17-Beta-estradiol inhibits transforming growth factor-beta signaling and function in breast cancer cells via activation of extracellular signal-regulated kinase through the G protein-coupled receptor 30.

PubMed

Kleuser, Burkhard; Malek, Daniela; Gust, Ronald; Pertz, Heinz H; Potteck, Henrik

2008-12-01

Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-beta plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-beta signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-beta signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-beta signaling network as well as TGF-beta functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-beta-estradiol (E2) to inhibit the TGF-beta pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-beta signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-beta signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-beta pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-beta signaling. These findings are of great clinical relevance, because down-regulation of TGF-beta signaling is associated with the development of breast cancer resistance in response to antiestrogens.

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.

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

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

PubMed

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

2008-01-01

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

Emergence, development and diversification of the TGF-beta signalling pathway within the animal kingdom.

PubMed

Huminiecki, Lukasz; Goldovsky, Leon; Freilich, Shiri; Moustakas, Aristidis; Ouzounis, Christos; Heldin, Carl-Henrik

2009-02-03

The question of how genomic processes, such as gene duplication, give rise to co-ordinated organismal properties, such as emergence of new body plans, organs and lifestyles, is of importance in developmental and evolutionary biology. Herein, we focus on the diversification of the transforming growth factor-beta (TGF-beta) pathway -- one of the fundamental and versatile metazoan signal transduction engines. After an investigation of 33 genomes, we show that the emergence of the TGF-beta pathway coincided with appearance of the first known animal species. The primordial pathway repertoire consisted of four Smads and four receptors, similar to those observed in the extant genome of the early diverging tablet animal (Trichoplax adhaerens). We subsequently retrace duplications in ancestral genomes on the lineage leading to humans, as well as lineage-specific duplications, such as those which gave rise to novel Smads and receptors in teleost fishes. We conclude that the diversification of the TGF-beta pathway can be parsimoniously explained according to the 2R model, with additional rounds of duplications in teleost fishes. Finally, we investigate duplications followed by accelerated evolution which gave rise to an atypical TGF-beta pathway in free-living bacterial feeding nematodes of the genus Rhabditis. Our results challenge the view of well-conserved developmental pathways. The TGF-beta signal transduction engine has expanded through gene duplication, continually adopting new functions, as animals grew in anatomical complexity, colonized new environments, and developed an active immune system.

TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

PubMed

Mori, Shigeo; Matsuzaki, Koichi; Yoshida, Katsunori; Furukawa, Fukiko; Tahashi, Yoshiya; Yamagata, Hideo; Sekimoto, Go; Seki, Toshihito; Matsui, Hirofumi; Nishizawa, Mikio; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2004-09-23

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

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

Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah

2011-11-18

Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology inmore » pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful

PKB/Akt modulates TGF-beta signalling through a direct interaction with Smad3.

PubMed

Remy, Ingrid; Montmarquette, Annie; Michnick, Stephen W

2004-04-01

Transforming growth factor beta (TGF-beta) has a major role in cell proliferation, differentiation and apoptosis in many cell types. Integration of the TGF-beta pathway with other signalling cascades that control the same cellular processes may modulate TGF-beta responses. Here we report the discovery of a new functional link between TGF-beta and growth factor signalling pathways, mediated by a physical interaction between the serine-threonine kinase PKB (protein kinase B)/Akt and the transcriptional activator Smad3. Formation of the complex is induced by insulin, but inhibited by TGF-beta stimulation, placing PKB-Smad3 at a point of convergence between these two pathways. PKB inhibits Smad3 by preventing its phosphorylation, binding to Smad4 and nuclear translocation. In contrast, Smad3 does not inhibit PKB. Inhibition of Smad3 by PKB occurs through a kinase-activity-independent mechanism, resulting in a decrease in Smad3-mediated transcription and protection of cells against TGF-beta-induced apoptosis. Consistently, knockdown of the endogenous PKB gene with small-interfering RNA (siRNA) has the opposite effect. Our results suggest a very simple mechanism for the integration of signals arising from growth-factor- and TGF-beta-mediated pathways.

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.

Disruption of transforming growth factor-beta signaling by curcumin induces gene expression of peroxisome proliferator-activated receptor-gamma in rat hepatic stellate cells.

PubMed

Zheng, Shizhong; Chen, Anping

2007-01-01

Activation of hepatic stellate cells (HSC), the major effectors of hepatic fibrogenesis, is coupled with sequential alterations in gene expression, including an increase in receptors for transforming growth factor-beta (TGF-beta) and a dramatic reduction in the peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The relationship between them remains obscure. We previously demonstrated that curcumin induced gene expression of PPAR-gamma in activated HSC, leading to reducing cell proliferation, inducing apoptosis and suppressing expression of extracellular matrix genes. The underlying molecular mechanisms are largely unknown. We recently observed that stimulation of PPAR-gamma activation suppressed gene expression of TGF-beta receptors in activated HSC, leading to the interruption of TGF-beta signaling. This observation supported our assumption of an antagonistic relationship between PPAR-gamma activation and TGF-beta signaling in HSC. In this study, we further hypothesize that TGF-beta signaling might negatively regulate gene expression of PPAR-gamma in activated HSC. The present report demonstrates that exogenous TGF-beta1 inhibits gene expression of PPAR-gamma in activated HSC, which is eliminated by the pretreatment with curcumin likely by interrupting TGF-beta signaling. Transfection assays further indicate that blocking TGF-beta signaling by dominant negative type II TGF-beta receptor increases the promoter activity of PPAR-gamma gene. Promoter deletion assays, site-directed mutageneses, and gel shift assays localize two Smad binding elements (SBEs) in the PPAR-gamma gene promoter, acting as curcumin response elements and negatively regulating the promoter activity in passaged HSC. The Smad3/4 protein complex specifically binds to the SBEs. Overexpression of Smad4 dose dependently eliminates the inhibitory effects of curcumin on the PPAR-gamma gene promoter and TGF-beta signaling. Taken together, these results demonstrate that the interruption of TGF-beta

Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor. beta

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

Kuratomi, Y.; Ono, M.; Yasutake, C.

1987-01-01

A mutant clone (MO-5) was originally isolated as a clone resistant to Na/sup +//K/sup +/ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF):binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-..beta.. efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing inmore » soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-..beta.. while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-..beta.. showed low colony formation capacity in soft agar in the absence of TGF-..beta... Colonies of MO-5 formed by TGF-..beta.. in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-..beta... Pretreatment of MO-5 with TGF-..beta.. induced secretion of TGF-..beta..-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-..beta..-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the transformed phenotype in MO-5 is discussed.« less

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.

Pin1 promotes transforming growth factor-beta-induced migration and invasion.

PubMed

Matsuura, Isao; Chiang, Keng-Nan; Lai, Chen-Yu; He, Dongming; Wang, Guannan; Ramkumar, Romila; Uchida, Takafumi; Ryo, Akihide; Lu, Kunping; Liu, Fang

2010-01-15

Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-beta responses. TGF-beta binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-beta-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-beta, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-beta-induced growth-inhibitory responses and a number of TGF-beta/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-beta-mediated migration and invasion. Accordingly, TGF-beta induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-beta-induced migration and invasion of cancer cells.

Effect of TGF-beta1 on MMP/TIMP and TGF-beta1 receptors in great saphenous veins and its significance on chronic venous insufficiency.

PubMed

Serralheiro, Pedro; Cairrão, Elisa; Maia, Cláudio J; João, Marina; Almeida, Carlos M Costa; Verde, Ignacio

2017-06-01

Objectives Transforming growth factor-beta1 (TGF-β1) may participate in local chronic inflammatory processes in varicose veins and in venous wall structure modifications through regulation of matrix metalloproteinases (MMP) and their inhibitors (tissue inhibitor of metalloproteinase (TIMP)). The aim of this study was to analyze the effect of TGF-β1 in the vein wall, namely on the gene expression of selected MMP, TIMP and TGF-β1 receptors. Methods Healthy vein samples were harvested from eight subjects who underwent coronary bypass graft surgery with great saphenous vein. Each vein sample was divided into two segments, which were cultivated separately in vitro (one of the segments had TGF-β1 added) and then submitted to gene expression analysis. Results In the TGF-β1 supplemented group, there was a general increase in the mean gene expression. Specifically, expression of MMP9, MMP12, TIMP1 and TIMP2 were statistically significant. Conclusion The results of this study demonstrate that the gene expression of MMP9, MMP12, TIMP1 and TIMP2 was influenced by the addition of TGF-β1. These results may be translated to chronic venous insufficiency framework and suggest involvement of TGF-β1 in the vein wall pathology.

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

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

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

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

c-Ski, Smurf2, and Arkadia as regulators of TGF-beta signaling: new targets for managing myofibroblast function and cardiac fibrosis.

PubMed

Cunnington, Ryan H; Nazari, Mansoreh; Dixon, Ian M C

2009-10-01

Recent studies demonstrate the critical role of the extracellular matrix in the organization of parenchymal cells in the heart. Thus, an understanding of the modes of regulation of matrix production by cardiac myofibroblasts is essential. Transforming growth factor beta (TGF-beta) signaling is transduced through the canonical Smad pathway, and the involvement of this pathway in matrix synthesis and other processes requires precise control. Inhibition of Smad signaling may be achieved at the receptor level through the targeting of the TGF-beta type I receptors with an inhibitory Smad7/Smurf2 complex, or at the transcriptional level through c-Ski/receptor-Smad/co-mediator Smad4 interactions. Conversely, Arkadia protein intensifies TGF-beta-induced effects by marking c-Ski and inhibitory Smad7 for destruction. The study of these TGF-beta mediators is essential for future treatment of fibrotic disease, and this review highlights recent relevant findings that may impact our understanding of cardiac fibrosis.

TGF-beta is specifically expressed in human dermal papilla cells and modulates hair folliculogenesis.

PubMed

Inoue, Keita; Aoi, Noriyuki; Yamauchi, Yuji; Sato, Takahiro; Suga, Hirotaka; Eto, Hitomi; Kato, Harunosuke; Tabata, Yasuhiko; Yoshimura, Kotaro

2009-01-01

Dermal papilla cells (DPCs) in the mammalian hair follicle have been shown to develop hair follicles through epithelial-mesenchymal interactions. A cell therapy to regenerate human hair is theoretically possible by expanding autologous human DPCs (hDPCs) and transplanting them into bald skin, though much remains to be overcome before clinical success. In this study, we compared gene signatures of hDPCs at different passages and human dermal fibroblasts, and found transforming growth factor (TGF)-beta(2) to be highly expressed in cultured hDPCs. Keratinocyte conditioned medium, which is known to help preserve the hair-inducing capacity of hDPCs, up-regulated TGF-beta(2) expression of hDPCs and also enhanced their alkaline phosphatase (ALP) activity, a known index for hair-inductive capacity. Through screening of components secreted from keratinocytes, the vitamin D(3) analogue was found to promote TGF-beta(2) expression and ALP activity of hDPCs. In animal hair folliculogenesis models using rat epidermis and expanded hDPCs, inhibition of TGF-beta(2) signalling at the ligand or receptor level significantly impaired hair folliculogenesis and maturation. These results suggest an important role for TGF-beta(2) in hair follicle morphogenesis and provide insights into the establishment of future cell therapies for hair regrowth by transplanting expanded DPCs.

Integrin-mediated transforming growth factor-beta activation regulates homeostasis of the pulmonary epithelial-mesenchymal trophic unit.

PubMed

Araya, Jun; Cambier, Stephanie; Morris, Alanna; Finkbeiner, Walter; Nishimura, Stephen L

2006-08-01

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

Redox-mediated activation of latent transforming growth factor-beta 1

NASA Technical Reports Server (NTRS)

Barcellos-Hoff, M. H.; Dix, T. A.; Chatterjee, A. (Principal Investigator)

1996-01-01

Transforming growth factor beta 1 (TGF beta) is a multifunctional cytokine that orchestrates response to injury via ubiquitous cell surface receptors. The biological activity of TGF beta is restrained by its secretion as a latent complex (LTGF beta) such that activation determines the extent of TGF beta activity during physiological and pathological events. TGF beta action has been implicated in a variety of reactive oxygen-mediated tissue processes, particularly inflammation, and in pathologies such as reperfusion injury, rheumatoid arthritis, and atherosclerosis. It was recently shown to be rapidly activated after in vivo radiation exposure, which also generates reactive oxygen species (ROS). In the present studies, the potential for redox-mediated LTGF beta activation was investigated using a cell-free system in which ROS were generated in solution by ionizing radiation or metal ion-catalyzed ascorbate reaction. Irradiation (100 Gray) of recombinant human LTGF beta in solution induced 26% activation compared with that elicited by standard thermal activation. Metal-catalyzed ascorbate oxidation elicited extremely efficient recombinant LTGF beta activation that matched or exceeded thermal activation. The efficiency of ascorbate activation depended on ascorbate concentrations and the presence of transition metal ions. We postulate that oxidation of specific amino acids in the latency-conferring peptide leads to a conformation change in the latent complex that allows release of TGF beta. Oxidative activation offers a novel route for the involvement of TGF beta in tissue processes in which ROS are implicated and endows LTGF beta with the ability to act as a sensor of oxidative stress and, by releasing TGF beta, to function as a signal for orchestrating the response of multiple cell types. LTGF beta redox sensitivity is presumably directed toward recovery of homeostasis; however, oxidation may also be a mechanism of LTGF beta activation that can be deleterious during

TGF-beta1 inhibits expression and activity of hENT1 in a nitric oxide-dependent manner in human umbilical vein endothelium.

PubMed

Vega, José L; Puebla, Carlos; Vásquez, Rodrigo; Farías, Marcelo; Alarcón, Julio; Pastor-Anglada, Marçal; Krause, Bernardo; Casanello, Paola; Sobrevia, Luis

2009-06-01

We studied whether transforming growth factor beta1 (TGF-beta1) modulates human equilibrative nucleoside transporters 1 (hENT1) expression and activity in human umbilical vein endothelial cells (HUVECs). hENT1-mediated adenosine transport and expression are reduced in gestational diabetes and hyperglycaemia, conditions associated with increased synthesis and release of nitric oxide (NO) and TGF-beta1 in this cell type. TGF-beta1 increases NO synthesis via activation of TGF-beta receptor type II (TbetaRII), and NO inhibits hENT1 expression and activity in HUVECs. HUVECs (passage 2) were used for experiments. Total and hENT1-mediated adenosine transport was measured in the absence or presence of TGF-beta1, NG-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor), and/or KT-5823 (protein kinase G inhibitor) in control cells and cells expressing a truncated form of TGF-beta1 receptor type II (TTbetaRII). Western blot and real-time PCR were used to determine hENT1 protein abundance and mRNA expression. SLC29A1 gene promoter and specific protein 1 (Sp1) transcription factor activity was assayed. Vascular reactivity was assayed in endothelium-intact or -denuded umbilical vein rings. TGF-beta1 reduced hENT1-mediated adenosine transport, hENT1 protein abundance, hENT1 mRNA expression, and SLC29A1 gene promoter activity, but increased Sp1 binding to DNA. TGF-beta1 effect was blocked by L-NAME and KT-5823 and mimicked by SNAP in control cells. However, TGF-beta1 was ineffective in cells expressing TTbetaRII or a mutated Sp1 consensus sequence. Vasodilatation in response to TGF-beta1 and S-(4-nitrobenzyl)-6-thio-inosine (an ENT inhibitor) was endothelium-dependent and blocked by KT-5823 and ZM-241385. hENT1 is down-regulated by activation of TbetaRII by TGF-beta1 in HUVECs, a phenomenon where NO and Sp1 play key roles. These findings comprise physiological mechanisms that could be important in diseases where TGF-beta

Induction of TGF-beta1 and TGF-beta1-dependent predominant Th17 differentiation by group A streptococcal infection.

PubMed

Wang, Beinan; Dileepan, Thamotharampillai; Briscoe, Sarah; Hyland, Kendra A; Kang, Johnthomas; Khoruts, Alexander; Cleary, P Patrick

2010-03-30

Recurrent group A Streptococcus (GAS) tonsillitis and associated autoimmune diseases indicate that the immune response to this organism can be ineffective and pathological. TGF-beta1 is recognized as an essential signal for generation of regulatory T cells (Tregs) and T helper (Th) 17 cells. Here, the impact of TGF-beta1 induction on the T-cell response in mouse nasal-associated lymphoid tissue (NALT) following intranasal (i.n.) infections is investigated. ELISA and TGF-beta1-luciferase reporter assays indicated that persistent infection of mouse NALT with GAS sets the stage for TGF-beta1 and IL-6 production, signals required for promotion of a Th17 immune response. As predicted, IL-17, the Th17 signature cytokine, was induced in a TGF-beta1 signaling-dependent manner in single-cell suspensions of both human tonsils and NALT. Intracellular cytokine staining and flow cytometry demonstrated that CD4(+) IL-17(+) T cells are the dominant T cells induced in NALT by i.n. infections. Moreover, naive mice acquired the potential to clear GAS by adoptive transfer of CD4(+) T cells from immunized IL-17A(+)/(+) mice but not cells from IL-17A(-)/(-) mice. These experiments link specific induction of TGF-beta1 by a bacterial infection to an in vivo Th17 immune response and show that this cellular response is sufficient for protection against GAS. The association of a Th17 response with GAS infection reveals a potential mechanism for destructive autoimmune responses in humans.

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

PubMed

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

2007-01-01

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

Inhibitory effect of transforming growth factor-. beta. (TGF-. beta. ) on insulin-like growth factor 1 (IGF-1)-induced proliferation and differentiation in primary cultures of pig preadipocytes

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

Richardson, R.L.; Hausman, G.J.; Gaskins, H.R.

1990-02-26

The influence of serum, IGF-1 and TGF-{beta} on the differentiation of preadipocytes was examined in primary cultures of porcine adipose tissue cells. In serum-supplemented or serum-free, IGF-1 (1 and 10 nM) had no effect on total cell number. However, IGF-1 (10nM) increased adipocyte number only in serum-supplemented (1% pig serum) cultures, whereas TGF-{beta} (15 pm) reduced the adipocyte number in the presence and absence of IGF-1. Replication of preadipocytes was analyzed with a ({sup 3}H) thymidine assay. Preadipocyte proliferation (cpm in adipocyte fraction) was increased by IGF-1 (10nM) only in cultures containing pig serum. TGF-{beta} had no effect on preadipocytemore » proliferation specifically, but slightly increased total ({sup 3}H) thymidine incorporation in cultures with serum. Glycerol phosphate dehydrogenase (GPDH) specific activity was decreased by adding TGF-{beta} to serum-free cultures but TGF-{beta} had little effect in serum-supplemented cultures. Cellular secretion of IGF-1 was decreased when TGF-{beta} was added to serum-free or serum-supplemented cultures. These studies indicate that TGF-{beta} does not inhibit adipocyte development in the initial growth phase, but may inhibit differentiation and/or hypertrophy at a later stage of development.« less

Transforming growth factor-beta 1 (TGF-beta1) promotes IL-2 mRNA expression through the up-regulation of NF-kappaB, AP-1 and NF-AT in EL4 cells.

PubMed

Han, S H; Yea, S S; Jeon, Y J; Yang, K H; Kaminski, N E

1998-12-01

Transforming growth factor beta1 (TGF-beta1) has been previously shown to modulate interleukin 2 (IL-2) secretion by activated T-cells. In the present studies, we determined that TGF-beta1 induced IL-2 mRNA expression in the murine T-cell line EL4, in the absence of other stimuli. IL-2 mRNA expression was significantly induced by TGF-beta1 (0.1-1 ng/ml) over a relatively narrow concentration range, which led to the induction of IL-2 secretion. Under identical condition, we examined the effect of TGF-beta1 on the activity of nuclear factor AT (NF-AT), nuclear factor kappaB (NF-kappaB), activator protein-1 (AP-1) and octamer, all of which contribute to the regulation of IL-2 gene expression. Electrophoretic mobility shift assays showed that TGF-beta1 markedly increased NF-AT, NF-kappaB and AP-1 binding to their respective cognate DNA binding sites, whereas octamer binding remained constant, as compared with untreated cells. Employing a reporter gene expression system with p(NF-kappaB)3-CAT, p(NF-AT)3-CAT and p(AP-1)3-CAT, TGF-beta1 treatment of transfected EL4 cells induced a dose-related increase in chloramphenicol acetyltransferase activity that correlated well with the DNA binding profile found in the electrophoretic mobility shift assay studies. These results show that TGF-beta1, in the absence of any additional stimuli, up-regulates the activity of key transcription factors involved in IL-2 gene expression, including NF-AT, NF-kappaB and AP-1, to help promote IL-2 mRNA expression by EL4 cells.

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

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.

Expression and structural features of endoglin (CD105), a transforming growth factor beta1 and beta3 binding protein, in human melanoma.

PubMed Central

Altomonte, M.; Montagner, R.; Fonsatti, E.; Colizzi, F.; Cattarossi, I.; Brasoveanu, L. I.; Nicotra, M. R.; Cattelan, A.; Natali, P. G.; Maio, M.

1996-01-01

Human endoglin (CD105) is a member of the transforming growth factor beta (TGF-beta) receptor family that binds TGF-beta1 and -beta3, but not TGF-beta2, on human endothelial cells. Immunohistochemical analyses demonstrated that CD105 is expressed on normal and neoplastic cells of the melanocytic lineage. The anti-CD105 MAb, MAEND3, stained 50, 25 and 34% of intradermal naevi, primary and metastatic melanomas investigated, respectively, and nine out of 12 melanoma cell lines. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that CD105 expressed by melanoma cells consists of a homodimeric protein with an apparent molecular weight of 180 and 95 kDa under non-reducing and reducing conditions. Cross-linking of 125I-labelled TGF-beta1 to melanoma cells, Mel 97, by disuccinimidyl suberate (DSS) demonstrated that CD105 expressed on pigmented cells binds TGF-beta1; the pattern of binding of TGF-beta1 to melanoma cells was found to be similar to that of human umbilical vein endothelial cells. The addition of exogenous, bioactive TGF-beta1 significantly (P<0.05) inhibited the growth of CD105-positive melanoma cells, Mel 97, but did not affect that of CD105-negative melanoma cells, F0-1. These data, altogether, demonstrate that CD105 is expressed on pigmented cells and might play a functionally relevant role in the biology of human melanoma cells by regulating their sensitivity to TGF-betas. Images Figure 1 Figure 3 Figure 4 PMID:8932339

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.

The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-beta, increases podocyte motility and albumin permeability.

PubMed

Lee, Eun Young; Chung, Choon Hee; Khoury, Charbel C; Yeo, Tet Kin; Pyagay, Petr E; Wang, Amy; Chen, Sheldon

2009-07-01

The role of monocyte chemoattractant protein-1 (MCP-1) in diabetic nephropathy is typically viewed through the lens of inflammation, but MCP-1 might exert noninflammatory effects on the kidney cells directly. Glomerular podocytes in culture, verified to express the marker nephrin, were exposed to diabetic mediators such as high glucose or angiotensin II and assayed for MCP-1. Only transforming growth factor-beta (TGF-beta) significantly increased MCP-1 production, which was prevented by SB431542 and LY294002, indicating that signaling proceeded through the TGF-beta type I receptor kinase and the phosphatidylinositol 3-kinase pathway. The TGF-beta-induced MCP-1 was found to activate the podocyte's cysteine-cysteine chemokine receptor 2 (CCR2) and, as a result, enhance the cellular motility, cause rearrangement of the actin cytoskeleton, and increase podocyte permeability to albumin in a Transwell assay. The preceding effects of TGF-beta were replicated by treatment with recombinant MCP-1 and blocked by a neutralizing anti-MCP-1 antibody or a specific CCR2 inhibitor, RS102895. In conclusion, this is the first description that TGF-beta signaling through PI3K induces the podocyte expression of MCP-1 that can then operate via CCR2 to increase cellular migration and alter albumin permeability characteristics. The pleiotropic effects of MCP-1 on the resident kidney cells such as the podocyte may exacerbate the disease process of diabetic albuminuria.

Modulation of TGF-beta signaling during progression of chronic liver diseases.

PubMed

Matsuzaki, Koichi

2009-01-01

A large body of work has established roles for epithelial cells as important mediators of progressive fibrosis and carcinogenesis. Transforming growth factor-beta (TGF-beta) and pro-inflammatory cytokines are important inducers of fibro-carcinogenesis. TGF-beta signaling involves phosphorylation of Smad3 at middle linker and/or C-terminal regions. Reversible shifting of Smad3-dependent signaling between tumor-suppression and oncogenesis in hyperactive Ras-expressing epithelial cells indicates that Smad3 phosphorylated at the C-terminal region (pSmad3C) transmits a tumor-suppressive TGF-beta signal, while oncogenic activities such as cell proliferation and invasion are promoted by Smad3 phosphorylated at the linker region (pSmad3L). Notably, pSmad3L-mediated signaling promotes extracellular matrix deposition by activated mesenchymal cells. During progression of chronic liver diseases, hepatic epithelial hepatocytes undergo transition from the tumor-suppressive pSmad3C pathway to the fibrogenic/oncogenic pSmad3L pathway, accelerating liver fibrosis and increasing risk of hepatocellular carcinoma. c-Jun N-terminal kinase activated by pro-inflammatory cytokines is mediating this perturbed hepatocytic TGF-beta signaling. Thus, TGF-beta signaling of hepatocytes affected by chronic inflammation offers a general framework for understanding the molecular mechanisms of human fibro-carcinogenesis during progression of chronic liver diseases.

Age-related changes in expression of transforming growth factor-beta and receptors in cells of intervertebral discs.

PubMed

Matsunaga, Shunji; Nagano, Satoshi; Onishi, Toshiyuki; Morimoto, Norio; Suzuki, Shusaku; Komiya, Setsuro

2003-01-01

The authors conducted a study to determine age-related changes in expression of transforming growth factor (TGF)-beta1, -beta2, -beta3, and Type I and Type II receptors in various cells in the nucleus pulposus and anulus fibrosus. Immunolocalization of TGFbetas and Type I and II receptors was examined during the aging process of cervical intervertebral discs in senescence-accelerated mice (SAM). The TGFbeta family has important roles for cellular function of various tissues. Its role in disc aging, however, is unknown. Detailed information on the temporal and spatial localization of TGFbetas and their receptors in discs is required before discussing introduction of them clinically into the intervertebral disc. Three groups of five SAM each were used. The groups of SAM were age 8, 24, and 50 weeks, respectively. Hematoxylin and eosin staining and immunohistochemical study involving specific antibodies for TGFbeta1, -beta2, -beta3, and Types I and II TGF receptors were performed. Intervertebral discs exhibited degenerative change with advancing age. The TGFbetas and their receptors were present in the fibrocartilaginous cells within the anulus fibrosus and notochord-like cells within the nucleus pulposus of young mice. Expression of TGFbetas and Type I and Type II receptors changed markedly in the cells within the anulus fibrosus during the aging process. The TGFbetas and their receptors were present in cells within the nucleus pulposus and the anulus fibrosus of young mice, and their expression decreased with age.

Orphan nuclear receptor small heterodimer partner inhibits transforming growth factor-beta signaling by repressing SMAD3 transactivation.

PubMed

Suh, Ji Ho; Huang, Jiansheng; Park, Yun-Yong; Seong, Hyun-A; Kim, Dongwook; Shong, Minho; Ha, Hyunjung; Lee, In-Kyu; Lee, Keesook; Wang, Li; Choi, Hueng-Sik

2006-12-22

Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling.

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 m

Role of ROS-mediated TGF beta activation in laser photobiomodulation

NASA Astrophysics Data System (ADS)

Arany, Praveen R.; Chen, Aaron Chih-Hao; Hunt, Tristan; Mooney, David J.; Hamblin, Michael

2009-02-01

The ability of laser light to modulate specific biological processes has been well documented but the precise mechanism mediating these photobiological interactions remains an area of intense investigation. We recently published the results of our clinical trial with 30 patients in an oral tooth-extraction wound healing model using a 904nm GaAs laser (Oralaser 1010, Oralia, Konstnaz, Germany), assessing healing parameters using routine histopathology and immunostaining (Arany et al Wound Rep Regen 2007, 15, 866). We observed a better organized healing response in laser irradiated oral tissues that correlated with an increased expression of TGF-beta1 immediately post laser irradiation. Our data suggested the source of latent TGF-beta1 might be from the degranulating platelets in the serum, an abundant source of in vivo latent TGF-beta, in the freshly wounded tissues. Further, we also demonstrated the ability of the low power near-infrared laser irradiation to activate the latent TGF-beta complexes in vitro at varying fluences from 10sec (0.1 J/cm2) to 600secs (6 J/cm2). Using serum we observed two isoforms, namely TGF-beta1 and TGF-beta3, were capable of being activated by laser irradiation using an isoform-specific ELISA and a reporter based (p3TP) assay system. We are presently pursuing the precise photomolecular mechanisms focusing on potential chromophores, wavelength and fluence parameters affecting the Latent TGF-beta activation process in serum. As ROS mediated TGF-beta activation has been previously demonstrated and we are also exploring the role of Laser generated-ROS in this activation process. In summary, we present evidence of a potential molecular mechanism for laser photobiomodulation in its ability to activate latent TGF-beta complexes.

TGF-.beta. antagonists as mitigators of radiation-induced tissue damage

DOEpatents

Barcellos-Hoff, Mary H.

1997-01-01

A method for treating tissue damage caused by radiation is described by use of a TGF-.beta. antagonist, such as an anti-TGF-.beta. antibody or a TGF-.beta. latency associated protein. It is administered not more than a week after exposure, and is particularly useful in mitigating the side effects of breast cancer therapy.

TGF-{beta} antagonists as mitigators of radiation-induced tissue damage

DOEpatents

Barcellos-Hoff, M.H.

1997-04-01

A method for treating tissue damage caused by radiation is described by use of a TGF-{beta} antagonist, such as an anti-TGF-{beta} antibody or a TGF-{beta} latency associated protein. It is administered not more than a week after exposure, and is particularly useful in mitigating the side effects of breast cancer therapy.

Equine endometrial fibrosis correlates with 11beta-HSD2, TGF-beta1 and ACE activities.

PubMed

Ganjam, V K; Evans, T J

2006-03-27

Endometrial periglandular fibrosis (EPF) contributes to embryonic and fetal loss in mares. Equine EPF correlates inversely with conception and successful gestation. In the modified Kenney endometrial biopsy classification system, EPF categories I, IIA, IIB, and III correspond to minimal, mild, moderate, and severe fibrosis (+/-inflammation), respectively. Paraffin sections of biopsy specimens were stained with H&E, and picrosirius red (specific for fibrillar collagens types I and III), to determine %EPCVF. Endometrial ACE-binding activity, TGF-beta1 and 11beta-HSD2 activities were also measured. Ultrastructural changes in EPF categories IIB and III endometria strongly suggested myofibroblastic transformation. ACE-binding activity was highest in EPF category IIB; however, endometrial TGF-beta1 and 11beta-HSD2 activities were significantly correlated to the severity of EPF (P<0.05). We conclude that, locally generated angiotensin II initiates the expression of TGF-beta1 resulting in myofibroblastic transformation. 11Beta-HSD2 in concert appears to modulate the severity of endometrial fibrosis.

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.

TGF-beta1 release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function

NASA Technical Reports Server (NTRS)

Lu, L.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

2001-01-01

BACKGROUND: Controlled release of transforming growth factor-beta1 (TGF-beta1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-beta1 delivery and the effects of released TGF-beta1 on the proliferation and differentiation of marrow stromal cells in vitro. METHODS: Recombinant human TGF-beta1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-beta1 was determined after 3 days in culture. The effects of TGF-beta1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period. RESULTS: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content resulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating that the activity of TGF-beta1 was retained during microparticle

Effect of transforming growth factor beta (TGF-β) receptor I kinase inhibitor on prostate cancer bone growth.

PubMed

Wan, Xinhai; Li, Zhi-Gang; Yingling, Jonathan M; Yang, Jun; Starbuck, Michael W; Ravoori, Murali K; Kundra, Vikas; Vazquez, Elba; Navone, Nora M

2012-03-01

Transforming growth factor beta 1 (TGF-β1) has been implicated in the pathogenesis of prostate cancer (PCa) bone metastasis. In this study, we tested the antitumor efficacy of a selective TGF-β receptor I kinase inhibitor, LY2109761, in preclinical models. The effect of LY2109761 on the growth of MDA PCa 2b and PC-3 human PCa cells and primary mouse osteoblasts (PMOs) was assessed in vitro by measuring radiolabeled thymidine incorporation into DNA. In vivo, the right femurs of male SCID mice were injected with PCa cells. We monitored the tumor burden in control- and LY2109761-treated mice with MRI analysis and the PCa-induced bone response with X-ray and micro-CT analyses. Histologic changes in bone were studied by performing bone histomorphometric evaluations. PCa cells and PMOs expressed TGF-β receptor I. TGF-β1 induced pathway activation (as assessed by induced expression of p-Smad2) and inhibited cell growth in PC-3 cells and PMOs but not in MDA PCa 2b cells. LY2109761 had no effect on PCa cells but induced PMO proliferation in vitro. As expected, LY2109761 reversed the TGF-β1-induced pathway activation and growth inhibition in PC-3 cells and PMOs. In vivo, LY2109761 treatment for 6weeks resulted in increased volume in normal bone and increased osteoblast and osteoclast parameters. In addition, LY2109761 treatment significantly inhibited the growth of MDA PCa 2b and PC-3 in the bone of SCID mice (p<0.05); moreover, it resulted in significantly less bone loss and change in osteoclast-associated parameters in the PC-3 tumor-bearing bones than in the untreated mice. In summary, we report for the first time that targeting TGF-β receptors with LY2109761 can control PCa bone growth while increasing the mass of normal bone. This increased bone mass in nontumorous bone may be a desirable side effect of LY2109761 treatment for men with osteopenia or osteoporosis secondary to androgen-ablation therapy, reinforcing the benefit of effectively controlling PCa growth

Effect of transforming growth factor beta (TGF-β) receptor I kinase inhibitor on prostate cancer bone growth

PubMed Central

Wan, Xinhai; Li, Zhi-Gang; Yingling, Jonathan M.; Yang, Jun; Starbuck, Michael W.; Ravoori, Murali K.; Kundra, Vikas; Vazquez, Elba; Navone, Nora M.

2012-01-01

Transforming growth factor beta 1 (TGF-β1) has been implicated in the pathogenesis of prostate cancer (PCa) bone metastasis. In this study, we tested the antitumor efficacy of a selective TGF-β receptor I kinase inhibitor, LY2109761, in preclinical models. The effect of LY2109761 on the growth of MDA PCa 2b and PC-3 human PCa cells and primary mouse osteoblasts (PMOs) was assessed in vitro by measuring radiolabeled thymidine incorporation into DNA. In vivo, the right femurs of male SCID mice were injected with PCa cells. We monitored the tumor burden in control- and LY2109761-treated mice with MRI analysis and the PCa-induced bone response with x-ray and micro-CT analyses. Histologic changes in bone were studied by performing bone histomorphometric evaluations. PCa cells and PMOs expressed TGF-β receptor I. TGF-β1 induced pathway activation (as assessed by induced expression of p-Smad2) and inhibited cell growth in PC-3 cells and PMOs but not in MDA PCa 2b cells. LY2109761 had no effect on PCa cells but induced PMO proliferation in vitro. As expected, LY2109761 reversed the TGF-β1–induced pathway activation and growth inhibition in PC-3 cells and PMOs. In vivo, LY2109761 treatment for 6 weeks resulted in increased volume in normal bone and increased osteoblast and osteoclast parameters. In addition, LY2109761 treatment significantly inhibited the growth of MDA PCa 2b and PC-3 in the bone of SCID mice (p < 0.05); moreover, it resulted in significantly less bone loss and change in osteoclast-associated parameters in the PC-3 tumor–bearing bones than in the untreated mice. In summary, we report for the first time that targeting TGF-β receptors with LY2109761 can control PCa bone growth while increasing the mass of normal bone. This increased bone mass in nontumorous bone may be a desirable side effect of LY2109761 treatment for men with osteopenia or osteoporosis secondary to androgen-ablation therapy, reinforcing the benefit of effectively controlling PCa

Losartan improves resistance artery lesions and prevents CTGF and TGF-beta production in mild hypertensive patients.

PubMed

Gómez-Garre, D; Martín-Ventura, J L; Granados, R; Sancho, T; Torres, R; Ruano, M; García-Puig, J; Egido, J

2006-04-01

Although structural and functional changes of resistance arteries have been proposed to participate in arterial hypertension (HTA) outcome, not all therapies may correct these alterations, even if they normalize the blood pressure (BP). The aim of this study was to investigate the mechanisms of the protection afforded by the angiotensin receptor antagonist losartan in resistance arteries from patients with essential HTA. In all, 22 untreated hypertensive patients were randomized to receive losartan or amlodipine for 1 year and the morphological characteristics of resistance vessels from subcutaneous biopsies were evaluated. Protein expression of connective tissue growth factor (CTGF), transforming growth factor beta (TGF-beta), and collagens III and IV was detected by immunohistochemistry. In comparison with normotensive subjects, resistance arteries from hypertensive patients showed a significant media:lumen (M/L) ratio increment and a higher protein expression of CTGF, TGF-beta, and collagens. After 1 year of treatment, both losartan and amlodipine similarly controlled BP. However, M/L only decreased in patients under losartan treatment, whereas in the amlodipine-treated group this ratio continued to increase significantly. The administration of losartan prevented significant increments in CTGF, TGF-beta, and collagens in resistance arteries. By contrast, amlodipine-treated patients showed a higher vascular CTGF, TGF-beta, and collagen IV staining than before treatment. Our results show that the administration of losartan, but not amlodipine, to hypertensive patients improves structural abnormalities and prevents the production of CTGF and TGF-beta in small arteries, despite similar BP lowering. These data may explain the molecular mechanisms of the better vascular protection afforded by drugs interfering with the renin-angiotensin system.

New insights into the dual role of TGF-beta | Center for Cancer Research

Cancer.gov

The dual role of TGF-beta in cancer continues to challenge investigators in the field. TGF-beta is a well-known factor associated with tumor suppression in normal cells and yet promotes tumor progression in advanced stages of cancer. For years, the mechanisms that underpin this conundrum have not been fully understood. Ying Zhang, Ph.D., senior investigator in the Laboratory of Cellular and Molecular Biology, has been exploring this problem by examining and characterizing several key molecules in the TGF-beta signaling pathway. Read more…

Elevated plasma TGF-beta1 in renal diseases: cause or consequence?

PubMed

Junker, U; Haufe, C C; Nuske, K; Rebstock, K; Steiner, T; Wunderlich, H; Junker, K; Reinhold, D

2000-07-01

We previously reported elevated levels of TGF-beta1 in patients with renal carcinoma. Certain aspects led us to ask whether they might be caused by chronic damage to the kidney(s). Here we report on an extended set of patients with various renal diseases, lung cancer, humoral immunodeficiency and controls. For latent TGF-beta1 in plasma, we find that the control, immunodeficiency, lung cancer and kidney transplant groups do not differ significantly (means, 7.0-8.8 ng/ml). Also, acute short-term renal stress (extracorporal lithotrypsy) does not lead to an increase of TGF-beta1. However, the pyelonephritis patients present with levels of 19.0 ng/ml, chronic extracorporal dialysis patients with 15.5 ng/ml, and renal cell carcinoma patients with 22.8 ng/ml. For active TGF-beta1 these findings are exactly recovered. For serum levels, only the renal carcinoma group presents with significantly elevated levels of TGF-beta1. Kidney transplantation seems to normalize TGF-beta1 levels, while in the kidney cancer patients surgery has an effect only in part of the group. We conclude that elevated plasma TGF-beta1 levels are common in at least two chronic renal disease conditions, and that it normalizes with restoration of renal function. It is tempting to speculate that chronic elevation of TGF-beta1 in these patients may be critically involved in these conditions predisposing to renal cancer. Copyright 2000 Academic Press.

