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-{alpha}-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-{alpha}-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.« less

In vitro and in vivo expression of interleukin-1alpha and tumor necrosis factor-alpha mRNA in pemphigus vulgaris: interleukin-1alpha and tumor necrosis factor-alpha are involved in acantholysis.

PubMed

Feliciani, C; Toto, P; Amerio, P; Pour, S M; Coscione, G; Shivji, G; Wang, B; Sauder, D N

2000-01-01

Keratinocyte-derived cytokines have been implicated in the pathogenesis of a number of skin diseases. In this study we examined the possible role of keratinocyte-derived cytokines in the development of acantholysis in pemphigus vulgaris. Nineteen patients with pemphigus vulgaris, demonstrating the characteristic clinical, pathologic, and immunopathologic findings were studied. In situ immunolabeling demonstrated the presence of two cytokines interleukin-1alpha and tumor necrosis factor-alpha, in lesional and perilesional areas. Results were confirmed by reverse transcriptase-polymerase chain reaction, demonstrating overexpression of both cytokines in vivo. To study the role of these cytokines in the pathogenesis of pemphigus vulgaris both in vitro and in vivo studies were performed. The results of the in vitro study demonstrated that pemphigus vulgaris IgG induced interleukin-1alpha and tumor necrosis factor-alpha mRNA in the skin. The potential pathogenic role of these mediators was demonstrated by a blocking study using antibodies against human interleukin-1alpha and tumor necrosis factor-alpha in keratinocytes cultures. A combination of anti-interleukin-1alpha and anti-tumor necrosis factor-alpha antibodies inhibited in vitro pemphigus vulgaris IgG induced acantholysis. To confirm the role of interleukin-1 and tumor necrosis factor-alpha in pemphigus, we utilized passive transfer studies using interleukin-1 deficient mice (ICE-/-, interleukin-1beta-/-) and tumor necrosis factor-alpha receptor deficient mice (TNFR1R2-/-). Both groups demonstrated a decreased susceptibility to the passive transfer of pemphigus. Our data support the role of cytokines interleukin-1 and tumor necrosis factor-alpha in the pathogenesis of pemphigus vulgaris.

Chorionic gonadotropin regulates the transcript level of VHL, p53, and HIF-2alpha in human granulosa lutein cells.

PubMed

Herr, D; Keck, C; Tempfer, C; Pietrowski, Detlef

2004-12-01

The ovarian corpus luteum plays a critical role in reproduction being the primary source of circulating progesterone. After ovulation the corpus luteum is build by avascular granulosa lutein cells through rapid vascularization regulated by gonadotropic hormones. The present study was performed to investigate whether this process might be influenced by the human chorionic gonadotropin (hCG)-dependent expression of different tumor suppressor genes and hypoxia dependent transcription factors. RNA was isolated from cultured granulosa lutein cells, transcribed into cDNA, and the transcript level of following genes were determined: RB-1, VHL, NF-1, NF-2, Wt-1, p53, APC, and hypoxia inducible factor-1 (HIF-1), -2, and -3alpha. Additionally, the influence of hCG on the expression of VHL, p53, and HIf2alpha were investigated. We demonstrate that in human granulosa lutein cells the tumor suppressor genes RB-1, VHL, NF-1, NF-2, Wt-1, p53, and APC and the hypoxia dependent transcription factors HIF-1alpha, -2alpha, and -3alpha are expressed. In addition, we showed that hCG regulates the expression of p53, VHL, and HIF-2alpha. Our results indicate that hCG may determine the growth and development of the corpus luteum by mediating hypoxic and apoptotic pathways in human granulosa lutein cells. Copyright 2004 Wiley-Liss, Inc.

High glucose concentrations attenuate hypoxia-inducible factor-1{alpha} expression and signaling in non-tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dehne, Nathalie, E-mail: dehne@biochem.uni-frankfurt.de; Hintereder, Gudrun, E-mail: Gudrun.Hintereder@kgu.de; Bruene, Bernhard, E-mail: bruene@pathobiochemie1.de

2010-04-15

Hypoxia-inducible factor (HIF) is the major transcription factor mediating adaption to hypoxia e.g. by enhancing glycolysis. In tumor cells, high glucose concentrations are known to increase HIF-1{alpha} expression even under normoxia, presumably by enhancing the concentration of tricarboxylic acid cycle intermediates, while reactions of non-tumor cells are not well defined. Therefore, we analyzed cellular responses to different glucose concentrations in respect to HIF activation comparing tumor to non-tumor cells. Using cells derived from non-tumor origin, we show that HIF-1{alpha} accumulation was higher under low compared to high glucose concentrations. Low glucose allowed mRNA expression of HIF-1 target genes like adrenomedullin.more » Transfection of C{sub 2}C{sub 12} cells with a HIF-1{alpha} oxygen-dependent degradation domaine-GFP fusion protein revealed that prolyl hydroxylase (PHD) activity is impaired at low glucose concentrations, thus stabilizing the fusion protein. Mechanistic considerations suggested that neither O{sub 2} redistribution nor an altered redox state explains impaired PHD activity in the absence of glucose. In order to affect PHD activity, glucose needs to be metabolized. Amino acids present in the medium also diminished HIF-1{alpha} expression, while the addition of fatty acids did not. This suggests that glucose or amino acid metabolism increases oxoglutarate concentrations, which enhances PHD activity in non-tumor cells. Tumor cells deprived of glutamine showed HIF-1{alpha} accumulation in the absence of glucose, proposing that enhanced glutaminolysis observed in many tumors enables these cells to compensate reduced oxoglutarate production in the absence of glucose.« less

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soeda, Junpei; Morgan, Maelle; McKee, Chad

Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells inmore » the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. {alpha}1 and {alpha}3-nAChR mRNA expression was significantly upregulated in NASH fibrosis compared to normal livers. Conclusion: Nicotine at levels in smokers' blood is pro-fibrogenic, through actions on hHSCs expressed nAChRs. Therefore, CS, via its nicotine content, may worsen liver fibrosis. Moreover, nicotinic receptor antagonists may have utility as novel anti-fibrotic agents.« less

Preventive effects of Schistosoma japonicum ova on trinitrobenzenesulfonic acid-induced colitis and bacterial translocation in mice.

PubMed

Zhao, Yuan; Zhang, Shuncai; Jiang, Li; Jiang, Jie; Liu, Hongchun

2009-11-01

To evaluate the preventive effects of Schistosoma japonicum ova on trinitrobenzenesulfonic acid (TNBS)-induced colitis and bacterial translocation in mice. BALB/c mice were randomly divided into three groups: control group; TNBS(+)Ova(-) group; and TNBS(+)Ova(+) group. Mice of the TNBS(+)Ova(+) group were exposed to 10 000 freeze-killed S. japonicum ova by i.p. injection on day 1 and day 11. On day 15, mice were challenged with TNBS to induce colitis. The following variables were assessed: colon pathological changes; serum expression of tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and interleukin-10 (IL-10); expression of Toll-like receptor 4 (TLR4) in colon; IFN-gamma, IL-10 and TLR4 mRNA expression in colon; and the bacterial translocation rate. Compared to TNBS(+)Ova(-) group, the colonic inflammation in the TNBS(+)Ova(+) group were relieved. A highly significant elevation of IFN-gamma and TNF-alpha were observed in the TNBS-induced colitis group. After exposure to the eggs, IFN-gamma was significantly decreased, while TNF-alpha was similar to that of the TNBS(+)ova(-) group. No obvious variation was seen in IL-10 expression in TNBS-induced colitis, compared to the controls. Exposure to the eggs led to a significant upregulation of IL-10 expression. TLR4 expression was elevated after injected with TNBS and was downregulated in the eggs group. Less intestinal bacterial translocation frequency was observed when exposed to eggs. S. japonicum ova can prevent the TNBS-induced colitis and reduce the bacterial translocation frequency in mice. The mechanisms were supposed to be due to the regulation of T-helper cell 1/2 balance and TLR4 expression.

Persisting mild hypothermia suppresses hypoxia-inducible factor-1alpha protein synthesis and hypoxia-inducible factor-1-mediated gene expression.

PubMed

Tanaka, Tomoharu; Wakamatsu, Takuhiko; Daijo, Hiroki; Oda, Seiko; Kai, Shinichi; Adachi, Takehiko; Kizaka-Kondoh, Shinae; Fukuda, Kazuhiko; Hirota, Kiichi

2010-03-01

The transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in regulating gene expression in response to hypoxia-ischemia. Ischemia causes the tissue not only to be hypoxic but also to be hypothermic because of the hypoperfusion under certain circumstances. On the other hand, the induced hypothermia is one of the most common therapeutic modalities to extend tolerance to hypoxia. Although hypoxia elicits a variety of cellular and systemic responses at different organizational levels in the body, little is known about how hypoxia-induced responses are affected by low temperature. We examined the influence of mild hypothermic conditions (28-32 degrees C) on HIF-1 in both in vitro and in vivo settings. In vitro experiments adopting cultured cells elucidated that hypoxia-induced HIF-1 activation was resistant to 4-h exposure to the low temperature. In contrast, exposure to the low temperature as long as 24 h suppressed HIF-1 activation and the subsequent upregulation of HIF-1 target genes such as VEGF or GLUT-1. HIF-1alpha protein stability in the cell was not affected by hypothermic treatment. Furthermore, intracellular ATP content was reduced under 1% O(2) conditions but was not largely affected by hypothermic treatment. The evidence indicates that reduction of oxygen consumption is not largely involved in suppression of HIF-1. In addition, we demonstrated that HIF-1 DNA-binding activity and HIF-1-dependent gene expressions induced under 10% O(2) atmosphere in mouse brain were not influenced by treatment under 3-h hypothermic temperature but were inhibited under 5-h treatment. On the other hand, we indicated that warming ischemic legs of mice for 24 h preserved HIF-1 activity. In this report we describe for the first time that persisting low temperature significantly reduced HIF-1alpha neosynthesis under hypoxic conditions, leading to a decrease in gene expression for adaptation to hypoxia in both in vitro and in vivo settings.

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Shuai; Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208; Lv, Jiaju

2012-03-30

Highlights: Black-Right-Pointing-Pointer Cyld deficiency suppresses pro-inflammatory phenotypic switch of VSMCs. Black-Right-Pointing-Pointer Cyld deficiency inhibits MAPK rather than NF-kB activity in inflamed VSMCs. Black-Right-Pointing-Pointer CYLD is up-regulated in the coronary artery with neointimal hyperplasia. -- Abstract: CYLD, a deubiquitinating enzyme (DUB), is a critical regulator of diverse cellular processes, ranging from proliferation and differentiation to inflammatory responses, via regulating multiple key signaling cascades such as nuclear factor kappa B (NF-{kappa}B) pathway. CYLD has been shown to inhibit vascular lesion formation presumably through suppressing NF-{kappa}B activity in vascular cells. However, herein we report a novel role of CYLD in mediating pro-inflammatory responsesmore » in vascular smooth muscle cells (VSMCs) via a mechanism independent of NF-{kappa}B activity. Adenoviral knockdown of Cyld inhibited basal and the tumor necrosis factor alpha (TNF{alpha})-induced mRNA expression of pro-inflammatory cytokines including monocyte chemotactic protein-1 (Mcp-1), intercellular adhesion molecule (Icam-1) and interleukin-6 (Il-6) in rat adult aortic SMCs (RASMCs). The CYLD deficiency led to increases in the basal NF-{kappa}B transcriptional activity in RASMCs; however, did not affect the TNF{alpha}-induced NF-{kappa}B activity. Intriguingly, the TNF{alpha}-induced I{kappa}B phosphorylation was enhanced in the CYLD deficient RASMCs. While knocking down of Cyld decreased slightly the basal expression levels of I{kappa}B{alpha} and I{kappa}B{beta} proteins, it did not alter the kinetics of TNF{alpha}-induced I{kappa}B protein degradation in RASMCs. These results indicate that CYLD suppresses the basal NF-{kappa}B activity and TNF{alpha}-induced I{kappa}B kinase activation without affecting TNF{alpha}-induced NF-{kappa}B activity in VSMCs. In addition, knocking down of Cyld suppressed TNF{alpha}-induced activation of mitogen activated protein kinases (MAPKs) including extracellular signal-activated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 in RASMCs. TNF{alpha}-induced RASMC migration and monocyte adhesion to RASMCs were inhibited by the Cyld knockdown. Finally, immunochemical staining revealed a dramatic augment of CYLD expression in the injured coronary artery with neointimal hyperplasia. Taken together, our results uncover an unexpected role of CYLD in promoting inflammatory responses in VSMCs via a mechanism involving MAPK activation but independent of NF-{kappa}B activity, contributing to the pathogenesis of vascular disease.« less

{beta}-Catenin regulates airway smooth muscle contraction.

PubMed

Jansen, Sepp R; Van Ziel, Anna M; Baarsma, Hoeke A; Gosens, Reinoud

2010-08-01

beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

PubMed

Murata, J; Ayukawa, K; Ogasawara, M; Watanabe, H; Saiki, I

1999-03-15

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lundasen, Thomas; Molecular Nutrition Unit, Department of Biosciences and Nutrition, NOVUM, Karolinska Institutet, Huddinge, SE-141 86 Stockholm; Hunt, Mary C.

The metabolic regulator fibroblast growth factor 21 (FGF21) has antidiabetic properties in animal models of diabetes and obesity. Using quantitative RT-PCR, we here show that the hepatic gene expression of FGF21 is regulated by the peroxisome proliferator-activated receptor alpha (PPAR{alpha}). Fasting or treatment of mice with the PPAR{alpha} agonist Wy-14,643 induced FGF21 mRNA by 10-fold and 8-fold, respectively. In contrast, FGF21 mRNA was low in PPAR{alpha} deficient mice, and fasting or treatment with Wy-14,643 did not induce FGF21. Obese ob/ob mice, known to have increased PPAR{alpha} levels, displayed 12-fold increased hepatic FGF21 mRNA levels. The potential importance of PPAR{alpha} formore » FGF21 expression also in human liver was shown by Wy-14,643 induction of FGF21 mRNA in human primary hepatocytes, and PPAR{alpha} response elements were identified in both the human and mouse FGF21 promoters. Further studies on the mechanisms of regulation of FGF21 by PPAR{alpha} in humans will be of great interest.« less

Hantaan virus nucleocapsid protein binds to importin alpha proteins and inhibits tumor necrosis factor alpha-induced activation of nuclear factor kappa B.

PubMed

Taylor, Shannon L; Frias-Staheli, Natalia; García-Sastre, Adolfo; Schmaljohn, Connie S

2009-02-01

Hantaviruses such as Hantaan virus (HTNV) and Andes virus cause two human diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, respectively. For both, disease pathogenesis is thought to be immunologically mediated and there have been numerous reports of patients with elevated levels of proinflammatory and inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), in their sera. Multiple viruses have developed evasion strategies to circumvent the host cell inflammatory process, with one of the most prevalent being the disruption of nuclear factor kappa B (NF-kappaB) activation. We hypothesized that hantaviruses might also moderate host inflammation by interfering with this pathway. We report here that the nucleocapsid (N) protein of HTNV was able to inhibit TNF-alpha-induced activation of NF-kappaB, as measured by a reporter assay, and the activation of endogenous p65, an NF-kappaB subunit. Surprisingly, there was no defect in the degradation of the inhibitor of NF-kappaB (IkappaB) protein, nor was there any alteration in the level of p65 expression in HTNV N-expressing cells. However, immunofluorescence antibody staining demonstrated that cells expressing HTNV N protein and a green fluorescent protein-p65 fusion had limited p65 nuclear translocation. Furthermore, we were able to detect an interaction between HTNV N protein and importin alpha, a nuclear import molecule responsible for shuttling NF-kappaB to the nucleus. Collectively, our data suggest that HTNV N protein can sequester NF-kappaB in the cytoplasm, thus inhibiting NF-kappaB activity. These findings, which were obtained using cells transfected with cDNA representing the HTNV N gene, were confirmed using HTNV-infected cells.

Expression of tumour necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy.

PubMed Central

Clausell, N.; de Lima, V. C.; Molossi, S.; Liu, P.; Turley, E.; Gotlieb, A. I.; Adelman, A. G.; Rabinovitch, M.

1995-01-01

BACKGROUND--The formation of coronary artery neointima experimentally induced in piglets after cardiac transplantation is related to an immune-inflammatory reaction associated with increased expression of T cells and inflammatory mediators (tumour necrosis factor alpha and interleukin 1 beta) and upregulation of fibronectin. In vivo blockade of tumour necrosis factor alpha in rabbits after cardiac transplantation results in reduced neointimal formation. The objective of this study was to investigate the hypothesis that coronary restenosis after atherectomy or percutaneous balloon angioplasty is associated with a similar inflammatory cascade initiated by mechanical injury. METHODS--Specimens taken at coronary atherectomy were analysed from 16 patients. Nine had had the procedure performed twice, firstly, to remove a primary lesion, and secondly, to remove a restenotic lesion. Seven had percutaneous balloon angioplasty after removal of restenotic tissue. Coronary atherectomy specimens were analysed by immunohistochemistry for the presence of T cells, macrophages, major histocompatibility complex II, interleukin 1 beta, tumour necrosis factor alpha, fibronectin, and the receptor for hyaluronan mediated motility. RESULTS--The groups were clinically and angiographically similar with equivalent lumens before and after atherectomy. Restenotic lesions had increased expression of tumour necrosis factor alpha and fibronectin compared with the primary lesions (P < 0.05 for both). There was also a trend towards a greater number of T cells and increased expression of interleukin 1 beta. CONCLUSIONS--Restenosis is associated with increased expression of tumour necrosis factor alpha and fibronectin, suggesting that an immune-inflammatory reaction probably contributes to neointimal formation and may represent a form of wound healing and repair secondary to mechanical injury. Images PMID:7626352

Human gingival fibroblasts express functional chemokine receptor CXCR6.

PubMed

Hosokawa, Y; Hosokawa, I; Ozaki, K; Nakae, H; Matsuo, T

2009-06-01

We have reported that CXCL16, a recently discovered transmembrane chemokine, is expressed in human gingival fibroblasts (HGF). However, it is not known whether HGF express CXCR6, the receptor for CXCL16, or CXCL16 affects HGF biology. We have shown that HGF expressed CXCR6 by reverse transcription-polymerase chain reaction and flow cytometric analysis. Moreover, we elucidated that tumour necrosis factor (TNF)-alpha and cytosine-guanine dinucleotide (CpG) DNA (Toll-like receptor-9 ligand) treatment enhanced CXCR6 expression by HGF. Interleukin (IL)-4, IL-13 and CpG DNA up-regulated CXCR6 expression by TNF-alpha-stimulated HGF. On the other hand, IL-1beta and interferon-gamma inhibited CXCR6 expression on TNF-alpha-treated HGF. CXCL16 treatment induced HGF proliferation and phosphorylation of extracellular regulated kinase (ERK) and protein kinase B (AKT) in HGF. In conclusion, HGF expressed CXCR6 functionally, because CXCL16 induced HGF proliferation and ERK and AKT phosphorylation in HGF. These results indicate that CXCL16 may play an important role in the pathogenesis and remodelling in periodontally diseased tissues.

[Effect of Xinmailong on hypoxia-inducible factor-1alpha expression in neonatal rats with asphyxia].

PubMed

Huang, Li-Xin; Wu, Xing-Heng

2009-08-01

Xinmailong, a compound extracted from Periplaneta americana, is used for the treatment of cardiovascular diseases. This study investigated the effects of Xinmailong on myocardial hypoxia-inducible factor-1alpha (HIF-1alpha) and plasma endothelin-1(ET-1) levels in neonatal rats with asphyxia and explored the protection mechanism of Xinmailong in hypoxia-ischemic myocardial injury. Seven-day-old Sprague-Dawley rats were randomly divided into three groups (n=30 each): sham-operated, asphyxia, Xinmailong-treated asphyxia. Each group was randomly subdivided into three groups according to the observed time points: 6 hrs, 24 hrs and 72 hrs. Xinmailong (5 mg/kg) was intraperitoneally injected to the rats in the Xinmailong-treated group five minutes before asphyxia. Myocardial HIF-1alpha expression, and plasma ET-1 and creatine kinase (CK) levels were measured. The histopathologic changes of the myocardium were observed by hematoxylin-eosin staining. Four rats died in the asphyxia group while only one died in the Xinmailong-treated group during the experiment. The plasma ET-1 and CK levels as well as myocardial HIF-1alpha expression increased at 6 hrs, reached a peak at 24 hrs, and declined at 72 hrs after asphyxia in the asphyxia group, being higher than that in the sham-operated group (P<0.01). Myocardial ischemia was observed in the three time points, and cell necrosis occurred at 24 hrs after asphyxia in the asphyxia group. Myocardial HIF-1alpha expression was positively correlated with plasma ET-1 levels (r=0.876, P<0.01). In the Xinmailong-treated group, plasma levels of CK and ET-1 as well as myocardial HIF-1alpha expression were significantly lower than those in the asphyxia group (P<0.01). Myocardial ischemia was alleviated and no cell necrosis was found in the Xinmailong-treated group. Asphyxia leads to increase in myocardial HIF-1alpha expression and plasma levels of ET-1 and CK. Xinmailong can reduce the myocardial expression of HIF-1alpha and decrease plasma ET-1 levels, thus alleviating hypoxia-ischemic myocardial injury.

Boric acid inhibits LPS-induced TNF-alpha formation through a thiol-dependent mechanism in THP-1 cells.

PubMed

Cao, Jun; Jiang, Liping; Zhang, Xiaomei; Yao, Xiaofeng; Geng, Chengyan; Xue, Xiangxin; Zhong, Laifu

2008-01-01

Oxidative stress plays an important role during inflammatory diseases and antioxidant administration to diminish oxidative stress may arrest inflammatory processes. Boron has been implicated to modulate certain inflammatory mediators and regulate inflammatory processes. Here we investigated the role of the tripeptide glutathione (GSH) in modulating the effects of boric acid (BA) on lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) formation in THP-1 monocytes. Interestingly, we found that BA had no significant effects on both TNF-alpha production and intracellular GSH contents, whereas it could inhibit LPS-induced TNF-alpha formation and ameliorated the d,l-buthionine-S,R-sulfoximine (BSO)-induced GSH depletion. Twenty-four hour incubation with BSO induced a decrease of the intracellular GSH and an increase of TNF-alpha. Treatment with N-acetyl-l-cysteine (NAC) did not significantly increase intracellular content of GSH but significantly reduced the secretion of TNF-alpha. BSO-pretreatment for 24h enhanced the LPS-induced secretion and mRNA expression of TNF-alpha further. BA inhibited LPS-stimulated TNF-alpha formation was also seen after GSH depletion by BSO. These results indicate that BA may have anti-inflammatory effect in the LPS-stimulated inflammation and the effect of BA on TNF-alpha secretion may be induced via a thiol-dependent mechanism.

Dietary α-Mangostin Provides Protective Effects against Acetaminophen-Induced Hepatotoxicity in Mice via Akt/mTOR-Mediated Inhibition of Autophagy and Apoptosis.

PubMed

Yan, Xiao-Tong; Sun, Yin-Shi; Ren, Shen; Zhao, Li-Chun; Liu, Wen-Cong; Chen, Chen; Wang, Zi; Li, Wei

2018-05-01

Acetaminophen overdose-induced hepatotoxicity is the most common cause of acute liver failure in many countries. Previously, alpha-mangostin (α-MG) has been confirmed to exert protective effects on a variety of liver injuries, but the protective effect on acetaminophen-induced acute liver injury (ALI) remains largely unknown. This work investigated the regulatory effect and underlying cellular mechanisms of α-MG action to attenuate acetaminophen-induced hepatotoxicity in mice. The increased serum aminotransferase levels and glutathione (GSH) content and reduced malondialdehyde (MDA) demonstrated the protective effect of α-MG against acetaminophen-induced hepatotoxicity. In addition, α-MG pretreatment inhibited increases in tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) caused by exposure of mice to acetaminophen. In liver tissues, α-MG inhibited the protein expression of autophagy-related microtubule-associated protein light chain 3 (LC3) and BCL2/adenovirus E1B protein-interacting protein 3 (BNIP3). Western blotting analysis of liver tissues also proved evidence that α-MG partially inhibited the activation of apoptotic signaling pathways via increasing the expression of Bcl-2 and decreasing Bax and cleaved caspase 3 proteins. In addition, α-MG could in part downregulate the increase in p62 level and upregulate the decrease in p-mTOR, p-AKT and LC3 II /LC3 I ratio in autophagy signaling pathways in the mouse liver. Taken together, our findings proved novel perspectives that detoxification effect of α-MG on acetaminophen-induced ALI might be due to the alterations in Akt/mTOR pathway in the liver.

Role of hypoxia-inducible factor-{alpha} in hepatitis-B-virus X protein-mediated MDR1 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Hyo-Kyung; Han, Chang Yeob; Cheon, Eun-Pa

2007-06-01

The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and induced the nuclear translocation of C/EBP{beta}. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1{alpha} siRNAmore » but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1{alpha} activation, and suggest HIF-1{alpha} for the therapeutic target of HBV-mediated chemoresistance.« less

Flavocoxid, a dual inhibitor of cyclooxygenase and 5-lipoxygenase, blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages.

PubMed

Altavilla, D; Squadrito, F; Bitto, A; Polito, F; Burnett, B P; Di Stefano, V; Minutoli, L

2009-08-01

The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated. LPS-stimulated (1 microg.mL(-1)) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32-128 microg.mL(-1)) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein kappaB-alpha (IkappaB-alpha) levels were evaluated by Western blot analysis. Nuclear factor kappaB (NF-kappaB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-alpha (TNF-alpha) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated. LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 microg.mL(-1)) significantly inhibited COX-2 (LPS = 18 +/- 2.1; flavocoxid = 3.8 +/- 0.9 integrated intensity), 5-LOX (LPS = 20 +/- 3.8; flavocoxid = 3.1 +/- 0.8 integrated intensity) and iNOS expression (LPS = 15 +/- 1.1; flavocoxid = 4.1 +/- 0.4 integrated intensity), but did not modify COX-1 expression. PGE(2) and LTB(4) levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IkappaB-alpha protein (LPS = 1.9 +/- 0.2; flavocoxid = 7.2 +/- 1.6 integrated intensity), blunted increased NF-kappaB binding activity (LPS = 9.2 +/- 2; flavocoxid = 2.4 +/- 0.7 integrated intensity) and the enhanced TNF-alpha mRNA levels (LPS = 8 +/- 0.9; flavocoxid = 1.9 +/- 0.8 n-fold/beta-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages. Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.

Inhibition of liver fibrosis by solubilized coenzyme Q10: Role of Nrf2 activation in inhibiting transforming growth factor-beta1 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Hoo-Kyun; Pokharel, Yuba Raj; Lim, Sung Chul

2009-11-01

Coenzyme Q10 (CoQ10), an endogenous antioxidant, is important in oxidative phosphorylation in mitochondria. It has anti-diabetic and anti-cardiovascular disease effects, but its ability to protect against liver fibrosis has not been studied. Here, we assessed the ability of solubilized CoQ10 to improve dimethylnitrosamine (DMN)-induced liver fibrogenesis in mice. DMN treatments for 3 weeks produced a marked liver fibrosis as assessed by histopathological examination and tissue 4-hydroxyproline content. Solubilized CoQ10 (10 and 30 mg/kg) significantly inhibited both the increases in fibrosis score and 4-hydroxyproline content induced by DMN. Reverse transcription-polymerase chain reaction and Western blot analyses revealed that solubilized CoQ10 inhibitedmore » increases in the transforming growth factor-beta1 (TGF-beta1) mRNA and alpha-smooth muscle actin (alpha-SMA) protein by DMN. Interestingly, hepatic glutamate-cysteine ligase (GCL) and glutathione S-transferase A2 (GSTA2) were up-regulated in mice treated with CoQ10. Solubilized CoQ10 also up-regulated antioxidant enzymes such as catalytic subunits of GCL and GSTA2 via activating NF-E2 related factor2 (Nrf2)/antioxidant response element (ARE) in H4IIE hepatoma cells. Moreover, CoQ10's inhibition of alpha-SMA and TGF-beta1 expressions disappeared in Nrf2-null MEF cells. In contrast, Nrf2 overexpression significantly decreased the basal expression levels of alpha-SMA and TGF-beta1 in Nrf2-null MEF cells. These results demonstrated that solubilized CoQ10 inhibited DMN-induced liver fibrosis through suppression of TGF-beta1 expression via Nrf2/ARE activation.« less

Influence of Panax ginseng on Alpha-Adrenergic Receptor of Benign Prostatic Hyperplasia

PubMed Central

Kim, Su Kang; Chung, Joo-Ho; Lee, Byung-Cheol; Lee, Sang Won; Lee, Kang Hyo

2014-01-01

Purpose Benign prostatic hyperplasia (BPH) is the most common prostate problem in older men. The present study aimed to investigate the inhibitory effect of Panax ginseng C.A. Meyer (P. ginseng) on a rat model of testosterone-induced BPH. Methods The rats were divided into 3 groups (each group, n=10): control, testosterone-induced BPH (20 mg/kg, subcutaneous injection), and P. ginseng (200 mg/kg, orally) groups. After 4 weeks, all animals were sacrificed to examine the blood biochemical profiles, prostate volume, weight, histopathological changes, alpha-1D adrenergic receptor (Adra1d) mRNA expression, and epidermal growth factor receptor (EGFR) and B-cell CLL/lymphoma 2 (BCL2) protein expression. Results The group treated with P. ginseng showed significantly lesser prostate size and weight than the testosterone-induced BPH group. In addition, P. ginseng decreased the mRNA expression of Adra1d as well as the expression of EGFR and BCL2 in prostate tissue. Conclusions These results suggest that P. ginseng may inhibit the alpha-1-adrenergic receptor to suppress the development of BPH. PMID:25558416

Tumour necrosis factor-alpha-induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus.

PubMed

Garcia, J A; Ferreira, H L; Vieira, F V; Gameiro, R; Andrade, A L; Eugênio, F R; Flores, E F; Cardoso, T C

2017-06-01

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine-derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine-derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor-alpha-induced protein 8 (TNFAIP8). CDV replication-induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV-induced cancer therapy. © 2015 John Wiley & Sons Ltd.

PI3K/Akt contributes to increased expression of Toll-like receptor 4 in macrophages exposed to hypoxic stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, So Young; Jeong, Eunshil; Joung, Sun Myung

2012-03-16

Highlights: Black-Right-Pointing-Pointer Hypoxic stress-induced TLR4 expression is mediated by PI3K/Akt in macrophages. Black-Right-Pointing-Pointer PI3K/Akt regulated HIF-1 activation leading to TLR4 expression. Black-Right-Pointing-Pointer p38 mitogen-activated protein kinase was not involved in TLR4 expression by hypoxic stress. Black-Right-Pointing-Pointer Sulforaphane suppressed hypoxia-mediated TLR4 expression by inhibiting PI3K/Akt. -- Abstract: Toll-like receptors (TLRs) play critical roles in triggering immune and inflammatory responses by detecting invading microbial pathogens and endogenous danger signals. Increased expression of TLR4 is implicated in aggravated inflammatory symptoms in ischemic tissue injury and chronic diseases. Results from our previous study showed that TLR4 expression was upregulated by hypoxic stress mediated bymore » hypoxia-inducible factor-1 (HIF-1) at a transcriptional level in macrophages. In this study, we further investigated the upstream signaling pathway that contributed to the increase of TLR4 expression by hypoxic stress. Either treatment with pharmacological inhibitors of PI3K and Akt or knockdown of Akt expression by siRNA blocked the increase of TLR4 mRNA and protein levels in macrophages exposed to hypoxia and CoCl{sub 2}. Phosphorylation of Akt by hypoxic stress preceded nuclear accumulation of HIF-1{alpha}. A PI3K inhibitor (LY294002) attenuated CoCl{sub 2}-induced nuclear accumulation and transcriptional activation of HIF-1{alpha}. In addition, HIF-1{alpha}-mediated upregulation of TLR4 expression was blocked by LY294002. Furthermore, sulforaphane suppressed hypoxia- and CoCl{sub 2}-induced upregulation of TLR4 mRNA and protein by inhibiting PI3K/Akt activation and the subsequent nuclear accumulation and transcriptional activation of HIF-1{alpha}. However, p38 was not involved in HIF-1{alpha} activation and TLR4 expression induced by hypoxic stress in macrophages. Collectively, our results demonstrate that PI3K/Akt contributes to hypoxic stress-induced TLR4 expression at least partly through the regulation of HIF-1 activation. These reveal a novel mechanism for regulation of TLR4 expression upon hypoxic stress and provide a therapeutic target for chronic diseases related to hypoxic stress.« less

Constitutive activation of NF-kappa B and secretion of interleukin-8 induced by the G protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus involve G alpha(13) and RhoA.

PubMed

Shepard, L W; Yang, M; Xie, P; Browning, D D; Voyno-Yasenetskaya, T; Kozasa, T; Ye, R D

2001-12-07

The Kaposi's sarcoma herpesvirus (KSHV) open reading frame 74 encodes a G protein-coupled receptor (GPCR) for chemokines. Exogenous expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi's sarcoma. We show here that expression of KSHV-GPCR in transfected cells results in constitutive transactivation of nuclear factor kappa B (NF-kappa B) and secretion of interleukin-8, and this response involves activation of G alpha(13) and RhoA. The induced expression of a NF-kappa B luciferase reporter was partially reduced by pertussis toxin and the G beta gamma scavenger transducin, and enhanced by co-expression of G alpha(13) and to a lesser extent, G alpha(q). These results indicate coupling of KSHV-GPCR to multiple G proteins for NF-kappa B activation. Expression of KSHV-GPCR led to stress fiber formation in NIH 3T3 cells. To examine the involvement of the G alpha(13)-RhoA pathway in KSHV-GPCR-mediated NF-kappa B activation, HeLa cells were transfected with KSHV-GPCR alone and in combination with the regulator of G protein signaling (RGS) from p115RhoGEF or a dominant negative RhoA(T19N). Both constructs, as well as the C3 exoenzyme from Clostritium botulinum, partially reduced NF-kappa B activation by KSHV-GPCR, and by a constitutively active G alpha(13)(Q226L). KSHV-GPCR-induced NF-kappa B activation is accompanied by increased secretion of IL-8, a function mimicked by the activated G alpha(13) but not by an activated G alpha(q)(Q209L). These results suggest coupling of KSHV-GPCR to the G alpha(13)-RhoA pathway in addition to other G proteins.

Involvement of estrogen receptor variant ER-alpha36, not GPR30, in nongenomic estrogen signaling.

PubMed

Kang, Lianguo; Zhang, Xintian; Xie, Yan; Tu, Yaping; Wang, Dong; Liu, Zhenming; Wang, Zhao-Yi

2010-04-01

Accumulating evidence suggested that an orphan G protein-coupled receptor (GPR)30, mediates nongenomic responses to estrogen. The present study was performed to investigate the molecular mechanisms underlying GPR30 function. We found that knockdown of GPR30 expression in breast cancer SK-BR-3 cells down-regulated the expression levels of estrogen receptor (ER)-alpha36, a variant of ER-alpha. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-alpha36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-alpha36 via an activator protein 1 binding site. Both 17beta-estradiol (E2) and G1, a compound reported to be a selective GPR30 agonist, increased the phosphorylation levels of the MAPK/ERK1/2 in SK-BR-3 cells, which could be blocked by an anti-ER-alpha36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-alpha36, such as transcription activation activity of a VP16-ER-alpha36 fusion protein and activation of the MAPK/ERK1/2 in ER-alpha36-expressing cells. ER-alpha36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca(2+) mobilization only in ER-alpha36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-alpha36 expression. The selective G protein-coupled receptor (GPR)30 agonist G1 actually interacts with ER-alpha36. Thus, the ER-alpha variant ER-alpha36, not GPR30, is involved in nongenomic estrogen signaling.

Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Eric; Jakinovich, Paul; Bae, Aekyung

Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1}more » knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a suppressor of PKC activity.« less

Inhibition of tumor necrosis factor-{alpha}-induced expression of adhesion molecules in human endothelial cells by the saponins derived from roots of Platycodon grandiflorum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Ji Young; Kim, Dong Hee; Kim, Hyung Gyun

2006-01-15

Adhesion molecules play an important role in the development of atherogenesis and are produced by endothelial cells after being stimulated with various inflammatory cytokines. This study examined the effect of saponins that were isolated from the roots of Platycodon grandiflorum A. DC (Campanulaceae), Changkil saponins (CKS), on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. CKS significantly inhibited the TNF{alpha}-induced increase in monocyte adhesion to endothelial cells as well as decreased the protein and mRNA expression levels of vascular adhesion molecule-1 and intercellular cell adhesion molecule-1 on endothelial cells. Furthermore, CKS significantly inhibited themore » TNF{alpha}-induced production of intracellular reactive oxygen species (ROS) and activation of NF-{kappa}B by preventing I{kappa}B degradation and inhibiting I{kappa}B kinase activity. Overall, CKS has anti-atherosclerotic and anti-inflammatory activity, which is least in part the result of it reducing the cytokine-induced endothelial adhesion to monocytes by inhibiting intracellular ROS production, NF-{kappa}B activation, and cell adhesion molecule expression in endothelial cells.« less

The potential antifibrotic impact of apocynin and alpha-lipoic acid in concanavalin A-induced liver fibrosis in rats: Role of NADPH oxidases 1 and 4.

PubMed

Fayed, Mostafa R; El-Naga, Reem N; Akool, El-Sayed; El-Demerdash, Ebtehal

2018-01-01

Liver fibrosis results from chronic inflammation that precipitates excessive accumulation of extracellular matrix. Oxidative stress is involved in its pathogenesis. This study aimed to elucidate the potential antifibrotic effect of the NADPH oxidase (NOX) inhibitor, apocynin against concanavalin A (ConA)-induced immunological model of liver fibrosis, and to investigate the ability of the antioxidant, alpha-lipoic acid (α-LA) to potentiate this effect. Rats were treated with apocynin and/or α-LA for six weeks. Hepatotoxicity indices, oxidative stress, insulin, NOXs, inflammatory and liver fibrosis markers were assessed. Treatment of animals with apocynin and α-LA significantly ameliorated the changes in liver functions and histopathological architecture induced by ConA. Liver fibrosis induced by ConA was evident where alpha-smooth muscle actin and transforming growth factor- beta1 were elevated, which was further confirmed by Masson's trichrome stain and increased hydroxyproline. Co-treatment with apocynin and α-LA significantly reduced their expression. Besides, apocynin and α-LA significantly ameliorated oxidative stress injury evoked by ConA, as evidenced by enhancing reduced glutathione content, antioxidant enzymes activities and decreasing lipid peroxides. ConA induced a significant elevation in serum insulin level and inflammatory markers; tumor necrosis factor-alpha, interleukin-6 and nuclear factor kappa b. Furthermore, the mRNA tissue expression of NOXs 1 and 4 was found to be elevated in the ConA group. All these elevations were significantly reduced by apocynin and α-LA co-treatment. These findings indicate that using apocynin and α-LA in combination possess marked antifibrotic effects, and that NOX enzymes are partially involved in the pathogenesis of ConA-induced liver fibrosis.

Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miettinen, Johanna A., E-mail: johanna.miettinen@oulu.fi; Pietilae, Mika; Salonen, Riikka J.

Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-{alpha}) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-{alpha} exposure on MSCs derived from human bone marrow. We found,more » as expected, that cell proliferation was significantly enhanced during TNF-{alpha} exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-{alpha} exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-{alpha} exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-{alpha} exposure, which might influence MSC differentiation stage and capacity.« less

The suppression of radiation-induced NF-{kappa}B activity by dexamethasone correlates with increased cell death in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nam, Seon Young; Chung, Hee-Yong

2005-10-21

In this study, we show that dexamethasone treatment increases ionizing radiation-induced cell death by inducing the inhibitory {kappa}B{alpha} (I{kappa}B{alpha}) pathway in mice. The effect of dexamethasone on radiation-induced cell death was assessed by changes in total spleen cellularity and bone marrow colony-forming unit-granulocyte-macrophage (CFU-GM) contents after total body irradiation. While in vivo treatment of mice with dexamethasone alone (1 mg/kg/day, for 2 days) failed to elicit cell death in spleen cells, the combined treatment with dexamethasone (1 mg/kg/day, for 2 days) and {gamma}-rays (1 or 5 Gy) caused a 50-80% reduction in total cellularity in spleen and CFU-GM contents inmore » bone marrow. These results demonstrate that dexamethasone has a synergistic effect on radiation-induced cellular damages in vivo. Immunoblot analysis showed that dexamethasone treatment significantly increases I{kappa}B{alpha} expression in the spleens of irradiated mice. In addition, the dexamethasone treatment significantly reduced radiation-induced nuclear translocation of the nucleus factor-{kappa}B in the spleens of irradiated mice. These results indicate that dexamethasone treatment in vivo may increase radiation-induced cell damages by increasing I{kappa}B{alpha} expression in hematopoietic organs such as spleen and bone marrow.« less

The effect of Saccharomyces boulardii on human colon cells and inflammation in rats with trinitrobenzene sulfonic acid-induced colitis.

PubMed

Lee, Sang Kil; Kim, Youn Wha; Chi, Sung-Gil; Joo, Yeong-Shil; Kim, Hyo Jong

2009-02-01

Saccharomyces boulardii (S. boulardii) has beneficial effects in the treatment of intestinal inflammation; however, little is known about the mechanisms by which these effects occur. We investigated the effects of S. boulardii on the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and interleukin-8 (IL-8), using human HT-29 colonocytes and a rat model of trinitrobenzene sulfonic acid (TNBS)-induced colitis. The effect of S. boulardii on gene expression was assessed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), and Northern blot and Western blot assays. Pharmacological inhibitors for various signaling pathways were used to determine the signaling pathways implicated in the S. boulardii regulation of PPAR-gamma and IL-8. We found that S. boulardii up-regulated and down-regulated PPAR-gamma and IL-8 expression at the transcription level, both in vitro and in vivo (P < 0.05, respectively). Saccharomyces boulardii blocked tumor necrosis factor-alpha (TNF-alpha) regulation of PPAR-gamma and IL-8 through disruption of TNF-alpha-mediated nuclear factor kappa B (NF-kappaB) activation. Furthermore, S. boulardii suppressed colitis and expression of pro-inflammatory cytokine genes in vivo (P < 0.05, respectively). Our study demonstrated that S. boulardii reduces colonic inflammation and regulates inflammatory gene expression.

Anti-inflammatory effects of compounds alpha-humulene and (-)-trans-caryophyllene isolated from the essential oil of Cordia verbenacea.

PubMed

Fernandes, Elizabeth S; Passos, Giselle F; Medeiros, Rodrigo; da Cunha, Fernanda M; Ferreira, Juliano; Campos, Maria M; Pianowski, Luiz F; Calixto, João B

2007-08-27

This study evaluated the anti-inflammatory properties of two sesquiterpenes isolated from Cordia verbenacea's essential oil, alpha-humulene and (-)-trans-caryophyllene. Our results revealed that oral treatment with both compounds displayed marked inhibitory effects in different inflammatory experimental models in mice and rats. alpha-humulene and (-)-trans-caryophyllene were effective in reducing platelet activating factor-, bradykinin- and ovoalbumin-induced mouse paw oedema, while only alpha-humulene was able to diminish the oedema formation caused by histamine injection. Also, both compounds had important inhibitory effects on the mouse and rat carrageenan-induced paw oedema. Systemic treatment with alpha-humulene largely prevented both tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) generation in carrageenan-injected rats, whereas (-)-trans-caryophyllene diminished only TNFalpha release. Furthermore, both compounds reduced the production of prostaglandin E(2) (PGE(2)), as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) expression, induced by the intraplantar injection of carrageenan in rats. The anti-inflammatory effects of alpha-humulene and (-)-trans-caryophyllene were comparable to those observed in dexamethasone-treated animals, used as positive control drug. All these findings indicate that alpha-humulene and (-)-trans-caryophyllene, derived from the essential oil of C. verbenacea, might represent important tools for the management and/or treatment of inflammatory diseases.

Granulocyte colony-stimulating-factor-induced psoriasiform dermatitis resembles psoriasis with regard to abnormal cytokine expression and epidermal activation.

PubMed

Mössner, R; Beckmann, I; Hallermann, C; Neumann, C; Reich, K

2004-06-01

Psoriasis is a chronic inflammatory skin disorder characterized by accumulation of Th1-type T cells and neutrophils, regenerative keratinocyte proliferation and differentiation, and enhanced epidermal production of antimicrobial peptides. The underlying cause is unknown, but there are some similarities with the immunologic defense program against bacteria. Development of psoriasiform skin lesions has been reported after administration of granulocyte colony-stimulating factor (G-CSF), a cytokine induced in monocytes by bacterial antigens. To further investigate the relation between this type of cytokine-induced dermatitis and psoriasis, we analyzed the cutaneous cytokine profile [tumor necrosis factor-alpha (TNF-alpha), interferon-gamma, transforming growth factor-beta1 (TGF-beta1), interleukin-10 (IL-10), IL-12p35 and p40, and IL-8] and expression of markers of epidermal activation [Ki-67, cytokeratin-16, major histocompatibility complex (MHC) class II, intercellular adhesion molecule-1 (ICAM-1)] in a patient who developed G-CSF-induced psoriasiform dermatitis by using quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistology. The histologic picture resembled psoriasis with regard to epidermal hyperparakeratosis and the accumulation of lymphocytes in the upper corium. CD8(+) T cells were found to infiltrate the epidermis which was associated with an aberrant expression of Ki-67, cytokeratin-16, MHC class II, and ICAM-1 on adjacent keratinocytes. As compared to normal skin (n = 7), there was an increased expression of TNF-alpha, IL-12p40, and IL-8, a decreased expression of TGF-beta1, and a lack of IL-10, similar to the findings in active psoriasis (n = 8). Therefore, G-CSF may cause a lymphocytic dermatitis that, similar to psoriasis, is characterized by a pro-inflammatory Th1-type cytokine milieu and an epidermal phenotype indicative of aberrant maturation and acquisition of non-professional immune functions.

[Regulatory role of hypoxia inducible factor-1 alpha in the changes of contraction of vascular smooth muscle cell induced by hypoxia].

PubMed

Zhang, Yuan; Liu, Liang-ming; Ming, Jia; Yang, Guang-ming; Chen, Wei

2007-11-01

To observe the regulatory role and mechanism of hypoxia inducible factor-1 alpha (HIF-1 alpha) in the contractile changes of vascular smooth muscle cell (VSMC) induced by hypoxia. Cells were divided into three groups: normal, hypoxia and oligomycin treated groups. VSMC and vascular endothelial cell (VEC) were co-cultured in Transwell models with the hypoxic time of 0, 0.5, 1, 2, 3, 4 and 6 hours respectively. The contractile response of VSMC to norepinephrine were determined by measuring the fluorescent infiltration rate in the lower chamber. The mRNA expression of HIF-1 alpha, endothelial-nitric oxide synthase (eNOS), inducible-nitric oxide synthase(iNOS), heme oxygenase-1 (HO-1) and cyclooxygenase-2 (COX-2) were determined by reverse transcription-polymerase chain reaction (RT-PCR). VSMC contraction was increased at the early stage of hypoxia with the 1.53-fold increase at 0.5 hour as compared to the normal group (P<0 .01), and decreased gradually at the prolonged period of hypoxia with the drop of 30% at 6 hours as compared to the normal group (P<0.05). Oligomycin treatment significantly inhibited the increase of VSMC contraction at early stage, while improved it at late hypoxic period with the 6 hours increase of 12.8% (P<0.05). HIF-1 alpha, iNOS, COX-2 and HO-1 mRNA exhibited a time-dependent increase following hypoxia, and peaked at 6, 2, 3 and 4 hours respectively, they were increased 1.62, 3.23, 2.26 and 2.86-folds as compared with normal group (all P<0.01). iNOS, COX-2 and HO-1 mRNA expression were fluctuated in the normal range following oligomycin administration (all P>0.05). Hypoxia can elicit a biphasic changes of VSMC contraction, and HIF-1 alpha seems to play an important role in the regulation of VSMC contraction induced by hypoxia by regulating eNOS, iNOS, COX-2 and HO-1 expression.

Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

PubMed Central

1994-01-01

Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect. PMID:7964480

Different protective actions of losartan and tempol on the renal inflammatory response to acute sodium overload.

PubMed

Rosón, María I; Della Penna, Silvana L; Cao, Gabriel; Gorzalczany, Susana; Pandolfo, Marcela; Toblli, Jorge E; Fernández, Belisario E

2010-07-01

The aim of this work was to study the role of local intrarenal angiotensin II (Ang II) and the oxidative stress in the up-regulation of pro-inflammatory cytokines expression observed in rats submitted to an acute sodium overload. Sprague-Dawley rats were infused for 2 h with isotonic saline solution (Control group) and with hypertonic saline solution alone (Na group), plus the AT1 receptor antagonist losartan (10 mg kg(-1) in bolus) (Na-Los group), or plus the superoxide dismutase mimetic tempol (0.5 mg min(-1) kg(-1)) (Na-Temp group). Mean arterial pressure, glomerular filtration rate, and fractional sodium excretion (FE(Na)) were measured. Ang II, NF-kappaB, hypoxia inducible factor-1 alpha (HIF-1 alpha), transforming growth factor beta1 (TGF-beta1), smooth muscle actin (alpha-SMA), endothelial nitric oxide synthase (eNOS), and RANTES renal expression was evaluated by immunohistochemistry. Ang II, NF-kappaB, and TGF-beta1 and RANTES early inflammatory markers were overexpressed in Na group, accompanied by enhanced HIF-1 alpha immunostaining, lower eNOS expression, and unmodified alpha-SMA. Losartan and tempol increased FE(Na) in sodium overload group. Although losartan reduced Ang II and NF-kappaB staining and increased eNOS expression, it did not restore HIF-1 alpha expression and did not prevent inflammation. Conversely, tempol increased eNOS and natriuresis, restored HIF-1 alpha expression, and prevented inflammation. Early inflammatory markers observed in rats with acute sodium overload is associated with the imbalance between HIF-1 alpha and eNOS expression. While both losartan and tempol increased natriuresis and eNOS expression, only tempol was effective in restoring HIF-1 alpha expression and down-regulating TGF-beta1 and RANTES expression. The protective role of tempol, but not of losartan, in the inflammatory response may be associated with its greater antioxidant effects. (c) 2010 Wiley-Liss, Inc.

Clotrimazole ameliorates intestinal inflammation and abnormal angiogenesis by inhibiting interleukin-8 expression through a nuclear factor-kappaB-dependent manner.

PubMed

Thapa, Dinesh; Lee, Jong Suk; Park, Su-Young; Bae, Yun-Hee; Bae, Soo-Kyung; Kwon, Jun Bum; Kim, Kyoung-Jin; Kwak, Mi-Kyoung; Park, Young-Joon; Choi, Han Gon; Kim, Jung-Ae

2008-11-01

Increased interleukin (IL)-8 plays an important role not only in activation and recruitment of neutrophils but also in inducing exaggerated angiogenesis at the inflamed site. In the present study, we investigated the fact that clotrimazole (CLT) inhibits intestinal inflammation, and the inhibitory action is mediated through suppression of IL-8 expression. In the trinitrobenzene sulfonic acid (TNBS)-induced rat colitis model, CLT dose-dependently protected from the TNBS-induced weight loss, colon ulceration, and myeloperoxidase activity increase. In the lesion site, CLT also suppressed the TNBS-induced angiogenesis, IL-8 expression, and nuclear factor (NF)-kappaB activation. In a cellular model of colitis using tumor necrosis factor (TNF)-alpha-stimulated HT29 colon epithelial cells, treatment with CLT significantly suppressed TNF-alpha-mediated IL-8 induction and NF-kappaB transcriptional activity revealed by a luciferase reporter gene assay. Furthermore, cotreatment with CLT and pyrrolidine dithiocarbamate, a NF-kappaB inhibitor, synergistically reduced the NF-kappaB transcriptional activity as well as IL-8 expression. In an in vitro angiogenesis assay, CLT suppressed IL-8-induced proliferation, tube formation, and invasion of human umbilical vein endothelial cells. The in vivo angiogenesis assay using chick chorioallantoic membrane also showed that CLT significantly inhibited the IL-8-induced formation of new blood vessels. Taken together, these results suggest that CLT may prevent the progression of intestinal inflammation by not only down-regulating IL-8 expression but also inhibiting the action of IL-8 in both colon epithelial and vascular endothelial cells during pathogenesis of intestinal inflammation.

Hypoxia-inducible factor 1-alpha release after intracoronary versus intramyocardial stem cell therapy in myocardial infarction.

PubMed

Gyöngyösi, Mariann; Hemetsberger, Rayyan; Posa, Aniko; Charwat, Silvia; Pavo, Noemi; Petnehazy, Ors; Petrasi, Zsolt; Pavo, Imre J; Hemetsberger, Hani; Benedek, Imre; Benedek, Teodora; Benedek, Istvan; Kovacs, Istvan; Kaun, Christoph; Maurer, Gerald

2010-04-01

We have investigated the effect of stem cell delivery on the release of hypoxia-inducible factor 1 alpha (HIF-1alpha) in peripheral circulation and myocardium in experimental myocardial ischemia. Closed-chest, reperfused myocardial infarction (MI) was created in domestic pigs. Porcine mesenchymal stem cells (MSCs) were cultured and delivered (9.8 +/- 1.2 x 10(6)) either percutaneously NOGA-guided transendocardially (Group IM) or intracoronary (Group IC) 22 +/- 4 days post-MI. Pigs without MSC delivery served as sham control (Group S). Plasma HIF-1alpha was measured at baseline, immediately post- and at follow-up (FUP; 2 h or 24 h) post-MSC delivery by ELISA kit. Myocardial HIF-1alpha expression of infarcted, normal myocardium, or border zone was determined by Western blot. Plasma level of HIF-1alpha increased immediately post-MI (from 278 +/- 127 to 631 +/- 375 pg/ml, p < 0.05). Cardiac delivery of MSCs elevated the plasma levels of HIF-1alpha significantly (p < 0.05) in groups IC and IM immediately post-MSC delivery, and returned to baseline level at FUP, without difference between the groups IC and IM. The myocardial tissue HIF-1alpha expression in the infarcted area was higher in Group IM than in Group IC or S (1,963 +/- 586 vs. 1,307 +/- 392 vs. 271 +/- 110 activity per square millimeter, respectively, p < 0.05), while the border zone contained similarly lower level of HIF-1alpha, but still significantly higher as compared with Group S. Trend towards increase in myocardial expression of HIF-1alpha was measured in Group IM at 24 h, in contrast to Group IC. In conclusion, both stem cell delivery modes increase the systemic and myocardial level of HIF-1alpha. Intramyocardial delivery of MSC seems to trigger the release of angiogenic HIF-1alpha more effectively than does intracoronary delivery.

Thalidomide in rat liver cirrhosis: blockade of tumor necrosis factor-alpha via inhibition of degradation of an inhibitor of nuclear factor-kappaB.

PubMed

Paul, Shelley Chireyath; Lv, Peng; Xiao, Yan-Jv; An, Ping; Liu, Shi-Quan; Luo, He-Sheng

2006-01-01

Thalidomide inhibited tumor necrosis factor-alpha (TNF-alpha) effectively in many trials. The aim of this study was to investigate the effect of thalidomide on the expression of nuclear factor-kappaB (NF-kappaB), inhibitor of NF-kappaB (IkappaB) and TNF-alpha in a rat model of liver cirrhosis. Liver cirrhosis was achieved by intraperitoneal injection of carbon tetrachloride thrice weekly, and thalidomide (10 or 100 mg/kg/day) was given daily by intragastric route for 8 weeks. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), prealbumin (PA), hyaluronic acid (HA) and laminin (LN), and hydroxyproline (HYP), NF-kappaBp65, alpha-smooth muscle actin (alpha-SMA) protein and TNF-alpha mRNA were studied in the liver, IkappaBalpha and TNF-alpha protein in the cytoplasm and NF-kappaBp65 protein in the nucleus. Compared with nontreated cirrhotic rats, the histopathology of rats given thalidomide (100 mg/kg) was significantly better. Serum ALT, AST, HA and LN and HYP content in the liver were significantly decreased and PA was elevated (p < 0.01) in this group; the expression of TNF-alpha mRNA and protein, NF-kappaBp65 and alpha-SMA were significantly decreased and IkappaBalpha protein was also elevated (p < 0.01). Thalidomide downregulates NF-kappaB-induced TNF-alpha and activates hepatic stellate cells (HSC) via inhibition of IkappaB degradation to prevent liver cirrhosis. Copyright 2006 S. Karger AG, Basel.

HIV-1, HTLV-I and the interleukin-2 receptor: insights into transcriptional control.

PubMed

Böhnlein, E; Lowenthal, J W; Wano, Y; Franza, B R; Ballard, D W; Greene, W C

1989-01-01

In this study, we present direct evidence for the binding of the inducible cellular protein, HIVEN86A, to a 12-bp element present in the IL-2R alpha promoter. This element shares significant sequence similarity with the NF-kappa B binding sites present in the HIV-1 and kappa immunoglobulin enhancers. Transient transfection studies indicate that this kappa B element is both necessary and sufficient to confer tax or mitogen inducibility to a heterologous promoter. As summarized schematically in Fig. 5, the findings suggest that the HIVEN86A protein may play a central role in the activation of cellular genes required for T-cell growth, specifically the IL-2R alpha gene. In addition, the induced HIVEN86A protein also binds to a similar sequence present in the HIV-1 LTR leading to enhanced viral gene expression and ultimately T-cell death. Thus, mitogen activation of the HIV-1 LTR appears to involve the same inducible transcription factor(s) that normally regulates IL-2R alpha gene expression and T-cell growth. These findings further underscore the importance of the state of T-cell activation in the regulation of HIV-1 replication. Our results also demonstrate that HIVEN86A is induced by the tax protein of HTLV-I. Thus, in HTLV-I infected cells, normally the tight control of the transient expression of the IL-2R alpha gene is lost. The constitutive high-level display of IL-2 receptors may play a role in leukemic transformation mediated by HTLV-I (ATL). Apparently by the same mechanism, the tax protein also activates the HIV-1 LTR through the induction of HIVEN86A.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiangiogenic activity of vitexicarpine in experimentally induced hepatocellular carcinoma: Impact on vascular endothelial growth factor pathway.

PubMed

Hassoun, Shimaa M; Abdel-Rahman, Noha; Eladl, Entsar I; El-Shishtawy, Mamdouh M

2017-06-01

Angiogenesis plays important roles in progression of hepatocellular carcinoma. The antiangiogenic mechanisms of vitexicarpine are not fully defined. Therefore, we conducted the following study to evaluate the antiangiogenic mechanism and antitumor activity of vitexicarpine in vivo model of hepatocellular carcinoma through modulation of vascular endothelial growth factor signaling pathway. Hepatocellular carcinoma was induced in Sprague Dawley rats by thioacetamide. Hepatocellular carcinoma was assessed by measuring serum alpha-fetoprotein and investigating liver sections stained with hematoxylin/eosin. Hepatocellular carcinoma rats were injected with vitexicarpine (150 mg/kg) for 2 weeks. Hepatic vascular endothelial growth factor was measured by enzyme-linked immunosorbent assay. Protein and expression of hepatic phospho-Ser473-AKT (p-AKT) and phospho-Tyr419-Src (p-Src) were determined. The apoptotic pathway was evaluated by assessment of protein expression of caspase-3. Vitexicarpine increased rats' survival time and decreased serum alpha-fetoprotein as well as it ameliorated fibrosis and massive hepatic tissue breakdown. It attenuated hepatocellular carcinoma-induced protein and gene expression of vascular endothelial growth factor, p-AKT, p-Src, and caspase-3. In conclusion, this study suggests that vitexicarpine possesses both antiangiogenic and antitumor activities through inhibition of vascular endothelial growth factor, p-AKT/AKT, and p-Src with subsequent inhibition of apoptotic pathway.

Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon-alpha and -gamma signal transduction pathways.

PubMed Central

Müller, M; Laxton, C; Briscoe, J; Schindler, C; Improta, T; Darnell, J E; Stark, G R; Kerr, I M

1993-01-01

Mutants in complementation group U3, completely defective in the response of all genes tested to interferons (IFNs) alpha and gamma, do not express the 91 and 84 kDa polypeptide components of interferon-stimulated gene factor 3 (ISGF3), a transcription factor known to play a primary role in the IFN-alpha response pathway. The 91 and 84 kDa polypeptides are products of a single gene. They result from differential splicing and differ only in a 38 amino acid extension at the C-terminus of the 91 kDa polypeptide. Complementation of U3 mutants with cDNA constructs expressing the 91 kDa product at levels comparable to those observed in induced wild-type cells completely restored the response to both IFN-alpha and -gamma and the ability to form ISGF3. Complementation with the 84 kDa component similarly restored the ability to form ISGF3 and, albeit to a lower level, the IFN-alpha response of all genes tested so far. It failed, however, to restore the IFN-gamma response of any gene analysed. The precise nature of the DNA motifs and combination of factors required for the transcriptional response of all genes inducible by IFN-alpha and -gamma remains to be established. The results presented here, however, emphasize the apparent general requirement of the 91 kDa polypeptide in the primary transcriptional response to both types of IFN. Images PMID:7693454

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castillo, Gaelle del; Murillo, Miguel M.; IDIBELL-Institut de Recerca Oncologica, Gran Via s/n, Km 2.7, 08907 L'Hospitalet, Barcelona

Transforming growth factor-beta (TGF-{beta}) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdes et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-{beta}-treated fetal hepatocytes: T{beta}T-FH). We prove that they secrete mitogenic and survival factors, as analyzed by themore » proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes T{beta}T-FH to die after serum withdrawal. T{beta}T-FH expresses high levels of transforming growth factor-alpha (TGF-{alpha}) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-{alpha} antibody restores the capacity of cells to die. TGF-{beta}, which is expressed by T{beta}T-FH, mediates up-regulation of TGF-{alpha} and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-{beta} confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Achanzar, William E.; Moyer, Carolyn F.; Marthaler, Laura T.

We previously reported prevention of urolithiasis and associated rat urinary bladder tumors by urine acidification (via diet acidification) in male rats treated with the dual peroxisome proliferator-activated receptor (PPAR){alpha}/{gamma} agonist muraglitazar. Because urine acidification could potentially alter PPAR signaling and/or cellular proliferation in urothelium, we evaluated urothelial cell PPAR{alpha}, PPAR{delta}, PPAR{gamma}, and epidermal growth factor receptor (EGFR) expression, PPAR signaling, and urothelial cell proliferation in rats fed either a normal or an acidified diet for 5, 18, or 33 days. A subset of rats in the 18-day study also received 63 mg/kg of the PPAR{gamma} agonist pioglitazone daily for themore » final 3 days to directly assess the effects of diet acidification on responsiveness to PPAR{gamma} agonism. Urothelial cell PPAR{alpha} and {gamma} expression and signaling were evaluated in the 18- and 33-day studies by immunohistochemical assessment of PPAR protein (33-day study only) and quantitative real-time polymerase chain reaction (qRT-PCR) measurement of PPAR-regulated gene expression. In the 5-day study, EGFR expression and phosphorylation status were evaluated by immunohistochemical staining and egfr and akt2 mRNA levels were assessed by qRT-PCR. Diet acidification did not alter PPAR{alpha}, {delta}, or {gamma} mRNA or protein expression, PPAR{alpha}- or {gamma}-regulated gene expression, total or phosphorylated EGFR protein, egfr or akt2 gene expression, or proliferation in urothelium. Moreover, diet acidification had no effect on pioglitazone-induced changes in urothelial PPAR{gamma}-regulated gene expression. These results support the contention that urine acidification does not prevent PPAR{gamma} agonist-induced bladder tumors by altering PPAR{alpha}, {gamma}, or EGFR expression or PPAR signaling in rat bladder urothelium.« less

Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor.

PubMed

Burkart, Anna D; Mukherjee, Abir; Mayo, Kelly E

2006-03-01

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

Acute exercise induces biphasic increase in respiratory mRNA in skeletal muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikeda, Shin-ichi; Kizaki, Takako; Haga, Shukoh

2008-04-04

Peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) promotes the expression of oxidative enzymes in skeletal muscle. We hypothesized that activation of the p38 MAPK (mitogen-activated protein kinase) in response to exercise was associated with exercise-induced PGC-1{alpha} and respiratory enzymes expression and aimed to demonstrate this under the physiological level. We subjected mice to a single bout of treadmill running and found that the exercise induced a biphasic increase in the expression of respiratory enzymes mRNA. The second phase of the increase was accompanied by an increase in PGC-1{alpha} protein, but the other was not. Administration of SB203580 (SB), an inhibitor ofmore » p38 MAPK, suppressed the increase in PGC-1{alpha} expression and respiratory enzymes mRNA in both phases. These data suggest that p38 MAPK is associated with the exercise-induced expression of PGC-1{alpha} and biphasic increase in respiratory enzyme mRNAs in mouse skeletal muscle under physiological conditions.« less

6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1alpha resulting in new vessel formation.

PubMed

Yoo, Y-G; Na, T-Y; Yang, W-K; Kim, H-J; Lee, I-K; Kong, G; Chung, J-H; Lee, M-O

2007-05-31

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. Previously, we reported that the orphan nuclear receptor Nur77 functions in stabilizing HIF-1alpha. Here, we demonstrate that 6-mercaptopurine (6-MP), an activator of the NR4A family members, enhances transcriptional activity of HIF-1. 6-MP enhanced the protein-level of HIF-1alpha as well as vascular endothelial growth factor (VEGF) in a dose- and time-dependent manner. The induction of HIF-1alpha was abolished by the transfection of either a dominant-negative Nur77 mutant or si-Nur77, indicating a critical role of Nur77 in the 6-MP action. The HIF-1alpha protein level remained up to 60 min in the presence of 6-MP when de novo protein synthesis was blocked by cycloheximide, suggesting that 6-MP induces stabilization of the HIF-1alpha protein. The fact that 6-MP decreased the association of HIF-1alpha with von Hippel-Lindau protein and the acetylation of HIF-1alpha, may explain how 6-MP induced stability of HIF-1alpha. Further, 6-MP induced the transactivation function of HIF-1alpha by recruiting co-activator cyclic-AMP-response-element-binding protein. Finally, 6-MP enhanced the expression of HIF-1alpha and VEGF, and the formation of capillary tubes in human umbilical vascular endothelial cells. Together, our results provide a new insight for 6-MP action in the stabilization of HIF-1alpha and imply a potential application of 6-MP in hypoxia-associated human vascular diseases.

The novel hypoxic cytotoxin, TX-2098 has antitumor effect in pancreatic cancer; possible mechanism through inhibiting VEGF and hypoxia inducible factor-1{alpha} targeted gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyake, Kotaro, E-mail: hif.panc@gmail.com; Nishioka, Masanori; Imura, Satoru

Tumor hypoxia has been considered to be a potential therapeutic target, because hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. In the present study, we investigated the antitumor effect of a novel hypoxic cytotoxin, 3-[2-hydroxyethyl(methyl)amino]-2-quinoxalinecarbonitrile 1,4-dioxide (TX-2098) in inhibiting the expression of hypoxia inducible factor-1{alpha} (HIF-1{alpha}), and consequently vascular endothelial cell growth factor (VEGF) expression in pancreatic cancer. The antitumor effects of TX-2098 under hypoxia were tested against various human pancreatic cancer cell lines using WST-8 assay. VEGF protein induced pancreatic cancer was determined on cell-free supernatant by ELISA. Moreover, nude mice bearingmore » subcutaneously (s.c.) or orthotopically implanted human SUIT-2 were treated with TX-2098. Tumor volume, survival and expression of HIF-1 and associated molecules were evaluated in treatment versus control groups. In vitro, TX-2098 inhibited the proliferation of various pancreatic cancer cell lines. In s.c model, tumors from nude mice injected with pancreatic cancer cells and treated with TX-2098 showed significant reductions in volume (P < 0.01 versus control). Quantitative real-time reverse transcription-PCR analysis revealed that TX-2098 significantly inhibited mRNA expression of the HIF-1 associated molecules, VEGF, glucose transporter 1 and Aldolase A (P < 0.01 versus control). These treatments also prolong the survival in orthotopic models. These results suggest that the effect of TX-2098 in pancreatic cancer might be correlated with the expression of VEGF and HIF-1 targeted molecules. -- Highlights: Black-Right-Pointing-Pointer We designed and synthesized novel hypoxic cytoxin, TX-2098. Black-Right-Pointing-Pointer TX-2098 inhibited the proliferation of human pancreatic cancer cells than TPZ. Black-Right-Pointing-Pointer TX-2098 reduced VEGF protein level than TPZ. Black-Right-Pointing-Pointer TX-2098 inhibited mRNA expression of VEGF, GLUT1 and Aldolase A, not HIF-1{alpha}. Black-Right-Pointing-Pointer TX-2098 improved the survival in orthotopic SUIT-2 xenograft model.« less

Involvement of matrix metalloproteinase-13 in stromal-cell-derived factor 1 alpha-directed invasion of human basal cell carcinoma cells.

