Transforming growth factor-alpha (TGF-alpha) in a semisolid dosage form: preservative and vehicle selection.

PubMed

Tan, E L; Shah, H S; Leister, K J; Kozick, L M; Pasciak, P; Vanderlaan, R K; Yu, C D; Patel, B

1993-08-01

The selection of an ideal semisolid vehicle for growth factors presents a challenge. Some antimicrobial agents are known to delay wound healing. The objective of this investigation was to identify appropriate preservatives and vehicles for TGF-alpha. Criteria for acceptance are noninterference with the mitogenic activity of TGF-alpha as well as adequate product preservation. Vehicles considered were o/w creams, ointments, and a gel. Combinations of six preservatives were tested. Selection was determined using both microbial preservative challenge and TGF-alpha mitogenic assay. In the former, 10 species of microorganisms were inoculated into formulation samples. At selected time intervals, it was determined whether colonies decreased, increased, or remained constant. In the mitogenic assay, samples of either preservatives or formulation prototypes were introduced to TGF-alpha-stimulated fibroblast cell cultures. Mitogenesis was determined by measuring 3H-dThd uptake into newly synthesized DNA. As preservatives, sorbic acid and quaternium-15 appear to satisfy both selection criteria. A thermosetting gel appears most promising as vehicle.

The effect of pasteurization on transforming growth factor alpha and transforming growth factor beta 2 concentrations in human milk.

PubMed

McPherson, R J; Wagner, C L

2001-01-01

Transforming growth factor alpha (TGF-alpha) and beta 2 (TGF-beta2) are present in human milk and are involved in growth differentiation and repair of neonatal intestinal epithelia. Heat treatment at 56 degrees C has been shown effective for providing safe banked donor milk, with good retention of other biologically active factors. The purpose of our study was to determine the effect of heat sterilization on TGF-alpha and TGF-beta2 concentrations in human milk. Twenty milk samples were collected from 20 lactating mothers in polypropylene containers and frozen at -20 degrees C for transport or storage. Before heat treatment by holder pasteurization, the frozen milk was thawed and divided into 1-mL aliquots. All samples were heated in an accurately regulated water bath until a holding temperature was achieved, then held for 30 minutes using constant agitation. Holding temperature ranged from 56.5 degrees C to 56.9 degrees C. The milk was then stored at 4 degrees C overnight for analysis the following day. The concentration of TGF-alpha was measured by radioimmunoassay. Mean concentration +/- SD of TGF-alpha in raw milk samples was 119+/-50 pg/mL, range 57 to 234. The mean concentration +/- SD of TGF-alpha in heat treated samples was 113+/-50 pg/mL, range 51 to 227. TGF-alpha concentration was minimally affected by pasteurization, with an overall loss of 6.1%. Of 19 samples, 4 had increased and 15 had decreased concentrations after pasteurization (mean percent SEM: 94%+/-7% of raw milk, range 72%+/-107%). The concentration of acid-activated TGF-beta2 was measured by enzyme-linked immunosorbent assay. Mean concentration +/- SD of TGF-beta2 in raw milk samples was 5624+/-5038 pg/mL, range 195 to 15480. The mean concentration +/- SD of TGF-beta2 in heat-treated samples was 5073+/-4646 pg/mL, range 181 to 15140. TGF-beta2 survived with relatively little loss (0.6%): of 18 samples, 11 had increased and 7 had decreased concentrations after pasteurization (mean percent +/- SEM: 99.4+/-6.7% of raw milk, range 79%-120%). In conclusion, both TGF-alpha and TGF-beta2 were well-preserved in whole milk after holder pasteurization at 56.5 degrees C. The relative increase in growth factor concentration in some of the samples may be attributable to the release of that factor from the cellular and/or fat compartments into the aqueous fraction of human milk. These findings have implications regarding use of donor milk as an alternate source of growth factors and cytokines for the newborn gut when mother's milk is unavailable.

Overexpression of TGF-alpha increases lung tissue hysteresivity in transgenic mice.

PubMed

Pillow, J J; Korfhagen, T R; Ikegami, M; Sly, P D

2001-12-01

Increased transforming growth factor (TGF)-alpha has been observed in neonatal chronic lung disease. Lungs of transgenic mice that overexpress TGF-alpha develop enlarged air spaces and pulmonary fibrosis compared with wild-type mice. We hypothesized that these pathological changes may alter the mechanical coupling of viscous and elastic forces within lung parenchyma. Respiratory impedance was measured in open-chested, tracheostomized adult wild-type and TGF-alpha mice by using the forced oscillation technique (0.25-19.63 Hz) delivered by flexiVent (Scireq, Montreal, PQ). Estimates of airway resistance (Raw), inertance (I), and the coefficients of tissue damping (G(L)) and tissue elastance (H(L)) were obtained by fitting a model to each impedance spectrum. Hysteresivity (eta) was calculated as G(L)/H(L). There was a significant increase in eta (P < 0.01) and a trend to a decrease in H(L) (P = 0.07) of TGF-alpha mice compared with the wild-type group. There was no significant change in Raw, I, or G(L). Structural abnormality present in the lungs of adult TGF-alpha mice alters viscoelastic coupling of the tissues, as evidenced by a change in eta.

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.

Study of the biologic behavior of odontogenic keratocyst and orthokeratinaized odontogenic cyst using TGF-alpha and P53 markers.

PubMed

Deyhimi, Parviz; Hashemzadeh, Zahra

2014-04-01

Odontogenic keratocyst (OKC) is an aggressive cyst, and its recurrence rate is higher than that of other odontogenic cysts. Orthokeratinized odontogenic cyst (OOC) is less aggressive than OKC, but bears the probability of carcinomatous changes. In this study, we evaluated the expression and intensity of P53 and TGF-alpha in order to compare the biologic behavior or probable carcinomatous changes of these two cysts. In this cross-sectional study, 15 OKC and 15 OOC were stained immunohistochemically for P53 and TGF-alpha using the Novolink polymer method. Then, all slides were examined by an optical microscope with 400× magnification, and the stained cells in the basal and parabasal layers were counted. Finally, the results were analyzed by the Mann-Whitney and Wilcoxon tests (P-value<0.05). The difference between the expression of P53 and TGF alpha in the basal layer of OKC and OOC was not statistically significant (P-value>0.05), but the expression of P53 and TGF-alpha in the parabasal layer in OKC was statistically higher compared to OOC (P<0.05). Considering the known role of P53 and TGF-alpha in malignant changes and the higher expression of P53 and TGF-alpha in OKC compared to those in OOC, the probability of carcinomatous changes was higher in OKC than in OOC. Copyright © 2013 Elsevier GmbH. All rights reserved.

Chronic arsenic exposure increases TGFalpha concentration in bladder urothelial cells of Mexican populations environmentally exposed to inorganic arsenic

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

Valenzuela, Olga L.; Germolec, Dori R.; Borja-Aburto, Victor H.

Inorganic arsenic (iAs) is a well-established carcinogen and human exposure has been associated with a variety of cancers including those of skin, lung, and bladder. High expression of transforming growth factor alpha (TGF-{alpha}) has associated with local relapses in early stages of urinary bladder cancer. iAs exposures are at least in part determined by the rate of formation and composition of iAs metabolites (MAs{sup III}, MAs{sup V}, DMAs{sup III}, DMAs{sup V}). This study examines the relationship between TGF-{alpha} concentration in exfoliated bladder urothelial cells (BUC) separated from urine and urinary arsenic species in 72 resident women (18-51 years old) frommore » areas exposed to different concentrations of iAs in drinking water (2-378 ppb) in central Mexico. Urinary arsenic species, including trivalent methylated metabolites were measured by hydride generation atomic absorption spectrometry method. The concentration of TGF-{alpha} in BUC was measured using an ELISA assay. Results show a statistically significant positive correlation between TGF-{alpha} concentration in BUC and each of the six arsenic species present in urine. The multivariate linear regression analyses show that the increment of TGF-{alpha} levels in BUC was importantly associated with the presence of arsenic species after adjusting by age, and presence of urinary infection. People from areas with high arsenic exposure had a significantly higher TGF-{alpha} concentration in BUC than people from areas of low arsenic exposure (128.8 vs. 64.4 pg/mg protein; p < 0.05). Notably, exfoliated cells isolated from individuals with skin lesions contained significantly greater amount of TGF-{alpha} than cells from individuals without skin lesions: 157.7 vs. 64.9 pg/mg protein (p = 0.003). These results suggest that TGF-{alpha} in exfoliated BUC may serve as a susceptibility marker of adverse health effects on epithelial tissue in arsenic-endemic areas.« 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.

Role of astrocytes in reproduction and neuroprotection.

PubMed

Mahesh, Virendra B; Dhandapani, Krishnan M; Brann, Darrell W

2006-02-26

Hypothalamic astrocytes secrete TGF-beta and 3 alpha,5 alpha-tetrahydro progesterone (3 alpha,5 alpha-THP) in culture. When the astrocyte-conditioned medium (ACM) was incubated with the hypothalamic cell line GT1-7, it resulted in the secretion of GnRH. Immunoneutralization with TGF-beta antibody or ultra-filteration with a 10 kDa cut off filter resulted in attenuation of the GnRH releasing ability of ACM, indicating that TGF-beta was a major factor involved with GnRH release. Treatment with estrogens increases TGF-beta secretion. These observations indicate a significant role of astrocytes in GnRH secretion. Serum-deprivation results in the death of GT1-7 neurons in culture and addition of ACM or TGF-beta to the culture, attenuates cell death. The mechanism of protection from cell death appears to involve phosphorylation of MKK4, JNK, c-Jun(Ser63), and enhancement of AP-1 binding. Co-administration of JNK inhibitors, but not MEK inhibitors attenuated ACM or TGF-beta-induced c-Jun(Ser63) phosphorylation and their neuroprotective effects. These studies suggest that astrocytes can protect neurons, at least in part, by the release of TGF-beta and activation of a c-Jun/AP-1 protective pathway.

Regulation of the membrane mucin Muc4 in corneal epithelial cells by proteosomal degradation and TGF-beta.

PubMed

Lomako, Joseph; Lomako, Wieslawa M; Carothers Carraway, Coralie A; Carraway, Kermit L

2010-04-01

MUC4 is a heterodimeric membrane mucin, composed of a mucin subunit ASGP-1 (MUC4alpha) and a transmembrane subunit ASGP-2 (MUC4beta), which has been implicated in the protection of epithelial cell surfaces. In the rat stratified corneal epithelium Muc4 is found predominantly in the most superficial cell layers. Since previous studies in other tissues have shown that Muc4 is regulated by TGF-beta via a proteosomal degradation mechanism, we investigated the regulation of corneal Muc4 in stratified cultures of corneal epithelial cells. Application of proteosome or processing inhibitors led to increases in levels of Muc4, particularly in the basal and intermediate levels of the stratified cultures. These changes were accompanied by increases in Muc4 ubiquitination, chaperone association and incorporation into intracellular aggresomes. In contrast, treatment with TGF-beta resulted in reduced levels of Muc4, which were reversed by proteosome inhibition. The results support a model in which Muc4 precursor is synthesized in all layers of the corneal epithelium, but Muc4 is degraded in basal and intermediate layers by a proteosomal mechanism at least partly dependent on TGF-beta inhibition of Muc4 processing. J. Cell. Physiol. 223: 209-214, 2010. (c) 2009 Wiley-Liss, Inc.

Streptococcal modulation of cellular invasion via TGF-beta1 signaling.

PubMed

Wang, Beinan; Li, Shaoying; Southern, Peter J; Cleary, Patrick P

2006-02-14

Group A Streptococcus (GAS) and other bacterial pathogens are known to interact with integrins as an initial step in a complex pathway of bacterial ingestion by host cells. Efficient GAS invasion depends on the interaction of bound fibronectin (Fn) with integrins and activation of integrin signaling. TGF-beta1 regulates expression of integrins, Fn, and other extracellular matrix proteins, and positively controls the integrin signaling pathway. Therefore, we postulated that TGF-beta1 levels could influence streptococcal invasion of mammalian cells. Pretreatment of HEp-2 cells with TGF-beta1 increased their capacity to ingest GAS when the bacteria expressed fibronectin-binding proteins (M1 or PrtF1). Western blots revealed significant induction of alpha5 integrin and Fn expression by HEp-2 cells in response to TGF-beta1. Increased ingestion of streptococci by these cells was blocked by a specific inhibitor of the TGF-beta1 receptor I and antibodies directed against alpha5 integrin and Fn, indicating that increased invasion depends on TGF-beta1 up-regulation of both the alpha5 integrin and Fn. The capacity of TGF-beta1 to up-regulate integrin expression and intracellular invasion by GAS was reproduced in primary human tonsil fibroblasts, which could be a source of TGF-beta1 in chronically infected tonsils. The relationship between TGF-beta1 and GAS invasion was strengthened by the observation that TGF-beta1 production was stimulated in GAS-infected primary human tonsil fibroblasts. These findings suggest a mechanism by which GAS induce a cascade of changes in mammalian tissue leading to elevated expression of the alpha5beta1 receptor, enhanced invasion, and increased opportunity for survival and persistence in their human host.

Up-regulation of Bcl-2 through hyperbaric pressure transfection of TGF-beta1 ameliorates ischemia-reperfusion injury in rat cardiac allografts.

PubMed

Grünenfelder, Jürg; Miniati, Douglas N; Murata, Seiichiro; Falk, Volkmar; Hoyt, E Grant; Robbins, Robert C

2002-02-01

Oxidative stress after ischemia-reperfusion of cardiac allografts leads to activation of cardiomyocytes and production of cytokines. Bcl-2, an inhibitor of the apoptotic pathway, also has strong antioxidant properties. Ischemia-reperfusion injury after transplantation leads to decreased bcl-2 and increased tumor necrosis factor (TNF)-alpha levels. Transforming growth factor (TGF)-beta1 is known to attenuate ischemia-reperfusion injury and inhibits apoptosis of myofibroblasts. We hypothesize that TGF-beta1, prevents bcl-2 cleavage and increased TNF-alpha production. Rat PVG donor hearts were heterotopically transplanted into ACI recipients. Donor hearts were procured and assigned to groups: (1) intracoronary TGF-beta1 (200 ng/ml) perfusion and pressure at 78 psi for 45 minutes (n = 4); (2) intracoronary TGF-beta1 perfusion and incubation for 45 minutes without pressure (n = 4), (3) saline perfusion and incubation for 45 minutes without pressure (n = 4). Hearts were procured 4 hours after transplantation and analyzed by reverse transcriptase-polymerase chain reaction for bcl-2 mRNA expression, ELISA for TNF-alpha, and for myeloperoxidase activity (MPO). Bcl-2 decreased in untreated animals (bcl-2:G3PDH ratio = 0.85 +/- 0.73 vs 1.16 +/- 0.11, not significant [NS]), whereas TNF-alpha increased to 669.99 +/- 127.09 vs 276.84 +/- 73.65 pg/mg total protein in controls (p < 0.003). In TGF-beta(1) pressure-treated hearts, bcl-2 was up-regulated (2.49 +/- 0.6 vs 1.16 +/- 0.11, controls, p < 0.005), whereas TNF-alpha was unchanged (396.1 +/- 100.38 vs 276.84 +/- 73.65 pg/mg, NS). Hearts treated with TGF-beta1 and pressure showed significant up-regulation of bcl-2 compared with hearts treated with TGF-beta1 without pressure (2.49 +/- 0.6 vs 1.17 +/- 0.6, p < 0.02). MPO showed no differences. Bcl-2 is down-regulated and TNF-alpha up-regulated in this model of ischemia-reperfusion injury. Furthermore, TGF-beta1 is linked to this process and ameliorates reperfusion injury by up-regulating bcl-2 and inhibiting TNF-alpha. Therapeutic overexpression of myocardial TGF-beta1 may be clinically useful to control ischemia-reperfusion injury associated with cardiac transplantation.

Autocrine-derived epidermal growth factor receptor ligands contribute to recruitment of tumor-associated macrophage and growth of basal breast cancer cells in vivo.

PubMed

Nickerson, Nicole K; Mill, Christopher P; Wu, Hsin-Jung; Riese, David J; Foley, John

2013-01-01

Epidermal growth factor receptor (EGFR) expression has been linked to progression of basal breast cancers. Many breast cancer cells harbor the EGFR and produce its family of ligands, suggesting they may participate in autocrine and paracrine signaling with cells of the tumor microenvironment. EGFR ligand expression was profiled in the basal breast cancer cell line MDA-231 where AREG, TGF-alpha, and HBEGF were the three ligands most highly expressed. Autocrine signaling was modulated through silencing or overexpression of these three ligands using lentiviral constructs and the impact measured using motility, proliferation, and cytokine expression assays. Changes in receptor phosphorylation and receptor turnover were examined. Knockdown of AREG or TGF-alpha in vitro resulted in decreased motility (p < 0.05) and decreased expression of macrophage chemoattractants. Overexpression of TGF-alpha increased motility and chemoattractant expression, whereas AREG did not. HBEGF modulation had no effect on any cellular behaviors. All the cells with altered ligand production were inoculated into female athymic nude mice to form mammary fat pad tumors, followed by immunohistochemical analysis for necrosis, angiogenesis, and macrophage recruitment. In vivo, knockdown of AREG or TGF-alpha increased survival (p < 0.001) while decreasing angiogenesis (p < 0.001), tumor growth (p < 0.001), and macrophage attraction (p < 0.001). Overexpression of AREG appeared to elicit a greater effect than TGF-alpha on mammary fat pad tumor growth by increasing angiogenesis (p < 0.001) and macrophage attraction to the tumor (p < 0.01). We propose these changes in mammary tumor growth were the result of increased recruitment of macrophages to the tumor by cells with altered autocrine EGFR signaling. We conclude that AREG and TGF-alpha were somewhat interchangeable in their effects on EGFR signaling; however, TGF-alpha had a greater effect in vitro and AREG had a greater effect in vivo.

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

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.

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.

Monocyte production of transforming growth factor beta in long-term hemodialysis: modulation by hemodialysis membranes.

PubMed

Mege, J L; Capo, C; Purgus, R; Olmer, M

1996-09-01

Cytokines are likely involved in hemodialysis-associated complications such as immunodeficiency and beta 2 microglobulin amyloidosis. Because transforming growth factors beta (TGF beta) exert immunosuppressive effects on lymphocytes, down-modulate monocyte functions, and promote fibrosis, we hypothesize that they participate in the deleterious effects of hemodialysis. We investigated the production of TGF beta 1 and TGF beta 2 by monocytes from controls and patients dialyzed with high-flux cellulose triacetate (CT) and polyacrylonitrile (PAN) membranes. The detection of both TGF beta s required an acidification step, suggesting that they are secreted as latent complexes. The spontaneous production of TGF beta 1 and TGF beta 2 was significantly higher in patients dialyzed with CT or PAN than in controls, but the oversecretion of TGF beta 1 was more sustained in CT-treated patients than in PAN-dialyzed patients. The production of interleukin-6 (IL-6) was increased in both patient groups as compared with controls. In contrast to TGF beta 1, the increase was greater in PAN-treated patients than in CT-treated patients, and the release of tumor necrosis factor alpha (TNF alpha) was increased only in PAN-treated patients. Taken together, our results show that hemodialysis is associated with the oversecretion of monocyte cytokines. Moreover, the type of dialysis membrane specifically affects the balance between the secretion of suppressive cytokines such as TGF beta and that of inflammatory cytokines such as IL-6 and TNF alpha.

Positive and Negative Regulation of Muscle Cell Identity by Members of the hedgehog and TGF-β Gene Families

PubMed Central

Du, Shao Jun; Devoto, Stephen H.; Westerfield, Monte; Moon, Randall T.

1997-01-01

We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-β signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-β superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-β signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-β signals exert competing positive and negative influences on the development of muscle pioneer cells. PMID:9314535

Improvement of macrophage dysfunction by administration of anti-transforming growth factor-beta antibody in EL4-bearing hosts.

PubMed

Maeda, H; Tsuru, S; Shiraishi, A

1994-11-01

An experimental therapy for improvement of macrophage dysfunction caused by transforming growth factor-beta (TGF-beta) was tried in EL4 tumor-bearing mice. TGF-beta was detected in cell-free ascitic fluid from EL4-bearers, but not in that from normal mice, by western blot analysis. The ascites also showed growth-suppressive activity against Mv1Lu cells, and the suppressive activity was potentiated by transient acidification. To investigate whether the functions of peritoneal macrophages were suppressed in EL4-bearers, the abilities to produce nitric oxide and tumor necrosis factor-alpha (TNF-alpha) upon lipopolysaccharide (LPS) stimulation were measured. Both abilities of macrophages in EL4-bearing mice were suppressed remarkably on day 9, and decreased further by day 14, compared with non-tumor-bearing controls. TGF-beta activity was abrogated by administration of anti-TGF-beta antibody to EL4-bearing mice. While a large amount of TGF-beta was detected in ascitic fluid from control EL4-bearers, little TGF-beta was detectable in ascites from EL4-bearers given anti-TGF-beta antibody. Furthermore, while control macrophages exhibited little or no production of nitric oxide and TNF-alpha on LPS stimulation in vitro, macrophages from EL4-bearers administered with anti-TGF-beta antibody showed the same ability as normal macrophages. These results clearly indicate that TGF-beta contributes to macrophage dysfunction and that the administration of specific antibody for TGF-beta reverses macrophage dysfunction in EL4-bearing hosts.

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.

RACK1 binds to Smad3 to modulate transforming growth factor-beta1-stimulated alpha2(I) collagen transcription in renal tubular epithelial cells.

PubMed

Okano, Kazuhiro; Schnaper, H William; Bomsztyk, Karol; Hayashida, Tomoko

2006-09-08

Although it is clear that transforming growth factor-beta1 (TGF-beta1) is critical for renal fibrogenesis, the complexity of the involved mechanisms is increasingly apparent. TGF-beta1 stimulates phosphorylation of Smad2/3 and activates other signaling molecules as well. The molecular link between these other kinases and Smads is not known. We sought new binding partners for Smad3 in renal cells and identified receptor for activated protein kinase C 1 (RACK1) as a novel binding partner of Smad3. The linker region of Smad3 and the tryptophan-aspartic acid repeat 6 and 7 of RACK1 are sufficient for the association. RACK1 also interacts with Smad3 in the human kidney epithelial cell line, HKC. Silencing RACK1 increases transcriptional activity of TGF-beta1-responsive promoter sequences of the Smad binding element (SBE), p3TP-Lux, and alpha2(I) collagen. Conversely, overexpressed RACK1 negatively modulates alpha2(I) collagen transcriptional activity in TGF-beta1-stimulated cells. RACK1 did not affect phosphorylation of Smad3 at the C terminus or in the linker region. However, RACK1 reduced direct binding of Smad3 to the SBE motif. Mutating a RACK1 tyrosine at residue 246, but not at 228, decreased the inhibitory effect of RACK1 on both alpha2(I) collagen promoter activity and Smad binding to SBE induced by TGF-beta1. These results suggest that RACK1 modulates transcription of alpha2(I) collagen by TGF-beta1 through interference with Smad3 binding to the gene promoter.

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 ...

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

Cytokines released from blood monocytes and expressed in mucocutaneous lesions of patients with paracoccidioidomycosis evaluated before and during trimethoprim-sulfamethoxazole treatment.

PubMed

Parise-Fortes, M R; Marques, S A; Soares, A M V C; Kurokawa, C S; Marques, M E A; Peracoli, M T S

2006-04-01

Mucocutaneous lesions in paracoccidioidomycosis are granulomatous and result from tissue responses to Paracoccidioides brasiliensis, the aetiological agent. In this study we investigate the expression of tumour necrosis factor (TNF)-alpha, interleukin (IL)-10 and transforming growth factor (TGF)-beta1 by immunohistochemistry in skin and mucosa lesions from patients with the chronic form of paracoccidioidomycosis, evaluated before and at day 20 of trimethoprim-sulfamethoxazole treatment. Cytokine production by peripheral blood monocytes was also studied by enzyme immunoassay. Intense immunostaining for TNF-alpha was detected in mononuclear cells that infiltrated granulomas in all skin and mucosa lesions before treatment simultaneously with low IL-10 granular deposits in these cells. At day 20 of treatment, there was reduced TNF-alpha and IL-10 deposition. Immunoreactive TGF-beta1 was observed diffusely in the dermis and generally in the cytoplasm of macrophages and giant cells, before treatment, and as increased TGF-beta1 deposits in the fibrosis area at day 20 of treatment. Peripheral blood monocytes from patients with paracoccidioidomycosis, evaluated before treatment, produced high endogenous levels of TNF-alpha, TGF-beta1 and IL-10 in relation to healthy controls. Lipopolysaccharide-stimulated monocytes from patients secreted lower levels of TNF-alpha in both periods of evaluation while no impairment in capacity of IL-10 and TGF-beta production was observed. Trimethoprim-sulfamethoxazole therapy was effective in decreasing fungal load in the lesions, allowing patient immune response to control the infection leading to the healing of the lesions.

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...

An endogenous tryptophan photo-product, FICZ, is potentially involved in photo-aging by reducing TGF-β-regulated collagen homeostasis.

PubMed

Murai, Mika; Tsuji, Gaku; Hashimoto-Hachiya, Akiko; Kawakami, Yoshihito; Furue, Masutaka; Mitoma, Chikage

2018-01-01

Persistent ultraviolet (UV) radiation in the form of sunlight causes photo-aging of the skin by reducing the production of type I collagen, the major constituent of the extracellular matrix of the dermis. Transforming growth factor (TGF)-β transforms dermal fibroblasts into α2-smooth muscle actin (ACTA2)-expressing myofibroblasts. Myofibroblasts produce a precursor form of type I collagen, type I procollagen (collagen I), consisting of pro-alpha1 (produced by the COL1A1 gene) and pro-alpha2 chains (produced by the COL1A2 gene). Smad2/3 is a key downstream molecule of TGF-β signaling. The mechanisms through which UV inhibits collagen I synthesis are not fully understood. 6-Formylindolo[3,2-b]carbazole (FICZ) is an endogenous tryptophan photo-metabolite generated by UV irradiation. FICZ is well known as a high-affinity ligand for aryl hydrocarbon receptor (AHR). However, the physiological roles of FICZ in photo-aging have yet to be addressed. To evaluate the effects of FICZ on the TGF-β-mediated ACTA2 and collagen I expression in normal human dermal fibroblasts (NHDFs). Quantitative real-time polymerase chain reaction and western blot analysis were performed to determine the expression of ACTA2, COL1A1, and COL1A2 in NHDFs with or without FICZ and TGF-β. The phosphorylated Smad2/3 (pSmad2/3) protein levels in cytoplasmic or nuclear portions were investigated by western blot analysis. Immunofluorescence staining was conducted to evaluate pSmad2/3 localization, and F-actin staining with phalloidin was performed to visualize actin polymerization in myofibroblasts. The actions of FICZ on the TGF-β-mediated collagen I expression and nuclear translocation of pSmad2/3 were analyzed in the presence of selective AHR antagonists or in AHR-knockdown NHDFs. We found that FICZ significantly inhibited the TGF-β-induced upregulation of mRNA and protein levels of ACTA2 and collagen I and actin polymerization in myofibroblasts. FICZ did not disturb the phosphorylation of Smad2/3. Notably, FICZ reduced the expression of pSmad2/3 in the nucleus, while it increased that in the cytoplasm, suggesting that it inhibits the nuclear translocation of pSmad2/3 induced by TGF-β. The inhibitory actions of FICZ on the TGF-β-mediated collagen I expression and nuclear translocation of pSmad2/3 were independent of AHR signaling. Another endogenous AHR agonist, kynurenine, also inhibited the TGF-β-mediated ACTA2 and collagen I upregulation in NHDFs in an AHR-independent manner; however, its effects were insignificant in comparison with those of FICZ. These findings suggest that the endogenous photo-product FICZ may be a key chromophore that involves in photo-aging. Downregulation of FICZ signaling is thus a potential strategy to protect against photo-aging. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Functions of TGF-β-exposed plasmacytoid dendritic cells.

PubMed

Saas, Philippe; Perruche, Sylvain

2012-01-01

Plasmacytoid dendritic cells (pDCs) belong to the family of dendritic cells and possess specific features that distinguish them from conventional dendritic cells. For instance, pDC are the main interferon-alpha-secreting cells. Plasmacytoid dendritic cells exert both proinflammatory and regulatory functions. This is attested by the involvement of pDC through interferon-alpha secretion in several autoimmune diseases, and by the implication of pDC in tolerance. The same is true for TGF-β that plays a dual role in inflammation. In this review, we discuss recent data on pDC and TGF-β interactions. As with many cell types, pDCs are able to respond to TGF-β using the classic Smad signaling pathway. In addition, pDCs are capable to secrete TGF-β, in particular in response to TGF-β exposure. Exposure of pDCs to TGF-β prevents type I interferon secretion in response to TLR7/9 ligands. In contrast, the consequences of TGF-β on the antigen-presenting cell capacities of pDC are less clear, since TGF-β-exposed pDCs may lead to both regulatory T-cell and interleukin-17-secreting cell polarization. Here, we discuss the factors that may influence this polarization. We also discuss how pDCs exposed to TGF-β may participate in tolerance induction and maintenance, or, on the contrary, in autoimmune diseases.

Dexamethasone antagonizes IL-4 and IL-10-induced release of IL-1RA by monocytes but augments IL-4-, IL-10-, and TGF-beta-induced suppression of TNF-alpha release.

PubMed

Joyce, D A; Steer, J H; Kloda, A

1996-07-01

The activities of monocyte-derived tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta are potentially modified by IL-1RA and soluble receptors for TNF (sTNF-R), which are themselves monocyte products. IL-4, IL-10, TGF-beta, and glucocorticoids (GC) all suppress the lipopolysaccharide (LPS)-stimulated release of TNF-alpha and IL-1beta but vary in their effects on IL-1RA and sTNF-R. This raises the prospect of interactions between the cytokines and glucocorticoids, which may be antagonistic or additive on IL-1 and TNF activity. We, therefore, studied the interactions of the GC dexamethasone (Dex) with IL-4, IL-10, and transforming growth factor (TGF)-beta on the release of TNF-alpha and IL-1RA by human monocytes and the monocytic THP-1 cell line. Low concentration of Dex (10(-8)-10(-7)M) acted additively with low concentrations of IL-4 (0.01-1 ng/ml), IL-10 (0.01-0.1 U/ml), or TGF-beta (0.01-1 ng/ml) to profoundly suppress LPS-stimulated release of TNF-alpha by whole blood and, to a lesser degree, THP-1 cells. Dex also suppressed spontaneous release of IL-1RA from PBMC and THP-1 cells, whereas IL-4 and IL-10, but not TGF-beta, stimulated release. Dex antagonized the enhanced release in IL-4 and IL-10-stimulated cultures. The capacity to stimulate release of IL-1RA may contribute to the anti-inflammatory potential of IL-4 and IL-10 in monocyte/macrophage-mediated disease. GC, therefore, do not uniquely enhance the suppressive functions of IL-4 and IL-10 on monokine activity. The therapeutic benefit of combinations of GC and IL-4, IL-10 or TGF-beta in disease may depend on the roles of the individual monokines and antagonists in pathogenesis.

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.

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

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.

PPARgamma agonists inhibit TGF-beta-PKA signaling in glomerulosclerosis.

PubMed

Zou, Rong; Xu, Gang; Liu, Xiao-cheng; Han, Min; Jiang, Jing-jing; Huang, Qian; He, Yong; Yao, Ying

2010-01-01

To study the probable mechanisms of the anti-glomerulosclerosis effects induced by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists in rat intraglomerular mesangial cells (MCs). Cells were transfected with the pTAL-PPRE-tk-Luc(+) plasmid and then treated with different concentrations of PPARgamma agonist, either troglitazone or telmisartan, for the indicated times. Promega luciferase assays were subsequently used for the detection of PPARgamma activation. Protein expression levels were assessed by Western blot, and PepTag assays were used for the non-radioactive detection of protein kinase A (PKA) activity. The deposition of alpha-smooth muscle actin (alpha-SMA) and p-cyclic AMP responsive element binding protein (pCREB) were analyzed by confocal laser scanning. Both troglitazone and telmisartan remarkably inhibit the PKA activation and pCREB expression that is stimulated by TGF-beta. The PPARgamma agonists also inhibited alpha-SMA and collagen IV protein expression by blocking PKA activation. PPARgamma ligands effectively suppress the activation of MCs and the accumulation of collagen IV stimulated by TGF-beta in vitro. The renal protection provided by PPARgamma agonists is partly mediated via their blockade of TGF-beta/PKA signaling.

Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition.

PubMed

Elberg, Gerard; Elberg, Dorit; Logan, Charlotte J; Chen, Lijuan; Turman, Martin A

2006-01-01

Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alphaSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-beta1 (TGF-beta1). We report herein the limitations of commonly used reference genes for mRNA quantitation. We determined the expression of alphaSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alphaSMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta1. Thus, depending on which reference gene was used, TGF-beta1 appeared to have different effects on expression of MKL1 and alphaSMA. RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta1 treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression. 2006 S. Karger AG, Basel.

Expression of TNF-alpha, TGF-beta, IP-10 and IL-10 mRNA in kidneys of hamsters infected with pathogenic Leptospira.

PubMed

Lowanitchapat, Alisa; Payungporn, Sunchai; Sereemaspun, Amornpun; Ekpo, Pattama; Phulsuksombati, Duangporn; Poovorawan, Yong; Chirathaworn, Chintana

2010-09-01

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. Although several components of this organism have been identified, the molecular mechanisms underlying pathogenesis of this infectious disease are still poorly understood. Besides, direct injury by microbial factors, cytokines produced in response to infection have been proposed to be involved in pathogenesis of leptospirosis. In this study, cytokine gene expression in kidneys was investigated. Hamsters were injected with pathogenic Leptospira interrogans serovar Pyrogenes and were sacrificed on days 3, 5 and 7 after infection. RNA was extracted from kidney tissues. Real-time PCR was performed to demonstrate expression of TNF-alpha, TGF-beta, IP-10 and IL-10 mRNA in kidneys. TNF-alpha, TGF-beta and IP-10 expression could be demonstrated since day 3 post-infection whereas IL-10 expression was detected later on day 5. Leptospira infection resulted in not only expression of a proinflammatory cytokine, TNF-alpha, but also a T cell chemokine, IP-10. Detection of IP-10 suggested the involvement of T cell recruitment in the immune response or pathology in infected kidneys. Expressions of anti-inflammatory cytokines, TGF-beta and IL-10 were also observed. However, the level of TGF-beta expression was prominent since day 3 post-infection whereas IL-10 expression was clearly observed on day 5. Further experiments will provide additional information whether there is a correlation between the expression of these cytokines and pathologies found in an affected organ. Copyright 2009 Elsevier Ltd. All rights reserved.

Cutting Edge: Active TGF-β1 Released from GARP/TGF-β1 Complexes on the Surface of Stimulated Human B Lymphocytes Increases Class-Switch Recombination and Production of IgA.

PubMed

Dedobbeleer, Olivier; Stockis, Julie; van der Woning, Bas; Coulie, Pierre G; Lucas, Sophie

2017-07-15

Production of active TGF-β is regulated at a posttranslational level and implies release of the mature cytokine dimer from the inactive, latent TGF-β precursor. There are several cell-type specific mechanisms of TGF-β activation. We identified a new mechanism operating on the surface of human regulatory T cells and involving membrane protein GARP, which binds latent TGF-β1. The paracrine activity of regulatory T cell-derived TGF-β1 contributes to immunosuppression and can be inhibited with anti-GARP Abs. Whether other immune cell types use surface GARP to activate latent TGF-β1 was not known. We show in this study that stimulated, human B lymphocytes produce active TGF-β1 from surface GARP/latent TGF-β1 complexes with isotype switching to IgA production. Copyright © 2017 by The American Association of Immunologists, Inc.

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.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

PubMed

Matsumoto, M; Imagawa, M; Aoki, Y

2000-07-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

PubMed Central

Matsumoto, M; Imagawa, M; Aoki, Y

2000-01-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression. PMID:10861232

Pregnancy Specific Glycoprotein 23 binds to CD151 and Induces the Secretion of IL-10 and TGF-beta1 in Murine Macrophages

DTIC Science & Technology

2007-07-11

levels of trophoblast-specific beta-1-globulin (SP1) and alpha -1- fetoprotein (AFP) in pregnant women with rheumatoid arthritis]. Cesk Gynekol, 1991...transforming growth factor-beta TNF-": tumor necrosis factor- alpha TXA: thromboxane A2 uNK: uterine natural killer cell 1 PART ONE...specific glycoprotein, pregnancy-associated plasma protein A, "- fetoprotein , as well as an array of cytokines, including IL-6, and TGF-! [95

SOCS3 promotes TLR4 response in macrophages by feedback inhibiting TGF-beta1/Smad3 signaling.

PubMed

Liu, Xia; Zhang, Yongliang; Yu, Yizhi; Yang, Xiao; Cao, Xuetao

2008-03-01

Endogenous transforming growth factor-beta1 (TGF-beta1) plays an important role in the negative regulation of toll-like receptor (TLR) signaling in a feedback manner. Suppressors of cytokine signaling 3 (SOCS3) has been shown to be induced by TGF-beta1 in osteoclast/macrophage, while the reports on the role of SOCS3 in regulating TLR4 signaling were controversial. The functional relationship between SOCS3 and TGF-beta1/Smad3 pathway in TLR4 response also remains unclear. In this study, we demonstrate that LPS-induced endogenous TGF-beta1 contributes to the inducible SOCS3 expression in macrophages. SOCS3 silencing could markedly decrease the LPS-induced production of TNF-alpha and IL-6 in macrophages. Interestingly, less decrease of LPS-induced TNF-alpha, IL-6 by SOCS3 silencing was observed in Smad3 null macrophages. Furthermore, we found SOCS3 could interact with Smad3, and inhibit Smad3 nuclear translocation and transcriptional activity. Therefore, our data demonstrate that SOCS3 is a positive regulator of TLR4 response by feedback inhibiting endogenous TGF-beta1/Smad3 signaling, thus outlining a new feedback regulatory manner for TLR4 response in macrophages.

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.

Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha6-integrin subunit.

PubMed

Kashimata, M; Gresik, E W

1997-02-01

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate branching morphogenesis of fetal mouse submandibular gland (SMG) rudiments in vitro. The EGF system (EGF, TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in the extracellular matrix. We show here that inhibition of EGFR tyrosine-kinase activity by a tyrphostin retards in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these mRNA were quantitatively determined by nuclease protection assays. These findings suggest that the EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of cultured SMG rudiments was inhibited by anti-alpha6 antibodies. Synthesis of alpha6-subunit in cultured SMGs, detected by metabolic labeling and immunoprecipitation, was increased by EGF and drastically reduced by tyrphostin. RT-PCR revealed that mRNAs for alpha6- and beta1- and beta4-integrin subunits are expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that 1) the EGF system is a physiologic regulator of development of fetal mouse SMG, and 2) one mechanism by which it acts may be by regulating expression of integrins, which in turn control interaction of epithelial cells with the extracellular matrix.

Effects of type I/type II interferons and transforming growth factor-beta on B-cell differentiation and proliferation. Definition of costimulation and cytokine requirements for immunoglobulin synthesis and expression.

PubMed

Estes, D M; Tuo, W; Brown, W C; Goin, J

1998-12-01

In this report, we sought to determine the role of selected type I interferons [interferon-alpha (IFN-alpha) and interferon-tau (IFN-tau)], IFN-gamma and transforming growth factor-beta (TGF-beta) in the regulation of bovine antibody responses. B cells were stimulated via CD40 in the presence or absence of B-cell receptor (BCR) cross-linking. IFN-alpha enhanced IgM, IgG2 and IgA responses but did not enhance IgG1 responses. BCR signalling alone was more effective at inducing IgG2 responses with IFN-alpha than dual cross-linking with CD40. Recombinant ovine IFN-tau was less effective at inducing IgG2 responses when compared with IFN-alpha, though IgA responses were similar in magnitude following BCR cross-linking. At higher concentrations, IFN-tau enhanced IgA responses greater than twofold over the levels observed with IFN-alpha. Previous studies have shown that addition of IFN-gamma to BCR or pokeweed mitogen-activated bovine B cells stimulates IgG2 production. However, following CD40 stimulation alone, IFN-gamma was relatively ineffective at stimulating high-rate synthesis of any non-IgM isotype. Dual cross-linking via CD40 and the BCR resulted in decreased synthesis of IgM with a concomitant increase in IgA and similar levels of IgG2 production to those obtained via the BCR alone. We also assessed the effects of endogenous and exogenous TGF-beta on immunoglobulin synthesis by bovine B cells. Exogenous TGF-beta stimulates both IgG2 and IgA production following CD40 and BCR cross-linking in the presence of IL-2. Blocking endogenous TGF-beta did not inhibit the up-regulation of IgG2 or IgA by interferons.

Deficiency in neuronal TGF-β signaling promotes neurodegeneration and Alzheimer’s pathology

PubMed Central

Tesseur, Ina; Zou, Kun; Esposito, Luke; Bard, Frederique; Berber, Elisabeth; Can, Judith Van; Lin, Amy H.; Crews, Leslie; Tremblay, Patrick; Mathews, Paul; Mucke, Lennart; Masliah, Eliezer; Wyss-Coray, Tony

2006-01-01

Alzheimer’s disease (AD) is characterized by progressive neurodegeneration and cerebral accumulation of the β-amyloid peptide (Aβ), but it is unknown what makes neurons susceptible to degeneration. We report that the TGF-β type II receptor (TβRII) is mainly expressed by neurons, and that TβRII levels are reduced in human AD brain and correlate with pathological hallmarks of the disease. Reducing neuronal TGF-β signaling in mice resulted in age-dependent neurodegeneration and promoted Aβ accumulation and dendritic loss in a mouse model of AD. In cultured cells, reduced TGF-β signaling caused neuronal degeneration and resulted in increased levels of secreted Aβ and β-secretase–cleaved soluble amyloid precursor protein. These results show that reduced neuronal TGF-β signaling increases age-dependent neurodegeneration and AD-like disease in vivo. Increasing neuronal TGF-β signaling may thus reduce neurodegeneration and be beneficial in AD. PMID:17080199

Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts.

PubMed

Teixeira-Salum, Tatiana Beber; Rodrigues, Denise Bertulucci Rocha; Gervásio, Aurélia M; Souza, Cássio J A; Rodrigues, Virmondes; Loyola, Adriano Motta

2010-03-01

Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL-4, TGF-beta, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters. Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme-linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical. TNF-alpha and IFN-gamma were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL-4 was reactive in 24% of cysts, and TGF-beta was positive in all samples. Patients with tenderness showed significantly higher levels of IFN-gamma and IL-4 (P < 0.05). Swelling was associated with high levels of TNF-alpha, IFN-gamma, and IL-4 (P < 0.05). Lesions presenting bone resorption were associated with high levels of NO (P < 0.05). Periapical granulomas display a regulatory environment characterized by high TGF-beta and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN-gamma, TNF-alpha, and IL-4.

Anti-inflammatory and neuroprotective effect of a phytoestrogen compound on rat microglia.

PubMed

Marotta, F; Mao, G S; Liu, T; Chui, D H; Lorenzetti, A; Xiao, Y; Marandola, P

2006-11-01

Ovariectomized Wistar rats received orally 15 mg/kg of a phytoestrogen compound (genistein, daidzein, glycitein, black cohosh, angelica sin., licorice, vitex agnus) for 2 weeks to test its ability to modulate inflammatory microglia response. Microglial proliferation was tested by trypan blue and by absorbance. Serial supernatant sampling was performed for 24 h to check TNF-alpha, IL-beta, IL-6, and TGF-beta. LPS caused a time course increase of all cytokines, with IL-beta and TNF-alpha peaking at the 12th hour, whereas IL-6 and TGF-beta peaked at the 24 h observation. Rats fed with the phytoestrogen displayed a significantly lower level of proinflammatory cytokines and a higher level of TGF-beta, as shown also by Western blot analysis. This finding may offer promise in the field of nutraceutical intervention.

Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication.

PubMed

Organ, Edward L; Nalbantyan, Christopher D; Nanney, Lillian B; Woodward, Stephen C; Sheng, Jinsong; Dubois, Raymond N; Price, James; Sutcliffe, Marilyn; Coffey, Robert J; Rubin, Donald H

2004-07-01

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.

RETINOIC ACID INDUCTION OF CLEFT PALATE IN EGF AND TGF-ALPHA KNOCKOUT MICE: STAGE SPECIFIC INFLUENCES OF GROWTH FACTOR EXPRESSION

EPA Science Inventory

ABBOTT, B. D., LEFFLER, K.E. AND BUCKALEW, A.R, Reproductive Toxicology Division, NHEERL, ORD, US EPA, Research Triangle Park, North Carolina. Retinoic acid induction of cleft palate (CP) in EGF and TGF knockout mice: Stage specific influences of growth factor expression.
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Galectin-1 suppresses alpha2(I) collagen through Smad3 in renal epithelial cells.

PubMed

Okano, K; Uchida, K; Nitta, K; Hayashida, T

2008-10-01

Transforming growth factor (TGF-beta1) promotes renal fibrogenesis through activation of Smads. Galectin-1 is reported to prevent experimental glomerulonephritis. Here we investigated the fact that transfected galectin-1 significantly suppressed the transcription of alpha2(I) collagen (COL1A2) in TGF-beta1- activated human renal epithelial cells. Conversely, galectin-1 silencing RNA reduced secretion of type I collagen by HKC cells. Galectin-1 significantly decreased activation of a TGF-beta1-responsive reporter construct and of a minimal reporter construct that contains four repeats of the Smad binding element (SBE). Galectin-1 had no effect on phosphorylation of Smad3 at the linker region and C-terminus, whereas it decreased affinity of Smad3 to the SBE. Additionally, the inhibitory effect of galectin-1 disappeared using a mutated reporter construct, 376 m-LUC, in which a potential Smad recognition site within the promoter is mutated. Taken together, the results suggest that galectin-1 decreases Smad3-complex from binding to the SBE, down-regulating transcription of COL1A2 in TGF-beta1-stimulated renal epithelial cells.

Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors.

PubMed Central

Paria, B C; Dey, S K

1990-01-01

We have established a model that shows cooperative interaction among preimplantation embryos and the role of growth factors on their development and growth. Two-cell mouse embryos cultured singly in 25-microliters microdrops had inferior development to blastocysts and lower cell numbers per blastocyst compared with those cultured in groups of 5 or 10. The inferior development of singly cultured embryos was markedly improved by addition of epidermal growth factor (EGF) or transforming growth factor alpha or beta 1 (TGF-alpha or TGF-beta 1) to the culture medium. The stage of embryonic development, primarily affected by these treatments, was between eight-cell/morula and blastocyst. Furthermore, blastocysts developed from eight-cell embryos cultured in groups or singly in the presence of EGF showed a higher incidence of zona hatching compared with those cultured singly in the absence of EGF. Detection of EGF receptors on the embryonic cell surface at eight-cell/morula and blastocyst stages suggests beneficial effects of EGF or TGF-alpha on preimplantation embryo development and blastocyst functions. Insulin-like growth factor I (IGF-I) had no influence on embryo development. To further document the cooperative interactions among embryos, the volume of the culture medium was doubled to 50 microliters. This increase in culture volume was even more detrimental to the development of singly cultured embryos. However, this detrimental effect was significantly reversed by EGF and reversed even more markedly by a combination of EGF and TGF-beta 1 but not by TGF-beta 1 alone. Although TGF-beta 1 plus IGF-I caused a modest improvement of embryo development, the response was not as great as shown by EGF alone. Furthermore, IGF-I had no additive effect on EGF-induced embryonic development. The study presents clear evidence that specific growth factors of embryonic and/or reproductive tract origin participate in preimplantation embryo development and blastocyst functions in an autocrine/paracrine manner. Images PMID:2352946

Secretory leukocyte protease inhibitor gene deletion alters bleomycin-induced lung injury, but not development of pulmonary fibrosis.

PubMed

Habgood, Anthony N; Tatler, Amanda L; Porte, Joanne; Wahl, Sharon M; Laurent, Geoffrey J; John, Alison E; Johnson, Simon R; Jenkins, Gisli

2016-06-01

Idiopathic pulmonary fibrosis is a progressive, fatal disease with limited treatment options. Protease-mediated transforming growth factor-β (TGF-β) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi(-/-) mice following injury. Slpi(-/-), and wild-type, mice received oropharyngeal administration of bleomycin (30 IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase (MMP)-2 and MMP-9 were measured. Lung fibrosis was determined by collagen subtype-specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-β activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-β activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi(-/-) animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-β activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1α1), III, alpha 1(Col3α1), IV, alpha 1(Col4α1) mRNA expression, and a significant increase in total lung collagen 28 days post bleomycin. In contrast Slpi(-/-) mice showed no significant increase of alveolar TGF-β activity following bleomycin, above their already elevated levels, although global TGF-β activity did increase. Slpi(-/-) mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-β activation, and inhibits sustained Col1α1, Col3α1, and Col4α1 gene expression following lung injury. However, these changes do not prevent the development of lung fibrosis. Overall, these data suggest that the absence of Slpi does not markedly modify the development of lung fibrosis following bleomycin-induced lung injury.

GARP is regulated by miRNAs and controls latent TGF-β1 production by human regulatory T cells.

PubMed

Gauthy, Emilie; Cuende, Julia; Stockis, Julie; Huygens, Caroline; Lethé, Bernard; Collet, Jean-François; Bommer, Guido; Coulie, Pierre G; Lucas, Sophie

2013-01-01

GARP is a transmembrane protein present on stimulated human regulatory T lymphocytes (Tregs), but not on other T lymphocytes (Th cells). It presents the latent form of TGF-β1 on the Treg surface. We report here that GARP favors the cleavage of the pro-TGF-β1 precursor and increases the amount of secreted latent TGF-β1. Stimulated Tregs, which naturally express GARP, and Th cells transfected with GARP secrete a previously unknown form of latent TGF-β1 that is disulfide-linked to GARP. These GARP/TGF-β1 complexes are possibly shed from the T cell surface. Secretion of GARP/TGF-β1 complexes was not observed with transfected 293 cells and may thus be restricted to the T cell lineage. We conclude that in stimulated human Tregs, GARP not only displays latent TGF-β1 at the cell surface, but also increases its secretion by forming soluble disulfide-linked complexes. Moreover, we identified six microRNAs (miRNAs) that are expressed at lower levels in Treg than in Th clones and that target a short region of the GARP 3' UTR. In transfected Th cells, the presence of this region decreased GARP levels, cleavage of pro-TGF-β1, and secretion of latent TGF-β1.

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.

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.

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

Molecular Basis for the Recognition and Cleavages of IGF-II, TGF-[alpha], and Amylin by Human Insulin-Degrading Enzyme

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

Guo, Qing; Manolopoulou, Marika; Bian, Yao

2010-02-11

Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-{beta}, peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulin-like growth factor (IGF)-II and transforming growth factor-{alpha} (TGF-{alpha}) over IGF-I and epidermal growth factor, respectively. Here, we used high-accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-{alpha}, amylin, reduced amylin, and amyloid-{beta} bymore » human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-{alpha} at 2.3 {angstrom} and IDE-amylin at 2.9 {angstrom}. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (amino acids 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and epidermal growth factor families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.« less

TERATOGENIC EFFECTS OF RETINOIC ACID ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR AND TRANSFORMING GROWTH FACTOR-ALPHA

EPA Science Inventory

Background: EGF and TGF regulate cell proliferation and differentiation in the embryo. The induction of cleft palate (CP) by all trans retinoic acid (RA) was associated with altered expression of TGF, EGF receptor and binding of EGF. The present study uses knockout (KO) mice to e...

TGF-β and BMP Signaling in Osteoblast Differentiation and Bone Formation

PubMed Central

Chen, Guiqian; Deng, Chuxia; Li, Yi-Ping

2012-01-01

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation. PMID:22298955

Proinflammatory and anti-inflammatory cytokines present in the acute phase of experimental colitis treated with Saccharomyces boulardii.

PubMed

Grijó, Nathália Nahas; Borra, Ricardo Carneiro; Sdepanian, Vera Lucia

2010-09-01

To study the proinflammatory and anti-inflammatory cytokines present in the acute phase of trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis treated with Saccharomyces boulardii. Thirty male Wistar rats were divided into three groups: (1) treated group--received Saccharomyces boulardii for 14 days; (2) non-treated group--received sodium chloride solution for 14 days; (3) control group. Colitis was induced on the seventh day of the study in the treated and the non-treated groups using TNBS (10 mg) dissolved in 50% ethanol. Quantification of cytokines, including interleukin (IL)-1beta (IL-1beta), IL-6, transforming growth factor-beta (TGF-beta), IL-10 and tumor necrosis factor-alpha (TNF-alpha), in the serum and colonic tissue collected on day 14 were carried out using an enzyme-linked immunosorbent assay (ELISA). The mean concentrations of TGF-beta in both the serum and the colonic tissue of the treated group were statistically higher than that of the control group. The mean concentration of TGF-beta in the colonic tissue of the non-treated group was also statistically higher than the control group. The group treated with Saccharomyces boulardii showed increased amounts of TGF-beta, an anti-inflammatory cytokine, during the acute phase of colitis. There were no differences in the amount of TNF-alpha, IL-1beta, IL-6, and IL-10 between the treated and the non-treated or the control groups during the acute phase of experimental colitis induced by TNBS.

Non-Smad TGF-β signaling components are possible biomarkers of tamoxifen resistance

NASA Astrophysics Data System (ADS)

Babyshkina, N.; Zavyalova, M.; Patalyak, S.; Dronova, T.; Slonimskaya, E.; Cherdyntseva, N.

2017-09-01

A crosstalk between the estrogen receptor alpha (ERα) and tyrosine kinase receptors contribute to endocrine resistance. We investigated the effect of the four Smad-independent TGF-β signaling components and the distribution pattern of ERα expression on the response to adjuvant tamoxifen treatment in 122 estrogen positive breast cancer patients. We identified a low mRNA expression of TGF-βR1 in tamoxifen resistant group patients (TR) in contrast to tamoxifen sensitive group (TS). Similarly, negative TGF-βR1 expression was significantly higher in TR patients than in TS patients. The expression of TGF-βR1 was strongly correlated with the distribution pattern of ERα expression, level of CD44+/CD24-/low cells and Akt (pS473) expression. The patients with a low mRNA expression of TGF-βR1 as well as with a negative TGF-βR1 expression had an unfavorable prognosis concerning progression-free survival. The expression of TGF-βR1 and the distribution pattern of ERα expression can be considered as additional molecular predictive markers for estrogen positive breast cancer patients treated with adjuvant tamoxifen.

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.

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.

LACK OF EXPRESSION OF EGF AND TGF-ALPHA IN THE FETAL MOUSE ALTERS FORMATION OF PROSTATIC EPITHELIAL BUDS AND INFLUENCES THE RESPONSE TO TCDD

EPA Science Inventory

Lack of Expression of EGF and TGF in the Fetal Mouse Alters Formation of Prostatic Epithelial Buds and Responsiveness to TCDD-Induced Impairment of Prostatic Bud Formation.

Barbara D. Abbott, Tien-Min Lin, Nathan T. Rasmussen, Robert W. Moore,
Ralph M. Albrecht, Judi...

Inhibition of arsenic induced-rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-{beta}/Smad activation

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

Pan Xinjuan; Dai Yujie; Li Xing

2011-08-01

Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30 ppm) with or without GSE (100 mg/kg, every other day by oral gavage) for 12 months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3more » phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-{beta}1, type I procollagen (Coll-I) and {alpha}-smooth muscle actin ({alpha}-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-{beta}1-induced transactivation of the TGF-{beta}-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-{beta}1-induced mRNA expression of Coll-I and {alpha}-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-{beta}/Smad activation. - Research Highlights: > GSE attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. > GSE reduced arsenic-mediated Smad2/3 phosphorylation and NADPH oxidase subunits (Nox2, Nox4 and p47phox). > Beneficial effects of GSE on As-induced liver injury was via inhibition of NADPH oxidase and TGF-{beta}/Smad activation.« less

Proliferation of NS0 cells in protein-free medium: the role of cell-derived proteins, known growth factors and cellular receptors.

PubMed

Spens, Erika; Häggström, Lena

2009-05-20

NS0 cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may open for the novel approaches to improve animal cell processes. The following proteins were identified in NS0 conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6, TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11, IL-25, MIF, TGF-beta and TNF-alpha as well as the known growth factors EGF, IGF-I/II, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NS0 cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NS0 cell membranes since TGF-beta(1) caused cell death, and IGF-I/II and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NS0 cells as demonstrated by siRNA gene silencing.

Regulatory T cell frequency in patients with melanoma with different disease stage and course, and modulating effects of high-dose interferon-alpha 2b treatment.

PubMed

Ascierto, Paolo A; Napolitano, Maria; Celentano, Egidio; Simeone, Ester; Gentilcore, Giusy; Daponte, Antonio; Capone, Mariaelena; Caracò, Corrado; Calemma, Rosa; Beneduce, Gerardo; Cerrone, Margherita; De Rosa, Vincenzo; Palmieri, Giuseppe; Castello, Giuseppe; Kirkwood, John M; Marincola, Francesco M; Mozzillo, Nicola

2010-08-16

High-dose interferon-alpha 2b (IFN-alpha 2b) is the only approved systemic therapy in the United States for the adjuvant treatment of melanoma. The study objective was to explore the immunomodulatory mechanism of action for IFN-alpha 2b by measuring serum regulatory T cell (Treg), serum transforming growth factor-beta (TGF-beta), interleukin (IL)-10, and autoantibody levels in patients with melanoma treated with the induction phase of the high-dose IFN-alpha 2b regimen. Patients with melanoma received IFN-alpha 2b administered intravenously (20 MU/m2 each day from day 1 to day 5 for 4 consecutive weeks). Serum Treg levels were measured as whole lymphocytes in CD4+ cells using flow cytometry while TGF-beta, IL-10, and autoantibody levels were measured using enzyme-linked immunosorbent assays. Twenty-two patients with melanoma received IFN-alpha 2b treatment and were evaluated for Treg levels. Before treatment, Treg levels were significantly higher in patients with melanoma when compared with data from 20 healthy subjects (P = 0.001; Mann-Whitney test). Although a trend for reduction of Treg levels following IFN-α 2b treatment was observed (average decrease 0.29% per week), statistical significance was not achieved. Subgroup analyses indicated higher baseline Treg levels for stage III versus IV disease (P = 0.082), early recurrence versus no recurrence (P = 0.017), deceased versus surviving patients (P = 0.021), and preoperative neoadjuvant versus postoperative adjuvant treatment groups (not significant). No significant effects were observed on the levels of TGF-beta, IL-10, and autoantibodies in patients with melanoma treated with IFN-alpha 2b. Patients with melanoma in this study showed increased basal levels of Treg that may be relevant to their disease and its progression. Treg levels shifted in patients with melanoma treated with IFN-alpha 2b, although no firm conclusions regarding the role of Tregs as a marker of treatment response or outcome can be made at present.

Attenuation of CCl4-Induced Hepatic Fibrosis in Mice by Vaccinating against TGF-β1

PubMed Central

Li, Shuang; Lv, Yifei; Su, Houqiang; Jiang, Huiping; Hao, Zhiming

2013-01-01

Transforming growth factor β1 (TGF-β1) is the pivotal pro-fibrogenic cytokine in hepatic fibrosis. Reducing the over-produced expression of TGF-β1 or blocking its signaling pathways is considered to be a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the feasibility of attenuating hepatic fibrosis by vaccination against TGF-β1 with TGF-β1 kinoids. Two TGF-β1 kinoid vaccines were prepared by cross-linking TGF-β1-derived polypeptides (TGF-β125–[41-65] and TGF-β130–[83-112]) to keyhole limpet hemocyanin (KLH). Immunization with the two TGF-β1 kinoids efficiently elicited the production of high-levels of TGF-β1-specific antibodies against in BALB/c mice as tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The antisera neutralized TGF-β1-induced growth-inhibition on mink lung epithelial cells (Mv1Lu) and attenuated TGF-β1-induced Smad2/3 phosphorylation, α-SMA, collagen type 1 alpha 2 (COL1A2), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in the rat hepatic stellate cell (HSC) line, HSC-T6. Vaccination against TGF-β1 with the kinoids significantly suppressed CCl4-induced collagen deposition and the expression of α-SMA and desmin, attenuated hepatocyte apoptosis and accelerated hepatocyte proliferation in BALB/c mice. These results demonstrated that immunization with the TGF-β1 kinoids efficiently attenuated CCl4-induced hepatic fibrosis and liver injury. Our study suggests that vaccination against TGF-β1 might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases. PMID:24349218

Attenuation by all-trans-retinoic acid of sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

PubMed

Tatsuta, M; Iishi, H; Baba, M; Hirasawa, R; Yano, H; Sakai, N; Nakaizumi, A

1999-02-01

The effect of prolonged administration of all-trans-retinoic acid (RA) on sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine, and the labelling and apoptotic indices and immunoreactivity of transforming growth factor (TGF) alpha in the gastric cancers was investigated in Wistar rats. After 25 weeks of carcinogen treatment, the rats were given chow pellets containing 10% sodium chloride and subcutaneous injections of RA at doses of 0.75 or 1.5 mg kg(-1) body weight every other day. In week 52, oral supplementation with sodium chloride significantly increased the incidence of gastric cancers compared with the untreated controls. Long-term administration of RA at both doses significantly reduced the incidence of gastric cancers, which was enhanced by oral administration of sodium chloride. RA at both doses significantly decreased the labelling index and TGF-alpha immunoreactivity of gastric cancers, which were enhanced by administration of sodium chloride, and significantly increased the apoptotic index of cancers, which was lowered by administration of sodium chloride. These findings suggest that RA attenuates gastric carcinogenesis, enhanced by sodium chloride, by increasing apoptosis, decreasing DNA synthesis, and reducing TGF-alpha expression in gastric cancers.

Boldine Improves Kidney Damage in the Goldblatt 2K1C Model Avoiding the Increase in TGF-β.

PubMed

Gómez, Gonzalo I; Velarde, Victoria

2018-06-25

Boldine, a major aporphine alkaloid found in the Chilean boldo tree, is a potent antioxidant. Oxidative stress plays a detrimental role in the pathogenesis of kidney damage in renovascular hypertension (RVH). The activation of the renin-angiotensin system (RAS) is crucial to the development and progression of hypertensive renal damage and TGF-β is closely associated with the activation of RAS. In the present study, we assessed the effect of boldine on the progression of kidney disease using the 2K1C hypertension model and identifying mediators in the RAS, such as TGF-β, that could be modulated by this alkaloid. Toward this hypothesis, rats ( n = 5/group) were treated with boldine (50 mg/kg/day, gavage) for six weeks after 2K1C surgery (pressure ≥ 180 mmHg). Kidney function was evaluated by measuring of proteinuria/creatininuria ratio (U prot/U Crea), oxidative stress (OS) by measuring thiobarbituric acid reactive substances (TBARS). The evolution of systolic blood pressure (SBP) was followed weekly. Alpha-smooth muscle actin (α-SMA) and Col III were used as markers of kidney damage; ED-1 and osteopontin (OPN) were used as markers of inflammation. We also explored the effect in RAS mediators, such as ACE-1 and TGF-β. Boldine treatment reduced the UProt/UCrea ratio, plasma TBARS, and slightly reduced SBP in 2K1C hypertensive rats, producing no effect in control animals. In 2K1C rats treated with boldine the levels of α-SMA, Col III, ED-1, and OPN were lower when compared to 2K1C rats. Boldine prevented the increase in ACE-1 and TGF-β in 2K1C rats, suggesting that boldine reduces kidney damage. These results suggest that boldine could potentially be used as a nutraceutic.

Regulation of GM-CSF-induced dendritic cell development by TGF-beta1 and co-developing macrophages.

PubMed

Yamaguchi, Y

1998-01-01

Using a culture system of bone marrow progenitor cells with GM-CSF and TGF-beta1, a study was performed to analyze the effect of TGF-beta1 on the development of dendritic cells (DC) and to elucidate the regulatory role of macrophages co-developing with dendritic cells. The results demonstrate that DC generated in the presence of TGF-beta1 were immature with respect to the expression of CD86, nonspecific esterase activity and cell shape. Such inhibitory effects of TGF-beta1 were dependent on FcR+ macrophages, which were depleted by panning. TGF-beta1 did not appear to inhibit the commitment of progenitor cells to the DC lineage. In addition, TGF-beta1 also acted directly on the intermediate stage of DC to prevent their over-maturation, which results in a preferential decrease in MHC class II, but not in CD86, in the presence of TNF-alpha. FcR+ suppressive macrophages were also shown to facilitate DC maturation when stimulated via FcR-mediated signals even in the presence of TGF-beta1. These results indicate that TGF-beta1 indirectly and directly regulate the development of DC and that co-developing macrophages have a regulatory role in DC maturation.

Fibronectin regulates the activation of THP-1 cells by TGF-beta1.

PubMed

Wang, A C; Fu, L

2001-03-01

To determine how fibronectin regulates the immunomodulatory effects of transforming growth factor (TGF)-beta on THP-1 cells. THP-1 monocytic cell line. THP-1 cells were primed for 48 h in the presence or absence of 250 pM TGF-beta1. Assays or assessments carried out, together with statistical test applied. We found that adherence to fibronectin dramatically modulates the effects of TGF-beta1 on the human monocytic cell line THP-1. TGF-beta did not significantly affect constitutive interleukin (IL)-8 secretion or IL-1beta-induced IL-8 secretion from suspended cells. In contrast, TGF-beta stimulated IL-8 secretion as well as augmented IL-1beta-induced IL-8 secretion from adherent cells. The differential effects of TGF-beta1 on IL-8 secretion from suspended and adherent cells could not be explained by differences in IL-1 receptor antagonist production. The effects of fibronectin on TGF-beta1 induced IL-8 secretion from THP-1 cells were mimicked by adhesion to immobilized anti-a4beta1 integrin antibody and to a fibronectin fragment containing the CS-1 domain. These results indicate that alpha4beta1-mediated adhesion to fibronectin may play a key role during inflammation by profoundly influencing the effects of TGF-beta1 on monocytes.

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.

Inhibition of the TGF-β receptor I kinase promotes hematopoiesis in MDS

PubMed Central

Zhou, Li; Nguyen, Aaron N.; Sohal, Davendra; Ying Ma, Jing; Pahanish, Perry; Gundabolu, Krishna; Hayman, Josh; Chubak, Adam; Mo, Yongkai; Bhagat, Tushar D.; Das, Bhaskar; Kapoun, Ann M.; Navas, Tony A.; Parmar, Simrit; Kambhampati, Suman; Pellagatti, Andrea; Braunchweig, Ira; Zhang, Ying; Wickrema, Amittha; Medicherla, Satyanarayana; Boultwood, Jacqueline; Platanias, Leonidas C.; Higgins, Linda S.; List, Alan F.; Bitzer, Markus

2008-01-01

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2, a downstream mediator of transforming growth factor–β (TGF-β) receptor I kinase (TBRI) activation, is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34+ cells, providing direct evidence of overactivation of TGF-β pathway in this disease. Suppression of the TGF-β signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor, SD-208, inhibits smad2 activation in hematopoietic progenitors, suppresses TGF-β–mediated gene activation in BM stromal cells, and reverses TGF-β–mediated cell-cycle arrest in BM CD34+ cells. Furthermore, SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-β1. Moreover, in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-β signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS. PMID:18474728

Transforming Growth Factor β-1 Stimulates Profibrotic Epithelial Signaling to Activate Pericyte-Myofibroblast Transition in Obstructive Kidney Fibrosis

PubMed Central

Wu, Ching-Fang; Chiang, Wen-Chih; Lai, Chun-Fu; Chang, Fan-Chi; Chen, Yi-Ting; Chou, Yu-Hsiang; Wu, Ting-Hui; Linn, Geoffrey R.; Ling, Hong; Wu, Kwan-Dun; Tsai, Tun-Jun; Chen, Yung-Ming; Duffield, Jeremy S.; Lin, Shuei-Liong

2014-01-01

Pericytes have been identified as the major source of precursors of scar-producing myofibroblasts during kidney fibrosis. The underlying mechanisms triggering pericyte-myofibroblast transition are poorly understood. Transforming growth factor β-1 (TGF-β1) is well recognized as a pluripotent cytokine that drives organ fibrosis. We investigated the role of TGF-β1 in inducing profibrotic signaling from epithelial cells to activate pericyte-myofibroblast transition. Increased expression of TGF-β1 was detected predominantly in injured epithelium after unilateral ureteral obstruction, whereas downstream signaling from the TGF-β1 receptor increased in both injured epithelium and pericytes. In mice with ureteral obstruction that were treated with the pan anti–TGF-β antibody (1D11) or TGF-β receptor type I inhibitor (SB431542), kidney pericyte-myofibroblast transition was blunted. The consequence was marked attenuation of fibrosis. In addition, epithelial cell cycle G2/M arrest and production of profibrotic cytokines were both attenuated. Although TGF-β1 alone did not trigger pericyte proliferation in vitro, it robustly induced α smooth muscle actin (α-SMA). In cultured kidney epithelial cells, TGF-β1 stimulated G2/M arrest and production of profibrotic cytokines that had the capacity to stimulate proliferation and transition of pericytes to myofibroblasts. In conclusion, this study identified a novel link between injured epithelium and pericyte-myofibroblast transition through TGF-β1 during kidney fibrosis. PMID:23142380

Phthalimide neovascular factor 1 (PNF1) modulates MT1-MMP activity in human microvascular endothelial cells.

PubMed

Wieghaus, Kristen A; Gianchandani, Erwin P; Neal, Rebekah A; Paige, Mikell A; Brown, Milton L; Papin, Jason A; Botchwey, Edward A

2009-07-01

We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode of action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray data set of PNF1-treated (vs. control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-alpha) and, subsequently, transforming growth factor-beta (TGF-beta) signaling networks by PNF1. Here we validate this microarray data set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this data set and identify three specific TGF-beta-induced genes with regulation by PNF1 conserved over multiple timepoints-amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)-that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1-48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode of action and important for successful promotion of the neovascularization that we have observed by the drug in vivo.

Growth and differentiation factor 11 (GDF11): Functions in the regulation of erythropoiesis and cardiac regeneration.

PubMed

Rochette, Luc; Zeller, Marianne; Cottin, Yves; Vergely, Catherine

2015-12-01

Members of the TGF-β superfamily transduce their signals through type I and II receptor serine/threonine kinases. The regulation of members of the TGF-β family is known to be complex, because many proteins able to bind the ligands and inhibit their activities have been identified. Growth and differentiation factor 11 (Gdf11) as activins belong to the TGF-β family. GDF11, like other members of the TGF-β superfamily, is produced from precursor proteins by proteolytic processing. The binding of activins to activin type IIA (ActRIIA) or type IIB (ActRIIB) receptors induces the recruitment and phosphorylation of an activin type I receptor which then phosphorylates the Smad2 and Smad3 intracellular signaling proteins. GDF11 signal through the ActRIIB pathway. Recent studies have reported that GDF11-ActRIIB-Smad2/3-dependent signaling is a key regulatory mechanism in proliferating erythroid precursors as it controls their late-stage maturation. The administration of GDF11 is effective in experimental cardiac hypertrophy, and the identification of GDF11 as a "rejuvenating factor" opens up perspectives for the treatment of age-related cardiac dysfunction. Recent studies of the heart indicate that exposure to young blood reverses age-related impairments. GDF11 could be one of the circulating molecules that influence the aging of different tissues. Is GDF11 an "elixir of youth"? Copyright © 2015 Elsevier Inc. All rights reserved.

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

Regulation of the expression of GARP/latent-TGF-β1 complexes on mouse T cells and their role in Regulatory T Cell and Th17 differentiation1

PubMed Central

Edwards, Justin P.; Fujii, Hodaka; Zhou, Angela X.; Creemers, John; Unutmaz, Derya; Shevach, Ethan M.

2013-01-01

GARP/LRRC32 has previously been defined as a marker of activated human regulatory T-cells (Tregs) that is responsible for surface localization of latent TGF-β1. We find that GARP and latent TGF-β1 are also found on mouse Tregs activated via TCR stimulation, but in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-β1 and TGF-β1 loading into GARP and is independent of furin-mediated processing of pro-TGF-β1 to latent TGF-β1. Specific deletion of GARP in CD4+ T cells results in lack of expression of latent-TGF-β1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of T conventional cells in vitro. Activated Tregs expressing GARP/latent-TGF-β1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17 producing cells and Treg is preferentially induced by Tregs expressing the latent-TGF-β1/GARP complex on their cell surface rather than by secreted latent-TGF-β1. PMID:23645881

Regulation of the expression of GARP/latent TGF-β1 complexes on mouse T cells and their role in regulatory T cell and Th17 differentiation.

PubMed

Edwards, Justin P; Fujii, Hodaka; Zhou, Angela X; Creemers, John; Unutmaz, Derya; Shevach, Ethan M

2013-06-01

GARP/LRRC32 was defined as a marker of activated human regulatory T cells (Tregs) that is responsible for surface localization of latent TGF-β1. We find that GARP and latent TGF-β1 are also found on mouse Tregs activated via TCR stimulation; however, in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus, and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-β1 and TGF-β1 loading into GARP and is independent of furin-mediated processing of pro-TGF-β1 to latent TGF-β1. Specific deletion of GARP in CD4(+) T cells results in lack of expression of latent TGF-β1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of conventional T cells in vitro. Activated Tregs expressing GARP/latent TGF-β1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17-producing cells and Tregs is caused preferentially by Tregs expressing the latent TGF-β1/GARP complex on their cell surface rather than by secreted latent TGF-β1.

[Effect of multi-glycoside of Tripterygium wilfordii Hook. f. in intervening TGF-beta1/Smad signaling pathway of adriamycin-induced nephropathy model rat].

PubMed

Wan, Yi-gang; Sun, Wei; Dou, Chen-hui

2011-04-01

To explore the potential molecular mechanisms of multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) for ameliorating glomerulosclerosis (GS) by observing its intervention effect on transforming growth factor (TGF)-beta1/Smad signaling pathway in adriamycin-induced nephropathy (ADRN) model rat. Fifteen female Sprague-Dawley (SD) rats were randomly divided into three groups, the sham-operation group (A), the untreated model group (B), and the GTW treated model group (C). Rats in Group B and C were made into ADRN model by right nephrectomy and intravenous injection of adriamycin (ADR, 0. 4 mL and 0. 2 mL respectively in 4 weeks). After the model was successfully established, rats in Group C were orally given GTW (50 mg/kg per day), while rats in Group B were intervened with distilled water. The intervention for two groups was 6 weeks. Rats' body weight were weighed and 24 h urinary protein excretion (Upro) detected by the end of the 2nd, 4th, 8th and 10th week. All rats were sacrificed at the end of 10th week after operation to withdraw blood and kidney tissue to examine serum biochemical parameters, glomerular morphological changes, alpha-smooth muscle actin (alpha-SMA), and collagen type I expression. Besides, the mRNA expressions of TGF-beta1, Smad3 and Smad7, as well as protein expressions of TGF-beta1, and phosphorylated Smad2/3 (p-Smad2/3) in glomeruli were detected by RT-PCR or Western blotting. As compared with Group B, in Group C, Upro and serum albumin were improved significantly, but no difference between groups was found in levels of blood urea nitrogen(BUN), serum creatinine(SCr), or hepatic cell injury. Mesangial cell proliferation, extracellular matrix (ECM) and collagen deposition were suppressed by GTW. Expressions of alpha-SMA and collagen type I decreased, and the characteristic changes of GS were attenuated. The mRNA expressions of TGF-P,31, Smad3 and protein expression of TGF-beta1, p-Smad2/3 in renal tissues were down-regulated, while the protein expression of Smad7 mRNA was up-regulated. GTW showed effect in ameliorating GS in vivo. It could reduce the ECM deposition and improve GS by way of intervening TGF-beta1/Smad signaling pathway in the kidney through regulating the mRNA or protein expressions of key signal molecules, such as Smad3 and p-Smad2/3.