Effect of angiotensin II receptor blocker on plasma levels of TGF-beta 1 and interstitial fibrosis in hypertensive kidney transplant patients.

PubMed

el-Agroudy, Amgad E; Hassan, Nabil A; Foda, Mohamed A; Ismail, Amani M; el-Sawy, Essam A; Mousa, Omar; Ghoneim, Mohamed A

2003-01-01

Transforming growth factor-beta1 (TGF-beta 1) is involved in the pathogenesis of chronic allograft nephropathy after kidney transplantation. The aim of the study was to evaluate the effect of the angiotensin receptor blocker losartan on TGF-beta 1 plasma levels and proteinuria in hypertensive transplant recipients. A total of 162 transplant recipients were included in the study. The patients were randomized into 3 groups: group 1 received losartan; group II received an angiotensin-converting enzyme inhibitor (captopril), and group III received a calcium channel blocker (amlodipine). All the parameters were recorded at the time of therapy initiation and at 1, 4 and 12 weeks and 12 months thereafter. Graft biopsy before the start and at the end of the study was done to evaluate histopathological progression. Blood pressure was controlled in the 3 groups; however, the need for other antihypertensive agents was significant in groups I and II. Treatment with losartan significantly decreased the plasma level of TGF-beta1, 24-hour urinary protein and serum uric acid (p < 0.05). No significant changes were seen in the hemoglobin or serum potassium levels. The rate of histopathological progression was significantly lower in the losartan group. No patient was discharged from the study due to side effects. After transplantation all drugs were able to control blood pressure with good safety and tolerability. The study demonstrates that ARB significantly decreases the plasma levels of TGF-beta1, proteinuria and uric acid. These results could play an important and decisive role in the treatment and prevention of chronic allograft nephropathy. Copyright 2003 S. Karger AG, Basel

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.

Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mediate TGF-{beta}- and myostatin-induced suppression of proliferation in porcine embryonic myogenic cell cultures

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

Kamanga-Sollo, E.; Pampusch, M.S.; White, M.E.

2005-11-15

We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-{beta} superfamily members myostatin and TGF-{beta}{sub 1} have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-{beta}{submore » 1} or myostatin significantly (P < 0.01) increases levels of IGFBP-3 and -5 mRNA. We have previously shown that immunoneutralization of IGFBP-3 decreases the proliferation-suppressing activity of TGF-{beta}{sub 1} and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P < 0.05) decreases the DNA synthesis-suppressing activity of these molecules. Simultaneous immunoneutralization of both IGFBP-3 and IGFBP-5 in TGF-{beta}{sub 1} or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-{beta}{sub 1} or myostatin treatment (P < 0.05). Even though immunoneutralization of IGFBP-3 and -5 increased DNA synthesis rates in TGF-{beta}{sub 1} or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-{beta} and myostatin to suppress proliferation of PEMC.« less

Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-beta signaling.

PubMed

Gao, Sheng; Alarcón, Claudio; Sapkota, Gopal; Rahman, Sadia; Chen, Pan-Yu; Goerner, Nina; Macias, Maria J; Erdjument-Bromage, Hediye; Tempst, Paul; Massagué, Joan

2009-11-13

TGF-beta induces phosphorylation of the transcription factors Smad2 and Smad3 at the C terminus as well as at an interdomain linker region. TGF-beta-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. Here, we identify Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain, Nedd4L specifically recognizes a TGF-beta-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 polyubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGF-beta-activated Smads and restricts the amplitude and duration of TGF-beta gene responses, and in mouse embryonic stem cells, it limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGF-beta signal transduction.

Reduced expression of TGF beta is associated with advanced disease in transitional cell carcinoma.

PubMed Central

Coombs, L. M.; Pigott, D. A.; Eydmann, M. E.; Proctor, A. J.; Knowles, M. A.

1993-01-01

The gene structure and expression of the related peptide regulatory factors TGF beta 1 and TGF beta 2 were studied in a panel of seven urothelial carcinoma cell lines and 40 transitional cell carcinomas. The latter comprised 15 grade 1, 18 grade 2 and 5 grade 3 tumours and two cases of carcinoma in situ. Control tissues included ten matched 'field' biopsies and 17 other biopsies including 11 biopsies of macroscopically normal urothelium, two of which were from patients with no history of bladder cancer. No amplification of rearrangements of either TGF beta 1 or TGF beta 2 were detected in any sample. A complex pattern of expression or the two genes was found in the urothelial cell lines. High, but variable levels of the 2.5 kb TGF beta 1 transcript were detected and lower and more variable levels of the three (4.1 kb, 5.1 kb and 6.5 kb) transcripts of TGF beta 2 were detected. Although those cell lines expressing most TGF beta 1 tended to express less TGF beta 2 transcript there was no clear-cut relationship. In comparison, no TGF beta 2 transcript was identified in any primary transitional cell carcinoma or control tissue. Markedly reduced or undetectable levels of TGF beta 1 transcript were detected in 4/15 (26%) grade 1, 5/18 (28%) grade 2 and 3/5 (60%) grade 3 tumours. There was no clear relationship to tumour stage, lymphocytic infiltration or stromal content of the tumours. Clinical review one year after the 2 year period of tumour collection showed that 6/9 (66%) of patients with tumours with reduced levels of transcript had died or had disease which was not controllable by local resection and 3/9 (33%) had developed tumour re-occurrences. In comparison, in the group with normal levels of expression of TGF beta 1, 3/18 (17%) had disease which was not controllable by local means, 9/18 (50%) had tumour re-occurrence and 6/18 (33%) had no evidence of disease. The association of reduced expression of TGF beta 1 and advanced disease was statistically significant

Butyrate modulates TGF-beta1 generation and function: potential renal benefit for Acacia(sen) SUPERGUM (gum arabic)?

PubMed

Matsumoto, N; Riley, S; Fraser, D; Al-Assaf, S; Ishimura, E; Wolever, T; Phillips, G O; Phillips, A O

2006-01-01

Anecdotal evidence suggests that high fibre supplementation of dietary intake may have health benefits in renal disease related to alterations in circulating levels of short-chain fatty acids. The aim of the study was to examine the hypothesis that dietary manipulation may increase serum butyrate and thus have potential beneficial effects in renal disease. We examined the effect of dietary supplementation with a gum arabic sample of standardized molecular characteristics, Acacia(sen) SUPERGUM EM2 (SUPERGUM), on systemic levels of butyrate in normal human subjects. In an in vitro study, we also examined the potential role of butyrate in modifying the generation of the profibrotic cytokine transforming growth factor-beta (TGF-beta1) by renal epithelial cells. Following 8 weeks of dietary supplementation with 25 g/day of SUPERGUM, there was a two-fold increase in serum butyrate (n=7, P=0.03). In vitro work demonstrated that exposure of renal epithelial cells to elevated concentrations of butyrate suppressed both basal and stimulated TGF-beta1 synthesis. The action of butyrate was mediated by suppression of the extracellular signal-regulated kinase/mitogen-activated protein kinase signalling pathway. In addition, butyrate exposures reduced the response of renal epithelial cells to TGF-beta1 as assessed by luciferase activity of a TGF-beta-responsive reporter construct. Attenuation of TGF-beta1 signalling was associated with reduced phosphorylation of Smad 3 and decreased trafficking of TGF-beta1 receptors into signalling, non-lipid raft-associated membrane fractions. In conclusion, the data demonstrate that dietary supplementation with SUPERGU increased serum butyrate, which at least in vitro has beneficial effects on renal pro-fibrotic cytokine generation.

Bmi-1 extends the life span of normal human oral keratinocytes by inhibiting the TGF-{beta} signaling

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

Kim, Reuben H., E-mail: rkim@dentistry.ucla.edu; UCLA Dental Research Institute, Los Angeles, CA 90095; UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA 90095

2010-10-01

We previously demonstrated that Bmi-1 extended the in vitro life span of normal human oral keratinocytes (NHOK). We now report that the prolonged life span of NHOK by Bmi-1 is, in part, due to inhibition of the TGF-{beta} signaling pathway. Serial subculture of NHOK resulted in replicative senescence and terminal differentiation and activation of TGF-{beta} signaling pathway. This was accompanied with enhanced intracellular and secreted TGF-{beta}1 levels, phosphorylation of Smad2/3, and increased expression of p15{sup INK4B} and p57{sup KIP2}. An ectopic expression of Bmi-1 in NHOK (HOK/Bmi-1) decreased the level of intracellular and secreted TGF-{beta}1 induced dephosphorylation of Smad2/3, andmore » diminished the level of p15{sup INK4B} and p57{sup KIP2}. Moreover, Bmi-1 expression led to the inhibition of TGF-{beta}-responsive promoter activity in a dose-specific manner. Knockdown of Bmi-1 in rapidly proliferating HOK/Bmi-1 and cancer cells increased the level of phosphorylated Smad2/3, p15{sup INK4B}, and p57{sup KIP2}. In addition, an exposure of senescent NHOK to TGF-{beta} receptor I kinase inhibitor or anti-TGF-{beta} antibody resulted in enhanced replicative potential of cells. Taken together, these data suggest that Bmi-1 suppresses senescence of cells by inhibiting the TGF-{beta} signaling pathway in NHOK.« less

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

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.

ALK and TGF-Beta Resistance in Breast Cancer

DTIC Science & Technology

2017-10-01

Award Number: W81XWH‐15‐1‐0650 TITLE: ALK and TGF-Beta Resistance in Breast Cancer PRINCIPAL INVESTIGATOR: Xin-Hua Feng CONTRACTING...and TGF-Beta Resistance in Breast Cancer 5b. GRANT NUMBER W81XWH‐15‐1‐0650 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Xin-Hua Feng...response is a hallmark in human cancer . However, the mechanisms underlying TGF- resistance in breast cancer have not been elucidated. Anaplastic

Synergy between TGF-beta 3 and NT-3 to promote the survival of spiral ganglia neurones in vitro.

PubMed

Marzella, P L; Clark, G M; Shepherd, R K; Bartlett, P F; Kilpatrick, T J

1998-01-09

Transforming growth factor-betas (TGF-betas) have been implicated in normal inner ear development and in promoting neuronal survival. Early rat post-natal spiral ganglion cells (SGC) in dissociated cell culture were used as a model of auditory innervation to test the trophic factors TGF-beta3 and neurotrophin-3 (NT-3) for their ability, individually or in combination, to promote neuronal survival. The findings from this study suggest that TGF-beta3 supports neuronal survival in a concentration-dependent manner. Moreover TGF-beta3 and NT-3-potentiated spiral ganglion neuronal survival in a synergistic fashion.

A critical role for transcription factor Smad4 in T cell function independent of transforming growth factor beta receptor signaling

PubMed Central

Gu, Ai-Di; Zhang, Song; Wang, Yunqi; Xiong, Hui; Curtis, Thomas A.; Wan, Yisong Y.

2014-01-01

Summary Transforming growth factor-beta (TGF-β) suppresses T cell function to maintain self-tolerance and to promote tumor immune evasion. Yet how Smad4, a transcription factor component of TGF-β signaling, regulates T cell function remains unclear. Here we have demonstrated an essential role for Smad4 in promoting T cell function during autoimmunity and anti-tumor immunity. Smad4 deletion rescued the lethal autoimmunity resulting from transforming growth factor-beta receptor (TGF-βR) deletion and compromised T-cell-mediated tumor rejection. While Smad4 was dispensable for T cell generation, homeostasis and effector function, it was essential for T cell proliferation following activation in vitro and in vivo. The transcription factor Myc was identified to mediate Smad4-controlled T cell proliferation. This study thus reveals a requirement of Smad4 for T-cell-mediated autoimmunity and tumor rejection, which is beyond the current paradigm. It highlights a TGF-βR-independent role for Smad4 in promoting T cell function, autoimmunity and anti-tumor immunity. PMID:25577439

PGE(2) inhibition of TGF-beta1-induced myofibroblast differentiation is Smad-independent but involves cell shape and adhesion-dependent signaling.

PubMed

Thomas, Peedikayil E; Peters-Golden, Marc; White, Eric S; Thannickal, Victor J; Moore, Bethany B

2007-08-01

Myofibroblasts are pathogenic in pulmonary fibrotic disease due to their exuberant production of matrix rich in collagen that interferes with gas exchange and the ability of these cells to contract and distort the alveolar space. Transforming growth factor-beta1 (TGF-beta1) is a well-known inducer of myofibroblast differentiation. TGF-beta1-induced transformation of fibroblasts to apoptosis-resistant myofibroblasts is adhesion-dependent and focal adhesion kinase (FAK)-mediated. Prostaglandin E(2) (PGE(2)) inhibits this differentiation via E prostanoid receptor 2 (EP2) signaling and cAMP elevation, but whether PGE(2) does so by interfering with TGF-beta1 signaling is unknown. Thus we examined the effects of PGE(2) in the presence and absence of TGF-beta1 stimulation on candidate signaling pathways in human lung fibroblasts. We now demonstrate that PGE(2) does not interfere with TGF-beta1-induced Smad phosphorylation or its translocation to the nucleus. Rather, PGE(2) has dramatic effects on cell shape and cytoskeletal architecture and disrupts the formation of appropriate focal adhesions. PGE(2) treatment diminishes TGF-beta1-induced phosphorylation of paxillin, STAT-3, and FAK and, in turn, limits activation of the protein kinase B (PKB/Akt) pathway. These alterations do not, however, result in increased apoptosis within the first 24 h of treatment. Interestingly, the effects of PGE(2) stimulation alone do not always mirror the effects of PGE(2) in the presence of TGF-beta1, indicating that the context for EP2 signaling is different in the presence of TGF-beta1. Taken together, our results demonstrate that PGE(2) has the potential to limit TGF-beta1-induced myofibroblast differentiation via adhesion-dependent, but Smad-independent, pathways.

Macrophage colony-stimulating factor accelerates wound healing and upregulates TGF-beta1 mRNA levels through tissue macrophages.

PubMed

Wu, L; Yu, Y L; Galiano, R D; Roth, S I; Mustoe, T A

1997-10-01

Macrophage colony-stimulating factor (M-CSF) is produced by many cell types involved in wound repair, yet it acts specifically on monocytes and macrophages. The monocyte-derived cell is thought to be important in wound healing, but the importance of the role of tissue macrophages in wound healing has not been well defined. Dermal ulcers were created in normal and ischemic ears of young rabbits. Either rhM-CSF (17 microg/wound) or buffer was applied to each wound. Wounds were bisected and analyzed histologically at Days 7 and 10 postwounding. The amounts of epithelial growth and granulation tissue deposition were measured in all wounds. The level of increase of TGF-beta1 mRNA level in M-CSF-treated wounds was examined using competitive RT-PCR. M-CSF increased new granulation tissue formation by 37% (N = 21, P < 0.01) and 50% (P < 0.01) after single and multiple treatments, respectively, in nonischemic wounds. TGF-beta1 mRNA levels in rhM-CSF-treated wounds increased 5.01-fold (N = 8) over vehicle-treated wounds under nonischemic conditions. In contrast, no effect could be detected in ischemic wounds treated with rhM-CSF, and these wounds only showed a 1.66-fold increase in TGF-beta1 mRNA levels when compared to ischemic wounds treated with vehicle alone. GAPDH, a housekeeping gene, showed no change. As mesenchymal cells lack receptors for M-CSF, the improved healing of wounds treated with topical rhM-CSF must reflect a generalized enhancement of activation and function of tissue macrophages, as demonstrated by upregulation of TGF-beta. The lack of effect under ischemic conditions suggests that either macrophage activity and/or response to M-CSF is adversely affected under those conditions; this may suggest the pathogenesis of impaired wound healing at the cellular level. Copyright 1997 Academic Press.

SNP analyses of growth factor genes EGF, TGF{beta}-1, and HGF reveal haplotypic association of EGF with autism

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

Toyoda, Takao; Thanseem, Ismail; Kawai, Masayoshi

Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-{beta} (TGF{beta}) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGF{beta}1, and HGF genes with autism, in amore » trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGF{beta}1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism.« less

New insights into the dual role of TGF-beta | Center for Cancer Research

Cancer.gov

The dual role of TGF-beta in cancer continues to challenge investigators in the field. TGF-beta is a well-known factor associated with tumor suppression in normal cells and yet promotes tumor progression in advanced stages of cancer. For years, the mechanisms that underpin this conundrum have not been fully understood. Ying Zhang, Ph.D., senior investigator in the Laboratory

Peroxisome proliferator-activated receptor gamma and transforming growth factor-beta pathways inhibit intestinal epithelial cell growth by regulating levels of TSC-22.

PubMed

Gupta, Rajnish A; Sarraf, Pasha; Brockman, Jeffrey A; Shappell, Scott B; Raftery, Laurel A; Willson, Timothy M; DuBois, Raymond N

2003-02-28

Peroxisome proliferator-activated receptor gamma (PPARgamma) and transforming growth factor-beta (TGF-beta) are key regulators of epithelial cell biology. However, the molecular mechanisms by which either pathway induces growth inhibition and differentiation are incompletely understood. We have identified transforming growth factor-simulated clone-22 (TSC-22) as a target gene of both pathways in intestinal epithelial cells. TSC-22 is member of a family of leucine zipper containing transcription factors with repressor activity. Although little is known regarding its function in mammals, the Drosophila homolog of TSC-22, bunched, plays an essential role in fly development. The ability of PPARgamma to induce TSC-22 was not dependent on an intact TGF-beta1 signaling pathway and was specific for the gamma isoform. Localization studies revealed that TSC-22 mRNA is enriched in the postmitotic epithelial compartment of the normal human colon. Cells transfected with wild-type TSC-22 exhibited reduced growth rates and increased levels of p21 compared with vector-transfected cells. Furthermore, transfection with a dominant negative TSC-22 in which both repressor domains were deleted was able to reverse the p21 induction and growth inhibition caused by activation of either the PPARgamma or TGF-beta pathways. These results place TSC-22 as an important downstream component of PPARgamma and TGF-beta signaling during intestinal epithelial cell differentiation.

GASDERMIN, suppressed frequently in gastric cancer, is a target of LMO1 in TGF-beta-dependent apoptotic signalling.

PubMed

Saeki, N; Kim, D H; Usui, T; Aoyagi, K; Tatsuta, T; Aoki, K; Yanagihara, K; Tamura, M; Mizushima, H; Sakamoto, H; Ogawa, K; Ohki, M; Shiroishi, T; Yoshida, T; Sasaki, H

2007-10-04

Defining apoptosis-regulatory cascades of the epithelium is important for understanding carcinogenesis, since cancer cells are considered to arise as a result of the collapse of the cascades. We previously reported that a novel gene GASDERMIN (GSDM) is expressed in the stomach but suppressed in gastric cancer cell lines. Furthermore, in this study, we demonstrated that GSDM is expressed in the mucus-secreting pit cells of the gastric epithelium and frequently silenced in primary gastric cancers. We found that GSDM has a highly apoptotic activity and its expression is regulated by a transcription factor LIM domain only 1 (LMO1) through a sequence to which Runt-related transcription factor 3 (RUNX3) binds, in a GSDM promoter region. We observed coexpression of GSDM with LMO1, RUNX3 and type II transforming growth factor-beta receptor (TGF-betaRII) in the pit cells, and found that TGF-beta upregulates the LMO1- and GSDM-expression in the gastric epithelial cell line and induces apoptosis, which was confirmed by the finding that the apoptosis induction is inhibited by suppression of each LMO1-, RUNX3- and GSDM expression, respectively. The present data suggest that TGF-beta, LMO1, possibly RUNX3, and GSDM form a regulatory pathway for directing the pit cells to apoptosis.

TGF-beta1 secretion of ROS-17/2.8 cultures on NiTi implant material.

PubMed

Kapanen, Anita; Kinnunen, Anne; Ryhänen, Jorma; Tuukkanen, Juha

2002-08-01

The biocompatibility of an orthopedic implant depends on the effect of the implant on bone-forming cells, osteoblasts. Changes in osteoblastic proliferation, maturation and differentiation are important events in ossification that enable monitoring the effect of the implant. Transforming growth factor-beta (TGF-beta) is known to suppress osteoblast proliferation and, on the other hand, to induce the maturation and differentiation of osteoblasts. Moreover, osteoblasts produce TGF-beta, which is embedded in the bone matrix and activated by bone-resorbing osteoclasts. TGF-beta inhibits osteoclastic activity. Here, we show for the first time the effect of nickel titanium shape memory metal (NiTi) on osteoblastic cytokine expression. In this study, we measured the levels of TGF-beta with enzyme-linked immunosorbent assay (ELISA) from a ROS-17/2.8 osteosarcoma cell line cultured on different metal alloy discs. ELISA results were proportioned to total DNA content of the samples. We compared NiTi, to stainless steel (Stst), pure titanium (Ti) and pure nickel (Ni). The TGF-beta1/DNA value in the NiTi group (0.0007 +/- 0.0003) was comparable with those seen in the Stst (0.0008 +/- 0.0001) and Ti (0.0007 +/- 0.0001) groups. The concentration in the Ni group was lower (0.0006 +/- 0.0003), though not statistically significantly so. In addition, the effect of surface roughness on TGF-beta1 production was studied. We compared three different grades of roughness in three differently hot-rolled alloys: NiTi. hot-rolled at 950 degrees C. Ti alloy hot-rolled at 850 degrees C (TiI) and the same Ti alloy hot-rolled at 1,050 degrees C (TiII). We found that increasing roughness of the NiTi surface increased the TGF-beta1 concentration. On the other hand, all roughness groups of TiII showed low levels of TGF-beta1. while a rough TiI surface induced similar TGF-beta1, expression as rough NiTi. Further, these same measurements made with interleukine 6 (IL-6) were found to be under the

Existence of a regulatory loop between MCP-1 and TGF-beta in glomerular immune injury.

PubMed

Wolf, Gunter; Jocks, Thomas; Zahner, Gunther; Panzer, Ulf; Stahl, Rolf A K

2002-11-01

Glomerular upregulation of monocyte chemotactic protein-1 (MCP-1), followed by an influx of monocytes resulting eventually in extracellular matrix deposition is a common sequel of many types of glomerulonephritis. However, it is not entirely clear how early expression of MCP-1 is linked to the later development of glomerulosclerosis. Because transforming growth factor-beta (TGF-beta) is a key regulator of extracellular matrix proteins, we hypothesized that there might be a regulatory loop between early glomerular MCP-1 induction and subsequent TGF-beta expression. To avoid interference with other cytokines that may be released from infiltrating monocytes, isolated rat kidneys were perfused with a polyclonal anti-thymocyte-1 antiserum (ATS) and rat serum (RS) as a complement source to induce glomerular injury. Renal TGF-beta protein and mRNA expressions were strongly stimulated after perfusion with ATS-RS. This effect was attenuated by coperfusion with a neutralizing anti-MCP-1 but was partly mimicked by perfusion with recombinant MCP-1 protein. On the other hand, renal MCP-1 expression and production were stimulated by administration of ATS-RS. Additional perfusion with an anti-TGF-beta antibody further aggravated this increase, whereas application of recombinant TGF-beta protein reduced MCP-1 formation. Our data demonstrate an intrinsic regulatory loop in which increased MCP-1 levels stimulate TGF-beta formation in resident glomerular cells in the absence of infiltrating immune competent cells.

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

Inhibition of Transforming Growth Factor-Beta1 SignalingAttenuates Ataxia Telangiectasia Mutated Activity in Response toGenotoxic Stress

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

Kirshner, Julia; Jobling, Michael F.; Pajares, Maria Jose

2006-01-01

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor {beta} (TGF{beta})-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF{beta} inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf{beta}I null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF{beta} type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced H2AX radiation-induced foci; and increased radiosensitivity compared with TGF{beta} competent cells.more » We determined that loss of TGF{beta} signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF{beta} restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf{beta}I, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF{beta} may be used to advantage in cancer therapy.« less

[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.

Sumoylation of Smad3 stimulates its nuclear export during PIASy-mediated suppression of TGF-{beta} signaling

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

Imoto, Seiyu; Ohbayashi, Norihiko; Ikeda, Osamu

2008-05-30

Sma- and MAD-related protein 3 (Smad3) plays crucial roles in the transforming growth factor-{beta} (TGF-{beta})-mediated signaling pathway, which produce a variety of cellular responses, including cell proliferation and differentiation. In our previous study, we demonstrated that protein inhibitor of activated STATy (PIASy) suppresses TGF-{beta} signaling by interacting with and sumoylating Smad3. In the present study, we examined the molecular mechanisms of Smad3 sumoylation during PIASy-mediated suppression of TGF-{beta} signaling. We found that small-interfering RNA-mediated reduction of endogenous PIASy expression enhanced TGF-{beta}-induced gene expression. Importantly, coexpression of Smad3 with PIASy and SUMO1 affected the DNA-binding activity of Smad3. Furthermore, coexpression ofmore » Smad3 with PIASy and SUMO1 stimulated the nuclear export of Smad3. Finally, fluorescence resonance energy transfer analyses revealed that Smad3 interacted with SUMO1 in the cytoplasm. These results suggest that PIASy regulates TGF-{beta}/Smad3-mediated signaling by stimulating sumoylation and nuclear export of Smad3.« less

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

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.

IGF-binding proteins mediate TGF-beta 1-induced apoptosis in bovine mammary epithelial BME-UV1 cells.

PubMed

Gajewska, Małgorzata; Motyl, Tomasz

2004-10-01

TGF-beta 1 is an antiproliferative and apoptogenic factor for mammary epithelial cells (MEC) acting in an auto/paracrine manner and thus considered an important local regulator of mammary tissue involution. However, the apoptogenic signaling pathway induced by this cytokine in bovine MEC remains obscure. The present study was focused on identification of molecules involved in apoptogenic signaling of transforming growth factor-beta 1 (TGF-beta 1) in the model of bovine mammary epithelial cell line (BME-UV1). Laser scanning cytometry (LSC), Western blot and electrophoretic mobility shift assay (EMSA) were used for analysis of expression and activity of TGF-beta 1-related signaling molecules. The earliest response occurring within 1-2 h after TGF-beta 1 administration was an induction and activation of R-Smads (Smad2 and Smad3) and Co-Smad (Smad4). An evident formation of Smad-DNA complexes began from 2nd hour after MEC exposure to TGF-beta 1. Similarly to Smads, proteins of AP1 complex: phosphorylated c-Jun and JunD appeared to be early reactive molecules; however, an increase in their expression was detected only in cytosolic fraction. In the next step, an increase of IGF binding protein-3 (IGFBP-3) and IGFBP-4 expression was observed from 6th hour followed by a decrease in the activity of protein kinase B (PKB/Akt), which occurred after 24 h of MEC exposure to TGF-beta 1. The decrease in PKB/Akt activity coincided in time with the decline of phosphorylated Bad expression (inactive form). Present study supported additional evidence that stimulation of insulin-like growth factor I (IGF-I) was associated with complete abrogation of TGF-beta 1-induced activation of Bad and Bax and in the consequence protection against apoptosis. In conclusion, apoptotic effect of TGF-beta 1 in bovine MEC is mediated by IGFBPs and occurs through IGF-I sequestration, resulting in inhibition of PKB/Akt-dependent survival pathway.

Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

PubMed

Hough, Chris; Radu, Maria; Doré, Jules J E

2012-01-01

The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

Opposite actions of transforming growth factor-beta 1 on the gene expression of atrial natriuretic peptide biological and clearance receptors in a murine thymic stromal cell line.

PubMed

Agui, T; Xin, X; Cai, Y; Shim, G; Muramatsu, Y; Yamada, T; Fujiwara, H; Matsumoto, K

1995-09-01

The regulation of the gene expression of the atrial natriuretic peptide receptor (ANPR) subtypes, ANPR-A, ANPR-B, and ANPR-C, was investigated in a murine thymic stromal cell line, MRL 104.8a. When MRL 104.8a cells were cultured with transforming growth factor (TGF)-beta1, [125I]ANP binding sites increased with increasing dose of TGF-beta1. These binding sites were identified as ANPR-C by a displacement experiment with ANPR-C-specific ligand, C-ANF, and by the affinity cross-linking of the [125I]ANP binding sites with a chemical cross-linker to determine the molecular weight of the ANPR. This augmentation of the ANPR-C expression was elucidated to occur at the transcriptional level by Northern blot experiment, comparison of the relative amounts of mRNA by reverse transcription (RT)-PCR, and in vitro nuclear transcription assay. Conversely, the expression of the ANP biological receptors, ANPR-A and ANPR-B, was shown to be down-regulated by TGF-beta1. These data suggest that TGF-beta1 regulates the gene expression of ANPRs in the thymic stromal cells and that ANP and TGF-beta1 might affect the thymic stromal cell functions.

[Effect of TGF-beta1 on embryo implantation and development in mice in vitro].

PubMed

Luo, Shan; Yin, Hai-ning; Li, Shang-wei

2010-03-01

To investigate the role of TGF-beta1 in embryo implantation and development in vitro in mice. Mouse embryos at 2-cell stage were cultured in the media of M16 with exposure to different levels of TGF-beta1 (0, 1, 10 and 50 ng/mL). The percentage of embryos reaching fixed stages (early blastocyst, expanding blastocyst and hatched blastocyst) was monitored 68 h and 92 h after the culture. The expanding blastocys cultured for 68 h in M16 without TGF-beta1 and those with 10 ng/mL of TGF-beta1 were transferred to pseudopregnant mice. On the 6th day post transfer, the successful rates of implantation were counted. The level of IL-10/IFN-gamma in the serum and maternal-fetus interface of the mice was detected by ELISA on the 6th day post transfer. TGF-beta1 improved embryo growth in vitro. TGF-beta1 at a level of 10 ng/mL had the maximum impact, with 15.6%, 68.09%, 1.42% of embryos reaching early, expanding, and hatched stage, respectively, 68 h after culture, and 6.38%, 28.37%, 53.19% of embryos reaching early, expanding, and hatched stage, respectively, 92 h after culture. The promoting effect declined when TGF-beta1 reached 50 ng/mL. The successful rate of implantation of embryos cultured in M16 with TGF-beta1 was significantly higher than those cultured in M16 without TGF-beta1 (35. 2% vs. 17.19%, P < 0.05). The embryos cultured in M16 with TGF-beta1 had significantly lower level of IFN-gamma in the maternal-fetus interface than those cultured in M16 without TGF-beta1 [(30.89 +/- 11.31) pg/mL vs. (43.23 +/- 18. 09) pg/mL, P < 0.053. TGF-beta1 at an appropriate dose improves embryo implantation in mice in vitro. The mechanism may involve the improvement of the quality of embryos and their development, and decrease of IFN-gamma synthesis in maternal-fetal interface, a chemical that could cause Th2 bias.

Cross-talk between Smad and p38 MAPK signalling in transforming growth factor {beta} signal transduction in human glioblastoma cells

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

Dziembowska, Magdalena; Danilkiewicz, Malgorzata; Wesolowska, Aleksandra

2007-03-23

Transforming growth factor-beta (TGF-{beta}) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation, and survival. Malignant tumour cells often do not respond to TGF-{beta} by growth inhibition, but retain responsiveness to cytokine in regulating extracellular matrix deposition, cell adhesion, and migration. We demonstrated that TGF-{beta}1 does not affect viability or proliferation of human glioblastoma T98G, but increases transcriptional responses exemplified by induction of MMP-9 expression. TGF-{beta} receptors were functional in T98G glioblastoma cells leading to SMAD3/SMAD4 nuclear translocation and activation of SMAD-dependent promoter. In parallel, a selective activation of p38 MAPK, and phosphorylation of its substrates: ATF2more » and c-Jun proteins were followed by a transient activation of AP-1 transcription factor. Surprisingly, an inhibition of p38 MAPK with a specific inhibitor, SB202190, abolished TGF-inducible activation of Smad-dependent promoter and decreased Smad2 phosphorylation. It suggests an unexpected interaction between Smad and p38 MAPK pathways in TGF-{beta}1-induced signalling.« less

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.

TGF-beta1 stimulates expression of the aromatase (CYP19) gene in human osteoblast-like cells and THP-1 cells.

PubMed

Shozu, M; Zhao, Y; Simpson, E R

2000-02-25

Recent evidence has shown that bone is not only a target of estrogen action but also a source of local estrogen production. Bone cells such as osteoblasts express aromatase (P450arom) and the expression of P450arom in osteoblasts is positively regulated in a tissue specific fashion, as in the case of other tissues which express P450arom. To clarify the physiological factors regulating expression of P450arom in bone, we tested TGF-beta1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-beta1 increased IL-1beta+DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-beta1 was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-beta1 enhanced DEX-induced aromatase activity almost linearly by 12 h and thereafter. Increased levels of P450arom transcripts were also demonstrated by RT-PCR at 3 h of TGF-beta1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-beta1. Increase in P450arom expression by TGF-beta1 was attributable to expression driven by promoter I.4. TGF-beta1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-beta1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter I.4 (P450-I.4/Luc). Two constructs (-410/+14 and-340/+14) that contain a functional glucocorticoid response element (GRE) and downstream sequence showed significant increase of luciferase activity in response to TGF-beta1. Deletion and mutation of the GRE in P450-I.4/Luc (-340/+14) abolished the TGF-beta1. The luciferase activity of a (GRE)(1)-SV40/Luc construct was also stimulated by TGF-beta1. These results indicate that TGF-beta1 increases the expression of P450arom at the

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

TGF-beta antisense oligonucleotides reduce mRNA expression of matrix metalloproteinases in cultured wound-healing-related cells.

PubMed

Philipp, Katrin; Riedel, Frank; Germann, Günter; Hörmann, Karl; Sauerbier, Michael

2005-02-01

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix. The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. We investigated the effect of TGF-beta antisense oligonucleotides on the mRNA expression of matrix metalloproteinases in cultured human keratinocytes, fibroblasts and endothelial cells using multiplex RT-PCR. The treatment of keratinocytes and fibroblasts with TGF-beta antisense oligonucleotides resulted in a significant decrease of expression of mRNA of MMP-1 and MMP-9 compared to controls. Accordingly, a decreased expression of MMP-1 mRNA in endothelial cells was detectable. Other MMPs were not affected. Affecting all dermal wound-healing-related cell types, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in chronic wounds. Pharmaceutical intervention in this area ultimately may help clinicians to proactively intervene in an effort to prevent normal wounds from becoming chronic.

c-Ski inhibits the TGF-beta signaling pathway through stabilization of inactive Smad complexes on Smad-binding elements.

PubMed

Suzuki, Hiroyuki; Yagi, Ken; Kondo, Miki; Kato, Mitsuyasu; Miyazono, Kohei; Miyazawa, Keiji

2004-06-24

c-Ski inhibits transforming growth factor-beta (TGF-beta) signaling through interaction with Smad proteins. c-Ski represses Smad-mediated transcriptional activation, probably through its action as a transcriptional co-repressor. c-Ski also inhibits TGF-beta-induced downregulation of genes such as c-myc. However, mechanisms for transcriptional regulation of target genes by c-Ski have not been fully determined. In this study, we examined how c-Ski inhibits both TGF-beta-induced transcriptional activation and repression. DNA-affinity precipitation analysis revealed that c-Ski enhances the binding of Smad2 and 4, and to a lesser extent Smad3, to both CAGA and TGF-beta1 inhibitory element probes. A c-Ski mutant, which is unable to interact with Smad4, failed to enhance the binding of Smad complex on these probes and to inhibit the Smad-responsive promoter. These results suggest that stabilization of inactive Smad complexes on DNA is a critical event in c-Ski-mediated inhibition of TGF-beta signaling.

Transforming growth factor-beta regulates mammary carcinoma cell survival and interaction with the adjacent microenvironment.

PubMed

Bierie, Brian; Stover, Daniel G; Abel, Ty W; Chytil, Anna; Gorska, Agnieszka E; Aakre, Mary; Forrester, Elizabeth; Yang, Li; Wagner, Kay-Uwe; Moses, Harold L

2008-03-15

Transforming growth factor (TGF)-beta signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-beta receptor (T beta RII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (T beta RII((fl/fl);PY) and T beta RII((fl/fl);PY;WC), respectively). The loss of TGF-beta signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-beta signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80(+) cell populations in T beta RII((fl/fl);PY;WC) mice when compared with T beta RII((fl/fl);PY) controls. The recruitment of F4/80(+) cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5(+) dNp63(+) cell population in primary T beta RII((fl/fl);PY;WC) tumors and corresponding pulmonary metastases, suggesting that loss of TGF-beta signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-beta signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo.

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

Transforming growth factor-beta and nitrates in epithelial ovarian cancer.

PubMed

Khalifa, A; Kassim, S K; Ahmed, M I; Fayed, S T

1999-12-01

The role of transforming growth factor-beta (TGF-beta) and nitric oxide (NO) in ovarian neoplasia is still not clear. We studied the expression of TGF-beta by enzyme immunoassay, and nitrates (as a stable end product of NO) in 127 ovarian tissues (36 normal, 37 benign, and 54 malignant). Ploidy status and synthetic phase fraction (SPF) were also assessed by flow cytometry. Mean ranks of TGF-beta, nitrate, and SPF were significant among different groups (X2 = 12.01, P = 0.0025, X2 = 67.42, P = 0.000, X2 = 9.06, P = 0.011 respectively). Nitrate mean ranks were significant among different FIGO stages of the disease (X2 = 17.6, P = 0.000). A significant correlation was shown between TGF-beta, and nitrate levels in all tissues (r = 0.24, P = 0.01), as well as in malignant tissues (r = 0.3, P = 0.026). Cutoff values were determined for both TGF-beta (290 pg/mg protein), and nitrates (310 nmole/mg non protein nitrogenous substances). At these cut-offs, nitrates showed a sensitivity of 93% and 84% specificity for malignant versus normal cases, while TGF-beta had 76% sensitivity, and 82.4% specificity for poor versus good outcome. Patients with epithelial ovarian cancer were followed up for a total of 40 months. Survival analysis showed that patients with TGF-beta above the cut-off had worse prognosis (X2 = 12.69, P = 0.004). The present results suggest that malignant transformation of ovarian tissues is associated with increased TGF-beta and NO production. NO level is related to the development and progression of epithelial ovarian cancer, while high levels of TGF-beta could be of prognostic significance.

c-Ski overexpression promotes tumor growth and angiogenesis through inhibition of transforming growth factor-beta signaling in diffuse-type gastric carcinoma.