PubMed

Chu, C-Y; Cha, S-T; Chang, C-C; Hsiao, C-H; Tan, C-T; Lu, Y-C; Jee, S-H; Kuo, M-L

2007-04-12

Basal cell carcinoma (BCC) is one of the most common skin neoplasms in humans and is usually characterized by local aggressiveness with little metastatic potential, although deep invasion, recurrence, and regional and distant metastases may occur. Here, we studied the mechanism of BCC invasion. We found that human BCC tissues and a BCC cell line had significant expression of CXCR4, which was higher in invasive than non-invasive BCC types. Further, of 19 recurrent tumors among 390 BCCs diagnosed during the past 12 years, 17/19 (89.5%) had high CXCR4 expression. We found that the CXCR4 ligand, stromal-cell-derived factor 1alpha (SDF-1alpha), directed BCC invasion and that this was mediated by time-dependent upregulation of mRNA expression and gelatinase activity of matrix metalloproteinase-13 (MMP-13). The transcriptional regulation of MMP-13 by SDF-1alpha was mediated by phosphorylation of extracellular signal-related kinase 1/2 and activation of the AP-1 component c-Jun. Finally, CXCR4-transfected BCC cells injected into nude mice induced aggressive BCCs that co-expressed CXCR4 and MMP-13. The identification of SDF-1alpha/CXCR4 as an important factor in BCC invasiveness may contribute insight into mechanisms involved in the aggressive potential of human BCC and may improve therapy for invasive BCCs.

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha.

PubMed

Gort, E H; van Haaften, G; Verlaan, I; Groot, A J; Plasterk, R H A; Shvarts, A; Suijkerbuijk, K P M; van Laar, T; van der Wall, E; Raman, V; van Diest, P J; Tijsterman, M; Vooijs, M

2008-03-06

Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that play a crucial role in oxygen homeostasis. Intratumoral hypoxia and genetic alterations lead to HIF activity, which is a hallmark of solid cancer and is associated with poor clinical outcome. HIF activity is regulated by an evolutionary conserved mechanism involving oxygen-dependent HIFalpha protein degradation. To identify novel components of the HIF pathway, we performed a genome-wide RNA interference screen in Caenorhabditis elegans, to suppress HIF-dependent phenotypes, like egg-laying defects and hypoxia survival. In addition to hif-1 (HIFalpha) and aha-1 (HIFbeta), we identified hlh-8, gska-3 and spe-8. The hlh-8 gene is homologous to the human oncogene TWIST1. We show that TWIST1 expression in human cancer cells is enhanced by hypoxia in a HIF-2alpha-dependent manner. Furthermore, intronic hypoxia response elements of TWIST1 are regulated by HIF-2alpha, but not HIF-1alpha. These results identify TWIST1 as a direct target gene of HIF-2alpha, which may provide insight into the acquired metastatic capacity of hypoxic tumors.

Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Qiang; The First Affiliated Hospital of Xiamen University, Xiamen; Jiang, Yuan

2011-06-17

Highlights: {yields} Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. {yields} FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. {yields} FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. {yields} FGFR4-ECD reduced tetracycline-induced fatty liver in mice. {yields} FGFR4-ECD partially restored tetracycline-repressed PPAR{alpha} expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whethermore » neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.« less

Epidermal growth factor receptor expression is related to post-mitotic events in cerebellar development: regulation by thyroid hormone.

PubMed

Carrasco, Emilce; Blum, Mariann; Weickert, Cynthia Shannon; Casper, Diana

2003-01-10

It has been established that thyroid hormone and neurotrophic factors both orchestrate developmental events in the brain. However, it is not clear how these two influences are related. In this study, we investigated the effects of thyroid hormone on cerebellar development and the coincident expression of transforming growth factor-alpha (TGF-alpha), a ligand in the epidermal growth factor (EGF) family, and the epidermal growth factor receptor (EGFR). Profiles of thyroid hormone expression were measured in postnatal animals and were found to peak at postnatal day 15 (P15). These levels dropped below detectable levels when mice were made hypothyroid with propylthiouracil (PTU). TGF-alpha and EGFR expression, as determined by RNAse protection assay, was maximal at P6 in normal animals, but remained low in hypothyroid animals, suggesting that thyroid hormone was responsible for their induction. In situ hybridization and immunohistochemical analysis of EGFR expression revealed that this receptor was present on granule cells within the inner zone of the external granule cell layer (EGL), suggesting that EGFR-ligands were not inducing granule cell proliferation. The persistence of EGFR expression on migrating granule cells and subsequent down-regulation of expression in the internal granule cell layer (IGL) implicates a role for EGFR-ligands in differentiation and/or migration. In hypothyroid animals, we observed a delayed progression of granule cell migration, consistent with the persistence of EGFR labeling in the EGL, and in the 'pile-up' of labeled cells at the interface between the molecular layer and the Purkinje cell layer. Taken together, these results implicate thyroid hormone in the coordinated expression of TGF-alpha and EGFR, which are positioned to play a role in post-mitotic developmental events in the cerebellum.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laouar, A.; Glesne, D.; Huberman, E.

The role of protein kinase C-{beta} (PKC-{beta}) in apoptosis induced by tumor necrosis factor (TNF)-{alpha} and anti-Fas monoclonal antibody (mAb) in the human myeloid HL-60 leukemia cell line was studied by using its variant HL-525, which is deficient in PKC-{beta}. In contrast to the parental HL-60 cells, HL-525 is resistant to TNF-{alpha}-induced apoptosis but sensitive to anti-Fas mAb-induced apoptosis. Both cell types expressed similar levels of the TNF-receptor I, whereas the Fas receptor was detected only in HL-525 cells. Transfecting the HL-525 cells with an expression vector containing PKC-{beta} reestablished their susceptibility to TNF-{alpha}-induced apoptosis. The apoptotic effect of TNF-{alpha}more » in HL-60 and the transfectants was abrogated by fumonisin, an inhibitor of ceramide generation, and by the peptide Ac-YVAD-BoMK, an inhibitor of caspase-1 and -4. Supplementing HL-525 cells with exogenous ceramides bypassed the PKC-{beta} deficiency and induced apoptosis, which was also restrained by the caspase-1 and -4 inhibitor. The apoptotic effect of anti-Fas mAb in HL-525 cells was abrogated by the antioxidants N-acetylcysteine and glutathione and by the peptide z-DEVD-FMK, an inhibitor of caspase-3 and -7. We suggest that TNF-{alpha}-induced apoptosis involves PKC-{beta} and then ceramide and, in turn, caspase-1 and/or -4, whereas anti-Fas mAb-induced apoptosis utilizes reactive oxygen intermediates and, in turn, caspase-3 and/or -7.« less

Thiazolidinediones inhibit REG I{alpha} gene transcription in gastrointestinal cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamauchi, Akiyo; Laboratory of Molecular Genetics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578; Department of Biochemistry, Nara Medical University, Kashihara 634-8521

2009-02-13

REG (Regenerating gene) I{alpha} protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPAR{gamma}-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG I{alpha} protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG I{alpha} gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPAR{gamma}, in PPAR{gamma}-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPAR{gamma}-antagonist GW9662. Although TZDs did not inhibit the REG I{alpha} gene promoter activity in PPAR{gamma}-non-expressingmore » cells, PPAR{gamma} overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG I{alpha} gene transcription through a PPAR{gamma}-dependent pathway. The TZD-induced REG I{alpha} mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG I{alpha} and PPAR{gamma}.« less

Molecular Signatures of Peripheral Blood Mononuclear Cells during Chronic Interferon-alpha Treatment: Relationship with Depression and Fatigue

PubMed Central

Felger, Jennifer C.; Cole, Steve W.; Pace, Thaddeus W. W.; Hu, Fang; Woolwine, Bobbi J.; Doho, Gregory H.; Raison, Charles L.; Miller, Andrew H.

2012-01-01

Background Interferon (IFN)-alpha treatment for infectious disease and cancer causes high rates of depression and fatigue, and has been used to investigate the impact of inflammatory cytokines on brain and behavior. However, little is known about the transcriptional impact of chronic IFN-alpha on immune cells in vivo and its relationship to IFN-alpha-induced behavioral changes. Methods Genome-wide transcriptional profiling was performed on peripheral blood mononuclear cells from 21 patients with chronic hepatitis C either awaiting IFN-alpha therapy (n=10) or at 12 weeks of IFN-alpha treatment (n=11). Results Significance analysis of microarray data identified 252 up-regulated and 116 down-regulated gene transcripts. Of up-regulated genes, 2'-5'-oligoadenylate synthetase 2 (OAS2), a gene linked to chronic fatigue syndrome (CFS), was the only gene that was differentially expressed in patients with IFN-alpha-induced depression/fatigue, and correlated with depression and fatigue scores at 12 weeks (r=0.80, p=0.003 and r=0.70, p=0.017, respectively). Promoter-based bioinformatic analyses linked IFN-alpha-related transcriptional alterations to transcription factors involved in myeloid differentiation, IFN-alpha signaling, AP1 and CREB/ATF pathways, which were derived primarily from monocytes and plasmacytoid dendritic cells. IFN-alpha-treated patients with high depression/fatigue scores demonstrated up-regulation of genes bearing promoter motifs for transcription factors involved in myeloid differentiation, IFN-alpha and AP1 signaling, and reduced prevalence of motifs for CREB/ATF, which has been implicated in major depression. Conclusions Depression and fatigue during chronic IFN-alpha administration were associated with alterations in the expression (OAS2) and transcriptional control (CREB/ATF) of genes linked to behavioral disorders including CFS and major depression, further supporting an immune contribution to these diseases. PMID:22152193

NMDA receptor dependent PGC-1alpha up-regulation protects the cortical neuron against oxygen-glucose deprivation/reperfusion injury.

PubMed

Luo, Yun; Zhu, Wenjing; Jia, Jia; Zhang, Chenyu; Xu, Yun

2009-09-01

The peroxisome proliferator activated receptor coactivator 1 alpha (PGC-1alpha) is a nuclear transcriptional coactivator that is widely expressed in the brain areas. Over-expression of PGC-1alpha can protect neuronal cells from oxidant-induced injury. The purpose of the current study is to investigate the role of PGC-1alpha in the oxygen (anoxia) deprivation (OGD) neurons. The PGC-1alpha mRNA and protein level between control and OGD neurons were examined by real-time PCR and Western blot. More PGC-1alpha expression was found in the OGD neurons compared with the normal group. Over-expression of PGC-1alpha suppressed cell apoptosis while inhibition of the PGC-1alpha expression induced cell apoptosis in OGD neurons. Furthermore, increase of PGC-1alpha resulted in activation of N-methyl-D-aspartate (NMDA) receptor, p38, and ERK mitogen-activated protein kinase (MAPK) pathway. The blocking of the NMDA receptor by its antagonists MK-801 reduced PGC-1alpha mRNA expression in OGD neurons, while NMDA itself can directly induce the expression of PGC-1alpha in neuronal cells. At the same time, PD98059 (ERK MAPK inhibitor) and SB203580 (P38 MAPK inhibitor) also prevented the up-regulation of PGC-1alpha in OGD neurons and MK801 can inhibit the expression of P38 and ERK MAPK. These data suggested that the expression of PGC-1alpha was up-regulated in OGD mice cortical neurons, which protected the neurons against OGD injury. Moreover, this effect was correlated to the NMDA receptor and the ERK and P38 MAPK pathway. The protective effect of PGC-1alpha on OGD cortical neurons may be useful for stroke therapy.

Hepatic effects of a methionine-choline-deficient diet in hepatocyte RXR{alpha}-null mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gyamfi, Maxwell Afari; Tanaka, Yuji; He Lin

Retinoid X receptor-{alpha} (RXR{alpha}) is an obligate partner for several nuclear hormone receptors that regulate important physiological processes in the liver. In this study the impact of hepatocyte RXR{alpha} deficiency on methionine and choline deficient (MCD) diet-induced steatosis, oxidative stress, inflammation, and hepatic transporters gene expression were examined. The mRNA of sterol regulatory element-binding protein (SREBP)-regulated genes, important for lipid synthesis, were not altered in wild type (WT) mice, but were increased 2.0- to 5.4-fold in hepatocyte RXR{alpha}-null (H-RXR{alpha}-null) mice fed a MCD diet for 14 days. Furthermore, hepatic mRNAs and proteins essential for fatty acid {beta}-oxidation were not alteredmore » in WT mice, but were decreased in the MCD diet-fed H-RXR{alpha}-null mice, resulting in increased hepatic free fatty acid levels. Cyp2e1 enzyme activity and lipid peroxide levels were induced only in MCD-fed WT mice. In contrast, hepatic mRNA levels of pro-inflammatory factors were increased only in H-RXR{alpha}-null mice fed the MCD diet. Hepatic uptake transporters Oatp1a1 and Oatp1b2 mRNA levels were decreased in WT mice fed the MCD diet, whereas the efflux transporter Mrp4 was increased. However, in the H-RXR{alpha}-null mice, the MCD diet only moderately decreased Oatp1a1 and induced both Oatp1a4 and Mrp4 gene expression. Whereas the MCD diet increased serum bile acid levels and alkaline phosphatase activity in both WT and H-RXR{alpha}-null mice, serum ALT levels were induced (2.9-fold) only in the H-RXR{alpha}-null mice. In conclusion, these data suggest a critical role for RXR{alpha} in hepatic fatty acid homeostasis and protection against MCD-induced hepatocyte injury.« less

A xanthine-based epithelium-dependent airway relaxant KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) increases respiratory performance and protects against tumor necrosis factor-alpha-induced tracheal contraction, involving nitric oxide release and expression of cGMP and protein kinase G.

PubMed

Lin, Rong-Jyh; Wu, Bin-Nan; Lo, Yi-Ching; An, Li-Mei; Dai, Zen-Kong; Lin, Young-Tso; Tang, Chao-Shun; Chen, Ing-Jun

2006-02-01

KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) was investigated in guinea pig tracheal smooth muscle. Intratracheal instillation of tumor necrosis factor (TNF)-alpha (0.01 mg/kg/300 microl) induced bronchoconstriction, increases of lung resistance, and decreases of dynamic lung compliance. Instillation of KMUP-3 (0.5-2.0 mg/kg) reversed this situation. In isolated trachea precontracted with carbachol, KMUP-3 (10-100 microM)-caused relaxations were attenuated by epithelium removal and by pretreatments with an inhibitor of K(+) channel, tetraethylammonium (10 mm); K(ATP) channel, glibenclamide (1 microM); voltage-dependent K(+) channel, 4-aminopyridine (100 microM); Ca(2+)-dependent K(+) channel, charybdotoxin (0.1 microM) or apamin (1 microM); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one (ODQ, 1 microM); nitric-oxide (NO) synthase, N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM); and adenylate cyclase, SQ 22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine] (100 microM). KMUP-3 (0.01-100 microM) induced increases of cGMP and cAMP in primary culture of tracheal smooth muscle cells (TSMCs). The increase in cGMP by KMUP-3 was reduced by ODQ and L-NAME; the increase in cAMP was reduced by SQ 22536. Western blot analysis indicated that KMUP-3 (1 microM) induced expression of protein kinase A (PKA)(ri) and protein kinase G (PKG)(1alpha 1beta) in TSMCs.SQ 22536 inhibited KMUP-3-induced expression of (PKA)(ri). On the contrary, ODQ inhibited KMUP-3-induced expression of PKG(1alpha 1beta) In epithelium-intact trachea, KMUP-3 increased the NO release. Activation of sGC, NO release, and inhibition of phosphodiesterases in TSMCs by KMUP-3 may result in increases of intracellular cGMP and cAMP, which subsequently activate PKG and PKA, efflux of K(+) ion, and associated reduction in Ca(2+) influx in vitro, indicating the action mechanism to protect against TNF-alpha-induced airway dysfunction in vivo.

Peroxisome proliferator-activated receptor-gamma co-activator 1alpha-mediated metabolic remodeling of skeletal myocytes mimics exercise training and reverses lipid-induced mitochondrial inefficiency.

PubMed

Koves, Timothy R; Li, Ping; An, Jie; Akimoto, Takayuki; Slentz, Dorothy; Ilkayeva, Olga; Dohm, G Lynis; Yan, Zhen; Newgard, Christopher B; Muoio, Deborah M

2005-09-30

Peroxisome proliferator-activated receptor-gamma co-activator 1alpha (PGC1alpha) is a promiscuous co-activator that plays a key role in regulating mitochondrial biogenesis and fuel homeostasis. Emergent evidence links decreased skeletal muscle PGC1alpha activity and coincident impairments in mitochondrial performance to the development of insulin resistance in humans. Here we used rodent models to demonstrate that muscle mitochondrial efficiency is compromised by diet-induced obesity and is subsequently rescued by exercise training. Chronic high fat feeding caused accelerated rates of incomplete fatty acid oxidation and accumulation of beta-oxidative intermediates. The capacity of muscle mitochondria to fully oxidize a heavy influx of fatty acid depended on factors such as fiber type and exercise training and was positively correlated with expression levels of PGC1alpha. Likewise, an efficient lipid-induced substrate switch in cultured myocytes depended on adenovirus-mediated increases in PGC1alpha expression. Our results supported a novel paradigm in which a high lipid supply, occurring under conditions of low PGC1alpha, provokes a disconnect between mitochondrial beta-oxidation and tricarboxylic acid cycle activity. Conversely, the metabolic remodeling that occurred in response to PGC1alpha overexpression favored a shift from incomplete to complete beta-oxidation. We proposed that PGC1alpha enables muscle mitochondria to better cope with a high lipid load, possibly reflecting a fundamental metabolic benefit of exercise training.

Peroxisome proliferator-activated receptor {alpha} agonist-induced down-regulation of hepatic glucocorticoid receptor expression in SD rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Xiang; Li Ming; Sun Weiping

2008-04-18

It was reported that glucocorticoid production was inhibited by fenofibrate through suppression of type-1 11{beta}-hydroxysteroid dehydrogenase gene expression in liver. The inhibition might be a negative-feedback regulation of glucocorticoid receptor (GR) activity by peroxisome proliferator-activated receptor alpha (PPAR{alpha}), which is quickly induced by glucocorticoid in the liver. However, it is not clear if GR expression is changed by fenofibrate-induced PPAR{alpha} activation. In this study, we tested this possibility in the liver of Sprague-Dawley rats. GR expression was reduced by fenofibrate in a time- and does-dependent manner. The inhibition was observed in liver, but not in fat and muscle. The corticosteronemore » level in the blood was increased significantly by fenofibrate. These effects of fenofibrate were abolished by PPAR{alpha} inhibitor MK886, suggesting that fenofibrate activated through PPAR{alpha}. In conclusion, inhibition of GR expression may represent a new molecular mechanism for the negative feedback regulation of GR activity by PPAR{alpha}.« less

Protein arginine methyltransferase 5 is an essential component of the hypoxia-inducible factor 1 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Ji-Hong; Choi, Yong-Joon; Cho, Chung-Hyun

2012-02-10

Highlights: Black-Right-Pointing-Pointer HIF-1{alpha} is expressed PRMT5-dependently in hypoxic cancer cells. Black-Right-Pointing-Pointer The HIF-1 regulation of hypoxia-induced genes is attenuated in PRMT5-knocked-down cells. Black-Right-Pointing-Pointer The de novo synthesis of HIF-1{alpha} depends on PRMT5. Black-Right-Pointing-Pointer PRMT5 is involved in the HIF-1{alpha} translation initiated by 5 Prime UTR of HIF-1{alpha} mRNA. -- Abstract: Protein arginine methyltransferase 5 (PRMT5) is an enzyme that transfers one or two methyl groups to the arginine residues of histones or non-histone proteins, and that plays critical roles in cellular processes as diverse as receptor signaling and gene expression. Furthermore, PRMT5 is highly expressed in tumors, where it maymore » be associated with tumor growth. Although much research has been conducted on PRMT5, little is known regarding its role in adaption to hypoxia. As hypoxia-inducible factor 1 (HIF-1) is a key player in hypoxic response, we examined the possible involvement of PRMT5 in the HIF-1 signaling pathway. Of the siRNAs targeting PRMT1-8, only PRMT5 siRNA attenuated the hypoxic induction of HIF-1{alpha} in A549 cells, and this result was reproducible in all three cancer cell lines examined. PRMT5 knock-down also repressed the promoter activities and the transcript levels of HIF-1-governed genes. Mechanistically, de novo synthesis of HIF-1{alpha} protein was reduced in PRMT5-knocked-down A549 cells, and this was rescued by PRMT5 restoration. In contrast, HIF-1{alpha} transcription, RNA processing, and protein stability were unaffected by PRMT5 knock-down. Furthermore, PRMT5 was found to be essential for the HIF-1{alpha} translation initiated by the 5 Prime UTR of HIF-1{alpha} mRNA. Given our results and previous reports, we believe that PRMT5 probably promotes tumor growth by stimulating cell proliferation and by participating in the construction of a tumor-favorable microenvironment via HIF-1 activation.« less

Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.

Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference mapmore » of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.« less

The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

PubMed

Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

2002-09-01

Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

Hepcidin regulation in wild-type and Hfe knockout mice in response to alcohol consumption: evidence for an alcohol-induced hypoxic response.

PubMed

Heritage, Mandy L; Murphy, Therese L; Bridle, Kim R; Anderson, Gregory J; Crawford, Darrell H G; Fletcher, Linda M

2009-08-01

Expression of Hamp1, the gene encoding the iron regulatory peptide hepcidin, is inappropriately low in HFE-associated hereditary hemochromatosis and Hfe knockout mice (Hfe(-/-)). Since chronic alcohol consumption is also associated with disturbances in iron metabolism, we investigated the effects of alcohol consumption on hepcidin mRNA expression in Hfe(-/-) mice. Hfe(-/-) and C57BL/6 (wild-type) mice were pair-fed either an alcohol liquid diet or control diet for up to 8 weeks. The mRNA levels of hepcidin and ferroportin were measured at the mRNA level by RT-PCR and protein expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was measured by western blot. Hamp1 mRNA expression was significantly decreased and duodenal ferroportin expression was increased in alcohol-fed wild-type mice at 8 weeks. Time course experiments showed that the decrease in hepcidin mRNA was not immediate, but was significant by 4 weeks. Consistent with the genetic defect, Hamp1 mRNA was decreased and duodenal ferroportin mRNA expression was increased in Hfe(-/-) mice fed on the control diet compared with wild-type animals and alcohol further exacerbated these effects. HIF-1alpha protein levels were elevated in alcohol-fed wild-type animals compared with controls. Alcohol may decrease Hamp1 gene expression independently of the HFE pathway possibly via alcohol-induced hypoxia.

Extract of corn silk (stigma of Zea mays) inhibits the tumour necrosis factor-alpha- and bacterial lipopolysaccharide-induced cell adhesion and ICAM-1 expression.

PubMed

Habtemariam, S

1998-05-01

Treatment of human endothelial cells with cytokines such as tumour necrosis factor-alpha (TNF) or E. coli lipopolysaccharide (LPS) induces the expression of several adhesion molecules and enhances leukocyte adhesion to endothelial cell surface. Interfering with this leukocyte adhesion or adhesion molecules upregulation is an important therapeutic target for the treatment of bacterial sepsis and various inflammatory diseases. In the course of screening marketed European anti-inflammatory herbal drugs for TNF antagonistic activity, a crude ethanolic extract of corn silk (stigma of Zea mays) exhibited significant activity. The extract at concentrations of 9-250 micrograms/ml effectively inhibited the TNF- and LPS-induced adhesiveness of EAhy 926 endothelial cells to monocytic U937 cells. Similar concentration ranges of corn silk extract did also block the TNF and LPS but not the phorbol 12-myristate 13-acetate-induced ICAM-1 expression on EAhy 926 endothelial cell surface. The extract did not alter the production of TNF by LPS-activated macrophages and failed to inhibit the cytotoxic activity of TNF. It is concluded that corn silk possesses important therapeutic potential for TNF- and LPS-mediated leukocyte adhesion and trafficking.

Gastroprotective effects of sulforaphane and thymoquinone against acetylsalicylic acid-induced gastric ulcer in rats.

PubMed

Zeren, Sezgin; Bayhan, Zulfu; Kocak, Fatma Emel; Kocak, Cengiz; Akcılar, Raziye; Bayat, Zeynep; Simsek, Hasan; Duzgun, Sukru Aydin

2016-06-15

Nonsteroidal anti-inflammatory drugs (NSAIDs) commonly cause gastric ulcers (GUs). We investigated the effects of sulforaphane (SF) and thymoquinone (TQ) in rats with acetylsalicylic acid (ASA)-induced GUs. Thirty-five male Wistar-Albino rats were divided into five groups: control; ASA; ASA with vehicle; ASA + SF; and ASA + TQ. Compounds were administered by oral gavage before GU induction. GUs were induced by intragastric administration of ASA. Four hours after GU induction, rats were killed and stomachs excised. Total oxidant status, total antioxidant status, total thiol, nitric oxide, asymmetric dimethylarginine, tumor necrosis factor-alpha levels, superoxide dismutase activity, and glutathione peroxidase activity in tissue were measured. Messenger RNA expression of dimethylarginine dimethylaminohydrolases, heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2, and nuclear factor kappa-light-chain-enhancer of activated B cells were analyzed. Renal tissues were evaluated by histopathologic and immunohistochemical means. SF and TQ reduced GU indices, apoptosis, total oxidant status, asymmetric dimethylarginine, and tumor necrosis factor-alpha levels, nuclear factor kappa-light-chain-enhancer of activated B cells, and inducible nitric oxide synthase expressions (P < 0.001, P = 0.001). Both examined compounds increased superoxide dismutase activity, glutathione peroxidase activity, total antioxidant status, total thiol, nitric oxide levels, endothelial nitric oxide synthase, dimethylarginine dimethylaminohydrolases, HO-1, nuclear factor erythroid 2-related factor 2, and HO-1 expressions (P < 0.001). These results suggest that pretreatment with SF or TQ can reduce ASA-induced GUs via anti-inflammatory, antioxidant, and antiapoptotic effects. These compounds may be useful therapeutic strategies to prevent the gastrointestinal adverse effects that limit nonsteroidal anti-inflammatory drugs use. Copyright © 2016 Elsevier Inc. All rights reserved.

Protein kinase C delta overexpressing transgenic mice are resistant to chemically but not to UV radiation-induced development of squamous cell carcinomas: a possible link to specific cytokines and cyclooxygenase-2.

PubMed

Aziz, Moammir H; Wheeler, Deric L; Bhamb, Bhushan; Verma, Ajit K

2006-01-15

Protein kinase C delta (PKCdelta), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the novel PKCs (delta, epsilon, and eta) expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately 8-fold) PKCdelta protein in basal epidermal cells and cells of the hair follicle are resistant to the development of both skin papillomas and squamous cell carcinoma (SCC) elicited by 7,12-dimethylbenz(a)anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion protocol. We now present that PKCdelta overexpression in transgenic mice failed to suppress the induction of SCC developed by repeated exposures to UV radiation (UVR), the environmental carcinogen linked to the development of human SCC. Both TPA and UVR treatment of wild-type mice (a) increased the expression of proliferating cell nuclear antigen (PCNA) and apoptosis; (b) stimulated the expression of cytokines tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte CSF (G-CSF); and (c) increased cyclooxygenase-2 (COX-2) expression and expression of phosphorylated Akt (p-Akt), p38, extracellular signal-regulated kinase-1 (ERK1), and ERK2. PKCdelta overexpression in transgenic mice enhanced TPA-induced but not UVR-induced apoptosis and suppressed TPA-stimulated but not UVR-stimulated levels of cell PCNA, cytokines (TNF-alpha, G-CSF, and GM-CSF), and the expression of COX-2, p-Akt, and p38. The results indicate that UVR-mediated signal transduction pathway to the induction of SCC does not seem to be sensitive to PKCdelta overexpression. The proapoptotic activity of PKCdelta coupled with its ability to suppress TPA-induced expression of proinflammatory cytokines, COX-2 expression, and the phosphorylation of Akt and p38 may play roles in the suppression of TPA-promoted development of SCC.

Contribution of HIF-1alpha or HIF-2alpha to erythropoietin expression: in vivo evidence based on chromatin immunoprecipitation.

PubMed

Yeo, Eun-Jin; Cho, Young-Suk; Kim, Myung-Suk; Park, Jong-Wan

2008-01-01

Circulating erythropoietin (EPO) is mainly produced by the kidneys and mediates erythrogenesis in bone marrow and nonhematopoietic cell survival. EPO is also produced in other tissues where it functions as a paracrine. Moreover, the hypoxic induction of EPO is known to be mediated by HIF-1alpha and HIF-2alpha, but it remains obscure as to which of these two mediators mainly contributes to EPO expression. Thus, we designed in vivo experiments to evaluate the contributions made by HIF-1alpha and HIF-2alpha to EPO expression. In mice exposed to mild whole body hypoxia, HIF-1alpha and HIF-2alpha were both induced in all tissues examined. However, EPO mRNA was expressed in kidney and brain, but not in liver and lung. Likewise, chromatin immunoprecipitation (CHIP) analyses demonstrated that HIF-1alpha or HIF-2alpha binding to the EPO gene increased under hypoxic conditions only in kidney and brain. A comparison of CHIP data and EPO mRNA levels suggested that, during mild hypoxia, renal EPO transcription is induced equally by HIF-1alpha and HIF-2alpha, but that brain EPO is mainly induced during hypoxia by HIF-2alpha. Thus, HIF-1alpha and HIF-2alpha appear to contribute to EPO expression tissue specifically.

Inhibition of dipeptidyl peptidase 4 regulates microvascular endothelial growth induced by inflammatory cytokines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takasawa, Wataru; Ohnuma, Kei; Hatano, Ryo

2010-10-08

Research highlights: {yields} TNF-{alpha} or IL-1{beta} induces EC proliferation with reduction of CD26 expression. {yields} CD26 siRNA or DPP-4 inhibition enhances TNF-{alpha} or IL-1{beta}-induced EC proliferation. {yields} Loss of CD26/DPP-4 enhances aortic sprouting induced by TNF-{alpha} or IL-1{beta}. {yields} Capillary formation induced by TNF-{alpha} or IL-1{beta} is enahced in the CD26{sup -/-} mice. -- Abstract: CD26/DPP-4 is abundantly expressed on capillary of inflamed lesion as well as effector T cells. Recently, CD26/dipeptidyl peptidase 4 (DPP-4) inhibition has been used as a novel oral therapeutic approach for patients with type 2 diabetes. While accumulating data indicate that vascular inflammation is amore » key feature of both micro- and macro-vascular complications in diabetes, the direct role of CD26/DPP-4 in endothelial biology is to be elucidated. We herein showed that proinflammatory cytokines such as tumor necrosis factor or interleukin-1 reduce expression of CD26 on microvascular endothelial cells, and that genetical or pharmacological inhibition of CD26/DPP-4 enhances endothelial growth both in vitro and in vivo. With DPP-4 inhibitors being used widely in the treatment of type 2 diabetes, our data strongly suggest that DPP-4 inhibition plays a pivotal role in endothelial growth and may have a potential role in the recovery of local circulation following diabetic vascular complications.« less

Bee venom inhibits hepatic fibrosis through suppression of pro-fibrogenic cytokine expression.

PubMed

Kim, Soo-Jung; Park, Ji-Hyun; Kim, Kyung-Hyun; Lee, Woo-Ram; Chang, Young-Chae; Park, Kwan-Kyu; Lee, Kwang-Gill; Han, Sang-Mi; Yeo, Joo-Hong; Pak, Sok Cheon

2010-01-01

Bee venom (BV) has a long tradition of use for the control of pain and inflammation in various chronic diseases. Carbon tetrachloride (CCl4) is known to induce hepatotoxicity after being metabolized to the highly reactive trichloromethyl free radical and its peroxy radical. The purpose of the current study was to examine whether BV regulates the pro-inflammation and fibrosis related genes against a mouse model of hepatic fibrosis induced by CCl4 and ethanol-treated hepatocytes (ETH). Test mice were administered with CCl4 (2 ml/mg) and hepatocytes were treated with 25 mM ethanol. BV was added to the final concentration of 0.05-0.5 mg/kg and 1-100 ng/ml for in vivo and in vitro testing, respectively. Fibrotic livers and ETH were used for the measurement of hepatocyte necrosis, pro-inflammatory cytokines and fibrogenic genes. BV suppressed CCl4-induced hepatocyte necrosis markers of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). It also inhibited the secretion of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. Moreover, BV inhibited CCl4-induced expression of transforming growth factor (TGF)-beta1, alpha-smooth muscle actin (SMA) and fibronectin. Similarly, ETH exhibited significant suppression of IL-1beta, TNF-alpha, TGF-beta1 and fibronectin when cultured with BV. These results suggest that BV possesses anti-fibrogenic properties that are mediated by the suppression of pro-inflammatory cytokines and fibrogenic gene expression. BV has substantial therapeutic potential for the treatment of fibrotic diseases.

Arsenite and its metabolites, MMA(III) and DMA(III), modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice.

PubMed

Medina-Díaz, I M; Estrada-Muñiz, E; Reyes-Hernández, O D; Ramírez, P; Vega, L; Elizondo, G

2009-09-01

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA(III) induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA(III) increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA(III) induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

Arsenite and its metabolites, MMA{sup III} and DMA{sup III}, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half themore » drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA{sup III} induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA{sup III} increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA{sup III} induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.« less

Insulin-like growth factor-binding protein-5 (IGFBP-5) inhibits TNF-{alpha}-induced NF-{kappa}B activity by binding to TNFR1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Jae Ryoung; Huh, Jae Ho; Lee, Yoonna

2011-02-25

Research highlights: {yields} Binding assays demonstrated that secreted- and cellular-IGFBP-5 interacted with TNFR1. {yields} The interaction between IGFBP-5 and TNFR1 was inhibited by TNF-{alpha} and was blocked TNF-{alpha}-activated NF-{kappa}B activity. {yields} IGFBP-5 interacted with TNFR1 through its N- and L-domains but the binding of L-domain to TNFR1 was blocked by TNF-{alpha}. {yields} Competition between the L-domain of IGFBP-5 and TNF-{alpha} blocked TNF-{alpha}-induced NF-{kappa}B activity. {yields} This study suggests that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-{alpha} inhibitor. -- Abstract: IGFBP-5 is known to be involved in various cell phenomena such as proliferation,more » differentiation, and apoptosis. However, the exact mechanisms by which IGFBP-5 exerts its functions are unclear. In this study, we demonstrate for the first time that IGFBP-5 is a TNFR1-interacting protein. We found that ectopic expression of IGFBP-5 induced TNFR1 gene expression, and that IGFBP-5 interacted with TNFR1 in both an in vivo and an in vitro system. Secreted IGFBP-5 interacted with GST-TNFR1 and this interaction was blocked by TNF-{alpha}, demonstrating that IGFBP-5 might be a TNFR1 ligand. Furthermore, conditioned media containing secreted IGFBP-5 inhibited PMA-induced NF-{kappa}B activity and IL-6 expression in U-937 cells. Coimmunoprecipitation assays of TNFR1 and IGFBP-5 wild-type and truncation mutants revealed that IGFBP-5 interacts with TNFR1 through its N- and L-domains. However, only the interaction between the L-domain of IGFBP-5 and TNFR1 was blocked by TNF-{alpha} in a dose-dependent manner, suggesting that the L-domain of IGFBP-5 can function as a TNFR1 ligand. Competition between the L-domain of IGFBP-5 and TNF-{alpha} resulted in inhibition of TNF-{alpha}-induced NF-{kappa}{Beta} activity. Taken together, our results suggest that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-{alpha} inhibitor.« less

Identification and characterization of an alternative promoter of the human PGC-1{alpha} gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshioka, Toyo; Inagaki, Kenjiro; Noguchi, Tetsuya, E-mail: noguchi@med.kobe-u.ac.jp

2009-04-17

The transcriptional regulator peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1{alpha} expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1{alpha} transcript (designated PGC-1{alpha}-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1{alpha}-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca{sup 2+}- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated proteinmore » kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1{alpha}-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1{alpha} expression in contracting muscle.« less

Tumor necrosis factor receptor-1 can function through a G alpha q/11-beta-arrestin-1 signaling complex.

PubMed

Kawamata, Yuji; Imamura, Takeshi; Babendure, Jennie L; Lu, Juu-Chin; Yoshizaki, Takeshi; Olefsky, Jerrold M

2007-09-28

Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNFalpha exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNFalpha are observed in obese and/or diabetic individuals. TNFalpha has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that beta-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNFalpha acutely stimulates tyrosine phosphorylation of G alpha(q/11) with an increase in G alpha(q/11) activity. Small interfering RNA-mediated knockdown of beta-arrestin-1 inhibits TNFalpha-induced tyrosine phosphorylation of G alpha(q/11) by interruption of Src kinase activation. TNFalpha stimulates lipolysis in 3T3-L1 adipocytes, and beta-arrestin-1 knockdown blocks the effects of TNFalpha to stimulate ERK activation and glycerol release. TNFalpha also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and beta-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNFalpha action; 1) the trimeric G-protein component G alpha(q/11) and the adapter protein beta-arrestin-1 can function as signaling molecules in the TNFalpha action cascade; 2) beta-arrestin-1 can couple TNFalpha stimulation to ERK activation and lipolysis; 3) beta-arrestin-1 and G alpha(q/11) can mediate TNFalpha-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression.

Induction of experimental bone metastasis in mice by transfection of integrin alpha 4 beta 1 into tumor cells.

PubMed Central

Matsuura, N.; Puzon-McLaughlin, W.; Irie, A.; Morikawa, Y.; Kakudo, K.; Takada, Y.

1996-01-01

Cell adhesion receptors (eg, integrins and CD44) play an important role in invasion and metastasis during tumor progression. The increase in integrin alpha 4 beta 1 expression on primary melanomas has been reported to significantly correlate with the development of metastases. alpha 4 beta 1 is a cell surface heterodimer that mediates cell-cell and cell-extracellular matrix interactions through adhesion to vascular cell adhesion molecule (VCAM)-1 and to the IIICS region of fibronectin. To test the effects of alpha 4 beta 1 expression on tumor cell metastasis, Chinese hamster ovary cells were transfected with human alpha 4 cDNA. Whereas alpha 4-negative Chinese hamster ovary cells developed only pulmonary metastasis, alpha 4-positive Chinese hamster ovary cells developed bone and pulmonary metastasis in 3 to 4 weeks when injected intravenously into nude mice. Bone metastasis was inhibited by antibody against alpha 4 or VCAM-1. Expression of alpha 3 beta 1, alpha 6 beta 1, or alpha V beta 1 did not induce bone metastasis. Expression of alpha 4 beta 1 also induced bone metastasis in K562 human erythroleukemia cells injected into SCID mice. These results demonstrate that alpha 4 beta 1 can induce tumor cell trafficking to bone, probably via interaction with VCAM-1 that is constitutively expressed on bone marrow stromal cells. Images Figure 1 Figure 3 PMID:8546226

The organic solute transporters alpha and beta are induced by hypoxia in human hepatocytes

PubMed Central

Schaffner, Carlos A; Mwinyi, Jessica; Gai, Zhibo; Thasler, Wolfgang E; Eloranta, Jyrki J; Kullak-Ublick, Gerd A

2015-01-01

Background & Aims The organic solute transporters alpha and beta (OSTα-OSTβ) form a heterodimeric transporter located at the basolateral membrane of intestinal epithelial cells and hepatocytes. Liver injury caused by ischaemia-reperfusion, cancer, inflammation or cholestasis can induce a state of hypoxia in hepatocytes. Here, we studied the effect of hypoxia on the expression of OSTα-OSTβ. Methods OSTα-OSTβ expression was measured in Huh7 cells and primary human hepatocytes (PHH) exposed to chenodeoxycholic acid (CDCA), hypoxia or both. OSTα-OSTβ promoter activity was analysed in luciferase reporter gene assays. Binding of hypoxia-inducible factor-1 alpha (HIF-1α) to the OSTα-OSTβ gene promoters was studied in electrophoretic mobility shift assays (EMSA). Results Expression of OSTα and OSTβ increased in PHH under conditions of hypoxia. Exposure of Huh7 cells or PHH to CDCA (50 μM) enhanced the effect of hypoxia on OSTα mRNA levels. In luciferase assays and EMSA, the inducing effect of low oxygen could be assigned to HIF-1α, which binds to hypoxia responsive elements (HRE) in the OSTα and OSTβ gene promoters. Site-directed mutagenesis of either the predicted HRE or the bile acid responsive FXR binding site abolished inducibility of the OSTα promoter, indicating that both elements need to be intact for induction by hypoxia and CDCA. In a rat model of chronic renal failure, the known increase in hepatic OSTα expression was associated with an increase in HIF-1α protein levels. Conclusion OSTα-OSTβ expression is induced by hypoxia. FXR and HIF-1α bind in close proximity to the OSTα gene promoter and produce synergistic effects on OSTα expression. PMID:24703425

F-prostanoid receptor regulation of fibroblast growth factor 2 signaling in endometrial adenocarcinoma cells.

PubMed

Sales, Kurt J; Boddy, Sheila C; Williams, Alistair R W; Anderson, Richard A; Jabbour, Henry N

2007-08-01

Prostaglandin (PG) F(2alpha) is a potent bioactive lipid in the female reproductive tract, and exerts its function after coupling with its heptahelical G-protein-coupled receptor [F-series-prostanoid (FP) receptor] to initiate cell signaling and target gene transcription. In the present study, we found elevated expression of fibroblast growth factor (FGF) 2, FGF receptor 1 (FGFR1), and FP receptor, colocalized within the neoplastic epithelial cells of endometrial adenocarcinomas. We investigated a role for PGF(2alpha)-FP receptor interaction in modulating FGF2 expression and signaling using an endometrial adenocarcinoma cell line stably expressing the FP receptor to the levels detected in endometrial adenocarcinomas (FPS cells) and endometrial adenocarcinoma tissue explants. PGF(2alpha)-FP receptor activation rapidly induced FGF2 mRNA expression, and elevated FGF2 protein expression and secretion into the culture medium in FPS cells and endometrial adenocarcinoma explants. The effect of PGF(2alpha) on the expression and secretion of FGF2 could be abolished by treatment of FPS cells and endometrial tissues with an FP receptor antagonist (AL8810) and inhibitor of ERK (PD98059). Furthermore, we have shown that FGF2 can promote the expression of FGF2 and cyclooxygenase-2, and enhance proliferation of endometrial adenocarcinoma cells via the FGFR1 and ERK pathways, thereby establishing a positive feedback loop to regulate neoplastic epithelial cell function in endometrial adenocarcinomas.

Effects of Subinhibitory Concentrations of Antibiotics on Alpha-Toxin (hla) Gene Expression of Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Isolates

PubMed Central

Ohlsen, Knut; Ziebuhr, Wilma; Koller, Klaus-Peter; Hell, Wolfgang; Wichelhaus, Thomas A.; Hacker, Jörg

1998-01-01

Concentrations of antibiotics below the MIC are able to modulate the expression of virulence-associated genes. In this study, the influence of subinhibitory doses of 31 antibiotics on the expression of the gene encoding the staphylococcal alpha-toxin (hla), a major virulence factor of Staphylococcus aureus, was investigated with a novel gene fusion protocol. The most striking observation was a strong induction of hla expression by subinhibitory concentrations of β-lactams and an almost complete inhibition of alpha-toxin expression by clindamycin. Whereas glycopeptide antibiotics had no effect, the macrolide erythromycin and several aminoglycosides reduced and fluoroquinolones slightly stimulated hla expression. Furthermore, Northern blot analysis of hla mRNA and Western blot (immunoblot) analysis of culture supernatants of both methicillin-sensitive and methicillin-resistant S. aureus strains revealed that methicillin-induced alpha-toxin expression is a common phenomenon of alpha-toxin-producing strains. Some methicillin-resistant S. aureus isolates produced up to 30-fold more alpha-toxin in the presence of 10 μg of methicillin per ml than in its absence. The results indicate that the novel gene fusion technique is a useful tool for studying the modulation of virulence gene expression by antibiotics. Moreover, the results suggest that the effects of certain antibiotics on virulence properties may be relevant for the management of S. aureus infections. PMID:9797209

TNF-{alpha} promotes cell survival through stimulation of K{sup +} channel and NF{kappa}B activity in corneal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Ling; Reinach, Peter; Lu, Luo

2005-11-15

Tumor necrosis factor (TNF-{alpha}) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-{alpha} also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-{alpha} stimulation induced activation of a voltage-gated K{sup +} channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-{alpha} on downstream events included NF{kappa}B nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-{alpha} induced increases inmore » p21 expression resulting in partial cell cycle attenuation in the G{sub 1} phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-{alpha}-induced K{sup +} channel activity effectively prevented NF{kappa}B nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-{alpha}. In conclusion, TNF-{alpha} promotes survival of HCE cells through sequential stimulation of K{sup +} channel and NF{kappa}B activities. This response to TNF-{alpha} is dependent on stimulating K{sup +} channel activity because following suppression of K{sup +} channel activity TNF-{alpha} failed to activate NF{kappa}B nuclear translocation and binding to nuclear DNA.« less

TGF-beta1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k.

PubMed

Lechuga, Carmen G; Hernández-Nazara, Zamira H; Domínguez Rosales, José-Alfredo; Morris, Elena R; Rincón, Ana Rosa; Rivas-Estilla, Ana María; Esteban-Gamboa, Andrés; Rojkind, Marcos

2004-11-01

Transforming growth factor-beta1 (TGF-beta1), the main cytokine involved in liver fibrogenesis, induces expression of the type I collagen genes in hepatic stellate cells by a transcriptional mechanism, which is hydrogen peroxide and de novo protein synthesis dependent. Our recent studies have revealed that expression of type I collagen and matrix metalloproteinase-13 (MMP-13) mRNAs in hepatic stellate cells is reciprocally modulated. Because TGF-beta1 induces a transient elevation of alpha1(I) collagen mRNA, we investigated whether this cytokine was able to induce the expression of MMP-13 mRNA during the downfall of the alpha1(I) collagen mRNA. In the present study, we report that TGF-beta1 induces a rapid decline in steady-state levels of MMP-13 mRNA at the time that it induces the expression of alpha1(I) collagen mRNA. This change in MMP-13 mRNA expression occurs within the first 6 h postcytokine administration and is accompanied by a twofold increase in gene transcription and a fivefold decrease in mRNA half-life. This is followed by increased expression of MMP-13 mRNA, which reaches maximal values by 48 h. Our results also show that this TGF-beta1-mediated effect is de novo protein synthesis-dependent and requires the activity of p38MAPK, phosphatidylinositol 3-kinase, AKT, and p70(S6k). Altogether, our data suggest that regulation of MMP-13 by TGF-beta1 is a complex process involving transcriptional and posttranscriptional mechanisms.

Quantitative immuno-electron microscopic analysis of depolarization-induced expression of PGC-1alpha in cultured rat visual cortical neurons.

PubMed

Meng, Hui; Liang, Huan Ling; Wong-Riley, Margaret

2007-10-17

Peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC- 1alpha) is a coactivator of nuclear receptors and other transcription factors that regulate several metabolic processes, including mitochondrial biogenesis, energy homeostasis, respiration, and gluconeogenesis. PGC-1alpha plays a vital role in stimulating genes that are important to oxidative metabolism and other mitochondrial functions in brown adipose tissue and skeleton muscles, but the significance of PGC-1alpha in the brain remains elusive. The goal of our present study was to determine by means of quantitative immuno-electron microscopy the expression of PGC-1alpha in cultured rat visual cortical neurons under normal conditions as well as after depolarizing stimulation for varying periods of time. Our results showed that: (a) PGC-1alpha was normally located in both the nucleus and the cytoplasm. In the nucleus, PGC-1alpha was associated mainly with euchromatin rather than heterochromatin, consistent with active involvement in transcription. In the cytoplasm, it was associated mainly with free ribosomes. (b) Neuronal depolarization by KCl for 0.5 h induced a significant increase in PGC-1alpha labeling density in both the nucleus and the cytoplasm (P<0.01). The heightened expression continued after 1 and 3 h of depolarizing treatment (P<0.01), but decreased from 5 h onward and returned to baseline level by 10 h. These results indicate that PGC-1alpha responds very early to increased neuronal activity by synthesizing more proteins in the cytoplasm and translocating them to the nucleus for gene activation. PGC-1alpha level in neurons is, therefore, tightly regulated by neuronal activity.

Tumor necrosis factor-alpha inhibits stem cell factor-induced proliferation of human bone marrow progenitor cells in vitro. Role of p55 and p75 tumor necrosis factor receptors.

PubMed Central

Rusten, L S; Smeland, E B; Jacobsen, F W; Lien, E; Lesslauer, W; Loetscher, H; Dubois, C M; Jacobsen, S E

1994-01-01

Stem cell factor (SCF), a key regulator of hematopoiesis, potently synergizes with a number of hematopoietic growth factors. However, little is known about growth factors capable of inhibiting the actions of SCF. TNF-alpha has been shown to act as a bidirectional regulator of myeloid cell proliferation and differentiation. This study was designed to examine interactions between TNF-alpha and SCF. Here, we demonstrate that TNF-alpha potently and directly inhibits SCF-stimulated proliferation of CD34+ hematopoietic progenitor cells. Furthermore, TNF-alpha blocked all colony formation stimulated by SCF in combination with granulocyte colony-stimulating factor (CSF) or CSF-1. The synergistic effect of SCF observed in combination with GM-CSF or IL-3 was also inhibited by TNF-alpha, resulting in colony numbers similar to those obtained in the absence of SCF. These effects of TNF-alpha were mediated through the p55 TNF receptor, whereas little or no inhibition was signaled through the p75 TNF receptor. Finally, TNF-alpha downregulated c-kit cell-surface expression on CD34+ bone marrow cells, and this was predominantly a p55 TNF receptor-mediated event as well. Images PMID:7518828

Increased expression of protein kinase A inhibitor alpha (PKI-alpha) and decreased PKA-regulated genes in chronic intermittent alcohol exposure.

PubMed

Repunte-Canonigo, Vez; Lutjens, Robert; van der Stap, Lena D; Sanna, Pietro Paolo

2007-03-23

Intermittent models of alcohol exposure that mimic human patterns of alcohol consumption produce profound physiological and biochemical changes and induce rapid increases in alcohol self-administration. We used high-density oligonucleotide microarrays to investigate gene expression changes during chronic intermittent alcohol exposure in three brain regions that receive mesocorticolimbic dopaminergic projections and that are believed to be involved in alcohol's reinforcing actions: the medial prefrontal cortex, the nucleus accumbens and the amygdala. An independent replication of the experiment was used for RT-PCR validation of the microarray results. The protein kinase A inhibitor alpha (PKI-alpha, Pkia), a member of the endogenous PKI family implicated in reducing nuclear PKA activity, was found to be increased in all three regions tested. Conversely, we observed a downregulation of the expression of several PKA-regulated transcripts in one or more of the brain regions studied, including the activity and neurotransmitter-regulated early gene (Ania) - 1, -3, -7, -8, the transcription factors Egr1 and NGFI-B (Nr4a1) and the neuropeptide NPY. Reduced expression of PKA-regulated genes in mesocorticolimbic projection areas may have motivational significance in the rapid increase in alcohol self-administration induced by intermittent alcohol exposure.

Characterization of the early pulmonary inflammatory response associated with PTFE fume exposure

NASA Technical Reports Server (NTRS)

Johnston, C. J.; Finkelstein, J. N.; Gelein, R.; Baggs, R.; Oberdorster, G.; Clarkson, T. W. (Principal Investigator)

1996-01-01

Heating of polytetrafluoroethylene (PTFE) has been described to release fumes containing ultrafine particles (approximately 18 nm diam). These fumes can be highly toxic in the respiratory tract inducing extensive pulmonary edema with hemorrhagic inflammation. Fischer-344 rats were exposed to PTFE fumes generated by temperatures ranging from 450 to 460 degrees C for 15 min at an exposure concentration of 5 x 10(5) particles/cm3, equivalent to approximately 50 micrograms/m3. Responses were examined 4 hr post-treatment when these rats demonstrated 60-85% neutrophils (PMNs) in their lung lavage. Increases in abundance for messages encoding the antioxidants manganese superoxide dismutase and metallothionein (MT) increased 15- and 40-fold, respectively. For messages encoding the pro- and anti-inflammatory cytokines: inducible nitric oxide synthase, interleukin 1 alpha, 1 beta, and 6 (IL-1 alpha, IL-1 beta, and IL-6), macrophage inflammatory protein-2, and tumor necrosis factor-alpha (TNF alpha) increases of 5-, 5-, 10-, 40-, 40-, and 15-fold were present. Vascular endothelial growth factor, which may play a role in the integrity of the endothelial barrier, was decreased to 20% of controls. In situ sections were hybridized with 33P cRNA probes encoding IL-6, MT, surfactant protein C, and TNF alpha. Increased mRNA abundance for MT and IL-6 was expressed around all airways and interstitial regions with MT and IL-6 demonstrating similar spatial distribution. Large numbers of activated PMNs expressed IL-6, MT, and TNF alpha. Additionally, pulmonary macrophages and epithelial cells were actively involved. These observations support the notion that PTFE fumes containing ultrafine particles initiate a severe inflammatory response at low inhaled particle mass concentrations, which is suggestive of an oxidative injury. Furthermore, PMNs may actively regulate the inflammatory process through cytokine and antioxidant expression.

Bradykinin-induced growth inhibition of normal rat kidney (NRK) cells is paralleled by a decrease in epidermal-growth-factor receptor expression.

PubMed Central

Van Zoelen, E J; Peters, P H; Afink, G B; Van Genesen, S; De Roos, D G; Van Rotterdam, W; Theuvenet, A P

1994-01-01

Normal rat kidney fibroblasts, grown to density arrest in the presence of epidermal growth factor (EGF), can be induced to undergo phenotypic transformation by treatment with transforming growth factor beta or retinoic acid. Here we show that bradykinin blocks this growth-stimulus-induced loss of density-dependent growth arrest by a specific receptor-mediated mechanism. The effects of bradykinin are specific, and are not mimicked by other phosphoinositide-mobilizing agents such as prostaglandin F2 alpha. Northern-blot analysis and receptor-binding studies demonstrate that bradykinin also inhibits the retinoic acid-induced increase in EGF receptor levels in these cells. These studies provide additional evidence that EGF receptor levels modulate EGF-induced expression of the transformed phenotype in these cells. Images Figure 5 PMID:8135739

Fibronectin-mediation cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation : the signaling role of protein kinase C-{beta}.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, B.; Laouar, A.; Huberman, E.

1998-05-08

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-betamore » -deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor {alpha}5{beta}1 integrin. HL-525 cells, which constitutively display high levels of surface {alpha}5{beta}1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that {alpha}5{beta}1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.« less

Elongation Factor 1 alpha interacts with phospho-Akt in breast cancer cells and regulates their proliferation, survival and motility.

PubMed

Pecorari, Luisa; Marin, Oriano; Silvestri, Chiara; Candini, Olivia; Rossi, Elena; Guerzoni, Clara; Cattelani, Sara; Mariani, Samanta A; Corradini, Francesca; Ferrari-Amorotti, Giovanna; Cortesi, Laura; Bussolari, Rita; Raschellà, Giuseppe; Federico, Massimo R; Calabretta, Bruno

2009-08-03

Akt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells. We confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1alpha. EF1alpha contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1alpha expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1alpha siRNAs with specific pAkt inhibitors whereas EF1alpha downregulation slightly attenuated the decreased invasion induced by Akt inhibitors. We show here that EF1alpha is a pAkt-interacting protein which regulates pAkt levels. Since EF1alpha is often overexpressed in breast cancer, the consequences of EF1alpha increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2.

Role of interleukin-1beta and tumor necrosis factor-alpha-dependent expression of cyclooxygenase-2 mRNA in thermal hyperalgesia induced by chronic inflammation in mice.

PubMed

Narita, M; Shimamura, M; Imai, S; Kubota, C; Yajima, Y; Takagi, T; Shiokawa, M; Inoue, T; Suzuki, M; Suzuki, T

2008-03-18

The present study investigated whether the endogenous pro-inflammatory cytokines [interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha)]-dependent expression of cyclooxygenase-2 (COX-2) mRNA within the spinal cord could be involved in the development of chronic inflammatory pain-like behaviors in mice. We demonstrated that the expression of COX-2 mRNA on the ipsilateral side of the spinal cord was significantly increased 6 h and 3 days after intraplantar injection of complete Freund's adjuvant (CFA), compared with the expression in saline-treated mice. In addition, the chronic pain-like behaviors following CFA injection were markedly suppressed by repeated intrathecal (i.t.) pre-treatment with the COX-2 inhibitor etodolac, but not with the COX-1 inhibitor mofezolac. The cytosolic level of the activated form of nuclear factor-kappa B (NF-kappaB), which is a major contributor to the induction of COX-2, on the ipsilateral side of the mouse spinal cord was also increased compared with that in the saline-treated mice. The key finding in the present study was that a single i.t. injection with either IL-1beta or TNF-alpha induced a marked increase in spinal COX-2 mRNA and persistent thermal hyperalgesia in mice. Furthermore, CFA-induced hypersensitivity to inflammatory pain was significantly reduced by repeated i.t. pre-injection of the recombinant Fc chimera of IL-1 receptor I or soluble TNF receptor I, which sequesters endogenous IL-1beta or TNF-alpha, respectively. In contrast, the expression of spinal COX-2 mRNA in CFA-treated mice was similar to that in saline-treated mice at 7 days after CFA injection. The present findings strongly indicate the early intrathecal use of the COX-2 inhibitor for the relief of chronic inflammatory pain. Furthermore, together with the result in a previous study that pro-inflammatory cytokines lead to stimulation of a NF-kappaB-dependent transcriptional pathway, these findings suggest that a spinal cytokine/NF-kappaB/COX-2 pathway may play an important role in the development, but not maintenance, of chronic pain following peripheral tissue inflammation.

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase.

PubMed

Sury, Matthias D; Frese-Schaper, Manuela; Mühlemann, Miranda K; Schulthess, Fabienne T; Blasig, Ingolf E; Täuber, Martin G; Shaw, Sidney G; Christen, Stephan

2006-11-01

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPAR{gamma}-LXR{alpha}-ABCA1 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yanni; Lai, Fangfang; Xu, Yang

2011-11-04

Highlights: Black-Right-Pointing-Pointer Using an ABCA1p-LUC HepG2 cell line, we found that MPA upregulated ABCA1 expression. Black-Right-Pointing-Pointer MPA induced ABCA1 and LXR{alpha} protein expression in HepG2 cells. Black-Right-Pointing-Pointer PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. Black-Right-Pointing-Pointer The effect of MPA upregulating ABCA1 was due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 pathway. -- Abstract: ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1.more » Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50 = 0.09 {mu}M). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPAR{gamma}; EC50 = 5.2-9.3 {mu}M). Liver X receptor {alpha} (LXR{alpha}) is a target gene of PPAR{gamma} and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXR{alpha} protein expression in HepG2 cells. Addition of PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPAR{gamma}. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism.« less

Expression of transforming growth factor-beta1, -beta2 and -beta3 in normal and diseased canine mitral valves.

PubMed

Aupperle, H; März, I; Thielebein, J; Schoon, H-A

2008-01-01

The pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, but activation and proliferation of valvular stromal cells (VSC) and their transdifferentiation into myofibroblast-like cells has been described. These alterations may be influenced by transforming growth factor-beta (TGF-beta), a cytokine involved in extracellular matrix (ECM) regulation and mesenchymal cell differentiation. The present study investigates immunohistochemically the expression of TGF-beta1, -beta2, -beta3 and smooth muscle alpha actin (alpha-SMA) in normal canine mitral valves (MVs) (n=10) and in the valves of dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal mitral valves there was no expression of alpha-SMA but VSC displayed variable expression of TGF-beta1 (10% of VSC labelled), TGF-beta2 (1-5% labelled) and TGF-beta3 (50% labelled). In mild CVD the affected atrialis contain activated and proliferating alpha-SMA-positive VSC, which strongly expressed TGF-beta1 and -beta3, but only 10% of these cells expressed TGF-beta2. In unaffected areas of the leaflet there was selective increase in expression of TGF-beta1 and -beta3. In advanced CVD the activated subendothelial VSC strongly expressed alpha-SMA, TGF-beta1 and -beta3. Inactive VSC within the centre of the nodules had much less labelling for TGF-beta1 and -beta3. TGF-beta1 labelling was strong within the ECM. These data suggest that TGF-beta plays a role in the pathogenesis of CVD by inducing myofibroblast-like differentiation of VSC and ECM secretion. Changed haemodynamic forces and expression of matrix metalloproteinases (MMPs) may in turn regulate TGF-beta expression.

Id-1 activation of PI3K/Akt/NFkappaB signaling pathway and its significance in promoting survival of esophageal cancer cells.

PubMed

Li, Bin; Cheung, Pak Yan; Wang, Xianghong; Tsao, Sai Wah; Ling, Ming Tat; Wong, Yong Chuan; Cheung, Annie L M

2007-11-01

Inhibitor of differentiation or DNA binding (Id-1) is a helix-loop-helix protein that is over-expressed in many types of cancer including esophageal cancer. This study aims to investigate its effects on the phosphatidylinositol-3-kinase (PI3K)/Akt/ nuclear factor kappa B (NFkappaB) signaling pathway and the significance in protecting esophageal cancer cells against apoptosis. We found elevated expression of phosphorylated forms of Akt, glycogen synthase kinase 3beta and inhibitor of kappa B, as well as increased nuclear translocation of NFkappaB subunit p65 and NFkappaB DNA-binding activity, in esophageal cancer cells with stable ectopic Id-1 expression. Transient transfection of Id-1 into HEK293 cells confirmed activation of PI3K/Akt/NFkappaB signaling and the effects were counteracted by the PI3K inhibitor LY294002. Treatment with tumor necrosis factor-alpha (TNF-alpha) elicited a significantly weaker apoptotic response, following a marked and sustained activation of Akt and NFkappaB in the Id-1-over-expressing cells, compared with the vector control. The effects of Id-1 on the PI3K/Akt/NFkappaB signaling pathway and apoptosis were reversed in esophageal cancer cells transfected with siRNA against Id-1. In addition, inhibition of PI3K or NFkappaB signaling using the PI3K inhibitor LY294002 or the NFkappaB inhibitor Bay11-7082 increased the sensitivity of Id-1-over-expressing esophageal cancer cells to TNF-alpha-induced apoptosis. Our results provide the first evidence that Id-1 induces the activation of PI3K/Akt/NFkappaB signaling pathway, and protects esophageal cancer cells from TNF-alpha-induced apoptosis in vitro. Inactivation of Id-1 may provide us with a novel strategy to improve the treatment and survival of patients with esophageal cancer.

Divergent effects of 17-{beta}-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-{alpha}-induced neointima formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nintasen, Rungrat; Multidisciplinary Cardiovascular Research Center; Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University

2012-04-20

Highlights: Black-Right-Pointing-Pointer TNF-{alpha} augments neointimal hyperplasia in human saphenous vein. Black-Right-Pointing-Pointer TNF-{alpha} induces detrimental effects on endothelial and smooth muscle cell function. Black-Right-Pointing-Pointer Estradiol exerts modulatory effects on TNF-induced vascular cell functions. Black-Right-Pointing-Pointer The modulatory effects of estradiol are discriminatory and cell-type specific. -- Abstract: Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-{alpha} (TNF-{alpha}). TNF-{alpha} can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protectivemore » effects of estradiol (E2). We therefore investigated the effects of TNF-{alpha} on human neointima formation and SMC/EC functions and any modulatory effects of E2. Saphenous vein (SV) segments were cultured in the presence of TNF-{alpha} (10 ng/ml), E2 (2.5 nM) or both in combination. Neointimal thickening was augmented by incubation with TNF-{alpha}, an effect that was abolished by co-culture with E2. TNF-{alpha} increased SV-SMC proliferation in a concentration-dependent manner that was optimal at 10 ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1-50 nM). Surprisingly, E2 itself at low concentrations (1 and 5 nM) stimulated SV-SMC proliferation to a level comparable to that of TNF-{alpha} alone. SV-EC migration was significantly impaired by TNF-{alpha} (42% of control), and co-culture with E2 partially restored the ability of SV-EC to migrate and repair the wound. In contrast, TNF-{alpha} increased SV-SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-{alpha} potently induced ICAM-1 and VCAM-1 expression in both SV-EC and SV-SMC. However there was no modulation by E2 in either cell-type. In conclusion, TNF-{alpha} induced SV neointima formation, increased SMC proliferation and migration, impaired SV-EC migration and increased expression of adhesion molecules. E2 exerted distinct cell-type and function-specific modulation, the mechanisms underlying which are worthy of further detailed study.« less

Curcumin inhibits interferon-{alpha} induced NF-{kappa}B and COX-2 in human A549 non-small cell lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jeeyun; Im, Young-Hyuck; Jung, Hae Hyun

2005-08-26

The A549 cells, non-small cell lung cancer cell line from human, were resistant to interferon (IFN)-{alpha} treatment. The IFN-{alpha}-treated A549 cells showed increase in protein expression levels of NF-{kappa}B and COX-2. IFN-{alpha} induced NF-{kappa}B binding activity within 30 min and this increased binding activity was markedly suppressed with inclusion of curcumin. Curcumin also inhibited IFN-{alpha}-induced COX-2 expression in A549 cells. Within 10 min, IFN-{alpha} rapidly induced the binding activity of a {gamma}-{sup 32}P-labeled consensus GAS oligonucleotide probe, which was profoundly reversed by curcumin. Taken together, IFN-{alpha}-induced activations of NF-{kappa}B and COX-2 were inhibited by the addition of curcumin in A549more » cells.« less

Liver X receptor alpha regulates fatty acid synthase expression in chicken.

PubMed

Demeure, O; Duby, C; Desert, C; Assaf, S; Hazard, D; Guillou, H; Lagarrigue, S

2009-12-01

Liver X receptor alpha (LXRalpha), also referred to as nuclear receptor subfamily 1, group H, member 3 is a member of the nuclear hormone receptor superfamily, and has recently been shown to act as a master transcription factor governing hepatic lipogenesis in mammals. Liver X receptor alpha directly regulates both the expression of other lipogenic transcription factors and the expression of lipogenic enzymes, thereby enhancing hepatic fatty acid synthesis (FASN). In birds, like in humans, fatty acid synthesis primarily occurs in the liver. Whether LXRalpha is involved in hepatic regulation of lipogenic genes remained to be investigated in this species. Here we show that fatty acid synthase and the expression of other lipogenic genes (sterol regulatory element binding protein 1 and steroyl coenzyme A desaturase 1) are induced in chicken hepatoma cells in response to a pharmacological liver X receptor agonist, T0901317. A detailed analysis of the chicken FASN promoter revealed a functional liver X response element. These data define the chicken FASN gene as a direct target of LXRalpha and further expand the role of LXRalpha as a regulator of lipid metabolism in this species.

Evidence for a role of TGF-beta1 in the expression and regulation of alpha-SMA in fetal growth restricted placentae.