L-Cysteine-induced up-regulation of the low-density lipoprotein receptor is mediated via a transforming growth factor-alpha signalling pathway.

PubMed

Tanaka, Yuma; Shimada, Masaya; Nagaoka, Satoshi

2014-02-14

Sulphur-containing amino acids regulate plasma cholesterol levels in animals and humans. However, their mechanism of action remains unclear. Low-density lipoprotein receptor (LDLR) plays an important role in cholesterol metabolism. We therefore investigated the effects of sulphur-containing amino acids on the expression of LDLR in hepatocytes. HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium with or without sulphur-containing amino acids and cysteine-containing compounds. We found that L-cysteine increased LDLR mRNA and enhanced LDLR gene promoter activity through the extracellular-signal-related kinase and p38 mitogen-activated protein kinase signalling pathways in HepG2 cells. Moreover, we observed that L-cysteine stimulated the release of transforming growth factor-alpha (TGF-α) and that TGF-α increased the LDLR mRNA levels. This study provides a report of the L-cysteine mediated up-regulation of the LDLR expression via TGF-α signalling pathway. Our findings provide insights into cholesterol homeostasis and amino acid signalling. Copyright © 2014 Elsevier Inc. All rights reserved.

Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein

PubMed Central

1995-01-01

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation. PMID:7593177

Effect of doxycycline on epithelial-mesenchymal transition via the p38/Smad pathway in respiratory epithelial cells.

PubMed

Shin, Jae-Min; Kang, Ju-Hyung; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2017-03-01

Doxycycline has antibacterial and anti-inflammatory effects, and it also suppresses collagen biosynthesis. This study aimed to confirm the effects and mechanism of doxycycline on transforming growth factor (TGF) beta 1 induced epithelial-mesenchymal transition and cell migration in A549 and primary nasal epithelial cells. A 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay and phalloidin-fluorescein isothiocyanate staining were used to evaluate cytotoxicity and cellular morphologic changes. Western blot and immunofluorescence staining were used to determine the expression levels of E-cadherin, vimentin, alpha-smooth muscle actin, fibronectin, phosphorylated Smad2/3, and mitogen-activated protein kinases. Scratch and transwell migration assays were used to assess cellular migration ability. Doxycycline (0-10 μg/mL) had no significant cytotoxic effects in A549 and primary nasal epithelial cells. Increased expression of mesenchymal markers, including vimentin, alpha-smooth muscle actin, and fibronectin in TGF beta 1 induced A549 cells were downregulated by doxycycline treatment. In contrast, E-cadherin expression was upregulated in TGF beta 1 induced A549 cells. An in vitro cell migration assay showed that doxycycline also inhibited the ability of TGF beta 1 induced migration. Doxycycline treatment suppressed the activation of Smad2/3 and p38, whereas its inhibitory effects were similar to each element-specific inhibitor in A549 and primary nasal epithelial cells. Doxycycline inhibited TGF beta 1 induced epithelial-to-mesenchymal transition and migration by targeting Smad2/3 and p38 signal pathways in respiratory epithelial cells.

Role of TGF Beta and PPAR Alpha Signaling Pathways in Radiation Response of Locally Exposed Heart: Integrated Global Transcriptomics and Proteomics Analysis.

PubMed

Subramanian, Vikram; Seemann, Ingar; Merl-Pham, Juliane; Hauck, Stefanie M; Stewart, Fiona A; Atkinson, Michael J; Tapio, Soile; Azimzadeh, Omid

2017-01-06

Epidemiological data from patients undergoing radiotherapy for thoracic tumors clearly show the damaging effect of ionizing radiation on cardiovascular system. The long-term impairment of heart function and structure after local high-dose irradiation is associated with systemic inflammatory response, contraction impairment, microvascular damage, and cardiac fibrosis. The goal of the present study was to investigate molecular mechanisms involved in this process. C57BL/6J mice received a single X-ray dose of 16 Gy given locally to the heart at the age of 8 weeks. Radiation-induced changes in the heart transcriptome and proteome were investigated 40 weeks after the exposure. The omics data were analyzed by bioinformatics tools and validated by immunoblotting. Integrated network analysis of transcriptomics and proteomics data elucidated the signaling pathways that were similarly affected at gene and protein level. Analysis showed induction of transforming growth factor (TGF) beta signaling but inactivation of peroxisome proliferator-activated receptor (PPAR) alpha signaling in irradiated heart. The putative mediator role of mitogen-activated protein kinase cascade linking PPAR alpha and TGF beta signaling was supported by data from immunoblotting and ELISA. This study indicates that both signaling pathways are involved in radiation-induced heart fibrosis, metabolic disordering, and impaired contractility, a pathophysiological condition that is often observed in patients that received high radiation doses in thorax.

Biomarkers of Renal Tumor Burden and Progression in TSC

DTIC Science & Technology

2013-09-01

found no significant increase in CKD in those TSC patients with cysts versus those with normal renal anatomy , excluding patients with PKD, 8 and...similarly no significant increase in CKD in patients with “undisturbed” AMLs versus those with cysts or normal renal anatomy . Biopsy of AMLs had no...SMDF (NRG1 Isoform), Soggy-1, Sonic Hedgehog (ShhN-terminal), SPARC, Spinesin, TACI (TNFRSF13B), Tarc, TCCR (WSX-1), TECK (CCL25), TFPI, TGF alpha, TGF

[The modern approach to wound treatment].

PubMed

Komarcević, A

2000-01-01

Wound healing is a complex process involving interactions among a variety of different cell types. The normal wound repair process consists of three phases--inflammation, proliferation, and remodeling that occur in a predictable series of cellular and biochemical events. Wounds are classified according to various criteria: etiology, lasting, morphological characteristics, communications with solid or hollow organs, the degree of contamination. In the last few years many authors use the Color Code Concept, which classifies wounds as red, yellow and black wounds. This paper presents conventional methods of local wound treatment (mechanical cleansing, disinfection with antiseptic solutions, wound debridement--surgical, biological and autolytic; wound closure, topical antibiotic treatment, dressing), as well as general measures (sedation, antitetanous and antibiotic protection, preoperative evaluation and correction of malnutrition, vasoconstriction, hyperglycemia and steroid use, appropriate surgical technique, and postoperative prevention of vasoconstriction through pain relief, warming and adequate volume resuscitation). Growth factors play a role in cell division, migration, differentiation, protein expression, enzyme production and have a potential ability to heal wounds by stimulating angiogenesis and cellular proliferation, affecting the production and the degradation of the extracellular matrix, and by being chemotactic for inflammatory cells and fibroblasts. There are seven major families of growth factors: epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), interleukins (ILs), and colony-stimulating factor (CSF). Acute wounds contain many growth factors that play a crucial role in the initial phases of wound healing. The events of early wound healing reflect a finely balanced environment leading to uncomplicated and rapid wound healing. Chronic wounds, for many reasons, have lost this fine balance. Multiple studies have evaluated the effect that exogenously applied growth factors have on the healing of chronic wounds. In the study conducted by Knighton and colleagues, topical application of mixture of various growth factors (PDGF, TGF-beta, PDAF, PF4, PDEGF) demonstrated increased wound healing over controls. Brown and associates demonstrated a decrease in skin graft donor site healing time of 1 day using topically applied EGF. Herndon and ass. used systemic growth hormone in burned children and reduction in healing time made a significant clinical difference by allowing earlier wound coverage and decreasing the duration of hospitalization. The TGF family of growth factors is believed to be primarily responsible for excessive scar formation, especially the beta 1 and beta 2 isoforms. TGF-beta 3 isoform has recently been described and may have an inhibitory function on scar formation by being a natural antagonist to the TGF-beta 1 and TGF-beta 2 isoforms. Cytokines, especially interferon-alpha (INF-alpha), INF-alpha, and INF-alpha 2b, may also reduce scar formation. These cytokines decrease the proliferation rate of fibroblasts and reduce the rate of collagen and fibronectin synthesis by reducing the production of mRNA. Expression of nitric oxide synthase (NOS) and heat shock proteins (HSP) have an important role in wound healing, as well as trace elements (zinc, copper, manganese). Applications of some drugs (antioxidants--asiaticoside, vitamin E and ascorbic acid; calcium D-pantothenate, exogenous fibronectin; antileprosy drugs--oil of hydnocarpus; alcoholic extract of yeast) accelerate wound healing. Thymic peptide thymosin beta 4 (T beta 4R) topically applicated, increases collagen deposition and angiogenesis and stimulates keratinocyte migration. Thymosin alpha 1 (T alpha 1R), peptide isolated from the thymus, is a potent chemoattractant which accelerates angiogenesis and wound healing. On the contrary, steroid drugs, hemorrhage and denervation of wounds have negative effect on the healing process.

Biogenesis of lysosomal enzymes in the alpha-glucosidase II-deficient modA mutant of Dictyostelium discoideum: retention of alpha-1,3-linked glucose on N-linked oligosaccharides delays intracellular transport but does not alter sorting of alpha-mannosidase or beta-glucosidase.

PubMed

Ebert, D L; Bush, J M; Dimond, R L; Cardelli, J A

1989-09-01

The endoplasmic reticulum-localized enzyme alpha-glucosidase II is responsible for removing the two alpha-1,3-linked glucose residues from N-linked oligosaccharides of glycoproteins. This activity is missing in the modA mutant strain, M31, of Dictyostelium discoideum. Results from both radiolabeled pulse-chase and subcellular fractionation experiments indicate that this deficiency did not prevent intracellular transport and proteolytic processing of the lysosomal enzymes, alpha-mannosidase and beta-glucosidase. However, the rate at which the glucosylated precursors left the rough endoplasmic reticulum was several-fold slower than the rate at which the wild-type precursors left this compartment. Retention of glucose residues did not disrupt the binding of the precursor forms of the enzymes with intracellular membranes, indicating that the delay in movement of proteins from the ER did not result from lack of association with membranes. However, the mutant alpha-mannosidase precursor contained more trypsin-sensitive sites than did the wild-type precursor, suggesting that improper folding of precursor molecules might account for the slow rate of transport to the Golgi complex. Percoll density gradient fractionation of extracts prepared from M31 cells indicated that the proteolytically processed mature forms of alpha-mannosidase and beta-glucosidase were localized to lysosomes. Finally, the mutation in M31 may have other, more dramatic, effects on the lysosomal system since two enzymes, N-acetylglucosaminidase and acid phosphatase, were secreted much less efficiently from lysosomal compartments by the mutant strain.

Glutamine inhibits CCl4 induced liver fibrosis in mice and TGF-β1 mediated epithelial-mesenchymal transition in mouse hepatocytes.

PubMed

Shrestha, Nirajan; Chand, Lokendra; Han, Myung Kwan; Lee, Seung Ok; Kim, Chan Young; Jeong, Yeon Jun

2016-07-01

Glutamine, traditionally a non-essential amino acid, now has been considered as essential in serious illness and injury. It is a major precursor for glutathione synthesis. However, the anti-fibrotic effect of glutamine and its molecular mechanism in experimental liver fibrosis have not been explored. In the present study we aimed to examine the potential role of glutamine in carbon tetrachloride (CCl4) induced liver fibrosis and TGF-β1 mediated epithelial mesenchymal transition (EMT) and apoptosis in mouse hepatocytes. Liver fibrosis was induced by intraperitoneal injection of CCl4 three times a week for 10 weeks. Glutamine treatment effectively attenuated liver injury and oxidative stress. Collagen content was significantly decreased in liver sections of glutamine treated mice compared to CCl4 model mice. Furthermore, glutamine decreased expression level of α-SMA and TGF-β in liver tissue. Our in vitro study showed that TGF-β1 treatment in hepatocytes resulted in loss of E-cadherin and increased expression of mesenchymal markers and EMT related transcription factor. In addition, TGF-β1 increased the expression of apoptotic markers. However, glutamine interestingly suppressed TGF-β1 mediated EMT and apoptosis. In conclusion, our results suggest that glutamine ameliorates CCl4 induced liver fibrosis and suppresses TGF-β1 induced EMT progression and apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hepatic apoptotic activity following transient normothermic inflow occlusion and reperfusion in the swine model.

PubMed

Helling, T S; Edwards, C A; Helling, T S; Chang, C C; Hodges, M C; Dhar, A; VanWay, C

1999-09-01

Accelerated hepatic apoptosis was first described in portal vein-ligated livers but has since been reported in a variety of liver injuries. Because porto-prival states can induce apoptosis we sought to determine whether transient ischemic periods followed by reperfusion would trigger such cell death. The cytokines TNF-alpha and TGF-beta are known to facilitate apoptosis and are released in response to a number of stimuli including ischemia. We also investigated alterations in plasma and tissue levels of these cytokines which might lend support to their role in increased apoptotic activity following ischemia/reperfusion. Female pigs were used as the experimental model. Inflow occlusion of portal and hepatic arterial blood was performed to a portion of the swine liver directing the entire splanchnic flow to the remaining hepatic lobes for a period of 2 h. The livers were then reperfused and plasma and tissue samples taken for determination of apoptotic activity utilizing cell death immunoperoxidase staining of 3'-OH DNA ends generated by fragmentation and ELISA assay of histone-associated DNA fragments. Plasma and tissue levels of TNF-alpha and plasma levels of TGF-beta were determined by ELISA assay. An increase in apoptotic activity following reperfusion was seen at Day 2 and Day 4 compared to preischemic values by the cell death stain. The ELISA cell death assay showed an increase in apoptotic activity at 60 min, Day 2, and Day 4. Moreover, the ELISA cell death assay showed enhanced apoptotic activity in "hyperperfused" hepatic lobes compared to preischemic, or resting, liver. This was also observed when compared to sham-operated animals. Surprisingly, there was no detectable increase in plasma TNF-alpha or TGF-beta levels following ischemia/reperfusion, although homogenized liver TNF-alpha levels were increased at 60 min and Day 2 following reperfusion. We conclude that transient hepatic inflow occlusion followed by reperfusion can induce increased apoptotic activity in the swine model. Furthermore, increased apoptotic activity also occurs in the hyperperfused liver raising the possibility of a locally active factor or global hepatic expression of receptor activity in response to ischemia/reperfusion. This period of ischemia/reperfusion did not produce a detectable increase in circulating cytokine levels, and accelerated apoptosis could not be linked to heightened TNF-alpha or TGF-beta plasma activity. Higher tissue levels of TNF-alpha could reflect enhanced binding to TNF cell surface receptors or amplified receptor expression. Copyright 1999 Academic Press.

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

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.

Correlation between spontaneous apoptosis and the expression of angiogenic factors in advanced gastric adenocarcinoma.

PubMed

Ikeguchi, M; Cai, J; Fukuda, K; Oka, S; Katano, K; Tsujitani, S; Maeta, M; Kaibara, N

2001-06-01

The aim of this study was to investigate whether angiogenic factors influence the occurrence of spontaneous apoptosis in advanced gastric cancer. The apoptotic indices (AIs) of 97 tumors from 97 patients with advanced gastric cancer (pT3, pN0, pM0, Stage II) were analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) method. Intratumoral microvessel densities (IMVDs) of tumors stained with anti-CD34 monoclonal antibody were quantified under x 200 magnification using computer-assisted image analysis. The expressions of angiogenic factors, such as vascular endothelial growth factor (VEGF), thymidine phosphorylase (dThdPase), transforming growth factor-alpha (TGF-alpha), and p53 were analyzed immunohistochemically and compared with IMVDs and AIs. The mean IMVD of the 97 tumors was 365/mm2 (range 147-990/mm2). The mean AI of tumors was 2.1% (range 0-11.3%). A significant inverse correlation between the AIs and the IMVDs was shown (p = -0.278, P = 0.0064). The mean IMVDs of tumors with high expressions of dThdPase, TGF-alpha, or p53 were significantly higher than those of tumors with low expressions of these factors. The mean AI of tumors with high expressions of dThdPase was significantly lower than that of tumors with low expressions of dThdPase (P = 0.023). However, no significant correlations were detected between AIs and the expression levels of VEGF, TGF-alpha, or p53. In gastric cancer, dThdPase may play an important role in tumor progression by increasing microvessels and by suppressing apoptosis of cancer cells.

A critical developmental role for tgfbr2 in myogenic cell lineages is revealed in mice expressing SM22-Cre, not SMMHC-Cre.

PubMed

Frutkin, Andrew D; Shi, Haikun; Otsuka, Goro; Levéen, Per; Karlsson, Stefan; Dichek, David A

2006-10-01

Smooth muscle cell (SMC)-specific deletion of transforming growth factor beta (TGF-beta) signaling would help elucidate the mechanisms through which TGF-beta signaling contributes to vascular development and disease. We attempted to generate mice with SMC-specific deletion of TGF-beta signaling by mating mice with a conditional ("floxed") allele for the type II TGF-beta receptor (tgfbr2flox) to mice with SMC-targeted expression of Cre recombinase. We bred male mice transgenic for smooth muscle myosin heavy chain (SMMHC)-Cre with females carrying tgfbr2flox. Surprisingly, SMMHC-Cre mice recombined tgfbr2flox at low levels in SMC and at high levels in the testis. Recombination of tgfbr2flox in testis correlated with high-level expression of SMMHC-Cre in testis and germline transmission of tgfbr2null. In contrast, mice expressing Cre from a SM22alpha promoter (SM22-Cre) efficiently recombined tgfbr2flox in vascular and visceral SMC and the heart, but not in testis. Use of the R26R reporter allele confirmed that Cre-mediated recombination in vascular SMC was inefficient for SMMHC-Cre mice and highly efficient for SM22-Cre mice. Breedings that introduced the SM22-Cre allele into tgfbr2flox/flox zygotes in order to generate adult mice that are hemizygous for SM22-Cre and homozygous for tgfbr2flox- and would have conversion of tgfbr2flox/flox to tgfbr2null/null in SMC-produced no live SM22-Cre : tgfbr2flox/flox pups (P<0.001). We conclude: (1) "SMC-targeted" Cre lines vary significantly in specificity and efficiency of Cre expression; (2) TGF-beta signaling in the subset of cells that express SM22alpha is required for normal development; (3) generation of adult mice with absent TGF-beta signaling in SMC remains a challenge.

Downregulation of miR-133 and miR-590 contributes to nicotine-induced atrial remodelling in canines.

PubMed

Shan, Hongli; Zhang, Yong; Lu, Yanjie; Zhang, Ying; Pan, Zhenwei; Cai, Benzhi; Wang, Ning; Li, Xuelian; Feng, Tieming; Hong, Yuan; Yang, Baofeng

2009-08-01

The present study was designed to decipher molecular mechanisms underlying nicotine's promoting atrial fibrillation (AF) by inducing atrial structural remodelling. The canine model of AF was successfully established by nicotine administration and rapid pacing. The atrial fibroblasts isolated from healthy dogs were treated with nicotine. The role of microRNAs (miRNAs) on the expression and regulation of transforming growth factor-beta1 (TGF-beta1), TGF-beta receptor type II (TGF-betaRII), and collagen production was evaluated in vivo and in vitro. Administration of nicotine for 30 days increased AF vulnerability by approximately eight- to 15-fold in dogs. Nicotine stimulated remarkable collagen production and atrial fibrosis both in vitro in cultured canine atrial fibroblasts and in vivo in atrial tissues. Nicotine produced significant upregulation of expression of TGF-beta1 and TGF-betaRII at the protein level, and a 60-70% decrease in the levels of miRNAs miR-133 and miR-590. This downregulation of miR-133 and miR-590 partly accounts for the upregulation of TGF-beta1 and TGF-betaRII, because our data established TGF-beta1 and TGF-betaRII as targets for miR-133 and miR-590 repression. Transfection of miR-133 or miR-590 into cultured atrial fibroblasts decreased TGF-beta1 and TGF-betaRII levels and collagen content. These effects were abolished by the antisense oligonucleotides against miR-133 or miR-590. The effects of nicotine were prevented by an alpha7 nicotinic acetylcholine receptor antagonist. We conclude that the profibrotic response to nicotine in canine atrium is critically dependent upon downregulation of miR-133 and miR-590.

Activation of cardiac fibroblasts by ethanol is blocked by TGF-β inhibition.

PubMed

Law, Brittany A; Carver, Wayne E

2013-08-01

Alcohol abuse is the second leading cause of dilated cardiomyopathy, a disorder specifically referred to as alcoholic cardiomyopathy (ACM). Rodent and human studies have revealed cardiac fibrosis to be a consequence of ACM, and prior studies by this laboratory have associated this occurrence with elevated transforming growth factor-beta (TGF-β) and activated fibroblasts (myofibroblasts). To date, there have been no other studies to investigate the direct effect of alcohol on the cardiac fibroblast. Primary rat cardiac fibroblasts were cultured in the presence of ethanol (EtOH) and assayed for fibroblast activation by collagen gel contraction, alpha-smooth muscle actin (α-SMA) expression, migration, proliferation, apoptosis, collagen I and III, and TGF-β expression. The TGF-β receptor type 1 inhibitor compound SB 431542 and a soluble recombinant TGF-βII receptor (RbII) were used to assess the role of TGF-β in the response of cardiac fibroblasts to EtOH. Treatment for cardiac fibroblasts with EtOH at concentrations of 100 mg/dl or higher resulted in fibroblast activation and fibrogenic activity after 24 hours including an increase in contraction, α-SMA expression, migration, and expression of collagen I and TGF-β. No changes in fibroblast proliferation or apoptosis were observed. Inhibition of TGF-β by SB 431542 and RbII attenuated the EtOH-induced fibroblast activation. EtOH treatment directly promotes cardiac fibroblast activation by stimulating TGF-β release from fibroblasts. Inhibiting the action of TGF-β decreases the fibrogenic effect induced by EtOH treatment. The results of this study support TGF-β to be an important component in cardiac fibrosis induced by exposure to EtOH. Copyright © 2013 by the Research Society on Alcoholism.

Activation of cardiac fibroblasts by ethanol is blocked by TGF-β inhibition

PubMed Central

Law, Brittany A.; Carver, Wayne E.

2013-01-01

Background Alcohol abuse is the second leading cause of dilated cardiomyopathy, a disorder specifically referred to as Alcoholic Cardiomyopathy (ACM). Rodent and human studies have revealed cardiac fibrosis to be a consequence of ACM and prior studies by this lab have associated this occurrence with elevated transforming growth factor-beta (TGF-β) and activated fibroblasts (myofibroblasts). To date there have been no other studies to investigate the direct effect of alcohol on the cardiac fibroblast. Methods Primary rat cardiac fibroblasts were cultured in the presence of ethanol and assayed for fibroblast activation by collagen gel contraction, alpha smooth muscle- actin (α-SMA) expression, migration, proliferation, apoptosis, collagen I & III and TGF-β expression. The TGF-β receptor type 1 inhibitor compound SB 431542 and a soluble recombinant TGF-βII receptor (RbII) were used to assess the role of of TGF-β in the response of cardiac fibroblasts to ethanol. Results Treatment of cardiac fibroblasts with ethanol at concentrations of 100 mg/dl or higher resulted in fibroblast activation and fibrogenic activity after 24 hours including an increase in contraction, α-SMA expression, migration, and expression of collagen I and TGF-β. No changes in fibroblast proliferation or apoptosis were observed. Inhibition of TGF-β by SB 431542 and RbII attenuated the ethanol-induced fibroblast activation. Conclusions Ethanol treatment directly promotes cardiac fibroblast activation by stimulating TGF-β release from fibroblasts. Inhibiting the action of TGF-β decreases the fibrogenic effect induced by ethanol treatment. The results of this study support TGF-β to be an important component in cardiac fibrosis induced by exposure to ethanol. PMID:23528014

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.

Identification and targeting of a TACE-dependent autocrine loopwhich predicts poor prognosis in breast cancer

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

Kenny, Paraic A.; Bissell, Mina J.

2005-06-15

The ability to proliferate independently of signals from other cell types is a fundamental characteristic of tumor cells. Using a 3D culture model of human breast cancer progression, we have delineated a protease-dependent autocrine loop which provides an oncogenic stimulus in the absence of proto-oncogene mutation. Inhibition of this protease, TACE/ADAM17, reverts the malignant phenotype by preventing mobilization of two crucial growth factors, Amphiregulin and TGF{alpha}. We show further that the efficacy of EGFR inhibitors is overcome by physiological levels of growth factors and that successful EGFR inhibition is dependent on reducing ligand bioavailability. Using existing patient outcome data, wemore » demonstrate a strong correlation between TACE and TGF{alpha} expression in human breast cancers that is predictive of poor prognosis.« less

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.

Anxiety-like behavior as an early endophenotype in the TgF344-AD rat model of Alzheimer's disease.

PubMed

Pentkowski, Nathan S; Berkowitz, Laura E; Thompson, Shannon M; Drake, Emma N; Olguin, Carlos R; Clark, Benjamin J

2018-01-01

Alzheimer's disease (AD) is characterized by progressive cognitive decline and the presence of aggregates of amyloid beta (plaques) and hyperphosphorylated tau (tangles). Early diagnosis through neuropsychological testing is difficult due to comorbidity of symptoms between AD and other types of dementia. As a result, there is a need to identify the range of behavioral phenotypes expressed in AD. In the present study, we utilized a transgenic rat (TgF344-AD) model that bears the mutated amyloid precursor protein as well as presenilin-1 genes, resulting in progressive plaque and tangle pathogenesis throughout the cortex. We tested young adult male and female TgF344-AD rats in a spatial memory task in the Morris water maze and for anxiety-like behavior in the elevated plus-maze. Results indicated that regardless of sex, TgF344-AD rats exhibited increased anxiety-like behavior in the elevated plus-maze, which occurred without significant deficits in the spatial memory. Together, these results indicate that enhanced anxiety-like behavior represents an early-stage behavioral marker in the TgF344-AD rat model. Copyright © 2017 Elsevier Inc. All rights reserved.

Bile alcohol metabolism in man. Conversion of 5beta-cholestane-3alpha, 7alpha,12alpha, 25-tetrol to cholic acid.

PubMed Central

Salen, G; Shefer, S; Setoguchi, T; Mosbach, E H

1975-01-01

To study the role of C25-HYDROXY BILE ALCOHOLS AS PRECURSORS OF CHOlic acid, [G-3-H]5beta-cholestane-3alpha,7alpha12alpha,25-tetrol was administered intravenously to two subjects with cerebrotendinous xanthomatosis (CTX) and two normal individuals. One day after pulse labeling, radioactivity was present in the cholic acid isolated from the bile and feces of the subjects with CTX and the bile of the normal individuals. In the two normal subjects, the sp act decay curves of [G-3-H]-cholic acid were exponential, and no traces of [G-3-H]-5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol were detected. In contrast, appreciable quantities of labeled 5beta-cholestane-3alpha,-7aopha,12alpha,25-tetrol were present in the bile and feces of the CTX subjects. The sp act vs. time curves of fecal [G-3-H]5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol and [G-3-H]-cholic acid showed a precursor-product relationship. Although these results suggest that 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol may be a precursor of cholic acid in man, the possibility that C26-hydroxy intermediates represent the normal pathway can not be excluded. PMID:1141434

Elevated serum cytokines correlated with altered behavior, serum cortisol rhythm, and dampened 24-hour rest-activity patterns in patients with metastatic colorectal cancer.

PubMed

Rich, Tyvin; Innominato, Pasquale F; Boerner, Julie; Mormont, M Christine; Iacobelli, Stefano; Baron, Benoit; Jasmin, Claude; Lévi, Francis

2005-03-01

Incapacitating symptom burden in cancer patients contributes to poor quality of life (QOL) and can influence treatment outcomes because of poor tolerance to therapy. In this study, the role of circulating cytokines in the production symptoms in cancer patients is evaluated. Eighty patients with metastatic colorectal cancer with either normal (group I, n = 40) or dampened (group II, n = 40) 24-hour rest/activity patterns measured by actigraphy were identified. Actigraphy patterns were correlated with QOL indices, serum cortisol obtained at 8:00 a.m. and 4:00 p.m. and with serum levels of transforming growth factor-alpha, tumor necrosis factor-alpha, and interleukin 6 (IL-6) obtained at 8:00 a.m. and analyzed in duplicate by ELISA. Cytokine levels and survival were also correlated. Group II patients had significantly higher pre treatment levels of all three cytokines, displayed significantly poorer emotional and social functioning, had higher fatigue, more appetite loss, and poorer performance status compared with group I patients. Transforming growth factor-alpha (TGF-alpha) and IL-6 were significantly increased in the patients with WHO performance status >1 and in those with appetite loss. Fatigue was significantly associated with elevated TGF-alpha only. IL-6 was increased in those patients with extensive liver involvement and multiple organ replacement, and it was significantly correlated with dampened cortisol rhythm. In a multivariate analysis, IL-6 was correlated with poor treatment outcome. Significant correlations were found between serum levels of TGF-alpha and IL-6, circadian patterns in wrist activity and serum cortisol and tumor-related symptoms in patients with metastatic colorectal cancer. These data support the hypothesis that some cancer patient's symptoms of fatigue, poor QOL, and treatment outcome are related to tumor or host generated cytokines and could reflect cytokine effects on the circadian timing system. This interplay between cytokine signaling pathways, the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, and efferent pathways of the suprachiasmatic nucleus that control circadian physiology, opens the way to new rational interventions for symptom management in cancer patients.

Linkage study of nonsyndromic cleft lip with or without cleft palate using candidate genes and mapped polymorphic markers

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

Stein, J.D.; Nelson, L.D.; Conner, B.J.

1994-09-01

Nonsyndromic cleft lip with or without cleft palate (CL(P)) involves fusion or growth failure of facial primordia during development. Complex segregation analysis of clefting populations suggest that an autosomal dominant gene may play a role in this common craniofacial disorder. We have ascertained 16 multigenerational families with CL(P) and tested linkage to 29 candidate genes and 139 mapped short tandem repeat markers. The candidate genes were selected based on their expression in craniofacial development or were identified through murine models. These include: TGF{alpha}, TGF{beta}1, TGF{beta}2, TGF{beta}3, EGF, EGFR, GRAS, cMyc, FGFR, Jun, JunB, PDFG{alpha}, PDGF{beta}, IGF2R, GCR Hox7, Hox8, Hox2B,more » twirler, 5 collagen and 3 extracellular matrix genes. Linkage was tested assuming an autosomal dominant model with sex-specific decreased penetrance. Linkage to all of the candidate loci was excluded in 11 families. RARA was tested and was not informative. However, haplotype analysis of markers flanking RARA on 17q allowed exclusion of this candidate locus. We have previously excluded linkage to 61 STR markers in 11 families. Seventy-eight mapped short tandem repeat markers have recently been tested in 16 families and 30 have been excluded. The remaining are being analyzed and an exclusion map is being developed based on the entire study results.« less

Alpha-lipoic acid improves subclinical left ventricular dysfunction in asymptomatic patients with type 1 diabetes.

PubMed

Hegazy, Sahar K; Tolba, Osama A; Mostafa, Tarek M; Eid, Manal A; El-Afify, Dalia R

2013-01-01

Oxidative stress plays an important role in the development of diabetic cardiomyopathy. Alpha-lipoic acid (ALA) is a powerful antioxidant that may have a protective role in diabetic cardiac dysfunction. We investigated the possible beneficial effect of alpha-lipoic acid on diabetic left ventricular (LV) dysfunction in children and adolescents with asymptomatic type 1 diabetes (T1D). Thirty T1D patients (aged 10-14) were randomized to receive insulin treatment (n = 15) or insulin plus alpha-lipoic acid 300 mg twice daily (n = 15) for four months. Age and sex matched healthy controls (n = 15) were also included. Patients were evaluated with conventional 2-dimensional echocardiographic examination (2D), pulsed tissue Doppler (PTD), and 2-dimensional longitudinal strain echocardiography (2DS) before and after therapy. Glutathione, malondialdhyde (MDA), nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), Fas ligand (Fas-L), matrix metalloproteinase 2 (MMP-2), and troponin-I were determined and correlated to echocardiographic parameters. Diabetic patients had significantly lower levels of glutathione and significantly higher MDA, NO, TNF-alpha, Fas-L, MMP-2, and troponin-I levels than control subjects. The expression of transforming growth factor beta (TGF-beta) mRNA in peripheral blood mononuclear cells was also increased in diabetic patients. Significant correlations of mitral e'/a' ratio and left ventricular global peak systolic strain with glutathione, MDA, NO, TNF-alpha, and Fas-L were observed in diabetic patients. Alpha-lipoic acid significantly increased glutathione level and significantly decreased MDA, NO, TNF-alpha, Fas-L, MMP-2, troponin-I levels, and TGF-beta gene expression. Moreover, alpha-lipoic acid significantly increased mitral e'/a' ratio and left ventricular global peak systolic strain in diabetic patients. These findings suggest that alpha-lipoic acid may have a role in preventing the development of diabetic cardiomyopathy in type 1 diabetes.

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).

Protein-expression profiles in mouse blood-plasma following acute whole-body exposure to (137)Cs gamma rays.

PubMed

Rithidech, Kanokporn Noy; Honikel, Louise; Rieger, Robert; Xie, Weiping; Fischer, Thomas; Simon, Sanford R

2009-05-01

To compare the pattern of protein-expression profiles in blood-plasma after exposure of CBA/CaJ mice to 0 or 3 Gy of (137)Cs gamma rays. Two-dimensional electrophoresis gel coupled with mass spectrometry was used to analyze blood-samples collected at days 2 and 7 post-irradiation. At each sacrifice-time, alterations in expression-level of protein spots between control- and exposed-groups were analyzed statistically by the PDQuest software using Student's t-test (at the significance level of p < 0.05). Mass spectrometry was used to identify the identity of protein-spots with significantly altered expression-level. At day 2, 18 proteins were significantly up-regulated in exposed-mice. These included: alpha-2-Heremans-Schmid (HS)-glycoprotein, apolipoprotein (Apo)-AII-precursor, Apo-E, beta-2-glycoprotein-I, clusterin, fibrinogen-alpha-chain, fibrinogen-gamma-polypeptide, fetuin-B, haptoglobin, high-molecular-weight (HMW)-kininogen (Kng), low-MW-Kng, Kng1-precursor, liver-carboxylesterase-I-precursor, major-urinary-protein-6-precursor, mannose-binding-protein-C-precursor, mannose-binding-lectin-C, and prothrombin-precursor. Gelsolin was detected in control-mice only. At day 7, high expression-levels of 14 proteins were detected in control-mice (i.e., alpha-1-antitrypsin-precursor, carboxylesterase-N, cholesterol-7-alpha-hydroxylase, contraspin, coagulation-factor-II, coagulation-factor-XIII, gelsolin, immunoglobulin-G-heavy-chain, neurexin, prothrombin-precursor, protein-phosphatase, putative-calcium-influx-channel, vitamin-D-binding-protein, and 1110018G07Rik); while 15 proteins were highly expressed in exposed-mice. These included: alpha-1-acid-glycoprotein, alpha-2-HS-glycoprotein, alpha-1-protease-inhibitor-2, ApoA-IV, ApoC-I, ApoH, beta-1-globin, clusterin, complement-component-3, fibrinogen-beta-chain, HMW-Kng, major-histocompatibility-complex-class-Ia-H2-K, serine-(cysteine)-proteinase-inhibitor, retinoblastoma-associated-protein-140, and vascular-cell-adhesion-molecule-1. Although different proteins (mostly involved in inflammatory responses) were detected in exposed-mice, alterations in expression-levels of clusterin, gelsolin, kininogen, and alpha-2-HS-glycoproteins were found at both times. Despite the need for validation, the results suggested that alterations in expression-levels of specific proteins may be indicative of radiation-exposure. The results also provided the important step in an eventual establishment of blood-based biomarkers of radiation-exposure in vivo.

Structure and activation of pro-activin A

PubMed Central

Wang, Xuelu; Fischer, Gerhard; Hyvönen, Marko

2016-01-01

Activins are growth factors with multiple roles in the development and homeostasis. Like all TGF-β family of growth factors, activins are synthesized as large precursors from which mature dimeric growth factors are released proteolytically. Here we have studied the activation of activin A and determined crystal structures of the unprocessed precursor and of the cleaved pro-mature complex. Replacing the natural furin cleavage site with a HRV 3C protease site, we show how the protein gains its bioactivity after proteolysis and is as active as the isolated mature domain. The complex remains associated in conditions used for biochemical analysis with a dissociation constant of 5 nM, but the pro-domain can be actively displaced from the complex by follistatin. Our high-resolution structures of pro-activin A share features seen in the pro-TGF-β1 and pro-BMP-9 structures, but reveal a new oligomeric arrangement, with a domain-swapped, cross-armed conformation for the protomers in the dimeric protein. PMID:27373274

Galectin-1 mediates TGF-β-induced transformation from normal fibroblasts into carcinoma-associated fibroblasts and promotes tumor progression in gastric cancer

PubMed Central

Zheng, Lingyan; Xu, Cong; Guan, Zhonghai; Su, Xingyun; Xu, Zhenzhen; Cao, Jiang; Teng, Lisong

2016-01-01

Rcinoma-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment. Cancer cells can induce the transformation from normal fibroblasts (NFs) into CAFs, reciprocally, CAFs promote tumor invasion and proliferation. TGF-β has been the mostly accepted factor to fuel NFs transformation into CAFs. Galectin-1 (Gal1) is highly upregulated in CAFs of multiple human cancers, and overexpression of Gal1 in CAFs promotes tumor progression. The effect of Gal1 on TGF-β-induced CAFs activation has not yet been established in gastric cancer (GC). In this study, we show that Gal1 expression in stroma is positively related to TGF-β in epithelial cells by retrospective analysis of GC patient samples. Meanwhile, conditioned media (CMs) from gastric cancer cells induce expression of both Gal1 and the CAFs marker alpha smooth muscle actin (α-SMA) in NFs via TGF-β secretion. Knockdown of Gal1 prevents TGF-β-induced the conversion of NFs to CAFs. CMs from fibroblasts overexpressing Gal1 inhibits cancer cells apoptosis, promotes migration and invasion in vitro. Thus, Gal1 is significantly involved in the development of tumor-promoting microenvironment by enhancing TGF-β signaling in a positive feedback loop. Targeting Gal1 in tumor stroma should be considered as a potential therapeutic target for GC. PMID:27186290

Effect of proinflammatory cytokines on PIGA- hematopoiesis.

PubMed

Kulkarni, Shashikant; Bessler, Monica

2003-09-01

Blood cells from patients with paroxysmal nocturnal hemoglobinuria lack glycosyl phosphatidylinositol (GPI)-linked proteins, due to a somatic mutation in the X-linked PIGA gene. It is believed that clonal expansion of PIGA- blood cells is due to a survival advantage in the hostile marrow environment of aplastic anemia. Here we investigated the effects of inhibitory cytokines in mice genetically engineered to have blood cells deficient in GPI-linked proteins. The effect of inhibitory cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], macrophage inflammatory protein-1 alpha [MIP-1alpha], and transforming growth factor-beta1 [TGF-beta1]) was investigated, using clonogenic assays, competitive repopulation, and in vivo induction of proinflammatory cytokines by double-stranded RNA. The expression of Fas on progenitor cells and its up-regulation by inhibitory cytokines were analyzed by flow cytometry. TNF-alpha, IFN-gamma, MIP-1alpha, and TGF-beta1 suppressed colony formation in a dose-dependent fashion that was similar for PIGA+ and PIGA- blood bone marrow cells. Competitive repopulation of bone marrow cells cultured in IFN-gamma and TNF-alpha resulted in a comparable ability of PIGA+ and PIGA- hematopoietic stem cells to reconstitute hematopoiesis. Fas expression was minimal on PIGA+ and PIGA- progenitor cells and was up-regulated to the same extent in response to IFN-gamma and TNF-alpha as assessed by Fas antibody-mediated apoptosis. Similarly, in vivo induction of proinflammatory cytokines by double-stranded RNA had no effect on the proportion of circulating PIGA- blood cells. These results indicate that PIGA+ and PIGA- hematopoietic progenitor cells respond similarly to inhibitory cytokines, suggesting that other factors are responsible for the clonal expansion of paroxysmal nocturnal hemoglobinuria cells.

Increased T cell recruitment to the CNS after amyloid beta 1-42 immunization in Alzheimer's mice overproducing transforming growth factor-beta 1.

PubMed

Buckwalter, Marion S; Coleman, Bronwen S; Buttini, Manuel; Barbour, Robin; Schenk, Dale; Games, Dora; Seubert, Peter; Wyss-Coray, Tony

2006-11-01

Immunotherapy targeting the amyloid beta (Abeta) peptide is a novel therapy under investigation for the treatment of Alzheimer's disease (AD). A clinical trial using Abeta(1-42) (AN1792) as the immunogen was halted as a result of development of meningoencephalitis in a small number of patients. The cytokine TGF-beta1 is a key modulator of immune responses that is increased in the brain in AD. We show here that local overexpression of TGF-beta1 in the brain increases both meningeal and parenchymal T lymphocyte number. Furthermore, TGF-beta1 overexpression in a mouse model for AD [amyloid precursor protein (APP) mice] leads to development of additional T cell infiltrates when mice were immunized at a young but not old age with AN1792. Notably, only mice overproducing both Abeta (APP mice) and TGF-beta1 experienced a rise in T lymphocyte number after immunization. One-third of infiltrating T cells were CD4 positive. We did not observe significant differences in B lymphocyte numbers in any of the genotypes or treatment groups. These results demonstrate that TGF-beta1 overproduction in the brain can promote T cell infiltration, in particular after Abeta(1-42) immunization. Likewise, levels of TGF-beta1 or other immune factors in brains of AD patients may influence the response to Abeta(1-42) immunization.

Effect of TGF-β1 Stimulation on the Secretome of Human Adipose-Derived Mesenchymal Stromal Cells.

PubMed

Rodríguez, Tania M; Saldías, Alejandro; Irigo, Marcelo; Zamora, Jorge Velasco; Perone, Marcelo J; Dewey, Ricardo A

2015-08-01

Adipose tissue is an attractive source of mesenchymal stromal cells (MSCs) owing to the relative ease of obtaining large volumes with more MSC abundance compared with other sources. Increasing evidence supports the fact that trophic factors secreted by MSCs play a pivotal therapeutic role. Several strategies in regenerative medicine use MSCs, mainly exploiting their immunosuppressive effect and homing capacity to sites of damage. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine that, depending on the cell niche, can display either anti-inflammatory or proinflammatory effects. TGF-β1 expression increases in various tissues with damage, especially when accompanied by inflammation. Thus, we analyzed the effect of TGF-β1 on the secretion by adipose-derived mesenchymal stromal cells (ASCs) of a panel of 80 cytokines/chemokines using an antibody array. To avoid a possible effect of fetal bovine serum (FBS) on ASCs secretion, we performed our analysis by culturing cells in FBS-free conditions, only supplemented with 0.1% of bovine serum albumin. We report the cytokine profile secreted by ASCs. We also found that TGF-β1 exposure modulates 8 chemokines and 18 cytokines, including TGF-β1 and -β2, and other important cytokines involved in immunosuppression, allergic responses, and bone resorption. ©AlphaMed Press.

Hydraulic pressure inducing renal tubular epithelial-myofibroblast transdifferentiation in vitro.

PubMed

Li, Fei-yan; Xie, Xi-sheng; Fan, Jun-ming; Li, Zi; Wu, Jiang; Zheng, Rong

2009-09-01

The effects of hydraulic pressure on renal tubular epithelial-myofibroblast transdifferentiation (TEMT) were investigated. We applied hydraulic pressure (50 cm H2O) to normal rat kidney tubular epithelial cells (NRK52E) for different durations. Furthermore, different pressure magnitudes were applied to cells. The morphology, cytoskeleton, and expression of myofibroblastic marker protein and transforming growth factor-beta1 (TGF-beta1) of NRK52E cells were examined. Disorganized actin filaments and formation of curling clusters in actin were seen in the cytoplasm of pressurized cells. We verified that de novo expression of alpha-smooth muscle actin induced by pressure, which indicated TEMT, was dependent on both the magnitude and duration of pressure. TGF-beta1 expression was significantly upregulated under certain conditions, which implies that the induction of TEMT by hydraulic pressure is related with TGF-beta1. We illustrate for the first time that hydraulic pressure can induce TEMT in a pressure magnitude- and duration-dependent manner, and that this TEMT is accompanied by TGF-beta1 secretion.

Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes.

PubMed

Qureshi, Hamid Yaqoob; Ricci, Gemma; Zafarullah, Muhammad

2008-09-01

Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

Influence of alpha-lipoic acid on nicotine-induced lung and liver damage in experimental rats.

PubMed

Ateyya, Hayam; Nader, Manar A; Attia, Ghalia M; El-Sherbeeny, Nagla A

2017-05-01

Nicotine mediates some of the injurious effects caused by consuming tobacco products. This work aimed at investigating the defensive role of alpha-lipoic acid (ALA) with its known antioxidant and antiinflammatory effect in nicotine-induced lung and liver damage. Rats were arranged into 4 groups: control, nicotine, ALA, and ALA-nicotine groups. Oxidative stress and antioxidant status were determined by assessing thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), and glutathione (GSH) levels in lung and liver. Liver enzymes and lipid profiles were measured and pulmonary and hepatic damage were assessed by histopathological examination. Also, serum levels of transforming growth factor beta 1 (TGF-β1) and vascular cell adhesion molecule 1 (VCAM-1) were determined. The results revealed an increase in TBARS in tissues and a reduction in both SOD and GSH activity in the nicotine-treated rats. Nicotine induced high levels of liver enzymes, TGF-β1, VCAM-1, and dyslipidemia with histopathological changes in the lung and liver. ALA administration along with nicotine attenuated oxidative stress and normalized the SOD and GSH levels, ameliorated dyslipidemia, and improved TGF-β1 and VCAM-1 with better histopathology of the lung and liver. The study data revealed that ALA may be beneficial in alleviating nicotine-induced oxidative stress, dyslipidemia, and both lung and liver damage.

The Molecular Epidemiology of Malaria in Western Kenya

DTIC Science & Technology

2002-09-01

including tumor necrosis factor alpha (TNF- α), interleukin-10 (IL-10), transforming growth factor beta (TGF-β), interleukin-6 (IL-6), and interferon gamma...Ricard S, Troesch A, Mallet C, Generenaz L, Evans A, Arveiler D, Luc G, Ruidavets JB, Poirier O. Polymorphisms of the transforming growth factor- beta 1...transforming growth factor- beta 1 and tumour necrosis factor-alpha genes: a technical report. Transpl Immunol 1998 6(3): 193-7. 36. Olomolaiye OO

1alpha,25-dihydroxyvitamin D3 potentiates the beneficial effects of allergen immunotherapy in a mouse model of allergic asthma: role for IL-10 and TGF-beta.

PubMed

Taher, Yousef A; van Esch, Betty C A M; Hofman, Gerard A; Henricks, Paul A J; van Oosterhout, Antoon J M

2008-04-15

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), a potent inhibitor of NF-kappaB expression, can prevent the maturation of dendritic cells in vitro leading to tolerogenic dendritic cells with increased potential to induce regulatory T cells. Herein, we investigated whether the combination of allergen immunotherapy with 1,25(OH)(2)D(3) potentiates the suppressive effects of immunotherapy and whether the immunoregulatory cytokines IL-10 and TGF-beta are involved in the effector phase. OVA-sensitized and challenged BALB/c mice displayed airway hyperresponsiveness (AHR) and increased serum OVA-specific IgE levels, bronchoalveolar lavage eosinophilia, and Th2 cytokine levels. In this model, the dose response of allergen immunotherapy 10 days before OVA inhalation challenge shows strong suppression of asthma manifestations at 1 mg of OVA, but partial suppression of bronchoalveolar lavage eosinophilia, IgE up-regulation, and no reduction of AHR at 100 microg. Interestingly, coadministration of 10 ng of 1,25(OH)(2)D(3) with 100 microg of OVA immunotherapy significantly inhibited AHR and potentiated the reduction of serum OVA-specific IgE levels, airway eosinophilia, and Th2-related cytokines concomitant with increased IL-10 levels in lung tissues and TGF-beta and OVA-specific IgA levels in serum. Similar effects on suboptimal immunotherapy were observed by inhibition of the NF-kappaB pathway using the selective IkappaB kinase 2 inhibitor PS-1145. The suppressive effects of this combined immunotherapy were partially reversed by treatment with mAb to either IL-10R or TGF-beta before OVA inhalation challenge but completely abrogated when both Abs were given. These data demonstrate that 1,25(OH)(2)D(3) potentiates the efficacy of immunotherapy and that the regulatory cytokines IL-10 and TGF-beta play a crucial role in the effector phase of this mouse model.

A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells.

PubMed

Peters, B P; Krzesicki, R F; Hartle, R J; Perini, F; Ruddon, R W

1984-12-25

Human choriocarcinoma cells (JAR) synthesize the alpha and beta subunits of the glycoprotein hormone chorionic gonadotropin (hCG) (R.W. Ruddon, C.A. Hanson, A. H. Bryan, G.J. Putterman, E.L. White, F. Perini, K. S. Meade, and P.H. Aldenderfer (1980) J. Biol. Chem. 255, 1000-1007). In addition to the hCG dimer (alpha beta), JAR cells secrete uncombined alpha and beta subunits into the culture medium (L.A. Cole, R.J. Hartle, J.A. Laferla, and R.W. Ruddon (1983) Endocrinology 113, 1176-1178). Pulse-chase studies with [35S]methionine or [3H]mannose were carried out in order to compare free alpha, free beta, and the alpha beta dimer with regard to the kinetics of synthesis, N-linked oligosaccharide processing, and secretion and to determine the kinetics of alpha-beta subunit combination. A panel of three antisera was used to immunoprecipitate directly the free subunits and the alpha beta dimer sequentially from the same cell lysates and culture media. The alpha subunit of hCG was synthesized in a slight molar excess (1.2-1.5-fold) over the beta subunit, and alpha beta dimer was rapidly formed by combination of the intracellular alpha and beta precursors. Dimer formation was already apparent in JAR cells following a 10-min biosynthetic labeling incubation with [35S]methionine. The combination of subunits ceased by 30 min of chase even though 51% of alpha and 44% of beta remained free within the cells. Combination of the alpha and beta precursors had occurred before their N-linked oligosaccharides were processed beyond the Man8GlcNAc2 structure. The initial trimming of glucosyl and mannosyl units from the high-mannose oligosaccharides of the hCG precursors occurred more rapidly for free alpha and CG-alpha than for free beta and CG-beta. JAR cells accumulated alpha precursors bearing mostly Man8GlcNAc2 units and beta precursors bearing Man8GlcNAc2 units that represent the substrates of the rate-limiting step in the secretory pathway. In spite of the fact that their N-linked oligosaccharides were trimmed at different rates, free alpha, free beta, and alpha beta dimer were all secreted into the medium at the same rate, with a half-time of 35 min. The secreted hCG forms were stable in the chase medium between 4 and 8h, indicating that extracellular degradation, combination of free subunits to form dimer, or dissociation of dimer to form free subunits did not occur.(ABSTRACT TRUNCATED AT 400 WORDS)

Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients.

PubMed

Yamamoto, N; Naraparaju, V R; Asbell, S O

1996-06-15

Serum vitamin D3-binding protein (Gc protein) can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor of the macrophage activating factor (MAF). Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high titered MAF, Gc-MAF. When peripheral blood monocytes/macrophages of 52 patients bearing various types of cancer were incubated with 100 pg/ml of GcMAF, the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of patient plasma Gc protein was found to be severely reduced in about 25% of this patient population. About 45% of the patients had moderately reduced MAF precursor activities. Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase detected in the patient's bloodstream. The source of the enzyme appeared to be cancerous cells. Radiation therapy decreased plasma alpha-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting alpha-N-acetylgalactosaminidase. Both alpha-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient bloodstream can serve as diagnostic and prognostic indices.

Human dendritic cells produce TGF-beta 1 under the influence of lung carcinoma cells and prime the differentiation of CD4+CD25+Foxp3+ regulatory T cells.

PubMed

Dumitriu, Ingrid E; Dunbar, Donald R; Howie, Sarah E; Sethi, Tariq; Gregory, Christopher D

2009-03-01

Dendritic cells (DCs) have a central role in the development of adaptive immune responses, including antitumor immunity. Factors present in the tumor milieu can alter the maturation of DCs and inhibit their capacity to activate T cells. Using gene expression analysis, we found that human DCs increased the expression of TGF-beta1 transcripts following culture with human lung carcinoma cells (LCCs). These DCs produced increased amounts of TGF-beta1 protein compared with DCs not exposed to tumor cells. LCCs also decreased the expression of CD86 and HLA-DR by immature DCs. Furthermore, LCCs decreased CD86 expression and the production of TNF-alpha and IL-12 p70 by mature DCs. Moreover, LCCs also converted mature DCs into cells producing TGF-beta1. These TGF-beta1-producing DCs were poor at eliciting the activation of naive CD4(+) T cells and sustaining their proliferation and differentiation into Th1 (IFN-gamma(+)) effectors. Instead, TGF-beta1-producing DCs demonstrated an increased ability to generate CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress the proliferation of T lymphocytes. These results identify a novel mechanism by which the function of human DCs is altered by tumor cells and contributes to the evasion of the immune response.

Stereospecific deuteration of alpha-furanosyl azomycin nucleosides: a model reaction for tritium radiolabeling.

PubMed

Kumar, Piyush; Emami, Saeed; McEwan, Alexander J B; Wiebe, Leonard I

2008-06-01

Stereospecific synthesis of 1-alpha-d-(2-deuteroribofuranosyl)-2-nitroimidazole (2'-[(2)H]-alpha-AZR) is reported. This, deuteration was independent of the configuration of C-2' -OH group (arabinose or ribose) in sugar moiety of starting molecules. Slightly better yield (>37%) of the deuterated product, 6, from arabinosyl precursor in comparison to corresponding ribose precursor (29%) was obtained which may reflect better stereochemical availability of C-2' -OH in arabinose during oxidation.

Association of transforming growth factor-beta1 gene polymorphism in the development of Epstein-Barr virus-related hematologic diseases.

PubMed

Hatta, Kanako; Morimoto, Akira; Ishii, Eiichi; Kimura, Hiroshi; Ueda, Ikuyo; Hibi, Shigeyoshi; Todo, Shinjiro; Sugimoto, Tohru; Imashuku, Shinsaku

2007-11-01

Epstein-Barr virus (EBV) is etiologically associated with various hematologic disorders, including primary acute infectious mononucleosis (IM), hemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV infection (CAEBV) and malignant lymphomas. Although cytokines play a central role in EBV-related immune responses, the exact mechanisms causing different clinical responses remain unclear. In this study, the pattern of cytokine gene polymorphisms was comparatively analyzed in EBV-related diseases. Eighty-nine patients with EBV-related disease were analyzed; 30 with IM, 28 with EBV-HLH and 31 with CAEBV. Eighty-one EBV-seropositive healthy adults were also used as controls. Associations with polymorphisms of various cytokines, including interleukin (IL)-1 alpha and IL-1 beta were evaluated. The gene polymorphisms were typed by polymerase chain reaction with sequence-specific primers. A significant difference of polymorphisms was found for transforming growth factor (TGF)-beta1; the frequency of TGF-beta1 codon 10 C allele was significantly higher in patients with EBV-related diseases than in controls (p<0.001). The difference was significant in patients with IM or HLH (p<0.001), but not in those with CAEBV (p=0.127), compared with controls. As regards other cytokines, the frequency of the IL-1 alpha -889 C allele was significantly lower in patients with IM than in controls (p<0.05). Our results suggests that TGF-beta1 codon 10 C allele plays a role in the development of EBV-related diseases and that the IL-1 alpha -889 C allele may be involved in response failure and sequential progression into the development of HLH.

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

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

Yano, Hiroyuki; Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593; Hamanaka, Ryoji

Highlights: Black-Right-Pointing-Pointer We examine how radiation affects the expression level and signal pathway of collagen. Black-Right-Pointing-Pointer TGF-{beta}1 mRNA is elevated earlier than those of collagen genes after irradiation. Black-Right-Pointing-Pointer Smad pathway mediates the expression of collagen in radiation induced fibrosis. Black-Right-Pointing-Pointer MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Realmore » time RT-RCR showed that both {alpha}1and {alpha}2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-{beta}1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-{beta} receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of {alpha}2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.« less

Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

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

Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah

Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cellsmore » by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.« less

Revisiting the liver in human yellow fever: virus-induced apoptosis in hepatocytes associated with TGF-beta, TNF-alpha and NK cells activity.

PubMed

Quaresma, Juarez A S; Barros, Vera L R S; Pagliari, Carla; Fernandes, Elaine R; Guedes, Fernanda; Takakura, Cleusa F H; Andrade, Heitor F; Vasconcelos, Pedro F C; Duarte, Maria I S

2006-02-05

Flavivirus infection as dengue and yellow fever persists as a terrible menace to pandemics, due to Aedes prevalence in the Americas. Yellow fever is characterized by hepatocyte damage, with steatosis, apoptosis and necrosis, mainly in the midzonal region of the liver, but the injury mechanism has not been studied at the light of recent knowledge, such as the advances in cell death mechanisms, inflammatory response and cytokine cell expression tools. We studied 53 human liver paraffin embedded blocks from patients who died with yellow fever, all with histological demonstration of higher prevalence of apoptosis over necrosis and mild disproportionate inflammatory response. Viral antigens were found most frequently in hepatocytes from the midzonal area than other lobule areas, as detected by specific immunohistochemistry. Infiltrating cell subpopulations showed mainly CD4+ T lymphocytes, with small numbers of CD8+ cytotoxic lymphocytes, CD20+ B lymphocytes, NKT+ cells and S100+ dendritic cells in the sites of inflammation, as compared to normal and leptospirosis liver blocks. Some cells expressed TNF-alpha and IFN-gamma, but a much more intense proportion of TGF-beta expressing cells were found, suggesting both a Th1 and Th3 patterns of immune response in yellow fever. Most affected hepatocyte presented apoptosis markers that appear at the cell death main pathway in this infection. Viral antigens, which production could interfere in hepatocyte biology, could induce the activation of apoptosis cascade, but TGF-beta was also an apoptosis promoter. Our finding supports the key effect of the yellow fever virus in hepatocyte injury, resulting in prevalence of apoptosis over necrosis, aside from a TGF-beta action induced by the inflammatory response.

Behavioural inflexibility in a comorbid rat model of striatal ischemic injury and mutant hAPP overexpression.

PubMed

Levit, Alexander; Regis, Aaron M; Garabon, Jessica R; Oh, Seung-Hun; Desai, Sagar J; Rajakumar, Nagalingam; Hachinski, Vladimir; Agca, Yuksel; Agca, Cansu; Whitehead, Shawn N; Allman, Brian L

2017-08-30

Alzheimer disease (AD) and stroke coexist and interact; yet how they interact is not sufficiently understood. Both AD and basal ganglia stroke can impair behavioural flexibility, which can be reliably modeled in rats using an established operant based set-shifting test. Transgenic Fischer 344-APP21 rats (TgF344) overexpress pathogenic human amyloid precursor protein (hAPP) but do not spontaneously develop overt pathology, hence TgF344 rats can be used to model the effect of vascular injury in the prodromal stages of Alzheimer disease. We demonstrate that the injection of endothelin-1 (ET1) into the dorsal striatum of TgF344 rats (Tg-ET1) produced an exacerbation of behavioural inflexibility with a behavioural phenotype that was distinct from saline-injected wildtype & TgF344 rats as well as ET1-injected wildtype rats (Wt-ET1). In addition to profiling the types of errors made, interpolative modeling using logistic exposure-response regression provided an informative analysis of the timing and efficiency of behavioural flexibility. During set-shifting, Tg-ET1 committed fewer perseverative errors than Wt-ET1. However, Tg-ET1 committed significantly more regressive errors and had a less efficient strategy change than all other groups. Thus, behavioural flexibility was more vulnerable to striatal ischemic injury in TgF344 rats. Copyright © 2017 Elsevier B.V. All rights reserved.

TGF-Alpha Expression During Breast Tumorigenesis

DTIC Science & Technology

1999-07-01

contributors to breast tumorigenesis. Therefore, we examined TGFa expression in normal and transformed epithelium. We also examined TOFa and EGFR protein in...patient samples it is difficult to determine what effect tissue culture has on normal T(3Fa protein synthesis and processing. TOFa protein expression

Mediation of wound-related Rous sarcoma virus tumorigenesis by TFG (transforming growth factor)-. beta

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

Sieweke, M.H.; Bissell, M.J.; Thompson, N.L.

1990-06-29

In Rous sarcoma virus (RSV)-infected chickens, wounding leads to tumor formation with nearly 100% frequency in tissues that would otherwise remain tumor-free. Identifying molecular mediators of this phenomenon should yield important clues to the mechanisms involved in RSV tumorigenesis. Immunohistochemical staining showed that TGF-{beta} is present locally shortly after wounding, but not in unwounded controls. In addition, subcutaneous administration of recombinant transforming growth factor {beta}1 (TGF-{beta}1) could substitute completely for wounding in tumor induction. A treatment protocol of four doses of 800 nanograms of TGF-{beta} resulted in v-src-expressing tumors with 100% frequency; four doses of only 10 nanograms still ledmore » to tumor formation in 80% of the animals. This effect was specific, as other growth factors with suggested roles in would healing did not elicit the same response. Epidermal growth factor (EGF) or TGF-{alpha} had no effect, and platelet-derived growth factor (PDGF) or insulin-like growth factor-1 (IGF-1) yielded only occasional tumors after longer latency. TGF-{beta} release during the would-healing response may thus be a critical event that creates a conducive environment for RSV tumorigenesis and may act as a cofactor for transformation in this system. 31 refs., 3 figs., 2 tabs.« less

Mesenchymal stromal cells express GARP/LRRC32 on their surface: effects on their biology and immunomodulatory capacity.

PubMed

Carrillo-Galvez, Ana Belén; Cobo, Marién; Cuevas-Ocaña, Sara; Gutiérrez-Guerrero, Alejandra; Sánchez-Gilabert, Almudena; Bongarzone, Pierpaolo; García-Pérez, Angélica; Muñoz, Pilar; Benabdellah, Karim; Toscano, Miguel G; Martín, Francisco; Anderson, Per

2015-01-01

Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-β1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-β1 to the cell surface of activated Foxp3(+) regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-β1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-β1 and reduced their proliferative capacity in a TGF-β1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker. © 2014 AlphaMed Press.

Induction of myofibroblastic differentiation in vitro by covalently immobilized transforming growth factor-beta(1).

PubMed

Metzger, Wolfgang; Grenner, Nadine; Motsch, Sandra E; Strehlow, Rothin; Pohlemann, Tim; Oberringer, Martin

2007-11-01

Growth factors are an important tool in tissue engineering. Bone morphogenetic protein-2 and transforming growth factor-beta(1) (TGF-beta(1)) are used to provide bioactivity to surgical implants and tissue substitute materials. Mostly growth factors are used in soluble or adsorbed form. However, simple adsorption of proteins to surfaces is always accompanied by reduced stability and undefined pharmacokinetics. This study aims to prove that TGF-beta(1) can be covalently immobilized to functionalized surfaces, maintaining its ability to induce myofibroblastic differentiation of normal human dermal fibroblasts. In vivo, fibroblasts differentiate to myofibroblasts (MFs) during soft tissue healing by the action of TGF-beta(1). As surfaces for our experiments, we used slides bearing aldehyde, epoxy, or amino groups. For our in vitro cell culture experiments, we used the expression of alpha-smooth muscle actin as a marker for MFs after immunochemical staining. Using the aldehyde and the epoxy slides, we were able to demonstrate the activity of immobilized TGF-beta(1) through a significant increase in MF differentiation rate. A simple immunological test was established to detect TGF-beta(1) on the surfaces. This technology enables the creation of molecular "landscapes" consisting of several factors arranged in a distinct spatial pattern and immobilized on appropriate surfaces.

Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.

PubMed

Amano, Satoshi; Ogura, Yuki; Akutsu, Nobuko; Nishiyama, Toshio

2007-02-01

Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

A transgenic Alzheimer rat with plaques, tau pathology, behavioral impairment, oligomeric Aβ and frank neuronal loss

PubMed Central

Cohen, Robert M.; Rezai-Zadeh, Kavon; Weitz, Tara M.; Rentsendorj, Altan; Gate, David; Spivak, Inna; Bholat, Yasmin; Vasilevko, Vitaly; Glabe, Charles G.; Breunig, Joshua J.; Rakic, Pasko; Davtyan, Hayk; Agadjanyan, Michael G.; Kepe, Vladimir; Barrio, Jorge; Bannykh, Serguei; Szekely, Christine A.; Pechnick, Robert N.; Town, Terrence

2013-01-01

Alzheimer’s disease (AD) is hallmarked by amyloid plaques, neurofibrillary tangles, and widespread cortical neuronal loss (Selkoe, 2001). The ‘amyloid cascade hypothesis’ posits that cerebral amyloid sets neurotoxic events into motion that precipitate Alzheimer dementia (Hardy and Allsop, 1991). Yet, faithful recapitulation of all AD features in widely used transgenic (Tg) mice engineered to overproduce Aβ peptides has been elusive. We have developed a Tg rat model (line TgF344-AD) expressing mutant human amyloid precursor protein (APPsw) and presenilin 1 (PS1ΔE9) genes, each independent causes of early-onset familial AD. TgF344-AD rats manifest age-dependent cerebral amyloidosis that precedes tauopathy, gliosis, apoptotic loss of neurons in the cerebral cortex and hippocampus, and cognitive disturbance. These results demonstrate progressive neurodegeneration of the Alzheimer type in these animals. The TgF344-AD rat fills a critical need for a next-generation animal model to enable basic and translational AD research. PMID:23575824

A transgenic Alzheimer rat with plaques, tau pathology, behavioral impairment, oligomeric aβ, and frank neuronal loss.

PubMed

Cohen, Robert M; Rezai-Zadeh, Kavon; Weitz, Tara M; Rentsendorj, Altan; Gate, David; Spivak, Inna; Bholat, Yasmin; Vasilevko, Vitaly; Glabe, Charles G; Breunig, Joshua J; Rakic, Pasko; Davtyan, Hayk; Agadjanyan, Michael G; Kepe, Vladimir; Barrio, Jorge R; Bannykh, Serguei; Szekely, Christine A; Pechnick, Robert N; Town, Terrence

2013-04-10

Alzheimer's disease (AD) is hallmarked by amyloid plaques, neurofibrillary tangles, and widespread cortical neuronal loss (Selkoe, 2001). The "amyloid cascade hypothesis" posits that cerebral amyloid sets neurotoxic events into motion that precipitate Alzheimer dementia (Hardy and Allsop, 1991). Yet, faithful recapitulation of all AD features in widely used transgenic (Tg) mice engineered to overproduce Aβ peptides has been elusive. We have developed a Tg rat model (line TgF344-AD) expressing mutant human amyloid precursor protein (APPsw) and presenilin 1 (PS1ΔE9) genes, each independent causes of early-onset familial AD. TgF344-AD rats manifest age-dependent cerebral amyloidosis that precedes tauopathy, gliosis, apoptotic loss of neurons in the cerebral cortex and hippocampus, and cognitive disturbance. These results demonstrate progressive neurodegeneration of the Alzheimer type in these animals. The TgF344-AD rat fills a critical need for a next-generation animal model to enable basic and translational AD research.

Transforming growth factor alpha is a critical mediator of radiation lung injury.

PubMed

Chung, Eun Joo; Hudak, Kathryn; Horton, Jason A; White, Ayla; Scroggins, Bradley T; Vaswani, Shiva; Citrin, Deborah

2014-09-01

Radiation fibrosis of the lung is a late toxicity of thoracic irradiation. Epidermal growth factor (EGF) signaling has previously been implicated in radiation lung injury. We hypothesized that TGF-α, an EGF receptor ligand, plays a key role in radiation-induced fibrosis in lung. Mice deficient in transforming growth factor (TGF-α(-/-)) and control C57Bl/6J (C57-WT) mice were exposed to thoracic irradiation in 5 daily fractions of 6 Gy. Cohorts of mice were followed for survival (n ≥ 5 per group) and tissue collection (n = 3 per strain and time point). Collagen accumulation in irradiated lungs was assessed by Masson's trichrome staining and analysis of hydroxyproline content. Cytokine levels in lung tissue were assessed with ELISA. The effects of TGF-α on pneumocyte and fibroblast proliferation and collagen production were analyzed in vitro. Lysyl oxidase (LOX) expression and activity were measured in vitro and in vivo. Irradiated C57-WT mice had a median survival of 24.4 weeks compared to 48.2 weeks for irradiated TGF-α(-/-) mice (P = 0.001). At 20 weeks after irradiation, hydroxyproline content was markedly increased in C57-WT mice exposed to radiation compared to TGF-α(-/-) mice exposed to radiation or unirradiated C57-WT mice (63.0, 30.5 and 37.6 μg/lung, respectively, P = 0.01). C57-WT mice exposed to radiation had dense foci of subpleural fibrosis at 20 weeks after exposure, whereas the lungs of irradiated TGF-α (-/-) mice were largely devoid of fibrotic foci. Lung tissue concentrations of IL-1β, IL-4, TNF-α, TGF-β and EGF at multiple time points after irradiation were similar in C57-WT and TGF-α(-/-) mice. TGF-α in lung tissue of C57-WT mice rose rapidly after irradiation and remained elevated through 20 weeks. TGF-α(-/-) mice had lower basal LOX expression than C57-WT mice. Both LOX expression and LOX activity were increased after irradiation in all mice but to a lesser degree in TGF-α(-/-) mice. Treatment of NIH-3T3 fibroblasts with TGF-α resulted in increases in proliferation, collagen production and LOX activity. These studies identify TGF-α as a critical mediator of radiation-induced lung injury and a novel therapeutic target in this setting. Further, these data implicate TGF-α as a mediator of collagen maturation through a TGF-β independent activation of lysyl oxidase.

The TGF-beta-pseudoreceptor BAMBI is strongly expressed in COPD lungs and regulated by nontypeable Haemophilus influenzae.

PubMed

Drömann, Daniel; Rupp, Jan; Rohmann, Kristina; Osbahr, Sinia; Ulmer, Artur J; Marwitz, Sebastian; Röschmann, Kristina; Abdullah, Mahdi; Schultz, Holger; Vollmer, Ekkehard; Zabel, Peter; Dalhoff, Klaus; Goldmann, Torsten

2010-05-31

Nontypeable Haemophilus influenzae (NTHI) may play a role as an infectious trigger in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available regarding the influence of acute and persistent infection on tissue remodelling and repair factors such as transforming growth factor (TGF)-beta. NTHI infection in lung tissues obtained from COPD patients and controls was studied in vivo and using an in vitro model. Infection experiments were performed with two different clinical isolates. Detection of NTHI was done using in situ hybridization (ISH) in unstimulated and in in vitro infected lung tissue. For characterization of TGF-beta signaling molecules a transcriptome array was performed. Expression of the TGF-pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) was analyzed using immunohistochemistry (IHC), ISH and PCR. CXC chemokine ligand (CXCL)-8, tumor necrosis factor (TNF)-alpha and TGF-beta expression were evaluated in lung tissue and cell culture using ELISA. In 38% of COPD patients infection with NTHI was detected in vivo in contrast to 0% of controls (p < 0.05). Transcriptome arrays showed no significant changes of TGF-beta receptors 1 and 2 and Smad-3 expression, whereas a strong expression of BAMBI with upregulation after in vitro infection of COPD lung tissue was demonstrated. BAMBI was expressed ubiquitously on alveolar macrophages (AM) and to a lesser degree on alveolar epithelial cells (AEC). Measurement of cytokine concentrations in lung tissue supernatants revealed a decreased expression of TGF-beta (p < 0.05) in combination with a strong proinflammatory response (p < 0.01). We show for the first time the expression of the TGF pseudoreceptor BAMBI in the human lung, which is upregulated in response to NTHI infection in COPD lung tissue in vivo and in vitro. The combination of NTHI-mediated induction of proinflammatory cytokines and inhibition of TGF-beta expression may influence inflammation induced tissue remodeling.

Transforming Growth Factor β1 Induces the Expression of Collagen Type I by DNA Methylation in Cardiac Fibroblasts

PubMed Central

Pan, Xiaodong; Chen, Zhongpu; Huang, Rong; Yao, Yuyu; Ma, Genshan

2013-01-01

Transforming growth factor-beta (TGF-β), a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1) expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs) were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression. PMID:23560091

Structural modification of serum vitamin D3-binding protein and immunosuppression in AIDS patients.

PubMed

Yamamoto, N; Naraparaju, V R; Srinivasula, S M

1995-11-01

A serum glycoprotein, vitamin D3-binding protein (Gc protein), can be converted by beta-galactosidase of stimulated B lymphocytes and sialidase of T lymphocytes to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is a precursor for MAF. Treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high-titered MAF (GcMAF). When peripheral blood monocytes/macrophages of 46 HIV-infected patients were treated with GcMAF (100 pg/ml), the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of plasma Gc protein was low in 16 (35%) of of these patients. Loss of the MAF precursor activity appeared to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase found in the patient blood stream. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Thus, precursor activity of Gc protein and alpha-N-acetylgalactosaminidase activity in patient blood can serve as diagnostic and prognostic indices.

Protective role for miR-9-5p in the fibrogenic transformation of human dermal fibroblasts.

PubMed

Miguel, Verónica; Busnadiego, Oscar; Fierro-Fernández, Marta; Lamas, Santiago

2016-01-01

Excessive accumulation of extracellular matrix (ECM) proteins is the hallmark of fibrotic diseases, including skin fibrosis. This response relies on the activation of dermal fibroblasts that evolve into a pro-fibrogenic phenotype. One of the major players in this process is the cytokine transforming growth factor-β (TGF-β). MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression affecting a wide range of pathophysiological events including fibrogenesis. MicroRNA-9-5p (miR-9-5p) has been shown to exert a protective role in lung and peritoneal fibrosis. This study aimed to evaluate the role of miR-9-5p in skin fibrosis. miR-9-5p is up-regulated in TGF-β1-treated human dermal fibroblasts (HDFs). In silico identification of miR-9-5p targets spotted the type II TGF-β receptor (TGFBR2) as a potential TGF-β signaling-related effector for this miRNA. Consistently, over-expression of miR-9-5p in HDFs down-regulated TGFBR2 at both the mRNA and protein levels and reduced the phosphorylation of Smad2 and the translocation of Smad2/3 to the nucleus. In keeping, over-expression of miR-9-5p significantly delayed TGF-β1-dependent transformation of dermal fibroblasts, decreasing the expression of ECM protein collagen, type I, alpha 1 (Col1α1), and fibronectin (FN), the amount of secreted collagen proteins, and the expression of the archetypal myofibroblast marker alpha-smooth muscle actin (α-SMA). By contrast, specific inhibition of miR-9-5p resulted in enhanced presence of fibrosis markers. The expression of miR-9-5p was also detected in the skin and plasma in the mouse model of bleomycin-induced dermal fibrosis. Using lentiviral constructs, we demonstrated that miR-9-5p over-expression was also capable of deterring fibrogenesis in this same model. miR-9-5p significantly prevents fibrogenesis in skin fibrosis. This is mediated by an abrogation of TGF-β-mediated signaling through the down-regulation of TGFBR2 expression in HDFs. These results may pave the way for future diagnostic or therapeutic developments for skin fibrosis based on miR-9-5p.

Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus.