PubMed

Kiyono, Kunihiko; Suzuki, Hiroshi I; Morishita, Yasuyuki; Komuro, Akiyoshi; Iwata, Caname; Yashiro, Masakazu; Hirakawa, Kosei; Kano, Mitsunobu R; Miyazono, Kohei

2009-10-01

c-Ski, originally identified as a proto-oncogene product, is an important negative regulator of transforming growth factor (TGF)-beta family signaling through interaction with Smad2, Smad3, and Smad4. High expression of c-Ski has been found in some cancers, including gastric cancer. We previously showed that disruption of TGF-beta signaling by dominant-negative TGF-beta type II receptor in a diffuse-type gastric carcinoma model accelerated tumor growth through induction of tumor angiogenesis by decreased expression of the anti-angiogenic factor thrombospondin (TSP)-1. Here, we examined the function of c-Ski in human diffuse-type gastric carcinoma OCUM-2MLN cells. Overexpression of c-Ski inhibited TGF-beta signaling in OCUM-2MLN cells. Interestingly, c-Ski overexpression resulted in extensive acceleration of the growth of subcutaneous xenografts in BALB/c nu/nu female mice (6 weeks of age). Similar to tumors expressing dominant-negative TGF-beta type II receptor, histochemical studies revealed less fibrosis and increased angiogenesis in xenografted tumors expressing c-Ski compared to control tumors. Induction of TSP-1 mRNA by TGF-beta was attenuated by c-Ski in vitro, and expression of TSP-1 mRNA was decreased in tumors expressing c-Ski in vivo. These findings suggest that c-Ski overexpression promotes the growth of diffuse-type gastric carcinoma through induction of angiogenesis.

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.

Linking TGF-beta-mediated Cdc25A inhibition and cytoskeletal regulation through RhoA/p160(ROCK) signaling.

PubMed

Brown, Kimberly; Bhowmick, Neil A

2004-04-01

Transforming growth factor-beta (TGF-beta) can mediate G(1)/S cell-cycle inhibition and changes in the cytoskeletal organization through multiple parallel downstream signaling pathways. Recent findings regarding TGF-beta-mediated cell-cycle checkpoint control and epithelial to mesenchymal transition have converged to the RhoA/p160(ROCK) signaling pathway. The activation of TGF-beta-mediated p160(ROCK)rapidly inhibits the Cdc25A phosphatase as a component of the G(1)/S checkpoint control at the time cytoskeletal re-organization occurs. This can be likened to the ability to preserve genomic integrity in circumstances of genotoxic stress. The inactivation of the RhoA/p160(ROCK) pathway may be a mechanism by which cancer cells bypass growth inhibition even in the presence of TGF-beta.

Contrasting effects of TGF-beta 1 and TNF-alpha on the development of dendritic cells from progenitors in mouse bone marrow.

PubMed

Yamaguchi, Y; Tsumura, H; Miwa, M; Inaba, K

1997-01-01

Dendritic cells (DC) are a distinct population of leukocytes and specialized antigen-presenting cells for T cell responses. Prior work has shown that GM-CSF can induce the development of large numbers of DC from proliferating progenitors in mouse bone marrow. We have monitored the effects of potentially enhancing and suppressive cytokines in these cultures. In this system, many immature DC develop from proliferating precursors during the first six days of culture, and between days 6-8 maturation of typical nonadherent and nonreplicating DC takes place. The maturation is accompanied by a large increase in the expression of major histocompatibilities complex class II (MHC II) and B7-2/CD86, and in mixed leukocyte reaction stimulating activity. Tumor necrosis factor-alpha (TNF-alpha), previously shown to be required for development of human DC, was found to enhance the maturation of mouse DC in the last two days of culture. Transforming growth factor-beta 1 (TGF-beta 1), on the other hand, almost totally blocked DC maturation, but it had to be given in the first six days of culture when the DC were actively proliferating. TGF-beta 1 did not block the production of immature, MHC II-positive but B7-2/CD86-negative DC. Maturation would take place between days 6-8 as long as the cultures were depleted of Fc-receptor-bearing cells, or if TNF-alpha were added. In both instances, maturation was not blocked even when TGF-beta 1 remained in the culture. We conclude that the development of DC, in response to GM-CSF, can be modified by other cytokines. TGF-beta 1 is suppressive but only indirectly via Fc-receptor-bearing suppressive cells, presumably suppressive macrophages, while TNF-alpha enhances the final maturation of DC.

High-Fluence Light-Emitting Diode-Generated Red Light Modulates the Transforming Growth Factor-Beta Pathway in Human Skin Fibroblasts.

PubMed

Mamalis, Andrew; Jagdeo, Jared

2018-05-24

Skin fibrosis is a significant medical problem with limited available treatment modalities. The key cellular characteristics include increased fibroblast proliferation, collagen production, and transforming growth factor-beta (TGF-B)/SMAD pathway signaling. The authors have previously shown that high-fluence light-emitting diode red light (HF-LED-RL) decreases cellular proliferation and collagen production. Herein, the authors investigate the ability of HF-LED-RL to modulate the TGF-B/SMAD pathway. Normal human dermal fibroblasts were cultured and irradiated with a commercially available hand-held LED array. After irradiation, cell lysates were collected and levels of pSMAD2, TGF-Beta 1, and TGF-Beta I receptor were measured using Western blot. High-fluence light-emitting diode red light decreased TGF-Beta 1 ligand (TGF-B1) levels after irradiation. 320 J/cm HF-LED-RL resulted in 59% TGF-B1 and 640 J/cm HF-LED-RL resulted in 54% TGF-B1, relative to controls. 640 J/cm HF-LED-RL resulted in 62% pSMAD2 0 hours after irradiation, 65% pSMAD2 2 hours after irradiation, and 95% 4 hours after irradiation, compared with matched controls. High-fluence light-emitting diode red light resulted in no significant difference in transforming growth factor-beta receptor I levels compared with matched controls. Skin fibrosis is a significant medical problem with limited available treatment modalities. Light-emitting diode-generated red light is a safe, economic, and noninvasive modality that has a body of in vitro evidence supporting the reduction of key cellular characteristics associated with skin fibrosis.

Suppression of transforming growth factor-beta-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway.

PubMed

Chen, R H; Su, Y H; Chuang, R L; Chang, T Y

1998-10-15

Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-beta1 (TGF-beta). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-beta-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-beta-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-beta-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-beta-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-beta was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-beta-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-beta-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.

MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

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

Li, Xiaoou; Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan; Liu, Lian

2014-11-28

Highlights: • Endogenous miR-26a inhibits TGF-beta 1 induced proliferation of lung fibroblasts. • miR-26a induces G1 arrest through directly targeting 3′-UTR of CCND2. • TGF indispensable receptor, TGF-beta R I, is regulated by miR-26a. • miR-26a acts through inhibiting TGF-beta 2 feedback loop to reduce TGF-beta 1. • Collagen type I and connective tissue growth factor are suppressed by miR-26a. - Abstract: MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship betweenmore » miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis.« less

Repression of TGF-beta signaling by the oncogenic protein SKI in human melanomas: consequences for proliferation, survival, and metastasis.

PubMed

Medrano, Estela E

2003-05-19

Transforming growth factor-beta (TGF-beta ) has dual and paradoxical functions as a tumor suppressor and promoter of tumor progression and metastasis. TGF-Ji-mediated growth inhibition is gradually lost during melanoma tumor progression, but there are no measurable defects at the receptor level. Furthermore, melanoma cells release high levels of TGF-beta to the microenvironment, which upon activation induces matrix deposition, angiogenesis, survival, and transition to more aggressive phenotypes. The SKI and SnoN protein family associate with and repress the activity of Smad2, Smad3, and Smad4, three members of the TGF-fl signaling pathway. SKI also facilitates cell-cycle progression by targeting the RB pathway by at least two ways: it directly associates with RB and represses its activity when expressed at high levels, and indirectly, it represses Smad-mediated induction of p21(Waf-1) This results in increased CDK2 activity, RB phosphorylation,and inactivation. Therefore, high levels of SKI result in lesions to the RB pathway in a manner similar to p16 (INK4a) loss. SKI mRNA and protein levels dramatically increase during human melanoma tumor progression. In addition,the SKI protein shifts from nuclear localization in intraepidermal melanoma cells to nuclear and cytoplasmic in invasive and metastatic melanomas. Here, I discuss the basis for repression of intracellular TGF-beta signaling by SKI, some additional activities of this protein, and propose that by disrupting multiple tumor suppressor pathways, SKI functions as a melanoma oncogene.

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.

The pleiotropic roles of transforming growth factor beta inhomeostasis and carcinogenesis of endocrine organs.

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

Fleisch, Markus C.; Maxwell, Christopher A.; Barcellos-Hoff,Mary-Helen

2006-01-13

Transforming growth factor beta (TGF-beta) is a ubiquitous cytokine that plays a critical role in numerous pathways regulating cellular and tissue homeostasis. TGF-beta is regulated by hormones and is a primary mediator of hormone response in uterus, prostate and mammary gland. This review will address the role of TGF-beta in regulating hormone dependent proliferation and morphogenesis. The subversion of TGF-beta regulation during the processes of carcinogenesis, with particular emphasis on its effects on genetic stability and epithelial to mesenchymal transition (EMT), will also be examined. An understanding of the multiple and complex mechanisms of TGF-beta regulation of epithelial function, andmore » the ultimate loss of TGF-beta function during carcinogenesis, will be critical in the design of novel therapeutic interventions for endocrine-related cancers.« less

The effect of TGF-beta2 on MMP-2 production and activity in highly metastatic human bladder carcinoma cell line 5637.

PubMed

Dehnavi, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ataei, Zahra; Aryan, Hajar

2009-06-01

Transforming growth factor-beta (TGF-beta) superfamily regulates matrix metalloproteinases (MMP), which intrinsically regulate various cell behaviors leading to metastasis. We investigated the effect of TGF-beta(2) on MMP-2 regulation in human bladder carcinoma cell line 5637. Zymography, ELISA, and real-time polymerase chain reaction revealed that TGF-beta(2) stimulated MMP-2 production, but the transcription of its gene remained unchanged. Wortmannin could not inhibit MMP-2 secretion and activity and conversely the amount of the protein and its enzymatic activity were increased. These data suggest that TGF-beta(2) increased MMP-2 at the posttranscriptional level and this upregulation was independent of phosphatidylinositol 3-kinase signaling pathway.

Low dose radiation interactions with the transformation growth factor (TFG)-beta pathway

NASA Astrophysics Data System (ADS)

Maslowski, Amy Jesse

A major limiting factor for long-term, deep-space missions is the radiation dose to astronauts. Because the dose to the astronauts is a mixed field of low- and high-LET radiation, there is a need to understand the effects of both radiation types on whole tissue; however, there are limited published data on the effects of high-LET (linear-energy-transfer) radiation on tissue. Thus, we designed a perfusion chamber system for rat trachea in order to mimic in vivo respiratory tissue. We successfully maintained the perfused tracheal tissue ex vivo in a healthy and viable condition for up to three days. In addition, this project studied the effects of high-LET Fe particles on the overall transformation growth factor (TGF)-beta response after TGF-beta inactivation and compared the results to the TGF-beta response post x-ray irradiation. It was found that a TGF-beta response could be measured in the perfused tracheal tissue, for x-ray and Fe particle irradiations, despite the high autofluorescent background intrinsic to tissue. However, after comparing the TGF-beta response of x-ray irradiation to High-Z-High-energy (HZE) irradiation, there was not a significant difference in radiation types. The TGF-beta response in x-ray and HZE irradiated perfusion chambers was also measured over time post irradiation. It was found that for 6 hour and 8 hour post irradiation, the TGF-beta response was higher for lower doses of radiation than for higher doses. This is in contrast to the 0 hour fixation which found the TGF-beta response to increase with increased dose. The inverse relationship found for 6 hour and 8 hour fixation times may indicate a threshold response for TGF-beta response; i.e., for low doses, a threshold of dose must be reached for an immediate TGF-beta response, otherwise the tissue responds more slowly to the irradiation damage. This result was unexpected and will require further investigation to determine if the threshold can be determined for the 250 kVp x-rays and

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

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.

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.

Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

NASA Technical Reports Server (NTRS)

Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

2002-01-01

Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures

PubMed Central

1987-01-01

In culture, vascular smooth muscle cells (SMC) grow in a "hill-and- valley" (multilayered) pattern of organization. We have studied the growth, behavioral organization, and biosynthetic phenotype of rat aortic SMC exposed to purified platelet-derived growth regulatory molecules. We show that multilayered growth is not a constitutive feature of cultured SMC, and that beta-type transforming growth factor (TGF-beta) is the primary determinant of multilayered growth and the hill-and-valley pattern of organization diagnostic for SMC in culture. TGF-beta inhibited, in a dose-dependent manner, the serum- or platelet- derived growth factor-mediated proliferation of these cells in two- dimensional culture, but only when cells were plated at subconfluent densities. The ability of TGF-beta to inhibit SMC growth was inversely correlated to plating cell density. When SMC were plated at monolayer density (5 X 10(4) cells/cm2) to allow maximal cell-to-cell contact, TGF-beta potentiated cell growth. This differential response of SMC to TGF-beta may contribute to the hill-and-valley pattern of organization. Unlike its effect on other cell types, TGF-beta did not enhance the synthesis of fibronectin or its incorporation into the extracellular matrix. However, the synthesis of a number of other secreted proteins was altered by TGF-beta treatment. SMC treated with TGF-beta for 4 or 8 h secreted markedly enhanced amounts of an Mr 38,000-D protein doublet whose synthesis is known to be increased by heparin (another inhibitor of SMC growth), suggesting metabolic similarities between heparin- and TGF-beta-mediated SMC growth inhibition. The data suggest that TGF-beta may play an important and complex regulatory role in SMC proliferation and organization during development and after vascular injury. PMID:3475277

Id-1 promotes TGF-{beta}1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

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

Di Kaijun; Wong, Y.C.; Wang Xianghong

Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-{beta}1 (transforming growth factor {beta}1). Here we demonstrate a novel role of Id-1 in promoting TGF-{beta}1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-{beta}1, which was associated with increased cell-substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-{beta}1-induced cell motility was mediated through activation ofmore » MEK-ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-{beta}1-treated cells through down-regulation of E-cadherin, redistribution of {beta}-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell-cell adhesion in response to TGF-{beta}1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-{beta}1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.« less

Hepatitis B virus X protein shifts human hepatic transforming growth factor (TGF)-beta signaling from tumor suppression to oncogenesis in early chronic hepatitis B.

PubMed

Murata, Miki; Matsuzaki, Koichi; Yoshida, Katsunori; Sekimoto, Go; Tahashi, Yoshiya; Mori, Shigeo; Uemura, Yoshiko; Sakaida, Noriko; Fujisawa, Junichi; Seki, Toshihito; Kobayashi, Kazuki; Yokote, Koutaro; Koike, Kazuhiko; Okazaki, Kazuichi

2009-04-01

Hepatitis B virus X (HBx) protein is suspected to participate in oncogenesis during chronic hepatitis B progression. Transforming growth factor beta (TGF-beta) signaling involves both tumor suppression and oncogenesis. TGF-beta activates TGF-beta type I receptor (TbetaRI) and c-Jun N-terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Reversible shifting of Smad3-mediated signaling between tumor suppression and oncogenesis in HBx-expressing hepatocytes indicated that TbetaRI-dependent pSmad3C transmitted a tumor-suppressive TGF-beta signal, while JNK-dependent pSmad3L promoted cell growth. We used immunostaining, immunoblotting, and in vitro kinase assay to compare pSmad3L- and pSmad3C-mediated signaling in biopsy specimens representing chronic hepatitis, cirrhosis, or hepatocellular carcinoma (HCC) from 90 patients chronically infected with hepatitis B virus (HBV) with signaling in liver specimens from HBx transgenic mice. In proportion to plasma HBV DNA levels, early chronic hepatitis B specimens showed prominence of pSmad3L in hepatocytic nuclei. HBx-activated JNK/pSmad3L/c-Myc oncogenic pathway was enhanced, while the TbetaRI/pSmad3C/p21(WAF1) tumor-suppressive pathway was impaired as human and mouse HBx-associated hepatocarcinogenesis progressed. Of 28 patients with chronic hepatitis B who showed strong oncogenic pSmad3L signaling, six developed HCC within 12 years; only one of 32 patients showing little pSmad3L developed HCC. In contrast, seven of 30 patients with little Smad3C phosphorylation developed HCC, while no patient who retained hepatocytic tumor-suppressive pSmad3C developed HCC within 12 years. HBx shifts hepatocytic TGF-beta signaling from the tumor-suppressive pSmad3C pathway to the oncogenic pSmad3L pathway in early carcinogenic process. Hepatocytic pSmad3L and pSmad3C assessment in HBV-infected liver specimens should prove

Transforming growth factor-beta1 accelerates resorption of a calcium carbonate biomaterial in periodontal defects.

PubMed

Koo, Ki-Tae; Susin, Cristiano; Wikesjö, Ulf M E; Choi, Seong-Ho; Kim, Chong-Kwan

2007-04-01

In a previous study, recombinant human transforming growth factor-beta1 (rhTGF-beta(1)) in a calcium carbonate carrier was implanted into critical-size, supraalveolar periodontal defects under conditions for guided tissue regeneration (GTR) to study whether rhTGF-beta(1) would enhance or accelerate periodontal regeneration. The results showed minimal benefits of rhTGF-beta(1), and a clear account for this could not be offered. One potential cause may be that the rhTGF-beta(1) formulation was biologically inactive. Several growth or differentiation factors have been suggested to accelerate degradation of biomaterials used as carriers. The objective of this study was to evaluate possible activity of rhTGF-beta(1) on biodegradation of the calcium carbonate carrier. rhTGF-beta(1) in a putty-formulated particulate calcium carbonate carrier was implanted into critical-size, supraalveolar periodontal defects under conditions for GTR in five beagle dogs. Contralateral defects received the calcium carbonate carrier combined with GTR without rhTGF-beta(1) (control). The animals were euthanized at week 4 post-surgery and block biopsies of the defect sites were collected for histologic and histometric analysis. Radiographs were obtained at defect creation and weeks 2 and 4 after defect creation. No statistically significant differences were observed in new bone formation (bone height and area) among the treatments. However, total residual carrier was significantly reduced in sites receiving rhTGF-beta(1) compared to control (P = 0.04). Similarly, carrier density was considerably reduced in sites receiving rhTGF-beta(1) compared to control; the difference was borderline statistically significant (P = 0.06). Within the limitations of the study, it may be concluded that rhTGF-beta(1) accelerates biodegradation of a particulate calcium carbonate biomaterial, indicating a biologic activity of the rhTGF-beta(1) formulation apparently not encompassing enhanced or accelerated

Fermentable metabolite of Zymomonas mobilis controls collagen reduction in photoaging skin by improving TGF-beta/Smad signaling suppression.

PubMed

Tanaka, Hiroshi; Yamaba, Hiroyuki; Kosugi, Nobuhiko; Mizutani, Hiroshi; Nakata, Satoru

2008-04-01

Solar ultraviolet (UV) irradiation causes damages on human skin and premature skin aging (photoaging). UV-induced reduction of type I collagen in dermis is widely considered primarily induction of wrinkled appearance of photoaging skin. Type I procollagen synthesis is reduced under UV irradiation by blocking transforming growth factor-beta (TGF-beta)/Smad signaling; more specifically, it is down-regulation of TGF-beta type II receptor (T beta RII). Therefore, preventing UV-induced loss of T beta RII results decreased type I collagen reduction in photoaging skin. Zymomonas mobilis is an alcohol fermentable, gram-negative facultative anaerobic bacterium whose effect on skin tissue is scarcely studied. We investigated the protective effects of fermentable metabolite of Z. mobilis (FM of Z. mobilis) against reduction of type I procollagen synthesis of UV-induced down-regulation of T beta RII in human dermal fibroblasts FM of Z. mobilis was obtained from lyophilization of bacterium culture supernatant. The levels of T beta RII and type I procollagen mRNA in human dermal fibroblasts were measured by quantitative real-time RT-PCR, and T beta RII protein levels were assayed by western blotting. T beta RII, type I procollagen, and type I collagen proteins in human dermal fibroblasts or hairless mouse skin were detected by immunostaining. FM of Z. mobilis inhibited down regulation of T beta RII mRNA, and protein levels in UVB irradiated human dermal fibroblasts consequently recover reduced type I procollagen synthesis. These results indicate UVB irradiation inhibits type I procollagen synthesis by suppression of TGF-beta/Smad signaling pathway, and FM of Z. mobilis has inhibitory effect on UVB-induced reduction of type I procollagen synthesis. While short period UVB irradiation decreased both T beta RII and type I procollagen protein levels in hairless mouse skin, topical application of FM of Z. mobilis prevented this decrease. Wrinkle formation in hairless mouse skin

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

NFI-Ski interactions mediate transforming growth factor beta modulation of human papillomavirus type 16 early gene expression.

PubMed

Baldwin, Amy; Pirisi, Lucia; Creek, Kim E

2004-04-01

Human papillomaviruses (HPVs) are present in virtually all cervical cancers. An important step in the development of malignant disease, including cervical cancer, involves a loss of sensitivity to transforming growth factor beta (TGF-beta). HPV type 16 (HPV16) early gene expression, including that of the E6 and E7 oncoprotein genes, is under the control of the upstream regulatory region (URR), and E6 and E7 expression in HPV16-immortalized human epithelial cells is inhibited at the transcriptional level by TGF-beta. While the URR contains a myriad of transcription factor binding sites, including seven binding sites for nuclear factor I (NFI), the specific sequences within the URR or the transcription factors responsible for TGF-beta modulation of the URR remain unknown. To identify potential transcription factors and binding sites involved in TGF-beta modulation of the URR, we performed DNase I footprint analysis on the HPV16 URR using nuclear extracts from TGF-beta-sensitive HPV16-immortalized human keratinocytes (HKc/HPV16) treated with and without TGF-beta. Differentially protected regions were found to be located around NFI binding sites. Electrophoretic mobility shift assays, using the NFI binding sites as probes, showed decreased binding upon TGF-beta treatment. This decrease in binding was not due to reduced NFI protein or NFI mRNA levels. Mutational analysis of individual and multiple NFI binding sites in the URR defined their role in TGF-beta sensitivity of the promoter. Overexpression of the NFI family members in HKc/HPV16 decreased the ability of TGF-beta to inhibit the URR. Since the oncoprotein Ski has been shown to interact with and increase the transcriptional activity of NFI and since cellular Ski levels are decreased by TGF-beta treatment, we explored the possibility that Ski may provide a link between TGF-beta signaling and NFI activity. Anti-NFI antibodies coimmunoprecipitated endogenous Ski in nuclear extracts from HKc/HPV16, confirming that NFI

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.

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

Controlled release of TGF-beta 1 from RADA self-assembling peptide hydrogel scaffolds

PubMed Central

Zhou, Ao; Chen, Shuo; He, Bin; Zhao, Weikang; Chen, Xiaojun; Jiang, Dianming

2016-01-01

Bioactive mediators, cytokines, and chemokines have an important role in regulating and optimizing the synergistic action of materials, cells, and cellular microenvironments for tissue engineering. RADA self-assembling peptide hydrogels have been proved to have an excellent ability to promote cell proliferation, wound healing, tissue repair, and drug delivery. Here, we report that D-RADA16 and L-RADA16-RGD self-assembling peptides can form stable second structure and hydrogel scaffolds, affording the slow release of growth factor (transforming growth factor cytokine-beta 1 [TGF-beta 1]). In vitro tests demonstrated that the plateau release amount can be obtained till 72 hours. Moreover, L-RADA16, D-RADA16, and L-RADA16-RGD self-assembling peptide hydrogels containing TGF-beta 1 were used for 3D cell culture of bone mesenchymal stem cells of rats for 2 weeks. The results revealed that these three RADA16 peptide hydrogels had a significantly favorable influence on proliferation of bone mesenchymal stem cells and hold some promise in slow and sustained release of growth factor. PMID:27703332

Smad2 and Smad3 phosphorylated at both linker and COOH-terminal regions transmit malignant TGF-beta signal in later stages of human colorectal cancer.

PubMed

Matsuzaki, Koichi; Kitano, Chiaki; Murata, Miki; Sekimoto, Go; Yoshida, Katsunori; Uemura, Yoshiko; Seki, Toshihito; Taketani, Shigeru; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2009-07-01

Transforming growth factor (TGF)-beta initially inhibits growth of mature epithelial cells. Later, however, autocrine TGF-beta signaling acts in concert with the Ras pathway to induce a proliferative and invasive phenotype. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also Ras-associated kinases, which differentially phosphorylate the mediators Smad2 and Smad3 to create distinct phosphorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C) and both linker and COOH-terminally phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). In this study, we investigated actions of pSmad2L/C and pSmad3L/C in cancer progression. TGF-beta inhibited cell growth by down-regulating c-Myc oncoprotein through the pSmad2C and pSmad3C pathway; TGF-beta signaling, in turn, enhanced cell growth by up-regulating c-Myc through the cyclin-dependent kinase (CDK) 4-dependent pSmad2L/C and pSmad3L/C pathways in cell nuclei. Alternatively, TbetaRI and c-Jun NH2-terminal kinase (JNK) together created cytoplasmic pSmad2L/C, which entered the nucleus and stimulated cell invasion, partly by up-regulating matrix metalloproteinase-9. In 20 clinical samples, pSmad2L/C and pSmad3L/C showed nuclear localization at invasion fronts of all TGF-beta-producing human metastatic colorectal cancers. In vitro kinase assay confirmed that nuclear CDK4 and cytoplasmic JNK obtained from the tumor tissue could phosphorylate Smad2 or Smad3 at their linker regions. We suggest that CDK4, together with JNK, alters tumor-suppressive TGF-beta signaling to malignant characteristics in later stages of human colorectal cancer. The linker phosphorylation of Smad2 and Smad3 may represent a target for intervention in human metastatic cancer.

Transforming growth factor-beta in the chicken fundal layers: an immunohistochemical study.

PubMed

Mathis, Ute; Schaeffel, Frank

2010-06-01

In the chicken model of myopia, it has first been shown that imposing defocus to the retina results in active remodelling of the sclera which, in turn, results in axial length changes of the eye. Transforming growth factor-beta (TGF-beta) is one of the scleral growth modulators but its cellular localization in the fundal layers, colocalization and function are not well known. The aim of the current study was to investigate the cellular distribution of the three isoforms TGF-beta1, 2 and 3 by immunohistochemical labelling. Furthermore, the effects of visual experience that induces refractive errors on TGF-beta2 labelling were examined. Transversal cryostat sections of the fundal layers were analyzed by indirect immunofluorescent labelling and cell counts. Visual experience was changed by having the chicks wear either diffusers, or positive or negative lenses of 7D power in front of the right eyes for various periods of time. Left eyes served as uncovered controls. All TGF-beta isoforms were localized in both scleral layers. In choroid, diffuse labelling of all isoforms was found. In retina, TGF-beta1 and 3 were detected in bipolar, amacrine and ganglion cells and TGF-beta2 in amacrine and ganglion cells. To further characterize these cells, double-labelling with known amacrine and bipolar cell markers was performed (calbindin, cellular retinoic acid binding protein (CRABP), Islet1, Lim3 and protein kinase C (PKC)). TGF-beta1, 2 and 3 could be colocalized with calbindin and CRABP in single amacrine cells. TGF-beta1-positive bipolar cells were immunoreactive to Lim3. TGF-beta1 and 3 were never colocalized with PKC in bipolar cells. Also, colocalization with peptides known to be involved in myopia development in chicks, such as glucagon, or vasointestinal polypeptide and the key enzyme for dopamine synthesis, tyrosine hydroxylase, was not observed. Lenses or diffusers, worn by the chicks for various periods of time, had no effect on TGF-beta2 immunoreactivity in

Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-beta1.

PubMed

Haydont, Valérie; Riser, Bruce L; Aigueperse, Jocelyne; Vozenin-Brotons, Marie-Catherine

2008-06-01

The fibrogenic differentiation of resident mesenchymal cells is a key parameter in the pathogenesis of radiation fibrosis and is triggered by the profibrotic growth factors transforming growth factor (TGF)-beta1 and CCN2. TGF-beta1 is considered the primary inducer of fibrogenic differentiation and is thought to control its long-term maintenance, whereas CCN2 is considered secondary effector of TGF-beta1. Yet, in long-term established fibrosis like that associated with delayed radiation enteropathy, in situ TGF-beta1 deposition is low, whereas CCN2 expression is high. To explore this apparent paradox, cell response to increasing doses of TGF-beta1 was investigated in cells modeling initiation and maintenance of fibrosis, i.e., normal and fibrosis-derived smooth muscle cells, respectively. Activation of cell-specific signaling pathways by low TGF-beta1 doses was demonstrated with a main activation of the Rho/ROCK pathway in fibrosis-derived cells, whereas the Smad pathway was mainly activated in normal cells. This leads to subsequent and cell-specific regulation of the CCN2 gene. These results suggested a specific profibrotic role of CCN2 in fibrosis-initiated cells. Furthermore, the modulation of CCN2 expression by itself and the combination of TGF-beta1 and CCN2 was investigated in fibrosis-derived cells. In fibrosis-initiated cells CCN2 triggered its autoinduction; furthermore, low concentration of TGF-beta1-potentiated CCN2 autoinduction. Our findings showed a differential requirement and action of TGF-beta1 in the fibrogenic response of normal vs. fibrosis-derived cells. This study defines a novel Rho/ROCK but Smad3-independent mode of TGF-beta signaling that may operate during the chronic stages of fibrosis and provides evidence of both specific and combinatorial roles of low TGF-beta1 dose and CCN2.

Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals.

PubMed Central

Diaz-Meco, M T; Dominguez, I; Sanz, L; Municio, M M; Berra, E; Cornet, M E; Garcia de Herreros, A; Johansen, T; Moscat, J

1992-01-01

Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has been established that the mechanism whereby TGF-beta 1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-beta 1 action. In the study reported here, we demonstrate that TGF-beta 1 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-beta 1. Images PMID:1309592

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.

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.

Transforming growth factor (TGF. beta. ) decreases the proliferation of human bone marrow fibroblasts by inhibiting the platelet-derived growth factor (PDGF) binding

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

Bryckaert, M.C.; Tobelem, G.; Lindroth, M.

1988-12-01

Human bone marrow fibroblasts were cultivated and characterized by immunofluorescent staining and electron microscopy. Their interactions with PDGF and TGF{beta} were studied. While a positive intracellular antifibronectin staining was observed, the cultured cells were not labeled with specific antibodies toward factor VIII von Willebrand factor (F VIII/vWF), desmin, and macrophage antigen. The binding of pure human PDGF to the cultured bone marrow fibroblasts was investigated. Addition of an excess of unlabeled PDGF decreased the binding to 75 and 80%, which means that the nonspecific binding represented 20-25% of total binding, whereas epidermal growth factor (EGF) had no effect. Two classesmore » of sites were detected by Scatchard analysis. The stimulation of DNA synthesis of PDGF was quantified by ({sup 3}H)thymidine incorporation. The results suggested that PDGF and TGF{beta} could modulate the growth of bone marrow fibroblasts.« less

Transforming growth factor-beta production in anti-glomerular basement membrane disease in the rabbit.

PubMed Central

Coimbra, T.; Wiggins, R.; Noh, J. W.; Merritt, S.; Phan, S. H.

1991-01-01

The purpose of this study was to assay for the presence of collagen synthesis stimulatory activity in the kidney during immune-induced renal injury that results in severe fibrosis in both glomerular and interstitial compartments. A model of antiglomerular basement (anti-GBM) disease in the rabbit was induced on day 0 by the injection of anti-GBM antibody and renal cortex tissues were then sampled at various time points. Only conditioned media prepared from diseased renal cortical samples showed collagen synthesis stimulatory activity when tested on rabbit mesangial cells. The activity had an estimated molecular weight range of 16 to 25 kd and was neutralized by antibody to transforming growth factor-beta (TGF-beta). A standard assay for TGF-beta using a mink lung epithelial cell line confirmed the increase in TGF-beta activity in conditioned media of diseased cortex from day 7 and day 14 animals, which was not significantly activated by previous acidification. This suggests that most of the TGF-beta present in renal conditioned media was in the active form. The increase in renal cortical secretion of active TGF-beta was accompanied by increases in renal cortical TGF-beta mRNA content on days 4 and 7 after induction, with subsequent return to control levels. A similar increase in TGF-beta activity was present in nonacidified conditioned media of purified glomeruli from diseased days 7 and 14 animals, which was also accompanied by significant increases in TGF-beta mRNA. However with acidification no significant differences were noted between control and diseased samples, suggesting the presence of substantial latent TGF-beta activity in control glomerular conditioned media. These same control-conditioned media contained inhibitor activity for added exogenous TGF-beta. These results support the conclusion that the association between increased TGF-beta secretion and increased renal cortical collagen synthesis in this model is consistent with a role for this cytokine

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.

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

A negative feedback control of transforming growth factor-beta signaling by glycogen synthase kinase 3-mediated Smad3 linker phosphorylation at Ser-204.

PubMed

Millet, Caroline; Yamashita, Motozo; Heller, Mary; Yu, Li-Rong; Veenstra, Timothy D; Zhang, Ying E

2009-07-24

Through the action of its membrane-bound type I receptor, transforming growth factor-beta (TGF-beta) elicits a wide range of cellular responses that regulate cell proliferation, differentiation, and apo ptosis. Many of these signaling responses are mediated by Smad proteins. As such, controlling Smad activity is crucial for proper signaling by TGF-beta and its related factors. Here, we show that TGF-beta induces phosphorylation at three sites in the Smad3 linker region in addition to the two C-terminal residues, and glycogen synthase kinase 3 is responsible for phosphorylation at one of these sites, namely Ser-204. Alanine substitution at Ser-204 and/or the neighboring Ser-208, the priming site for glycogen synthase kinase 3 in vivo activity, strengthened the affinity of Smad3 to CREB-binding protein, suggesting that linker phosphorylation may be part of a negative feedback loop that modulates Smad3 transcriptional activity. Thus, our findings reveal a novel aspect of the Smad3 signaling mechanism that controls the final amplitude of cellular responses to TGF-beta.

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

Differential regulation of immature articular cartilage compressive moduli and Poisson's ratios by in vitro stimulation with IGF-1 and TGF-beta1.

PubMed

Williams, Gregory M; Dills, Kristin J; Flores, Christian R; Stender, Michael E; Stewart, Kevin M; Nelson, Lauren M; Chen, Albert C; Masuda, Koichi; Hazelwood, Scott J; Klisch, Stephen M; Sah, Robert L

2010-09-17

Mechanisms of articular cartilage growth and maturation have been elucidated by studying composition-function dynamics during in vivo development and in vitro culture with stimuli such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta 1 (TGF-beta1). This study tested the hypothesis that IGF-1 and TGF-beta1 regulate immature cartilage compressive moduli and Poisson's ratios in a manner consistent with known effects on tensile properties. Bovine calf articular cartilage from superficial-articular (S) and middle-growth (M) regions were analyzed fresh or following culture in medium with IGF-1 or TGF-beta1. Mechanical properties in confined (CC) and unconfined (UCC) compression, cartilage matrix composition, and explant size were assessed. Culture with IGF-1 resulted in softening in CC and UCC, increased Poisson's ratios, substantially increased tissue volume, and accumulation of glycosaminoglycan (GAG) and collagen (COL). Culture with TGF-beta1 promoted maturational changes in the S layer, including stiffening in CC and UCC and increased concentrations of GAG, COL, and pyridinoline crosslinks (PYR), but little growth. Culture of M layer explants with TGF-beta1 was nearly homeostatic. Across treatment groups, compressive moduli in CC and UCC were positively related to GAG, COL, and PYR concentrations, while Poisson's ratios were negatively related to concentrations of these matrix components. Thus, IGF-1 and TGF-beta1 differentially regulate the compressive mechanical properties and size of immature articular cartilage in vitro. Prescribing tissue growth, maturation, or homeostasis by controlling the in vitro biochemical environment with such growth factors may have applications in cartilage repair and tissue engineering.

Reversal of acute and chronic synovial inflammation by anti-transforming growth factor beta.

PubMed

Wahl, S M; Allen, J B; Costa, G L; Wong, H L; Dasch, J R

1993-01-01

Transforming growth factor beta (TGF-beta) induces leukocyte recruitment and activation, events central to an inflammatory response. In this study, we demonstrate that antagonism of TGF-beta with a neutralizing antibody not only blocks inflammatory cell accumulation, but also tissue pathology in an experimental model of chronic erosive polyarthritis. Intraarticular injection of monoclonal antibody 1D11.16, which inhibits both TGF-beta 1 and TGF-beta 2 bioactivity, into animals receiving an arthropathic dose of bacterial cell walls significantly inhibits arthritis. Inhibition was observed with a single injection of 50 micrograms antibody, and a 1-mg injection blocked acute inflammation > 75% compared with the contralateral joints injected with an irrelevant isotype control antibody (MOPC21) as quantitated by an articular index (AI = 0.93 +/- 0.23 for 1D11.16, and AI = 4.0 +/- 0 on day 4; p < 0.001). Moreover, suppression of the acute arthritis achieved with a single injection of antibody was sustained into the chronic, destructive phase of the disease (on day 18, AI = 0.93 +/- 0.07 vs. AI = 2.6 +/- 0.5; p < 0.01). The decreased inflammatory index associated with anti-TGF-beta treatment was consistent with histopathologic and radiologic evidence of a therapeutic response. These data implicate TGF-beta as a profound agonist not only in the early events responsible for synovial inflammation, but also in the chronicity of streptococcal cell wall fragment-induced inflammation culminating in destructive pathology. Interrupting the cycle of leukocyte recruitment and activation with TGF-beta antagonists may provide a mechanism for resolution of chronic destructive lesions.

Reversal of acute and chronic synovial inflammation by anti- transforming growth factor beta

PubMed Central

1993-01-01

Transforming growth factor beta (TGF-beta) induces leukocyte recruitment and activation, events central to an inflammatory response. In this study, we demonstrate that antagonism of TGF-beta with a neutralizing antibody not only blocks inflammatory cell accumulation, but also tissue pathology in an experimental model of chronic erosive polyarthritis. Intraarticular injection of monoclonal antibody 1D11.16, which inhibits both TGF-beta 1 and TGF-beta 2 bioactivity, into animals receiving an arthropathic dose of bacterial cell walls significantly inhibits arthritis. Inhibition was observed with a single injection of 50 micrograms antibody, and a 1-mg injection blocked acute inflammation > 75% compared with the contralateral joints injected with an irrelevant isotype control antibody (MOPC21) as quantitated by an articular index (AI = 0.93 +/- 0.23 for 1D11.16, and AI = 4.0 +/- 0 on day 4; p < 0.001). Moreover, suppression of the acute arthritis achieved with a single injection of antibody was sustained into the chronic, destructive phase of the disease (on day 18, AI = 0.93 +/- 0.07 vs. AI = 2.6 +/- 0.5; p < 0.01). The decreased inflammatory index associated with anti-TGF-beta treatment was consistent with histopathologic and radiologic evidence of a therapeutic response. These data implicate TGF-beta as a profound agonist not only in the early events responsible for synovial inflammation, but also in the chronicity of streptococcal cell wall fragment-induced inflammation culminating in destructive pathology. Interrupting the cycle of leukocyte recruitment and activation with TGF-beta antagonists may provide a mechanism for resolution of chronic destructive lesions. PMID:8418203

Regeneration of hyaline articular cartilage with irradiated transforming growth factor beta1-producing fibroblasts.