PubMed

Todros, T; Marzioni, D; Lorenzi, T; Piccoli, E; Capparuccia, L; Perugini, V; Cardaropoli, S; Romagnoli, R; Gesuita, R; Rolfo, A; Paulesu, L; Castellucci, M

2007-01-01

There is evidence that alpha-smooth muscle actin (alpha-SMA) is a protein that plays a pivotal role in the production of contractile forces and it is induced by transforming growth factor-beta1 (TGF-beta1). We have analysed the expression of alpha-SMA, TGF-beta1, its receptor RI and the activator phospho-Smad2 in (a) fetal growth restriction pre-eclamptic placentae characterised by early onset and absence of end diastolic velocities in the umbilical arteries (FGR-AED) and (b) control placentae accurately matched for gestational age. The study was performed by immunohistochemical, quantitative Western blotting, ELISA, RT-PCR and in vitro analyses. We found that TGF-beta1 stimulates alpha-SMA production in chorionic villi cultured in vitro. In addition, we observed that in vivo TGF-beta1 concentration is significantly higher in FGR-AED placental samples than in control placentae and that this growth factor could have a paracrine action on villous stroma myofibroblasts expressing TGF-beta1 receptors and phospho-Smad2. Indeed, we report that alpha-SMA undergoes a redistribution in FGR-AED placental villous tree, i.e. we show that alpha-SMA is enhanced in medium and small stem villi and significantly decreased in the peripheral villi. Our data allow us to consider TGF-beta1 and alpha-SMA as key molecules related to FGR-AED placental villous tree phenotypic changes responsible for increased impedance to blood flow observable in this pathology.

3',5'-cyclic adenosine monophosphate response element binding protein up-regulated cytochrome P450 lanosterol 14alpha-demethylase expression involved in follicle-stimulating hormone-induced mouse oocyte maturation.

PubMed

Ning, Gang; Ouyang, Hong; Wang, Songbo; Chen, Xiufen; Xu, Baoshan; Yang, Jiange; Zhang, Hua; Zhang, Meijia; Xia, Guoliang

2008-07-01

Cytochrome P450 lanosterol 14alpha-demethylase (CYP51) is a key enzyme in sterols and steroids biosynthesis that can induce meiotic resumption in mouse oocytes. The present study investigated the expression mechanism and function of CYP51 during FSH-induced mouse cumulus oocyte complexes (COCs) meiotic resumption. FSH increased cAMP-dependent protein kinase (PKA) RIIbeta level and induced cAMP response element-binding protein (CREB) phosphorylation and CYP51 expression in cumulus cells before oocyte meiotic resumption. Moreover, CYP51 and epidermal growth factor (EGF)-like factor [amphiregulin (AR)] expression were blocked by (2)-naphthol-AS-Ephosphate (KG-501) (a drug interrupting the formation of CREB functional complex). KG-501 and RS21607 (a specific inhibitor of CYP51 activity) inhibited oocyte meiotic resumption, which can be partially rescued by progesterone. These two inhibitors also inhibited FSH-induced MAPK phosphorylation. EGF could rescue the suppression by KG-501 but not RS21607. Furthermore, type II PKA analog pairs, N(6)-monobutyryl-cAMP plus 8-bromo-cAMP, increased PKA RIIbeta level and mimicked the action of FSH, including CREB phosphorylation, AR and CYP51 expression, MAPK activation, and oocyte maturation. All these data suggest that CYP51 plays a critical role in FSH-induced meiotic resumption of mouse oocytes. CYP51 and AR gene expression in cumulus cells are triggered by FSH via a type II PKA/CREB-dependent signal pathway. Our study also implicates that CYP51 activity in cumulus cells participates in EGF receptor signaling-regulated oocyte meiotic resumption.

Fatty acid homeostasis and induction of lipid regulatory genes in skeletal muscles of peroxisome proliferator-activated receptor (PPAR) alpha knock-out mice. Evidence for compensatory regulation by PPAR delta.

PubMed

Muoio, Deborah M; MacLean, Paul S; Lang, David B; Li, Shi; Houmard, Joseph A; Way, James M; Winegar, Deborah A; Corton, J Christopher; Dohm, G Lynis; Kraus, William E

2002-07-19

Ablation of peroxisome proliferator activated receptor (PPAR) alpha, a lipid-activated transcription factor that regulates expression of beta-oxidative genes, results in profound metabolic abnormalities in liver and heart. In the present study we used PPAR alpha knockout (KO) mice to determine whether this transcription factor is essential for regulating fuel metabolism in skeletal muscle. When animals were challenged with exhaustive exercise or starvation, KO mice exhibited lower serum levels of glucose, lactate, and ketones and higher nonesterified fatty acids than wild type (WT) littermates. During exercise, KO mice exhausted earlier than WT and exhibited greater rates of glycogen depletion in liver but not skeletal muscle. Fatty acid oxidative capacity was similar between muscles of WT and KO when animals were fed and only 28% lower in KO muscles when animals were starved. Exercise-induced regulation and starvation-induced regulation of pyruvate-dehydrogenase kinase 4 and uncoupling protein 3, two classical and robustly responsive PPAR alpha target genes, were similar between WT and KO in skeletal muscle but markedly different between genotypes in heart. Real time quantitative PCR analyses showed that unlike in liver and heart, in mouse skeletal muscle PPAR delta is severalfold more abundant than either PPAR alpha or PPAR gamma. In both human and rodent myocytes, the highly selective PPAR delta agonist GW742 increased fatty acid oxidation about 2-fold and induced expression of several lipid regulatory genes, including pyruvate-dehydrogenase kinase 4 and uncoupling protein 3, responses that were similar to those elicited by the PPAR alpha agonist GW647. These results show redundancy in the functions of PPARs alpha and delta as transcriptional regulators of fatty acid homeostasis and suggest that in skeletal muscle high levels of the delta-subtype can compensate for deficiency of PPAR alpha.

Alterations in the mitochondrial regulatory pathways constituted by the nuclear co-factors PGC-1alpha or PGC-1beta and mitofusin 2 in skeletal muscle in type 2 diabetes.

PubMed

Zorzano, Antonio; Hernández-Alvarez, María Isabel; Palacín, Manuel; Mingrone, Geltrude

2010-01-01

Muscle mitochondrial metabolism is regulated by a number of factors, many of which are responsible for the transcription of nuclear genes encoding mitochondrial proteins such as PPARdelta, PGC-1alpha or PGC-1beta. Recent evidence indicates that proteins participating in mitochondrial dynamics also regulate mitochondrial metabolism. Thus, in cultured cells the mitochondrial fusion protein mitofusin 2 (Mfn2) stimulates respiration, substrate oxidation and the expression of subunits involved in respiratory complexes. Mitochondrial dysfunction has been reported in skeletal muscle of type 2 diabetic patients. Reduced mitochondrial mass and defective activity has been proposed to explain this dysfunction. Alterations in mitochondrial metabolism may be crucial to account for some of the pathophysiological traits that characterize type 2 diabetes. Skeletal muscle of type 2 diabetic patients shows reduced expression of PGC-1alpha, PGC-1beta, and Mfn2. In addition, a differential response to bilio-pancreatic diversion-induced weight loss in non-diabetic and type 2 diabetic patients has been reported. While non-diabetic morbidly obese subjects showed an increased expression of genes encoding Mfn2, PGC-1alpha, PGC-1beta, PPARdelta or SIRT1 in response to bariatric surgery-induced weight loss, no effect was detected in type 2 diabetic patients. These observations suggest the existence of a heritable component responsible for the abnormal control of the expression of genes encoding for modulators of mitochondrial biogenesis/metabolism, and which may participate in the development of the disease. Copyright © 2010 Elsevier B.V. All rights reserved.

The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.

PubMed

Yang, Chih-Wei; Wu, Mai-Szu; Pan, Ming-Jeng; Hsieh, Wang-Ju; Vandewalle, Alain; Huang, Chiu-Ching

2002-08-01

Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

A DC-81-indole conjugate agent suppresses melanoma A375 cell migration partially via interrupting VEGF production and stromal cell-derived factor-1{alpha}-mediated signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsieh, Ming-Chu; Hu, Wan-Ping; Yu, Hsin-Su

2011-09-01

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrialmore » membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1{alpha} (SDF-1{alpha}) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF-1{alpha}-enhanced melanoma migration. IN4CPBD not only abolished the SDF-1{alpha}-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF-1{alpha}-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency. - Research Highlights: > A novel carboxylate-PBD hybrid as anti-melanoma drug. > IN4CPBD interrupts melanoma cell cycle progression and induces apoptosis. > IN4CPBD suppresses SDF-1{alpha}-enhanced signaling and melanoma migration. > IN4CPBD abolishes angiogenic factor production and chemotactic effect of SDF-1{alpha}. > This drug is clinically applicable to melanoma therapy.« less

TNF-alpha-induced c-Fos generation in the nucleus of the solitary tract is blocked by NBQX and MK-801.

PubMed

Emch, G S; Hermann, G E; Rogers, R C

2001-11-01

Previous studies have shown that identified neurons of the nucleus of the solitary tract (NST) are excited by the cytokine tumor necrosis factor-alpha (TNF-alpha). Vagal afferent connections with the NST are predominantly glutaminergic. Therefore, we hypothesized that TNF-alpha effects on NST neurons may be via modulation of glutamate neurotransmission. The present study used activation of the immediate early gene product c-Fos as a marker for neuronal activation in the NST. c-Fos expression was evaluated after microinjections of TNF-alpha in the presence or absence of either the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) or the N-methyl-D- aspartate (NMDA) antagonist MK-801. To assess the specificity of the interaction between TNF-alpha and glutamate, c-Fos expression was also evaluated after injection of oxytocin (OT) (which has a direct excitatory effect in this area of the brain stem) in the presence and absence of NBQX or MK-801. c-Fos labeling was significantly increased in the NST after TNF-alpha exposure. Coinjection of either NBQX or MK-801 with TNF-alpha prevented significant c-Fos induction in the NST. Microinjections of OT also induced significant NST c-Fos elevation, but this expression was unaffected by coinjection of either antagonist with OT. These data lead us to conclude that TNF-alpha activation of NST neurons depends on glutamate and such an interaction is not generalized to all agonists that act on the NST.

Wogonin suppresses TNF-{alpha}-induced MMP-9 expression by blocking the NF-{kappa}B activation via MAPK signaling pathways in human aortic smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Syng-Ook; Jeong, Yun-Jeong; Yu, Mi Hee

2006-12-08

Matrix metalloproteinase-9 (MMP-9) plays a major role in the pathogenesis of atherosclerosis and restenosis by regulating both migration and proliferation of vascular smooth muscle cells (VSMC) after an arterial injury. In this study, we examined the inhibitory effect of three major flavonoids in Scutellariae Radix, baicalin, baicalein, and wogonin, on TNF-{alpha}-induced MMP-9 expression in human aortic smooth muscle cells (HASMC). Wogonin, but not baicalin and baicalein, significantly and selectively suppressed TNF-{alpha}-induced MMP-9 expression in HASMC. Reporter gene, electrophoretic mobility shift, and Western blotting assays showed that wogonin inhibits MMP-9 gene transcriptional activity by blocking the activation of NF-{kappa}B via MAPKmore » signaling pathways. Moreover, the Matrigel migration assay showed that wogonin reduced TNF-{alpha}-induced HASMC migration. These results suggest that wogonin effectively suppresses TNF-{alpha}-induced HASMC migration through the selective inhibition of MMP-9 expression and represents a potential agent for the prevention of vascular disorders related to the migration of VSMC.« less

A possible mechanism of maxillofacial abscess formation: involvement of Porphyromonas endodontalis lipopolysaccharide via the expression of inflammatory cytokines.

PubMed

Murakami, Y; Hanazawa, S; Tanaka, S; Iwahashi, H; Yamamoto, Y; Fujisawa, S

2001-12-01

In a previous study, we developed a specific monoclonal antibody against Porphyromonas endodontalis lipopolysaccharide, and demonstrated that this lipopolysaccharide was detected in bacterially infected root canal fluid. We suggest here that P. endodontalis lipopolysaccharide in the infectious materials plays a stimulatory role in maxillofacial abscess formation via the expression of inflammatory cytokines. Our epidemiological study showed that this lipopolysaccharide was detected in significant levels the infectious material of patients with periapical periodontitis and odontogenic abscesses. Interestingly, infectious material-induced expression of tumor necrosis factor-alpha, interleukin-1beta, or neutrophil chemoattractant KC genes in mouse macrophages, was significantly neutralized by monoclonal antibody against the lipopolysaccharide. In addition, we also detected a significant amount of tumor necrosis factor-alpha in the infectious material. These results suggest that P. endodontalis lipopolysaccharide plays an important role in the pathogenic mechanism of maxillofacial abscess formation via the expression of inflammatory cytokines.

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

PubMed

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

Protective role of Smad6 in inflammation-induced valvular cell calcification

PubMed Central

Li, Xin; Lim, Jina J.; Lu, Jinxiu; Pedego, Taylor M.; Demer, Linda; Tintut, Yin

2016-01-01

Calcific aortic vascular and valvular disease (CAVD) is associated with hyperlipidemia, the effects of which occur through chronic inflammation. Evidence suggests that inhibitory small mothers against decapentaplegic (I-Smads; Smad6 and 7) regulate valve embryogenesis and may serve as a mitigating factor in CAVD. However, whether I-Smads regulate inflammation-induced calcific vasculopathy is not clear. Therefore, we investigated the role of I-Smads in atherosclerotic calcification. Results showed that expression of Smad6, but not Smad7, was reduced in aortic and valve tissues of hyperlipidemic compared with normolipemic mice, while expression of tumor necrosis factor alpha (TNF-a) was upregulated. To test whether the effects are in response to inflammatory cytokines, we isolated murine aortic valve leaflets and cultured valvular interstitial cells (mVIC) from the normolipemic mice. By immunochemistry, mVICs were strongly positive for vimentin, weakly positive for smooth muscle alpha actin, and negative for an endothelial cell marker. TNF-a upregulated alkaline phosphatase (ALP) activity and matrix mineralization in mVICs. By gene expression analysis, TNF-a significantly upregulated bone morphogenetic protein 2 (BMP-2) expression while downregulating Smad6 expression. Smad7 expression was not significantly affected. To further test the role of Smad6 on TNF-a-induced valvular cell calcification, we knocked down Smad6 expression using lentiviral transfection. In cells transfected with Smad6 shRNA, TNF-a further augmented ALP activity, expression of BMP-2, Wnt- and redox-regulated genes, and matrix mineralization compared with the control cells. These findings suggest that TNF-a induces valvular and vascular cell calcification, in part, by specifically reducing the expression of a BMP-2 signaling inhibitor, Smad6. PMID:25864564

CPT1{alpha} over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jambor de Sousa, Ulrike L.; Koss, Michael D.; Fillies, Marion

2005-12-16

To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1{alpha} (CPT1{alpha}). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1{alpha} transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1{alpha} over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1{alpha} over-expressing cells in a concentration-dependent manner. Both, PA and CPT1{alpha} over-expression increased cell death. Interestingly,more » PA reduced total cell number only in cells over-expressing CPT1{alpha}, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo.« less

Interleukin 1 alpha-induced expression of manganous superoxide dismutase reduces myocardial reperfusion injury in the rat.

PubMed

Nogae, C; Makino, N; Hata, T; Nogae, I; Takahashi, S; Suzuki, K; Taniguchi, N; Yanaga, T

1995-10-01

We investigated the effects of pretreatment with interleukin (IL)-1 alpha on the expression of manganous (Mn) superoxide dismutase (SOD) mRNA and reperfusion-induced arrhythmias and the size of myocardial infarct in rats. Male Wistar rats received 10 mg intraperitoneal injections of human recombinant IL-1 alpha. Their hearts were thereafter isolated at 6, 12, 24, 36 h. A Northern analysis showed that Mn-SOD mRNA was mainly expressed in the heart and slightly in kidney, but not in any other organs. The expression of Mn-SOD mRNA peaked at 6 h after the injection of IL-1 alpha. The Mn-SOD protein content was most increased 12 h after injection. In the isolated heart model, the rats were pretreated with IL-1 alpha 24 h earlier and their hearts were perfused by the Langendorff method. After 20 min of ischemia which was induced by a ligation of a coronary artery, reperfusion-induced arrhythmias were observed. There were no significant differences in the incidence of ventricular arrhythmias between the IL-1 alpha pretreated and the untreated hearts. IL-1 alpha pretreatment significantly reduced the mean duration of the ventricular arrhythmias and also delayed the onset of arrhythmias. The effect of IL-1 alpha pretreatment was also investigated in a 30-min model of ischemia followed by a 3-min reperfusion in anesthetized rats. The infarct size expressed as a percentage of the area at risk was significantly reduced in the IL-1 alpha pretreated hearts compared with the untreated hearts. The left ventricular systolic pressure increased significantly in rat hearts pretreated with IL-1 alpha. Our results therefore showed that the pretreatment with IL-1 alpha induced the overexpression of Mn-SOD mRNA in the rat hearts and also suggested that pretreatment with IL-1 alpha 24 h before ischemia reduced the risk of ischemia-reperfusion injury.

Effect of pro-inflammatory mediators on membrane-associated mucins expressed by human ocular surface epithelial cells.

PubMed

Albertsmeyer, Ann-Christin; Kakkassery, Vinodh; Spurr-Michaud, Sandra; Beeks, Olivia; Gipson, Ilene K

2010-03-01

Membrane-associated mucins are altered on the ocular surface in non-Sjögren's dry eye. This study sought to determine if inflammatory mediators, present in tears of dry eye patients, regulate membrane-associated mucins MUC1 and -16 at the level of gene expression, protein biosynthesis and/or ectodomain release. A human corneal limbal epithelial cell line (HCLE), which produces membrane-associated mucins, was used. Cells were treated with interleukin (IL)-6, -8, or -17, tumor necrosis factor-alpha (TNF-alpha), and Interferon-gamma (IFN-gamma), or a combination of TNF-alpha and IFN-gamma, or IFN-gamma and IL-17, for 1, 6, 24, or 48 h. Presence of receptors for these mediators was verified by RT-PCR. Effects of the cytokines on expression levels of MUC1 and -16 were determined by real-time PCR, and on mucin protein biosynthesis and ectodomain release in cell lysates and culture media, respectively, by immunoblot analysis. TNF-alpha and IFN-gamma each significantly induced MUC1 expression, cellular protein content and ectodomain release over time. Combined treatment with the two cytokines was not additive. By comparison, one of the inflammatory mediators, IFN-gamma, affected all three parameters-gene expression, cellular protein, and ectodomain release-for MUC16. Combined treatment with TNF-alpha and IFN-gamma showed effects similar to IFN-gamma alone, except that ectodomain release followed that of TNF-alpha, which induced MUC16 ectodomain release. In conclusion, inflammatory mediators present in tears of dry eye patients can affect MUC1 and -16 on corneal epithelial cells and may be responsible for alterations of surface mucins in dry eye.

Increased proliferation of endothelial cells with overexpression of soluble TNF-alpha receptor I gene.

PubMed

Sugano, Masahiro; Tsuchida, Keiko; Tomita, Hideharu; Makino, Naoki

2002-05-01

Vascular endothelial growth factor (VEGF) can overcome a potential anti-angiogenic effect of TNF-alpha by inhibiting endothelial apoptosis induced by this cytokine. Soluble TNF-alpha receptor I (sTNFRI) is an extracellular domain of TNFRI and antagonizes the activity of TNF-alpha. Here we report that sTNFRI is able to stimulate the growth of endothelial cells not by antagonizing TNF-alpha. Exogenously added recombinant human sTNFRI stimulated significantly more cell growth of human umbilical venous endothelial cells (HUVEC) with a low dose (50-200 pg/ml) compared with smooth muscle cells. In contrast, monoclonal antibody against TNF-alpha did not stimulate growth of human HUVEC. The sTNFRI expression plasmid (pcDNA3.1 plasmid) was introduced into the cell culture using OPTI-MEM, lipofectin and transferrin. Growth of HUVEC transfected with sTNFRI vector also increased significantly compared with those transfected with control vector. HUVEC transfected with sTNFRI vector increased the extracellular domain of TNFRI mRNA levels, but did not affect the intracellular domain of TNFRI mRNA levels. Accumulation of sTNFRI significantly increased in conditioned medium from HUVEC transfected with sTNFRI vector compared with those transfected with control vector. HUVEC transfected with sTNFRI vector not only increased sTNFRI but also prevented shedding of sTNFRI from TNFRI. The TNF-alpha -induced internucleosomic fragmentation was also significantly prevented in HUVEC transfected with sTNFRI vector compared with those transfected with control vector. These results suggest that instead of growth factors such as VEGF, local transfection of the sTNFRI gene may have potential therapeutic value in vascular diseases in which TNF-alpha is also usually highly expressed.

Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamagishi, Nobuyuki; Goto, Kazumasa; Nakagawa, Satomi

2010-09-10

Hsp105{alpha} and Hsp105{beta} are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105{alpha} is expressed constitutively in the cytoplasm of cells, while Hsp105{beta}, an alternatively spliced form of Hsp105{alpha}, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105{beta} but also Hsp105{alpha} accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105{alpha} and Hsp105{beta} with changes in the nuclearmore » localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105{alpha} and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105{alpha} and Hsp105{beta} on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105{alpha} and Hsp105{beta} suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.« less

Stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Porcile, Carola; Bajetto, Adriana; Barbieri, Federica

2005-08-15

Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1{alpha} treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro.more » In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1{alpha} induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important 'cross-talk' between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.« less

Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue.

PubMed

Mydlo, J H; Michaeli, J; Cordon-Cardo, C; Goldenberg, A S; Heston, W D; Fair, W R

1989-06-15

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.

Anti-fibrotic effects of thalidomide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats.

PubMed

Chong, Lee-Won; Hsu, Yi-Chao; Chiu, Yung-Tsung; Yang, Kuo-Ching; Huang, Yi-Tsau

2006-05-01

Tumor necrosis factor-alpha (TNF-alpha) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, alpha-N-phthalidoglutarimide, (C(13)H(10)N(2))(4), has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-alpha effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or TNF-alpha. The inhibitory effects of thalidomide on the NFkappaB signaling cascade and fibrosis markers including alpha-smooth muscle actin (alpha-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats, which were randomly assigned to 1 of 4 groups: vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100-800 nM) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IKKalpha expression and IkappaBalpha phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-beta1-induced alpha-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89 +/- 0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56 +/- 0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition, real-time PCR analysis indicated that hepatic mRNA expressions of TGF-beta1, alpha-SMA, collagen 1alpha2, TNF-alpha and iNOS genes were attenuated by thalidomide treatment. In conclusion, our results showed that thalidomide inhibited activation of HSC-T6 cells by TNF-alpha and ameliorated liver fibrosis in DMN-intoxicated rats.

CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents.

PubMed

Zhang, Feng; Shu, Jin-Ling; Li, Ying; Wu, Yu-Jing; Zhang, Xian-Zheng; Han, Le; Tang, Xiao-Yu; Wang, Chen; Wang, Qing-Tong; Chen, Jing-Yu; Chang, Yan; Wu, Hua-Xun; Zhang, Ling-Ling; Wei, Wei

2017-01-01

Paeoniflorin-6'- O -benzene sulfonate (code: CP-25) was the chemistry structural modifications of Paeoniflorin (Pae). CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF) or Tumor necrosis factor alpha (TNF-alpha). CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19 + B cells, CD19 + CD20 + B cells, CD19 + CD27 + B cells and CD19 + CD20 + CD27 + B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

Tilmicosin and tylosin have anti-inflammatory properties via modulation of COX-2 and iNOS gene expression and production of cytokines in LPS-induced macrophages and monocytes.

PubMed

Cao, Xing-Yuan; Dong, Mei; Shen, Jian-Zhong; Wu, Bei-Bei; Wu, Cong-Ming; Du, Xiang-Dang; Wang, Zhuo; Qi, Yi-Tao; Li, Bing-Yu

2006-05-01

Macrolides have been reported to modify the host immune and inflammatory responses both in vivo and in vitro. We examined the in vitro effect of the macrolides tilmicosin and tylosin, which are only used in the veterinary clinic, on the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and cytokines by lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and mouse peripheral blood mononuclear cells (PBMCs). Compared with 5 microg/mL, tilmicosin and tylosin concentrations of 10 microg/mL and 20 microg/mL significantly decreased the production of 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), PGE(2), NO, tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-6, and increased IL-10 production. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression were also significantly reduced. These results support the opinion that macrolides may exert an anti-inflammatory effect through modulating the synthesis of several mediators and cytokines involved in the inflammatory process.

In vitro screening of 200 pesticides for agonistic activity via mouse peroxisome proliferator-activated receptor (PPAR){alpha} and PPAR{gamma} and quantitative analysis of in vivo induction pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takeuchi, Shinji; Matsuda, Tadashi; Kobayashi, Satoshi

2006-12-15

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors and key regulators of lipid metabolism and cell differentiation. However, there have been few studies reporting on a variety of environmental chemicals, which may interact with these receptors. In the present study, we characterized mouse PPAR{alpha} and PPAR{gamma} agonistic activities of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 11 acid amides, 7 triazines, 8 ureas and 44 others) by in vitro reporter gene assays using CV-1 monkey kidney cells. Three of the 200 pesticides, diclofop-methyl, pyrethrins and imazalil, which have different chemical structures, showed PPAR{alpha}-mediatedmore » transcriptional activities in a dose-dependent manner. On the other hand, none of the 200 pesticides showed PPAR{gamma} agonistic activity at concentrations {<=} 10{sup -5} M. To investigate the in vivo effects of diclofop-methyl, pyrethrins and imazalil, we examined the gene expression of PPAR{alpha}-inducible cytochrome P450 4As (CYP4As) in the liver of female mice intraperitoneally injected with these compounds ({<=} 300 mg/kg). RT-PCR revealed significantly high induction levels of CYP4A10 and CYP4A14 mRNAs in diclofop-methyl- and pyrethrins-treated mice, whereas imazalil induced almost no gene expressions of CYP4As. In particular, diclofop-methyl induced as high levels of CYP4A mRNAs as WY-14643, a potent PPAR{alpha} agonist. Thus, most of the 200 pesticides tested do not activate PPAR{alpha} or PPAR{gamma} in in vitro assays, but only diclofop-methyl and pyrethrins induce PPAR{alpha} agonistic activity in vivo as well as in vitro.« less

Effects of the antirheumatic remedy hox alpha--a new stinging nettle leaf extract--on matrix metalloproteinases in human chondrocytes in vitro.

PubMed

Schulze-Tanzil, G; de, Souza P; Behnke, B; Klingelhoefer, S; Scheid, A; Shakibaei, M

2002-04-01

Inflammatory joint diseases are characterized by enhanced extracellular matrix degradation which is predominantly mediated by cytokine-stimulated upregulation of matrix metalloproteinase (MMP) expression. Besides tumour necrosis factor-alpha (TNF-alpha), Interleukin-1beta (IL-1beta) produced by articular chondrocytes and synovial macrophages, is the most important cytokine stimulating MMP expression under inflammatory conditions. Blockade of these two cytokines and their downstream effectors are suitable molecular targets of antirheumatic therapy. Hox alpha is a novel stinging nettle (Urtica dioica/Urtica urens) leaf extract used for treatment of rheumatic diseases. The aim of the present study was to clarify the effects of Hox alpha and the monosubstance 13-HOTrE (13-Hydroxyoctadecatrienic acid) on the expression of matrix metalloproteinase-1, -3 and -9 proteins (MMP-1, -3, -9). Human chondrocytes were cultured on collagen type-II-coated petri dishes, exposed to IL-1beta and treated with or without Hox alpha and 13-HOTrE. A close analysis by immunofluorescence microscopy and western blot analysis showed that Hox alpha and 13-HOTrE significantly suppressed IL-1beta-induced expression of matrix metalloproteinase-1, -3 and -9 proteins on the chondrocytes in vitro. The potential of Hox alpha and 13-HOTrE to suppress the expression of matrix metalloproteinases may explain the clinical efficacy of stinging nettle leaf extracts in treatment of rheumatoid arthritis. These results suggest that the monosubstance 13-HOTrE is one of the more active antiinflammatory substances in Hox alpha and that Hox alpha may be a promising remedy for therapy of inflammatory joint diseases.

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression.

PubMed

Gee, Katrina; Lim, Wilfred; Ma, Wei; Nandan, Devki; Diaz-Mitoma, Francisco; Kozlowski, Maya; Kumar, Ashok

2002-11-15

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.

Reduced O2 concentration during CAM development--its effect on angiogenesis and gene expression in the broiler embryo CAM.

PubMed

Druyan, S; Levi, E

2012-01-01

Hypoxia during embryogenesis may induce changes in the development of some physiological regulatory systems, thereby causing permanent phenotypic changes in the embryo. Various levels of hypoxia at different time points during embryogenesis were found to affect both anatomical and physiological morphogenesis. These changes and adaptations depended on the timing, intensity, and duration of the hypoxic exposure and, moreover, were regulated by differential expression of developmentally important genes, mostly expressed in a stage- and time-dependent manner. Eggs incubated in a 17%-oxygen atmosphere for 12 h/d from E5 through E12 exhibited a clear and significant increase in the vascular area of the chorioallantoic membrane (CAM); an increase that was already significant within 12 h after the end of the 1st hypoxic exposures (E6). We used the combination of the genes, β-actin, RPLP0 and HPRT as a reference for gene expression profiling, in studying the expression levels of hypoxia-inducible factor 1-alpha (HIF1α), vascular endothelial growth factor alpha-2 (VEGF α 2), vascular endothelial growth factor receptor 2 (KDR), matrix metalloproteinase-2 (MMP2), and fibroblast growth factor 2 (FGF2), under normal and hypoxic conditions. In general, expression of all five investigated genes throughout the embryonic day of development had similar patterns of hypoxia-induced alterations. In E5.5 embryos, expression of HIF1α, MMP2, VEGFα2, and KDR was significantly higher in hypoxic embryos than in controls. In E6 embryos expression of HIF1α, VEGFα2, and FGF2 was significantly higher in hypoxic embryos than in controls. From E6.5 onward expression levels of the examined genes did not show any differences between hypoxic and control embryos. It can be concluded that in this experimental model, exposing broiler embryos to 17% O(2) from E5 to E7 induced significant angiogenesis, as expressed by the above genes. Further studies to examine whether this early exposure to hypoxic condition affects the chick's ability to withstand a post-hatch hypoxic environment is still required. Copyright © 2012 Elsevier B.V. All rights reserved.

c-FLIP is involved in erythropoietin-mediated protection of erythroid-differentiated cells from TNF-alpha-induced apoptosis.

PubMed

Vittori, Daniela; Vota, Daiana; Callero, Mariana; Chamorro, María E; Nesse, Alcira

2010-05-04

The TNF-alpha (tumour necrosis factor) affects a wide range of biological activities, such as cell proliferation and apoptosis. Cell life or death responses to this cytokine might depend on cell conditions. This study focused on the modulation of factors that would affect the sensitivity of erythroid-differentiated cells to TNF-alpha. Hemin-differentiated K562 cells showed higher sensitivity to TNF-induced apoptosis than undifferentiated cells. At the same time, hemin-induced erythroid differentiation reduced c-FLIP (cellular FLICE-inhibitory protein) expression. However, this negative effect was prevented by prior treatment with Epo (erythropoietin), which allowed the cell line to maintain c-FLIP levels. On the other hand, erythroid-differentiated UT-7 cells - dependent on Epo for survival - showed resistance to TNF-alpha pro-apoptotic action. Only after the inhibition of PI3K (phosphatidylinositol-3 kinase)-mediated pathways, which was accompanied by negative c-FLIP modulation and increased erythroid differentiation, were UT-7 cells sensitive to TNF-alpha-triggered apoptosis. In summary, erythroid differentiation might deregulate the balance between growth promotion and death signals induced by TNF-alpha, depending on cell type and environmental conditions. The role of c-FLIP seemed to be critical in the protection of erythroid-differentiated cells from apoptosis or in the determination of their sensitivity to TNF-mediated programmed cell death. Epo, which for the first time was found to be involved in the prevention of c-FLIP down-regulation, proved to have an anti-apoptotic effect against the pro-inflammatory factor. The identification of signals related to cell life/death switching would have significant implications in the control of proliferative diseases and would contribute to the understanding of mechanisms underlying the anaemia associated with inflammatory processes.

Regulation of hypoxia-inducible factor-1alpha by nitric oxide through mitochondria-dependent and -independent pathways.

PubMed Central

Mateo, Jesús; García-Lecea, Marta; Cadenas, Susana; Hernández, Carlos; Moncada, Salvador

2003-01-01

Nitric oxide (NO) has been reported both to promote and to inhibit the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1). In order to avoid the pitfalls associated with the use of NO donors, we have developed a human cell line (Tet-iNOS 293) that expresses the inducible NO synthase (iNOS) under the control of a tetracycline-inducible promoter. Using this system to generate finely controlled amounts of NO, we have demonstrated that the stability of the alpha-subunit of HIF-1 is regulated by NO through two separate mechanisms, only one of which is dependent on a functional respiratory chain. HIF-1alpha is unstable in cells maintained at 21% O(2), but is progressively stabilized as the O(2) concentration decreases, resulting in augmented HIF-1 DNA-binding activity. High concentrations of NO (>1 microM) stabilize HIF-1alpha at all O(2) concentrations tested. This effect does not involve the respiratory chain, since it is preserved in cells lacking functional mitochondria (rho(0)-cells) and is not reproduced by other inhibitors of the cytochrome c oxidase. By contrast, lower concentrations of NO (<400 nM) cause a rapid decrease in HIF-1alpha stabilized by exposure of the cells to 3% O(2). This effect of NO is dependent on the inhibition of mitochondrial respiration, since it is mimicked by other inhibitors of mitochondrial respiration, including those not acting at cytochrome c oxidase. We suggest that, although stabilization of HIF-1alpha by high concentrations of NO might have implications in pathophysiological processes, the inhibitory effect of lower NO concentrations is likely to be of physiological relevance. PMID:14531732

Selective inhibition of iNOS attenuates trauma-hemorrhage/resuscitation-induced hepatic injury.

PubMed

Kan, Wen-Hong; Hsu, Jun-Te; Schwacha, Martin G; Choudhry, Mashkoor A; Raju, Raghavan; Bland, Kirby I; Chaudry, Irshad H

2008-10-01

Although trauma-hemorrhage produces tissue hypoxia, systemic inflammatory response and organ dysfunction, the mechanisms responsible for these alterations are not clear. Using a potent selective inducible nitric oxide (NO) synthase inhibitor, N-[3-(aminomethyl) benzyl]acetamidine (1400W), and a nonselective NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), we investigated whether inducible NO synthase plays any role in producing hepatic injury, inflammation, and changes of protein expression following trauma-hemorrhage. To investigate this, male Sprague-Dawley rats were subjected to midline laparotomy and hemorrhagic shock (mean blood pressure 35-40 mmHg for approximately 90 min) followed by fluid resuscitation. Animals were treated with either vehicle (DMSO) or 1400W (10 mg/kg body wt ip), or L-NAME (30 mg/kg iv), 30 min before resuscitation and killed 2 h after resuscitation. Trauma-hemorrhage/resuscitation induced a marked hypotension and increase in markers of hepatic injury (i.e., plasma alpha-glutathione S-transferase, tissue myeloperoxidase activity, and nitrotyrosine formation). Hepatic expression of iNOS, hypoxia-inducible factor-1alpha, ICAM-1, IL-6, TNF-alpha, and neutrophil chemoattractant (cytokine-induced neutrophil chemoattractant-1 and macrophage inflammatory protein-2) protein levels were also markedly increased following trauma-hemorrhage/resuscitation. Administration of the iNOS inhibitor 1400W significantly attenuated hypotension and expression of these mediators of hepatic injury induced by trauma-hemorrhage/resuscitation. However, administration of L-NAME could not attenuate hepatic dysfunction and tissue injury mediated by trauma-hemorrhage, although it improved mean blood pressure as did 1400W. These results indicate that increased expression of iNOS following trauma-hemorrhage plays an important role in the induction of hepatic damage under such conditions.

Shifts in renin-angiotensin system components, angiogenesis, and oxidative stress-related protein expression in the lamina cribrosa region of streptozotocin-induced diabetic mice.

PubMed

Qian, Xiaobing; Lin, Leilei; Zong, Yao; Yuan, Yongguang; Dong, Yanmin; Fu, Yue; Shao, Wanwen; Li, Yujie; Gao, Qianying

2018-03-01

This study aimed to analyse shifts in renin-angiotensin system (RAS) components, angiogenesis, and oxidative stress-related protein expression in the lamina cribrosa (LC) region in streptozotocin (STZ)-induced diabetic mice. Six months after diabetes induction, the retinal vessels of male C57BL/6 J mice were observed by colour photography, fundus fluorescein angiography (FFA), and immunofluorescent staining following incubation with CD31. Immunofluorescence for glial fibrillary acidic protein (GFAP), alpha-smooth muscle actin (α-SMA),and NG2 was also performed. Angiotensin-converting enzyme 1 (ACE1), angiotensin II type I receptor (AT1R), renin, hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), and haeme oxygenase 1 (HO-1) expression levels were confirmed by immunohistochemical and western blotting analyses. Compared with control mice, diabetic mice had significantly higher blood glucose concentrations (p < 0.001) and significantly lower body weights (p < 0.001). Colour photography and FFA did not reveal any vessel abnormalities in the diabetic mice; however, immunostaining of whole-mount retinas revealed an increased number of retinal vessels. Furthermore, histopathological staining showed significant reduction in the whole retinal thickness. GFAP expression was slightly higher, whereas fewer NG2 + pericytes were observed in diabetic mice than in control mice. ACE1, AT1R, renin, HIF-1α, VEGF, VEGFR2, and HO-1 expression were up-regulated in the LC of the STZ-induced diabetic mice. Collectively, ACE 1, AT1R, HIF-1α, VEGF, VEGFR2, and HO-1 activation in the LC region in diabetic mice may be involved in diabetes via the RAS and induction of angiogenesis and oxidative stress.

α-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes

PubMed Central

Potapovich, AI; Pastore, S; Kostyuk, VA; Lulli, D; Mariani, V; De Luca, C; Dudich, EI; Korkina, LG

2009-01-01

Background and purpose: The immunomodulatory effects of α-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. Experimental approach: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFκB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFκB DNA binding activity was measured by specific assays. Nitric oxide and H2O2 production and redox status were assessed by fluorescent probe and biochemical methods. Key results: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H2O2 and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFκB was activated by AFP alone or by its combination with UVA. Conclusions and implications: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin. PMID:19785658

Alpha-lipoic acid improves high-fat diet-induced hepatic steatosis by modulating the transcription factors SREBP-1, FoxO1 and Nrf2 via the SIRT1/LKB1/AMPK pathway.

PubMed

Yang, Yi; Li, Wang; Liu, Yang; Sun, Yuning; Li, Yan; Yao, Qing; Li, Jianning; Zhang, Qian; Gao, Yujing; Gao, Ling; Zhao, Jiajun

2014-11-01

Understanding the mechanism by which alpha-lipoic acid supplementation has a protective effect upon nonalcoholic fatty liver disease in vivo and in vitro may lead to targets for preventing hepatic steatosis. Male C57BL/6J mice were fed a normal diet, high-fat diet or high-fat diet supplemented with alpha-lipoic acid for 24 weeks. HepG2 cells were incubated with normal medium, palmitate or alpha-lipoic acid. The lipid-lowering effects were measured. The protein expression and distribution were analyzed by Western blot, immunoprecipitation and immunofluorescence, respectively. We found that alpha-lipoic acid enhanced sirtuin 1 deacetylase activity through liver kinase B1 and stimulated AMP-activated protein kinase. By activating the sirtuin 1/liver kinase B1/AMP-activated protein kinase pathway, the translocation of sterol regulatory element-binding protein-1 into the nucleus and forkhead box O1 into the cytoplasm was prevented. Alpha-lipoic acid increased adipose triacylglycerol lipase expression and decreased fatty acid synthase abundance. In in vivo and in vitro studies, alpha-lipoic acid also increased nuclear NF-E2-related factor 2 levels and downstream target amounts via the sirtuin 1 pathway. Alpha-lipoic acid eventually reduced intrahepatic and serum triglyceride content. The protective effects of alpha-lipoic acid on hepatic steatosis appear to be associated with the transcription factors sterol regulatory element-binding protein-1, forkhead box O1 and NF-E2-related factor 2. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin in germinating rice seeds.

PubMed

Nanjo, Yohei; Asatsuma, Satoru; Itoh, Kimiko; Hori, Hidetaka; Mitsui, Toshiaki; Fujisawa, Yukiko

2004-06-01

Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of alpha-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of alpha-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of alpha-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of 45Ca2+ into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of alpha-amylase II-4 effectively. While the gibberellin-induced expression of alpha-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein.

Probiotic lactobacillus and estrogen effects on vaginal epithelial gene expression responses to Candida albicans.

PubMed

Wagner, R Doug; Johnson, Shemedia J

2012-06-20

Vaginal epithelial cells have receptors, signal transduction mechanisms, and cytokine secretion capabilities to recruit host defenses against Candida albicans infections. This research evaluates how probiotic lactobacilli affect the defensive epithelial response. This study used quantitative reverse transcription-polymerase chain reaction assay (qRT-PCR), flow cytometry, and a multiplex immunoassay to observe changes in the regulation of gene expression related to cytokine responses in the VK2 (E6/E7) vaginal epithelial cell line treated with 17β-estradiol, exposed to probiotic Lactobacillus rhamnosus GR-1® and Lactobacillus reuteri RC-14® and challenged with C. albicans. Data were statistically evaluated by repeated measures analysis of variance and paired t-tests where appropriate. C. albicans induced mRNA expression of genes related to inflammatory cytokine responses associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signal transduction pathways. 17β-estradiol suppressed expression of interleukin-1α (IL-1α), IL-6, IL-8, and tumor necrosis factor alpha (TNFα) mRNA. Probiotic lactobacilli suppressed C. albicans-induced nuclear factor-kappa B inhibitor kinase kinase alpha (Iκκα), Toll-like receptor-2 (TLR2), TLR6, IL-8, and TNFα, also suggesting inhibition of NF-κB signaling. The lactobacilli induced expression of IL-1α, and IL-1β mRNA, which was not inhibited by curcumin, suggesting that they induce an alternate inflammatory signal transduction pathway to NF-κB, such as the mitogen activated protein kinase and activator protein-1 (MAPK/AP-1) signal transduction pathway. Curcumin inhibited IL-13 secretion, suggesting that expression of this cytokine is mainly regulated by NF-κB signaling in VK2 cells. The results suggest that C. albicans infection induces pro-inflammatory responses in vaginal epithelial cells, and estrogen and lactobacilli suppress expression of NF-κB-related inflammatory genes. Probiotic lactobacilli may induce IL-1α and IL-1β expression by an alternate signal transduction pathway, such as MAPK/AP-1. Activation of alternate signaling mechanisms by lactobacilli to modify epithelial cell cytokine production may be a mechanism for probiotic modulation of morbidity in vulvovaginal candidiasis.

Therapeutic and prophylactic thalidomide in TNBS-induced colitis: synergistic effects on TNF-alpha, IL-12 and VEGF production.

PubMed

Carvalho, Ana Teresa; Souza, Heitor; Carneiro, Antonio Jose; Castelo-Branco, Morgana; Madi, Kalil; Schanaider, Alberto; Silv, Flavia; Pereira Junior, Fernando Antonio; Pereira, Marcia G; Tortori, Claudio; Dines, Ilana; Carvalho, Jane; Rocha, Eduardo; Elia, Celeste

2007-04-21

To evaluated the therapeutic and prophylactic effect of thalidomide on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Thalidomide has been reported to downregulate the expression of tumor necrosis factor alpha (TNF-alpha), IL-12, and vascular endothelial growth factor (VEGF), hallmarks of intestinal inflammation in Crohnos disease (CD). Male Wistar rats were divided in five groups of ten animals each. Four groups received a rectal infusion of TNBS in ethanol. The first group was sacrificed 7 d after colitis induction. The second and third groups received either thalidomide or placebo by gavage and were sacrificed at 14 d. The fourth group received thalidomide 6 h before TNBS administration, and was sacrificed 7 d after induction. The fifth group acted as the control group and colitis was not induced. Histological inflammatory scores of the colon were performed and lamina propria CD4+ T cells, macrophages, and VEGF+ cells were detected by immunohistochemistry. TNF-alpha and IL-12 were quantified in the supernatant of organ cultures by ELISA. Significant reduction in the inflammatory score and in the percentage of VEGF+ cells was observed in the group treated with thalidomide compared with animals not treated with thalidomide. Both TNF-alpha and IL-12 levels were significantly reduced among TNBS induced colitis animals treated with thalidomide compared with animals that did not receive thalidomide. TNF-alpha levels were also significantly reduced among the animals receiving thalidomide prophylaxis compared with untreated animals with TNBS-induced colitis. Intestinal levels of TNF-alpha and IL-12 were significantly correlated with the inflammatory score and the number of VEGF+ cells. Thalidomide significantly attenuates TNBS-induced colitis by inhibiting the intestinal production of TNF-alpha, IL-12, and VEGF. This effect may support the use of thalidomide as an alternate approach in selected patients with CD.

The G protein-coupled receptor 30 is up-regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) in breast cancer cells and cardiomyocytes.

PubMed

Recchia, Anna Grazia; De Francesco, Ernestina Marianna; Vivacqua, Adele; Sisci, Diego; Panno, Maria Luisa; Andò, Sebastiano; Maggiolini, Marcello

2011-03-25

GPR30, also known as GPER, has been suggested to mediate rapid effects induced by estrogens in diverse normal and cancer tissues. Hypoxia is a common feature of solid tumors involved in apoptosis, cell survival, and proliferation. The response to low oxygen environment is mainly mediated by the hypoxia-inducible factor named HIF-1α, which activates signaling pathways leading to adaptive mechanisms in tumor cells. Here, we demonstrate that the hypoxia induces HIF-1α expression, which in turn mediates the up-regulation of GPER and its downstream target CTGF in estrogen receptor-negative SkBr3 breast cancer cells and in HL-1 cardiomyocytes. Moreover, we show that HIF-1α-responsive elements located within the promoter region of GPER are involved in hypoxia-dependent transcription of GPER, which requires the ROS-induced activation of EGFR/ERK signaling in both SkBr3 and HL-1 and cells. Interestingly, the apoptotic response to hypoxia was prevented by estrogens through GPER in SkBr3 cells. Taken together, our data suggest that the hypoxia-induced expression of GPER may be included among the mechanisms involved in the anti-apoptotic effects elicited by estrogens, particularly in a low oxygen microenvironment.

Chemopreventive effect of selenium and Chinese medicinal herbs on N-nitrosobis(2-oxopropyl)amine-induced hepatocellular carcinoma in Syrian hamsters.

PubMed

Lee, Chang-Yin; Hsu, Yi-Chao; Wang, Jir-You; Chen, Chien-Chih; Chiu, Jen-Hwey

2008-07-01

Oxidative DNA damage by reactive oxygen species is involved in the process of liver carcinogenesis. To test the hypothesis that a remedy containing Scutellaria baicalensis Georgi (Sb) and Bupleurum scorzonerifolfium Willd (Bs) (Sb/Bs remedy) modulates hepatic neoplastic growth, BOP (N-nitrosobis(2-oxopropyl)amine)-induced liver cancers in hamsters were established. Parameters such as survival rate, tumour area, tumour foci, 8-hydroxydeoxyguanosine (8-OHdG), caspase-3, transforming growth factor (TGF-beta1) and tumour necrosis factor-alpha (TNF-alpha) were measured after Sb/Bs remedy treatment during BOP-induced carcinogenesis. The results showed that the Sb/Bs remedy and its constituents Sb and Bs suppressed the tumour area in BOP-induced liver tumours. Because selenium (Sel) is toxic at a high dose (10 mg/kg), with a low survival rate (0%), the combination of Sb/Bs remedy and low-dose Sel (1 mg/kg) was found to decrease the tumour area and the number of tumour foci while increasing serum TNF-alpha and TGF-beta1, but not IL-6 levels. Besides, the Sb/Bs remedy, when combined with low-dose Sel, not only decreased the expression of 8-OHdG and increased caspase-3 expression within the glutathione S-transferase placental form-positive tumour foci but also increased tumour apoptosis in BOP-induced hamsters. We conclude that low-dose Sel has a chemoprevention effect on BOP-induced liver tumours and such an effect was more enhanced when combined with Sb/Bs treatment.

Anti-obesogenic effects of WY14643 (PPAR-alpha agonist): Hepatic mitochondrial enhancement and suppressed lipogenic pathway in diet-induced obese mice.

PubMed

Veiga, Flavia Maria Silva; Graus-Nunes, Francielle; Rachid, Tamiris Lima; Barreto, Aline Barcellos; Mandarim-de-Lacerda, Carlos Alberto; Souza-Mello, Vanessa

2017-09-01

Non-alcoholic fatty liver disease (NAFLD) presents with growing prevalence worldwide, though its pharmacological treatment remains to be established. This study aimed to evaluate the effects of a PPAR-alpha agonist on liver tissue structure, ultrastructure, and metabolism, focusing on gene and protein expression of de novo lipogenesis and gluconeogenesis pathways, in diet-induced obese mice. Male C57BL/6 mice (three months old) received a control diet (C, 10% of lipids, n = 10) or a high-fat diet (HFD, 50% of lipids, n = 10) for ten weeks. These groups were subdivided to receive the treatment (n = 5 per group): C, C-alpha (PPAR-alpha agonist, 2.5 mg/kg/day mixed in the control diet), HFD and HFD-alpha group (PPAR-alpha agonist, 2.5 mg/kg/day mixed in the HFD). The effects were compared with biometrical, biochemical, molecular biology and transmission electron microscopy (TEM) analyses. HFD showed greater body mass (BM) and insulinemia than C, both of which were tackled by the treatment in the HFD-alpha group. Increased hepatic protein expression of glucose-6-phosphatase, CHREBP and gene expression of PEPCK in HFD points to increased gluconeogenesis. Treatment rescued these parameters in the HFD-alpha group, eliciting a reduced hepatic glucose output, confirmed by the smaller GLUT2 expression in HFD-alpha than in HFD. Conversely, favored de novo lipogenesis was found in the HFD group by the increased expression of PPAR-gamma, and its target gene SREBP-1, FAS and GK when compared to C. The treatment yielded a marked reduction in the expression of all lipogenic factors. TEM analyses showed a greater numerical density of mitochondria per area of tissue in treated than in untreated groups, suggesting an increase in beta-oxidation and the consequent NAFLD control. PPAR-alpha activation reduced BM and treated insulin resistance (IR) and NAFLD by increasing the number of mitochondria and reducing hepatic gluconeogenesis and de novo lipogenesis protein and gene expressions in a murine obesity model. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Purinergic signaling is required for fluid shear stress-induced NF-{kappa}B translocation in osteoblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Genetos, Damian C., E-mail: dgenetos@ucdavis.edu; Karin, Norman J.; Geist, Derik J.

2011-04-01

Fluid shear stress regulates gene expression in osteoblasts, in part by activation of the transcription factor NF-{kappa}B. We examined whether this process was under the control of purinoceptor activation. MC3T3-E1 osteoblasts under static conditions expressed the NF-{kappa}B inhibitory protein I{kappa}B{alpha} and exhibited cytosolic localization of NF-{kappa}B. Under fluid shear stress, I{kappa}B{alpha} levels decreased, and concomitant nuclear localization of NF-{kappa}B was observed. Cells exposed to fluid shear stress in ATP-depleted medium exhibited no significant reduction in I{kappa}B{alpha}, and NF-{kappa}B remained within the cytosol. Similar results were found using oxidized ATP or Brilliant Blue G, P2X{sub 7} receptor antagonists, indicating that themore » P2X{sub 7} receptor is responsible for fluid shear-stress-induced I{kappa}B{alpha} degradation and nuclear accumulation of NF-{kappa}B. Pharmacologic blockage of the P2Y6 receptor also prevented shear-induced I{kappa}B{alpha} degradation. These phenomena involved neither ERK1/2 signaling nor autocrine activation by P2X{sub 7}-generated lysophosphatidic acid. Our results suggest that fluid shear stress regulates NF-{kappa}B activity through the P2Y{sub 6} and P2X{sub 7} receptor.« less

Analysis of Subcellular RNA Fractions Revealed a Transcription-Independent Effect of Tumor Necrosis Factor Alpha on Splicing, Mediated by Spt5.

PubMed

Diamant, Gil; Eisenbaum, Tal; Leshkowitz, Dena; Dikstein, Rivka

2016-05-01

The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) modulates the expression of many genes, primarily through activation of NF-κB. Here, we examined the global effects of the elongation factor Spt5 on nascent and mature mRNAs of TNF-α-induced cells using chromatin and cytosolic subcellular fractions. We identified several classes of TNF-α-induced genes controlled at the level of transcription, splicing, and chromatin retention. Spt5 was found to facilitate splicing and chromatin release in genes displaying high induction rates. Further analysis revealed striking effects of TNF-α on the splicing of 25% of expressed genes; the vast majority were not transcriptionally induced. Splicing enhancement of noninduced genes by TNF-α was transient and independent of NF-κB. Investigating the underlying basis, we found that Spt5 is required for the splicing facilitation of the noninduced genes. In line with this, Spt5 interacts with Sm core protein splicing factors. Furthermore, following TNF-α treatment, levels of RNA polymerase II (Pol II) but not Spt5 are reduced from the splicing-induced genes, suggesting that these genes become enriched with a Pol II-Spt5 form. Our findings revealed the Pol II-Spt5 complex as a highly competent coordinator of cotranscriptional splicing. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Macrophage activation by factors released from acetaminophen-injured hepatocytes: Potential role of HMGB1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dragomir, Ana-Cristina; Laskin, Jeffrey D.; Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu

2011-06-15

Toxic doses of acetaminophen (AA) cause hepatocellular necrosis. Evidence suggests that activated macrophages contribute to the pathogenic process; however, the factors that activate these cells are unknown. In these studies, we assessed the role of mediators released from AA-injured hepatocytes in macrophage activation. Treatment of macrophages with conditioned medium (CM) collected 24 hr after treatment of mouse hepatocytes with 5 mM AA (CM-AA) resulted in increased production of reactive oxygen species (ROS). Macrophage expression of heme oxygenase-1 (HO-1) and catalase mRNA was also upregulated by CM-AA, as well as cyclooxygenase (COX)-2 and 12/15-lipoxygenase (LOX). CM-AA also upregulated expression of themore » proinflammatory chemokines, MIP-1{alpha} and MIP-2. The effects of CM-AA on expression of COX-2, MIP-1{alpha} and MIP-2 were inhibited by blockade of p44/42 MAP kinase, suggesting a biochemical mechanism mediating macrophage activation. Hepatocytes injured by AA were found to release HMGB1, a potent macrophage activator. This was inhibited by pretreatment of hepatocytes with ethyl pyruvate (EP), which blocks HMGB1 release. EP also blocked CM-AA induced ROS production and antioxidant expression, and reduced expression of COX-2, but not MIP-1{alpha} or MIP-2. These findings suggest that HMGB1 released by AA-injured hepatocytes contributes to macrophage activation. This is supported by our observation that expression of the HMGB1 receptor RAGE is upregulated in macrophages in response to CM-AA. These data indicate that AA-injured hepatocytes contribute to the inflammatory environment in the liver through the release of mediators such as HMGB1. Blocking HMGB1/RAGE may be a useful approach to limiting classical macrophage activation and AA-induced hepatotoxicity. - Research Highlights: > These studies analyze macrophage activation by mediators released from acetaminophen-damaged hepatocytes. > Factors released from acetaminophen-injured hepatocytes induce macrophage ROS production and expression of COX-2, chemokines, and RAGE. > Hepatocyte-mediated macrophage activation involves p44/42 MAP kinase signaling. > HMGB1 is released from acetaminophen-injured hepatocytes and contributes to macrophage activation.« less

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells.

PubMed

Zhao, Dezheng; Zhan, Yanai; Koon, Hon Wai; Zeng, Huiyan; Keates, Sarah; Moyer, Mary P; Pothoulakis, Charalabos

2004-10-15

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.

Distinct expression pattern of IFN-alpha and TNF-alpha in juvenile idiopathic arthritis synovial tissue.

PubMed

Gattorno, M; Chicha, L; Gregorio, A; Ferlito, F; Rossi, F; Jarrossay, D; Lanzavecchia, A; Martini, A; Manz, M G

2007-04-01

Recent laboratory and clinical data suggest that two prototype autoimmune diseases, systemic lupus erythematosus and rheumatoid arthritis are mainly driven by distinct cytokines, interferon (IFN)-alpha and tumour necrosis factor (TNF)-alpha, respectively. We here investigated the presence and characteristics of natural type I IFN-producing cells (IPCs), as well as IFN-alpha and TNF-alpha expression at sites of inflammation in juvenile idiopathic arthritis (JIA). Peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MNCs) (n = 25 each) from JIA patients with active disease were studied. IPCs were identified as BCDA-2(+)CD123(+)HLA-DR(+)CD45RA(+) cells, and dendritic cells (DCs) as CD11c(+)CD14(-/low)lin(-) cells by flow cytometry. IPCs and DCs were analysed for Toll-like receptor-7 and -9 mRNA expression by real-time polymerase chain reaction. IFN-alpha was measured by enzyme-linked immunosorbent assay in serum, SF and in supernatants of influenza virus-infected, cultured IPCs. Synovial tissues of n = 6 additional JIA patients were analysed by immunohistochemistry using mAbs against CD123, IFN-alpha, TNF-alpha, CD3, CD19 and CD138. IPCs were enriched in SF MNCs compared with PB MNCs in all JIA patients. Influenza-induced, but no spontaneous IFN-alpha release was detected from SF IPCs, and serum and SF IFN-alpha levels were not elevated. Nonetheless, in synovial tissue IFN-alpha producing cells accumulated at inflammatory lymph-follicular-like structures, while TNF-alpha producing cells were mostly found at the lining and sublining layers. These data suggest that besides TNF-alpha-expressing cells, IFN-alpha-producing IPCs are involved in initiation, maintenance or regulation of the inflammatory response in JIA.

Regulation of matrix metalloproteinase-9 expression between gingival fibroblast cells from old and young rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Su-Jung; Chung, Yong-Koo; Chung, Tae-Wook

2009-01-09

Gingival fibroblast cells (rGF) from aged rats have an age-related decline in proliferative capacity compared with young rats. We investigated G1 phase cell cycle regulation and MMP-9 expression in both young and aged rGF. G1 cell cycle protein levels and activity were significantly reduced in response to interleukin-1{beta} (IL-1{beta}) stimulation with increasing in vitro age. Tumor necrosis factor-{alpha} (TNF-{alpha})-induced matrix metalloproteinase-9 (MMP-9) expression was also decreased in aged rGF in comparison with young rGF. Mutational analysis and gel shift assays demonstrated that the lower MMP-9 expression in aged rGF is associated with lower activities of transcription factors NF-{kappa}B and AP-1.more » These results suggest that cell cycle dysregulation and down-regulation of MMP-9 expression in rGF may play a role in gingival remodeling during in vitro aging.« less

HIV turns plasmacytoid dendritic cells (pDC) into TRAIL-expressing killer pDC and down-regulates HIV coreceptors by Toll-like receptor 7-induced IFN-alpha.

PubMed

Hardy, Andrew W; Graham, David R; Shearer, Gene M; Herbeuval, Jean-Philippe

2007-10-30

Plasmacytoid dendritic cells (pDC) are key players in viral immunity and produce IFN-alpha after HIV-1 exposure, which in turn regulates TNF-related apoptosis-inducing ligand (TRAIL) expression by CD4(+) T cells. We show here that infectious and noninfectious HIV-1 virions induce activation of pDC into TRAIL-expressing IFN-producing killer pDC (IKpDC). IKpDC expressed high levels of activation markers (HLA-DR, CD80, CD83, and CD86) and the migration marker CCR7. Surprisingly, CXCR4 and CCR5 were down-regulated on IKpDC. We also show that HIV-1-induced IKpDC depended on Toll-like receptor 7 (TLR7) activation. HIV-1 or TLR7 agonistexposed IKpDC induced apoptosis of the CD4(+) T cell line SupT1 via the TRAIL pathway. Furthermore, IFN-alpha produced after HIV-induced TLR7 stimulation was responsible for TRAIL expression and the down-regulation of both CXCR4 and CCR5 by IKpDC. In contrast, activation and migration markers were not regulated by IFN-alpha. Finally, IFN-alpha increased the survival of IKpDC. We characterized a subset of pDC with a killer activity that is activated by endosomal-associated viral RNA and not by infection.

Identification of a novel compound that inhibits iNOS and COX-2 expression in LPS-stimulated macrophages from Schisandra chinensis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, You Jin; Park, Sun Young; Kim, Sun Gun

2010-01-22

A novel {alpha}-iso-cubebenol, which has anti-inflammatory effects in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, was isolated from the fruits of Schisandra chinensis. {alpha}-iso-cubebenol inhibited LPS-induced nitric oxide (NO) and prostaglandin E{sub 2} (PGE{sub 2}) production. Consistent with these findings, {alpha}-iso-cubebenol also reduced the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 at the protein and mRNA levels in a concentration-dependent manner. {alpha}-iso-cubebenol also inhibited LPS-induced nuclear translocation of the NF-{kappa}B p65 subunit. Furthermore, {alpha}-iso-cubebenol suppressed the phosphorylation of ERK, JNK, and p38 kinase induced by LPS. Since the novel {alpha}-iso-cubebenol blocked the production of several pro-inflammatory mediators induced by LPSmore » in macrophages, the molecule can be useful material for the development of anti-inflammatory agents against bacterial infections or endotoxin.« less

CXCR4 and SDF1 expression in human meningiomas: a proliferative role in tumoral meningothelial cells in vitro.

PubMed

Bajetto, Adriana; Barbieri, Federica; Pattarozzi, Alessandra; Dorcaratto, Alessandra; Porcile, Carola; Ravetti, Jean Louis; Zona, Gianluigi; Spaziante, Renato; Schettini, Gennaro; Florio, Tullio

2007-01-01

Chemokines participate in cellular processes associated with tumor proliferation, migration, and angiogenesis. We previously demonstrated that stromal cell-derived factor 1 (SDF1) exerts a mitogenic activity in glioblastomas through the activation of its receptor CXCR4. Here we studied the expression of this chemokine in human meningiomas and its possible role in cell proliferation. Reverse transcriptase-PCR analysis for CXCR4 and SDF1 was performed on 55 human meningiomas (47 WHO grade I, 5 WHO II, and 3 WHO III). Immunolabeling for CXCR4 and SDF1 was performed on paraffin-embedded sections of these tumors. [(3)H]Thymidine uptake and Western blot analyses were performed on primary meningeal cell cultures of tumors to evaluate the proliferative activity of human SDF1alpha (hSDF1alpha) in vitro and the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) activation in this process. CXCR4 mRNA was expressed by 78% of the tumor specimens and SDF1 mRNA by 53%. CXCR4 and SDF1 were often detected in the same tumor tissues and colocalized with epithelial membrane antigen immunostaining. In 9 of 12 primary cultures from meningiomas, hSDF1alpha induced significant cell proliferation that was strongly reduced by the mitogen-activated protein kinase kinase inhibitor PD98059, involving ERK1/2 activation in the proliferative signal of hSDF1alpha. In fact, CXCR4 stimulation led to ERK1/2 phosphorylation/activation. In addition, the hSDF1alpha-induced cell proliferation was significantly correlated with the MIB1 staining index in the corresponding surgical specimen. In conclusion, we found that human meningiomas express CXCR4 and SDF1 and that hSDF1alpha induces proliferation in primary meningioma cell cultures through the activation of ERK1/2.

Chronic hypoxia-induced alteration of presynaptic protein profiles and neurobehavioral dysfunction are averted by supplemental oxygen in Lymnaea stagnalis.

PubMed

Fei, G-H; Feng, Z-P

2008-04-22

Chronic hypoxia causes neural dysfunction. Oxygen (O(2)) supplements have been commonly used to increase the O(2) supply, yet the therapeutic benefit of this treatment remains controversial due to a lack of cellular and molecular evidence. In this study, we examined the effects of short-burst O(2) supplementation on neural behavior and presynaptic protein expression profiles in a simple chronic hypoxia model of snail Lymnaea stagnalis. We reported that hypoxia delayed the animal response to light stimuli, suppressed locomotory activity, induced expression of stress-response proteins, hypoxia inducible factor-1alpha (HIF-1alpha) and heat shock protein 70 (HSP70), repressed syntaxin-1 (a membrane-bound presynaptic protein) and elevated vesicle-associated membrane protein-1 (VAMP-1) (a vesicle-bound presynaptic protein) level. O(2) supplements relieved suppression of neural behaviors, and corrected hypoxia-induced protein alterations in a dose-dependent manner. The effectiveness of supplemental O(2) was further evaluated by determining time courses for recovery of neural behaviors and expression of stress response proteins and presynaptic proteins after relief from hypoxia conditions. Our findings suggest that O(2) supplement improves hypoxia-induced adverse alterations of presynaptic protein expression and neurobehaviors, however, the optimal level of O(2) required for improvement is protein specific and system specific.

Interferon-alpha and interferon-gamma sensitize human tenon fibroblasts to mitomycin-C.

PubMed

Wang, Xiao Yang; Crowston, Jonathan G; Zoellner, Hans; Healey, Paul R

2007-08-01

To investigate the effect of interferon (IFN)-alpha and IFN-gamma pretreatment on mitomycin C (MMC)-induced cell death in human Tenon fibroblasts (HTFs) and the mechanisms by which IFN-alpha and IFN-gamma modulate the susceptibility of HTFs to MMC. HTFs were pretreated with IFN-alpha and IFN-gamma for 48 hours before 5-minute application of 0.4 mg/mL MMC. Cell death after 48 hours was determined by Annexin V/propidium iodide (PI) staining and lactate dehydrogenase (LDH) release assay. Fas, Fas-ligand, and Bcl-2 expression were determined by flow cytometry. Fas associated death domain (FADD), Bax, cytochrome c, and caspase expression were determined by Western blot analysis and immunofluorescence staining. MMC treatment increased cell death and upregulated Fas and FADD expression, but had no effect on Fas-Ligand, Bax, Bcl-2, or cytochrome c. Neither IFN-alpha nor IFN-gamma alone induced HTF death, but each increased cell death 2 days after MMC treatment in a dose-dependent fashion. Combination IFN-alpha and IFN-gamma had a synergistic effect. IFN-alpha and IFN-gamma pretreatment increased Fas expression. Fas upregulation was associated with increased sensitivity to MMC. IFN pretreatment increased procaspase-8, procaspase-9, and procaspase-3 expression, and caspase-3 activation. Caspase-8, caspase-3, and broad caspase inhibitors, but not caspase-9 inhibitor, inhibited MMC-induced cell death in nonpretreated and IFN-pretreated cells. IFN-alpha and IFN-gamma enhance the susceptibility of HTFs to MMC-induced cell death through a Fas-mediated and a caspase-3-dependent pathway. Pretreatment with IFN primed HTFs to MMC, providing a potential means for initially slowing the healing response with IFN and subsequently terminating fibroblast activity through MMC-induced cell death.

Retention of prolyl hydroxylase PHD2 in the cytoplasm prevents PHD2-induced anchorage-independent carcinoma cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jokilehto, Terhi; Turku Graduate School of Biomedical Sciences, Turku; Hoegel, Heidi

2010-04-15

Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1{alpha} and -2{alpha}). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells.more » The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1{alpha} or -2{alpha} expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.« less

Alterations of hypoxia-inducible factor-1 alpha in the hippocampus of mice acutely and repeatedly exposed to hypoxia.

PubMed

Shao, Guo; Gao, Cui-Ying; Lu, Guo-Wei

2005-01-01

This work aims at investigating the effects of hypoxic preconditioning on hypoxia-inducible factor-1 alpha (HIF-1alpha) expression in the hippocampus of mice during acute and repeated hypoxic exposures. The mice were randomly divided into three groups and exposed, respectively, to hypoxia for 4 runs (group H4), 1 run (group H1), and 0 run (group H0). Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation were used to examine the HIF-1alpha responses in the mouse hippocampus following exposure to hypoxia. The tolerance of mice to hypoxia increased significantly following acute and repetitive exposure to autoprogressive hypoxia. Total mRNA, total protein, and nuclear protein were extracted from the hippocampus for RT-PCR, Western blot, and EMSA, respectively. The HIF-1alpha mRNA levels were found to be increased in group H1 and decreased in group H4. The HIF-1alpha protein levels and HIF-1 DNA-binding activities were increased in group H1 and markedly increased in group H4. One of the HIF-1 target genes, vascular endothelial growth factor, increased in group H4. HIF-1 activation is thought to be involved in the protection of the brain of hypoxic preconditioned mice. Copyright 2005 S. Karger AG, Basel

G protein-coupled receptor 30 expression is up-regulated by EGF and TGF alpha in estrogen receptor alpha-positive cancer cells.