PubMed

Yamamoto, N; Naraparaju, V R; Moore, M; Brent, L H

1997-03-01

A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus.

Glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae: absence of ceramides from complete precursor glycolipids.

PubMed Central

Sipos, G; Puoti, A; Conzelmann, A

1994-01-01

Glycosylphosphatidylinositol (GPI) anchoring of membrane proteins occurs through two distinct steps, namely the assembly of a precursor glycolipid and its subsequent transfer onto newly synthesized proteins. To analyze the structure of the yeast precursor glycolipid we made use of the pmi40 mutant that incorporates very high amounts of [3H]mannose. Two very polar [3H]mannose-labeled glycolipids named CP1 and CP2 qualified as GPI precursor lipids since their carbohydrate head group, Man alpha 1,2(X-->PO4-->6)Man alpha 1,2Man alpha 1,6Man alpha-GlcN-inositol (with X most likely being ethanolamine) comprises the core structure which is common to all GPI anchors described so far. CP1 predominates in cells grown at 24 degrees C whereas CP2 is induced by stress conditions. The apparent structural identity of the head groups suggests that CP1 and CP2 contain different lipid moieties. The lipid moieties of both CP1 and CP2 can be removed by mild alkaline hydrolysis although the protein-bound GPI anchors made by the pmi40 cells under identical labeling conditions contain mild base resistant ceramides. These findings imply that the ceramide moiety found on the majority of yeast GPI anchored proteins is added through a lipid remodeling step that occurs after the addition of the GPI precursor glycolipids to proteins. Images PMID:8026463

Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

PubMed

Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

2005-04-01

After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

Characteristic cytokine generation patterns in cancer cells and infiltrating lymphocytes in oral squamous cell carcinomas and the influence of chemoradiation combined with immunotherapy on these patterns.

PubMed

Yamamoto, Tetsuya; Kimura, Tsuyoshi; Ueta, Eisaku; Tatemoto, Yukihiro; Osaki, Tokio

2003-01-01

Cytokines produced by tumor cells and tumor-infiltrating lymphocytes (TIL) appear to regulate tumor cell growth and the cytotoxic activity of TIL. The objectives of the present study were to investigate cytokine generation patterns in tumor cells and TIL and to examine the influence of cancer therapy on this cytokine production and the cytotoxic activity of TIL. We determined the levels of cytokines produced by tumor cells and TIL in vitro and measured the cytotoxic activity of TIL against Daudi cells in patients with oral squamous cell carcinoma (OSC) before and 1 week after the start of concomitant chemo-radio-immunotherapy. Before the therapy, OSC cells generated higher levels of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) than did oral keratinocytes isolated from the noninflamed gingivae of healthy individuals, but both kinds of cells generated similar levels of interleukin (IL)-1beta and IL-6. Compared with peripheral blood mononuclear cells (PBMC) of the patients, TIL produced higher levels of IL-1beta, IL-6, IL-10, TNF-alpha and TGF-beta, whereas their production of IL-12 and interferon-gamma (IFN-gamma) was only slightly higher than that in PBMC. After 1 week of therapy, the cytokine production by OSC cells had largely decreased, while the production of TNF-alpha, IFN-gamma, TGF-beta and IL-12 by TIL had increased greatly, although other cytokine levels were almost constant during the investigations. The cytotoxic activity of TIL was higher than that of PBMC before the therapy, and this activity was strongly increased by 1 week of therapy. These results suggest that the cytokine productivities of TIL and tumor cells differ from those of PBMC and normal keratinocytes, respectively, and that chemo-radio-immunotherapy modulates in situ cytokine generation, which is advantageous for inhibition of tumor cell growth and activation of TIL. Copyright 2003 S. Karger AG, Basel

[Effects of comprehensive therapy on serum SPARC levels in ankylosing spondylitis patients accompanied with osteoporosis].

PubMed

Xu, Jing-ren; Lin, Yan; Zhang, Chun-Yan; Li, Wei-Min; Guo, Chen-Jun; Ye, Lei

2013-04-01

To observe the effects of comprehensive therapy on serum secreted protein acidic and rich in cysteine (SPARC) levels in ankylosing spondylitis (AS) patients accompanied with osteoporosis (OP), and to explore the possible mechanisms for SPARC in AS patients accompanied with osteoporosis. Totally 48 AS patients accompanied with OP (Group A) were treated with massage, intravenous infusion of Cervus and Cucumis Polypeptide Injection, and Bushen Quhan Zhiwang Decoction (BQZD) for 3 months. At the same time, 45 normal healthy subjects were recruited as the normal control group (Group B). Serum SPARC levels were measured by ELISA in Group A before and after comprehensive therapy and in those of Group B. The levels of bone mineral density of femoral neck (FN BMD), bone mineral density of 2 -4 lumbar spine (L2-4 BMD), bone specific alkaline phosphatase (BSAP), tumor necrosis factor alpha (TNF-alpha), and transforming growth factor beta-1 (TGF-beta1) were detected. Meanwhile, Bath AS disease activity index (BASDAI) and Bath AS functional index (BASFI) were detected in Group A before and after treatment. The correlations between the aforesaid indices and serum SPARC levels were analyzed. Serum SPARC levels were significantly lower in those of Group A than in those of Group B (175. 30 +/- 72.04 micro/L vs 190. 52 +/- 86. 13 microg/ L, P <0. 01). Serum SPARC levels in those of Group A were negatively correlated with TNF-alpha (r = -0.261, P <0.01), positively with L2-4 BMD, TGF-beta1, and BSAP (r =0.437,0.256, 0.385, P <0.05, P <0.01). L2-4BMD and BSAP were independently predictors of serum SPARC in patients of Group A. After comprehensive therapy, the levels of TNF-alpha, BASDAI, and BASFI obviously decreased, TGF-beta1, BSAP, L2-4 BMD, and FN BMD obviously increased (P <0. 05, P <0. 01). The serum SPARC levels also significantly increased (188.32 +/- 87.50 microg/L, P <0. 05). Comprehensive therapy could effectively improve the bone metabolism, clinical symptoms and the activity function of joints, and elevate serum SPARC levels.

In vitro biosynthesis of 17 alpha,20 alpha,20 beta-dihydroxy-4-pegnen-3-one by the ovaries, testes, and head kidneys of the Atlantic salmon Salmo salar

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

Sangalang, G.B.; Freeman, H.C.

Ovaries, testes, and head kidneys of sexually mature Atlantic salmon, Salmo salar, biosynthesized 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHP) from equimolar amounts of (/sup 3/H)pregnenolone plus (4-/sup 14/C)progesterone in vitro. The /sup 3/H:/sup 14/C isotope ratios of steroid metabolites indicated that the biosynthetic pathways to 17 alpha,20 beta-diOHP in the testes differed from those observed in the ovaries and head kidneys. (4-/sup 14/C)progesterone appeared to be the principal precursor of 17 alpha,20 beta-diOHP in the testes, whereas both precursors were efficiently biotransformed to 17 alpha,20 beta-diOPH in the ovaries and head kidneys. 17 alpha-Hydroxy-4-pregnen-3-one (17 alpha-OHP) was the immediate precursormore » to 17 alpha,20 beta-diOHP in all tissues. However, appreciable amounts of 17 alpha,20 beta-diOHP accumulated in vitro in the testes only in the presence of exogenous (/sup 14/C)progesterone. Incubation of the testes, ovaries, and head kidneys with (/sup 14/C)pregnenolone resulted in high yields of 17 alpha,20 beta-diOHP in the ovaries and head kidneys but no detectable amounts of the steroid in the testes. The results confirm that progesterone is the favored precursor to 17 alpha,20 beta-diOHP in the testes. The results also suggest that the head kidneys may be an excellent cellular source of 17 alpha,20 beta-diOHP in both male and female. Atlantic salmon and may play an important role in the sexual maturation process in this fish. It is suggested that biosynthetic control mechanism affecting 17 alpha,20 beta-diOHP synthesis and/or spermiation and ovulation may differ in male and female Atlantic salmon.« less

The reduction in inflammation and impairment in wound healing by using strontium chloride hexahydrate.

PubMed

Berksoy Hayta, Sibel; Durmuş, Kasim; Altuntaş, Emine Elif; Yildiz, Esin; Hisarciklıo, Mehmet; Akyol, Melih

2018-03-01

Numerous growth factors, cytokine, mitogen and chemotactic factors are involved in wound healing. Even though inflammation is important for the stimulation of proliferative phase, excessive inflammation also causes impairment in wound healing. Strontium salts suppress keratinocyte-induced TNF-alpha and interleukin-1 and interleukin-6 in in vitro cultures. This study was conducted to determine the effects of administration of topical strontium chloride hexahydrate on wound healing through TNF-alpha and TGF-beta in surgical wound healing model of in-vivo rat skin. Twenty-four rats were used in the study. After approximately 2 cm cutaneous-subcutaneous incision was horizontally carried out on the mid-neckline of the rats, the incision was again closed using 2.0 vicryl. The rats were assigned into three groups including eight rats in each group. Placebo emollient ointment and also the ointments, which were containing 5% and 10% strontium chloride hexahydrate and were prepared at the same base with placebo ointment, were administered to the groups by a blind executor twice a day for a week. At the end of seventh day, the rats were sacrificed and cutaneous and subcutaneous tissue of their wound site was resected for histopathological examination. Scoring of histopathological wound healing and scoring of tissue TNF-alpha and TGF-beta level with immunohistochemical staining were performed. The groups, to which both 5% and 10% strontium chloride hexahydrate was administered, had lower immunohistochemical TNF-alpha levels and histopathological wound scores compared to controls, which was statistically significant (p < 0.05). Strontium chloride hexahydrate can lead to impairment in wound healing by suppressing inflammation through TNF-alpha.

Mouse macrophages primed with alendronate down-regulate monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) production in response to Toll-like receptor (TLR) 2 and TLR4 agonist via Smad3 activation.

PubMed

Masuda, Takahiro; Deng, Xue; Tamai, Riyoko

2009-08-01

Alendronate is one of the nitrogen-containing bisphosphonates (NBPs) used as anti-bone resorptive drugs. However, NBPs have inflammatory side effects including osteomyelitis and osteonecrosis of the jaw. In the present study, we examined the effects of alendronate on chemokine production by the macrophage-like cell line, J774.1, when incubated with Pam(3)CSK(4) (a Toll-like receptor (TLR) 2 agonist) and Lipid A (a TLR4 agonist). Pretreatment of J774.1 cells with alendronate decreased the production of TLR ligand-induced monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) but did not influence nuclear factor-kappaB (NF-kappaB) activation. While this agent induced caspase-8 activation, a caspase-8 inhibitor did not affect the decrease in MCP-1 production by alendronate and TLR ligands. Thus, the alendronate-mediated decrease in chemokine production was independent of NF-kappaB and caspase-8 activation. Although transforming growth factor-beta1 (TGF-beta1) is known to inhibit chemokine production by various cell types via Smad3 activation, pretreatment with alendronate did not increase TGF-beta1 production by J774.1 cells incubated in the presence or absence of TLR ligands. However, alendronate directly activated Smad3. These results suggest that by down-regulating MCP-1 and MIP-1alpha production via Smad3, long-term use of alendronate might inhibit normal activation and migration of osteoclasts and cause osteonecrosis.

Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae.

PubMed

Mayrhofer, Severine; Pöggeler, Stefanie

2005-04-01

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.

Chronic arsenic exposure increases TGFalpha concentration in bladder urothelial cells of Mexican populations environmentally exposed to inorganic arsenic☆

PubMed Central

Valenzuela, Olga L.; Germolec, Dori R.; Borja-Aburto, Víctor H.; Contreras-Ruiz, José; García-Vargas, Gonzalo G.; Del Razo, Luz M.

2009-01-01

Inorganic arsenic (iAs) is a well-established carcinogen and human exposure has been associated with a variety of cancers including those of skin, lung, and bladder. High expression of transforming growth factor alpha (TGF-α) has associated with local relapses in early stages of urinary bladder cancer. iAs exposures are at least in part determined by the rate of formation and composition of iAs metabolites (MAsIII, MAsV, DMAsIII, DMAsV). This study examines the relationship between TGF-α concentration in exfoliated bladder urothelial cells (BUC) separated from urine and urinary arsenic species in 72 resident women (18-51 years old) from areas exposed to different concentrations of iAs in drinking water (2-378 ppb) in central Mexico. Urinary arsenic species, including trivalent methylated metabolites were measured by hydride generation atomic absorption spectrometry method. The concentration of TGF-α in BUC was measured using an ELISA assay. Results show a statistically significant positive correlation between TGF-α concentration in BUC and each of the six arsenic species present in urine. The multivariate linear regression analyses show that the increment of TGF-α levels in BUC was importantly associated with the presence of arsenic species after adjusting by age, and presence of urinary infection. People from areas with high arsenic exposure had a significantly higher TGF-α concentration in BUC than people from areas of low arsenic exposure (128.8 vs. 64.4 pg/mg protein; p<0.05). Notably, exfoliated cells isolated from individuals with skin lesions contained significantly greater amount of TGF-α than cells from individuals without skin lesions: 157.7 vs. 64.9 pg/mg protein (p=0.003). These results suggest that TGF-α in exfoliated BUC may serve as a susceptibility marker of adverse health effects on epithelial tissue in arsenic-endemic areas. PMID:17267001

Regulatory T cells generated during cytomegalovirus in vitro stimulation of mononuclear cells from HIV-infected individuals on HAART correlate with decreased lymphocyte proliferation

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

Jesser, Renee D.; Li, Shaobing; Weinberg, Adriana

2006-09-01

HIV-infected patients fail to fully recover cell-mediated immunity despite HAART. To identify regulatory factors, we studied the phenotype and function of in vitro cytomegalovirus (CMV)-stimulated T cells from HAART recipients. CFSE-measured proliferation showed CD4{sup +} and CD8{sup +} cells dividing in CMV-stimulated cultures. Compared with healthy controls, CMV-stimulated lymphocytes from HAART recipients had lower {sup 3}H-thymidine incorporation; lower IFN{gamma} and TNF{alpha} production; higher CD4{sup +}CD27{sup -}CD28{sup -} and CD8{sup +}CD27{sup -}CD28{sup -} frequencies; lower CD4{sup +}CD25{sup hi}; and higher FoxP3 expression in CD8{sup +}CD25{sup hi} cells. CMV-specific proliferation correlated with higher IFN{gamma}, TNF{alpha} and IL10 levels and higher CD4{sup +}perforin{supmore » +} and CD8{sup +}perforin{sup +} frequencies. Decreased proliferation correlated with higher CD4{sup +}CD27{sup -}CD28{sup -} frequencies and TGF{beta}1 production, which also correlated with each other. Anti-TGF{beta}1 neutralizing antibodies restored CMV-specific proliferation in a dose-dependent fashion. In HIV-infected subjects, decreased proliferation correlated with higher CMV-stimulated CD8{sup +}CD25{sup hi} frequencies and their FoxP3 expression. These data indicate that FoxP3- and TGF{beta}1-expressing regulatory T cells contribute to decreased immunity in HAART recipients.« less

Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses in human mesangial cells.

PubMed

Hayashida, Tomoko; Decaestecker, Mark; Schnaper, H William

2003-08-01

Transforming growth factor beta (TGF-beta) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF-beta1 activated extracellular signal-regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant-negative ERK blockade reduced TGF-beta1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C-terminal SS(P)XS(P) motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 microM) or U0126 (25 microM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK-dependent activation of Smad2/3 occurs only in certain cell types. TGF-beta stimulated phosphorylation of an expressed Smad3A construct, with a mutated C-terminal SS(P)XS(P) motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced alpha2(I) collagen promoter activity, an effect blocked by co-transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF-beta1 on collagen promoter activity were additive. These results indicate that ERK-dependent R-Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.

Analysis of the therapeutic gain in the treatment of human osteosarcoma microcolonies in vitro with 211At-labelled monoclonal antibody.

PubMed

Larsen, R H; Bruland, O S; Hoff, P; Alstad, J; Rofstad, E K

1994-06-01

Microcolonies were obtained by culturing cells of two human osteosarcoma lines (OHS and KPDX) and one human melanoma line (WIX-c) for either 24 or 72 h. The microcolonies were treated with either alpha-particle radiation emitted by the 211At-labelled monoclonal antibody (MAb) TP-3 or external beam X-rays. Survival of microcolonies was assayed by colony formation. Therapeutic gain factor (TGF) values were calculated for two survival levels, 50% and 20% microcolony regeneration (i.e. at least one cell in 50% or 20% of the colonies survived the treatments). The TGF values were affected by the specific activity of the 211At-MAb conjugate, the antigen expression of the cells and the size and growth pattern of the microcolonies. Treatment with 211At-TP-3 gave TGF values that varied from 1.3 +/- 0.4 to 4.5 +/- 0.7 (mean +/- s.e.). The antigen-rich OHS cell line had on average 1.6 times higher TGF than the antigen-poor KPDX cell line. The TGF increased significantly with colony size for the densely packed colonies of the KPDX cell line but not for the OHS cell line, which had colonies with cells growing in a more scattered pattern. Control experiments with the two non-specific 211At forms, free 211At and 211At-labelled bovine serum albumin, gave TGF values from 0.6 +/- 0.1 to 1.0 +/- 0.3. This study suggests that in vivo evaluation of 211At-MAbs using relevant tumour models is desirable.

Analysis of the therapeutic gain in the treatment of human osteosarcoma microcolonies in vitro with 211At-labelled monoclonal antibody.

PubMed Central

Larsen, R. H.; Bruland, O. S.; Hoff, P.; Alstad, J.; Rofstad, E. K.

1994-01-01

Microcolonies were obtained by culturing cells of two human osteosarcoma lines (OHS and KPDX) and one human melanoma line (WIX-c) for either 24 or 72 h. The microcolonies were treated with either alpha-particle radiation emitted by the 211At-labelled monoclonal antibody (MAb) TP-3 or external beam X-rays. Survival of microcolonies was assayed by colony formation. Therapeutic gain factor (TGF) values were calculated for two survival levels, 50% and 20% microcolony regeneration (i.e. at least one cell in 50% or 20% of the colonies survived the treatments). The TGF values were affected by the specific activity of the 211At-MAb conjugate, the antigen expression of the cells and the size and growth pattern of the microcolonies. Treatment with 211At-TP-3 gave TGF values that varied from 1.3 +/- 0.4 to 4.5 +/- 0.7 (mean +/- s.e.). The antigen-rich OHS cell line had on average 1.6 times higher TGF than the antigen-poor KPDX cell line. The TGF increased significantly with colony size for the densely packed colonies of the KPDX cell line but not for the OHS cell line, which had colonies with cells growing in a more scattered pattern. Control experiments with the two non-specific 211At forms, free 211At and 211At-labelled bovine serum albumin, gave TGF values from 0.6 +/- 0.1 to 1.0 +/- 0.3. This study suggests that in vivo evaluation of 211At-MAbs using relevant tumour models is desirable. PMID:8198960

Vasodilator-stimulated phosphoprotein promotes activation of hepatic stellate cells by regulating Rab11-dependent plasma membrane targeting of transforming growth factor beta receptors.

PubMed

Tu, Kangsheng; Li, Jiachu; Verma, Vikas K; Liu, Chunsheng; Billadeau, Daniel D; Lamprecht, Georg; Xiang, Xiaoyu; Guo, Luyang; Dhanasekaran, Renumathy; Roberts, Lewis R; Shah, Vijay H; Kang, Ningling

2015-01-01

Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF-β) stimulation, hepatic stellate cells (HSCs), which are liver-specific pericytes, transdifferentiate into tumor-associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator-stimulated phosphoprotein (VASP) of HSCs regulated the TGF-β-mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up-regulation of VASP and alpha-smooth muscle actin (α-SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF-β-mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF-β receptor II (TβRII) and Rab11, a Ras-like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11-dependent targeting of TβRII to the plasma membrane, thereby desensitizing HSCs to TGF-β1 stimulation. Our study demonstrates a requirement of VASP for TGF-β-mediated HSC activation in the tumor microenvironment by regulating Rab11-dependent recycling of TβRII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver. © 2014 by the American Association for the Study of Liver Diseases.

Simulation of TGF-Beta Activation by Low-Dose HZE Radiation in a Cell Culture

NASA Technical Reports Server (NTRS)

Plante, Ianik; Cucinotta, Francis A.

2009-01-01

High charge (Z) and energy (E) (HZE) nuclei comprised in the galactic cosmic rays are main contributors to space radiation risk. They induce many lesions in living matter such as non-specific oxidative damage and the double-strand breaks (DSBs), which are considered key precursors of early and late effects of radiation. There is increasing evidence that cells respond collectively rather than individually to radiation, suggesting the importance of cell signaling1. The transforming growth factor (TGF ) is a signaling peptide that is expressed in nearly all cell type and regulates a large array of cellular processes2. TGF have been shown to mediate cellular response to DNA damage3 and to induce apoptosis in non-irradiated cells cocultured with irradiated cells4. TFG molecules are secreted by cells in an inactive complex known as the latency-associated peptide (LAP). TGF is released from the LAP by a conformational change triggered by proteases, thrombospondin-1, integrins, acidic conditions and .OH radical5. TGF then binds to cells receptors and activates a cascade of events mediated by Smad proteins6, which might interfere with the repair of DNA. Meanwhile, increasingly sophisticated Brownian Dynamics (BD) algorithms have appeared recently in the literature7 and can be applied to study the interaction of molecules with receptors. These BD computer models have contributed to the elucidation of signal transduction, ligand accumulation and autocrine loops in the epidermal growth factor (EGF) and its receptor (EFGR) system8. To investigate the possible roles of TGF in an irradiated cell culture, our Monte-Carlo simulation codes of the radiation track structure9 will be used to calculate the activation of TFG triggered by .OH produced by low doses of HZE ions. The TGF molecules will then be followed by a BD algorithm in a medium representative of a cell culture to estimate the number of activated receptors.

Toward a reliable distinction between patients with mild cognitive impairment and Alzheimer-type dementia versus major depression.

PubMed

Post, Anke; Ackl, Nibal; Rücker, Monika; Schreiber, Yvonne; Binder, Elisabeth B; Ising, Marcus; Sonntag, Annette; Holsboer, Florian; Almeida, Osborne F X

2006-05-01

Cerebrospinal fluid (CSF) levels of soluble amyloid precursor protein (sAPP) and its alpha-secreted form (alpha-sAPP) were investigated as a means to distinguish between individuals with mild cognitive impairment (MCI) and Alzheimer-type dementia (DAT) and those with major depressive episode (MDE) showing secondary memory deficits. Twenty-seven patients with MCI, 32 with probable DAT, and 24 with MDE attending a memory clinic were studied. Cerebrospinal fluid levels of sAPP/amyloid precursor-like protein 2 (APLP2) and alpha-sAPP were detected by Western blotting. Patients with MDE had the highest CSF levels of total sAPP/APLP2 as compared with MCI and DAT patients (p < .001); sAPP/APLP2 levels were higher in MCI than in DAT subjects. Whereas alpha-sAPP levels did not differ between the MCI and DAT groups, median levels of this peptide were significantly lower in MCI and DAT versus MDE patients. Soluble amyloid precursor protein/APLP2 and alpha-sAPP concentrations in CSF can differentiate between DAT and MCI versus MDE, facilitating early ameliorative interventions and appropriate treatment regimens.

Increased neuronal beta-amyloid precursor protein expression in human temporal lobe epilepsy: association with interleukin-1 alpha immunoreactivity.

PubMed

Sheng, J G; Boop, F A; Mrak, R E; Griffin, W S

1994-11-01

Levels of immunoreactive beta-amyloid precursor protein and interleukin-1 alpha were found to be elevated in surgically resected human temporal lobe tissue from patients with intractable epilepsy compared with postmortem tissue from neurologically unaffected patients (controls). In tissue from epileptics, the levels of the 135-kDa beta-amyloid precursor protein isoform were elevated to fourfold (p < 0.05) those of controls and those of the 130-kDa isoform to threefold (p < 0.05), whereas those of the 120-kDa isoform (p > 0.05) were not different from control values. beta-Amyloid precursor protein-immunoreactive neurons were 16 times more numerous, and their cytoplasm and proximal processes were more intensely immunoreactive in tissue sections from epileptics than controls (133 +/- 12 vs. 8 +/- 3/mm2; p < 0.001). However, neither beta-amyloid precursor protein-immunoreactive dystrophic neurites nor beta-amyloid deposits were found in this tissue. Interleukin-1 alpha-immunoreactive cells (microglia) were three times more numerous in epileptics than in controls (80 +/- 8 vs. 25 +/- 5/mm2; p < 0.001), and these cells were often found adjacent to beta-amyloid precursor protein-immunoreactive neuronal cell bodies. Our findings, together with functions established in vitro for interleukin-1, suggest that increased expression of this protein contributes to the increased levels of beta-amyloid precursor protein in epileptics, thus indicating a potential role for both of these proteins in the neuronal dysfunctions, e.g., hyperexcitability, characteristic of epilepsy.

Stereoselective Synthesis of [alpha, alpha][superscript ']-Biprolines

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

Vartak, Ashish P.; Young, Jr., Victor G.; Johnson, Rodney L.

2010-11-10

A means to induce dehydrodimerization of Seebach's oxazolidinone (5), the stereochemical outcome of which is entirely temperature dependent, is described. The resultant dimers 3 and 4 are precursors to (R,R)-alpha,alpha'-biproline (1) and meso-alpha,alpha'-biproline (2), respectively. An organohypobromite and an iminium halide are proposed to serve as electrophiles in the reaction with the enolate of 5 to give 3 and 4, respectively.

Immunostaining and transcriptional enhancement of interleukin-1 receptor type I in the rat dental follicle.

PubMed

Wise, G E; Zhao, L

1997-05-01

Interleukin-1alpha (IL-1alpha) enhances the gene expression of colony-stimulating factor-one (CSF-1) in dental follicle cells. In turn, CSF-1 appears to be a critical molecule in stimulating the cellular events of eruption that require the presence of the follicle. Chronologically, the maximal transcription and translation of CSF-1 in the follicle occurs early postnatally, followed by a decline later. Thus, in this study, immunostaining for the interleukin-1 receptor type I (IL-1RI) was used to determine if it paralleled the CSF-1 localization and chronology. The results showed that IL-1RI is primarily localized in the dental follicle, with maximal immunostaining early postnatally and a greatly reduced staining by day 10. In conjunction with this, molecules that enhance the gene expression of IL-1alpha epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1) were also shown to enhance the expression of IL-1RI, but IL-1alpha did not increase the gene expression of IL-1RI. After injections of EGF at different times postnatally the mRNA of IL-1RI increased over comparable controls. Between days 2 and 5 the IL-1RI mRNA in the follicle decreased. In combination the results suggest that, as the expression of IL-1alpha is enhanced in the stellate reticulum either by EGF or TGF-beta1, these two molecules could also enhance the expression of IL-1RI in the dental follicle such that more receptors would be available to respond to the increased IL-1alpha secreted. The maximal presence of the receptors (IL-1RI) in the dental follicle early postnatally, followed by their subsequent decline, parallels the rise and fall of CSF-1 in the follicle. Thus, regulation of the IL-1RI and IL-1RI gene expression might be a means of regulating changes in CSF-1 in the follicle.

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

Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells.

PubMed

Peng, L A; Schousboe, A; Hertz, L

1991-01-01

Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transmination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.

The 53-kDa proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillus niger has Taka-amylase-like activity.

PubMed

Ravi-Kumar, K; Venkatesh, K S; Umesh-Kumar, S

2007-04-01

The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent alpha-glucosidase inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of alpha-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions.

Progressive loss of sensitivity to growth control by retinoic acid and transforming growth factor-beta at late stages of human papillomavirus type 16-initiated transformation of human keratinocytes.

PubMed

Creek, K E; Geslani, G; Batova, A; Pirisi, L

1995-01-01

Retinoids (vitamin A and its natural and synthetic derivatives) have shown potential as chemopreventive agents, and diets poor in vitamin A and/or its precursor beta-carotene have been linked to an increased risk of cancer at several sites including the cervix. Human papillomavirus (HPV) plays an important role in the etiology of cervical cancer. We have developed an in vitro model of cancer progression using human keratinocytes (HKc) immortalized by HPV16 DNA (HKc/HPV16). Although immortal, early passage HKc/HPV16, like normal HKc, require epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation and undergo terminal differentiation in response to serum and calcium. However, following prolonged culture, growth factor independent HKc/HPV16 lines that no longer require EGF and BPE can be selected (HKc/GFI). Further selection of HKc/GFI produces lines that are resistant to serum- and calcium- induced terminal differentiation (HKc/DR). HKc/DR, but not early passage HKc/HPV16, are susceptible to malignant conversion following transfection with viral Harvey ras or Herpes simplex virus type II DNA. We have investigated the sensitivity of low to high passage HKc/HPV16 and HKc/GFI to growth control by all-trans-retinoic acid (RA, an active metabolite of vitamin A). Early passage HKc/HPV16 are very sensitive to growth inhibition by RA, and in these cells RA decreases the expression of the HPV16 oncogenes E6 and E7. However, as the cells progress in culture they lose their sensitivity to RA. Growth inhibition by RA may be mediated through the cytokine transforming growth factor-beta (TGF-beta), a potent inhibitor of epithelial cell proliferation. RA treatment of HKc/HPV16 and HKc/GFI results in a dose-and time-dependent induction (maximal of 3-fold) in secreted levels of TGF-beta. Also, Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced TGF-beta 1 and TGF-beta 2 expression about 3- and 50-fold, respectively. We next studied the effect of TGF-beta 1 and TGF-beta 2 on the proliferation of early to late passage HKc/HPVa6, HKc/GFI and HKc/DR. While early passage HKc/HPV16 were as sensitive as normal HKc to growth inhibition by TGF-beta 1 and TGF-beta 2, the cells became increasingly resistant to TGF-beta during in vitro progression, with the proliferation of HKc/DR being virtually unaffected by TGF-beta 1 or TGF-beta 2 treatment. Overall, loss of growth inhibition by RA parallels loss of TGF-beta sensitivity.(ABSTRACT TRUNCATED AT 400 WORDS)

DACH1 inhibits transforming growth factor-beta signaling through binding Smad4.

PubMed

Wu, Kongming; Yang, Ying; Wang, Chenguang; Davoli, Maria A; D'Amico, Mark; Li, Anping; Cveklova, Kveta; Kozmik, Zbynek; Lisanti, Michael P; Russell, Robert G; Cvekl, Ales; Pestell, Richard G

2003-12-19

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.

Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis.

PubMed

Apte, M V; Haber, P S; Darby, S J; Rodgers, S C; McCaughan, G W; Korsten, M A; Pirola, R C; Wilson, J S

1999-04-01

The pathogenesis of pancreatic fibrosis is unknown. In the liver, stellate cells play a major role in fibrogenesis by synthesising increased amounts of collagen and other extracellular matrix (ECM) proteins when activated by profibrogenic mediators such as cytokines and oxidant stress. To determine whether cultured rat pancreatic stellate cells produce collagen and other ECM proteins, and exhibit signs of activation when exposed to the cytokines platelet derived growth factor (PDGF) or transforming growth factor beta (TGF-beta). Cultured pancreatic stellate cells were immunostained for the ECM proteins procollagen III, collagen I, laminin, and fibronectin using specific polyclonal antibodies. For cytokine studies, triplicate wells of cells were incubated with increasing concentrations of PDGF or TGF-beta. Cultured pancreatic stellate cells stained strongly positive for all ECM proteins tested. Incubation of cells with 1, 5, and 10 ng/ml PDGF led to a significant dose related increase in cell counts as well as in the incorporation of 3H-thymidine into DNA. Stellate cells exposed to 0.25, 0.5, and 1 ng/ml TGF-beta showed a dose dependent increase in alpha smooth muscle actin expression and increased collagen synthesis. In addition, TGF-beta increased the expression of PDGF receptors on stellate cells. Pancreatic stellate cells produce collagen and other extracellular matrix proteins, and respond to the cytokines PDGF and TGF-beta by increased proliferation and increased collagen synthesis. These results suggest an important role for stellate cells in pancreatic fibrogenesis.

Hydrogen Rich Water Attenuates Renal Injury and Fibrosis by Regulation Transforming Growth Factor-β Induced Sirt1.

PubMed

Xing, Zhaoyu; Pan, Wanma; Zhang, Jing; Xu, Xianlin; Zhang, Xuemei; He, Xiaozhou; Fan, Min

2017-01-01

The current research was designed to study the role of hydrogen in renal fibrosis and the renal epithelial to mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1). Hydrogen rich water (HW) was used to treat animal and cell models. Unilateral ureteral obstruction (UUO) was performed on Balb/c mice to create a model of renal fibrosis. Human kidney proximal tubular epithelial cells (HK-2 cells) were treated with TGF-β1 for 36 h to induce EMT. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured to test renal function, in addition, kidney histology and immunohistochemical staining of alpha-smooth muscle actin (α-SMA) positive cells was performed to examine the morphological changes. The treatment with UUO induced a robust fibrosis of renal interstitium, shrink of glomerulus and partial fracture of basement membrane. Renal function was also impaired in the experimental group with UUO, with an increase of Scr and BUN in serum. After that, Western-blot was performed to examine the expression of α-SMA, fibronectin, E-cadherin, Smad2 and Sirtuin-1 (Sirt1). The treatment with HW attenuated the development of fibrosis and deterioration of renal function in UUO model. In HK-2 cells, the pretreatment of HW abolished EMT induced by TGF-β1. The down-regulation the expression of Sirt1 induced by TGF-β1 which was dampened by the treatment with HW. Sirtinol, a Sirt1 inhibitor, reversed the effect of HW on EMT induced by TGF-β1. HW can inhibit the development of fibrosis in kidney and prevents HK-2 cells from undergoing EMT which is mediated through Sirt1, a downstream molecule of TGF-β1.

MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-β signalling pathway

PubMed Central

Lynch, Jennifer; Fay, Joanna; Meehan, Maria; Bryan, Kenneth; Watters, Karen M.; Murphy, Derek M.; Stallings, Raymond L.

2012-01-01

Transforming growth factor-β (TGF-β) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-β non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-β pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-β signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-β non-canonical signaling by miR-335, which in turn is downregulated by MYCN. PMID:22382496

Beneficial effects of enalapril on chlorhexidine digluconate-induced liver peritoneal fibrosis in rats.

PubMed

Lee, Chung-Jen; Subeq, Yi-Maun; Lee, Ru-Ping; Ke, Chen-Yen; Lin, Nien-Tsung; Hsu, Bang-Gee

2011-08-31

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 enalapril on chlorhexidine digluconate-induced liver PF by decreasing transforming growth factor-β1 (TGF-β1) production in rats. 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 one week. Rats received daily intravenous injections of low dose enalapril (1 mg/kg), or high dose enalapril (2.5 mg/kg), for one week. After 7 days, conventional 4.25% Dianeal (30 ml) was administered via a PD catheter with a dwell time of 4 h and assessment of peritoneal function. At the end of dialysis, the rats were sacrificed and liver peritoneum was harvested for microscopic examination and immunohistochemistry. There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D₄/P₄(urea) level was reduced, the D₄/D₀ glucose level, serum and the dialysate TGF-β1 level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, collagen and vascular endothelial growth factor (VEGF) were elevated in the PF group compared with the vehicle group. High dose of enalapril decreased the serum and dialysate TGF-β1 levels, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen and VEGF-positive cells in the liver peritoneum. Low dose of enalapril did not protect against chlorhexidine digluconate-induced PF in the rat. Enalapril protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-β1 production.

Comparison of potentials between stem cells isolated from human anterior cruciate ligament and bone marrow for ligament tissue engineering.

PubMed

Cheng, Ming-Te; Liu, Chien-Lin; Chen, Tain-Hsiung; Lee, Oscar K

2010-07-01

We have previously isolated and identified stem cells from human anterior cruciate ligament (ACL). The purpose of this study was to evaluate the differences in proliferation, differentiation, and extracellular matrix (ECM) formation abilities between bone marrow stem cells (BMSCs) and ACL-derived stem cells (LSCs) from the same donors when cultured with different growth factors, including basic fibroblast growth factor (bFGF), epidermal growth factor, and transforming growth factor-beta 1 (TGF-beta1). Ligament tissues and bone marrow aspirate were obtained from patients undergoing total knee arthroplasty and ACL reconstruction surgeries. Proliferation, colony formation, and population doubling capacity as well as multilineage differentiation potentials of LSCs and BMSCs were compared. Gene expression and ECM production for ligament engineering were also evaluated. It was found that BMSCs possessed better osteogenic differentiation potential than LSCs, while similar adipogenic and chondrogenic differentiation abilities were observed. Proliferation rates of both LSCs and BMSCs were enhanced by bFGF and TGF-beta1. TGF-beta1 treatment significantly increased the expression of type I collagen, type III collagen, fibronectin, and alpha-smooth muscle actin in LSCs, but TGF-beta1 only upregulated type I collagen and tenascin-c in BMSCs. Protein quantification further confirmed the results of differential gene expression and suggested that LSCs and BMSCs increase ECM production upon TGF-beta1 treatment. In summary, in comparison with BMSCs, LSCs proliferate faster and maintain an undifferentiated state with bFGF treatment, whereas under TGF-beta1 treatment, LSCs upregulate major tendinous gene expression and produce a robust amount of ligament ECM protein, making LSCs a potential cell source in future applications of ACL tissue engineering.

TGF-beta in human milk is associated with wheeze in infancy.

PubMed

Oddy, Wendy H; Halonen, Marilyn; Martinez, F D; Lohman, I Carla; Stern, Debra A; Kurzius-Spencer, Margaret; Guerra, Stefano; Wright, Anne L

2003-10-01

Cytokines secreted in human milk might play important roles in newborn health and in the development of infant immune responses. We investigated the relationship of the concentration and dose of cytokines in human milk to infant wheeze at 1 year of age. Our objective was to test whether the cytokines in milk could account for some of the apparent protective effect of breast-feeding against wheeze in the first year of life. Data on breast-feeding and infant wheeze were collected prospectively from birth to 1 year from 243 mothers participating in the Infant Immune Study in Tucson, Arizona. Breast milk samples obtained at a mean age of 11 days postpartum were assayed by means of ELISA for concentrations of TGF-beta1, IL-10, TNF-alpha, and the soluble form of CD14. The dose of each cytokine was assessed for a relationship with wheeze in bivariate and logistic regression analyses. Increasing duration of breast-feeding was significantly associated with a decreased prevalence of wheeze (P =.039). There was wide variability in levels of each cytokine in milk, as well as variability between women in the amount of each cytokine produced. There was a significant inverse association between the dose of TGF-beta1 received through milk with the percentage of wheeze (P =.017), and the relationship was linear (P =.006). None of the other cytokines showed a linear relationship with wheeze. In multivariate analyses the risk of wheeze was significantly decreased (odds ratio, 0.22; 95% CI 0.05-0.89; P =.034) with increasing TGF-beta1 dose (long breast-feeding and medium-high TGF-beta1 level compared with short breast-feeding and low TGF-beta. This analysis shows that the dose of TGF-beta1 received from milk has a significant relationship with infant wheeze, which might account for at least some of the protective effect of breast-feeding against wheeze.

Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-{beta} responsiveness

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

Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua

2008-03-10

Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-{beta} (TGF-{beta}) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-{beta}, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-{beta}. To explore this notion, we characterized TGF-{beta}-induced activation of fibroblasts from CCN2-null (CCN2{sup -/-}) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-{beta} signal transduction and regulation of collagen genemore » expression were examined in CCN2{sup -/-} MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2{sup -/-} MEFs was markedly reduced compared to wild type MEFs, TGF-{beta}-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2{sup -/-} MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-{beta}-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.« less

Valsartan decreases TGF-β1 production and protects against chlorhexidine digluconate-induced liver peritoneal fibrosis in rats.

PubMed

Subeq, Yi-Maun; Ke, Chen-Yen; Lin, Nien-Tsung; Lee, Chung-Jen; Chiu, Yi-Han; Hsu, Bang-Gee

2011-02-01

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 valsartan on chlorhexidine digluconate-induced PF by decreasing TGF-β1 production in rats. PF was induced in Sprague-Dawley rats by daily administration of 0.5 ml 0.1% chlorhexidine digluconate in normal saline via peritoneal dialysis (PD) tube for 1 week. Rats received daily intravenous injections of low dose valsartan (1 mg/kg) or high dose valsartan (3 mg/kg) 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 D₄/P(4Urea) level was reduced, the D₄/D₀ glucose level, serum and dialysate transforming growth factor-β1 (TGF-β1) level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, collagen, and vascular endothelial growth factor (VEGF) were elevated in the PF group compared with the vehicle group. High dose of valsartan decreased the serum and dialysate TGF-β1 level, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen, and VEGF-positive cells in liver peritoneum. The low dose of valsartan did not protect against chlorhexidine digluconate-induced PF in rat. Valsartan protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-β1 production. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

Phthalimide neovascular factor 1 (PNF1) modulates MT1-MMP activity in human microvascular endothelial cells

PubMed Central

Wieghaus, Kristen A.; Gianchandani, Erwin P.; Neal, Rebekah A.; Paige, Mikell A.; Brown, Milton L.; Papin, Jason A.; Botchwey, Edward A.

2009-01-01

We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode-of-action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray dataset of PNF1-treated (versus control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-α) and, subsequently, transforming growth factor-beta (TGF-β) signaling networks by PNF1. Here we validate this microarray data-set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this dataset and identify three specific TGF-β-induced genes with regulation by PNF1 conserved over multiple timepoints—amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)—that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1–48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode-of-action and important for successful promotion of the neovascularization that we have observed by the drug in vivo. PMID:19326468

Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model.

PubMed

Cashman, J D; Clark-Lewis, I; Eaves, A C; Eaves, C J

1999-12-01

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice transplanted with human cord blood or adult marrow cells and injected 6 weeks posttransplant with 2 daily doses of transforming growth factor-beta(1) (TGF-beta(1)), monocyte chemoattractant protein-1 (MCP-1), or a nonaggregating form of macrophage inflammatory protein-1alpha (MIP-1alpha) showed unique patterns of inhibition of human progenitor proliferation 1 day later. TGF-beta(1) was active on long-term culture initiating cells (LTC-IC) and on primitive erythroid and granulopoietic colony-forming cells (HPP-CFC), but had no effect on mature CFC. MCP-1 inhibited the cycling of both types of HPP-CFC but not LTC-IC. MIP-1alpha did not inhibit either LTC-IC or granulopoietic HPP-CFC but was active on erythroid HPP-CFC and mature granulopoietic CFC. All of these responses were independent of the source of human cells transplanted. LTC-IC of either human cord blood or adult marrow origin continue to proliferate in NOD/SCID mice for many weeks, although the turnover of all types of human CFC in mice transplanted with adult human marrow (but not cord blood) is downregulated after 6 weeks. Interestingly, administration of either MIP-1beta, an antagonist of both MIP-1alpha and MCP-1 or MCP-1(9-76), an antagonist of MCP-1 (and MCP-2 and MCP-3), into mice in which human marrow-derived CFC had become quiescent, caused the rapid reactivation of these progenitors in vivo. These results provide the first definition of stage-specific inhibitors of human hematopoietic progenitor cell cycling in vivo. In addition they show that endogenous chemokines can contribute to late graft failure, which can be reversed by the administration of specific antagonists.

Correlation of serum MMP3 and other biomarkers with clinical outcomes in patients with ankylosing spondylitis: A pilot study

USDA-ARS?s Scientific Manuscript database

The studies aimed to assess a set of biomarkers for their correlations with disease activity/severity of patients with ankylosing spondylitis (AS). A total of 24 AS patients were treated with etanercept and prospectively followed for 12 weeks. Serum levels of TNF-alpha, IFN-gamma, TGF-beta, IL6, IL1...

Low molecular weight fucoidan and its fractions inhibit renal epithelial mesenchymal transition induced by TGF-β1 or FGF-2.

PubMed

Li, Xinpeng; Li, Xiaohong; Zhang, Quanbin; Zhao, Tingting

2017-12-01

We investigated the renal protective effects of low molecular weight fucoidan (LMWF) and its two fractions (F0.5 and F1.0), which were extracted from Laminaria japonica, on the epithelial-mesenchymal transition (EMT) induced by transforming growth factor beta 1 (TGF-β1) and fibroblast growth factor 2 (FGF-2) in HK-2 human renal proximal tubular cells. Cell morphology and EMT markers (fibronectin and alpha-smooth muscle actin) demonstrated that cells treated with TGF-β1 or FGF-2 developed EMT to a significant extent. Treatment with LMWF or its fractions markedly attenuated the EMT and decreased expression of the EMT markers. The F1.0 fraction, the sulfated fucan fraction, was found to be the main active component of LMWF, and heparanase (HPSE) was a key factor in renal tubular epithelial trans-differentiation. The F1.0 fraction inhibited elevated HPSE and matrix metallopeptidase 9 expression, thereby attenuating the progress of EMT. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

Selective deletion of apolipoprotein E in astrocytes ameliorates the spatial learning and memory deficits in Alzheimer's disease (APP/PS1) mice by inhibiting TGF-β/Smad2/STAT3 signaling.

PubMed

Zheng, Jin-Yu; Sun, Jian; Ji, Chun-Mei; Shen, Lin; Chen, Zhong-Jun; Xie, Peng; Sun, Yuan-Zhao; Yu, Ru-Tong

2017-06-01

Astrocytes and apolipoprotein E (apoE) play critical roles in cognitive function, not only under physiological conditions but also in some pathological situations, particularly in the pathological progression of Alzheimer's disease (AD). The regulatory mechanisms underlying the effect of apoE, derived from astrocytes, on cognitive deficits during AD pathology development are unclear. In this study, we generated amyloid precursor protein/apoE knockout (APP/apoE KO ) and APP/glial fibrillary acidic protein (GFAP)-apoE KO mice (the AD mice model used in this study was based on the APP-familial Alzheimer disease overexpression) to investigate the role of apoE, derived from astrocytes, in AD pathology and cognitive function. To explore the mechanism, we investigated the amyloidogenic process related transforming growth factor β/mothers against decapentaplegic homolog 2/signal transducer and activator of transcription 3 (TGF-β/Smad2/STAT3) signaling pathway and further confirmed by administering TGF-β-overexpression adeno-associated virus (specific to astrocytes) to APP/GFAP-apoE KO mice and TGF-β-inhibition adeno-associated virus (specific to astrocytes) to APP/WT mice. Whole body deletion of apoE significantly ameliorated the spatial learning and memory impairment, reduced amyloid β-protein production and inhibited astrogliosis in APP/apoE KO mice, as well as specific deletion apoE in astrocytes in APP/GFAP-apoE KO mice. Moreover, amyloid β-protein accumulation was increased due to promotion of amyloidogenesis of APP, and astrogliosis was upregulated by activation of TGF-β/Smad2/STAT3 signaling. Furthermore, the overexpression of TGF-β in astrocytes in APP/GFAP-apoE KO mice abrogated the effects of apoE knockout. In contrast, repression of TGF-β in astrocytes of APP/WT mice exerted a therapeutic effect similar to apoE knockout. These data suggested that apoE derived from astrocytes contributes to the risk of AD through TGF-β/Smad2/STAT3 signaling activation. These findings enhance our understanding of the role of apoE, derived from astrocytes, in AD and suggest it to be a potential biomarker and therapeutic target for AD. Copyright © 2017 Elsevier Inc. All rights reserved.

Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes.

PubMed

Duperray, A; Berthier, R; Chagnon, E; Ryckewaert, J J; Ginsberg, M; Plow, E; Marguerie, G

1987-06-01

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.

[The role of apoptosis of granulosa cells in follicular atresia].

PubMed

Grotowski, W; Lecybył, R; Warenik-Szymankiewicz, A; Trzeciak, W H

1997-07-01

Apoptosis plays an important role in the process of morphogenesis and embryogenesis. Its increase or inhibition is an etiopathological factor in many different diseases. It has recently been shown that apoptosis of granulosa cells is one of the main mechanisms responsible for follicular atresia. There are many other factors influencing the process of granulosa cells apoptosis, among them the most important are: RnGH, FSH, LH, sex hormones (estrogens and androgens), growth factors and their receptors (EGF/TGF-alpha, FGF, IGF-1) and cytokines (e.g. TNF-alpha). The article presents data concerning the regulatory mechanisms of granulosa cells apoptosis in the ovary.

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

The use of low molecular weight heparin-pluronic nanogels to impede liver fibrosis by inhibition the TGF-β/Smad signaling pathway.

PubMed

Lee, Ju-Hee; Lee, Hyunseung; Joung, Yoon Ki; Jung, Kyung Hee; Choi, Jong-Hoon; Lee, Don-Haeng; Park, Ki Dong; Hong, Soon-Sun

2011-02-01

Low molecular weight heparin (LH) has been reported to have anti-fibrotic and anti-cancer effects. To enhance the efficacy and minimize adverse effects of LH, a low molecular weight heparin-pluronic nanogel (LHP) was synthesized by conjugating carboxylated pluronic F127 to LH. The LHP reduced anti-coagulant activity by about 33% of the innate activity. Liver fibrosis was induced by the injection of 1% dimethylnitrosamine (DMN) in rats, and LH or LHP (1000 IU/kg body weight) was treated once daily for 4 weeks. LHP administration prevented DMN-mediated liver weight loss and decreased the values of aspartate transaminase, alanine transaminase, total bilirubin, and direct bilirubin. LHP markedly reduced the fibrotic area compared to LH. Also, LHP potently inhibited mRNA or protein expression of alpha-smooth muscle actin, collagen type I, matrix metalloproteinase-2, and tissue inhibitor of metalloproteinase-1 compared to LH, in DMN-induced liver fibrosis. In addition, LHP decreased the expression of transforming growth factor-β(1) (TGF-β(1)), p-Smad 2, and p-Smad 3, which are all important molecules of the TGF-β/Smad signaling pathway. The results support an LHP shows anti-fibrotic effect in the liver via inhibition of the TGF-β/Smad pathway as well as by the elimination of the extracellular matrix. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

Factors regulating collagen synthesis and degradation during second-intention healing of wounds in the thoracic region and the distal aspect of the forelimb of horses.

PubMed

Schwartz, Anne J; Wilson, David A; Keegan, Kevin G; Ganjam, Venkataseshu K; Sun, Yao; Weber, Karl T; Zhang, Jiakun

2002-11-01

To determine significant molecular and cellular factors responsible for differences in second-intention healing in thoracic and metacarpal wounds of horses. 6 adult mixed-breed horses. A full-thickness skin wound on the metacarpus and another such wound on the pectoral region were created, photographed, and measured, and tissue was harvested from these sites weekly for 4 weeks. Gene expression of type-I collagen, transforming growth factor (TGF)-beta1, matrix metalloproteinase (MMP)-1, and tissue inhibitor of metalloproteinase (TIMP)-1 were determined by quantitative in situ hybridization. Myofibroblasts were detected by immunohistochemical labeling with alpha-smooth muscle actin (alpha-SMA). Collagen accumulation was detected by use of picrosirius red staining. Tissue morphology was examined by use of H&E staining. Unlike thoracic wounds, forelimb wounds enlarged during the first 2 weeks. Myofibroblasts, detected by week 1, remained abundant with superior organization in thoracic wounds. Type-I collagen mRNA accumulated progressively in both wounds. More type-I collagen and TGF-beta1 mRNA were seen in forelimb wounds. Volume of MMP-1 mRNA decreased from day 0 in both wounds. By week 3, TIMP-1 mRNA concentration was greater in thoracic wounds. Greater collagen synthesis in metacarpal than thoracic wounds was documented by increased concentrations of myofibroblasts, type-I collagen mRNA,TGF-beta1 mRNA, and decreased collagen degradation (ie, MMP-1). Imbalanced collagen synthesis and degradation likely correlate with development of exuberant granulation tissue, delaying healing in wounds of the distal portions of the limbs. Factors that inhibit collagen synthesis or stimulate collagenase may provide treatment options for horses with exuberant granulation tissue.

Decreased interleukin-7 and transforming growth factor-beta1 levels in blister fluids as compared to the respective serum levels in patients with bullous pemphigoid. Opposite behavior of TNF-alpha, interleukin-4 and interleukin-10.

PubMed

Giacalone, B; D'Auria, L; Bonifati, C; Ferraro, C; Riccardi, E; Mussi, A; D'Agosto, G; Cordiali-Fei, P; Ameglio, F

1998-08-01

This study analyzes both the blister fluid (BF) and serum levels of IL-7 and TGF-beta1 in samples from 18 patients affected with bullous pemphigoid (BP). These cytokines clearly present lower concentrations (P<0.001) in BFs than in the sera (1/20 and 1/2, respectively). In contrast, TNF-alpha, IL-10 and IL-4 present increased amounts in BFs that were 12, 12 and 17-fold, respectively. Eighteen sera (and 10 suction BF) from normal individuals were also employed as control. Normal sera presented significantly lower serum IL-7 concentrations than BP, while no significant TGF-beta1 variations were observed between normal and pathologic serum samples. In addition, the serum levels detected in BP patients were significantly correlated with disease intensity (r=0.64, P=0.003, evaluated as the number of blisters/erosions for each patient) as well as with the peripheral B-lymphocyte counts (r=0.80, P<0.001) and antibodies directed against the basement membrane zone (r=0.65, P<0.005). Although a clear explanation of this phenomenon is lacking, the data presented in this report agree with a strong decrease of IL-7 production at the local level (keratinocyte is known to produce IL-7 and the latter is known to be down-regulated by IL-10, and in other models also by TGF-beta1 and IL-4, whose levels are elevated in BP BFs) as opposed to an increased peripheral release of the same modulator. The IL-7 reduction may have a biological relevance in controlling a chronic, progressive disease.

Strikingly higher interleukin (IL)-1alpha, IL-1beta and soluble interleukin-1 receptor antagonist (sIL-1RA) but similar IL-2, sIL-2R, IL-3, IL-4, IL-6, sIL-6R, IL-10, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta and interferon IFN-gamma urine levels in healthy females compared to healthy males: protection against urinary tract injury?

PubMed

Sadeghi, M; Daniel, V; Naujokat, C; Weimer, R; Opelz, G

2005-11-01

The aim of this prospective study was to examine gender-related differences of cytokines in the plasma and urine of healthy individuals that might provide a clue concerning the lower rate of chronic renal diseases in females. Soluble interleukin-1 receptor antagonist (sIL-1RA), interleukin (IL)-1alpha, IL-1beta, IL-2, sIL-2R, IL-3, IL-4, IL-6, sIL-6R, IL-10, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta(2) and interferon (IFN)-gamma were determined using standard enzyme-linked immunosorbent assay (ELISA). Cytokine levels were determined in simultaneously obtained plasma and urine samples of 18 male and 28 female healthy members of our laboratory staff. Urine cytokine levels were studied three times at 1-month intervals. All individuals had a negative urine nitrite test and showed no symptoms of urinary tract infection (UTI). Plasma levels of all studied cytokines were similar in males and females (P = n.s.). However, females had significantly higher urine IL-1alpha (P < 0.0001; P < 0.0001; P < 0.0001) and sIL-1RA (P = 0.0001; P = 0.0003; P = 0.0002) than males at three and higher IL-1beta at one of the three investigations (P = 0.098; P = 0.003; P = 0.073). Urine levels of the other cytokines were similar in males and females. Higher urine levels of IL-1alpha, IL-1beta and sIL-1RA in females may result from stimulation of cells in the urinary tract. Increased sIL-1RA might block T lymphocyte activation. The elevated cytokines may play a role in the protection of the female urinary tract from certain renal diseases, such as pyelonephritis and other inflammatory and sclerotic kidney diseases.

Efficient production of hyperpolarized bicarbonate by chemical reaction on a DNP precursor to measure pH.

PubMed

Ghosh, Rajat K; Kadlecek, Stephen J; Pourfathi, Mehrdad; Rizi, Rahim R

2015-11-01

To produce hyperpolarized bicarbonate indirectly via chemical reaction from a hyperpolarized precursor and utilize it for the simultaneous regional measurement of metabolism and pH. Alpha keto carboxylic acids are first hyperpolarized by dissolution dynamic nuclear polarization (DNP). These precursor molecules are rapidly reacted with hydrogen peroxide (H2O2) to decarboxylate the species, resulting in new target molecules. Unreacted H2O2 is removed from the system by reaction with sulfite. Interrogation of the ratio of dissolved carbon dioxide (CO2) to bicarbonate can be used to determine pH. Conversion of hyperpolarized alpha keto acids to bicarbonate and CO2 results in a minimal loss of the spin order. The reaction can be conducted to completion within seconds and preserves the nuclear spin polarization. Through a rapid chemical reaction, we can conserve the nuclear spin order of a DNP precursor to generate multiple hyperpolarized bioprobes otherwise unamenable to polarization. This indirect technique for the production of hyperpolarized agents can be applied to different precursor compounds to generate additional novel probes. © 2014 Wiley Periodicals, Inc.

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.

Stromal Tissue Rigidity Promotes Mesenchymal Stem Cell-Mediated Corneal Wound Healing Through the Transforming Growth Factor β Signaling Pathway.

PubMed

Yang, Yun-Hsiang; Hsieh, Ting-Lieh; Ji, Andrea Tung-Qian; Hsu, Wei-Tse; Liu, Chia-Yu; Lee, Oscar Kuang-Sheng; Ho, Jennifer Hui-Chun

2016-10-01

The healing of a corneal epithelial defect is essential for preventing infectious corneal ulcers and subsequent blindness. We previously demonstrated that mesenchymal stem cells (MSCs) in the corneal stroma, through a paracrine mechanism, yield a more favorable therapeutic benefit for corneal wound re-epithelialization than do MSCs in the corneal epithelium. In this study, MSCs were grown on a matrix with the rigidity of the physiological human vitreous (1 kPa), corneal epithelium (8 kPa), or corneal stroma (25 kPa) for investigating the role of corneal tissue rigidity in MSC functions regarding re-epithelialization promotion. MSC growth on a 25-kPa dish significantly promoted the wound healing of human corneal epithelial (HCE-T) cells. Among growth factors contributing to corneal epithelial wound healing, corneal stromal rigidity selectively enhanced transforming growth factor-beta (TGF-β) secretion from MSCs. Inhibitors of TGF-β pan receptor, TGF-β receptor 1, and Smad2 dose dependently abrogated MSC-mediated HCE-T wound healing. Furthermore, MSCs growth on a matrix with corneal stromal rigidity enhanced the ability of themselves to promote corneal re-epithelialization by activating matrix metalloproteinase (MMP) expression and integrin β1 production in HCE-T cells through TGF-β signaling pathway activation. Smad2 activation resulted in the upregulation of MMP-2 and -13 expression in HCE-T cells, whereas integrin β1 production favored a Smad2-independent TGF-β pathway. Altogether, we conclude that corneal stromal rigidity is a critical factor for MSC-induced promotion of corneal re-epithelialization. The activation of the TGF-β signaling pathway, which maintains the balance between integrin and MMP expression, in HCE-T cells is the major pathway responsible for MSC-mediated wound healing. Stem Cells 2016;34:2525-2535. © 2016 AlphaMed Press.

Prolonged use of alendronate alters the biology of cranial repair in estrogen-deficient rats' associated simultaneous immunohistochemical expression of TGF-β1+, α-ER+, and BMPR1B.

PubMed

Giovanini, Allan Fernando; de Sousa Passoni, Giuliene Nunes; Göhringer, Isabella; Deliberador, Tatiana Miranda; Zielak, João Cesar; Storrer, Carmem Lucia Muller; Costa-Casagrande, Thais Andrade; Scariot, Rafaela

2018-06-01

TGF-β1 is a cytokine that may induce both osteoneogenesis through Runx-2 or fibrosis via the transcription of α-smooth muscle actin (α-SMA). Because it has been previously known that alendronate increases the level of TGF-β1 and that under the usual condition of bone metabolism the estrogen may prevent the fibrotic effect of TGF-β1, the aim of this study was to evaluate if alendronate alters the cellular differentiation process post calvarial surgery in estrogen-deficient specimens. A transosseous defect that was 5 mm in diameter was created on the calvarium of each of 32 female rats with previous ovarian-salpingo-oophorectomy. All defects were treated with autografts, and 16 rats received the administration of 1 mg/kg of alendronate three times a week until euthanasia on the 15th and 60th day post surgery. Histomorphometric and immunohistochemical analyses of the expression of TGF-β1, estrogen receptor alpha nuclear (α-ER), α-SMA, BMPR1B, and Runx-2 were performed, and ELISA was used to measure the level of estrogen. All animals demonstrated low levels of estrogen post ovarian-salpingo-oophorectomy. The histological results demonstrated larger bone matrix deposition in specimens treated with alendronate on the 15th day post surgery. The result was associated with a higher co-expression of TGF-β1, BMPR1B, and Runx-2 when compared with the control group. In addition, on the 60th day post surgery, the increase of bone matrix deposition from 15th to 60th day was discrete in specimens treated with alendronate compared with the control group. This result coincided with the intense simultaneous expression of TGF-β1, α-ER, and α-SMA, whereas the expression of BMPR1B and Runx-2 decreased. The prolonged administration of alendronate altered the cranial repair in ovarian-salpingo-oophorectomized specimens due to the simultaneous occurrence of low estrogen and the presence of TGF-β1+/α-ER+ inducing the presence of α-SMA + , whereas BMPR1B and Runx-2 were suppressed. The prolonged administration of alendronate alters osteoneogenesis and induces an unusual microenvironment in the bone that seems to imitate the physiological tissue damage that culminates in the loss of the functional layer of endometrium.

Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.

PubMed

Crosas-Molist, Eva; Meirelles, Thayna; López-Luque, Judit; Serra-Peinado, Carla; Selva, Javier; Caja, Laia; Gorbenko Del Blanco, Darya; Uriarte, Juan José; Bertran, Esther; Mendizábal, Yolanda; Hernández, Vanessa; García-Calero, Carolina; Busnadiego, Oscar; Condom, Enric; Toral, David; Castellà, Manel; Forteza, Alberto; Navajas, Daniel; Sarri, Elisabet; Rodríguez-Pascual, Fernando; Dietz, Harry C; Fabregat, Isabel; Egea, Gustavo

2015-04-01

Marfan's syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-β signaling. TGF-β is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-β signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan's syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-β pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. In Marfan VSMC, both in tissue and in culture, there are variable TGF-β-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan's syndrome aneurysm formation. © 2015 American Heart Association, Inc.

Lipid peroxidation products do not activate hepatic stellate cells.

PubMed

Fang, Hsun-Lang; Lin, Wen-Chuan

2008-11-20

Lipid peroxidation (LPO) is known to be associated with liver fibrosis in chronic liver injury. However, direct effects of the products of LPO on liver fibrogenesis are still not clear. In this study, we examined the LPO products, such as malondiladehyde (MDA), 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), and 15-keto-13,14-dihydro-PGF(2alpha) (15-keto-PGF(2alpha)), on the activation of hepatic stellate cells (HSCs) in vivo and in vitro. Carbon tetrachloride (CCl(4)) was given orally to rats twice a week for 8 weeks. Corn oil was given daily to rats for 8 weeks. CCl(4) induced both free-radical-medicated and cyclooxygenase-2-dependent LPO. Free radical-medicated LPO showed an increase with corn oil treatment, whereas no effect was reflected on COX-2-dependent LPO. CCl(4) induced liver fibrosis in rats, but no liver fibrosis was observed in rats treated with corn oil. In vitro studies demonstrated that MDA, 8-iso-PGF(2alpha) and 15-keto-PGF(2alpha), did not activate HSCs, which were preactivated or not preactivated by TGF-beta1. Our results clearly indicate that LPO products, such as MDA, 8-iso-PGF(2alpha) and 15-keto-PGF(2alpha), cannot directly activate HSCs.

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.

The Dean and Betty Gallo Prostate Cancer Center

DTIC Science & Technology

2004-07-01

8217 Deleted: Completed 1998), / . Deleted: Clin Cancer Res, 1997 ,phase II study of 13 cis retinoic Completed: Serum TGF- beta and IGF- 30 DiPaola...t , sensitive to activation of both ER alpha and beta . Further laboratory studies by DiPaola and collaborators Deleted: identified additional... Beta -catenin regulates Cripto- and Wnt3- dependent gene expression programs in mouse axis and mesoderm formation. Development, 130: 6283-6294. 2003

Regulation of IL-10 expression by upstream stimulating factor (USF-1) in glioma-associated microglia.

PubMed

Zhang, Leying; Handel, Michelle Van; Schartner, Jill M; Hagar, Aaron; Allen, Grant; Curet, Marjorie; Badie, Behnam

2007-03-01

Understanding the local CNS immune response to neoplasms is essential in the development of immune-based treatments for malignant brain tumors. Using rodent glioma models, we have recently found tumor-associated microglia/macrophages (MG/MP) to be less responsive to known MG/MP activators such as CpG, LPS and IFN-gamma. To understand the mechanism of MG/MP suppression, nuclear extracts from rodent intracranial C6 gliomas, C6 glioma-associated MG/MP, normal brain, and normal MG/MP were obtained and studied using Electrophoretic Mobility Shift Assay (EMSA). Among the nuclear factors studied (AP-1, IRF, USF-1 and Stat-1) only USF-1, which is constitutively expressed in most cells, was down-regulated in tumor-associated MG/MP, but not normal MG/MP. Because tumor-associated MG/MP had higher expression of IL-10 (but not TNF-alpha or TGF-beta), we evaluated the role of USF-1 on IL-10 expression. siRNA mediated inhibition of USF-1 expression in primary MG/MP cultures resulted in up-regulation of IL-10 mRNA but not TNF-alpha or TGF-beta. These findings suggest that USF-1 may play a role in IL-10 regulation in MG/MP in brain tumors.

The noradrenaline precursor L-threo-3,4-dihydroxyphenylserine exhibits antinociceptive activity via central alpha-adrenoceptors in the mouse.

PubMed Central

Kawabata, A.; Kasamatsu, K.; Umeda, N.; Takagi, H.

1994-01-01

1. Systemic (s.c. or p.o.) administration of L-threo-3,4-dihydroxyphenylserine (droxidopa, L-threo-DOPS; L-DOPS), a noradrenaline precursor, at a dose-range of 100-800 mg kg-1, produced naloxone-resistant antinociception in a dose-dependent manner in the mouse, as assessed by the tail flick test, kaolin-induced writhing test and formalin-induced nociception test. 2. Antinociception elicited by L-DOPS (400 mg kg-1, s.c.) was not affected by s.c. injection of benserazide, a peripherally preferential L-aromatic amino acid decarboxylase inhibitor, but was suppressed by its intracerebroventricular (i.c.v.) injection. 3. I.c.v. or intrathecal (i.t.) administration of the non-selective alpha-blocker, phentolamine, significantly reduced L-DOPS-induced antinociception. 4. I.c.v. administration of the alpha 1-blocker, prazosin, but not the alpha 2-blocker, yohimbine, abolished the antinociceptive effects of L-DOPS. In contrast, both blockers, when administered i.t., exhibited significant inhibitory effects. 5. These results suggest that systemic L-DOPS produces opioid-independent antinociception, mediated by supraspinal alpha 1-adrenoceptors and by spinal alpha 1- and alpha 2-adrenoceptors and may predict additional therapeutic applications of L-DOPS as an analgesic. PMID:7911717

Biochemical changes in response to intensive resistance exercise training in the elderly.

PubMed

Bautmans, Ivan; Njemini, Rose; Vasseur, Sabine; Chabert, Hans; Moens, Lisa; Demanet, Christian; Mets, Tony

2005-01-01

It is assumed that low-grade inflammation, characterized by increased circulating IL-6 and TNF-alpha, is related to the development of sarcopenia. Physical exercise, especially high intensity resistance training, has been shown to be effective in restoring the strength deficit in the elderly. Intensive exercise is accompanied by significant release of IL-6 and TNF-alpha into the blood circulation, but does not result in muscle wasting. Exercise-induced changes in heat-shock protein (Hsp), responsible for cellular protection during stressful situations, might interfere with the acute phase reaction and muscle adaptation. To investigate if intensive strength training in elderly persons induces changes in Hsp70 expression, and if these changes are related to changes in the acute phase reaction or muscle adaptation. 31 elderly persons (aged 68.4+/-5.4 years) performed 6 weeks' intensive strength training. At baseline and after 6 weeks, muscle strength, functional performance (physical activity profile, 6-min walk, 30- second chair stand, grip strength, chair sit & reach and back scratch), linear isokinetic leg extension, circulating IL-6, TNF-alpha, IL-10 and TGF-beta, and Hsp70 in monocytes (M) and lymphocytes (L) immediately after sampling (IAS), after incubation at 37 and 42 degrees C were determined. In 12 participants, cytokines were determined in untrained and trained conditions before and after a single training session. After 6 weeks' training, muscle strength and functional performance improved significantly, together with decreased Hsp70 IAS and Hsp70 37 degrees C and increased Hsp70 42 degrees C (all p<0.05). Strength gains correlated positively with baseline Hsp70 37 degrees C and training-induced changes of Hsp70 42 degrees C in M and L. In an untrained condition, training induced an increase of IL-6 (p<0.05) and a tendency of IL-10 to decrease (p=0.06). In a trained condition the decrease of IL-10 disappeared. Baseline physical activity and 6-min walk distance correlated negatively with circulating IL-6 (p<0.05); except for a negative correlation between TGF-beta and Hsp70 37 degrees C L (p<0.05), no significant relationships were found between cytokines and Hsp70. After the training program, Hsp70 37 degrees C was negatively related to circulating TNF-alpha, IL-10 and TGF-beta. Strength training in the elderly induces changes in Hsp70 expression, associated to strength gains and circulating cytokines. Copyright (c) 2005 S. Karger AG, Basel.

Microwave sintering of sol-gel derived abrasive grain

DOEpatents

Plovnick, Ross; Celikkaya, Ahmet; Blake, Rodger D.

1997-01-01

A method is provided for making microwave-sintered, free flowing alpha alumina-based ceramic abrasive grain, under conditions effective to couple microwaves with calcined alpha alumina-based abrasive gain precursor and sinter it at a temperature of at least about 1150.degree. C.

Comparisons of the effects of TCDD and hydrocortisone on growth factor expression provide insight into their interaction in the embryonic mouse palate

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

Abbott, B.D.; Harris, M.W.; Birnbaum, L.S.

Cleft palate (CP) can be induced in embryonic mice by a wide range of compounds, including glucocorticoids and 2,3,7,8-tyetrachlorodibenzo-p-dioxin (TCDD). Hydrocortisone (HC), a glucocorticoid, retards embryonic growth producing small palatal shelves, while TCDD exposure blocks the fusion of normally sized shelves. TCDD induction of CP involves altered differentiation of the medial epithelial cells. Recent studies indicate that growth factors such as EGF, TGF-alpha, TGF-beta1, and TGF-beta2 are involved in palatogenesis, regulating proliferation, differentiation, and extracellular matrix production. A synergism has been observed between HC and TCDD in which doses too low to induce CP alone are able to produce >90%more » incidence when coadministered. In the present study a standard teratology protocol was performed in C57BL/6N mice to examine the synergism at doses lower than those previously published. Data from the study indicate synergistic interactions at doses as low as 3 micrograms TCDD/kg + 1 mg HC/kg. This extreme sensitivity suggests the involvement of a receptor-mediated mechanism possibly resulting in altered regulation of gene expression. (Copyright (c) 1992 Wiley-Liss, Inc.)« less

αvβ6 Integrin Regulates Renal Fibrosis and Inflammation in Alport Mouse

PubMed Central

Hahm, Kyungmin; Lukashev, Matvey E.; Luo, Yi; Yang, William J.; Dolinski, Brian M.; Weinreb, Paul H.; Simon, Kenneth J.; Chun Wang, Li; Leone, Diane R.; Lobb, Roy R.; McCrann, Donald J.; Allaire, Normand E.; Horan, Gerald S.; Fogo, Agnes; Kalluri, Raghu; Shield, Charles F.; Sheppard, Dean; Gardner, Humphrey A.; Violette, Shelia M.

2007-01-01

The transforming growth factor (TGF)-β-inducible integrin αvβ6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-β. Herein, we show that αvβ6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture’s syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of αvβ6 in renal disease, we studied the effects of function-blocking αvβ6 monoclonal antibodies (mAbs) and genetic ablation of the β6 subunit on kidney fibrosis in Col4A3−/− mice, a mouse model of Alport syndrome. Expression of αvβ6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with αvβ6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in β6-deficient Alport mice. Transcript profiling of kidney tissues showed that αvβ6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-β RII treatment, suggesting shared regulatory functions of αvβ6 and TGF-β. These findings demonstrate that αvβ6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target. PMID:17200187

Endocan, TGF-beta, and ADMA as Risk Factors for Endothelial Dysfunction and Possible Vascular Disease in Patients with Subclinical Hypothyroidism.

PubMed

Arpaci, Dilek; Karakece, Engin; Tocoglu, Aysel Gurkan; Ergenc, Hasan; Gurol, Gonul; Ciftci, Ihsan Hakki; Tamer, Ali

2016-12-01

Although the relationship between atherosclerosis and overt hypothyroidism has been confirmed, it remains controversial in cases of subclinical hypothyroidism. Higher TSH and similar T4 suggest differences in set-points or differences due to diagnostic limitations regarding subclinical hypothyroidism. Endothelial dysfunction (ED) is a marker rather than a precursor of cardiovascular disease. Asymmetric dimethylarginine (ADMA) and endocan are known as novel markers of ED in various diseases. Transforming growth factor-beta (TGF-β) has a protective role against autoimmune diseases such as thyroiditis. This study aimed to determine the relationships between serum ADMA, endocan, TGF-β, and the high-sensitivity C-reactive protein (hs-CRP) levels, a proven indicator of ED, in patients with SH. Thirty-five patients with SH and 21 age- and sex-matched euthyroid subjects were included in the study. The levels of TSH, FT4, lipid parameters, endocan, ADMA, TGF-β, and hs-CRP were measured. No significant differences in age or sex were found between the patient and control groups (p=0.294 and 0.881, respectively). Mean TSH level was higher in the patient group (p=0.005), whereas mean fT4 level was similar in two groups (p=0.455). The average hs-CRP, endocan, TGF-β l level in the patient group was higher than control group (p=0.001; P=0.012; P=0.025; P<0.01 respectively). A positive correlation was found between the endocan and ADMA levels (r=0.760, p=0.000). ADMA levels also were positively correlated with hs-CRP. Both the TSH and low-density lipoprotein cholesterol (LDL-C) levels were positively correlated with the hs-CRP level. Subclinical hypothyroidism is associated with increased levels of serum endocan, ADMA, and TGF-β, which are new markers for ED. In particular, ADMA was correlated with both endocan and hs-CRP levels. These findings are suggestive for increased risk of ED and subsequent development of atherosclerosis in patients with SH. © 2016 by the Association of Clinical Scientists, Inc.

Biosynthesis of trichothecenes and apotrichothecenes.

PubMed

Zamir, L O; Nikolakakis, A; Sauriol, F; Mamer, O

1999-05-01

Fusarium culmorum produces two major trichothecenes, 3-acetyldeoxynivalenol and sambucinol, and some minor apotrichothecenes. It was desired to investigate if during their biosynthesis a C-11-keto intermediate was involved. To verify this postulate, trichodiene, a known precursor to trichothecenes, was synthesized with two deuteriums at C-11 and one at C-15. It was then fed to F. culmorum cultures, and the derived metabolites were purified and analyzed. The results ruled out the involvement of an 11-keto intermediate but revealed two novel apotrichothecenes. The characterization of their structures suggested that one of the 2-hydroxy-11alpha-apotrichothecene stereoisomers (2alpha or 2beta) could be converted to sambucinol. These apotrichothecenes were therefore synthesized labeled specifically with two deuteriums at C-4 and C-15 and fed to F. culmorum cultures. Indeed, the result established for the first time that 2alpha-hydroxy-11alpha-apotrichothecene was a precursor to sambucinol. A biosynthetic scheme for the production of trichothecenes and apotrichothecenes is described.