PubMed

Song, Sun U; Hong, Young-Jin; Oh, In-Suk; Yi, Youngsuk; Choi, Kyoung Baek; Lee, Jung Woo; Park, Kwang-Won; Han, Jeoung-Uk; Suh, Jun-Kyu; Lee, Kwan Hee

2004-01-01

The regeneration of hyaline articular cartilage by cell-mediated gene therapy using transforming growth factor beta(1) (TGF-beta(1))-producing fibroblasts (NIH 3T3-TGF-beta(1)) has been reported previously. In this study, we investigated whether TGF-beta(1)-producing fibroblasts irradiated with a lethal dose of radiation are still capable of inducing the regeneration of hyaline articular cartilage. NIH 3T3TGF-beta(1) fibroblasts were exposed to doses of 20, 40, or 80 Gy, using a irradiator, and then injected into artificially made partial defects on the femoral condyle of rabbit knee joints. The rabbits were killed 3 or 6 weeks postinjection and hyaline articular cartilage regeneration was evaluated by histological and immunohistochemical staining (n = 5 per each group). Irradiated NIH 3T3-TGFbeta(1) fibroblasts started to die rapidly 3 days after irradiation; moreover, the kinetics of their viability were similar regardless of the radiation intensity. TGF-beta1 expression, measured by ELISA, showed that the TGF-beta(1) protein produced from the irradiated cells peaked 5 days after irradiation and thereafter declined rapidly. Complete filling of the defect with reparative tissue occurred in all the groups, although variations were observed in terms of the nature of the repair tissue. Histological and immunohistochemical staining of the repair tissue showed that the tissue newly formed by irradiated NIH 3T3-TGF-beta(1) fibroblasts after exposure to 20 Gy had hyaline cartilage-like characteristics, as was observed in the nonirradiated controls. On the other hand, the repair tissue formed by NIH 3T3-TGF-beta(1) fibroblasts irradiated with 40 or 80 Gy showed more fibrous cartilage-like tissue. These results suggest that TGF-beta(1)-producing fibroblasts irradiated up to a certain level of lethal dose (i.e., 20 Gy) are able to induce normal-appearing articular cartilage in vivo. Therefore, irradiated heterologous cell-mediated TGF-beta(1) gene therapy may be clinically

Hematological and TGF-beta variations after whole-body proton irradiation

NASA Technical Reports Server (NTRS)

Kajioka, E. H.; Andres, M. L.; Mao, X. W.; Moyers, M. F.; Nelson, G. A.; Gridley, D. S.

2000-01-01

The acute effects of proton whole-body irradiation on five bone-marrow-derived cell types and transforming growth factor-beta 1 (TGF-beta 1) were examined and compared to the effects of photons (60Co). C57BL/6 mice were exposed to 3 Gy (0.4 Gy/min) protons at spread-out Bragg peak (SOBP), protons at entry (E), or 60Co and euthanized on days 0.5-17 thereafter. 60Co-irradiated animals had decreased erythrocytes, hemoglobin and hematocrit at 12 hours post-exposure; depression was not noted in proton (SOBP or E)-irradiated groups until day 4. Significantly decreased leukocyte counts were observed at this same time in all irradiated groups, with lymphocyte loss being greater than that of monocytes, and the depression was generally maintained. In contrast, the levels of neutrophils and thrombocytes fluctuated, especially during the first week; significant differences were noted among irradiated groups in neutrophil levels. Plasma TGF-beta 1 was elevated on day 7 in the 60Co, but not proton, irradiated mice. Collectively, the data show that dramatic and persistent changes occurred in all irradiated groups. However, few differences in assay results were seen between animals exposed to protons (SOBP or E) or photons, as well as between the groups irradiated with either of the two regions of the proton Bragg curve.

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.

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

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

PubMed

Mathieu, C; Jozan, S; Mazars, P; Côme, M G; Moisand, A; Valette, A

1995-01-01

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

A Polymorphism Within the Promoter of the TGF{beta}1 Gene Is Associated With Radiation Sensitivity Using an Objective Radiologic Endpoint

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

Kelsey, Chris R., E-mail: kelse003@mc.duke.edu; Jackson, Lauren; Langdon, Scott

2012-02-01

Purpose: To evaluate whether single nucleotide polymorphisms (SNPs) in the transforming growth factor-{beta}1 (TGF{beta}1) gene are associated with radiation sensitivity using an objective radiologic endpoint. Methods and Materials: Preradiation therapy and serial postradiation therapy single photon emission computed tomography (SPECT) lung perfusion scans were obtained in patients undergoing treatment for lung cancer. Serial blood samples were obtained to measure circulating levels of TGF{beta}1. Changes in regional perfusion were related to regional radiation dose yielding a patient-specific dose-response curve, reflecting the patient's inherent sensitivity to radiation therapy. Six TGF{beta}1 SNPs (-988, -800, -509, 869, 941, and 1655) were assessed using high-resolutionmore » melting assays and DNA sequencing. The association between genotype and slope of the dose-response curve, and genotype and TGF{beta}1 ratio (4-week/preradiation therapy), was analyzed using the Kruskal-Wallis test. Results: 39 white patients with preradiation therapy and {>=}6-month postradiation therapy SPECT scans and blood samples were identified. Increasing slope of the dose-response curve was associated with the C(-509)T SNP (p = 0.035), but not the other analyzed SNPs. This SNP was also associated with higher TGF{beta}1 ratios. Conclusions: This study suggests that a polymorphism within the promoter of the TGF{beta}1 gene is associated with increased radiation sensitivity (defined objectively by dose-dependent changes in SPECT lung perfusion).« less

Functional cloning of the proto-oncogene brain factor-1 (BF-1) as a Smad-binding antagonist of transforming growth factor-beta signaling.

PubMed

Rodriguez, C; Huang, L J; Son, J K; McKee, A; Xiao, Z; Lodish, H F

2001-08-10

Using the plasminogen activator inhibitor (PAI) promoter to drive the expression of a reporter gene (mouse CD2), we devised a system to clone negative regulators of the transforming growth factor-beta (TGF-beta) signaling pathway. We infected a TGF-beta-responsive cell line (MvLu1) with a retroviral cDNA library, selecting by fluorescence-activated cell sorter single cells displaying low PAI promoter activity in response to TGF-beta. Using this strategy we cloned the proto-oncogene brain factor-1 (BF-1). BF-1 represses the PAI promoter in part by associating with both unphosphorylated Smad3 (in the cytoplasm) and phosphorylated Smad3 (in the nucleus), thus preventing its binding to DNA. BF-1 also associates with Smad1, -2, and -4; the Smad MH2 domain binds to BF-1, and the C-terminal segment of BF-1 is uniquely and solely required for binding to Smads. Further, BF-1 represses another TGF-beta-induced promoter (p15), it up-regulates a TGF-beta-repressed promoter (Cyclin A), and it reverses the growth arrest caused by TGF-beta. Our results suggest that BF-1 is a general inhibitor of TGF-beta signaling and as such may play a key role during brain development.

Down-regulation of connective tissue growth factor by inhibition of transforming growth factor beta blocks the tumor-stroma cross-talk and tumor progression in hepatocellular carcinoma.

PubMed

Mazzocca, Antonio; Fransvea, Emilia; Dituri, Francesco; Lupo, Luigi; Antonaci, Salvatore; Giannelli, Gianluigi

2010-02-01

Tumor-stroma interactions in hepatocellular carcinoma (HCC) are of key importance to tumor progression. In this study, we show that HCC invasive cells produce high levels of connective tissue growth factor (CTGF) and generate tumors with a high stromal component in a xenograft model. A transforming growth factor beta (TGF-beta) receptor inhibitor, LY2109761, inhibited the synthesis and release of CTGF, as well as reducing the stromal component of the tumors. In addition, the TGF-beta-dependent down-regulation of CTGF diminished tumor growth, intravasation, and metastatic dissemination of HCC cells by inhibiting cancer-associated fibroblast proliferation. By contrast, noninvasive HCC cells were found to produce low levels of CTGF. Upon TGF-beta1 stimulation, noninvasive HCC cells form tumors with a high stromal content and CTGF expression, which is inhibited by treatment with LY2109761. In addition, the acquired intravasation and metastatic spread of noninvasive HCC cells after TGF-beta1 stimulation was blocked by LY2109761. LY2109761 interrupts the cross-talk between cancer cells and cancer-associated fibroblasts, leading to a significant reduction of HCC growth and dissemination. Interestingly, patients with high CTGF expression had poor prognosis, suggesting that treatment aimed at reducing TGF-beta-dependent CTGF expression may offer clinical benefits. Taken together, our preclinical results indicate that LY2109761 targets the cross-talk between HCC and the stroma and provide a rationale for future clinical trials.

[The effect of overdose fluoride on the expression of TGF-beta1 in rat's dental pulps].

PubMed

Wu, Yu; He, Ke-xing; Yang, Yi-ping; Cao, Yang; Liang, Zhi-feng

2006-04-01

To observe the effect of overdose fluoride on the expression of TGF-beta1 in rat's dental pulps. 20 wister rats were divided into two groups. In the control group, equal dose distilled water were given to the rats. In the experimental group, 20 mg.kg(-1).d(-1) NaF were given. After 8 weeks of treatment, immunohistochemical staining was adopted for detection of the expression of TGF-beta1 in dental pulps of the rats. SPSS10.0 software package was used for Student's t test. Image analysis results showed that the expression of TGF-beta1 in the dental pulp and inner dentin were inhibited in the experimental group as compared with the control group (P<0.01). The overdose fluoride will inhibit the secretion of TGF-beta1, which leads to abnormal development of the teeth.

Preliminary crystallographic analysis of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-[beta] receptor promoter DNA

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

Agarkar, Vinod B.; Babayeva, Nigar D.; Rizzino, Angie

2010-10-08

Ets proteins are transcription factors that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. Like other Ets-family members, Elf3 functions as a sequence-specific DNA-binding transcriptional factor. A mouse Elf3 C-terminal fragment (amino-acid residues 269-371) containing the DNA-binding domain has been crystallized in complex with mouse type II TGF-{beta} receptor promoter (TR-II) DNA. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 42.66, b = 52, c = 99.78 {angstrom}, and diffracted to a resolution of 2.2 {angstrom}.

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

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.

Opposite Smad and chicken ovalbumin upstream promoter transcription factor inputs in the regulation of the collagen VII gene promoter by transforming growth factor-beta.

PubMed

Calonge, María Julia; Seoane, Joan; Massagué, Joan

2004-05-28

A critical component of the epidermal basement membrane, collagen type VII, is produced by keratinocytes and fibroblasts, and its production is stimulated by the cytokine transforming growth factor-beta (TGF-beta). The gene, COL7A1, is activated by TGF-beta via Smad transcription factors in cooperation with AP1. Here we report a previously unsuspected level of complexity in this regulatory process. We provide evidence that TGF-beta may activate the COL7A1 promoter by two distinct inputs operating through a common region of the promoter. One input is provided by TGF-beta-induced Smad complexes via two Smad binding elements that function redundantly depending on the cell type. The second input is provided by relieving the COL7A1 promoter from chicken ovalbumin upstream promoter transcription factor (COUP-TF)-mediated transcriptional repression. We identified COUP-TFI and -TFII as factors that bind to the TGF-beta-responsive region of the COL7A1 promoter in an expression library screening. COUP-TFs bind to a site between the two Smad binding elements independently of Smad or AP1 and repress the basal and TGF-beta-stimulated activities of this promoter. We provide evidence that endogenous COUP-TF activity represses the COL7A1 promoter. Furthermore, we show that TGF-beta addition causes a rapid and profound down-regulation of COUP-TF expression in keratinocytes and fibroblasts. The results suggest that TGF-beta signaling may exert tight control over COL7A1 by offsetting the balance between opposing Smad and COUP-TFs.

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.

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.

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.

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.

Evaluation of TGF beta1 expression and comparison the thickness of different aorta layers in experimental diabetes.

PubMed

Cuce, G; Kalkan, S S; Esen, H H

2011-01-01

It was aimed to investigate the effects of experimental diabetes on TGF beta1 expression and tunica intima and media thickness in abdominal and thoracic aorta. Fourteen three months old female rats were divided into two groups, non-diabetic and streptozotocin (STZ) induced diabetic group. Hematoxylin-Eosin and Verhoeff's Van Gieson elastic staining and TGF beta1 immunohistochemistry staining were performed. Abdominal and thoracic intima and media thickness of aortas were measured with the oculometer. Evaluation of intima and media thickness measurements showed no significant statistical differences between non-diabetic and diabetic groups. TGF beta1 expression increased significantly in thoracic diabetic (TD) group. The 60 day duration of diabetes is not sufficiently enough time for the development of pathological changes that could lead to thickening in aortic intima-media layers. TGF beta1 expression was negative in the abdominal aorta that can predispose to the development of atherosclerosis, which could develop overtime. This finding may be interpreted as an appropriate basis for the development of atherosclerosis. In the thoracic aorta TGF beta1 may coordinate cellular events such as tissue repair (Fig. 5, Ref. 23).

TGF beta and IL13 in Schistosomiasis mansoni associated pulmonary arterial hypertension; a descriptive study with comparative groups.

PubMed

Ferreira, Rita de Cassia dos Santos; Montenegro, Silvia Maria Lucena; Domingues, Ana Lucia Coutinho; Bandeira, Angela Pontes; Silveira, Carlos Antonio da Mota; Leite, Luiz Arthur Calheiros; Pereira, Clara de Almeida; Fernandes, Izolda Moura; Mertens, Alessandra Brainer; Almeida, Milena Oliveira

2014-05-21

It is suggested that interleukin (IL)-13 and transforming growth factor (TGF)-beta play a role in the pulmonary vascular changes found in animal models of schistosomiasis. The aim of this study was to assess and compare the serum levels of total TGF-beta and IL-13 of patients with schistosomiasis with pulmonary arterial hypertension (PAH) and patients with schistosomiasis without PAH. 34 patients from the schistosomiasis outpatient clinic of the Hospital das Clinicas, Recife, Pernambuco, Brazil, without PAH assessed by echocardiography and 34 patients from the Reference Centre of Pulmonary Hypertension of Pronto Socorro Cardiológico de Pernambuco, Recife, Brazil with PAH, confirmed by right heart catheterization, were enrolled on the study. Both groups presented with schistosomal periportal fibrosis after abdominal ultrasound. Serum levels of TGF-beta1 and IL-13 were determined by ELISA. Student t test to independent samples, Mann-Whitney test to nonparametric variables, Pearson correlation test for correlation analyses and Fisher Chi-squared test to compare categorical analyses were used. The median value of TGF-beta1 was significantly higher in patients with PAH (22496.9 pg/ml, interquartile range [IR] 15936.7 - 32087.8) than in patients without PAH (13629.9 pg/ml, IR: 10192.2- 22193.8) (p = 0.006). There was no difference in the median value of IL-13 in the group with Sch-PAH compared to patients without Sch-PAH (p > 0.05). Our results suggest that TGF-beta possibly plays a role in the pathogenesis of schistosomiasis-associated PAH.

Bucindolol, a nonselective beta 1- and beta 2-adrenergic receptor antagonist, decreases beta-adrenergic receptor density in cultured embryonic chick cardiac myocyte membranes.

PubMed

Asano, K; Zisman, L S; Yoshikawa, T; Headley, V; Bristow, M R; Port, J D

2001-06-01

Bucindolol and carvedilol, nonselective beta1- and beta2-adrenergic receptor antagonists, have been widely used in clinical therapeutic trials of congestive heart failure. The aim of the current study was to investigate long-term effects of bucindolol or carvedilol on beta-adrenergic receptor protein and gene expression in cardiac myocytes. Embryonic chick cardiac myocytes were cultured and incubated with bucindolol (1 microM), carvedilol (1 microM), or norepinephrine (1 microM) for 24 h. 125I-iodocyanopindolol binding assays demonstrated that incubation with norepinephrine or bucindolol, but not carvedilol, significantly decreased beta-adrenergic receptor density in crude membranes prepared from the myocytes. Neither bucindolol nor carvedilol significantly stimulated adenylyl cyclase activity in membranes from drug-untreated cells. Unlike by norepinephrine, the receptor density reduction by bucindolol incubation was not accompanied by a change in beta1-adrenergic receptor messenger RNA abundance. A decrease in membrane beta-adrenergic receptor density without a change in cognate messenger RNA abundance was also observed in hamster DDT1 MF2 cell line incubated with bucindolol (1 microM, 24 h). We conclude that incubation with bucindolol, but not carvedilol, results in true reduction of beta-adrenergic receptor density in chick cardiac myocyte membranes by mechanisms that are distinct from those responsible for receptor density reduction by the agonist norepinephrine.

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

Betaglycan expression is transcriptionally up-regulated during skeletal muscle differentiation. Cloning of murine betaglycan gene promoter and its modulation by MyoD, retinoic acid, and transforming growth factor-beta.

PubMed

Lopez-Casillas, Fernando; Riquelme, Cecilia; Perez-Kato, Yoshiaki; Ponce-Castaneda, M Veronica; Osses, Nelson; Esparza-Lopez, Jose; Gonzalez-Nunez, Gerardo; Cabello-Verrugio, Claudio; Mendoza, Valentin; Troncoso, Victor; Brandan, Enrique

2003-01-03

Betaglycan is a membrane-anchored proteoglycan co-receptor that binds transforming growth factor beta (TGF-beta) via its core protein and basic fibroblast growth factor through its glycosaminoglycan chains. In this study we evaluated the expression of betaglycan during the C(2)C(12) skeletal muscle differentiation. Betaglycan expression, as determined by Northern and Western blot, was up-regulated during the conversion of myoblasts to myotubes. The mouse betaglycan gene promoter was cloned, and its sequence showed putative binding sites for SP1, Smad3, Smad4, muscle regulatory factor elements such as MyoD and MEF2, and retinoic acid receptor. Transcriptional activity of the mouse betaglycan promoter reporter was also up-regulated in differentiating C(2)C(12) cells. We found that MyoD, but not myogenin, stimulated this transcriptional activity even in the presence of high serum. Betaglycan promoter activity was increased by RA and inhibited by the three isoforms of TGF-beta. On the other hand, basic fibroblast growth factor, BMP-2, and hepatocyte growth factor/scatter factor, which are inhibitors of myogenesis, had little effect. In myotubes, up-regulated betaglycan was also detectable by TGF-beta affinity labeling and immunofluorescence microscopy studies. The latter indicated that betaglycan was localized both on the cell surface and in the ECM. Forced expression of betaglycan in C(2)C(12) myoblasts increases their responsiveness to TGF-beta2, suggesting that it performs a TGF-beta presentation function in this cell lineage. These results indicate that betaglycan expression is up-regulated during myogenesis and that MyoD and RA modulate its expression by a mechanism that is independent of myogenin.

Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling.

PubMed

Chen, Weijun; Lam, Suvana S; Srinath, Hema; Schiffer, Celia A; Royer, William E; Lin, Kai

2007-04-13

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.

Increased expression of c-Ski as a co-repressor in transforming growth factor-beta signaling correlates with progression of esophageal squamous cell carcinoma.

PubMed

Fukuchi, Minoru; Nakajima, Masanobu; Fukai, Yasuyuki; Miyazaki, Tatsuya; Masuda, Norihiro; Sohda, Makoto; Manda, Ryokuhei; Tsukada, Katsuhiko; Kato, Hiroyuki; Kuwano, Hiroyuki

2004-03-01

Transforming growth factor-beta (TGF-beta) regulates cell growth inhibition, and inactivation of the TGF-beta signaling pathway contributes to tumor development. In our previous study, altered expression of TGF-beta, TGF-beta-specific receptors and Smad4 was shown to correlate with tumor progression in esophageal squamous cell carcinoma (SCC). These components, however, were maintained normally in some patients with esophageal SCC. In our study, the mechanism by which aggressive esophageal SCC maintains these components was investigated, with particular emphasis on the participation of c-Ski and SnoN as transcriptional co-repressors in TGF-beta signaling. Immunohistochemistry for c-Ski and SnoN was carried out on surgical specimens obtained from 80 patients with esophageal SCC. The expression of c-Ski and SnoN was also studied in 6 established cell lines derived from esophageal SCC and compared to an immortalized human esophageal cell line by Western blotting. High levels of expression of c-Ski, detected immunohistologically, were found to correlate with depth of invasion (p = 0.0080) and pathologic stage (p = 0.0447). There was, however, no significant correlation between expression of SnoN and clinicopathologic characteristics. A significant correlation between c-Ski and TGF-beta expression was observed. Moreover, in patients with TGF-beta negative expression, the survival rates of patients with c-Ski positive expression were significantly lower than those of patients with c-Ski negative expression (p = 0.0486). c-Ski was expressed at a high level in 5 of 6 cell lines derived from esophageal SCC compared to immortalized esophageal keratinocytes. Furthermore, the cyclin-dependent kinase (CDK) inhibitor, p21 that was up-regulated by TGF-beta signaling was expressed at a low level in the 5 cell lines. The expression of c-Ski protein as a transcriptional co-repressor in TGF-beta signaling seems to be correlated with tumor progression of esophageal SCC. Copyright 2003

Small C-terminal domain phosphatases dephosphorylate the regulatory linker regions of Smad2 and Smad3 to enhance transforming growth factor-beta signaling.

PubMed

Wrighton, Katharine H; Willis, Danielle; Long, Jianyin; Liu, Fang; Lin, Xia; Feng, Xin-Hua

2006-12-15

Transforming growth factor-beta (TGF-beta) controls a diverse set of cellular processes, and its canonical signaling is mediated via TGF-beta-induced phosphorylation of receptor-activated Smads (2 and 3) at the C-terminal SXS motif. We recently discovered that PPM1A can dephosphorylate Smad2/3 at the C-terminal SXS motif, implicating a critical role for phosphatases in regulating TGF-beta signaling. Smad2/3 activity is also regulated by phosphorylation in the linker region (and N terminus) by a variety of intracellular kinases, making it a critical platform for cross-talk between TGF-beta and other signaling pathways. Using a functional genomic approach, we identified the small C-terminal domain phosphatase 1 (SCP1) as a specific phosphatase for Smad2/3 dephosphorylation in the linker and N terminus. A catalytically inactive SCP1 mutant (dnSCP1) had no effect on Smad2/3 phosphorylation in vitro or in vivo. Of the other FCP/SCP family members SCP2 and SCP3, but not FCP1, could also dephosphorylate Smad2/3 in the linker/N terminus. Depletion of SCP1/2/3 enhanced Smad2/3 linker phosphorylation. SCP1 increased TGF-beta-induced transcriptional activity in agreement with the idea that phosphorylation in the Smad2/3 linker must be removed for a full transcriptional response. SCP1 overexpression also counteracts the inhibitory effect of epidermal growth factor on TGF-beta-induced p15 expression. Taken together, this work identifies the first example of a Smad2/3 linker phosphatase(s) and reveals an important new substrate for SCPs.

Effect of transforming growth factor-beta1 on decorin expression and muscle morphology during chicken embryonic and posthatch growth and development.

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, as well as a regulator of extracellular matrix (ECM) production. Decorin, a member of the small leucine-rich ECM proteoglycans, binds to TGF-beta1 and modulates TGF-beta1-dependent cell growth stimulation or inhibition. The expression of decorin can be regulated by TGF-beta1 during muscle proliferation and differentiation. How TGF-beta1 affects decorin and muscle growth, however, has not been well documented in vivo. The present study investigated the effect of TGF-beta1 on decorin expression and intracellular connective tissue development during skeletal muscle growth. Exogenous TGF-beta1 significantly decreased the number of myofibers in a given area at both 1 d and 6 wk posthatch. The TGF-beta1-treated muscle had a significant decrease in decorin mRNA expression at embryonic day (ED) 10, whereas protein amounts decreased at 17 ED and 1 d posthatch compared to the control muscle. Decorin was localized in both the endomysium and perimysium in the control pectoralis major muscle. Transforming growth factor-beta1 reduced decorin in both the endomysium and perimysium from 17 ED to 6 wk posthatch. Compared to the control muscle, the perimysium space in the pectoralis major muscle was dramatically decreased by TGF-beta1 during embryonic development through posthatch growth. Because decorin regulates collagen fibrillogenesis, a major component of the ECM, the reduction of decorin by TGF-beta1 treatment may cause the irregular formation of collagen fibrils, leading to the decrease in endomysium and perimysium space. The results from the current study suggest that the effect of TGF-beta1 on decorin expression and localization was likely associated with altered development of the perimysium and the regulation of muscle fiber development.

Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA.

PubMed

Zhao, Bryan M; Hoffmann, F Michael

2006-09-01

Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.

Up-regulated transcriptional repressors SnoN and Ski bind Smad proteins to antagonize transforming growth factor-beta signals during liver regeneration.

PubMed

Macias-Silva, Marina; Li, Wei; Leu, Julia I; Crissey, Mary Ann S; Taub, Rebecca

2002-08-09

Transforming growth factor-beta (TGF-beta) functions as an antiproliferative factor for hepatocytes. However, for unexplained reasons, hepatocytes become resistant to TGF-beta signals and can proliferate despite the presence of TGF-beta during liver regeneration. TGF-beta is up-regulated during liver regeneration, although it is not known whether it is active or latent. TGF-beta activity may be examined by assessing Smad activation, a downstream signaling pathway. Smad pathway activation during liver regeneration induced by partial hepatectomy or CC4 injury was examined by assessing the levels of phospho-Smad2 and Smad2-Smad4 complexes. We found that Smad proteins were slightly activated in quiescent liver, but that their activation was further enhanced in regenerating liver. Interestingly, TGF-beta/Smad pathway inhibitors (SnoN and Ski) were up-regulated during regeneration, and notably, SnoN was induced mainly in hepatocytes. SnoN and Ski are transcriptional repressors that may render some cells resistant to TGF-beta via binding Smad proteins. Complexes between SnoN, Ski, and the activated Smad proteins were detected from 2 to 120 h during the major proliferative phase in regenerating liver. Inhibitory complexes decreased after liver mass restitution (5-15 days), suggesting that persistently activated Smad proteins might participate in returning the liver to a quiescent state. Our data show that active TGF-beta/Smad signals are present during regeneration and suggest that SnoN/Ski induction might explain hepatocyte resistance to TGF-beta during the proliferative phase.

Protective effects of melittin on transforming growth factor-{beta}1 injury to hepatocytes via anti-apoptotic mechanism

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

Lee, Woo-Ram; Park, Ji-Hyun; Kim, Kyung-Hyun

Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). Melittin has multiple effects, including anti-bacterial, anti-viral and anti-inflammatory, in various cell types. However, the anti-apoptotic mechanisms of melittin have not been fully elucidated in hepatocytes. Apoptosis contributes to liver inflammation and fibrosis. Knowledge of the apoptotic mechanisms is important to develop new and effective therapies for treatment of cirrhosis, portal hypertension, liver cancer, and other liver diseases. In the present study, we investigated the anti-apoptotic effect of melittin on transforming growth factor (TGF)-{beta}1-induced apoptosis in hepatocytes. TGF-{beta}1-treated hepatocytesmore » were exposed to low doses (0.5 and 1 {mu}g/mL) and high dose (2 {mu}g/mL) of melittin. The low doses significantly protected these cells from DNA damage in TGF-{beta}1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-{beta}1-induced apoptotic activation of the Bcl-2 family and caspase family of proteins, which resulted in the inhibition of poly-ADP-ribose polymerase (PARP) cleavage. These results demonstrate that TGF-{beta}1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-{beta}1-induced injury to hepatocytes via the mitochondrial pathway. These results suggest that an optimal dose of melittin can serve to protect cells against TGF-{beta}1-mediated injury. - Highlights: > We investigated the anti-apoptotic effect of melittin on TGF-{beta}1-induced hepatocyte. > TGF-{beta}1 induces hepatocyte apoptosis. > TGF-{beta}1-treated hepatocytes were exposed to low doses and high dose of melittin. > Optimal dose of melittin exerts anti-apoptotic effects to hepatocytes.« less

N-terminal tyrosine phosphorylation of caveolin-2 negates anti-proliferative effect of transforming growth factor beta in endothelial cells

PubMed Central

Abel, Britain; Willoughby, Cara; Jang, Sungchan; Cooper, Laura; Xie, Leike; Vo-Ransdell, Chi; Sowa, Grzegorz

2012-01-01

Here we show that tyrosine phosphorylation of caveolin-2 (Cav-2) negatively regulates the anti-proliferative function of transforming growth factor beta (TGF-beta) in endothelial cells. In contrast to wild-type-Cav-2, retroviral re-expression of Y19/27F-Cav-2 in Cav-2 knockout endothelial cells did not affect anti-proliferative effect of TGF-beta compared to empty vector. Conversely, although less effective than wild-type, re-expression of S23/36A-Cav-2 reduced the effect of TGF-beta compared to empty vector. This differential effect of tyrosine and serine phosphorylation mutants of Cav-2 correlated with TGF-beta-induced Smad3 phosphorylation and transcriptional activation of plasminogen activator inhibitor-1. Thus tyrosine-phosphorylated Cav-2 counteracts anti-proliferative effect of TGF-beta in endothelial cells. PMID:22819829

Angiotensin II Receptor Antagonism Reduces Transforming Growth Factor Beta and Smad Signaling in Thoracic Aortic Aneurysm

PubMed Central

Nataatmadja, Maria; West, Jennifer; Prabowo, Sulistiana; West, Malcolm

2013-01-01

ABSTRACT Background The expression of transforming growth factor beta (TGF-β) and Smad3 regulates extracellular matrix homeostasis and inflammation in aortic aneurysms. The expression of Smad3 depends on signaling by angiotensin II (AngII) receptor pathways through TGF-β receptor–dependent and –independent pathways. Methods To determine the expression of AngII type 1 (AT1R) and type 2 receptors (AT2R), TGF-β, and Smad3 in thoracic aortic aneurysms, we performed immunohistochemistry testing on tissue and cultured cells derived from subjects with Marfan syndrome (MFS) and bicuspid aortic valve (BAV) malformation and from normal aortas of subjects who were organ donors. Results MFS and BAV aneurysm tissue showed enhanced accumulation of TGF-β and Smad3 in vascular smooth muscle cells (VSMCs) and in inflammatory cells in the subintimal layer and tunica media. The normal aortic wall exhibited minimal TGF-β and Smad3 staining. Cultured VSMCs from MFS and BAV samples showed nuclear Smad3 and strong cytoplasmic TGF-β expression in the cytoplasmic vesicles. In control cells, Smad3 was located mainly in the cytoplasm, and weak cytoplasmic TGF-β was distributed with a pattern similar to that of the aneurysm-derived cells. Compared to normal aorta cells, AT1R and AT2R expression was increased in both aneurysm types. Treatment of cultured VSMCs with the AT1R antagonist losartan caused both reduced TGF-β vesicle localization and nuclear expression of Smad3. Conclusions Increased TGF-β and Smad3 expression in aneurysm tissue and cultured VSMCs is consistent with aberrant TGF-β expression and the activation of Smad3 signaling. Losartan-mediated reduction in TGF-β expression and the cytoplasmic localization of Smad3 support a role for AT1R antagonism in the inhibition of aneurysm progression. PMID:23532685

The neurotrophins act synergistically with LIF and members of the TGF-beta superfamily to promote the survival of spiral ganglia neurons in vitro.

PubMed

Marzella, P L; Gillespie, L N; Clark, G M; Bartlett, P F; Kilpatrick, T J

1999-12-01

A number of growth factor families have been implicated in normal inner ear development, auditory neuron survival and protection. Several growth factors, including transforming growth factor-beta5 (TGF-beta5) and TGF-beta3, neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) were tested for their ability, individually or in combination, to promote auditory neuron survival in dissociated cell cultures of early rat post-natal spiral ganglion cells (SGCs). The results indicate that at discrete concentrations all growth factors act in an additive fashion and some in synergy when promoting neuronal survival. These findings support the hypothesis that growth factors from different families may be interdependent when sustaining neuronal integrity.

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.

Microarray identifies ADAM family members as key responders to TGF-beta1 in alveolar epithelial cells.

PubMed

Keating, Dominic T; Sadlier, Denise M; Patricelli, Andrea; Smith, Sinead M; Walls, Dermot; Egan, Jim J; Doran, Peter P

2006-09-01

The molecular mechanisms of Idiopathic Pulmonary Fibrosis (IPF) remain elusive. Transforming Growth Factor beta 1(TGF-beta1) is a key effector cytokine in the development of lung fibrosis. We used microarray and computational biology strategies to identify genes whose expression is significantly altered in alveolar epithelial cells (A549) in response to TGF-beta1, IL-4 and IL-13 and Epstein Barr virus. A549 cells were exposed to 10 ng/ml TGF-beta1, IL-4 and IL-13 at serial time points. Total RNA was used for hybridisation to Affymetrix Human Genome U133A microarrays. Each in vitro time-point was studied in duplicate and an average RMA value computed. Expression data for each time point was compared to control and a signal log ratio of 0.6 or greater taken to identify significant differential regulation. Using normalised RMA values and unsupervised Average Linkage Hierarchical Cluster Analysis, a list of 312 extracellular matrix (ECM) proteins or modulators of matrix turnover was curated via Onto-Compare and Gene-Ontology (GO) databases for baited cluster analysis of ECM associated genes. Interrogation of the dataset using ontological classification focused cluster analysis revealed coordinate differential expression of a large cohort of extracellular matrix associated genes. Of this grouping members of the ADAM (A disintegrin and Metalloproteinase domain containing) family of genes were differentially expressed. ADAM gene expression was also identified in EBV infected A549 cells as well as IL-13 and IL-4 stimulated cells. We probed pathologenomic activities (activation and functional activity) of ADAM19 and ADAMTS9 using siRNA and collagen assays. Knockdown of these genes resulted in diminished production of collagen in A549 cells exposed to TGF-beta1, suggesting a potential role for these molecules in ECM accumulation in IPF.

The corepressor CtBP interacts with Evi-1 to repress transforming growth factor beta signaling.

PubMed

Izutsu, K; Kurokawa, M; Imai, Y; Maki, K; Mitani, K; Hirai, H

2001-05-01

Evi-1 is a zinc finger nuclear protein whose inappropriate expression leads to leukemic transformation of hematopoietic cells in mice and humans. This was previously shown to block the antiproliferative effect of transforming growth factor beta (TGF-beta). Evi-1 represses TGF-beta signaling by direct interaction with Smad3 through its first zinc finger motif. Here, it is demonstrated that Evi-1 represses Smad-induced transcription by recruiting C-terminal binding protein (CtBP) as a corepressor. Evi-1 associates with CtBP1 through one of the consensus binding motifs, and this association is required for efficient inhibition of TGF-beta signaling. A specific inhibitor for histone deacetylase (HDAc) alleviates Evi-1-mediated repression of TGF-beta signaling, suggesting that HDAc is involved in the transcriptional repression by Evi-1. This identifies a novel function of Evi-1 as a member of corepressor complexes and suggests that aberrant recruitment of corepressors is one of the mechanisms for Evi-1-induced leukemogenesis.

Latent transforming growth factor beta1 activation in situ: quantitative and functional evidence after low-dose gamma-irradiation

NASA Technical Reports Server (NTRS)

Ehrhart, E. J.; Segarini, P.; Tsang, M. L.; Carroll, A. G.; Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)

1997-01-01

The biological activity of transforming growth factor beta1 (TGF-beta) is controlled by its secretion as a latent complex in which it is noncovalently associated with latency-associated peptide (LAP). Activation is the extracellular process in which TGF-beta is released from LAP, and is considered to be a primary regulatory control. We recently reported rapid and persistent changes in TGF-beta immunoreactivity in conjunction with extracellular matrix remodeling in gamma-irradiated mouse mammary gland. Our hypothesis is that these specific changes in immunoreactivity are indicative of latent TGF-beta activation. In the present study, we determined the radiation dose response and tested whether a functional relationship exists between radiation-induced TGF-beta and collagen type III remodeling. After radiation exposures as low as 0.1 Gy, we detected increased TGF-beta immunoreactivity in the mammary epithelium concomitant with decreased LAP immunostaining, which are events consistent with activation. Quantitative image analysis demonstrated a significant (P=0.0005) response at 0.1 Gy without an apparent threshold and a linear dose response to 5 Gy. However, in the adipose stroma, loss of LAP demonstrated a qualitative threshold at 0.5 Gy. Loss of LAP paralleled induction of collagen III immunoreactivity in this tissue compartment. We tested whether TGF-beta mediates collagen III expression by treating animals with TGF-beta panspecific monoclonal antibody, 1D11.16, administered i.p. shortly before irradiation. Radiation-induced collagen III staining in the adipose stroma was blocked in an antibody dose-dependent manner, which persisted through 7 days postirradiation. RNase protection assay revealed that radiation-induced elevation of total gland collagen III mRNA was also blocked by neutralizing antibody treatment. These data provide functional confirmation of the hypothesis that radiation exposure leads to latent TGF-beta activation, support our interpretation of the

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.

TGF{beta}1 polymorphisms and late clinical radiosensitivity in patients treated for gynecologic tumors

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

Ruyck, Kim de; Van Eijkeren, Marc; Claes, Kathleen

2006-07-15

Purpose: To investigate the association between six transforming growth factor {beta}1 gene (TGF{beta}1) polymorphisms (-1.552delAGG, -800G>A, -509C>T, Leu10Pro, Arg25Pro, Thr263Ile) and the occurrence of late normal tissue reactions after gynecologic radiotherapy (RT). Methods and Materials: Seventy-eight women with cervical or endometrial cancer and 140 control individuals were included in the study. According to the Common Terminology Criteria for Adverse Events version 3.0 (CTCAEv3.0) scale, 25 patients showed late adverse RT reactions (CTC2+), of whom 11 had severe complications (CTC3+). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single base extension and genotyping assays were performed to examine the polymorphic sites inmore » TGF{beta}1. Results: Homozygous variant -1.552delAGG, -509TT, and 10Pro genotypes were associated with the risk of developing late severe RT reactions. Triple (variant) homozygous patients had a 3.6 times increased risk to develop severe RT reactions (p = 0.26). Neither the -800A allele, nor the 25Pro allele or the 263Ile allele were associated with clinical radiosensitivity. There was perfect linkage disequilibrium (LD) between the -1.552delAGG and the -509C>T polymorphisms, and tight LD between the -1.552/-509 and the Leu10Pro polymorphisms. Haplotype analysis revealed two major haplotypes but could not distinguish radiosensitive from nonradiosensitive patients. Conclusions: The present study shows that homozygous variant TGF{beta}1 -1.552delAGG, -509TT, and 10Pro genotypes may be associated with severe clinical radiosensitivity after gynecologic RT.« less

Increased expression of transforming growth factor beta s after acute oedematous pancreatitis in rats suggests a role in pancreatic repair.