PubMed

Vivacqua, Adele; Lappano, Rosamaria; De Marco, Paola; Sisci, Diego; Aquila, Saveria; De Amicis, Francesca; Fuqua, Suzanne A W; Andò, Sebastiano; Maggiolini, Marcello

2009-11-01

In the present study, we evaluated the regulation of G protein-coupled receptor (GPR)30 expression in estrogen receptor (ER)-positive endometrial, ovarian, and estrogen-sensitive, as well as tamoxifen-resistant breast cancer cells. We demonstrate that epidermal growth factor (EGF) and TGF alpha transactivate the GPR30 promoter and accordingly up-regulate GPR30 mRNA and protein levels only in endometrial and tamoxifen-resistant breast cancer cells. These effects exerted by EGF and TGF alpha were dependent on EGF receptor (EGFR) expression and activation and involved phosphorylation of the Tyr(1045) and Tyr(1173) EGFR sites. Using gene-silencing experiments and specific pharmacological inhibitors, we have ascertained that EGF and TGF alpha induce GPR30 expression through the EGFR/ERK transduction pathway, and the recruitment of c-fos to the activator protein-1 site located within GPR30 promoter sequence. Interestingly, we show that functional cross talk of GPR30 with both activated EGFR and ER alpha relies on a physical interaction among these receptors, further extending the potential of estrogen to trigger a complex stimulatory signaling network in hormone-sensitive tumors. Given that EGFR/HER2 overexpression is associated with tamoxifen resistance, our data may suggest that ligand-activated EGFR could contribute to the failure of tamoxifen therapy also by up-regulating GPR30, which in turn could facilitates the action of estrogen. In addition, important for resistance is the ability of tamoxifen to bind to and activate GPR30, the expression of which is up-regulated by EGFR activation. Our results emphasize the need for new endocrine agents able to block widespread actions of estrogen without exerting any stimulatory activity on transduction pathways shared by the steroid and growth factor-signaling networks.

Anti-nociceptive effect of thalidomide on zymosan-induced experimental articular incapacitation.

PubMed

Vale, Mariana L; Cunha, Fernando Q; Brito, Gerly A C; Benevides, Verônica M; Ferreira, Sérgio H; Poole, Stephen; Ribeiro, Ronaldo A

2006-05-01

The anti-nociceptive effect of thalidomide on zymosan-induced articular knee joint incapacitation in rats was investigated. Thalidomide (5-45 mg/kg), given 30 min before but not 2 h after the intra-articular injection of zymosan, inhibited the nociceptive response in a dose-dependent manner. Furthermore, thalidomide pretreatment significantly reduced the concentration of tumor necrosis factor-alpha (TNF-alpha, -68.4%) in the exudate of zymosan-injected joints, but not those of interleukin-1beta, interleukin-6, CINC-1 or interleukin-10. The expression of TNF-alpha, determined by immunohistochemical staining, in synovial tissues obtained from articular joints injected with zymosan was also inhibited by thalidomide pretreatment. The anti-nociceptive effect of thalidomide was not reversed by the co-administration of an opioid receptor antagonist, naloxone, suggesting that endogenous opioids do not mediate the anti-nociceptive effect of thalidomide in this model. In conclusion, the anti-nociceptive activity of thalidomide in zymosan-induced articular incapacitation is associated with the inhibition of TNF-alpha by resident synovial cells.

The VP35 protein of Ebola virus impairs dendritic cell maturation induced by virus and lipopolysaccharide.

PubMed

Jin, Huali; Yan, Zhipeng; Prabhakar, Bellur S; Feng, Zongdi; Ma, Yijie; Verpooten, Dustin; Ganesh, Balaji; He, Bin

2010-02-01

Ebola virus causes rapidly progressive haemorrhagic fever, which is associated with severe immuosuppression. In infected dendritic cells (DCs), Ebola virus replicates efficiently and inhibits DC maturation without inducing cytokine expression, leading to impaired T-cell proliferation. However, the underlying mechanism remains unclear. In this study, we report that Ebola virus VP35 impairs the maturation of mouse DCs. When expressed in mouse immature DCs, Ebola virus VP35 prevents virus-stimulated expression of CD40, CD80, CD86 and major histocompatibility complex class II. Further, it suppresses the induction of cytokines such as interleukin (IL)-6, IL-12, tumour necrosis factor alpha and alpha/beta interferon (IFN-alpha/beta). Notably, Ebola VP35 attenuates the ability of DCs to stimulate the activation of CD4(+) T cells. Addition of type I IFN to mouse DCs only partially reverses the inhibitory effects of VP35. Moreover, VP35 perturbs mouse DC functions induced by lipopolysaccharide, an agonist of Toll-like receptor 4. Deletion of the amino terminus abolishes its activity, whereas a mutation in the RNA binding motif has no effect. Our work highlights a critical role of VP35 in viral interference in DC function with resultant deficiency in T-cell function, which may contribute to the profound virulence of Ebola virus infection.

Transitions towards either slow-oxidative or fast-glycolytic phenotype can be induced in the murine WTt myogenic cell line.

PubMed

Peltzer, J; Carpentier, G; Martelly, I; Courty, J; Keller, A

2010-09-01

Contraction and energy metabolism are functions of skeletal muscles co-regulated by still largely unknown signals. To help elucidating these interconnecting pathways, we are developing new cellular models that will allow to control the switch from a neonatal to an adult slow-oxidative or fast-glycolytic phenotype of myofibers, during in vitro differentiation. Thus, our purpose was to direct the differentiation of the newly characterized WTt clone, from a mixed towards either fast or slow phenotype, by modifying amounts of two transcription factors respectively involved in control of glycolytic and oxidative energy metabolism, namely HIF-1alpha and PPARdelta. Our data support the idea that HIF-1alpha protein stabilization would favor expression of fast phenotypic markers, accompanied or not by a decreased expression of slow markers, depending on treatment conditions. Conversely, PPARdelta over-expression appears to enhance the slow-oxidative phenotype of WTt myotubes. Furthermore, we have observed that expression of PGC-1alpha, a coregulator of PPAR, is also modified in this cell line upon conditions that stabilize HIF-1alpha protein. This observation points to the existence of a regulatory link between pathways controlled by the two transcription factors HIF-1alpha and PPARdelta. Therefore, these cells should be useful to analyze the balance between oxidative and glycolytic energy production as a function of phenotypic transitions occurring during myogenic maturation. The newly characterized murine WTt clone will be a good tool to investigate molecular mechanisms implicating HIF-1alpha and PPARdelta in the coordinated metabolic and contractile regulations involved in myogenesis. (c) 2010 Wiley-Liss, Inc.

Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways.

PubMed

Twu, Cheryl; Liu, Nancy Q; Popik, Waldemar; Bukrinsky, Michael; Sayre, James; Roberts, Jaclyn; Rania, Shammas; Bramhandam, Vishnu; Roos, Kenneth P; MacLellan, W Robb; Fiala, Milan

2002-10-29

We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-alpha. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-alpha and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways.

PCNA mono-ubiquitination and activation of translesion DNA polymerases by DNA polymerase {alpha}.

PubMed

Suzuki, Motoshi; Niimi, Atsuko; Limsirichaikul, Siripan; Tomida, Shuta; Miao Huang, Qin; Izuta, Shunji; Usukura, Jiro; Itoh, Yasutomo; Hishida, Takashi; Akashi, Tomohiro; Nakagawa, Yoshiyuki; Kikuchi, Akihiko; Pavlov, Youri; Murate, Takashi; Takahashi, Takashi

2009-07-01

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

Spleen tyrosine kinase mediates high glucose-induced transforming growth factor-{beta}1 up-regulation in proximal tubular epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Won Seok; Chang, Jai Won; Han, Nam Jeong

The role of spleen tyrosine kinase (Syk) in high glucose-induced intracellular signal transduction has yet to be elucidated. We investigated whether Syk is implicated in high glucose-induced transforming growth factor-{beta}1 (TGF-{beta}1) up-regulation in cultured human proximal tubular epithelial cells (HK-2 cell). High glucose increased TGF-{beta}1 gene expression through Syk, extracellular signal-regulated kinase (ERK), AP-1 and NF-{kappa}B. High glucose-induced AP-1 DNA binding activity was decreased by Syk inhibitors and U0126 (an ERK inhibitor). Syk inhibitors suppressed high glucose-induced ERK activation, whereas U0126 had no effect on Syk activation. High glucose-induced NF-{kappa}B DNA binding activity was also decreased by Syk inhibitors. Highmore » glucose increased nuclear translocation of p65 without serine phosphorylation of I{kappa}B{alpha} and without degradation of I{kappa}B{alpha}, but with an increase in tyrosine phosphorylation of I{kappa}B{alpha} that may account for the activation of NF-{kappa}B. Both Syk inhibitors and Syk-siRNA attenuated high glucose-induced I{kappa}B{alpha} tyrosine phosphorylation and p65 nuclear translocation. Depletion of p21-activated kinase 2 (Pak2) by transfection of Pak2-siRNA abolished high glucose-induced Syk activation. In summary, high glucose-induced TGF-{beta}1 gene transcription occurred through Pak2, Syk and subsequent ERK/AP-1 and NF-{kappa}B pathways. This suggests that Syk might be implicated in the diabetic kidney disease.« less

Expression of alpha-expansin and expansin-like genes in deepwater rice.

PubMed

Lee, Yi; Kende, Hans

2002-11-01

Previously, we have studied the expression and regulation of four alpha- and 14 beta-expansin genes in deepwater rice (Oryza sativa). We now report on the structure, expression, and regulation of 22 additional alpha-expansin (Os-EXP) genes, four expansin-like (Os-EXPL) genes, and one expansin-related (Os-EXPR) gene, which have recently been identified in the expressed sequence tag and genomic databases of rice. Alpha-expansins are characterized by a series of conserved Cys residues in the N-terminal half of the protein, a histidine-phenylalanine-aspartate (HFD) motif in the central region, and a series of tryptophan residues near the carboxyl terminus. Of the 22 additional alpha-expansin genes, five are expressed in internodes and leaves, three in coleoptiles, and nine in roots, with high transcript levels in the growing regions of these organs. Transcripts of five alpha-expansin genes were found in roots only. Expression of five alpha-expansin genes was induced in the internode by treatment with gibberellin (GA) and by wounding. The wound response resulted from excising stem sections or from piercing pinholes into the stem of intact plants. EXPL proteins lack the HFD motif and have two additional Cys residues in their C- and N-terminal regions. The positions of conserved tryptophan residues at the C-terminal region are different from those of alpha- and beta-expansins. Expression of the Os-EXPL3 gene is correlated with elongation and slightly induced by applied GA. However, the expression of the Os-EXPL1 and Os-EXPL2 genes showed limited correlation with cell elongation and was not induced by GA. We found no expression of the Os-EXPR1 gene in the organs examined.

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

PubMed

Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

2005-08-01

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Negative regulatory role of PI3-kinase in TNF-induced tumor necrosis.

PubMed

Matschurat, Susanne; Blum, Sabine; Mitnacht-Kraus, Rita; Dijkman, Henry B P M; Kanal, Levent; De Waal, Robert M W; Clauss, Matthias

2003-10-20

Tissue factor is the prime initiator of blood coagulation. Expression of tissue factor in tumor endothelial cells leads to thrombus formation, occlusion of vessels and development of hemorrhagic infarctions in the tumor tissue, often followed by regression of the tumor. Tumor cells produce endogenous vascular endothelial growth factor (VEGF), which sensitizes endothelial cells for systemically administered tumor necrosis factor alpha (TNF alpha) and synergistically enhances the TNF-induced expression of tissue factor. We have analyzed the pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells (HUVECs) after combined stimulation with TNF and VEGF. By using specific low molecular weight inhibitors, we demonstrated that protein kinase C (PKC), p44/42 and p38 mitogen-activated protein (MAP) kinases, and stress-activated protein kinase (JNK) are essentially involved in the induction of tissue factor. In contrast, the application of wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, led to strongly enhanced expression of tissue factor in TNF- and VEGF-treated cells, implicating a negative regulatory role for PI3-kinase. In vivo, the application of wortmannin promoted the formation of TNF-induced hemorrhages and intratumoral necroses in murine meth A tumors. The co-injection of wortmannin lowered the effective dose of applied TNF. Therefore, it is conceivable that the treatment of TNF-sensitive tumors with a combination of TNF and wortmannin will ensure the selective damage of the tumor endothelium and minimize the risk of systemic toxicity of TNF. TNF-treatment in combination with specific inhibition of PI3-kinase is a novel concept in anti-cancer therapy. Copyright 2003 Wiley-Liss, Inc.

Cinnamaldehyde inhibits the tumor necrosis factor-{alpha}-induced expression of cell adhesion molecules in endothelial cells by suppressing NF-{kappa}B activation: Effects upon I{kappa}B and Nrf2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao, B.-C.; Hsieh, C.-W.; Liu, Y.-C.

The production of adhesion molecules and subsequent attachment of leukocytes to endothelial cells (ECs) are critical early events in atherogenesis. These adhesion molecules thus play an important role in the development of this disease. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of cinnamaldehyde, a Cinnamomum cassia Presl-specific diterpene. In our current study, we have examined the effects of both cinnamaldehyde and extracts of C. cassia on cytokine-induced monocyte/human endothelial cell interactions. We find that these compounds inhibit the adhesion of TNF{alpha}-induced monocytes to endothelial cells and suppress the expression of the cell adhesion molecules, VCAM-1 and ICAM-1, atmore » the transcriptional level. Moreover, in TNF{alpha}-treated ECs, the principal downstream signal of VCAM-1 and ICAM-1, NF-{kappa}B, was also found to be abolished in a time-dependent manner. Interestingly, cinnamaldehyde exerts its anti-inflammatory effects by blocking the degradation of the inhibitory protein I{kappa}B-{alpha}, but only in short term pretreatments, whereas it does so via the induction of Nrf2-related genes, including heme-oxygenase-1 (HO-1), over long term pretreatments. Treating ECs with zinc protoporphyrin, a HO-1 inhibitor, partially blocks the anti-inflammatory effects of cinnamaldehyde. Elevated HO-1 protein levels were associated with the inhibition of TNF{alpha}-induced ICAM-1 expression. In addition to HO-1, we also found that cinnamaldehyde can upregulate Nrf2 in nuclear extracts, and can increase ARE-luciferase activity and upregulate thioredoxin reductase-1, another Nrf2-related gene. Moreover, cinnamaldehyde exposure rapidly reduces the cellular GSH levels in ECs over short term treatments but increases these levels after 9 h exposure. Hence, our present findings indicate that cinnamaldehyde suppresses TNF-induced singling pathways via two distinct mechanisms that are activated by different pretreatment periods.« less

[Locally administered lentivirus-mediated siRNA inhibits wear debris-induced inflammation].

PubMed

Peng, Xiao-chun; Zhang, Xian-long; Tao, Kun; Cheng, Tao; Zhu, Jun-feng; Zeng, Bing-fang

2009-03-01

To determine the safety and efficacy of local administration of lentivirus-mediated small interfering RNA (siRNA) targeting tumor necrosis factor-alpha (TNF-alpha) in murine air pouch model. From May 2007 to April 2008 a siRNA targeting TNF-alpha and a missense siRNA were designed, and recombine lentivirus which coexpressed the green fluorescent protein (GFP) as a marker gene was constructed. Air pouches were established and stimulated by Ti-6Al-4V particles. Pouches were divided into 3 groups randomly. Lentivirus-mediated siRNA targeting TNF-alpha (TNF-alpha group) or lentivirus-mediated missense siRNA (MS group), or virus-free saline (control group) were injected into pouches respectively. Pouch membrane, peripheral blood, heart, liver, spleen, kidney, lung and brain were harvested at 28 d after transfection, and assayed for markers of inflammation using histological, molecular, immunological techniques and Xenogen in vivo imaging system (IVIS) 50 vivo bioluminescent assay system. Xenogen IVIS 50 vivo image revealed strong expression of GFP localized in pouch areas and no expression in other parts of mice both in TNF-alpha group and MS group at 4 weeks after transfection, while no expression of GFP was found in control group. By RT-PCR and ELISA, the mRNA and protein levels of TNF-alpha in TNF-alpha group decreased by 81.6% and 82.6% respectively compared to control group (P < 0.01), and decreased by 78.9% and 84.0% respectively compared to MS group (P < 0.01), whereas TNF-alpha level in peripheral blood, heart, liver, spleen, kidney, lung and brain remained invariant (P > 0.05). Less inflammatory responses (thinner pouch membrane and decreased cellular infiltration) were observed in TNF-alpha group. Efficient local delivery of lentivirus-mediated siRNA targeting TNF-alpha into modified murine air pouch can inhibit debris-induced inflammation effectively, with no systemic adverse effects.

Paeonol attenuates TNBS-induced colitis by inhibiting NF-{kappa}B and STAT1 transactivation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishiguro, Kazuhiro; Ando, Takafumi; Maeda, Osamu

2006-11-15

Paeonol, a major phenolic component of Moutan Cortex, is known to have anti-inflammatory activity. However, the effect of Paeonol on colitis has not been evaluated and the molecular mechanism of its anti-inflammatory action remains unknown. The aim of this study was to determine if Paeonol enema attenuates trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. We also investigated the effects of Paeonol in colon cancer-derived CW-2 cells and T cell leukemia-derived Jurkat cells treated with tumor necrosis factor {alpha} (TNF{alpha}) and/or interferon {gamma} (IFN{gamma}), which play critical roles in TNBS-induced colitis. Paeonol enema attenuated TNBS-induced colitis judging by body weigh reduction,more » colon length and histological score. Myeloperoxidase activity and inducible nitric oxide synthase (iNOS) production in the colon were also reduced with Paeonol enema. In CW-2 cells, Paeonol inhibited iNOS protein and mRNA expression induced by costimulation of TNF{alpha} and IFN{gamma}. Furthermore, Paeonol reduced TNF{alpha}-induced NF-{kappa}B transactivation and IFN{gamma}-induced STAT1 transactivation in CW-2 cells and also in Jurkat cells. These findings suggest that Paeonol enema may be useful for the treatment of colitis.« less

Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-{kappa}B translocation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jawan, Bruno; Kao, Y.-H.; Department of Biological Sciences, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung 804, Taiwan

Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 {mu}M after 48 h incubation. Pretreatment with 100 {mu}M PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstratedmore » that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-{alpha}, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and I{kappa}B{alpha}, as well as the nuclear translocation of NF-{kappa}B primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-{kappa}B nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers.« less

Expression of CD73/ecto-5'-nucleotidase on human gingival fibroblasts and contribution to the inhibition of interleukin-1alpha-induced granulocyte-macrophage colony stimulating factor production.

PubMed

Nemoto, Eiji; Kunii, Ryotaro; Tada, Hiroyuki; Tsubahara, Taisuke; Ishihata, Hiroshi; Shimauchi, Hidetoshi

2004-02-01

CD73/5'-nucleotidase (5'-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5'-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. We demonstrated that CD73/5'-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5'-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5'-NT, adenosine 5'-[alpha,beta-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5'-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N6-benzyl-5'-N-ethylcarboxamidoadenosine) A3 receptor agonist > or = (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine > or = (N6-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine), A2 [3,7-dimethyl-1-(2-propargyl)xanthine], A2A[8-(3-chlorostyryl)caffeine], and A2B (alloxazine) antagonists. The CD73/5'-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5'-AMP but not adenosine. These findings suggested that CD73/5'-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5'-AMP to adenosine.

Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4alpha and PGC1alpha.

PubMed

Dietrich, Christoph G; Martin, Ina V; Porn, Anne C; Voigt, Sebastian; Gartung, Carsten; Trautwein, Christian; Geier, Andreas

2007-09-01

Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4alpha/PGC-1alpha-pathway. Because HNF4alpha has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4alpha is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1alpha in electrophoretic mobility shift assays remained unchanged, HNF4alpha binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4alpha and PGC-1alpha. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4alpha, PGC-1alpha, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4alpha/PGC-1alpha pathway.

Expression of TNF-alpha and immunohistochemical distribution of hepatic macrophage surface markers in carbon tetrachloride-induced chronic liver injury in rats.

PubMed

Orfila, C; Lepert, J C; Alric, L; Carrera, G; Beraud, M; Vinel, J P; Pipy, B

1999-10-01

In liver injury induced by carbon tetrachloride, secondary hepatic injury occurs from inflammatory processes originating from products released by activated Kupffer cells, which play a central role in hepatic inflammation. The purpose of our study was to demonstrate, in rats, the relationships between a function of the hepatic macrophages, TNF-alpha production and the state of activation of these cells, characterized by their phenotype, in the different phases of the process and development of fibrosis in a carbon tetrachloride-induced cirrhosis model. The immunohistochemical localization of proinflammatory cytokine TNF-alpha and surface surface makers (ED1 and ED2) was studied in hepatitis and cirrhosis in response to 3 and 9 weeks ingestion of carbon tetrachloride. After carbon tetrachloride ingestion, accompanying the increased necrosis, immunohistochemical analysis of liver tissue sections demonstrated the significantly increased number of cells expressing ED1, ED2 and TNF-alpha, compared to normal. The number of cells expressing the surface phenotypic markers of liver macrophages increased and this change was concomitantly associated with an increased cellular expression of TNF-alpha. Local macrophage proliferation and influx of newly recruited blood monocytes resulted in an increase of the macrophage population. The populational changes involved difference in functional activity and enhanced TNF-alpha expression. This cytokine expressed in the carbon tetrachloride-induced inflammatory process is associated with the development of fibrosis and may contribute to disease severity.

BZLF1, an Epstein-Barr virus immediate-early protein, induces p65 nuclear translocation while inhibiting p65 transcriptional function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morrison, Thomas E.; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; Kenney, Shannon C.

We have previously demonstrated that the Epstein-Barr virus immediate-early BZLF1 protein interacts with, and is inhibited by, the NF-{kappa}B family member p65. However, the effects of BZLF1 on NF-{kappa}B activity have not been intensively studied. Here we show that BZLF1 inhibits p65-dependent gene expression. BZLF1 inhibited the ability of IL-1, as well as transfected p65, to activate the expression of two different NF-{kappa}B-responsive genes, ICAM-1 and I{kappa}B-{alpha}. BZLF1 also reduced the constitutive level of I{kappa}B-{alpha} protein in HeLa and A549 cells, and increased the amount of nuclear NF-{kappa}B to a similar extent as tumor necrosis factor-alpha (TNF-{alpha}) treatment. In spitemore » of this BZLF1-associated increase in the nuclear form of NF-{kappa}B, BZLF1 did not induce binding of NF-{kappa}B to NF-{kappa}B responsive promoters (as determined by chromatin immunoprecipitation assay) in vivo, although TNF-{alpha} treatment induced NF-{kappa}B binding as expected. Overexpression of p65 dramatically inhibited the lytic replication cycle of EBV in 293-EBV cells, confirming that NF-{kappa}B also inhibits BZLF1 transcriptional function. Our results are consistent with a model in which BZLF1 inhibits the transcriptional function of p65, resulting in decreased transcription of I{kappa}B-{alpha}, decreased expression of I{kappa}B-{alpha} protein, and subsequent translocation of NF-{kappa}B to the nucleus. This nuclear translocation of NF-{kappa}B may promote viral latency by negatively regulating BZLF1 transcriptional activity. In situations where p65 activity is limiting in comparison to BZLF1, the ability of BZLF1 to inhibit p65 transcriptional function may protect the virus from the host immune system during the lytic form of infection.« less

Suppressive effect of zinc ion on iNOS expression induced by interferon-gamma or tumor necrosis factor-alpha in murine keratinocytes.

PubMed

Yamaoka, J; Kume, T; Akaike, A; Miyachi, Y

2000-05-01

Zinc, an essential metal, is a critical component of zinc binding proteins such as zinc fingers, zinc enzymes and metallothioneins. Recently, evidence for its anti-inflammatory property in skin has been accumulating, as shown in the treatment of acne, alopecia and zinc deficiency. In cutaneous inflammations, a large amount of nitric oxide (NO) is produced through induction of inducible nitric oxide synthase (iNOS) under the influence of proinflammatory cytokines, resulting in tissue damages in skin, as clarified in other organs. Therefore, we asked if the effect of zinc on NO production and/or on iNOS expression in keratinocytes may explain the anti-inflammatory property of zinc in skin. Accordingly, we sought to determine in this study whether zinc ion may have effect on IFN-gamma or TNF-alpha induced NO production and iNOS expression in cultured murine keratinocytes. Ten microM of zinc ion remarkably suppressed cytokine-induced NO production in keratinocytes. Furthermore, zinc ion also suppressed cytokine-induced iNOS expression in the protein level as well as in the messenger RNA level. These results suggest the possibility that the suppressive effect of zinc ion on cytokine-induced NO production in keratinocytes may be in part implicated in the anti-inflammatory property of zinc in some of skin disorders.

Urokinase plasminogen activator mRNA is induced by IL-1alpha and TNF-alpha in in vitro acantholysis.

PubMed

Feliciani, Claudio; Toto, Paola; Wang, Binghe; Sauder, Daniel N; Amerio, Pierluigi; Tulli, Antonio

2003-08-01

The role of urokinase type plasminogen activator (uPA) has been well documented in the pathogenesis of pemphigus vulgaris (PV). Activation of plasminogen into active serine protease plasmin initiates extracellular proteolysis leading to acantholysis but the mechanisms underlying this process are not clearly understood. We have previously shown that keratinocyte derived cytokines IL-1alpha and TNF-alpha are involved in PV-induced acantholysis. In the present study we sought to examine whether keratinocyte-derived IL-1alpha and TNF-alpha are correlated with uPA induction in keratinocytes during acantholysis. Normal human keratinocytes were incubated with diluted PV serum. mRNAs for IL-1alpha, TNF-alpha and uPA were examined with RT-PCR at various time points and acantholysis was measured. IL-1alpha, TNF-alpha and uPA mRNAs were all induced in keratinocytes following PV serum stimulation; IL-1alpha/TNF-alpha mRNAs' expression was earlier than the expression of uPA mRNA. To further examine the role of IL-1alpha, TNF-alpha and uPA in acantholysis, we performed antibody blocking studies. Anti-IL-1alpha, anti-TNF-alpha and anti-uPA antibodies suppressed acantholysis by 76%, 80% and 90%, respectively. In addition, anti-IL-1alpha and anti-TNF-alpha antibodies inhibited uPA mRNA induction, whereas anti-uPA antibodies did not alter IL-1alpha/TNF-alpha mRNAs' expression. Our results confirm the role of uPA in acantholysis and suggest an involvement of IL-1alpha/TNF-alpha in uPA induction.

Leptin acts on neoplastic behavior and expression levels of genes related to hypoxia, angiogenesis, and invasiveness in oral squamous cell carcinoma.

PubMed

Sobrinho Santos, Eliane Macedo; Guimarães, Talita Antunes; Santos, Hércules Otacílio; Cangussu, Lilian Mendes Borborema; de Jesus, Sabrina Ferreira; Fraga, Carlos Alberto de Carvalho; Cardoso, Claudio Marcelo; Santos, Sérgio Henrique Souza; de Paula, Alfredo Maurício Batista; Gomez, Ricardo Santiago; Guimarães, André Luiz Sena; Farias, Lucyana Conceição

2017-05-01

Leptin, one of the main hormones controlling energy homeostasis, has been associated with different cancer types. In oral cancer, its effect is not well understood. We investigated, through in vitro and in vivo assays, whether leptin can affect the neoplastic behavior of oral squamous cell carcinoma. Expression of genes possibly linked to the leptin pathway was assessed in leptin-treated oral squamous cell carcinoma cells and also in tissue samples of oral squamous cell carcinoma and oral mucosa, including leptin, leptin receptor, hypoxia-inducible factor 1-alpha, E-cadherin, matrix metalloproteinase-2, matrix metalloproteinase-9, Col1A1, Ki67, and mir-210. Leptin treatment favored higher rates of cell proliferation and migration, and reduced apoptosis. Accordingly, leptin-treated oral squamous cell carcinoma cells show decreased messenger RNA caspase-3 expression, and increased levels of E-cadherin, Col1A1, matrix metalloproteinase-2, matrix metalloproteinase-9, and mir-210. In tissue samples, hypoxia-inducible factor 1-alpha messenger RNA and protein expression of leptin and leptin receptor were high in oral squamous cell carcinoma cases. Serum leptin levels were increased in first clinical stages of the disease. In animal model, oral squamous cell carcinoma-induced mice show higher leptin receptor expression, and serum leptin level was increased in dysplasia group. Our findings suggest that leptin seems to exert an effect on oral squamous cell carcinoma cells behavior and also on molecular markers related to cell proliferation, migration, and tumor angiogenesis.

Corticosteroids reduce IL-6 in ASM cells via up-regulation of MKP-1.

PubMed

Quante, Timo; Ng, Yee Ching; Ramsay, Emma E; Henness, Sheridan; Allen, Jodi C; Parmentier, Johannes; Ge, Qi; Ammit, Alaina J

2008-08-01

The mechanisms by which corticosteroids reduce airway inflammation are not completely understood. Traditionally, corticosteroids were thought to inhibit cytokines exclusively at the transcriptional level. Our recent evidence, obtained in airway smooth muscle (ASM), no longer supports this view. We have found that corticosteroids do not act at the transcriptional level to reduce TNF-alpha-induced IL-6 gene expression. Rather, corticosteroids inhibit TNF-alpha-induced IL-6 secretion by reducing the stability of the IL-6 mRNA transcript. TNF-alpha-induced IL-6 mRNA decays at a significantly faster rate in ASM cells pretreated with the corticosteroid dexamethasone (t(1/2) = 2.4 h), compared to vehicle (t(1/2) = 9.0 h; P < 0.05) (results are expressed as decay constants [k] [mean +/- SEM] and half-life [h]). Interestingly, the underlying mechanism of inhibition by corticosteroids is via the up-regulation of an endogenous mitogen-activated protein kinase (MAPK) inhibitor, MAPK phosphatase-1 (MKP-1). Corticosteroids rapidly up-regulate MKP-1 in a time-dependent manner (44.6 +/- 10.5-fold increase after 24 h treatment with dexamethasone; P < 0.05), and MKP-1 up-regulation was temporally related to the inhibition of TNF-alpha-induced p38 MAPK phosphorylation. Moreover, TNF-alpha acts via a p38 MAPK-dependent pathway to stabilize the IL-6 mRNA transcript (TNF-alpha, t(1/2) = 9.6 h; SB203580 + TNF-alpha, t(1/2) = 1.5 h), exogenous expression of MKP-1 significantly inhibits TNF-alpha-induced IL-6 secretion and MKP-1 siRNA reverses the inhibition of TNF-alpha-induced IL-6 secretion by dexamethasone. Taken together, these results suggest that corticosteroid-induced MKP-1 contributes to the repression of IL-6 secretion in ASM cells.

Differential regulation of the cell cycle by alpha1-adrenergic receptor subtypes.

PubMed

Gonzalez-Cabrera, Pedro J; Shi, Ting; Yun, June; McCune, Dan F; Rorabaugh, Boyd R; Perez, Dianne M

2004-11-01

Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.

Critical role of cyclooxygenase-2 activation in pathogenesis of hydronephrosis caused by lactational exposure of mice to dioxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishimura, Noriko; Matsumura, Fumio; Vogel, Christopher F.A.

2008-09-15

Congenital hydronephrosis is a serious disease occurring among infants and children. Besides the intrinsic genetic factors, in utero exposure to a xenobiotic, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been suggested to induce hydronephrosis in rodents owing to anatomical obstruction in the ureter. Here, we report that hydronephrosis induced in mouse pups exposed lactationally to TCDD is not associated with anatomical obstruction, but with abnormal alterations in the subepithelial mesenchyma of the ureter. In the kidneys of these pups, the expressions of a battery of inflammatory cytokines including monocyte chemoattractant protein (MCP)-1, tumor necrosis factor {alpha} (TNF{alpha}) and interleukin (IL) -1{beta} were up-regulated asmore » early as postnatal day (PND) 7. The amounts of cyclooxygenase (COX) -2 mRNA and protein as well as prostaglandin E2 (PGE{sub 2}) were conspicuously up-regulated in an arylhydrocarbon-receptor-dependent manner in the TCDD-induced hydronephrotic kidney, with a subsequent down-regulation of the gene expressions of Na{sup +} and K{sup +} transporters, NKCC2 and ROMK. Daily administration of a COX-2 selective inhibitor to newborns until PND 7 completely abrogated the TCDD-induced PGE{sub 2} synthesis and gene expressions of inflammatory cytokines and electrolyte transporters, and eventually prevented the onset of hydronephrosis. These findings suggest an essential role of COX-2 in mediating the TCDD action of inducing hydronephrosis through the functional impairment rather than the anatomical blockade of the ureter.« less

Protection against dexamethasone-induced muscle atrophy is related to modulation by testosterone of FOXO1 and PGC-1{alpha}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Weiping, E-mail: weiping.qin@mssm.edu; Department of Medicine, Mount Sinai School of Medicine, NY; Pan, Jiangping

Research highlights: {yields} In rat gastrocnemius muscle, dexamethasone reduced PGC-1{alpha} cellular and nuclear levels without altering mRNA levels for this factor. {yields} Dexamethasone reduced phosphorylating of p38 MAPK, which stabilizes PGC-1{alpha} and promotes its nuclear entry. {yields} Co-administration of testosterone with dexamethasone increased cellular and nuclear levels of PGC-1{alpha} protein without changing its mRNA levels. {yields} Co-administration of testosterone restored p38 MAPK levels to those of controls. -- Abstract: Glucocorticoid-induced muscle atrophy results from muscle protein catabolism and reduced protein synthesis, associated with increased expression of two muscle-specific ubiquitin ligases (MAFbx and MuRF1), and of two inhibitors of protein synthesis,more » REDD1 and 4EBP1. MAFbx, MuRF1, REDD1 and 4EBP1 are up-regulated by the transcription factors FOXO1 and FOXO3A. The transcriptional co-activator PGC-1{alpha} has been shown to attenuate many forms of muscle atrophy and to repress FOXO3A-mediated transcription of atrophy-specific genes. Dexamethasone-induced muscle atrophy can be prevented by testosterone, which blocks up-regulation by dexamethasone of FOXO1. Here, an animal model of dexamethasone-induced muscle atrophy was used to further characterize effects of testosterone to abrogate adverse actions of dexamethasone on FOXO1 levels and nuclear localization, and to determine how these agents affect PGC-1{alpha}, and its upstream activators, p38 MAPK and AMPK. In rat gastrocnemius muscle, testosterone blunted the dexamethasone-mediated increase in levels of FOXO1 mRNA, and FOXO1 total and nuclear protein. Dexamethasone reduced total and nuclear PGC-1{alpha} protein levels in the gastrocnemius; co-administration of testosterone with dexamethasone increased total and nuclear PGC-1{alpha} levels above those present in untreated controls. Testosterone blocked dexamethasone-induced decreases in activity of p38 MAPK in the gastrocnemius muscle. Regulation of FOXO1, PGC-1{alpha} and p38 MAPK by testosterone may represent a novel mechanism by which this agent protects against dexamethasone-induced muscle atrophy.« less

Protein Kinase CK2 Regulates Cytoskeletal Reorganization during Ionizing Radiation-Induced Senescence of Human Mesenchymal Stem Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Daojing; Jang, Deok-Jin

2009-08-21

Human mesenchymal stem cells (hMSC) are critical for tissue regeneration. How hMSC respond to genotoxic stresses and potentially contribute to aging and cancer remain underexplored. We demonstrated that ionizing radiation induced cellular senescence of hMSC over a period of 10 days, showing a critical transition between day 3 and day 6. This was confirmed by senescence-associated beta-galactosidase (SA-{beta}-gal) staining, protein expression profiles of key cell cycle regulators (retinoblastoma (Rb) protein, p53, p21{sup waf1/Cip1}, and p16{sup INK4A}), and senescence-associated secretory phenotypes (SASPs) (IL-8, IL-12, GRO, and MDC). We observed dramatic cytoskeletal reorganization of hMSC through reduction of myosin-10, redistribution of myosin-9,more » and secretion of profilin-1. Using a SILAC-based phosphoproteomics method, we detected significant reduction of myosin-9 phosphorylation at Ser1943, coinciding with its redistribution. Importantly, through treatment with cell permeable inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT)), and gene knockdown using RNA interference, we identified CK2, a kinase responsible for myosin-9 phosphorylation at Ser1943, as a key factor contributing to the radiation-induced senescence of hMSC. We showed that individual knockdown of CK2 catalytic subunits CK2{alpha} and CK2{alpha}{prime} induced hMSC senescence. However, only knockdown of CK2{alpha} resulted in morphological phenotypes resembling those of radiation-induced senescence. These results suggest that CK2{alpha} and CK2{alpha}{prime} play differential roles in hMSC senescence progression, and their relative expression might represent a novel regulatory mechanism for CK2 activity.« less

Enhancement of neurite outgrowth in PC12 cells stimulated with cyclic AMP and NGF by 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), novel lipophilic ascorbate derivatives.

PubMed

Zhou, Xiaohua; Tai, Akihiro; Yamamoto, Itaru

2003-03-01

It has been shown that ascorbate (AsA) and its stable derivative, ascorbic acid 2-O-alpha-glucoside (AA-2G), do not elicit neurite outgrowth in PC12 cells. However, these ascorbates are synergistically enhanced by both dibutyryl cyclic AMP (Bt(2)cAMP)- and nerve growth factor (NGF)-induced neurite outgrowth in this model. In the present study, the effects of a series of novel lipophilic ascorbate derivatives, 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), on neurite outgrowth induced by Bt(2)cAMP and NGF were examined in PC12 cells. We found that all the tested acylated ascorbate derivatives enhanced neurite formation induced by both agents in a dose-dependent manner. Of the 6-Acyl-AA-2G derivatives, 6-octanoyl ascorbic acid 2-O-alpha-glucoside (6-Octa-AA-2G) enhanced the Bt(2)cAMP-induced phosphorylated MAPK p44 and p42 expression. A alpha-glucosidase inhibitor, castanospermine, completely abrogated the promotion of neurite outgrowth and MAPK expression by 6-Octa-AA-2G. Addition of 6-Octa-AA-2G (0.5 mM) to PC12 cells caused a rapid and significant increase in intracellular AsA content, which reached a maximum and was maintained from 12 to 24 h after the culture. These findings suggest that 6-Acyl-AA-2G is rapidly hydrolyzed to AsA within the cell and enhances neurite differentiation through the interaction with the inducer-activated MAPK pathway.

Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Wei, E-mail: detachedy@yahoo.com.cn; Sun, Ting; Cao, Jianping

2012-05-01

Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1{alpha} (HIF-1{alpha}) and miR-210 expression and cell arrest in the G{sub 0}/G{sub 1} phase inmore » all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G{sub 0}/G{sub 1} phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: Black-Right-Pointing-Pointer miR-210 downregulation radiosensitized hypoxic hepatoma. Black-Right-Pointing-Pointer AIFM3 was identified as a direct target gene of miR-210. Black-Right-Pointing-Pointer miR-210 might be a therapeutic target to hypoxic hepatoma.« less

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

PubMed

Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

2009-12-01

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast cell-mediated allergic reactions.

Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2alpha during inflammatory conditions.

PubMed

Sugita, Sunao; Horie, Shintaro; Nakamura, Orie; Maruyama, Kazuichi; Takase, Hiroshi; Usui, Yoshihiko; Takeuchi, Masaru; Ishidoh, Kazumi; Koike, Masato; Uchiyama, Yasuo; Peters, Christoph; Yamamoto, Yoshimi; Mochizuki, Manabu

2009-10-15

Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4(+) T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2alpha, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2alpha's role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-beta, but not IFN-gamma inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2alpha in RPE. Similarly, CTLA-2alpha via RPE was able to promote TGF-beta production by the CD4(+) T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-beta and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4(+) T cells from EAU in CathL knockout mice or rCTLA-2alpha from EAU animals were found to contain a high population of forkhead box p3(+) T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2alpha, thereby enabling the bystander T cells to be converted into Treg cells via TGF-beta promotion.

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

PubMed

Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

1992-08-05

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Membrane remodeling, an early event in benzo[alpha]pyrene-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tekpli, Xavier; Rissel, Mary; Huc, Laurence

2010-02-15

Benzo[alpha]pyrene (B[alpha]P) often serves as a model for mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). Our previous work suggested a role of membrane fluidity in B[alpha]P-induced apoptotic process. In this study, we report that B[alpha]P modifies the composition of cholesterol-rich microdomains (lipid rafts) in rat liver F258 epithelial cells. The cellular distribution of the ganglioside-GM1 was markedly changed following B[alpha]P exposure. B[alpha]P also modified fatty acid composition and decreased the cholesterol content of cholesterol-rich microdomains. B[alpha]P-induced depletion of cholesterol in lipid rafts was linked to a reduced expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Aryl hydrocarbon receptor (AhR) and B[alpha]P-related H{submore » 2}O{sub 2} formation were involved in the reduced expression of HMG-CoA reductase and in the remodeling of membrane microdomains. The B[alpha]P-induced membrane remodeling resulted in an intracellular alkalinization observed during the early phase of apoptosis. In conclusion, B[alpha]P altered the composition of plasma membrane microstructures through AhR and H{sub 2}O{sub 2} dependent-regulation of lipid biosynthesis. In F258 cells, the B[alpha]P-induced membrane remodeling was identified as an early apoptotic event leading to an intracellular alkalinization.« less

15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-{alpha} by decreasing levels of acetylated histone H3 and H4 at its promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Engdahl, Ryan; Monroy, M. Alexandra; Temple University School of Medicine, Department of Anatomy and Cell Biology, 3400 North Broad Street, Philadelphia, PA 19140

2007-07-20

Prostaglandin metabolite 15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPAR{gamma}. We investigated the ability of 15d-PGJ2 to inhibit TNF-{alpha} gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 {mu}M) inhibited LPS-stimulated TNF-{alpha} mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPAR{gamma} ligand, GW1929, failed to inhibit LPS-induced TNF-{alpha} mRNA expression nor did a PPAR{gamma} antagonist, GW9662, alter the repression of TNF-{alpha} mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPAR{gamma}-independent inhibition of TNF-{alpha} mRNA in THP-1more » cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-{alpha} promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-{alpha} promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-{alpha} promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-{alpha} promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-{alpha} promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-{alpha} transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter.« less

Hypothermia increases interleukin-6 and interleukin-10 in juvenile endotoxemic mice.

PubMed

Stewart, Corrine R; Landseadel, Jessica P; Gurka, Matthew J; Fairchild, Karen D

2010-01-01

To develop a juvenile mouse model to establish effects of in vivo hypothermia on expression of the inflammation-modulating cytokines tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and interleukin-10. Although induced hypothermia is neuroprotective in some patients, the mechanisms of protection are not well understood and concerns remain over potential detrimental effects, particularly in the setting of infection. We previously showed that in vitro hypothermia increases production of tumor necrosis factor-alpha and interleukin-1beta in lipopolysaccharide-treated monocytes. : Laboratory investigation. Research laboratory. Juvenile (4-wk) male C57BL/6 mice. : Mice were given chlorpromazine to suspend thermoregulation and lipopolysaccharide to stimulate cytokine production. Core temperature was maintained at 32 degrees C or 37 degrees C for 6 hrs by adjusting environmental temperature. In separate experiments, lipopolysaccharide-treated mice were kept in a cooling chamber without chlorpromazine treatment. Plasma and organs were collected for cytokine quantitation. Chlorpromazine-treated hypothermic mice had 2.3-fold and 1.8-fold higher plasma interleukin-6 and interleukin-10 levels at 6 hrs compared with identically treated normothermic mice (p < .05), whereas plasma tumor necrosis factor-alpha and interleukin-1beta were not significantly different at 2 hrs or 6 hrs. Liver tumor necrosis factor-alpha and interleukin-6 were significantly higher in hypothermic vs. normothermic mice, but lung and brain cytokines were not different. Lipopolysaccharide-treated mice kept in a cooling chamber without chlorpromazine treatment developed varying degrees of hypothermia with associated increases in plasma interleukin-6 and interleukin-10. A nonspecific marker of stress (plasma corticosterone) was not affected by hypothermia in lipopolysaccharide-treated mice. Further studies are necessary to determine the mechanism and physiologic consequences of augmented systemic interleukin-6 and interleukin-10 expression during induced hypothermia.

Tat-APE1/ref-1 protein inhibits TNF-{alpha}-induced endothelial cell activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Yun Jeong; Lee, Ji Young; Joo, Hee Kyoung

2008-03-28

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-{alpha}-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1 h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-{alpha}-induced monocyte adhesion and vascular cell adhesion molecule-1 expressionmore » in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.« less

Receptor-selective retinoids implicate retinoic acid receptor alpha and gamma in the regulation of bmp-2 and bmp-4 in F9 embryonal carcinoma cells.

PubMed

Rogers, M B

1996-01-01

The effect of retinoids on malignant cells and embryos indicates that retinoids influence the expression of growth factors or alter the response of cells to growth factors. The bone morphogenetic proteins, Bmp-2 and Bmp-4, are candidates for such growth factors because retinoic acid (RA) treatment of F9 embryonal carcinoma cells induced Bmp-2 mRNA, while simultaneously repressing Bmp-4 levels. Also, recombinant Bmp-2 affected the growth and differentiation of these cells. Regulation of each gene was concentration dependent and required continuous RA treatment. The short half-lives of the Bmp-2 (75 +/- 11 min) and Bmp-4 (70 +/- 4 min) mRNAs suggest that their abundance is primarily controlled at the transcriptional level. To determine which RA receptor (RAR) controls bmp-2 and bmp-4 expression, F9 cells were exposed to various receptor-selective retinoids. RAR alpha- and gamma-selective retinoids induced Bmp-2 and repressed Bmp-4 equally as well as all-trans RA. In contrast, a RAR beta-selective retinoid had little effect on Bmp-2 induction but repressed Bmp-4. A RAR alpha-selective antagonist inhibited all-trans RA stimulation of Bmp-2, although not as dramatically as a RAR beta gamma-selective antagonist. No differences were observed between Bmp levels in all-trans RA and 9-cis RA-treated cells, indicating that the RXRs play little part in controlling these genes. The results are consistent with RAR alpha and gamma-controlled Bmp-2 and Bmp-4 regulation.

Comparison of prostaglandin F2alpha, bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression.

PubMed

Liang, Yanbin; Li, Chen; Guzman, Victor M; Evinger, Albert J; Protzman, Charles E; Krauss, Achim H-P; Woodward, David F

2003-07-18

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.

Tocotrienols suppress proinflammatory markers and cyclooxygenase-2 expression in RAW264.7 macrophages.

PubMed

Yam, Mun-Li; Abdul Hafid, Sitti Rahma; Cheng, Hwee-Ming; Nesaretnam, Kalanithi

2009-09-01

Tocotrienols are powerful chain breaking antioxidant. Moreover, they are now known to exhibit various non-antioxidant properties such as anti-cancer, neuroprotective and hypocholesterolemic functions. This study was undertaken to investigate the anti-inflammatory effects of tocotrienol-rich fraction (TRF) and individual tocotrienol isoforms namely delta-, gamma-, and alpha-tocotrienol on lipopolysaccharide-stimulated RAW264.7 macrophages. The widely studied vitamin E form, alpha-tocopherol, was used as comparison. Stimulation of RAW264.7 with lipopolysaccharide induced the release of various inflammatory markers. 10 mcirog/ml of TRF and all tocotrienol isoforms significantly inhibited the production of interleukin-6 and nitric oxide. However, only alpha-tocotrienol demonstrated a significant effect in lowering tumor necrosis factor-alpha production. Besides, TRF and all tocotrienol isoforms except gamma-tocotrienol reduced prostaglandin E(2) release. It was accompanied by the down-regulation of cyclooxygenase-2 gene expression by all vitamin E forms except alpha-tocopherol. Collectively, the data suggested that tocotrienols are better anti-inflammatory agents than alpha-tocopherol and the most effective form is delta-tocotrienol.

Noradrenaline inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin 6 production in human whole blood.

PubMed Central

van der Poll, T; Jansen, J; Endert, E; Sauerwein, H P; van Deventer, S J

1994-01-01

Sepsis and lipopolysaccharide (LPS) trigger the systemic release of both cytokines and catecholamines. Cytokines are known to be capable of eliciting a stress hormone response in vivo. The present study sought insight into the effect of noradrenaline on LPS-induced release of tumor necrosis factor alpha (TNF) and interleukin 6 (IL-6) in human whole blood. Whole blood was incubated with LPS for 4 h at 37 degrees C in the presence and absence of noradrenaline and/or specific alpha and beta antagonists and agonists. Noradrenaline caused a dose-dependent inhibition of LPS-induced TNF and IL-6 production. This effect could be completely prevented by addition of the specific beta 1, antagonist metoprolol, while it was not affected by the alpha antagonist phentolamine. Specific beta-adrenergic stimulation by isoprenaline mimicked the inhibiting effect of noradrenaline on LPS-evoked cytokine production, whereas alpha-adrenergic stimulation by phenylephrine had no effect. Fluorescence-activated cell sorter analysis demonstrated that beta-adrenergic stimulation had no effect on LPS binding to and internalization into mononuclear cells or on the expression of CD14, the major receptor for LPS on mononuclear cells. In acute sepsis, enhanced release of noradrenaline may be part of a negative feedback mechanism meant to inhibit ongoing TNF and IL-6 production. PMID:8168970

Triiodothyronine (T3) induces HIF1A and TGFA expression in MCF7 cells by activating PI3K.

PubMed

Moretto, Fernanda Cristina Fontes; De Sibio, Maria Teresa; Luvizon, Aline Carbonera; Olimpio, Regiane Marques Castro; de Oliveira, Miriane; Alves, Carlos Augusto Barnabe; Conde, Sandro José; Nogueira, Célia Regina

2016-06-01

High expression levels of hypoxia inducing factor 1 alpha are related to mammary carcinogenesis. In previous studies, we demonstrated that expression of transforming growth factor alpha increases upon treatment with triiodothyronine, but this expression does not occur in cellular models that do not express the estrogen receptor, or when cells are co-treated with the anti-estrogen, tamoxifen. The aim of this study was to determine the effect of the hormone triiodothyronine on the expression of the genes HIF1A and TGFA in the breast cancer cell line MCF7. The cell line was subjected to treatment with triiodothyronine at the supraphysiological dose of 10(-8)M for 10min, 30min, 1h, and 4h in the presence or absence of actinomycin D, the gene expression inhibitor, cycloheximide, the protein synthesis inhibitor, and LY294002, the phosphoinositide 3 kinase inhibitor. HIF1A and TGFA mRNA expression was analyzed by reverse transcription polymerase chain reaction. For data analysis, we used analysis of variance complemented by Tukey test and an adopted minimum of 5% significance. We found that HIF1A and TGFA expression increased in the presence of triiodothyronine at all times studied. HIF1A expression decreased in triiodothyronine-treated cells when gene transcription was also inhibited; however, TGFA expression decreased after 10 and 30min of treatment even when transcription was not inhibited. We found that activation of PI3K was necessary for triiodothyronine to modulate HIF1A and TGFA expression. Copyright © 2016 Elsevier Inc. All rights reserved.

Inflammation modulates the expression of the intestinal mucins MUC2 and MUC4 in gastric tumors.

PubMed

Mejías-Luque, R; Lindén, S K; Garrido, M; Tye, H; Najdovska, M; Jenkins, B J; Iglesias, M; Ernst, M; de Bolós, C

2010-03-25

Infection of gastric mucosa by Helicobacter pylori induces an inflammatory response with increased levels of proinflammatory cytokines. Among them, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 induce the activation of signaling pathways that regulate genes expression, such as MUC2 and MUC4 intestinal mucins ectopically detected in gastric tumors. This study evaluated if the predominant inflammatory cell type correlates with MUC2 and MUC4 expression in human intestinal gastric tumors (n=78). In addition, we analyzed the regulatory effects of the associated inflammatory signaling pathways on their expression in gastric cancer cell lines, and in a mouse model with hyperactivated STAT3 signaling pathway. Tumors with predominant lymphoplasmocytic infiltrate (chronic inflammation), presented higher levels of MUC2 and were more differentiated than tumors with predominant polymorphonuclear infiltrate (acute inflammation). These differences can be attributed to specific cytokines, because TNF-alpha and IL-1beta induced MUC2 but no MUC4 expression in gastric cancer cell lines. The two groups of tumors expressed similar levels of MUC4 that correlated with the expression of STAT3 transcription factor, implicated in the activation of genes through the IL-6 pathway. In gastric tissues from gp130(+/+), gp130(Y757F/Y757F) and gp130(Y757F/Y757F) Stat3(-/+) mice, Muc2 was not detected, whereas Muc4 was found in the gastric tumors developed in the gp130(Y757F/Y757F) mice, with hyperactivated STAT3. These data indicate that the signaling pathways associated with the inflammatory response can modulate the expression of MUC2 and MUC4 intestinal mucin genes, in human and mouse gastric tumors.

Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-alpha and translocating PKC-delta.

PubMed

Zhang, Xian-Ming; Chen, Jia; Xia, Yu-Gui; Xu, Qiang

2005-03-01

In our previous study, quercetin was found to induce apoptosis of murine melanoma B16-BL6 cells. The cellular and molecular mechanism of quercetin-induced apoptosis was investigated in the present study. Nuclear morphology was determined by fluorescence microscopy. DNA fragmentation was analyzed by electrophoresis and quantified by the diphenylamine method. The transmembrane potential of mitochondria was measured by flow cytometry. Bcl-2, Bcl-X(L), PKC-alpha, PKC-beta, and PKC-delta were detected by Western blotting. Caspase activity was determined spectrophotometrically. Quercetin induced the condensation of nuclei of B16-BL6 cells in a dose-dependent pattern as visualized by Hoechst 33258 and propidium iodide dying. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly enhanced apoptosis induced by quercetin, while doxorubicin, a PKC inhibitor, markedly decreased it. Both PMA and doxorubicin showed a consistent effect on the fragmentation of nuclear DNA caused by various dosages of quercetin. Quercetin dose-dependently led to loss of the mitochondrial membrane potential, which was also significantly reinforced or antagonized by PMA and doxorubicin, respectively. Moreover, PMA showed reinforcement, while doxorubicin showed significant antagonization, of the quercetin-mediated decrease in the expression of Bcl-2. Quercetin promoted caspase-3 activity in a dose-dependent manner, which was also regulated by PMA and doxorubicin with a pattern similar to that seen in their effect on apoptosis, mitochondrial membrane potential and Bcl-2 expression, but none of these were directly affected by PMA and doxorubicin. Free fatty acid and chlorpromazine, a PKC activator and inhibitor, respectively, did not interfere with these effects of quercetin. B16-BL6 cells expressed PKC-alpha, PKC-beta, and PKC-delta. Quercetin dose-dependently inhibited the expression of PKC-alpha but not that of PKC-beta and PKC-delta. Doxorubicin almost completely blocked the effect of quercetin on the expression of PKC-alpha. Quercetin was also involved in the translocation of PKC-delta from the cytosol to the nucleus. PMA enhanced the effect of quercetin on the translocation of PKC-delta. These results indicate that quercetin induced apoptosis of murine melanoma B16-BL6 cells by injuring their mitochondria, increasing the activity of caspase-3, inhibiting the expression of Bcl-2 and PKC-alpha, and inducing the translocation of PKC-delta. Doxorubicin inhibited these effects of quercetin by blocking the decreased expression of PKC-alpha induced by quercetin while PMA increased these effects by enhancing the translocation of PKC-delta induced by quercetin.

Expression of alpha-AR subtypes in T lymphocytes and role of the alpha-ARs in mediating modulation of T cell function.

PubMed

Bao, Jing-Yin; Huang, Yan; Wang, Feng; Peng, Yu-Ping; Qiu, Yi-Hua

2007-01-01

Previous work in our laboratory has shown that alpha-adrenoreceptors (alpha-ARs) and beta-ARs exist on lymphocytes from functional profile, and that the receptors mediate the regulation of lymphocyte function by catecholamines. In the present study, we directly examined the expression of alpha-AR subtypes, alpha(1)-AR and alpha(2)-AR mRNAs, in T lymphocytes and explored the roles of the alpha-AR subtypes and intracellular signal transduction mechanisms linked to the receptors in mediating the modulation of T lymphocyte function. T lymphocytes from mesenteric lymph nodes of rats were purified by using a nylon wool column. Reverse transcription polymerase chain reaction was used to detect the expression of alpha(1)-AR and alpha(2)-AR mRNAs in the freshly isolated T cells and the mitogen concanavalin A (Con A)-activated lymphocytes. Colorimetric methylthiazoletetrazolium assay was employed to measure lymphocyte proliferation induced by Con A. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels in the Con A-stimulated lymphocyte culture supernatants were examined by enzyme-linked immunosorbent assay. T cells expressed both alpha(1)-AR and alpha(2)-AR mRNAs. The expression of both alpha(1)-AR and alpha(2)-AR mRNAs was significantly higher in the Con A-activated lymphocytes than in the resting lymphocytes. Phenylephrine, a selective alpha(1)-AR agonist, had no evident effect on lymphocyte proliferation nor on IFN-gamma and IL-4 production induced by Con A. However, the selective alpha(2)-AR agonist clonidine attenuated Con A-induced lymphocyte proliferation as well as IFN-gamma and IL-4 production. The inhibited lymphocyte proliferation and IFN-gamma and IL-4 production by clonidine were blocked by yohimbine, an alpha(2)-AR antagonist. Either phospholipase C inhibitor U-73122 or protein kinase C inhibitor chelerythrine partially prevented the suppressive effect of clonidine on Con A-stimulated lymphocyte proliferation and IL-4 production. T lymphocytes express both alpha(1)-ARs and alpha(2)-ARs, but only the alpha(2)-ARs participate in the suppressive modulation of lymphocyte proliferation and cytokine production in vitro. The inhibitory effect of alpha(2)-AR stimulation on lymphocyte function is partially mediated via the phospholipase C-protein kinase C pathway. (c) 2008 S. Karger AG, Basel.

Induction of indolamine 2,3-dioxygenase and kynurenine 3-monooxygenase in rat brain following a systemic inflammatory challenge: a role for IFN-gamma?

PubMed

Connor, Thomas J; Starr, Neasa; O'Sullivan, Joan B; Harkin, Andrew

2008-08-15

Inflammation-mediated dysregulation of the kynurenine pathway has been implicated as a contributor to a number of major brain disorders. Consequently, we examined the impact of a systemic inflammatory challenge on kynurenine pathway enzyme expression in rat brain. Indoleamine 2,3-dioxygenase (IDO) expression was induced in cortex and hippocampus following systemic lipopolysaccharide (LPS) administration. Whilst IDO expression was paralleled by increased circulating interferon (IFN)-gamma concentrations, IFN-gamma expression in the brain was only modestly altered following LPS administration. In contrast, induction of IDO was associated with increased central tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 expression. Similarly, in cultured glial cells LPS-induced IDO expression was accompanied by increased TNF-alpha and IL-6 expression, whereas IFN-gamma was not detectable. These findings indicate that IFN-gamma is not required for LPS-induced IDO expression in brain. A robust increase in kynurenine-3-monooxygenase (KMO) expression was observed in rat brain 24h post LPS, without any change in kynurenine aminotransferase II (KAT II) expression. In addition, we report that constitutive expression of KAT II is approximately 8-fold higher than KMO in cortex and 20-fold higher in hippocampus. Similarly, in glial cells constitutive expression of KAT II was approximately 16-fold higher than KMO, and expression of KMO but not KAT II was induced by LPS. These data are the first to demonstrate that a systemic inflammatory challenge stimulates KMO expression in brain; a situation that is likely to favour kynurenine metabolism in a neurotoxic direction. However, our observation that expression of KAT II is much higher than KMO in rat brain is likely to counteract potential neurotoxicity that could arise from KMO induction following an acute inflammation.

Hepatocyte nuclear factor-4 alpha in noise-induced cochlear neuropathy.

PubMed

Groth, Jane Bjerg; Kao, Shyan-Yuan; Briët, Martijn C; Stankovic, Konstantina M

2016-12-01

Noise-induced hearing loss (NIHL) is a problem of profound clinical significance and growing magnitude. Alarmingly, even moderate noise levels, previously assumed to cause only temporary shifts in auditory thresholds ("temporary" NIHL), are now known to cause cochlear synaptopathy and subsequent neuropathy. To uncover molecular mechanisms of this neuropathy, a network analysis of genes reported to have significantly altered expression after temporary threshold shift-inducing noise exposure was performed. The transcription factor Hepatocyte Nuclear Factor-4 alpha (HNF4α), which had not previously been studied in the context of cochlear response to noise, was identified as a hub of a top-ranking network. Hnf4α expression and localization using quantitative RT-PCR and in situ hybridization, respectively, were described in adolescent and adult mice exposed to neuropathic noise levels in adolescence. Isoforms α3 and α12 in the cochlea were also identified. At every age examined, Hnf4α mRNA expression in the cochlear apex was similar to expression in the base. Hnf4α expression was evident in select cochlear cells, including spiral ganglion neurons (SGNs) and hair cells, and was significantly upregulated from 6 to 70 weeks of age, especially in SGNs. This age-related Hnf4α upregulation was inhibited by neuropathic noise exposure in adolescence. Hnf4α silencing with shRNA transfection into auditory neuroblast cells (VOT-33) reduced cell viability, as measured with the MTT assay, suggesting that Hnf4α may be involved in SGN survival. Our results motivate future studies of HNF4α in cochlear pathophysiology, especially because HNF4α mutations and polymorphisms are associated with human diseases that may include hearing loss. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1374-1386, 2016. © 2016 Wiley Periodicals, Inc.

Alpha-Synuclein Expression Restricts RNA Viral Infections in the Brain.

PubMed

Beatman, Erica L; Massey, Aaron; Shives, Katherine D; Burrack, Kristina S; Chamanian, Mastooreh; Morrison, Thomas E; Beckham, J David

2015-12-30

We have discovered that native, neuronal expression of alpha-synuclein (Asyn) inhibits viral infection, injury, and disease in the central nervous system (CNS). Enveloped RNA viruses, such as West Nile virus (WNV), invade the CNS and cause encephalitis, yet little is known about the innate neuron-specific inhibitors of viral infections in the CNS. Following WNV infection of primary neurons, we found that Asyn protein expression is increased. The infectious titer of WNV and Venezuelan equine encephalitis virus (VEEV) TC83 in the brains of Asyn-knockout mice exhibited a mean increase of 10(4.5) infectious viral particles compared to the titers in wild-type and heterozygote littermates. Asyn-knockout mice also exhibited significantly increased virus-induced mortality compared to Asyn heterozygote or homozygote control mice. Virus-induced Asyn localized to perinuclear, neuronal regions expressing viral envelope protein and the endoplasmic reticulum (ER)-associated trafficking protein Rab1. In Asyn-knockout primary neuronal cultures, the levels of expression of ER signaling pathways, known to support WNV replication, were significantly elevated before and during viral infection compared to those in Asyn-expressing primary neuronal cultures. We propose a model in which virus-induced Asyn localizes to ER-derived membranes, modulates virus-induced ER stress signaling, and inhibits viral replication, growth, and injury in the CNS. These data provide a novel and important functional role for the expression of native alpha-synuclein, a protein that is closely associated with the development of Parkinson's disease. Neuroinvasive viruses such as West Nile virus are able to infect neurons and cause severe disease, such as encephalitis, or infection of brain tissue. Following viral infection in the central nervous system, only select neurons are infected, implying that neurons exhibit innate resistance to viral infections. We discovered that native neuronal expression of alpha-synuclein inhibited viral infection in the central nervous system. When the gene for alpha-synuclein was deleted, mice exhibited significantly decreased survival, markedly increased viral growth in the brain, and evidence of increased neuron injury. Virus-induced alpha-synuclein localized to intracellular neuron membranes, and in the absence of alpha-synuclein expression, specific endoplasmic reticulum stress signaling events were significantly increased. We describe a new neuron-specific inhibitor of viral infections in the central nervous system. Given the importance of alpha-synuclein as a cause of Parkinson's disease, these data also ascribe a novel functional role for the native expression of alpha-synuclein in the CNS. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Biological responses to PDGF-BB versus PDGF-DD in human mesangial cells.

PubMed

van Roeyen, C R C; Ostendorf, T; Denecke, B; Bokemeyer, D; Behrmann, I; Strutz, F; Lichenstein, H S; LaRochelle, W J; Pena, C E; Chaudhuri, A; Floege, J

2006-04-01

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor.

A new mechanism for DNA alterations induced by alpha particles such as those emitted by radon and radon progeny.

PubMed Central

Lehnert, B E; Goodwin, E H

1997-01-01

The mechanism(s) by which alpha (alpha) particles like those emitted from inhaled radon and radon progeny cause their carcinogenic effects in the lung remains unclear. Although direct nuclear traversals by alpha-particles may be involved in mediating these outcomes, increasing evidence indicates that a particles can cause alterations in DNA in the absence of direct hits to cell nuclei. Using the occurrence of excessive sister chromatid exchanges (SCE) as an index of DNA damage in human lung fibroblasts, we investigated the hypothesis that alpha-particles may induce DNA damage through the generation of extracellular factors. We have found that a relatively low dose of alpha-particles can result in the generation of extracellular factors, which, upon transfer to unexposed normal human cells, can cause excessive SCE to an extent equivalent to that observed when the cells are directly irradiated with the same irradiation dose. A short-lived, SCE-inducing factor(s) is generated in alpha-irradiated culture medium containing serum in the absence of cells. A more persistent SCE-inducing factor(s), which can survive freeze-thaw and is heat labile is produced by fibroblasts after exposure to the alpha-particles. These results indicate that the initiating target for alpha-particle-induced genetic changes can be larger than a cell's nucleus or even a whole cell. How transmissible factors like those observed here in vitro may extend to the in vivo condition in the context of a-particle-induced carcinogenesis in the respiratory tract remains to be determined. PMID:9400706

Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

DOEpatents

Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

2003-04-01

The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

Evidence for actin cytoskeleton-dependent and -independent pathways for RelA/p65 nuclear translocation in endothelial cells.

PubMed

Fazal, Fabeha; Minhajuddin, Mohd; Bijli, Kaiser M; McGrath, James L; Rahman, Arshad

2007-02-09

Activation of the transcription factor NF-kappaB involves its release from the inhibitory protein IkappaBalpha in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-kappaB remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor alpha, respectively. We demonstrate that thrombin alters the actin cytoskeleton in endothelial cells and interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevents thrombin-induced NF-kappaB activation and consequently, expression of its target gene, ICAM-1. The blockade of NF-kappaB activation occurs downstream of IkappaBalpha degradation and is associated with impaired RelA/p65 nuclear translocation. Importantly, thrombin induces association of RelA/p65 with actin and this interaction is sensitive to stabilization/destabilization of the actin filaments. In parallel studies, stabilizing or destabilizing the actin filaments fails to inhibit RelA/p65 nuclear accumulation and ICAM-1 expression by tumor necrosis factor alpha, consistent with its inability to induce actin filament formation comparable with thrombin. Thus, these studies reveal the existence of actin cytoskeleton-dependent and -independent pathways that may be engaged in a stimulus-specific manner to facilitate RelA/p65 nuclear import and thereby ICAM-1 expression in endothelial cells.

Ginkgolide B preconditioning protects neurons against ischaemia-induced apoptosis.

PubMed

Wu, Xiaomei; Qian, Zhongming; Ke, Ya; Du, Fang; Zhu, Li

2009-01-01

Ischaemic preconditioning (IP) has been reported to protect the brain against subsequent lethal ischaemia, but it has not been used clinically to prevent ischaemic injury because of safety concerns. The aim of the present study was to see whether Ginkgolide B (GB) is capable of preconditioning as IP to protect neurons against ischaemic injury; if so, which mechanism is involved. Cultured mouse cortical neurons at day 8 were pre-treated with GB (120 micromol/l) for 24 hrs or exposed to short-term ischaemia (1 hr) followed by 24-hr normal culture to induce IP before being treated with severe ischaemia (5 hrs). GB and IP significantly increased cell viability, expression of hypoxia-inducible factor-1 alpha (HIF-1alpha), erythropoietin (EPO), phosphorylated Bad at serine 136 (136p-Bad) and phosphorylated glycogen synthase kinase- 3beta at serine 9 (p-GSK-3beta), and decreased the percentage of apoptotic cells and the level of active caspase-3 in severely ischaemic neurons. Moreover, LY294002 that is a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) significantly reduced the enhanced expression of HIF-1alpha, EPO and 136p-Bad induced by GB and IP. These results suggest that GB, like IP in neurons, is capable of preconditioning against ischaemia-induced apoptosis, the mechanism of which may involve the PI3K signalling pathway.

Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.

PubMed

Kaminski, Alexander; Ma, Nan; Donndorf, Peter; Lindenblatt, Nicole; Feldmeier, Gregor; Ong, Lee-Lee; Furlani, Dario; Skrabal, Christian A; Liebold, Andreas; Vollmar, Brigitte; Steinhoff, Gustav

2008-01-01

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1alpha)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1alpha enhanced c-kit+-cell migration. In vivo, SDF-1alpha alone triggered endothelial rolling-not firm adherence-of c-kit+ cells in WT mice. While TNF-alpha alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1alpha+TNF-alpha, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1alpha mediates firm adhesion c-kit+ cells only in the presence of TNF-alpha stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.

Effect of thalidomide on endotoxin-induced decreases in activity and expression of hepatic cytochrome P450 3A2.

PubMed

Ueyama, Jun; Nadai, Masayuki; Zhao, Ying Lan; Kanazawa, Hiroaki; Takagi, Kenji; Kondo, Takaaki; Takagi, Kenzo; Wakusawa, Shinya; Abe, Fumie; Saito, Hiroko; Miyamoto, Ken-Ichi; Hasegawa, Takaaki

2008-08-01

Thalidomide has been reported to inhibit the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) that are involved in the down-regulation of hepatic cytochrome P450 (CYP) induced by endotoxin. In the present study, we investigated the effects of thalidomide on endotoxin-induced decreases in the activity and expression of hepatic CYP3A2 in rats. Thalidomide (50 mg/kg) was administered orally 22 h and 2 h before intraperitoneal injection of endotoxin (1 mg/kg). Twenty-four hours after the injection of endotoxin, antipyrine clearance experiments were conducted, in which the rats were sacrificed and protein levels of hepatic CYP3A2 were measured. There were no significant differences in the histopathological changes in the liver between the endotoxin-treated and endotoxin plus thalidomide-treated rats. Thalidomide had no effect on the systemic clearance of antipyrine, which is a proper indicator for hepatic CYP3A2 activity, whereas it enhanced endotoxin-induced decrease in the systemic clearance of antipyrine. Western blot analysis revealed that thalidomide had no effect on the protein levels of hepatic CYP3A2, whereas it enhanced the down-regulation of hepatic CYP3A2 by endotoxin. However, there were no significant differences in the concentrations of TNF-alpha and NO in plasma between the endotoxin-treated and endotoxin plus thalidomide-treated rats. The present findings suggest that thalidomide enhances endotoxin-induced decreases in the activity and expression of hepatic CYP3A2.

Protective effect of a coffee preparation (Nescafe pure) against carbon tetrachloride-induced liver fibrosis in rats.

PubMed

Shi, Hongyang; Dong, Lei; Zhang, Yong; Bai, Yanhua; Zhao, Juhui; Zhang, Li

2010-06-01

We examined the effects of a coffee preparation on liver fibrosis induced by carbon tetrachloride (CCl(4)) and explored the possible mechanisms. Rats were divided randomly into four groups: control, CCl(4), and two coffee preparation groups. Except for the control group, liver fibrosis was induced in male Sprague-Dawley (SD) rats by subcutaneous injection with 40% CCl(4) twice a week for 8 weeks. At the same time, a coffee preparation (300 mg/kg and 150 mg/kg) was administered to the two coffee preparation groups intragastrically once daily. Upon pathological examination, a coffee preparation treatment significantly reduced liver damage and symptoms of liver fibrosis. The mRNA expression of collagen I, collagen III, bcl-2, vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGF-beta1) were markedly increased by CCl(4) treatment but suppressed by a coffee preparation treatment. Whereas compared with the CCl(4) group, the mRNA expression of Bax was increased in the coffee preparation group. The protein expression of Bax and bcl-2 were confirmed by western blot. Intragastric administration of a coffee preparation reduced the protein expression of alpha-smooth muscle actin (alpha-SMA) and the glucose-regulated proteins (GRP) 78 and 94 in rats increased by CCl(4). Our data indicate that a coffee preparation can efficiently inhibit CCl(4)-induced liver fibrosis in rats. The coffee preparation may therefore be a potential functional food for preventing liver fibrosis. Copyright 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells.

PubMed

Kawasaki, Kengo; Muroyama, Koutarou; Yamamoto, Norio; Murosaki, Shinji

2015-01-01

The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.

Combined effect of substance P and curcumin on cutaneous wound healing in diabetic rats.

PubMed

Kant, Vinay; Kumar, Dinesh; Prasad, Raju; Gopal, Anu; Pathak, Nitya N; Kumar, Pawan; Tandan, Surender K

2017-05-15

Our earlier studies demonstrated that topically applied substance P (SP) or curcumin on excision skin wound accelerated the wound healing in streptozotocin-induced diabetic rats. In the present study, we aimed to evaluate the wound healing potential of combination of SP and curcumin in diabetic rats. Open cutaneous excision wound was created on the back of each of the 60 diabetic rats. Wound-inflicted rats were equally divided into three groups namely, control, gel treated, and SP + curcumin treated. Normal saline, pluronic gel, and SP (0.5 × 10 -6 M) + curcumin (0.15%) were topically applied once daily for 19 d to these control, gel-treated, and SP + curcumin groups, respectively. SP + curcumin combination significantly accelerated wound closure and decreased messenger RNA expressions of tumor necrosis factor-alpha, interleukin-1beta, and matrix metalloproteinase-9, whereas the combination markedly increased the expressions of interleukin-10, vascular endothelial growth factor, transforming growth factor-beta1, hypoxia-inducible factor 1-alpha, stromal cell-derived factors-1alpha, heme oxygenase-1 and endothelial nitric oxide synthase, and activities of superoxide dismutase, catalase, and glutathione peroxidase in granulation-healing tissue, compared with control and gel-treated groups. In combination group, granulation tissue was better, as was evidenced by improved fibroblast proliferation, collagen deposition, microvessel density, growth-associated protein 43-positive nerve fibers, and thick regenerated epithelial layer. The combination of SP and curcumin accelerated wound healing in diabetic rats and both the drugs were compatible at the doses used in this study. Copyright © 2017 Elsevier Inc. All rights reserved.

Reduced transcript stabilization restricts TNF-alpha expression in RAW264.7 macrophages infected with pathogenic mycobacteria: evidence for an involvement of lipomannan.

PubMed

Basler, Tina; Holtmann, Helmut; Abel, Jens; Eckstein, Torsten; Baumer, Wolfgang; Valentin-Weigand, Peter; Goethe, Ralph

2010-01-01

Despite the critical role that TNF-alpha plays in the containment of mycobacterial infection, the mechanisms involved in regulation of its expression by mycobacteria are poorly defined. We addressed this question by studying MAP, which causes a chronic enteritis in ruminants and is linked to human Crohn's disease. We found that in MAP infected macrophages, TNF-alpha gene expression was substantially lower than in macrophages infected with nonpathogenic MS or stimulated with LPS. TNF-alpha transcriptional one could not fully explain the differential TNF-alpha mRNA expression, suggesting that there must be a substantial contribution by post-transcriptional mechanisms.Accordingly, we found reduced TNF-alpha mRNA stability in MAP-infected macrophages. Further comparison of MAP- and MS-infected macrophages revealed that lower TNF-alpha mRNA stability combined with lower mRNA and protein expression in MAP-infected macrophages correlated with lower p38 MAPK phosphorylation. These findings were independent of viability of MAP and MS. We demonstrate that the major mycobacterial cell-wall lipoglycan LM of MAP and MS induced TNF-alpha mRNA transcription,but only the MS-LM induced p38 MAPK-dependent transcript stabilization. Overall, our data suggest that pathogenic mycobacteria cause weak p38 and TNF-alpha mRNA stabilization as a result of their structural cell-wall components such as LM and thereby, restrict TNF-alpha expression in macrophages.

Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

NASA Technical Reports Server (NTRS)

Pavalko, F. M.; Chen, N. X.; Turner, C. H.; Burr, D. B.; Atkinson, S.; Hsieh, Y. F.; Qiu, J.; Duncan, R. L.

1998-01-01

Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Zhichao; Yu, Xuemei; Wu, Weibin

2012-07-13

Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated thatmore » the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.« less

Functional coupling of rat myometrial alpha 1-adrenergic receptors to Gh alpha/tissue transglutaminase 2 during pregnancy.

PubMed

Dupuis, Morgan; Lévy, Arlette; Mhaouty-Kodja, Sakina

2004-04-30

Gh alpha protein, which exhibits both transglutaminase and GTPase activities, represents a new class of GTP-binding proteins. In the present study, we characterized Gh alpha in rat uterine smooth muscle (myometrium) and followed its expression during pregnancy by reverse transcription-PCR and Western blot. We also measured transglutaminase and GTP binding functions and used a smooth muscle cell line to evaluate the role of Gh alpha in cell proliferation. The results show that pregnancy is associated with an up-regulation of Gh alpha expression at both the mRNA and protein level. Gh alpha induced during pregnancy is preferentially localized to the plasma membrane. This was found associated with an increased ability of plasma membrane preparations to catalyze Ca(2+)-dependent incorporation of [(3)H]putrescine into casein in vitro. In the cytosol, significant changes in the level of immunodetected Gh alpha and transglutaminase activity were seen only at term. Activation of alpha1-adrenergic receptors (alpha1-AR) enhanced photoaffinity labeling of plasma membrane Gh alpha. Moreover, the level of alpha1-AR-coupled Gh alpha increased progressively with pregnancy, which parallels the active period of myometrial cell proliferation. Overexpression of wild type Gh alpha in smooth muscle cell line DDT1-MF2 increased alpha1-AR-induced [(3)H]thymidine incorporation. A similar response was obtained in cells expressing the transglutaminase inactive mutant (C277S) of Gh alpha. Together, these findings underscore the role of Gh alpha as signal transducer of alpha1-AR-induced smooth muscle cell proliferation. In this context, pregnant rat myometrium provides an interesting physiological model to study the mechanisms underlying the regulation of the GTPase function of Gh alpha

Nuclear receptor ERR alpha and coactivator PGC-1 beta are effectors of IFN-gamma-induced host defense.

PubMed

Sonoda, Junichiro; Laganière, Josée; Mehl, Isaac R; Barish, Grant D; Chong, Ling-Wa; Li, Xiangli; Scheffler, Immo E; Mock, Dennis C; Bataille, Alain R; Robert, Francois; Lee, Chih-Hao; Giguère, Vincent; Evans, Ronald M

2007-08-01

Macrophage activation by the proinflammatory cytokine interferon-gamma (IFN-gamma) is a critical component of the host innate response to bacterial pathogenesis. However, the precise nature of the IFN-gamma-induced activation pathway is not known. Here we show using genome-wide expression and chromatin-binding profiling that IFN-gamma induces the expression of many nuclear genes encoding mitochondrial respiratory chain machinery via activation of the nuclear receptor ERR alpha (estrogen-related receptor alpha, NR3B1). Studies with macrophages lacking ERR alpha demonstrate that it is required for induction of mitochondrial reactive oxygen species (ROS) production and efficient clearance of Listeria monocytogenes (LM) in response to IFN-gamma. As a result, mice lacking ERR alpha are susceptible to LM infection, a phenotype that is localized to bone marrow-derived cells. Furthermore, we found that IFN-gamma-induced activation of ERR alpha depends on coactivator PGC-1 beta (peroxisome proliferator-activated receptor gamma coactivator-1 beta), which appears to be a direct target for the IFN-gamma/STAT-1 signaling cascade. Thus, ERR alpha and PGC-1 beta act together as a key effector of IFN-gamma-induced mitochondrial ROS production and host defense.

Gypenoside XLIX, a naturally occurring gynosaponin, PPAR-alpha dependently inhibits LPS-induced tissue factor expression and activity in human THP-1 monocytic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Tom Hsun-Wei; Van Hoan Tran; Roufogalis, Basil D.

2007-01-01

Tissue factor (TF) is involved not only in the progression of atherosclerosis and other cardiovascular diseases, but is also associated with tumor growth, metastasis, and angiogenesis and hence may be an attractive target for directed cancer therapeutics. Gynostemma pentaphyllum (GP) is widely used in the treatment of various cardiovascular diseases including atherosclerosis, as well as cancers. Gypenoside (Gyp) XLIX, a dammarane-type glycoside, is one of the prominent components in GP. We have recently reported Gyp XLIX to be a potent peroxisome proliferator-activated receptor (PPAR)-alpha activator. Here we demonstrate that Gyp XLIX (0-300 {mu}M) concentration dependently inhibited TF promoter activity aftermore » induction by the inflammatory stimulus lipopolysaccharide (LPS) in human monocytic THP-1 cells transfected with promoter reporter constructs pTF-LUC. Furthermore, Gyp XLIX inhibited LPS-induced TF mRNA and protein overexpression in THP-1 monocyte cells. Its inhibition of LPS-induced TF hyperactivity was further confirmed by chromogenic enzyme activity assay. The activities of Gyp XLIX reported in this study were similar to those of Wy-14643, a potent synthetic PPAR-alpha activator. Furthermore, the Gyp XLIX-induced inhibitory effect on TF luciferase activity was completely abolished in the presence of the PPAR-alpha selective antagonist MK-886. The present findings suggest that Gyp XLIX inhibits LPS-induced TF overexpression and enhancement of its activity in human THP-1 monocytic cells via PPAR-alpha-dependent pathways. The data provide new insights into the basis of the use of the traditional Chinese herbal medicine G. pentaphyllum for the treatment of cardiovascular and inflammatory diseases, as well as cancers.« less

Monoclonal antibodies to alphaVbeta3 (7E3 and LM609) inhibit sickle red blood cell-endothelium interactions induced by platelet-activating factor.

PubMed

Kaul, D K; Tsai, H M; Liu, X D; Nakada, M T; Nagel, R L; Coller, B S

2000-01-15

Abnormal interaction of sickle red blood cells (SS RBC) with the vascular endothelium has been implicated as a factor in the initiation of vasoocclusion in sickle cell anemia. Both von Willebrand factor (vWf) and thrombospondin (TSP) play important roles in mediating SS RBC-endothelium interaction and can bind to the endothelium via alphaVbeta3 receptors. We have used monoclonal antibodies (MoAb) directed against alphaVbeta3 and alphaIIbbeta3 (GPIIb/IIIa) integrins to dissect the role of these integrins in SS RBC adhesion. The murine MoAb 7E3 inhibits both alphaVbeta3 and alphaIIbbeta3 (GPIIb/IIIa), whereas MoAb LM609 selectively inhibits alphaVbeta3, and MoAb 10E5 binds only to alphaIIbbeta3. In this study, we have tested the capacity of these MoAbs to block platelet-activating factor (PAF)-induced SS RBC adhesion in the ex vivo mesocecum vasculature of the rat. Infusion of washed SS RBC in preparations treated with PAF (200 pg/mL), with or without a control antibody, resulted in extensive adhesion of these cells in venules, accompanied by frequent postcapillary blockage and increased peripheral resistance units (PRU). PAF also caused increased endothelial surface and interendothelial expression of endothelial vWf. Importantly, pretreatment ofthe vasculature with either MoAb 7E3 F(ab')(2) or LM609, but not 10E5 F(ab')(2), after PAF almost completely inhibited SS RBC adhesion in postcapillary venules, the sites of maximal adhesion and frequent blockage. The inhibition of adhesion with 7E3 or LM609 was accompanied by smaller increases in PRU and shorter pressure-flow recovery times. Thus, blockade of alphaVbeta3 may constitute a potential therapeutic approach to prevent SS RBC-endothelium interactions under flow conditions. (Blood. 2000;95:368-374)

Suppressed heat shock protein response in the kidney of exercise-trained diabetic rats.

PubMed

Lappalainen, J; Oksala, N K J; Laaksonen, D E; Khanna, S; Kokkola, T; Kaarniranta, K; Sen, C K; Atalay, M

2018-07-01

Impaired expression of heat shock proteins (HSPs) and increased oxidative stress may contribute to the pathophysiology of diabetes by disrupted tissue protection. Acute exercise induces oxidative stress, whereas exercise training up-regulates endogenous antioxidant defenses and HSP expression. Although diabetic nephropathy is a major contributor to diabetic morbidity, information regarding the effect of HSPs on kidney protection is limited. This study evaluated the effects of eight-week exercise training on kidney HSP expression and markers of oxidative stress at rest and after acute exercise in rats with or without streptozotocin-induced diabetes. Induction of diabetes increased DNA-binding activity of heat shock factor-1, but decreased the expression of HSP72, HSP60, and HSP90. The inflammatory markers IL-6 and TNF-alpha were increased in the kidney tissue of diabetic animals. Both exercise training and acute exercise increased HSP72 and HSP90 protein levels only in non-diabetic rats. On the other hand, exercise training appeared to reverse the diabetes-induced histological changes together with decreased expression of TGF-beta as a key inducer of glomerulosclerosis, and decreased levels of IL-6 and TNF-alpha. Notably, HSP72 and TGF-beta were negatively correlated. In conclusion, impaired HSP defense seems to contribute to kidney injury vulnerability in diabetes and exercise training does not up-regulate kidney HSP expression despite the improvements in histopathological and inflammatory markers. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Partial Oxygen Pressure Affects the Expression of Prognostic Biomarkers HIF-1 Alpha, Ki67, and CK20 in the Microenvironment of Colorectal Cancer Tissue.

PubMed

Zhang, Lirong; Hu, Yu; Xi, Ning; Song, Jie; Huang, Wenjing; Song, Shanshan; Liu, Yiting; Liu, Xianying; Xie, Yingjun

2016-01-01

Hypoxia is prognostically important in colorectal cancer (CRC) therapy. Partial oxygen pressure (pO 2 ) is an important parameter of hypoxia. The correlation between pO 2 levels and expression levels of prognostic biomarkers was measured in CRC tissues. Human CRC tissues were collected and pO 2 levels were measured by OxyLite. Three methods for tissue fixation were compared, including formalin, Finefix, and Finefix-plus-microwave. Immunohistochemistry (IHC) staining was conducted by using the avidin-biotin complex technique for detecting the antibodies to hypoxia inducible factor-1 (HIF-1) alpha, cytokeratin 20 (CK20), and cell proliferation factor Ki67. The levels of pO 2 were negatively associated with the size of CRC tissues. Finefix-plus-microwave fixation has the potential to replace formalin. Additionally, microwave treatment improved Finefix performance in tissue fixation and protein preservation. The percentage of positive cells and gray values of HIF-1 alpha, CK20, and Ki67 were associated with CRC development ( P < 0.05). The levels of pO 2 were positively related with the gray values of Ki67 and negatively related with the values of HIF-1 alpha and CK20 ( P < 0.05). Thus, the levels of microenvironmental pO 2 affect the expression of predictive biomarkers HIF-1 alpha, CK20, and Ki67 in the development of CRC tissues.

Tumor necrosis factor-alpha converting enzyme in the human placenta throughout gestation.

PubMed

Hung, Tai-Ho; Chen, Szu-Fu; Hsieh, Ching-Chang; Hsu, Jenn-Jeih; Li, Meng-Jen; Yeh, Yi-Lin; Hsieh, T'sang-T'ang

2008-02-01

Ectodomain shedding of epidermal growth factor receptor ligands such as transforming growth factor- alpha (TGF-alpha), heparin-binding epidermal growth factor-like growth factor (HBEGF), and amphiregulin (AREG) is considered to be important during implantation. Tumor necrosis factor-alpha converting enzyme (TACE) has been suggested as the major sheddase for these molecules. The objectives of this study are (1) to characterize the expression of TACE in the human placenta throughout gestation; (2) to determine the association between the expression of TACE with TGF-alpha, HBEGF, and AREG; (3) to ascertain whether TACE mediates TGF-alpha, HBEGF, and AREG shedding; and (4) to examine the effect of hypoxia on the expression of TACE. By analyzing a total of 55 villous samples representing different gestational ages, the authors found that TACE was continuously expressed in the placentas throughout gestation and that the levels of TACE were positively correlated with the levels of TGF-alpha, HBEGF, and AREG. Preadministration of a TACE inhibitor in villous explant cultures or transfection of cytotrophoblastic cells with TACE-specific small interference RNA decreased the shedding of HBEGF and AREG. Moreover, hypoxia (2% O(2)) caused an increase in the levels of TACE mRNA and protein in villous explants and primary cytotrophoblastic cells in vitro. These results indicate that oxygen regulates the expression of TACE and that TACE may be important for placental development during human pregnancy.

Glioma-secreted soluble factors stimulate microglial activation: The role of interleukin-1β and tumor necrosis factor-α.

PubMed

Hwang, Ji-Sun; Jung, Eun-Hye; Kwon, Mi-Youn; Han, Inn-Oc

2016-09-15

We aimed to elucidate the effect of soluble factors secreted by glioma on microglial activation. Conditioned medium (CM) from glioma cells, CRT-MG and C6, significantly induced nitric oxide (NO) production and stimulated the mRNA expression of inducible NO synthase (iNOS), interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-α) and cyclooxygenase 2 (COX-2) in BV2 cells. Glioma CM stimulated p38 mitogen-activated protein kinase (MAPK) phosphorylation, and a p38 MAPK inhibitor, SB203580, suppressed CM-induced NO production in BV2 cells. In addition, CM stimulated nuclear factor-kappaB (NF-κB) DNA binding and transcriptional activity, which was repressed by SB203580. Gliomas displayed higher mRNA expression and release of TNF-α and IL-1β than primary astrocyte cells. Neutralization of TNF-α and IL-1β in C6-CM using a neutralizing antibody inhibited NO/iNOS expression in BV-2 cells. These results indicate potential contribution of diffusible tumor-derived factors to regulate microglial activation and subsequent tumor microenvironment. Copyright © 2016. Published by Elsevier B.V.

RIP1-mediated mitochondrial dysfunction and ROS production contributed to tumor necrosis factor alpha-induced L929 cell necroptosis and autophagy.

PubMed

Ye, Yuan-Chao; Wang, Hong-Ju; Yu, Lu; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2012-12-01

Tumor necrosis factor alpha (TNFα) induces necroptosis and autophagy; however, the detailed molecular mechanism is not fully understood. In this study, we found that TNFα administration caused mitochondrial dysfunction and reactive oxygen species (ROS) production, which led to necroptosis and autophagy in murine fibrosarcoma L929 cells. Notably, the RIP1 (serine-threonine kinase receptor-interacting protein 1, a main adaptor protein of necroptosis) specific inhibitor necrostatin-1 (Nec-1) recovered mitochondrial dysfunction and ROS production due to TNFα administration. Moreover, pan-caspase inhibitor z-VAD-fmk (zVAD) increased RIP1 expression and exacerbated TNFα-induced mitochondrial dysfunction and ROS production, indicating that RIP1 led to mitochondrial dysfunction and ROS production. In addition, cytochrome c release from mitochondria was accompanied with TNFα administration, and Nec-1 blocked the release of cytochrome c upon TNFα administration, while zVAD enhanced the release. These further suggested that RIP1 induced mitochondrial dysfunction accompanied with cytochrome c release. Furthermore, autophagy inhibitor 3-methyladenine (3MA) did not affect RIP1 expression as well as mitochondrial dysfunction and ROS production. Together with our previous publication that autophagy was a downstream consequence of necroptosis, we concluded that TNFα induced mitochondrial dysfunction accompanied with ROS production and cytochrome c release via RIP1, leading to necroptosis and resulting autophagic cell death. Copyright © 2012 Elsevier B.V. All rights reserved.

Interferon-alpha-induced changes in metallothionein expression in liver biopsies from patients with chronic hepatitis C.

PubMed

Nagamine, Takeaki; Suzuki, Keiji; Kondo, Toshihiko; Nakazato, Kyomi; Kakizaki, Satoru; Takagi, Hitoshi; Nakajima, Katuyuki

2005-08-01

An association between reactive oxygen species and liver damage has been postulated in the course of hepatitis C virus (HCV) infection. Metallothionein (MT), induced by HCV core protein and interferon (IFN), plays a role in scavenging free radicals. MT expression in liver biopsies obtained from 21 patients with chronic HCV infection before and after IFN-alpha therapy was investigated. Changes in Knodell histological activity index (HAI) scores, MT protein levels (immunohistochemistry), MT-I and MT-II messenger (m)RNA expression levels (in situ hybridization) and proliferating cell nuclear antigen (PCNA) labelling index were determined and compared in serial liver specimens. MT staining was clustered around the portal tracts with inflammatory cells and fibrosis. The pattern of MT protein before IFN-alpha therapy was similar in all patients, but was higher in IFN-sustained responders than in nonresponders after IFN-alpha therapy. HAI scores and PCNA labelling indexes were significantly reduced after IFN-alpha therapy. MT-II mRNA expression correlated positively with PCNA index before therapy and with HAI scores after therapy (P<0.05). No correlation was found between MT-I mRNA and HAI scores or PCNA index. The findings indicate that IFN-alpha-induced hepatic MT may participate in the therapeutic effects of IFN-alpha for HCV. In addition, MT-II mRNA expression may be involved in cell proliferation in the livers of patients with chronic HCV infection.

Hepatocyte nuclear factor 4alpha contributes to thyroid hormone homeostasis by cooperatively regulating the type 1 iodothyronine deiodinase gene with GATA4 and Kruppel-like transcription factor 9.

PubMed

Ohguchi, Hiroto; Tanaka, Toshiya; Uchida, Aoi; Magoori, Kenta; Kudo, Hiromi; Kim, Insook; Daigo, Kenji; Sakakibara, Iori; Okamura, Masashi; Harigae, Hideo; Sasaki, Takeshi; Osborne, Timothy F; Gonzalez, Frank J; Hamakubo, Takao; Kodama, Tatsuhiko; Sakai, Juro

2008-06-01

Type 1 iodothyronine deiodinase (Dio1), a selenoenzyme catalyzing the bioactivation of thyroid hormone, is highly expressed in the liver. Dio1 mRNA and enzyme activity levels are markedly reduced in the livers of hepatocyte nuclear factor 4alpha (HNF4alpha)-null mice, thus accounting for its liver-specific expression. Consistent with this deficiency, serum T4 and rT3 concentrations are elevated in these mice compared with those in HNF4alpha-floxed control littermates; however, serum T3 levels are unchanged. Promoter analysis of the mouse Dio1 gene demonstrated that HNF4alpha plays a key role in the transactivation of the mouse Dio1 gene. Deletion and substitution mutation analyses demonstrated that a proximal HNF4alpha site (direct repeat 1 [TGGACAAAGGTGC]; HNF4alpha-RE) is crucial for transactivation of the mouse Dio1 gene by HNF4alpha. Mouse Dio1 is also stimulated by thyroid hormone signaling, but a direct role for thyroid hormone receptor action has not been reported. We also showed that thyroid hormone-inducible Krüppel-like factor 9 (KLF9) stimulates the mouse Dio1 promoter very efficiently through two CACCC sequences that are located on either side of HNF4alpha-RE. Furthermore, KLF9 functions together with HNF4alpha and GATA4 to synergistically activate the mouse Dio1 promoter, suggesting that Dio1 is regulated by thyroid hormone in the mouse through an indirect mechanism requiring prior KLF9 induction. In addition, we showed that physical interactions between the C-terminal zinc finger domain (Cf) of GATA4 and activation function 2 of HNF4alpha and between the basic domain adjacent to Cf of GATA4 and a C-terminal domain of KLF9 are both required for this synergistic response. Taken together, these results suggest that HNF4alpha regulates thyroid hormone homeostasis through transcriptional regulation of the mouse Dio1 gene with GATA4 and KLF9.

Molecular evidence for the existence of lipopolysaccharide-induced TNF-alpha factor (LITAF) and Rel/NF-kB pathways in disk abalone (Haliotis discus discus).

PubMed

De Zoysa, Mahanama; Nikapitiya, Chamilani; Oh, Chulhong; Whang, Ilson; Lee, Jae-Seong; Jung, Sung-Ju; Choi, Cheol Young; Lee, Jehee

2010-01-01

The lipopolysaccharide-induced TNF-alpha factor (LITAF) and Rel family nuclear factor kappaB (Rel/NF-kB) are two important transcription factors which play major roles in the regulating inflammatory cytokine, apoptosis and immune related genes. Here, we report the discovery of disk abalone LITAF (AbLITAF) and Rel/NF-kB (AbRel/NF-kB) homologues and their immune responses. Full-length cDNA of AbLITAF consists of 441 bp open reading frame (ORF) that translates into putative peptide of 147 aa. Analysis of AbLITAF sequence showed it has characteristic LITAF (Zn(+2)) binding domain with two CXXC motifs. Phylogenetic analysis results further revealed that AbLITAF is a member of LITAF family. AbRel/NF-kB is 584 aa protein that contains several characteristic motifs including Rel homology domain (RHD), Rel protein signature, DNA binding motif, nuclear localization signal (NLS) and transcription factor immunoglobulin - like fold (TIG) similar to their invertebrate and vertebrate counterparts. Tissue specific analysis results showed that both AbLITAF and AbRel/NF-kB mRNA was expressed ubiquitously in all selected tissues in constitutive manner. However, constitutive expression of AbLITAF was higher than AbRel/NF-kB in all tissues except mantle. Upon immune challenge by bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) and viral hemoragic septicemia virus (VHSV), AbLITAF showed the significant up-regulation in gills while AbRel/NF-kB transcription was not change significantly. Based on transcriptional response against immune challenge, we could suggest that regulation of TNF-alpha expression may have occurred mainly by LITAF activation rather than NF-kB in disk abalone. The cumulative data from other molluscs and our data with reference to TNF-alpha, LITAF and Rel/NF-kB from disk abalone provide strong evidence that LITAF and NF-kB are independent pathways likely to occur throughout the Phylum mollusca. 2010 Elsevier Ltd. All rights reserved.

c-erbA and v-erbA modulate growth and gene expression of a mouse glial precursor cell line.

PubMed

Iglesias, T; Llanos, S; López-Barahona, M; Pérez-Aranda, A; Rodríguez-Peña, A; Bernal, J; Höhne, A; Seliger, B; Muñoz, A

1994-07-01

The c-erbA alpha protooncogene coding for the thyroid hormone (T3) receptor (TR alpha 1) and the viral, mutated v-erbA oncogene were expressed in an immortal mouse glial cell line (B3.1) using retroviral vectors. c-erbA alpha expression led to a decrease in cell proliferation in high and low serum conditions, both in the presence and in the absence of T3. In serum-free medium, c-erbA-expressing cells (B3.1 + TR alpha 1) were completely arrested, whereas cells expressing v-erbA (B3.1 + v-erbA) showed a higher DNA synthesis rate than normal B3.1 cells. Although proliferation of all three cell types was stimulated by platelet-derived growth factor and basic fibroblast growth factor, differences were also observed in the response to these agents. B3.1 + TR alpha 1 cells were more sensitive to platelet-derived growth factor than B3.1 and B3.1 + v-erbA cells. In contrast, B3.1 cells responded to basic fibroblast growth factor better than B3.1 + TR alpha 1 or B3.1 + v-erbA cells. Insulin-like growth factor I potentiated the action of platelet-derived growth factor and basic fibroblast growth factor. Again, different responses to treatment with insulin-like growth factor I alone were observed; B3.1 + TR alpha 1 cells did not respond to it, whereas B3.1 + v-erbA cells showed a dramatic stimulation by this agent. Interestingly, in the presence of T3, the blockade in B3.1 + TR alpha 1 cell proliferation was accompanied by the down-regulation of the typical astrocytic genes, glial fibrillary acidic protein and vimentin. These hormone effects were not found in v-erbA-expressing cells. In addition, v-erbA inhibited the basal expression of the cyclic nucleotide phosphodiesterase gene, an oligodendrocytic marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiatherosclerotic effects of Artemisia princeps Pampanini cv. Sajabal in LDL receptor deficient mice.

PubMed

Han, Jong-Min; Kim, Min-Jung; Baek, Seung-Hwa; An, Sojin; Jin, Yue-Yan; Chung, Hae-Gon; Baek, Nam-In; Choi, Myung-Sook; Lee, Kyung-Tae; Jeong, Tae-Sook

2009-02-25

Antiatherosclerotic effects of ethanolic extracts of Artemisia princeps Pampanini cv. Sajabal (ESJ) were investigated in low-density lipoprotein receptor deficient (LDLR(-/-)) mice. The Western diet-induced high levels of total cholesterol and triglyceride were similar in the ESJ and control groups. However, circulating oxidized LDL was significantly decreased in the ESJ group (p < 0.05). ESJ also markedly decreased aortic expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta), and reduced the aortic lesion formation and macrophage accumulation by 36.7% (p < 0.05) and 43% (p < 0.01) in the control group, respectively. Additionally, ESJ inhibited atherogenic properties with cytokine-induced surface expression of cell adhesion molecules, chemokines, and monocyte adhesion to the human umbilical vein endothelial cells (HUVECs), and simultaneously suppressed nuclear factor-kappaB (NF-kappaB) activation. These results suggest that ethanolic extracts of Artemisia princeps Pampanini cv. Sajabal contributes to the antiatherosclerotic and anti-inflammatory activities in LDLR(-/-) mice.

Insulin induces alpha1B-adrenergic receptor phosphorylation and desensitization.

PubMed

García-Sáinz, J Adolfo; Romero-Avila, M Teresa; Molina-Muñoz, Tzindilú; Medina, Luz del Carmen

2004-09-03

The ability of insulin to induce alpha1B-adrenoceptor phosphorylation and desensitization was tested in two model systems: rat-1 cells that stably express alpha1B-adrenoceptors, through transfection, and endogenously express insulin receptors and DDT1 MF2 cells that endogenously express both receptors. Insulin induced concentration-dependent increases in the phosphorylation state of the adrenergic receptors in the two models with similar EC50 values (0.5-2 nM). The effect was rapid in the two systems but it was sustained in rat-1 cells and transient in DDT1 MF2 cells. In both cell lines, the insulin-mediated phosphorylation of alpha1B-adrenoceptors was blocked by wortmannin and LY 294002, and by staurosporine and bisindolylmaleimide I, indicating that the effect involved phosphoinositide 3-kinase and protein kinase C activities. The adrenoceptor phosphorylation induced by insulin was associated to desensitization as evidences by a diminished elevation of intracellular calcium in response to noradrenaline. Inhibitors of phosphoinositide 3-kinase and protein kinase C blocked the functional desensitization induced by insulin.

Alpha-latrotoxin induces exocytosis by inhibition of voltage-dependent K+ channels and by stimulation of L-type Ca2+ channels via latrophilin in beta-cells.

PubMed

Lajus, Sophie; Vacher, Pierre; Huber, Denise; Dubois, Mathilde; Benassy, Marie-Noëlle; Ushkaryov, Yuri; Lang, Jochen

2006-03-03

The spider venom alpha-latrotoxin (alpha-LTX) induces massive exocytosis after binding to surface receptors, and its mechanism is not fully understood. We have investigated its action using toxin-sensitive MIN6 beta-cells, which express endogenously the alpha-LTX receptor latrophilin (LPH), and toxin-insensitive HIT-T15 beta-cells, which lack endogenous LPH. alpha-LTX evoked insulin exocytosis in HIT-T15 cells only upon expression of full-length LPH but not of LPH truncated after the first transmembrane domain (LPH-TD1). In HIT-T15 cells expressing full-length LPH and in native MIN6 cells, alpha-LTX first induced membrane depolarization by inhibition of repolarizing K(+) channels followed by the appearance of Ca(2+) transients. In a second phase, the toxin induced a large inward current and a prominent increase in intracellular calcium ([Ca(2+)](i)) reflecting pore formation. Upon expression of LPH-TD1 in HIT-T15 cells just this second phase was observed. Moreover, the mutated toxin LTX(N4C), which is devoid of pore formation, only evoked oscillations of membrane potential by reversible inhibition of iberiotoxin-sensitive K(+) channels via phospholipase C, activated L-type Ca(2+) channels independently from its effect on membrane potential, and induced an inositol 1,4,5-trisphosphate receptor-dependent release of intracellular calcium in MIN6 cells. The combined effects evoked transient increases in [Ca(2+)](i) in these cells, which were sensitive to inhibitors of phospholipase C, protein kinase C, or L-type Ca(2+) channels. The latter agents also reduced toxin-induced insulin exocytosis. In conclusion, alpha-LTX induces signaling distinct from pore formation via full-length LPH and phospholipase C to regulate physiologically important K(+) and Ca(2+) channels as novel targets of its secretory activity.

Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

PubMed

Wang, Xiao Yang; Crowston, Jonathan G; White, Andrew J R; Zoellner, Hans; Healey, Paul R

2014-08-01

The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression. © 2013 Royal Australian and New Zealand College of Ophthalmologists.

Differential effect of 1{alpha},25-dihydroxyvitamin D{sub 3} on Hsp28 and PKC{beta} gene expression in the phorbol ester-resistant human myeloid HL-525 leukemic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Yong J.; Galoforo, S.S.; Berns, C.M.

We investigated the effect of 1{alpha},25-dihydroxyvitamin D{sub 3} [1,25-(OH){sub 2}D{sub 3}] on the expression of the 28-kDa heat shock protein gene (hsp28) and the protein kinase C beta gene (PKC{beta}) in the human myeloid HL-60 leukemic cell variant HL-525, which is resistance to phorbol ester-induced macrophage differentiation. Northern and Western blot analysis showed little or no hsp28 gene expression in the HL-60 cell variant, HL-205, which is susceptible to such differentiation, while a relatively high basal level of hps28 gene expression was observed in the HL-525 cells. However, both cell lines demonstrated heat shock-induced expression of this gene. During treatmentmore » with 50-300 nM 1,25-(OH){sub 2}D{sub 3}, a marked reduction of hsp28 gene expression was not associated with heat shock transcription factor-heat shock element (HSF-HSE) binding activity. Our results suggest that the differential effect of 1,25-(OH){sub 2}D{sub 3} on hsp28 and PKC{beta} gene expression is due to the different sequence composition of the vitamin D response element in the in the promoter region as well as an accessory factor for each gene or that 1,25-(OH){sub 2}D{sub 3} increases PKC{beta} gene expression, which in turn negatively regulates the expression of the hsp28 gene, or vice versa.« less

Involvement of tumour necrosis factor-alpha-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells.

PubMed

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-07-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257-264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand-tumour necrosis factor-alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses.

Targeted delivery of siRNA to macrophages for anti-inflammatory treatment.

PubMed

Kim, Sang-Soo; Ye, Chunting; Kumar, Priti; Chiu, Isaac; Subramanya, Sandesh; Wu, Haoquan; Shankar, Premlata; Manjunath, N

2010-05-01

Inflammation mediated by tumor necrosis factor-alpha (TNF-alpha) and the associated neuronal apoptosis characterizes a number of neurologic disorders. Macrophages and microglial cells are believed to be the major source of TNF-alpha in the central nervous system (CNS). Here, we show that suppression of TNF-alpha by targeted delivery of small interfering RNA (siRNA) to macrophage/microglial cells dramatically reduces lipopolysaccharide (LPS)-induced neuroinflammation and neuronal apoptosis in vivo. Because macrophage/microglia express the nicotinic acetylcholine receptor (AchR) on their surface, we used a short AchR-binding peptide derived from the rabies virus glycoprotein (RVG) as a targeting ligand. This peptide was fused to nona-D-arginine residues (RVG-9dR) to enable siRNA binding. RVG-9dR was able to deliver siRNA to induce gene silencing in macrophages and microglia cells from wild type, but not AchR-deficient mice, confirming targeting specificity. Treatment with anti-TNF-alpha siRNA complexed to RVG-9dR achieved efficient silencing of LPS-induced TNF-alpha production by primary macrophages and microglia cells in vitro. Moreover, intravenous injection with RVG-9dR-complexed siRNA in mice reduced the LPS-induced TNF-alpha levels in blood as well as in the brain, leading to a significant reduction in neuronal apoptosis. These results demonstrate that RVG-9dR provides a tool for siRNA delivery to macrophages and microglia and that suppression of TNF-alpha can potentially be used to suppress neuroinflammation in vivo.

Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium

PubMed Central

1996-01-01

Mature adult parenchymal hepatocytes, typically of restricted capacity to proliferate in culture, can now enter into clonal growth under the influence of hepatocyte growth factor (scatter factor) (HGF/SF), epidermal growth factor (EGF), and transforming growth factor alpha (TGFalpha) in the presence of a new chemically defined medium (HGM). The expanding populations of hepatocytes lose expression of hepatocyte specific genes (albumin, cytochrome P450 IIB1), acquire expression of markers expressed by bile duct epithelium (cytokeratin 19), produce TGFalpha and acidic FGF and assume a very simplified morphologic phenotype by electron microscopy. A major change associated with this transition is the decrease in ratio between transcription factors C/EBPalpha and C/EBPbeta, as well as the emergence in the proliferating hepatocytes of transcription factors AP1, NFkappaB. The liver associated transcription factors HNFI, HNF3, and HNF4 are preserved throughout this process. After population expansion and clonal growth, the proliferating hepatocytes can return to mature hepatocyte phenotype in the presence of EHS gel (Matrigel). This includes complete restoration of electron microscopic structure and albumin expression. The hepatocyte cultures however can instead be induced to form acinar/ductular structures akin to bile ductules (in the presence of HGF/SF and type I collagen). These transformations affect the entire population of the hepatocytes and occur even when DNA synthesis is inhibited. Similar acinar/ductular structures are seen in embryonic liver when HGF/SF and its receptor are expressed at high levels. These findings strongly support the hypothesis that mature hepatocytes can function as or be a source of bipotential facultative hepatic stem cells (hepatoblasts). These studies also provide evidence for the growth factor and matrix signals that govern these complex phenotypic transitions of facultative stem cells which are crucial for recovery from acute and chronic liver injury. PMID:8601590

The viral protein A238L inhibits TNF-alpha expression through a CBP/p300 transcriptional coactivators pathway.

PubMed

Granja, Aitor G; Nogal, Maria L; Hurtado, Carolina; Del Aguila, Carmen; Carrascosa, Angel L; Salas, María L; Fresno, Manuel; Revilla, Yolanda

2006-01-01

African swine fever virus (ASFV) is able to inhibit TNF-alpha-induced gene expression through the synthesis of A238L protein. This was shown by the use of deletion mutants lacking the A238L gene from the Vero cell-adapted Ba71V ASFV strain and from the virulent isolate E70. To further analyze the molecular mechanism by which the viral gene controls TNF-alpha, we have used Jurkat cells stably transfected with the viral gene to identify the TNF-alpha regulatory elements involved in the induction of the gene after stimulation with PMA and calcium ionophore. We have thus identified the cAMP-responsive element and kappa3 sites on the TNF-alpha promoter as the responsible of the gene activation, and demonstrate that A238L inhibits TNF-alpha expression through these DNA binding sites. This inhibition was partially reverted by overexpression of the transcriptional factors NF-AT, NF-kappaB, and c-Jun. Furthermore, we present evidence that A238L inhibits the activation of TNF-alpha by modulating NF-kappaB, NF-AT, and c-Jun trans activation through a mechanism that involves CREB binding protein/p300 function, because overexpression of these transcriptional coactivators recovers TNF-alpha promoter activity. In addition, we show that A238L is a nuclear protein that binds to the cyclic AMP-responsive element/kappa3 complex, thus displacing the CREB binding protein/p300 coactivators. Taken together, these results establish a novel mechanism in the control of TNF-alpha gene expression by a viral protein that could represent an efficient strategy used by ASFV to evade the innate immune response.

Carbachol inhibits TNF-α-induced endothelial barrier dysfunction through alpha 7 nicotinic receptors.

PubMed

Li, Yu-zhen; Liu, Xiu-hua; Rong, Fei; Hu, Sen; Sheng, Zhi-yong

2010-10-01

To test whether carbachol can influence endothelial barrier dysfunction induced by tumor necrosis factor (TNF)-α and whether the alpha 7 nicotinic receptor can mediate this process. Rat cardiac microvascular endothelial cells were exposed to carbachol followed by TNF-α treatment in the presence or the absence of α-bungarotoxin (an antagonist of the alpha 7 nicotinic receptor). Permeability of endothelial cells cultured on Transwell filters was assayed using FITC-albumin. F-actin was stained with FITC- phalloidin. Expression of vascular endothelial cadherin, intercellular adhesion molecule 1 (ICAM-1), phosphor-ERK1/2 and phosphor-JNK was detected using Western blot. Carbachol (2 μmol/L-2 mmol/L) prevented increase in endothelial cell permeability induced by TNF-α (500 ng/mL) in a dose-dependent manner. Further, it attenuated the down-regulation of vascular endothelial cadherin and the up-regulation of ICAM-1 induced by TNF-α. In addition, treatment of endothelial cells with carbachol decreased phosphor-ERK1/2 and phosphor-JNK. These effects of carbachol were blocked by α-bungarotoxin 3 μg/mL. These data suggest that the inhibitory effect of carbachol on TNF-α-induced endothelial barrier dysfunction mediated by the alpha 7 nicotinic receptor.

Carbachol inhibits TNF-α-induced endothelial barrier dysfunction through alpha 7 nicotinic receptors

PubMed Central

Li, Yu-zhen; Liu, Xiu-hua; Rong, Fei; Hu, Sen; Sheng, Zhi-yong

2010-01-01

Aim: To test whether carbachol can influence endothelial barrier dysfunction induced by tumor necrosis factor (TNF)-α and whether the alpha 7 nicotinic receptor can mediate this process. Methods: Rat cardiac microvascular endothelial cells were exposed to carbachol followed by TNF-α treatment in the presence or the absence of α-bungarotoxin (an antagonist of the alpha 7 nicotinic receptor). Permeability of endothelial cells cultured on Transwell filters was assayed using FITC-albumin. F-actin was stained with FITC- phalloidin. Expression of vascular endothelial cadherin, intercellular adhesion molecule 1 (ICAM-1), phosphor-ERK1/2 and phosphor-JNK was detected using Western blot. Results: Carbachol (2 μmol/L-2 mmol/L) prevented increase in endothelial cell permeability induced by TNF-α (500 ng/mL) in a dose-dependent manner. Further, it attenuated the down-regulation of vascular endothelial cadherin and the up-regulation of ICAM-1 induced by TNF-α. In addition, treatment of endothelial cells with carbachol decreased phosphor-ERK1/2 and phosphor-JNK. These effects of carbachol were blocked by α-bungarotoxin 3 μg/mL. Conclusion: These data suggest that the inhibitory effect of carbachol on TNF-α-induced endothelial barrier dysfunction mediated by the alpha 7 nicotinic receptor. PMID:20871620

Activation of peroxisome proliferator-activated receptor-alpha protects the heart from ischemia/reperfusion injury.

PubMed

Yue, Tian-li; Bao, Weike; Jucker, Beat M; Gu, Juan-li; Romanic, Anne M; Brown, Peter J; Cui, Jianqi; Thudium, Douglas T; Boyce, Rogely; Burns-Kurtis, Cynthia L; Mirabile, Rosanna C; Aravindhan, Karpagam; Ohlstein, Eliot H

2003-11-11

Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) is expressed in the heart and regulates genes involved in myocardial fatty acid oxidation (FAO). The role of PPAR-alpha in acute ischemia/reperfusion myocardial injury remains unclear. The coronary arteries of male mice were ligated for 30 minutes. After reperfusion for 24 hours, ischemic and infarct sizes were determined. A highly selective and potent PPAR-alpha agonist, GW7647, was administered by mouth for 2 days, and the third dose was given 1 hour before ischemia. GW7647 at 1 and 3 mg x kg(-1) x d(-1) reduced infarct size by 28% and 35%, respectively (P<0.01), and myocardial contractile dysfunction was also improved. Cardioprotection by GW7647 was completely abolished in PPAR-alpha-null mice. Ischemia/reperfusion downregulated mRNA expression of cardiac PPAR-alpha and FAO enzyme genes, decreased myocardial FAO enzyme activity and in vivo cardiac fat oxidation, and increased serum levels of free fatty acids. All of these changes were reversed by GW7647. Moreover, GW7647 attenuated ischemia/reperfusion-induced release of multiple proinflammatory cytokines and inhibited neutrophil accumulation and myocardial expression of matrix metalloproteinases-9 and -2. Furthermore, GW7647 inhibited nuclear factor-kappaB activation in the heart, accompanied by enhanced levels of inhibitor-kappaBalpha. Activation of PPAR-alpha protected the heart from reperfusion injury. This cardioprotection might be mediated through metabolic and antiinflammatory mechanisms. This novel effect of the PPAR-alpha agonist could provide an added benefit to patients treated with PPAR-alpha activators for dyslipidemia.

Antioxidants and NOX1/NOX4 inhibition blocks TGFβ1-induced CCN2 and α-SMA expression in dermal and gingival fibroblasts

PubMed Central

Murphy-Marshman, Hannah; Quensel, Katherine; Shi-wen, Xu; Barnfield, Rebecca; Kelly, Jacalyn; Peidl, Alex; Stratton, Richard J.

2017-01-01

TGFbeta induces fibrogenic responses in fibroblasts. Reactive oxygen species (ROS)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) may contribute to fibrogenic responses. Here, we examine if the antioxidant N-acetylcysteine (NAC), the NOX inhibitor diphenyleneiodonium (DPI) and the selective NOX1/NOX4 inhibitor GKT-137831 impairs the ability of TGFbeta to induce profibrotic gene expression in human gingival (HGF) and dermal (HDF) fibroblasts. We also assess if GKT-137831 can block the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts. We use real-time polymerase chain reaction and Western blot analysis to evaluate whether NAC and DPI impair the ability of TGFbeta1 to induce expression of fibrogenic genes in fibroblasts. The effects of GKT-137831 on TGFbeta-induced protein expression and the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts were tested using Western blot and collagen gel contraction analyses. In HDF and HGF, TGFbeta1 induces CCN2, CCN1, endothelin-1 and alpha-smooth muscle actin (SMA) in a fashion sensitive to NAC. Induction of COL1A1 mRNA was unaffected. Similar results were seen with DPI. NAC and DPI impaired the ability of TGFbeta1 to induce protein expression of CCN2 and alpha-SMA in HDF and HGF. GKT-137831 impaired TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF. In lesional SSc dermal fibroblasts, GKT-137831 reduced alpha-SMA and CCN2 protein overexpression and collagen gel contraction. These results are consistent with the hypothesis that antioxidants or NOX1/4 inhibition may be useful in blocking profibrotic effects of TGFbeta on dermal and gingival fibroblasts and warrant consideration for further development as potential antifibrotic agents. PMID:29049376

Reversal of ethanol-induced hepatotoxicity by cinnamic and syringic acids in mice.

PubMed

Yan, Sheng-Lei; Wang, Zhi-Hong; Yen, Hsiu-Fang; Lee, Yi-Ju; Yin, Mei-Chin

2016-12-01

Ethanol was used to induce acute hepatotoxicity in mice. Effects of cinnamic acid (CA) and syringic acid (SA) post-intake for hepatic recovery from alcoholic injury was investigated. Ethanol treated mice were supplied by CA or SA at 40 or 80 mg/kg BW/day for 5 days. Results showed that ethanol stimulated protein expression of CYP2E1, p47 phox , gp91 phox , cyclooxygenase-2 and nuclear factor kappa B in liver. CA or SA post-intake restricted hepatic expression of these molecules. Ethanol suppressed nuclear factor erythroid 2-related factor (Nrf2) expression, and CA or SA enhanced Nrf2 expression in cytosolic and nuclear fractions. Ethanol increased the release of reactive oxygen species, oxidized glutathione, interleukin-6, tumor necrosis factor-alpha, nitric acid and prostaglandin E 2 . CA or SA lowered hepatic production of these oxidative and inflammatory factors. Histological data revealed that ethanol administration caused obvious foci of inflammatory cell infiltration, and CA or SA post-intake improved hepatic inflammatory infiltration. These findings support that cinnamic acid and syringic acid are potent nutraceutical agents for acute alcoholic liver disease therapy. However, potential additive or synergistic benefits of cinnamic and syringic acids against ethanol-induced hepatotoxicity need to be investigated. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-{kappa}B in human aortic smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manea, Adrian, E-mail: adrian.manea@icbp.ro; Tanase, Laurentia I.; Raicu, Monica

Inflammation-induced changes in the activity and expression of NADPH oxidases (Nox) play a key role in atherogenesis. The molecular mechanisms of Nox regulation are scantily elucidated. Since nuclear factor-{kappa}B (NF-{kappa}B) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-{kappa}B signaling in the regulation of Nox1 and Nox4 transcription in human aortic smooth muscle cells (SMCs). Cultured cells were exposed to tumor necrosis factor-{alpha} (TNF{alpha}), a potent inducer of both Nox and NF-{kappa}B, up to 24 h. Lucigenin-enhanced chemiluminescence and dichlorofluorescein assays, real-time polymerase chain reaction, and Western blot analysismore » showed that inhibition of NF-{kappa}B pathway reduced significantly the TNF{alpha}-dependent up-regulation of Nox-derived reactive oxygen species production, Nox1 and Nox4 expression. In silico analysis indicated the existence of typical NF-{kappa}B elements in the promoters of Nox1 and Nox4. Transient overexpression of p65/NF-{kappa}B significantly increased the promoter activities of both isoforms. Physical interaction of p65/NF-{kappa}B proteins with the predicted sites was demonstrated by chromatin immunoprecipitation assay. These findings demonstrate that NF-{kappa}B is an essential regulator of Nox1- and Nox4-containing NADPH oxidase in SMCs. Elucidation of the complex relationships between NF-{kappa}B and Nox enzymes may lead to a novel pharmacological strategy to reduce both inflammation and oxidative stress in atherosclerosis and its associated complications.« less

A nuclear factor kappa B-derived inhibitor tripeptide inhibits UVB-induced photoaging process.

PubMed

Oh, Jee Eun; Kim, Min Seo; Jeon, Woo-Kwang; Seo, Young Kwon; Kim, Byung-Chul; Hahn, Jang Hee; Park, Chang Seo

2014-12-01

Ultraviolet (UV) irradiation on the skin induces photoaging which is characterized by keratinocyte hyperproliferation, generation of coarse wrinkles, worse of laxity and roughness. Upon UV irradiation, nuclear factor kappa B (NF-κB) is activated which plays a key role in signaling pathway leading to inflammation cascade and this activation stimulates expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1alpha (IL-1α) and a stress response gene cyclooxygenase-2 (COX-2). In addition, activation of NF-κB up-regulates the expression of matrix metalloprotease-1 (MMP-1) and consequently collagen in dermis is degraded. In this study, the effects of a NF-κB-derived inhibitor tripeptide on the UVB-induced photoaging and inflammation were investigated in vitro and in vivo. A NF-κB-derived inhibitor tripeptide (NF-κB-DVH) was synthesized based on the sequence of dimerization region of the subunit p65 of NF-κB. Its inhibitory activity was confirmed using chromatin immunoprecipitation assay and in situ proximate ligation assay. The effects of anti-photoaging and anti-inflammation were analyzed by Enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunochemistry. NF-κB-DVH significantly decreased UV-induced expression of TNF-α, IL-1α, MMP-1 and COX-2 while increased production of type I procollagen. Results showed NF-κB-DVH had strong anti-inflammatory activity probably by inhibiting NF-κB activation pathway and suggested to be used as a novel agent for anti-photoaging. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

PubMed

Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

2003-09-01

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

Molecular mechanisms of topical anti-inflammatory effects of lipoxin A(4) in endotoxin-induced uveitis.

PubMed

Medeiros, Rodrigo; Rodrigues, Gustavo Büchele; Figueiredo, Cláudia Pinto; Rodrigues, Eduardo Büchele; Grumman, Astor; Menezes-de-Lima, Octavio; Passos, Giselle Fazzioni; Calixto, João Batista

2008-07-01

Lipoxin A(4) (LXA(4)) is a lipid mediator that plays an important role in inflammation resolution. We assessed the anti-inflammatory effect of LXA(4) on endotoxin-induced uveitis (EIU) in rats. The inflammatory cell number and levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)), and protein, as well as expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF), in the anterior chamber of the eye were determined 24 h after lipopolysaccharide (LPS; 200 mug/paw) intradermal injection. The immunohistochemical reactivities of nuclear factor-kappaB (NF-kappaB) and c-Jun were also examined. Topical LXA(4) (1-10 ng/eye) pretreatment decreased the number of inflammatory cells and the protein leakage into the aqueous humor (AqH). In addition, topical LXA(4) (10 ng/eye) inhibited the LPS-induced production of IL-1beta, TNF-alpha, and PGE(2), and expression of COX-2 and VEGF. A decreased activation of NF-kappaB and c-Jun was also found in LXA(4)-treated eyes. It is very interesting that an anti-inflammatory effect was achieved even when LXA(4) (10 ng/eye) was applied topically after LPS challenge, as indicated by the reduction in the cellular and protein extravasations into the AqH. Moreover, topical treatment of corticosteroid prednisolone (200 mug/eye) beginning before or after LPS injection reduced all of the molecular and biochemical alterations promoted on EIU rats in an efficacy similar to that of LXA(4). Together, the present results provide clear evidence that pharmacological activation of LXA(4) signaling pathway potently reduces the EIU in rats. Therefore, LXA(4) stable analogs could represent promising agents for the management of ocular inflammatory diseases.

SHH-dependent knockout of HIF-1 alpha accelerates the degenerative process in mouse intervertebral disc.

PubMed

Wu, W J; Zhang, X K; Zheng, X F; Yang, Y H; Jiang, S D; Jiang, L S

2013-01-01

Hypoxia-inducible factor-1alpha (HIF-1 alpha) has been reported to have an important role in the metabolism and synthesis of extracellular matrix of the nucleus pulposus cells (NPCs) and was assumed to be involved in the process of intervertebral disc degeneration. The objective of this study was to investigate the role of HIF-1alpha in disc degeneration in vivo using a conditional HIF-1alpha knockout (KO) mouse model. ShhCre transgenic mice were mated with HIF-1 alpha fl/fl mice to generate conditional HIF-1alpha KO mice (HIF-1alpha fl/fl-ShhCre+). Three mice of each genotype (Wide-type and HIF-1alpha KO) at the age of 3 days, 6, and 12 weeks were sacrificed after genotyping. Five lumbar disc samples were harvested from each mouse, with a total of 45 disc samples for each genotype. In situ hybridization and immunohistochemical analysis were used to check the efficacy of HIF-1alpha knockout. Histological grading of the disc degeneration was performed according to the classification system proposed by Boos et al. Picro-sirius red staining, Safranine O/fast green staining and immunohistochemical study were used to evaluate the expression of aggrecan, type-II collagen and vascular endothelial growth factor (VEGF). Histologic analysis revealed more NPC deaths and signs of degeneration in HIF-1alpha KO mice and the degeneration scores of HIF-1alpha KO mice were significantly higher than those of the Wide-type mice at the age of 6 weeks and 12 weeks. There were less expressions of aggrecan, type-II collagen and VEGF in the intervertebral discs of HIF1-alpha KO mice than in those of wild-type mice. Taken together, the results of our study indicated that HIF-1alpha is a pivotal contributor to NPC survival and the homeotasis of extracellular matrix through the HIF-1alpha/VEGF signaling pathway, and plays an important role in the development of disc degeneration.

Upregulation of estrogen receptor expression in the uterus of ovariectomized B6C3F1 mice and Ishikawa cells treated with bromoethane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aoyama, Hiroaki; Couse, John F.; Hewitt, Sylvia C.

2005-12-15

In a 2-year NTP bioassay, Bromoethane (BE) was found to induce endometrial neoplasms in the uterus of B6C3F1 mice [; ]. In women, hormonal influences, such as 'unopposed' estrogenic stimulus, have been implicated as important etiologic factors in uterine cancer. BE, however, does not affect the serum concentrations of sex hormones in female B6C3F1 mice [] and the mechanism of BE-induced uterine carcinogenesis still remains unclear. In the present study, we examined the estrogenic effects of BE on the uterus of ovariectomized B6C3F1 mice and on Ishikawa cells. Groups of 6 mice were given daily s.c. injections of 0, 100,more » 500 or 1000 mg BE/kg for 3 consecutive days. Mice treated with 17{beta}-estradiol served as positive controls. Mice were necropsied 24 h after the final injection, and uteri were weighed and examined histologically and immunohistochemically along with the vagina. Changes observed in the estrogen-treated mice included increased uterine weights, edema and inflammation of the endometrium, increased epithelial layers of the uterine and vaginal lumens and keratinization of the vaginal epithelium. In the BE-treated mice, no such changes occurred; however, immunohistochemical staining of the uterus revealed a significant increase in immunoexpression of the estrogen receptor alpha (ER{alpha}) in the two higher dose groups. Analysis of mRNA also showed slightly increased uterine ER{alpha} expression in these groups. Upregulated expression of ER{alpha} was confirmed in BE-treated Ishikawa cells, in which Western blotting analyses identified an intense signal at approximately 66 kDa, which is consistent with ER{alpha}. These data suggest that upregulated expression of ER{alpha} may be important in the induction of endometrial neoplasms in BE-treated mice.« less

Synthesis of Toll-like receptor 4 in Kupffer cells and its role in alcohol-induced liver disease.

PubMed

Zuo, Guoqing; Gong, Jianping; Liu, Chang'an; Wu, Chuanxin; Li, Shengwei; Dai, Lili

2003-02-01

To observe the synthesis of Toll-like receptor (TLR) 4 protein and its mRNA expression in Kupffer cells (KCs) and evaluate the role of TLR 4 in liver injury to rats through alcohol-induced liver disease. Twenty-eight Wistar rats were divided into two groups: ethanol-fed (group E) and control (group C). Group E rats were given ethanol at a dose of 5 - 12 g x kg(-1) x d(-1), while group C received dextrose. Animals from both groups were killed at 4 and 8 weeks. The KCs were isolated and synthesis of TLR 4 protein was determined by laser scanning confocal microscopy. TLR 4 mRNA expression in KCs was determined by reverse transcription polymerase chain reaction (RT-PCR) analysis. The levels of endotoxin, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in plasma were determined. Changes in liver pathology were observed. Laser scanning confocal microscopy showed that the intensity of fluorescence of TLR 4 protein in group E was stronger than group C. Ethanol administration led to a significant increase in TLR 4 mRNA expression in group E compared with group C (P < 0.05). The concentrations of plasma endotoxin, TNF-alpha and IL-6 were higher in group E than in group C (P < 0.05). Liver sections from rats in group E demonstrated marked pathological changes. Ethanol administration can lead to the synthesis of TLR 4 protein and its gene expression in KCs, indicating that TLR 4 may play a major role in the development of alcohol-induced liver injury.

Over-expression of C/EBP-{alpha} induces apoptosis in cultured rat hepatic stellate cells depending on p53 and peroxisome proliferator-activated receptor-{gamma}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Xueqing; Huang Guangcun; Mei Shuang

2009-03-06

Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-{alpha} (C/EBP-{alpha}) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-{alpha} gene (Ad-C/EBP-{alpha}) could induce HSCs apoptosis in a dose- and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-{alpha} resulted in the up-regulation of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) andmore » P53, while P53 expression was regulated by PPAR-{gamma}. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-{gamma} and p53 in the process of apoptosis triggered by C/EBP-{alpha} in HSCs.« less

Role of CD14 in responses to clinical isolates of Escherichia coli: effects of K1 capsule expression.

PubMed

Metkar, Shalaka; Awasthi, Shanjana; Denamur, Erick; Kim, Kwang Sik; Gangloff, Sophie C; Teichberg, Saul; Haziot, Alain; Silver, Jack; Goyert, Sanna M

2007-11-01

Severe bacterial infections leading to sepsis or septic shock can be induced by bacteria that utilize different factors to drive pathogenicity and/or virulence, leading to disease in the host. One major factor expressed by all clinical isolates of gram-negative bacteria is lipopolysaccharide (LPS); a second factor expressed by some Escherichia coli strains is a K1 polysaccharide capsule. To determine the role of the CD14 LPS receptor in the pathogenic effects of naturally occurring E. coli, the responses of CD14-/- and CD14+/+ mice to three different isolates of E. coli obtained from sepsis patients were compared; two isolates express both smooth LPS and the K1 antigen, while the third isolate expresses only LPS and is negative for K1. An additional K1-positive isolate obtained from a newborn with meningitis and a K1-negative isogenic mutant of this strain were also used for these studies. CD14-/- mice were resistant to the lethal effects of the K1-negative isolates. This resistance was accompanied by significantly lower levels of systemic tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) in these mice than in CD14+/+ mice, enhanced clearance of the bacteria, and significantly fewer additional gross symptoms. In contrast, CD14-/- mice were as sensitive as CD14+/+ mice to the lethal effects of the K1-positive isolates, even though they had significantly lower levels of TNF-alpha and IL-6 than CD14+/+ mice. These studies show that different bacterial isolates can use distinctly different mechanisms to cause disease and suggest that new, nonantibiotic therapeutics need to be directed against multiple targets.

[EFFECT OF VITAMIN C ON APOPTOSIS OF NUCLEUS PULPOSUS CELLS INDUCED BY TUMOR NECROSIS FACTOR a AND SERUM DEPRIVATION].

PubMed

Dai, Libing; Liu, Zhihe; Liang, Weiguo; Yao, Yicun; Xu, Jiakel; Ye, Dongping; Zou, Longqiang; Shen, Yan

2015-04-01

To explore the effect of Vitamin C (Vit C) on the apoptosis of human nucleus pulposus (NP) cells induced by tumor necrosis factor a (TNF-alpha) and serum deprivation. The NP cells were isolated from patients undergoing spine corrective operation by collagenase trypsin. The experiment was divided into 3 groups: Vit C group (group A), TNF-alpha group (group B), and serum deprivation group (group C). Group A was reassigned to Al subgroup (basic medium), A2 subgroup (100 pg/mL Vit C), and A3 subgroup (200 pg/mL Vit C). Group B was reassigned to B0 subgroup (control group), Bi subgroup (100 ng/mL TNF-alpha), B2 subgroup (100 microg/mL Vit C+100 ng/mL TNF-alpha), and B3 subgroup (200 microg/mL Vit C+100 ng/mL TNF-alpha). Group C was reassigned to C0 subgroup (Control group), C1 subgroup (2% FBS), C2 subgroup (2% FBS+100 microg/mL Vit C), and C3 subgroup (2% FBS+200 microg/mL Vit C). After application of 100 pg/mL or 200 microg/mL Vit C for 24 hours, NP cells were stimulated by TNF-alpha and serum deprivation, then the apoptosis rate of NP cells was detected by a flow cytometry, and the gene expressions of the extracellular matrix of NP cells (collagen type I, collagen type II, aggrecan, and Sox9) and apoptosis related genes (p53, FAS, and Caspase 3) were detected by real-time fluoroscent quantitative PCR. Results Group A: Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 of NP cells in A2 and A3 subgroups when compared with Al subgroup (P<0.05), but there was no significant difference between A2 subgroup and A3 subgroup (P>0.05); Vit C could promote the expressions of the extracellular matrix (collagen type I, collagen type II, aggrecan, and Sox9) of NP cells in a concentration dependent manner (P<0.05). Group B: TNF-alpha significantly increased the apoptosis rate and the gene expressions of p53, FAS, and Caspase 3 in B1 subgroup when compared with B0 subgroup (P<0.05); however, Vit C significantly increased the apoptosis rate and the gene expressions in B2 subgroup, and significantly decreased them in B3 subgroup when compared with B1 subgroup (P<0.05). Group C: 2% FBS significantly increased the apoptosis rate of NP cells and significantly reduced the gene expressions of p53, FAS, and Caspase 3 in C1 subgroup when compared with C0 subgroup (P<0.05); Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 in C3 subgroup, but it could significantly increase them in C2 subgroup when compared with C1 subgroup (P<0.05). Vit C can promote the synthesis and secretion of extracellular matrix of NP cells. 200 microg/mL Vit C may delay the apoptosis induced by TNF-alpha and serum deprivation, indicating the potential therapeutic effect of Vit C on intervertebral disc degeneration.

Antioxidant and anti-inflammatory activities of alpha lipoic acid protect against indomethacin-induced gastric ulcer in rats.