Maternal obesity induces fibrosis in fetal myocardium of sheep

PubMed Central

Huang, Yan; Yan, Xu; Zhao, Jun X.; Zhu, Mei J.; McCormick, Richard J.; Ford, Stephen P.; Nathanielsz, Peter W.; Ren, Jun

2010-01-01

Maternal obesity (MO) has harmful effects on both fetal development and subsequent offspring health. The impact of MO on fetal myocardium development has received little attention. Fibrogenesis is regulated by the transforming growth factor-β (TGF-β)/p38 signaling pathway. Using the well-established model of MO in pregnant sheep, we evaluated the effect of MO on TGF-β/p38 and collagen accumulation in fetal myocardium. Nonpregnant ewes were assigned to a control diet [Con, fed 100% of National Research Council (NRC) nutrient recommendations] or obesogenic diet (OB, fed 150% of NRC recommendations) from 60 days before conception. Fetal ventricular muscle was sampled at 75 and 135 days of gestation (dG). At 75 dG, the expression of precursor TGF-β was 39.9 ± 9.9% higher (P < 0.05) in OB than Con fetal myocardium, consistent with the higher content of phosphorylated Smad3 in OB myocardium. The phosphorylation of p38 tended to be higher in OB myocardium (P = 0.08). In addition, enhanced Smad complexes were bound to Smad-binding elements in 75 dG OB fetal myocardium measured by DNA mobility shift assay (130.2 ± 26.0% higher, P < 0.05). Similar elevation of TGF-β signaling was observed in OB fetal myocardium at 135 dG. Total collagen concentration in OB was greater than Con fetal myocardium (2.42 ± 0.16 vs. 1.87 ± 0.04%, P < 0.05). Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 were higher in the Con group compared with OB sheep (43.86 ± 16.01 and 37.23 ± 7.97% respectively, P < 0.05). In summary, MO results in greater fetal heart connective tissue accumulation associated with an upregulated TGF-β/p38 signaling pathway at late gestation; such changes would be expected to negatively impact offspring heart function. PMID:20876759

Chemogenetic locus coeruleus activation restores reversal learning in a rat model of Alzheimer's disease.

PubMed

Rorabaugh, Jacki M; Chalermpalanupap, Termpanit; Botz-Zapp, Christian A; Fu, Vanessa M; Lembeck, Natalie A; Cohen, Robert M; Weinshenker, David

2017-11-01

See Grinberg and Heinsen (doi:10.1093/brain/awx261) for a scientific commentary on this article. Clinical evidence suggests that aberrant tau accumulation in the locus coeruleus and noradrenergic dysfunction may be a critical early step in Alzheimer’s disease progression. Yet, an accurate preclinical model of these phenotypes that includes early pretangle tau accrual in the locus coeruleus, loss of locus coeruleus innervation and deficits locus coeruleus/norepinephrine modulated behaviours, does not exist, hampering the identification of underlying mechanisms and the development of locus coeruleus-based therapies. Here, a transgenic rat (TgF344-AD) expressing disease-causing mutant amyloid precursor protein (APPsw) and presenilin-1 (PS1ΔE9) was characterized for histological and behavioural signs of locus coeruleus dysfunction reminiscent of mild cognitive impairment/early Alzheimer’s disease. In TgF344-AD rats, hyperphosphorylated tau was detected in the locus coeruleus prior to accrual in the medial entorhinal cortex or hippocampus, and tau pathology in the locus coeruleus was negatively correlated with noradrenergic innervation in the medial entorhinal cortex. Likewise, TgF344-AD rats displayed progressive loss of hippocampal norepinephrine levels and locus coeruleus fibres in the medial entorhinal cortex and dentate gyrus, with no frank noradrenergic cell body loss. Cultured mouse locus coeruleus neurons expressing hyperphosphorylation-prone mutant human tau had shorter neurites than control neurons, but similar cell viability, suggesting a causal link between pretangle tau accrual and altered locus coeruleus fibre morphology. TgF344-AD rats had impaired reversal learning in the Morris water maze compared to their wild-type littermates, which was rescued by chemogenetic locus coeruleus activation via designer receptors exclusively activated by designer drugs (DREADDs). Our results indicate that TgF344-AD rats uniquely meet several key criteria for a suitable model of locus coeruleus pathology and dysfunction early in Alzheimer’s disease progression, and suggest that a substantial window of opportunity for locus coeruleus/ norepinephrine-based therapeutics exists.

Human umbilical cord blood-derived mesenchymal stem cells attenuate hyperoxia-induced lung injury in neonatal rats.

PubMed

Chang, Yun Sil; Oh, Wonil; Choi, Soo Jin; Sung, Dong Kyung; Kim, Soo Yoon; Choi, Eun Yang; Kang, Saem; Jin, Hye Jin; Yang, Yoon Sun; Park, Won Soon

2009-01-01

Recent evidence suggests mesenchymal stem cells (MSCs) can downmodulate bleomycin-induced lung injury, and umbilical cord blood (UCB) is a promising source for human MSCs. This study examined whether intratracheal or intraperitoneal transplantation of human UCB-derived MSCs can attenuate hyperoxia-induced lung injury in immunocompetent newborn rats. Wild-type Sprague-Dawley rats were randomly exposed to 95% oxygen or air from birth. In the transplantation groups, a single dose of PKH26-labeled human UCB-derived MSCs was administered either intratracheally (2 x 10(6) cells) or intraperitoneally (5 x 10(5) cells) at postnatal day (P) 5. At P14, the harvested lungs were examined for morphometric analyses of alveolarization and TUNEL staining, as well as the myeoloperoxidase activity, the level of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and transforming growth factor (TGF)-beta mRNA, alpha-smooth muscle actin (SMA) protein, and collagen levels. Differentiation of MSCs to the respiratory epithelium was also evaluated both in vitro before transplantation and in vivo after transplantation. Despite one fourth dosage of MSCs, significantly more PKH26-labeled donor cells were recovered with intratracheal administration than with intraperitoneal administration both during normoxia and hyperoxia. The hyperoxia-induced increase in the number of TUNEL-positive cells, myeloperoixdase activity, and the level of IL-6 mRNA were significantly attenuated with both intratracheal and intraperitoneal MSCs transplantation. However, the hyperoxia-induced impaired alveolarization and increased the level of TNF-alpha and TGF-beta mRNA, alpha-SMA protein, and collagen were significantly attenuated only with intratracheal MSCs transplantation. MSCs differentiated into respiratory epithelium in vitro and a few PKH26-positive donor cells were colocalized with pro surfactant protein C in the damaged lungs. In conclusion, intratracheal transplantation of human UCB-derived MSCs is more effective than intraperitoneal transplantation in attenuating the hyperoxia-induced lung injury in neonatal rats.

Contributions of toluene and alpha-pinene to SOA formed in an irradiated toluene/alpha-pinene/NO(x)/ air mixture: comparison of results using 14C content and SOA organic tracer methods.

PubMed

Offenberg, John H; Lewis, Charles W; Lewandowski, Michael; Jaoui, Mohammed; Kleindienst, Tadeusz E; Edney, Edward O

2007-06-01

An organic tracer method, recently proposed for estimating individual contributions of toluene and alpha-pinene to secondary organic aerosol (SOA) formation, was evaluated by conducting a laboratory study where a binary hydrocarbon mixture, containing the anthropogenic aromatic hydrocarbon, toluene, and the biogenic monoterpene, alpha-pinene, was irradiated in air in the presence of NO(x) to form SOA. The contributions of toluene and alpha-pinene to the total SOA concentration, calculated using the organic tracer method, were compared with those obtained with a more direct 14C content method. In the study, SOA to SOC ratios of 2.07 +/- 0.08 and 1.41 +/- 0.04 were measured for toluene and (alpha-pinene SOA, respectively. The individual tracer-based SOA contributions of 156 microg m(-3) for toluene and 198 microg m(-)3 for alpha-pinene, which together accounted for 82% of the gravimetrically determined total SOA concentration, compared well with the 14C values of 182 and 230 microg m(-3) measured for the respective SOA precursors. While there are uncertainties associated with the organic tracer method, largely due to the chemical complexity of SOA forming chemical mechanisms, the results of this study suggest the organic tracer method may serve as a useful tool for determining whether a precursor hydrocarbon is a major SOA contributor.

Expression of cutaneous lymphocyte-associated antigen (CLA) in tonsillar T-cells and its induction by in vitro stimulation with alpha-streptococci in patients with pustulosis palmaris et plantaris (PPP).

PubMed

Nozawa, Hayabusa; Kishibe, Kan; Takahara, Miki; Harabuchi, Yasuaki

2005-07-01

Pustulosis palmaris et plantaris (PPP) is known to be a one of the tonsil-related diseases because tonsillectomy is quite effective in curing this condition. However etiological association between tonsils and PPP have not fully clarified yet. Cutaneous lymphocyte-associated antigen (CLA) is known to be a specific homing receptor that facilitates T-cell migration into skin. In this study, we investigated the expression of CLA on T-cells in tonsil, peripheral blood, and skin from patients with PPP. Two-color flow cytometric and two-color immunohistological analyses revealed that the numbers of CLA/CD3 double-positive cells in freshly isolated tonsillar mononuclear cells (TMC) and in tonsillar tissues were significantly higher in patients with PPP than in patients without PPP (P<0.01, each). In vitro stimulus with alpha-streptococcal antigens enhanced CLA expression of tonsillar T-cells and TGF-beta production of TMC in patients with PPP (P<0.01, each), but did not in patients without PPP. In peripheral blood from PPP patients, the number of the CLA/CD3 double-positive cells significantly decreased at 6 months after tonsillectomy (P<0.05). The CLA/CD3 double-positive cells and the postcapillary venule that expressed with a ligand of CLA, E-selectin, were found more frequently in the plantar skin from patients with PPP as compared to that from healthy volunteers (P<0.01, each). These data suggest that a novel immune response to alpha-streptococci may enhance CLA expression on tonsillar T-cells through TGF-beta production in patients with PPP, resulting in moving of CLA-positive tonsillar T-cells to skin and tissue damages. This may play a key role in pathogenesis of PPP.

Identification of biomarkers in human head and neck tumor cell lines that predict for in vitro sensitivity to gefitinib.

PubMed

Hickinson, D Mark; Marshall, Gayle B; Beran, Garry J; Varella-Garcia, Marileila; Mills, Elizabeth A; South, Marie C; Cassidy, Andrew M; Acheson, Kerry L; McWalter, Gael; McCormack, Rose M; Bunn, Paul A; French, Tim; Graham, Alex; Holloway, Brian R; Hirsch, Fred R; Speake, Georgina

2009-06-01

Potential biomarkers were identified for in vitro sensitivity to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in head and neck cancer. Gefitinib sensitivity was determined in cell lines, followed by transcript profiling coupled with a novel pathway analysis approach. Eleven cell lines were highly sensitive to gefitinib (inhibitor concentration required to give 50% growth inhibition [GI(50)] < 1 microM), three had intermediate sensitivity (GI(50) 1-7 microM), and six were resistant (GI(50) > 7 microM); an exploratory principal component analysis revealed a separation between the genomic profiles of sensitive and resistant cell lines. Subsequently, a hypothesis-driven analysis of Affymetrix data (Affymetrix, Inc., Santa Clara, CA, USA) revealed higher mRNA levels for E-cadherin (CDH1); transforming growth factor, alpha (TGF-alpha); amphiregulin (AREG); FLJ22662; EGFR; p21-activated kinase 6 (PAK6); glutathione S-transferase Pi (GSTP1); and ATP-binding cassette, subfamily C, member 5 (ABCC5) in sensitive versus resistant cell lines. A hypothesis-free analysis identified 46 gene transcripts that were strongly differentiated, seven of which had a known association with EGFR and head and neck cancer (human EGF receptor 3 [HER3], TGF-alpha, CDH1, EGFR, keratin 16 [KRT16], fibroblast growth factor 2 [FGF2], and cortactin [CTTN]). Polymerase chain reaction (PCR) and enzyme-linked immunoabsorbant assay analysis confirmed Affymetrix data, and EGFR gene mutation, amplification, and genomic gain correlated strongly with gefitinib sensitivity. We identified biomarkers that predict for in vitro responsiveness to gefitinib, seven of which have known association with EGFR and head and neck cancer. These in vitro predictive biomarkers may have potential utility in the clinic and warrant further investigation.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

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

Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.

Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression hostmore » offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.« less

Liver regeneration after partial hepatectomy in rat is more impaired in a steatotic liver induced by dietary fructose compared to dietary fat

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

Tanoue, Shirou; Uto, Hirofumi, E-mail: hirouto@m2.kufm.kagoshima-u.ac.jp; Kumamoto, Ryo

Highlights: {yields} Hepatic steatosis in rats fed a high fructose diet was less severe than that in rats fed a high fat diet. {yields} Liver regeneration was more impaired in rats fed a high fructose diet than in rats fed a high fat diet. {yields} Dysregulation of genes associated with metabolism may contribute to impairment of liver regeneration. {yields} Regulation of the TGF-{beta}1 level after partial hepatectomy may be impaired in rats fed a high fructose diet. -- Abstract: Hepatic steatosis (HS) has a negative effect on liver regeneration, but different pathophysiologies of HS may lead to different outcomes. Malemore » Sprague-Dawley rats were fed a high fructose (66% fructose; H-fruc), high fat (54% fat; H-fat), or control chow diet for 4 weeks. Based on hepatic triglyceride content and oil red O staining, HS developed in the H-fruc group, but was less severe compared to the H-fat group. Hepatic mRNA expression levels of fatty acid synthase and fructokinase were increased and those of carnitine palmitoyltransferase-1 and peroxisome proliferator-activated receptor-{alpha} were decreased in the H-fruc group compared to the H-fat group. Liver regeneration after 70% partial hepatectomy (PHx) was evaluated by measuring the increase in postoperative liver mass and PCNA-positive hepatocytes, and was impaired in the H-fruc group compared to the H-fat and control groups on days 3 and 7. Serum levels of tumor necrosis factor-{alpha}, interleukin-6 and hepatocyte growth factor did not change significantly after PHx. In contrast, serum TGF-{beta}1 levels were slightly but significantly lower in the control group on day 1 and in the H-fat group on day 3 compared to the level in each group on day 0, and then gradually increased. However, the serum TGF-{beta}1 level did not change after PHx in the H-fruc group. These results indicate that impairment of liver regeneration after PHx in HS is related to the cause, rather than the degree, of steatosis. This difference may result from altered metabolic gene expression profiles and potential dysregulation of TGF-{beta}1 expression.« less

Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

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

Thompson, Pamela D.; Sakwe, Amos; Koumangoye, Rainelli

2014-02-15

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels ofmore » AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head and neck squamous cell carcinoma cell lines synthesize and secret AHSG. • AHSG depleted cell lines are significantly inhibited in their ability to proliferate, adhere, migrate, invade and protect MMP-9. • Human AHSG and bovine fetuin-A are functionally equivalent in regards to growth promotion of cancer cell lines.« less

Mechanisms of Angiogenesis and Lymphangiogenesis in TSC Skin Tumors

DTIC Science & Technology

2011-09-16

associated macrophage PKD Polycystic kidney disease TGF-β Transforming growth factor-β pS6 Phospho ribosomal protein S6 TNF-α Tumor necrosis factor...blood supply. They are found in the skin and other tissues except the central nervous system, bone marrow and avascular structures including epidermis...273:8413-8418 232. Cha HS, Bae EK, Koh JH, Chai JY, Jeon CH, Ahn KS, Kim J, Koh EM: Tumor necrosis factor-alpha induces vascular endothelial

Interferon-gamma interferes with transforming growth factor-beta signaling through direct interaction of YB-1 with Smad3.

PubMed

Higashi, Kiyoshi; Inagaki, Yutaka; Fujimori, Ko; Nakao, Atsuhito; Kaneko, Hideo; Nakatsuka, Iwao

2003-10-31

Transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) exert antagonistic effects on collagen synthesis in human dermal fibroblasts. We have recently shown that Y box-binding protein YB-1 mediates the inhibitory effects of IFN-gamma on alpha2(I) procollagen gene (COL1A2) transcription through the IFN-gamma response element located between -161 and -150. Here we report that YB-1 counter-represses TGF-beta-stimulated COL1A2 transcription by interfering with Smad3 bound to the upstream sequence around -265 and subsequently by interrupting the Smad3-p300 interaction. Western blot and immunofluorescence analyses using inhibitors for Janus kinases or casein kinase II suggested that the casein kinase II-dependent signaling pathway mediates IFN-gamma-induced nuclear translocation of YB-1. Down-regulation of endogenous YB-1 expression by double-stranded YB-1-specific RNA abrogated the transcriptional repression of COL1A2 by IFN-gamma in the absence and presence of TGF-beta. In transient transfection assays, overexpression of YB-1 in human dermal fibroblasts exhibited antagonistic actions against TGF-beta and Smad3. Physical interaction between Smad3 and YB-1 was demonstrated by immunoprecipitation-Western blot analyses, and electrophoretic mobility shift assays using the recombinant Smad3 and YB-1 proteins indicated that YB-1 forms a complex with Smad3 bound to the Smad-binding element. Glutathione S-transferase pull-down assays showed that YB-1 binds to the MH1 domain of Smad3, whereas the central and carboxyl-terminal regions of YB-1 were required for its interaction with Smad3. YB-1 also interferes with the Smad3-p300 interaction by its preferential binding to p300. Altogether, the results provide a novel insight into the mechanism by which IFN-gamma/YB-1 counteracts TGF-beta/Smad3. They also indicate that IFN-gamma/YB-1 inhibits COL1A2 transcription by dual actions: via the IFN-gamma response element and through a cross-talk with the TGF-beta/Smad signaling pathway.

Ursolic acid suppresses TGF-β1-induced quiescent HSC activation and transformation by inhibiting NADPH oxidase expression and Hedgehog signaling

PubMed Central

Yu, Shan-Shan; Chen, Biao; Huang, Chen-Kai; Zhou, Juan-Juan; Huang, Xin; Wang, An-Jiang; Li, Bi-Min; He, Wen-Hua; Zhu, Xuan

2017-01-01

Activation of quiescent hepatic stellate cells (q-HSCs) and their transformation to myofibroblasts (MFBs) is a key event in liver fibrosis. Hedgehog (Hh) signaling stimulates q-HSCs to differentiate into MFBs, and NADPH oxidase (NOX) may be involved in regulating Hh signaling. The author's preliminary study demonstrated that ursolic acid (UA) selectively induces apoptosis in activated HSCs and inhibits their proliferation in vitro via negative regulation of NOX activity and expression. However, the effect of UA on q-HSCs remains to be elucidated. The present study aimed to investigate the effect of UA on q-HSC activation and HSC transformation and to observe alterations in the NOX and Hh signaling pathways during q-HSC activation. q-HSC were isolated from adult male Sprague-Dawley rats. Following culture for 3 days, the cells were treated with or without transforming growth factor-β1 (TGF-β1; 5 µg/l); intervention groups were pretreated with UA (40 µM) or diphenyleneiodonium chloride (DPI; 10 µM) for 30 min prior to addition of TGF-β1. mRNA and protein expression of NOX and Hh signaling components and markers of q-HSC activation were examined by western blotting and reverse transcription-polymerase chain reaction. TGF-β1 induced activation of q-HSCs, with increased expression of α-smooth muscle actin (α-SMA) and type I collagen. In addition, expression of NOX subunits (gp91phox, p67phox, p22phox, and Rac1) and Hh signaling components, including sonic Hh, sterol-4-alpha-methyl oxidase, and Gli family zinc finger 2, were upregulated in activated HSCs. Pretreatment of q-HSCs with UA or DPI prior to TGF-β1 significantly downregulated expression of NOX subunits and Hh signaling components and additionally inhibited expression of α-SMA and type I collagen, thereby preventing transformation to MFBs. UA inhibited TGF-β1-induced activation of q-HSCs and their transformation by inhibiting expression of NOX subunits and the downstream Hh pathway. PMID:29042951

Role of bone marrow cells in the development of pancreatic fibrosis in a rat model of pancreatitis induced by a choline-deficient/ethionine-supplemented diet

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

Akita, Shingo; Kubota, Koji; Kobayashi, Akira, E-mail: kbys@shinshu-u.ac.jp

Highlights: Black-Right-Pointing-Pointer BMC-derived PSCs play a role in a rat CDE diet-induced pancreatitis model. Black-Right-Pointing-Pointer BMC-derived PSCs contribute mainly to the early stage of pancreatic fibrosis. Black-Right-Pointing-Pointer BMC-derived activated PSCs can produce PDGF and TGF {beta}1. -- Abstract: Bone marrow cell (BMC)-derived myofibroblast-like cells have been reported in various organs, including the pancreas. However, the contribution of these cells to pancreatic fibrosis has not been fully discussed. The present study examined the possible involvement of pancreatic stellate cells (PSCs) originating from BMCs in the development of pancreatic fibrosis in a clinically relevant rat model of acute pancreatitis induced by amore » choline-deficient/ethionine-supplemented (CDE) diet. BMCs from female transgenic mice ubiquitously expressing green fluorescent protein (GFP) were transplanted into lethally irradiated male rats. Once chimerism was established, acute pancreatitis was induced by a CDE diet. Chronological changes in the number of PSCs originating from the donor BMCs were examined using double immunofluorescence for GFP and markers for PSCs, such as desmin and alpha smooth muscle actin ({alpha}SMA), 1, 3 and 8 weeks after the initiation of CDE feeding. We also used immunohistochemical staining to evaluate whether the PSCs from the BMCs produce growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF) {beta}1. The percentage of BMC-derived activated PSCs increased significantly, peaking after 1 week of CDE treatment (accounting for 23.3 {+-} 0.9% of the total population of activated PSCs) and then decreasing. These cells produced both PDGF and TGF{beta}1 during the early stage of pancreatic fibrosis. Our results suggest that PSCs originating from BMCs contribute mainly to the early stage of pancreatic injury, at least in part, by producing growth factors in a rat CDE diet-induced pancreatitis model.« less

Incorporating Single-nucleotide Polymorphisms Into the Lyman Model to Improve Prediction of Radiation Pneumonitis

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

Tucker, Susan L., E-mail: sltucker@mdanderson.org; Li Minghuan; Xu Ting

2013-01-01

Purpose: To determine whether single-nucleotide polymorphisms (SNPs) in genes associated with DNA repair, cell cycle, transforming growth factor-{beta}, tumor necrosis factor and receptor, folic acid metabolism, and angiogenesis can significantly improve the fit of the Lyman-Kutcher-Burman (LKB) normal-tissue complication probability (NTCP) model of radiation pneumonitis (RP) risk among patients with non-small cell lung cancer (NSCLC). Methods and Materials: Sixteen SNPs from 10 different genes (XRCC1, XRCC3, APEX1, MDM2, TGF{beta}, TNF{alpha}, TNFR, MTHFR, MTRR, and VEGF) were genotyped in 141 NSCLC patients treated with definitive radiation therapy, with or without chemotherapy. The LKB model was used to estimate the risk ofmore » severe (grade {>=}3) RP as a function of mean lung dose (MLD), with SNPs and patient smoking status incorporated into the model as dose-modifying factors. Multivariate analyses were performed by adding significant factors to the MLD model in a forward stepwise procedure, with significance assessed using the likelihood-ratio test. Bootstrap analyses were used to assess the reproducibility of results under variations in the data. Results: Five SNPs were selected for inclusion in the multivariate NTCP model based on MLD alone. SNPs associated with an increased risk of severe RP were in genes for TGF{beta}, VEGF, TNF{alpha}, XRCC1 and APEX1. With smoking status included in the multivariate model, the SNPs significantly associated with increased risk of RP were in genes for TGF{beta}, VEGF, and XRCC3. Bootstrap analyses selected a median of 4 SNPs per model fit, with the 6 genes listed above selected most often. Conclusions: This study provides evidence that SNPs can significantly improve the predictive ability of the Lyman MLD model. With a small number of SNPs, it was possible to distinguish cohorts with >50% risk vs <10% risk of RP when they were exposed to high MLDs.« less

Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis.

PubMed

Neef, Markus; Ledermann, Monika; Saegesser, Hans; Schneider, Vreni; Reichen, Juerg

2006-12-01

Mammalian target of rapamycin (mTOR) signalling is central in the activation of hepatic stellate cells (HSCs), the key source of extracellular matrix (ECM) in fibrotic liver. We tested the therapeutic potential of the mTOR inhibitor rapamycin in advanced cirrhosis. Cirrhosis was induced by bile duct-ligation (BDL) or thioacetamide injections (TAA). Rats received oral rapamycin (0.5 mg/kg/day) for either 14 or 28 days. Untreated BDL and TAA-rats served as controls. Liver function was quantified by aminopyrine breath test. ECM and ECM-producing cells were quantified by morphometry. MMP-2 activity was measured by zymography. mRNA expression of procollagen-alpha1, transforming growth factor-beta1 (TGF-beta1) and beta2 was quantified by RT-PCR. Fourteen days of rapamycin improved liver function. Accumulation of ECM was decreased together with numbers of activated HSCs and MMP-2 activity in both animal models. TGF-beta1 mRNA was downregulated in TAA, TGF-beta2 mRNA was downregulated in BDL. 28 days of rapamycin treatment entailed a survival advantage of long-term treated BDL-rats. Low-dose rapamycin treatment is effectively antifibrotic and attenuates disease progression in advanced fibrosis. Our results warrant the clinical evaluation of rapamycin as an antifibrotic drug.

Simultaneous improvement in production of microalgal biodiesel and high-value alpha-linolenic acid by a single regulator acetylcholine.

PubMed

Parsaeimehr, Ali; Sun, Zhilan; Dou, Xiao; Chen, Yi-Feng

2015-01-01

Photoautotrophic microalgae are a promising avenue for sustained biodiesel production, but are compromised by low yields of biomass and lipids at present. We are developing a chemical approach to improve microalgal accumulation of feedstock lipids as well as high-value alpha-linolenic acid which in turn might provide a driving force for biodiesel production. We demonstrate the effectiveness of the small bioactive molecule "acetylcholine" on accumulation of biomass, total lipids, and alpha-linolenic acid in Chlorella sorokiniana. The effectiveness exists in different species of Chlorella. Moreover, the precursor and analogs of acetylcholine display increased effectiveness at higher applied doses, with maximal increases by 126, 80, and 60% over controls for biomass, total lipids, and alpha-linolenic acid, respectively. Production of calculated biodiesel was also improved by the precursor and analogs of acetylcholine. The biodiesel quality affected by changes in microalgal fatty acid composition was addressed. The chemical approach described here could improve the lipid yield and biodiesel production of photoautotrophic microalgae if combined with current genetic approaches.

Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins.

PubMed

Stojanovski, Diana; Guiard, Bernard; Kozjak-Pavlovic, Vera; Pfanner, Nikolaus; Meisinger, Chris

2007-12-03

The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the beta-barrel-specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a beta-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane alpha-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of alpha-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to beta-barrel proteins but also includes the majority of alpha-helical Tom proteins.

Enantiomeric Excesses of Acid Labile Amino Acid Precursors of the Murchison Meteorite

NASA Astrophysics Data System (ADS)

Pizzarello, Sandra

1998-10-01

Amino acids present in carbonaceous chondrite are extracted in water in part as free compounds and in approximately equal part as acid labile precursors. On the assumption that they would be free of contamination, the precursors of two Murchison amino acids that have terrestrial occurrence, alanine and glutamic acid, have been targeted for analysis of their enantiomeric ratios. Pyroglutamic acid, the precursor of glutamic acid, was found with an L-enantiomeric excess comparable to that of the free acid, while alanine's precursor, N-acetyl alanine, appears approximately racemic. Also alpha-imino propioacetic acid, a proposed end product of alanine synthesis in the meteorite, was analyzed and found racemic.

Enantiomeric Excesses of Acid Labile Amino Acid Precursors of the Murchison Meteorite

NASA Technical Reports Server (NTRS)

Pizzarello, Sandra

1998-01-01

Amino acids present in carbonaceous chondrite are extracted in water in part as free compounds and in approximately equal part as acid labile precursors. On the assumption that they would be free of contamination, the precursors of two Murchison amino acids that have terrestrial occurrence, alanine and glutamic acid, have been targeted for analysis of their enantiomeric ratios. Pyroglutamic acid, the precursor of glutamic acid, was found with an L-enantiomeric excess comparable to that of the free acid, while alanine's precursor, N-acetyl alanine, appears approximately racemic. Also alpha-imino propioacetic acid, a proposed end product of alanine synthesis in the meteorite, was analyzed and found racemic.

Reducing endoglin activity limits calcineurin and TRPC-6 expression and improves survival in a mouse model of right ventricular pressure overload.

PubMed

Kapur, Navin K; Qiao, Xiaoying; Paruchuri, Vikram; Mackey, Emily E; Daly, Gerard H; Ughreja, Kishan; Ughreja, Keshan; Morine, Kevin J; Levine, Jonathan; Aronovitz, Mark J; Hill, Nicholas S; Jaffe, Iris Z; Letarte, Michelle; Karas, Richard H

2014-07-11

Right ventricular (RV) failure is a major cause of mortality worldwide and is often a consequence of RV pressure overload (RVPO). Endoglin is a coreceptor for the profibrogenic cytokine, transforming growth factor beta 1 (TGF-β1). TGF-β1 signaling by the canonical transient receptor protein channel 6 (TRPC-6) was recently reported to stimulate calcineurin-mediated myofibroblast transformation, a critical component of cardiac fibrosis. We hypothesized that reduced activity of the TGF-β1 coreceptor, endoglin, limits RV calcineurin expression and improves survival in RVPO. We first demonstrate that endoglin is required for TGF-β1-mediated calcineurin/TRPC-6 expression and up-regulation of alpha-smooth muscle antigen (α-SMA), a marker of myofibroblast transformation, in human RV fibroblasts. Using endoglin haploinsufficient mice (Eng(+/-)) we show that reduced endoglin activity preserves RV function, limits RV fibrosis, and attenuates activation of the calcineurin/TRPC-6/α-SMA pathway in a model of angio-obliterative pulmonary hypertension. Next, using Eng(+/-) mice or a neutralizing antibody (Ab) against endoglin (N-Eng) in wild-type mice, we show that reduced endoglin activity improves survival and attenuates RV fibrosis in models of RVPO induced by pulmonary artery constriction. To explore the utility of targeting endoglin, we observed a reversal of RV fibrosis and calcineurin levels in wild-type mice treated with a N-Eng Ab, compared to an immunoglobulin G control. These data establish endoglin as a regulator of TGF-β1 signaling by calcineurin and TRPC-6 in the RV and identify it as a potential therapeutic target to limit RV fibrosis and improve survival in RVPO, a common cause of death in cardiac and pulmonary disease. © 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

[Can the concentration of immunomodulating factors in mature breastmilk be associated with food allergy in breastfed children?].

PubMed

Hogendorf, Anna; Stańczyk-Przyłuska, Anna; Siniewicz-Luzeńczyk, Katarzyna; Wiszniewska, Magdalena; Arendarczyk, Jerzy; Banasik, Małgorzata; Fendler, Wojciech; Kowalski, Marek; Zeman, Krzysztof

2012-01-01

The aim of the study was to determine the concentration of such immunomodulating factors as transforming growth factor beta1 (TGF-β1), interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-α) in mature human milk and to relate the levels of the above mentioned cytokines in mature breast milk to the occurence of food allergy in children during the first 24 months of life. Data on breastfeeding, symptoms of food allergy in children and breast milk samples were collected prospectively from birth to 24 months of age from 84 mothers participating in the Polish birth cohort of "EuroPrevall" study, in the years 2005-2007. Cytokine levels were measured in the whey with commercial enzyme-linked immunosorbent assays (ELISA) kits. Ten out of the eighty four (11.9%) participating children had positive SPT and/or sIgE to food antigens. In 7 out of 84 (8.4%) children DBPCFC confirmed the diagnosis of food allergy. The median concentration of TGF-β1 was 21.94 pg/ml (range 10.47-83.19), TNF-α 1.46 pg/ml (range 0.35-16.50), IL- 101.83 pg/ml (range 0.58-31.04). There was a positive correlation between the concentration of IL-10 and TGF-β1. The level of TNF-α correlated positively with the duration of lactation (p=0.04). There was no significant difference between the concentration of IL-10, TGF-β1, TNF-α, in the mature breast milk of mothers of children with symptoms of allergy and positive SPT and/or sIgE, mothers of children with positive DBPCFC and in the milk of mothers of control children. There was no significant difference between the concentration of IL-10, TGF-β1, TNF-α, in the mature breast milk of mothers of children with food allergy and in the breast milk of mothers of control children.

Inflammatory microenvironment and tumor necrosis factor alpha as modulators of periostin and CCN2 expression in human non-healing skin wounds and dermal fibroblasts.

PubMed

Elliott, Christopher G; Forbes, Thomas L; Leask, Andrew; Hamilton, Douglas W

2015-04-01

Non-healing skin wounds remain a significant clinical burden, and in recent years, the regulatory role of matricellular proteins in skin healing has received significant attention. Periostin and CCN2 are both upregulated at day 3 post-wounding in murine skin, where they regulate aspects of the proliferative phase of repair including mesenchymal cell infiltration and myofibroblast differentiation. In this study, we examined 1) the wound phenotype and expression patterns of periostin and CCN2 in non-healing skin wounds in humans and 2) the regulation of their expression in wound fibroblasts by tumor necrosis factor α (TNFα) and transforming growth factor-β1 (TGF-β1). Chronic skin wounds had a pro-inflammatory phenotype, characterized by macrophage infiltration, TNFα immunoreactivity, and neutrophil infiltration. Periostin, but not CCN2, was significantly suppressed in non-healing wound edge tissue at the mRNA and protein level compared with non-involved skin. In vitro, human wound edge fibroblasts populations were still able to proliferate and contract collagen gels. Compared to cells from non-involved skin, periostin and α-SMA mRNA levels increased significantly in the presence of TGF-β1 in wound cells and were significantly decreased by TNFα, but not those of Col1A2 or CCN2. In the presence of both TGF-β1 and TNFα, periostin and α-SMA mRNA levels were significantly reduced compared to TGF-β1 treated wound cells. Effects of TGF-β1 and TNFα on gene expression were also more pronounced in wound edge cells compared to non-involved fibroblasts. We conclude that variations in the expression of periostin and CCN2, are related to an inflammatory microenvironment and the presence of TNFα in human chronic wounds. Copyright © 2015. Published by Elsevier B.V.

Expression of proteins in serum, synovial fluid, synovial membrane, and articular cartilage samples obtained from dogs with stifle joint osteoarthritis secondary to cranial cruciate ligament disease and dogs without stifle joint arthritis.

PubMed

Garner, Bridget C; Kuroki, Keiichi; Stoker, Aaron M; Cook, Cristi R; Cook, James L

2013-03-01

To identify proteins with differential expression between healthy dogs and dogs with stifle joint osteoarthritis secondary to cranial cruciate ligament (CCL) disease. Serum and synovial fluid samples obtained from dogs with stifle joint osteoarthritis before (n = 10) and after (8) surgery and control dogs without osteoarthritis (9) and archived synovial membrane and articular cartilage samples obtained from dogs with stifle joint osteoarthritis (5) and dogs without arthritis (5). Serum and synovial fluid samples were analyzed via liquid chromatography-tandem mass spectrometry; results were compared against a nonredundant protein database. Expression of complement component 3 in archived tissue samples was determined via immunohistochemical methods. No proteins had significantly different expression between serum samples of control dogs versus those of dogs with stifle joint osteoarthritis. Eleven proteins (complement component 3 precursor, complement factor I precursor, apolipoprotein B-100 precursor, serum paraoxonase and arylesterase 1, zinc-alpha-2-glycoprotein precursor, serum amyloid A, transthyretin precursor, retinol-binding protein 4 precursor, alpha-2-macroglobulin precursor, angiotensinogen precursor, and fibronectin 1 isoform 1 preproprotein) had significantly different expression (> 2.0-fold) between synovial fluid samples obtained before surgery from dogs with stifle joint osteoarthritis versus those obtained from control dogs. Complement component 3 was strongly expressed in all (5/5) synovial membrane samples of dogs with stifle joint osteoarthritis and weakly expressed in 3 of 5 synovial membrane samples of dogs without stifle joint arthritis. Findings suggested that the complement system and proteins involved in lipid and cholesterol metabolism may have a role in stifle joint osteoarthritis, CCL disease, or both.

DMH1, a Highly Selective Small Molecule BMP Inhibitor Promotes Neurogenesis of hiPSCs: Comparison of PAX6 and SOX1 Expression during Neural Induction

PubMed Central

2012-01-01

Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-β1 branches of the TGF-β signaling pathways by the endogenous antagonist, Noggin, and the small molecule SB431542, respectively, induces efficient neuralization of hiPSCs, a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost, consistent activity, and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1, a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration, we could selectively modulate the number of SOX1 expressing cells, whereas PAX6, another neural precursor marker, remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations, therefore, suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons, a subset of which also express tyrosine hydroxylase. Thus, the combined use of DMH1, a highly specific BMP-pathway inhibitor, and SB431542, a TGF-β1-pathway specific inhibitor, provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors. PMID:22860217

Calcium alpha-ketoglutarate administration to malnourished hemodialysis patients improves plasma arginine concentrations.

PubMed

Riedel, E; Hampl, H; Steudle, V; Nündel, M

1996-01-01

Calcium alpha-ketoglutarate administration to 24 malnourished hemodialysis patients for 1 year leads to a significant increase in plasma concentrations of L-arginine from 53.6 +/- 18.3 (compared to a healthy control group: 87.5 +/- 27.3) to 71.1 +/- 15.9 mumol/l (p < 0.05). Furthermore, concentrations in plasma of proline and histidine, precursors of glutamate biosynthesis, are increased; inorganic phosphate and urea are significantly decreased in hemodialysis patients after 1 year of calcium alpha-ketoglutarate administration.

Molecular characterization of variant alpha-subunit of electron transfer flavoprotein in three patients with glutaric acidemia type II--and identification of glycine substitution for valine-157 in the sequence of the precursor, producing an unstable mature protein in a patient.

PubMed Central

Indo, Y; Glassberg, R; Yokota, I; Tanaka, K

1991-01-01

In our previous study of eight glutaric acidemia type II (GAII) fibroblast lines by using [35S]methionine labeling and immunoprecipitation, three of them had a defect in the synthesis of the alpha-subunit of electron transfer flavoprotein (alpha-ETF) (Ikeda et al. 1986). In one of them (YH1313) the labeling of the mature alpha-ETF was barely detectable, while that of the precursor (p) was stronger. In another (YH605) no synthesis of immunoreactive p alpha-ETF was detectable. In the third cell line (YH1391) the rate of variant p alpha-ETF synthesis was comparable to normal, but its electrophoretic mobility was slightly faster than normal. In the present study, the northern blot analysis revealed that all three mutant cell lines contained p alpha-ETF mRNA and that their size and amount were comparable to normal. In immunoblot analysis, both alpha- and beta-ETF bands were barely detectable in YH1313 and YH605 but were detectable in YH1391 in amounts comparable to normal. Sequencing of YH1313 p alpha-ETF cDNA via PCR identified a transversion of T-470 to G. We then devised a simple PCR method for the 119-bp section (T-443/G-561) for detecting this mutation. In the upstream primer, A-466 was artificially replaced with C, to introduce a BstNI site into the amplified copies in the presence of G-470 from the variant sequence. The genomic DNA analysis using this method demonstrated that YH1313 was homozygous for T----G-470 transversion. It was not detected either in two other alpha-ETF-deficient GAII or in seven control cell lines. The alpha-ETF cDNA sequence in YH605 was identical to normal. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:1882842

The selective solubilization of different murine splenocyte membrane fractions with lubrol WX and triton X-100 distinguishes two forms of Ia antigens. A cell surface (alpha, beta) and an intracellular (alpha, Ii, beta).

PubMed

Moosic, J P; Sung, E; Nilson, A; Jones, P P; McKean, D J

1982-08-25

The selective solubilization of different murine lymphocyte membrane compartments with several nonionic detergents was used to study the subcellular distribution of two distinct forms of lymphocyte cell recognition structures (Ia antigens). Ia antigens were isolated with a monoclonal anti-Ia immunoadsorbent from murine splenocytes that had been solubilized with four different nonionic detergents. Analyses of the immunoprecipitates indicated that Lubrol WX was selectively solubilizing a subpopulation of Ia consisting of mature highly glycosylated alpha and beta polypeptides which were not associated with Ii polypeptide. A second Ia subpopulation consisting of less glycosylated cytoplasmic precursor alpha and beta polypeptides associated with Ii polypeptide was immunoprecipitated from the Lubrol WX-insoluble material after solubilizing this material with Triton X-100. Comparable results were obtained when HLA-DR antigens were similarly isolated from cultured human lymphoblastoid cells. This selective solubilization phenomenon was not unique to Ia antigens. Only mature highly glycosylated H-2K molecules were immunoprecipitated from the Lubrol WX-soluble material while the less glycosylated precursor H-2K molecules were immunoprecipitated from the Triton X-100-solubilized Lubrol-insoluble material. These data directly demonstrate that the Ii polypeptide is exclusively associated with the intracellular Ia antigen cytoplasmic precursor molecules. These data also indicate that, under the conditions used in these experiments, Lubrol WX does not completely solubilize integral membrane proteins that have previously been shown to be associated with the rough endoplasmic reticulum.

Apelin attenuates TGF-β1-induced epithelial to mesenchymal transition via activation of PKC-ε in human renal tubular epithelial cells.

PubMed

Wang, Li-Yan; Diao, Zong-Li; Zheng, Jun-Fang; Wu, Yi-Ru; Zhang, Qi-Dong; Liu, Wen-Hu

2017-10-01

Epithelial to mesenchymal transition (EMT), a process whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of renal fibrosis. Apelin, a bioactive peptide, has recently been recognized to protect against renal profibrotic activity, but the underlying mechanism has not yet been elucidated. In this study, we investigated the regulation of EMT in the presence of apelin-13 in vitro. Expression of the mesenchymal marker alpha-smooth muscle actin (α-SMA) and the epithelial marker E-cadherin was examined by immunofluorescence and western blotting in transforming growth factor beta 1 (TGF-β1)-stimulated human proximal tubular epithelial cells. Expression of extracellular matrix, fibronectin and collagen-I was examined by quantitative real-time PCR and ELISA. F13A, an antagonist of the apelin receptor APJ, and small interfering RNA targeting protein kinase C epsilon (PKC-ε) were used to explore the relevant signaling pathways. Apelin attenuated TGF-β1-induced EMT, and inhibited the EMT-associated increase in α-SMA, loss of E-cadherin, and secretion of extracellular matrix. Moreover, apelin activated PKC-ε in tubular epithelial cells, which in turn decreased phospho-Smad2/3 levels and increased Smad-7 levels. APJ inhibition or PKC-ε deletion diminished apelin-induced modulation of Smad signaling and suppression of tubular EMT. Our findings identify a novel PKC-ε-dependent mechanism in which apelin suppresses TGF-β1-mediated activation of Smad signaling pathways and thereby inhibits tubular EMT. These results suggest that apelin may be a new agent that can suppress renal fibrosis and retard chronic kidney disease progression. Copyright © 2017 Elsevier Inc. All rights reserved.

The emergence of non-cytolytic NK1.1+ T cells in the long-term culture of murine tumour-infiltrating lymphocytes: a possible role of transforming growth factor-beta.

PubMed

Tamada, K; Harada, M; Ito, O; Takenoyama, M; Mori, T; Matsuzaki, G; Nomoto, K

1996-12-01

The mechanism by which murine tumour-infiltrating lymphocytes (TIL) decreased their anti-tumour activity during an in vitro culture with interleukin-2 (IL-2) was investigated. A phenotype analysis revealed that the TIL cultured for 7 days (TIL-d7) were exclusively NKI.1- CD4- CD8+ CD3+ cells and that this population was replaced by natural killer (NK)1.1+ CD4- CD8 CD3+ cells by day 27 (TIL-d27) during the culture of TIL. The TIL-d7 cells showed a cytolytic activity against B16 melanoma, whereas the TIL-d27 cells had lost this activity, suggesting that the decrease in the anti tumour effect of TIL during the culture with IL-2 was due to their populational change. Analysis on the characteristics of the TIL-d27 cells revealed that they expressed skewed T-cell receptor (TCR) V beta 5 and increased mRNA expression of V alpha 14. In addition, they expressed transforming growth factor beta (TGF-beta) mRNA. Interestingly, TGF-beta augmented the proliferation of TIL-d27 cells under the presence of IL-2, but suppressed that of TIL-d7 cells. Moreover, the proliferation of TIL-d27 cells was suppressed by anti-TGF-beta monoclonal antibody. Collectively, these results suggest that, in contrast to its suppressive effect on anti-tumour effector T cells. TGF-beta could be an autocrine growth factor for NKL1.1+ T cells and thereby induce non-cytolytic NK1.1+ T cells in the long-term culture of TIL.

The emergence of non-cytolytic NK1.1+ T cells in the long-term culture of murine tumour-infiltrating lymphocytes: a possible role of transforming growth factor-beta.

PubMed Central

Tamada, K; Harada, M; Ito, O; Takenoyama, M; Mori, T; Matsuzaki, G; Nomoto, K

1996-01-01

The mechanism by which murine tumour-infiltrating lymphocytes (TIL) decreased their anti-tumour activity during an in vitro culture with interleukin-2 (IL-2) was investigated. A phenotype analysis revealed that the TIL cultured for 7 days (TIL-d7) were exclusively NKI.1- CD4- CD8+ CD3+ cells and that this population was replaced by natural killer (NK)1.1+ CD4- CD8 CD3+ cells by day 27 (TIL-d27) during the culture of TIL. The TIL-d7 cells showed a cytolytic activity against B16 melanoma, whereas the TIL-d27 cells had lost this activity, suggesting that the decrease in the anti tumour effect of TIL during the culture with IL-2 was due to their populational change. Analysis on the characteristics of the TIL-d27 cells revealed that they expressed skewed T-cell receptor (TCR) V beta 5 and increased mRNA expression of V alpha 14. In addition, they expressed transforming growth factor beta (TGF-beta) mRNA. Interestingly, TGF-beta augmented the proliferation of TIL-d27 cells under the presence of IL-2, but suppressed that of TIL-d7 cells. Moreover, the proliferation of TIL-d27 cells was suppressed by anti-TGF-beta monoclonal antibody. Collectively, these results suggest that, in contrast to its suppressive effect on anti-tumour effector T cells. TGF-beta could be an autocrine growth factor for NKL1.1+ T cells and thereby induce non-cytolytic NK1.1+ T cells in the long-term culture of TIL. Images Figure 4 Figure 6 PMID:9014832

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

Jin Lim, Min; Ahn, Jiyeon; Youn Yi, Jae

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-β1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expressionmore » level and nuclear accumulation of Gal-1. The TGF-β1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-β1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases. - Highlights: • Galectin-1 (Gal-1) promotes TGF-β-induced fibroblast differentiation via activation of PI3-kinase and p38 MAPK. • Gal-1 binds to Smad2 and phosphorylated Smad2. • GAl-1 may be a new therapeutic target for attenuating lung fibrotic process.« less

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

PDGFBB promotes PDGFR{alpha}-positive cell migration into artificial bone in vivo

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

Yoshida, Shigeyuki; Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582; Iwasaki, Ryotaro

2012-05-18

Highlights: Black-Right-Pointing-Pointer We examined effects of PDGFBB in PDGFR{alpha} positive cell migration in artificial bones. Black-Right-Pointing-Pointer PDGFBB was not expressed in osteoblastic cells but was expressed in peripheral blood cells. Black-Right-Pointing-Pointer PDGFBB promoted PDGFR{alpha} positive cell migration into artificial bones but not osteoblast proliferation. Black-Right-Pointing-Pointer PDGFBB did not inhibit osteoblastogenesis. -- Abstract: Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure.more » Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor {alpha} (PDGFR{alpha})-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGF{beta}) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.« less

Decarboxylase inhibition and blood pressure reduction by alpha-methyl-3,4-dihydroxy-DL-phenylalanine.

PubMed

OATES, J A; GILLESPIE, L; UDENFRIEND, S; SJOERDSMA, A

1960-06-24

alpha-Methyl-3,4-dihydroxy-DLphenylalanine has been found to be an effective inhibitor of aromatic amino acid decarboxylation in man. This was shown by decreased formation of serotonin, tryptamine, and tyramine from the precursor amino acids. Reduction of amine biosynthesis is associated with lowering of blood pressure in hypertensive patients and a transient sedative effect.

HPLC Analysis of [Alpha]- and [Beta]-Acids in Hops

ERIC Educational Resources Information Center

Danenhower, Travis M.; Force, Leyna J.; Petersen, Kenneth J.; Betts, Thomas A.; Baker, Gary A.

2008-01-01

Hops have been used for centuries to impart aroma and bitterness to beer. The cones of the female hop plant contain both essential oils, which include many of the fragrant components of hops, and a collection of compounds known as [alpha]- and [beta]-acids that are the precursors to bittering agents. In order for brewers to predict the ultimate…

The nuclear protein PH5P of the inter-alpha-inhibitor superfamily: a missing link between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and a novel actor of DNA repair?

PubMed

Jean, L; Risler, J L; Nagase, T; Coulouarn, C; Nomura, N; Salier, J P

1999-03-05

Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair.

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.

Curcumin protects against radiation-induced acute and chronic cutaneous toxicity in mice and decreases mRNA expression of inflammatory and fibrogenic cytokines

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

Okunieff, Paul; Xu Jianhua; Hu Dongping

2006-07-01

Purpose: To determine whether curcumin ameliorates acute and chronic radiation skin toxicity and to examine the expression of inflammatory cytokines (interleukin [IL]-1, IL-6, IL-18, IL-1Ra, tumor necrosis factor [TNF]-{alpha}, and lymphotoxin-{beta}) or fibrogenic cytokines (transforming growth factor [TGF]-{beta}) during the same acute and chronic phases. Methods and Materials: Curcumin was given intragastrically or intraperitoneally to C3H/HeN mice either: 5 days before radiation; 5 days after radiation; or both 5 days before and 5 days after radiation. The cutaneous damage was assessed at 15-21 days (acute) and 90 days (chronic) after a single 50 Gy radiation dose was given to themore » hind leg. Skin and muscle tissues were collected for measurement of cytokine mRNA. Results: Curcumin, administered before or after radiation, markedly reduced acute and chronic skin toxicity in mice (p < 0.05). Additionally, curcumin significantly decreased mRNA expression of early responding cytokines (IL-1 IL-6, IL-18, TNF-{alpha}, and lymphotoxin-{beta}) and the fibrogenic cytokine, TGF-{beta}, in cutaneous tissues at 21 days postradiation. Conclusion: Curcumin has a protective effect on radiation-induced cutaneous damage in mice, which is characterized by a downregulation of both inflammatory and fibrogenic cytokines in irradiated skin and muscle, particularly in the early phase after radiation. These results may provide the molecular basis for the application of curcumin in clinical radiation therapy.« less

Circulating neuroactive C21- and C19-steroids in young men before and after ejaculation.

PubMed

Stárka, L; Hill, M; Havlíková, H; Kancheva, L; Sobotka, V

2006-01-01

Twelve neuroactive and neuroprotective steroids, androgens and androgen precursors i.e. 3alpha,17beta-dihydroxy-5alpha-androstane, 3alpha-hydroxy-5alpha-androstan-17-one, 3alpha-hydroxy-5beta-androstan-17-one, androst-5-ene-3beta,17beta-diol, 3beta,17alpha-dihydroxy-pregn-5-en-20-one (17alpha-hydroxy-pregnenolone), 3beta-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA), testosterone, androst-4-ene-3,17-dione (androstenedione), 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone), 3beta-hydroxy-pregn-5-en-20-one (pregnenolone), 7alpha-hydroxy-DHEA, and 7beta-hydroxy-DHEA were measured using the GC-MS system in young men before and after ejaculation provoked by masturbation. The circulating level of 17alpha-hydroxypregnenolone increased significantly, whereas the other circulating steroids were not changed at all. This fact speaks against the hypothesis that a drop in the level of neuroactive steroids, e.g. allopregnanolone may trigger the orgasm-related increase of oxytocin, reported by other authors.

Thalidomide and lenalidomide extend survival in a transgenic mouse model of amyotrophic lateral sclerosis.

PubMed

Kiaei, Mahmoud; Petri, Susanne; Kipiani, Khatuna; Gardian, Gabrielle; Choi, Dong-Kug; Chen, Junyu; Calingasan, Noel Y; Schafer, Peter; Muller, George W; Stewart, Charles; Hensley, Kenneth; Beal, M Flint

2006-03-01

Accumulating evidence suggests that inflammation plays a major role in the pathogenesis of motor neuron death in amyotrophic lateral sclerosis (ALS). Important mediators of inflammation such as the cytokine tumor necrosis factor-alpha (TNF-alpha) and its superfamily member fibroblast-associated cell-surface ligand (FasL) have been implicated in apoptosis. We found increased TNF-alpha and FasL immunoreactivity in lumbar spinal cord sections of ALS patients and G93A transgenic mice. Both increased TNF-alpha and FasL immunostaining in the lumbar spinal cord of the G93A SOD1 transgenic mice occurred at 40-60 d, well before the onset of symptoms and loss of motor neurons. We tested the neuroprotective effect of thalidomide and its analog lenalidomide, pharmacological agents that inhibit the expression of TNF-alpha and other cytokines by destabilizing their mRNA. Treatment with either thalidomide or lenalidomide attenuated weight loss, enhanced motor performance, decreased motor neuron cell death, and significantly increased the life span in G93A transgenic mice. Treated G93A mice showed a reduction in TNF-alpha and FasL immunoreactivity as well as their mRNA in the lumbar spinal cord. Both compounds also reduced interleukin (IL)-12p40, IL-1alpha, and IL-1beta and increased IL-RA and TGF-beta1 mRNA. Therefore, both thalidomide and lenalidomide bear promise as therapeutic interventions for the treatment of ALS.

TNF-alpha and IL-10 gene polymorphisms show a weak association with pemphigus vulgaris in the Slovak population.

PubMed

Javor, J; Chmurova, N; Parnicka, Z; Ferencik, S; Grosse-Wilde, H; Buc, M; Svecova, D

2010-01-01

Pemphigus vulgaris is a rare chronic autoimmune disease of skin and mucous membranes, with several cytokines participating in its development. The role of their gene polymorphisms in susceptibility to the disease is, however, not fully understood. The aim of our case-control study was to investigate whether some of 22 single nucleotide polymorphisms (SNPs) in 13 cytokine genes (IL-1alpha, IL-1beta, IL-1RI, IL-1Ra, IL-4Ralpha, IL-12, IFN-gamma, TGF-beta1, TNF-alpha, IL-2, IL-4, IL-6 and IL-10) are associated with pemphigus vulgaris in the Slovak population. DNA samples were obtained from 34 pemphigus vulgaris patients and 140 healthy controls of Slovak origin. Cytokine gene SNPs were determined using the polymerase chain reaction with sequence-specific primers (PCR-SSP) method. Results We found a weak association between pemphigus vulgaris and polymorphic variants in TNF-alpha and IL-10 genes only, with haplotypes TNF-alpha-308G/-238G and IL-10 -1082A/-819C/-592C being significantly overrepresented in pemphigus vulgaris patients (TNF-alpha GG: 94.12% vs. 82.86%, P = 0.0216; IL-10 ACC: 44.12% vs. 30.00%, P = 0.0309). Our preliminary results suggest that certain TNF-alpha and IL-10 gene polymorphisms might contribute to genetic susceptibility to pemphigus vulgaris; however, their overall impact on disease development will be rather limited.

LC-MS-Guided Isolation of Penicilfuranone A: A New Antifibrotic Furancarboxylic Acid from the Plant Endophytic Fungus Penicillium sp. sh18.

PubMed

Wang, Wei-Guang; Li, Ao; Yan, Bing-Chao; Niu, Shu-Bin; Tang, Jian-Wei; Li, Xiao-Nian; Du, Xue; Challis, Gregory L; Che, Yongsheng; Sun, Han-Dong; Pu, Jian-Xin

2016-01-22

Penicilfuranone A (1), a novel furancarboxylic acid, and its proposed biosynthetic precursor, gregatin A (2), were isolated from the cultures of the fungus Penicillium sp. sh18 endophytic to the stems of Isodon eriocalyx var. laxiflora guided by HPLC-MS. X-ray crystallography was applied to the structure determination of furancarboxylic acid for the first time, allowing unambiguous assignment of 1. Penicilfuranone A displays a significant antifibrotic effect in activated hepatic stellate cells via negative regulation of transforming growth factor-β (TGF-β)/Smad signaling.

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.

[Alpha-melanocyte-stimulating hormone. From bench to bedside].

PubMed

Böhm, M; Luger, T A

2010-06-01

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a tridecapeptide that is produced by the skin itself from the precursor proopiomelanocortin. It crucially mediates ultraviolet light-induced tanning after binding to melanocortin-1 receptors (MC-1R) expressed on the surface of epidermal melanocytes. The potent pigment-inducing and also cytoprotective actions of alpha-MSH are the rationale for the performance of first phase II clinical trials with Nle4-D-Phe7-alpha-MSH (NDP-alpha-MSH), a subcutaneously administered synthetic and superpotent alpha-MSH analogue, in patients with photodermatoses such as erythropoietic protoporphyria. Since alpha-MSH has shown promising anti-inflammatory and antifibrotic properties in numerous preclinical studies, it will be most interesting to evaluate these effects in further clinical pilot studies with NDP-alpha-MSH. In addition to alpha-MSH analogues, truncated tripeptides such as KDPT which do not bind to MC-1R but have sustained anti-inflammatory properties are currently emerging as another novel therapeutic strategy in dermatology.

Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjögren's syndrome keratoconjunctivitis sicca.

PubMed

Pflugfelder, S C; Jones, D; Ji, Z; Afonso, A; Monroy, D

1999-09-01

To compare epidermal growth factor (EGF) concentration in tear fluid and levels of inflammatory cytokines in the conjunctival epithelium of patients with Sjögren's syndrome keratoconjunctivitis sicca with those of normal controls. Schirmer 1 tear testing, corneal fluorescein staining and conjunctival impression cytology for quantitation of goblet cell density were performed in ten patients with Sjögren's syndrome-associated keratoconjunctivitis sicca and ten asymptomatic normal controls. ELISA was used to detect the concentration of EGF in tear fluid and interleukin 6 in lysates of conjunctival cytology specimens obtained from all subjects. The levels of RNA transcripts encoding inflammatory cytokines [interleukin 1alpha_(IL-1alpha), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor alpha_(TNF-alpha), and transforming growth factor beta1 (TGF-beta1)] as well as a housekeeping gene (G3PDH) were evaluated in conjunctival cytology specimens taken from all subjects by semiquantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR). Decreased tear fluid EGF concentration was noted in Sjögren's syndrome patients (mean 0.68 +/- 0.59 ng/ml) compared to controls (mean 1.66 +/- 0.45 ng/ml, P = 0.004). Significantly increased levels of IL-1alpha, IL-6, IL-8, TNF-alpha and TGF-beta1 RNA transcripts were found in the conjunctival epithelium of Sjögren's syndrome patients compared to controls (P < 0.05), while the level of G3PDH was similar in both groups. The concentration of IL-6 protein was significantly higher in Sjögren's syndrome conjunctiva samples (P = 0.012). Tear EGF concentration correlated with Schirmer 1 scores (rho 0.767, P < 0.001), corneal fluorescein staining scores (rho -0.562, P = 0.01), conjunctival goblet cell density (rho 0.661, P = 0.001) and the levels of IL-1alpha_and IL-8 RNA in the conjunctival epithelium (rho -0.677 and -0.747, respectively, P = 0.001). Both IL-1alpha_and IL-8 RNA in the conjunctival epithelium increased as Schirmer 1 scores decreased (P

Mechanisms for c-myc Induced Mouse Mammary Gland Carcinogenesis and for the Synergistic Role of TGF(alpha) in the Process

DTIC Science & Technology

2001-07-01

1997 Glucose deprivation- induced cytotoxicity in drug resistant genomic status of the c-myc locus in infiltrating ductal human breast carcinoma MCF-7...AD Award Number: DAMD17-00-1-0270 TITLE: Mechanisms for c-myc Induced Mouse Mammary Gland Carcinogenesis and for the Synergistic Role of TGFOX in the...AND SUBTITLE 5. FUNDING NUMBERS Mechanisms for c-myc Induced Mouse Mammary Gland DAMD17-00-1-0270 Carcinogenesis and for the Synergistic Role of TGFa in

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.

Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes.

PubMed

Zhao, Guoying; Karageorgos, Litsa; Hutchinson, Rhonda G; Hopwood, John J; Hemsley, Kim

2010-05-01

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) in which an absence of sulfamidase results in incomplete degradation and subsequent accumulation of its substrate, heparan sulfate. Most neurodegenerative LSD remain untreatable. However, therapy options, such as gene, enzyme end cell therapy, are under investigation. Previously, we have constructed an embryonic stem (ES) cell line (NS21) that over-expresses human sulphamidase as a potential treatment for murine MPS IIIA. In the present study the sulfatase-modifying factor I (SUMF1) and enhanced green fluorescence protein (eGFP) genes were co-introduced under a cytomegalovirus (CMV) promoter into NS21 cells, to enhance further sulfamidase activity and provide a marker for in vivo cell tracking, respectively. eGFP was also introduced under the control of the human elongation factor-1alpha (hEF-1alpha) promoter to compare the stability of transgene expression. During differentiation of ES cells into glial precursors, SUMF1 was down-regulated and was hardly detectable by day 18 of differentiation. Likewise, eGFP expression was heterogeneous and highly unstable. Use of a human EF-1alpha promoter resulted in more homogeneous eGFP expression, with approximately 50% of cells eGFP positive following differentiation into glial precursors. Compared with NS21 cells, the outgrowth of eGFP-expressing cells was not as confluent when differentiated into glial precursors. Our data suggest that SUMF1 enhances sulfamidase activity in ES cells, hEF-1alpha is a stronger promoter than CMV for ES cells and over-expression of eGFP may affect cell growth and contribute to unstable gene expression.

SMAD3 Negatively Regulates Serum Irisin and Skeletal Muscle FNDC5 and Peroxisome Proliferator-activated Receptor γ Coactivator 1-α (PGC-1α) during Exercise*

PubMed Central

Tiano, Joseph P.; Springer, Danielle A.; Rane, Sushil G.

2015-01-01

Beige adipose cells are a distinct and inducible type of thermogenic fat cell that express the mitochondrial uncoupling protein-1 and thus represent a powerful target for treating obesity. Mice lacking the TGF-β effector protein SMAD3 are protected against diet-induced obesity because of browning of their white adipose tissue (WAT), leading to increased whole body energy expenditure. However, the role SMAD3 plays in WAT browning is not clearly understood. Irisin is an exercise-induced skeletal muscle hormone that induces WAT browning similar to that observed in SMAD3-deficient mice. Together, these observations suggested that SMAD3 may negatively regulate irisin production and/or secretion from skeletal muscle. To address this question, we used wild-type and SMAD3 knock-out (Smad3−/−) mice subjected to an exercise regime and C2C12 myotubes treated with TGF-β, a TGF-β receptor 1 pharmacological inhibitor, adenovirus expressing constitutively active SMAD3, or siRNA against SMAD3. We find that in Smad3−/− mice, exercise increases serum irisin and skeletal muscle FNDC5 (irisin precursor) and its upstream activator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) to a greater extent than in wild-type mice. In C2C12 myotubes, TGF-β suppresses FNDC5 and PGC-1α mRNA and protein levels via SMAD3 and promotes SMAD3 binding to the FNDC5 and PGC-1α promoters. These data establish that SMAD3 suppresses FNDC5 and PGC-1α in skeletal muscle cells. These findings shed light on the poorly understood regulation of irisin/FNDC5 by demonstrating a novel association between irisin and SMAD3 signaling in skeletal muscle. PMID:25648888

Solar H-alpha features with hot onsets. III. Long fibrils in Lyman-alpha and with ALMA

NASA Astrophysics Data System (ADS)

Rutten, R. J.

2017-02-01

In H-alpha most of the solar surface is covered by dense canopies of long opaque fibrils, but predictions for quiet-Sun observations with ALMA have ignored this fact. Comparison with Ly-alpha suggests that the extraordinary opacity of H-alpha fibrils is caused by hot precursor events. Application of a recipe that assumes momentary Saha-Boltzmann extinction during their hot onset to millimeter wavelengths suggests that ALMA will observe H-alpha-like fibril canopies, not acoustic shocks underneath, and will yield data more interesting than if these canopies were transparent. An additional file is available at the end of the PDF file of this article.This study is offered as compliment to M.W.M. de Graauw. Our ways, objects, instruments and spectral domains parted after the 1970 eclipse but converge here.

Structure-based design of benzimidazole sugar conjugates: synthesis, SAR and in vivo anti-inflammatory and analgesic activities.

PubMed

El-Nezhawy, Ahmed O H; Gaballah, Samir T; Radwan, Mohamed A A; Baiuomy, Ayman R; Abdel-Salam, Omar M E

2009-11-01

A series of 2-methyl-N-substituted-benzimidazoles, bearing hydroxypyrrolidinon-5-yl or hydroxypyrrolidin-2-yl, 2,3:5,6-di-O-isopropylidene-alpha-D-mannofuranoside, 2,3,5,6-tetrahydroxy-alpha-D-mannofuranoside, 1:2,5:6-di-O-isopropylidene-alpha-D-gluco-furanose,3-O-benzyl-6,7-dideoxy-1:2-O-isopropylidene-alpha-D-xylo-heptofuranos-5-ulose, 3-O-benzyl-6,7-dideoxy-1,2-dihydroxy-alpha-D-xylo-heptofuranos-5-ulose, 1,2,5,6-tetrahydroxy-alpha-D-glucofuranose sugar moieties, were obtained in good yields from 2-methyl N-(trichloroacetamidomethyl)benzimidazole as a donor and carbohydrate residues as acceptor precursors in the presence of catalytic amount of trimethylsilyl trifluoromethanesulfonate (TMSOTf) as Lewis acid. Compounds 6, 7, 10, 13, 15, and 16 showed significant anti-inflammatory and analgesic activities.

Gender-specific effects of endogenous testosterone: female alpha-estrogen receptor-deficient C57Bl/6J mice develop glomerulosclerosis.

PubMed

Elliot, S J; Berho, M; Korach, K; Doublier, S; Lupia, E; Striker, G E; Karl, M

2007-08-01

Young female mice on a C57Bl/6J (B6) background are considered glomerulosclerosis (GS)-resistant but aging B6 mice develop mild GS. Estrogen deficiency accelerates while estrogen replacement retards GS in young sclerosis-prone oligosyndactyly mutant mice on an ROP background. To explore the effects of sex hormones on glomerular structure and function in the context of gender and genetic background, we studied mice in which the estrogen-receptor (ER) genes alpha- or -beta were deleted (alpha- or betaER knockout (KO)) and crossed into the B6 background. We also studied ovariectomized (Ovx) B6 mice given testosterone. Male and female betaERKO and male alphaERKO mice had no glomerular dysfunction at 9 months of age; however, alphaERKO female mice displayed albuminuria and GS. Ovx prevented glomerular dysfunction in alphaERKO female mice by eliminating endogenous testosterone production while exogenous testosterone induced GS in Ovx B6 mice. Androgen receptor (AR) expression and function was found in microdissected glomeruli and cultured mesangial cells. Testosterone compared to placebo increased both AR expression and TGF-beta1 mRNA levels in glomeruli isolated from female B6 mice. Estrogen deficiency had no deleterious effects on the glomeruli in B6 mice. Our study shows that genetic traits strongly influence the GS-promoting effects of estrogen deficiency while testosterone induces GS in a gender-specific manner.

Cytokine gene expression in skin of susceptible guinea-pig infected with Treponema pallidum.

PubMed Central

Wicher, V; Scarozza, A M; Ramsingh, A I; Wicher, K

1998-01-01

Using a semi-quantitative multiplex reverse transcription-polymerase chain reaction assay, we examined cytokine mRNA expression for interleukin-1alpha (IL-1alpha), IL-2, IL-10, IL-12p40, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) in skin samples obtained from C4-deficient (C4D) guinea-pigs inoculated intradermally with virulent Treponema pallidum (VTP). Controls included unmanipulated animals, guinea-pigs injected with T. pallidum-free rabbit inflammatory testicular fluid (ITF) alone, or mixed with heat-killed organisms (HKTP). The expression of IL-1alpha, IL-12p40, and TNF-alpha mRNA [T helper type 1 (Th1)] remained within the normal range in both infected and control animals throughout the experimental period. However, a significant increase (P<0.05) in IL-10 mRNA (Th2) was found exclusively in the VTP-inoculated animals from 3 to 30 days post-infection. Another unique characteristic of the inflammatory response in infected guinea-pigs was the appearance, between 11 and 30 days post-inoculation, of a substantial number of eosinophils in addition to infiltrating mononuclear cells. The results showed a local Th2 response which is consistent with an inadequate immune response. This is reflected by the lengthy and incomplete clearance of the pathogen from the local site of entry and the chronic infection of distant organs. Images Figure 1 Figure 4 PMID:9824482

Developmental roles of the BMP1/TLD metalloproteinases.

PubMed

Ge, Gaoxiang; Greenspan, Daniel S

2006-03-01

The astacin family (M12A) of the metzincin subclan MA(M) of metalloproteinases has been detected in developing and mature individuals of species that range from hydra to humans. Functions of this family of metalloproteinase vary from digestive degradation of polypeptides, to biosynthetic processing of extracellular proteins, to activation of growth factors. This review will focus on a small subgroup of the astacin family; the bone morphogenetic protein 1 (BMP1)/Tolloid (TLD)-like metalloproteinases. In vertebrates, the BMP1/TLD-like metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating mineralization of the ECM of hard tissues. Roles in ECM formation include: processing of the C-propeptides of procollagens types I-III, to yield the major fibrous components of vertebrate ECM; proteolytic activation of the enzyme lysyl oxidase, necessary to formation of covalent cross-links in collagen and elastic fibers; processing of NH2-terminal globular domains and C-propeptides of types V and XI procollagen chains to yield monomers that are incorporated into and control the diameters of collagen type I and II fibrils, respectively; processing of precursors for laminin 5 and collagen type VII, both of which are involved in securing epidermis to underlying dermis; and maturation of small leucine-rich proteoglycans. The BMP1/TLD-related metalloproteinases are also capable of activating the vertebrate transforming growth factor-beta (TGF-beta)-like "chalones" growth differentiation factor 8 (GDF8, also known as myostatin), and GDF11 (also known as BMP11), involved in negative feedback inhibition of muscle and neural tissue growth, respectively; by freeing them from noncovalent latent complexes with their cleaved prodomains. BMP1/TLD-like proteinases also liberate the vertebrate TGF-beta-like morphogens BMP2 and 4 and their invertebrate ortholog decapentaplegic, from latent complexes with the vertebrate extracellular antagonist chordin and its invertebrate ortholog short gastrulation (SOG), respectively. The result is formation of the BMP signaling gradients that form the dorsal-ventral axis in embryogenesis. Thus, BMP1/TLD-like proteinases appear to be key to regulating and orchestrating formation of the ECM and signaling by various TGF-beta-like proteins in morphogenetic and homeostatic events. Copyright 2006 Wiley-Liss, Inc.

[State of mitochondrial respiration and calcium capacity in livers of rats with different resistance to hypoxia after injections of L-arginine].

PubMed

Kurhaliuk, N M

2001-01-01

In experiments on rats with different resistance to hypoxia are investigated processes of mitochondrial respiration, oxidative phosphorylation and calcium capacity in liver under precursor nitric oxide L-arginine (600 mg/kg) and blockator nitric oxide synthase L-NNA (35 mg/kg) injections. We are used next substrates of oxidation: 0.35 mM succinate, 1 mM alpha-ketoglutarate, 1 mM alpha-ketoglutarate and 2 mM malonic acid. Increasing of ADP-stimulation respiration states under exogenous L-arginine injection, decreasing efficacy of respiration processes (respiration control on Chance and ADP/O) under such substrates oxidation, testify to oxide energy support decreasing and reversing nitric oxide inhibit in such conditions. This will be used as mechanism cell regulation succinate dehydrogenase activity. It has shown that L-arginine injection increase calcium mitochondrial capacity low resistance to hypoxia rats using substrates of oxidation succinate and alpha-ketoglutarate to control meanings of high resistance rats. Effects of nitric oxide precursor influence on this processes limit NO-synthase inhibitor L-NNA.

Prebiotic syntheses of vitamin coenzymes: II. Pantoic acid, pantothenic acid, and the composition of coenzyme A

NASA Technical Reports Server (NTRS)

Miller, S. L.; Schlesinger, G.

1993-01-01

Pantoic acid can by synthesized in good prebiotic yield from isobutyraldehyde or alpha-ketoisovaleric acid + H2CO + HCN. Isobutyraldehyde is the Strecker precursor to valine and alpha-ketoisovaleric acid is the valine transamination product. Mg2+ and Ca2+ as well as several transition metals are catalysts for the alpha-ketoisovaleric acid reaction. Pantothenic acid is produced from pantoyl lactone (easily formed from pantoic acid) and the relatively high concentrations of beta-alanine that would be formed on drying prebiotic amino acid mixtures. There is no selectivity for this reaction over glycine, alanine, or gamma-amino butyric acid. The components of coenzyme A are discussed in terms of ease of prebiotic formation and stability and are shown to be plausible choices, but many other compounds are possible. The gamma-OH of pantoic acid needs to be capped to prevent decomposition of pantothenic acid. These results suggest that coenzyme A function was important in the earliest metabolic pathways and that the coenzyme A precursor contained most of the components of the present coenzyme.

Gene expression profiles in chondrosarcoma cells subjected to cyclic stretching and hydrostatic pressure. A cDNA array study.

PubMed

Karjalainen, Hannu M; Sironen, Reijo K; Elo, Mika A; Kaarniranta, Kai; Takigawa, Masaharu; Helminen, Heikki J; Lammi, Mikko J

2003-01-01

Mechanical forces have a profound effect on cartilage tissue and chondrocyte metabolism. Strenuous loading inhibits the cellular metabolism, while optimal level of loading at correct frequency raises an anabolic response in chondrocytes. In this study, we used Atlas Human Cancer cDNA array to investigate mRNA expression profiles in human chondrosarcoma cells stretched 8% for 6 hours at a frequency of 0.5 Hz. In addition, cultures were exposed to continuous and cyclic (0.5 Hz) 5 MPa hydrostatic pressure. Cyclic stretch had a more profound effect on the gene expression profiles than 5 MPa hydrostatic pressure. Several genes involved with the regulation of cell cycle were increased in stretched cells, as well as mRNAs for PDGF-B, glucose-1-phosphate uridylyltransferase, Tiam1, cdc37 homolog, Gem, integrin alpha6, and matrix metalloproteinase-3. Among down-regulated genes were plakoglobin, TGF-alpha, retinoic acid receptor-alpha and Wnt8b. A smaller number of changes was detected after pressure treatments. Plakoglobin was increased under cyclic and continuous 5 MPa hydrostatic pressure, while mitogen-activated protein kinase-9, proliferating cell nuclear antigen, Rad6, CD9 antigen, integrins alphaE and beta8, and vimentin were decreased. Cyclic and continuous pressurization induces a number of specific changes. In conclusion, a different set of genes were affected by three different types of mechanical stimuli applied on chondrosarcoma cells.