PubMed Central

Riesle, E; Friess, H; Zhao, L; Wagner, M; Uhl, W; Baczako, K; Gold, L I; Korc, M; Büchler, M W

1997-01-01

BACKGROUND: Transforming growth factor beta isoforms (TGF beta s) belong to a family of multifunctional regulators of cellular growth and differentiation. They are mitogenic and chemotactic for fibroblasts and are potent stimulators of extracellular matrix production (collagen) and deposition. Upregulation of TGF beta transcription has been reported for several in vivo systems during repair after injury. AIMS: To study the expression of the three mammalian isoforms of TGF beta (TGF beta 1-3) and their relation to collagen expression as a marker for fibroblast response in acute oedematous pancreatitis in rats. METHODS: Using northern blot analysis and immunohistochemistry, the expression and localisation of TGF beta isoforms, collagen, and amylase were analysed during the course of acute oedematous pancreatitis in rats, experimentally induced by intravenous caerulein infusion. RESULTS: Induction of acute pancreatitis resulted in a biphasic peak pattern of expression of TGF beta 1, beta 2, and beta 3 mRNA, with a pronounced increase from day 1 to day 3 (sixfold, 2.5-fold, fivefold, respectively) and again from day 5 to day 7 (three-fold, 2.3-fold, 3.5-fold, respectively). The temporal changes in TGF beta mRNA identically paralleled the expression in collagen mRNA. In contrast, amylase mRNA expression, used as a general indicator of acinar cell integrity, was slightly decreased after induction of acute pancreatitis. Immunohistochemical analysis of pancreatitis tissue showed that increased expression of TGF beta s was mainly present in the pancreatic acinar and ductal cells; this was evident within one day after pancreatitis induction. CONCLUSION: Overexpression of TGF beta s after induction of acute pancreatitis suggests a role for these proteins in pancreatic repair and remodelling. The increased levels of TGF beta s may help suppress immune activation, and may contribute to the increase in the extracellular matrix including collagen and to the repair of the

Prostaglandin E1 inhibits collagen expression in anti-thymocyte antibody-induced glomerulonephritis: possible role of TGF beta.

PubMed

Schneider, A; Thaiss, F; Rau, H P; Wolf, G; Zahner, G; Jocks, T; Helmchen, U; Stahl, R A

1996-07-01

To test whether or not prostaglandins mediate extracellular matrix formation in immune-mediated glomerular disease, rats with anti-thymocyte antibody-induced glomerulonephritis were treated with prostaglandin E1 (PGE1) (250 micrograms/twice daily/s.c.). Glomerular expression of collagen types III and IV was assessed by Northern blotting, immunohistology and Western blotting. Proliferation of glomerular cells was evaluated by staining for the proliferating cell nuclear antigen (PCNA) and consecutive cell counting. At day five after induction of the disease, glomerular mRNA levels of collagen types III and IV were three- to fivefold higher compared with non-nephritic controls. Similarly glomerular deposition of these collagens was markedly increased when assessed by immunohistology. The treatment of nephritic rats with PGE1 reduced the increased glomerular mRNA levels as well as the protein concentration and the deposition of extracellular collagens. The number of PCNA positive cells which was significantly higher in nephritic rats when compared with control animals (24 hr, nephritis 2.53 +/- 0.33 and Control 0.26 +/- 0.06, P = 0.011; 5 days, nephritis 5.10 +/- 1.13 and Control 0.75 +/- 0.08, cells per glomerular cross section, P = 0.03) was reduced by PGE1 (24 hr, nephritis+PGE1 0.44 +/- 0.30, P = 0.0001; 5 days, nephritis +/- PGE1 1.91 +/- 1.84 cells per glomerular cross section, P = 0.001). Prostaglandin E1 also ameliorated the glomerular infiltration of monocytes at 24 hours (nephritis 4.36 +/- 2.82, nephritis + PGE1 2.20 +/- 1.82, cells per glomerular cross section) and five days (nephritis 1.51 +/- 0.58, nephritis+PGE1 1.12 +/- 0.61, cells per glomerular cross section). To further characterize possible mechanisms by which PGE1 reduces extracellular matrix deposition, the glomerular expression of transforming growth factor (TGF-beta), and interleukin 1 beta (IL-1 beta) was assessed by Northern blotting. Nephritic glomeruli showed increased mRNA levels of TGF-beta

3-Phosphoinositide-dependent PDK1 negatively regulates transforming growth factor-beta-induced signaling in a kinase-dependent manner through physical interaction with Smad proteins.

PubMed

Seong, Hyun-A; Jung, Haiyoung; Kim, Kyong-Tai; Ha, Hyunjung

2007-04-20

We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-beta signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-beta treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-beta-induced transcription, as well as TGF-beta-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-beta. Taken together, our results suggest that PDK1 negatively regulates TGF-beta-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1.

Transforming growth factor-beta stimulates wound healing and modulates extracellular matrix gene expression in pig skin. I. Excisional wound model.

PubMed

Quaglino, D; Nanney, L B; Kennedy, R; Davidson, J M

1990-09-01

The effect of transforming growth factor-beta 1 (TGF-beta 1) on matrix gene expression has been investigated during the process of wound repair, where the formation of new connective tissue represents a critical step in restoring tissue integrity. Split-thickness excisional wounds in the pig were studied by in situ hybridization in order to obtain subjective findings on the activity and location of cells involved in matrix gene expression after the administration of recombinant TGF-beta 1. Data focus on the stimulatory role of this growth factor in granulation tissue formation, on the enhanced mRNA content of collagen types I and III, fibronectin, TGF-beta 1 itself, and on the reduction in stromelysin mRNA, suggesting that increased matrix formation measured after treatment with TGF-beta 1 is due to fibroplasia regulated by the abundance of mRNAs for several different structural, matrix proteins as well as inhibition of proteolytic phenomena elicited by metalloproteinases. These studies reveal elastin mRNA early in the repair process, and elastin mRNA expression is enhanced by administration of TGF-beta 1. Moreover, we show that TGF-beta 1 was auto-stimulating in wounds, accounting, at least in part, for the persistent effects of single doses of this multipotential cytokine.

The TGF-beta-induced gene product, betaig-h3: its biological implications in peritoneal dialysis.

PubMed

Park, Sun-Hee; Choi, Soon-Youn; Kim, Mi-Hyung; Oh, Eun-Joo; Ryu, Hye Myung; Kim, Chan-Duck; Kim, In-San; Kim, Yong-Lim

2008-01-01

TGF-beta is involved in peritoneal changes during long-term peritoneal dialysis (PD). TGF-beta induces betaig-h3 in several cell lines, and betaig-h3 may be a marker for biologically active TGF-beta. However, no study has reported induction of betaig-h3 in human peritoneal mesothelial cells (HPMCs) or its involvement in PD-related peritoneal membrane changes. We used cultured HPMCs to investigate the biological roles of betaig-h3 during mesothelial cell injury and repair, employing the adhesion, spreading, scratching and cell migration assays. Changes in betaig-h3 expression after high glucose exposure in vivo were also evaluated using an animal chronic PD model. In vitro, TGF-beta1 induced betaig-h3 in cultured HPMCs, and betaig-h3-mediated mesothelial cell adhesion occurred via alphavbeta3 integrin. betaig-h3 enhanced mesothelial cell adhesion and migration and, in part, wound healing during mesothelial cell injury. The animal study demonstrated that compared to the control group, betaig-h3 concentrations in the dialysate effluent increased in the dialysis group with alterations in peritoneal structure and function during PD, and betaig-h3 positively correlated with peritoneal solute transport. Immunohistochemical and immunoblotting results showed that betaig-h3 localizes in the mesothelium and submesothelial matrix of the parietal peritoneum, and in the vascular endothelium of omentum. betaig-h3 protein expression was higher in the dialysis group. In vitro, betaig-h3 induced by TGF-beta1 in HPMCs improved adhesion and migration of HPMCs during wound healing. In the chronic infusion model of PD, betaig-h3 played a role in the functional deterioration of the peritoneal membrane, which is associated with fibrosis.

TGF-beta1 inhibits Cx43 expression and formation of functional syncytia in cultured smooth muscle cells from human detrusor.

PubMed

Neuhaus, Jochen; Heinrich, Marco; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe

2009-02-01

Human detrusor smooth muscle cells (hBSMCs) are coupled by connexin 43 (Cx43)-positive gap junctions to form functional syncytia. Gap junctional communication likely is necessary for synchronised detrusor contractions and is supposed to be altered in voiding disturbances. Other authors have shown that the pleiotropic cytokine TGF-beta1 upregulates Cx43 expression in human aortic smooth muscle cells. In this study, we examined the TGF-beta1 effects on Cx43 expression in cultured hBSMCs. hBSMC cultures, established from patients undergoing cystectomy, were treated with recombinant human TGF-beta1. Cx43 expression was then examined by Western blotting, real-time PCR, and immunocytochemistry. Dye-injection experiments were used to study the size of functional syncytia. Dye-coupling experiments revealed stable formation of functional syncytia in passaged cell cultures (P1-P4). Stimulation with TGF-beta1 led to significant reduction of Cx43 immunoreactivity and coupling. Cx43 protein expression was significantly downregulated and Cx43 mRNA was only 30% of the control level. Interestingly, low phosphorylation species of Cx43 were particularly affected. Our experiments demonstrated a significant down regulation of connexin 43 by TGF-beta1 in cultured hBSMCs. These findings support the view that TGF-beta1 is involved in the pathophysiology of urinary bladder dysfunction.

Dual blockade of aldosterone and angiotensin II additively suppresses TGF-beta and NADPH oxidase in the hypertensive kidney.

PubMed

Onozato, Maristela Lika; Tojo, Akihiro; Kobayashi, Naohiko; Goto, Atsuo; Matsuoka, Hiroaki; Fujita, Toshiro

2007-05-01

Angiotensin II blockade and spironolactone effectively reduces proteinuria in humans. To clarify the mechanisms of the beneficial effect of blockade of both aldosterone and angiotensin II, we associated the aldosterone antagonist eplerenone to an angiotensin-converting enzyme inhibitor (ACEI) and examined the effect on renal transforming growth factor (TGF)-beta expression and oxidative stress by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the Dahl salt-sensitive rat with heart failure (DSHF). Dahl salt-resistant control rats and DSHF rats were fed with 8% NaCl diet and at 11 weeks the DSHF rats were treated with vehicle, eplerenone (Epl), trandolapril or a combination of both drugs for 7 weeks. DSHF rats showed increased NADPH oxidase and decreased superoxide dismutase (SOD) resulting in increased oxidative stress. ACEI and Epl reduced NADPH oxidase showing an additive effect in their combination; ACEI increased manganese SOD (MnSOD) and Epl increased MnSOD, copper-zinc SOD and catalase, resulting in the lowest levels of oxidative stress with the combination therapy. Glomerulosclerosis and proteinuria were increased in the DSHF rats, and Epl suppressed them more effectively than ACEI to levels not different from the combination of both, showing a positive correlation with NADPH oxidase expression and TGF-beta. Renal TGF-beta was specifically suppressed with Epl The association of Epl to ACEI is beneficial due to further reduction of NADPH oxidase and specific inhibition of TGF-beta resulting in improvement of renal damage.

TGF-{beta} signals the formation of a unique NF1/Smad4-dependent transcription repressor-complex in human diploid fibroblasts

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

Luciakova, Katarina, E-mail: katarina.luciakova@savba.sk; Kollarovic, Gabriel; Kretova, Miroslava

2011-08-05

Highlights: {yields} TGF-{beta} induces the formation of unique nuclear NF1/Smad4 complexes that repress expression of the ANT-2 gene. {yields} Repression is mediated through an NF1-dependent repressor element in the promoter. {yields} The formation of NF1/Smad4 complexes and the repression of ANT2 are prevented by inhibitors of p38 kinase and TGF-{beta} RI. {yields} NF1/Smad complexes implicate novel role for NF1 and Smad proteins in the regulation of growth. -- Abstract: We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G.more » Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123-130]. In the present study, we show that TGF-{beta}, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-{beta} is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-{beta} are prevented by inhibitors of TGF-{beta} RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-{beta} and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-{beta} signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state.« less

[Association between Fok I vitamin D receptor (VDR) gene polymorphism and plasmatic concentrations of transforming growth factor-beta1 and interferon gamma in type 1 diabetes mellitus].

PubMed

López, Tatiana; García, Diego; Angel, Bárbara; Carrasco, Elena; Codner, Ethel; Ugarte, Francisca; Pérez-Bravo, Francisco

2008-02-02

In order to assess whether Fok I vitamin D receptor gene (VDR) polymorphism is involved in the genetic susceptibility of type 1 diabetes, a case-control study was conducted and VDR genotypes were related to serum concentrations of 25(OH) vitamin D and cytokines transforming growth factor beta1 (TGF-beta1) and interferon gamma (INF-gamma). 151 incident cases of type 1 diabetes and 182 non related healthy controls from Santiago were studied for VDR polymorphisms in peripheral blood DNA. Exon 2 (Fok I) segments were amplified by polimerase chain reaction and analyzed by means of restriction fragment length polymorphism to determine each corresponding genotype. Differences for allele, genotype and serological markers as 25(OH) vitamin D, TGF-beta1 and INF-gamma levels distribution between patients and controls were analyzed. Fok I polymorphism distribution analysis showed no differences between patients and controls. Among diabetics, higher levels of TGF-beta1 (median, 282.6 pg/ml; range, 131.8-3,031.4) were observed compared with healthy children (median, 232.2 pg/ml; range, 135.7-506.5) (p < 0.0038). Similar results were observed for INF-gamma concentrations (median, 121.1 pg/ml, and range, 5.3-228.8, in cases, and median, 89.6 pg/ml, and range, 10.9-117.2 in controls) (p < 0.0004). The diabetic carriers of the ff genotype showed low levels of 25(OH) vitamin D compared with the carriers of the F allele: mean (standard deviation), 23.1 (5.9) versus 27.9 (10.3) ng/ml (p < 0.03). A similar result was observed for TGF-beta1 concentrations in diabetic carriers of ff genotype and patients carriers of the F allele (298.5 versus 276.6; p < 0.05). Fok I polymorphism of VDR could have a marginal role in the immunologic disturbance in type 1 diabetes.

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.

Identification and expression of Smads associated with TGF-beta/activin/nodal signaling pathways in the rainbow trout (Oncorhynuchus mykiss)

USDA-ARS?s Scientific Manuscript database

The Smad proteins are essential components of the TGF-beta/activin/nodal family signaling pathway. We report the identification and characterization of transcripts representing 3 receptor Smads (Smad2a, Smad2b, Smad3), 2 common Smads (Smad4a, Smad4b) and one inhibitory Smad (Smad7). Phylogenetic an...

Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma.

PubMed

Laouar, Yasmina; Sutterwala, Fayyaz S; Gorelik, Leonid; Flavell, Richard A

2005-06-01

Interferon-gamma and interleukin 12 produced by the innate arm of the immune system are important regulators of T helper type 1 (T(H)1) cell development, but signals that negatively regulate their expression remain controversial. Here we show that transforming growth factor-beta (TGF-beta) controlled T(H)1 differentiation through the regulation of interferon-gamma produced by natural killer (NK) cells. Blockade of TGF-beta signaling in NK cells caused the accumulation of a large pool of NK cells secreting copious interferon-gamma, responsible for T(H)1 differentiation and protection from leishmania infection. In contrast, blockade of TGF-beta signaling in dendritic cells did not affect dendritic cell homeostasis or interleukin 12 production, thus indicating a previously undescribed demarcation of the function of TGF-beta in NK cells versus dendritic cells.

[Effects of Valeriana officinalis var. latifolia on expression of transforming growth factor beta 1 in hypercholesterolemic rats].

PubMed

Si, Xiao-yun; Jia, Ru-han; Huang, Cong-xin; Ding, Guo-hua; Liu, Hong-yan

2003-09-01

To evaluate the effect of Valeriana officinalis var latifolia(VOL) on expression of transforming growth factor beta 1 (TGF-beta 1) in hypercholesterolemic rats and study its possible mechanisms. Dietary-induced hypercholesterolemia was induced in male Wistar rats by given 4% cholesterol and 1% cholic acid diet for 16 weeks. Changes of serum lipid, urinary albumin, renal function and Mesangial matrix index were assessed. Moreover, immunohistochemical stain for TGF-beta 1 and type IV collagen were performed. VOL could reduce the serum levels of total cholesterol, low density lipoprotein, urinary albumin and serum creatinine. Light microscopy and immunohistochemical stain revealed that in the same time of lowing serum lipid, Mesangial matrix index was significantly reduced, accompanied by decreased expression of TGF-beta 1 and type IV collagen. VOL has the protective effect on lipid-induced nephropathy, and the inhibition of TGF-beta 1 expression might be the mechanism of VOL on renal protection.

Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta 1-producing fibroblasts.

PubMed

Lee, K H; Song, S U; Hwang, T S; Yi, Y; Oh, I S; Lee, J Y; Choi, K B; Choi, M S; Kim, S J

2001-09-20

Transforming growth factor beta (TGF-beta) has been considered as a candidate for gene therapy of orthopedic diseases. The possible application of cell-mediated TGF-beta gene therapy as a new treatment regimen for degenerative arthritis was investigated. In this study, fibroblasts expressing active TGF-beta 1 were injected into the knee joints of rabbits with artificially made cartilage defects to evaluate the feasibility of this therapy for orthopedic diseases. Two to 3 weeks after the injection there was evidence of cartilage regeneration, and at 4 to 6 weeks the cartilage defect was completely filled with newly grown hyaline cartilage. Histological analyses of the regenerated cartilage suggested that it was well integrated with the adjacent normal cartilage at the sides of the defect and that the newly formed tissue was indeed hyaline cartilage. Our findings suggest that cell-mediated TGF-beta 1 gene therapy may be a novel treatment for orthopedic diseases in which hyaline cartilage damage has occurred.

Bone sialoprotein mediates the tumor cell-targeted prometastatic activity of transforming growth factor beta in a mouse model of breast cancer.

PubMed

Nam, Jeong-Seok; Suchar, Adam M; Kang, Mi-Jin; Stuelten, Christina H; Tang, Binwu; Michalowska, Aleksandra M; Fisher, Larry W; Fedarko, Neal S; Jain, Alka; Pinkas, Jan; Lonning, Scott; Wakefield, Lalage M

2006-06-15

Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.

Generational Analysis Reveals that TGF-Beta1 Inhibits the Rate of Angiogenesis in Vivo by Selective Decrease in the Number of New Vessels

NASA Technical Reports Server (NTRS)

Parsons-Wingerter, Patricia; Elliott, Katherine E.; Farr, Andrew G.; Radhakrishnan, Krishnan; Clark, John I.; Sage, E. Helene

2000-01-01

Quantitative analysis of vascular generational branching demonstrated that transforming growth factor-beta1 (TGF-beta1), a multifunctional cytokine and angiogenic regulator, strongly inhibited angiogenesis in the arterial tree of the developing quail chorioallantoic membrane (CAM) by inhibition of the normal increase in the number of new, small vessels. The cytokine was applied uniformly in solution at embryonic day 7 (E7) to the CAMs of quail embryos cultured in petri dishes. After 24 h the rate of arterial growth was inhibited by as much as 105% as a function of increasing TGF-beta1 concentration. Inhibition of the rate of angiogenesis in the arterial tree by TGF-beta1 relative to controls was measured in digital images by three well-correlated, computerized methods. The first computerized method, direct measurement by the computer code VESGEN of vascular morphological parameters according to branching generations G(sub 1) through G(sub greater than or equal to 5), revealed that TGF-beta1 selectively inhibited the increase in the number density of small vessels, N(sub v greater than or equal to 5), (382 plus or minus 85 per square centimeter) for specimens treated with 1 microgram TGF-beta1/CAM for 24 h, compared to 583 plus or minus 99 per square centimeter for controls), but did not significantly affect other parameters such as average vessel length or vessel diameter. The second and third methods, the fractal dimension (D(sub f)) and grid intersection (rho (sub v)), are statistical descriptors of spatial pattern and density. According to D(sub f) and rho(sub v), arterial density increased in control specimens from 1.382 plus or minus 0.007 and 662 plus or minus 52 per square centimeters at E7 (0 h) to 1.439 plus or minus 0.013 and 884 plus or minus 55 per square centimeters at E8 (24 h), compared to 1.379 plus or minus 0.039 and 650 plus or minus 111 per square centimeter for specimens treated with 1 microgram TGF-beta1/CAM for 24 h. TGF-beta1 therefore

A role for human MUC4 mucin gene, the ErbB2 ligand, as a target of TGF-beta in pancreatic carcinogenesis.

PubMed

Jonckheere, Nicolas; Perrais, Michaël; Mariette, Christophe; Batra, Surinder K; Aubert, Jean-Pierre; Pigny, Pascal; Van Seuningen, Isabelle

2004-07-29

MUC4: encodes a large transmembrane mucin that is overexpressed in pancreatic adenocarcinomas. The molecular mechanisms responsible for that altered pattern of expression are unknown. TGF-beta, a pleiotropic cytokine, regulates numerous genes involved in pancreatic carcinogenesis via activation of the Smads proteins and MUC4 promoter is rich in Smad-binding elements. Our aim was to study whether the regulation of MUC4 expression by TGF-beta in pancreatic cancer cells was strictly dependent on Smad4 activity. Three pancreatic cancer cell lines, CAPAN-1 (MUC4+/Smad4-), CAPAN-2 (MUC4+/Smad4+) and PANC-1 (MUC4-/Smad4+), were used. By RT-PCR, transfection assays and immunohistochemistry, we show that (i) both MUC4 mRNA and apomucin expression are upregulated by TGF-beta, (ii) Smad2 positively cooperates with Smad4 to activate the promoter, (iii) activation of Smad4 by exogenous TGF-beta induces Smad4 binding to the promoter, (iv) Smad7 and c-ski both inhibit activation by Smad4. When Smad4 is mutated and inactive, TGF-beta activates MUC4 expression via MAPK, PI3K and PKA signaling pathways. Absence of expression in PANC-1 cells is due to histone deacetylation. Altogether, these results indicate that upregulation of MUC4 by TGF-beta is restricted to well-differentiated pancreatic cancer cells, and point out a novel mechanism for TGF-beta as a key molecule in targeting MUC4 overexpression in pancreatic adenocarcinomas.

Functional properties of an isolated. cap alpha beta. heterodimeric human placenta insulin-like growth factor 1 receptor complex

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

Feltz, S.M.; Swanson, M.L.; Wemmie, J.A.

1988-05-03

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta..more » heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.« less

Cloning and sequence analysis of the human brain beta-adrenergic receptor. Evolutionary relationship to rodent and avian beta-receptors and porcine muscarinic receptors.

PubMed

Chung, F Z; Lentes, K U; Gocayne, J; Fitzgerald, M; Robinson, D; Kerlavage, A R; Fraser, C M; Venter, J C

1987-01-26

Two cDNA clones, lambda-CLFV-108 and lambda-CLFV-119, encoding for the beta-adrenergic receptor, have been isolated from a human brain stem cDNA library. One human genomic clone, LCV-517 (20 kb), was characterized by restriction mapping and partial sequencing. The human brain beta-receptor consists of 413 amino acids with a calculated Mr of 46480. The gene contains three potential glucocorticoid receptor-binding sites. The beta-receptor expressed in human brain was homology with rodent (88%) and avian (52%) beta-receptors and with porcine muscarinic cholinergic receptors (31%), supporting our proposal [(1984) Proc. Natl. Acad. Sci. USA 81, 272 276] that adrenergic and muscarinic cholinergic receptors are structurally related. This represents the first cloning of a neurotransmitter receptor gene from human brain.

CDK2 phosphorylation of Smad2 disrupts TGF-beta transcriptional regulation in resistant primary bone marrow myeloma cells.

PubMed

Baughn, Linda B; Di Liberto, Maurizio; Niesvizky, Ruben; Cho, Hearn J; Jayabalan, David; Lane, Joseph; Liu, Fang; Chen-Kiang, Selina

2009-02-15

Resistance to growth suppression by TGF-beta1 is common in cancer; however, mutations in this pathway are rare in hematopoietic malignancies. In multiple myeloma, a fatal cancer of plasma cells, malignant cells accumulate in the TGF-beta-rich bone marrow due to loss of both cell cycle and apoptotic controls. Herein we show that TGF-beta activates Smad2 but fails to induce cell cycle arrest or apoptosis in primary bone marrow myeloma and human myeloma cell lines due to its inability to activate G(1) cyclin-dependent kinase (CDK) inhibitors (p15(INK4b), p21(CIP1/WAF1), p27(KIP1), p57(KIP2)) or to repress c-myc and Bcl-2 transcription. Correlating with aberrant activation of CDKs, CDK-dependent phosphorylation of Smad2 on Thr(8) (pT8), a modification linked to impaired Smad activity, is elevated in primary bone marrow myeloma cells, even in asymptomatic monoclonal gammopathy of undetermined significance. Moreover, CDK2 is the predominant CDK that phosphorylates Smad2 on T8 in myeloma cells, leading to inhibition of Smad2-Smad4 association that precludes transcriptional regulation by Smad2. Our findings provide the first direct evidence that pT8 Smad2 couples dysregulation of CDK2 to TGF-beta resistance in primary cancer cells, and they suggest that disruption of Smad2 function by CDK2 phosphorylation acts as a mechanism for TGF-beta resistance in multiple myeloma.

Mucosal tolerance to experimental autoimmune myasthenia gravis is associated with down-regulation of AChR-specific IFN-gamma-expressing Th1-like cells and up-regulation of TGF-beta mRNA in mononuclear cells.

PubMed

Ma, C G; Zhang, G X; Xiao, B G; Wang, Z Y; Link, J; Olsson, T; Link, H

1996-02-13

Oral and nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freund's adjuvant (CFA) resulted in prevention or marked decrease of the severity of experimental autoimmune myasthenia gravis (EAMG) and suppression of AChR-specific B-cell responses and of AChR-reactive T-cell function. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radiolabeled cDNA oligonucleotide proves was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine interferon-gamma (IFN-gamma), the B cell-stimulating interleukin-4 (IL-4), and the immunosuppressive transforming growth factor-beta (TGF-beta). Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-gamma, IL-4, and TGF-beta mRNA-expressing cells, compared to control rats receiving PBS orally or nasally and injected with CFA only. Oral and nasal tolerance was accompanied by decreased numbers of AChR-reactive IFN-gamma and IL-4 mRNA-expressing cells and strong up-regulation of TGF-beta mRNA-positive cells in lymphoid organs when compared to nontolerized EAMG control rats. The results suggest that IFN-gamma and IL-4 are central effector molecules in the development of EAMG and that TGF-beta plays an important role in tolerance induction to EAMG.

Interaction with beta-arrestin determines the difference in internalization behavor between beta1- and beta2-adrenergic receptors.

PubMed

Shiina, T; Kawasaki, A; Nagao, T; Kurose, H

2000-09-15

The beta(1)-adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. As beta-arrestin is important for internalization, we examine the interaction of beta-arrestin with beta(1)AR with three different methods: intracellular trafficking of beta-arrestin, binding of in vitro translated beta-arrestin to intracellular domains of beta(1)- and beta(2)ARs, and inhibition of betaAR-stimulated adenylyl cyclase activities by beta-arrestin. The green fluorescent protein-tagged beta-arrestin 2 translocates to and stays at the plasma membrane by beta(2)AR stimulation. Although green fluorescent protein-tagged beta-arrestin 2 also translocates to the plasma membrane, it returns to the cytoplasm 10-30 min after beta(1)AR stimulation. The binding of in vitro translated beta-arrestin 1 and beta-arrestin 2 to the third intracellular loop and the carboxyl tail of beta(1)AR is lower than that of beta(2)AR. The fusion protein of beta-arrestin 1 with glutathione S-transferase inhibits the beta(1)- and beta(2)AR-stimulated adenylyl cyclase activities, although inhibition of the beta(1)AR-stimulated activity requires a higher concentration of the fusion protein than that of the beta(2)AR-stimulated activity. These results suggest that weak interaction of beta(1)AR with beta-arrestins explains the resistance to agonist-induced internalization. This is further supported by the finding that beta-arrestin can induce internalization of beta(1)AR when beta-arrestin 1 does not dissociate from beta(1)AR by fusing to the carboxyl tail of beta(1)AR.

In vivo IL-10 and TGF-beta production by PBMC from long-term kidney transplant recipients with excellent graft function: a possible feedback mechanism participating in immunological stability.

PubMed

Alberú, Josefina; Richaud-Patin, Yvonne; Vázquez-Lavista, Luis Gabriel; de Leo, Claudia; Guzmán-Rodríguez, Hugo; Mancilla, Eduardo; Correa-Rotter, Ricardo; Chew-Wong, Alfredo; Llorente, Luis

2004-04-01

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) are Th2-derived multifunctional cytokines that exhibit potent immunoregulatory and anti-inflammatory properties which might prolong graft survival. The aim of this study was to explore whether spontaneous production of IL-10 and TGF-beta by blood mononuclear cells correlates with excellent long-term graft function. A cross-sectional study was carried out in 32 kidney transplant recipients, without albuminuria, treated with azathioprine and prednisone. Spontaneous IL-10 and TGF-beta were measured by enzyme-linked immunosorbent assay in supernatants from 24 h cultured unstimulated peripheral blood mononuclear cells. Both cytokines were also determined in 10 healthy kidney donors. There was no correlation between IL-10 or TGF-beta with any variable tested, namely age, SCr, histocompatibility, and post-transplant follow-up. In vivo IL-10 production displayed a statistical trend to be higher in transplant recipients than in controls (362.3 +/- 465, range 12.5-1929.3 pg/ml, and 189 +/- 170, range 4.17-485.7 pg/ml, respectively; p = 0.08), whereas no difference was observed in TGF-beta among the same groups (134.7 +/- 79.2, range 68-421 pg/ml, and 121.4 +/- 25.8, range 75-151 pg/ml, respectively). Interestingly, a statistically significant inverse correlation was observed between IL-10 and TGF-beta in kidney transplant recipients (p = 0.03). The higher IL-10 production observed in long-term kidney transplant recipients supports the notion that this cytokine contributes in decreasing allogenic immune responses and allows prolongation of allograft survival. The balance between TGF-beta and IL-10 may be of paramount importance in graft acceptance.

Bioactive molecules in milk and their role in health and disease: the role of transforming growth factor-beta.

PubMed

Donnet-Hughes, A; Duc, N; Serrant, P; Vidal, K; Schiffrin, E J

2000-02-01

Human breast milk is rich in nutrients, hormones, growth factors and immunoactive molecules, which influence the growth, development and immune status of the newborn infant. Although several of these factors are also present in bovine milk, the greater susceptibility of the formula-fed infant to infection and disease and the development of allergy is often attributed to the reduced level of protective factors in milk formulas. Nevertheless, modifying manufacturing processes may preserve the biological activity of some bioactive molecules in end products. Transforming growth factor (TGF)-beta is one such molecule. TGF-beta is a polypeptide, which has been described in both human and bovine milk. It is implicated in many processes, including epithelial cell growth and differentiation, development, carcinogenesis and immune regulation. The present article discusses the biological activity of TGF-beta2 that has been preserved and activated in a cow's milk-based product. More specifically, it addresses possible mechanisms of action in the intestinal lumen and speculates on how milk products containing naturally occurring TGF-beta2 could be exploited in functional foods for the infant or as therapies for specific intestinal diseases.

TGF-beta: a new role for an old AktTOR.

PubMed

Goraksha-Hicks, Pankuri; Rathmell, Jeffrey C

2009-07-01

Nutrient overabundance is known to promote cellular hypertrophy, a significant pathological event in diseases like diabetes and cancer, although mechanisms have remained unclear. In this issue of Developmental Cell, Wu and Derynck provide a new model that links metabolism and cell growth by demonstrating that hyperglycemia can increase TGF-beta-dependent activation of the mTOR pathway to promote cellular hyperplasia.

Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta 1.

PubMed Central

Liu, C; Adamson, E; Mercola, D

1996-01-01

The early growth response 1 (EGR-1) gene product is a transcription factor with role in differentiation and growth. We have previously shown that expression of exogenous EGR-1 in various human tumor cells unexpectedly and markedly reduces growth and tumorigenicity and, conversely, that suppression of endogenous Egr-1 expression by antisense RNA eliminates protein expression, enhances growth, and promotes phenotypic transformation. However, the mechanism of these effects remained unknown. The promoter of human transforming growth factor beta 1 (TGF-beta 1) contains two GC-rich EGR-1 binding sites. We show that expression of EGR-1 in human HT-1080 fibrosarcoma cells uses increased secretion of biologically active TGF-beta 1 in direct proportion (rPearson = 0.96) to the amount of EGR-1 expressed and addition of recombinant human TGF-beta 1 is strongly growth-suppressive for these cells. Addition of monoclonal anti-TGF-beta 1 antibodies to EGR-1-expressing HT-1080 cells completely reverses the growth inhibitory effects of EGR-1. Reporter constructs bearing the EGR-1 binding segment of the TGF-beta 1 promoter was activated 4- to 6-fold relative to a control reporter in either HT-1080 cells that stably expressed or parental cells cotransfected with an EGR-1 expression vector. Expression of delta EGR-1, a mutant that cannot interact with the corepressors, nerve growth factor-activated factor binding proteins NAB1 and NAB2, due to deletion of the repressor domain, exhibited enhanced transactivation of 2- to 3.5-fold over that of wild-type EGR-1 showing that the reporter construct reflected the appropriate in vivo regulatory context. The EGR-1-stimulated transactivation was inhibited by expression of the Wilms tumor suppressor, a known specific DNA-binding competitor. These results indicate that EGR-1 suppresses growth of human HT-1080 fibrosarcoma cells by induction of TGF-beta 1. Images Fig. 1 Fig. 5 PMID:8876223

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.

Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

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

Bahouth, S.W.; Malbon, C.C.

1987-05-01

Xenopus laevis oocytes are a useful transcription and expression system for DNA and RNA, respectively. Total cellular RNA was extracted from mouse lymphoma S49 cells and poly(A)/sup +/mRNA prepared by affinity chromatography of RNA on oligo(dT) cellulose. The membranes of S49 cells contain beta-adrenergic receptors that display pharmacological characteristics of beta/sub 2/-subtype. Xenopus laevis oocytes were injected with 50 ng of mRNA/oocyte. Expression of beta-adrenergic receptors in oocytes incubated for 30 hr after microinjection was assessed in membranes by radioligand binding using (/sup 3/H) dihydroalprenolol. The injected oocytes displayed 0.34 fmol receptor/oocyte as compared to 0.02 fmol receptor/oocyte in themore » control oocytes. The affinity of beta-adrenergic receptors in injected oocytes for this radioligand was 2 nM, a value similar to the affinity of beta-adrenergic receptors for DHA in S49 cell membranes. The potency of beta-adrenergic agonists in competing for DHA binding to oocytes membranes was isoproterenol > epinephrine > norepineprine, indicating that the expressed beta-adrenergic receptors were of the beta/sub 2/-subtype. The K/sub I/ of these agonists for the beta-adrenergic receptor in oocyte membranes was 0.03, 0.15 and 1.2 ..mu..M, respectively. The role of post-translational modification in dictating receptor subtype is analyzed using mRNA of beta/sub 1/- as well as beta/sub 2/-adrenergic receptors.« less

Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma.

PubMed

Matsuzaki, Koichi; Murata, Miki; Yoshida, Katsunori; Sekimoto, Go; Uemura, Yoshiko; Sakaida, Noriko; Kaibori, Masaki; Kamiyama, Yasuo; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi; Seki, Toshihito

2007-07-01

Many patients with chronic hepatitis caused by hepatitis C virus (HCV) infection develop liver fibrosis with high risk for hepatocellular carcinoma (HCC), but the mechanism underling this process is unclear. Conversely, transforming growth factor beta (TGF-beta) activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), which convert the mediator Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Whereas the TbetaRI/pSmad3C pathway suppresses epithelial cell growth by upregulating p21(WAF1) transcription, JNK/pSmad3L-mediated signaling promotes extracellular matrix deposition, partly, by upregulating plasminogen activator inhibitor 1 (PAI-1). We studied the domain-specific Smad3 phosphorylation in biopsy specimens representing chronic hepatitis, cirrhosis, or HCC from 100 patients chronically infected with HCV, and correlated Smad3 phosphorylation with clinical course. As HCV-infected livers progressed from chronic hepatitis through cirrhosis to HCC, hepatocytic pSmad3L/PAI-1 increased with fibrotic stage and necroinflammatory grade, and pSmad3C/p21(WAF1) decreased. Of 14 patients with chronic hepatitis C with strong hepatocytic pSmad3L positivity, 8 developed HCC within 12 years; only 1 of 12 showing little pSmad3L positivity developed HCC. We further sought molecular mechanisms in vitro. JNK activation by the pro-inflammatory cytokine interleukin-1beta stimulated the pSmad3L/PAI-1 pathway in facilitating hepatocytic invasion, in the meantime reducing TGF-beta-dependent tumor-suppressive activity by the pSmad3C/p21(WAF1) pathway. These results indicate that chronic inflammation associated with HCV infection shifts hepatocytic TGF-beta signaling from tumor-suppression to fibrogenesis, accelerating liver fibrosis and increasing risk for HCC.

Requirement for the SnoN oncoprotein in transforming growth factor beta-induced oncogenic transformation of fibroblast cells.

PubMed

Zhu, Qingwei; Pearson-White, Sonia; Luo, Kunxin

2005-12-01

Transforming growth factor beta (TGF-beta) was originally identified by virtue of its ability to induce transformation of the AKR-2B and NRK fibroblasts but was later found to be a potent inhibitor of the growth of epithelial, endothelial, and lymphoid cells. Although the growth-inhibitory pathway of TGF-beta mediated by the Smad proteins is well studied, the signaling pathway leading to the transforming activity of TGF-beta in fibroblasts is not well understood. Here we show that SnoN, a member of the Ski family of oncoproteins, is required for TGF-beta-induced proliferation and transformation of AKR-2B and NRK fibroblasts. TGF-beta induces upregulation of snoN expression in both epithelial cells and fibroblasts through a common Smad-dependent mechanism. However, a strong and prolonged activation of snoN transcription that lasts for 8 to 24 h is detected only in these two fibroblast lines. This prolonged induction is mediated by Smad2 and appears to play an important role in the transformation of both AKR-2B and NRK cells. Reduction of snoN expression by small interfering RNA or shortening of the duration of snoN induction by a pharmacological inhibitor impaired TGF-beta-induced anchorage-independent growth of AKR-2B cells. Interestingly, Smad2 and Smad3 play opposite roles in regulating snoN expression in both fibroblasts and epithelial cells. The Smad2/Smad4 complex activates snoN transcription by direct binding to the TGF-beta-responsive element in the snoN promoter, while the Smad3/Smad4 complex inhibits it through a novel Smad inhibitory site. Mutations of Smad4 that render it defective in heterodimerization with Smad3, which are found in many human cancers, convert the activity of Smad3 on the snoN promoter from inhibitory to stimulatory, resulting in increased snoN expression in cancer cells. Thus, we demonstrate a novel role of SnoN in the transforming activity of TGF-beta in fibroblasts and also uncovered a mechanism for the elevated SnoN expression in

TGF-beta induces connexin43 gene expression in normal murine mammary gland epithelial cells via activation of p38 and PI3K/AKT signaling pathways.

PubMed

Tacheau, Charlotte; Fontaine, Juliette; Loy, Jennifer; Mauviel, Alain; Verrecchia, Franck

2008-12-01

One of the shared physiological roles between TGF-beta and connexin family members is to inhibit epithelial cell cycle progression and consequently, to provide protection against malignant transformation. Herein, we demonstrated that TGF-beta1 induces the expression of connexin43 (Cx43) in normal murine mammary gland (NMuMG) cell lines at the protein and mRNA levels, and transcriptionally. Using overexpression of a truncated dominant-negative form of Cx43, we determined that the modulation of gap junctional communication by TGF-beta1 plays a key role in the control of NMuMG cells proliferation by TGF-beta1. In addition, using overexpression of truncated dominant-negative forms of either Smad2 or Smad3, and MDA-MB-468 human breast carcinoma cells deficient for Smad4, we determined that the Smad cascade is not implicated in TGF-beta1 effect on Cx43 expression. Using specific pharmacologic inhibitors for JNK, ERK, p38, and PI3K/AKT signaling pathways, we demonstrated the cooperative role of p38 and PI3K/AKT signaling in TGF-beta1-induced Cx43 expression and gap junctional communication. Furthermore, transfection of a c-jun antisense expression vector significantly prevented TGF-beta1-induced Cx43 gene expression demonstrating the involvement of c-Jun/AP-1 pathway together with p38 and PI3K/AKT pathways in mediating TGF-beta1-induced Cx43 gene expression.