PubMed

Gomaa, Asmaa M S; Abd El-Mottaleb, Nashwa A; Aamer, Hazem A

2018-05-01

Little is known about the role of tumor necrosis factor-alpha (TNF-α), plasminogen activator inhibitor-1 (PAI-1), and inducible nitric oxide synthase (iNOS) in the gastric ulcer and the effect of alpha lipoic acid (ALA) in their modulation. Hence, this experimental study was designed to assess the possible protective effect of ALA against indomethacin (IND)-induced gastric ulcer in rats, as well as to determine the possible underlying mechanisms with a special focus on TNF-α, PAI-1, and iNOS. Adult male rats (n = 28) were divided into four equal groups: the control group received distilled water, the vehicle group received 0.5% carboxymethylcellulose, the ulcer group received a single oral dose of IND (50 mg/kg) and the ALA-treated group received ALA (100 mg/kg) orally for 3 days before ulcer induction. Four hours after IND administration, all rats were sacrificed. The ulcer index, and gastric tissue homogenate contents of total antioxidant capacity (TAC), malondialdehyde (MDA), TNF-α, and PAI-1 were evaluated. Immunohistochemical evaluation of iNOS protein expression and histopathological examination of gastric tissue were investigated. The results revealed that ALA pretreatment significantly decreased the ulcer index, the gastric levels of MDA, TNF-α, PAI-1, and iNOS protein expression while increased the gastric levels of TAC as well as improved the histopathological appearance of gastric tissues. In conclusion, ALA ameliorated the IND-induced gastric ulceration. This could be attributed to its antioxidant and anti-inflammatory activities via suppression of TNF-α-induced elevation of both PAI-1 level and iNOS expression in the gastric tissue. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Increased serum IL-6, TNF-alpha and IL-10 levels in patients with bullous pemphigoid: relationships with disease activity.

PubMed

D'Auria, L; Mussi, A; Bonifati, C; Mastroianni, A; Giacalone, B; Ameglio, F

1999-01-01

The present report analyzes the serum levels of three cytokines, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in 15 patients with bullous pemphigoid (BP) (compared with 20 healthy controls) to evaluate a possible involvement of these biological modulators in the clinical expression of this disease. BP is a rare bullous disease of autoimmune origin with evidence of inflammatory processes that cause skin lesions with local increase of various pro-inflammatory mediators. Determination of cytokine concentrations were obtained employing commercially available ELISA kits. The sera of BP patients showed increased levels of these three cytokines (P < 0.01). When the number of skin lesions (blisters and/or erosion) of each patient, employed as a marker of disease activity, was correlated with the serum levels of IL-6 and TNF-alpha, significant correlations were found (IL-6: P < 0.01 and TNF-alpha: P < 0.01, respectively), suggesting a possible role of these mediators in the development of BP blisters. The serum levels of IL-6 also correlated (P = 0.01 with those of serum C reactive protein (CRP), an acute-phase protein induced by IL-6 in hepatocytes. In addition, serum TNF-alpha and sE-selectin (an adhesion molecule previously reported to be increased by this cytokine) levels were also correlated (P < 0.05). On the basis of these data, it may be indicated that at least IL-6 and TNF-alpha are associated with the clinical expression of BP and that the endothelial activation (possibly induced by the TNF-alpha activity), seems to be an important phase of this dermatosis.

Targeted inhibition of p38alpha MAPK suppresses tumor-associated endothelial cell migration in response to hypericin-based photodynamic therapy.

PubMed

Hendrickx, Nico; Dewaele, Michael; Buytaert, Esther; Marsboom, Glenn; Janssens, Stefan; Van Boven, Maurits; Vandenheede, Jackie R; de Witte, Peter; Agostinis, Patrizia

2005-11-25

Photodynamic therapy (PDT) is an established anticancer modality and hypericin is a promising photosensitizer for the treatment of bladder tumors. We show that exposure of bladder cancer cells to hypericin PDT leads to a rapid rise in the cytosolic calcium concentration which is followed by the generation of arachidonic acid by phospholipase A2 (PLA2). PLA2 inhibition significantly protects cells from the PDT-induced intrinsic apoptosis and attenuates the activation of p38 MAPK, a survival signal mediating the up-regulation of cyclooxygenase-2 that converts arachidonic acid into prostanoids. Importantly, inhibition of p38alpha MAPK blocks the release of vascular endothelial growth factor and suppresses tumor-promoted endothelial cell migration, a key step in angiogenesis. Hence, targeted inhibition of p38alpha MAPK could be therapeutically beneficial to PDT, since it would prevent COX-2 expression, the inducible release of growth and angiogenic factors by the cancer cells, and cause an increase in the levels of free arachidonic acid, which promotes apoptosis.

Production of interleukin-1alpha by human endometrial stromal cells is triggered during menses and dysfunctional bleeding and is induced in culture by epithelial interleukin-1alpha released upon ovarian steroids withdrawal.

PubMed

Pretto, Chrystel M; Gaide Chevronnay, Héloïse P; Cornet, Patricia B; Galant, Christine; Delvaux, Denis; Courtoy, Pierre J; Marbaix, Etienne; Henriet, Patrick

2008-10-01

Endometrial breakdown during menstruation and dysfunctional bleeding is triggered by the abrupt expression of matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1). The paracrine induction of MMP-1 in stromal cells via epithelium-derived IL-1alpha is repressed by ovarian steroids. However, the control by estradiol (E) and progesterone (P) of endometrial IL-1alpha expression and bioactivity remains unknown. Variations of endometrial IL-1alpha mRNA and protein along the menstrual cycle and during dysfunctional bleeding were determined using RT-PCR, in situ hybridization, and immunolabeling. The mechanism of EP control was analyzed using culture of explants, laser capture microdissection, and purified cells. Data were compared with expression changes of IL-1beta and IL-1 receptor antagonist. IL-1alpha is synthesized by epithelial cells throughout the cycle but E and/or P prevents its release. In contrast, endometrial stromal cells produce IL-1alpha only at menses and during irregular bleeding in areas of tissue breakdown. Stromal expression of IL-1alpha, like that of MMP-1, is repressed by P (alone or with E) but triggered by epithelium-derived IL-1alpha released upon EP withdrawal. Our experiments in cultured endometrium suggest that IL-1alpha released by epithelial cells triggers the production of IL-1alpha by stromal cells in a paracrine amplification loop to induce MMP-1 expression during menstruation and dysfunctional bleeding. All three steps of this amplification cascade are repressed by EP.

The A3 adenosine receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-kappaB signaling pathway in synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats.

PubMed

Ochaion, A; Bar-Yehuda, S; Cohen, S; Amital, H; Jacobson, K A; Joshi, B V; Gao, Z G; Barer, F; Patoka, R; Del Valle, L; Perez-Liz, G; Fishman, P

2008-08-15

The A(3) adenosine receptor (A(3)AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis. In this study we present a novel A(3)AR agonist, CF502, with high affinity and selectivity at the human A(3)AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-kappaB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100 microg/kg). As is typical of other G-protein coupled receptors, the A(3)AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IkappaB kinase (IKK), I kappa B (IkappaB), NF-kappaB and tumor necrosis factor-alpha (TNF-alpha) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3beta), beta-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-kappaB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A(3)AR agonists for the treatment of rheumatoid arthritis.

Progressive exercise preconditioning protects against circulatory shock during experimental heatstroke.

PubMed

Hung, Ching-Hsia; Chang, Nen-Chung; Cheng, Bor-Chih; Lin, Mao-Tsun

2005-05-01

Heat shock protein (HSP) 72 expression protects against arterial hypotension in rat heatstroke. HSP72 can also be induced in multiple organs, including hearts from rats with endurance exercise. We validated the hypothesis that progressive exercise preconditioning may confer cardiovascular protection during heatstroke by inducing the overexpression of HSP72 in multiple organs. To deal with the matter, we assessed the effects of heatstroke on mean arterial pressure, heart rate, cardiac output, stroke volume, total peripheral vascular resistance, colonic temperature, blood gases, and serum or tissue levels of tumor necrosis factor-alpha (TNF-alpha) in urethane-anesthetized rats pretreated without or with progressive exercise training for 1, 2, or 3 weeks. In addition, HSP72 expression in multiple organs was determined in different groups of animals. Heatstroke was induced by exposing the rats to a high blanket temperature (43 degrees C); the moment at which mean arterial pressure decreased from the peak value was taken as the time of heatstroke onset. Previous exercise training for 3 weeks, but not 1 or 2 weeks, conferred significant protection against hyperthermia, arterial hypotension, decreased cardiac output, decreased stroke volume, decreased peripheral vascular resistance, and increased levels of serum or tissue TNF-alpha during heatstroke and correlated with overexpression of HSP72 in multiple organs, including heart, liver, and adrenal gland. However, 10 days after 3 weeks of progressive exercise training, when HSP72 expression in multiple organs returned to basal values, the beneficial effects exerted by 3 weeks of exercise training were no longer observed. These results strongly suggest that HSP72 preconditioning with progressive exercise training protects against hyperthermia, circulatory shock, and TNF-alpha overproduction during heatstroke.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Danno, Hirosuke; Ishii, Kiyo-aki; Nakagawa, Yoshimi

To elucidate the physiological role of CREBH, the hepatic mRNA and protein levels of CREBH were estimated in various feeding states of wild and obesity mice. In the fast state, the expression of CREBH mRNA and nuclear protein were high and profoundly suppressed by refeeding in the wild-type mice. In ob/ob mice, the refeeding suppression was impaired. The diet studies suggested that CREBH expression was activated by fatty acids. CREBH mRNA levels in the mouse primary hepatocytes were elevated by addition of the palmitate, oleate and eicosapenonate. It was also induced by PPAR{alpha} agonist and repressed by PPAR{alpha} antagonist. Luciferasemore » reporter gene assays indicated that the CREBH promoter activity was induced by fatty acids and co-expression of PPAR{alpha}. Deletion studies identified the PPRE for PPAR{alpha} activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay confirmed that PPAR{alpha} directly binds to the PPRE. Activation of CREBH at fasting through fatty acids and PPAR{alpha} suggest that CREBH is involved in nutritional regulation.« less

Induction of VEGF expression by alpha-tocopherol and alpha-tocopheryl phosphate via PI3Kgamma/PKB and hTAP1/SEC14L2-mediated lipid exchange

USDA-ARS?s Scientific Manuscript database

In several studies, vitamin E has been observed to influence angiogenesis and vasculogenesis. We recently showed that the phosphorylated form of alpha-tocopherol (alphaT), alpha-tocopheryl phosphate (alphaTP), increases the expression of the vascular endothelial growth factor (VEGF). Thus, alphaTP m...

PPAR alpha and PPAR gamma coactivation rapidly induces Egr-1 in the nuclei of the dorsal and ventral urinary bladder and kidney pelvis urothelium of rats.

PubMed

Egerod, Frederikke Lihme; Svendsen, Jette Eldrup; Hinley, Jennifer; Southgate, Jennifer; Bartels, Annette; Brünner, Nils; Oleksiewicz, Martin B

2009-12-01

To facilitate studies of the rat bladder carcinogenicity of dual-acting PPAR alpha+gamma agonists, we previously identified the Egr-1 transcription factor as a candidate carcinogenicity biomarker and developed rat models based on coadministration of commercially available specific PPAR alpha and PPAR gamma agonists. Immunohistochemistry for Egr-1 with a rabbit monoclonal antibody demonstrated that male vehicle-treated rats exhibited minimal urothelial expression and specifically, no nuclear signal. In contrast, Egr-1 was induced in the nuclei of bladder, as well as kidney pelvis, urothelia within one day (2 doses) of oral dosing of rats with a combination of 8 mg/kg rosiglitazone and 200 mg/kg fenofibrate (specific PPAR gamma and PPAR alpha agonists, respectively). These findings were confirmed by Western blotting using a different Egr-1 antibody. Egr-1 was induced to similar levels in the dorsal and ventral bladder urothelium, arguing against involvement of urinary solids. Egr-1 induction sometimes occurred in a localized fashion, indicating physiological microheterogeneity in the urothelium. The rapid kinetics supported that Egr-1 induction occurred as a result of pharmacological activation of PPAR alpha and PPAR gamma, which are coexpressed at high levels in the rat urothelium. Finally, our demonstration of a nuclear localization supports that the Egr-1 induced by PPAR alpha and PPAR gamma coactivation in the rat urothelium may be biologically active.

Silencing alpha-fetoprotein inhibits VEGF and MMP-2/9 production in human hepatocellular carcinoma cell.

PubMed

Meng, Wenbo; Li, Xun; Bai, Zhongtian; Li, Yan; Yuan, Jinqiu; Liu, Tao; Yan, Jun; Zhou, Wence; Zhu, Kexiang; Zhang, Hui; Li, Yumin

2014-01-01

Alpha-fetoprotein not only serves as a diagnostic marker for liver cancer, but also posses a variety of biological functions. However, the role of Alpha-fetoprotein on tumor angiogenesis and cell invasion remains incompletely understood. In this study, we aimed to evaluate if Alpha-fetoprotein can regulate the major angiogenic factors and matrix metalloproteinases in human liver cancer cells. Alpha-fetoprotein silencing was achieved by Stealth RNAi. Expression of Alpha-fetoprotein was examined by a full-automatic electrochemistry luminescence immunity analyzer. Expression of VEGF, VEGFR-2, MMP-9, and MMP-2 was examined by Western blot and immunocytochemistry. Apoptosis was detected by TUNEL assay. Angiogenesis was detected by in vitro angiogenesis assay kit. Silencing of Alpha-fetoprotein led to an increased apoptosis, which was associated with a decreased expression of vascular endothelial growth factor, vascular endothelial growth factor receptor 2, matrix metalloproteinases-2/9. These results suggest that Alpha-fetoprotein may play a regulatory role on angiogenesis and cell invasion during liver cancer development.

Inhibition of integrin-linked kinase blocks podocyte epithelial-mesenchymal transition and ameliorates proteinuria.

PubMed

Kang, Young Sun; Li, Yingjian; Dai, Chunsun; Kiss, Lawrence P; Wu, Chuanyue; Liu, Youhua

2010-08-01

Proteinuria is a primary clinical symptom of a large number of glomerular diseases that progress to end-stage renal failure. Podocyte dysfunctions play a fundamental role in defective glomerular filtration in many common forms of proteinuric kidney disorders. Since binding of these cells to the basement membrane is mediated by integrins, we determined the role of integrin-linked kinase (ILK) in podocyte dysfunction and proteinuria. ILK expression was induced in mouse podocytes by various injurious stimuli known to cause proteinuria including TGF-beta1, adriamycin, puromycin, and high ambient glucose. Podocyte ILK was also found to be upregulated in human proteinuric glomerular diseases. Ectopic expression of ILK in podocytes decreased levels of the epithelial markers nephrin and ZO-1, induced mesenchymal markers such as desmin, fibronectin, matrix metalloproteinase-9 (MMP-9), and alpha-smooth muscle actin (alpha-SMA), promoted cell migration, and increased the paracellular albumin flux across podocyte monolayers. ILK also induced Snail, a key transcription factor mediating epithelial-mesenchymal transition (EMT). Blockade of ILK activity with a highly selective small molecule inhibitor reduced Snail induction and preserved podocyte phenotypes following TGF-beta1 or adriamycin stimulation. In vivo, this ILK inhibitor ameliorated albuminuria, repressed glomerular induction of MMP-9 and alpha-SMA, and preserved nephrin expression in murine adriamycin nephropathy. Our results show that upregulation of ILK is a convergent pathway leading to podocyte EMT, migration, and dysfunction. ILK may be an attractive target for therapeutic intervention of proteinuric kidney diseases.

Aliskiren ameliorates chlorhexidine digluconate-induced peritoneal fibrosis in rats.

PubMed

Ke, Chun-Yen; Lee, Chia-Chi; Lee, Chung-Jen; Subeq, Yi-Maun; Lee, Ru-Ping; Hsu, Bang-Gee

2010-04-01

Peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The present study was designed to investigate the protective effects of aliskiren on chlorhexidine digluconate-induced PF in rats. The PF was induced in Sprague-Dawley rats by daily administration of 0.5 mL 0.1% chlorhexidine digluconate in normal saline via PD tube for 1 week. Rats received daily intravenous injections of low-dose aliskiren (1 mg kg(-1)) or high-dose aliskiren (10 mg kg(-1)) for 1 week. After 7 days, conventional 4.25% Dianeal (30 mL) was administered via a PD catheter with a dwell time of 4 h and assessed of peritoneal function. At the end of dialysis, rats were sacrificed and the liver peritoneum was harvested for microscopically and immunohistochemistry. There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D(4)/P(4) urea level was reduced, the D(4)/D(0) glucose level, serum and dialysate transforming growth factor-beta1 (TGF-beta1) level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-beta1, alpha-smooth muscle actin (alpha-SMA), fibronectin, collagen, and vascular endothelial growth factor (VEGF) were elevated in the PS group compared with the vehicle group. Aliskiren decreased the serum and dialysate TGF-beta1 level, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-beta1, alpha-SMA, fibronectin, collagen, and VEGF-positive cells in liver peritoneum. Moreover, high-dose aliskiren had better protective effects against PF than low dose in rats. Aliskiren protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-beta1 production.

Role for transforming growth factor-beta1 in alport renal disease progression.

PubMed

Sayers, R; Kalluri, R; Rodgers, K D; Shield, C F; Meehan, D T; Cosgrove, D

1999-11-01

Alport syndrome results from mutations in either the alpha3(IV), alpha4(IV), or alpha5(IV) collagen genes. The disease is characterized by a progressive glomerulonephritis usually associated with a high-frequency sensorineural hearing loss. A mouse model for an autosomal form of Alport syndrome [collagen alpha3(IV) knockout] was produced and characterized. In this study, the model was exploited to demonstrate a potential role for transforming growth factor-beta1 (TGF-beta1) in Alport renal disease pathogenesis. Kidneys from normal and Alport mice, taken at different stages during the course of renal disease progression, were analyzed by Northern blot, in situ hybridization, and immunohistology for expression of TGF-beta1 and components of the extracellular matrix. Normal and Alport human kidney was examined for TGF-beta1 expression using RNase protection. The mRNAs encoding TGF-beta1 (in both mouse and human), entactin, fibronectin, and the collagen alpha1(IV) and alpha2(IV) chains were significantly induced in total kidney as a function of Alport renal disease progression. The induction of these specific mRNAs was observed in the glomerular podocytes of animals with advanced disease. Type IV collagen, laminin-1, and fibronectin were markedly elevated in the tubulointerstitium at 10 weeks, but not at 6 weeks, suggesting that elevated expression of specific mRNAs on Northern blots reflects events associated with tubulointerstitial fibrosis. The concomitant accumulation of mRNAs encoding TGF-beta1 and extracellular matrix components in the podocytes of diseased kidneys may reflect key events in Alport renal disease progression. These data suggest a role for TGF-beta1 in both glomerular and tubulointerstitial damage associated with Alport syndrome.

PGC-1alpha increases skeletal muscle lactate uptake by increasing the expression of MCT1 but not MCT2 or MCT4.

PubMed

Benton, Carley R; Yoshida, Yuko; Lally, James; Han, Xiao-Xia; Hatta, Hideo; Bonen, Arend

2008-09-17

We examined the relationship between PGC-1alpha protein; the monocarboxylate transporters MCT1, 2, and 4; and CD147 1) among six metabolically heterogeneous rat muscles, 2) in chronically stimulated red (RTA) and white tibialis (WTA) muscles (7 days), and 3) in RTA and WTA muscles transfected with PGC-1alpha-pcDNA plasmid in vivo. Among rat hindlimb muscles, there was a strong positive association between PGC-1alpha and MCT1 and CD147, and between MCT1 and CD147. A negative association was found between PGC-1alpha and MCT4, and CD147 and MCT4, while there was no relationship between PGC-1alpha or CD147 and MCT2. Transfecting PGC-1alpha-pcDNA plasmid into muscle increased PGC-1alpha protein (RTA +23%; WTA +25%) and induced the expression of MCT1 (RTA +16%; WTA +28%), but not MCT2 and MCT4. As a result of the PGC-1alpha-induced upregulation of MCT1 and its chaperone CD147 (+29%), there was a concomitant increase in the rate of lactate uptake (+20%). In chronically stimulated muscles, the following proteins were upregulated, PGC-1alpha in RTA (+26%) and WTA (+86%), MCT1 in RTA (+61%) and WTA (+180%), and CD147 in WTA (+106%). In contrast, MCT4 protein expression was not altered in either RTA or WTA muscles, while MCT2 protein expression was reduced in both RTA (-14%) and WTA (-10%). In these studies, whether comparing oxidative capacities among muscles or increasing their oxidative capacities by PGC-1alpha transfection and chronic muscle stimulation, there was a strong relationship between the expression of PGC-1alpha and MCT1, and PGC-1alpha and CD147 proteins. Thus, MCT1 and CD147 belong to the family of metabolic genes whose expression is regulated by PGC-1alpha in skeletal muscle.

Activin A stimulates IkappaB-alpha/NFkappaB and RANK expression for osteoclast differentiation, but not AKT survival pathway in osteoclast precursors.

PubMed

Sugatani, T; Alvarez, U M; Hruska, K A

2003-09-01

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor-kappaB (NFkappaB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IkappaB-alpha, and stimulates nuclear translocation of NFkappaB and receptor activator of nuclear factor-kappaB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IkappaB-alpha and induced nuclear translocation of phosphorylated-NFkappaB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL- and M-CSF-stimulated nuclear translocation of NFkappaB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M-CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFkappaB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis. Copyright 2003 Wiley-Liss, Inc.

Alpha-Tocopheryl phosphate induces VEGF expression via CD36/PI3Kgamma in THP-1 monocytes

USDA-ARS?s Scientific Manuscript database

The CD36 scavenger receptor binds several ligands and mediates ligand uptake and ligand-dependent signal transduction and gene expression, events that may involve CD36 internalization. Here we show that CD36 internalization in THP-1 monocytes is triggered by alpha-tocopherol (alpha-T) and more stron...

TNF{alpha} and IL-1{beta} are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Wenwen; Zheng, Xuexing; Department of Anesthesiology, University of Miami Miller School of Medicine, Miami, FL 33136

2012-04-20

Highlights: Black-Right-Pointing-Pointer LPS induces proinflammatory cytokine release in HAPI cells. Black-Right-Pointing-Pointer JNK pathway is dependent on TLR4 signaling to release cytokines. Black-Right-Pointing-Pointer NF-{kappa}B pathway is dependent on Nod1 signaling to release cytokines. -- Abstract: A growing body of evidence recently suggests that glial cell activation plays an important role in several neurodegenerative diseases and neuropathic pain. Microglia in the central nervous system express toll-like receptor 4 (TLR4) that is traditionally accepted as the primary receptor of lipopolysaccharide (LPS). LPS activates TLR4 signaling pathways to induce the production of proinflammatory molecules. In the present studies, we verified the LPS signaling pathwaysmore » using cultured highly aggressively proliferating immortalized (HAPI) microglial cells. We found that HAPI cells treated with LPS upregulated the expression of TLR4, phospho-JNK (pJNK) and phospho-NF-{kappa}B (pNF-{kappa}B), TNF{alpha} and IL-1{beta}. Silencing TLR4 with siRNA reduced the expression of pJNK, TNF{alpha} and IL-1{beta}, but not pNF-{kappa}B in the cells. Inhibition of JNK with SP600125 (a JNK inhibitor) decreased the expression of TNF{alpha} and IL-1{beta}. Unexpectedly, we found that inhibition of Nod1 with ML130 significantly reduced the expression of pNF-{kappa}B. Inhibition of NF-{kappa}B also reduced the expression of TNF{alpha} and IL-1{beta}. Nod1 ligand, DAP induced the upregulation of pNF-{kappa}B which was blocked by Nod1 inhibitor. These data indicate that LPS-induced pJNK is TLR4-dependent, and that pNF-{kappa}B is Nod1-dependent in HAPI cells treated with LPS. Either TLR4-JNK or Nod1-NF-{kappa}B pathways is involved in the expression of TNF{alpha} and IL-1{beta}.« less

The peptide NDP-MSH induces phenotype changes in the heart that resemble ischemic preconditioning.

PubMed

Catania, Anna; Lonati, Caterina; Sordi, Andrea; Leonardi, Patrizia; Carlin, Andrea; Gatti, Stefano

2010-01-01

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a pro-opiomelanocortin (POMC)-derived peptide that exerts multiple protective effects on host cells. Previous investigations showed that treatment with alpha-MSH or synthetic melanocortin agonists reduces heart damage in reperfusion injury and transplantation. The aim of this preclinical research was to determine whether melanocortin treatment induces preconditioning-like cardioprotection. In particular, the plan was to assess whether melanocortin administration causes phenotype changes similar to those induced by repetitive ischemic events. The idea was conceived because both ischemic preconditioning and melanocortin signaling largely depend on cAMP response element binding protein (CREB) phosphorylation. Rats received single i.v. injections of 750microg/kg of the alpha-MSH analogue Nle(4),DPhe(7)-alpha-MSH (NDP-MSH) or saline and were sacrificed at 0.5, 1, 3, or 5h. Western blot analysis showed that rat hearts expressed melanocortin 1 receptor (MC1R) protein. Treatment with NDP-MSH was associated with early and marked increase in interleukin 6 (IL-6) mRNA. This was followed by signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling 3 (SOCS3). There were no changes in expression of other cytokines of the IL-6 family. Expression of IL-10, IL-1beta, and TNF-alpha was likewise unaltered. In hearts of rats treated with NDP-MSH there was increased expression of the orphan nuclear receptor Nur77. The data indicate that NDP-MSH induces phenotype changes that closely resemble ischemic preconditioning and likely contribute to its established protection against reperfusion injury. In addition, the increased expression of Nur77 and SOCS3 could be part of a broader anti-inflammatory effect.

Synthetic serine elastase inhibitor reduces cigarette smoke-induced emphysema in guinea pigs.

PubMed

Wright, Joanne L; Farmer, Stephen G; Churg, Andrew

2002-10-01

To test whether a serine elastase inhibitor could prevent or reduce emphysema, we exposed guinea pigs to cigarette smoke acutely, or daily for 6 months, and treated some animals with the neutrophil elastase inhibitor ZD0892. Acute smoke exposure increased lavage neutrophils and increased desmosine and hydroxyproline, measures of elastin and collagen breakdown; all these measures were reduced by ZD0892. Long-term smoke exposure produced emphysema and increases in lavage neutrophils, desmosine, hydroxyproline, and plasma tumor necrosis factor alpha (TNF-alpha). ZD0892 treatment returned lavage neutrophils, desmosine, and hydroxyproline levels to control values, and decreased airspace enlargement by 45% and TNF-alpha by 30%. Animals exposed to smoke for 4 months and then to smoke plus ZD0892 for 2 months were not protected against emphysema. Mice exposed to smoke showed increases in gene expression of neutrophil chemoattractant macrophage inflammatory protein-2, macrophage chemoattractant protein-1, and TNF-alpha at 2 hours along with increased plasma TNF-alpha; ZD0892 prevented the increases in macrophage inflammatory protein-2 and macrophage chemoattractant protein-1 expression and reduced plasma TNF-alpha levels to baseline. These data demonstrate that a serine elastase inhibitor ameliorates the inflammatory and destructive effects of cigarette smoke, and that these effects are mediated in part by neutrophils and by smoke-driven TNF-alpha production.

Decursin inhibits induction of inflammatory mediators by blocking nuclear factor-kappaB activation in macrophages.

PubMed

Kim, Jung-Hee; Jeong, Ji-Hye; Jeon, Sung-Tak; Kim, Ho; Ock, Jiyeon; Suk, Kyoungho; Kim, Sang-In; Song, Kyung-Sik; Lee, Won-Ha

2006-06-01

In the course of screening inhibitors of matrix metalloproteinase (MMP)-9 induction in macrophages, we isolated decursin, a coumarin compound, from the roots of Angelicae gigas. As a marker for the screening and isolation, we tested expression of MMP-9 in RAW264.7 cells and THP-1 cells after treatment with bacterial lipopolysaccharide (LPS), the TLR-4 ligand. Decursin suppressed MMP-9 expression in cells stimulated by LPS in a dose-dependent manner at concentrations below 60 microM with no sign of cytotoxicity. The suppressive effect of decursin was observed not only in cells stimulated with ligands for TLR4, TLR2, TLR3, and TLR9 but also in cells stimulated with interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha, indicating that the molecular target of decursin is common signaling molecules induced by these stimulants. In addition to the suppression of MMP-9 expression, decursin blocked nitric oxide production and cytokine (IL-8, MCP-1, IL-1beta, and TNF-alpha) secretion induced by LPS. To find out the molecular mechanism responsible for the suppressive effect of decursin, we analyzed signaling molecules involved in the TLR-mediated activation of MMP-9 and cytokines. Decursin blocked phosphorylation of IkappaB and nuclear translocation of NF-kappaB in THP-1 cells activated with LPS. Furthermore, expression of a luciferase reporter gene under the promoter containing NF-kappaB binding sites was blocked by decursin. These data indicate that decursin is a novel inhibitor of NF-kappaB activation in signaling induced by TLR ligands and cytokines.

By Different Cellular Mechanisms, Lymphatic Vessels Sprout by Endothelial Cell Recruitment Whereas Blood Vessels Grow by Vascular Expansion

NASA Technical Reports Server (NTRS)

Parsons-Wingerter, Patricia; McKay, Terri L.; Leontiev, Dmitry; Condrich, Terence K.; DiCorleto, Paul E.

2005-01-01

The development of effective vascular therapies requires the understanding of all modes of vessel formation contributing to vasculogenesis, angiogenesis (here termed hemangiogenesis) and lymphangiogenesis. We show that lymphangiogenesis proceeds by blind-ended vessel sprouting via recruitment of isolated endothelial progenitor cells to the tips of growing vessels, whereas hemangiogenesis occurs by non-sprouting vessel expansion from the capillary network, during middevelopment in the quail chorioallantoic membrane (CAM). Blood vessels expanded out of capillaries that displayed transient expression of alpha smooth muscle actin (alphaSMA), accompanied by mural recruitment of migratory progenitor cells expressing SMA. Lymphatics and blood vessels were identified by confocal/fluorescence microscopy of vascular endothelial growth factor (VEGF) receptors VEGFR-1 and VEGFR-2, alphaSMA (expressed on CAM blood vessels but not on lymphatics), homeobox transcription factor Prox-1 (specific to CAM lymphatic endothelium), and the quail hematopoetic/vascular marker, QH-1. Expression of VEGFR-1 was highly restricted to blood vessels (primarily capillaries). VEGFR-2 was expressed intensely in isolated hematopoietic cells, lymphatic vessels and moderately in blood vessels. Prox-1 was absent from endothelial progenitor cells prior to lymphatic recruitment. Although vascular endothelial growth factor-165 (VEGF(sub 165)) is a key regulator of numerous cellular processes in hemangiogenesis and vasculogenesis, the role of VEGF(sub 165) in lymphangiogenesis is less clear. Exogenous VEGF(sub 165) increased blood vessel density without changing endogenous modes of vascular/lymphatic vessel formation or marker expression patterns. However, VEGF(sub 165) did increase the frequency of blood vascular anastomoses and strongly induced the antimaturational dissociation of lymphatics from blood vessels, with frequent formation of homogeneous lymphatic networks.

Leptin interferes with the effects of the antiestrogen ICI 182,780 in MCF-7 breast cancer cells.

PubMed

Garofalo, Cecilia; Sisci, Diego; Surmacz, Eva

2004-10-01

Obesity is a risk factor for breast cancer development in postmenopausal women and correlates with shorter disease-free and overall survival in breast cancer patients, regardless of menopausal status. Adipose tissue is a major source of leptin, a cytokine regulating energy balance and controlling different processes in peripheral tissues, including breast cancer cell growth. Here, we investigated whether leptin can counteract antitumorigenic activities of the antiestrogen ICI 182,780 in breast cancer cells. Mitogenic response to leptin and the effects of leptin on ICI 182,780-dependent growth inhibition were studied in MCF-7 estrogen receptor alpha-positive breast cancer cells. The expression of leptin receptor and the activation of signaling pathways were studied by Western immunoblotting. The interference of leptin with ICI 182,780-induced estrogen receptor alpha degradation was probed by Western immunoblotting, fluorescence microscopy, and pulse-chase experiments. Leptin effects on estrogen receptor alpha-dependent transcription in the presence and absence of ICI 182,780 were studied by luciferase reporter assays and chromatin immunoprecipitation. MCF-7 cells were found to express the leptin receptor and respond to leptin with cell growth and activation the signal transducers and activators of transcription 3, extracellular signal-regulated kinase-1/2, and Akt/GSK3/pRb pathways. The exposure of cells to 10 nmol/L ICI 182,780 blocked cell proliferation, induced rapid estrogen receptor alpha degradation, inhibited nuclear estrogen receptor alpha expression, and reduced estrogen receptor alpha-dependent transcription from estrogen response element-containing promoters. All of these effects of ICI 182,780 were significantly attenuated by simultaneous treatment of cells with 100 ng/mL leptin. Leptin interferes with the effects of ICI 182,780 on estrogen receptor alpha in breast cancer cells. Thus, high leptin levels in obese breast cancer patients might contribute to the development of antiestrogen resistance.

Cykotine mRNA expression in mouse retina after laser injury by reverse transcriptase-polymerase chain reaction (RT-PCR)

NASA Astrophysics Data System (ADS)

Schuschereba, Steven T.; Bowman, Phillip D.; Ujimore, Veronica; Hoxie, Stephen W.; Pizarro, Jose M.; Cross, Michael E.; Lund, David J.

1996-04-01

The purpose of this study was to identify cytokines produced by the retina after laser injury. With the aid of a scanning laser ophthalmoscope (SLO), right eyes of mice received lesions from a continuous wave argon laser. Left eyes served as unirradiated controls. At 2, 4, 6, 12, 24, and 48 hr after laser irradiation groups of 3 mice were euthanized and retinas fixed for histology or isolated for RNA. Messenger RNA (mRNA) was reverse-transcribed into complementary DNA (cDNA) and subjected to polymerase chain reaction for the following cytokines: tumor necrosis factor-(alpha) (TNF-(alpha) ), interleukin-1(alpha) /(Beta) (IL- 1(alpha) /(Beta) ), interleukin-6 (IL-6), transforming growth factor-(Beta) 1 (TGF- (Beta) 1), macrophage colony stimulating factor (M-CSF), inducible nitric oxide synthase (iNOS), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH). Histologically, lesions were confined to the photoreceptors, retinal pigment epithelium, and choroid. In laser-injured retinas, mRNA levels were elevated for IL-1(alpha) , TGF-(Beta) 1, iNOS, and G3PDH, but not TNF-(alpha) , IL-1(Beta) , or IL-6. It appears that the retina, in response to laser injury, upregulates a select number of cytokines in a time-course dependent fashion.

Notch1 stimulation induces a vascularization switch with pericyte-like cell differentiation of glioblastoma stem cells.

PubMed

Guichet, Pierre-Olivier; Guelfi, Sophie; Teigell, Marisa; Hoppe, Liesa; Bakalara, Norbert; Bauchet, Luc; Duffau, Hugues; Lamszus, Katrin; Rothhut, Bernard; Hugnot, Jean-Philippe

2015-01-01

Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study, we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, and HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signaling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration, which was accompanied by the sharp reduction in neural stem cell transcription factor expression (ASCL1, OLIG2, and SOX2), while HEY1/2, KLF9, and SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRβ, and α-smooth muscle actin [αSMA]) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (heparin binding epidermal growth factor [HB-EGF], IL8, and PLGF), matrix metalloproteinases (MMP9), and adhesion proteins (vascular cell adhesion molecule 1 [VCAM1], intercellular adhesion molecule 1 [ICAM1], and integrin alpha 9 [ITGA9]). In xenotransplantation experiments, contrasting with the infiltrative and poorly vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly disseminating but highly vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties. © 2014 AlphaMed Press.

Reduction of PTP1B induces differential expression of PI3-kinase (p85alpha) isoforms.

PubMed

Rondinone, Cristina M; Clampit, Jill; Gum, Rebecca J; Zinker, Bradley A; Jirousek, Michael R; Trevillyan, James M

2004-10-15

Protein tyrosine phosphatase 1B (PTP1B) inhibition increases insulin sensitivity and normalizes blood glucose levels in animals. The molecular events associated with PTP1B inhibition that increase insulin sensitivity remain controversial. Insulin resistant, diabetic ob/ob mice, dosed with PTP1B antisense for 3 weeks exhibited a decrease in PTP1B protein levels and a change in the expression level of p85alpha isoforms in liver, characterized by a reduction in p85alpha and an upregulation of the p50alpha and p55alpha isoforms. Transfection of mouse hepatocytes with PTP1B antisense caused a downregulation PTP1B and p85alpha protein levels. Furthermore, transfection of mouse hepatocytes with PTP1B siRNA downregulated p85alpha protein expression and enhanced insulin-induced PKB phosphorylation. Treatment of mouse hepatocytes with p85alpha antisense oligonucleotide caused a reduction of p85alpha and an increase in p50alpha and p55alpha isoforms and enhanced insulin-stimulated PKB activation. These results demonstrate that PTP1B inhibition causes a direct differential regulation of p85alpha isoforms of PI3-kinase in liver and that reduction of p85alpha may be one mechanism by which PTP1B inhibition improves insulin sensitivity and glucose metabolism in insulin-resistant states. Copyright 2004 Elsevier Inc.

Natural killer cells promote tissue injury and systemic inflammatory responses during fatal Ehrlichia-induced toxic shock-like syndrome.

PubMed

Stevenson, Heather L; Estes, Mark D; Thirumalapura, Nagaraja R; Walker, David H; Ismail, Nahed

2010-08-01

Human monocytotropic ehrlichiosis is caused by Ehrlichia chaffeensis, a Gram-negative bacterium lacking lipopolysaccharide. We have shown that fatal murine ehrlichiosis is associated with CD8(+)T cell-mediated tissue damage, tumor necrosis factor-alpha, and interleukin (IL)-10 overproduction, and CD4(+)Th1 hyporesponsiveness. In this study, we examined the relative contributions of natural killer (NK) and NKT cells in Ehrlichia-induced toxic shock. Lethal ehrlichial infection in wild-type mice induced a decline in NKT cell numbers, and late expansion and migration of activated NK cells to the liver, a main infection site that coincided with development of hepatic injury. The spatial and temporal changes in NK and NKT cells in lethally infected mice correlated with higher NK cell cytotoxic activity, higher expression of cytotoxic molecules such as granzyme B, higher production of interferon-gamma and tumor necrosis factor-alpha, increased hepatic infiltration with CD8alphaCD11c(+) dendritic cells and CD8(+)T cells, decreased splenic CD4(+)T cells, increased serum concentrations of IL-12p40, IL-18, RANTES, and monocyte chemotactic protein-1, and elevated production of IL-18 by liver mononuclear cells compared with nonlethally infected mice. Depletion of NK cells prevented development of severe liver injury, decreased serum levels of interferon-gamma, tumor necrosis factor-alpha, and IL-10, and enhanced bacterial elimination. These data indicate that NK cells promote immunopathology and defective anti-ehrlichial immunity, possibly via decreasing the protective immune response mediated by interferon-gamma producing CD4(+)Th1 and NKT cells.

Apigenin protects against alcohol-induced liver injury in mice by regulating hepatic CYP2E1-mediated oxidative stress and PPARα-mediated lipogenic gene expression.

PubMed

Wang, Feng; Liu, Jin-Cheng; Zhou, Rui-Jun; Zhao, Xi; Liu, Mei; Ye, Hua; Xie, Mei-Lin

2017-09-25

Alcohol is a major cause of liver injury, and there are currently no ideal pharmacological reagents that can prevent or reverse this disease. Apigenin is one of the most common flavonoids present in numerous plants and has many beneficial effects. But whether or not apigenin may protect against alcohol-induced liver injury remains unknown. Our aim was to examine the effect and potential mechanisms. The experimental mice were given 56% erguotou wine or simultaneously given apigenin 150-300 mg/kg by gavage for 30 days. The results showed that in the apigenin-treated mice, the expression of hepatic cytochrome P450 2E1 (CYP2E1) and nuclear factor kappa B proteins as well as contents of hepatic malondialdehyde and tumor necrosis factor-alpha were reduced, while the levels of hepatic reduced glutathione, glutathione reductase, glutathione peroxidase, and glutathione S-transferase were increased, especially in the 300 mg/kg group. A significant change in hepatic steatosis was also observed in the apigenin 300 mg/kg group. Apigenin pretreatment could increase the expression of hepatic peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase-1 proteins, and decrease the expression of hepatic sterol regulatory element binding protein-1c, fatty acid synthase, and diacylglycerol acyltransferase proteins. These findings demonstrated that apigenin might exert a protective effect on alcohol-induced liver injury, and its mechanisms might be related to the regulations of hepatic CYP2E1-mediated oxidative stress and PPARα-mediated lipogenic gene expression. Copyright © 2017 Elsevier B.V. All rights reserved.

Glycyrrhetinic acid suppressed NF-κB activation in TNF-α-induced hepatocytes.

PubMed

Chen, Hong-Jhang; Kang, Shih-Pei; Lee, I-Jung; Lin, Yun-Lian

2014-01-22

Tumor necrosis factor-alpha (TNF-α) is a crucial inflammatory cytokine when hepatocytes are damaged. Glycyrrhiza uralensis Fisch. (Chinese licorice) has been widely used in Chinese herbal prescriptions for the treatment of liver diseases and as a food additive. Nuclear factor-kappa B (NF-κB) reporter gene assay in TNF-α-induced HepG2 was used as a screening platform. IκBα phosphorylation and p65 translocation were measured by Western blotting, and nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression were further confirmed in rat primary hepatocytes. Results showed that TNF-α enhanced NF-κB activity was significantly attenuated by glycyrrhetinic acid in a concentration-dependent manner in the NF-κB reporter gene assay. Glycyrrhetinic acid decreased the gene expression of iNOS through inhibited IκBα phosphorylation and p65 translocation in protein level. Furthermore, NO production and iNOS expression were reduced by glycyrrhetinic acid in TNF-α-induced rat primary hepatocytes. These results suggest that glycyrrhetinic acid may provide hepatoprotection against chronic liver inflammation through attenuating NF-κB activation to alleviate the inflammation.

Pomegranate protects liver against cecal ligation and puncture-induced oxidative stress and inflammation in rats through TLR4/NF-κB pathway inhibition.

PubMed

Makled, Mirhan N; El-Awady, Mohammed S; Abdelaziz, Rania R; Atwan, Nadia; Guns, Emma T; Gameil, Nariman M; Shehab El-Din, Ahmed B; Ammar, Elsayed M

2016-04-01

Acute liver injury secondary to sepsis is a major challenge in intensive care unit. This study was designed to investigate potential protective effects of pomegranate against sepsis-induced acute liver injury in rats and possible underlying mechanisms. Pomegranate was orally given (800mg/kg/day) for two weeks before sepsis induction by cecal ligation and puncture (CLP). Pomegranate improved survival and attenuated liver inflammatory response, likely related to downregulation of mRNA expression of toll like recptor-4, reduced nuclear translocation and DNA binding activity of proinflammatory transcription factor NF-κB subunit p65, decreased mRNA and protein expression of tumor necrosis factor-alpha and reduction in myeloperoxidase activity and mRNA expression. Pomegranate also decreased CLP-induced oxidative stress as reflected by decreased malondialdehyde content, and increased reduced glutathione level and superoxide dismutase activity. These results confirm the antiinflammatory and antioxidant effects of pomegranate in CLP-induced acute liver injury mediated through inhibiting TLR4/NF-κB pathway, lipid peroxidation and neutrophil infiltration. Copyright © 2016 Elsevier B.V. All rights reserved.

TNF-{alpha} similarly induces IL-6 and MCP-1 in fibroblasts from colorectal liver metastases and normal liver fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, Lars, E-mail: lars.mueller@uksh-kiel.de; Seggern, Lena von; Schumacher, Jennifer

2010-07-02

Cancer-associated fibroblasts (CAFs) represent the predominant cell type of the neoplastic stroma of solid tumors, yet their biology and functional specificity for cancer pathogenesis remain unclear. We show here that primary CAFs from colorectal liver metastases express several inflammatory, tumor-enhancing factors, including interleukin (IL)-6 and monocyte-chemoattractant protein (MCP)-1. Both molecules were intensely induced by TNF-{alpha} on the transcript and protein level, whereas PDGF-BB, TGF-{beta}1 and EGF showed no significant effects. To verify their potential specialization for metastasis progression, CAFs were compared to fibroblasts from non-tumor liver tissue. Interestingly, these liver fibroblasts (LFs) displayed similar functions. Further analyses revealed a comparablemore » up-regulation of intercellular adhesion molecule-1 (ICAM-1) by TNF-{alpha}, and of alpha-smooth muscle actin, by TGF-{beta}1. Moreover, the proliferation of both cell types was induced by PDGF-BB, and CAFs and LFs displayed an equivalent migration towards HT29 colon cancer cells in Boyden chamber assays. In conclusion, colorectal liver metastasis may be supported by CAFs and resident fibroblastic cells competent to generate a prometastatic microenvironment through inflammatory activation of IL-6 and MCP-1.« less

Immunosuppressive effects of factor IX products: an in vitro study.

PubMed

Grosset, A B; McGregor, J R; Samlowski, W E; Rodgers, G M

1999-11-01

The effects of a recombinant factor IX product (BeneFix), and of five plasma-derived factor IX products, AlphaNine, Immunine, Konyne, Mononine and Replinine on in vitro peripheral blood mononuclear cell (PBMC) immune function were compared in a blinded study. We assessed the effects of these products on Con-A-induced lymphocyte proliferation and interleukin-2 and interleukin-10 secretion, expression of lymphocyte activation markers, and nitric oxide secretion by stimulated mouse peritoneal macrophages. At 1 mL-1 for 48 h, Konyne reduced Con-A-induced mitogenesis by 50% (P < 0.05); AlphaNine, Mononine and BeneFix had no effect. At 10 IU mL-1, Con-A-induced mi- togenesis was at control levels with Mononine and BeneFix, but was reduced to <15% (P < 0.05) with each of the other products. IL-2 and IL-10 secretion by Con-A-stimulated lymphocytes was also markedly depressed by all the products tested except Mononine and BeneFix. Dialysis of these products did not substantially affect these results. Flow cytometric analysis of lymphocyte activation markers following Con-A stimulation showed that Konyne also decreased IL-2 receptor alpha and beta chain (CD25 and CD122) induction on PBMC. Konyne also inhibited nitric oxide secretion to levels <18% of controls. These results indicate that certain factor IX products, including some of purported higher purity, substantially depress in vitro immune function. The importance of these findings to in vivo immune function in haemophilia B patients remains to be established.

The IL-6/sIL-6R treatment of a malignant melanoma cell line enhances susceptibility to TNF-{alpha}-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagley, Yadav; Yoo, Yung-Choon; Seo, Han Geuk

2007-03-23

Melanoma is an intractable tumor that has shown very impressive and promising response to local administration of high dose recombinant TNF-{alpha} in combination with IFN-{gamma} in clinical studies. In this study, we investigated the effect of IL-6/sIL-6R on TNF-{alpha}-resistant B16/F10.9 melanoma cells. A low dose of TNF-{alpha} or IL-6/sIL-6R had minimal affect on the cell growth. However, the highly active fusion protein of sIL-6R and IL-6 (IL6RIL6), covalently linked by a flexible peptide, sensitized TNF-{alpha}-resistant F10.9 melanoma cells to TNF-{alpha}-induced apoptosis. Stimulation of the cells with IL6RIL6 plus TNF-{alpha} resulted in both the activation of caspase-3 and the reduction ofmore » bcl-2 expression. Flow cytometry analysis showed that IL6RIL6-upregulated TNF-R55 and TNF-R75 expression, suggesting an increase in TNF-{alpha} responsiveness by IL6RIL6 resulting from the induction of TNF receptors. Moreover, exposure of F10.9 cells to neutralizing antibody to TNF-R55 significantly inhibited IL6RIL6/TNF-{alpha}-induced cytotoxicity. These results suggest that the IL6/sIL6R/gp130 system, which sensitizes TNF-{alpha}-resistant melanoma cells to TNF-{alpha}-induced apoptosis, may provide a new target for immunotherapy.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Anna D.; Malur, Anagha; Barna, Barbara P.

Peroxisome proliferator-activated receptor-gamma (PPAR{gamma}) is a nuclear transcription factor involved in lipid metabolism that is constitutively expressed in the alveolar macrophages of healthy individuals. PPAR{gamma} has recently been implicated in the catabolism of surfactant by alveolar macrophages, specifically the cholesterol component of surfactant while the mechanism remains unclear. Studies from other tissue macrophages have shown that PPAR{gamma} regulates cholesterol influx, efflux, and metabolism. PPAR{gamma} promotes cholesterol efflux through the liver X receptor-alpha (LXR{alpha}) and ATP-binding cassette G1 (ABCG1). We have recently shown that macrophage-specific PPAR{gamma} knockout (PPAR{gamma} KO) mice accumulate cholesterol-laden alveolar macrophages that exhibit decreased expression of LXR{alpha} andmore » ABCG1 and reduced cholesterol efflux. We hypothesized that in addition to the dysregulation of these cholesterol efflux genes, the expression of genes involved in cholesterol synthesis and influx was also dysregulated and that replacement of PPAR{gamma} would restore regulation of these genes. To investigate this hypothesis, we have utilized a Lentivirus expression system (Lenti-PPAR{gamma}) to restore PPAR{gamma} expression in the alveolar macrophages of PPAR{gamma} KO mice. Our results show that the alveolar macrophages of PPAR{gamma} KO mice have decreased expression of key cholesterol synthesis genes and increased expression of cholesterol receptors CD36 and scavenger receptor A-I (SRA-I). The replacement of PPAR{gamma} (1) induced transcription of LXR{alpha} and ABCG1; (2) corrected suppressed expression of cholesterol synthesis genes; and (3) enhanced the expression of scavenger receptors CD36. These results suggest that PPAR{gamma} regulates cholesterol metabolism in alveolar macrophages.« less

Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice.

PubMed

Most, D; Efron, D T; Shi, H P; Tantry, U S; Barbul, A

2001-10-01

Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test. Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05). Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These data further delineate the modulatory effect of iNOS and nitric oxide in healing skin grafts.

Modulation of Cytokine-Induced Cyclooxygenase 2 Expression by PPARG Ligands Through NFκB Signal Disruption in Human WISH and Amnion Cells1

PubMed Central

Ackerman, William E.; Zhang, Xiaolan L.; Rovin, Brad H.; Kniss, Douglas A.

2006-01-01

Cyclooxygenase (COX) activity increases in the human amnion in the settings of term and idiopathic preterm labor, contributing to the generation of uterotonic prostaglandins (PGs) known to participate in mammalian parturition. Augmented COX activity is highly correlated with increased COX2 (also known as prostaglandin-endoperoxide synthase 2, PTGS2) gene expression. We and others have demonstrated an essential role for nuclear factor κB (NFκB) in cytokine-driven COX2 expression. Peroxisome proliferator-activated receptor gamma (PPARG), a member of the nuclear hormone receptor superfamily, has been shown to antagonize NFκB activation and inflammatory gene expression, including COX2. We hypothesized that PPARG activation might suppress COX2 expression during pregnancy. Using primary amnion and WISH cells, we evaluated the effects of pharmacological (thiazolidinediones) and putative endogenous (15-deoxy-Δ12,14-prostaglandin J2, 15d-PGJ2) PPARG ligands on cytokine-induced NFκB activation, COX2 expression, and PGE2 production. We observed that COX2 expression and PGE2 production induced by tumor necrosis factor alpha (TNF) were significantly abrogated by 15d-PGJ2. The thiazolidinediones rosiglitazone (ROSI) and troglitazone (TRO) had relatively little effect on cytokine-induced COX2 expression except at high concentrations, at which these agents tended to increase COX2 abundance relative to cells treated with TNF alone. Interestingly, treatment with ROSI, but not TRO, led to augmentation of TNF-stimulated PGE2 production. Mechanistically, we observed that 15d-PGJ2 markedly diminished cytokine-induced activity of the NFκB transcription factor, whereas thiazolidinediones had no discernable effect on this system. Our data suggest that pharmacological and endogenous PPARG ligands use both receptor-dependent and -independent mechanisms to influence COX2 expression. PMID:15843495

Stimulation of EphB2/ephrin-B1 signalling by tumour necrosis factor alpha in human dental pulp stem cells.

PubMed

Zhu, Lifang; Dissanayaka, Waruna Lakmal; Green, David William; Zhang, Chengfei

2015-04-01

The aim of this study was to investigate whether in vitro stimulation of dental pulp stem cells (DPSCs) by tumour necrosis factor alpha (TNF-α) would induce secretion of EphB2/ephrin-B1 signalling. Dental pulp stem cells isolated from human dental pulp were treated with TNF-α (5-100 ng/ml) over 2-48 h. EphB2/ephrin-B1 mRNA and protein levels were measured by real-time polymerase chain reaction (RT-PCR) and western blot analysis respectively. Additionally, DPSCs were pre-incubated with TNF-α receptor neutralizing antibodies or infected with nuclear factor-kappa B (NF-ĸB) inhibitor, p38 MAPK inhibitor, Jun N-terminal kinase (JNK) inhibitor and MEK inhibitor before TNF-α treatment. Results were analysed by one-way ANOVA. Tumour necrosis factor alpha increased EphB2 mRNA expression in DPSCs at concentrations up to 20 ng/ml and ephrin-B1 at concentrations up to 40 ng/ml (P < 0.05). Its mRNA expression reached maximum at 24 h when treated with TNF-α at 20 ng/ml (P < 0.05). EphB2/ephrin-B1 protein expression levels were high at 16 and 24 h as shown by western blotting. Neutralizing antibodies for TNFR1/2 receptors down-regulated EphB2/ephrin-B1 mRNA expression (P < 0.05) and ephrin-B1 protein expression, but not EphB2 protein expression. JNK-inhibitor inhibited EphB2 mRNA expression only (P < 0.05). EphB2/ephrin-B1 were invoked in DPSCs with TNF-α treatment via the JNK-dependent pathway, but not NF-ĸB, p38 MAPK or MEK signalling. © 2015 John Wiley & Sons Ltd.

TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF)

EPA Science Inventory

TITLE:
TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF). AUTHORS (ALL): Abbott, Barbara D.1; Best, Deborah S.1; Narotsky, Michael G.1. SPONSOR NAME: None INSTITUTIONS (ALL): 1. Repro Tox ...

Alpha-lipoic acid blocks HIV-1 LTR-dependent expression of hygromycin resistance in THP-1 stable transformants.

PubMed

Merin, J P; Matsuyama, M; Kira, T; Baba, M; Okamoto, T

1996-09-23

Gene expression of human immunodeficiency virus (HIV) depends on a host cellular transcription factors including nuclear factor-kappaB (NF-kappaB). The involvement of reactive oxygen intermediates (ROI) has been implicated as intracellular messengers in the inducible activation of NF-kappaB. In this study, we compared the efficacy of two antioxidants, alpha-lipoic acid (LA) and N-acetylcysteine (NAC), which are widely recognized NF-kappaB inhibitors. Here, we demonstrate that LA has a more potent activity in inhibiting NF-KappaB-mediated gene expression in THP-1 cells that have been stably transfected with a plasmid bearing a hygromycin B resistance gene under the control of HIV-1 long terminal repeat (LTR) promoter. The spontaneous activation of NF-kappaB in this cell culture system leads to expression of the hygromycin phosphotransferase gene hence rendering the cells resistance to hygromycin B. In this study, the effect of the test compounds against transcriptional activity of HIV-1 LTR was evaluated based on the degree of cellular toxicity due to the inhibitory activity on the expression of hygromycin B resistance gene in the presence of hygromycin B. We also found that 0.2 mM LA could cause 40% reduction in the HIV-1 expression from the TNF-alpha-stimulated OM 10.1, a cell line latently infected with HIV-1. On the other hand, 10 mM NAC was required to elicit the same effect. Furthermore, the initiation of HIV-1 induction by TNF-alpha was completely abolished by 1 mM LA. These findings confirm the involvement of ROI in NF-kappaB-mediated HIV gene expression as well as the efficacy of LA as a therapeutic regimen for HIV infection and acquired immunodeficiency syndrome (AIDS). Moreover, this study validates the applicability of our present assay system which we primarily designed for the screening of candidate drugs against HIV-1 gene expression.

Eupatolide inhibits lipopolysaccharide-induced COX-2 and iNOS expression in RAW264.7 cells by inducing proteasomal degradation of TRAF6.

PubMed

Lee, Jongkyu; Tae, Nara; Lee, Jung Joon; Kim, Taeho; Lee, Jeong-Hyung

2010-06-25

Inula britannica is a traditional medicinal plant used to treat bronchitis, digestive disorders, and inflammation in Eastern Asia. Here, we identified eupatolide, a sesquiterpene lactone from I. britannica, as an inhibitor of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression. Eupatolide inhibited the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) as well as iNOS and COX-2 protein expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Eupatolide dose-dependently decreased the mRNA levels and the promoter activities of COX-2 and iNOS in LPS-stimulated RAW264.7 cells. Moreover, eupatolide significantly suppressed the LPS-induced expression of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) reporter genes. Pretreatment of eupatolide inhibited LPS-induced phosphorylation and degradation of I kappaB alpha, and phosphorylation of RelA/p65 on Ser-536 as well as the activation of mitogen-activated protein kinases (MAPKs) and Akt in LPS-stimulated RAW264.7 cells. Eupatolide induced proteasomal degradation of tumor necrosis factor receptor-associated factor-6 (TRAF6), and subsequently inhibited LPS-induced TRAF6 polyubiquitination. These results suggest that eupatolide blocks LPS-induced COX-2 and iNOS expression at the transcriptional level through inhibiting the signaling pathways such as NF-kappaB and MAPKs via proteasomal degradation of TRAF6. Taken together, eupatolide may be a novel anti-inflammatory agent that induces proteasomal degradation of TRAF6, and a valuable compound for modulating inflammatory conditions. (c) 2010 Elsevier B.V. All rights reserved.

The MC160 Protein Expressed by the Dermatotropic Poxvirus Molluscum Contagiosum Virus Prevents Tumor Necrosis Factor Alpha-Induced NF-κB Activation via Inhibition of I Kappa Kinase Complex Formation

PubMed Central

Nichols, Daniel Brian; Shisler, Joanna L.

2006-01-01

The pluripotent cytokine tumor necrosis factor alpha (TNF-α) binds to its cognate TNF receptor I (TNF-RI) to stimulate inflammation via activation of the NF-κB transcription factor. To prevent the detrimental effects of TNF-α in keratinocytes infected with the molluscum contagiosum virus (MCV), this poxvirus is expected to produce proteins that block at least one step of the TNF-RI signal transduction pathway. One such product, the MC160 protein, is predicted to interfere with this cellular response because of its homology to other proteins that regulate TNF-RI-mediated signaling. We report here that expression of MC160 molecules did significantly reduce TNF-α-mediated NF-κB activation in 293T cells, as measured by gene reporter and gel mobility shift assays. Since we observed that MC160 decreased other NF-κB activation pathways, namely those activated by receptor-interacting protein, TNF receptor-associated factor 2, NF-κB-inducing kinase, or MyD88, we hypothesized that the MC160 product interfered with I kappa kinase (IKK) activation, an event common to multiple signal transduction pathways. Indeed, MC160 protein expression was associated with a reduction in in vitro IKK kinase activity and IKK subunit phosphorylation. Further, IKK1-IKK2 interactions were not detected in MC160-expressing cells, under conditions demonstrated to induce IKK complex formation, but interactions between the MC160 protein and the major IKK subunits were undetectable. Surprisingly, MC160 expression correlated with a decrease in IKK1, but not IKK2 levels, suggesting a mechanism for MC160 disruption of IKK1-IKK2 interactions. MCV has probably retained its MC160 gene to inhibit NF-κB activation by interfering with signaling via multiple biological mediators. In the context of an MCV infection in vivo, MC160 protein expression may dampen the cellular production of proinflammatory molecules and enhance persistent infections in host keratinocytes. PMID:16378960

The nuclear IkappaB protein IkappaBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria.

PubMed

Hirotani, Tomonori; Lee, Pui Y; Kuwata, Hirotaka; Yamamoto, Masahiro; Matsumoto, Makoto; Kawase, Ichiro; Akira, Shizuo; Takeda, Kiyoshi

2005-03-15

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMphi). CLPMphi of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMphi gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMphi. These genes included nuclear IkappaB proteins such as Bcl-3 and IkappaBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-alpha production, we analyzed the role of IkappaBNS in macrophages. Lentiviral introduction of IkappaBNS resulted in impaired LPS-induced IL-6 production, but not TNF-alpha production in the murine macrophage cell line RAW264.7. IkappaBNS expression led to constitutive and intense DNA binding of NF-kappaB p50/p50 homodimers. IkappaBNS was recruited to the IL-6 promoter, but not to the TNF-alpha promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IkappaBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-alpha. Thus, IkappaBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IkappaB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.

The immunosuppressant drug, thalidomide, improves hepatic alterations induced by a high-fat diet in mice.

PubMed

Pinto, Livia de Fraia; Compri, Cecília Melleti; Fornari, João Victor; Bartchewsky, Waldemar; Cintra, Dennys Eduardo; Trevisan, Miriam; Carvalho, Patrícia de Oliveira; Ribeiro, Marcelo Lima; Velloso, Licio A; Saad, Mario J; Pedrazzoli, José; Gambero, Alessandra

2010-04-01

Pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-alpha, are known to be involved in the establishment of insulin resistance. Insulin resistance plays a key role in the development of obesity-related pathologies, such as type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). The state of chronic inflammation associated with obesity led us to hypothesize that TNF-alpha blockade may have an effect on experimentally obese animals. We studied the effects of thalidomide, an immunosuppressant and anti-TNF-alpha drug, on hepatic alterations that were induced by a high-fat diet (HFD) in mice. Obesity was induced in Swiss mice using a HFD for 12 weeks. Thalidomide-treated animals received thalidomide i.p. (100 mg/kg/day, 10 days). Glucose, aspartate aminotransferases and alanine aminotransferases levels were assessed in the blood. Insulin and glucose tolerance tests were performed. The liver was excised for histological, triglyceride, gene and protein expression analyses. We found improvements in both the basal glucose blood levels and the response to insulin administration in the treated animals. The molecular analysis of insulin signalling revealed a restoration of the hepatic insulin receptor substrate (IRS)-1 and AKT phosphorylation. The hepatic expression of TNF-alpha was inhibited and the levels correlated with a significant reduction in the steatosis area. Other hepatic inflammatory markers, such as iNOS and suppressor of cytokine signalling (SOCS-3), were also reduced. We suggest that immunosuppressant drugs that target TNF-alpha and that may also contribute to reductions in the inflammatory markers that are associated with obesity could be a therapeutic option in NAFLD and type 2 diabetes.

Essential oil from leaves of Liquidambar formosana ameliorates inflammatory response in lipopolysaccharide-activated mouse macrophages.

PubMed

Hua, Kuo-Feng; Yang, Tzu-Jung; Chiu, Huan-Wen; Ho, Chen-Lung

2014-06-01

The essential oil from Liquidambar formosana leaves (EOLF) was demonstrated to exhibit anti-inflammatory activity in mouse macrophages. EOLF reduced nitrite oxide generation, secretion levels of tumor necrosis factor-alpha and interleukin-6, and expression levels of prointerleukin-beta, inducible nitric oxide synthase, and cyclooxygenase-2 in lipopolysaccharide (LPS)-activated mouse macrophages. EOLF also reduced NLRP3 inflammasome-derived interleukin-1beta secretion. The underlying mechanisms for the EOLF-mediated anti-inflammatory activity were (1) reduction of LPS-induced reactive oxygen species generation; (2) reduction of LPS-induced activation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 MAP kinase; (3) reduction of LPS-induced nuclear factor-kappaBeta activation. Furthermore, 25 compounds were identified in the EOLF using GC-FID and GC-MS and the major compounds were terpinen-4-ol (32.0%), beta-pinene (18.0%), gamma-terpinene (13.8%), and alpha-terpinene (9.7%). We found that LPS-induced nitrite oxide generation was inhibited significantly by terpinen-4-ol. Our results indicated that EOLF has anti-inflammatory activity and may provide a molecular rationale for future therapeutic interventions in immune modulation.

C-terminal cleavage of DeltaNp63alpha is associated with TSA-induced apoptosis in immortalized corneal epithelial cells.

PubMed

Robertson, Danielle M; Ho, Su-Inn; Cavanagh, H Dwight

2010-08-01

In the central human corneal epithelium, loss of DeltaNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for DeltaNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for DeltaNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line. For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual DeltaNp63 isoforms, DeltaNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 muM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous DeltaNp63alpha, DeltaNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. Transient expression of DeltaNp63-EGFP alpha and beta isoforms resulted in the formation of a smaller isoform similar in size to DeltaNp63gamma-EGFP. FRAP demonstrated that DeltaNp63alpha-EGFP has greater immobile fraction than beta or gamma. TSA induced caspase-mediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous DeltaNp63alpha and p53. TSA upregulated DeltaNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of DeltaNp63alpha-EGFP. siRNA knockdown of DeltaNp63alpha correlated with a reduction in p53 independently of TSA. DeltaNp63alpha is the dominant active isoform in corneal epithelial cell nuclei. Loss of DeltaNp63alpha occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins.

Inhibition of LPS binding to MD-2 co-receptor for suppressing TLR4-mediated expression of inflammatory cytokine by 1-dehydro-10-gingerdione from dietary ginger

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Sun Hong; Kyeong, Min Sik; Hwang, Yuri

Highlights: Black-Right-Pointing-Pointer 1-Dehydro-10-gingerdione (1D10G) from ginger inhibits LPS binding to MD-2. Black-Right-Pointing-Pointer 1D10G suppresses MyD88- or TRIF-dependent signaling in LPS-activated macrophages. Black-Right-Pointing-Pointer 1D10G down-regulates the expression of NF-{kappa}B-, AP1- or IRF3-target genes. Black-Right-Pointing-Pointer MD-2 is a molecular target in the anti-inflammatory action of 1D10G. -- Abstract: Myeloid differentiation protein 2 (MD-2) is a co-receptor of toll-like receptor 4 (TLR4) for innate immunity. Here, we delineated a new mechanism of 1-dehydro-10-gingerdione (1D10G), one of pungent isolates from ginger (Zingiber officinale), in the suppression of lipopolysaccharide (LPS)-induced gene expression of inflammatory cytokines. 1D10G inhibited LPS binding to MD-2 with higher affinity thanmore » gingerol and shogaol from dietary ginger. Moreover, 1D10G down-regulated TLR4-mediated expression of nuclear factor-{kappa}B (NF-{kappa}B) or activating protein 1 (AP1)-target genes such as tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin-1{beta}, as well as those of interferon (IFN) regulatory factor 3 (IRF3)-target IFN-{beta} gene and IFN-{gamma} inducible protein 10 (IP-10) in LPS-activated macrophages. Taken together, MD-2 is a molecular target in the anti-inflammatory action of 1D10G.« less

Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor.

PubMed

Barrow, Alexander D; Edeling, Melissa A; Trifonov, Vladimir; Luo, Jingqin; Goyal, Piyush; Bohl, Benjamin; Bando, Jennifer K; Kim, Albert H; Walker, John; Andahazy, Mary; Bugatti, Mattia; Melocchi, Laura; Vermi, William; Fremont, Daved H; Cox, Sarah; Cella, Marina; Schmedt, Christian; Colonna, Marco

2018-01-25

Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRβ signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular cloning of alpha-amylases from cotton boll weevil, Anthonomus grandis and structural relations to plant inhibitors: an approach to insect resistance.

PubMed

Oliveira-Neto, Osmundo B; Batista, João A N; Rigden, Daniel J; Franco, Octávio L; Falcão, Rosana; Fragoso, Rodrigo R; Mello, Luciane V; dos Santos, Roseane C; Grossi-de-Sá, Maria F

2003-01-01

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of alpha-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two alpha-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5' and 3' RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several alpha-amylase inhibitors from plants were assayed against A. grandis alpha-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and alpha-AI1 inhibitors on A. grandis alpha-amylase activity. This work suggests that genetic engineering of cotton to express alpha-amylase inhibitors may offer a novel route to A. grandis resistance.

Distinct mechanisms for N-acetylcysteine inhibition of cytokine-induced E-selectin and VCAM-1 expression.