Enhanced susceptibility to periodontitis in an animal model of depression: reversed by chronic treatment with the anti-depressant tianeptine.

PubMed

Breivik, Torbjørn; Gundersen, Yngvar; Myhrer, Trond; Fonnum, Frode; Osmundsen, Harald; Murison, Robert; Gjermo, Per; von Hörsten, Stephan; Opstad, Per Kristian

2006-07-01

To test the hypothesis that the olfactory bulbectomy model of depression in rats could influence susceptibility to ligature-induced periodontitis, and that chronic treatment with the anti-depressant drug tianeptine could attenuate this effect. Tianeptine was given twice daily (10 mg/kg, i.p.) during the entire experiment, starting 29 days before induction of olfactory bulbectomy and periodontitis. Olfactory bulbectomized (OB) rats and sham-operated rats were given saline in a similar manner. Periodontal disease was assessed when the ligatures had been in place for 21 days. Two hours before decapitation, rats were injected with lipopolysaccharide (LPS;100 microg/kg, i.p.) to induce a robust immune and stress response. Compared with sham-operated controls, OB rats developed significantly more periodontal bone loss, exhibited characteristic behavioural responses in a novel open field test, and showed a decreased expression of glucocorticoid receptors (GRs) in the hippocampus. LPS provoked a significantly larger increase in circulating levels of the stress hormone corticosterone and the cytokine transformation growth factor (TGF)-1beta but smaller tumour necrosis factor (TNF)-alpha levels. Tianeptine treatment of OB rats significantly inhibited peridodontal bone loss, normalized behavioural responses, enhanced TGF-1beta levels, and abolished TNF-alpha decrease, but did not attenuate the increased corticosterone response and the decreased hippocampal GR expression. These experimental results are consistent with an emerging literature showing that life stress, anxiety, depression, pathological grief, and poor coping behaviour may dysregulate regulatory mechanisms within the brain involved in immune regulation, and thereby alter immune responses and influence the susceptibility/resistance to inflammatory disorders.

Clonal population of adult stem cells: life span and differentiation potential.

PubMed

Seruya, Mitchel; Shah, Anup; Pedrotty, Dawn; du Laney, Tracey; Melgiri, Ryan; McKee, J Andrew; Young, Henry E; Niklason, Laura E

2004-01-01

Adult stem cells derived from bone marrow, connective tissue, and solid organs can exhibit a range of differentiation potentials. Some controversy exists regarding the classification of mesenchymal stem cells as bona fide stem cells, which is in part derived from the limited ability to propagate true clonal populations of precursor cells. We isolated putative mesenchymal stem cells from the connective tissue of an adult rat (rMSC), and generated clonal populations via three rounds of dilutional cloning. The replicative potential of the clonal rMSC line far exceeded Hayflick's limit of 50-70 population doublings. The high capacity for self-renewal in vitro correlated with telomerase activity, as demonstrated by telomerase repeat amplification protocol (TRAP) assay. Exposure to nonspecific differentiation culture medium revealed multilineage differentiation potential of rMSC clones. Immunostaining confirmed the appearance of mesodermal phenotypes, including adipocytes possessing lipid-rich vacuoles, chondrocytes depositing pericellular type II collagen, and skeletal myoblasts expressing MyoD1. Importantly, the spectrum of differentiation capability was sustained through repeated passaging. Furthermore, serum-free conditions that led to high-efficiency smooth muscle differentiation were identified. rMSCs plated on collagen IV-coated surfaces and exposed to transforming growth factor-beta1 (TGF-beta1) differentiated into a homogeneous population expressing alpha-actin and calponin. Hence, clonogenic analysis confirmed the presence of a putative MSC population derived from the connective tissue of rat skeletal muscle. The ability to differentiate into a smooth muscle cell (SMC) phenotype, combined with a high proliferative capacity, make such a connective tissue-derived MSC population ideal for applications in vascular tissue construction.

Lice induced immuno-oxidative wreckage of goats.

PubMed

Ajith, Y; Dimri, U; Singh, Shanker K; Gopalakrishnan, A; Devi, Gopinath; Verma, M R; Joshi, Vivek; Alam, Shahjahan

2017-08-15

The present study aimed to evaluate the immuno-oxidative patho-biology of lice infestation in goats. Sixty goats were divided into five groups; sucking lice (Linognathus africanus) infested (Group 1, n=12), chewing lice (Bovicola caprae) infested-mild (Group 2, n=12), chewing lice (B. caprae) infested-moderate (Group 3, n=12), chewing lice (B. caprae) infested-severe (Group 4, n=12) and healthy control (Group 5, n=12). To assess the pathological changes, markers of oxidative stress (lipid peroxidation-LPO, reduced glutathione-GSH, superoxide dismutase-SOD, Catalase-CAT and total antioxidant capacity-TAC), the markers of immune status (Tumour necrosis factor alpha- TNF-α, Interleukin-10- IL-10, Transforming growth factor beta 1- TGF-β1, ratios of TNF-α/IL-10 and TNF-α/TGF-β1) and hemato-biochemical status were evaluated. Significant anemia, hypoglycemia, hypoproteinemia and hypoalbuminemia were observed in caprine pediculosis irrespective of the type of lice infested. Remarkably increased oxidative stress was observed in chewing lice infested goats and no significant changes in oxidative stress markers were observed in sucking lice infested goats. TGF-β mediated suppression of Th1 and Th2 immune responses was observed in sucking lice infested goats; whereas, a Th2 cytokine dominant inflammatory response was observed in chewing lice infested goats. From the present study, it may be concluded that sucking lice infestation produces remarkable immunosuppression and chewing lice infestation produces significant oxidative stress and inflammatory responses in goats. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluoride potentiates tubulointerstitial nephropathy caused by unilateral ureteral obstruction.

PubMed

Kido, Takamasa; Tsunoda, Masashi; Sugaya, Chiemi; Hano, Hiroshi; Yanagisawa, Hiroyuki

2017-12-01

The contamination of ground water by fluoride has been reported worldwide. Most fluoride (approximately 70%) is filtered by the kidneys; humans or experimental animals with renal damage therefore may be more affected by fluoride exposure than those with normal kidney function. Tubulointerstitial fibrosis, which involves macrophage-promoted extracellular matrix production and myofibroblast migration, can be induced in rats by unilateral ureteral obstruction (UUO). We examined the effects of fluoride exposure on tubulointerstitial fibrosis in the obstructed kidney of UUO rats. The left ureters of 6-week-old male rats were ligated using silk sutures. Fluoride was then administered for 2 weeks at doses of 0, 75, and 150ppm in the drinking water. Real-time polymerase chain reaction was performed to analyze transforming growth factor beta 1 (TGF-β 1 ) transcription; histological and immunohistochemical staining were used to identify positive areas within the renal cortex and staining-positive cells by image analysis. Significant increases were observed in the obstructed kidneys of UUO rats exposed to 150ppm fluoride (compared to 0ppm) for areas or number of cells that stained with Masson trichrome or with antibodies against collagen type I, alpha-smooth muscle actin (α-SMA, a myofibroblast marker), ED1, ED2, and ED3 (macrophage markers), and TGF-β 1 . Taken together, these observations suggested that fluoride exacerbates tuburointerstitial nephropathy resulting from UUO, and that this effect occurs via activation of the M2 macrophage-TGF-β1-fibroblast/myofibroblast-collagen synthesis pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Correlation between Fibrillin-1 Degradation and mRNA Downregulation and Myofibroblast Differentiation in Cultured Human Dental Pulp Tissue

PubMed Central

Yoshiba, Nagako; Yoshiba, Kunihiko; Ohkura, Naoto; Takei, Erika; Edanami, Naoki; Oda, Youhei; Hosoya, Akihiro; Nakamura, Hiroaki; Okiji, Takashi

2015-01-01

Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-β1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-β signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-β1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing. PMID:25805839

Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

PubMed

Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

2016-01-01

BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

Nonneutral plasma diagnostic commissioning for the ALPHA Antihydrogen experiment

NASA Astrophysics Data System (ADS)

Konewko, S.; Friesen, T.; Tharp, T. D.; Alpha Collaboration

2017-10-01

The ALPHA experiment at CERN creates antihydrogen by mixing antiproton and positron plasmas. Diagnostic measurements of the precursor plasmas are performed using a diagnostic suite, colloquially known as the ``stick.'' This stick has a variety of sensors and is able to move to various heights to align the desired diagnostic with the beamline. A cylindrical electrode, a faraday cup, an electron gun, and a microchannel-plate detector (MCP) are regularly used to control and diagnose plasmas in ALPHA. We have designed, built, and tested a new, upgraded stick which includes measurement capabilities in both beamline directions.

Molecular mechanisms of cardiac electromechanical remodeling during Chagas disease: Role of TNF and TGF-β.

PubMed

Cruz, Jader Santos; Machado, Fabiana Simão; Ropert, Catherine; Roman-Campos, Danilo

2017-02-01

Chagas disease is caused by the trypanosomatid Trypanosoma cruzi, which chronically causes heart problems in up to 30% of infected patients. Chagas disease was initially restricted to Latin America. However, due to migratory events, this disease may become a serious worldwide health problem. During Chagas disease, many patients die of cardiac arrhythmia despite the apparent benefits of anti-arrhythmic therapy (e.g., amiodarone). Here, we assimilate the cardiac form of Chagas disease to an inflammatory cardiac disease. Evidence from the literature, mostly provided using experimental models, supports this view and argues in favor of new strategies for treating cardiac arrhythmias in Chagas disease by modulating cytokine production and/or action. But the complex nature of myocardial inflammation underlies the need to better understand the molecular mechanisms of the inflammatory response during Chagas disease. Here, particular attention has been paid to tumor necrosis factor alpha (TNF) and transforming growth factor beta (TGF-β) although other cytokines may be involved in the chagasic cardiomyopathy. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Convenient approaches to synthesis of furanoid sugar-aza-crown ethers from C-ribosyl azido aldehyde via a reductive amination/amidation.

PubMed

Hsieh, Yu-Chi; Chir, Jiun-Ly; Zou, Wei; Wu, Hsiu-Han; Wu, An-Tai

2009-05-26

A short and highly efficient route to the alpha-anomer of a furanoid sugar-aza-crown ether was developed by a one-pot reductive amination of an alpha-anomer C-ribosyl azido aldehyde. In addition, the beta-anomer furanoid sugar-aza-crown ether was synthesized from a linear disaccharide precursor via amidation and then followed by microwave-assisted amide reduction.

Loss of miR-29 in Myoblasts Contributes to Dystrophic Muscle Pathogenesis

PubMed Central

Wang, Lijun; Zhou, Liang; Jiang, Peiyong; Lu, Leina; Chen, Xiaona; Lan, Huiyao; Guttridge, Denis C; Sun, Hao; Wang, Huating

2012-01-01

microRNAs (miRNAs) are noncoding RNAs that regulate gene expression in post-transcriptional fashion, and emerging studies support their importance in a multitude of physiological and pathological processes. Here, we describe the regulation and function of miR-29 in Duchenne muscular dystrophy (DMD) and its potential use as therapeutic target. Our results demonstrate that miR-29 expression is downregulated in dystrophic muscles of mdx mice, a model of DMD. Restoration of its expression by intramuscular and intravenous injection improved dystrophy pathology by both promoting regeneration and inhibiting fibrogenesis. Mechanistic studies revealed that loss of miR-29 in muscle precursor cells (myoblasts) promotes their transdifferentiation into myofibroblasts through targeting extracellular molecules including collagens and microfibrillar-associated protein 5 (Mfap5). We further demonstrated that miR-29 is under negative regulation by transforming growth factor-β (TGF-β) signaling. Together, these results not only identify TGF-β–miR-29 as a novel regulatory axis during myoblasts conversion into myofibroblasts which constitutes a novel contributing route to muscle fibrogenesis of DMD but also implicate miR-29 replacement therapy as a promising treatment approach for DMD. PMID:22434133

Interleukin-1, tumor necrosis factor-alpha, and transforming growth factor-beta 1 and integrative meniscal repair: influences on meniscal cell proliferation and migration

PubMed Central

2011-01-01

Introduction Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-β1 (TGF-β1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-β1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair. Methods A micro-wound assay was used to assess meniscal cell migration and proliferation in response to the following treatments for 0, 24, or 48 hours: 0 to 10 ng/mL IL-1, TNF-α, or TGF-β1, in the presence or absence of 10% serum. Proliferated and total cells were fluorescently labeled and imaged using confocal laser scanning microscopy and the number of proliferated, migrated, and total cells was determined in the micro-wound and edges of each image. Meniscal cell proliferation was also assessed throughout meniscal repair model explants treated with 0 or 10 ng/mL IL-1, TNF-α, or TGF-β1 for 14 days. At the end of the culture period, biomechanical testing and histological analyses were also performed. Statistical differences were assessed using an ANOVA and Newman-Keuls post hoc test. Results IL-1 and TNF-α decreased cell proliferation in both cell and tissue models of meniscal repair. In the presence of serum, TGF-β1 increased outer zone cell proliferation in the micro-wound and in the cross section of meniscal repair model explants. Both IL-1 and TNF-α decreased the integrative shear strength of repair and extracellular matrix deposition in the meniscal repair model system, while TGF-β1 had no effect on either measure. Conclusions Meniscal cell proliferation in vivo may be diminished following joint injury due to the up-regulation of inflammatory cytokines, thereby limiting native cellular repair of meniscal lesions. Therefore, therapies that can promote meniscal cell proliferation have promise to enhance meniscal repair and improve tissue engineering strategies. PMID:22087734

Anti-cytokine autoantibodies in autoimmunity: preponderance of neutralizing autoantibodies against interferon-alpha, interferon-omega and interleukin-12 in patients with thymoma and/or myasthenia gravis.

PubMed

Meager, A; Wadhwa, M; Dilger, P; Bird, C; Thorpe, R; Newsom-Davis, J; Willcox, N

2003-04-01

We have screened for spontaneous anticytokine autoantibodies in patients with infections, neoplasms and autoimmune diseases, because of their increasingly reported co-occurrence. We tested for both binding and neutralizing autoantibodies to a range of human cytokines, including interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-alpha2 (IFN-alpha2), IFN-omega, IFN-beta, IFN-gamma, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta-1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF), in plasmas or sera. With two notable exceptions described below, we found only occasional, mostly low-titre, non-neutralizing antibodies, mainly to GM-CSF; also to IL-10 in pemphigoid. Strikingly, however, high-titre, mainly IgG, autoantibodies to IFN-alpha2, IFN-omega and IL-12 were common at diagnosis in patients with late-onset myasthenia gravis (LOMG+), thymoma (T) but no MG (TMG-) and especially with both thymoma and MG together (TMG+). The antibodies recognized other closely related type I IFN-alpha subtypes, but rarely the distantly related type I IFN-beta, and never (detectably) the unrelated type II IFN-gamma. Antibodies to IL-12 showed a similar distribution to those against IFN-alpha2, although prevalences were slightly lower; correlations between individual titres against each were so modest that they appear to be entirely different specificities. Neither showed any obvious correlations with clinical parameters including thymoma histology and HLA type, but they did increase sharply if the tumours recurred. These antibodies neutralized their respective cytokine in bioassays in vitro; although they persisted for years severe infections were surprisingly uncommon, despite the immunosuppressive therapy also used in most cases. These findings must hold valuable clues to autoimmunizing mechanisms in paraneoplastic autoimmunity.

cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei.

PubMed

Hwang, Jae-Ho; Yokoyama, Yoshihiro; Mizuta, Shoshi; Yoshinaka, Reiji

2006-05-01

A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain in an elasmobranch.

Biological Role and Therapeutic Targeting of TGF-β3 in Glioblastoma.

PubMed

Seystahl, Katharina; Papachristodoulou, Alexandros; Burghardt, Isabel; Schneider, Hannah; Hasenbach, Kathy; Janicot, Michel; Roth, Patrick; Weller, Michael

2017-06-01

Transforming growth factor (TGF)-β contributes to the malignant phenotype of glioblastoma by promoting invasiveness and angiogenesis and creating an immunosuppressive microenvironment. So far, TGF-β 1 and TGF-β 2 isoforms have been considered to act in a similar fashion without isoform-specific function in glioblastoma. A pathogenic role for TGF-β 3 in glioblastoma has not been defined yet. Here, we studied the expression and functional role of endogenous and exogenous TGF-β 3 in glioblastoma models. TGF-β 3 mRNA is expressed in human and murine long-term glioma cell lines as well as in human glioma-initiating cell cultures with expression levels lower than TGF-β 1 or TGF-β 2 in most cell lines. Inhibition of TGF-β 3 mRNA expression by ISTH2020 or ISTH2023, two different isoform-specific phosphorothioate locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, blocks downstream SMAD2 and SMAD1/5 phosphorylation in human LN-308 cells, without affecting TGF-β 1 or TGF-β 2 mRNA expression or protein levels. Moreover, inhibition of TGF-β 3 expression reduces invasiveness in vitro Interestingly, depletion of TGF-β 3 also attenuates signaling evoked by TGF-β 1 or TGF-β 2 In orthotopic syngeneic (SMA-560) and xenograft (LN-308) in vivo glioma models, expression of TGF-β 3 as well as of the downstream target, plasminogen-activator-inhibitor (PAI)-1 , was reduced, while TGF-β 1 and TGF-β 2 levels were unaffected following systemic treatment with TGF-β 3 -specific antisense oligonucleotides. We conclude that TGF-β 3 might function as a gatekeeper controlling downstream signaling despite high expression of TGF-β 1 and TGF-β 2 isoforms. Targeting TGF-β 3 in vivo may represent a promising strategy interfering with aberrant TGF-β signaling in glioblastoma. Mol Cancer Ther; 16(6); 1177-86. ©2017 AACR . ©2017 American Association for Cancer Research.

Excess 25-hydroxyvitamin D3 exacerbates tubulointerstitial injury in mice by modulating macrophage phenotype.

PubMed

Kusunoki, Yasuo; Matsui, Isao; Hamano, Takayuki; Shimomura, Akihiro; Mori, Daisuke; Yonemoto, Sayoko; Takabatake, Yoshitsugu; Tsubakihara, Yoshiharu; St-Arnaud, René; Isaka, Yoshitaka; Rakugi, Hiromi

2015-11-01

Vitamin D hydroxylated at carbon 25 (25(OH)D) is generally recognized as a precursor of active vitamin D. Despite its low affinity for the vitamin D receptor (VDR), both deficient and excessive 25(OH)D levels are associated with poor clinical outcomes. Here we studied direct effects of 25(OH)D3 on the kidney using 25(OH)D-1α-hydroxylase (CYP27B1) knockout mice. The effects of 25(OH)D3 on unilateral ureteral obstruction were analyzed as proximal tubular cells and macrophages are two major cell types that take up 25(OH)D and contribute to the pathogenesis of kidney injury. Excess 25(OH)D3 in obstructed mice worsened oxidative stress and tubulointerstitial fibrosis, whereas moderate levels of 25(OH)D3 had no effects. The exacerbating effects of excess 25(OH)D3 were abolished in CYP27B1/VDR double-knockout mice and in macrophage-depleted CYP27B1 knockout mice. Excess 25(OH)D3 upregulated both M1 marker (TNF-α) and M2 marker (TGF-β1) levels of kidney-infiltrating macrophages. In vitro analyses verified that excess 25(OH)D3 directly upregulated TNF-α and TGF-β1 in cultured macrophages but not in tubular cells. TNF-α and 25(OH)D3 cooperatively induced oxidative stress by upregulating iNOS in tubular cells. Aggravated tubulointerstitial fibrosis in mice with excess 25(OH)D3 indicated that macrophage-derived TGF-β1 also had a key role in the pathogenesis of surplus 25(OH)D3. Thus, excess 25(OH)D3 worsens tubulointerstitial injury by modulating macrophage phenotype.

Signaling through the TGF Beta-Activin Receptors ALK4/5/7 Regulates Testis Formation and Male Germ Cell Development

PubMed Central

Stringer, Jessica M.; van den Bergen, Jocelyn A.; Wilhelm, Dagmar; Sinclair, Andrew H.; Western, Patrick S.

2013-01-01

The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfβ family members has indicated that Tgfβ's are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activin's, TGFβ's, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFβ signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFβ receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFβ and Activin signaling during testis formation and male germ cell development. PMID:23342175

Bacterially activated B-cells drive T cell differentiation towards Tr1 through PD-1/PD-L1 expression.

PubMed

Said, Sawsan Sudqi; Barut, Guliz Tuba; Mansur, Nesteren; Korkmaz, Asli; Sayi-Yazgan, Ayca

2018-04-01

Regulatory B cells (Bregs) play a crucial role in immunological tolerance primarily through the production of IL-10 in many diseases including autoimmune disorders, allergy, infectious diseases, and cancer. To date, various Breg subsets with overlapping phenotypes have been identified. However, the roles of Bregs in Helicobacter infection are largely unknown. In the present study, we investigate the phenotype and function of Helicobacter -stimulated B cells. Our results demonstrate that Helicobacter felis -stimulated IL-10- producing B cells (Hf stim - IL-10 + B) are composed of B10 and Transitional 2 Marginal Zone Precursor (T2-MZP) cells with expression of CD9, Tim-1, and programmed death 1 (PD-1). On the other hand, Helicobacter felis -stimulated IL-10- nonproducing B (Hf stim - IL-10 - B) cells are mainly marginal zone (MZ) B cells that express PD-L1 and secrete TGF-β, IL-6, and TNF-α, and IgM and IgG2b. Furthermore, we show that both Hf stim - IL-10 + B cells and Hf stim - IL-10 - B cells induce CD49b + LAG-3 + Tr1 cells. Here, we describe a novel mechanism for PD-1/PD-L1- driven B cell-dependent Tr1 cell differentiation. Finally, we explore the capability of Hf stim - IL-10 - B cells to induce Th17 cell differentiation, which we find to be dependent on TGF-β. Taken together, the current study demonstrates that Hf stim - B cells induce Tr1 cells through the PD-1/PD-L1 axis and Th17 cells by secreting TGF-β. Copyright © 2018 Elsevier Ltd. All rights reserved.

Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer

PubMed Central

Bardeesy, Nabeel; Cheng, Kuang-hung; Berger, Justin H.; Chu, Gerald C.; Pahler, Jessica; Olson, Peter; Hezel, Aram F.; Horner, James; Lauwers, Gregory Y.; Hanahan, Douglas; DePinho, Ronald A.

2006-01-01

SMAD4 is inactivated in the majority of pancreatic ductal adenocarcinomas (PDAC) with concurrent mutational inactivation of the INK4A/ARF tumor suppressor locus and activation of the KRAS oncogene. Here, using genetically engineered mice, we determined the impact of SMAD4 deficiency on the development of the pancreas and on the initiation and/or progression of PDAC—alone or in combination with PDAC-relevant mutations. Selective SMAD4 deletion in the pancreatic epithelium had no discernable impact on pancreatic development or physiology. However, when combined with the activated KRASG12D allele, SMAD4 deficiency enabled rapid progression of KRASG12D-initiated neoplasms. While KRASG12D alone elicited premalignant pancreatic intraepithelial neoplasia (PanIN) that progressed slowly to carcinoma, the combination of KRASG12D and SMAD4 deficiency resulted in the rapid development of tumors resembling intraductal papillary mucinous neoplasia (IPMN), a precursor to PDAC in humans. SMAD4 deficiency also accelerated PDAC development of KRASG12D INK4A/ARF heterozygous mice and altered the tumor phenotype; while tumors with intact SMAD4 frequently exhibited epithelial-to-mesenchymal transition (EMT), PDAC null for SMAD4 retained a differentiated histopathology with increased expression of epithelial markers. SMAD4 status in PDAC cell lines was associated with differential responses to transforming growth factor-β (TGF-β) in vitro with a subset of SMAD4 wild-type lines showing prominent TGF-β-induced proliferation and migration. These results provide genetic confirmation that SMAD4 is a PDAC tumor suppressor, functioning to block the progression of KRASG12D-initiated neoplasms, whereas in a subset of advanced tumors, intact SMAD4 facilitates EMT and TGF-β-dependent growth. PMID:17114584

Effect of Escherichia coli and Lactobacillus rhamnosus on Macrophage Inflammatory Protein 3α, Tumor Necrosis Factor Alpha, and Transforming Growth Factor β Release by Polarized Rat Uterine Epithelial Cells in Culture

PubMed Central

Crane-Godreau, Mardi A.; Wira, Charles R.

2004-01-01

Entry of bacteria from the vagina into the uterus raises the question of uterine epithelial cell (UEC) signaling in response to the presence of bacteria. Our model system helps to define microbially elicited UEC basolateral cytokine release, important in regulating underlying stromal immune cell protection. UECs from adult rats were grown in cell culture inserts to establish a confluent polarized monolayer as was determined by transepithelial resistance (TER). Polarized epithelial cell cultures were treated apically with live or heat-killed Escherichia coli or Lactobacillus rhamnosus prior to collection of basolateral media after 24 h of incubation. Coculture of polarized UECs with live E. coli had no effect on epithelial cell TER. In response to exposure to live E. coli, epithelial cell basolateral release of macrophage inflammatory protein 3α (MIP3α) and tumor necrosis factor alpha (TNF-α) increased at a time when basolateral release of biologically active transforming growth factor β (TGF-β) decreased. Incubation of UECs with heat-killed E. coli resulted in an increased basolateral release of MIP3α and TNF-α, without affecting TER or TGF-β. In contrast to E. coli, live or heat-killed L. rhamnosus had no effect on TER or cytokine release. These studies indicate that polarized rat UECs respond to gram-negative E. coli by releasing the cytokines MIP3α and TNF-α, signals important to both the innate and adaptive immune systems. These findings suggest that UEC responses to bacteria are selective and important in initiating and regulating immune protection in the female reproductive tract. PMID:15039305

The CC chemokine eotaxin/CCL11 has a selective profibrogenic effect on human lung fibroblasts.

PubMed

Puxeddu, Ilaria; Bader, Reem; Piliponsky, Adrian Martin; Reich, Reuven; Levi-Schaffer, Francesca; Berkman, Neville

2006-01-01

Eotaxin/CCL11 plays an important role in asthma. It acts through the chemokine receptor CCR3 expressed on hematopoietic and nonhematopoietic cells in the lung. To determine whether eotaxin/CCL11 modulates lung and bronchial fibroblast properties and thereby might contribute to airway remodeling. CCR3 expression was characterized on a lung fibroblast line (MRC-5; flow cytometry, fluorescent microscopy, RT-PCR, and Northern blotting), on primary bronchial fibroblasts (flow cytometry), and on fibroblasts in human lung tissue (confocal laser microscopy). The effects of eotaxin/CCL11 on lung fibroblast migration (Boyden chamber), proliferation (tritiated thymidine incorporation), alpha-smooth muscle actin expression (ELISA), 3-dimensional collagen gel contraction (floating gel), pro-alpha1(I) collagen mRNA (Northern blotting), total collagen synthesis (tritiated proline incorporation), matrix metalloproteinase activity (gelatin zymography), and TGF-beta(1) release (ELISA) were evaluated. The contribution of eotaxin/CCL11/CCR3 binding on lung fibroblasts was also investigated by neutralizing experiments. CCR3 is constitutively expressed in cultured lung and primary bronchial fibroblasts and colocalizes with specific surface markers for human fibroblasts in lung tissue. Eotaxin/CCL11 selectively modulates fibroblast activities by increasing their proliferation, matrix metalloproteinase 2 activity, and collagen synthesis but not their differentiation into myofibroblasts, contractility in collagen gel, or TGF-beta(1) release. Eotaxin/CCL11 enhances migration of lung fibroblasts in response to nonspecific chemoattractants, and this effect is completely inhibited by anti-CCR3-neutralizing antibodies. These data demonstrate that eotaxin/CCL11 has a direct and selective profibrogenic effect on lung and bronchial fibroblasts, providing a novel mechanism whereby eotaxin/CCL11 can participate in airway remodeling in asthma.

Inhibition of pancreatic stellate cell activity by adipose-derived stem cells.

PubMed

Yu, Fu-Xiang; Su, Long-Feng; Dai, Chun-Lei; Wang, Yang; Teng, Yin-Yan; Fu, Jun-Hui; Zhang, Qi-Yu; Tang, Yin-He

2015-04-01

Pancreatic stellate cells (PSCs) play a critical role in the development of pancreatic fibrosis. In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory effects of adipose-derived stem cells (ADSCs) on activation and proliferation of PSCs. Pancreatic tissue was obtained from Sprague-Dawley rats for PSCs isolation. Transwell cell cultures were adopted for co-culture of ADSCs and PSCs. PSCs proliferation and apoptosis were determined using CCK-8 and flow cytometry, respectively. alpha-SMA expressions were analyzed using Western blotting. The levels of cytokines [nerve growth factor (NGF), interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1)] in conditioned medium were detected by ELISA. Gene expression (MMP-2, MMP-9 and TIMP-1) was analyzed using qRT-PCR. This method produced 17.6+/-6.5X10(3) cells per gram of the body weight with a purity of 90%-95% and a viability of 92%-97%. Co-culture of PSCs with ADSCs significantly inhibited PSCs proliferation and induced PSCs apoptosis. Moreover, alpha-SMA expression was significantly reduced in PSCs+ADSCs compared with that in PSC-only cultures, while expression of fibrinolytic proteins (e.g., MMP-2 and MMP-9) was up-regulated and anti-fibrinolytic protein (TIMP-1) was down-regulated. In addition, NGF expression was up-regulated, but IL-10 and TGF-beta1 expressions were down-regulated in the co-culture conditioned medium compared with those in the PSC-only culture medium. This study provided an easy and reliable technique to isolate PSCs. The data demonstrated the inhibitory effects of ADSCs on the activation and proliferation of PSCs in vitro.

Detrimental effects of nicotine on thioacetamide-induced liver injury in mice.

PubMed

Zhou, Zixiong; Park, Surim; Kim, Jong Won; Zhao, Jing; Lee, Moo-Yeol; Choi, Kyung Chul; Lim, Chae Woong; Kim, Bumseok

2017-09-01

Nicotine exerts a number of physiological effects. The purpose of this study was to determine the effects of nicotine on thioacetamide (TAA)-induced liver fibrosis in mice. For in vivo experiments, hepatic fibrosis was induced by TAA (0.25 g/kg, i.p.) three times a week for 6 weeks. Mice of TAA treated groups were administered daily with distilled water and nicotine (50 or 100 μg/mL) via gastrogavage throughout the experimental period. For in vitro experiments, HepG2 (human liver cancer cell line) and LX-2 (human hepatic stellate cell line) were used to determine oxidative stress and fibrosis, respectively. Compared to control groups, TAA treated groups had significantly differences in serum alanine transferase and aspartate aminotransferase levels and nicotine accentuated liver injury. Moreover, nicotine increased the mRNA levels of TAA-induced transforming growth factor-β (TGF-β) and collagen type I alpha 1 in the liver. Nicotine also increased TAA-induced oxidative stress. Histological examination confirmed that nicotine aggravated the degree of fibrosis caused by TAA treatment. Additionally, nicotine enhanced hepatic stellate cell activation via promoting the expression of α-smooth muscle actin. Oral administration of nicotine significantly aggravated TAA-induced hepatic fibrosis in mice through enhancing TGF-β secretion and TAA-induced oxidative stress. The increase in TGF-β levels might be associated with the strengthening of oxidative processes, subsequently leading to increased hepatic stellate cell activation and extracellular matrix deposition. These results suggest that patients with liver disease should be advised to abandon smoking since nicotine may exacerbate hepatic fibrosis.

Developmental regulation by cytokines of bone marrow-derived dendritic cells and epidermal Langerhans cells.

PubMed

Yamaguchi, Y

1998-01-01

Dendritic cells (DC) are specialized antigen-presenting cells involved in T cell-mediated immune responses. Differentiation and functional maturation of the DC are now known to be regulated by various cytokines, including TGF-beta1. The experiments of this study examined the effect of other cytokines, such as IL-4, IL-10 and IL-6, on the differentiation and maturation of bone marrow (BM)-derived DC (BM-DC) and epidermal Langerhans cells (LC). When IL-6 or IL-10 was added to cultures of BM cells in the presence of GM-CSF, both cytokines, as in the case of TGF-beta1, suppressed the maturation of DC in terms of the expression of adhesion and costimulatory molecules and T cell-stimulating activity. In contrast, IL-4 was not suppressive but rather supportive for the differentiation of DC. However, these suppressive cytokines hardly counteracted the maturation-inducing activity of TNF-alpha when added to cultures of immature DC. In addition, they appeared to block the overmaturation of DC, which is characterized by a loss of MHC class II molecules. Regarding LC maturation in epidermal cell cultures, IL-6 and IL-10 were inhibitory for the expression of CD86 and CD80 in a dose-dependent fashion. Unlike BM-DC, LC maturation was slightly enhanced by TGF-beta1. The protein antigen-presentation by LC to Th1 clone was not affected by IL-6, but slightly reduced by IL-10. These results suggest that each cytokine contributes to regulate the differentiation and maturation of DC at a different developmental stage.

Inhibition of HSP70 and a Collagen-Specific Molecular Chaperone (HSP47) Expression in Rat Osteoblasts by Microgravity

NASA Technical Reports Server (NTRS)

Kumei, Yasuhiro; Morita, Sadao; Shimokawa, Hitoyata; Ohya, Kei'ichi; Akiyama, Hideo; Hirano, Masahiko; Sams, Clarence F.; Whitson, Peggy A.

2003-01-01

Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

Peptidome Analysis Reveals Novel Serum Biomarkers for Children with Autism Spectrum Disorder in China.

PubMed

Yang, Juan; Chen, Yanni; Xiong, Xiaofan; Zhou, Xiaobo; Han, Lin; Ni, Ei; Wang, Wenjing; Wang, Xiaofei; Zhao, Lingyu; Shao, Dongdong; Huang, Chen

2018-05-13

Autism spectrum disorder (ASD) is a neurological and developmental disorder that begins early in childhood and lasts throughout one's life. Early diagnosis is essential for ASD since early treatment can enable children with ASD to make significant gains in language and social skills, but remains challenging since there are currently no specific biomarkers of ASD. The study aimed to identify serum biomarkers for ASD. Serum of Han Chinese children with ASD (n = 68) and age-matched healthy controls (n = 80) was analyzed using magnetic bead-based separation combined with mass spectrum. Eight potential ASD serum biomarker peaks (m/z: 3886.69, 7775.12, 2381.71, 6638.63, 3319.17, 894.34, 4968.59, and 5910.53) with higher expression in ASD group were further identified as peptide regions of Plasma Serine Protease Inhibitor Precursor (SERPINA5), Platelet Factor 4 (PF4), Fatty Acid Binding Protein 1(FABP1), Apolipoprotein C-I Precursor (APOC1), Alpha-fetoprotein Precursor (AFP), Carboxypeptidase B2 (CPB2), Trace Amine-associated Receptor 6 (TAAR6) and Isoform1 of Fibrinogen Alpha Chain Precursor (FGA). The expression of identified proteins was validated by enzyme-linked immunosorbent assay (ELISA). Our findings reveal the exceptional disease etiology of ASD from a serum proteomic perspective, and the identified proteins might be potential biomarkers for ASD diagnosis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Nilotinib ameliorates lipopolysaccharide-induced acute lung injury in rats

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

El-Agamy, Dina S., E-mail: dinaagamy1@yahoo.com

The present study aimed to investigate the effect of the new tyrosine kinase inhibitor, nilotinib on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats and explore its possible mechanisms. Male Sprague-Dawley rats were given nilotinib (10 mg/kg) by oral gavage twice daily for 1 week prior to exposure to aerosolized LPS. At 24 h after LPS exposure, bronchoalveolar lavage fluid (BALF) samples and lung tissue were collected. The lung wet/dry weight (W/D) ratio, protein level and the number of inflammatory cells in the BALF were determined. Optical microscopy was performed to examine the pathological changes in lungs. Malondialdehyde (MDA) content,more » superoxidase dismutase (SOD) and reduced glutathione (GSH) activities as well as nitrite/nitrate (NO{sub 2}{sup -}/NO{sub 3}{sup -}) levels were measured in lung tissues. The expression of inflammatory cytokines, tumor necrosis factor-{alpha} (TNF-{alpha}), transforming growth factor-{beta}{sub 1} (TGF-{beta}{sub 1}) and inducible nitric oxide synthase (iNOS) were determined in lung tissues. Treatment with nilotinib prior to LPS exposure significantly attenuated the LPS-induced pulmonary edema, as it significantly decreased lung W/D ratio, protein concentration and the accumulation of the inflammatory cells in the BALF. This was supported by the histopathological examination which revealed marked attenuation of LPS-induced ALI in nilotinib treated rats. In addition, nilotinib significantly increased SOD and GSH activities with significant decrease in MDA content in the lung. Nilotinib also reduced LPS mediated overproduction of pulmonary NO{sub 2}{sup -}/NO{sub 3}{sup -} levels. Importantly, nilotinib caused down-regulation of the inflammatory cytokines TNF-{alpha}, TGF-{beta}{sub 1} and iNOS levels in the lung. Taken together, these results demonstrate the protective effects of nilotinib against the LPS-induced ALI. This effect can be attributed to nilotinib ability to counteract the inflammatory cells infiltration and hence ROS generation and regulate cytokine effects. - Research highlights: > The protective effects of nilotinib against LPS-induced ALI in rats were studied. > Nilotinib showed potent anti-inflammatory activity as it attenuated PMN infiltration and hence ROS generation. > In addition, nilotinib caused down-regulation of proinflammatory cytokine production.« less

Preterm human milk contains a large pool of latent TGF-β, which can be activated by exogenous neuraminidase

PubMed Central

Namachivayam, Kopperuncholan; Blanco, Cynthia L.; Frost, Brandy L.; Reeves, Aaron A.; Jagadeeswaran, Ramasamy; MohanKumar, Krishnan; Safarulla, Azif; Mandal, Partha; Garzon, Steven A.; Raj, J. Usha

2013-01-01

Human milk contains substantial amounts of transforming growth factor (TGF)-β, particularly the isoform TGF-β2. We previously showed in preclinical models that enterally administered TGF-β2 can protect against necrotizing enterocolitis (NEC), an inflammatory bowel necrosis of premature infants. In this study we hypothesized that premature infants remain at higher risk of NEC than full-term infants, even when they receive their own mother's milk, because preterm human milk contains less bioactive TGF-β than full-term milk. Our objective was to compare TGF-β bioactivity in preterm vs. full-term milk and identify factors that activate milk-borne TGF-β. Mothers who delivered between 23 0/7 and 31 6/7 wk or at ≥37 wk of gestation provided milk samples at serial time points. TGF-β bioactivity and NF-κB signaling were measured using specific reporter cells and in murine intestinal tissue explants. TGF-β1, TGF-β2, TGF-β3, and various TGF-β activators were measured by real-time PCR, enzyme immunoassays, or established enzymatic activity assays. Preterm human milk showed minimal TGF-β bioactivity in the native state but contained a large pool of latent TGF-β. TGF-β2 was the predominant isoform of TGF-β in preterm milk. Using a combination of several in vitro and ex vivo models, we show that neuraminidase is a key regulator of TGF-β bioactivity in human milk. Finally, we show that addition of bacterial neuraminidase to preterm human milk increased TGF-β bioactivity. Preterm milk contains large quantities of TGF-β, but most of it is in an inactive state. Addition of neuraminidase can increase TGF-β bioactivity in preterm milk and enhance its anti-inflammatory effects. PMID:23558011

Nutritional regulation of hepatic heme biosynthesis and porphyria through PGC-1alpha.

PubMed

Handschin, Christoph; Lin, Jiandie; Rhee, James; Peyer, Anne-Kathrin; Chin, Sherry; Wu, Pei-Hsuan; Meyer, Urs A; Spiegelman, Bruce M

2005-08-26

Inducible hepatic porphyrias are inherited genetic disorders of enzymes of heme biosynthesis. The main clinical manifestations are acute attacks of neuropsychiatric symptoms frequently precipitated by drugs, hormones, or fasting, associated with increased urinary excretion of delta-aminolevulinic acid (ALA). Acute attacks are treated by heme infusion and glucose administration, but the mechanisms underlying the precipitating effects of fasting and the beneficial effects of glucose are unknown. We show that the rate-limiting enzyme in hepatic heme biosynthesis, 5-aminolevulinate synthase (ALAS-1), is regulated by the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). Elevation of PGC-1alpha in mice via adenoviral vectors increases the levels of heme precursors in vivo as observed in acute attacks. The induction of ALAS-1 by fasting is lost in liver-specific PGC-1alpha knockout animals, as is the ability of porphyrogenic drugs to dysregulate heme biosynthesis. These data show that PGC-1alpha links nutritional status to heme biosynthesis and acute hepatic porphyria.

Progressive pulmonary fibrosis is mediated by TGF-β isoform 1 but not TGF-β3

PubMed Central

Ask, Kjetil; Bonniaud, Philippe; Maass, Katja; Eickelberg, Oliver; Margetts, Peter J; Warburton, David; Groffen, John; Gauldie, Jack; Kolb, Martin

2008-01-01

Tissue repair is a well orchestrated biological process involving numerous soluble mediators, and an imbalance between these factors may result in impaired repair and fibrosis. Transforming growth factor (TGF) β is a key profibrotic element in this process and it is thought that its three isoforms act in a similar way. Here, we report that TGF-β3 administered to rat lungs using transient overexpression initiates profibrotic effects similar to those elicited by TGF-β1, but causes less severe and progressive changes. The data suggest that TGF-β3 does not lead to inhibition of matrix degradation in the same way as TGF-β1, resulting in non-fibrotic tissue repair. Further, TGF-β3 is able to downregulate TGF-β1 induced gene expression, suggesting a regulatory role of TGF-β3. TGF-β3 overexpression results in an upregulation of Smad proteins similar to TGF-β1, but is less efficient in inducing the ALK 5 and TGF-β type II receptor (TβRII). We provide evidence that this difference may contribute to the progressive nature of TGF-β1 induced fibrotic response, in contrast to the limited fibrosis observed following TGF-β3 overexpression. TGF-β3 is important in “normal wound healing”, but is outbalanced by TGF-β1 in “fibrotic wound healing” in the lung. PMID:17931953

Methods for synthesizing alane without the formation of adducts and free of halides

DOEpatents

Zidan, Ragaiy; Knight, Douglas A; Dinh, Long V

2013-02-19

A process is provided to synthesize an alane without the formation of alane adducts as a precursor. The resulting product is a crystallized .alpha.-alane and is a highly stable product and is free of halides.

Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair.

PubMed

Kim, Han-Seop; Lee, Jungwoon; Lee, Da Yong; Kim, Young-Dae; Kim, Jae Yun; Lim, Hyung Jin; Lim, Sungmin; Cho, Yee Sook

2017-06-06

Schwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSCs), using combined sequential treatment with inhibitors of the TGF-β and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Heparan sulfates and the decrease of N-glycans promote early adipogenic differentiation rather than myogenesis of murine myogenic progenitor cells.

PubMed

Grassot, Vincent; Bouchatal, Amel; Da Silva, Anne; Chantepie, Sandrine; Papy-Garcia, Dulce; Maftah, Abderrahman; Gallet, Paul-François; Petit, Jean-Michel

In vitro, extracted muscle satellite cells, called myogenic progenitor cells, can differentiate either in myotubes or preadipocytes, depending on environmental factors and the medium. Transcriptomic analyses on glycosylation genes during satellite cells differentiation into myotubes showed that 31 genes present a significant variation of expression at the early stages of murine myogenic progenitor cells (MPC) differentiation. In the present study, we analyzed the expression of 383 glycosylation related genes during murine MPC differentiation into preadipocytes and compared the data to those previously obtained during their differentiation into myotubes. Fifty-six glycosylation related genes are specifically modified in their expression during early adipogenesis. The variations correspond mainly to: a decrease of N-glycans, and of alpha (2,3) and (2,6) linked sialic acids, and to a high level of heparan sulfates. A high amount of TGF-β1 in extracellular media during early adipogenesis was also observed. It seems that the increases of heparan sulfates and TGF-β1 favor pre-adipogenic differentition of MPC and possibly prevent their myogenic differentiation. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

The effects of dexamethasone, ascorbic acid, and β-glycerophosphate on osteoblastic differentiation by regulating estrogen receptor and osteopontin expression.

PubMed

Park, Jun-Beom

2012-03-01

Ascorbic acid (AA), β-glycerophosphate (GP), and dexamethasone (DEX) are the compounds known to favor the expression of the osteoblastic phenotype in several bone cell systems. In this report, the combination effects of differentiation agents on osteoprecursor cells were evaluated. The effect on cell proliferation was determined by a cell viability test with morphologic analysis. Differentiation and mineralization were evaluated using an alkaline phosphatase activity test and alizarin red-S staining. Protein expressions related to bone formation, such as transforming growth factor-beta (TGF-β), estrogen receptor-alpha (ER-α), and osteopontin (OPN) were evaluated by using a Western blot analysis. AA and GP provided an inductive effect for differentiation of osteoprecusor cells, while short-term application of DEX seemed to lead to a dose-dependent increase of cellular differentiation. Long-term use of DEX seemed to reduce mineralization. These effects may seem to be regulated by the expression of ER-α, OPN, and TGF-β. Further studies related to this mechanism within the in vivo model may be necessary to ascertain greater detail. Copyright © 2012 Elsevier Inc. All rights reserved.

Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

PubMed

He, Ying-Ying; Lee, Wei-Hui; Zhang, Yun

2004-09-01

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice.

Effect of alpha-tocopherol, pyridoxine and dexpanthenol on the stress in crease of nonesterified fatty acids levels in the brain.

PubMed

Chmela, Z; Sklenovský, A; Dostálová, K; Rypka, M

1993-01-01

The supposed antistress effect of vitamins-alpha-tocopherol, pyridoxine and dexpanthenol (pantothenic acid precursor)--was followed on the model of nociceptive stress in laboratory rats. The decrease of the stress enhancement of nonesterified fatty acids (NEFA), estimated in the brain cortex, hypothalamus and the brain stem, was taken for the indicator of the antistress effect. Nonesterified fatty acids were determined with the help of gas chromatography following the separation performed by thin layer chromatographic method. Five-day application of alpha-tocopherol acetate (per os, 300 mg.kg-1) led to a decrease of the stress enhancement of arachidonic acid level in the brain stem.

Saponification of esters of chiral alpha-amino acids anchored through their amine function on solid support.

PubMed

Cantel, Sonia; Desgranges, Stéphane; Martinez, Jean; Fehrentz, Jean-Alain

2004-06-01

Anchoring an alpha-amino acid residue by its amine function onto a solid support is an alternative to develop chemistry on its carboxylic function. This strategy can involve the use of amino-acid esters as precursors of the carboxylic function. A complete study on the Wang-resin was performed to determine the non racemizing saponification conditions of anchored alpha-amino esters. The use of LiOH, NaOH, NaOSi(Me)3, various solvents and temperatures were tested for this reaction. After saponification and cleavage from the support, samples were examined through their Marfey's derivatives by reversed phase HPLC to evaluate the percentage of racemization.

Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing HPV-16 E6/E7 is Related to Reduced Macrophage Inflammatory Protein-3Alpha Production

DTIC Science & Technology

2005-01-01

high-risk human papillomavirus ( HPV ) types, particularly type 16 and 18, contributes to 90% of cervical cancer cases. HPV infects cutaneous or mucosal...been implicated in cervical/ anogenital cancer and oral squamous cell carcinomas (41). The mucosal lesions caused by HPVs often resolve over time, and a...Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing HPV -16 E6/E7 is Related to Reduced Macrophage

The MAP kinase pathway is involved in odontoblast stimulation via p38 phosphorylation.

PubMed

Simon, Stephane; Smith, Anthony J; Berdal, Ariane; Lumley, Philip J; Cooper, Paul R

2010-02-01

We have previously shown that the p38 gene is highly expressed in odontoblasts during active primary dentinogenesis, but is drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on these observations, we hypothesized that p38 expression might be upregulated, and the protein activated by phosphorylation, when odontoblasts are stimulated such as during tertiary reactionary dentinogenesis. We stimulated immortalized, odontoblast-like MDPC-23 cells, alone or in combination, with heat-inactivated Streptococcus mutans, EDTA-extracted dentine matrix proteins (DMPs), or growth factors, including transforming growth factor (TGF)-beta1, tumor necrosis factor-alpha (TNF-alpha), and adrenomedullin (ADM). We used ELISA to measure the resulting phosphorylation of the p38 protein, as well as its degree of nuclear translocation. Our results suggest that the p38-MAPKinase pathway is activated during odontoblast stimulation in tertiary dentinogenesis by both p38 phosphorylation and enhanced nuclear translocation. Data indicate that odontoblast behaviour therefore potentially recapitulates that during active primary dentinogenesis. Copyright 2010 American Association of Endodontists. All rights reserved.

Monoamine oxidase and head-twitch response in mice. Mechanisms of alpha-methylated substrate derivatives.

PubMed

Nakagawasai, Osamu; Arai, Yuichiro; Satoh, Shin-etsu; Satoh, Nobunori; Neda, Mitsuro; Hozumi, Masato; Oka, Ryusho; Hiraga, Hajime; Tadano, Takeshi

2004-01-01

It is well known that head-twitch response (HTR) in mice represents hallucinations, since administration of lysergic acid diethylamide (LSD) produces hallucinations in humans, and the HTR in mice is induced by administration of LSD as a hallucinogen. The HTR is produced by excitation of 5-hydroxytryptamine (5-HT)2A receptors. In this paper, we review the mechanisms of HTR induced by various drugs such as 5-HT precursor, 5-HT receptor agonist, 5-HT releaser, hallucinogenic compounds, benzodiazepins and cannabinoid. The response induced by HTR-inducers is significantly enhanced by combined treatment with a non-selective form of monoamine oxidase (MAO) inhibitor. Thus, the relationship between MAO activity and HTR caused by these drugs (especially, alpha-methylated analogous compounds which 5-fluoro-alpha-methyltryptamine, 6-fluoro-alpha-methyltryptamine and p-hydroxyamphetamine) is presented in detail.

Loss of c-myc repression coincides with ovarian cancer resistance to transforming growth factor beta growth arrest independent of transforming growth factor beta/Smad signaling.

PubMed

Baldwin, Rae Lynn; Tran, Hang; Karlan, Beth Y

2003-03-15

Many epithelial carcinomas, including ovarian, are refractory to the antiproliferative effects of transforming growth factor (TGF) beta. In some cancers, TGF-beta resistance has been linked to TGF-beta receptor II (TbetaR-II) and Smad4 mutations; however, in ovarian cancer, the mechanism of resistance remains unclear. Primary ovarian epithelial cell cultures were used as a model system to determine the mechanisms of TGF-beta resistance. To simulate in vivo responses to TGF-beta, primary cultures derived from normal human ovarian surface epithelium (HOSE) and from ovarian carcinomas (CSOC) were grown on collagen I gel, the predominant matrix molecule in the ovarian tumor milieu. When treated with 5 ng/ml TGF-beta for 72 h, HOSE (n = 11) proliferation was inhibited by 20 +/- 21% on average. In contrast, CSOC (n = 10) proliferation was stimulated 5 +/- 10% in response to TGF-beta (a statistically significant difference in response when compared with HOSE; P = 0.001). To dissect the TGF-beta/Smad signaling pathway we used a quantitative RNase protection assay (RPA) for measuring mRNA levels of TGF-beta pathway components in 20 HOSE and 20 CSOC cultures. Basal mRNA levels of TGF-beta receptors I and II, downstream signaling components Smad2, 3, 4, 6, 7, and the transcriptional corepressors Ski and SnoN did not show a statistically significant difference between HOSE and CSOC, and cannot explain their differential susceptibility to TGF-beta-induced cell cycle arrest. To assess functional differences of the TGF-beta pathway in TGF-beta-sensitive HOSE and TGF-beta-resistant CSOC, we measured Smad2/4 and 3/4 complex induction after TGF-beta treatment. HOSE and CSOC showed equivalent Smad2/4 and 3/4 complex induction after TGF-beta exposure for 0, 0.5, 2, and 4 h. It has been proposed that SnoN and Ski are corepressors of the TGF-beta/Smad pathway and undergo TGF-beta-induced degradation followed by reinduction of SnoN mRNA. However, our data show equivalent SnoN degradation in HOSE and CSOC, and equivalent SnoN mRNA induction after TGF-beta treatment. Surprising, TGF-beta-induced Ski degradation was not observed in HOSE or CSOC, suggesting that Ski may not function as a TGF-beta/Smad corepressor in ovarian epithelial cells. These data implied that the TGF-beta/Smad pathway remains functional in CSOC, although CSOC cells are resistant to antimitogenic TGF-beta effects. CSOC resistance to TGF-beta coincided with the loss of c-myc down-regulation. These data suggest that TGF-beta/Smad signaling is blocked downstream of Smad complex formation or that an alternate signaling pathway other than TGF-beta/Smad may transmit TGF-beta-induced cell cycle arrest in the ovarian epithelium.

An Anacardiaceae preparation reduces the expression of inflammation-related genes in murine macrophages.

PubMed

Leiro, J; García, D; Arranz, J A; Delgado, R; Sanmartín, M L; Orallo, F

2004-08-01

This study investigated the effects of an aqueous extract of the stem bark of Mangifera indica L. (Anacardiaceae; Vimang), which contains a defined mixture of components including polyphenols (principally mangiferin, MA), triterpenes, phytosteroids, fatty acids and microelements, on expression of inflammation mediators in inflammatory murine macrophages after stimulation in vitro with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). In vitro treatment with Vimang at 4 microg/ml reduced levels of NOS-2 mRNA and NOS-2, while treatment at 40 microg/ml also reduced levels of COX-2 mRNA, COX-2, and prostaglandin E2 (PGE2). Results suggested that MA is involved in these effects. In vitro treatment with Vimang at 40 microg/ml also inhibited mRNA levels of the proinflammatory cytokines interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha) and colony-stimulating factor (GM-CSF), but did not affect mRNA levels of IL-6 or tumor growth factor-beta (TGF-beta). Extracellular release of TNF-alpha by inflammatory macrophages was inhibited by in vitro treatment with Vimang at the same concentrations that showed inhibition of TNF-alpha mRNA levels. The inhibition of TNF-alpha production appears to be at least partially attributable to MA. Vimang at 4 microg/ml decreased mRNA levels of nuclear factor-kappaB (NF-kappaB) but did not affect expression of the NF-kappaB inhibitor (IkappaB). These data indicate that the potent anti-inflammatory effects of Vimang are due to selective modulation of the expression of inflammation-related genes, leading to attenuation of macrophage activation.

THERMODYNAMIC MODELING OF LIQUID AEROSOLS CONTAINING DISSOLVED ORGANICS AND ELECTROLYTES

EPA Science Inventory

Many tropospheric aerosols contain large fractions of soluble organic material, believed to derive from the oxidation of precursors such alpha-pinene. The chemical composition of aerosol organic matter is complex and not yet fully understood.

The key properties of solu...

The spark discharge synthesis of amino acids from various hydrocarbons

NASA Technical Reports Server (NTRS)

Ring, D.; Miller, S. L.

1984-01-01

The spark discharge synthesis of amino acids using an atmosphere of CH4+N2+H2O+NH3 has been investigated with variable pNH3. The amino acids produced using higher hydrocarbons (ethane, ethylene, acetylene, propane, butane, and isobutane) instead of CH4 were also investigated. There was considerable range in the absolute yields of amino acids, but the yields relative to glycine (or alpha-amino-n-butyric acid) were more uniform. The relative yields of the C3 to C6 aliphatic alpha-amino acids are nearly the same (with a few exceptions) with all the hydrocarbons. The glycine yields are more variable. The precursors to the C3-C6 aliphatic amino acids seem to be produced in the same process, which is separate from the synthesis of glycine precursors. It may be possible to use these relative yields as a signature for a spark discharge synthesis provided corrections can be made for subsequent decomposition events (e.g. in the Murchison meteorite).

Hydrothermal synthesis of porous triphasic hydroxyapatite/(alpha and beta) tricalcium phosphate.

PubMed

Vani, R; Girija, E K; Elayaraja, K; Prakash Parthiban, S; Kesavamoorthy, R; Narayana Kalkura, S

2009-12-01

A novel, porous triphasic calcium phosphate composed of nonresorbable hydroxyapatite (HAp) and resorbable tricalcium phosphate (alpha- and beta-TCP) has been synthesized hydrothermally at a relatively low temperature. The calcium phosphate precursor for hydrothermal treatment was prepared by gel method in the presence of ascorbic acid. XRD, FT-IR, Raman analyses confirmed the presence of HAp/TCP. The surface area and average pore size of the samples were found to be 28 m2/g and 20 nm, respectively. The samples were found to be bioactive in simulated body fluid (SBF).

Bone sialoprotein stimulates focal adhesion-related signaling pathways: role in migration and survival of breast and prostate cancer cells.

PubMed

Gordon, Jonathan A R; Sodek, Jaro; Hunter, Graeme K; Goldberg, Harvey A

2009-08-15

Bone sialoprotein (BSP) is a secreted glycoprotein found in mineralized tissues however, BSP is aberrantly expressed in a variety of osteotropic tumors. Elevated BSP expression in breast and prostate primary carcinomas is directly correlated with increased bone metastases and tumor progression. In this study, the intracellular signaling pathways responsible for BSP-induced migration and tumor survival were examined in breast and prostate cancer cells (MDA-MB-231, Hs578T and PC3). Additionally, the effects of exogenous TGF-beta1 and EGF, cytokines associated with tumor metastasis and present in high-levels in the bone microenvironment, were examined in BSP-expressing cancer cells. Expression of BSP but not an integrin-binding mutant (BSP-KAE) in tumor cell lines resulted in increased levels of alpha(v)-containing integrins and number of mature focal adhesions. Adhesion of cells to recombinant BSP or the expression of BSP stimulated focal adhesion kinase and ERK phosphorylation, as well as activated AP-1-family proteins. Activation of these pathways by BSP expression increased the expression of the matrix metalloproteinases MMP-2, MMP-9, and MMP-14. The BSP-mediated activation of the FAK-associated pathway resulted in increased cancer cell invasion in a Matrigel-coated Boyden-chamber assay and increased cell survival upon withdrawal of serum. Addition of EGF or TGF-beta1 to the BSP-expressing cell lines significantly increased ERK phosphorylation, AP-1 activation, MMP-2 expression, cell migration and survival compared to untreated cells expressing BSP. This study thus defines the cooperative mechanisms by which BSP can enhance specific factors associated with a metastatic phenotype in tumor cell lines, an effect that is increased by circulating TGF-beta1 and EGF. (c) 2009 Wiley-Liss, Inc.

Activin receptor-like kinase 5 inhibition reverses impairment of endothelial cell viability by endogenous islet mesenchymal stromal cells.

PubMed

Clarkin, Claire E; King, Aileen J; Dhadda, Paramjeet; Chagastelles, Pedro; Nardi, Nance; Wheeler-Jones, Caroline P; Jones, Peter M

2013-03-01

Following islet transplantation, islet graft revascularization is compromised due to loss of endothelial cells (ECs) during islet culture. TGF-β signaling pathways are essential for vascular homeostasis but their importance for islet EC function is unclear. We have identified a population of multipotent mesenchymal stromal cells (MSCs) within islets and investigated how modulation of TGF-β signaling by these cells influences islet EC viability. Cultured islets exhibited reduced expression of EC markers (VEGFR2, VE-cadherin and CD31), which was associated with diminished but sustained expression of endoglin a marker of both ECs and MSCs. Double fluorescent labeling of islets in situ with the EC marker CD31 disclosed a population of CD31-negative cells which were positive for endoglin. In vitro coculture of microvascular ECs with endoglin-positive, CD31-negative islet MSCs reduced VEGFR2 protein expression, disrupted EC angiogenic behavior, and increased EC detachment. Medium conditioned by islet MSCs significantly decreased EC viability and increased EC caspase 3/7 activity. EC:MSC cocultures showed enhanced Smad2 phosphorylation consistent with altered ALK5 signaling. Pharmacological inhibition of ALK5 activity with SB431542 (SB) improved EC survival upon contact with MSCs, and SB-treated cultured islets retained EC marker expression and sensitivity to exogenous VEGF164 . Thus, endoglin-expressing islet MSCs influence EC ALK5 signaling in vitro, which decreases EC viability, and changes in ALK5 activity in whole cultured islets contribute to islet EC loss. Modifying TGF-β signaling may enable maintenance of islet ECs during islet isolation and thus improve islet graft revascularization post-transplantation. Copyright © 2013 AlphaMed Press.

Silymarin and caffeine combination ameliorates experimentally-induced hepatic fibrosis through down-regulation of LPAR1 expression.

PubMed

Eraky, Salma M; El-Mesery, Mohamed; El-Karef, Amro; Eissa, Laila A; El-Gayar, Amal M

2018-05-01

Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson's trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-β1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-β1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-β1, and CTGF. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Mesenchymal stem cells cancel azoxymethane-induced tumor initiation.

PubMed

Nasuno, Masanao; Arimura, Yoshiaki; Nagaishi, Kanna; Isshiki, Hiroyuki; Onodera, Kei; Nakagaki, Suguru; Watanabe, Shuhei; Idogawa, Masashi; Yamashita, Kentaro; Naishiro, Yasuyoshi; Adachi, Yasushi; Suzuki, Hiromu; Fujimiya, Mineko; Imai, Kohzoh; Shinomura, Yasuhisa

2014-04-01

The role of mesenchymal stem cells (MSCs) in tumorigenesis remains controversial. Therefore, our goal was to determine whether exogenous MSCs possess intrinsic antineoplastic or proneoplastic properties in azoxymethane (AOM)-induced carcinogenesis. Three in vivo models were studied: an AOM/dextran sulfate sodium colitis-associated carcinoma model, an aberrant crypt foci model, and a model to assess the acute apoptotic response of a genotoxic carcinogen (AARGC). We also performed in vitro coculture experiments. As a result, we found that MSCs partially canceled AOM-induced tumor initiation but not tumor promotion. Moreover, MSCs inhibited the AARGC in colonic epithelial cells because of the removal of O(6)-methylguanine (O(6) MeG) adducts through O(6) MeG-DNA methyltransferase activation. Furthermore, MSCs broadly affected the cell-cycle machinery, potentially leading to G1 arrest in vivo. Coculture of IEC-6 rat intestinal cells with MSCs not only arrested the cell cycle at the G1 phase, but also induced apoptosis. The anti-carcinogenetic properties of MSCs in vitro required transforming growth factor (TGF)-β signaling because such properties were completely abrogated by absorption of TGF-β under indirect coculture conditions. MSCs inhibited AOM-induced tumor initiation by preventing the initiating cells from sustaining DNA insults and subsequently inducing G1 arrest in the initiated cells that escaped from the AARGC. Furthermore, tumor initiation perturbed by MSCs might potentially dysregulate WNT and TGF-β-Smad signaling pathways in subsequent tumorigenesis. Obtaining a better understanding of MSC functions in colon carcinogenesis is essential before commencing the broader clinical application of promising MSC-based therapies for cancer-prone patients with inflammatory bowel disease. © AlphaMed Press.

Advanced glycation end products and the progressive course of renal disease.

PubMed

Heidland, A; Sebekova, K; Schinzel, R

2001-10-01

In experimental and human diabetic nephropathy (DN), it has been shown that advanced glycation end products (AGEs), in particular, carboxymethyl-lysine and pentosidine, accumulate with malondialdehyde in glomerular lesions in relation to disease severity and in the presence of an upregulated receptor for AGE (RAGE) in podocytes. Toxic effects of AGEs result from structural and functional alterations in plasma and extracellular matrix (ECM) proteins, in particular, from cross-linking of proteins and interaction of AGEs with their receptors and/or binding proteins. In mesangial and endothelial cells, the AGE-RAGE interaction caused enhanced formation of oxygen radicals with subsequent activation of nuclear factor-kappaB and release of pro-inflammatory cytokines (interleukin-6, tumor necrosis factor-alpha), growth factors (transforming growth factor-beta1 [TGF-beta1], insulin-like growth factor-1), and adhesion molecules (vascular cell adhesion molecule-1, intercellular adhesion molecule-1). In tubular cells, incubation with AGE albumin was followed by stimulation of the mitogen-activating protein (MAP) kinase pathway and its downstream target, the activating protien-1 (AP-1) complex, TGF-beta1 overexpression, enhanced protein kinase C activity, decreased cell proliferation, and impaired protein degradation rate, in part caused by decreased cathepsin activities. The pathogenic relevance of AGEs was further verified by in vivo experiments in euglycemic rats and mice by the parenteral administration of AGE albumin, leading in the glomeruli to TGF-beta1 overproduction, enhanced gene expression of ECM proteins, and morphological lesions similar to those of DN. Evidence for the pathogenic relevance of AGEs in DN also comes from experimental studies in which the formation and/or action of AGEs was modulated by aminoguanidine, OPB-9195, pyridoxamine, soluble RAGEs, serine protease trypsin, and antioxidants, resulting in improved cell and/or renal function.

Linagliptin potentiates the effect of l-dopa on the behavioural, biochemical and immunohistochemical changes in experimentally-induced Parkinsonism: Role of toll-like receptor 4, TGF-β1, NF-κB and glucagon-like peptide 1.

PubMed

Kabel, Ahmed M; Omar, Mohamed S; Alhadhrami, A; Alharthi, Salman S; Alrobaian, Majed M

2018-05-01

Our aim was to assess the effect of different doses of linagliptin with or without l-dopa/Carbidopa on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism in mice. Eighty Balb/c mice were divided into 8 equal groups: Control; MPTP; MPTP + l-dopa/Carbidopa; MPTP + linagliptin 3 mg/kg/day; MPTP + linagliptin 10 mg/kg/day; MPTP + Carboxymethyl cellulose; MPTP + l-dopa/Carbidopa + linagliptin 3 mg/kg/day and MPTP + l-dopa/Carbidopa + linagliptin 10 mg/kg/day. Striatal dopamine, tumor necrosis factor alpha (TNF-α), interleukin 10 (IL-10), transforming growth factor beta 1 (TGF-β1), toll-like receptor 4 (TLR4), antioxidant enzymes, adenosine triphosphate (ATP), glucagon-like peptide-1 (GLP-1), receptors of advanced glycation end products (RAGE), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase-1 (HO-1), mitochondrial complex I activity, catalepsy and total swim scores were measured. Also, the substantia nigra was subjected to immunohistochemical examination. The combination of l-dopa/Carbidopa and linagliptin in a dose-dependent manner resulted in significant improvement of the behavioural changes, striatal dopamine, antioxidant parameters, Nrf2/HO-1 content, GLP-1, ATP and mitochondrial complex I activity with significant decrease in striatal RAGE, TGF-β1, TNF-α, IL-10, TLR4 and alleviated the immunohistochemical changes better than the groups that received either l-dopa/Carbidopa or linagliptin alone. The combination of l-dopa/Carbidopa and linagliptin might represent a promising therapeutic modality for management of parkinsonism. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparison between tocotrienol and omeprazole on gastric growth factors in stress-exposed rats.

PubMed

Nur Azlina, Mohd Fahami; Qodriyah, Hj Mohd Saad; Chua, Kien Hui; Kamisah, Yusof

2017-08-28

To investigate and compare the effects of tocotrienol and omeprazole on gastric growth factors in rats exposed to water-immersion restraint stress (WIRS). Twenty-eight male Wistar rats were randomly assigned to four groups of seven rats. The two control groups were administered vitamin-free palm oil (vehicle) and the two treatment groups were given omeprazole (20 mg/kg) or tocotrienol (60 mg/kg) by oral gavage. After 28 d of treatment, rats from one control group and both treated groups were subjected to WIRS one time for 3.5 h. Gastric lesions were measured and gastric tissues were obtained to measure vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor-alpha (TGF-α) mRNA expression. Rats exposed to WIRS for 3.5 h demonstrated the presence of considerable ulcers in the form of gastric erosion. The lesion index in the stressed control (S) group was increased ( P < 0.001) compared to the tocotrienol treated and omeprazole treated groups. Stress led to a decrease in gastric VEGF ( P < 0.001), bFGF ( P < 0.001) and TGF-α ( P < 0.001) mRNA levels and caused an increase in EGF mRNA ( P < 0.001) that was statistically significant compared to the non-stressed control group. Although both treatment agents exerted similar ulcer reducing ability, only treatment with tocotrienol led to increased expression of VEGF ( P = 0.008), bFGF ( P = 0.001) and TGF-α ( P = 0.002) mRNA. Tocotrienol provides gastroprotective effects in WIRS-induced ulcers. Compared to omeprazole, tocotrienol exerts a similar protective effect, albeit through multiple mechanisms of protection, particularly through up-regulation of growth factors that assist in repair of gastric tissue injuries.

Secretomes reveal several novel proteins as well as TGF-β1 as the top upstream regulator of metastatic process in breast cancer.

PubMed

Erin, Nuray; Ogan, Nur; Yerlikaya, Azmi

2018-03-20

Metastatic breast cancer is resistant to many conventional treatments and novel therapeutic targets are needed. We previously isolated subsets of 4T1 murine breast cancer cells which metastasized to liver (4TLM), brain (4TBM), and heart (4THM). Among these cells, 4TLM is the most aggressive one, demonstrating mesenchymal phenotype. Here we compared secreted proteins from 4TLM, 4TBM, and 4THM cells and compared with that of hardly metastatic 67NR cells to detect differentially secreted factors involved in organ-specific metastasis. Label-free LC-MS/MS proteomic technique was used to detect the differentially secreted proteins. Eighty-five of over 500 secreted proteins were significantly altered in metastatic breast cancer cells. Differential expression of several proteins such as fibulin-4, Bone Morphogenetic Protein 1, TGF-β1 MMP-3, MMP-9, and Thymic Stromal Lymphopoietin were further verified using ELISA or Western blotting. Many of these identified proteins were also present in human metastatic breast carcinomas. Annexin A1 and A5, laminin beta 1, Neutral alpha-glucosidase AB were commonly found at least in three out of six studies examined here. Ingenuity Pathway Analysis showed that proteins differentially secreted from metastatic cells are involved primarily in carcinogenesis and TGF-β1 is the top upstream regulator in all metastatic cells. Cells metastasized to different organs displayed significant differences in several of secreted proteins. Proteins differentially altered were fibronectin, insulin-like growth factor-binding protein 7, and Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1. On the other hand, many exosomal proteins were also common to all metastatic cells, demonstrating involvement of key universal factors in distant metastatic process.

Peripheral blood mononuclear cells from patients with rheumatoid arthritis spontaneously secrete vascular endothelial growth factor (VEGF): specific up-regulation by tumour necrosis factor-alpha (TNF-α) in synovial fluid

PubMed Central

BOTTOMLEY, MJ; WEBB, NJA; WATSON, CJ; HOLT, PJL; FREEMONT, AJ; BRENCHLEY, PEC

1999-01-01

This study was designed to investigate VEGF production from peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) compared with healthy controls and to identify the predominant cellular source in PBMC isolated from RA patients. The regulation of PBMC VEGF production by cytokines and synovial fluid (SF) was studied. PBMC were isolated from RA patients and healthy controls and stimulated with lipopolysaccharide (LPS), IL-1β, IL-4, IL-6, IL-8, IL-10, TNF-α and transforming growth factor-beta (TGF-β) isoforms for varying time points up to 72 h at 37°C/5% CO2. The effect of SF on VEGF secretion by PBMC was also studied. Supernatant VEGF levels were measured using a flt-1 receptor capture ELISA. RA patients had significantly higher spontaneous production of VEGF compared with controls, and monocytes were identified as the predominant cellular source. RA PBMC VEGF production was up-regulated by TGF-β isoforms and TNF-α and down-regulated by IL-4 and IL-10, with no effect observed with IL-1β, IL-6 and IL-8. Antibody blocking experiments confirmed that TNF-α and not TGF-β isoforms in SF increased VEGF secretion by RA PBMC. These results emphasize the importance of monocytes as a source of VEGF in the pathophysiology of RA. Several cytokines known to be present in SF can modulate the level of VEGF secretion, but the predominant effect of SF in VEGF up-regulation is shown to be dependent on TNF-α. PMID:10403932

Grape seed extract ameliorates bleomycin-induced mouse pulmonary fibrosis.

PubMed

Liu, Qi; Jiang, Jun-Xia; Liu, Ya-Nan; Ge, Ling-Tian; Guan, Yan; Zhao, Wei; Jia, Yong-Liang; Dong, Xin-Wei; Sun, Yun; Xie, Qiang-Min

2017-05-05

Pulmonary fibrosis is common in a variety of inflammatory lung diseases, such as interstitial pneumonia, chronic obstructive pulmonary disease, and silicosis. There is currently no effective clinical drug treatment. It has been reported that grape seed extracts (GSE) has extensive pharmacological effects with minimal toxicity. Although it has been found that GSE can improve the lung collagen deposition and fibrosis pathology induced by bleomycin in rat, its effects on pulmonary function, inflammation, growth factors, matrix metalloproteinases and epithelial-mesenchymal transition remain to be researched. In the present study, we studied whether GSE provided protection against bleomycin (BLM)-induced mouse pulmonary fibrosis. ICR strain mice were treated with BLM in order to establish pulmonary fibrosis models. GSE was given daily via intragastric administration for three weeks starting at one day after intratracheal instillation. GSE at 50 or 100mg/kg significantly reduced BLM-induced inflammatory cells infiltration, proinflammatory factor protein expression, and hydroxyproline in lung tissues, and improved pulmonary function in mice. Additionally, treatment with GSE also significantly impaired BLM-induced increases in lung fibrotic marker expression (collagen type I alpha 1 and fibronectin 1) and decreases in an anti-fibrotic marker (E-cadherin). Further investigation indicated that the possible molecular targets of GSE are matrix metalloproteinases-9 (MMP-9) and TGF-β1, given that treatment with GSE significantly prevented BLM-induced increases in MMP-9 and TGF-β1 expression in the lungs. Together, these results suggest that supplementation with GSE may improve the quality of life of lung fibrosis patients by inhibiting MMP-9 and TGF-β1 expression in the lungs. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of [6]-gingerol on myofibroblast differentiation in transforming growth factor beta 1-induced nasal polyp-derived fibroblasts.

PubMed

Park, Sook A; Park, Il-Ho; Cho, Jung-Sun; Moon, You-Mi; Lee, Seung Hoon; Kim, Tae Hoon; Lee, Sang Hag; Lee, Heung-Man

2012-01-01

[6]-Gingerol is one of the major pungent principles of ginger and has diverse effects, including anti-inflammatory, and antioxidative effects. Reactive oxygen species (ROS) are released during the phenotypic transformation of fibroblasts to myofibroblasts, a process that is involved in the growth of nasal polyps by inducing extracellular matrix (ECM) accumulation. The purpose of this study was to determine the effect of [6]-gingerol on myofibroblast differentiation and collagen production of nasal polyp-derived fibroblasts (NPDFs) and to determine if the effect of [6]-gingerol is linked to an antioxidant effect. NPDFs were incubated and treated with transforming growth factor (TGF) beta 1. The ROS generated by NPDFs were determined using 2″,7″-dichlorfluorescein-diacetate. The fluorescence was captured by a fluorescent microscope and measured using a fluorometer. The expression of alpha-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein and pSmad2/3 was determined by immunofluorescence microscopy and or Western blotting. The amount of total soluble collagen production was analyzed by the SirCol collagen dye-binding assay. TGF-beta 1 stimulation increased ROS production by NPDFs. [6]-Gingerol decreased the production of ROS in TGF-beta 1-induced NPDFs. Myofibroblast differentiation, collagen production, and phosphorylation of Smad2/3 were prevented by [6]-gingerol and inhibition of ROS generation with antioxidant such as diphenyliodonium, N-acetylcysteine, and ebselen. These results suggest the possibility that [6]-gingerol may play an important role in inhibiting the production of the ECM in the development of nasal polyps through an antioxidant effect.