Role of estrogen receptors alpha, beta and GPER1/GPR30 in pancreatic beta-cells.

PubMed

Nadal, Angel; Alonso-Magdalena, Paloma; Soriano, Sergi; Ripoll, Cristina; Fuentes, Esther; Quesada, Ivan; Ropero, Ana Belen

2011-01-01

Estrogen receptors (ER) are emerging as important molecules involved in the adaptation of beta-cells to insulin resistance. The onset of type 2 diabetes is marked by insulin secretory dysfunction and decreased beta-cell mass. During pregnancy, puberty and obesity there is increased metabolic demand and insulin resistance is developed. This metabolic state increases the demand on beta-cells to augment insulin biosynthesis and release. In this respect, ERalpha is directly implicated in the E2-regulation of insulin content and secretion, while ERbeta is in the E2-potentiation of glucose-induced insulin release. Both receptors develop their actions within the physiological range of E2. In addition, the G protein-coupled estrogen receptor (GPER1/GPR30) seems to be implicated in the E2-regulation of stimulus-secretion coupling in the three cell types of the islet. The increased demand of insulin production for long time may lead to beta-cell stress and apoptosis. ERalpha, ERbeta and GPER1/GPR30 are involved in preventing beta-cell apoptosis, impeding the loss of critical beta-cell mass. Therefore, estrogen receptors may play an essential role in the adaptation of the pancreas to insulin resistant periods.

Targeting Transforming Growth Factor Beta to Enhance the Fracture Resistance of Bone

DTIC Science & Technology

2013-01-01

Transforming Growth Factor Beta to Enhance the Fracture Resistance of Bone is to determine whether the suppression of TGF-β activity improves the fracture...effect primarily occurred in the old rats. Effect of TGF-β suppression on fracture resistance in female mice Since the suppression of TGF-β activity by...treated mice. This suggests that 1D11 treatment depleted the osteoprogenitor pool to some extent as inhibition of TGF-β activity in vivo may favor

Association between Plasma Levels of Transforming Growth Factor-beta1, IL-23 and IL-17 and the Severity of Autism in Egyptian Children

ERIC Educational Resources Information Center

Hashim, Haitham; Abdelrahman, Hadeel; Mohammed, Doaa; Karam, Rehab

2013-01-01

It has been recently shown that dysregulation of transforming growth factor-beta1 (TGF-beta1), IL-23 and IL-17 has been identified as a major factor involved in autoimmune disorders. Based on the increasing evidence of immune dysfunction in autism the aim of this study was to measure serum levels of TGF-beta1, IL-23 and IL-17 in relation to the…

Low breast milk TGF-beta2 is induced by Lactobacillus reuteri supplementation and associates with reduced risk of sensitization during infancy.

PubMed

Böttcher, Malin Fagerås; Abrahamsson, Thomas Robert; Fredriksson, Mats; Jakobsson, Ted; Björkstén, Bengt

2008-09-01

The immunological composition of breast milk differs between mothers. The reasons for these differences and the consequences for the breast-fed infants are poorly understood. The aim of this study was to evaluate the effect of probiotic Lactobacillus reuteri supplementation on the immunological composition of breast milk in relation to sensitization and eczema in the babies. Total IgA, secretory IgA (SIgA), TGF-beta1, TGF-beta2, IL-10, TNF, soluble CD14 (sCD14), and Na/K ratios were analyzed in colostrum and mature milk obtained from women treated with L. reuteri (n = 54) or placebo (n = 55) from gestational week 36 until delivery. Bacteriological analyses of L. reuteri were performed in faecal samples of the mothers. The infants were followed prospectively for 2 yr regarding development of eczema and sensitization as defined by a positive skin prick test and/or circulating allergen-specific IgE antibodies at 6, 12, and 24 months of age. Supplementation of L. reuteri during pregnancy was associated with low levels of TGF-beta2 and slightly increased levels of IL-10 in colostrum. For TGF-beta2, this association was most pronounced in mothers with detectable L. reuteri in faeces. Infants receiving breast milk with low levels of TGF-beta2 were less likely to become sensitized during their first 2 yr of life. A similar trend was observed for development of IgE-associated eczema. The levels of total IgA, SIgA, TGF-beta1, TNF, sCD14, and Na/K ratios in breast milk were not affected by the intake of L. reuteri. None of these parameters correlated with sensitization or development of eczema in the infant, except for high Na/K ratios that associated with increased risk of sensitization. Supplementation with L. reuteri during late pregnancy reduces breast milk levels of TGF-beta2, and low levels of this cytokine are associated with less sensitization and possibly less IgE-associated eczema in breast-fed infants.

Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor.

PubMed

Xu, W; Angelis, K; Danielpour, D; Haddad, M M; Bischof, O; Campisi, J; Stavnezer, E; Medrano, E E

2000-05-23

The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type beta transforming growth factor (TGF-beta) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-beta. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-beta-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-beta-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-beta-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-beta-mediated growth inhibition in a prostate-derived epithelial cell line.

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

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.

Involvement of H- and N-Ras isoforms in transforming growth factor-{beta}1-induced proliferation and in collagen and fibronectin synthesis

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

Martinez-Salgado, Carlos; Fuentes-Calvo, Isabel; Instituto 'Reina Sofia' de Investigacion Nefrologica, Universidad de Salamanca, 37007 Salamanca

2006-07-01

Transforming growth factor {beta}1 (TGF-{beta}1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-{beta} and Ras signaling pathways are closely related: TGF-{beta}1 overcomes Ras mitogenic effects and Ras counteracts TGF-{beta} signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-{beta}1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras {sup -/-}/N-ras {sup -/-}) isoforms andmore » from heterozygote mice (H-ras {sup +/-}/N-ras {sup +/-}). ECM synthesis is increased in basal conditions in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts, this increase being higher after stimulation with TGF-{beta}1. TGF-{beta}1-induced fibroblast proliferation is smaller in H-ras {sup -/-}/N-ras {sup -/-} than in H-ras {sup +/-}/N-ras {sup +/-} fibroblasts. Erk activation is decreased in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.« less

Can transforming growth factor-beta1 and retinoids modify the activity of estradiol and antiestrogens in MCF-7 breast cancer cells?.

PubMed

Czeczuga-Semeniuk, Ewa; Anchim, Tomasz; Dziecioł, Janusz; Dabrowska, Milena; Wołczyński, Sławomir

2004-01-01

Retinoic acid and transforming growth factor-beta (TGF-beta) affect differentiation, proliferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [3H]thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-beta1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-beta1 concentrations, a marked reduction in the stimulatory action of estradiol (10(-9) and 10(-8) M) was observed whereas in combination with tamoxifen (10(-7) and 10(-6) M) only 30 ng/ml TGF-beta1 caused a statistically significant reduction to approximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-beta1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 +/- 19% (control =100%) by 3 ng/ml TGF-beta1, and this dose was used throughout. It was found that addition of TGF-beta1 and isotretinoin to the culture did not decrease proliferation, while TGF-beta1 and tretinoin at low concentrations (3 x 10(-8) and 3 x 10(-7) M) reduced the percentage of proliferating cells by approximately 30% (67+/-8% and 67+/-5%, P<0.05 compared to values in the tretinoin group). Both retinoids also led to a statistically significant decrease in the stimulatory effect of 10(-9) M estradiol, attenuated by TGF-beta1. In addition, the retinoids in combination with TGF-beta1 and tamoxifen (10(-6) M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the

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.

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.

PDGF-beta receptor expression and ventilatory acclimatization to hypoxia in the rat.

PubMed

Alea, O A; Czapla, M A; Lasky, J A; Simakajornboon, N; Gozal, E; Gozal, D

2000-11-01

Activation of platelet-derived growth factor-beta (PDGF-beta) receptors in the nucleus of the solitary tract (nTS) modulates the late phase of the acute hypoxic ventilatory response (HVR) in the rat. We hypothesized that temporal changes in PDGF-beta receptor expression could underlie the ventilatory acclimatization to hypoxia (VAH). Normoxic ventilation was examined in adult Sprague-Dawley rats chronically exposed to 10% O(2), and at 0, 1, 2, 7, and 14 days, Northern and Western blots of the dorsocaudal brain stem were performed for assessment of PDGF-beta receptor expression. Although no significant changes in PDGF-beta receptor mRNA occurred over time, marked attenuation of PDGF-beta receptor protein became apparent after day 7 of hypoxic exposure. Such changes were significantly correlated with concomitant increases in normoxic ventilation, i.e., with VAH (r: -0.56, P < 0.005). In addition, long-term administration of PDGF-BB in the nTS via osmotic pumps loaded with either PDGF-BB (n = 8) or vehicle (Veh; n = 8) showed that although no significant changes in the magnitude of acute HVR occurred in Veh over time, the typical attenuation of HVR by PDGF-BB decreased over time. Furthermore, PDGF-BB microinjections did not attenuate HVR in acclimatized rats at 7 and 14 days of hypoxia (n = 10). We conclude that decreased expression of PDGF-beta receptors in the dorsocaudal brain stem correlates with the magnitude of VAH. We speculate that the decreased expression of PDGF-beta receptors is mediated via internalization and degradation of the receptor rather than by transcriptional regulation.

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.

Analysis of common transforming growth factor beta-1 gene polymorphisms in gastric and duodenal ulcer disease: pilot study.

PubMed

Polonikov, Alexey V; Ivanov, Vladimir P; Belugin, Dmitry A; Khoroshaya, Irina V; Kolchanova, Inessa O; Solodilova, Mariya A; Tutochkina, Margarita P; Stepchenko, Alexander A

2007-04-01

Transforming growth factor-beta1 (TGF-beta1) has been shown to be an important cytokine that plays a role in cell proliferation, differentiation, tissue injury repair and ulcer healing. The purpose of this pilot study was to investigate if common polymorphisms Leu10Pro, Arg25Pro and C-509T within the TGF-beta1 gene are associated with susceptibility to gastric and duodenal ulcer disease in Russians. Blood samples from 377 unrelated patients with gastric and duodenal ulcer disease and 226 sex- and age-matched healthy controls were used to determine TGF-beta1 gene polymorphisms by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Leu10Pro substitution in the signal peptide of TGF-beta1 has been found to be associated with susceptibility to gastric ulcer (odds ratio [OR] 1.76, 95% confidence interval [CI] 1.12-2.77). A genotype combination of 10Leu/Leu x 25Arg/Arg x -509C/C was also associated with susceptibility to gastric ulcer disease (OR 1.81, P = 0.01). In addition, the frequency of a combination of genotypes 10Pro/Pro x 25Arg/Pro x -509C/T was statistically lower in patients with duodenal ulcer than in controls (OR 0.42, P = 0.05). A significant difference (P = 0.04) in the distribution of rare haplotypes of the TGF-beta1 gene between patients with duodenal ulcer and healthy controls has been found. Polymorphism Leu10Pro was in positive linkage disequilibrium with C-509T polymorphism (coefficient D = 0.191; P < 0.0001). These findings indicate that the Leu10Pro and C-509T polymorphisms may be involved in the modulation of expression of the TGF-beta1 gene, and therefore a predisposition to peptic ulcer disease could be linked to particular alleles of this gene. In particular, a possible role of TGF-beta1 in the pathogenesis of gastric ulcer disease is discussed.

Differential role of Sloan–Kettering Institute (Ski) protein in Nodal and transforming growth factor-beta (TGF-β)-induced Smad signaling in prostate cancer cells

PubMed Central

Khan, Shafiq A.

2012-01-01

Transforming growth factor-beta (TGF-β) signaling pathways contain both tumor suppressor and tumor promoting activities. We have demonstrated that Nodal, another member of the TGF-β superfamily, and its receptors are expressed in prostate cancer cells. Nodal and TGF-β exerted similar biological effects on prostate cells; both inhibited proliferation in WPE, RWPE1 and DU145 cells, whereas neither had any effect on the proliferation of LNCaP or PC3 cells. Interestingly, Nodal and TGF-β induced migration in PC3 cells, but not in DU145 cells. TGF-β induced predominantly phosphorylation of Smad3, whereas Nodal induced phosphorylation of only Smad2. We also determined the expression and differential role of Ski, a corepressor of Smad2/3, in Nodal and TGF-β signaling in prostate cancer cells. Similar levels of Ski mRNA were found in several established prostate cell lines; however, high levels of Ski protein were only detected in prostate cancer cells and prostate cancer tissue samples. Exogenous Nodal and TGF-β had no effects on Ski mRNA levels. On the other hand, TGF-β induced a rapid degradation of Ski protein mediated by the proteasomal pathway, whereas Nodal had no effect on Ski protein. Reduced Ski levels correlated with increased basal and TGF-β-induced Smad2/3 phosphorylation. Knockdown of endogenous Ski reduced proliferation in DU145 cells and enhanced migration of PC3 cells. We conclude that high levels of Ski expression in prostate cancer cells may be responsible for repression of TGF-β and Smad3 signaling, but Ski protein levels do not influence Nodal and Smad2 signaling. PMID:22843506

ALK and TGF-Beta Resistance in Breast Cancer

DTIC Science & Technology

2017-10-01

and H.F. Lodish, Role of transforming growth factor beta in human disease. N Engl J Med, 2000. 342(18): p. 1350-8. 3. Massague, J., S.W. Blain, and... Transforming growth factor-beta signaling in normal and malignant hematopoiesis. Leukemia, 2003. 17(9): p. 1731-7. 5. Lehman, H.L., et al., Modeling and

Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus.

PubMed

Pierreux, C E; Nicolás, F J; Hill, C S

2000-12-01

Smad4 plays a pivotal role in all transforming growth factor beta (TGF-beta) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-beta signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-beta signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-beta signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm.

TGF-beta(1) gene-race interactions for resting and exercise blood pressure in the HERITAGE Family Study.

PubMed

Rivera, M A; Echegaray, M; Rankinen, T; Pérusse, L; Rice, T; Gagnon, J; Leon, A S; Skinner, J S; Wilmore, J H; Rao, D C; Bouchard, C

2001-10-01

We examined the possible association between a transforming growth factor (TGF)-beta(1) gene polymorphism in codon 10 and blood pressure (BP) at rest, in acute response to exercise in the pretrained (sedentary) and trained states, as well as in its training response (Delta) to 20 wk of endurance exercise. Subjects were 257 black and 480 white, healthy sedentary normotensive subjects from the HERITAGE Family Study. The polymorphism was detected by polymerase chain reaction and digestion with the Msp A1 I endonuclease yielding a wild (leucine-10) and a mutant (proline-10) allele. Resting and exercise [50 W plus 60, 80, and 100% maximal oxygen consumption (VO(2)(max))] BP were determined before and after training. Significant (P < 0.05) race-genotype interactions were found for systolic (S) BP in both the sedentary and trained states. Among whites but not in blacks, the TGF-beta(1) genotypes were significantly (P < 0.05) associated with sedentary-state SBP at rest, at 50 W, and at 60 and 100% VO(2)(max)as well as with trained-state SBP at rest and at 80 and 100% VO(2)(max). The leucine-10 homozygotes had significantly (P < 0.05) lower SBP than proline-10 homozygotes. DeltaBP was not significantly associated with genotype. These results support the hypothesis of an association between the TGF-beta(1) marker in codon 10 and SBP at rest and in response to acute exercise in whites but not in blacks.

Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

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

Calabrese, G.; Sallese, M.; Stornaiuolo, A.

1994-09-01

Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment ofmore » the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.« less

Molecular role of TGF-beta, secreted from a new type of CD4+ suppressor T cell, NY4.2, in the prevention of autoimmune IDDM in NOD mice.

PubMed

Han, H S; Jun, H S; Utsugi, T; Yoon, J W

1997-06-01

A new type of CD4+ T cell clone (NY4.2) isolated from pancreatic islet-infiltrated lymphocytes of acutely diabetic non-obese diabetic (NOD) mice prevents the development of insulin-dependent diabetes mellitus (IDDM) in NOD mice, as well as the recurrence of autoimmune diabetes in syngeneic islet-transplanted NOD mice. It has been demonstrated that the cytokine TGF-beta, secreted from the cells of this clone, is the substance which prevents autoimmune IDDM. This investigation was initiated to determine the molecular role TGF-beta plays in the prevention of autoimmune IDDM by determining its effect on IL-2-induced signal transduction in Con A-activated NOD mouse splenocytes and HT-2 cells. First, we determined whether TGF-beta, secreted from NY4.2 T cells, inhibits IL-2-dependent T cell proliferation in HT-2 cells (IL-2-dependent T cell line) and NOD splenocytes. We found that TGF-beta suppresses IL-2-dependent T cell proliferation. Second, we determined whether TGF-beta inhibits the activation of Janus kinases (JAKs), as well as signal transducers and activators of transcription (STAT) proteins, involved in an IL-2-induced signalling pathway that normally leads to the proliferation of T cells. We found that TGF-beta inhibited tyrosine phosphorylation of JAK1, JAK3, STAT3 and STAT5 in Con A blasts from NOD splenocytes and HT-2 cells. Third, we examined whether TGF-beta inhibits the cooperation between STAT proteins and mitogen-activated protein kinase (MAPK), especially extracellular signal-regulated kinase 2 (ERK2). We found that TGF-beta inhibited the association of STAT3 and STAT5 with ERK2 in Con A blasts from NOD splenocytes and HT-2 cells. On the basis of these observations, we conclude that TGF-beta may interfere with signal transduction via inhibition of the IL-2-induced JAK/STAT pathway and inhibition of the association of STAT proteins with ERK2 in T cells from NOD splenocytes, resulting in the inhibition of IL-2-dependent T cell proliferation. TGF-beta

Productive interaction between transmembrane mutants of the bovine papillomavirus E5 protein and the platelet-derived growth factor beta receptor.

PubMed

Lai, Char-Chang; Edwards, Anne P B; DiMaio, Daniel

2005-02-01

The bovine papillomavirus E5 protein is a 44-amino-acid transmembrane protein that transforms cells by binding to the transmembrane region of the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in sustained receptor signaling. However, there are published reports that certain mutants with amino acid substitutions in the membrane-spanning segment of the E5 protein transform cells without activating the PDGF beta receptor. We re-examined several of these transmembrane mutants, and here we present five lines of evidence that these mutants do in fact activate the PDGF beta receptor, resulting in cellular signaling and transformation.

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

PubMed Central

Jhappan, C; Geiser, A G; Kordon, E C; Bagheri, D; Hennighausen, L; Roberts, A B; Smith, G H; Merlino, G

1993-01-01

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland. Images PMID:8491177

Microvascular pericytes express platelet-derived growth factor-beta receptors in human healing wounds and colorectal adenocarcinoma.

PubMed Central

Sundberg, C.; Ljungström, M.; Lindmark, G.; Gerdin, B.; Rubin, K.

1993-01-01

The expression of platelet-derived growth factor- beta (PDGF-beta) receptors in the microvasculature of human healing wounds and colorectal adenocarcinoma was investigated. Frozen sections were subjected to double immunofluorescence staining using monoclonal antibodies (MAbs) specific for pericytes (MAb 225.28 recognizing the high-molecular weight-melanoma-associated antigen, expressed by activated pericytes during angiogenesis), endothelial cells (MAb PAL-E), laminin, as well as PDGF-beta receptors (MAb PDGFR-B2) and its ligand PDGF-B chain (MAb PDGF 007). Stained sections were analyzed by computer-aided imaging processing that allowed for a numerical quantification of the degree of colocalization of the investigated antigens. An apparent background colocalization, varying between 23 and 35%, between markers for cells not expected to co-localize was recorded. This background could be due to limitations of camera resolution, to out-of-focus fluorescence, and to interdigitations of the investigated structures. In all six tumor specimens, co-localization of PDGF-beta receptors and PAL-E was not different from the background co-localization, whereas that of PDGF-beta receptors and high-molecular weight-melanoma-associated antigen was significantly higher with mean values between 57 and 71%. Qualitatively, the same pattern was obtained in the two investigated healing wounds. PDGF-B chain did not co-localize with either PAL-E or high-molecular weight-melanoma-associated antigen, but PDGF-B chain-expressing cells were, however, frequently found juxtaposed to the microvasculature. The expression of PDGF-beta receptors on pericytes in activated microvessels and the presence of PDGF-B chain-expressing cells in close proximity to the microvasculature of healing wounds and colorectal adenocarcinoma is compatible with a role for PDGF in the physiology of the microvasculature in these conditions. Images Figure 1 p1381-a Figure 3 Figure 4 PMID:8238254

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.

Connective Tissue Disorders and Cardiovascular Complications: The indomitable role of Transforming Growth Factor-beta signaling

PubMed Central

Wheeler, Jason B.; Ikonomidis, John S.; Jones, Jeffrey A.

2015-01-01

Marfan Syndrome (MFS) and Loeys-Dietz Syndrome (LDS) represent heritable connective tissue disorders that cosegregate with a similar pattern of cardiovascular defects (thoracic aortic aneurysm, mitral valve prolapse/regurgitation, and aortic dilatation with regurgitation). This pattern of cardiovascular defects appears to be expressed along a spectrum of severity in many heritable connective tissue disorders and raises suspicion of a relationship between the normal development of connective tissues and the cardiovascular system. Given the evidence of increased transforming growth factor-beta (TGF-β) signaling in MFS and LDS, this signaling pathway may represent the common link in this relationship. To further explore this hypothetical link, this chapter will review the TGF-β signaling pathway, heritable connective tissue syndromes related to TGF-β receptor (TGFBR) mutations, and discuss the pathogenic contribution of TGF-β to these syndromes with a primary focus on the cardiovascular system. PMID:24443024

Transcriptional activation of mouse mast cell Protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor.

PubMed

Funaba, Masayuki; Ikeda, Teruo; Murakami, Masaru; Ogawa, Kenji; Tsuchida, Kunihiro; Sugino, Hiromu; Abe, Matanobu

2003-12-26

Previous studies have revealed that activin A and transforming growth factor-beta1 (TGF-beta1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-beta1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-beta pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-beta signaling in a tissue-specific manner.

Regulation of natriuretic peptide receptor A and B expression by transforming growth factor-beta 1 in cultured aortic smooth muscle cells.

PubMed

Fujio, N; Gossard, F; Bayard, F; Tremblay, J

1994-06-01

Two types of natriuretic peptide receptors (NPR-A and NPR-B) are membrane guanylate cyclases whose relative expression varies in different tissues. Because natriuretic peptides have been shown to inhibit aortic smooth muscle proliferation, we investigated the regulation of NPR-A and NPR-B in these cells under different proliferative conditions. NPR subtype mRNA levels were measured by our newly developed quantitative reverse transcription-polymerase chain reaction assay using mutated NPR-A and NPR-B cRNA as internal standards. The functional impact of their expression was determined by atrial natriuretic peptide (ANP)- and C-type natriuretic peptide (CNP)-induced stimulation of cyclic GMP production. In the intact aorta, NPR-B mRNA levels were found to be 10-fold higher than those of NPR-A. This dominance was further amplified (1000-fold) in long-term cultures (10 to 15 passages) of aortic smooth muscle cells (ASMC). Higher cyclic GMP production with CNP than with ANP was observed in cultured ASMC from Wistar-Kyoto (WKY) rats. Similar stimulation by the two agonists was noted in spontaneously hypertensive rat (SHR) cells, paralleled by a 10-fold increase in NPR-A mRNA levels and ANP stimulation of cyclic GMP in hypertensive cells. The present study also evaluated NPR-A and NPR-B mRNA control by transforming growth factor-beta 1 (TGF-beta 1), an important regulator of cell proliferation that is overexpressed in SHR ASMC. TGF-beta 1 decreased both NPR-A and NPR-B mRNA levels with a predominant effect in SHR cells at high cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Axin and GSK3- control Smad3 protein stability and modulate TGF- signaling.

PubMed

Guo, Xing; Ramirez, Alejandro; Waddell, David S; Li, Zhizhong; Liu, Xuedong; Wang, Xiao-Fan

2008-01-01

The broad range of biological responses elicited by transforming growth factor-beta (TGF-beta) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. Here we show that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-beta (GSK3-beta). Smad3 physically interacts with Axin and GSK3-beta only in the absence of TGF-beta. Reduction in the expression or activity of Axin/GSK3-beta leads to increased Smad3 stability and transcriptional activity without affecting TGF-beta receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-beta. Mechanistically, Axin facilitates GSK3-beta-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-beta, and suggest a new function of the Axin/GSK3-beta complex in modulating critical TGF-beta/Smad3-regulated processes during development and tumor progression.

Effect of doxycycline on transforming growth factor-beta-1-induced matrix metalloproteinase 2 expression, migration, and collagen contraction in nasal polyp-derived fibroblasts.

PubMed

Shin, Jae-Min; Park, Joo-Hoo; Kang, Byungjin; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2016-11-01

It is well known that doxycycline has antibacterial and anti-inflammatory effects. In this study, we aimed to investigate the effects of doxycycline on the transforming growth factor (TGF) beta 1-induced matrix metalloproteinase (MMP) 2 expression, migration, and collagen contraction, and to determine its molecular mechanism on nasal polyp-derived fibroblasts (NPDF). NPDFs were isolated from the nasal polyps of six patients. Doxycycline was used to pretreat TGF-beta-1-induced NPDFs and ex vivo organ cultures of nasal polyps. Cytotoxicity was evaluated by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Smad2/3 is one of the major transcription factors of TGF-beta signaling. The expression levels of MMP2 and Smad2/3 were measured by using Western blotting, reverse transcription-polymerase chain reaction, and immunofluorescence staining. The enzymic activity of MMP2 was analyzed by using gelatin zymography. Fibroblast migration was evaluated by using transwell migration assays. Contractile activity was measured by a collagen gel contraction assay. The expression level of MMP2 in nasal polyp tissues increased in comparison with inferior turbinate tissues. TGF-beta-1-induced NPDFs were not affected by doxycycline (0-40 μg/mL). The expression levels of MMP2 and activation of Smad2/3 in TGF-beta-1-induced NPDFs and in organ cultures of nasal polyps were significantly downregulated with doxycycline pretreatment. Doxycycline also reduced TGF-beta-1-induced fibroblast migration and collagen contraction in NPDFs. Doxycycline inhibited TGF-beta-1-induced MMP2 expression, migration, and collagen contraction via the Smad2/3 signal pathways in NPDFs.

Small Molecular Inhibitor of Transforming Growth Factor-{beta} Protects Against Development of Radiation-Induced Lung Injury

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

Anscher, Mitchell S.; Thrasher, Bradley; Zgonjanin, Larisa

2008-07-01

Purpose: To determine whether an anti-transforming growth factor-{beta} (TGF-{beta}) type 1 receptor inhibitor (SM16) can prevent radiation-induced lung injury. Methods and Materials: One fraction of 28 Gy or sham radiotherapy (RT) was administered to the right hemithorax of Sprague-Dawley rats. SM16 was administered in the rat chow (0.07 g/kg or 0.15 g/kg) beginning 7 days before RT. The rats were divided into eight groups: group 1, control chow; group 2, SM16, 0.07 g/kg; group 3, SM16, 0.15 g/kg; group 4, RT plus control chow; group 5, RT plus SM16, 0.07 g/kg; group 6, RT plus SM16, 0.15 g/kg; group 7,more » RT plus 3 weeks of SM16 0.07 g/kg followed by control chow; and group 8, RT plus 3 weeks of SM16 0.15 g/kg followed by control chow. The breathing frequencies, presence of inflammation/fibrosis, activation of macrophages, and expression/activation of TGF-{beta} were assessed. Results: The breathing frequencies in the RT plus SM16 0.15 g/kg were significantly lower than the RT plus control chow from Weeks 10-22 (p <0.05). The breathing frequencies in the RT plus SM16 0.07 g/kg group were significantly lower only at Weeks 10, 14, and 20. At 26 weeks after RT, the RT plus SM16 0.15 g/kg group experienced a significant decrease in lung fibrosis (p = 0.016), inflammatory response (p = 0.006), and TGF-{beta}1 activity (p = 0.011). No significant reduction was found in these measures of lung injury in the group that received SM16 0.7g/kg nor for the short-course (3 weeks) SM16 at either dose level. Conclusion: SM16 at a dose of 0.15 g/kg reduced functional lung damage, morphologic changes, inflammatory response, and activation of TGF-{beta} at 26 weeks after RT. The data suggest a dose response and also suggest the superiority of long-term vs. short-term dosing.« less

Opposite effects of dihydrosphingosine 1-phosphate and sphingosine 1-phosphate on transforming growth factor-beta/Smad signaling are mediated through the PTEN/PPM1A-dependent pathway.

PubMed

Bu, Shizhong; Kapanadze, Bagrat; Hsu, Tien; Trojanowska, Maria

2008-07-11

Transforming growth factor-beta (TGF-beta) is an important regulator of physiological connective tissue biosynthesis and plays a central role in pathological tissue fibrosis. Previous studies have established that a biologically active lipid mediator, sphingosine 1-phosphate (S1P), mimics some of the profibrotic functions of TGF-beta through cross-activation of Smad signaling. Here we report that another product of sphingosine kinase, dihydrosphingosine 1-phosphate (dhS1P), has an opposite role in the regulation of TGF-beta signaling. In contrast to S1P, dhS1P inhibits TGF-beta-induced Smad2/3 phosphorylation and up-regulation of collagen synthesis. The effects of dhS1P require a lipid phosphatase, PTEN, a key modulator of cell growth and survival. dhS1P stimulates phosphorylation of the C-terminal domain of PTEN and its subsequent translocation into the nucleus. We demonstrate a novel function of nuclear PTEN as a co-factor of the Smad2/3 phosphatase, PPM1A. Complex formation of PTEN with PPM1A does not require the lipid phosphatase activity but depends on phosphorylation of the serine/threonine residues located in the C-terminal domain of PTEN. Upon complex formation with PTEN, PPM1A is protected from degradation induced by the TGF-beta signaling. Consequently, overexpression of PTEN abrogates TGF-beta-induced Smad2/3 phosphorylation. This study establishes a novel role for nuclear PTEN in the stabilization of PPM1A. PTEN-mediated cross-talk between the sphingolipid and TGF-beta signaling pathways may play an important role in physiological and pathological TGF-beta signaling.

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

TGF-beta-induced apoptosis in human thyrocytes is mediated by p27kip1 reduction and is overridden in neoplastic thyrocytes by NF-kappaB activation.

PubMed

Bravo, Susana B; Pampín, Sandra; Cameselle-Teijeiro, José; Carneiro, Carmen; Domínguez, Fernando; Barreiro, Francisco; Alvarez, Clara V

2003-10-30

Millions of people worldwide suffer goiter, a proliferative disease of the follicular cells of the thyroid that may become neoplastic. Thyroid neoplasms have low proliferative index, low apoptotic index and a high incidence of metastasis. TGF-beta is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF-beta in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Grave's disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF-beta induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF-beta induced apoptosis in normal thyrocyte cultures and all neoplastic cultures except the metastatic cultures. This apoptosis was mediated by a reduction in p27kip1 levels, inducing cell-cycle initiation. Antisense p27 expression induced apoptosis in the absence of TGF-beta. By contrast, in cells in which p27 was overexpressed, TGF-beta had a survival effect. In growth medium, a net survival effect occurs in neoplastic thyrocytes only, not normal thyrocytes, due to activation of the NF-kappaB survival program. Together, these findings suggest that (a) thyroid neoplasms are due to reduced apoptosis, not increased division, in line with the low proliferative index of these pathologies, and (b) TGF-beta induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-kappaB program.

Glycosaminoglycan and transforming growth factor beta1 changes in human plasma and urine during the menstrual cycle, in vitro fertilization treatment, and pregnancy.

PubMed

De Muro, Pierina; Capobianco, Giampiero; Formato, Marilena; Lepedda, Antonio Junior; Cherchi, Gian Mario; Gordini, Laila; Dessole, Salvatore

2009-07-01

To evaluate transforming growth factor beta1 (TGF-beta1) and glycosaminoglycans (GAG) changes in human plasma and urine during the menstrual cycle, IVF-ET, and pregnancy. Prospective clinical study. University hospital. Thirteen women with apparently normal menstrual cycle (group 1); 18 women undergoing IVF-ET (group 2); and 14 low-risk pregnant women (group 3). We assayed plasma and urine concentrations of TGF-beta1, urine content, and distribution of GAG. Blood and urine samples were collected during days 2 to 3, 12 to 13, and 23 to 24 in group 1; in group 2, samples were obtained at menstrual phase, oocyte pick-up day, and 15 days after ET; in group 3, samples were obtained during gestational weeks 10-12, 22-24, and 30-32 and 1 month after delivery. Changes in TGF-beta1 and GAG content. The mean value of total urinary trypsin inhibitor/chondroitin sulfate (UTI/CS) showed a distinct peak at day 12 of the menstrual cycle in the fertile women in whom we monitored the ovulatory period. In the IVF-ET group, GAG distribution and TGF-beta1 levels showed significant differences during the cycle. We observed increased levels of plasma TGF-beta1 15 days after ET. A significant increase of total UTI/CS value with increasing gestation was detected. Transforming growth factor beta1 and GAG levels could represent an additional tool to monitor reproductive events and could be useful, noninvasive markers of ovulation and ongoing pregnancy.

Regulatory CD8{sup +} T cells induced by exposure to all-trans retinoic acid and TGF-{beta} suppress autoimmune diabetes

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

Kishi, Minoru; Yasuda, Hisafumi, E-mail: yasuda@med.kobe-u.ac.jp; Abe, Yasuhisa

Antigen-specific regulatory CD4{sup +} T cells have been described but there are few reports on regulatory CD8{sup +} T cells. We generated islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific regulatory CD8{sup +} T cells from 8.3-NOD transgenic mice. CD8{sup +} T cells from 8.3-NOD splenocytes were cultured with IGRP, splenic dendritic cells (SpDCs), TGF-{beta}, and all-trans retinoic acid (ATRA) for 5 days. CD8{sup +} T cells cultured with either IGRP alone or IGRP and SpDCs in the absence of TGF-{beta} and ATRA had low Foxp3{sup +} expression (1.7 {+-} 0.9% and 3.2 {+-} 4.5%, respectively). In contrast, CD8{sup +} T cellsmore » induced by exposure to IGRP, SpDCs, TGF-{beta}, and ATRA showed the highest expression of Foxp3{sup +} in IGRP-reactive CD8{sup +} T cells (36.1 {+-} 10.6%), which was approximately 40-fold increase compared with that before induction culture. CD25 expression on CD8{sup +} T cells cultured with IGRP, SpDCs, TGF-{beta}, and ATRA was only 7.42%, whereas CD103 expression was greater than 90%. These CD8{sup +} T cells suppressed the proliferation of diabetogenic CD8{sup +} T cells from 8.3-NOD splenocytes in vitro and completely prevented diabetes onset in NOD-scid mice in cotransfer experiments with diabetogenic splenocytes from NOD mice in vivo. Here we show that exposure to ATRA and TGF-{beta} induces CD8{sup +}Foxp3{sup +} T cells ex vivo, which suppress diabetogenic T cells in vitro and in vivo.« less

Transforming growth factor beta induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis.

PubMed

Fantini, M C; Becker, C; Tubbe, I; Nikolaev, A; Lehr, H A; Galle, P; Neurath, M F

2006-05-01

The imbalance between effector and regulatory T cells plays a central role in the pathogenesis of inflammatory bowel diseases. In addition to the thymus, CD4+CD25+ regulatory T cells can be induced in the periphery from a population of CD25- T cells by treatment with transforming growth factor beta (TGF-beta). Here, we analysed the in vivo function of TGF-beta induced regulatory T (Ti-Treg) cells in experimental colitis. Ti-Treg cells were generated in cell culture in the presence or absence of TGF-beta and tested for their regulatory potential in experimental colitis using the CD4+CD62L+ T cell transfer model. Ti-Treg cells significantly suppressed Th1 mediated colitis on CD4+CD62L+ T cell transfer in vivo, as shown by high resolution endoscopy, histology, immunohistochemistry, and cytokine analysis. Further analysis of in vivo and in vitro expanded Ti-Treg cells showed that exogenous interleukin 2 (IL-2) was crucial for survival and expansion of these cells. Our data suggest that regulatory Ti-Treg cells expand by TGF-beta and exogenous IL-2 derived from effector T cells at the site of inflammation. In addition to Tr1 and thymic CD4+CD25+ T cells, peripheral Ti-Treg cells emerge as a class of regulatory T cells with therapeutic potential in T cell mediated chronic intestinal inflammation.

Potentiating role of interleukin-1beta (IL-1beta) and IL-1beta type 1 receptors in the medial hypothalamus in defensive rage behavior in the cat.

PubMed

Hassanain, M; Bhatt, S; Zalcman, S; Siegel, A

2005-06-28

Recently, this laboratory provided evidence that interleukin-1beta (IL-1beta), an immune and brain-derived cytokine, microinjected into the medial hypothalamus, potentiates defensive rage behavior in the cat elicited from the midbrain periaqueductal gray (PAG), and that such effects are blocked by a 5-HT2 receptor antagonist. Since this finding represents the first time that a brain cytokine has been shown to affect defensive rage behavior, the present study replicated and extended these findings by documenting the specific potentiating role played by IL-1beta Type 1 receptor (IL-1RI), and the anatomical relationship between IL-1beta and 5-HT2 receptors in the medial hypothalamus. IL-1beta (10 ng) microinjected into the medial hypothalamus induced two separate phases of facilitation, one at 60 min and another at 180 min, post-injection. In turn, these effects were blocked with pretreatment of the selective IL-1 Type I receptor antagonist (IL-1ra) (10 ng), demonstrating the selectivity of the effects of IL-1beta on medial hypothalamic neurons upon PAG-elicited defensive rage behavior. The next stage of the study utilized immunohistochemical methods to demonstrate that IL-1beta and 5-HT2 receptors were present on the same neurons within regions of the medial hypothalamus where IL-1beta and the IL-1beta receptor antagonists were administered. This provided anatomical evidence suggesting a relationship between IL-1RI and 5-HT2 receptors in the medial hypothalamus that is consistent with the previous pharmacological observations in our laboratory. The overall findings show that activation of IL-1RI in the medial hypothalamus potentiates defensive rage behavior in the cat and that these effects may also be linked to the presence of 5-HT2 receptors on the same groups of neurons in this region of hypothalamus.

Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta.

PubMed Central

Perez, J R; Higgins-Sochaski, K A; Maltese, J Y; Narayanan, R

1994-01-01

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion. Images PMID:8035811

On the role of transforming growth factor-beta in the growth inhibitory effects of retinoic acid in human pancreatic cancer cells.