PubMed

Faruqi, R M; Poptic, E J; Faruqi, T R; De La Motte, C; DiCorleto, P E

1997-08-01

We have examined the effects of N-acetyl-L-cysteine (NAC), a well-characterized, thiol-containing antioxidant, on agonist-induced monocytic cell adhesion to endothelial cells (EC). NAC inhibited interleukin-1 (IL-1 beta)-induced, but not basal, adhesion with 50% inhibition at approximately 20 mM. Monocytic cell adhesion to EC in response to tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA) was similarly inhibited by NAC. Unlike published studies with pyrrolidinedithiocarbamate, which specifically inhibited vascular cell adhesion molecule 1 (VCAM-1), NAC inhibited IL-1 beta-induced mRNA and cell surface expression of both E-selectin and VCAM-1. NAC had no effect on the half-life of E-selectin or VCAM-1 mRNA. Although NAC reduced nuclear factor-kappa B (NF-kappa B) activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding to the consensus NF-kappa B binding sites of the VCAM-1 gene (VCAM-NF-kappa B), the antioxidant had no appreciable effect when an oligomer corresponding to the consensus NF-kappa B binding site of the E-selectin gene (E-selectin-NF-kappa B) was used. Because NF-kappa B has been reported to be redox sensitive, we studied the effects of NAC on the EC redox environment. NAC caused an expected dramatic increase in the reduced glutathione (GSH) levels in EC. In vitro studies demonstrated that whereas the binding affinity of NF-kappa B to the VCAM-NF-kappa B oligomer peaked at a GSH-to-oxidized glutathione (GSSG) ratio of approximately 200 and decreased at higher ratios, the binding to the E-selectin-NF-kappa B oligomer appeared relatively unaffected even at ratios > 400, i.e., those achieved in EC treated with 40 mM NAC. These results suggest that NF-kappa B binding to its consensus sequences in the VCAM-1 and E-selectin gene exhibits marked differences in redox sensitivity, allowing for differential gene expression regulated by the same transcription factor. Our data also demonstrate that NAC increases the GSH-to-GSSG ratio within the EC suggesting one possible mechanism through which this antioxidant inhibits agonist-induced monocyte adhesion to EC.

The role of tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) in mediating autophagy in myositis skeletal muscle: A potential non-immune mechanism of muscle damage

PubMed Central

Alger, Heather M.; Raben, Nina; Pistilli, Emidio; Francia, Dwight; Rawat, Rashmi; Getnet, Derese; Ghimbovschi, Svetlana; Chen, Yi-Wen; Lundberg, Ingrid E.; Nagaraju, Kanneboyina

2011-01-01

Objective Multinucleated cells are relatively resistant to classical apoptosis, and the factors initiating cell-death and damage in myositis are not well defined. We hypothesized that non-immune autophagic cell death may play a role in muscle fiber damage. Recent literature indicates that tumor necrosis factor-alpha-related apoptosis inducing ligand (TRAIL) may induce both NFκB (nuclear factor kappa-light chain enhancer of activated B cells) activation and autophagic cell death in other systems. Here, we have investigated its role in cell death and pathogenesis in vitro and in vivo using myositis (human and mouse) muscle tissues. Methods Gene expression profiling indicated that expression of TRAIL and several autophagy markers was specifically upregulated in myositis muscle tissue; these results were confirmed by immunohistochemistry and immunoblotting. We also analyzed TRAIL-induced cell death (apoptosis and autophagy) and NFκB activation in vitro in cultured cells. Results TRAIL was expressed predominantly in muscle fibers of myositis, but not in biopsies from normal or other dystrophic-diseased muscle. Autophagy markers were upregulated in human and mouse models of myositis. TRAIL expression was restricted to regenerating/atrophic areas of muscle fascicles, blood vessels, and infiltrating lymphocytes. TRAIL induced NFκB activation and IκB degradation in cultured cells that are resistant to TRAIL-induced apoptosis but undergo autophagic cell death. Conclusion Our data demonstrate that TRAIL is expressed in myositis muscle and may mediate both activation of NFκB and autophagic cell death in myositis. Thus, this non-immune pathway may be an attractive target for therapeutic intervention in myositis. PMID:21769834

No Detectable Hypoxia in Malignant Salivary Gland Tumors: Preliminary Results

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wijffels, Karien; Hoogsteen, Ilse J.; Lok, Jasper

2009-04-01

Purpose: Hypoxia is detected in most solid tumors and is associated with malignant progression and adverse treatment outcomes. However, the oxygenation status of malignant salivary gland tumors has not been previously studied. The aim of this study was to investigate the potential clinical relevance of hypoxia in this tumor type. Methods and Materials: Twelve patients scheduled for surgical resection of a salivary gland tumor were preoperatively injected with the hypoxia marker pimonidazole and the proliferation marker iododeoxyuridine. Tissue samples of the dissected tumor were immunohistochemically stained for blood vessels, pimonidazole, carbonic anhydrase-IX, glucose transporters-1 and -3 (Glut-1, Glut-3), hypoxia-inducible factor-1{alpha},more » iododeoxyuridine, and epidermal growth factor receptor. The tissue sections were quantitatively assessed by computerized image analysis. Results: The tissue material from 8 patients was of sufficient quality for quantitative analysis. All tumors were negative for pimonidazole binding, as well as for carbonic anhydrase-IX, Glut-1, Glut-3, and hypoxia-inducible factor-1{alpha}. The vascular density was high, with a median value of 285 mm{sup -2} (range, 209-546). The iododeoxyuridine-labeling index varied from <0.1% to 12.2% (median, 2.2%). Epidermal growth factor receptor expression levels were mostly moderate to high. In one-half of the cases, nuclear expression of epidermal growth factor receptor was observed. Conclusion: The absence of detectable pimonidazole binding, as well as the lack of expression of hypoxia-associated proteins in all tumors, indicates that malignant salivary gland tumors are generally well oxygenated. It is unlikely that hypoxia is a relevant factor for their clinical behavior and treatment responsiveness.« less

De-phosphorylation of TR{alpha}-1 by p44/42 MAPK inhibition enhances T{sub 3}-mediated GLUT5 gene expression in the intestinal cell line Caco-2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mochizuki, Kazuki; Sakaguchi, Naomi; Takabe, Satsuki

2007-08-10

Thyroid hormone and p44/42 MAPK inactivation are important in intestinal differentiation. We demonstrated not only that treatment with p44/42 MAPK inhibitor U0126 in intestinal cell line Caco-2 cells reduced the phosphorylation of serine and threonine residues of TR{alpha}-1, but also that T{sub 3} and U0126 synergistically induced GLUT5 gene expression. EMSA demonstrated that the binding activity of TR{alpha}-1-RXR heterodimer on GLUT5-TRE in nuclear proteins of Caco-2 cells was synergistically enhanced by co-incubation in vitro with T{sub 3} and CIAP, which strongly de-phosphorylates proteins. ChIP and transfection assays revealed that co-treatment of T{sub 3} and U0126 induces TR{alpha}-1-RXR binding to GLUT5-TREmore » on the human GLUT5 enhancer region, and recruitment of the transcriptional complex in cells. These results suggest that inactivation of p44/42 MAPK enhances T{sub 3}-induced GLUT5 gene expression in Caco-2 cells through increasing TR{alpha}-1 transactivity and binding activity to the GLUT5-TRE, probably due to de-phosphorylation of TR{alpha}-1.« less

Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeon, Youngsic; Jung, Yujung; Kim, Min Cheol

2014-08-08

Highlights: • SHQA increases PPARα/γ transactivation and inhibits MMP-2/-9 expression. • SHQA inhibits TNFα-induced AP-1 and MAPK signaling. • SHQA inhibits TNFα-induced p65 translocation and IκBα phosphorylation. • SHQA inhibits TNFα-induced AP-1 and NF-κB signaling via PPARα. - Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-agingmore » and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ’s role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα.« less

Up-regulation of hypoxia-inducible factor (HIF)-1α and HIF-target genes in cortical neurons by the novel multifunctional iron chelator anti-Alzheimer drug, M30.

PubMed

Avramovich-Tirosh, Y; Bar-Am, O; Amit, T; Youdim, M B H; Weinreb, O

2010-06-01

Based on a multimodal drug design paradigm, we have synthesized a multifunctional non-toxic, brain permeable iron chelator, M30, possessing the neuroprotective propargylamine moiety of the anti-Parkinsonian drug, rasagiline (Azilect) and antioxidant-iron chelator moiety of an 8-hydroxyquinoline derivative of our iron chelator, VK28. M30 was recently found to confer potential neuroprotective effects in vitro and in various preclinical neurodegenerative models and regulate the levels and processing of the Alzheimer's amyloid precursor protein and its toxic amyloidogenic derivative, Abeta. Here, we show that M30 activates the hypoxia-inducible factor (HIF)-1alpha signaling pathway, thus promoting HIF-1alpha mRNA and protein expression levels, as well as increasing transcription of HIF-1alpha-dependent genes, including vascular endothelial growth factor, erythropoietin, enolase-1, p21 and tyrosine hydroxylase in rat primary cortical cells. In addition, M30 also increased the expression levels of the transcripts of brain derived neurotrophic factor (BDNF) and growth-associated protein-43 (GAP-43). Regarding aspects of relevance to Alzheimer's disease (AD), western blotting analysis of glycogen synthase kinase- 3beta (GSK-3beta) signaling pathway revealed that M30 enhanced the levels of phospho-AKT (Ser473) and phospho- GSK-3beta (Ser9) and attenuated Tau phosphorylation. M30 was also shown to protect cultured cortical neurons against Abeta(25-35) toxicity. All these multimodal pharmacological activities of M30 might be beneficial for its potent efficacy in the prevention and treatment of neurodegenerative conditions, such as Parkinson's disease and AD in which oxidative stress and iron-mediated toxicity are involved.

Oncolytic effects of adenovirus mutant capable of replicating in hypoxic and normoxic regions of solid tumor.

PubMed

Cho, Won-Kyung; Seong, Young Rim; Lee, Yeune Hee; Kim, Min Ji; Hwang, Kyung-Sun; Yoo, Jinsang; Choi, Seeyoung; Jung, Cho-Rok; Im, Dong-Soo

2004-11-01

Solid tumors contain normoxic and hypoxic regions depending on the distance from the capillary. Normal cells may also be exposed to hypoxia under certain physiological conditions. Tumor hypoxia has been shown to associate strongly with tumor propagation and malignant progression. Hypoxia-inducible factor (HIF)-1alpha is stable under hypoxia and induces transcription of target genes by binding to the hypoxia-response element (HRE). Here we investigated the oncolytic effects of a novel adenovirus mutant with a deleted E1B55 gene (Ad.Delta55.HRE), in which the expression of E1A, which is essential for adenoviral replication, is regulated under the control of an HRE-expression system. Ad.Delta55.HRE expressed E1A under normoxia and more E1A under hypoxia and exhibited oncolytic effects on various cultured tumor cells, but its cytotoxic effect is relatively attenuated in normal fibroblast cells under normoxic and hypoxic conditions. Ad.Delta55.HRE lysed Huh-7 hepatoma cells stably expressing HIF-1alpha more effectively compared to parental cells. Ad.Delta55.HRE treatment exhibited significant antitumor activity in PC-3 prostate- and MDA-MB-435 breast tumor-bearing nude mice in which HIF-1alpha protein was immunohistochemically detected. The E1A and hexon proteins of adenovirus were immunostained in MDA-MB-435 xenografts after Ad.Delta55.HRE treatment, suggestive of viral replication. Our results suggest that Ad.Delta55.HRE may be useful for the treatment of solid tumors.

Nitric oxide provokes tumor necrosis factor-alpha expression in adult feline myocardium through a cGMP-dependent pathway.

PubMed

Kalra, D; Baumgarten, G; Dibbs, Z; Seta, Y; Sivasubramanian, N; Mann, D L

2000-09-12

The mechanism(s) responsible for the persistent coexpression of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in the failing heart is unknown. To determine whether NO was sufficient to provoke TNF-alpha biosynthesis, we examined the effects of an NO donor, S-nitroso-N-acetyl penicillamine (SNAP), in buffer-perfused Langendorff hearts. SNAP (1 micromol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF-alpha mRNA and protein biosynthesis in adult cat hearts. The effects of SNAP were completely abrogated by a NO quenching agent, 2-(4-carboxyphenyl)-4, 4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), and mimicked by sodium nitroprusside. Electrophoretic mobility shift assays demonstrated that SNAP treatment led to the rapid induction of nuclear factor kappa-beta (NF-kappaB) but not AP-1. The importance of the cGMP pathway in terms of mediating NO-induced TNF-alpha biosynthesis was shown by studies that demonstrated that 8-bromo-cGMP mimicked the effects of SNAP and that the effects of SNAP could be completely abrogated using a cGMP antagonist, 1H-(1,2, 4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or protein kinase G antagonist (Rp-8-Br-cGMPS). SNAP and 8-Br-cGMP were both sufficient to lead to the site-specific phosphorylation (serine 32) and degradation of IkappaBalpha in isolated cardiac myocytes. Finally, protein kinase G was sufficient to directly phosphorylate IkappaBalpha on serine 32, a critical step in the activation of NF-kappaB. These studies show that NO provokes TNF-alpha biosynthesis through a cGMP-dependent pathway, which suggests that the coincident expression of TNF-alpha and NO may foster self-sustaining positive autocrine/paracrine feedback inflammatory circuits within the failing heart.

The angiogenic factor CCN1 promotes adhesion and migration of circulating CD34+ progenitor cells: potential role in angiogenesis and endothelial regeneration.

PubMed

Grote, Karsten; Salguero, Gustavo; Ballmaier, Matthias; Dangers, Marc; Drexler, Helmut; Schieffer, Bernhard

2007-08-01

Tissue regeneration involves the formation of new blood vessels regulated by angiogenic factors. We reported recently that the expression of the angiogenic factor CCN1 is up-regulated under various pathophysiologic conditions within the cardiovascular system. Because CD34+ progenitor cells participate in cardiovascular tissue regeneration, we investigated whether CCN1-detected for the first time in human plasma-promotes the recruitment of CD34+ progenitor cells to endothelial cells, thereby enhancing endothelial proliferation and neovascularization. In this study, we demonstrated that CCN1 and supernatants from CCN1-stimulated human CD34+ progenitor cells promoted proliferation of endothelial cells and angiogenesis in vitro and in vivo. In addition, CCN1 induced migration and transendothelial migration of CD34+ cells and the release of multiple growth factors, chemokines, and matrix metalloproteinase-9 (MMP-9) from these cells. Moreover, the CCN1-specific integrins alpha(M)beta(2) and alpha(V)beta(3) are expressed on CD34+ cells and CCN1 stimulated integrin-dependent signaling. Furthermore, integrin antagonists (RGD-peptides) suppressed both binding of CCN1 to CD34+ cells and CCN1-induced adhesion of CD34+ cells to endothelial cells. These data suggest that CCN1 promotes integrin-dependent recruitment of CD34+ progenitor cells to endothelial cells, which may contribute to paracrine effects on angiogenesis and tissue regeneration.

Erythropoietin protects myocardin-expressing cardiac stem cells against cytotoxicity of tumor necrosis factor-{alpha}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madonna, Rosalinda; Institute of Cardiology, and Center of Excellence on Aging, 'G. d'Annunzio' University, Chieti; Shelat, Harnath

2009-10-15

Cardiac stem cells are vulnerable to inflammation caused by infarction or ischemic injury. The growth factor, erythropoietin (Epo), ameliorates the inflammatory response of the myocardium to ischemic injury. This study was designed to assess the role of Epo in regulation of expression and activation of the cell death-associated intracellular signaling components in cardiac myoblasts stimulated with the proinflammatory cytokine tumor necrosis factor (TNF)-{alpha}. Cardiac myoblasts isolated from canine embryonic hearts characterized by expression of myocardin A, a promyogenic transcription factor for cardiovascular muscle development were pretreated with Epo and then exposed to TNF-{alpha}. Compared to untreated cells, the Epo-treated cardiacmore » myoblasts exhibited better morphology and viability. Immunoblotting revealed lower levels of active caspase-3 and reductions in iNOS expression and NO production in Epo-treated cells. Furthermore, Epo pretreatment reduced nuclear translocation of NF-{kappa}B and inhibited phosphorylation of inhibitor of kappa B (I{kappa}B) in TNF-{alpha}-stimulated cardiac myoblasts. Thus, Epo protects cardiac myocyte progenitors or myoblasts against the cytotoxic effects of TNF-{alpha} by inhibiting NF-{kappa}B-mediated iNOS expression and NO production and by preventing caspase-3 activation.« less

Thioredoxin-1 Protects Spinal Cord from Demyelination Induced by Methamphetamine through Suppressing Endoplasmic Reticulum Stress and Inflammation

PubMed Central

Yang, Lihua; Guo, Yinli; Huang, Mengbin; Wu, Xiaoli; Li, Xiang; Chen, Guobing; Li, Ye; Bai, Jie

2018-01-01

Methamphetamine (METH) is a psychostimulant abused around the world. Emerging evidence indicates that METH causes brain damage. However, there are very few reports on METH-induced demyelination. Thioredoxin-1 (Trx-1) is a redox regulating protein and plays the roles in protecting neurons from various stresses. However, whether Trx-1 resists demyelination induced by METH has not been reported. In this study, we found that METH-induced thin myelin sheaths in spinal cord, whereas Trx-1 overexpression transgenic (TG) mice restored the myelin sheaths thickness. The expressions of myelin-associated glycoprotein, myelin basic protein, and cyclin-dependent kinase 5 were decreased by METH, whereas these alterations were blocked in Trx-1 TG mice. The expressions of procaspase-12 and procaspase-3 were decreased by METH, the expression of calpain1 was increased by METH, whereas the alterations were suppressed in Trx-1 TG mice. As same as, the expressions of the extracellular signal-regulated kinase, nuclear factor κB, tumor necrosis factor-alpha, and interleukin-1beta were induced by METH, which were suppressed in Trx-1 TG mice. These data suggest that Trx-1 may play a critical role in resisting the METH-mediated demyelination in spinal cord through regulating endoplasmic reticulum stress and inflammation pathways. PMID:29467717

Pioglitazone inhibits LOX-1 expression in human coronary artery endothelial cells by reducing intracellular superoxide radical generation.

PubMed

Mehta, Jawahar L; Hu, Bo; Chen, Jiawei; Li, Dayuan

2003-12-01

LOX-1, a novel lectin-like receptor for oxidized LDL (ox-LDL), is expressed in response to ox-LDL, angiotensin II (Ang II), tumor necrosis factor (TNF)-alpha, and other stress stimuli. It is highly expressed in atherosclerotic tissues. Peroxisome proliferator-activated receptor (PPAR)-gamma ligands, such as pioglitazone, exert antiatherosclerotic effects. This study examined the regulation of LOX-1 expression in human coronary artery endothelial cells (HCAECs) by pioglitazone. Fourth generation HCAECs were treated with ox-LDL, Ang II, or TNF-alpha with or without pioglitazone pretreatment. All 3 stimuli upregulated LOX-1 expression (mRNA and protein). Pioglitazone, in a concentration-dependent manner, reduced LOX-1 expression (P<0.01 versus ox-LDL, Ang II, or TNF-alpha alone). Ox-LDL, Ang II, and TNF-alpha each enhanced intracellular superoxide radical generation, and pioglitazone pretreatment reduced superoxide generation (P<0.01 versus ox-LDL, Ang II, or TNF-alpha). Furthermore, all 3 stimuli upregulated the expression of the transcription factors nuclear factor-kappaB and activator protein-1 (determined by electrophoretic mobility shift assay), and pioglitazone pretreatment reduced this expression (P<0.01 versus ox-LDL, Ang II, or TNF-alpha). To determine the biological significance of pioglitazone-mediated downregulation of LOX-1, we studied monocyte adhesion to ox-LDL-treated HCAECs. Pioglitazone reduced the adhesion of monocytes to activated HCAECs in a fashion similar to that produced by antisense to LOX-1 mRNA. These observations suggest that the PPAR-gamma ligand pioglitazone reduces intracellular superoxide radical generation and subsequently reduces the expression of transcription factors, expression of the LOX-1 gene, and monocyte adhesion to activated endothelium. The salutary effect of PPAR-gamma ligands in atherogenesis may involve the inhibition of LOX-1 and the adhesion of monocytes to endothelium.

Naringin Decreases TNF-α and HMGB1 Release from LPS-Stimulated Macrophages and Improves Survival in a CLP-Induced Sepsis Mice

PubMed Central

Hong, Sang Bum; Lee, Kyung Jin

2016-01-01

Naringin, a flavanone glycoside extracted from various plants, has a wide range of pharmacological effects. In the present study, we investigated naringin’s mechanism of action and its inhibitory effect on lipopolysaccharide-induced tumor necrosis factor-alpha and high-mobility group box 1 expression in macrophages, and on death in a cecal ligation and puncture induced mouse model of sepsis. Naringin increased heme oxygenase 1 expression in peritoneal macrophage cells through the activation of adenosine monophosphate-activated protein kinase, p38, and NF-E2-related factor 2. Inhibition of heme oxygenase 1 abrogated the naringin’s inhibitory effect on high-mobility group box 1 expression and NF-kB activation in lipopolysaccharide-stimulated macrophages. Moreover, mice pretreated with naringin (200 mg/kg) exhibited decreased sepsis-induced mortality and lung injury, and alleviated lung pathological changes. However, the naringin’s protective effects on sepsis-induced lung injury were eliminated by zinc protoporphyrin, a heme oxygenase 1 competitive inhibitor. These results revealed the mechanism underlying naringin’s protective effect in inflammation and may be beneficial for the treatment of sepsis. PMID:27716835

Butter feeding enhances TNF-alpha production from macrophages and lymphocyte adherence in murine small intestinal microvessels.

PubMed

Fujiyama, Yoichi; Hokari, Ryota; Miura, Soichiro; Watanabe, Chikako; Komoto, Shunsuke; Oyama, Tokushige; Kurihara, Chie; Nagata, Hiroshi; Hibi, Toshifumi

2007-11-01

Dietary fat is known to modulate immune functions. Intake of an animal fat-rich diet has been linked to increased risk of inflammation; however, little is known about how animal fat ingestion directly affects intestinal immune function. The objective of this study was to assess the effect of butter feeding on lymphocyte migration in intestinal mucosa and the changes in adhesion molecules and cytokines involved in this effect. T-lymphocytes isolated from the spleen were fluorescence-labeled and injected into recipient mice. Butter was administered into the duodenum, and villus microvessels of the small intestinal mucosa were observed under an intravital microscope. mRNA expression of adhesion molecules and cytokines in the intestinal mucosa were determined by quantitative PCR. The effect of butter feeding on tumor necrosis factor (TNF)-alpha mRNA expression of intestinal macrophages was also determined. Intraluminal butter administration significantly increased lymphocyte adherence to intestinal microvessels accompanied by increases in expression levels of adhesion molecules ICAM-1, MAdCAM-1 and VCAM-1. This accumulation was significantly attenuated by anti-MAdCAM-1 and anti-ICAM-1 antibodies. Butter administration significantly increased TNF-alpha in the lamina proprial macrophages but not interleukin-6. Anti-TNF-alpha treatment attenuated the enhanced expression of adhesion molecules induced by butter administration. T-lymphocyte adherence to microvessels of the small intestinal mucosa was significantly enhanced after butter ingestion. This enhancement is due to increase in expression levels of adhesion molecules of the intestinal mucosa, which is mediated by TNF-alpha from macrophages in the intestinal lamina propria.

The yeast genome may harbor hypoxia response elements (HRE).

PubMed

Ferreira, Túlio César; Hertzberg, Libi; Gassmann, Max; Campos, Elida Geralda

2007-01-01

The hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor activated when cells are submitted to hypoxia. The heterodimer is composed of two subunits, HIF-1alpha and the constitutively expressed HIF-1beta. During normoxia, HIF-1alpha is degraded by the 26S proteasome, but hypoxia causes HIF-1alpha to be stabilized, enter the nucleus and bind to HIF-1beta, thus forming the active complex. The complex then binds to the regulatory sequences of various genes involved in physiological and pathological processes. The specific regulatory sequence recognized by HIF-1 is the hypoxia response element (HRE) that has the consensus sequence 5'BRCGTGVBBB3'. Although the basic transcriptional regulation machinery is conserved between yeast and mammals, Saccharomyces cerevisiae does not express HIF-1 subunits. However, we hypothesized that baker's yeast has a protein analogous to HIF-1 which participates in the response to changes in oxygen levels by binding to HRE sequences. In this study we screened the yeast genome for HREs using probabilistic motif search tools. We described 24 yeast genes containing motifs with high probability of being HREs (p-value<0.1) and classified them according to biological function. Our results show that S. cerevisiae may harbor HREs and indicate that a transcription factor analogous to HIF-1 may exist in this organism.

Tumor suppression function of the Big-h3 gene in radiation carcinogenesis

NASA Astrophysics Data System (ADS)

Zhao, Y.; Piao, C.; Hei, T.

Interaction between cell and extracellular matrix (ECM) plays a crucial role in tumor invasiveness and metastasis. Using an immortalized human bronchial epithelial (BEP2D) cell model, we show here that expression of Big-h3 gene, a secreted adhesion molecule induced by transforming growth factor- beta (TGF-beta ), is markedly decreased in independently generated, high LET radiation-induced tumor cell lines (TL1-TL5) relative to parental BEP2D cells. Expression of this gene was restored to control level in fusion cell lines between the tumorigenic and parental BEP2D cells that were no longer tumorigenic in nude mice. Transfection of Big-h3 gene into tumor cells resulted in a significant reduction of tumor growth. While integrin receptor alpha 5/beta 1 was overexpressed in tumor cells, its expression was corrected to the level of control BEP2D cells after Big-h3 transfection. These data suggest that Big-h3 is involved in tumor progression by regulating integrin receptor alpha 5/beta 1. . WWee We further show that down regulation of Big-h3 results from loss of expression of TGFbeta1 in tumor cells. The findings provide strong evidence that the Big-h3 gene has tumor suppressor function in radiation induced tumorigenic human bronchial epithelial cells and suggest a potential target for interventional therapy.

Nuclear translocation of HIF-1α induced by influenza A (H1N1) infection is critical to the production of proinflammatory cytokines.

PubMed

Guo, Xinkun; Zhu, Zhaoqin; Zhang, Wanju; Meng, Xiaoxiao; Zhu, Yong; Han, Peng; Zhou, Xiaohui; Hu, Yunwen; Wang, Ruilan

2017-05-24

Infection with the influenza A (H1N1) virus is a major challenge for public health because it can cause severe morbidity and even mortality in humans. The over-secretion of inflammatory cytokines (cytokine storm) is considered to be a key contributor to the severe pneumonia caused by H1N1 infection. It has been reported that hypoxia-inducible factor 1-alpha (HIF-1α) is associated with the production of proinflammatory molecules, but whether HIF-1α participates in the acute inflammatory responses against H1N1 infection is still unclear. To investigate the role of HIF-1α in H1N1 infection, the expression and nuclear translocation of HIF-1α in A549 and THP-1 cell lines infected with H1N1 virus were observed. The results showed that without altering the intracellular mRNA or protein expression of HIF-1α, H1N1 infection only induced nuclear translocation of HIF-1α under normal oxygen concentrations. The use of 2-methoxyestradiol (2ME2), a HIF-1α inhibitor that blocks HIF-1α nuclear accumulation, in H1N1-infected cells decreased the mRNA and protein expression of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 and increased the levels of IL-10. In contrast, H1N1-infected cells under hypoxic conditions had increased HIF-1α nuclear accumulation, increased expression of TNF-α and IL-6 and decreased levels of IL-10. In conclusion, our data implied that in vitro H1N1 infection induced nuclear translocation of HIF-1α without altering the expression of HIF-1α, which may promote the secretion of proinflammatory cytokines during H1N1 infection.

Chronic liver injury in mice promotes impairment of skin barrier function via tumor necrosis factor-alpha.

PubMed

Yokoyama, Satoshi; Hiramoto, Keiichi; Koyama, Mayu; Ooi, Kazuya

2016-09-01

Alcohol is frequently used to induce chronic liver injury in laboratory animals. Alcohol causes oxidative stress in the liver and increases the expression of inflammatory mediators that cause hepatocellular damage. However, during chronic liver injury, it is unclear if/how these liver-derived factors affect distal tissues, such as the skin. The purpose of this study was to evaluate skin barrier function during chronic liver injury. Hairless mice were administered 5% or 10% ethanol for 8 weeks, and damages to the liver and skin were assessed using histological and protein-analysis methods, as well as by detecting inflammatory mediators in the plasma. After alcohol administration, the plasma concentration of the aspartate and alanine aminotransferases increased, while albumin levels decreased. In mice with alcohol-induced liver injury, transepidermal water loss was significantly increased, and skin hydration decreased concurrent with ceramide and type I collagen degradation. The plasma concentrations of [Formula: see text]/[Formula: see text] and tumor necrosis factor-alpha (TNF-α) were significantly increased in mice with induced liver injury. TNF receptor (TNFR) 2 expression was upregulated in the skin of alcohol-administered mice, while TNFR1 levels remained constant. Interestingly, the impairment of skin barrier function in mice administered with 10% ethanol was ameliorated by administering an anti-TNF-α antibody. We propose a novel mechanism whereby plasma TNF-α, via TNFR2 alone or with TNFR1, plays an important role in skin barrier function during chronic liver disease in these mouse models.

Curcumin attenuates inflammatory response in IL-1beta-induced human synovial fibroblasts and collagen-induced arthritis in mouse model.

PubMed

Moon, Dong-Oh; Kim, Mun-Ok; Choi, Yung Hyun; Park, Yung-Min; Kim, Gi-Young

2010-05-01

Curcumin, a major component of turmeric, has been shown to exhibit anti-oxidant and anti-inflammatory activities. The present study was performed to determine whether curcumin is efficacious against both collagen-induced arthritis (CIA) in mice and IL-1beta-induced activation in fibroblast-like synoviocytes (FLSs). DBA/1 mice were immunized with bovine type II collagen (CII) and treated with curcumin every other day for 2weeks after the initial immunization. For arthritis, we evaluated the incidence of disease and used an arthritis index based on paw thickness. In vitro proliferation of CII- or concanavalin A-induced splenic T cells was examined using IFN-gamma production. Pro-inflammatory cytokines TNF-alpha and IL-1beta were examined in the mouse ankle joint and serum IgG1 and IgG2a isotypes were analyzed. The expression levels of prostaglandin E(2) (PGE(2)), cyclooxygenase-2 (COX-2), and matrix metalloproteinases (MMPs) in human FLSs were also determined. The results showed that compared with untreated CIA mice, curcumin-treated mice downregulated clinical arthritis score, the proliferation of splenic T cells, expression levels of TNF-alpha and IL-1beta in the ankle joint, and expression levels of IgG2a in serum. Additionally, by altering nuclear factor (NF)-kappaB transcription activity in FLSs, curcumin inhibited PGE(2) production, COX-2 expression, and MMP secretion. These results suggest that curcumin can effectively suppress inflammatory response by inhibiting pro-inflammatory mediators and regulating humoral and cellular immune responses. Copyright 2010 Elsevier B.V. All rights reserved.

New therapeutic aspect for carvedilol: Antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamdy, Nadia; El-Demerdash, Ebtehal, E-mail: ebtehal_dm@yahoo.com

2012-06-15

Portal hypertension is a common complication of chronic liver diseases associated with liver fibrosis and cirrhosis. At present, beta-blockers such as carvedilol remain the medical treatment of choice for protection against variceal bleeding and other complications. Since carvedilol has powerful antioxidant properties we assessed the potential antifibrotic effects of carvedilol and the underlying mechanisms that may add further benefits for its clinical usefulness using a chronic model of carbon tetrachloride (CCl4)-induced hepatotoxicity. Two weeks after CCl4 induction of chronic hepatotoxicity, rats were co-treated with carvedilol (10 mg/kg, orally) daily for 6 weeks. It was found that treatment of animals withmore » carvedilol significantly counteracted the changes in liver function and histopathological lesions induced by CCl4. Also, carvedilol significantly counteracted lipid peroxidation, GSH depletion, and reduction in antioxidant enzyme activities; glutathione-S-transferase and catalase that was induced by CCl4. In addition, carvedilol ameliorated the inflammation induced by CCl4 as indicated by reducing the serum level of acute phase protein marker; alpha-2-macroglobulin and the liver expression of nuclear factor-kappa B (NF-κB). Finally, carvedilol significantly reduced liver fibrosis markers including hydroxyproline, collagen accumulation, and the expression of the hepatic stellate cell (HSC) activation marker; alpha smooth muscle actin. In conclusion, the present study provides evidences for the promising antifibrotic effects of carvedilol that can be explained by amelioration of oxidative stress through mainly, replenishment of GSH, restoration of antioxidant enzyme activities and reduction of lipid peroxides as well as amelioration of inflammation and fibrosis by decreasing collagen accumulation, acute phase protein level, NF-κB expression and finally HSC activation. -- Highlights: ► Carvedilol is a beta blocker with antioxidant and antifibrotic properties. ► It restores GSH and antioxidant enzyme activities and reduces lipid peroxidation. ► It ameliorates inflammation and nuclear factor kappa-B expression. ► It ameliorates fibrosis by decreasing collagen accumulation and HSC activation.« less

Effect of peroxisome proliferator-activated receptor alpha activators on tumor necrosis factor expression in mice during endotoxemia.

PubMed

Hill, M R; Clarke, S; Rodgers, K; Thornhill, B; Peters, J M; Gonzalez, F J; Gimble, J M

1999-07-01

Inflammatory mediators orchestrate the host immune and metabolic response to acute bacterial infections and mediate the events leading to septic shock. Tumor necrosis factor (TNF) has long been identified as one of the proximal mediators of endotoxin action. Recent studies have implicated peroxisome proliferator-activated receptor alpha (PPARalpha) as a potential target to modulate regulation of the immune response. Since PPARalpha activators, which are hypolipidemic drugs, are being prescribed for a significant population of older patients, it is important to determine the impact of these drugs on the host response to acute inflammation. Therefore, we examined the role of PPARalpha activators on the regulation of TNF expression in a mouse model of endotoxemia. CD-1 mice treated with dietary fenofibrate or Wy-14,643 had fivefold-higher lipopolysaccharide (LPS)-induced TNF plasma levels than LPS-treated control-fed animals. Higher LPS-induced TNF levels in drug-fed animals were reflected physiologically in significantly lower glucose levels in plasma and a significantly lower 50% lethal dose than those in LPS-treated control-fed animals. Utilizing PPARalpha wild-type (WT) and knockout (KO) mice, we showed that the effect of fenofibrate on LPS-induced TNF expression was indeed mediated by PPARalpha. PPARalpha WT mice fed fenofibrate also had a fivefold increase in LPS-induced TNF levels in plasma compared to control-fed animals. However, LPS-induced TNF levels were significantly decreased and glucose levels in plasma were significantly increased in PPARalpha KO mice fed fenofibrate compared to those in control-fed animals. Data from peritoneal macrophage studies indicate that Wy-14,643 modestly decreased TNF expression in vitro. Similarly, overexpression of PPARalpha in 293T cells decreased activity of a human TNF promoter-luciferase construct. The results from these studies suggest that any anti-inflammatory activity of PPARalpha in vivo can be masked by other systemic effects of PPARalpha activators.

Air pollution alters brain and pituitary endothelin-1 and inducible nitric oxide synthase gene expression.

PubMed

Thomson, Errol M; Kumarathasan, Prem; Calderón-Garcidueñas, Lilian; Vincent, Renaud

2007-10-01

Recent work suggests that air pollution is a risk factor for cerebrovascular and neurodegenerative disease. Effects of inhaled pollutants on the production of vasoactive factors such as endothelin (ET) and nitric oxide (NO) in the brain may be relevant to disease pathogenesis. Inhaled pollutants increase circulating levels of ET-1 and ET-3, and the pituitary is a potential source of plasma ET, but the effects of pollutants on the expression of ET and NO synthase genes in the brain and pituitary are not known. In the present study, Fischer-344 rats were exposed by nose-only inhalation to particles (0, 5, 50mg/m3 EHC-93), ozone (0, 0.4, 0.8 ppm), or combinations of particles and ozone for 4 h. Real-time reverse transcription polymerase chain reaction was used to measure mRNA levels in the cerebral hemisphere and pituitary 0 and 24 h post-exposure. Ozone inhalation significantly increased preproET-1 but decreased preproET-3 mRNAs in the cerebral hemisphere, while increasing mRNA levels of preproET-1, preproET-3, and the ET-converting enzyme (ECE)-1 in the pituitary. Inducible NO synthase (iNOS) was initially decreased in the cerebral hemisphere after ozone inhalation, but increased 24 h post-exposure. Particles decreased tumour necrosis factor (TNF)-alpha mRNA in the cerebral hemisphere, and both particles and ozone decreased TNF-alpha mRNA in the pituitary. Our results show that ozone and particulate matter rapidly modulate the expression of genes involved in key vasoregulatory pathways in the brain and pituitary, substantiating the notion that inhaled pollutants induce cerebrovascular effects.

Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice.

PubMed

Lin, Jiandie; Wu, Pei-Hsuan; Tarr, Paul T; Lindenberg, Katrin S; St-Pierre, Julie; Zhang, Chen-Yu; Mootha, Vamsi K; Jäger, Sibylle; Vianna, Claudia R; Reznick, Richard M; Cui, Libin; Manieri, Monia; Donovan, Mi X; Wu, Zhidan; Cooper, Marcus P; Fan, Melina C; Rohas, Lindsay M; Zavacki, Ann Marie; Cinti, Saverio; Shulman, Gerald I; Lowell, Bradford B; Krainc, Dimitri; Spiegelman, Bruce M

2004-10-01

PGC-1alpha is a coactivator of nuclear receptors and other transcription factors that regulates several metabolic processes, including mitochondrial biogenesis and respiration, hepatic gluconeogenesis, and muscle fiber-type switching. We show here that, while hepatocytes lacking PGC-1alpha are defective in the program of hormone-stimulated gluconeogenesis, the mice have constitutively activated gluconeogenic gene expression that is completely insensitive to normal feeding controls. C/EBPbeta is elevated in the livers of these mice and activates the gluconeogenic genes in a PGC-1alpha-independent manner. Despite having reduced mitochondrial function, PGC-1alpha null mice are paradoxically lean and resistant to diet-induced obesity. This is largely due to a profound hyperactivity displayed by the null animals and is associated with lesions in the striatal region of the brain that controls movement. These data illustrate a central role for PGC-1alpha in the control of energy metabolism but also reveal novel systemic compensatory mechanisms and pathogenic effects of impaired energy homeostasis.

The role of lipopolysaccharide in infectious bone resorption of periapical lesion.

PubMed

Hong, Chi-Yuan; Lin, Sze-Kwan; Kok, Sang-Heng; Cheng, Shih-Jung; Lee, Ming-Shu; Wang, Tong-Mei; Chen, Chuan-Shuo; Lin, Li-Deh; Wang, Juo-Song

2004-03-01

The role of lipopolysaccharide (LPS) in periapical lesion-induced bone resorption was investigated. Polymyxin B (PMB), a specific inhibitor of LPS, was evaluated to treat the apical lesion. Lipopolysaccharide isolated from two common endodontic pathogens, Fusobacterium nucleatum and Porphyromonas endodontalis, stimulated mouse macrophage (J774) to release interleukin-1alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) in a time-dependent manner. Combination of LPS further enhanced the stimulation. PMB inhibited these effects significantly. LPS also stimulated matrix metalloproteinase-1 (MMP-1) gene expression in J774, whereas anti-IL-1 alpha and anti-TNF-alpha antibodies, as well as PMB, diminished this effect. A disease model of periapical lesion was established in Wistar rat. Administration of PMB reduced the extent of lesion-associated bone resorption by 76% to approximately 80%, and simultaneously reduced the numbers of MMP-1-producing macrophages. It is suggested that LPS released from the infected root canal triggers the synthesis of IL-1 alpha and TNF-alpha from macrophages. These pro-inflammatory cytokines up-regulate the production of MMP-1 by macrophages to promote periapical bone resorption.

Early immune response and regulation of IL-2 receptor subunits

NASA Technical Reports Server (NTRS)

Hughes-Fulford, Millie; Sugano, Eiko; Schopper, Thomas; Li, Chai-Fei; Boonyaratanakornkit, J. B.; Cogoli, Augusto

2005-01-01

Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and MAPK pathways plays a role in early T-cell activation and induction of IL-2, IL-2R(alpha) and IFN(gamma) gene expression.

Early immune response and regulation of IL-2 receptor subunits.

PubMed

Hughes-Fulford, Millie; Sugano, Eiko; Schopper, Thomas; Li, Chai-Fei; Boonyaratanakornkit, J B; Cogoli, Augusto

2005-09-01

Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and MAPK pathways plays a role in early T-cell activation and induction of IL-2, IL-2R(alpha) and IFN(gamma) gene expression.

PPAR-alpha agonist treatment increases trefoil factor family-3 expression and attenuates apoptosis in the liver tissue of bile duct-ligated rats.

PubMed

Karakan, Tarkan; Kerem, Mustafa; Cindoruk, Mehmet; Engin, Doruk; Alper, Murat; Akın, Okan

2013-01-01

Peroxisome proliferators-activated receptor alpha activation modulates cholesterol metabolism and suppresses bile acid synthesis. The trefoil factor family comprises mucin-associated proteins that increase the viscosity of mucins and help protect epithelial linings from insults. We evaluated the effect of short-term administration of fenofibrate, a peroxisome proliferators activated receptor alpha agonist, on trefoil factor family-3 expression, degree of apoptosis, generation of free radicals, and levels of proinflammatory cytokines in the liver tissue of bile duct-ligated rats. Forty male Wistar rats were randomly divided into four groups: 1 = sham operated, 2 = bile duct ligation, 3 = bile duct-ligated + vehicle (gum Arabic), and 4 = bile duct-ligated + fenofibrate (100 mg/kg/day). All rats were sacrificed on the 7 th day after obtaining blood samples and liver tissue. Liver function tests, tumor necrosis factor-alpha and interleukin 1 beta in serum, and trefoil factor family-3 mRNA expression, degree of apoptosis (TUNEL) and tissue malondialdehyde (malondialdehyde, end-product of lipid peroxidation by reactive oxygen species) in liver tissue were evaluated. Fenofibrate administration significantly reduced serum total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, and tumor necrosis factor-alpha and interleukin-1β levels. Apoptosis and malondialdehyde were significantly reduced in the fenofibrate group. Trefoil factor family-3 expression increased with fenofibrate treatment in bile duct-ligated rats. The peroxisome proliferators-activated receptor alpha agonist fenofibrate significantly increased trefoil factor family-3 expression and decreased apoptosis and lipid peroxidation in the liver and attenuated serum levels of proinflammatory cytokines in bile duct-ligated rats. Further studies are needed to determine the protective role of fenofibrate in human cholestatic disorders.

Fusion protein of CDR mimetic peptide with Fc inhibit TNF-alpha induced cytotoxicity.

PubMed

Qin, Weisong; Feng, Jiannan; Li, Yan; Lin, Zhou; Shen, Beifen

2006-02-01

The variable regions of antibodies play central roles in the binding with antigens. Based on the model of a tumour necrosis factor-alpha (TNF-alpha) neutralizing monoclonal antibody (named as Z12) with TNF-alpha, heavy chain CDR2 (HCDR2) and light chain CDR3 (LCDR3) of Z12 were found to be the most responsible to bind with TNF-alpha. A mimetic peptide (PT) was designed based on the sequence derived from HCDR2 and LCDR3. Fusion protein PT-Fc was constructed by linking PT with Fc of human IgG1 through a flexible linker (GGGGGS). The primary structural characteristics of Fc and PT-Fc were analyzed, including the flexibility, hydrophilicity and epitopes. It was demonstrated that PT and Fc in the fusion protein possessed bio-function properly and non-interfering with each other. Furthermore, PT-Fc was expressed in Escherichia coli by fusion with thioredoxin (Trx). After trx-PT-Fc was cleaved with recombinant enterokinase, PT-Fc was obtained. The results of in vitro cytotoxic assays showed that both PT and PT-Fc could efficiently inhibit TNF-alpha induced apoptosis on L929 cells. At the same micromole concentration, the inhibition activity of PT-Fc was significantly higher than PT.

Integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansing, Hope A.; Sarkeshik, Ali; Yates, John R.

2011-03-11

Research highlights: {yields} Proteomics of clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} receptors in oral carcinoma. {yields} p130Cas, Dek, Src and talin regulate oral carcinoma invasion. {yields} p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin. -- Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomicsmore » screen of proteins recruited to clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta} or {alpha}{sub 6}{beta} receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.« less

Etanercept Promotes Bone Formation via Suppression of Dickkopf-1 Expression in Rats with Collagen-Induced Arthritis

PubMed Central

Tanida, Atsushi; Kishimoto, Yuji; Okano, Toru; Hagino, Hiroshi

2013-01-01

Background Various clinical reports suggest etanercept (ETN) has some efficacy in bone formation in rheumatoid arthritis (RA). To examine this effect, we investigated the gene expression of cytokines relevant to osteoblast/osteoclast differentiation, and evaluated histomorphometric findings in mature rats with collagen-induced arthritis (CIA). Methods Total RNA was extracted from knee joints with CIA after ETN or placebo administration. Subsequently, realtime-PCR was carried out to quantify the mRNAs encoding Wnt-1, Dickkopf-1 (DKK-1), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegelin (OPG) and TNF (tumor necrosis factor)-alpha. In histomorphometric analysis, the infiltrating pannus volume and pannus surface, and the following items in contact with pannus surface were measured: osteoclast number, osteoid surface, osteoid volume and labeling surface. These were evaluated in the distal femur with CIA with or without ETN administration. Results TNF-alpha, RANKL and OPG mRNA expressions, linked to osteoclastogenesis, were not significantly different with or without ETN administration. ETN administration significantly increased Wnt-1 mRNA expression, the osteoblast promoter, and decreased DKK-1 mRNA expression, the Wnt signal inhibitor. In histomorphometric analysis, pannus volume, pannus surface and osteoclast number, parameters of bone destruction, were not significantly different among groups. Osteoid volume, osteoid surface and labeling surface, parameters of bone formation, increased significantly with ETN administration. Conclusion Our results suggest that ETN suppresses DDK-1 expression, and, as a result, Wnt expression is promoted and osteoblastogenesis becomes more active, independent of the regulation of osteoclast activity. Marked bone formation is attributed to the fact that ETN directly promotes osteoblastogenesis, not as a result of suppressing osteoclastogenesis. PMID:24031147

Transcriptional Response of Human Cells to Microbeam Irradiation with 2.1 MeV Alpha Particles

NASA Astrophysics Data System (ADS)

Hellweg, C. E.; Bogner, S.; Spitta, L.; Arenz, A.; Baumstark-Khan, C.; Greif, K. D.; Giesen, U.

Within the next decades an increasing number of human beings in space will be simultaneously exposed to different stimuli especially microgravity and radiation To assess the risks for humans during long-duration space missions the complex interplay of these parameters at the cellular level must be understood Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors Activation of the Nuclear Factor kappa B NF- kappa B pathway as a possible anti-apoptotic route represents such an important cellular stress response A screening assay for detection of NF- kappa B-dependent gene activation using the destabilized variant of Enhanced Green Fluorescent Protein d2EGFP as reporter protein had been developed It consists of Human Embryonic Kidney HEK 293 Cells stably transfected with a receptor-reporter-construct carrying d2EGFP under the control of a NF- kappa B response element Clones positive for Tumor Necrosis Factor alpha TNF- alpha inducible d2EGFP expression were selected as cellular reporters Irradiation was performed either with X-rays 150 kV 19 mA at DLR Cologne or with 2 1 MeV alpha particles LET sim 160 keV mu m at PTB Braunschweig After irradiation the following biological endpoints were determined i cell survival via the colony forming ability test ii time-dependent activation of NF- kappa B dependent d2EGFP gene expression using flow cytometry iii quantitative RT-PCR

Hypoxia-inducible factor regulates alphavbeta3 integrin cell surface expression.

PubMed

Cowden Dahl, Karen D; Robertson, Sarah E; Weaver, Valerie M; Simon, M Celeste

2005-04-01

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of alpha and beta aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt(-/-) and Hifalpha(-/-) TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin alphavbeta3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O(2)). Culturing B16F0 melanoma cells at 1.5% O(2) resulted in increased alphavbeta3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O(2) tension influence placental invasion and tumor migration by increasing cell surface expression of alphavbeta3 integrin.

MicroRNA-300 targets hypoxia inducible factor-3 alpha to inhibit tumorigenesis of human non-small cell lung cancer.

PubMed

Zhang, Y; Guo, Y; Yang, C; Zhang, S; Zhu, X; Cao, L; Nie, W; Yu, H

2017-01-01

Non-small cell lung cancer (NSCLC) is one of the most deadly human cancers. MicroRNA-300 acts as both tumor promoter and suppressor in different types of cancer. Here, we try to identify the function of microRNA-300 in human NSCLC. We compared MicroRNA-300 levels between tumor tissues versus paired adjacent non-tumor lung tissues from NSCLC patients, and in NSCLC versus normal lung cell lines. Effects of microRNA-300 on cell proliferation, invasion and migration were examined in vitro, and on tumor growth in vivo using a xenograft mouse model. Potential mRNA targets of microRNA-300 were predicted and underlying mechanism was explored. MicroRNA-300 expression was lower in both NSCLC tissues and cell lines. Overexpression of microRNA-300 inhibited proliferation, invasion and migration of NSCLC cells in vitro, and tumor growth in vivo. MicroRNA-300 could directly bind to the 3'-UTR of hypoxia inducible factor-3 alpha (HIF3α) mRNA, and inhibit both its mRNA and protein expressions. Restoring HIF3α expression could rescue the inhibitory effects of microRNA-300 on tumorigenesis of NSCLC both in vitro and in vivo. MicroRNA-300 is a tumor suppressor microRNA in NSCLC by downregulating HIF3α expression. Both microRNA-300 and HIF3α may serve as potential therapeutic targets in NSCLC treatment.

[Salidroside inhibits hypoxia-induced phenotypic modulation of corpus cavernosum smooth muscle cells in vitro].

PubMed

Chen, Gang; Huang, Xiao-Jun; Lü, Bo-Dong; Chen, Shi-Tao; Zhang, Shi-Geng; Yang, Ke-Bing

2013-08-01

To explore the effects of salidroside on the phenotypic modulation of corpus cavernosum smooth muscle cells (CCSMC) in hypoxic SD rats. CCSMCs were cultured in vitro and identified by immunohistochemistry. The cells were divided into six groups: normal control (21% O2), hypoxia (1% O2), hypoxia + salidroside 1 mg/L, hypoxia + salidroside 3 mg/L, hypoxia + salidroside 5 mg/L and hypoxia + PGE1 0.4 microg/L, and then cultured for 48 hours. The relative expressions of alpha-actin and osteopontin (OPN) in each group were determined by RT-PCR. The in vitro cultured CCSMCs grew well, with anti-alpha-smooth muscle actin monoclonal antibodies immunohistochemically positive. The relative expression of alpha-actin was markedly decreased while that of OPN remarkably increased in the hypoxia group as compared with the normal control group (P < 0.01). The hypoxia + salidroside 5 mg/L group showed a significantly higher expression of alpha-actin and lower expression of OPN than the hypoxia group (P < 0.01), but exhibited no significant differences from the hypoxia + PGE group (P > 0.05). Hypoxia can reduce the relative expression level of alpha-actin and increase that of OPN in the CCSMCs of SD rats, namely, induce their phenotypic modulation from the contraction to the non-contraction type. Salidroside can restrain hypoxia-induced phenotypic modulation of CCSMCs, and its inhibitory effect at 5 mg/L is similar to that of PGE1.

Control of erythropoietin gene expression and its use in medicine.

PubMed

Jelkmann, Wolfgang

2007-01-01

Erythropoietin (EPO) gene expression is under the control of inhibitory (GATA-2, NF-kappaB) and stimulatory (hypoxia-inducible transcription factor [HIF]-2, hepatocyte nuclear factor [HNF]-4alpha [alpha]) transcription factors. EPO deficiency is the main cause of the anemia in chronic kidney disease (CKD) and a contributing factor in the anemias of inflammation and cancer. Small, orally active compounds capable of stimulating endogenous EPO production are in preclinical or clinical trials for treatment of anemia. These agents include stabilizers of the HIFs that bind to the EPO enhancer and GATA inhibitors which prevent GATA from suppressing the EPO promoter. While HIF stabilizing drugs may prove useful as inexpensive second-line choices, at present, their side effects--particularly tumorigenicity--preclude their use as first-choice therapy. As an alternative, EPO gene therapy has been explored in animal studies and in trials on CKD patients. Here, a major problem is immunogenicity of ex vivo transfected implanted cells and of the recombinant protein produced after ex vivo or in vivo EPO complementary DNA (cDNA) transfer. Recombinant human EPO (rhEPO) engineered in Chinese hamster ovary (CHO) cell cultures (epoetin alpha and epoetin beta [beta]) and its hyperglycosylated analogue darbepoetin alpha are established and safe drugs to avoid allogeneic red blood cell transfusion. Gene-activated EPO (epoetin delta [delta]) from human fibrosarcoma cells (HT-1080) has recently been launched for use in CKD. It is important to know the basics of the technologies, production processes, and structural properties of the novel anti-anemic strategies and drugs.

Regulation of LPS-induced mRNA expression of pro-inflammatory cytokines via alteration of NF-κB activity in mouse peritoneal macrophages exposed to fluoride.

PubMed

Tian, Yuhu; Huo, Meijun; Li, Guangsheng; Li, Yanyan; Wang, Jundong

2016-10-01

F toxicity to immune system, especially to macrophage, has been studied a lot recently. Nuclear factor-kappa B (NF-κB), as a transcription factor, plays a central role in immune and inflammatory responses via the regulation of downstream gene expression. Recent studies indicated that fluoride effect on inflammatory cytokine secretion, however, the molecular mechanism was less understood. In our study, peritoneal macrophages (PMs) were divided several groups and were administrated sodium fluoride (NaF, 50, 100, 200, 400, 800 μM) and/or lipopolysaccharide (LPS, 30 ng/mg). The mRNA expression of p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in macrophages exposed to fluoride was determined by quantitative real-time RT-PCR respectively. The translocation of NF-κB from cytoplasm to nucleus, which in a way reflects NF-κB activity, was demonstrated by Immunofluorescence and ELISA. Our results showed that fluoride had a dose-dependent effect on NF-κB activity, which coincided with LPS-induced mRNA expression of its downstream genes, iNOS and IL-1β. Fluoride alone causes no effect on gene expression. However, the mRNA expression of TNF-α showed non-NF-κB-dependent manner. Therefore, we come to the conclusion that fluoride can regulate LPS-induced mRNA expression of iNOS and IL-1β via NF-κB pathway in mouse peritoneal macrophages. Copyright © 2016 Elsevier Ltd. All rights reserved.

Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells.

PubMed

Moulding, D A; Giles, R V; Spiller, D G; White, M R; Tidd, D M; Edwards, S W

2000-09-01

Mcl-1 is a member of the Bcl-2 protein family, which has been shown to delay apoptosis in transfection and/or overexpression experiments. As yet no gene knockout mice have been engineered, and so there is little evidence to show that loss of Mcl-1 expression is sufficient to trigger apoptosis. U937 cells constitutively express the antiapoptotic protein Bcl-2; but during differentiation, in response to the phorbol ester PMA (phorbol 12 beta-myristate 13 alpha-acetate), Mcl-1 is transiently induced. The purpose of this investigation was to determine the functional role played by Mcl-1 in this differentiation program. Mcl-1 expression was specifically disrupted by chimeric methylphosphonate/phosphodiester antisense oligodeoxynucleotides to just 5% of control levels. The depletion of Mcl-1 messenger RNA (mRNA) and protein was both rapid and specific, as indicated by the use of control oligodeoxynucleotides and analysis of the expression of other BCL2 family members and PMA-induced tumor necrosis factor-alpha (TNF-alpha). Specific depletion of Mcl-1 mRNA and protein, in the absence of changes in cellular levels of Bcl-2, results in a rapid entry into apoptosis. Levels of the proapoptotic protein Bax remained unchanged during differentiation, while Bak expression doubled within 24 hours. Apoptosis was detected within 4 hours of Mcl-1 antisense treatment by a variety of parameters including a novel live cell imaging technique allowing correlation of antisense treatment and apoptosis in individual cells. The induction of Mcl-1 is required to prevent apoptosis during differentiation of U937 cells, and the constitutive expression of Bcl-2 is unable to compensate for the loss of Mcl-1. (Blood. 2000;96:1756-1763)

Hepatic Leukemia Factor Promotes Resistance To Cell Death: Implications For Therapeutics and Chronotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J.

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation.« less

Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia.

PubMed

Jiang, Jin-Yi; Cheung, Carmen K M; Wang, Yifang; Tsang, Benjamin K

2003-01-01

Mammalian ovarian follicular development and atresia is closely regulated by the cross talk of cell death and cell survival signals, which include endocrine hormones (gonadotropins) and intra-ovarian regulators (gonadal steroids, cytokines and growth factors). The fate of the follicle is dependent on a delicate balance in the expression and actions of factors promoting follicular cell proliferation, growth and differentiation and of those inducing programmed cell death (apoptosis). As an important endocrine hormone, FSH binds to its granulosa cell receptors and promotes ovarian follicle survival and growth not only by stimulating proliferation and estradiol secretion of these cells, but also inhibiting the apoptosis by up-regulating the expression of intracellular anti-apoptotic proteins, such as XIAP and FLIP. In addition, intra-ovarian regulators, such as TGF-alpha and TNF-alpha, also play an important role in the control of follicular development and atresia. In response to FSH, Estradiol-17 beta synthesized from the granulosa cells stimulates thecal expression of TGF-alpha, which in turn increases granulosa cell XIAP expression and proliferation. The death receptor and ligand, Fas and Fas ligand, are expressed in granulosa cells following gonadotropin withdrawal, culminating in caspase-mediated apoptosis and follicular atresia. In contrast, TNF-alpha has both survival and pro-apoptotic function in the follicle, depending on the receptor subtype activated, but has been shown to promote granulosa cell survival by increasing XIAP and FLIP expression via the IkappaB-NFkappaB pathway. The pro-apoptotic action of TNF-alpha is mediated through the activation of caspases, via its receptor- (i.e. Caspases-8 and -3) and mitochrondria- (i.e. Caspase-9 and -3) death pathways. In the present manuscript, we have reviewed the actions and interactions of gonadotropins and intra-ovarian regulators in the control of granulosa cell fate and ultimately follicular destiny. We have highlighted the role and regulation of granulosa cell XIAP and FLIP expression, as well as their interactions with the death signaling pathways in the maintenance of granulosa cell survival during follicular development. We have provided strong evidence for these intracellular survival factors as key determinants for ovarian follicular destiny (growth versus atresia), the expression of which is regulated by a highly integrated endocrine, paracrine and autocrine mechanism. Further studies in these aspects will lead to a better understanding of the molecular and cellular regulation of follicular development and atresia, and provide invaluable insight into novel strategies in assisted reproduction in human infertility as well as in increasing reproductive efficiency in livestock industries.

Hydrostatic pressure influences HIF-2 alpha expression in chondrocytes.

PubMed

Inoue, Hiroaki; Arai, Yuji; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Tsuchida, Shinji; Matsuki, Tomohiro; Ueshima, Keiichirou; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

2015-01-05

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.

Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow

PubMed Central

Snykers, Sarah; Vanhaecke, Tamara; De Becker, Ann; Papeleu, Peggy; Vinken, Mathieu; Van Riet, Ivan; Rogiers, Vera

2007-01-01

Background The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated. Results Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3β, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1α, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)α, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity. Conclusion hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells. PMID:17407549

Immunocytochemical localization of cytokines and inducible nitric oxide synthase (iNOS) in oral mucosa and lymph nodes of patients with paracoccidioidomycosis.

PubMed

Neworal, E P M; Altemani, A; Mamoni, R L; Noronha, I L; Blotta, M H S L

2003-03-07

Paracoccidioidomycosis (PCM) is a deep mycosis caused by Paracoccidioides brasiliensis, with high incidence in Brazil. In order to examine the immune response in lesional tissue from patients with PCM, we analyzed cytokines as well as the phenotype of the cell infiltrate. Paraffin-embedded tissue from the oral mucosa of eight patients with the localized adult form (AF) of PCM and from the lymph nodes of 10 patients with the juvenile form (JF) of PCM was analyzed by immunohistochemistry to detect tumor necrosis factor-alpha (TNF-alpha), inducible nitric oxide synthase (iNOS), transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10). Most of the inflammatory cells in the lymph nodes were CD68+ (macrophages, epithelioid and giant cells), while a mixed infiltrate with macrophages, plasma cells and neutrophils was detected in the oral mucosa. TNF-alpha as well as iNOS expression was similar in lymph nodes and oral mucosa, whereas TGF-beta and IL-10 were observed in a larger number of macrophages, epithelioid and giant cells in the lymph nodes, where numerous yeast cells were visualized. The higher expression of anti-inflammatory cytokines (IL-10 and TGF-beta) in lesions of patients with the JF of PCM (lymph nodes) may represent a mechanism by which the fungus evades the host immune response, contributing to a more severe and disseminated form of the disease.

Hyperglycemia induces mixed M1/M2 cytokine profile in primary human monocyte-derived macrophages.

PubMed

Moganti, Kondaiah; Li, Feng; Schmuttermaier, Christina; Riemann, Sarah; Klüter, Harald; Gratchev, Alexei; Harmsen, Martin C; Kzhyshkowska, Julia

2017-10-01

Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytokine profile that can support of diabetes-associated inflammation and development of vascular complications. Copyright © 2016 Elsevier GmbH. All rights reserved.

Noradrenaline and alpha-adrenergic signaling induce the hsp70 gene promoter in mollusc immune cells.

PubMed

Lacoste, A; De Cian, M C; Cueff, A; Poulet, S A

2001-10-01

Expression of heat shock proteins (hsp) is a homeostatic mechanism induced in both prokaryotic and eukaryotic cells in response to metabolic and environmental insults. A growing body of evidence suggests that in mammals, the hsp response is integrated with physiological responses through neuroendocrine signaling. In the present study, we have examined the effect of noradrenaline (NA) on the hsp70 response in mollusc immune cells. Oyster and abalone hemocytes transfected with a gene construct containing a gastropod hsp70 gene promoter linked to the luciferase reporter-gene were exposed to physiological concentrations of NA, or to various alpha- and beta-adrenoceptor agonists and antagonists. Results show that NA and alpha-adrenergic stimulations induced the expression of luciferase in transfected mollusc immunocytes. Furthermore, exposure of hemocytes to NA or to the alpha-adrenoceptor agonist phenylephrine (PE) resulted in the expression of the inducible isoform of the hsp70 protein. Pertussis toxin (PTX), the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor calphostin C, the Ca(2+)-dependent PKC inhibitor Gö 6976 and the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 blocked the PE-mediated induction of the hsp70 gene promoter. These results suggest that alpha-adrenergic signaling induces the transcriptionnal upregulation of hsp70 in mollusc hemocytes through a PTX-sensitive G-protein, PLC, Ca(2+)-dependent PKC and PI 3-kinase. Thus, a functional link exists between neuroendocrine signaling and the hsp70 response in mollusc immune cells.

G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy.

PubMed

White, James P; Wrann, Christiane D; Rao, Rajesh R; Nair, Sreekumaran K; Jedrychowski, Mark P; You, Jae-Sung; Martínez-Redondo, Vicente; Gygi, Steven P; Ruas, Jorge L; Hornberger, Troy A; Wu, Zhidan; Glass, David J; Piao, Xianhua; Spiegelman, Bruce M

2014-11-04

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 (PGC-1α4) is a protein isoform derived by alternative splicing of the PGC1α mRNA and has been shown to promote muscle hypertrophy. We show here that G protein-coupled receptor 56 (GPR56) is a transcriptional target of PGC-1α4 and is induced in humans by resistance exercise. Furthermore, the anabolic effects of PGC-1α4 in cultured murine muscle cells are dependent on GPR56 signaling, because knockdown of GPR56 attenuates PGC-1α4-induced muscle hypertrophy in vitro. Forced expression of GPR56 results in myotube hypertrophy through the expression of insulin-like growth factor 1, which is dependent on Gα12/13 signaling. A murine model of overload-induced muscle hypertrophy is associated with increased expression of both GPR56 and its ligand collagen type III, whereas genetic ablation of GPR56 expression attenuates overload-induced muscle hypertrophy and associated anabolic signaling. These data illustrate a signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise.

G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy

PubMed Central

White, James P.; Wrann, Christiane D.; Rao, Rajesh R.; Nair, Sreekumaran K.; Jedrychowski, Mark P.; You, Jae-Sung; Martínez-Redondo, Vicente; Gygi, Steven P.; Ruas, Jorge L.; Hornberger, Troy A.; Wu, Zhidan; Glass, David J.; Piao, Xianhua; Spiegelman, Bruce M.

2014-01-01

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 (PGC-1α4) is a protein isoform derived by alternative splicing of the PGC1α mRNA and has been shown to promote muscle hypertrophy. We show here that G protein-coupled receptor 56 (GPR56) is a transcriptional target of PGC-1α4 and is induced in humans by resistance exercise. Furthermore, the anabolic effects of PGC-1α4 in cultured murine muscle cells are dependent on GPR56 signaling, because knockdown of GPR56 attenuates PGC-1α4–induced muscle hypertrophy in vitro. Forced expression of GPR56 results in myotube hypertrophy through the expression of insulin-like growth factor 1, which is dependent on Gα12/13 signaling. A murine model of overload-induced muscle hypertrophy is associated with increased expression of both GPR56 and its ligand collagen type III, whereas genetic ablation of GPR56 expression attenuates overload-induced muscle hypertrophy and associated anabolic signaling. These data illustrate a signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise. PMID:25336758

Estrogen receptor 1 (ESR1) regulates VEGFA in adipose tissue.

PubMed

Fatima, L A; Campello, R S; Santos, R de Souza; Freitas, H S; Frank, A P; Machado, U F; Clegg, D J

2017-12-01

Vascular endothelial growth factor A (VEGFA) is a key factor in the regulation of angiogenesis in adipose tissue. Poor vascularization during adipose tissue proliferation causes fibrosis and local inflammation, and is associated with insulin resistance. It is known that 17-beta estradiol (E2) regulates adipose tissue function and VEGFA expression in other tissues; however, the ability of E2 to regulate VEGFA in adipose tissue is currently unknown. In this study, we showed that, in 3T3-L1 cells, E2 and the estrogen receptor 1 (ESR1) agonist PPT induced VEGFA expression, while ESR1 antagonist (MPP), and selective knockdown of ESR1 using siRNA decreased VEGFA and prevented the ability of E2 to modulate its expression. Additionally, we found that E2 and PPT induced the binding of hypoxia inducible factor 1 alpha subunit (HIF1A) in the VEGFA gene promoter. We further found that VEGFA expression was lower in inguinal and gonadal white adipose tissues of ESR1 total body knockout female mice compared to wild type mice. In conclusion, our data provide evidence of an important role for E2/ESR1 in modulating adipose tissue VEGFA, which is potentially important to enhance angiogenesis, reduce inflammation and improve adipose tissue function.

Endocannabinoids participate in placental apoptosis induced by hypoxia inducible factor-1.

PubMed

Abán, C; Martinez, N; Carou, C; Albamonte, I; Toro, A; Seyahian, A; Franchi, A; Leguizamón, G; Trigubo, D; Damiano, A; Farina, M

2016-10-01

During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed towards the relevance of endocannabinoids (ECs) and hypoxia as modulators of trophoblast cell death. However, the relation between these factors is still unknown. In this report, we evaluated the participation of ECs in placental apoptosis induced by cobalt chloride (CoCl2), a hypoxia mimicking agent that stabilizes the expression of hypoxia inducible factor-1 alpha (HIF-1α). We found that HIF-1α stabilization decreased FAAH mRNA and protein levels, suggesting an increase in ECs tone. Additionally, CoCl2 incubation and Met-AEA treatment reduced cell viability and increased TUNEL-positive staining in syncytiotrophoblast layer. Immunohistochemical analysis demonstrated Bax and Bcl-2 protein expression in the cytoplasm of syncytiotrophoblast. Finally, HIF-1α stabilization produced an increase in Bax/Bcl-2 ratio, activation of caspase 3 and PARP cleavage. All these changes in apoptotic parameters were reversed with AM251, a CB1 antagonist. These results demonstrate that HIF-1α may induce apoptosis in human placenta via intrinsic pathway by a mechanism that involves activation of CB1 receptor suggesting a role of the ECs in this process.

Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha.

PubMed

Leibovich, S J; Polverini, P J; Shepard, H M; Wiseman, D M; Shively, V; Nuseir, N

Macrophages are important in the induction of new blood vessel growth during wound repair, inflammation and tumour growth. We show here that tumour necrosis factor-alpha (TNF-alpha), a secretory product of activated macrophages that is believed to mediate tumour cytotoxicity, is a potent inducer of new blood vessel growth (angiogenesis). In vivo, TNF-alpha induces capillary blood vessel formation in the rat cornea and the developing chick chorioallantoic membrane at very low doses. In vitro, TNF-alpha stimulates chemotaxis of bovine adrenal capillary endothelial cells and induces cultures of these cells grown on type-1 collagen gels to form capillary-tube-like structures. The angiogenic activity produced by activated murine peritoneal macrophages is completely neutralized by a polyclonal antibody to TNF-alpha, suggesting immunological features are common to TNF-alpha and the protein responsible for macrophage-derived angiogenic activity. In inflammation and wound repair, TNF-alpha could augment repair by stimulating new blood vessel growth; in tumours, TNF-alpha might both stimulate tumour development by promoting vessel growth and participate in tumour destruction by direct cytotoxicity.

Inflammation induction of Dickkopf-1 mediates chondrocyte apoptosis in osteoarthritic joint.

PubMed

Weng, L-H; Wang, C-J; Ko, J-Y; Sun, Y-C; Su, Y-S; Wang, F-S

2009-07-01

Dysregulated Wnt signaling appears to modulate chondrocyte fate and joint disorders. Dickkopf-1 (DKK1) regulates the pathogenesis of skeletal tissue by inhibiting Wnt actions. This study examined whether DKK1 expression is linked to chondrocyte fate in osteoarthritis (OA). Articular cartilage specimens harvested from nine patients with knee OA and from six controls with femoral neck fracture were assessed for DKK1, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), Bad, Bax, Bcl2 and caspase-3 expression by real time-polymerase chain reaction (RT-PCR) and immunohistochemistry. Apoptotic chondrocytes were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL) and 4', 6-dianidino-2-phenylindole dihydrochloride (DAPI) staining. Human chondrocyte cultures were treated with recombinant IL-1beta and monoclonal DKK1 antibody to determine whether DKK1 impairs chondrocyte survival. Expression of DKK1 correlated with inflammatory cytokine levels (IL-1beta and TNF-alpha expressions), proapoptosis regulators (Bad and caspase-3 expressions) and TUNEL staining in OA cartilage tissues. The IL-1beta induced expressions of DKK1, Bax, Bad and caspase-3-dependent apoptosis of chondrocyte cultures. Neutralization of DKK1 by monoclonal DKK1 antibody significantly abrogated IL-1beta-mediated caspase-3 cleavage and apoptosis and reversed chondrocyte proliferation. Recombinant DKK1 treatment impaired chondrocyte growth and promoted apoptosis. By suppressing nuclear beta-catenin accumulation and Akt phosphorylation, DKK1 mediated IL-1beta promotion of chondrocyte apoptosis. Chondrocyte apoptosis correlates with joint OA. Expression of DKK1 contributes to cartilage deterioration and is a potent factor in OA pathogenesis. Attenuating DKK1 may reduce cartilage deterioration in OA.

Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases.

PubMed

Yoshizumi, Masanori; Abe, Jun-Ichi; Tsuchiya, Koichiro; Berk, Bradford C; Tamaki, Toshiaki

2003-03-01

Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent findings suggest a steady laminar blood flow decreases EC apoptosis and inhibits TNF-mediated EC activation. EC apoptosis or activation is suggested to be involved in plaque erosion, which may lead to platelet aggregation. TNF-alpha regulates gene expression in ECs, in part, by stimulating mitogen-activated protein (MAP) kinases, which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAP kinases in ECs. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm(2)) on TNF-alpha-stimulated activity of three MAP kinases in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. TNF-alpha activated ERK1/2, JNK, and p38 maximally at 15 min in HUVEC. Pre-exposing HUVEC for 10 min to flow inhibited TNF-alpha activation of JNK, but showed no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, a specific ERK1/2 inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Transfection studies with dominant-negative constructs of the protein kinase MEK5 suggested an important role for big mitogen-activated protein kinase 1 (BMK1) in flow-mediated regulation of EC activation by TNF-alpha. Understanding the mechanisms by which steady laminar flow regulates JNK activation by cytokines may provide insight into the atheroprotective mechanisms induced by laminar blood flow.

Method for using a yeast alpha-amylase promoter

DOEpatents

Gao, Johnway; Skeen, Rodney S.; Hooker, Brian S.; Anderson, Daniel B.

2003-04-22

The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

Lipopolysaccharide-induced carotid body inflammation in cats: functional manifestations, histopathology and involvement of tumour necrosis factor-alpha.

PubMed

Fernández, Ricardo; González, Sergio; Rey, Sergio; Cortés, Paula P; Maisey, Kevin R; Reyes, Edison-Pablo; Larraín, Carolina; Zapata, Patricio

2008-07-01

In the absence of information on functional manifestations of carotid body (CB) inflammation, we studied an experimental model in which lipopolysaccharide (LPS) administration to pentobarbitone-anaesthetized cats was performed by topical application upon the CB surface or by intravenous infusion (endotoxaemia). The latter caused: (i) disorganization of CB glomoids, increased connective tissue, and rapid recruitment of polymorphonuclear cells into the vascular bed and parenchyma within 4 h; (ii) increased respiratory frequency and diminished ventilatory chemoreflex responses to brief hypoxia (breathing 100% N(2) for 10 s) and diminished ventilatory chemosensory drive (assessed by 100% O(2) tests) during normoxia and hypoxia; (iii) tachycardia, increased haematocrit and systemic hypotension in response to LPS i.v.; and (iv) increased basal frequency of carotid chemosensory discharges during normoxia, but no change in maximal chemoreceptor responses to brief hypoxic exposures. Lipopolysaccharide-induced tachypnoea was prevented by prior bilateral carotid neurotomy. Apoptosis was not observed in CBs from cats subjected to endotoxaemia. Searching for pro-inflammatory mediators, tumour necrosis factor-alpha (TNF-alpha) was localized by immunohistochemistry in glomus and endothelial cells; reverse transcriptase-polymerase chain reaction revealed that the CB expresses the mRNAs for both type-1 (TNF-R1) and type-2 TNF-alpha receptors (TNF-R2); Western blot confirmed a band of the size expected for TNF-R1; and histochemistry showed the presence of TNF-R1 in glomus cells and of TNF-R2 in endothelial cells. Experiments in vitro showed that the frequency of carotid nerve discharges recorded from CBs perfused and superfused under normoxic conditions was not significantly modified by TNF-alpha, but that the enhanced frequency of chemosensory discharges recorded along responses to hypoxic stimulation was transiently diminished in a dose-dependent manner by TNF-alpha injections. The results suggest that the CB may operate as a sensor for immune signals, that the CB exhibits histological features of acute inflammation induced by LPS, that TNF-alpha may participate in LPS-induced changes in chemosensory activity and that some pathophysiological reactions to high levels of LPS in the bloodstream may originate from changes in CB function.

Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

PubMed

Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A; Manna, Sunil K

2010-02-19

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

Staurosporine, but not Ro 31-8220, induces interleukin 2 production and synergizes with interleukin 1alpha in EL4 thymoma cells.

PubMed

Mahon, T M; Matthews, J S; O'Neill, L A

1997-07-01

Protein kinase C (PKC) has been implicated in interleukin 1 (IL1) signal transduction in a number of cellular systems, either as a key event in IL1 action or as a negative regulator. Here we have examined the effects of two PKC inhibitors, staurosporine and the more selective agent Ro 31-8220, on IL1 responses in the murine thymoma line EL4.NOB-1. A 1 h pulse of staurosporine was found to strongly potentiate the induction of IL2 by IL1alpha in these cells. In contrast, neither a pulse nor prolonged incubation with Ro 31-8220 affected the response to IL1alpha. Both agents blocked the response to PMA, however. A 1 h pulse of staurosporine was also found to induce IL2 production on its own, activate the transcription factor nuclear factor kappaB (NFkappaB) and increase the expression of a NFkappaB-linked reporter gene. It synergized with IL1alpha in all of these responses. Ro 31-8220 was again without effect, although both staurosporine and Ro 31-8220 blocked the activation of NFkappaB by PMA. Finally, staurosporine caused the translocation of PKC-alpha and -epsilon, and to a lesser extent PKC-beta, but not PKC-θ or -zeta, from the cytosol to the membrane, although a similar effect was observed with Ro 31-8220. The results suggest that PKC is not involved in IL1alpha signalling in EL4 cells. Furthermore, the potentiating effect of staurosporine on IL1alpha action does not involve PKC inhibition, and is likely to be at the level of NFkappaB activation.

The permissive effect of zinc deficiency on uroguanylin and inducible nitric oxide synthase gene upregulation in rat intestine induced by interleukin 1alpha is rapidly reversed by zinc repletion.

PubMed

Cui, Li; Blanchard, Raymond K; Cousins, Robert J

2003-01-01

Deficient intake of zinc from the diet upregulates both uroguanylin (UG) and inducible nitric oxide synthase (iNOS) expression in rats. Because these changes influence intestinal fluid secretion and intestinal cell pathophysiology, they relate to the incidence of diarrheal disease and its reversal by zinc as well as intestinal inflammation in general. A model of moderate zinc deficiency in rats, which changes molecular indices of zinc deficiency, was used to further explore the effects of the proinflammatory cytokine interleukin (IL)-1alpha and zinc repletion on these changes. IL-1alpha has been shown to have a role in the intestinal inflammation that occurs with bacterial infection. Our results showed a permissive effect of zinc deficiency on both UG and iNOS expression. Specifically, UG expression was responsive to zinc deficiency and IL-1alpha challenge, which were additive when combined, whereas iNOS expression was upregulated by IL-1alpha only during the deficiency. Immunohistochemistry showed that the increase in UG was limited to enterocytes of the upper villus but, in contrast, the increase in iNOS was principally in cells of the lamina propria of IL-1alpha-treated rats. Cells exhibiting UG upregulation did not co-express serotonin. Repletion with zinc reversed upregulation of the iNOS gene within 1 d, whereas UG upregulation required 3-4 d to return to normal. This differential response to repletion suggests that mechanisms of UG and iNOS dysregulation are different. Dysregulation of both genes may contribute to the severity of zinc-responsive diarrheal disease and intestinal inflammatory disease.

Differential expression of largemouth bass (Micropterus salmoides) estrogen receptor isotypes alpha, beta, and gamma by estradiol.

PubMed

Sabo-Attwood, Tara; Kroll, Kevin J; Denslow, Nancy D

2004-04-15

The expression levels of three estrogen receptor (ER) isotypes alpha, beta, and gamma were quantified in female largemouth bass (Micropterus salmoides) (LMB) liver, ovary, brain, and pituitary tissues. ER alpha and beta expression predominated in the liver, while ERs beta and gamma predominated in the other tissues. Temporally in females, ER alpha was highly up-regulated, ER gamma was slightly up-regulated, and ER beta levels remained unchanged in the liver when plasma 17-beta estradiol (E2) and vitellogenin (Vtg) levels were elevated in the spring. In ovarian tissue from these same fish, all three ERs were maximally expressed in the fall, during early oocyte development and prior to peak plasma E2 levels. When males were injected with E2, ER alpha was highly inducible, ER gamma was moderately up-regulated, and ER beta levels were not affected. None of the ER isotypes were induced by E2 in gonadal tissues. These results combined suggest that the ERs themselves are not regulated in the same manner by E2, and furthermore, do not contribute equally to the transcriptional regulation of genes involved in fish reproduction such as Vtg.

Exercise-induced changes in tumour LDH-B and MCT1 expression are modulated by oestrogen-related receptor alpha in breast cancer-bearing BALB/c mice

PubMed Central

Aveseh, Malihe; Nikooie, Rohollah; Aminaie, Mohsen

2015-01-01

Several factors, including overexpression of lactate dehydrogenase (LDH) and monocarboxylate transporters (MCTs), promote an aerobic lactate production that allows some cancer cells to sustain higher proliferation rates in hostile environments outside the cell. To elucidate the effect of endurance training on the metabolic phenotype of solid tumours, we focused on the tumour expression of LDH-A, LDH-B, MCT1, MCT4, oestrogen-related receptor alpha (ERRα) and LDH isozymes in control (C), trained (T), control+XCT790 (CX) and trained+XCT790 (TX) mice. First, we found that the metabolically altered tumours from the trained animals exhibited lower values for lactate concentration than the control group. The decreased lactate concentration was associated with a shift in the tumour LDH isozyme profile towards LDH-1. These exercise-induced changes were also associated with decreases in the expression of the tumour MCT1, ERRα and CD147 in the trained animals. Secondly, the inhibition of ERRα by treatment of MC4-L2 human breast cancer cells with XCT790 (inverse agonist ligand of ERRα) before injection into the animals not only increased LDH-B expression in the tumour, but also decreased MCT1 expression in the CX group in comparison to the C group. The effects of ERRα inhibition were not additive to the training effects on the expressions of MCT1 and LDH-B in the solid tumours. In conclusion, our results suggest that exercise-induced suppression of ERRα expression modulates alterations in solid tumour expression of LDH-B and MCT1 and contributes towards the prevention of tumour development. PMID:25907793

Homeobox A7 stimulates breast cancer cell proliferation by up-regulating estrogen receptor-alpha

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yu; Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4; Cheng, Jung-Chien

2013-11-01

Highlights: •HOXA7 regulates MCF7 cell proliferation. •HOXA7 up-regulates ERα expression. •HOXA7 mediates estrogen-induced MCF7 cell proliferation. -- Abstract: Breast cancer is the most common hormone-dependent malignancy in women. Homeobox (HOX) transcription factors regulate many cellular functions, including cell migration, proliferation and differentiation. The aberrant expression of HOX genes has been reported to be associated with human reproductive cancers. Estradiol (E2) and its nuclear receptors, estrogen receptor (ER)-alpha and ER-beta, are known to play critical roles in the regulation of breast cancer cell growth. However, an understanding of the potential relationship between HOXA7 and ER in breast cancer cells is limited.more » In this study, our results demonstrate that knockdown of HOXA7 in MCF7 cells significantly decreased cell proliferation and ERα expression. In addition, HOXA7 knockdown attenuated E2-induced cell proliferation as well as progesterone receptor (PR) expression. The stimulatory effects of E2 on cell proliferation and PR expression were abolished by co-treatment with ICI 182780, a selective ERα antagonist. In contrast, overexpression of HOXA7 significantly stimulated cell proliferation and ERα expression. Moreover, E2-induced cell proliferation, as well as PR expression, was enhanced by the overexpression of HOXA7. Neither knockdown nor overexpression of HOXA7 affected the ER-beta levels. Our results demonstrate a novel mechanistic role for HOXA7 in modulating breast cancer cell proliferation via regulation of ERα expression. This finding contributes to our understanding of the role HOXA7 plays in regulating the proliferation of ER-positive cancer cells.« less

Expression and in vitro regulation of integrins by normal human urothelial cells.

PubMed

Southgate, J; Kennedy, W; Hutton, K A; Trejdosiewicz, L K

1995-08-01

Integrins are thought to be essential adhesion receptors for the maintenance of tissue histioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed alpha 2 beta 1 and alpha 3 beta 1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, alpha 6 beta 4 and occasionally alpha v beta 4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca++ serum-free medium, the monolayer cultures expressed alpha 2 beta 1, alpha 3 beta 1 and alpha 5 beta 1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas alpha 6 beta 4 was distributed in a random pattern over the substratum. Increasing exogenous Ca++ concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, alpha 6 beta 4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of alpha 6 beta 4 in high Ca++ media, and also of alpha 3 and alpha 5, but not alpha 2, subunits. These results suggest that alpha 2 beta 1 and alpha 3 beta 1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas alpha 6 beta 4 is the principal integrin involved in substratum adhesion.

Mechanisms of intracellular calcium homeostasis in developing and mature bovine corpora lutea.

PubMed

Wright, Marietta F; Bowdridge, Elizabeth; McDermott, Erica L; Richardson, Samuel; Scheidler, James; Syed, Qaisar; Bush, Taylor; Inskeep, E Keith; Flores, Jorge A

2014-03-01

Although calcium (Ca(2+)) is accepted as an intracellular mediator of prostaglandin F2 alpha (PGF2alpha) actions on luteal cells, studies defining mechanisms of Ca(2+) homeostasis in bovine corpora lutea (CL) are lacking. The increase in intracellular Ca(2+) concentration ([Ca(2+)]i) induced by PGF2alpha in steroidogenic cells from mature CL is greater than in those isolated from developing CL. Our hypothesis is that differences in signal transduction associated with developing and mature CL contribute to the increased efficacy of PGF2alpha to induce a Ca(2+) signal capable of inducing regression in mature CL. To test this hypothesis, major genes participating in Ca(2+) homeostasis in the bovine CL were identified, and expression of mRNA, protein, or activity, in the case of phospholipase Cbeta (PLCbeta), in developing and mature bovine CL was compared. In addition, we examined the contribution of external and internal Ca(2+) to the PGF2alpha stimulated rise in [Ca(2+)]i in LLCs isolated from developing and mature bovine CL. Three differences were identified in mechanisms of calcium homeostasis between developing and mature CL, which could account for the lesser increase in [Ca(2+)]i in response to PGF2alpha in developing than in mature CL. First, there were lower concentrations of inositol 1,4,5-trisphosphate (IP3) after similar PGF2alpha challenge, indicating reduced phospholipase C beta (PLCbeta) activity, in developing than mature CL. Second, there was an increased expression of sorcin (SRI) in developing than in mature CL. This cytoplasmic Ca(2+) binding protein modulates the endoplasmic reticulum (ER) Ca(2+) release channel, ryanodine receptor (RyR), to be in the closed configuration. Third, there was greater expression of ATP2A2 or SERCA, which causes calcium reuptake into the ER, in developing than in mature CL. Developmental differences in expression detected in whole CL were confirmed by Western blots using protein samples from steroidogenic cells isolated from developing and mature CL. Localization of these genes in steroidogenic luteal cells was confirmed by immunohistochemistry. Therefore, it is concluded that the cellular mechanisms that allow PGF2alpha to induce a calcium signal of greater magnitude in mature than in developing CL involve 1) greater PLCbeta activity with enhanced generation of IP3, 2) an enhanced Ca(2+) release from the ER via unrestrained RYR2 due to a decrease in SRI expression, and 3) a reduction in calcium reuptake to the ER due to lower expression of ATP2A2. Accordingly, the increase in [Ca(2+)]i induced by PGF2alpha in mature large steroidogenic cells had less dependency from extracellular calcium than in those isolated from immature CL.

Signal-transducing mechanisms of ketamine-caused inhibition of interleukin-1{beta} gene expression in lipopolysaccharide-stimulated murine macrophage-like Raw 264.7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, T.-L.; Chang, C.-C.; Lin, Y.-L.

2009-10-01

Ketamine may affect the host immunity. Interleukin-1{beta} (IL-1{beta}), IL-6, and tumor necrosis factor-{alpha} (TNF-{alpha}) are pivotal cytokines produced by macrophages. This study aimed to evaluate the effects of ketamine on the regulation of inflammatory cytokine gene expression, especially IL-1{beta}, in lipopolysaccharide (LPS)-activated murine macrophage-like Raw 264.7 cells and its possible signal-transducing mechanisms. Administration of Raw 264.7 cells with a therapeutic concentration of ketamine (100 {mu}M), LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. Exposure to 100 {mu}M ketamine decreased the binding affinity of LPS and LPS-binding protein but didmore » not affect LPS-induced RNA and protein synthesis of TLR4. Treatment with LPS significantly increased IL-1{beta}, IL-6, and TNF-{alpha} gene expressions in Raw 264.7 cells. Ketamine at a clinically relevant concentration did not affect the synthesis of these inflammatory cytokines, but significantly decreased LPS-caused increases in these cytokines. Immunoblot analyses, an electrophoretic mobility shift assay, and a reporter luciferase activity assay revealed that ketamine significantly decreased LPS-induced translocation and DNA binding activity of nuclear factor-kappa B (NF{kappa}B). Administration of LPS sequentially increased the phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK. However, a therapeutic concentration of ketamine alleviated such augmentations. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA reduced cellular TLR4 amounts and ameliorated LPS-induced RAS activation and IL-1{beta} synthesis. Co-treatment with ketamine and TLR4 siRNA synergistically ameliorated LPS-caused enhancement of IL-1{beta} production. Results of this study show that a therapeutic concentration of ketamine can inhibit gene expression of IL-1{beta} possibly through suppressing TLR4-mediated signal-transducing phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK and subsequent translocation and transactivation of NF{kappa}B.« less

Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNF{alpha} in human epidermal keratinocytes (HaCaT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Onda, Kenji; Nagashima, Masahiro; Kawakubo, Yo

2006-12-08

Glucocorticoids (GCs) are essential drugs administered topically or systematically for the treatment of autoimmune skin diseases such as pemphigus. However, a certain proportion of patients does not respond well to GCs. Although studies on the relationship between cytokines and GC insensitivity in local tissues have attracted attention recently, little is known about the underlying mechanism(s) for GC insensitivity in epidermal keratinocytes. Here, we report that tumor necrosis factor (TNF) {alpha} reduces GC-induced transactivation of endogenous genes as well as a reporter plasmid which contains GC responsive element (GRE) in human epidermal keratinocyte cells (HaCaT). The GC insensitivity by TNF{alpha} wasmore » not accompanied by changes in mRNA expressions of GR isoforms ({alpha} or {beta}). However, we observed that mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors (PD98059 and U0126) significantly sensitized the GC-induced transactivation of anti-inflammatory genes (glucocorticoid-induced leucine zipper (GILZ) and mitogen-activated protein kinase phosphatase (MKP)-1) and FK506 binding protein (FKBP) 51 gene in the presence of TNF{alpha}. Additionally, we observed that TNF{alpha} reduced prednisolone (PSL)-dependent nuclear translocation of GR, which was restored by pre-treatment of MEK-1 inhibitors. This is the first study demonstrating a role of the MEK-1/ERK cascade in TNF{alpha}-mediated GC insensitivity. Our data suggest that overexpression of TNF{alpha} leads to topical GC insensitivity by reducing GR nuclear translocation in keratinocytes, and our findings also suggest that inhibiting the MEK-1/ERK cascade may offer a therapeutic potential for increasing GC efficacy in epidermis where sufficient inflammatory suppression is required.« less

Human T-cell lymphotropic virus type 1-infected T lymphocytes impair catabolism and uptake of glutamate by astrocytes via Tax-1 and tumor necrosis factor alpha.

PubMed

Szymocha, R; Akaoka, H; Dutuit, M; Malcus, C; Didier-Bazes, M; Belin, M F; Giraudon, P

2000-07-01

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of a chronic progressive myelopathy called tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). In this disease, lesions of the central nervous system (CNS) are associated with perivascular infiltration by lymphocytes. We and others have hypothesized that these T lymphocytes infiltrating the CNS may play a prominent role in TSP/HAM. Here, we show that transient contact of human or rat astrocytes with T lymphocytes chronically infected by HTLV-1 impairs some of the major functions of brain astrocytes. Uptake of extracellular glutamate by astrocytes was significantly decreased after transient contact with infected T cells, while the expression of the glial transporters GLAST and GLT-1 was decreased. In two-compartment cultures avoiding direct cell-to-cell contact, similar results were obtained, suggesting possible involvement of soluble factors, such as cytokines and the viral protein Tax-1. Recombinant Tax-1 and tumor necrosis factor alpha (TNF-alpha) decreased glutamate uptake by astrocytes. Tax-1 probably acts by inducing TNF-alpha, as the effect of Tax-1 was abolished by anti-TNF-alpha antibody. The expression of glutamate-catabolizing enzymes in astrocytes was increased for glutamine synthetase and decreased for glutamate dehydrogenase, the magnitudes of these effects being correlated with the level of Tax-1 transcripts. In conclusion, Tax-1 and cytokines produced by HTLV-1-infected T cells impair the ability of astrocytes to manage the steady-state level of glutamate, which in turn may affect neuronal and oligodendrocytic functions and survival.

Sulforaphane Inhibits Lipopolysaccharide-Induced Inflammation, Cytotoxicity, Oxidative Stress, and miR-155 Expression and Switches to Mox Phenotype through Activating Extracellular Signal-Regulated Kinase 1/2-Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway in Murine Microglial Cells.

PubMed

Eren, Erden; Tufekci, Kemal Ugur; Isci, Kamer Burak; Tastan, Bora; Genc, Kursad; Genc, Sermin

2018-01-01

Sulforaphane (SFN) is a natural product with cytoprotective, anti-inflammatory, and antioxidant effects. In this study, we evaluated the mechanisms of its effects on lipopolysaccharide (LPS)-induced cell death, inflammation, oxidative stress, and polarization in murine microglia. We found that SFN protects N9 microglial cells upon LPS-induced cell death and suppresses LPS-induced levels of secreted pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. SFN is also a potent inducer of redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), which is responsible for the transcription of antioxidant, cytoprotective, and anti-inflammatory genes. SFN induced translocation of Nrf2 to the nucleus via extracellular signal-regulated kinase 1/2 (ERK1/2) pathway activation. siRNA-mediated knockdown study showed that the effects of SFN on LPS-induced reactive oxygen species, reactive nitrogen species, and pro-inflammatory cytokine production and cell death are partly Nrf2 dependent. Mox phenotype is a novel microglial phenotype that has roles in oxidative stress responses. Our results suggested that SFN induced the Mox phenotype in murine microglia through Nrf2 pathway. SFN also alleviated LPS-induced expression of inflammatory microRNA, miR-155. Finally, SFN inhibits microglia-mediated neurotoxicity as demonstrated by conditioned medium and co-culture experiments. In conclusion, SFN exerts protective effects on microglia and modulates the microglial activation state.

Sulforaphane Inhibits Lipopolysaccharide-Induced Inflammation, Cytotoxicity, Oxidative Stress, and miR-155 Expression and Switches to Mox Phenotype through Activating Extracellular Signal-Regulated Kinase 1/2–Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway in Murine Microglial Cells

PubMed Central

Eren, Erden; Tufekci, Kemal Ugur; Isci, Kamer Burak; Tastan, Bora; Genc, Kursad; Genc, Sermin

2018-01-01

Sulforaphane (SFN) is a natural product with cytoprotective, anti-inflammatory, and antioxidant effects. In this study, we evaluated the mechanisms of its effects on lipopolysaccharide (LPS)-induced cell death, inflammation, oxidative stress, and polarization in murine microglia. We found that SFN protects N9 microglial cells upon LPS-induced cell death and suppresses LPS-induced levels of secreted pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. SFN is also a potent inducer of redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), which is responsible for the transcription of antioxidant, cytoprotective, and anti-inflammatory genes. SFN induced translocation of Nrf2 to the nucleus via extracellular signal-regulated kinase 1/2 (ERK1/2) pathway activation. siRNA-mediated knockdown study showed that the effects of SFN on LPS-induced reactive oxygen species, reactive nitrogen species, and pro-inflammatory cytokine production and cell death are partly Nrf2 dependent. Mox phenotype is a novel microglial phenotype that has roles in oxidative stress responses. Our results suggested that SFN induced the Mox phenotype in murine microglia through Nrf2 pathway. SFN also alleviated LPS-induced expression of inflammatory microRNA, miR-155. Finally, SFN inhibits microglia-mediated neurotoxicity as demonstrated by conditioned medium and co-culture experiments. In conclusion, SFN exerts protective effects on microglia and modulates the microglial activation state. PMID:29410668

DNA damage-inducible genes as biomarkers for exposures to environmental agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, N.F.; Carpenter, T.R.; Jaramillo, R.J.

1997-06-01

A biodosimetric approach to determine alpha-particle dose to the respiratory tract epithelium from known exposures to radon has been developed in the rat. Cytotoxicity assays have been used to obtain dose-conversion factors for cumulative exposures typical of those encountered by underground uranium miners. However, this approach is not sensitive enough to derive close-conversion factors for indoor radon exposures. The expression of DNA damage-inducible genes is being investigated as a biomarker of exposure to radon progeny. Exposure of cultures of A549 cells to alpha particles resulted in an increase in the protein levels of the DNA damage-inducible genes, p53, Cip 1,more » and Gadd45. These protein changes were associated with a transient arrest of cells passing through the cell cycle. This arrest was typified by an increase in the number of cells in the G{sub 1} and G{sub 2} phases and a decrease in the number of cells in the S phase. The effect of inhaled alpha particles (radon progeny) in rats was examined in the epithelial cells of the lateral wall of the anterior nasal cavity. Exposures to radon progeny resulted in a significant increase in the number of cells in the G{sub 1} phase and a decrease in the number of cells in the S phase. These cell-cycle changes were concomitant with an increase in the number of cells containing DNA strand breaks. In addition to ionizing radiation, A549 cells were exposed to 4-nitroquinoline-1-oxide, methyl methanesulphonate, crocidolite asbestos, and glass microfiber. These studies showed that physical and chemical agents induce different expression patterns of p53, Cip 1, and Gadd153 proteins and they could be used to discriminate between toxic and nontoxic materials such as asbestos and glass microfiber. The measurement of gene expression in A549 cells may provide a means to identify a broad spectrum of physical and chemical toxicants encountered in the environment. 9 figs., 42 refs.« less

TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction.

PubMed

Raval, Gira; Biswas, Soumika; Rayman, Patricia; Biswas, Kaushik; Sa, Gaurisankar; Ghosh, Sankar; Thornton, Mark; Hilston, Cynthia; Das, Tanya; Bukowski, Ronald; Finke, James; Tannenbaum, Charles S

2007-05-15

Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kouchi, Zen, E-mail: zkouchi@toyaku.ac.jp; Fujiwara, Yuki; Yamaguchi, Hideki

2011-05-20

Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however,more » its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.« less

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways.

PubMed

Guillermet-Guibert, J; Saint-Laurent, N; Davenne, L; Rochaix, P; Cuvillier, O; Culler, M D; Pradayrol, L; Buscail, L; Susini, C; Bousquet, C

2007-02-01

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.

The fibroblast growth factor receptor (FGFR) agonist FGF1 and the neural cell adhesion molecule-derived peptide FGL activate FGFR substrate 2alpha differently.

PubMed

Chen, Yongshuo; Li, Shizhong; Berezin, Vladimir; Bock, Elisabeth

2010-07-01

Activation of fibroblast growth factor (FGF) receptors (FGFRs) both by FGFs and by the neural cell adhesion molecule (NCAM) is crucial in the development and function of the nervous system. We found that FGFR substrate 2alpha (FRS2alpha), Src homologous and collagen A (ShcA), and phospholipase-Cgamma (PLCgamma) were all required for neurite outgrowth from cerebellar granule neurons (CGNs) induced by FGF1 and FGL (an NCAM-derived peptide agonist of FGFR1). Like FGF1, FGL induced tyrosine phosphorylation of FGFR1, FRS2alpha, ShcA, and PLCgamma in a time- and dose-dependent manner. However, the activation of FRS2alpha by FGL was significantly lower than the activation by FGF1, indicating a differential signaling profile induced by NCAM compared with the cognate growth factor.

Inhibitory effects of hepatocyte growth factor and interleukin-6 on transforming growth factor-beta1 mediated vocal fold fibroblast-myofibroblast differentiation.

PubMed

Vyas, Bimal; Ishikawa, Keiko; Duflo, Suzy; Chen, Xia; Thibeault, Susan L

2010-05-01

The role of myofibroblasts in vocal fold scarring has not been extensively studied, partly because of the lack of a robust in vitro model. The objective of this investigation was to develop and characterize a myofibroblast in vitro model that could be utilized to investigate the molecular mechanism of myofibroblast differentiation and function in injured vocal fold tissue. Differentiation of human primary vocal fold fibroblasts (hVFFs) to myofibroblasts was stimulated with 5, 10, or 20 ng/mL of recombinant transforming growth factor-beta1 (TGF-beta1). Cultures were analyzed by immunofluorescence and Western blotting, with an alpha-smooth muscle actin (alpha-SMA) antibody used as a myofibroblast marker. Normal rabbit vocal folds were treated with 10 ng/mL of TGF-beta1 for 7 days for in vivo corroboration. The effects of interleukin-6 (IL-6) and hepatocyte growth factor (HGF) on myofibroblast differentiation were studied with Western blots. The hVFFs demonstrated positive alpha-SMA labeling in cells stimulated by 10 and 20 ng/mL TGF-beta1, indicating that hVFFs were capable of differentiation to myofibroblasts. Transforming growth factor-beta1 induced the largest increase in alpha-SMA at 10 ng/mL on day 5 of treatment. Both HGF and IL-6 suppressed the expression of TGF-beta1-induced alpha-SMA. Our work characterizes a useful in vitro model of TGF-beta1-mediated vocal fold fibroblast-myofibroblast differentiation. The extent of differentiation appears to be attenuated by HGF, suggesting a potential mechanism to support prior work indicating that HGF plays a protective role in reducing scar formation in vocal fold injuries. Paradoxically, IL-6, which has been shown to play a profibrotic role in dermal studies, also attenuated the TGF-beta1 response.

Inhibition mechanism of compound ethanol extracts from wuweizi (fructus schisandrae chinensis) on renal interstitial fibrosis in diabetic nephropathy model mice.

PubMed

Zhang, Yanqiu; Zhang, Daning; Zhang, Mianzhi

2012-12-01

To evaluate inhibition effect and mechanism of compound ethanol extracts from Wuweizi (Fructus Schisandrae Chinensis), Chuanxiong (Rhizoma Chuanxiong) and Muli (Cocha Ostreae) (FRC) on glomerular and tubular interstitial fibrosis in streptozocin (STZ)-induced diabetic nephropathy (ND) model mice. Twenty-seven male C57BL/6 mice were divided randomly into 3 groups: nondibetic (ND), STZ-induced diabetic (D), and STZ-induced diabetic that were treated with 5 g x kg(-1) x day(-1) of FRC by oral gavage (D(FRC)), with 9 in each group. The protein expressions of E-cadherin, alpha-smooth muscle actin (alpha-SMA), Plasminogen Activator Inhibitor-1 (PAL-1) in renal tissues were investigated by Western blotting. The expressions of fibronectin (FN) and alpha-SMA were detected by immunohistochemical method. The morphological changes of renal tissues were observed under a microscope. Renal tissues in the D(FRC) group showed a lessened degree of fibrosis. Meanwhile, the expressions of FN, alpha-SMA and PAI-1 were significantly lower in the D(FRC) group than those in the D group (all P < 0.05). FRC can ameliorate the DN in the C57BL/6 mice, and its mechanism may relate to inhibition on the epithelial to mesenchymal transdifferentiation, endothelial-myofibroblast transition and PAL-1 expression.

ROLES OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF-A) IN MEDIATION OF DIOXIN (TCDD)-INDUCED DELAYS IN DEVELOPMENT OF THE MOUSE MAMMARY GLAND

EPA Science Inventory

Roles of Epidermal Growth Factor (EGF) and Transforming Growth Factor-alpha (TGF-a) in Mediation of Dioxin (TCDD)-Induced Delays in Development of the Mouse Mammary Gland.
Suzanne E. Fenton, Barbara Abbott, Lamont Bryant, and Angela Buckalew. U.S. EPA, NHEERL, Reproductive Tox...

Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye.

PubMed

Min, Ji Hwan; Lee, Chul Hee; Ji, Yong Woo; Yeo, Areum; Noh, Hyemi; Song, Insil; Kim, Eung Kweon; Lee, Hyung Keun

2016-01-01

By using hypoxia-inducible factor-1 alpha conditional knockout (HIF-1α CKO) mice and a dry eye (DE) mouse model, we aimed to determine the role played by delta-like ligand 4 (Dll4)/Notch signaling and HIF-1α in the lymphangiogenesis of lacrimal glands (LGs). C57BL/6 mice were housed in a controlled-environment chamber for DE induction. During DE induction, the expression level of Dll4/Notch signaling and lymphangiogenesis in LGs was measured by quantitative RT-PCR, immunoblot, and immunofluorescence staining. Next, lymphangiogenesis was measured after Dll4/Notch signal inhibition by anti-Dll4 antibody or γ-secretase inhibitor. Using HIF-1α CKO mice, the expression of Dll4/Notch signaling and lymphangiogenesis in LGs of DE-induced HIF-1α CKO mice were assessed. Additionally, the infiltration of CD45+ cells in LGs was assessed by immunohistochemical (IHC) staining and flow cytometry for each condition. DE significantly upregulated Dll4/Notch and lymphangiogenesis in LGs. Inhibition of Dll4/Notch significantly suppressed lymphangiogenesis in LGs. Compared to wild-type (WT) mice, DE induced HIF-1α CKO mice showed markedly low levels of Dll4/Notch and lymphangiogenesis. Inhibition of lymphangiogenesis by Dll4/Notch suppression resulted in increased CD45+ cell infiltration in LGs. Likewise, CD45+ cells infiltrated more in the LGs of HIF-1α CKO DE mice than in non-DE HIF-1α CKO mice. Dll4/Notch signaling and HIF-1α are closely related to lymphangiogenesis in DE-induced LGs. Lymphangiogenesis stimulated by Dll4/Notch and HIF-1α may play a role in protecting LGs from DE-induced inflammation by aiding the clearance of immune cells from LGs.

E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis.

PubMed

Jung, Cho-Rok; Hwang, Kyung-Sun; Yoo, Jinsang; Cho, Won-Kyung; Kim, Jin-Man; Kim, Woo Ho; Im, Dong-Soo

2006-07-01

The von Hippel-Lindau tumor suppressor, pVHL, forms part of an E3 ubiquitin ligase complex that targets specific substrates for degradation, including hypoxia-inducible factor-1alpha (HIF-1alpha), which is involved in tumor progression and angiogenesis. It remains unclear, however, how pVHL is destabilized. Here we show that E2-EPF ubiquitin carrier protein (UCP) associates with and targets pVHL for ubiquitin-mediated proteolysis in cells, thereby stabilizing HIF-1alpha. UCP is detected coincidently with HIF-1alpha in human primary liver, colon and breast tumors, and metastatic cholangiocarcinoma and colon cancer cells. UCP level correlates inversely with pVHL level in most tumor cell lines. In vitro and in vivo, forced expression of UCP boosts tumor-cell proliferation, invasion and metastasis through effects on the pVHL-HIF pathway. Our results suggest that UCP helps stabilize HIF-1alpha and may be a new molecular target for therapeutic intervention in human cancers.

Proteomic analysis of trichloroethylene-induced alterations in expression, distribution, and interactions of SET/TAF-Iα and two SET/TAF-Iα-binding proteins, eEF1A1 and eEF1A2, in hepatic L-02 cells.

PubMed

Hong, Wen-Xu; Yang, Liang; Chen, Moutong; Yang, Xifei; Ren, Xiaohu; Fang, Shisong; Ye, Jinbo; Huang, Haiyan; Peng, Chaoqiong; Zhou, Li; Huang, Xinfeng; Yang, Fan; Wu, Desheng; Zhuang, Zhixiong; Liu, Jianjun

2012-09-01

Emerging evidence indicates that trichloroethylene (TCE) exposure causes severe hepatotoxicity. However, the mechanisms of TCE hepatotoxicity remain unclear. Recently, we reported that TCE exposure up-regulated the expression of the oncoprotein SET/TAF-Iα and SET knockdown attenuated TCE-induced cytotoxicity in hepatic L-02 cells. To decipher the function of SET/TAF-Iα and its contributions to TCE-induced hepatotoxicity, we employed a proteomic analysis of SET/TAF-Iα with tandem affinity purification to identify SET/TAF-Iα-binding proteins. We identified 42 novel Gene Ontology co-annotated SET/TAF-Iα-binding proteins. The identifications of two of these proteins (eEF1A1, elongation factor 1-alpha 1; eEF1A2, elongation factor 1-alpha 2) were confirmed by Western blot analysis and co-immunoprecipitation (Co-IP). Furthermore, we analyzed the effects of TCE on the expression, distribution and interactions of eEF1A1, eEF1A2 and SET in L-02 cells. Western blot analysis reveals a significant up-regulation of eEF1A1, eEF1A2 and two isoforms of SET, and immunocytochemical analysis reveals that eEF1A1 and SET is redistributed by TCE. SET is redistributed from the nucleus to the cytoplasm, while eFE1A1 is translocated from the cytoplasm to the nucleus. Moreover, we find by Co-IP that TCE exposure significantly increases the interaction of SET with eEF1A2. Our data not only provide insights into the physiological functions of SET/TAF-Iα and complement the SET interaction networks, but also demonstrate that TCE exposure induces alterations in the expression, distribution and interactions of SET and its binding partners. These alterations may constitute the mechanisms of TCE cytotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

PDC-TREM, a plasmacytoid dendritic cell-specific receptor, is responsible for augmented production of type I interferon.

PubMed

Watarai, Hiroshi; Sekine, Etsuko; Inoue, Sayo; Nakagawa, Ryusuke; Kaisho, Tsuneyasu; Taniguchi, Masaru

2008-02-26

Type I interferons (IFNs) derived from plasmacytoid dendritic cells (PDCs) are critical for antiviral responses; however, the mechanisms underlying their production remain unclear. We have identified a receptor, PDC-TREM, which is associated with Plexin-A1 (PlxnA1) on the PDC cell surface and is preferentially expressed after TLR-stimulation. Limited TLR signals induced PDC-TREM expression but failed to induce IFN-alpha production. However, when coupled with Sema6D, a ligand for Plexin-A1, limited TLR-stimulation resulted in PDC-TREM-mediated DAP12-dependent phosphorylation of phosphoinositide 3-kinase (PI3K) and extracellular regulated kinase (Erk) 1/2 at 6-9 h, and IFN-alpha was produced. Inhibition of PDC-TREM expression by pdctrem-shRNA, blocking of PDC-TREM-binding with PlxnA1 by PDC-TREM mAb, and DAP12 deficiency all resulted in greatly reduced PDC-TREM-dependent activation of signaling molecules and IFN-alpha production. Thus, PDC-TREM is responsible for IFN-alpha production, whereas TLR signals are essential for PDC-TREM expression.

Divergent effects of estradiol and the estrogen receptor-alpha agonist PPT on eating and activation of PVN CRH neurons in ovariectomized rats and mice.

PubMed

Thammacharoen, Sumpun; Geary, Nori; Lutz, Thomas A; Ogawa, Sonoko; Asarian, Lori

2009-05-01

Eating is modulated by estradiol in females of many species and in women. To further investigate the estrogen receptor mechanism mediating this effect, ovariectomized rats and mice were treated with estradiol benzoate or the estrogen receptor-alpha (ER-alpha)-selective agonist PPT. PPT inhibited eating in rats much more rapidly than estradiol (approximately 2-6 h versus >24 h). In contrast, the latencies to vaginal estrus after PPT and estradiol were similar (>24 h). PPT also inhibited eating within a few hours in wild-type mice, but failed to inhibit eating in transgenic mice deficient in ER-alpha (ERalphaKO mice). PPT, but not estradiol, induced the expression of c-Fos in corticotrophin-releasing hormone (CRH)-expressing cells of the paraventricular nucleus (PVN) of the hypothalamus within 90-180 min in rats. Both PPT and estradiol reduced c-Fos expression in an ER-alpha-containing area of the nucleus of the solitary tract. The anomalously rapid eating-inhibitory effect of PPT suggests that PPT's neuropharmacological effect differs from estradiol's, perhaps because PPT differentially activates membrane versus nuclear ER-alpha or because PPT activates non-ER-alpha membrane estrogen receptors in addition to ER-alpha. The failure of PPT to inhibit eating in ERalphaKO mice, however, indicates that ER-alpha is necessary for PPT's eating-inhibitory action and that any PPT-induced activation of non-ER-alpha estrogen receptors is not sufficient to inhibit eating. Finally, the rapid induction of c-Fos in CRH-expressing cells in the PVN by PPT suggests that PPT elicits a neural response that is similar to that elicited by stress or aversive emotional stimuli.

Comparative gene expression profiles induced by PPAR{gamma} and PPAR{alpha}/{gamma} agonists in rat hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogue, Alexandra; Universite de Rennes 1, 35065 Rennes Cedex; Biologie Servier, 45520 Gidy

2011-07-01

Species-differential toxic effects have been described with PPAR{alpha} and PPAR{gamma} agonists between rodent and human liver. PPAR{alpha} agonists (fibrates) are potent hypocholesterolemic agents in humans while they induce peroxisome proliferation and tumors in rodent liver. By contrast, PPAR{gamma} agonists (glitazones) and even dual PPAR{alpha}/{gamma} agonists (glitazars) have caused idiosyncratic hepatic and nonhepatic toxicities in human without evidence of any damage in rodent during preclinical studies. The mechanisms involved in such differences remain largely unknown. Several studies have identified the major target genes of PPAR{alpha} agonists in rodent liver while no comprehensive analysis has been performed on gene expression changes inducedmore » by PPAR{gamma} and dual PPAR{alpha}/{gamma} agonists. Here, we investigated transcriptomes of rat hepatocytes after 24 h treatment with two PPAR{gamma} (troglitazone and rosiglitazone) and two PPAR{alpha}/{gamma} (muraglitazar and tesaglitazar) agonists. Although, hierarchical clustering revealed a gene expression profile characteristic of each PPAR agonist class, only a limited number of genes was specifically deregulated by glitazars. Functional analyses showed that many genes known as PPAR{alpha} targets were also modulated by both PPAR{gamma} and PPAR{alpha}/{gamma} agonists and quantitative differences in gene expression profiles were observed between these two classes. Moreover, most major genes modulated in rat hepatocytes were also found to be deregulated in rat liver after tesaglitazar treatment. Taken altogether, these results support the conclusion that differential toxic effects of PPAR{alpha} and PPAR{gamma} agonists in rodent liver do not result from transcriptional deregulation of major PPAR target genes but rather from qualitative and/or quantitative differential responses of a small subset of genes.« less

6-Shogaol suppressed lipopolysaccharide-induced up-expression of iNOS and COX-2 in murine macrophages.

PubMed

Pan, Min-Hsiung; Hsieh, Min-Chi; Hsu, Ping-Chi; Ho, Sheng-Yow; Lai, Ching-Shu; Wu, Hou; Sang, Shengmin; Ho, Chi-Tang

2008-12-01

Ginger, the rhizome of Zingiber officinale, is a traditional medicine with carminative effect, antinausea, anti-inflammatory, and anticarcinogenic properties. In this study, we investigated the inhibitory effects of 6-shogaol and a related compound, 6-gingerol, on the induction of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) in murine RAW 264.7 cells activated with LPS. Western blotting and reverse transcription-PCR analyses demonstrated that 6-shogaol significantly blocked protein and mRNA expression of inducible NOS (iNOS) and COX-2 in LPS-induced macrophages. The in vivo anti-inflammatory activity was evaluated by a topical 12-O-tetradecanoylphorbol 13-acetate (TPA) application to mouse skin. When applied topically onto the shaven backs of mice prior to TPA, 6-shogaol markedly inhibited the expression of iNOS and COX-2 proteins. Treatment with 6-shogaol resulted in the reduction of LPS-induced nuclear translocation of nuclear factor-kappaB (NF kappaB) subunit and the dependent transcriptional activity of NF kappaB by blocking phosphorylation of inhibitor kappaB (I kappaB)alpha and p65 and subsequent degradation of I kappaB alpha. Transient transfection experiments using NF kappaB reporter constructs indicated that 6-shogaol inhibits the transcriptional activity of NF kappaB in LPS-stimulated mouse macrophages. We found that 6-shogaol also inhibited LPS-induced activation of PI3K/Akt and extracellular signal-regulated kinase 1/2, but not p38 mitogen-activated protein kinase (MAPK). Taken together, these results show that 6-shogaol downregulates inflammatory iNOS and COX-2 gene expression in macrophages by inhibiting the activation of NF kappaB by interfering with the activation PI3K/Akt/I kappaB kinases IKK and MAPK.

Naked gene therapy of hepatocyte growth factor for dextran sulfate sodium-induced colitis in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanbe, Takamasa; Murai, Rie; Mukoyama, Tomoyuki

Ulcerative colitis (UC) is progressive and relapsing disease. To explore the therapeutic effects of naked gene therapy of hepatocyte growth factor (HGF) on UC, the SR{alpha} promoter driving HGF gene was intrarectally administered to the mice in which colitis was induced by dextran sulfate sodium (DSS). Expression of the transgene was seen in surface epithelium, lamina propria, and muscularis mucosae. The HGF-treated mice showed reduced colonic mucosal damage and increased body weights, compared with control mice (P < 0.01 and P < 0.05, respectively). The HGF-treated mice displayed increased number of PCNA-positive cells and decreased number of apoptotic cells thanmore » in control mice (P < 0.01, each). Phosphorylated AKT was dramatically increased after HGF gene administration, however, phosphorylated ERK1/2 was not altered. Microarray analysis revealed that HGF induced expression of proliferation- and apoptosis-associated genes. These data suggest that naked HGF gene delivery causes therapeutic effects through regulation of many downstream genes.« less

Dissociation between PGC-1alpha and GLUT-4 expression in skeletal muscle of rats fed a high-fat diet.

PubMed

Higashida, Kazuhiko; Higuchi, Mitsuru; Terada, Shin

2009-12-01

It has recently been reported that a 4-wk high-fat diet gradually increases skeletal muscle peroxisome proliferator activated receptor (PPAR) gamma coactivator-1alpha (PGC-1alpha) protein content, which has been suggested to regulate GLUT-4 gene transcription. However, it has not been reported that a high-fat diet enhances GLUT-4 mRNA expression and protein content in skeletal muscle, suggesting that an increase in PGC-1alpha protein content is not sufficient to induce muscle GLUT-4 biogenesis in a high-fat fed animal. Therefore, we first evaluated the relationship between PGC-1alpha and GLUT-4 expression in skeletal muscle of rats fed a high-fat diet for 4 wk. The PGC-1alpha protein content in rat epitrochlearis muscle significantly increased by twofold after the 4-wk high-fat diet feeding. However, the high-fat diet had no effect on GLUT-4 protein content and induced a 30% decrease in GLUT-4 mRNA expression in rat skeletal muscle (p<0.05). To clarify the mechanism by which a high-fat diet downregulates GLUT-4 mRNA expression, we next examined the effect of PPARdelta activation, which is known to occur in response to a high-fat diet, on GLUT-4 mRNA expression in L6 myotubes. Incubation with 500 nM GW501516 (PPARdelta activator) for 24 h significantly decreased GLUT-4 mRNA in L6 myotubes. Taken together, these findings suggest that a high-fat diet downregulates GLUT-4 mRNA, possibly through the activation of PPARdelta, despite an increase in PGC-1alpha protein content in rat skeletal muscle, and that a posttranscriptional regulatory mechanism maintains GLUT-4 protein content in skeletal muscle of rats fed a high-fat diet.

Expression of nuclear proto-oncogenes in isoproterenol-induced cardiac hypertrophy.

PubMed

Brand, T; Sharma, H S; Schaper, W

1993-11-01

Rat hearts infused with the beta-adrenergic agonist isoproterenol were examined for the expression of several nuclear proto-oncogenes (c-fos, fosB, c-jun, junB, and junD) and the immediate early gene Egr-1. During the first 24 h after the start of infusion, a strong but transient expression of c-fos was observed. Expression of c-jun and junD were not elevated whereas junB was. By using specific antagonists to the alpha- (prazosin) and beta-adrenergic receptor (propranolol), a beta-adrenoceptor-specific blockade of the isoproterenol-mediated nuclear response was demonstrated. In situ hybridization localized c-fos expression to cardiac myocytes. Labelling was distributed focally in the left and right ventricles, and was strong and homogeneous in the atria. In contrast to beta-adrenergic stimulation, alpha-adrenoceptor stimulation with phenylephrine and norepinephrine caused the induction of c-jun and Egr-1 in addition to the proto-oncogenes induced by isoproterenol. Thus distinct programs of early response gene expression were expressed in response to alpha- versus beta-adrenergic stimulation.

[Experimental study of IFN-alpha and IFN-gamma on reversing ATRA-resistance in MR2 cell line].

PubMed

He, Peng-Cheng; Zhang, Mei; Li, Jing; Cao, Yun-Xin; Cai, Rui-Bo; Liu, Ya-Lin

2007-03-01

To explore the possibility and the possible mechanism of reversing ATRA-resistance in MR2 cells by using IFN-alpha and IFN-gamma in combination with all-trans retinoic acid (ATRA). After MR2 cells(ATRA-resistance cell line) were treated with IFN-alpha, IFN-gamma and ATRA alone or IFN-alpha and IFN-gamma in combination with ATRA respectively, the cell proliferation was tested by MTT colorimetry, the cell differentiation was tested through light microscope, by NBT test and flow cytometry (FCM). The expression of promyelocytic leukemia (PML) protein was observed by indirect immunofluorescence staining. Both IFN-alpha and IFN-gamma could inhibit the proliferation of MR2 cells. The effects were more obviously in both IFN-alpha+ATRA group and IFN-gamma+ATRA group. But there were no significant difference between either IFN-alpha group and IFN-gamma group or IFN-alpha+ATRA group and IFN-gamma+ATRA group (P>0.05). Both IFN could also induce the differentiation of MR2 cells. The effects of IFN-alpha+ATRA group and IFN-gamma+ATRA group were more obvious. However, the differentiation of MR2 cells induced by IFN-gamma+ATRA group was more higher than that by IFN-alpha+ATRA group (P<0.05). Both IFN could induce the expression of PML protein. The reversing effcet of IFN-gamma+ATRA group on ATRA-resistence in MR2 cells are more powerful than that of IFN-alpha+ATRA group, which may be related to the different signal transduction pathway of IFN-alpha and IFN-gamma.

Role of tumor necrosis factor-alpha and platelet-activating factor in neoangiogenesis induced by synovial fluids of patients with rheumatoid arthritis.

PubMed

Lupia, E; Montrucchio, G; Battaglia, E; Modena, V; Camussi, G

1996-08-01

The aim of the present study was to investigate in vivo in a mouse model the stimulation of neoangiogenesis by synovial fluids of patients with rheumatoid arthritis (RA) and to determine the role of tumor necrosis factor (TNF)-alpha and platelet-activating factor (PAF) in the formation of new vessels. Angiogenesis was studied in a mouse model in which Matrigel, injected subcutaneously, was used as a vehicle for the delivery of potential angiogenic stimuli. Synovial fluids of patients with RA but not with osteoarthritis (OA) were shown to induce neoangiogenesis. Since synovial fluid of patients with RA contained significantly higher levels of TNF-alpha-like bioactivity and of PAF than that of patients with OA, the role of these mediators was evaluated by using an anti-TNF-alpha neutralizing monoclonal antibody (mAb) and a PAF receptor antagonist, WEB 2170. When added to Matrigel, anti-TNF-alpha mAb and particularly WEB 2170 significantly reduced neoangiogenesis induced by synovial fluids of RA patients. Moreover, PAF extracted and purified from synovial fluid induced angiogenesis. These results suggest that the neoangiogenesis observed in rheumatoid synovitis may be due, at least in part, to the angiogenic effect of locally produced TNF-alpha and PAF.

NBBA, a synthetic small molecule, inhibits TNF-{alpha}-induced angiogenesis by suppressing the NF-{kappa}B signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Nam Hee; Jung, Hye Jin; Shibasaki, Futoshi

2010-01-15

Nuclear factor-{kappa}B (NF-{kappa}B) is a crucial transcription factor that contributes to cancer development by regulating a number of genes involved in angiogenesis and tumorigenesis. Here, we describe (Z)-N-(3-(7-nitro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)benzylidene) propan-2-amine oxide (NBBA) as a new anti-angiogenic small molecule that targets NF-{kappa}B activity. NBBA showed stronger growth inhibition on human umbilical vein endothelial cells (HUVECs) than on the cancer cell lines we tested. Moreover, NBBA inhibited tumor necrosis factor-alpha (TNF-{alpha})-induced tube formation and invasion of HUVECs. In addition, NBBA suppressed the neovascularization of chorioallantonic membrane from growing chick embryos in vivo. To address the mode of action of the compound, the effectmore » of NBBA on TNF-{alpha}-induced NF-{kappa}B transcription activity was investigated. NBBA suppressed TNF-{alpha}-induced c-Jun N-terminal kinase phosphorylation, which resulted in suppression of transcription of NF-{kappa}B and its target genes, including interleukin-8, interleukin-1{alpha}, and epidermal growth factor. Collectively, these results demonstrated that NBBA is a new anti-angiogenic small molecule that targets the NF-{kappa}B signaling pathway.« less

Alpha-1-acid glycoprotein, its local production and immunopathological participation in experimental pulmonary tuberculosis.

PubMed

Martìnez Cordero, E; Gonzàlez, M M; Aguilar, L D; Orozco, E H; Hernàndez Pando, R

2008-05-01

Alpha-1-acid glycoprotein (AGP) is one of the major acute-phase proteins (APPs). Hepatic production and serum concentrations increase in response to systemic injury, inflammation, or infection. We reported previously that expression of the AGP gene is induced in the liver during experimental pulmonary tuberculosis. Since AGP may also be produced at the infection site and has some immunomodulatory properties, we used a model of progressive pulmonary tuberculosis in Balb/c mice to study the kinetics of AGP production in the lung and its influence on immunopathology. We found that AGP was produced in the lung during experimental tuberculosis. Alveolar macrophages and type II pneumocytes were the most important cellular sources during early infection (days 1-14). From day 21 postinfection, during the progressive phase of the infection, foamy macrophages located in pneumonic areas were the most important source of AGP and 10-fold higher concentrations were found on day 60. In a second part of the study, AGP was inactivated during the progressive phase by the administration of specific blocking antibodies. In comparison with control infected animals, tuberculous mice treated with blocking AGP antibodies showed higher expression of interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in association with significantly reduced bacillary loads and tissue damage. Thus, AGP is produced in the lung during experimental pulmonary tuberculosis and it has immunomodulatory activities, suppressing cell-mediated immunity and facilitating growth of bacilli and disease progression.

Trimethyltin-activated cyclooxygenase stimulates tumor necrosis factor-alpha release from glial cells through reactive oxygen species.

PubMed

Viviani, B; Corsini, E; Pesenti, M; Galli, C L; Marinovich, M

2001-04-15

Exposure of a primary culture of glial cells to the classical neurotoxicant trimethyltin (TMT) results in the release of prostaglandin (PG)E(2) and tumor necrosis factor (TNF)-alpha. Prior treatment of glial cells with either the nonspecific inhibitor of cyclooxygenase and lypoxygenase eicosatetraynoic acid (ETYA) or the cyclooxygenase inhibitor indomethacin completely prevented TMT-induced PGE(2) production and TNF-alpha release, suggesting a role for cyclooxygenase metabolites in TMT-induced TNF-alpha release. Exposure of glial cells to increasing concentrations of PGE(2) or other prostanoids did not increase TNF-alpha synthesis, while the presence of exogenous PGE(2) during treatment of glial cells with TMT actually suppressed TNF-alpha release. The activation of arachidonic acid metabolism produces reactive oxygen species (ROS). Scavenging of ROS by means of the antioxidant trolox prevented the TMT-induced release of TNF-alpha from glial cells, while indomethacin was found to suppress ROS formation induced by 1 microM TMT in glial cells. These results suggest that activation of arachidonic acid metabolism causes TNF-alpha release through the production of ROS rather than PGE(2). Indeed, PGE(2) may exert negative feedback on the release of TNF-alpha. Copyright 2001 Academic Press.

Induction of the nuclear factor HIF-1{alpha} in acetaminophen toxicity: Evidence for oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, Laura P.; Donahower, Brian; Burke, Angela S.

2006-04-28

Hypoxia inducible factor (HIF) controls the transcription of genes involved in angiogenesis, erythropoiesis, glycolysis, and cell survival. HIF-1{alpha} levels are a critical determinant of HIF activity. The induction of HIF-1{alpha} was examined in the livers of mice treated with a toxic dose of APAP (300 mg/kg IP) and sacrificed at 1, 2, 4, 8, and 12 h. HIF-1{alpha} was induced at 1-12 h and induction occurred prior to the onset of toxicity. Pre-treatment of mice with N-acetylcysteine (1200 mg/kg IP) prevented toxicity and HIF-1{alpha} induction. In further studies, hepatocyte suspensions were incubated with APAP (1 mM) in the presence ofmore » an oxygen atmosphere. HIF-1{alpha} was induced at 1 h, prior to the onset of toxicity. Inclusion of cyclosporine A (10 {mu}M), an inhibitor of mitochondrial permeability transition, oxidative stress, and toxicity, prevented the induction of HIF-1{alpha}. Thus, HIF-1{alpha} is induced before APAP toxicity and can occur under non-hypoxic conditions. The data suggest a role for oxidative stress in the induction of HIF-1{alpha} in APAP toxicity.« less

CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.

PubMed

Kasinrerk, W; Baumruker, T; Majdic, O; Knapp, W; Stockinger, H

1993-01-15

In this paper we demonstrate that granulocyte-macrophage CSF (GM-CSF) specifically induces the expression of CD1 molecules, CD1a, CD1b and CD1c, upon human monocytes. CD1 molecules appeared upon monocytes on day 1 of stimulation with rGM-CSF, and expression was up-regulated until day 3. Monocytes cultured in the presence of LPS, FMLP, PMA, recombinant granulocyte-CSF, rIFN-gamma, rTNF-alpha, rIL-1 alpha, rIL-1 beta, and rIL-6 remained negative. The induction of CD1 molecules by rGM-CSF was restricted to monocytes, since no such effect was observed upon peripheral blood granulocytes, PBL, and the myeloid cell lines Monomac1, Monomac6, MV4/11, HL60, U937, THP1, KG1, and KG1A. CD1a mRNA was detectable in rGM-CSF-induced monocytes but not in those freshly isolated. SDS-PAGE and immunoblotting analyses of CD1a mAb VIT6 immunoprecipitate from lysate of rGM-CSF-activated monocytes revealed an appropriate CD1a polypeptide band of 49 kDa associated with beta 2-microglobulin. Expression of CD1 molecules on monocytes complements the distribution of these structures on accessory cells, and their specific induction by GM-CSF strengthens the suggestion that CD1 is a family of crucial structures required for interaction between accessory cells and T cells.

Enhanced anti-inflammatory potential of cinnamate-zinc layered hydroxide in lipopolysaccharide-stimulated RAW 264.7 macrophages

PubMed Central

Adewoyin, Malik; Mohsin, Sumaiyah Megat Nabil; Arulselvan, Palanisamy; Hussein, Mohd Zobir; Fakurazi, Sharida

2015-01-01

Background Cinnamic acid (CA) is a phytochemical originally derived from Cinnamomum cassia, a plant with numerous pharmacological properties. The intercalation of CA with a nanocarrier, zinc layered hydroxide, produces cinnamate-zinc layered hydroxide (ZCA), which has been previously characterized. Intercalation is expected to improve the solubility and cell specificity of CA. The nanocarrier will also protect CA from degradation and sustain its release. The aim of this study was to assess the effect of intercalation on the anti-inflammatory capacity of CA. Methods In this study, the anti-inflammatory activity of ZCA was investigated and compared with that of nonintercalated CA. Evaluations were based on the capacity of ZCA and CA to modulate the release of nitric oxide, prostaglandin E2, interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), IL-1β, and IL-10 in lipopolysaccharide-induced RAW 264.7 cells. Additionally, the expression of proinflammatory enzymes, ie, cyclooxygenase-2, inducible nitric oxide synthase, and nuclear factor kappa B (NF-κB), were examined. Results Although both ZCA and CA downregulated nitric oxide, prostaglandin E2, tumor necrosis factor alpha, IL-1β, and IL-6, ZCA clearly displayed better activity. Similarly, expression of cyclooxygenase-2 and inducible nitric oxide synthase were inhibited in samples treated with ZCA and CA. The two compounds effectively inactivated the transcription factor NF-κB, but the anti-inflammatory cytokine, IL-10, was significantly upregulated by ZCA only. Conclusion The present findings suggest that ZCA possesses better anti-inflammatory potential than CA, while zinc layered hydroxide had little or no effect, and these results were comparable with the positive control. PMID:25995619

Anti-endotoxic shock effects of cyproheptadine in rats.

PubMed

Wang, Lizan; Zhang, Qingzhu; Hu, Xiuzhou; Lun, Ning; Wang, Baosheng; Zhu, Fanhe

2002-03-01

To investigate the antagonistic effect and mechanism of the effect of cyproheptadine (Cyp) on endotoxic shock in rats. Endotoxic shock was produced in rats by i.v. injection of lipopolysaccharides (LPS) (5 mg/kg). Tumor necrosis factor (TNF(alpha)) mRNA expression was assessed by Northern blot. Plasma TNF(alpha) content was measured by radioimmunoassay. Plasma superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured. The intracellular free calcium concentration ([Ca(2+)](i)) in single endothelial cells was determined by laser scanning confocal microscopy (LSCM). Cyp 5 mg/kg injected immediately after i.v. LPS raised the mean arterial blood pressure (MABP) of shocked rats and improved their 24 h survival rate. Meanwhile, Cyp markedly decreased TNF(alpha) mRNA levels in rat liver (18 +/- 10 vs. LPS + saline 38 +/- 10, P < 0.01) as well as plasma TNF(alpha) content [(7.8 +/- 2.4) microg/L vs. LPS + saline (21.5 +/- 3.2) microg/L, P < 0.01)]. It enhanced plasma SOD activity [(1037.2 +/- 112.8) NU/L vs LPS + saline (615.4 +/- 92.6) NU/L, P < 0.01], reduced the MDA content [(5.2 +/- 1.1) micromol/L vs. LPS + saline (9.8 +/- 1.5) micromol/L, P < 0.01], and inhibited TNF(alpha)-induced [Ca(2+)](i) elevation. Cyp exerts an anti-endotoxic shock effect by inhibiting TNF(alpha) gene expression, enhancing SOD activity, reducing lipid peroxidation, and preventing [Ca(2+)](i) overload.

Phosphorylation of tyrosine 720 in the platelet-derived growth factor alpha receptor is required for binding of Grb2 and SHP-2 but not for activation of Ras or cell proliferation.

PubMed Central

Bazenet, C E; Gelderloos, J A; Kazlauskas, A

1996-01-01

Following binding of platelet-derived growth factor (PDGF), the PDGF alpha receptor (alphaPDGFR) becomes tyrosine phosphorylated and associates with a number of signal transduction molecules, including phospholipase Cgamma-1 (PLCgamma-1), phosphatidylinositol 3-kinase (PI3K), the phosphotyrosine phosphatase SHP-2, Grb2, and Src. Here, we present data identifying a novel phosphorylation site in the kinase insert domain of the alphaPDGFR at tyrosine (Y) 720. We replaced this residue with phenylalanine and expressed the mutated receptor (F720) in Patch fibroblasts that do not express the alphaPDGFR. Characterization of the F720 mutant indicated that binding of two proteins, SHP-2 and Grb2, was severely impaired, whereas PLCgamma-1 and PI3K associated to wild-type levels. In addition, mutating Y720 to phenylalanine dramatically reduced PDGF-dependent tyrosine phosphorylation of SHP-2. Since Y720 was required for recruitment of two proteins, we investigated the mechanism by which these two proteins associated with the alphaPDGFR. SHP-2 bound the alphaPDGFR directly, whereas Grb2 associated indirectly, most probably via SHP-2, as Grb2 and SHP-2 coimmunoprecipitated when SHP-2 was tyrosine phosphorylated. We also compared the ability of the wild-type and F720 alphaPDGFRs to mediate a number of downstream events. Preventing the alphaPDGFR from recruiting SHP-2 and Grb2 did not compromise PDGF-AA-induced activation of Ras, initiation of DNA synthesis, or growth of cells in soft agar. We conclude that phosphorylation of the alphaPDGFR at Y720 is required for association of SHP-2 and Grb2 and tyrosine phosphorylation of SHP-2; however, these events are not required for the alphaPDGFR to activate Ras or initiate a proliferative response. In addition, these findings reveal that while SHP-2 binds to both of the receptors, it binds in different locations: to the carboxy terminus of the betaPDGFR but to the kinase insert of the alphaPDGFR. PMID:8943348