DPP and DSP are Necessary for Maintaining TGF-β1 Activity in Dentin

PubMed Central

Yamakoshi, Y.; Kinoshita, S.; Izuhara, L.; Karakida, T.; Fukae, M.; Oida, S.

2014-01-01

Porcine dentin sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentin. It is processed by proteases into 3 independent proteins: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We fractionated DPP and DSP along with TGF-β activity by ion exchange (IE) chromatography from developing pig molars and measured their alkaline phosphatase (ALP)-stimulating activity in human periodontal (HPDL) cells with or without TGF-β receptor inhibitor. We then purified TGF-β-unbound or -bound DPP and DSP by reverse-phase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-β isoform bound to DPP and DSP was identified as being TGF-β1 by both ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-β1 (CF-hTGF-β1) with TGF-β-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-β1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-β1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-β to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-β1. We conclude that both DPP and DSP help retain TGF-β1 activity in porcine dentin. PMID:24799420

Maternal breast milk transforming growth factor beta and feeding intolerance in preterm infants

PubMed Central

Frost, Brandy L.; Jilling, Tamas; Lapin, Brittany; Maheshwari, Akhil; Caplan, Michael S.

2015-01-01

Background Feeding intolerance occurs commonly in the NICU. Breast milk contains a large pool of transforming growth factor-beta (TGF-beta). Few studies describe TGF-beta levels in preterm milk, and the relationship to feeding intolerance (FI) remains unexplored. We measured TGF-beta levels in preterm breast milk to investigate a correlation with FI in preterm infants. Methods Prospective observational trial of 100 mother-infant pairs, enrolling infants born below 32 weeks gestation and less than 1500 grams, and mothers who planned to provide breast milk. TGF-beta levels were measured using ELISA. Infant charts were reviewed for outcomes. Results TGF-beta declined postnatally, most elevated in colostrum (p<0.01). TGF-beta 2 levels were higher than TGF-beta 1 at all time points (p<0.01). Colostrum TGF-beta levels correlated inversely with birth weight (p<0.01) and gestational age (p<0.05). One week TGF-beta 2 levels were reduced in growth-restricted infants with FI (p<0.01). Of infants with NEC, TGF-beta 2 levels appeared low, but small sample size precluded meaningful statistical comparisons. Conclusions TGF-beta levels decline temporally in preterm milk. TGF-beta 1 colostrum levels correlate inversely with birth weight and gestational age. TGF-beta 2 may play a role in FI in growth-restricted infants. The relationship of TGF-beta 2 and NEC merits future investigation. PMID:24995914

Kefir induces cell-cycle arrest and apoptosis in HTLV-1-negative malignant T-lymphocytes

PubMed Central

Maalouf, Katia; Baydoun, Elias; Rizk, Sandra

2011-01-01

Background: Adult lymphoblastic leukemia (ALL) is a malignancy that occurs in white blood cells. The overall cure rate in children is 85%, whereas it is only 40% in adults. Kefir is an important probiotic that contains many bioactive ingredients, which give it unique health benefits. It has been shown to control several cellular types of cancer. Purpose: The present study investigates the effect of a cell-free fraction of kefir on CEM and Jurkat cells, which are human T-lymphotropic virus type I (HTLV-1)-negative malignant T-lymphocytes. Methods: Cells were incubated with different kefir concentrations. The cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir on the proliferation of CEM and Jurkat cells was then assessed. The levels of transforming growth factor-alpha (TGF-α), transforming growth factor- beta1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), and MMP-9 mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, the growth inhibitory effects of kefir on cell-cycle progression/apoptosis were assessed by Cell Death Detection (ELISA) and flow cytometry. Results: The maximum cytotoxicity recorded after 48-hours treatment with 80 μg/μL kefir was only 42% and 39% in CEM and Jurkat cells, respectively. The percent reduction in proliferation was very significant, and was dose-, and time-dependent. In both cell lines, kefir exhibited its antiproliferative effect by downregulating TGF-α and upregulating TGF-β1 mRNA expression. Upon kefir treatment, a marked increase in cell-cycle distribution was noted in the preG1 phase of CEM and Jurkat cells, indicating the proapoptotic effect of kefir, which was further confirmed by Cell Death Detection ELISA. However, kefir did not affect the mRNA expression of metalloproteinases needed for the invasion of leukemic cell lines. Conclusion: In conclusion, kefir is effective in inhibiting proliferation and inducing apoptosis of HTLV-1-negative malignant T-lymphocytes. Therefore, further in vivo investigation is highly recommended. PMID:21448298

TGF-β but not BMP signaling induces prechondrogenic condensation through ATP oscillations during chondrogenesis.

PubMed

Kwon, Hyuck Joon

2012-08-10

Although both TGF-β and BMP signaling enhance expression of adhesion molecules during chondrogenesis, TGF-β but not BMP signaling can initiate condensation of uncondensed mesenchymal cells. However, it remains unclear what causes the differential effects between TGF-β and BMP signaling on prechondrogenic condensation. Our previous report demonstrated that ATP oscillations play a critical role in prechondrogenic condensation. Thus, the current study examined whether ATP oscillations are associated with the differential actions of TGF-β and BMP signaling on prechondrogenic condensation. The result revealed that while both TGF-β1 and BMP2 stimulated chondrogenic differentiation, TGF-β1 but not BMP2 induced prechondrogenic condensation. It was also found that TGF-β1 but not BMP2 induced ATP oscillations and inhibition of TGF-β but not BMP signaling prevented insulin-induced ATP oscillations. Moreover, blockage of ATP oscillations inhibited TGF-β1-induced prechondrogenic condensation. In addition, TGF-β1-driven ATP oscillations and prechondrogenic condensation depended on Ca(2+) influx via voltage-dependent calcium channels. This study suggests that Ca(2+)-driven ATP oscillations mediate TGF-β-induced the initiation step of prechondrogenic condensation and determine the differential effects between TGF-β and BMP signaling on chondrogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

Role of GARP in the activation of latent TGF-β1.

PubMed

Stockis, Julie; Dedobbeleer, Olivier; Lucas, Sophie

2017-09-26

TGF-β1, 2 and 3 cytokines are involved in many cellular processes including cell proliferation, differentiation, migration and survival. Whereas TGF-β2 and 3 play important roles in embryonic development, TGF-β1 is mostly implicated in controlling immune responses after birth. The production of TGF-β1 is a tightly regulated process, occurring mostly at a post-translational level. Virtually all cells produce the latent, inactive form of TGF-β1. In latent TGF-β1, the mature TGF-β1 dimer is non-covalently associated to the Latency Associated Peptide, or LAP, which prevents binding to the TGF-β1 receptor. Activation of the cytokine implies release of mature TGF-β1 from LAP. Only a few cell types activate latent TGF-β1, via mechanisms that are cell type specific. Proteins such as integrins, proteases and thrombospondin-1 activate TGF-β1 in epithelial cells, fibroblasts and dendritic cells. More recently, the protein GARP was shown to be involved in TGF-β1 activation by regulatory T cells (Treg), a subset of CD4 + T lymphocytes specialized in suppression of immune responses. GARP is a transmembrane protein that binds latent-TGF-β1 and tethers it on the Treg surface. The role of GARP was studied mostly in Tregs, and this was recently reviewed in L. Sun, H. Jin and H. Li, Oncotarget, 2016, 7, 42826-42836. However, GARP is also expressed in non-immune cells. This review focuses on the roles of GARP in latent TGF-β1 activation by immune and non-immune cells.

Treatment with unsaponifiable extracts of avocado and soybean increases TGF-beta1 and TGF-beta2 levels in canine joint fluid.

PubMed

Altinel, Levent; Saritas, Z Kadir; Kose, Kamil Cagri; Pamuk, Kamuran; Aksoy, Yusuf; Serteser, Mustafa

2007-02-01

Avocado and soya unsaponifiables (ASU) are plant extracts used as a slow-acting antiarthritic agent. ASU stimulate the synthesis of matrix components by chondrocytes, probably by increasing the production of transforming growth factor-beta (TGF-beta). TGF-beta is expressed by chondrocytes and osteoblasts and is present in cartilage matrix. This study investigates the effect of ASU treatment on the levels of two isoforms of TGFbeta, TGF-beta1 and TGF-beta2, in the knee joint fluid using a canine model. Twenty-four outbred dogs were divided into three groups. The control animals were given a normal diet, while the treated animals were given 300 mg ASU every three days or every day. Joint fluid samples were obtained prior to treatment, and at the end of every month (up to three months). TGF-beta1 and TGF-beta2 levels were measured using a quantitative sandwich enzyme immunoassay technique. ASU treatment caused an increase in TGF-beta1 and TGF-beta2 levels in the joint fluid when compared to controls. The different doses did not cause a significant difference in joint fluid TGF levels. TGF-beta1 levels in the treated animals reached maximum values at the end of the second month and then decreased after the third month, while TGF-beta2 levels showed a marginal increase during the first two months, followed by a marked increase at the end of the third month. In conclusion, ASU increased both TGF-beta1 and TGF-beta2 levels in knee joint fluid.

The effect of CT26 tumor-derived TGF-β on the balance of tumor growth and immunity.

PubMed

Owyang, Stephanie Y; Zhang, Min; Walkup, Grace A; Chen, Grace E; Grasberger, Helmut; El-Zaatari, Mohamad; Kao, John Y

2017-11-01

TGF-β is an important target for many cancer therapies under development. In addition to suppressing anti-tumor immunity, it has pleiotropic direct pro- and anti- tumor effects. The actions of increased endogenous TGF-β production remain unclear, and may affect the outcomes of anti-TGF-β cancer therapy. We hypothesize that tumor-derived TGF-β (td-TGF-β) plays an important role in maintaining tumor remission by controlling tumor proliferation in vivo, and that decreasing td-TGF-β in the tumor microenvironment will result in tumor progression. The aim of this study was to examine the effect of TGF-β in the tumor microenvironment on the balance between its anti-proliferative and immunosuppressive effects. A murine BALB/c spontaneous colon adenocarcinoma cell line (CT26) was genetically engineered to produce increased active TGF-β (CT26-TGF-β), a dominant-negative soluble TGF-β receptor (CT26-TGF-β-R), or the empty neomycin cassette as control (CT26-neo). In vitro proliferation rates were measured. For in vivo studies, the three cell lines were injected into syngeneic BALB/c mice, and tumor growth was measured over time. Immunodeficient BALB/c nude mice were used to investigate the role of T and B cells. In vitro, CT26-TGF-β-R and CT26-TGF-β cells showed increased and suppressed proliferation, respectively, compared to control (CT26-neo), confirming TGF-β has direct anti-tumor effects. In vivo, we found that CT26-TGF-β-R cells displayed slower growth compared to control, likely secondary to reduced suppression of anti-tumor immunity, as this effect was ablated in immunodeficient BALB/c nude mice. However, CT26-TGF-β cells (excess TGF-β) exhibited rapid early growth compared to control, but later failed to progress. The same pattern was shown in immunodeficient BALB/c nude mice, suggesting the effect on tumor growth is direct, with minimal immune system involvement. There was minimal effect on systemic antitumor immunity as determined by peripheral antigen-specific splenocyte type 1 cytokine production and tumor growth rate of CT26-neo on the contralateral flank of the same mice. Although TGF-β has opposing effects on tumor growth, this study showed that excessive td-TGF-β in the tumor microenvironment renders the tumor non-proliferative. Depleting excess td-TGF-β may release this endogenous tumor suppressive mechanism, thus triggering the progression of the tumor. Therefore, our findings support cautions against using anti-TGF-β strategies in treating cancer, as this may tip the balance of anti-immunity vs. anti-tumor effects of TGF-β, leading to tumor progression instead of remission. Copyright © 2017 European Federation of Immunological Societies. All rights reserved.

Asymmetric conjugate 1,4-addition of arylboronic acids to alpha, beta-unsaturated esters catalyzed by Rhodium(I)/(S)-binap

PubMed

Sakuma; Sakai; Itooka; Miyaura

2000-09-22

Arylboronic acids underwent the conjugate 1,4-addition to alpha, beta-unsaturated esters to give beta-aryl esters in high yields in the presence of a rhodium(I) catalyst. The addition of arylboronic acids to isopropyl crotonate resulted in high yields and high enantioselectivity exceeding 90% ee in the presence of 3 mol % of Rh(acac)(C(2)H(4))(2) and (S)-binap at 100 degrees C. The rhodium/(S)-binap complex provided (R)-3-phenylbutanoate in the addition of phenylboronic acid to benzyl crotonate. The effects on the enantioselectivity of chiral phosphine ligands, rhodium precursors, and substituents on alpha,beta-unsaturated esters are discussed, as well as the mechanistic aspect of the catalytic cycle.

Aqueous chemical growth of alpha-Fe2O3-alpha-Cr203 nanocompositethin films

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

Vayssieres, Lionel; Guo, Jinghua; Nordgren, Joseph

2001-06-30

We are reporting here on the inexpensive fabrication and optical properties of an iron(III) oxide chromium(III) oxide nanocomposite thin film of corundum crystal structure. Its novel and unique-designed architecture consists of uniformed, well-defined and oriented nanorods of Hematite (alpha-Fe2O3) of 50 nm in diameter and 500nm in length and homogeneously distributed nonaggregated monodisperse spherical nanoparticles of Eskolaite (alpha-Cr2O3) of 250 nm in diameter. This alpha-Fe2O3 alpha-Cr2O3 nanocomposite thin film is obtained by growing, directly onto transparent polycrystalline conducting substrate, an oriented layer of hematite nanorods and growing subsequently, the eskolaite layer. The synthesis is carried out by a template-free, low-temperature,more » multilayer thin film coating process using aqueous solution of metal salts as precursors. Almost 100 percent of the light is absorbed by the composite film between 300 and 525 nm and 40 percent at 800 nm which yields great expectations as photoanode materials for photovoltaic cells and photocatalytic devices.« less

Cytokines and bullous pemphigoid.

PubMed

D'Auria, L; Cordiali Fei, P; Ameglio, F

1999-06-01

This report reviews the data presented in the literature concerning the presence and levels of different cytokines in sera, lesional tissue or blister fluids of patients with bullous pemphigoid. The list of cytokines analysed includes 21 molecules: interleukins (IL)-1 => 8, IL-10 => 13, IL-15, granulocyte-monocyte-colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), oncostatin-M (OSM), regulated upon activation normal T cell expressed and presumably secreted (RANTES), transforming growth factor-beta 1 (TGF-beta 1), tumor necrosis factor-alpha (TNF-alpha) and vascular endothelial growth factor (VEGF). Basic information regarding the functions of these cytokines and their possible involvement in the pathogenetic steps of the disease, such as autoantigen expression, autoantibody induction, complement activation, local cell recruitment and stimulation, resident cell activation, release of various effector molecules and tissue damage are also reported. A specific function for each cytokine in bullous pemphigoid induction cannot be still defined, however, the literature attributes a major role to IL-1, IL-4, IL-5, IL-6, IL-8 and IFN-gamma. On the basis of significant (direct or inverse) correlations found between disease intensity and the blister fluid/serum levels, the following cytokines IL-7, IL-15, RANTES, VEGF and TNF-alpha, besides those previously mentioned, may also be involved in this disease.

Cytokine pattern in blister fluid and serum of patients with bullous pemphigoid: relationships with disease intensity.

PubMed

Ameglio, F; D'Auria, L; Bonifati, C; Ferraro, C; Mastroianni, A; Giacalone, B

1998-04-01

Few and contrasting data are available in the literature concerning the levels of various cytokines in blister fluid (BF) and in the serum of patients affected with bullous pemphigoid (BP). Using commercially available ELISA kits, this study reports the levels of 11 cytokines detected both in BF and sera of 15 BP patients and compares them with those of 15 control subjects' sera. Generally, no significant differences were observed in BP and control sera. In contrast, interleukin (IL) 1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) showed increased BF levels as compared with BP sera. Two cytokines, IL-11 and IL-12 did not show significant differences between BP BF and sera, while an opposite behaviour was observed for transforming growth factor beta 1 (TGF-beta 1), whose serum levels were higher than the concentrations in BF. Using the number of lesions of the patients as a possible disease intensity marker, significant correlations were found with the BF levels of IL-1 beta, IL-8, TNF-alpha and, most closely, IL-5. These data may have pathogenetic relevance and suggest the possibility that these biological modulators may be used as a quantitative marker of disease intensity.

Chronic aspiration of gastric fluid induces the development of obliterative bronchiolitis in rat lung transplants.

PubMed

Li, B; Hartwig, M G; Appel, J Z; Bush, E L; Balsara, K R; Holzknecht, Z E; Collins, B H; Howell, D N; Parker, W; Lin, S S; Davis, R D

2008-08-01

Long-term survival of a pulmonary allograft is currently hampered by obliterative bronchiolitis (OB), a form of chronic rejection that is unique to lung transplantation. While tracheobronchial aspiration from gastroesophageal reflux disease (GERD) has clinically been associated with OB, no experimental model exists to investigate this problem. Using a WKY-to-F344 rat orthotopic left lung transplant model, the effects of chronic aspiration on pulmonary allograft were evaluated. Recipients received cyclosporine with or without 8 weekly aspirations of gastric fluid into the allograft. Six (66.7%) of 9 allografts with aspiration demonstrated bronchioles with surrounding monocytic infiltrates, fibrosis and loss of normal lumen anatomy, consistent with the development of OB. In contrast, none of the allografts without aspiration (n = 10) demonstrated these findings (p = 0.002). Of the grafts examined grossly, 83% of the allografts with chronic aspiration but only 20% without aspiration appeared consolidated (p = 0.013). Aspiration was associated with increased levels of IL-1 alpha, IL-1 beta, IL-6, IL-10, TNF-alpha and TGF-beta in BAL and of IL-1 alpha, IL-4 and GM-CSF in serum. This study provides experimental evidence linking chronic aspiration to the development of OB and suggests that strategies aimed at preventing aspiration-related injuries might improve outcomes in clinical lung transplantation.

Neutrophil chemotaxis in response to TGF-beta isoforms (TGF-beta 1, TGF-beta 2, TGF-beta 3) is mediated by fibronectin.

PubMed

Parekh, T; Saxena, B; Reibman, J; Cronstein, B N; Gold, L I

1994-03-01

TGF-beta isoforms regulate numerous cellular functions including cell growth and differentiation, the cellular synthesis and secretion of extracellular matrix proteins, such as fibronectin (Fn), and the immune response. We have previously shown that TGF-beta 1 is the most potent chemoattractant described for human peripheral blood neutrophils (PMNs), suggesting that TGF-beta s may play a role in the recruitment of PMNs during the initial phase of the inflammatory response. In our current studies, we demonstrate that the maximal chemotactic response was attained near 40 fM for all mammalian TGF-beta isoforms. However, there was a statistically significant difference in migratory distance of the PMNs: TGF-beta 2 (556 microM) > TGF-beta 3 (463 microM) > TGF-beta 1 (380 microM) (beta 2: beta 3, p < or = 0.010; beta 3: beta 1, p < or = 0.04; beta 2: beta 1, p < or = 0.0012). A mAb to the cell binding domain (CBD) of Fn inhibited the chemotactic response to TGF-beta 1 and TGF-beta 3 by 63% and to TGF-beta 2 by 70%, whereas the response to FMLP, a classic chemoattractant, was only inhibited by 18%. In contrast, a mAb to a C-terminal epitope of Fn did not retard migration (< 1.5%). The Arg-gly-Asp-ser tetrapeptide inhibited chemotaxis by approximately the same extent as the anti-CBD (52 to 83%). Furthermore, a mAb against the VLA-5 integrin (VLA-5; Fn receptor) also inhibited TGF-beta-induced chemotaxis. These results indicate that chemotaxis of PMNs in response to TGF-beta isoforms is mediated by the interaction of the Arg-gly-Asp-ser sequence in the CBD of Fn with an integrin on the PMN cell surface, primarily the VLA-5 integrin. TGF-beta isoforms also elicited the release of cellular Fn from PMNs; we observed a 2.3-fold increase in Fn (389 to 401 ng/ml) in the supernatants of TGF-beta-stimulated PMNs compared with unstimulated cells (173.6 ng/ml). The concentration of TGF-beta required to cause maximal release of Fn from PMNs (4000 fM) is a concentration at which TGF-beta is no longer chemotactic, suggesting that PMNs only use Fn that is constitutively expressed for migration. At higher concentrations of TGF-beta, the Fn released may accumulate basal to the cell, ultimately retarding cellular migration and modulating the chemotactic response.

Membrane protein GARP is a receptor for latent TGF-beta on the surface of activated human Treg.

PubMed

Stockis, Julie; Colau, Didier; Coulie, Pierre G; Lucas, Sophie

2009-12-01

Human Treg and Th clones secrete the latent form of TGF-beta, in which the mature TGF-beta protein is bound to the latency-associated peptide (LAP), and is thereby prevented from binding to the TGF-beta receptor. We previously showed that upon TCR stimulation, human Treg clones but not Th clones produce active TGF-beta and bear LAP on their surface. Here, we show that latent TGF-beta, i.e. both LAP and mature TGF-beta, binds to glycoprotein A repetitions predominant (GARP), a transmembrane protein containing leucine rich repeats, which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF-beta mediated by binding to GARP may be necessary for the ability of Treg to activate TGF-beta upon TCR stimulation. However, it is not sufficient as lentiviral-mediated expression of GARP in human Th cells induces binding of latent TGF-beta to the cell surface, but does not result in the production of active TGF-beta upon stimulation of these Th cells.

Role of T Cell TGF-β Signaling in Intestinal Cytokine Responses and Helminthic Immune Modulation

PubMed Central

Ince, M. Nedim; Elliott, David E.; Setiawan, Tommy; Metwali, Ahmed; Blum, Arthur; Chen, Hung-lin; Urban, Joseph F.; Flavell, Richard A.; Weinstock, Joel V.

2010-01-01

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL-10 or TGF-β that are important in suppressing colitis. Helminths induce mucosal T cell IL-10 secretion and regulate lamina propria mononuclear cell Th1 cytokine generation in an IL-10 dependent manner in wild-type mice. Helminths also stimulate mucosal TGF-β release. As TGF-β exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF-β signaling in helminthic modulation of intestinal immunity. T cell TGF-β signaling is interrupted in TGF-βRII DN mice by T cell-specific over-expression of a dominant negative TGF-β receptor II. We studied lamina propria mononuclear cell responses in wild-type and TGF-βRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF-β signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL-10 secretion requires intact T cell TGF-β signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF-βRII DN mice. Thus, T cell TGF-β signaling is essential for helminthic stimulation of mucosal IL-10 production, helminthic modulation of intestinal interferon-γ generation and H. polygyrus-mediated suppression of chronic colitis. PMID:19544487

Tocopherol production in plant cell cultures.

PubMed

Caretto, Sofia; Nisi, Rossella; Paradiso, Annalisa; De Gara, Laura

2010-05-01

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, essential dietary components for mammals and exclusively synthesized by photosynthetic organisms. Of the four forms (alpha, beta, gamma and delta), alpha-tocopherol is the major vitamin E form present in green plant tissues, and has the highest vitamin E activity. Synthetic alpha-tocopherol, being a racemic mixture of eight different stereoisomers, always results less effective than the natural form (R,R,R) alpha-tocopherol. This raises interest in obtaining this molecule from natural sources, such as plant cell cultures. Plant cell and tissue cultures are able to produce and accumulate valuable metabolites that can be used as food additives, nutraceuticals and pharmaceuticals. Sunflower cell cultures, growing under heterotrophic conditions, were exploited to establish a suitable in vitro production system of natural alpha-tocopherol. Optimization of culture conditions, precursor feeding and elicitor application were used to improve the tocopherol yields of these cultures. Furthermore, these cell cultures were useful to investigate the relationship between alpha-tocopherol biosynthesis and photomixotrophic culture conditions, revealing the possibility to enhance tocopherol production by favouring sunflower cell photosynthetic properties. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.

Cytokine profile in canine immune-mediated polyarthritis and osteoarthritis.

PubMed

Hegemann, N; Wondimu, A; Kohn, B; Brunnberg, L; Schmidt, M F G

2005-01-01

The purpose of this study was to determine the cytokine profile in 21 dogs with canine immune-mediated polyarthritis (IMA) and 15 dogs with osteoarthritis (OA) caused by cranial cruciate ligament rupture (CCLR). The mRNA expression of interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon (IFN)-gamma, transforming growth factor (TGF)-beta, and tumour necrosis factor (TNF)-alpha were analysed in synovial fluid by semi-quantitative RT-PCR, while TNF-alpha protein was determined by L929 cytotoxicity assay. The frequency of lymphocytes was analysed using FACScan. Both disorders reveal a similar cytokine expression pattern, except for significant lower IL-1beta expression in OA. Th1 cytokines IL-2 and IFN-gamma were detected, while IL-4 was nearly absent in IMA and OA. Furthermore, the bioassay demonstrates a significantly higher production of TNF-alpha in synovial fluid of dogs with IMA, compared to dogs with OA (p < 0.05). The frequency of CD4+, CD8+ and MHC class II+ cells was relatively higher in synovial fluids compared to peripheral blood in IMA. These findings reveal that the difference between the cytokine pattern of canine IMA and OA seems to be rather quantitative than qualitative. Both joint disorders show predominance of pro-inflammatory cytokines and absence of TH2 cytokine expression, indicating the potential of IL-4 for a gene therapeutic approach.

Endotoxin-Induced Endothelial Fibrosis Is Dependent on Expression of Transforming Growth Factors β1 and β2

PubMed Central

Echeverría, César; Montorfano, Ignacio; Tapia, Pablo; Riedel, Claudia; Cabello-Verrugio, Claudio

2014-01-01

During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the ECs. We previously reported that endotoxins induce the conversion of ECs into activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their morphology and their protein expression pattern, thereby suppressing endothelial markers and upregulating fibrotic proteins. The most commonly used fibrotic inducers are transforming growth factor β1 (TGF-β1) and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced endothelial fibrosis remains unknown. We have shown that the endotoxin-induced endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of TGF-β1 and TGF-β2. Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the endothelial marker VE-cadherin and in the fibrotic proteins α-SMA and fibronectin. Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis. To the best of our knowledge, this is the first report showing that endotoxin induces endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular dysfunction. These findings contribute to understanding the molecular mechanism of endotoxin-induced endothelial fibrosis, which could be useful in the treatment of inflammatory diseases. PMID:24935972

Endotoxin-induced endothelial fibrosis is dependent on expression of transforming growth factors β1 and β2.

PubMed

Echeverría, César; Montorfano, Ignacio; Tapia, Pablo; Riedel, Claudia; Cabello-Verrugio, Claudio; Simon, Felipe

2014-09-01

During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the ECs. We previously reported that endotoxins induce the conversion of ECs into activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their morphology and their protein expression pattern, thereby suppressing endothelial markers and upregulating fibrotic proteins. The most commonly used fibrotic inducers are transforming growth factor β1 (TGF-β1) and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced endothelial fibrosis remains unknown. We have shown that the endotoxin-induced endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of TGF-β1 and TGF-β2. Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the endothelial marker VE-cadherin and in the fibrotic proteins α-SMA and fibronectin. Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis. To the best of our knowledge, this is the first report showing that endotoxin induces endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular dysfunction. These findings contribute to understanding the molecular mechanism of endotoxin-induced endothelial fibrosis, which could be useful in the treatment of inflammatory diseases. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Smad7 induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis.

PubMed

Halder, Sunil K; Beauchamp, R Daniel; Datta, Pran K

2005-07-01

Smad proteins play a key role in the intracellular signaling of the transforming growth factor beta (TGF-beta) superfamily of extracellular polypeptides that initiate signaling to regulate a wide variety of biological processes. The inhibitory Smad, Smad7, has been shown to function as intracellular antagonists of TGF-beta family signaling and is upregulated in several cancers. To determine the effect of Smad7-mediated blockade of TGF-beta signaling, we have stably expressed Smad7 in a TGF-beta-sensitive, well-differentiated, and non-tumorigenic cell line, FET, that was derived from human colon adenocarcinoma. Smad7 inhibits TGF-beta-induced transcriptional responses by blocking complex formation between Smad 2/3 and Smad4. While Smad7 has no effect on TGF-beta-induced activation of p38 MAPK and ERK, it blocks the phosphorylation of Akt by TGF-beta and enhances TGF-beta-induced phosphorylation of c-Jun. FET cells expressing Smad7 show anchorage-independent growth and enhance tumorigenicity in athymic nude mice. Smad7 blocks TGF-beta-induced growth inhibition by preventing TGF-beta-induced G1 arrest. Smad7 inhibits TGF-beta-mediated downregulation of c-Myc, CDK4, and Cyclin D1, and suppresses the expression of p21(Cip1). As a result, Smad7 inhibits TGF-beta-mediated downregulation of Rb phosphorylation. Furthermore, Smad7 inhibits the apoptosis of these cells. Together, Smad7 may increase the tumorigenicity of FET cells by blocking TGF-beta-induced growth inhibition and by inhibiting apoptosis. Thus, this study provides a mechanism by which a portion of human colorectal tumors may become refractory to tumor-suppressive actions of TGF-beta that might result in increased tumorigenicity.

Immunohistochemical detection of active transforming growth factor-beta in situ using engineered tissue

NASA Technical Reports Server (NTRS)

Barcellos-Hoff, M. H.; Ehrhart, E. J.; Kalia, M.; Jirtle, R.; Flanders, K.; Tsang, M. L.; Chatterjee, A. (Principal Investigator)

1995-01-01

The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein.

Regulation of the Bioavailability of TGF-β and TGF-β-Related Proteins

PubMed Central

Robertson, Ian B.; Rifkin, Daniel B.

2016-01-01

The bioavailability of members of the transforming growth factor β (TGF-β) family is controlled by a number of mechanisms. Bona fide TGF-β is sequestered into the matrix in a latent state and must be activated before it can bind to its receptors. Here, we review the molecules and mechanisms that regulate the bioavailability of TGF-β and compare these mechanisms with those used to regulate other TGF-β family members. We also assess the physiological significance of various latent TGF-β activators, as well as other extracellular modulators of TGF-β family signaling, by examining the available in vivo data from knockout mouse models and other biological systems. PMID:27252363

Studies of the Outer Membrane Proteins of Campylobacter Jejuni for Vaccine Development

DTIC Science & Technology

1991-11-26

Mycobacterium tuberculosis, and M.leprae (66%) and mitochondrial protein p1 precursor of human and Chinese hamster cells (64%), and rubisco subunit binding...175) SAWG--DIgNIISDAP’KXVGRXgVITVK (202) 64% Rubisco subunit binding-protein alpha subunit of wheat (151) SAGN--OELIZGANADAIDOGPOVVLStE (178) 57

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

PubMed

Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-05-01

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

Sphingosine mediates the immediate negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian cardiac myocyte.

PubMed

Oral, H; Dorn, G W; Mann, D L

1997-02-21

To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-alpha (TNF-alpha), we examined sphingosine levels in diluent and TNF-alpha-stimulated cardiac myocytes. TNF-alpha stimulation of adult feline cardiac myocytes provoked a rapid (<15 min) increase in the hydrolysis of [14C]sphingomyelin in cell-free extracts, as well as an increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-alpha-stimulated cardiac myocytes showed that TNF-alpha stimulation produced a rapid (<30 min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-alpha on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-alpha in isolated contracting myocytes; time course studies showed that exogenous D-sphingosine produced abnormalities in cell shortening that were maximal at 5 min. Finally, blocking sphingosine production using an inhibitor of ceramidase, n-oleoylethanolamine, completely abrogated the negative inotropic effects of TNF-alpha in isolated contracting cardiac myocytes. Additional studies employing biologically active ceramide analogs and sphingosine 1-phosphate suggested that neither the immediate precursor of sphingosine nor the immediate metabolite of sphingosine, respectively, were likely to be responsible for the immediate negative inotropic effects of TNF-alpha. Thus, these studies suggest that sphingosine mediates the immediate negative inotropic effects of TNF-alpha in isolated cardiac myocytes.

Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis.

PubMed

Conery, Andrew R; Cao, Yanna; Thompson, E Aubrey; Townsend, Courtney M; Ko, Tien C; Luo, Kunxin

2004-04-01

Transforming growth factor beta (TGF-beta) induces both apoptosis and cell-cycle arrest in some cell lines, but only growth arrest in others. It is not clear how this differential response to TGF-beta is specified. Smad proteins are critical mediators of TGF-beta signalling. After stimulation by TGF-beta, Smad2 and Smad3 become phosphorylated by the activated TGF-beta receptor kinases, oligomerize with Smad4, translocate to the nucleus and regulate the expression of TGF-beta target genes. Here we report that the sensitivity to TGF-beta induced apoptosis is regulated by crosstalk between the Akt/PKB serine/threonine kinase and Smad3 through a mechanism that is independent of Akt kinase activity. Akt interacts directly with unphosphorylated Smad3 to sequester it outside the nucleus, preventing its phosphorylation and nuclear translocation. This results in inhibition of Smad3-mediated transcription and apoptosis. Furthermore, the ratio of Smad3 to Akt correlates with the sensitivity of cells to TGF-beta induced apoptosis. Alteration of this ratio changes the apoptotic, but not the growth-inhibitory, responses of cells to TGF-beta. These findings identify an important determinant of sensitivity to TGF-beta-induced apoptosis that involves crosstalk between the TGF-beta and phosphatidylinositol-3-OH kinase (PI(3)K) pathways.

TGF-β concentrations and activity are down-regulated in the aqueous humor of patients with neovascular age-related macular degeneration.

PubMed

Tosi, Gian Marco; Neri, Giovanni; Caldi, Elena; Fusco, Fiorella; Bacci, Tommaso; Tarantello, Antonio; Nuti, Elisabetta; Marigliani, Davide; Baiocchi, Stefano; Traversi, Claudio; Barbarino, Marcella; Eandi, Chiara M; Parolini, Barbara; Mundo, Lucia; Santucci, Annalisa; Orlandini, Maurizio; Galvagni, Federico

2018-05-23

Controversy still exists regarding the role of the TGF-β in neovascular age-related macular degeneration (nAMD), a major cause of severe visual loss in the elderly in developed countries. Here, we measured the concentrations of active TGF-β1, TGF-β2, and TGF-β3 by ELISA in the aqueous humor of 20 patients affected by nAMD, who received 3 consecutive monthly intravitreal injections of anti-VEGF-A antibody. Samples were collected at baseline (before the first injection), month 1 (before the second injection), and month 2 (before the third injection). The same samples were used in a luciferase-based reporter assay to test the TGF-β pathway activation. Active TGF-β1 concentrations in the aqueous humor were below the minimum detectable dose. Active TGF-β2 concentrations were significantly lower at baseline and at month 1, compared to controls. No significant differences in active TGF-β3 concentration were found among the sample groups. Moreover, TGF-β pathway activation was significantly lower at baseline compared to controls. Our data corroborate an anti-angiogenic role for TGF-β2 in nAMD. This should be considered from the perspective of a therapy using TGF-β inhibitors.

Transforming growth factor-{beta}-inducible phosphorylation of Smad3.

PubMed

Wang, Guannan; Matsuura, Isao; He, Dongming; Liu, Fang

2009-04-10

Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.

Two pheromone precursor genes are transcriptionally expressed in the homothallic ascomycete Sordaria macrospora.

PubMed

Pöggeler, S

2000-06-01

In order to analyze the involvement of pheromones in cell recognition and mating in a homothallic fungus, two putative pheromone precursor genes, named ppg1 and ppg2, were isolated from a genomic library of Sordaria macrospora. The ppg1 gene is predicted to encode a precursor pheromone that is processed by a Kex2-like protease to yield a pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae. The ppg2 gene encodes a 24-amino-acid polypeptide that contains a putative farnesylated and carboxy methylated C-terminal cysteine residue. The sequences of the predicted pheromones display strong structural similarity to those encoded by putative pheromones of heterothallic filamentous ascomycetes. Both genes are expressed during the life cycle of S. macrospora. This is the first description of pheromone precursor genes encoded by a homothallic fungus. Southern-hybridization experiments indicated that ppg1 and ppg2 homologues are also present in other homothallic ascomycetes.

Specific lysine labeling by 18OH- during alkaline cleavage of the alpha-1-antitrypsin-trypsin complex.

PubMed Central

Cohen, A B; Gruenke, L D; Craig, J C; Geczy, D

1977-01-01

alpha-1-Antitrypsin is a serum protein that inhibits many proteolytic enzymes. Recently, it was suggested that the alpha-1-antitrypsin-trypsin complex is an acyl ester analogous to the acyl intermediate that forms between trypsin and its substrates. In previous work we showed that the alpha-1-antitrypsin-trypsin complex can be split at high pH, releasing a component of alpha-1-antitrypsin. This component had a new carboxyl-terminal lysine, and it had lost a peptide of about 4000 daltons. In order to determine whether the alpha-1-antitrypsin is bound to trypsin through the new carboxy-terminal lysine, as would be expected if the above hypothesis is correct, we split the complex in the presence of 18OH-. When the new carboxy-terminal lysine was cleaved with carboxypeptidase B, singly labeled, doubly labeled, and unlabeled lysine were recovered. These data support the hypothesis that the alpha-1-antitrypsin-trypsin complex is an acyl ester or a tetrahedral precursor that is transformed into the acyl ester form at high pH. If other enzymes are bound by a similar mechanism, the methods used may be useful in determining which amino acids on alpha-1-antitrypsin bind covalently to each enzyme. PMID:303770

Reciprocal regulation of TGF-β and reactive oxygen species: A perverse cycle for fibrosis

PubMed Central

Liu, Rui-Ming; Desai, Leena P.

2015-01-01

Transforming growth factor beta (TGF-β) is the most potent pro-fibrogenic cytokine and its expression is increased in almost all of fibrotic diseases. Although signaling through Smad pathway is believed to play a central role in TGF-β's fibrogenesis, emerging evidence indicates that reactive oxygen species (ROS) modulate TGF-β's signaling through different pathways including Smad pathway. TGF-β1 increases ROS production and suppresses antioxidant enzymes, leading to a redox imbalance. ROS, in turn, induce/activate TGF-β1 and mediate many of TGF-β's fibrogenic effects, forming a vicious cycle (see graphic flow chart on the right). Here, we review the current knowledge on the feed-forward mechanisms between TGF-β1 and ROS in the development of fibrosis. Therapeutics targeting TGF-β-induced and ROS-dependent cellular signaling represents a novel approach in the treatment of fibrotic disorders. PMID:26496488

The Phosphatidylinositol 3-Kinase/Akt Pathway Regulates Transforming Growth Factor-β Signaling by Destabilizing Ski and Inducing Smad7*

PubMed Central

Band, Arja M.; Björklund, Mia; Laiho, Marikki

2009-01-01

Ski is an oncoprotein that negatively regulates transforming growth factor (TGF)-β signaling. It acts as a transcriptional co-repressor by binding to TGF-β signaling molecules, Smads. Efficient TGF-β signaling is facilitated by rapid proteasome-mediated degradation of Ski by TGF-β. Here we report that Ski is phosphorylated by Akt/PKB kinase. Akt phosphorylates Ski on a highly conserved Akt motif at threonine 458 both in vitro and in vivo. The phosphorylation of Ski at threonine 458 is induced by Akt pathway activators including insulin, insulin-like growth factor-1, and hepatocyte growth factor. The phosphorylation of Ski causes its destabilization and reduces Ski-mediated inhibition of expression of another negative regulator of TGF-β, Smad7. Induction of Smad7 levels leads to inactivation of TGF-β receptors and TGF-β signaling cascade, as indicated by reduced induction of TGF-β target p15. Therefore, Akt modulates TGF-β signaling by temporarily adjusting the levels of two TGF-β pathway negative regulators, Ski and Smad7. These novel findings demonstrate that Akt pathway activation directly impacts TGF-β pathway. PMID:19875456

The concomitant apoptosis and EMT underlie the fundamental functions of TGF-β.

PubMed

Song, Jianguo; Shi, Weiwei

2018-01-01

TGF-β's multipotent cellular effects and their relations are critical for TGF-β's pathophysiological functions. However, these effects may appear to be paradoxical in understanding TGF-β's functions. Apoptosis and epithelial-mesenchymal transition (EMT) are two fundamental events that are deeply linked to various physiological and disease-related processes. These two major cellular fates are subtly regulated and can be potently stimulated by TGF-β, which profoundly contribute to the biological roles of TGF-β. Moreover, these two events are also indirectly and directly correlated with TGF-β-mediated growth inhibition and are relevant to the current understanding of the roles of TGF-β in tumorigenesis and cancer progression. Although TGF-β-induced apoptosis and EMT can be singly independent cellular events, they can also be mutually exclusive but interrelated concomitant events in various cases. Thus, the modulation of apoptosis and EMT is essential for the seemingly paradoxical functions of TGF-β. However, the concomitant effect of TGF-β on apoptosis and EMT, the balance and regulated alterations of them are still been ignored or underestimated. This review focuses on the TGF-β-induced concomitant apoptosis and EMT. We aim to provide an insight in understanding their significance, balance, and modulation in TGF-β-mediated biological functions. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Platelet TGF-β1 contributions to plasma TGF-β1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload

PubMed Central

Meyer, Alexander; Wang, Wei; Qu, Jiaxiang; Croft, Lori; Degen, Jay L.; Coller, Barry S.

2012-01-01

Circulating platelets contain high concentrations of TGF-β1 in their α-granules and release it on platelet adhesion/activation. We hypothesized that uncontrolled in vitro release of platelet TGF-β1 may confound measurement of plasma TGF-β1 in mice and that in vivo release and activation may contribute to cardiac pathology in response to constriction of the transverse aorta, which produces both high shear and cardiac pressure overload. Plasma TGF-β1 levels in blood collected from C57Bl/6 mice by the standard retro-bulbar technique were much higher than those obtained when prostaglandin E1 was added to inhibit release or when blood was collected percutaneously from the left ventricle under ultrasound guidance. Even with optimal blood drawing, plasma TGF-β1 was lower in mice rendered profoundly thrombocytopenic or mice with selectively low levels of platelet TGF-β1 because of megakaryocytespecific disruption of their TGF-β1 gene (Tgfb1flox). Tgfb1flox mice were also partially protected from developing cardiac hypertrophy, fibrosis, and systolic dysfunction in response to transverse aortic constriction. These studies demonstrate that plasma TGF-β1 levels can be assessed accurately, but it requires special precautions; that platelet TGF-β1 contributes to plasma levels of TGF-β1; and that platelet TGF-β1 contributes to the pathologic cardiac changes that occur in response to aortic constriction. PMID:22134166

4-O-Methylhonokiol Protects HaCaT Cells from TGF-β1-Induced Cell Cycle Arrest by Regulating Canonical and Non-Canonical Pathways of TGF-β Signaling.

PubMed

Kim, Sang-Cheol; Kang, Jung-Il; Hyun, Jin-Won; Kang, Ji-Hoon; Koh, Young-Sang; Kim, Young-Heui; Kim, Ki-Ho; Ko, Ji-Hee; Yoo, Eun-Sook; Kang, Hee-Kyoung

2017-07-01

4- O -methylhonokiol, a neolignan compound from Magnolia Officinalis , has been reported to have various biological activities including hair growth promoting effect. However, although transforming growth factor-β (TGF-β) signal pathway has an essential role in the regression induction of hair growth, the effect of 4- O -methylhonokiol on the TGF-β signal pathway has not yet been elucidated. We thus examined the effect of 4- O -methylhonokiol on TGF-β-induced canonical and noncanonical pathways in HaCaT human keratinocytes. When HaCaT cells were pretreated with 4- O -methylhonokiol, TGF-β1-induced G1/G0 phase arrest and TGF-β1-induced p21 expression were decreased. Moreover, 4- O -methylhonokiol inhibited nuclear translocation of Smad2/3, Smad4 and Sp1 in TGF-β1-induced canonical pathway. We observed that ERK phosphorylation by TGF-β1 was significantly attenuated by treatment with 4- O -methylhonokiol. 4- O -methylhonokiol inhibited TGF-β1-induced reactive oxygen species (ROS) production and reduced the increase of NADPH oxidase 4 (NOX4) mRNA level in TGF-β1-induced noncanonical pathway. These results indicate that 4- O -methylhonokiol could inhibit TGF-β1-induced cell cycle arrest through inhibition of canonical and noncanonical pathways in human keratinocyte HaCaT cell and that 4- O -methylhonokiol might have protective action on TGF-β1-induced cell cycle arrest.

The role of transforming growth factor β in T helper 17 differentiation.

PubMed

Zhang, Song

2018-04-23

T helper 17 (Th17) cells play critical roles in inflammatory and autoimmune diseases. The lineage-specific transcription factor RORγt is the key regulator for Th17 cell fate commitment. A substantial number of studies have established the importance of transforming growth factor β (TGF-β) -dependent pathways in inducing RORγt expression and Th17 differentiation. TGF-β superfamily members TGF-β 1 , TGF-β 3 or activin A, in concert with interleukin-6 or interleukin-21, differentiate naive T cells into Th17 cells. Alternatively, Th17 differentiation can occur through TGF-β-independent pathways. However, the mechanism of how TGF-β-dependent and TGF-β-independent pathways control Th17 differentiation remains controversial. This review focuses on the perplexing role of TGF-β in Th17 differentiation, depicts the requirement of TGF-β for Th17 development, and underscores the multiple mechanisms underlying TGF-β-promoted Th17 generation, pathogenicity and plasticity. With new insights and comprehension from recent findings, this review specifically tackles the involvement of the canonical TGF-β signalling components, SMAD2, SMAD3 and SMAD4, summarizes diverse SMAD-independent mechanisms, and highlights the importance of TGF-β signalling in balancing the reciprocal conversion of Th17 and regulatory T cells. Finally, this review includes discussions and perspectives and raises important mechanistic questions about the role of TGF-β in Th17 generation and function. © 2018 John Wiley & Sons Ltd.

Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines.

PubMed

Dzieran, Johanna; Fabian, Jasmin; Feng, Teng; Coulouarn, Cédric; Ilkavets, Iryna; Kyselova, Anastasia; Breuhahn, Kai; Dooley, Steven; Meindl-Beinker, Nadja M

2013-01-01

Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-β functions for the design of efficient TGF-β directed therapy in liver cancer.

[Bushen Huoxue Fang promotes the apoptosis of epithelial cells in the prostatic ductal system of rats with benign prostatic hyperplasia].

PubMed

Sun, Jie; Li, Qiu-Fen; Tian, Dai-Zhi; Jiang, Shao-Bo; Wu, Xian-De; Qiu, Shun-An; Ren, Xiao-Gang; Li, Yu-Bing

2014-09-01

To investigate the effects of Bushen Huoxue Fang (BSHX) on the apoptosis of epithelial cells in the prostatic ductal system of rats with benign prostatic hyperplasia (BPH) and its possible action mechanism. One hundred 3- month-old male Wistar rats were randomly divided into four groups of equal number (control, castrated, BPH model, and BSHX). BPH models were made by subcutaneous injection of testosterone following castration; the rats in the BSHX group were treated intragastrically with BSHX at 2.34 g/ml after modeling, while those in the other two groups with equal volume of saline, all for 37 days. On the 38th day, all the rats were sacrificed and their prostates harvested for detection of the distribution of TGF-beta1 and alpha-actin and the count of positive cells in the prostatic ductal system by immunohistochemical staining. The apoptosis rate of epithelial cells in the prostatic ductal system was determined by TUNEL assay. The expression of TGF-beta1 was significantly increased in the rats of the BSHX group as compared with the BPH models in both the proximal prostatic duct ([15.28 +/- 4.30]% vs [36.42 +/- 8.10]%, P < 0.01) and the distal prostatic duct ([4.42 +/- 2.07]% vs [8.71 +/- 2.28 ]%, P < 0.05), while the expression of alpha-actin in the proximal duct was remarkably higher in the BSHX-treated rats than in the models ([28.14 +/- 7.43]% vs [18.28 +/- 4.07]%, P < 0.01), but lower than in the control animals ([33.57 +/- 6.85]%, P < 0.05). Compared with the control group, the BPH models and BSHX-treated rats both exhibited markedly decreased apoptosis of epithelial cells in the proximal prostatic duct ([39.42 +/- 9.20]% vs [3.86 +/- 1.34]%, P < 0.01, and [31.14 +/- 5.64]%, P < 0.01) and distal prostatic duct ([17.60 +/- 4.86]% vs [3.07 +/- 1.14]%, P < 0.01, and [12.37 +/- 2.25]%, P < 0.05). The apoptosis rate of epithelial cells in the prostatic ductal system was significantly higher in the BSHX-treated rats than in the BPH models (P < 0.01). By upregulating the expression of TGF-beta, BSHX can suppress the reduction of smooth muscle cells in the proximal prostatic duct, promote the apoptosis of prostatic epithelial cells, and thus effectively inhibit benign prostatic hyperplasia.

Heat shock protein 22 (HSPB8) limits TGF-β-stimulated migration of osteoblasts.

PubMed

Yamamoto, Naohiro; Tokuda, Haruhiko; Kuroyanagi, Gen; Kainuma, Shingo; Matsushima-Nishiwaki, Rie; Fujita, Kazuhiko; Kozawa, Osamu; Otsuka, Takanobu

2016-11-15

Heat shock proteins (HSPs) are induced in response to various physiological and environmental conditions such as chemical and heat stress, and recognized to function as molecular chaperones. HSP22 (HSPB8), a low-molecular weight HSP, is ubiquitously expressed in many cell types. However, the precise role of HSP22 in bone metabolism remains to be clarified. In the present study, we investigated whether HSP22 is implicated in the transforming growth factor-β (TGF-β)-stimulated migration of osteoblast-like MC3T3-E1 cells. Although protein levels of HSP22 were clearly detected in unstimulated MC3T3-E1 cells, TGF-β failed to induce the protein levels. The TGF-β-stimulated migration was significantly up-regulated by knockdown of HSP22 expression. The cell migration stimulated by platelet-derived growth factor-BB was also enhanced by HSP22 knockdown. SB203580, an inhibitor of p38 mitogen-activated protein kinase, PD98059, an inhibitor of MEK1/2, or SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase had no effects on the TGF-β-induced migration. SIS3, a specific inhibitor of TGF-β-dependent Smad3 phosphorylation, significantly reduced the migration with or without TGF-β stimulation. Smad2, Smad3, Smad4 or Smad7 was not coimmunoprecipitated with HSP22. On the other hand, the TGF-β-induced Smad2 phosphorylation was enhanced by HSP22 down-regulation. The protein levels of TGF-β type II receptor (TGF-β RII) but not TGF-β type I receptor (TGF-β RI) was significantly up-regulated in HSP22 knockdown cells compared with those in the control cells. However, the levels of TGF-β RII mRNA in HSP22 knockdown cells were little different from those of the control cells. Neither TGF-β RI nor TGF-β RII was coimmunoprecipitated with HSP22. SIS3 reduced the amplification by HSP22 knockdown of the TGF-β-stimulated cell migration almost to the basal level. Our results strongly suggest that HSP22 functions as a negative regulator in the TGF-β-stimulated migration of osteoblasts via suppression of the Smad-dependent pathway, resulting from modulating the protein levels of TGF-β RII. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Involvement of the TGFβ1- ILK-Akt signaling pathway in the effects of hesperidin in type 2 diabetic nephropathy.

PubMed

Zhang, YingHui; Wang, Bing; Guo, Feng; Li, ZhiZhen; Qin, GuiJun

2018-06-14

Diabetic nephropathy is one of the manifestations of systemic microangiopathy in diabetes. Hesperetin, a natural flavanone glycoside compound in citrus fruits, has been demonstrated to exert hypoglycemic effects and protect kidney in experimental diabetic animals. The current study was aimed to investigate the mechanisms underlying the hypoglycemic effects of hesperetin in high-fat/streptozocin (STZ)-induced diabetic nephropathy. The results showed that mice in whom hesperetin was administered for 4 weeks attenuated the increased fasting blood glucose and impaired glucose tolerance ability that was observed in high-fat/STZ mice. In addition, we found that hesperetin ameliorated the abnormalities of biochemical parameters in serum, liver, and kidney of mice with diabetic nephropathy. Hesperetin also rescued the irregular distortions in glomerular basement membrane and expanded mesangial regions. Moreover, hesperetin repaired the function of podocyte by increasing renal nephrin expression and decreasing renal alpha-smooth muscle actin expression. Furthermore, hesperetin inhibited the expression of transforming growth factor-β1 (TGF-β1) and its downstream effectors integrin-linked kinase (ILK) and Akt. In conclusion, our study implies that hesperetin produced protective effects in diabetic nephropathy possibly by suppressing TGF-β1-ILK-Akt signaling. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

TGF-{beta} receptors, in a Smad-independent manner, are required for terminal skeletal muscle differentiation

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

Droguett, Rebeca; Cabello-Verrugio, Claudio; Santander, Cristian

2010-09-10

Skeletal muscle differentiation is strongly inhibited by transforming growth factor type {beta} (TGF-{beta}), although muscle formation as well as regeneration normally occurs in an environment rich in this growth factor. In this study, we evaluated the role of intracellular regulatory Smads proteins as well as TGF-{beta}-receptors (TGF-{beta}-Rs) during skeletal muscle differentiation. We found a decrease of TGF-{beta} signaling during differentiation. This phenomenon is explained by a decline in the levels of the regulatory proteins Smad-2, -3, and -4, a decrease in the phosphorylation of Smad-2 and lost of nuclear translocation of Smad-3 and -4 in response to TGF-{beta}. No changemore » in the levels and inhibitory function of Smad-7 was observed. In contrast, we found that TGF-{beta}-R type I (TGF-{beta}-RI) and type II (TGF-{beta}-RII) increased on the cell surface during skeletal muscle differentiation. To analyze the direct role of the serine/threonine kinase activities of TGF-{beta}-Rs, we used the specific inhibitor SB 431542 and the dominant-negative form of TGF-{beta}-RII lacking the cytoplasmic domain. The TGF-{beta}-Rs were important for successful muscle formation, determined by the induction of myogenin, creatine kinase activity, and myosin. Silencing of Smad-2/3 expression by specific siRNA treatments accelerated myogenin, myosin expression, and myotube formation; although when SB 431542 was present inhibition in myosin induction and myotube formation was observed, suggesting that these last steps of skeletal muscle differentiation require active TGF-{beta}-Rs. These results suggest that both down-regulation of Smad regulatory proteins and cell signaling through the TGF-{beta} receptors independent of Smad proteins are essential for skeletal muscle differentiation.« less

Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells.

PubMed Central

Riser, B. L.; Cortes, P.; Heilig, C.; Grondin, J.; Ladson-Wofford, S.; Patterson, D.; Narins, R. G.

1996-01-01

Glomerular distention from increased intraglomerular pressure stretches mesangial cells (MCs). Stretching MCs in culture stimulates extracellular matrix accumulation, suggesting that this may be a mechanism for glomerular hypertension-associated glomerulosclerosis. We examined whether mechanical stretching serves as a stimulus for the synthesis and activation of the prosclerotic molecule transforming growth factor (TGF)-beta, thus providing a potential system for auto-induction of extracellular matrix. Rat MCs cultured on flexible-bottom plates were subjected to cyclic stretching for up to 3 days and then assayed for TGF-beta mRNA, secretion of TGF-beta, and localization of active TGF-beta by immunostaining. MCs contained mRNA for all three mammalian isoforms of TGF-beta. Cyclic stretching for 36 hours increased TGF-beta1 and TGF-beta3 mRNA levels approximately twofold, without altering the levels of TGF-beta2 mRNA. This was followed at 48 to 72 hours by the increased secretion of both latent and active TGF-beta1. Latent, but not active, TGF-beta3 secretion also increased whereas the levels of TGF-beta2 were unaffected by mechanical force. The stretching force in this system is unequally distributed over the culture membrane. Localization of active TGF-beta by immunostaining demonstrated that the quantity of cell-associated cytokine across the culture was directly proportional to the zonal amplitude of the stretching force. These results demonstrate that stretching force stimulates MCs to selectively release and activate TGF-beta1. This mechanical induction of TGF-beta1 may help explain the increased extracellular matrix associated with intraglomerular hypertension. Images Figure 1 Figure 3 PMID:8669477

Caveolae are negative regulators of transforming growth factor-beta1 signaling in ureteral smooth muscle cells.

PubMed

Stehr, Maximilian; Estrada, Carlos R; Khoury, Joseph; Danciu, Theodora E; Sullivan, Maryrose P; Peters, Craig A; Solomon, Keith R; Freeman, Michael R; Adam, Rosalyn M

2004-12-01

The mechanisms underlying ureteral cell regulation are largely unknown. Previous studies have identified lipid rafts/caveolae as regulators of growth stimulatory signals in ureteral smooth muscle cells (USMCs). In this study we determined whether growth inhibitory signaling by transforming growth factor-beta1 (TGF-beta1) is also regulated by caveolae in USMC. Expression of components of the TGF-beta1 signaling axis in USMCs was determined by immunoblot and mRNA analyses. Growth regulatory activity of TGF-beta1 was assessed by H-thymidine incorporation. In select experiments caveolae were disrupted reversibly by cholesterol depletion and replenishment prior to TGF-beta1 treatment. TGF-beta1-responsive gene expression was evaluated using the TGF-beta1 responsive promoter-reporter construct 3TP-Lux. USMCs expressed TGF-beta1, types I and II TGF-beta1 receptors, and the effector Smad-2. TGF-beta1 potently inhibited DNA synthesis in USMCs (IC50 60 pM). TGF-beta1 mediated DNA synthesis inhibition was potentiated following the disruption of caveolae by cholesterol depletion. This effect was reversible with membrane cholesterol restoration. TGF-beta1 stimulated gene activity was augmented by caveolae disruption, while caveolae reformation returned promoter activity to baseline levels. TGF-beta1 is a potent growth inhibitor of USMCs and its activity can be enhanced by caveolae ablation. These findings suggest a role for TGF-beta1 in the growth regulation of normal ureteral cells and implicate caveolar membrane domains in the negative regulation of TGF-beta1 signaling. These studies may be relevant to ureteral pathologies that are characterized by smooth muscle dysplasia.

Down-regulation of TGF-b1, TGF-b receptor 2, and TGF-b-associated microRNAs, miR-20a and miR-21, in skin lesions of sulfur mustard-exposed Iranian war veterans.

PubMed

Valizadeh, Mohadeseh; Mirzaei, Behnaz; Tavallaei, Mahmood; Noorani, Mohammad Reza; Amiri, Mojtaba; Soroush, Mohammad Reza; Mowla, Seyed Javad

2015-01-01

Sulfur mustard (SM) affects divergent cellular pathways including cell cycle, apoptosis, necrosis, and inflammatory responses. SM-induced lesions in skin include late-onset hyper-pigmentation, xerosis, and atrophy. It seems that TGF-b signaling pathway is a major player for SM pathogenesis. Here, we have employed a real-time polymerase chain reaction (PCR) approach to evaluate the expression alterations of all TGF-b variants and their receptors in skin biopsies obtained from 10 Iran-Iraq war veterans. Using specific LNA primers, the expression alteration of a TGF-bR2 regulator, miR-20a, and TGF-b downstream target, miR-21, was also assessed in the same samples Our real-time PCR data revealed a significant down-regulation of TGF-b1 and TGF-bR2, the major mediators of TGF-b signaling pathway, in skin biopsies of SM-exposed patients (p = 0.0015 and p = 0.0115, respectively). Down-regulation of TGF-b signaling pathway seems to contribute in severe inflammation observed in SM-exposed patients' tissues. MiR-20a and miR-21, as two important TGF-b associated microRNAs (miRNAs), were also down-regulated in SM-exposed skin lesions, compared to those of control group (p = 0.0003). Based on our findings, these miRNAs could be directly or indirectly involve in the pathogenesis of SM. Altogether, our data suggest the suitability of TGF-b1, TGF-bR2, as well as miR-20a and miR-21 as potential biomarkers for diagnosis and treatment of SM-exposed patients.

An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

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

Kim, Sun Kyung; Barron, Lindsey; Hinck, Cynthia S.

The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor (TβRI) but dispensable for binding the other receptor requiredmore » for TGF-β signaling, the TGF-β type II receptor (TβRII), as an alternative therapeutic modality for blocking TGF-β signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-β monomers and bound TβRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-β signaling with a Ki of 20–70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for TβRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TβRI, enabled it to bind endogenous TβRII but prevented it from binding and recruiting TβRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-β signaling and may inform similar modifications of other TGF-β family members.« less

Anticipation and consumption of food each increase the concentration of neuroactive steroids in rat brain and plasma.

PubMed

Pisu, Maria Giuseppina; Floris, Ivan; Maciocco, Elisabetta; Serra, Mariangela; Biggio, Giovanni

2006-09-01

Stressful stimuli and anxiogenic drugs increase the plasma and brain concentrations of neuroactive steroids. Moreover, in rats trained to consume their daily meal during a fixed period, the anticipation of food is associated with changes in the function of various neurotransmitter systems. We have now evaluated the effects of anticipation and consumption of food in such trained rats on the plasma and brain concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) and 3alpha,21-dihydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH DOC), two potent endogenous positive modulators of type A receptors for gamma-aminobutyric acid (GABA). The abundance of these neuroactive steroids was increased in both the cerebral cortex and plasma of the rats during both food anticipation and consumption. In contrast, the concentration of their precursor, progesterone, was increased in the brain only during food consumption, whereas it was increased in plasma only during food anticipation. Intraperitoneal administration of the selective agonist abecarnil (0.1 mg/kg) 40 min before food presentation prevented the increase in the brain levels of 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC during food anticipation but not that associated with consumption. The change in emotional state associated with food anticipation may thus result in an increase in the plasma and brain levels of 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC in a manner sensitive to the activation of GABA(A) receptor-mediated neurotransmission. A different mechanism, insensitive to activation of such transmission, may underlie the changes in the concentrations of these neuroactive steroids during food consumption.

Release of active TGF-β1 from the latent TGF-β1/GARP complex on T regulatory cells is mediated by integrin β8.

PubMed

Edwards, Justin P; Thornton, Angela M; Shevach, Ethan M

2014-09-15

Activated T regulatory cells (Tregs) express latent TGF-β1 on their cell surface bound to GARP. Although integrins have been implicated in mediating the release of active TGF-β1 from the complex of latent TGF-β1 and latent TGF-β1 binding protein, their role in processing latent TGF-β1 from the latent TGF-β1/GARP complex is unclear. Mouse CD4(+)Foxp3(+) Treg, but not CD4(+)Foxp3(-) T cells, expressed integrin β8 (Itgb8) as detected by quantitative RT-PCR. Itgb8 expression was a marker of thymically derived (t)Treg, because it could not be detected on Foxp3(+)Helios(-) Tregs or on Foxp3(+) T cells induced in vitro. Tregs from Itgb8 conditional knockouts exhibited normal suppressor function in vitro and in vivo in a model of colitis but failed to provide TGF-β1 to drive Th17 or induced Treg differentiation in vitro. In addition, Itgb8 knockout Tregs expressed higher levels of latent TGF-β1 on their cell surface consistent with defective processing. Thus, integrin αvβ8 is a marker of tTregs and functions in a cell intrinsic manner in mediating the processing of latent TGF-β1 from the latent TGF-β1/GARP complex on the surface of tTregs.

TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

PubMed

Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

2010-01-01

Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

Evaluation of the transforming growth factor-beta activity in normal and dry eye human tears by CCL-185 cell bioassay.

PubMed

Zheng, Xiaofen; De Paiva, Cintia S; Rao, Kavita; Li, De-Quan; Farley, William J; Stern, Michael; Pflugfelder, Stephen C

2010-09-01

To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye. Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay. The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in normal controls (4129.3 +/- 1342.9 pg/mL) (P < 0.05). Among patients with DE, TGF-beta bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-beta in DE tears and 37.6% TGF-beta in normal tears were found to be biologically active. The CCL-185 cell assay was found to be a suitable tool for assessing TGF-beta activity in human tears. Tear TGF-beta bioactivity increases in DE, particularly in Sjögren syndrome, where elevated levels of TGF-beta1 transcripts in the conjunctival epithelium have been previously detected.

Dependence of humoral hypercalcemia of malignancy on parathyroid hormone-related protein expression in the canine anal sac apocrine gland adenocarcinoma (CAC-8) nude mouse model.

PubMed

Gröne, A; Weckmann, M T; Blomme, E A; Capen, C C; Rosol, T J

1998-09-01

Circulating parathyroid hormone-related protein (PTHrP) is the primary humoral factor in dogs with spontaneous humoral hypercalcemia of malignancy (HHM) and adenocarcinomas derived from apocrine glands of the anal sac. A canine apocrine adenocarcinoma model of HHM in nude mice (CAC-8) was developed and characterized. After 32 passages in vivo, a spontaneous variant of the tumor (CAC-8 Lo Ca) that has altered cellular morphology and that fails to induce HHM in tumor-bearing nude mice has been discovered. The hypercalcemic and nonhypercalcemic tumor lines were compared by tumor weight, effect on body weight, serum calcium concentration, plasma PTHrP concentration, histopathology, expression of PTHrP protein by radioimmunoassay and immunohistochemistry, and expression of PTHrP mRNA by in situ hybridization and northern blot analysis. Messenger RNA expression for other factors and cytokines known to alter PTHrP secretion or bone resorption in vivo, including tumor necrosis factor alpha (TNF alpha), interleukin (IL)-1, IL-6, and transforming growth factor beta (TGF beta), were also measured in the adenocarcinomas. There was no significant difference in weight of individual tumors. Nude mice bearing the CAC-8 (Lo Ca) tumor maintained normal body weight as compared with non-tumor-bearing control mice. In contrast, mice with the CAC-8 (Hi Ca) tumor had markedly decreased body weights. The CAC-8 (Hi Ca) tumor-bearing mice had severe hypercalcemia (mean = 13.4 mg/dl) and increased plasma concentrations of PTHrP (30.4 pM), whereas the CAC-8 (Lo Ca) tumor-bearing mice had a mean serum calcium concentration of 10.1 mg/dl and mildly increased PTHrP concentrations (5.7 pM) as compared with control mice (9.0 mg/dl and 1.0 pM, respectively). The original tumor (CAC-8 [Hi Ca]) is a well-differentiated adenocarcinoma, whereas the variant tumor (CAC-8 [Lo Ca]) is a solid carcinoma with both polygonal and spindle-shaped cells. The CAC-8 (Lo Ca) tumor had decreased PTHrP mRNA expression and protein synthesis. Messenger RNA expression of TGF beta, TNF alpha, IL-1, and IL-6 was similar in both tumors and was consistent with the central role of PTHrP in the induction of hypercalcemia in this animal model.

Effect of transforming growth factor-beta1 on embryonic and posthatch muscle growth and development in normal and low score normal chicken.

PubMed

Li, X; Velleman, S G

2009-02-01

During skeletal muscle development, transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. The TGF-beta1 signal is carried by Smad proteins into the cell nucleus, inhibiting the expression of key myogenic regulatory factors including MyoD and myogenin. However, the molecular mechanism by which TGF-beta1 inhibits muscle cell proliferation and differentiation has not been well documented in vivo. The present study investigated the effect of TGF-beta1 on in vivo skeletal muscle growth and development. A chicken line, Low Score Normal (LSN) with reduced muscling and upregulated TGF-beta1 expression, was used and compared to a normal chicken line. The injection of TGF-beta1 at embryonic day (ED) 3 significantly reduced the pectoralis major (p. major) muscle weight in the normal birds at 1 wk posthatch, whereas no significant difference was observed in the LSN birds. The difference between normal and LSN birds in response to TGF-beta1 is likely due to different levels of endogenous TGF-beta1 where the LSN birds have increased TGF-beta1 expression in their p. major muscle at both 17 ED and 6 wk posthatch. Smad3 expression was reduced by TGF-beta1 from 10 ED to 1 wk posthatch in normal p. major muscle. Unlike Smad3, Smad7 expression was not significantly affected by TGF-beta1 until posthatch in both normal and LSN p. major muscle. Expression of MyoD was reduced 35% by TGF-beta1 during embryonic development in normal p. major muscle, whereas LSN p. major muscle showed a delayed decrease at 1 d posthatch in MyoD expression in response to the TGF-beta1 treatment. Myogenin expression was reduced 29% by TGF-beta1 after hatch in normal p. major muscle. In LSN p. major muscle, TGF-beta1 treatment significantly decreased myogenin expression by 43% at 1 d posthatch and 32% at 1 wk posthatch. These data suggested that TGF-beta1 reduced p. major muscle growth by inhibiting MyoD and myogenin expression during both embryonic and posthatch development. Furthermore, TGF-beta1 also reduced the expression of the cell adhesion receptor beta1 integrin subunit during embryonic and posthatch muscle growth in normal and LSN chickens. Therefore, the reduction of beta1 integrin in response to TGF-beta1 is also associated with decreased posthatch muscle growth. The results from this study indicate that TGF-beta1 inhibits skeletal muscle growth by regulating MyoD and myogenin expression. These data also suggest that a beta1 integrin-mediated alternative pathway is likely involved in the TGF-beta1-induced reduction of muscle growth.

Excretion profile of boldenone and its metabolites after oral administration to veal calves.

PubMed

Ferretti, G; Palleschi, L; Marchiafava, C; delli Quadri, F; Fantozzi, L; Ferranti, C; Cammarata, P; Macrì, A; Montesissa, C; Draisci, R

2007-04-25

The residue profiles of boldenone (17beta-Bol), its epimer (17alpha-Bol) and the related compound androsta-1,4-diene-3,17-dione (ADD), were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male calves orally treated with boldenone, boldenone esters, and/or ADD. In all the experiments with the administered steroids residues of 17alpha-Bol decreased rapidly after end of treatment; detectable amounts of 17alpha-Bol were however noticed along the withdrawal observation period after end of treatment. Differently, residues of 17beta-Bol were detectable only shortly after administration. This in vivo research concerning oral treatments of cattle with boldenone related substances proves ADD to be a very active boldenone precursor in bovine animals.

Transforming growth factor-β decreases side population cells in hepatocellular carcinoma in vitro.

PubMed

Kim, Jong Bin; Lee, Seulki; Kim, Hye Ri; Park, Seo-Young; Lee, Minjong; Yoon, Jung-Hwan; Kim, Yoon Jun

2018-06-01

Hepatocellular carcinoma (HCC) can result from hepatitis B or C infection, fibrosis or cirrhosis. Transforming growth factor-β (TGF-β) is one of the main growth factors associated with fibrosis or cirrhosis progression in the liver, but its role is controversial in hepatocarcinogenesis. In the present study, the effect of TGF-β on the HCC Huh-7 and Huh-Bat cell lines was evaluated. To study the effect of TGF-β, Huh-7 and Huh-Bat cells were treated with TGF-β and a TGF-β receptor inhibitor (SB431542). Cell survival, cell cycle, numbers of side population (SP) cells and expression of the cancer stem cell marker cluster of differentiation (CD)133, epithelial-mesenchymal transition markers (E-cadherin, α-smooth muscle actin and vimentin) and TGF-β-regulated proteins [phospho-c-Jun N-terminal kinase (p-JNK), p-c-Jun and p-smad2] were investigated. TGF-β treatment resulted in decreased cell survival with a targeted effect on SP cells. Expression of CD133 and vimentin was upregulated by treatment with the TGF-β receptor antagonist SB431542, but not with TGF-β. By contrast, TGF-β induced accumulation of cells at G0/G1, and upregulated expression of p-JNK, p-c-Jun and p-smad2. However, these effects were blocked when cells were treated with TGF-β plus SB431542, indicating the specificity of the TGF-β effect. The present results indicated that TGF-β has anticancer effects mediated by survival inhibition of cancer stem cells, which may be developed as a novel therapy for HCC.

4-O-Methylhonokiol Protects HaCaT Cells from TGF-β1-Induced Cell Cycle Arrest by Regulating Canonical and Non-Canonical Pathways of TGF-β Signaling

PubMed Central

Kim, Sang-Cheol; Kang, Jung-Il; Hyun, Jin-Won; Kang, Ji-Hoon; Koh, Young-Sang; Kim, Young-Heui; Kim, Ki-Ho; Ko, Ji-Hee; Yoo, Eun-Sook; Kang, Hee-Kyoung

2017-01-01

4-O-methylhonokiol, a neolignan compound from Magnolia Officinalis, has been reported to have various biological activities including hair growth promoting effect. However, although transforming growth factor-β (TGF-β) signal pathway has an essential role in the regression induction of hair growth, the effect of 4-O-methylhonokiol on the TGF-β signal pathway has not yet been elucidated. We thus examined the effect of 4-O-methylhonokiol on TGF-β-induced canonical and noncanonical pathways in HaCaT human keratinocytes. When HaCaT cells were pretreated with 4-O-methylhonokiol, TGF-β1-induced G1/G0 phase arrest and TGF-β1-induced p21 expression were decreased. Moreover, 4-O-methylhonokiol inhibited nuclear translocation of Smad2/3, Smad4 and Sp1 in TGF-β1-induced canonical pathway. We observed that ERK phosphorylation by TGF-β1 was significantly attenuated by treatment with 4-O-methylhonokiol. 4-O-methylhonokiol inhibited TGF-β1-induced reactive oxygen species (ROS) production and reduced the increase of NADPH oxidase 4 (NOX4) mRNA level in TGF-β1-induced noncanonical pathway. These results indicate that 4-O-methylhonokiol could inhibit TGF-β1-induced cell cycle arrest through inhibition of canonical and noncanonical pathways in human keratinocyte HaCaT cell and that 4-O-methylhonokiol might have protective action on TGF-β1-induced cell cycle arrest. PMID:28190316

Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma

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

Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-jun

TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-βmore » signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. - Highlights: • TLX knockdown enhances TGF-β dependent Smad signaling in glioblastoma cells • TLX knockdown increases the protein level of TGF-β receptor II. • TLX stabilizes and retains Smurf1 in the cytoplasm. • TLX enhances Smurf1-dependent ubiquitination and degradation of TGF-β receptor II.« less

Osteoblast gene expression is differentially regulated by TGF-beta isoforms.

PubMed

Fagenholz, P J; Warren, S M; Greenwald, J A; Bouletreau, P J; Spector, J A; Crisera, F E; Longaker, M T

2001-03-01

The transforming growth factor beta (TGF-beta) superfamily encompasses a number of important growth factors including several TGF-beta isoforms, the bone morphogenetic proteins, activins, inhibins, and growth and differentiation factors. TGF-beta 1, -beta 2, and -beta 3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and bone repair. Numerous studies suggest that each isoform has unique in vivo functions; however, the effects of these TGF-beta isoforms on osteoblast gene expression and maturation have never been directly compared. In the current study, we treated undifferentiated neonatal rat calvaria osteoblast-enriched cell cultures with 2.5 ng/ml of each TGF-beta isoform and analyzed gene expression at 0, 3, 6, and 24 hours. We demonstrated unique isoform-specific regulation of endogenous TGF-beta 1 and type I collagen mRNA transcription. To assess the effects of extended TGF-beta treatment on osteoblast maturation, we differentiated osteoblast cultures in the presence of 2.5 ng/ml of each TGF-beta isoform. Analysis of collagen I, alkaline phosphatase, and osteocalcin demonstrated that each TGF-beta isoform uniquely suppressed the transcription of these osteoblast differentiation markers. Interestingly, TGF-beta isoform treatment increased osteopontin expression in primary osteoblasts after 4 and 10 days of differentiation. To our knowledge, these data provide the first direct comparison of the effects of the TGF-beta isoforms on osteoblast gene expression in vitro. Furthermore, these data suggest that TGF-beta isoforms may exert their unique in vivo effects by differentially regulating osteoblast cytokine secretion, extracellular matrix production, and the rate of cellular maturation.