PubMed

Singh, Brahmchetna; Murphy, Richard F; Ding, Xian-Zhong; Roginsky, Alexandra B; Bell, Richard H; Adrian, Thomas E

2007-12-24

Retinoids are potent growth inhibitory and differentiating agents in a variety of cancer cell types. We have shown that retinoids induce growth arrest in all pancreatic cancer cell lines studied, regardless of their p53 and differentiation status. However, the mechanism of growth inhibition is not known. Since TGF-beta2 is markedly induced by retinoids in other cancers and mediates MUC4 expression in pancreatic cancer cells, we investigated the role of TGF-beta in retinoic acid-mediated growth inhibition in pancreatic cancer cells. Retinoic acid markedly inhibited proliferation of two cell lines (Capan-2 and Hs766T) in a concentration and time-dependent manner. Retinoic acid increased TGF-beta2 mRNA content and secretion of the active and latent forms of TGF-beta2 (measured by ELISA and bioassay). The concentrations of active and TGF-beta2 secreted in response to 0.1 - 10 muM retinoic acid were between 1-5 pM. TGF-beta2 concentrations within this range also inhibited proliferation. A TGF-beta neutralizing antibody blocked the growth inhibitory effects of retinoic acid in Capan-2 cells and partially inhibitory the effects in Hs766T cells. These findings indicate that TGF-beta can cause growth inhibition of pancreatic cancer cells, in a p53-independent manner. Furthermore, it demonstrates the fundamental role of TGF-beta in growth inhibition in response to retinoic acid treatment is preserved in vitro.

Effects of hydrostatic pressure on cytoskeleton and BMP-2, TGF-beta, SOX-9 production in rat temporomandibular synovial fibroblasts.

PubMed

Wu, M-J; Gu, Z-Y; Sun, W

2008-01-01

Recent experimental evidence has suggested that pressure may play an important role in the pathogenesis of arthritic diseases such as temporomandibular disorders (TMDs), rheumatic diseases and osteoarthritis. This study examines the effects of hydrostatic pressure (HP) on cytoskeleton and protein production of bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta) and the SRY HMG box related gene 9 (SOX-9) in synovial fibroblasts (SFs) of rat temporomandibular joint (TMJ). SFs derived from rat TMJ were grown to confluence in Dulbecco's modified Eagle medium supplemented with 15% fetal calf serum. The monolayer of SFs was subjected to different HPs (0, 30, 60, and 90kPa) by an in-house designed pressure chamber for 12h. Changes of cell morphology were observed by fluorescent microscope. Production of TGF-beta, BMP-2 and SOX-9 was examined by immunocytochemical assay and western blot. Compared with the untreated control, the cellular actin configuration of SFs became elongated and more intense F-actin stress fiber staining was observed after HP loading. Exposure of SFs to HP for 12h resulted in significant up-regulation of BMP-2 by 46, 54, and 66% at 30, 60, and 90kPa, respectively, whilst TGF-beta increased by 11, 19, and 28% at 30, 60, and 90kPa, respectively. HP also induced the increase of SOX-9 by 72% at 30kPa and 83% at 60kPa, but only 54% at 90kPa. The obtained data suggest that HP induced the alteration of cytoskeleton and bone-morphogenetic-related proteins' production of SFs, which may influence the pathological condition of TMDs.

14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1.

PubMed

Hong, Hye-Young; Jeon, Woo-Kwang; Bae, Eun-Jin; Kim, Shin-Tae; Lee, Ho-Jae; Kim, Seong-Jin; Kim, Byung-Chul

2010-03-01

The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 zeta and 14-3-3 sigma on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-beta1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-beta1-mediated growth inhibition displayed increased expression of 14-3-3 zeta and decreased expression of 14-3-3 sigma compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 sigma or 14-3-3 zeta, we showed that 14-3-3 sigma is required for TGF-beta1-mediated growth inhibition whereas 14-3-3 zeta negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 zeta increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-beta1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 zeta phosphorylation sites in Smad3 markedly reduced the 14-3-3 zeta-mediated inhibition of TGF-beta1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 zeta in the suppression of TGF-beta1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 sigma or 14-3-3 zeta contributes to TGF-beta1 resistance in cancer cells.

Tumor evasion of the immune system by converting CD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta.

PubMed

Liu, Victoria C; Wong, Larry Y; Jang, Thomas; Shah, Ali H; Park, Irwin; Yang, Ximing; Zhang, Qiang; Lonning, Scott; Teicher, Beverly A; Lee, Chung

2007-03-01

CD4+CD25+ T regulatory (T(reg)) cells were initially described for their ability to suppress autoimmune diseases in animal models. An emerging interest is the potential role of T(reg) cells in cancer development and progression because they have been shown to suppress antitumor immunity. In this study, CD4+CD25- T cells cultured in conditioned medium (CM) derived from tumor cells, RENCA or TRAMP-C2, possess similar characteristics as those of naturally occurring T(reg) cells, including expression of Foxp3, a crucial transcription factor of T(reg) cells, production of low levels of IL-2, high levels of IL-10 and TGF-beta, and the ability to suppress CD4+CD25- T cell proliferation. Further investigation revealed a critical role of tumor-derived TGF-beta in converting CD4+CD25- T cells into T(reg) cells because a neutralizing Ab against TGF-beta, 1D11, completely abrogated the induction of T(reg) cells. CM from a nontumorigenic cell line, NRP-152, or irradiated tumor cells did not convert CD4+CD25- T cells to T(reg) cells because they produce low levels of TGF-beta in CM. Finally, we observed a reduced tumor burden in animals receiving 1D11. The reduction in tumor burden correlated with a decrease in tumor-derived TGF-beta. Treatment of 1D11 also reduced the conversion of CD4+ T cells into T(reg) cells and subsequent T(reg) cell-mediated suppression of antitumor immunity. In summary, we have demonstrated that tumor cells directly convert CD4+CD25- T cells to T(reg) cells through production of high levels of TGF-beta, suggesting a possible mechanism through which tumor cells evade the immune system.

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.

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

Substance P-induced trafficking of beta-arrestins. The role of beta-arrestins in endocytosis of the neurokinin-1 receptor.

PubMed

McConalogue, K; Déry, O; Lovett, M; Wong, H; Walsh, J H; Grady, E F; Bunnett, N W

1999-06-04

Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and beta-arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of beta-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or beta-arrestin1. SP induced (a) striking and rapid (<1 min) translocation of beta-arrestin1 from the cytosol to the plasma membrane, which preceded NK1R endocytosis; (b) redistribution of the NK1R and beta-arrestin1 into the same endosomes containing SP and the transferrin receptor (2-10 min); (c) prolonged colocalization of the NK1R and beta-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and beta-arrestin1 in the cytosol (4-6 h). SP stimulated a similar redistribution of immunoreactive beta-arrestin1 and beta-arrestin2. In contrast, SP did not affect Galphaq/11 distribution, which remained at the plasma membrane. Expression of the dominant negative beta-arrestin319-418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of beta-arrestins to the plasma membrane, where they participate in NK1R endocytosis. beta-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and beta-arrestins return to the cytosol.

Inverse expression of estrogen receptor-beta and nuclear factor-kappaB in urinary bladder carcinogenesis.

PubMed

Kontos, Stylianos; Kominea, Athina; Melachrinou, Maria; Balampani, Eleni; Sotiropoulou-Bonikou, Georgia

2010-09-01

To investigate the expression of nuclear factor-kappaB (NF-kappaB) and estrogen receptor-beta (ER-beta) signalling pathways in bladder urothelial carcinoma according to clinicopathological features, in order to elucidate their role during carcinogenesis. Immunohistochemical methodology was carried out on formalin-fixed, paraffin-embedded sections from urinary bladder carcinomas of 140 patients (94 males and 46 females) who underwent transurethral resection of bladder neoplasms. Correlations between ER-beta and NF-kappaB, and tumor grade and T-stage were evaluated, along with demographic data, sex and age. A significant decrease in ER-beta expression in the nucleus of bladder cells during loss of cell differentiation (r(s) = -0.61, P-value < 0.001, test of trend P-value = 0.003) and in muscle invasive carcinomas (T2-T4; test of trend P-value < 0.001) was found. p65 Subunit of NF-kappaB was expressed in the nucleus and in the cytoplasm of bladder epithelial cells. A strong positive association between tumor grade and nuclear expression of NF-kappaB was shown. No correlation between NF-kappaB, nuclear or cytoplasmic staining, with T-stage was observed. An inverse correlation between ER-beta and nuclear p65 immunoreactivity was observed (r(s) = -0.45, P-value < 0.001). There was no correlation with demographic data. Our immunohistochemical study suggests the possible inverse regulation of NF-kappaB and ER-beta transcription factor during bladder carcinogenesis. Selective ER-beta agonists and agents, inhibitors of NF-kappaB, might represent a possible new treatment strategy for bladder urothelial tumors.

Impact of transforming growth factor-beta1 on atrioventricular node conduction modification by injected autologous fibroblasts in the canine heart.

PubMed

Bunch, T Jared; Mahapatra, Srijoy; Bruce, G Keith; Johnson, Susan B; Miller, Dylan V; Horne, Benjamin D; Wang, Xiao-Li; Lee, Hon-Chi; Caplice, Noel M; Packer, Douglas L

2006-05-30

Atrioventricular (AV) nodal ablation for management of atrial fibrillation (AF) is irreversible and requires permanent pacemaker implantation. We hypothesized that as an alternative, implantation of autologous fibroblasts in the perinodal region would focally modify AV nodal conduction and that this modulation would be enhanced by pretreatment with transforming growth factor-beta1 (TGF-beta1), a stimulant of fibroblasts. Skin biopsies were taken from 12 mongrel dogs, and derived fibroblasts were dissociated and grown in culture for 2 weeks. Multiple injections (0.25 mL) were made through an 8F NOGA catheter along the fast/slow AV nodal pathways as guided by an electroanatomic mapping system. Seven dogs received fibroblasts alone (1x10(6) cells/mL), 7 dogs received TGF-beta1 (5 microg), 4 dogs received fibroblasts and TGF-beta1 (1x10(6) cells/mL+5 microg), and 4 dogs received saline only. AV node function was assessed at baseline and after 4 weeks. Saline (80 mL) with assigned therapy (0.25 mL per injection) was injected into the peri-AV nodal region in each dog. At baseline, the AH interval (66+/-3 ms) and the average RR interval (331+/-17 ms) in pacing-induced AF were similar in each cohort. The increase in AH interval in normal sinus rhythm was longer after fibroblast (23+/-4 versus 5+/-5 ms; P=0.05) and fibroblast plus TGF-beta1 (50+/-5 versus 5+/-5 ms; P<0.001) injections than with saline alone, with similar findings during high right atrium and distal coronary sinus pacing. The AH interval was not significantly increased after TGF-beta1 injections. The AH interval was significantly longer after fibroblast plus TGF-beta1 injections than with either therapy (TGF-beta1 or fibroblasts) alone. The RR interval during AF was increased in dogs that received fibroblasts alone (110+/-36 versus -41+/-34 ms) and to a greater extent with the addition of TGF-beta1 (294+/-108 versus -41+/-34 ms). No AV block was seen in any cohort at 4 weeks. Labeled fibroblasts that

Hepatocyte growth factor and transforming growth factor beta regulate 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in rat hepatocyte primary cultures.

PubMed Central

Joaquin, M; Rosa, J L; Salvadó, C; López, S; Nakamura, T; Bartrons, R; Gil, J; Tauler, A

1996-01-01

Hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta) are believed to be of major importance for hepatic regeneration after liver damage. We have studied the effect of these growth factors on fructose 2,6-bisphosphate (Fru-2,6-P2) levels and the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) in rat hepatocyte primary cultures. Our results demonstrate that HGF activates the expression of the 6PF2K/Fru-2,6-BPase gene by increasing the levels of its mRNA. As a consequence of this activation, the amount of 6PF2K/Fru-2,6-BPase protein and 6-phosphofructo-2-kinase activity increased, which was reflected by a rise in Fru-2,6-P2 levels. In contrast, TGF-beta decreased the levels of 6PF2K/Fru-2,6-BPase mRNA, which led to a decrease in the amount of 6PF2K/Fru-2,6-BPase protein and Fru-2,6-P2. The different actions of HGF and TGF-beta on 6PF2K/Fru-2,6-BPase gene expression are concomitant with their effect on cell proliferation. Here we show that, in the absence of hormones, primary cultures of hepatocytes express the F-type isoenzyme. In addition, HGF increases the expression of this isoenzyme, and dexamethasone activates the L-type isoform. HGF and TGF-beta were able to inhibit this activation. PMID:8660288

Increase Concentration of Transforming Growth Factor Beta (TGF-β) in Breast Milk of Mothers With Psychological Disorders.

PubMed

Shariat, Mamak; Abedinia, Nasrin; Rezaei, Nima; Farrokhzad, Nahid

2017-07-01

Several studies have shown an imbalance between proinflammatory and anti-inflammatory cytokines in depression and anxiety disorders. However, less attention has been paid to the role of cytokines in psychological disorder in mothers who breastfeed. This study looks at whether concentration levels of TGF-β2 are altered in anxious and depressive breastfeeding mothers. This study checked the concentration level of TGF-B2 in relation with psychological symptoms on 110 breastfeeding mothers; based on random sampling method with using of Beck Depression Inventory (BDI), General Health Questionnaire (GHQ) and Spielberger Stress Scale (STAI) in 2015 also TGF-β2 was measured in breast milk using ELISA. We used of Pearson Correlation Method, independent t-test and one-way analysis of variance (ANOVA) to analyze the data. Psychological symptoms (Anxiety and depression) showed positive correlation with TGF-Beta level in which relationships were significant (P=0.01). Psychological problems may be uniquely associated with the level of TGF-β in breast milk. More attention should be paid to the mental health of mothers during breastfeeding, and more research needs to be done in this subject to clarify the relationship between psychological variables with the level of TGF-β in breast milk.

Transforming growth factor-beta1 transcriptionally activates CD34 and prevents induced differentiation of TF-1 cells in the absence of any cell-cycle effects.

PubMed

Marone, M; Scambia, G; Bonanno, G; Rutella, S; de Ritis, D; Guidi, F; Leone, G; Pierelli, L

2002-01-01

A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor beta1 (TGF-beta1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-beta1 maintains human CD34(+) hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-beta1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-beta1 upregulates the human CD34 antigen in the CD34(+) cell lines TF-1 and KG-1a, but not in the more differentiated CD34(-) cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-beta1 did not effect cell growth, but induced transcriptional activation of full-length CD34 and prevented differentiation induced by differentiating agents. This effect was associated with nuclear translocation of Smad-2, activation of TAK-1, and with a dramatic decrease in p38 phosphorylation. In other systems TGF-beta1 has been shown to activate a TGF-beta-activated kinase 1 (TAK1), which in turn, activates p38. The specific inhibitor of p38 phosphorylation, SB202190, also increased CD34 RNA expression, indicating the existence of a link between p-38 inhibition by TGF-beta1 and CD34 overexpression. Our data demonstrate that TGF-beta1 transcriptionally activates CD34 and prevents differentiation of TF-1 cells by acting independently through the Smad, TAK1 and p38 pathways, and thus provide important clues for the understanding of hematopoietic development and a potential tool to modify response of hematopoietic cells to mitogens or differentiating agents.

A Poised Chromatin Platform for TGF-[beta] Access to Master Regulators

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

Xi, Qiaoran; Wang, Zhanxin; Zaromytidou, Alexia-Ileana

2012-02-07

Specific chromatin marks keep master regulators of differentiation silent yet poised for activation by extracellular signals. We report that nodal TGF-{beta} signals use the poised histone mark H3K9me3 to trigger differentiation of mammalian embryonic stem cells. Nodal receptors induce the formation of companion Smad4-Smad2/3 and TRIM33-Smad2/3 complexes. The PHD-Bromo cassette of TRIM33 facilitates binding of TRIM33-Smad2/3 to H3K9me3 and H3K18ac on the promoters of mesendoderm regulators Gsc and Mixl1. The crystal structure of this cassette, bound to histone H3 peptides, illustrates that PHD recognizes K9me3, and Bromo binds an adjacent K18ac. The interaction between TRIM33-Smad2/3 and H3K9me3 displaces the chromatin-compactingmore » factor HP1, making nodal response elements accessible to Smad4-Smad2/3 for Pol II recruitment. In turn, Smad4 increases K18 acetylation to augment TRIM33-Smad2/3 binding. Thus, nodal effectors use the H3K9me3 mark as a platform to switch master regulators of stem cell differentiation from the poised to the active state.« less

The role of peroxisome proliferator-activated receptor-{beta}/{delta} in epidermal growth factor-induced HaCaT cell proliferation

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

Liang Pengfei; Jiang Bimei; Yang Xinghua

2008-10-15

Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) expression and activation is involved in the cell proliferation. However, little is known about the role of PPAR{beta}/{delta} in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPAR{beta}/{delta} mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, anmore » EGF receptor (EGFR) special inhibitor, caused attenuation of PPAR{beta}/{delta} protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPAR{beta}/{delta} binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPAR{beta}/{delta} caused selectively inhibition of PPAR{beta}/{delta} protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPAR{beta}/{delta}, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPAR{beta}/{delta} up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPAR{beta}/{delta} promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPAR{beta}/{delta} expression in a c-Jun-dependent manner and PPAR{beta}/{delta} plays a vital role in EGF-stimulated proliferation of HaCaT cells.« less

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

Receptor-binding region in human choriogonadotropin/lutropin. beta. subunit

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

Keutmann, H.T.; Charlesworth, M.C.; Mason, K.A.

1987-04-01

Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. The authors prepared a series of peptides representing the intercysteine loop sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) ..beta.. subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCG..beta..-(38-57) and hLH..beta..-(38-57) peptides inhibited binding of /sup 125/I-labeled hCG half-maximally at 1.51 x 10/sup -4/ and 2.03 x 10/sup -5/ M, respectively,more » while other peptide hormones and fragments from elsewhere in the ..beta.. subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 x 10/sup -5/ M (hCG) and 2.18 x 10/sup -5/ M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCG..beta..-(38-57) peptide, native hCG and ..beta.. subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. These results indicate that the 38-57 region of ..beta.. subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones.« less

Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta.

PubMed

Okuyama, H; Shimahara, Y; Kawada, N; Seki, S; Kristensen, D B; Yoshizato, K; Uyama, N; Yamaoka, Y

2001-07-27

Redox-regulated processes are important elements in various cellular functions. Reducing agents, such as N-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-beta, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-beta receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that "redox therapy" is promising for fibrosis-related disease.

Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growth factor beta receptor activation and cell transformation.

PubMed

Klein, O; Polack, G W; Surti, T; Kegler-Ebo, D; Smith, S O; DiMaio, D

1998-11-01

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF beta receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF beta receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF beta receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF beta receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF beta receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF beta receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF beta receptor. On the basis of molecular modeling analysis and the known chemical properties of the

Nebivolol: the somewhat-different beta-adrenergic receptor blocker.

PubMed

Münzel, Thomas; Gori, Tommaso

2009-10-13

Although its clinical use in Europe dates almost 10 years, nebivolol is a beta-blocker that has been only recently introduced in the U.S. market. Like carvedilol, nebivolol belongs to the third generation of beta-blockers, which possess direct vasodilator properties in addition to their adrenergic blocking characteristics. Nebivolol has the highest beta(1)-receptor affinity among beta-blockers and, most interestingly, it substantially improves endothelial dysfunction via its strong stimulatory effects on the activity of the endothelial nitric oxide synthase and via its antioxidative properties. Because impaired endothelial activity is attributed a major causal role in the pathophysiology of hypertension, coronary artery disease, and congestive heart failure, the endothelium-agonistic properties of nebivolol suggest that this drug might provide additional benefit beyond beta-receptor blockade. Although lesser beta-blocker-related side effects have been reported in patients with chronic obstructive pulmonary disease or impotence taking nebivolol, side effects and contraindications overlap those of other beta-blockers. Clinically, this compound has been proven to have antihypertensive and anti-ischemic effects as well as beneficial effects on hemodynamics and prognosis in patients with chronic congestive heart failure. Further studies are now necessary to compare the benefit of nebivolol with that of other drugs in the same class and, most importantly, its prognostic impact in patients with hypertension.

Transforming growth factor-beta inhibits the expression of clock genes.

PubMed

Gast, Heidemarie; Gordic, Sonja; Petrzilka, Saskia; Lopez, Martin; Müller, Andreas; Gietl, Anton; Hock, Christoph; Birchler, Thomas; Fontana, Adriano

2012-07-01

Disturbances of sleep-wake rhythms are an important problem in Alzheimer's disease (AD). Circadian rhythms are regulated by clock genes. Transforming growth factor-beta (TGF-β) is overexpressed in neurons in AD and is the only cytokine that is increased in cerebrospinal fluid (CSF). Our data show that TGF-β2 inhibits the expression of the clock genes Period (Per)1, Per2, and Rev-erbα, and of the clock-controlled genes D-site albumin promoter binding protein (Dbp) and thyrotroph embryonic factor (Tef). However, our results showed that TGF-β2 did not alter the expression of brain and muscle Arnt-like protein-1 (Bmal1). The concentrations of TGF-β2 in the CSF of 2 of 16 AD patients and of 1 of 7 patients with mild cognitive impairment were in the dose range required to suppress the expression of clock genes. TGF-β2-induced dysregulation of clock genes may alter neuronal pathways, which may be causally related to abnormal sleep-wake rhythms in AD patients. © 2012 New York Academy of Sciences.

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.

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-beta, and H2O2.

PubMed

Lehmann, Petra; Rank, Petra; Hallfeldt, Klaus L J; Krebs, Bjarne; Gärtner, Roland

2006-08-01

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), as well as H2O2 and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-beta (5 ng/mL), or H2O2 (100 muM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. Asignificant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 microM), thyroidstimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 microM iodine, EGF (5 ng/mL) and TGF-beta (5 ng/mL), or H2O2 (100 microM) (p < 0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H2O2-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4 +/- 0.2 to 2.25 +/- 0.4 mU/microg DNA with 10 nMselenite and 2.6 + 0.4 mU/microg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-beta, EGF, iodide, and even H2O2.

Prevention of TGF-beta-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways.

PubMed

Lin, Sue-Jane; Chang, Chungming; Ng, Ah-Kau; Wang, Shu-Han; Li, Jia-Je; Hu, Cheng-po

2007-09-01

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-beta (TGF-beta). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-beta-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-beta-induced apoptosis.

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

PubMed

Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

1992-08-05

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Extracorporeal shock waves promote healing of collagenase-induced Achilles tendinitis and increase TGF-beta1 and IGF-I expression.

PubMed

Chen, Yeung-Jen; Wang, Ching-Jen; Yang, Kuender D; Kuo, Yur-Ren; Huang, Hui-Chen; Huang, Yu-Ting; Sun, Yi-Chih; Wang, Feng-Sheng

2004-07-01

Extracorporeal shock waves (ESW) have recently been used in resolving tendinitis. However, mechanisms by which ESW promote tendon repair is not fully understood. In this study, we reported that an optimal ESW treatment promoted healing of Achilles tendintis by inducing TGF-beta1 and IGF-I. Rats with the collagenease-induced Achilles tendinitis were given a single ESW treatment (0.16 mJ/mm(2) energy flux density) with 0, 200, 500 and 1000 impulses. Achilles tendons were subjected to biomechanical (load to failure and stiffness), biochemical properties (DNA, glycosaminoglycan and hydroxyproline content) and histological assessment. ESW with 200 impulses restored biomechanical and biochemical characteristics of healing tendons 12 weeks after treatment. However, ESW treatments with 500 and 1000 impulses elicited inhibitory effects on tendinitis repair. Histological observation demonstrated that ESW treatment resolved edema, swelling, and inflammatory cell infiltration in injured tendons. Lesion site underwent intensive tenocyte proliferation, neovascularization and progressive tendon tissue regeneration. Tenocytes at the hypertrophied cellular tissue and newly developed tendon tissue expressed strong proliferating cell nuclear antigen (PCNA) after ESW treatment, suggesting that physical ESW could increase the mitogenic responses of tendons. Moreover, the proliferation of tenocytes adjunct to hypertrophied cell aggregate and newly formed tendon tissue coincided with intensive TGF-beta1 and IGF-I expression. Increasing TGF-beta1 expression was noted in the early stage of tendon repair, and elevated IGF-I expression was persisted throughout the healing period. Together, low-energy shock wave effectively promoted tendon healing. TGF-beta1 and IGF-I played important roles in mediating ESW-stimulated cell proliferation and tissue regeneration of tendon.

Fragrant dioxane derivatives identify beta1-subunit-containing GABAA receptors.

PubMed

Sergeeva, Olga A; Kletke, Olaf; Kragler, Andrea; Poppek, Anja; Fleischer, Wiebke; Schubring, Stephan R; Görg, Boris; Haas, Helmut L; Zhu, Xin-Ran; Lübbert, Hermann; Gisselmann, Günter; Hatt, Hanns

2010-07-30

Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line.

PubMed

Kimmich, Tanja; Brüning, Ansgar; Käufl, Stephanie D; Makovitzky, Josef; Kuhn, Christina; Jeschke, Udo; Friese, Klaus; Mylonas, Ioannis

2010-08-01

Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in

A quantitative real-time RT-PCR assay to measure TGF-beta mRNA and its correlation with hematologic, plasma chemistry and organo-somatic indices responses in triamcinolone-treated Atlantic menhaden, Brevoortia tyrannus.

PubMed

Johnson, A K; Harms, C A; Levine, J F; Law, J McHugh

2006-01-01

A quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay was developed to measure transforming growth factor-beta (TGF-beta) in Atlantic menhaden (Brevoortia tyrannus), an estuarine-dependent species plagued by ulcerative skin lesions in the estuaries along the eastern United States. Atlantic menhaden were acclimated in a closed system for two weeks prior to initiation of the study. The synthetic glucocorticoid, triamcinolone acetonide (10mg/kg body weight) was administered by intracoelomic injection and its effect on the splenic mononuclear cell TGF-beta mRNA transcription, liver-somatic index, spleno-somatic index, hematology, and plasma chemistry were compared to untreated fish at 48 and 96h post-treatment. Triamcinolone-treated Atlantic menhaden showed suppression of TGF-beta mRNA production, neutrophilia, monocytosis, lymphopenia, and an increase in blood glucose concentrations. The health indices used in this study may help us interpret some of the changes observed during the development of ulcerative skin lesions in wild-caught menhaden.

Effect of the association of IGF-I, IGF-II, bFGF, TGF-beta1, GM-CSF, and LIF on the development of bovine embryos produced in vitro.

PubMed

Neira, J A; Tainturier, D; Peña, M A; Martal, J

2010-03-15

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-beta1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-beta1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P<0.05) on Day 8 after in vitro fertilization and similar results to use of SOF+10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-beta1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the

Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES✩

PubMed Central

Weltzin, Maegan M; Huang, Yanzhou; Schulte, Marvin K

2013-01-01

A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5–10 mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2− interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors. PMID:22732654

Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES.

PubMed

Weltzin, Maegan M; Huang, Yanzhou; Schulte, Marvin K

2014-06-05

A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5-10mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2- interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors. Copyright © 2012 Elsevier B.V. All rights reserved.

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

PubMed

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

2007-09-28

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

[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

Lymphotoxin beta receptor (Lt betaR): dual roles in demyelination and remyelination and successful therapeutic intervention using Lt betaR-Ig protein.

PubMed

Plant, Sheila R; Iocca, Heather A; Wang, Ying; Thrash, J Cameron; O'Connor, Brian P; Arnett, Heather A; Fu, Yang-Xin; Carson, Monica J; Ting, Jenny P-Y

2007-07-11

Inflammation mediated by macrophages is increasingly found to play a central role in diseases and disorders that affect a myriad of organs, prominent among these are diseases of the CNS. The neurotoxicant-induced, cuprizone model of demyelination is ideally suited for the analysis of inflammatory events. Demyelination on exposure to cuprizone is accompanied by predictable microglial activation and astrogliosis, and, after cuprizone withdrawal, this activation reproducibly diminishes during remyelination. This study demonstrates enhanced expression of lymphotoxin beta receptor (Lt betaR) during the demyelination phase of this model, and Lt betaR is found in areas enriched with microglial and astroglial cells. Deletion of the Lt betaR gene (Lt betaR-/-) resulted in a significant delay in demyelination but also a slight delay in remyelination. Inhibition of Lt betaR signaling by an Lt betaR-Ig fusion decoy protein successfully delayed demyelination in wild-type mice. Unexpectedly, this Lt betaR-Ig decoy protein dramatically accelerated the rate of remyelination, even after the maximal pathological disease state had been reached. This strongly indicates the beneficial role of Lt betaR-Ig in the delay of demyelination and the acceleration of remyelination. The discrepancy between remyelination rates in these systems could be attributed to developmental abnormalities in the immune systems of Lt betaR-/- mice. These findings bode well for the use of an inhibitory Lt betaR-Ig as a candidate biological therapy in demyelinating disorders, because it is beneficial during both demyelination and remyelination.

The role of transforming growth factor-beta in PEG-rHuMGDF-induced reversible myelofibrosis in rats.

PubMed

Yanagida, M; Ide, Y; Imai, A; Toriyama, M; Aoki, T; Harada, K; Izumi, H; Uzumaki, H; Kusaka, M; Tokiwa, T

1997-12-01

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) injected at a suprapharmacologic dose (100 microg/kg) daily for 5 d in normal rats caused marked increases in marrow megakaryocytes and platelet counts at 6-8 d followed by gradual decreases to control levels at 10-20 d. Interestingly, in addition to the expected thrombopoiesis, PEG-rHuMGDF was associated with myelofibrosis with a predominance of reticulin fibres at day 10 followed by complete normalization by day 20. At 6-8 d, the levels of transforming growth factor-beta1 (TGF-beta1) in the extracellular fluid of the marrow, the platelet poor plasma, and the platelet extract were increased 23-, 7- and 2-fold, respectively. The elevated levels of TGF-beta1 were gradually reduced to baseline levels at 13-20 d in accordance with the normalization of myelofibrosis and thrombopoiesis. An ultrastructural analysis showed that large fragments of megakaryocytes were deposited in the marrow parenchyma of PEG-rHuMGDF-treated rats at day 6. PEG-rHuMGDF administration at pharmacologic doses (1 and 10 microg/kg) did not induce the deposition of reticulin fibres in the marrow. These findings suggest that TGF-beta1 leaked from megakaryocytes is involved in the development of the PEG-rHuMGDF-induced myelofibrosis and that this is a reversible process related to the regulation of the excess production of platelets.

Characterization and inhibition of beta-adrenergic receptor kinase in intact myocytes.

PubMed

Laugwitz, K L; Kronsbein, K; Schmitt, M; Hoffmann, K; Seyfarth, M; Schömig, A; Ungerer, M

1997-08-01

beta-Adrenergic receptor kinase (beta ARK) phosphorylates and thereby inactivates agonist-occupied beta-adrenergic receptors (beta AR). beta ARK is thought to play an important role in the regulation of cardiac function. Therefore, we studied beta ARK activation and its inhibition in intact smooth muscle cells and in cardiomyoblasts. beta AR agonist-stimulated translocation of beta ARK was monitored by immunofluorescence labelling with specific antibodies and confocal laser scanning microscopy in DDT-MF 2 hamster smooth muscle cells and in H9c2 rat cardiomyoblasts. In unstimulated cells. beta ARK was mainly located in the cytosol. After beta AR agonist stimulation, the beta ARK signal was partially translocated to the membranes. Liposomal gene transfer of the COOH-terminus of beta ARK ('beta ARKmini') as a beta ARK inhibitor led to functional expression of this protein in both cell lines with high efficiency. Western blots with beta ARK antibodies showed a gene concentration-dependent immunoreactivity of the 'beta ARKmini' protein. 'beta ARKmini'-transfected myocytes demonstrated reduced membrane targeting of the beta ARK immuno-fluorescence signal. Additionally, the effect of 'beta ARKmini' on beta AR-induced desensitization of myocytic cAMP accumulation was investigated. In control cells, desensitization with isoproterenol led to a subsequent reduction of beta AR-induced cAMP accumulation. In 'beta ARKmini'-transfected myocytes, this beta AR-induced desensitization was significantly diminished, whereas normal beta AR-induced cAMP accumulation was unaffected. A gene concentration of 2 micrograms 'beta ARKmini' DNA/100,000 cardiomyoblasts, and of 0.7 microgram 'beta ARKmini' DNA/100,000 DDT-MF2 smooth muscle cells led to approximately 5.9- and approximately 5.6-fold overexpressions of 'beta ARKmini' vs. native beta ARK, respectively. These gene doses proved sufficient to attenuate beta-adrenergic desensitization significantly. (1) beta ARK translocation was

The role of estrogen receptor {beta} (ER{beta}) in malignant diseases-A new potential target for antiproliferative drugs in prevention and treatment of cancer

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

Warner, Margaret; Center for Nuclear Receptors and Cell Signaling, Department of Biochemistry and Cell Biology, University of Houston, Houston, TX; Gustafsson, Jan-Ake, E-mail: jan-ake.gustafsson@mednut.ki.se

2010-05-21

The discovery of ER{beta} in the middle of the 1990s represents a paradigm shift in our understanding of estrogen signaling. It has turned out that estrogen action is not mediated by one receptor, ER{alpha}, but by two balancing factors, ER{alpha} and ER{beta}, which are often antagonistic to one another. Excitingly, ER{beta} has been shown to be widespread in the body and to be involved in a multitude of physiological and pathophysiological events. This has led to a strong interest of the pharmaceutical industry to target ER{beta} by drugs against various diseases. In this review, focus is on the role ofmore » ER{beta} in malignant diseases where the anti proliferative activity of ER{beta} gives hope of new therapeutic approaches.« less

Beta2- and beta3-adrenergic receptor polymorphisms and exercise hemodynamics in postmenopausal women.

PubMed

McCole, Steve D; Shuldiner, Alan R; Brown, Michael D; Moore, Geoffrey E; Ferrell, Robert E; Wilund, Kenneth R; Huberty, Andrea; Douglass, Larry W; Hagberg, James M

2004-02-01

We sought to determine whether common genetic variations at the beta2 (beta2-AR, Gln27Glu) and beta3 (beta3-AR, Trp64Arg) adrenergic receptor gene loci were associated with cardiovascular (CV) hemodynamics during maximal and submaximal exercise. CV hemodynamics were assessed in 62 healthy postmenopausal women (20 sedentary, 22 physically active, and 20 endurance athletes) during treadmill exercise at 40, 60, 80, and 100% maximal O2 uptake using acetylene rebreathing to quantify cardiac output. The beta2-AR genotype and habitual physical activity (PA) levels interacted to significantly associate with arteriovenous O2 difference (a-vDO2) during submaximal exercise (P = 0.05), with the highest submaximal exercise a-vDO2 in sedentary women homozygous for the beta2-AR Gln allele and no genotype-dependent differences in submaximal exercise a-vDO2 in physically active and athletic women. The beta2-AR genotype also was independently associated with a-vDO2 during submaximal (P = 0.004) and approximately 100% maximal O2 uptake exercise (P = 0.006), with a 1.2-2 ml/100 ml greater a-vDO2 in the Gln/Gln than in the Glu/Glu genotype women. The beta3-AR genotype, independently or interacting with habitual PA levels, was not significantly associated with any CV hemodynamic variables during submaximal or maximal exercise. Thus it appears that the beta2-AR genotype, both independently and interacting with habitual PA levels, is significantly associated with a-vDO2 during exercise in postmenopausal women, whereas the beta3-AR genotype does not appear to be associated with any maximal or submaximal exercise CV hemodynamic responses in postmenopausal women.

The Roles of TGF-Beta and TGF-Beta Signaling Receptors in Breast Carcinogenesis.

DTIC Science & Technology

1997-07-01

phosphorylation of these molecules in a normal mammary epithelial cell line. Subsequently, we have focused on the functional role of Smad3 and Smad4 as...serine residues in the C-terminal portion of Smadl and Smad2, though the corresponding highly conserved sites in Smad3 and Smad5 most likely serve the...far, it appears that Smad2 and Smad3 , which share 92% sequence identity, are likely mediators for the TGF-B signal, whereas Smadl and Smad5, which

Proliferation of Estrogen Receptor alpha Positive Mammary Epithelial Cells is Restrained by TGFbeta1 in Adult Mice

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

Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.

2005-03-03

Transforming growth factor {beta}1 (TGF{beta}1) is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor {alpha} (ER{alpha}) cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF{beta}1 is necessary for the quiescence of ER{alpha}-positive population, we examined mouse mammary epithelial gland at estrus. Approximately 35% of cells showed TGF{beta}1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF{beta} signaling is autocrine. Furthermore, nuclear Smad co-localized with nuclear ER{alpha}. To test whether TGF{beta} was functional, we examined genetically engineered mice with different levels of TGF{beta}1. ER{alpha}more » co-localization with markers of proliferation (i.e. Ki-67 or BrdU) at estrus was significantly increased in the mammary glands of Tgf{beta}1 C57/bl/129SV heterozygote mice. This relationship was maintained following pregnancy, but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF{beta}1 via the MMTV promoter suppressed proliferation of ER{alpha} positive cells. Thus, TGF{beta}1 activation functionally restrains ER{alpha} positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF{beta}1 dysregulation may promote proliferation of ER{alpha} positive cells associated with breast cancer risk in humans.« less

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.

Ski co-repressor complexes maintain the basal repressed state of the TGF-beta target gene, SMAD7, via HDAC3 and PRMT5.

PubMed

Tabata, Takanori; Kokura, Kenji; Ten Dijke, Peter; Ishii, Shunsuke

2009-01-01

The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co-repressors and interact with other co-repressors such as N-CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c-Myb. In particular, the inhibition of TGF-beta-induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF-beta target gene, in TGF-beta-untreated HepG2 cells. Knockdown of these components using siRNA led to up-regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF-beta-responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase.

Analysis of hydrophobic interactions of antagonists with the beta2-adrenergic receptor.

PubMed

Novoseletsky, V N; Pyrkov, T V; Efremov, R G

2010-01-01

The adrenergic receptors mediate a wide variety of physiological responses, including vasodilatation and vasoconstriction, heart rate modulation, and others. Beta-adrenergic antagonists ('beta-blockers') thus constitute a widely used class of drugs in cardiovascular medicine as well as in management of anxiety, migraine, and glaucoma. The importance of the hydrophobic effect has been evidenced for a wide range of beta-blocker properties. To better understand the role of the hydrophobic effect in recognition of beta-blockers by their receptor, we carried out a molecular docking study combined with an original approach to estimate receptor-ligand hydrophobic interactions. The proposed method is based on automatic detection of molecular fragments in ligands and the analysis of their interactions with receptors separately. A series of beta-blockers, based on phenylethanolamines and phenoxypropanolamines, were docked to the beta2-adrenoceptor binding site in the crystal structure. Hydrophobic complementarity between the ligand and the receptor was calculated using the PLATINUM web-server (http://model.nmr.ru/platinum). Based on the analysis of the hydrophobic match for molecular fragments of beta-blockers, we have developed a new scoring function which efficiently predicts dissociation constant (pKd) with strong correlations (r(2) approximately 0.8) with experimental data.

Transforming growth factor beta mediates the progesterone suppression of an epithelial metalloproteinase by adjacent stroma in the human endometrium.