TGF-β promotes glioma cell growth via activating Nodal expression through Smad and ERK1/2 pathways

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

Sun, Jing; Liu, Su-zhi; Lin, Yan

Highlights: •TGF-β promoted Nodal expression in glioma cells. •TGF-β promoted Nodal expression via activating Smad and ERK1/2 pathways. •TGF-β promotes glioma cell growth via activating Nodal expression. -- Abstract: While there were certain studies focusing on the mechanism of TGF-β promoting the growth of glioma cells, the present work revealed another novel mechanism that TGF-β may promote glioma cell growth via enhancing Nodal expression. Our results showed that Nodal expression was significantly upregulated in glioma cells when TGF-β was added, whereas the TGF-β-induced Nodal expression was evidently inhibited by transfection Smad2 or Smad3 siRNAs, and the suppression was especially significantmore » when the Smad3 was downregulated. Another, the attenuation of TGF-β-induced Nodal expression was observed with blockade of the ERK1/2 pathway also. Further detection of the proliferation, apoptosis, and invasion of glioma cells indicated that Nodal overexpression promoted the proliferation and invasion of tumor cells and inhibited their apoptosis, resembling the effect of TGF-β addition. Downregulation of Nodal expression via transfection Nodal-specific siRNA in the presence of TGF-β weakened the promoting effect of the latter on glioma cells growth, and transfecting Nodal siRNA alone in the absence of exogenous TGF-β more profoundly inhibited the growth of glioma cells. These results demonstrated that while both TGF-β and Nodal promoted glioma cells growth, the former might exert such effect by enhancing Nodal expression, which may form a new target for glioma therapy.« less

The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy

PubMed Central

Ohsawa, Yutaka; Takayama, Kentaro; Nishimatsu, Shin-ichiro; Okada, Tadashi; Fujino, Masahiro; Fukai, Yuta; Murakami, Tatsufumi; Hagiwara, Hiroki; Itoh, Fumiko; Tsuchida, Kunihiro; Hayashi, Yoshio; Sunada, Yoshihide

2015-01-01

Myostatin, a muscle-specific transforming growth factor-β (TGF-β), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-β family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-β1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings. PMID:26226340

The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy.

PubMed

Ohsawa, Yutaka; Takayama, Kentaro; Nishimatsu, Shin-ichiro; Okada, Tadashi; Fujino, Masahiro; Fukai, Yuta; Murakami, Tatsufumi; Hagiwara, Hiroki; Itoh, Fumiko; Tsuchida, Kunihiro; Hayashi, Yoshio; Sunada, Yoshihide

2015-01-01

Myostatin, a muscle-specific transforming growth factor-β (TGF-β), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-β family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-β1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings.

Lupinus albus, a novel vegetable extract with metalloproteinase inhibitory properties: a potential periodontal therapy.

PubMed

Gaultier, Frédérick; Ejeil, Anne-Laure; Dridi, Sophie-Myriam; Piccardi, Nathalie; Piccirilli, Antoine; Msika, Philippe; Pellat, Bernard; Godeau, Gaston; Gogly, Bruno

2005-08-01

In this study we examine the properties of a vegetable extract from seeds of Lupinus albus (LU 105). In previous works we demonstrated that LU 105 reduced the expression, by gingival fibroblasts, of both matrix metalloproteinase (MMP)-2 and MMP-9. We decided to study the impact of LU 105 on cell proliferation and morphology. Using organ culture media we also studied the MMP and tissue inhibitors of metalloproteinases (timp) expression AND THE cytokines secretion. Healthy and inflamed gingival biopsies were placed in appendage culture with or without LU 105. The organ culture media were analyzed using Western blottings (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, TIMP-1, and TIMP-2) and gelatine zymography. A reverse transcription polymerase chain reaction (RT-PCR) was also performed on healthy and inflamed gingival biopsies, which were maintained in culture with or without LU 105 0.1%. Then, we decided to determine the amount of cytokines present in the organ culture media such as interleukin (IL)-1 beta, IL-4, IL-6, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha. When gingival biopsies derived from inflamed tissues were cultured with LU 105 0.1% in the culture media, the MMP and TIMP expression and activity decreased significantly when compared to cultures without LU 105. Moreover, we did not note any statistical difference in the cell proliferation compared with human gingival fibroblast cultures without LU 105. Furthermore, IL-1 beta, IL-6, TGF-beta, and TNF-alpha amounts in the culture media decreased significantly, whereas IL-4 increased significantly when LU 105 0.1% was added to the culture media. LU 105, a novel metalloproteinase inhibitor with few consequences on cell proliferation and morphology, is a vegetable extract with potential clinical capacity. J Periodontol 2005;76:1329-1338.

Inflammatory cytokine gene variants in coronary artery disease patients in Greece.

PubMed

Manginas, Athanassios; Tsiavou, Anastasia; Chaidaroglou, Antigoni; Giamouzis, Grigorios; Degiannis, Dimitrios; Panagiotakos, Demosthenis; Cokkinos, Dennis V

2008-12-01

Abundant evidence supports the central role of inflammatory cytokines in immune responses mediating the pathogenesis of atherosclerosis, coronary artery disease, and its complications, such as myocardial infarction and unstable angina. We investigated the association of genetic polymorphisms of the inflammatory cytokines, IL-10, TGF-beta1, IFN-gamma, IL-6, and TNF-alpha with the clinical presentation of coronary artery disease in 26 patients with stable angina, 45 patients with unstable angina and 58 patients who had experienced nonfatal myocardial infarction. Genotyping was performed by the sequence-specific primer polymerase chain reaction method. A significant difference in the frequencies of -174G/C IL-6 alleles was observed, with the low in-vitro producing -174*C allele predominating in patients with myocardial infarction, compared with stable angina and unstable angina patients, after the analysis of genotypes (P=0.024 and 0.022, respectively), phenotypes [P=0.0099, odds ratio (OR)=0.271, 95% confidence interval (CI)=0.1012-0.7292; P=0.03, OR=0.40, respectively] and haplotypes (P=0.007, OR=3.028, 95% CI=1.347-6.806; P=0.0096, OR=2.368, 95% CI=1.262-4.444; respectively). In addition, a predominance of the -1082ACC/ATA IL-10 genotype in the myocardial infarction group compared with the unstable angina group and the -874 A/A IFN-gamma genotype in the stable angina group compared with the unstable angina and the myocardial infarction group, was found. No significant differences in the distribution of genotypes, phenotypes and haplotypes in the three study groups, for the TNF-alpha-308 A/G and TGF-beta1-codon 25 G/C, codon 10 T/C polymorphisms were detected. Our data provide evidence that the IL-6-174G/C polymorphism may be involved in the pathogenesis of coronary artery disease, contributing to genetic susceptibility for myocardial infarction.

TGF-β in inflammatory bowel disease: a key regulator of immune cells, epithelium, and the intestinal microbiota.

PubMed

Ihara, Sozaburo; Hirata, Yoshihiro; Koike, Kazuhiko

2017-07-01

Inflammatory bowel disease (IBD) is defined as chronic intestinal inflammation, and includes ulcerative colitis and Crohn's disease. Multiple factors are involved in the pathogenesis of IBD, and the condition is characterized by aberrant mucosal immune reactions to intestinal microbes in genetically susceptible hosts. Transforming growth factor-β (TGF-β) is an immune-suppressive cytokine produced by many cell types and activated by integrins. Active TGF-β binds to its receptor and regulates mucosal immune reactions through the TGF-β signaling pathway. Dysregulated TGF-β signaling is observed in the intestines of IBD patients. TGF-β signal impairment in specific cell types, such as T-cells and dendritic cells, results in spontaneous colitis in mouse models. In addition, specific intestinal microbes contribute to immune homeostasis by modulating TGF-β production. In this review, we describe the role of TGF-β in intestinal immunity, focusing on immune cells, epithelium, and intestinal microbes. In addition, we present potential therapeutic strategies for IBD that target TGF-β.

TGF Afterglows: A New Radiation Mechanism From Thunderstorms

NASA Astrophysics Data System (ADS)

Rutjes, C.; Diniz, G.; Ferreira, I. S.; Ebert, U.

2017-10-01

Thunderstorms are known to create terrestrial gamma ray flashes (TGFs) which are microsecond-long bursts created by runaway of thermal electrons from propagating lightning leaders, as well as gamma ray glows that possibly are created by relativistic runaway electron avalanches (RREA) that can last for minutes or more and are sometimes terminated by a discharge. In this work we predict a new intermediate thunderstorm radiation mechanism, which we call TGF afterglow, as it is caused by the capture of photonuclear neutrons produced by a TGF. TGF afterglows are milliseconds to seconds long; this duration is caused by the thermalization time of the intermediate neutrons. TGF afterglows indicate that the primary TGF has produced photons in the energy range of 10-30 MeV; they are nondirectional in contrast to the primary TGF. Gurevich et al. might have reported TGF afterglows in 2011.

CXXC5 suppresses hepatocellular carcinoma by promoting TGF-β-induced cell cycle arrest and apoptosis.

PubMed

Yan, Xiaohua; Wu, Jingyi; Jiang, Quanlong; Cheng, Hao; Han, Jing-Dong J; Chen, Ye-Guang

2018-02-01

Evading TGF-β-mediated growth inhibition is often associated with tumorigenesis in liver, including hepatocellular carcinoma (HCC). To better understand the functions and the underlying molecular mechanisms of TGF-β in HCC initiation and progression, we carried out transcriptome sequencing (RNA-Seq) to identify the target genes of TGF-β. CXXC5, a member of the CXXC-type zinc finger domain-containing protein family, was identified as a novel TGF-β target gene in Hep3B HCC cells. Knockdown of CXXC5 attenuated the expression of a substantial portion of TGF-β target genes and ameliorated TGF-β-induced growth inhibition or apoptosis of Hep3B cells, suggesting that CXXC5 is required for TGF-β-mediated inhibition of HCC progression. Analysis of the TCGA database indicated that CXXC5 expression is reduced in the majority of HCC tissue samples in comparison to that in normal tissues. Furthermore, CXXC5 associates with the histone deacetylase HDAC1 and competes its interaction with Smad2/3, thereby abolishing the inhibitory effect of HDAC1 on TGF-β signaling. These observations together suggest that CXXC5 may act as a tumor suppressor by promoting TGF-β signaling via a positive feedback loop, and reveal a strategy for HCC to bypass TGF-β-mediated cytostasis by disrupting the positive feedback regulation. Our findings shed new light on TGF-β signaling regulation and demonstrate the function of CXXC5 in HCC development.

Transforming growth factor-β2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans

PubMed Central

Namachivayam, Kopperuncholan; Coffing, Hayley P.; Sankaranarayanan, Nehru Viji; Jin, Yingzi; MohanKumar, Krishnan; Frost, Brandy L.; Blanco, Cynthia L.; Patel, Aloka L.; Meier, Paula P.; Garzon, Steven A.; Desai, Umesh R.

2015-01-01

Human milk contains biologically important amounts of transforming growth factor-β2 isoform (TGF-β2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-β2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-β bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-β2 (rTGF-β2) to milk prior to feeding. Milk-borne TGF-β bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-β2 were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-β2 (20–40 nM) to human preterm milk samples failed to increase TGF-β bioactivity in milk. Milk-borne TGF-β2 was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-β2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-κB activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-β2 and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk. PMID:26045614

Release of active TGF-β1 from the Latent TGF-β1/GARP complex on T regulatory cells is mediated by Integrin β81

PubMed Central

Edwards, Justin P.; Thornton, Angela M.; Shevach, Ethan M.

2014-01-01

Activated T regulatory cells (Treg) express latent TGF-β1 on their cell surface bound to GARP. Although integrins have been implicated in mediating the release of active TGF-β1 from the complex of latent TGF-β1 and latent TGF-β1 binding protein, their role in processing latent TGF-β1 from the latent TGF-β1/GARP complex is unclear. Mouse CD4+Foxp3+ Treg, but not CD4+Foxp3− T cells, expressed integrin β8 (Itgb8) as detected by qRT-PCR. Itgb8 expression was a marker of thymically-derived (t)Treg, as it could not be detected on Foxp3+Helios− Tregs or on Foxp3+ T cells induced in vitro. Tregs from Itgb8 conditional knockouts exhibited normal suppressor function in vitro and in vivo in a model of colitis, but failed to provide TGF-β1 to drive Th17 or iTreg differentiation in vitro. In addition, Itgb8 knockout Tregs expressed higher levels of latent TGF-β1 on their cell surface consistent with defective processing. Thus, integrin αvβ8 is a marker of tTregs and functions in a cell intrinsic manner in mediating the processing of latent TGF-β1 from the latent TGF-β1/GARP complex on the surface of tTregs. PMID:25127859

The insulin response integrates increased TGF-β signaling through Akt-induced enhancement of cell surface delivery of TGF-β receptors

PubMed Central

Budi, Erine H.; Muthusamy, Baby Periyanayaki; Derynck, Rik

2015-01-01

Increased activity of transforming growth factor β (TGF-β), which binds to and stimulates cell surface receptors, contributes to cancer progression and fibrosis by driving epithelial cells toward a migratory mesenchymal phenotype and increasing the abundance of extracellular matrix proteins. The abundance of TGF-β receptors at the cell surface determines cellular responsiveness to TGF-β, which is often produced by the same cells that have the receptors, and thus serves as an autocrine signal. We found that Akt-mediated phosphorylation of AS160, a RabGAP [guanosine triphosphatase (GTPase)-activating protein] promoted the translocation of TGF-β receptors from intracellular stores to the plasma membrane of mouse embryonic fibroblasts (MEFs) and NMuMG epithelial cells. Consequently, insulin, which is commonly used to treat hyperglycemia and activates Akt signaling, increased the amount of TGF-β receptors at the cell surface, thereby enhancing TGF-β responsiveness. This insulin-induced increase in autocrine TGF-β signaling contributed to insulin-induced gene expression responses, attenuated the epithelial phenotype, and promoted the migration of NMuMG cells. Furthermore, the enhanced delivery of TGF-β receptors at the cell surface enabled insulin to increase TGF-β-induced gene responses. The enhancement of TGF-β responsiveness in response to Akt activation may help to explain the biological effects of insulin, the progression of cancers in which Akt is activated, and the increased incidence of fibroses in diabetes. PMID:26420907

Novel amine-based presursor compounds and composite membranes thereof

DOEpatents

Lee, Eric K. L.; Tuttle, Mark E.

1989-01-01

Novel amine-based precursor compounds comprising the condensation products of dialkylenetriamine and alpha, beta-unsaturated acid halides are disclosed, as well as composite membranes containing such compounds, the membranes being useful in RO-type processes for desalination and the removal of low molecular weight organic compounds such as phenols and carboxylic acids.

Aspartic proteases involved in Alzheimer's disease.

PubMed

Schmidt, Boris

2003-05-09

Alzheimer's disease afflicts every tenth human aged over 65. Despite the dramatic progress that has been made in understanding the disease, the exact cause of Alzheimer's disease is still unknown. Most gene mutations associated with Alzheimer's disease point at the same culprits: amyloid precursor protein and ultimately amyloid beta. The enigmatic proteases alpha-,beta-, and gamma-secretase are the three executioners of amyloid precursor protein processing, and disruption of their delicate balance is suspected to result in Alzheimer's disease. Significant progress has been made in the selective control of these proteases, regardless of the availability of structural information. Not even the absence of a robust cell-free assay for gamma-secretase could hamper the identification of nonpeptidic inhibitors of this enzyme for long. Within five years, four distinctly different structural moieties were developed and the first drug candidates are in clinical trials. Unfortunately, selective inhibition of amyloid beta formation remains a crucial issue because fundamental fragments of the gamma-secretase complex are important for other signaling events. This problem makes beta-secretase inhibition and alpha-secretase induction even more appealing.

Monoterpene oxidation in an oxidative flow reactor: SOA yields and the relationship between bulk gas-phase properties and organic aerosol growth

NASA Astrophysics Data System (ADS)

Friedman, B.; Link, M.; Farmer, D.

2016-12-01

We use an oxidative flow reactor (OFR) to determine the secondary organic aerosol (SOA) yields of five monoterpenes (alpha-pinene, beta-pinene, limonene, sabinene, and terpinolene) at a range of OH exposures. These OH exposures correspond to aging timescales of a few hours to seven days. We further determine how SOA yields of beta-pinene and alpha-pinene vary as a function of seed particle type (organic vs. inorganic) and seed particle mass concentration. We hypothesize that the monoterpene structure largely accounts for the observed variance in SOA yields for the different monoterpenes. We also use high-resolution time-of-flight chemical ionization mass spectrometry to calculate the bulk gas-phase properties (O:C and H:C) of the monoterpene oxidation systems as a function of oxidant concentrations. Bulk gas-phase properties can be compared to the SOA yields to assess the capability of the precursor gas-phase species to inform the SOA yields of each monoterpene oxidation system. We find that the extent of oxygenated precursor gas-phase species corresponds to SOA yield.

Tumour necrosis factor-alpha impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1.

PubMed

Keohane, Aoife; Ryan, Sinead; Maloney, Eimer; Sullivan, Aideen M; Nolan, Yvonne M

2010-01-01

Tumour necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine, which influences neuronal survival and function yet there is limited information available on its effects on hippocampal neural precursor cells (NPCs). We show that TNFalpha treatment during proliferation had no effect on the percentage of proliferating cells prepared from embryonic rat hippocampal neurosphere cultures, nor did it affect cell fate towards either an astrocytic or neuronal lineage when cells were then allowed to differentiate. However, when cells were differentiated in the presence of TNFalpha, significantly reduced percentages of newly born and post-mitotic neurons, significantly increased percentages of astrocytes and increased expression of TNFalpha receptors, TNF-R1 and TNF-R2, as well as expression of the anti-neurogenic Hes1 gene, were observed. These data indicate that exposure of hippocampal NPCs to TNFalpha when they are undergoing differentiation but not proliferation has a detrimental effect on their neuronal lineage fate, which may be mediated through increased expression of Hes1. Copyright 2009 Elsevier Inc. All rights reserved.

Past and Present Insights on Alpha-linolenic Acid and the Omega-3 Fatty Acid Family.

PubMed

Stark, Aliza H; Reifen, Ram; Crawford, Michael A

2016-10-25

Alpha-linolenic acid (ALA) is the parent essential fatty acid of the omega-3 family. This family includes docosahexaenoic acid (DHA), which has been conserved in neural signaling systems in the cephalopods, fish, amphibian, reptiles, birds, mammals, primates, and humans. This extreme conservation, in spite of wide genomic changes of over 500 million years, testifies to the uniqueness of this molecule in the brain and affirms the importance of omega-3 fatty acids. While DHA and its close precursor, eicosapentaenoic acids (EPA), have received much attention by the research community, ALA, as the precursor of both, has been considered of little interest. There are many papers on ALA requirements in experimental animals. Unlike humans, rats and mice can readily convert ALA to EPA and DHA, so it is unclear whether the effect is solely due to the conversion products or to ALA itself. The intrinsic role of ALA has yet to be defined. This paper will discuss both recent and historical findings related to this distinctive group of fatty acids, and will highlight the physiological significance of the omega-3 family.

Metformin is a novel suppressor for transforming growth factor (TGF)-β1

NASA Astrophysics Data System (ADS)

Xiao, Han; Zhang, Jianshu; Xu, Zhonghe; Feng, Yenan; Zhang, Mingliang; Liu, Jianli; Chen, Ruifei; Shen, Jing; Wu, Jimin; Lu, Zhizhen; Fang, Xiaohong; Li, Jingyuan; Zhang, Youyi

2016-06-01

Metformin is a widely used first-line antidiabetic drug that has been shown to protect against a variety of specific diseases in addition to diabetes, including cardiovascular disorders, polycystic ovary syndrome, and cancer. However, the precise mechanisms underlying the diverse therapeutic effects of metformin remain elusive. Here, we report that transforming growth factor-β1 (TGF-β1), which is involved in the pathogenesis of numerous diseases, is a novel target of metformin. Using a surface plasmon resonance-based assay, we identified the direct binding of metformin to TGF-β1 and found that metformin inhibits [125I]-TGF-β1 binding to its receptor. Furthermore, based on molecular docking and molecular dynamics simulations, metformin was predicted to interact with TGF-β1 at its receptor-binding domain. Single-molecule force spectroscopy revealed that metformin reduces the binding probability but not the binding force of TGF-β1 to its type II receptor. Consequently, metformin suppresses type II TGF-β1 receptor dimerization upon exposure to TGF-β1, which is essential for downstream signal transduction. Thus, our results indicate that metformin is a novel TGF-β suppressor with therapeutic potential for numerous diseases in which TGF-β1 hyperfunction is indicated.

PA700, the regulatory complex of the 26S proteasome, interferes with alpha-synuclein assembly.

PubMed

Ghee, Medeva; Melki, Ronald; Michot, Nadine; Mallet, Jacques

2005-08-01

Parkinson's disease is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway accompanied by the presence of intracellular cytoplasmic inclusions, termed Lewy bodies. Fibrillized alpha-synuclein forms the major component of Lewy bodies. We reported a specific interaction between rat alpha-synuclein and tat binding protein 1, a subunit of PA700, the regulatory complex of the 26S proteasome. It has been demonstrated that PA700 prevents the aggregation of misfolded, nonubiquinated substrates. In this study, we examine the effect of PA700 on the aggregation of wild-type and A53T mutant alpha-synuclein. PA700 inhibits both wild-type and A53T alpha-synuclein fibril formation as measured by Thioflavin T fluorescence. Using size exclusion chromatography, we present evidence for a stable PA700-alpha-synuclein complex. Sedimentation analyses reveal that PA700 sequesters alpha-synuclein in an assembly incompetent form. Analysis of the morphology of wild-type and A53T alpha-synuclein aggregates during the course of fibrillization by electron microscopy demonstrate the formation of amyloid-like fibrils. Secondary structure analyses of wild-type and A53T alpha-synuclein assembled in the presence of PA700 revealed a decrease in the overall amount of assembled alpha-synuclein with no significant change in protein conformation. Thus, PA700 acts on alpha-synuclein assembly and not on the structure of fibrils. We hypothesize that PA700 sequesters alpha-synuclein oligomeric species that are the precursors of the fibrillar form of the protein, thus preventing its assembly into fibrils.

TGF-β signaling is often attenuated during hepatotumorigenesis, but is retained for the malignancy of hepatocellular carcinoma cells.

PubMed

Mu, Xiaoxin; Lin, Shu; Yang, Junhua; Chen, Chen; Chen, Yun; Herzig, Maryanne C; Washburn, Kenneth; Halff, Glenn A; Walter, Christi A; Sun, Beicheng; Sun, Lu-Zhe

2013-01-01

The role of transforming growth factor-beta (TGF-β) signaling in hepatocarcinogenesis remains controversial. We aimed to reveal TGF-β signaling status in human and murine tissues of hepatocellular carcinoma (HCC) and the mechanisms that mediate TGF-β's role in regulating HCC malignancy. Here, TGF-β pathway component expression and activation in human and murine HCC tissues were measured with quantitative RT-PCR and Western blotting assays. The role of TGF-β receptor and Smad signaling in the growth and survival of several HCC cell lines was determined with several in vitro and in vivo approaches. We found that TGF-β receptor II (TβRII) expression was downregulated in two different HCC patient cohorts. Consistently, Smad3 phosphorylation was also downregulated in HCC tissues in comparison to that in adjacent normal tissues. Interestingly, many HCC cell lines were sensitive to TGF-β and growth-inhibited by exogenous TGF-β. However, stable knockdown of TβRII inhibited cell growth on plastic and in soft agar, and induced apoptosis resulting in suppressed subcutaneous tumor growth and metastatic potential in vivo. Furthermore, knockdown of Smad4 also led to a significant inhibition of growth on plastic and in soft agar with concomitant increase of apoptosis, PTEN expression, and reduced nuclear accumulation of linker region-phosphorylated Smad3. Taken together, TGF-β signaling pathway plays a dichotomous role in hepatocellular carcinogenesis. It appears to suppress HCC development, but is retained for HCC cell survival and malignancy. Furthermore, Smad4 can mediate both growth inhibitory activity induced by exogenous TGF-β and the survival activity induced by autocrine TGF-β revealing a delicate selection of the two opposing activities of TGF-β during HCC evolution.

TGF-β1 accelerates the DNA damage response in epithelial cells via Smad signaling

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

Lee, Jeeyong; Kim, Mi-Ra; Kim, Hyun-Ji

The evidence suggests that transforming growth factor-beta (TGF-β) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-β1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-β1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-β1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (γH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-β1 pretreatment. However, theymore » soon thereafter exhibited downregulation in TGF-β1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-β type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-β1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-β1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression. -- Highlights: •TGF-β1 pretreatment accelerates γ-radiation-induced DNA damage response. •TGF-β1-accelerated DNA damage response is dependent on Smad signaling and DNA Ligase IV. •TGF-β1 pretreatment protects epithelial cells from γ-radiation in vivo.« less

Silibinin inhibits triple negative breast cancer cell motility by suppressing TGF-β2 expression.

PubMed

Kim, Sangmin; Han, Jeonghun; Jeon, Myeongjin; You, Daeun; Lee, Jeongmin; Kim, Hee Jung; Bae, Sarang; Nam, Seok Jin; Lee, Jeong Eon

2016-08-01

Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that regulates many biological events including cell motility and angiogenesis. Here, we investigated the role of elevated TGF-β2 level in triple negative breast cancer (TNBC) cells and the inhibitory effect of silibinin on TGF-β2 action in TNBC cells. Breast cancer patients with high TGF-β2 expression have a poor prognosis. The levels of TGF-β2 expression increased significantly in TNBC cells compared with those in non-TNBC cells. In addition, cell motility-related genes such as fibronectin (FN) and matrix metalloproteinase-2 (MMP-2) expression also increased in TNBC cells. Basal FN, MMP-2, and MMP-9 expression levels decreased in response to LY2109761, a dual TGF-β receptor I/II inhibitor, in TNBC cells. TNBC cell migration also decreased in response to LY2109761. Furthermore, we observed that TGF-β2 augmented the FN, MMP-2, and MMP-9 expression levels in a time- and dose-dependent manner. In contrast, TGF-β2-induced FN, MMP-2, and MMP-9 expression levels decreased significantly in response to LY2109761. Interestingly, we found that silibinin decreased TGF-β2 mRNA expression level but not that of TGF-β1 in TNBC cells. Cell migration as well as basal FN and MMP-2 expression levels decreased in response to silibinin. Furthermore, silibinin significantly decreased TGF-β2-induced FN, MMP-2, and MMP-9 expression levels and suppressed the lung metastasis of TNBC cells. Taken together, these results suggest that silibinin suppresses metastatic potential of TNBC cells by inhibiting TGF-β2 expression in TNBC cells. Thus, silibinin may be a promising therapeutic drug to treat TNBC.

TGF-β1 stimulates movement of renal proximal tubular epithelial cells in a three-dimensional cell culture via an autocrine TGF-β2 production.

PubMed

Luo, Deyi; Guan, Qiunong; Wang, Kunjie; Nguan, Christopher Y C; Du, Caigan

2017-01-01

TGF-βs are multifunctional cytokines, but their roles in human renal homeostasis are not fully understood. This study investigated the role of TGF-β1 in the movement of human renal proximal tubular epithelial cells (PTECs) in a three-dimensional (3D) model. HKC-8 cells, a human PTEC line, were grown in a 3D collagen culture system. Cell movement was observed under a microscope. The gene expression was examined using PCR Arrays or qRT-PCR, and protein levels by Western blot. Here, we showed that the tight junction structure formed between adjacent cells of a HKC-8 cell colony in 3D cultures, and TGF-β1 stimulated their movement, evidenced by the appearance of fingerlike pseudopodia in the leader cells at the edge of the colonies. The cell movement of these human PTECs was correlated with up-regulation of both MMP2 and MMP9 and down-regulation or inactivation of PLAUR and PTK2B. Analysis of TGF-β signaling targets confirmed autocrine production of TGF-β2 and its cleaving enzyme furin as well as SNAI1 by TGF-β1stimulation. Knockdown of TGF-β2 expression disrupted TGF-β1-stimulated PTEC invasiveness, which was correlated with the down-regulation of MMP2 and MMP9. In conclusion, the activation of TGF-β receptor autocrine signaling by up-regulated TGF-β2 may play a pivotal role in TGF-β1-induced human PTEC movement, which could be mediated at least by both MMP2 and MMP9. Copyright © 2016 Elsevier Inc. All rights reserved.

Different roles of TGF-β in the multi-lineage differentiation of stem cells

PubMed Central

Wang, Ming-Ke; Sun, Hui-Qin; Xiang, Ying-Chun; Jiang, Fan; Su, Yong-Ping; Zou, Zhong-Min

2012-01-01

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells. Transforming growth factor β (TGF-β) family is a superfamily of growth factors, including TGF-β1, TGF-β2 and TGF-β3, bone morphogenetic proteins, activin/inhibin, and some other cytokines such as nodal, which plays very important roles in regulating a wide variety of biological processes, such as cell growth, differentiation, cell death. TGF-β, a pleiotropic cytokine, has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells, through the Smad pathway, non-Smad pathways including mitogen-activated protein kinase pathways, phosphatidylinositol-3-kinase/AKT pathways and Rho-like GTPase signaling pathways, and their cross-talks. For instance, it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells, immature cardiomyocytes, chondrocytes, neurocytes, hepatic stellate cells, Th17 cells, and dendritic cells. However, TGF-β inhibits the differentiation of stem cells into myotubes, adipocytes, endothelial cells, and natural killer cells. Additionally, TGF-β can provide competence for early stages of osteoblastic differentiation, but at late stages TGF-β acts as an inhibitor. The three mammalian isoforms (TGF-β1, 2 and 3) have distinct but overlapping effects on hematopoiesis. Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology, and will facilitate both basic research and clinical applications of stem cells. In this article, we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells. PMID:22993659

RAGE and TGF-β1 Cross-Talk Regulate Extracellular Matrix Turnover and Cytokine Synthesis in AGEs Exposed Fibroblast Cells

PubMed Central

Serban, Andreea Iren; Stanca, Loredana; Geicu, Ovidiu Ionut; Munteanu, Maria Cristina; Dinischiotu, Anca

2016-01-01

AGEs accumulation in the skin affects extracellular matrix (ECM) turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE) has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE’s proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications. PMID:27015414

RAGE and TGF-β1 Cross-Talk Regulate Extracellular Matrix Turnover and Cytokine Synthesis in AGEs Exposed Fibroblast Cells.

PubMed

Serban, Andreea Iren; Stanca, Loredana; Geicu, Ovidiu Ionut; Munteanu, Maria Cristina; Dinischiotu, Anca

2016-01-01

AGEs accumulation in the skin affects extracellular matrix (ECM) turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE) has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE's proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications.

Tannic acid attenuates TGF-β1-induced epithelial-to-mesenchymal transition by effectively intervening TGF-β signaling in lung epithelial cells.

PubMed

Pattarayan, Dhamotharan; Sivanantham, Ayyanar; Krishnaswami, Venkateshwaran; Loganathan, Lakshmanan; Palanichamy, Rajaguru; Natesan, Subramanian; Muthusamy, Karthikeyan; Rajasekaran, Subbiah

2018-03-01

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and an irreversible lung disorder characterized by the accumulation of fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) is thought to be one of the possible sources for a substantial increase in the number of fibroblasts/myofibroblasts in IPF lungs. Tannic acid (TA), a natural dietary polyphenolic compound has been shown to possess diverse pharmacological effects. However, whether TA can inhibit TGF-β1-mediated EMT in lung epithelial cells remains enigmatic. Both the human adenocarcinomic alveolar epithelial (A549) and normal bronchial epithelial (BEAS-2B) cells were treated with TGF-β1 with or without TA. Results showed that TA addition, markedly inhibited TGF-β1-induced EMT as assessed by reduced expression of N-cadherin, type-1-collagen, fibronectin, and vimentin. Furthermore, TA inhibited TGF-β1-induced cell proliferation through inducing cell cycle arrest at G0/G1 phase. TGF-β1-induced increase in the phosphorylation of Smad (Smad2 and 3), Akt as well as that of mitogen activated protein kinase (ERK1/2, JNK1/2, and p38) mediators was effectively inhibited by TA. On the other hand, TA reduced the TGF-β1-induced increase in TGF-β receptors expression. Using molecular docking approach, FTIR, HPLC and Western blot analyses, we further identified the direct binding of TA to TGF-β1. Finally, we conclude that TA might directly interact with TGF-β1, thereby repressing TGF-β signaling and subsequent EMT process in lung epithelial cells. Further animal studies are needed to clarify its potential therapeutic benefit in pulmonary fibrosis. © 2017 Wiley Periodicals, Inc.

Deuterium Enrichment of Amino and Hydroxy Acids Found in the Murchison Meteorite: Constraints on Parent Body Conditions

NASA Technical Reports Server (NTRS)

Lerner, Narcinda R.; Chang, Sherwood (Technical Monitor)

1997-01-01

The alpha-amino and alpha-hydroxy acids found in the Murchison carbonaceous chondrite are deuterium enriched. These compounds are thought to have originated from common deuterium enriched carbonyl precursors, by way of a Strecker synthesis which took place in a solution of HCN, NH3, and carbonyl compounds during the period of aqueous alteration of the meteorite parent body. However, the hydroxy acids found on Murchison are less deuterium enriched than the amino acids. With the objective of determining if the discrepancy in deuterium enrichment between the amino acids and the hydroxy acids found on Murchison is consistent with their formation in a Strecker synthesis, we have measured the deuterium content of alpha-amino and alpha-hydroxy acids produced in solutions of deuterated carbonyl compounds, KCN and NH4Cl, and also in mixtures of such solutions and Allende dust at 263 K and 295 K. Retention of the isotopic signature of the starting carbonyl by both alpha amino acids and alpha hydroxy acids is more dependent upon temperature, concentration and pH than upon the presence of meteorite dust in the solution. The constraints these observations place on Murchison parent body conditions will be discussed.

Potential of targeting TGF-β for organ transplant patients

PubMed Central

Iwashima, Makio; Love, Robert

2015-01-01

TGF-β was originally considered as an immunoregulatory cytokine, but accumulating data demonstrate that it also plays a critical role in development of effector immunity. Since TGF-β has a potent ability to alter immune responses, modulation of the TGF-β pathway for treatment of transplantation patients could be effective if carried out in a target selective manner. This review will focus on the role of TGF-β in T cell differentiation and discuss the prospect of TGF-β as the therapeutic target of lung transplantation acceptance. PMID:23464518

Activation of bean (Phaseolus vulgaris) [alpha]-amylase inhibitor requires proteolytic processing of the proprotein

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

Pueyo, J.J.; Hunt, D.C.; Chrispeels, M.J.

Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[submore » r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.« less

Prolonged peritoneal gene expression using a helper-dependent adenovirus.

PubMed

Liu, Limin; Shi, Chang-Xin; Ghayur, Ayesha; Zhang, Claire; Su, Je Yen; Hoff, Catherine M; Margetts, Peter J

2009-01-01

Encapsulating peritoneal sclerosis (EPS) is a rare complication of peritoneal dialysis. The causes of EPS are not well defined and are likely multifactorial. A suitable animal model would facilitate research into the pathophysiology and treatment of EPS. We developed a helper-dependent adenovirus that expresses both green fluorescent protein (GFP) and active transforming growth factor-beta (TGF-beta1; HDAdTGF-beta1). Mice were administered HDAdTGF-beta1 via intraperitoneal injection and the response was compared with mice administered either first-generation adenovirus expressing TGF-beta1 (AdTGF-beta1) or control adenovirus (AdGFP). HDAdTGF-beta1-treated mice continued to express the GFP reporter transgene to day 74, the end of the observation period. Transgene expression lasted less than 28 days in the animals treated with first-generation adenoviruses. Animals treated with first-generation AdTGF-beta1 demonstrated submesothelial thickening and angiogenesis at day 7, with almost complete resolution by day 28. The HDAdTGF-beta1-treated mice demonstrated progressive peritoneal fibrosis with adhesion formation and encapsulation of bowels. Weight gain was significantly reduced in animals treated with HDAdTGF-beta1 compared to both the control-treated animals and the AdTGF-beta1-treated animals. Inflammation was not a major component of the fibroproliferative response. Peritoneal administration of a first-generation AdTGF-beta1 leads to transient gene expression, resulting in a resolving fibrotic response and histology similar to that seen in simple peritoneal sclerosis. Prolonged TGF-beta1 expression induced by the helper-dependent HDAdTGF-beta1 led to changes in peritoneal morphology resembling EPS. This suggests that TGF-beta1 may be a contributing factor in both simple peritoneal sclerosis and EPS. This model will be useful for elucidation of the mechanism of EPS and evaluation of potential treatment.

Internode length in Pisum. Gene na may block gibberellin synthesis between ent-7. cap alpha. -hydroxykaurenoic acid and biggerellin A/sub 12/-aldehyde. [Pisum sativum

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

Ingram, T.J.; Reid, J.B.

1987-04-01

The elongation response of the gibberellin (GA) deficient genotypes na, ls, and lh of peas (Pisum sativum L.) to a range of GA-precursors was examined. Plants possessing gene na did not respond to precursors in the GA biosynthetic pathway prior to GA/sub 12/-aldehyde. In contrast, plants possessing lh and ls responded as well as wild-type plants (dwarfed with AMO-1618) to these compounds. The results suggest that GA biosynthesis is blocked prior to ent-kaurene in the lh and ls mutants and between ent-7..cap alpha..-hydroxykaurenoic acid and GA/sub 12/-aldehyde in the na mutant. Feeds of ent(/sup 3/H)kaurenoic acid and (/sup 2/H)GA/sub 12/-aldehydemore » to a range of genotypes supported the above conclusions. The na line WL1766 was shown by gas chromatography-mass spectrometry (GC-MS) to metabolize(/sup 2/H)GA/sub 12/-aldehyde to a number of (/sup 2/H)C/sub 19/-GAs including GA/sub 1/. However, there was no indication in na genotypes for the metabolism of ent-(/sup 3/H)kaurenoic acid to these GAs. In contrast, the expanding shoot tissue of all Na genotypes examined metabolized ent-(/sup 3/H)kaurenoic acid to radioactive compounds that co-chromatographed with GA/sub 1/, GA/sub 8/, GA/sub 20/, and GA/sub 29/. However, insufficient material was present for unequivocal identification of the metabolites. The radioactive profiles from HPLC of extracts of the node treated with ent-(/sup 3/H)kaurenoic acid were similar for both Na and na plants and contained ent-16..cap alpha..,17-dihydroxykaurenoic acid and ent-6..cap alpha..,7..cap alpha..,16..beta..,17-tetrahydroxykaurenoic acid (both characterized by GC-MS), suggesting that the metabolites arose from side branches of the main GA-biosynthetic pathway. Thus, both Na and na plants appear capable of ent-7..cap alpha..-hydroxylation.« less

TGF-β Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival

PubMed Central

Gingery, Anne; Bradley, Elizabeth W.; Pederson, Larry; Ruan, Ming; Horwood, Nikki J.; Oursler, Merry Jo

2008-01-01

To better understand the roles of TGF-β in bone metabolism, we investigated osteoclast survival in response TGF-β and found that TGF-β inhibited apoptosis. We examined the receptors involved in promotion of osteoclast survival and found that the canonical TGF-β receptor complex is involved in the survival response. The upstream MEK kinase TAK1 was rapidly activated following TGF-β treatment. Since osteoclast survival involves MEK, AKT, and NFκB activation, we examined TGF-β effects on activation of these pathways and observed rapid phosphorylation of MEK, AKT, IKK, IκB, and NFκB. The timing of activation coincided with SMAD activation and dominant negative SMAD expression did not inhibit NFκB activation, indicating that kinase pathway activation is independent of SMAD signaling. Inhibition of TAK1, MEK, AKT, NIK, IKK, or NFκB repressed TGF-β-mediated osteoclast survival. Adenoviral-mediated TAK1 or MEK inhibition eliminated TGF-β-mediated kinase pathway activation and constitutively active AKT expression overcame apoptosis induction following MEK inhibition. TAK1/MEK activation induces pro-survival BclXL expression and TAK1/MEK and SMAD pathway activation induces pro-survival Mcl-1 expression. These data show that TGF-β-induced NFκB activation is through TAK1/MEK-mediated AKT activation, which is essential for TGF-β to support of osteoclast survival. PMID:18586026

Asporin and transforming growth factor-beta gene expression in osteoblasts from subchondral bone and osteophytes in osteoarthritis.

PubMed

Sakao, Kei; Takahashi, Kenji A; Arai, Yuji; Saito, Masazumi; Honjyo, Kuniaki; Hiraoka, Nobuyuki; Kishida, Tsunao; Mazda, Osam; Imanishi, Jiro; Kubo, Toshikazu

2009-11-01

To clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism. Osteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-beta1, TGF-beta2, TGF-beta3, and Runx2 was analyzed using real-time RT-PCR. Expression of ASPN, TGF-beta1, and TGF-beta3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-beta1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage. The increased ratio of ASPN mRNA to TGF-beta1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-beta1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

Transforming growth factor-β synthesized by stromal cells and cancer cells participates in bone resorption induced by oral squamous cell carcinoma

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

Nakamura, Ryosuke; Department of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo; Kayamori, Kou

Transforming growth factor beta (TGF-β) plays a significant role in the regulation of the tumor microenvironment. To explore the role of TGF-β in oral cancer-induced bone destruction, we investigated the immunohistochemical localization of TGF-β and phosphorylated Smad2 (p-Smad2) in 12 surgical specimens of oral squamous cell carcinoma (OSCC). These studies revealed TGF-β and p-Smad2 expression in cancer cells in all tested cases. Several fibroblasts located between cancer nests and resorbing bone expressed TGF-β in 10 out of 12 cases and p-Smad2 in 11 out of 12 cases. Some osteoclasts also exhibited p ∼ Smad2 expression. The OSCC cell line, HSC3, and themore » bone marrow-derived fibroblastic cell line, ST2, synthesized substantial levels of TGF-β. Culture media derived from HSC3 cells could stimulate Tgf-β1 mRNA expression in ST2 cells. Recombinant TGF-β1 could stimulate osteoclast formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264 cells. TGF-β1 could upregulate the expression of p-Smad2 in RAW264 cells, and this action was suppressed by the addition of a neutralizing antibody against TGF-β or by SB431542. Transplantation of HSC3 cells onto the calvarial region of athymic mice caused bone destruction, associated with the expression of TGF-β and p-Smad2 in both cancer cells and stromal cells. The bone destruction was substantially inhibited by the administration of SB431542. The present study demonstrated that TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC-induced bone destruction. - Highlights: • Cancer cell, fibroblastic cells, and osteoclasts at bone resorbing area by oral cancer exhibited TGF-β and p-Smad2. • TGF-β1 stimulated osteoclastogenesis induced by RAKL in RAW264 cell. • Xenograft model of oral cancer-induced bone resorption was substantially inhibited by SB431542. • TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC-induced bone destruction.« less

Sustainability of CD24 expression, cell proliferation and migration, cisplatin-resistance, and caspase-3 expression during mesenchymal-epithelial transition induced by the removal of TGF-β1 in A549 lung cancer cells.

PubMed

Kim, Seong-Kwan; Park, Jin-A; Zhang, Dan; Cho, Sang-Hyun; Yi, Hee; Cho, Soo-Min; Chang, Byung-Joon; Kim, Jin-Suk; Shim, Jae-Han; Abd El-Aty, A M; Shin, Ho-Chul

2017-08-01

Epithelial-mesenchymal transition (EMT) is a notable mechanism underlying cancer cell metastasis. Transforming growth factor β1 (TGF-β1) has been used to induce EMT; however, there is a lack of information regarding the role of TGF-β1 in mesenchymal-epithelial transition (MET). In the present study, EMT was induced in A549 lung cancer cells using TGF-β1 (TGF-β1-treated group) and MET was induced sequentially from the TGF-β1-treated group by removing the TGF-β1 (MET/return group). Untreated A549 lung cancer cells were used as a control. Characteristic features, including cancer stem cell markers [cluster of differentiation (CD)24, CD44 and CD133], cell proliferation and migration and diverse intracellular mechanisms, were observed in all groups. Using western blot analysis, the TGF-β1-treated group demonstrated increased vimentin and reduced E-cadherin expression, whereas the MET/return group demonstrated the opposite trend. Among cancer stem cell markers, the population of CD24 low cells was reduced in the TGF-β1-treated group. Furthermore, the G2/M phase cell cycle population, cisplatin-sensitivity, and cell proliferation and migration ability were increased in the TGF-β1-treated group. These features were unaltered in the MET/return group when compared to the TGF-β1-treated group. Immunoblotting revealed an increase in the levels of SMAD3, phosphorylated SMAD3, phosphorylated extracellular signal-regulated kinase and caspase-3, and a decrease in active caspase-3 levels in the TGF-β1-treated group. Increased caspase-3 and reduced active caspase-3 levels were observed in the MET/return group, similar to those in the TGF-β1-treated group; however, levels of other signalling proteins were unchanged compared with the control group. EMT induced by TGF-β1 was not preserved; however, stemness-associated properties (CD24 expression, caspase-3 expression, cell proliferation and cisplatin-resistance) were sustained following removal of TGF-β1.

Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells.

PubMed

Huygens, Caroline; Liénart, Stéphanie; Dedobbeleer, Olivier; Stockis, Julie; Gauthy, Emilie; Coulie, Pierre G; Lucas, Sophie

2015-08-14

Production of active TGF-β1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-β1, favors its cleavage into latent inactive TGF-β1, induces the secretion and surface presentation of GARP·latent TGF-β1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-β1 complexes regulate TGF-β1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-β1, secretion of soluble latent TGF-β1, and surface presentation of GARP·TGF-β1 complexes by Tregs but does not contribute to TGF-β1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-β1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein.

PubMed

Stroschein, S L; Wang, W; Zhou, S; Zhou, Q; Luo, K

1999-10-22

Smad proteins mediate transforming growth factor-beta (TGF-beta) signaling to regulate cell growth and differentiation. The SnoN oncoprotein was found to interact with Smad2 and Smad4 and to repress their abilities to activate transcription through recruitment of the transcriptional corepressor N-CoR. Immediately after TGF-beta stimulation, SnoN is rapidly degraded by the nuclear accumulation of Smad3, allowing the activation of TGF-beta target genes. By 2 hours, TGF-beta induces a marked increase in SnoN expression, resulting in termination of Smad-mediated transactivation. Thus, SnoN maintains the repressed state of TGF-beta-responsive genes in the absence of ligand and participates in negative feedback regulation of TGF-beta signaling.

Association of β-catenin with P-Smad3 but not LEF-1 dissociates in vitro profibrotic from anti-inflammatory effects of TGF-β1.

PubMed

Tian, Xinrui; Zhang, Jianlin; Tan, Thian Kui; Lyons, J Guy; Zhao, Hong; Niu, Bo; Lee, So Ra; Tsatralis, Tania; Zhao, Ye; Wang, Ya; Cao, Qi; Wang, Changqi; Wang, Yiping; Lee, Vincent W S; Kahn, Michael; Zheng, Guoping; Harris, David C H

2013-01-01

Transforming growth factor β1 (TGF-β1) is known to be both anti-inflammatory and profibrotic. Cross-talk between TGF-β/Smad and Wnt/β-catenin pathways in epithelial-mesenchymal transition (EMT) suggests a specific role for β-catenin in profibrotic effects of TGF-β1. However, no such mechanistic role has been demonstrated for β-catenin in the anti-inflammatory effects of TGF-β1. In the present study, we explored the role of β-catenin in the profibrotic and anti-inflammatory effects of TGF-β1 by using a cytosolic, but not membrane, β-catenin knockdown chimera (F-TrCP-Ecad) and the β-catenin/CBP inhibitor ICG-001. TGF-β1 induced nuclear Smad3/β-catenin complex, but not β-catenin/LEF-1 complex or TOP-flash activity, during EMT of C1.1 (renal tubular epithelial) cells. F-TrCP-Ecad selectively degraded TGF-β1-induced cytoplasmic β-catenin and blocked EMT of C1.1 cells. Both F-TrCP-Ecad and ICG-001 blocked TGF-β1-induced Smad3/β-catenin and Smad reporter activity in C1.1 cells, suggesting that TGF-β1-induced EMT depends on β-catenin binding to Smad3, but not LEF-1 downstream of Smad3, through canonical Wnt. In contrast, in J774 macrophages, the β-catenin level was low and was not changed by interferon-γ (IFN-γ) or lipopolysaccharide (LPS) with or without TGF-β1. TGF-β1 inhibition of LPS-induced TNF-α and IFN-γ-stimulated inducible NO synthase (iNOS) expression was not affected by F-TrCP-Ecad, ICG-001 or by overexpression of wild-type β-catenin in J774 cells. Inhibition of β-catenin by either F-TrCP-Ecad or ICG-001 abolished LiCl-induced TOP-flash, but not TGF-β1-induced Smad reporter, activity in J774 cells. These results demonstrate for the first time that β-catenin is required as a co-factor of Smad in TGF-β1-induced EMT of C1.1 epithelial cells, but not in TGF-β1 inhibition of macrophage activation. Targeting β-catenin may dissociate the TGF-β1 profibrotic and anti-inflammatory effects.

GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP3+ regulatory T cells.

PubMed

Tran, Dat Q; Andersson, John; Wang, Rui; Ramsey, Heather; Unutmaz, Derya; Shevach, Ethan M

2009-08-11

TGF-beta family members are highly pleiotropic cytokines with diverse regulatory functions. TGF-beta is normally found in the latent form associated with latency-associated peptide (LAP). This latent complex can associate with latent TGFbeta-binding protein (LTBP) to produce a large latent form. Latent TGF-beta is also found on the surface of activated FOXP3(+) regulatory T cells (Tregs), but it is unclear how it is anchored to the cell membrane. We show that GARP or LRRC32, a leucine-rich repeat molecule of unknown function, is critical for tethering TGF-beta to the cell surface. We demonstrate that platelets and activated Tregs co-express latent TGF-beta and GARP on their membranes. The knockdown of GARP mRNA with siRNA prevented surface latent TGF-beta expression on activated Tregs and recombinant latent TGF-beta1 is able to bind directly with GARP. Confocal microscopy and immunoprecipitation strongly support their interactions. The role of TGF-beta on Tregs appears to have dual functions, both for Treg-mediated suppression and infectious tolerance mechanism.

GARP (LRRC32) is essential for the surface expression of latent TGF-β on platelets and activated FOXP3+ regulatory T cells

PubMed Central

Tran, Dat Q.; Andersson, John; Wang, Rui; Ramsey, Heather; Unutmaz, Derya; Shevach, Ethan M.

2009-01-01

TGF-β family members are highly pleiotropic cytokines with diverse regulatory functions. TGF-β is normally found in the latent form associated with latency-associated peptide (LAP). This latent complex can associate with latent TGFβ-binding protein (LTBP) to produce a large latent form. Latent TGF-β is also found on the surface of activated FOXP3+ regulatory T cells (Tregs), but it is unclear how it is anchored to the cell membrane. We show that GARP or LRRC32, a leucine-rich repeat molecule of unknown function, is critical for tethering TGF-β to the cell surface. We demonstrate that platelets and activated Tregs co-express latent TGF-β and GARP on their membranes. The knockdown of GARP mRNA with siRNA prevented surface latent TGF-β expression on activated Tregs and recombinant latent TGF-β1 is able to bind directly with GARP. Confocal microscopy and immunoprecipitation strongly support their interactions. The role of TGF-β on Tregs appears to have dual functions, both for Treg-mediated suppression and infectious tolerance mechanism. PMID:19651619

TGF-beta1 expression in EL4 lymphoma cells overexpressing growth hormone.

PubMed

Farmer, John T; Weigent, Douglas A

2006-03-01

Our previous studies show that growth hormone overexpression (GHo) upregulates the expression of the IGF-1R and IGF-2R resulting in the protection of the EL4 lymphoma cell line from apoptosis. In this study, we report that GHo also increases TGF-beta1 protein expression measured by luciferase promoter assay, Western analysis, and ELISA. Further, the data show that antibody to TGF-betaR2 decreases TGF-beta1 promoter activity to the level of vector alone control cells. GHo cells treated with (125)I-rh-latent TGF-beta1 showed increased activation of latent TGF-beta1 as measured by an increase in the active 24kDa, TGF-beta1 compared to vector alone control cells. The ability of endogenous GH to increase TGF-beta1 expression is blocked in EL4 cells by antisense but not sense oligodeoxynucleotides or in cells cultured with antibody to growth hormone (GH). The data suggest that endogenous GH may protect from apoptosis through the IGF-1R receptor while limiting cellular growth through increased expression and activation of TGF-beta1.

TGF-β Tumor Suppression through a Lethal EMT.

PubMed

David, Charles J; Huang, Yun-Han; Chen, Mo; Su, Jie; Zou, Yilong; Bardeesy, Nabeel; Iacobuzio-Donahue, Christine A; Massagué, Joan

2016-02-25

TGF-β signaling can be pro-tumorigenic or tumor suppressive. We investigated this duality in pancreatic ductal adenocarcinoma (PDA), which, with other gastrointestinal cancers, exhibits frequent inactivation of the TGF-β mediator Smad4. We show that TGF-β induces an epithelial-mesenchymal transition (EMT), generally considered a pro-tumorigenic event. However, in TGF-β-sensitive PDA cells, EMT becomes lethal by converting TGF-β-induced Sox4 from an enforcer of tumorigenesis into a promoter of apoptosis. This is the result of an EMT-linked remodeling of the cellular transcription factor landscape, including the repression of the gastrointestinal lineage-master regulator Klf5. Klf5 cooperates with Sox4 in oncogenesis and prevents Sox4-induced apoptosis. Smad4 is required for EMT but dispensable for Sox4 induction by TGF-β. TGF-β-induced Sox4 is thus geared to bolster progenitor identity, whereas simultaneous Smad4-dependent EMT strips Sox4 of an essential partner in oncogenesis. Our work demonstrates that TGF-β tumor suppression functions through an EMT-mediated disruption of a lineage-specific transcriptional network. Copyright © 2016 Elsevier Inc. All rights reserved.

TGF-β Tumor Suppression Through A Lethal EMT

PubMed Central

David, Charles J.; Huang, Yun-Han; Chen, Mo; Su, Jie; Zou, Yilong; Bardeesy, Nabeel; Iacobuzio-Donahue, Christine A.; Massagué, Joan

2016-01-01

TGF-β signaling can be pro-tumorigenic or tumor suppressive. We investigated this duality in pancreatic ductal adenocarcinoma (PDA), which, with other gastrointestinal cancers, exhibits frequent inactivation of the TGF-β mediator Smad4. We show that TGF-β induces an epithelial-mesenchymal transition (EMT), generally considered a pro-tumorigenic event. However, in TGF-β sensitive PDA cells, EMT becomes lethal by converting TGF-β-induced Sox4 from an enforcer of tumorigenesis into a promoter of apoptosis. This is the result of an EMT-linked remodeling of the cellular transcription factor landscape, including the repression of the gastrointestinal lineage-master regulator Klf5. Klf5 cooperates with Sox4 in oncogenesis and prevents Sox4-induced apoptosis. Smad4 is required for EMT but dispensable for Sox4 induction by TGF-β. TGF-β-induced Sox4 is thus geared to bolster progenitor identity, while simultaneous Smad4-dependent EMT strips Sox4 of an essential partner in oncogenesis. Our work demonstrates that TGF-β tumor suppression functions through an EMT-mediated disruption of a lineage-specific transcriptional network. PMID:26898331

TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

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

Ebi, Masahide; Kataoka, Hiromi, E-mail: hkataoka@med.nagoya-cu.ac.jp; Shimura, Takaya

2010-11-19

Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cellmore » growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGF{beta} enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells. Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGF{beta} might be an important pathway of gastric cancer cell proliferation by TGF{beta}.« less

Recognition of unusual presentation of natural killer cell leukemia.

PubMed

Gardiner, C M; Reen, D J; O'Meara, A

1995-10-01

Expansion of the natural killer (NK) subset of lymphocytes represents a rare leukemia phenotype with variations in clinical presentation, morphology, surface phenotype, and effector function. This paper reports on a 5-year-old male patient who had an unusual presentation of an NK cell leukemia that was initially diagnosed as neuroblastoma. A bone marrow (BM) aspirate showed clumps of undifferentiated cells with the following phenotype: CD56bright+, CD33dim+, CD45-, CD2-, CD19-, CD16-, and CD57-. Cytochemistry was noncontributory. The patient, having failed to respond to conventional neuroblastoma chemotherapy, was subsequently diagnosed as having NK cell leukemia based on functional in vitro assays. The patient responded to acute lymphoblastic leukemia (ALL) chemotherapy but relapsed 4 weeks into treatment and eventually died 25 weeks after initial presentation. The cell surface phenotype observed is consistent with a rare NK cell subset, the biology of which has not been well defined. Freshly isolated BM cells killed K562 cells in a conventional 51Cr-release assay. Both interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) induced LAK activity against the Daudi cell line. IL-2 induced proliferation of the leukemic cells. TNF-alpha, IFN-gamma, IL-6, IL-1ra, and TGF-beta levels were assessed and found to be concentrated in BM, in contrast to plasma samples. TNF-alpha was present at a high concentration in BM (150.9 pg/ml), probably a reflection of the associated disease pathology of severe bone pain and pyrexia. In summary, this paper details clinical and laboratory investigations of a leukemia of a rare NK cell subset.

Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma.

PubMed

Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-Jun; Yoshida, Takeshi; Funa, Keiko

2016-05-01

TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. Copyright © 2016. Published by Elsevier Inc.

Relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-beta1 levels.

PubMed

Hueso, Miguel; Navarro, Estanis; Moreso, Francesc; Beltrán-Sastre, Violeta; Ventura, Francesc; Grinyó, Josep M; Serón, Daniel

2006-05-27

Transforming growth factor (TGF)-beta(1) is increased in allograft rejection and its production is associated with single nucleotide polymorphisms (SNPs). The contribution of SNPs at codons 10 and 25 of the TGF-beta(1) gene to renal allograft damage was assessed in 6-month protocol biopsies and their association with TGF-beta(1) production. TGF-beta(1) genotypes were evaluated by polymerase chain reaction (PCR)/restriction fragment length polymorphism. Intragraft TGF-beta(1) messenger RNA (mRNA) was measured by real-time PCR and TGF-beta(1) plasma levels were assessed by enzyme-linked immunosorbent assay. Eighty consecutive patients were included. Allele T at codon 10 (risk ratio, 6.7; P = 0.02) and an episode of acute rejection before protocol biopsy (risk ratio, 6.2; P = 0.01) were independent predictors of subclinical rejection (SCR). TGF-beta(1) plasma levels, but not those of TGF-beta(1) mRNA, were increased in patients with SCR (2.59 ng/mL +/- 0.91 [n = 22] vs. 2.05 ng/mL +/- 0.76 [n = 43]; P = 0.01). There was no association between allele T and TGF-beta(1) plasma or intragraft levels. Allele T at codon 10 of the TGF-beta(1) gene is associated with a higher incidence of SCR.

Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages

NASA Technical Reports Server (NTRS)

Chong, H.; Vodovotz, Y.; Cox, G. W.; Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)

1999-01-01

Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

The antifibrotic effects of TGF-{beta}1 siRNA on hepatic fibrosis in rats

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

Lang, Qing; Liu, Qi; Xu, Ning

2011-06-10

Highlights: {yields} We constructed CCL4 induced liver fibrosis model successfully. {yields} We proofed that the TGF-{beta}1 siRNA had a definite therapy effect to CCL4 induced liver fibrosis. {yields} The therapy effect of TGF-{beta}1 siRNA had dose-dependent. -- Abstract: Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-{beta}1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-{beta}1 in hepatic fibrosis and its mechanismmore » in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-{beta}1 siRNA 0.125 mg/kg treatment group, TGF-{beta}1 siRNA 0.25 mg/kg treatment group and TGF-{beta}1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-{beta}1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-{beta}1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-{beta}1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-{beta}1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-{beta}1 siRNA negative control group and the TGF-{beta}1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-{beta}1, type I collagen and type III collagen (P < 0.01). Conclusions: Using siRNA to target TGF-{beta}1 can inhibit the expression of TGF-{beta}1 and attenuate rat hepatic fibrosis induced by a high-fat diet and CCL4. A possible mechanism is through the down-regulation of TGF-{beta}1 expression, which could inhibit HSC activation, as well as the proliferation and collagen production of collagen reducing, so that collagen deposition in the liver is reduced.« less

Inhibition of the αvβ6 integrin leads to limited alteration of TGF-α-induced pulmonary fibrosis

PubMed Central

Madala, Satish K.; Korfhagen, Thomas R.; Schmidt, Stephanie; Davidson, Cynthia; Edukulla, Ramakrishna; Ikegami, Machiko; Violette, Shelia M.; Weinreb, Paul H.; Sheppard, Dean

2014-01-01

A number of growth factors and signaling pathways regulate matrix deposition and fibroblast proliferation in the lung. The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-β (TGF-β) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGF-α) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGF-α specifically in the lung epithelium develop progressive fibrosis accompanied with cachexia, changes in lung mechanics, and marked pleural thickening. Although recent studies demonstrate that EGFR activation modulates the fibroproliferative effects involved in the pathogenesis of TGF-β induced pulmonary fibrosis, in converse, the direct role of EGFR induction of the TGF-β pathway in the lung is unknown. The αvβ6 integrin is an important in vivo activator of TGF-β activation in the lung. Immunohistochemical analysis of αvβ6 protein expression and bronchoalveolar analysis of TGF-β pathway signaling indicates activation of the αvβ6/TGF-β pathway only at later time points after lung fibrosis was already established in the TGF-α model. To determine the contribution of the αvβ6/TGF-β pathway on the progression of established fibrotic disease, TGF-α transgenic mice were administered Dox for 4 wk, which leads to extensive fibrosis; these mice were then treated with a function-blocking anti-αvβ6 antibody with continued administration of Dox for an additional 4 wk. Compared with TGF-α transgenic mice treated with control antibody, αvβ6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the β6 integrin, TGF-α transgenic mice were mated with β6-null mice and the degree of fibrosis was compared in adult mice following 8 wk of Dox administration. Genetic ablation of the β6 integrin attenuated histological and physiological changes in the lungs of TGF-α transgenic mice although a significant degree of fibrosis still developed. In summary, inhibition of the β6 integrin led to a modest, albeit significant, effect on pleural thickening and lung function decline observed with TGF-α-induced pulmonary fibrosis. These data support activation of the αvβ6/TGF-β pathway as a secondary effect contributing to TGF-α-induced pleural fibrosis and suggest a complex contribution of multiple mediators to the maintenance of progressive fibrosis in the lung. PMID:24508732

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

PubMed

Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

2009-08-07

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

Comparative Effects of Triflusal, S-Adenosylmethionine, and Dextromethorphan over Intestinal Ischemia/Reperfusion Injury

PubMed Central

Cámara-Lemarroy, Carlos R.; Guzmán-de la Garza, Francisco J.; Cordero-Pérez, Paula; Alarcón-Galván, Gabriela; Torres-Gonzalez, Liliana; Muñoz-Espinosa, Linda E.; Fernández-Garza, Nancy E.

2011-01-01

Ischemia/reperfusion (I/R) is a condition that stimulates an intense inflammatory response. No ideal treatment exists. Triflusal is an antiplatelet salicylate derivative with anti-inflammatory effects. S-adenosylmethionine is a metabolic precursor for glutathione, an endogenous antioxidant. Dextromethorphan is a low-affinity N-methyl-D-aspartate receptor inhibitor. There is evidence that these agents modulate some of the pathways involved in I/R physiopathology. Intestinal I/R was induced in rats by clamping the superior mesenteric artery for 60 minutes, followed by 60 minutes of reperfusion. Rats either received saline or the drugs studied. At the end of the procedure, serum concentrations of tumor necrosis factor-alpha (TNF-alpha), malonaldehyde (MDA), and total antioxidant capacity (TAC) were determined and intestinal morphology analyzed. I/R resulted in tissue damage, serum TNF-alpha and MDA elevations, and depletion of TAC. All drugs showed tissue protection. Only triflusal reduced TNF-alpha levels. All drugs lowered MDA levels, but only triflusal and S-adenosylmethionine maintained the serum TAC. PMID:22125445

The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan.

PubMed

Tsui, Ping; Higazi, Daniel R; Wu, Yanli; Dunmore, Rebecca; Solier, Emilie; Kasali, Toyin; Bond, Nicholas J; Huntington, Catherine; Carruthers, Alan; Hood, John; Borrok, M Jack; Barnes, Arnita; Rickert, Keith; Phipps, Sandrina; Shirinian, Lena; Zhu, Jie; Bowen, Michael A; Dall'Acqua, William; Murray, Lynne A

2017-01-01

Excessive transforming growth factor (TGF)-β is associated with pro-fibrotic responses in lung disease, yet it also plays essential roles in tissue homeostasis and autoimmunity. Therefore, selective inhibition of excessive and aberrant integrin-mediated TGF-β activation via targeting the α-v family of integrins is being pursued as a therapeutic strategy for chronic lung diseases, to mitigate any potential safety concerns with global TGF-β inhibition. In this work, we reveal a novel mechanism of inhibiting TGF-β activation utilized by an αvβ8 targeting antibody, 37E1B5. This antibody blocks TGF-β activation while not inhibiting cell adhesion. We show that an N-linked complex-type Fab glycan in H-CDR2 of 37E1B5 is directly involved in the inhibition of latent TGF-β activation. Removal of the Fab N-glycosylation site by single amino acid substitution, or removal of N-linked glycans by enzymatic digestion, drastically reduced the antibody's ability to inhibit latency-associated peptide (LAP) and αvβ8 association, and TGF-β activation in an αvβ8-mediated TGF-β signaling reporter assay. Our results indicate a non-competitive, allosteric inhibition of 37E1B5 on αvβ8-mediated TGF-β activation. This unique, H-CDR2 glycan-mediated mechanism may account for the potent but tolerable TGF-b activation inhibition and lack of an effect on cellular adhesion by the antibody.

Transforming growth factor-beta inhibits human antigen-specific CD4+ T cell proliferation without modulating the cytokine response.

PubMed

Tiemessen, Machteld M; Kunzmann, Steffen; Schmidt-Weber, Carsten B; Garssen, Johan; Bruijnzeel-Koomen, Carla A F M; Knol, Edward F; van Hoffen, Els

2003-12-01

Transforming growth factor (TGF)-beta has been demonstrated to play a key role in the regulation of the immune response, mainly by its suppressive function towards cells of the immune system. In humans, the effect of TGF-beta on antigen-specific established memory T cells has not been investigated yet. In this study antigen-specific CD4(+) T cell clones (TCC) were used to determine the effect of TGF-beta on antigen-specific proliferation, the activation status of the T cells and their cytokine production. This study demonstrates that TGF-beta is an adequate suppressor of antigen-specific T cell proliferation, by reducing the cell-cycle rate rather than induction of apoptosis. Addition of TGF-beta resulted in increased CD69 expression and decreased CD25 expression on T cells, indicating that TGF-beta is able to modulate the activation status of in vivo differentiated T cells. On the contrary, the antigen-specific cytokine production was not affected by TGF-beta. Although TGF-beta was suppressive towards the majority of the T cells, insensitivity of a few TCC towards TGF-beta was also observed. This could not be correlated to differential expression of TGF-beta signaling molecules such as Smad3, Smad7, SARA (Smad anchor for receptor activation) and Hgs (hepatocyte growth factor-regulated tyrosine kinase substrate). In summary, TGF-beta has a pronounced inhibitory effect on antigen-specific T cell proliferation without modulating their cytokine production.

Transforming growth factor-β (TGF-β) expression is increased in the subsynovial connective tissues of patients with idiopathic carpal tunnel syndrome.

PubMed

Chikenji, Takako; Gingery, Anne; Zhao, Chunfeng; Passe, Sandra M; Ozasa, Yasuhiro; Larson, Dirk; An, Kai-Nan; Amadio, Peter C

2014-01-01

Non-inflammatory fibrosis of the subsynovial connective tissue (SSCT) is a hallmark of carpal tunnel syndrome (CTS). The etiology of this finding and its relationship to the development of CTS remain poorly understood. Recent studies have found that transforming growth factor-β (TGF-β) plays a central role in fibrosis. The purpose of this study was to investigate the expression of TGF-β and connective tissue growth factor (CTGF), a downstream mediator of TGF-β, in the pathogenesis of CTS. We compared SSCT specimens from 26 idiopathic CTS patients with specimens from 10 human cadaver controls with no previous diagnosis of CTS. Immunohistochemistry was performed to determine levels TGF-β1, CTGF, collagen 1(Col1) and collagen 3 (Col3) expression. TGF-β1 (p < 0.01), CTGF (p < 0.01), and Col3 (p < 0.01) were increased in SSCT of CTS patients compared with control tissue. In addition, a strong positive correlation was found between TGF-β1 and CTGF, (R(2) = 0.80, p < 0.01) and a moderate positive correlation between Col3 and TGF-β1 (R(2) = 0.49, p < 0.01). These finding suggest that there is an increased expression of TGF-β and CTGF, a TGF-β regulated protein, and that this TGF-β activation may be responsible for SSCT fibrosis in CTS patients. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

TGF-β1 stimulates migration of type II endometrial cancer cells by down-regulating PTEN via activation of SMAD and ERK1/2 signaling pathways.

PubMed

Xiong, Siyuan; Cheng, Jung-Chien; Klausen, Christian; Zhao, Jianfang; Leung, Peter C K

2016-09-20

PTEN acts as a tumor suppressor primarily by antagonizing the PI3K/AKT signaling pathway. PTEN is frequently mutated in human cancers; however, in type II endometrial cancers its mutation rate is very low. Overexpression of TGF-β1 and its receptors has been reported to correlate with metastasis of human cancers and reduced survival rates. Although TGF-β1 has been shown to regulate PTEN expression through various mechanisms, it is not yet known if the same is true in type II endometrial cancer. In the present study, we show that treatment with TGF-β1 stimulates the migration of two type II endometrial cancer cell lines, KLE and HEC-50. In addition, TGF-β1 treatment down-regulates both mRNA and protein levels of PTEN. Overexpression of PTEN or inhibition of PI3K abolishes TGF-β1-stimulated cell migration. TGF-β1 induces SMAD2/3 phosphorylation and knockdown of common SMAD4 inhibits the suppressive effects of TGF-β1 on PTEN mRNA and protein. Interestingly, TGF-β1 induces ERK1/2 phosphorylation and pre-treatment with a MEK inhibitor attenuates the suppression of PTEN protein, but not mRNA, by TGF-β1. This study provides important insights into the molecular mechanisms mediating TGF-β1-induced down-regulation of PTEN and demonstrates an important role of PTEN in the regulation of type II endometrial cancer cell migration.

TGF-beta1 modulates focal adhesion kinase expression in rat intestinal epithelial IEC-6 cells via stimulatory and inhibitory Smad binding elements.

PubMed

Walsh, Mary F; Ampasala, Dinakar R; Rishi, Arun K; Basson, Marc D

2009-02-01

TGF-beta and FAK modulate cell migration, differentiation, proliferation and apoptosis, and TGF-beta promotes FAK transcription in intestinal epithelial cells via Smad-dependent and independent pathways. We utilized a 1320 bp FAK promoter-luciferase construct to characterize basal and TGF-beta-mediated FAK gene transcription in IEC-6 cells. Inhibiting JNK or Akt negated TGF-beta-stimulated promoter activity; ERK inhibition did not block the TGF-beta effect but increased basal activity. Co-transfection with Co-Smad4 enhanced the TGF-beta response while the inhibitory Smad7 abolished it. Serial deletions sequentially removing the four Smad binding elements (SBE) in the 5' untranslated region of the promoter revealed that the two most distal SBE's are positive regulators while SBE3 exerts a negative influence. Mutational deletion of two upstream p53 sites enhanced basal but did not affect TGF-beta-stimulated increases in promoter activity. TGF-beta increased DNA binding of Smad4, phospho-Smad2/3 and Runx1/AML1a to the most distal 435 bp containing 3 SBE and 2 AML1a sites by ChIP assay. However, although point mutation of SBE1 ablated the TGF-beta-mediated rise in SV40-promoter activity, mutation of AML1a sites did not. TGF-beta regulation of FAK transcription reflects a complex interplay between positive and negative non-Smad signals and SBE's, the last independent of p53 or AML1a.

Possible role of TIEG1 as a feedback regulator of myostatin and TGF-{beta} in myoblasts

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

Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke

2010-03-19

Myostatin and TGF-{beta} negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-{beta} signaling remains unclear. TGF-{beta} inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-{beta} signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-{beta} signaling using C2C12 myoblasts. Myostatin and TGF-{beta} induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated atmore » a late stage of differentiation. In contrast, TGF-{beta} enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-{beta} in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-{beta}. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-{beta} that prevents excess action in myoblasts.« less

Noncanonical transforming growth factor β signaling in scleroderma fibrosis

PubMed Central

Trojanowska, Maria

2014-01-01

Purpose of review Persistent transforming growth factor β (TGF-β) signaling is the major factor contributing to scleroderma (SSc) fibrosis. This review will summarize recent progress on the noncanonical TGF-β signaling pathways and their role in SSc fibrosis. Recent findings Canonical TGF-β signaling involves activation of the TGF-β receptors and downstream signal transducers Smad2/3. The term noncanonical TGF-β signaling includes a variety of intracellular signaling pathways activated by TGF-β independently of Smad2/3 activation. There is evidence that these pathways play important role in SSc fibrosis. In a subset of SSc fibroblasts, a multiligand receptor complex consisting of TGF-β and CCN2 receptors drives constitutive activation of the Smad1 pathway. CCN2 is also a primary effector of this pathway, thus establishing an autocrine loop that amplifies TGF-β signaling. SSc fibroblasts also demonstrate reduced expression of endogenous antagonists of TGF-β signaling including transcriptional repressors, Friend leukemia integration-1 and perixosome proliferator-activated receptor-γ, as well as inhibitor of Smad3 phosphorylation, PTEN. PTEN is a key mediator of the cross-talk between the sphingosine kinase and the TGF-β pathways. Summary Discovery of the role of noncanonical TGF-β signaling in fibrosis offers new molecular targets for the antifibrotic therapies. Due to the heterogeneous nature of SSc, knowledge of these pathways could help to tailor the therapy to the individual patient depending on the activation status of a specific profibrotic pathway. PMID:19713852

Role of TGF-β signaling in curcumin-mediated inhibition of tumorigenicity of human lung cancer cells

PubMed Central

Datta, Raktima; Halder, Sunil K.

2014-01-01

Purpose Curcumin has been shown to have potent anti-cancer activities like inhibition of cell proliferation, induction of apoptosis, and suppression of angiogenesis. Transforming growth factor-β (TGF-β) signaling plays a complex role in tumor suppression and promotion depending on the tumor type and stage. However, the effect of curcumin on TGF-β signaling in cancer cells and the role of TGF-β signaling in curcumin-induced anticancer activities have not been determined. Here, we investigate the role of curcumin on TGF-β signaling, and whether TGF-β signaling is involved in the antitumor activities of curcumin. Methods Human non-small cell lung cancer (NSCLC) cell lines, ACC-LC-176 (without TGF-β signaling), H358, and A549 (with TGF-β signaling) were treated with curcumin to determine cell growth, apoptosis, and tumorigenicity. Antitumor activities of curcumin were determined using these cell lines and an in vivo mouse model. We also tested the effect of curcumin on TGF-β/Smad signaling by western blotting and by luciferase assays. Results Curcumin inhibited cell growth and induced apoptosis of all three NSCLC cell lines in vitro and in vivo. It significantly reduced subcutaneous tumor growth by these three cell lines irrespective of TGF-β signaling status. Curcumin inhibited TGF-β-induced Smad2/3 phosphorylation and transcription in H358 and A549 cells, but not in ACC-LC-176 cells. Conclusions Curcumin reduces tumorigenicity of human lung cancer cells in vitro and in vivo by inhibiting cell proliferation and promoting apoptosis. These results suggest that TGF-β signaling is not directly involved in curcumin-mediated growth inhibition, induction of apoptosis, and inhibition of tumorigenicity. PMID:23224523

Smad phosphoisoform signaling specificity: the right place at the right time.

PubMed

Matsuzaki, Koichi

2011-11-01

Transforming growth factor (TGF)-β antagonizes mitogenic Ras signaling during epithelial regeneration, but TGF-β and Ras act synergistically in driving tumor progression. Insights into these apparently contradictory effects have come from recent detailed analyses of the TGF-β signaling process. Here, we summarize the different modes of TGF-β/Ras signaling in normal epithelium and neoplasms and show how perturbation of TGF-β signaling by Ras may contribute to a shift from tumor-suppressive to protumorigenic TGF-β activity during tumor progression. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad homology (MH) 1 and MH2 domains. TGF-β Type I receptor and Ras-associated kinases differentially phosphorylate Smad2 and Smad3 to create C-terminally (C), linker (L) or dually (L/C) phosphorylated (p) isoforms. In epithelial homeostasis, TGF-β-mediated pSmad3C signaling opposes proliferative responses induced by mitogenic signals. During carcinogenesis, activation of cytoplasmic Ras-associated kinases including mitogen-activated protein kinase confers a selective advantage on benign tumors by shifting Smad3 signaling from a tumor-suppressive pSmad3C to an oncogenic pSmad3L pathway, leading to carcinoma in situ. Finally, at the edges of advanced carcinomas invading adjacent tissues, nuclear Ras-associated kinases such as cyclin-dependent kinases, together with cytoplasmic kinases, alter TGF-β signals to more invasive and proliferative pSmad2L/C and pSmad3L/C signaling. Taken together, TGF-β signaling specificity arises from spatiotemporal dynamics of Smad phosphoisoforms. Based on these findings, we have reason to hope that pharmacologic inhibition of linker phosphorylation might suppress progression to human advanced carcinomas by switching from protumorigenic to tumor-suppressive TGF-β signaling.

Differential role of PTEN in transforming growth factor β (TGF-β) effects on proliferation and migration in prostate cancer cells.

PubMed

Kimbrough-Allah, Mawiyah N; Millena, Ana C; Khan, Shafiq A

2018-04-01

Transforming growth factor-β (TGF-β) acts as a tumor suppressor in normal epithelial cells but as a tumor promoter in advanced prostate cancer cells. PI3-kinase pathway mediates TGF-β effects on prostate cancer cell migration and invasion. PTEN inhibits PI3-kinase pathway and is frequently mutated in prostate cancers. We investigated possible role(s) of PTEN in TGF-β effects on proliferation and migration in prostate cancer cells. Expression of PTEN mRNA and proteins were determined using RT-PCR and Western blotting in RWPE1 and DU145 cells. We also studied the role of PTEN in TGF-β effects on cell proliferation and migration in DU145 cells after transient silencing of endogenous PTEN. Conversely, we determined the role of PTEN in cell proliferation and migration after over-expression of PTEN in PC3 cells which lack endogenous PTEN. TGF-β1 and TGF-β3 had no effect on PTEN mRNA levels but both isoforms increased PTEN protein levels in DU145 and RWPE1 cells indicating that PTEN may mediate TGF-β effects on cell proliferation. Knockdown of PTEN in DU145 cells resulted in significant increase in cell proliferation which was not affected by TGF-β isoforms. PTEN overexpression in PC3 cells inhibited cell proliferation. Knockdown of endogenous PTEN enhanced cell migration in DU145 cells, whereas PTEN overexpression reduced migration in PC3 cells and reduced phosphorylation of AKT in response to TGF-β. We conclude that PTEN plays a role in inhibitory effects of TGF-β on cell proliferation whereas its absence may enhance TGF-β effects on activation of PI3-kinase pathway and cell migration. © 2018 Wiley Periodicals, Inc.

Leiomyoma-derived transforming growth factor-β impairs bone morphogenetic protein-2-mediated endometrial receptivity.

PubMed

Doherty, Leo F; Taylor, Hugh S

2015-03-01

To determine whether transforming growth factor (TGF)-β3 is a paracrine signal secreted by leiomyoma that inhibits bone morphogenetic protein (BMP)-mediated endometrial receptivity and decidualization. Experimental. Laboratory. Women with symptomatic leiomyomas. Endometrial stromal cells (ESCs) and leiomyoma cells were isolated from surgical specimens. Leiomyoma-conditioned media (LCM) was applied to cultured ESC. The TGF-β was blocked by two approaches: TGF-β pan-specific antibody or transfection with a mutant TGF-β receptor type II. Cells were then treated with recombinant human BMP-2 to assess BMP responsiveness. Expression of BMP receptor types 1A, 1B, 2, as well as endometrial receptivity mediators HOXA10 and leukemia inhibitory factor (LIF). Enzyme-linked immunosorbent assay showed elevated TGF-β levels in LCM. LCM treatment of ESC reduced expression of BMP receptor types 1B and 2 to approximately 60% of pretreatment levels. Preincubation of LCM with TGF-β neutralizing antibody or mutant TGF receptor, but not respective controls, prevented repression of BMP receptors. HOXA10 and LIF expression was repressed in recombinant human BMP-2 treated, LCM exposed ESC. Pretreatment of LCM with TGF-β antibody or transfection with mutant TGF receptor prevented HOXA10 and LIF repression. Leiomyoma-derived TGF-β was necessary and sufficient to alter endometrial BMP-2 responsiveness. Blockade of TGF-β prevents repression of BMP-2 receptors and restores BMP-2-stimulated expression of HOXA10 and LIF. Blockade of TGF signaling is a potential strategy to improve infertility and pregnancy loss associated with uterine leiomyoma. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Altered TGF-β endocytic trafficking contributes to the increased signaling in Marfan syndrome.

PubMed

Siegert, Anna-Maria; Serra-Peinado, Carla; Gutiérrez-Martínez, Enric; Rodríguez-Pascual, Fernando; Fabregat, Isabel; Egea, Gustavo

2018-02-01

The main cardiovascular alteration in Marfan syndrome (MFS) is the formation of aortic aneurysms in which augmented TGF-β signaling is reported. However, the primary role of TGF-β signaling as a molecular link between the genetic mutation of fibrillin-1 and disease onset is controversial. The compartmentalization of TGF-β endocytic trafficking has been shown to determine a signaling response in which clathrin-dependent internalization leads to TGF-β signal propagation, and caveolin-1 (CAV-1) associated internalization leads to signal abrogation. We here studied the contribution of endocytic trafficking compartmentalization to increased TGF-β signaling in vascular smooth muscle cells (VSMC) from MFS patients. We examined molecular components involved in clathrin- (SARA, SMAD2) and caveolin-1- (SMAD7, SMURF2) dependent endocytosis. Marfan VSMC showed higher recruitment of SARA and SMAD2 to membranes and their increased interaction with TGF-β receptor II, as well as higher colocalization of SARA with the early endosome marker EEA1. We assessed TGF-β internalization using a biotinylated ligand (b-TGF-β), which colocalized equally with either EEA1 or CAV-1 in VSMC from Marfan patients and controls. However, in Marfan cells, colocalization of b-TGF-β with SARA and EEA1 was increased and accompanied by decreased colocalization with CAV-1 at EEA1-positive endosomes. Moreover, Marfan VSMC showed higher transcriptional levels and membrane enrichment of RAB5. Our results indicate that increased RAB5-associated SARA localization to early endosomes facilitates its TGF-β receptor binding and phosphorylation of signaling mediator SMAD2 in Marfan VSMC. This is accompanied by a reduction of TGF-β sorting into multifunctional vesicles containing cargo from both internalization pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

PubMed Central

Kaur, Jasvir; Reinhardt, Dieter P.

2012-01-01

Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

TGF-β1 downregulates StAR expression and decreases progesterone production through Smad3 and ERK1/2 signaling pathways in human granulosa cells.

PubMed

Fang, Lanlan; Chang, Hsun-Ming; Cheng, Jung-Chien; Leung, Peter C K; Sun, Ying-Pu

2014-11-01

Regulation of progesterone production in granulosa cells is important for normal reproductive functions. Steroidogenic acute regulatory protein (StAR) is recognized as the key regulatory protein involved in the rate-limiting step of steroidogenesis. TGF-β1 protein is detected in human follicular fluid, and TGF-β1 and its receptors are expressed in human granulosa cells. However, the functional role of TGF-β1 in the regulation of StAR expression and progesterone production in human granulosa cells remains unknown. Our objective was to investigate the effects of TGF-β1 on StAR expression and progesterone production in human granulosa cells. SVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization and immortalized with SV40 large T antigen. SVOG cells were used to investigate the effects of TGF-β1 on StAR expression and progesterone production at an academic research center. Levels of mRNA and protein were examined by RT-qPCR and western blotting, respectively. The accumulation levels of progesterone were measured by enzyme-linked immunosorbent assay (ELISA). TGF-β1 treatment downregulated StAR expression and decreased progesterone production. The suppressive effects of TGF-β1 on StAR expression and progesterone production were abolished by the inhibition of TGF-β type I receptor. In addition, treatment with TGF-β1 activated the Smad2/3 and ERK1/2 signaling pathways. The inhibition of the Smad3 and ERK1/2 signaling pathways attenuated the TGF-β1-induced downregulation of StAR expression and progesterone production. TGF-β1 downregulated StAR expression and decreased progesterone production by activating the Smad3 and ERK1/2 signaling pathways in human granulosa cells.

Rho/Rock cross-talks with transforming growth factor-β/Smad pathway participates in lung fibroblast-myofibroblast differentiation.

PubMed

Ji, Hong; Tang, Haiying; Lin, Hongli; Mao, Jingwei; Gao, Lili; Liu, Jia; Wu, Taihua

2014-11-01

The differentiation of fibroblasts, which are promoted by transforming growth factor-β (TGF-β)/Smad, is involved in the process of pulmonary fibrosis. The Rho/Rho-associated coiled-coil-forming protein kinase (Rock) pathway may regulate the fibroblast differentiation and myofibroblast expression of α-smooth muscle actin (α-SMA), however, the mechanism is not clear. The aim of the present study was to evaluate the role of Rho/Rock and TGF-β/Smad in TGF-β1-induced lung fibroblasts differentiation. Human embryonic lung fibroblasts were stimulated by TGF-β1, Y-27632 (inhibitor of Rho/Rock signaling) and staurosporine (inhibitor of TGF-β/Smad signaling). The α-SMA expression, cell cycle progression, content of the extracellular matrix (ECM) in cell culture supernatants and the expression of RhoA, RhoC, Rock1 and Smad2 were detected. The results demonstrated that α-SMA-positive cells significantly increased following TGF-β1 stimulation. Rho/Rock and TGF-β/Smad inhibitors suppressed TGF-β1-induced lung fibroblast differentiation. The inhibitors increased G 0 /G 1 and decreased S and G 2 /M percentages. The concentrations of the ECM proteins in the supernatant were significantly increased by TGF-β1 stimulation, whereas they were decreased by inhibitor stimulation. RhoA, RhoC, Rock1, Smad2 and tissue inhibitor of metalloproteinase-1 were upregulated by TGF-β1 stimulation. The Rho/Rock inhibitor downregulated Smad2 expression and the TGF-β/Smad inhibitor downregulated RhoA, RhoC and Rock1 expression. Therefore, the Rho/Rock pathway and Smad signaling were involved in the process of lung fibroblasts transformation, induced by TGF-β1, to myofibroblasts. The two pathways may undergo cross-talk in the lung fibroblasts differentiation in vitro .

Isogermacrene A, a proposed intermediate in sesquiterpene biosynthesis.

PubMed

Hackl, Thomas; König, Wilfried A; Muhle, Hermann

2004-08-01

In the essential oil of the liverwort Saccogyna viticulosa, collected on the island of Madeira, the new sesquiterpene hydrocarbons isogermacrene A (5) and its Cope rearrangement product iso-beta-elemene (6) were identified. 5 is proposed to act as the biogenetic precursor of several new sesquiterpenes identified in the volatiles of S. viticulosa. These include iso-alpha-humulene, alpha-gorgonene, gorgona-1,4(15), 11-triene and gorgon- 11-en-4-ol. In addition, the Cope product of zierene, isozierene, allo-aromadendra-4(15),10(14)-diene, aromadendra-4(15),10(14)-dien-1-ol and a prenylguaiane diterpene alcohol, named viticulol, were identified as new natural products.

Total synthesis of (+/-)-taiwaniaquinol B via a domino intramolecular friedel-crafts acylation/carbonyl alpha-tert-alkylation reaction.

PubMed

Fillion, Eric; Fishlock, Dan

2005-09-28

The first synthesis of taiwaniaquinol B, a 6-nor-5(6-->7)abeoabietane-type diterpenoid exhibiting the uncommon fused 6-5-6 tricyclic carbon skeleton, was accomplished in 15 steps. A Lewis acid-promoted tandem intramolecular Friedel-Crafts/carbonyl alpha-tert-alkylation reaction was exploited as the core strategy for the synthesis of the sterically congested 1-indanone-containing tricyclic structure. This multiple carbon-carbon bond forming reaction exploits the unique reactivity of Meldrum's acid. The facile precursor synthesis makes this a useful methodology for the expedient modification and assembly of sterically congested 1-indanone-containing ring systems.

Mucin1 mediates autocrine transforming growth factor beta signaling through activating the c-Jun N-terminal kinase/activator protein 1 pathway in human hepatocellular carcinoma cells.

PubMed

Li, Qiongshu; Liu, Guomu; Shao, Dan; Wang, Juan; Yuan, Hongyan; Chen, Tanxiu; Zhai, Ruiping; Ni, Weihua; Tai, Guixiang

2015-02-01

In a previous study, we observed by global gene expression analysis that oncogene mucin1 (MUC1) silencing decreased transforming growth factor beta (TGF-β) signaling in the human hepatocellular carcinoma (HCC) cell line SMMC-7721. In this study, we report that MUC1 overexpression enhanced the levels of phosphorylated Smad3 linker region (p-Smad3L) (Ser-213) and its target gene MMP-9 in HCC cells, suggesting that MUC1 mediates TGF-β signaling. To investigate the effect of MUC1 on TGF-β signaling, we determined TGF-β secretion in MUC1 gene silencing and overexpressing cell lines. MUC1 expression enhanced not only TGF-β1 expression at the mRNA and protein levels but also luciferase activity driven by a TGF-β promoter, as well as elevated the activation of c-Jun N-terminal kinase (JNK) and c-Jun, a member of the activation protein 1 (AP-1) transcription factor family. Furthermore, pharmacological reduction of TGF-β receptor (TβR), JNK and c-Jun activity inhibited MUC1-induced autocrine TGF-β signaling. Moreover, a co-immunoprecipitation assay showed that MUC1 directly bound and activated JNK. In addition, both MUC1-induced TGF-β secretion and exogenous TGF-β1 significantly increased Smad signaling and cell migration, which were markedly inhibited by either TβR inhibitor or small interfering RNA silencing of TGF-β1 gene in HCC cells. The high correlation between MUC1 and TGF-β1 or p-Smad3L (Ser-213) expression was shown in tumor tissues from HCC patients by immunohistochemical staining analysis. Collectively, these results indicate that MUC1 mediates autocrine TGF-β signaling by activating the JNK/AP-1 pathway in HCC cells. Therefore, MUC1 plays a key role in HCC progression and could serve as an attractive target for HCC therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

High LET Radiation Can Enhance TGF(Beta) Induced EMT and Cross-Talk with ATM Pathways

NASA Technical Reports Server (NTRS)

Wang, Minli; Hada, Megumi; Huff, Janice; Pluth, Janice M.; Anderson, Janniffer; ONeill, Peter; Cucinotta, Francis A.

2010-01-01

The TGF(Beta) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation in mammary epithelial cells. We investigated possible interactions between the TGF(Beta) and ATM pathways following simulated space radiation using hTERT immortalized human esophageal epithelial cells (EPC-hTERT), mink lung epithelial cells (Mv1lu), and several human fibroblast cell lines. TGF(Beta) is a key modulator of the Epithelial-Mesenchymal Transition (EMT), important in cancer progression and metastasis. The implication of EMT by radiation also has several lines of developing evidence, however is poorly understood. The identification of TGF(Beta) induced EMT can be shown in changes to morphology, related gene over expression or down regulation, which can be detected by RT-PCR, and immunostaining and western blotting. In this study, we have observed morphologic and molecular alternations consistent with EMT after Mv1lu cells were treated with TGF(Beta) High LET radiation enhanced TGF(Beta) mediated EMT with a dose as low as 0.1Gy. In order to consider the TGF(Beta) interaction with ATM we used a potent ATM inhibitor Ku55933 and investigated gene expression changes and Smad signaling kinetics. Ku559933 was observed to reverse TGF(Beta) induced EMT, while this was not observed in dual treated cells (radiation+TGF(Beta)). In EPC-hTERT cells, TGF(Beta) alone was not able to induce EMT after 3 days of application. A combined treatment with high LET, however, significantly caused the alteration of EMT markers. To study the function of p53 in the process of EMT, we knocked down P53 through RNA interference. Morphology changes associated with EMT were observed in epithelial cells with silenced p53. Our study indicates: high LET radiation can enhance TGF(Beta) induced EMT; while ATM is triggering the process of TGF(Beta)-induced EMT, p53 might be an essential repressor for EMT phenotypes.

Analysis of interleukin (IL)-1 beta and transforming growth factor (TGF)-beta-induced signal transduction pathways in IL-2 and TGF-beta secretion and proliferation in the thymoma cell line EL4.NOB-1.

PubMed

Siese, A; Jaros, P P; Willig, A

1999-02-01

In the present study we investigated the interleukin (IL)-1beta and transforming growth factor-beta1 (TGF-beta1)-mediated proliferation, and production of IL-2 and TGF-beta, in the murine T-cell line, EL4.NOB-1. This cell line is resistant to TGF-beta concerning growth arrest but not autoinduction or suppression of IL-1-induced IL-2 production. When cocultured with IL-1beta, TGF-beta showed growth-promoting activity that could be antagonized by adding the phosphatidyl choline-dependent phospholipase C (PC-PLC) inhibitor, D609. Using specific enzyme inhibitors of protein kinases (PK) C and A, mitogen-activated protein kinase (MAPK), phospholipase A2 (PLA2), phosphatidylinositol-dependent (PI)-PLC and PC-PLC, we showed that IL-1beta-induced IL-2 synthesis was dependent on all investigated kinases and phospholipases, except PC-PLC. TGF-beta1 was able to inhibit IL-2 synthesis by the activation of PKA and MAPK. The same kinases are involved in TGF-beta autoinduction that is accompanied by a secretion of the active but not the latent growth factor and is antagonized by IL-1beta. Addition of the PI-PLC inhibitor, ET 18OCH3, or the PLA2 inhibitor (quinacrine) alone, resulted in secretion of latent TGF-beta and, in the case of ET 18OCH3, active TGF-beta. These data implicate a role for PI-PLC and PLA2 in the control of latency and secretion. Analysis of specific tyrosine activity and c-Fos expression showed synergistic but no antagonistic effects. These events are therefore not involved in IL- and TGF-beta-regulated IL-2 and TGF-beta production, but might participate in IL-1/TGF-beta-induced growth promotion.

Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-β

PubMed Central

Alcaraz, Lindsay B.; Exposito, Jean-Yves; Chuvin, Nicolas; Pommier, Roxane M.; Cluzel, Caroline; Martel, Sylvie; Sentis, Stéphanie; Bartholin, Laurent; Lethias, Claire

2014-01-01

Transforming growth factor β (TGF-β) isoforms are secreted as inactive complexes formed through noncovalent interactions between the bioactive TGF-β entity and its N-terminal latency-associated peptide prodomain. Extracellular activation of the latent TGF-β complex is a crucial step in the regulation of TGF-β function for tissue homeostasis. We show that the fibrinogen-like (FBG) domain of the matrix glycoprotein tenascin-X (TNX) interacts physically with the small latent TGF-β complex in vitro and in vivo, thus regulating the bioavailability of mature TGF-β to cells by activating the latent cytokine into an active molecule. Activation by the FBG domain most likely occurs through a conformational change in the latent complex and involves a novel cell adhesion–dependent mechanism. We identify α11β1 integrin as a cell surface receptor for TNX and show that this integrin is crucial to elicit FBG-mediated activation of latent TGF-β and subsequent epithelial-to-mesenchymal transition in mammary epithelial cells. PMID:24821840

Mammary Gland Involution Provides a Unique Model to Study the TGF-β Cancer Paradox

PubMed Central

Guo, Qiuchen; Betts, Courtney; Pennock, Nathan; Mitchell, Elizabeth; Schedin, Pepper

2017-01-01

Transforming Growth Factor-β (TGF-β) signaling in cancer has been termed the “TGF-β paradox”, acting as both a tumor suppresser and promoter. The complexity of TGF-β signaling within the tumor is context dependent, and greatly impacted by cellular crosstalk between TGF-β responsive cells in the microenvironment including adjacent epithelial, endothelial, mesenchymal, and hematopoietic cells. Here we utilize normal, weaning-induced mammary gland involution as a tissue microenvironment model to study the complexity of TGF-β function. This article reviews facets of mammary gland involution that are TGF-β regulated, namely mammary epithelial cell death, immune activation, and extracellular matrix remodeling. We outline how distinct cellular responses and crosstalk between cell types during physiologically normal mammary gland involution contribute to simultaneous tumor suppressive and promotional microenvironments. We also highlight alternatives to direct TGF-β blocking anti-cancer therapies with an emphasis on eliciting concerted microenvironmental-mediated tumor suppression. PMID:28098775

Mutations in the TGF-β Repressor SKI Cause Shprintzen-Goldberg Syndrome with Aortic Aneurysm

PubMed Central

Doyle, Alexander J.; Doyle, Jefferson J.; Bessling, Seneca L.; Maragh, Samantha; Lindsay, Mark E.; Schepers, Dorien; Gillis, Elisabeth; Mortier, Geert; Homfray, Tessa; Sauls, Kimberly; Norris, Russell A.; Huso, Nicholas D.; Leahy, Dan; Mohr, David W.; Caulfield, Mark J.; Scott, Alan F.; Destrée, Anne; Hennekam, Raoul C.; Arn, Pamela H.; Curry, Cynthia J.; Van Laer, Lut; McCallion, Andrew S.; Loeys, Bart L.; Dietz, Harry C.

2012-01-01

Increased transforming growth factor beta (TGF-β) signaling has been implicated in the pathogenesis of syndromic presentations of aortic aneurysm, including Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS)1-4. However, the location and character of many of the causal mutations in LDS would intuitively infer diminished TGF-β signaling5. Taken together, these data have engendered controversy regarding the specific role of TGF-β in disease pathogenesis. Shprintzen-Goldberg syndrome (SGS) has considerable phenotypic overlap with MFS and LDS, including aortic aneurysm6-8. We identified causative variation in 10 patients with SGS in the proto-oncogene SKI, a known repressor of TGF-β activity9,10. Cultured patient dermal fibroblasts showed enhanced activation of TGF-β signaling cascades and increased expression of TGF-β responsive genes. Morpholino-induced silencing of SKI paralogs in zebrafish recapitulated abnormalities seen in SGS patients. These data support the conclusion that increased TGF-β signaling is the mechanism underlying SGS and contributes to multiple syndromic presentations of aortic aneurysm. PMID:23023332

Fate of grape flavor precursors during storage on yeast lees.

PubMed

Loscos, Natalia; Hernández-Orte, Purificación; Cacho, Juan; Ferreira, Vicente

2009-06-24

The effect of the addition of a grape flavor precursor extract to a grape juice, before or after fermentation with three different Saccharomyces cerevisiae yeast strains, on the evolution of the wine aroma composition during a 9-month aging period on yeast lees has been studied. Wine aroma compounds were determined by gas chromatography-mass spectrometry after alcoholic fermentation and after 3 and 9 months of storage. The aging of wine on lees caused important changes in the aroma profiles of wines, making the concentrations of three terpenes, norisoprenoids (except beta-damascenone and beta-ionone), 4-allyl-2,6-dimethoxyphenol, ethyl vanillate, syringaldehyde, and ethyl cinnamate increase, whereas the concentrations of most of the rest of compounds tended to decrease. Lees are responsible for the observed increasing trends, except for linalool and alpha-terpineol, and also for a large part of the observed decrements. As expected, the addition of precursors brings about an increment in the levels of most terpenes, norisoprenoids, vanillins, and ethyl cinnamate, and it is after an aging time when differences linked to the level of precursors in the must become more evident. The timing of the addition of precursors has a minor influence, except for beta-damascenone, vanillin, and syringaldehyde, for which supplementation after fermentation is more effective. It has also been observed that the precursor fraction makes the levels of vinylphenols decrease. Finally, it has also been found that lees from different yeast strains may have a slightly different abilities to release volatile compounds derived from precursors.

[Role of Ski/SnoN protein in the regulation of TGF-beta signal pathway].

PubMed

Lu, Zhao-hui; Chen, Jie

2003-04-01

TGF-beta signal pathway plays an important role in the cell growth, differentiation, formation of extracellular matrix, embryo development and carcinogenesis, etc. However, the regulation of TGF-beta pathway is not totally understood. In 1999, three independent research groups found that Ski/SnoN protein could inhibit the TGF-beta mediated transcription by recruiting N-CoR, a transcription co-repressor. Later studies suggested that TGF-beta and SMADs degraded the Ski/SnoN protein by mediating ubiquitin linkage, showing negative feedback regulation. The important findings in Ski/SnoN laid the theoretical foundation for demonstrating the function of TGF-beta signal pathway.

TGF-β1 activates the canonical NF-κB signaling to promote cell survival and proliferation in dystrophic muscle fibroblasts in vitro

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

Ma, Zhen-Yu; Department of Neurology, The Second Affiliated Hospital, Guangzhou Medical University, No.250 Changgang East Road, Guangzhou 510260, Guangdong Province; Zhong, Zhi-Gang

Activated fibroblasts continue to proliferate at injury sites, leading to progressive muscular fibrosis in Duchenne muscular dystrophy (DMD). TGF-β1 is a dominant profibrotic mediator thought to play a critical role in muscle fibrosis; however, the implicated mechanisms are not fully understood. Here we showed that TGF-β1 increased the resistance to apoptosis and stimulated cell cycle progression in dystrophic muscle fibroblasts under serum deprivation conditions in vitro. TGF-β1 treatment activated the canonical NF-κB pathway; and we found that pharmacological inhibition of IKKβ with IMD-0354 and RelA gene knockdown with siRNA attenuated these effects of TGF-β1 on dystrophic muscle fibroblasts. Collectively, our datamore » suggest that TGF-β1 prevents apoptosis and cell cycle arrest in dystrophic muscle fibroblasts through the canonical NF-κB signaling pathway. - Highlights: • TGF-β1 promotes survival and proliferation in dystrophic muscle fibroblasts. • TGF-β1 activated the canonical NF-κB pathway in dystrophic muscle fibroblasts. • Canonical NF-κB pathway mediates these effects of TGF-β1.« less

Influence of thyroid hormones and transforming growth factor-β1 on cystatin C concentrations.

PubMed

Kotajima, N; Yanagawa, Y; Aoki, T; Tsunekawa, K; Morimura, T; Ogiwara, T; Nara, M; Murakami, M

2010-01-01

Serum cystatin C concentrations are reported to increase in the hyperthyroid state. Serum concentrations of cystatin C and transforming growth factor-β1 (TGF-β1) were measured in patients with thyroid dysfunction, and the effects of 3,5,3'-tri-iodothyronine (T(3)) and TGF-β1 on cystatin C production in human hepatoblastoma (Hep G2) cells were studied. Serum concentrations of cystatin C and TGF-β1 were significantly higher in patients with Graves' disease compared with control subjects. Significantly positive correlations were observed between thyroid hormones and cystatin C, thyroid hormones and TGF-β1, and TGF-β1 and cystatin C in patients with thyroid dysfunction. Serum concentrations of cystatin C and TGF-β1 decreased after treatment for hyperthyroidism. Cystatin C mRNA levels and cystatin C secretion were increased by T(3) and TGF-β1 in cultured Hep G2 cells. These results suggest that serum cystatin C concentrations increase in patients with hyperthyroidism. The mechanisms for this may involve elevation of serum TGF-β1 levels and the stimulatory effects of T(3) and TGF-β1 on cystatin C production.

Tyrosine sulfation in precursors of collagen V.

PubMed

Fessler, L I; Brosh, S; Chapin, S; Fessler, J H

1986-04-15

Radioactive labeling of p-collagens V, collagens V, and, to a small extent, of procollagen V occurred when [35S]sulfate was incubated with tendons or primary tendon cell cultures, or blood vessels and crops of 17- to 19-day-old chick embryos, or with lung slices from neonatal rats. Most or all of this label is in the form of 1 or more sulfated tyrosine residues/chain of p alpha 1(V), alpha 1(V), p alpha 1'(V), alpha 1'(V), p alpha 2(V), and alpha 2(V), and it remains attached through purification by dialysis, ammonium sulfate precipitation, CsCl-GdnCl2 equilibrium buoyant density and velocity sedimentations, ion-exchange chromatography, and sodium dodecyl sulfate gel electrophoresis. Radioactive tyrosine sulfate was identified in alkaline hydrolysates of these collagen V chains, after labeling the tissues with either [35S]sulfate or [3H]tyrosine, by electrophoretic and chromatographic comigration with a tyrosine sulfate standard. Tunicamycin A1, which inhibits the attachment of N-linked complex carbohydrate, did not interfere with the sulfation process. The tyrosine sulfate is located in a noncollagenous domain, which is probably adjacent to the amino end of the collagen helix, and is retained throughout the physiological proteolytic processing of procollagens V. After digestion with Staphylococcus aureus V8 protease, 35S-labeled p alpha 1(V) and alpha 1(V) chains gave the same map of labeled peptides, and this differed from the map given by p alpha 1'(V) and alpha 1'(V) chains. Little sulfation of p alpha 2(V) and alpha 2(V) chains occurs. The implications of these observations for the structure and properties of procollagens V and their derivatives are considered.

Effect of swainsonine on the processing of the asparagine-linked carbohydrate chains of alpha 1-antitrypsin in rat hepatocytes. Evidence for the formation of hybrid oligosaccharides.

PubMed

Gross, V; Tran-Thi, T A; Vosbeck, K; Heinrich, P C

1983-03-25

The biosynthesis of the proteinase inhibitor alpha 1-antitrypsin has been studied in rat hepatocyte primary cultures. Newly synthesized alpha 1-antitrypsin was found in hepatocytes as a glycoprotein of an apparent molecular weight of 49,000 carrying oligosaccharide side chains of the high mannose type. In the hepatocyte medium a secreted alpha 1-antitrypsin of an apparent molecular weight of 54,000 could be identified as a glycoprotein with carbohydrate chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the two forms of alpha 1-antitrypsin. When the hepatocytes were treated with swainsonine, an intracellular form of alpha 1-antitrypsin with an apparent molecular weight of 49,000 indistinguishable from that of control cells was found. However, the alpha 1-antitrypsin secreted from swainsonine-treated hepatocytes was different from that present in control media. It was characterized by a lower apparent molecular weight (51,000), a higher amount of [3H]mannose incorporation, half as much incorporation of [3H]galactose, and the same amount of [3H]fucose incorporation compared to alpha 1-antitrypsin of control media. In contrast to the 54,000 complex type alpha 1-antitrypsin from control media the 51,000 alpha 1-antitrypsin from the medium of swainsonine-treated cells was found to be susceptible to the action of endoglucosaminidase H, even when fucose was attached to the proximal GlcNAc residue. alpha 1-Antitrypsin secreted from swainsonine-treated cells combines features usually associated with either high mannose or complex type oligosaccharides and therefore represents a hybrid structure. In spite of its effect on the carbohydrate part of alpha 1-antitrypsin swainsonine did not impair the secretion of the incompletely processed glycoprotein.

PGE2 contributes to TGF-β induced T regulatory cell function in human non-small cell lung cancer

PubMed Central

Baratelli, Felicita; Lee, Jay M; Hazra, Saswati; Lin, Ying; Walser, Tonya C; Schaue, Dorthe; Pak, Peter S; Elashoff, David; Reckamp, Karen; Zhang, Ling; Fishbein, Michael C; Sharma, Sherven; Dubinett, Steven M

2010-01-01

CD4+CD25bright regulatory T cells (Treg) play an important role in cancer-mediated immunosuppression. We and others have previously shown that prostaglandin E2 (PGE2) and transforming growth factor beta (TGF-β) induce CD4+CD25brightFOXP3+Treg. Based on these studies, we investigated the requirement for PGE2 in Treg induction by TGF-β. TGF-β stimulation of human CD4+ T cells induced COX-2-dependent production of PGE2. PGE2-neutralizing antibody treatment significantly reduced the suppressive function of TGF-β-induced Treg (TGF-β-Treg) in vitro. TGF-β concentration measured in the plasma of non-small cell lung cancer (NSCLC) patients directly correlated with the frequency of circulating CD4+CD25brightFOXP3+T cells. Flow cytometry analysis showed increased FOXP3 expression in circulating CD4+CD25+HLA-DR- cells of lung cancer patients compared to control subjects. Immunohistochemical analysis revealed co-expression of TGF-β, COX-2, and FOXP3 in serial sections from resected lung tumor tissues. All together these observations suggest interplay between TGF-β and COX-2 in the induction of Treg activities. Interrupting TGF-β and PGE2 signaling may be important in therapeutic interventions that aim to limit Tregfunction in lung cancer. PMID:20733946

Effects of transforming growth factor-β1 treatment on muscle regeneration and adipogenesis in glycerol-injured muscle.

PubMed

Mahdy, Mohamed A A; Warita, Katsuhiko; Hosaka, Yoshinao Z

2017-11-01

Transforming growth factor (TGF)-β1 is associated with fibrosis in many organs. Recent studies demonstrated that delivery of TGF-β1 into chemically injured muscle enhances fibrosis. In this study, we investigated the effects of exogenous TGF-β1 on muscle regeneration and adipogenesis in glycerol-injured muscle of normal mice. Tibialis anterior (TA) muscles were injured by glycerol injection. TGF-β1 was either co-injected with glycerol, as an 'early treatment' group, or injected at day 4 after glycerol, as a 'late treatment' group and the TA muscles were collected at day 7 after initial injury. Myotube density was significantly lower in the early treatment group than in the glycerol-injured group (without TGF-β1 treatment). Moreover, the Oil red O-positive area was significantly smaller in the early treatment group than in the late treatment group and glycerol-injured group. Furthermore, TGF-β1 treatment increased endomysial fibrosis and induced immunostaining of α-smooth muscle actin. The greater inhibitory effects of early TGF-β1 treatment than that of late TGF-β1 treatment during regeneration in glycerol-injured muscle suggest a more potent effect of TGF-β1 on the initial stage of muscle regeneration and adipogenesis. Combination of TGF-β1 with glycerol might be an alternative to enhance muscle fibrosis for future studies. © 2017 Japanese Society of Animal Science.

Effects of TGF-β1 on the Proliferation and Apoptosis of Human Cervical Cancer Hela Cells In Vitro.

PubMed

Tao, Ming-Zhu; Gao, Xia; Zhou, Tie-Jun; Guo, Qing-Xi; Zhang, Qiang; Yang, Cheng-Wan

2015-12-01

To investigate the effects of TGF-β1 on the proliferation and apoptosis of cervical cancer Hela cells in vitro. Human cervical cancer Hela cells were cultured in vitro and divided into the experimental and control groups. In the experimental groups, Hela cells were stimulated with different concentrations of TGF-β1 (0.01, 0.1, 1, and 10 ng/mL), while Hela cells cultured in serum-free medium without TGF-β1 were used as controls. The CCK8 method was adopted to detect the effect of TGF-β1 on Hela cell proliferation, and flow cytometry was used to determine cell apoptosis 72 h after TGF-β1 treatment. Compared with the control group, the CCK-8 tests showed that different concentrations of TGF-β1 had no obvious effect on Hela cell proliferation 24 h after treatment (P > 0.05). However, upon 48 or 72 h of treatment, TGF-β1 significantly inhibited the proliferation of Hela cells in a time- and dose-dependent manner (P < 0.05). The flow cytometry results indicated that TGF-β1 influenced the apoptosis of human cervical cancer Hela cells in a dose-dependent manner after 72 h of treatment (P < 0.05). TGF-β1 significantly inhibited the growth and induced the apoptosis of human cervical Hela cells in vitro.

Role of transforming growth factor-beta (TGF) beta in the physiopathology of rheumatoid arthritis.

PubMed

Gonzalo-Gil, Elena; Galindo-Izquierdo, María

2014-01-01

Transforming growth factor-beta (TGF-β) is a cytokine with pleiotropic functions in hematopoiesis, angiogenesis, cell proliferation, differentiation, migration and apoptosis. Although its role in rheumatoid arthritis is not well defined, TGF-β activation leads to functional immunomodulatory effects according to environmental conditions. The function of TGF-β in the development of arthritis in murine models has been extensively studied with controversial results. Recent findings point to a non-relevant role for TGF-β in a mice model of collagen-induced arthritis. The study of TGF-β on T-cell responses has shown controversial results as an inhibitor or promoter of the inflammatory response. This paper presents a review of the role of TGF-β in animal models of arthritis. Copyright © 2013 Elsevier España, S.L. All rights reserved.

E-selectin ligand 1 regulates bone remodeling by limiting bioactive TGF-β in the bone microenvironment.

PubMed

Yang, Tao; Grafe, Ingo; Bae, Yangjin; Chen, Shan; Chen, Yuqing; Bertin, Terry K; Jiang, Ming-Ming; Ambrose, Catherine G; Lee, Brendan

2013-04-30

TGF-β is abundantly produced in the skeletal system and plays a crucial role in skeletal homeostasis. E-selectin ligand-1 (ESL-1), a Golgi apparatus-localized protein, acts as a negative regulator of TGF-β bioavailability by attenuating maturation of pro-TGF-β during cartilage homeostasis. However, whether regulation of intracellular TGF-β maturation by ESL-1 is also crucial during bone homeostasis has not been well defined. Here, we show that Esl-1(-/-) mice exhibit a severe osteopenia with elevated bone resorption and decreased bone mineralization. In primary culture, Esl-1(-/-) osteoclast progenitors show no difference in osteoclastogenesis. However, Esl-1(-/-) osteoblasts show delayed differentiation and mineralization and stimulate osteoclastogenesis more potently in the osteoblast-osteoclast coculture, suggesting that ESL-1 primarily acts in osteoblasts to regulate bone homeostasis. In addition, Esl-1(-/-) calvaria exhibit an elevated mature TGF-β/pro-TGF-β ratio, with increased expression of TGF-β downstream targets (plasminogen activator inhibitor-1, parathyroid hormone-related peptide, connective tissue growth factor, and matrix metallopeptidase 13, etc.) and a key regulator of osteoclastogenesis (receptor activator of nuclear factor κB ligand). Moreover, in vivo treatment with 1D11, a pan-TGF-β antibody, significantly improved the low bone mass of Esl-1(-/-) mice, suggesting that elevated TGF-β signaling is the major cause of osteopenia in Esl-1(-/-) mice. In summary, our study identifies ESL-1 as an important regulator of bone remodeling and demonstrates that the modulation of TGF-β maturation is pivotal in the maintenance of a homeostatic bone microenvironment and for proper osteoblast-osteoclast coupling.

Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

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

Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun

2014-08-08

Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuousmore » TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.« less

Transforming growth factor β induces bone marrow mesenchymal stem cell migration via noncanonical signals and N-cadherin.

PubMed

Dubon, Maria Jose; Yu, Jinyeong; Choi, Sanghyuk; Park, Ki-Sook

2018-01-01

Transforming growth factor-beta (TGF-β) induces the migration and mobilization of bone marrow-derived mesenchymal stem cells (BM-MSCs) to maintain bone homeostasis during bone remodeling and facilitate the repair of peripheral tissues. Although many studies have reported the mechanisms through which TGF-β mediates the migration of various types of cells, including cancer cells, the intrinsic cellular mechanisms underlying cellular migration, and mobilization of BM-MSCs mediated by TGF-β are unclear. In this study, we showed that TGF-β activated noncanonical signaling molecules, such as Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), focal adhesion kinase (FAK), and p38, via TGF-β type I receptor in human BM-MSCs and murine BM-MSC-like ST2 cells. Inhibition of Rac1 by NSC23766 and Src by PP2 resulted in impaired TGF-β-mediated migration. These results suggested that the Smad-independent, noncanonical signals activated by TGF-β were necessary for migration. We also showed that N-cadherin-dependent intercellular interactions were required for TGF-β-mediated migration using functional inhibition of N-cadherin with EDTA treatment and a neutralizing antibody (GC-4 antibody) or siRNA-mediated knockdown of N-cadherin. However, N-cadherin knockdown did not affect the global activation of noncanonical signals in response to TGF-β. Therefore, these results suggested that the migration of BM-MSCs in response to TGF-β was mediated through N-cadherin and noncanonical TGF-β signals. © 2017 Wiley Periodicals, Inc.

SMAD4 feedback regulates the canonical TGF-β signaling pathway to control granulosa cell apoptosis.

PubMed

Du, Xing; Pan, Zengxiang; Li, Qiqi; Liu, Honglin; Li, Qifa

2018-02-02

Canonical TGF-β signals are transduced from the cell surface to the cytoplasm, and then translocated into the nucleus, a process that involves ligands (TGF-β1), receptors (TGFBR2/1), receptor-activated SMADs (SMAD2/3), and the common SMAD (SMAD4). Here we provide evidence that SMAD4, a core component of the canonical TGF-β signaling pathway, regulates the canonical TGF-β signaling pathway in porcine granulosa cells (GCs) through a feedback mechanism. Genome-wide analysis and qRT-PCR revealed that SMAD4 affected miRNA biogenesis in GCs. Interestingly, TGFBR2, the type II receptor of the canonical TGF-β signaling pathway, was downregulated in SMAD4-silenced GCs and found to be a common target of SMAD4-inhibited miRNAs. miR-425, the most significantly elevated miRNA in SMAD4-silenced GCs, mediated the SMAD4 feedback regulation of the TGF-β signaling pathway. This was accomplished through a direct interaction between the transcription factor SMAD4 and the miR-425 promoter, and a direct interaction between miR-425 and the TGFBR2 3'-UTR. Furthermore, miR-425 enhanced GC apoptosis by targeting TGFBR2 and the canonical TGF-β signaling pathway, which was rescued by SMAD4 and TGF-β1. Overall, our findings demonstrate that a positive feedback mechanism exists within the canonical TGF-β signaling pathway. This study also provides new insights into mechanism underlying the canonical TGF-β signaling pathway, which regulates GC function and follicular development.

Activation of TGF-β1-CD147 positive feedback loop in hepatic stellate cells promotes liver fibrosis.

PubMed

Li, Hai-Yan; Ju, Di; Zhang, Da-Wei; Li, Hao; Kong, Ling-Min; Guo, Yanhai; Li, Can; Wang, Xi-Long; Chen, Zhi-Nan; Bian, Huijie

2015-11-12

Activation of hepatic stellate cells (HSCs) by transforming growth factor-β1 (TGF-β1) initiates HBV-associated fibrogenesis. The mechanism of TGF-β1 modulating HSC activation is not fully uncovered. We hypothesized a positive feedback signaling loop of TGF-β1-CD147 promoting liver fibrogenesis by activation of HSCs. Human HSC cell line LX-2 and spontaneous liver fibrosis model derived from HBV transgenic mice were used to evaluate the activation of molecules in the signaling loop. Wound healing and cell contraction assay were performed to detect the CD147-overexpressed HSC migration and contraction. The transcriptional regulation of CD147 by TGF-β1/Smad4 was determined using dual-luciferase reporter assay and chromatin immunoprecipitation. We found that a positive reciprocal regulation between TGF-β1 and CD147 mediated HSC activation. CD147 over-expression promoted HSC migration and accelerated TGF-β1-induced cell contraction. Phosphorylation of Smad2 and Smad3 in cooperation with Smad4 mediated the TGF-β1-regulated CD147 expression. Smad4 activated the transcription by direct interaction with CD147 promoter. Meanwhile, CD147 modulated the activated phenotype of HSCs through the ERK1/2 and Sp1 which up-regulated α-SMA, collagen I, and TGF-β1 synthesis. These findings indicate that TGF-β1-CD147 loop plays a key role in regulating the HSC activation and combination of TGF-β receptor inhibitor and anti-CD147 antibody might be promised to reverse fibrogenesis.

Activation of TGF-β1-CD147 positive feedback loop in hepatic stellate cells promotes liver fibrosis

PubMed Central

Li, Hai-Yan; Ju, Di; Zhang, Da-Wei; Li, Hao; Kong, Ling-Min; Guo, Yanhai; Li, Can; Wang, Xi-Long; Chen, Zhi-Nan; Bian, Huijie

2015-01-01

Activation of hepatic stellate cells (HSCs) by transforming growth factor-β1 (TGF-β1) initiates HBV-associated fibrogenesis. The mechanism of TGF-β1 modulating HSC activation is not fully uncovered. We hypothesized a positive feedback signaling loop of TGF-β1-CD147 promoting liver fibrogenesis by activation of HSCs. Human HSC cell line LX-2 and spontaneous liver fibrosis model derived from HBV transgenic mice were used to evaluate the activation of molecules in the signaling loop. Wound healing and cell contraction assay were performed to detect the CD147-overexpressed HSC migration and contraction. The transcriptional regulation of CD147 by TGF-β1/Smad4 was determined using dual-luciferase reporter assay and chromatin immunoprecipitation. We found that a positive reciprocal regulation between TGF-β1 and CD147 mediated HSC activation. CD147 over-expression promoted HSC migration and accelerated TGF-β1-induced cell contraction. Phosphorylation of Smad2 and Smad3 in cooperation with Smad4 mediated the TGF-β1-regulated CD147 expression. Smad4 activated the transcription by direct interaction with CD147 promoter. Meanwhile, CD147 modulated the activated phenotype of HSCs through the ERK1/2 and Sp1 which up-regulated α-SMA, collagen I, and TGF-β1 synthesis. These findings indicate that TGF-β1-CD147 loop plays a key role in regulating the HSC activation and combination of TGF-β receptor inhibitor and anti-CD147 antibody might be promised to reverse fibrogenesis. PMID:26559755

TGF-β1 stimulates migration of type II endometrial cancer cells by down-regulating PTEN via activation of SMAD and ERK1/2 signaling pathways

PubMed Central

Xiong, Siyuan; Cheng, Jung-Chien; Klausen, Christian; Zhao, Jianfang; Leung, Peter C.K.

2016-01-01

PTEN acts as a tumor suppressor primarily by antagonizing the PI3K/AKT signaling pathway. PTEN is frequently mutated in human cancers; however, in type II endometrial cancers its mutation rate is very low. Overexpression of TGF-β1 and its receptors has been reported to correlate with metastasis of human cancers and reduced survival rates. Although TGF-β1 has been shown to regulate PTEN expression through various mechanisms, it is not yet known if the same is true in type II endometrial cancer. In the present study, we show that treatment with TGF-β1 stimulates the migration of two type II endometrial cancer cell lines, KLE and HEC-50. In addition, TGF-β1 treatment down-regulates both mRNA and protein levels of PTEN. Overexpression of PTEN or inhibition of PI3K abolishes TGF-β1-stimulated cell migration. TGF-β1 induces SMAD2/3 phosphorylation and knockdown of common SMAD4 inhibits the suppressive effects of TGF-β1 on PTEN mRNA and protein. Interestingly, TGF-β1 induces ERK1/2 phosphorylation and pre-treatment with a MEK inhibitor attenuates the suppression of PTEN protein, but not mRNA, by TGF-β1. This study provides important insights into the molecular mechanisms mediating TGF-β1-induced down-regulation of PTEN and demonstrates an important role of PTEN in the regulation of type II endometrial cancer cell migration. PMID:27542208

Hotspot ignition using a Z-pinch precursor plasma in a magneto-inertial ICF scheme

NASA Astrophysics Data System (ADS)

Chittenden, J. P.; Vincent, P.; Jennings, C. A.; Ciardi, A.

2006-01-01

Precursor plasma flow is a common feature of wire array Z-pinches. The precursor flow represents a fraction of the mass of the array which arrives on the axis early in time and remains confined at high density by the inertia of further material bombarding the axis. Later on, the main implosion of the Z-pinch then compresses this precursor to substantially higher density. We show that if the same system can be generated with a Deuterium-Tritium plasma then the precursor provides an ideal target for a cylindrical magneto-inertial ICF scheme. The implosion of the DT Z-pinch produces a dense, low temperature shell which compressively heats the precursor target to high temperatures and tamps its expansion. The azimuthal magnetic field in the hotspot is sufficient to reduce the Larmor radius for the alpha particles to much less than the hotspot size, which dramatically reduces the pR required for ignition. A computational analysis of this approach is presented, including a study of the thermonuclear burn wave propagation. The robustness of the scheme with respect to instabilities, confinement time and drive parameters is examined. The results indicate that a high energy gain can be achieved using Z-pinches with 50-100 MA currents and a few hundred nanosecond rise-times. This work was partially supported by the U.S. Department of Energy through cooperative agreement DE-FC03-02NA00057.

The Transforming Growth Factor-β1 (TGF-β1) Gene Polymorphisms (TGF-β1 T869C and TGF-β1 T29C) and Susceptibility to Postmenopausal Osteoporosis

PubMed Central

Sun, Jiajia; Zhang, Chi; Xu, Lei; Yang, Mingyuan; Yang, Huilin

2015-01-01

Abstract The aim of the present study was to integrate all the eligible studies and investigate whether the transforming growth factor-β1 (TGF-β1) gene polymorphisms (TGF-β1 T869C and TGF-β1 T29C) are correlated with postmenopausal osteoporosis (PMOP) risk. PMOP is a common skeletal disease and several genetic factors play an important role in the development and progression of PMOP. Significant associations between TGF-β1 gene polymorphisms (TGF-β1 T869C and TGF-β1 T29C) and PMOP risk have been reported; however, some of these results are controversial. A systematic online search was performed using PubMed, EMBASE, Web of Science, and the Cochrane Library to identify case–control studies investigating the relationship between TGF-β1 T869C and TGF-β1 T29C polymorphisms and the susceptibility of PMOP. The pooled odds ratio (OR) with 95% confidence interval (95% CI) was calculated to assess the associations, and subgroup meta-analyses were performed according to the ethnicity of the study populations. Eight studies involving 1851 cases and 2247 controls met the inclusion criteria after assessment by 2 reviewers. Overall, there were significant associations between TGF-β1 T869C and TGF-β1 T29C polymorphisms and PMOP (TGF-β1 T869C—C vs T: OR = 1.18, 95% CI = 1.02–1.36, P = 0.030; CC vs TT: OR = 1.38, 95% CI = 1.01–1.88, P = 0.042; CC vs CT/TT: OR = 1.39, 95% CI = 1.09–1.76, P = 0.008; TGF-β1 T29C—CT vs TT: OR = 1.25, 95% CI = 1.02–1.53, P = 0.032; CT/CC vs TT: OR = 1.37, 95% CI = 1.02–1.84, P = 0.035). In the subgroup analysis of ethnicity, significant association was observed between TGF-β1 T869C polymorphism and PMOP risk in Asian population (C vs T: OR = 1.18, 95% CI = 1.01–1.38, P = 0.043; CC vs TT: OR = 1.41, 95% CI = 1.01–1.97, P = 0.047; CT/CC vs TT: OR = 1.31, 95% CI = 1.03–1.66, P = 0.026; CC vs CT/TT: OR = 1.35, 95% CI = 1.03–1.75, P = 0.028); however, there was no significant association between TGF-β1 T869C polymorphism and PMOP risk in Caucasian population. With regard to TGF-β1 T29C polymorphism, significant association was also observed in Asian population (CT vs TT: OR = 1.37, 95% CI = 1.07–1.75, P = 0.013; CT/CC vs TT: OR = 1.54, 95% CI = 1.16–2.05, P = 0.003), while there was no significant association in Caucasian population. The TGF-β1 T869C and TGF-β1 T29C polymorphisms may be involved in susceptibility to PMOP, particular in Asian patients. PMID:25634187

Gravity and Skeletal Growth

NASA Technical Reports Server (NTRS)

Morey-Holton, Emily; Turner, Russell T.

1999-01-01

Two simultaneous experiments were performed using 5-week-old male Sprague Dawley rats; in one study, the rats were flown in low earth orbit; in the other study, the hindlimbs of the growing rats were elevated to prevent weight bearing. Following 9 d of unloading, weight bearing was restored for 4, 28, and 76 hrs. Afterwards, additional hindlimb unloading experiments were performed to evaluate the skeletal response to 0, 2, 4, 6, 8, 10, 12, 16, and 24 hrs of restored weight bearing following 7 d of unloading. Cancellous and cortical bone histomorphometry were evaluated in the left tibia at the proximal metaphysis and in the left femur at mid-diaphysis, respectively. Steady-state mRNA levels for bone matrix proteins and skeletal signaling peptides were determined in total cellular RNA extracted from trabeculae from the right proximal tibiametaphysis and periosteum from the right femur. Spaceflight and hindlimb unloading each resulted in cancellous osteopenia, as well as a tendency towards decreased periosteal bone formation. Both models for skeletal unloading resulted in site specific reductions in mRNA levels for transforming growth factor-beta (sub 1) (TGF-beta) osteocalcin (OC), and prepro-alpha (I) subunit of type 1 collagen (collagen) and little or no changes in mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAP) and insulin-like growth factor I (IGF-I). Restoration of normal weight bearing resulted in transient increases in mRNA levels for the bone matrix proteins and TGF-beta in the proximal metaphysis and periosteum and no changes in either GAP or IGF-I mRNA levels. The timecourse for the response differed between the two skeletal compartments; the tibial metaphysis responded much more quickly to reloading. These results suggest that the skeletal adaptation to acute physiological changes in mechanical usage are mediated, in part, by changes in mRNA levels for bone matrix proteins and TGF-beta.

Cav-1 deficiency promotes liver fibrosis in carbon tetrachloride (CCl4)-induced mice by regulation of oxidative stress and inflammation responses.

PubMed

Ji, De-Gang; Zhang, Yan; Yao, Song-Mei; Zhai, Xu-Jie; Zhang, Li-Rong; Zhang, Yao-Zhong; Li, Hui

2018-06-01

Caveolin-1 (Cav-1), as a membrane protein involved in the formation of caveolae, binds steroid receptors and endothelial nitric oxide synthase, limiting its translocation and activation. In the present study, we investigated the role of Cav-1 in the progression of hepatic fibrosis induced by carbon tetrachloride (CCl 4 ) in murine animals. Therefore, the wild type (WT) and Cav-1-knockout (Cav-1 -/- ) mice were used in our study and subjected to CCl 4 . The results indicated that CCl 4 induced the decrease of Cav-1 expression in liver tissue samples. And Cav-1 -/- intensified CCl 4 -triggered hepatic injury, evidenced by the stronger hepatic histological alterations, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and liver terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. CCl 4 led to oxidative stress, supported by the reduced superoxide dismutase (SOD) activity and glutathione (GSH) levels, as well as enhanced malondialdehyde (MDA) and O 2 - levels in liver samples. And the process was intensified by Cav-1 -/- . Additionally, CCl 4 -caused hepatic inflammation was aggregated by Cav-1 -/- via further increasing the secretion of pro-inflammatory cytokines. Moreover, CCl 4 -caused fibrosis was strengthened by Cav-1 -/- , which was evidenced by the up-regulation of α-smooth muscle actin (α-SMA), collagen alpha 1 type 1 (Col1A1), lysyl oxidase (Lox) and transforming growth factor-β1 (TGF-β1) in liver tissues. Similar results were observed in TGF-β1-stimulated hepatic stellate cells (HSCs) and LX-2 cells without Cav-1 expressions that in vitro, suppressing Cav-1 further accelerated TGF-β1-induced oxidative stress, inflammation and fibrosis development. In conclusion, our results indicated that Cav-1 played an important role in CCl 4 -induced hepatic injury, which may be used as potential therapeutic target for hepatic fibrosis treatment. Copyright © 2018. Published by Elsevier Masson SAS.

Temporal growth factor release from platelet-rich plasma, trehalose lyophilized platelets, and bone marrow aspirate and their effect on tendon and ligament gene expression.

PubMed

McCarrel, Taralyn; Fortier, Lisa

2009-08-01

Platelet-rich plasma (PRP) has generated substantial interest for tendon and ligament regeneration because of the high concentrations of growth factors in platelet alpha-granules. This study compared the temporal release of growth factors from bone marrow aspirate (BMA), PRP, and lyophilized platelet product (PP), and measured their effects on tendon and ligament gene expression. Blood and BMA were collected and processed to yield PRP and plasma. Flexor digitorum superficialis tendon (FDS) and suspensory ligament (SL) explants were cultured in 10% plasma in DMEM (control), BMA, PRP, or PP. TGF-beta1 and PDGF-BB concentrations were determined at 0, 24, and 96 h of culture using ELISA. Quantitative RT-PCR for collagen types I and III (COL1A1, COL3A1), cartilage oligomeric matrix protein (COMP), decorin, and matrix metalloproteinases-3 and 13 (MMP-3, MMP-13) was performed. TGF-beta1 and PDGF-BB concentrations were highest in PRP and PP. Growth factor quantity was unchanged in BMA, increased in PRP, and decreased in PP over 4 days. TGF-beta1 and platelet concentrations were positively correlated. Lyophilized PP and PRP resulted in increased COL1A1:COL3A1 ratio, increased COMP, and decreased MMP-13 expression. BMA resulted in decreased COMP and increased MMP-3 and MMP-13 gene expression. Platelet concentration was positively correlated with COL1A1, ratio of COL1A1:COL3A1, and COMP, and negatively correlated with COL3A1, MMP-13, and MMP-3. White blood cell concentration was positively correlated with COL3A1, MMP3, and MMP13, and negatively correlated with a ratio of COL1A1:COL3A1, COMP, and decorin. These findings support further in vivo investigation of PRP and PP for treatment of tendonitis and desmitis. Copyright 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Effects of transforming growth factor-beta1 on cell motility, collagen gel contraction, myofibroblastic differentiation, and extracellular matrix expression of human adipose-derived stem cell.

PubMed

Kakudo, Natsuko; Kushida, Satoshi; Suzuki, Kenji; Ogura, Tsunetaka; Notodihardjo, Priscilla Valentin; Hara, Tomoya; Kusumoto, Kenji

2012-12-01

Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, and their usefulness in plastic surgery has garnered attention in recent years. Although, there have been expectations that ASCs might function in wound repair and regeneration, no studies to date have examined the role of ASCs in the mechanism that promotes wound-healing. Transforming growth factor-beta1 (TGF-β1) is a strong candidate cytokine for the triggering of mesenchymal stem cell migration, construction of extracellular matrices, and differentiation of ASCs into myofibroblasts. Cell proliferation, motility, and differentiation, as well as extracellular matrix production, play an important role in wound-healing. We have evaluated the capacity of ASCs to proliferate and their potential to differentiate into phenotypic myofibroblasts, as well as their cell motility and collagen gel contraction ability, when cultured with TGF-β1. Cell motility was analyzed using a wound-healing assay. ASCs that differentiated into myofibroblasts expressed the gene for alpha-smooth muscle actin, and its protein expression was detected immunohistochemically. The extracellular matrix expression in ASCs was evaluated using real-time RT-PCR. Based on the results, we conclude that human ASCs have the potential for cell motility, extracellular matrix gene expression, gel contraction, and differentiation into myofibroblasts and, therefore, may play an important role in the wound-healing process.

COMBINED ALISKIREN AND AMLODIPINE REDUCE ALBUMINURIA VIA REDUCTION IN RENAL INFLAMMATION IN DIABETIC RATS

PubMed Central

Matavelli, Luis C.; Huang, Jiqian; Siragy, Helmy M.

2011-01-01

SUMMARY We hypothesized that compared to hydrochlorothiazide (HCTZ), the renin inhibitor aliskiren (ALISK) or amlodipine (AMLO) and their combination reduce albuminuria via reduction in renal inflammation, independent of BP changes. We studied normal and streptozotocin induced diabetic (DM) Sprague-Dawley rats treated for 6 weeks with vehicle, ALISK, HCTZ or AMLO individually and combined and evaluated effects of treatments on BP, urine albumin to creatinine ratio (UACR), renal interstitial fluid (RIF) levels of angiotensin II (Ang II), tumor necrosis factor-alpha (TNFα) and interleukin-6 (IL-6), and renal expression of TNFα, IL-6, transforming growth factor-beta1 (TGF-β1) and nuclear factor-kappa B (NF-κB). There were no differences in BP between treatments. Only ALISK and its combinations reduced RIF Ang II. UACR increased in DM rats and decreased with ALISK alone or combined with HCTZ or AMLO. HCTZ or AMLO individually and combined did not influence UACR. RIF TNFα and IL-6, and the renal expression of TNFα, IL-6, TGF-β1 and NF-κB were increased in DM rats. These renal inflammatory markers were reduced only with ALISK or AMLO individually or combined with other treatments. We conclude that ALISK alone and combined with HCTZ or AMLO reduced albuminuria in diabetes via reduction in renal inflammation, independent of BP changes. PMID:22075749

Anthelmintic Therapy Modifies the Systemic and Mycobacterial Antigen-Stimulated Cytokine Profile in Helminth-Latent Mycobacterium tuberculosis Coinfection.

PubMed

Anuradha, Rajamanickam; Munisankar, Saravanan; Bhootra, Yukthi; Dolla, Chandrakumar; Kumaran, Paul; Nutman, Thomas B; Babu, Subash

2017-04-01

Helminth infections are known to modulate cytokine responses in latent tuberculosis (LTB). However, very few studies have examined whether this modulation is reversible upon anthelmintic therapy. We measured the systemic and mycobacterial (TB) antigen-stimulated levels of type 1, type 2, type 17, and regulatory cytokines in individuals with LTB and with or without coexistent Strongyloides stercoralis infection before and after anthelmintic therapy. Our data reveal that individuals with LTB and coexistent S. stercoralis infection have significantly lower levels of systemic and TB antigen-stimulated type 1 (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and type 17 (IL-17A and/or IL-17F) cytokines and significantly higher levels of systemic but not TB antigen-stimulated type 2 (IL-4 and IL-5) and regulatory (transforming growth factor beta [TGF-β]) cytokines. Anthelmintic therapy resulted in significantly increased systemic levels of type 1 and/or type 17 cytokines and in significantly decreased systemic levels of type 2 and regulatory (IL-10 and TGF-β) cytokines. In addition, anthelmintic therapy resulted in significantly increased TB antigen-stimulated levels of type 1 cytokines only. Our data therefore confirm that the modulation of systemic and TB antigen-stimulated cytokine responses in S. stercoralis -LTB coinfection is reversible (for the most part) by anthelmintic treatment. Copyright © 2017 American Society for Microbiology.

Anticoccidial activities of Chitosan on Eimeria papillata-infected mice.

PubMed

Abdel-Latif, Mahmoud; Abdel-Haleem, Heba M; Abdel-Baki, Abdel-Azeem S

2016-07-01

Eimeria spp. multiply within the intestinal tract causing severe inflammatory responses. Chitosan (CS), meanwhile, has been shown to exhibit anti-inflammatory activities in different experimental models. Here, we investigated the effect of CS on the outcome of inflammation caused by Eimeria papillata in the mouse intestine. Investigations were undertaken into the oocyst output in feces and developmental stages and goblet cells in intestinal tissue. Assays for lipid peroxidation, nitric oxide (NO), and myeloperoxidase (MPO) were also performed. T cells in intestinal tissue were counted using immunohistochemistry while total IgA in serum or intestinal wash was assayed using ELISA. In addition, mRNA expression of tumor necrosis factor alpha (TNF-α), transforming growth factor β (TGF-β), interleukin (IL)-10, and IL-4 were detected using real-time PCR. The data indicated a reduction in both oocyst output and in the number of parasite developmental stages following CS treatment, while the goblet cell hypoplasia in infected mice was also inhibited. CS decreased lipid peroxidation, NO, and MPO but did not alter the T cell count or IgA levels in comparison to the infected group. The expression of TNF-α and TGF-β decreased but IL-10 and IL-4 increased after CS treatment in comparison to the non-treated infected group. In conclusion, CS showed anti-inflammatory and protective effects against E. papillata infection.

TGF-β in pancreatic cancer initiation and progression: two sides of the same coin.

PubMed

Shen, Wei; Tao, Guo-Qing; Zhang, Yu; Cai, Bing; Sun, Jian; Tian, Zhi-Qiang

2017-01-01

Pancreatic cancer is highly lethal malignant tumor with characterised rapid progression, invasiveness and resistance to radiochemotherapy. Transforming growth factor-β (TGF-β) signaling plays a dual role in both pro-tumorigenic and tumor suppressive of pancreatic cancer, depending on tumor stage and microenvironment. TGF-β signaling components alteration are common in pancreatic cancer, and its leading role in tumor formation and metastases has received increased attention. Many therapies have investigated to target TGF-β signaling in the preclinical and clinical setting. In this review, we highlight the dual roles of TGF-β and touch upon the perspectives on therapeutic target of TGF-β signaling in pancreatic cancer.

Phospho-control of TGF-β superfamily signaling

PubMed Central

Wrighton, Katharine H; Lin, Xia; Feng, Xin-Hua

2010-01-01

Members of the transforming growth factor-β (TGF-β) family control a broad range of cellular responses in metazoan organisms via autocrine, paracrine, and endocrine modes. Thus, aberrant TGF-β signaling can play a key role in the pathogenesis of several diseases, including cancer. TGF-β signaling pathways are activated by a short phospho-cascade, from receptor phosphorylation to the subsequent phosphorylation and activation of downstream signal transducers called R-Smads. R-Smad phosphorylation state determines Smad complex assembly/disassembly, nuclear import/export, transcriptional activity and stability, and is thus the most critical event in TGF-β signaling. Dephosphorylation of R-Smads by specific phosphatases prevents or terminates TGF-β signaling, highlighting the need to consider Smad (de)phosphorylation as a tightly controlled and dynamic event. This article illustrates the essential roles of reversible phosphorylation in controlling the strength and duration of TGF-β signaling and the ensuing physiological responses. PMID:19114991

KSHV LANA inhibits TGF-β signaling through epigenetic silencing of the TGF-β type II receptor

PubMed Central

Di Bartolo, Daniel L.; Cannon, Mark; Liu, Yi-Fang; Renne, Rolf; Chadburn, Amy; Boshoff, Chris

2008-01-01

Signaling through the transforming growth factor–β (TGF-β) pathway results in growth inhibition and induction of apoptosis in various cell types. We show that this pathway is blocked in Kaposi sarcoma herpesvirus (KSHV)–infected primary effusion lymphoma through down-regulation of the TGF-β type II receptor (TβRII) by epigenetic mechanisms. Our data also suggest that KSHV infection may result in lower expression of TβRII in Kaposi sarcoma and multicentric Castleman disease. KSHV-encoded LANA associates with the promoter of TβRII and leads to its methylation and to the deacetylation of proximal histones. Reestablishment of signaling through this pathway reduces viability of these cells, inferring that KSHV-mediated blockage of TGF-β signaling plays a role in the establishment and progression of KSHV-associated neoplasia. These data suggest a mechanism whereby KSHV evades both the antiproliferative effects of TGF-β signaling by silencing TβRII gene expression and immune recognition by suppressing TGF-β–responsive immune cells through the elevated secretion of TGF-β1. PMID:18199825

A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System.

PubMed

Qin, Yan; Garrison, Brian S; Ma, Wenjiang; Wang, Rui; Jiang, Aiping; Li, Jing; Mistry, Meeta; Bronson, Roderick T; Santoro, Daria; Franco, Charlotte; Robinton, Daisy A; Stevens, Beth; Rossi, Derrick J; Lu, Chafen; Springer, Timothy A

2018-06-12

Extracellular proTGF-β is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-β is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVβ8-dependent TGF-β activation. Lrrc33 -/- mice lack CNS vascular abnormalities associated with deficiency in TGF-β-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33 -/- brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33 -/- microglia in the same brain suggest that there is little spreading of TGF-β activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-β provide localized and selective activation of TGF-β. Copyright © 2018 Elsevier Inc. All rights reserved.

The oncoprotein Ski acts as an antagonist of transforming growth factor-beta signaling by suppressing Smad2 phosphorylation.

PubMed

Prunier, Celine; Pessah, Marcia; Ferrand, Nathalie; Seo, Su Ryeon; Howe, Philip; Atfi, Azeddine

2003-07-11

The phosphorylation of Smad2 and Smad3 by the transforming growth factor (TGF)-beta-activated receptor kinases and their subsequent heterodimerization with Smad4 and translocation to the nucleus form the basis for a model how Smad proteins work to transmit TGF-beta signals. The transcriptional activity of Smad2-Smad4 or Smad3-Smad4 complexes can be limited by the corepressor Ski, which is believed to interact with Smad complexes on TGF-beta-responsive promoters and represses their ability to activate TGF-beta target genes by assembling on DNA a repressor complex containing histone deacetylase. Here we show that Ski can block TGF-beta signaling by interfering with the phosphorylation of Smad2 and Smad3 by the activated TGF-beta type I receptor. Furthermore, we demonstrate that overexpression of Ski induces the assembly of Smad2-Smad4 and Smad3-Smad4 complexes independent of TGF-beta signaling. The ability of Ski to engage Smad proteins in nonproductive complexes provides new insights into the molecular mechanism used by Ski for disabling TGF-beta signaling.

Characterization of a possible amyloidogenic precursor in glutamine-repeat neurodegenerative diseases.

PubMed

Armen, Roger S; Bernard, Brady M; Day, Ryan; Alonso, Darwin O V; Daggett, Valerie

2005-09-20

Several neurodegenerative diseases are linked to expanded repeats of glutamine residues, which lead to the formation of amyloid fibrils and neuronal death. The length of the repeats correlates with the onset of Huntington's disease, such that healthy individuals have <38 residues and individuals with >38 repeats exhibit symptoms. Because it is difficult to obtain atomic-resolution structural information for poly(l-glutamine) (polyQ) in aqueous solution experimentally, we performed molecular dynamics simulations to investigate the conformational behavior of this homopolymer. In simulations of 20-, 40-, and 80-mer polyQ, we observed the formation of the "alpha-extended chain" conformation, which is characterized by alternating residues in the alpha(L) and alpha(R) conformations to yield a sheet. The structural transition from disordered random-coil conformations to the alpha-extended chain conformation exhibits modest length and temperature dependence, in agreement with the experimental observation that aggregation depends on length and temperature. We propose that fibril formation in polyQ may occur through an alpha-sheet structure, which was proposed by Pauling and Corey. Also, we propose an atomic-resolution model of how the inhibitory peptide QBP1 (polyQ-binding peptide 1) may bind to polyQ in an alpha-extended chain conformation to inhibit fibril formation.

Biosynthesis of 3-acetyldeoxynivalenol and sambucinol. Identification of the two oxygenation steps after trichodiene.

PubMed

Zamir, L O; Nikolakakis, A; Huang, L; St-Pierre, P; Sauriol, F; Sparace, S; Mamer, O

1999-04-30

The first two oxygenation steps post-trichodiene in the biosyntheses of the trichothecenes 3-acetyldeoxynivalenol and sambucinol were investigated. The plausible intermediates 2-hydroxytrichodiene (2alpha- and 2beta-) and 12,13-epoxytrichodiene and the dioxygenated compounds 12,13-epoxy-9,10-trichoene-2-ol (2alpha- and 2beta-) were prepared specifically labeled with stable isotopes. They were then fed separately and/or together to Fusarium culmorum cultures, and the derived trichothecenes were isolated, purified, and analyzed. The stable isotopes enable easy localization of the labels in the products by 2H NMR, 13C NMR, and mass spectrometry. We found that 2alpha-hydroxytrichodiene is the first oxygenated step in the biosynthesis of both 3-acetyldeoxynivalenol and sambucinol. The stereoisomer 2beta-hydroxytrichodiene and 12,13-epoxytrichodiene are not biosynthetic intermediates and have not been isolated as metabolites. We also demonstrated that the dioxygenated 12, 13-epoxy-9,10-trichoene-2alpha-ol is a biosynthetic precursor to trichothecenes as had been suggested in a preliminary work. Its stereoisomer was not found in the pathway. A further confirmation of our results was the isolation of both oxygenated trichodiene derivatives 2alpha-hydroxytrichodiene and 12,13-epoxy-9, 10-trichoene-2alpha-ol as natural metabolites in F. culmorum cultures.

Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation

PubMed Central

Chak, Kayam; Roy-Chaudhuri, Biswajoy; Kim, Hak Kyun; Kemp, Kayla C; Kay, Mark A

2016-01-01

MicroRNA-21 (miR-21) is consistently up-regulated in various neurological disorders, including epilepsy. Here, we show that the biogenesis of miR-21 is altered following pilocarpine status epilepticus (SE) with an increase in precursor miR-21 (pre-miR-21) in rats. We demonstrate that pre-miR-21 has an energetically favorable site overlapping with the miR-21 binding site and competes with mature miR-21 for binding in the 3′UTR of TGFBR2 mRNA, but not NT-3 mRNA in vitro. This binding competition influences miR-21-mediated repression in vitro and correlates with the increase in TGFBR2 and decrease in NT-3 following SE. Polysome profiling reveals co-localization of pre-miR-21 in the ribosome fraction with translating mRNAs in U-87 cells. The current work suggests that pre-miR-21 may post-transcriptionally counteract miR-21-mediated suppression following SE and could potentially lead to prolonged TGF-β receptor expression impacting epileptogenesis. The study further supports that the ratio of the pre to mature miRNA may be important in determining the regulatory effects of a miRNA gene. PMID:27725160

TGF-β Signaling Regulates Pancreatic β-Cell Proliferation through Control of Cell Cycle Regulator p27 Expression

PubMed Central

Suzuki, Tomoyuki; Dai, Ping; Hatakeyama, Tomoya; Harada, Yoshinori; Tanaka, Hideo; Yoshimura, Norio; Takamatsu, Tetsuro

2013-01-01

Proliferation of pancreatic β-cells is an important mechanism underlying β-cell mass adaptation to metabolic demands. Increasing β-cell mass by regeneration may ameliorate or correct both type 1 and type 2 diabetes, which both result from inadequate production of insulin by β-cells of the pancreatic islet. Transforming growth factor β (TGF-β) signaling is essential for fetal development and growth of pancreatic islets. In this study, we exposed HIT-T15, a clonal pancreatic β-cell line, to TGF-β signaling. We found that inhibition of TGF-β signaling promotes proliferation of the cells significantly, while TGF-β signaling stimulation inhibits proliferation of the cells remarkably. We confirmed that this proliferative regulation by TGF-β signaling is due to the changed expression of the cell cycle regulator p27. Furthermore, we demonstrated that there is no observed effect on transcriptional activity of p27 by TGF-β signaling. Our data show that TGF-β signaling mediates the cell-cycle progression of pancreatic β-cells by regulating the nuclear localization of CDK inhibitor, p27. Inhibition of TGF-β signaling reduces the nuclear accumulation of p27, and as a result this inhibition promotes proliferation of β-cells. PMID:23720603