PubMed Central

Bruner, K L; Rodgers, W H; Gold, L I; Korc, M; Hargrove, J T; Matrisian, L M; Osteen, K G

1995-01-01

Unlike most normal adult tissues, cyclic growth and tissue remodeling occur within the uterine endometrium throughout the reproductive years. The matrix metalloproteinases (MMPs), a family of structurally related enzymes that degrade specific components of the extracellular matrix are thought to be the physiologically relevant mediators of extracellular matrix composition and turnover. Our laboratory has identified MMPs of the stromelysin family in the cycling human endometrium, implicating these enzymes in mediating the extensive remodeling that occurs in this tissue. While the stromelysins are expressed in vivo during proliferation-associated remodeling and menstruation-associated endometrial breakdown, none of the stromelysins are expressed during the progesterone-dominated secretory phase of the cycle. Our in vitro studies of isolated cell types have confirmed progesterone suppression of stromal MMPs, but a stromal-derived paracrine factor was found necessary for suppression of the epithelial-specific MMP matrilysin. In this report, we demonstrate that transforming growth factor beta (TGF-beta) is produced by endometrial stroma in response to progesterone and can suppress expression of epithelial matrilysin independent of progesterone. Additionally, we find that an antibody directed against the mammalian isoforms of TGF-beta abolishes progesterone suppression of matrilysin in stromal-epithelial cocultures, implicating TGF-beta as the principal mediator of matrilysin suppression in the human endometrium. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7638197

Mutual antagonism of TGF-beta and Interleukin-2 in cell survival and lineage commitment of induced regulatory T cells

PubMed Central

Tischner, D; Wiegers, G J; Fiegl, H; Drach, M; Villunger, A

2012-01-01

Transforming growth factor beta (TGF-β)- and Interleukin-2 (IL-2)-mediated signaling enables the generation and expansion of induced regulatory T (iTreg) cells that carry high hopes for the treatment of chronic inflammatory and autoimmune diseases. Knowledge about factors stabilizing their lineage commitment and lifespan, however, is limited. Here, we investigated the behavior of iTreg cells, derived from apoptosis-defective mouse mutants, during activated cell autonomous cell death, triggered by cytokine-deprivation, or activation-induced cell death (AICD) after restimulation of the T-cell receptor, and compared these responses with those of effector T cells. We observed that iTreg cells were much more sensitive to IL-2-deprivation but poorly susceptible to AICD. In fact, when apoptosis was compromised, T-cell receptor (TCR)-religation resulted in methylation-independent, ERK- and PI3K/mTOR-mediated loss of Foxp3 expression, impaired suppressive capacity and effector cytokine production. Although iTreg cells prevented colitis induction they rapidly lost Foxp3-GFP expression and gained ability to produce effector cytokines thereby imposing Th1 cell fate on resident effector cells. Surprisingly, iTreg cell conversion itself was limited by TGF-β-mediated Bim/Bcl2L11-dependent apoptosis. Hence, the very same cytokine that drives the generation of iTreg cells can trigger their demise. Our results provide novel insights in iTreg cell biology that will assist optimization of iTreg-based therapy. PMID:22322859

Activated type I TGFbeta receptor (Alk5) kinase confers enhancedsurvival to mammary epithelial cells and accelerates mammary tumorprogression

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

Muraoka-Cook, Rebecca S.; Shin, Incheol; Yi, Jae Youn

2005-01-02

The transforming growth factor-betas (TGF{beta}s) are members of a large superfamily of pleiotropic cytokines that also includes the activins and the bone morphogenetic proteins (BMPs). Members of the TGF{beta} family regulate complex physiological processes such cell proliferation, differentiation, adhesion, cell-cell and cell-matrix interactions, motility, and cell death, among others (Massague, 1998). Dysregulation of TGF{beta} signaling contributes to several pathological processes including cancer, fibrosis, and auto-immune disorders (Massague et al., 2000). The TGF{beta}s elicit their biological effects by binding to type II and type I transmembrane receptor serine-threonine kinases (T{beta}RII and T{beta}RI) which, in turn, phosphorylated Smad 2 and Smad 3.more » Phosphorylated Smad 2/3 associate with Smad 4 and, as a heteromeric complex, translocate to the nucleus where they regulate gene transcription. The inhibitory Smad7 down regulates TGF{beta} signaling by binding to activated T{beta}RI and interfering with its ability to phosphorylate Smad 2/3 (Derynck and Zhang, 2003; Shi and Massague, 2003). Signaling is also regulated by Smad proteolysis. TGF{beta} receptor-mediated activation results in multi-ubiquitination of Smad 2 in the nucleus and subsequent degradation of Smad 2 by the proteasome (Lo and Massague, 1999). Activation of TGF{beta} receptors also induces mobilization of a Smad 7-Smurf complex from the nucleus to the cytoplasm; this complex recognizes the activated receptors and mediates their ubiquitination and internalization via caveolin-rich vesicles, leading to termination of TGF{beta} signaling (Di Guglielmo et al., 2003). Other signal transducers/pathways have been implicated in TGF{beta} actions. These include the extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), protein phosphatase PP2A, phosphatidylinositol-3 kinase (PI3K), and the family of Rho GTPases [reviewed

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.

Anxiety and beta-adrenergic receptor function in a normal population.

PubMed

Kang, Eun-Ho; Yu, Bum-Hee

2005-06-01

Many studies have shown a close relationship between anxiety and beta-adrenergic receptor function in patients with anxiety disorders. This study examined the relationship between beta-adrenergic receptor function and anxiety levels in a normal population. Subjects for this study included 36 men and 44 women between the ages of 20 and 40 years whose Body Mass Index (BMI) was between 18 and 26. All of them were healthy subjects who had no previous history of medical or psychiatric illnesses. The authors measured the Spielberger State-Trait Anxiety Inventory (STAI), Beck Depression Inventory (BDI), and Chronotropic 25 Dose (CD25) of isoproterenol, previously developed to assess in vivo beta-adrenergic receptor sensitivity. We also examined correlations between log normalized CD25 and mood states. The mean of CD25 was 2.64+/-1.37 mug and the mean of CD25 in men was significantly higher (i.e., lower beta-adrenergic receptor sensitivity) than that of women (3.26+/-1.35 vs. 2.14+/-1.17 microg; t = 3.99, p < 0.001). CD25 showed negative correlations with STAI state anxiety (r = -0.344, p = 0.002), trait anxiety (r = -0.331, p = 0.003), and BDI (r = -0.283, p = 0.011). CD25 was positively correlated with BMI (r = 0.423, p < 0.001) and age (r = 0.271, p = 0.015). In stepwise multiple regression analyses, 34% of the variance in CD25 was accounted for by sex, state anxiety, and BMI. The sensitivity of beta-adrenergic receptors increased as anxiety levels became higher in a normal population. Thus, the relationship between anxiety and beta-adrenergic receptor function in healthy subjects may be different from that of patients with anxiety disorders.

Effect of botulinum toxin type A on transforming growth factor beta1 in fibroblasts derived from hypertrophic scar: a preliminary report.

PubMed

Xiao, Zhibo; Zhang, Fengmin; Lin, Weibin; Zhang, Miaobo; Liu, Ying

2010-08-01

Hypertrophic scar is a common dermal disease. Numerous treatments are currently available but they do not always yield excellent therapeutic results. Hence, alternatives are needed. Recent basic and clinical research has shown that botulinum toxin type A (BTXA) has antihypertrophic scar properties but the molecular mechanism for this action is unknown. The aim of this study was to explore the effect of BTXA on transforming growth factor beta1 (TGF-beta1) in fibroblasts derived from hypertrophic scar and further elucidate its actual mechanism. Fibroblasts were isolated from tissue specimens of hypertrophic scar. Fibroblasts were treated with BTXA and the difference in proliferation between treated and nontreated cells was analyzed through the MTT method from the first to the fifth day after treatment. Proteins of TGF-beta1 were checked using ELISA in fibroblasts with BTXA and without BTXA from the first to the fifth day. The growth of the fibroblast treated with BTXA was obviously slower than that of the fibroblast without BTXA treatment (p < 0.01), which showed that BTXA effectively inhibited the growth of fibroblasts. Proteins of TGF-beta1 between fibroblasts with BTXA and fibroblasts without BTXA are statistically significant (p < 0.01). These results suggest that BTXA effectively inhibited the growth of fibroblasts derived from hypertrophic scar and in turn caused a decrease in TGF-beta1 protein, indicating that BTXA-based therapies for hypertrophic scar are promising and worth investigating further.

17 beta-estradiol and tamoxifen upregulate estrogen receptor beta expression and control podocyte signaling pathways in a model of type 2 diabetes.

PubMed

Catanuto, Paola; Doublier, Sophie; Lupia, Enrico; Fornoni, Alessia; Berho, Mariana; Karl, Michael; Striker, Gary E; Xia, Xiaomei; Elliot, Sharon

2009-06-01

Diabetic nephropathy remains one of the most important causes of end-stage renal disease. This is particularly true for women from racial/ethnic minorities. Although administration of 17beta-estradiol to diabetic animals has been shown to reduce extracellular matrix deposition in glomeruli and mesangial cells, effects on podocytes are lacking. Given that podocyte injury has been implicated as a factor leading to the progression of proteinuria and diabetic nephropathy, we treated db/db mice, a model of type 2 diabetic glomerulosclerosis, with 17beta-estradiol or tamoxifen to determine whether these treatments reduce podocyte injury and decrease glomerulosclerosis. We found that albumin excretion, glomerular volume, and extracellular matrix accumulation were decreased in these mice compared to placebo treatment. Podocytes isolated from all treatment groups were immortalized and these cell lines were found to express the podocyte markers WT-1, nephrin, and the TRPC6 cation channel. Tamoxifen and 17beta-estradiol treatment decreased podocyte transforming growth factor-beta mRNA expression but increased that of the estrogen receptor subtype beta protein. 17beta-estradiol, but not tamoxifen, treatment decreased extracellular-regulated kinase phosphorylation. These data, combined with improved albumin excretion, reduced glomerular size, and decreased matrix accumulation, suggest that both 17beta-estradiol and tamoxifen may protect podocytes against injury and therefore ameliorate diabetic nephropathy.

A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition.

PubMed

Yang, Junwei; Dai, Chunsun; Liu, Youhua

2005-01-01

Hepatocyte growth factor (HGF) is a potent antifibrotic cytokine that blocks tubular epithelial to mesenchymal transition (EMT) induced by TGF-beta1. However, the underlying mechanism remains largely unknown. This study investigated the signaling events that lead to HGF blockade of the TGF-beta1-initiated EMT. Incubation of human kidney epithelial cells HKC with HGF only marginally affected the expression of TGF-beta1 and its type I and type II receptors, suggesting that disruption of TGF-beta1 signaling likely plays a critical role in mediating HGF inhibition of TGF-beta1 action. However, HGF neither affected TGF-beta1-induced Smad-2 phosphorylation and its subsequent nuclear translocation nor influenced the expression of inhibitory Smad-6 and -7 in tubular epithelial cells. HGF specifically induced the expression of Smad transcriptional co-repressor SnoN but not Ski and TG-interacting factor at both mRNA and protein levels in HKC cells. SnoN physically interacted with activated Smad-2 by forming transcriptionally inactive complex and overrode the profibrotic action of TGF-beta1. In vivo, HGF did not affect Smad-2 activation and its nuclear accumulation in tubular epithelium, but it restored SnoN protein abundance in the fibrotic kidney in obstructive nephropathy. Hence, HGF blocks EMT by antagonizing TGF-beta1's action via upregulating Smad transcriptional co-repressor SnoN expression. These findings not only identify a novel mode of interaction between the signals activated by HGF receptor tyrosine kinase and TGF-beta receptor serine/threonine kinases but also illustrate the feasibility of confining Smad activity as an effective strategy for blocking renal fibrosis.

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor.

PubMed

Kim, Jaewon; Lee, Jihun; Brych, Stephen R; Logan, Timothy M; Blaber, Michael

2005-02-01

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.

Augmented cell survival in eutopic endometrium from women with endometriosis: Expression of c-myc, TGF-beta1 and bax genes

PubMed Central

Johnson, M Cecilia; Torres, Marisa; Alves, Alessandra; Bacallao, Ketty; Fuentes, Ariel; Vega, Margarita; Boric, M Angélica

2005-01-01

Background Endometriosis is a common gynaecological disorder characterized by the presence of endometrial tissue outside of the uterus. The fragments in normal menstruation are composed of necrotic and living cells, which do not survive in ectopic locations because of programmed cell death. The aim of this study was to evaluate if the balance between cell proliferation and apoptosis is changed in eutopic endometrium from women with endometriosis throughout the menstrual cycle by studying bax (pro-apoptotic), c-myc (regulator of cell cycle) and TGF-beta1 (involved in cell differentiation) genes. Methods Eutopic endometrium was obtained from: 30 women with endometriosis (32.8 +/- 5 years) and 34 fertile eumenorrheic women (36 +/- 5.3 years). We analyzed apoptosis (TUNEL: DNA fragmentation); cell proliferation (immunohistochemistry (IHC) for Ki67); c-myc, bax and TGF-beta1 mRNA abundance (RT-PCR) and TGF-beta1 protein (IHC) in endometrial explants. Results Cell proliferation strongly decreased from proliferative to late secretory phases in glands, but not in stroma, in both endometria. Positive staining in glands and stroma from proliferative endometrium with endometriosis was 1.9- and 2.2-fold higher than control endometrium, respectively (p < 0.05). Abundance of c-myc mRNA was 65% higher in proliferative endometrium from endometriosis than normal tissue (p < 0.05). TGF-beta1 (mRNA and protein) augmented during mid secretory phase in normal endometrium, effect not observed in endometrium with endometriosis. In normal endometrium, the percentage of apoptotic epithelial and stromal cells increased more than 30-fold during late secretory phase. In contrast, in endometrium from endometriosis, not only this increase was not observed, besides bax mRNA decreased 63% versus normal endometrium (p < 0.05). At once, in early secretory phase, apoptotic stromal cells increased 10-fold with a concomitant augment of bax mRNA abundance (42%) in endometria from endometriosis (p < 0

Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro.

PubMed

Miyanishi, Keita; Trindade, Michael C D; Lindsey, Derek P; Beaupré, Gary S; Carter, Dennis R; Goodman, Stuart B; Schurman, David J; Smith, R Lane

2006-06-01

This study examined the effects of intermittent hydrostatic pressure (IHP) and transforming growth factor-beta 3 on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. Chondrogenic gene expression was determined by quantifying mRNA signal levels for SOX9, a transcription factor critical for cartilage development and the cartilage matrix proteins, aggrecan and type II collagen. Extracellular matrix production was determined by weight and histology. IHP was applied to hMSCs in pellet culture at a level of 10 MPa and a frequency of 1 Hz for 4 h per day for periods of 3, 7, and 14 days. hMSCs responded to addition of TGF-beta 3 (10 ng/mL) with a greater than 10-fold increase (p < 0.01) in mRNA levels for each, SOX9, type II collagen, and aggrecan during a 14-day culture period. Applying IHP in the presence of TGF-beta 3 further increased the mRNA levels for these proteins by 1.9-, 3.3-, and 1.6-fold, respectively, by day 14. Chondrogenic mRNA levels were increased with just exposure to IHP. Extracellular matrix deposition of type II collagen and aggrecan increased in the pellets as a function of treatment conditions and time of culture. This study demonstrated adjunctive effects of IHP on TGF-beta 3-induced chondrogenesis and suggests that mechanical loading can facilitate articular cartilage tissue engineering.

Chronic angiotensin-(1-7) administration improves vascular remodeling after angioplasty through the regulation of the TGF-beta/Smad signaling pathway in rabbits.

PubMed

Zeng, Wutao; Chen, Weiyan; Leng, Xiuyu; He, Jian Gui; Ma, Hong

2009-11-06

Angiotensin-(1-7) [ANG-(1-7)] has been reported to attenuate neointimal formation after vascular injury and stent implantation in rats, but the mechanism remains mostly unresolved. Interestingly, the levels of circulating transforming growth factor-beta1 (TGF-beta1) after myocardial infarction were suppressed by ANG-(1-7), which suggests a possible downstream target for the anti-remodeling action of ANG-(1-7). Our study focused on the effects of ANG-(1-7) on vascular remodeling, including neointimal formation and collagen synthesis, and determining whether or not these effects were dependent upon the TGF-beta signaling pathway. Thirty-two New Zealand white rabbits underwent sham surgery or angioplasty in abdominal aorta. The animals were divided into four groups, which were sham, control, ANG-(1-7), and ANG-(1-7)+A-779. Subsequently, an osmotic minipump was implanted to deliver saline, ANG-(1-7) (576 microg kg(-1)d(-1)) or ANG-(1-7)+A-779 (576 microg kg(-1)d(-1)) for 4 weeks. The ANG-(1-7) group displayed a significant reduction in neointimal thickness (207.51+/-16.70 microm vs. 448.08+/-15.30 microm, P<0.001), neointimal area (0.266+/-0.009 mm(2) vs. 0.408+/-0.002 mm(2), P<0.001), and restenosis rate (28.13+/-2.74% vs. 40.13+/-2.74%, P<0.001) when compared to the control group. ANG-(1-7) also inhibited collagen synthesis by significantly decreasing the mRNA expression of Collagen I and Collagen III (vs. 0.2190+/-0.0036 vs. 0.3852+/-0.0212, P<0.001 and 1.1328+/-0.0554 vs. 1.7378+/-0.1164, P<0.001, respectively). Furthermore, the expression of TGF-beta1 and phosphor-Smad2 (p-Smad2) were significantly suppressed by ANG-(1-7) (vs. 1.21+/-0.07 vs. 1.54+/-0.08, P<0.001 and 0.31+/-0.01 vs. 0.43+/-0.02, P<0.001, respectively), but no effect on p38 phosphorylation was observed. [d-Ala(7)]-ANG-(1-7) (A-779), showed a tendency to attenuate the anti-remodeling effects of ANG-(1-7). ANG-(1-7) decreases the amount of vascular remodeling, including a reduction in neointimal

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.

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

PubMed

Segars, J H; Marks, M S; Hirschfeld, S; Driggers, P H; Martinez, E; Grippo, J F; Brown, M; Wahli, W; Ozato, K

1993-04-01

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.

Species differences in the localization and number of CNS beta adrenergic receptors: Rat versus guinea pig

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

Booze, R.M.; Crisostomo, E.A.; Davis, J.N.

1989-06-01

The localization and number of beta adrenergic receptors were directly compared in the brains of rats and guinea pigs. The time course of association and saturability of (125I)cyanopindolol (CYP) binding to slide-mounted tissue sections was similar in rats (Kd = 17 pM) and guinea pigs (Kd = 20 pM). The beta-1 and beta-2 receptor subtypes were examined through the use of highly selective unlabeled receptor antagonists, ICI 118,551 (50 nM) and ICI 89,406 (70 nM). Dramatic species differences between rats and guinea pigs were observed in the neuroanatomical regional localization of the beta adrenergic receptor subtypes. For example, in themore » thalamus prominent beta-1 and beta-2 receptor populations were identified in the rat; however, the entire thalamus of the guinea pig had few, if any, beta adrenergic receptors of either subtype. Hippocampal area CA1 had high levels of beta-2 adrenergic receptors in both rats and guinea pigs but was accompanied by a widespread distribution of beta-2 adrenergic receptors only in rats. Quantitative autoradiographic analyses of 25 selected neuroanatomical regions (1) confirmed the qualitative differences in CNS beta adrenergic receptor localization, (2) determined that guinea pigs had significantly lower levels of beta adrenergic receptors than rats and (3) indicated a differential pattern of receptor subtypes between the two species. Knowledge of species differences in receptor patterns may be useful in designing effective experiments as well as in exploring the relationships between receptor and innervation patterns. Collectively, these data suggest caution be used in extrapolation of the relationships of neurotransmitters and receptors from studies of a single species.« less

Transforming Growth Factor-Beta (TGF-β) Signaling in Paravertebral Muscles in Juvenile and Adolescent Idiopathic Scoliosis

PubMed Central

Kwiecien, Magdalena

2014-01-01

Most researchers agree that idiopathic scoliosis (IS) is a multifactorial disease influenced by complex genetic and environmental factors. The onset of the spinal deformity that determines the natural course of the disease, usually occurs in the juvenile or adolescent period. Transforming growth factors β (TGF-βs) and their receptors, TGFBRs, may be considered as candidate genes related to IS susceptibility and natural history. This study explores the transcriptional profile of TGF-βs, TGFBRs, and TGF-β responsive genes in the paravertebral muscles of patients with juvenile and adolescent idiopathic scoliosis (JIS and AIS, resp.). Muscle specimens were harvested intraoperatively and grouped according to the side of the curve and the age of scoliosis onset. The results of microarray and qRT-PCR analysis confirmed significantly higher transcript abundances of TGF-β2, TGF-β3, and TGFBR2 in samples from the curve concavity of AIS patients, suggesting a difference in TGF-β signaling in the pathogenesis of juvenile and adolescent curves. Analysis of TGF-β responsive genes in the transcriptomes of patients with AIS suggested overrepresentation of the genes localized in the extracellular region of curve concavity: LTBP3, LTBP4, ITGB4, and ITGB5. This finding suggests the extracellular region of paravertebral muscles as an interesting target for future molecular research into AIS pathogenesis. PMID:25313366

FXIIIA and TGF-beta over-expression produces normal musculo-skeletal phenotype in TG2-/- mice.

PubMed

Tarantino, U; Oliva, F; Taurisano, G; Orlandi, A; Pietroni, V; Candi, E; Melino, G; Maffulli, N

2009-04-01

Transglutaminase (TGs) enzymes and proteins crosslinking have for long time been implicated in the formation of hard tissue development, matrix maturation and mineralization. Among the TGs family members, in the context of connective tissue formation, TG2 and Factor XIII are expressed in cartilage by hypertrophic chondrocytes. Here, we analyse the morphological consequences of TG2 deficiency, during the development of skeletal elements. When TG2 is absent, there are not gross abnormalities in the development of the skeletal system, probably from compensatory mechanisms resulting in increased expression of FXIIIA and TGF-beta 1. In vivo other TGs may be involved in promoting chondrocytes and osteoblast differentiation and matrix mineralisation.

Chronic Toll-like receptor 4 stimulation in skin induces inflammation, macrophage activation, transforming growth factor beta signature gene expression, and fibrosis

PubMed Central

2014-01-01

Introduction The crucial role of innate immunity in the pathogenesis of systemic sclerosis (SSc) is well established, and in the past few years the hypothesis that Toll-like receptor 4 (TLR4) activation induced by endogenous ligands is involved in fibrogenesis has been supported by several studies on skin, liver, and kidney fibrosis. These findings suggest that TLR4 activation can enhance transforming growth factor beta (TGF-β) signaling, providing a potential mechanism for TLR4/Myeloid differentiation factor 88 (MyD88)-dependent fibrosis. Methods The expression of TLR4, CD14 and MD2 genes was analyzed by real-time polymerase chain reaction from skin biopsies of 24 patients with diffuse cutaneous SSc. In order to investigate the effects of the chronic skin exposure to endotoxin (Lipopolysaccharide (LPS)) in vivo we examined the expression of inflammation, TGF-β signaling and cellular markers genes by nanostring. We also identified cellular subsets by immunohistochemistry and flow cytometry. Results We found that TLR4 and its co-receptors, MD2 and CD14, are over-expressed in lesional skin from patients with diffuse cutaneous SSc, and correlate significantly with progressive or regressive skin disease as assessed by the Delta Modified Rodnan Skin Score. In vivo, a model of chronic dermal LPS exposure showed overexpression of proinflammatory chemokines, recruitment and activation of macrophages, and upregulation of TGF-β signature genes. Conclusions We delineated the role of MyD88 as necessary for the induction not only for the early phase of inflammation, but also for pro-fibrotic gene expression via activation of macrophages. Chronic LPS exposure might be a model of early stage of SSc when inflammation and macrophage activation are important pathological features of the disease, supporting a role for innate immune activation in SSc skin fibrosis. PMID:24984848

Chronic Toll-like receptor 4 stimulation in skin induces inflammation, macrophage activation, transforming growth factor beta signature gene expression, and fibrosis.

PubMed

Stifano, Giuseppina; Affandi, Alsya J; Mathes, Allison L; Rice, Lisa M; Nakerakanti, Sashidhar; Nazari, Banafsheh; Lee, Jungeun; Christmann, Romy B; Lafyatis, Robert

2014-07-01

The crucial role of innate immunity in the pathogenesis of systemic sclerosis (SSc) is well established, and in the past few years the hypothesis that Toll-like receptor 4 (TLR4) activation induced by endogenous ligands is involved in fibrogenesis has been supported by several studies on skin, liver, and kidney fibrosis. These findings suggest that TLR4 activation can enhance transforming growth factor beta (TGF-β) signaling, providing a potential mechanism for TLR4/Myeloid differentiation factor 88 (MyD88)-dependent fibrosis. The expression of TLR4, CD14 and MD2 genes was analyzed by real-time polymerase chain reaction from skin biopsies of 24 patients with diffuse cutaneous SSc. In order to investigate the effects of the chronic skin exposure to endotoxin (Lipopolysaccharide (LPS)) in vivo we examined the expression of inflammation, TGF-β signaling and cellular markers genes by nanostring. We also identified cellular subsets by immunohistochemistry and flow cytometry. We found that TLR4 and its co-receptors, MD2 and CD14, are over-expressed in lesional skin from patients with diffuse cutaneous SSc, and correlate significantly with progressive or regressive skin disease as assessed by the Delta Modified Rodnan Skin Score. In vivo, a model of chronic dermal LPS exposure showed overexpression of proinflammatory chemokines, recruitment and activation of macrophages, and upregulation of TGF-β signature genes. We delineated the role of MyD88 as necessary for the induction not only for the early phase of inflammation, but also for pro-fibrotic gene expression via activation of macrophages. Chronic LPS exposure might be a model of early stage of SSc when inflammation and macrophage activation are important pathological features of the disease, supporting a role for innate immune activation in SSc skin fibrosis.

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

Transient receptor potential channel M5 and phospholipaseC-beta2 colocalizing in zebrafish taste receptor cells.

PubMed

Yoshida, Yuki; Saitoh, Kana; Aihara, Yoshiko; Okada, Shinji; Misaka, Takumi; Abe, Keiko

2007-10-08

In mammals, transient receptor potential (TRP) channel M5 (TRPM5) is coexpressed with phospholipaseC-beta2 (PLC-beta2) in the taste receptor cells, and both PLC-beta2 and TRPM5 are essential elements in the signal transduction of sweet, bitter and umami stimuli. In this study, we identified the zebrafish homologue of TRPM5 (zfTRPM5) and examined its expression in the gustatory system by in-situ hybridization. Using a transgenic zebrafish line that expressed green fluorescent protein under the control of the PLC-beta2 promoter, we showed that zfTRPM5 is expressed in green fluorescent protein-labeled cells of the taste buds. These results demonstrate that zfTRPM5 and PLC-beta2 colocalize in zebrafish taste receptor cells, suggesting their crucial roles in taste signaling via the fish taste receptors.

Mood states, sympathetic activity, and in vivo beta-adrenergic receptor function in a normal population.

PubMed

Yu, Bum-Hee; Kang, Eun-Ho; Ziegler, Michael G; Mills, Paul J; Dimsdale, Joel E

2008-01-01

The purpose of this study was to examine the relationship between mood states and beta-adrenergic receptor function in a normal population. We also examined if sympathetic nervous system activity is related to mood states or beta-adrenergic receptor function. Sixty-two participants aged 25-50 years were enrolled in this study. Mood states were assessed using the Profile of Mood States (POMS). Beta-adrenergic receptor function was determined using the chronotropic 25 dose isoproterenol infusion test. Level of sympathetic nervous system activity was estimated from 24-hr urine norepinephrine excretion. Higher tension-anxiety, depression-dejection, and anger-hostility were related to decreased beta-adrenergic receptor sensitivity (i.e., higher chronotropic 25 dose values), but tension-anxiety was the only remaining independent predictor of beta-adrenergic receptor function after controlling for age, gender, ethnicity, and body mass index (BMI). Urinary norepinephrine excretion was unrelated to either mood states or beta-adrenergic receptor function. These findings replicate previous reports that anxiety is related to decreased (i.e., desensitized) beta-adrenergic receptor sensitivity, even after controlling for age, gender, ethnicity, and body mass index.

Adenovirus E1A functions as a cofactor for retinoic acid receptor beta (RAR beta) through direct interaction with RAR beta.

PubMed

Folkers, G E; van der Saag, P T

1995-11-01

Transcription regulation by DNA-bound activators is thought to be mediated by a direct interaction between these proteins and TATA-binding protein (TBP), TFIIB, or TBP-associated factors, although occasionally cofactors or adapters are required. For ligand-induced activation by the retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimer, the RAR beta 2 promoter is dependent on the presence of E1A or E1A-like activity, since this promoter is activated by retinoic acid only in cells expressing such proteins. The mechanism underlying this E1A requirement is largely unknown. We now show that direct interaction between RAR and E1A is a requirement for retinoic acid-induced RAR beta 2 activation. The activity of the hormone-dependent activation function 2 (AF-2) of RAR beta is upregulated by E1A, and an interaction between this region and E1A was observed, but not with AF-1 or AF-2 of RXR alpha. This interaction is dependent on conserved region III (CRIII), the 13S mRNA-specific region of E1A. Deletion analysis within this region indicated that the complete CRIII is needed for activation. The putative zinc finger region is crucial, probably as a consequence of interaction with TBP. Furthermore, the region surrounding amino acid 178, partially overlapping with the TBP binding region, is involved in both binding to and activation by AF-2. We propose that E1A functions as a cofactor by interacting with both TBP and RAR, thereby stabilizing the preinitiation complex.

Pregnancy outcome in dairy and beef cattle after artificial insemination and treatment with seminal plasma or transforming growth factor beta-1.

PubMed

Odhiambo, J F; Poole, D H; Hughes, L; Dejarnette, J M; Inskeep, E K; Dailey, R A

2009-09-01

Reduced capability of the uterus to support pregnancy in the absence of its interaction with secretions from male accessory glands has been demonstrated in rodents and to some extent in pigs. However, in cattle, the role of postmating inflammatory response on pregnancy success has not been studied. The current study examined the influence of uterine presensitization with seminal antigens at breeding on pregnancy outcome in cows. Lactating beef (n=1090) and dairy (n=800) cows received 0.5 mL seminal plasma (SP), 40 ng recombinant human transforming growth factor-beta1 (rhTGF-beta1), or 0.5 mL bovine serum albumin (BSA), or were left untreated before or at insemination. Semen was deposited into the anterior cervix using a second insemination gun. Pregnancy was diagnosed at 35 to 40 d postinsemination by transrectal ultrasonography or from records of calves born the subsequent calving season. Pregnancy rates in beef cows did not differ among treatments but differed among trials (69.8%, 52.5% vs. 40.3%; P<0.05). In trials where average pregnancy rates were below 50%, treatments with TGF-beta1 but not SP tended (P<0.07) to increase pregnancy rates in beef cows. In dairy cows, SP and TGF-beta1 improved pregnancy outcome by 10 percentage points, but these increments did not achieve statistical significance. In conclusion, this study did not find any conclusive evidence for the effect of TGF-beta1 or seminal plasma on pregnancy outcome in lactating dairy or beef cows but realized marginal improvements when pregnancy rates were below 50% (compromised fertility).

Effect of transforming growth factor-beta and growth differentiation factor-5 on proliferation and matrix production by human bone marrow stromal cells cultured on braided poly lactic-co-glycolic acid scaffolds for ligament tissue engineering.

PubMed

Jenner, J M G Th; van Eijk, F; Saris, D B F; Willems, W J; Dhert, W J A; Creemers, Laura B

2007-07-01

Tissue engineering of ligaments based on biomechanically suitable biomaterials combined with autologous cells may provide a solution for the drawbacks associated with conventional graft material. The aim of the present study was to investigate the contribution of recombinant human transforming growth factor beta 1 (rhTGF-beta1) and growth differentiation factor (GDF)-5, known for their role in connective tissue regeneration, to proliferation and matrix production by human bone marrow stromal cells (BMSCs) cultured onto woven, bioabsorbable, 3-dimensional (3D) poly(lactic-co-glycolic acid) scaffolds. Cells were cultured for 12 days in the presence or absence of these growth factors at different concentrations. Human BMSCs attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I and collagen III. No differentiation was demonstrated toward cartilage or bone tissue. The addition of rhTGF-beta1 (1-10 ng/mL) and GDF-5 (10-100 ng/mL) increased cell content (p < 0.05), but only TGF-beta1 also increased total collagen production (p < 0.05) and collagen production per cell, which is a parameter indicating differentiation. In conclusion, stimulation with rhTGF-beta1, and to a lesser extent with GDF-5, can modulate human BMSCs toward collagenous soft tissue when applied to a 3D hybrid construct. The use of growth factors could play an important role in the improvement of ligament tissue engineering.

Clostridium perfringens enterotoxin is a superantigen reactive with human T cell receptors V beta 6.9 and V beta 22

PubMed Central

1992-01-01

Candidate superantigens were screened for their ability to induce lysis of human histocompatibility leukocyte antigen class II-positive targets by human CD8+ influenza-specific cytotoxic T cell (CTL) lines. Clostridium perfringens enterotoxin (CPET) induced major histocompatibility complex unrestricted killing by some but not all CTL lines. Using "anchored" polymerase chain reactions, CPET was shown to selectively stimulate peripheral blood lymphocytes bearing T cell receptor V beta 6.9 and V beta 22 in five healthy donors. V beta 24, V beta 21, V beta 18, V beta 5, and V beta 6.1-5 appeared to be weakly stimulated. Antigen processing was not required for CPET to induce proliferation. Like the staphylococcal enterotoxins, CPET is a major cause of food poisoning. These data suggest that superantigenic and enterotoxigenic properties may be closely linked. PMID:1512551

A Synopsis of Factors Regulating Beta Cell Development and Beta Cell Mass

PubMed Central

Prasadan, Krishna; Shiota, Chiyo; Xiangwei, Xiao; Ricks, David; Fusco, Joseph; Gittes, George

2016-01-01

The insulin-secreting beta cells in the endocrine pancreas regulate blood glucose levels, and loss of functional beta cells leads to insulin deficiency, hyperglycemia (high blood glucose) and diabetes mellitus. Current treatment strategies for type-1 (autoimmune) diabetes are islet transplantation, which has significant risks and limitations, or normalization of blood glucose with insulin injections, which is clearly not ideal. The type-1 patients can lack insulin counter-regulatory mechanism; therefore, hypoglycemia is a potential risk. Hence, a cell-based therapy offers a better alternative for the treatment of diabetes. Past research was focused on attempting to generate replacement beta cells from stem cells, however, recently there has been an increasing interest in identifying mechanisms that will lead to the conversion of pre-existing differentiated endocrine cells into beta cells. The goal of this review is to provide an overview of several of the key factors that regulate new beta cell formation (neogenesis) and beta cell proliferation. PMID:27105622

Effects of Transforming Growth Factor Beta 1 in Cerebellar Development: Role in Synapse Formation

PubMed Central

Araujo, Ana P. B.; Diniz, Luan P.; Eller, Cristiane M.; de Matos, Beatriz G.; Martinez, Rodrigo; Gomes, Flávia C. A.

2016-01-01

Granule cells (GC) are the most numerous glutamatergic neurons in the cerebellar cortex and represent almost half of the neurons of the central nervous system. Despite recent advances, the mechanisms of how the glutamatergic synapses are formed in the cerebellum remain unclear. Among the TGF-β family, TGF-beta 1 (TGF-β1) has been described as a synaptogenic molecule in invertebrates and in the vertebrate peripheral nervous system. A recent paper from our group demonstrated that TGF-β1 increases the excitatory synapse formation in cortical neurons. Here, we investigated the role of TGF-β1 in glutamatergic cerebellar neurons. We showed that the expression profile of TGF-β1 and its receptor, TβRII, in the cerebellum is consistent with a role in synapse formation in vitro and in vivo. It is low in the early postnatal days (P1–P9), increases after postnatal day 12 (P12), and remains high until adulthood (P30). We also found that granule neurons express the TGF-β receptor mRNA and protein, suggesting that they may be responsive to the synaptogenic effect of TGF-β1. Treatment of granular cell cultures with TGF-β1 increased the number of glutamatergic excitatory synapses by 100%, as shown by immunocytochemistry assays for presynaptic (synaptophysin) and post-synaptic (PSD-95) proteins. This effect was dependent on TβRI activation because addition of a pharmacological inhibitor of TGF-β, SB-431542, impaired the formation of synapses between granular neurons. Together, these findings suggest that TGF-β1 has a specific key function in the cerebellum through regulation of excitatory synapse formation between granule neurons. PMID:27199658

Transforming growth factor-beta and interleukin-10 in breast milk and development of atopic diseases in infants.

PubMed

Rigotti, E; Piacentini, G L; Ress, M; Pigozzi, R; Boner, A L; Peroni, D G

2006-05-01

Precise relationship between breastfeeding and infant allergy is poorly understood. Objective Aim was to quantify TGF-beta(1) and IL-10 in colostrum and mature milk from allergic and non-allergic mothers and to verify relationship with allergic disease development. Mothers (13 allergics, nine controls) of 22 newborns participated to prospective study on development of children atopy. Colostrum and mature milk were assayed for TGF-beta(1) and IL-10 by ELISA. Children underwent paediatrician evaluation at 6 months of life. Data are presented as median values and range. A significant difference in concentration of TGF-beta(1) between colostrum (330, range 0-3400 pg/mL) and mature milk (215, range 0-2400 pg/mL) was observed in samples from allergic mothers (P=0.015). In mature milk TGF-beta(1) was significantly lower in allergic (215, range 0-2400 pg/mL) than in non-allergic mothers (1059, range 0-6250 pg/mL) (P=0.015). IL-10 was weakly expressed without significant differences between allergic (4.8, range 0-42 and 9.5, range 0-42 pg/mL in colostrum and in mature milk) and non-allergic mothers (0, range 0-42 pg/mL in colostrum and 0, range 0-42 pg/mL in mature milk). After 6 months 46% infants from allergic mothers, but none from controls, presented atopic dermatitis. TGF-beta(1) was significantly less secreted in mature milk of allergic mothers, while no difference in IL-10 was found. Particular cytokine patterns in milk could influence development of atopic diseases. Further immunological studies in this field are necessary.

IGF-1-dependent subunit communication of the IGF-1 holoreceptor: Interactions between. alpha. beta. heterodimeric receptor halves

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

Wilden, P.A.; Treadway, J.L.; Morrison, B.D.

1989-12-12

Examination of {sup 125}I-IGF-1 affinity cross-linking and {beta}-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated {alpha}{beta} heterodimeric IGF-1 receptors into an {alpha}{sub 2}{beta}{sub 2} heterotetrameric state, in a similar manner to that observed for the insulin receptor. The formation of the {alpha}{sub 2}{beta}{sub 2} heterotetrameric IGF-1 receptor complex from the partially purified {alpha}{beta} heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified {alpha}{beta} heterodimers into an {alpha}{sub 2}{beta}{sub 2} heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulatemore » the protein kinase activity of the purified {alpha}{beta} heterodimeric insulin receptor complex. Incubation of the {alpha}{sub 2}{beta}{sub 2} heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter {sup 125}I-IGF-1 binding or IGF-1 stimulation of protein kinase activity. However, IAN treatment of the {alpha}{beta} heterodimeric IGF-1 receptors inhibited the IGF-1 dependent covalent formation of the disulfide-linked {alpha}{sub 2}{beta}{sub 2} heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated {alpha}{beta} heterodimeric IGF-1 receptor complexes into a disulfide-linked {alpha}{sub 2}{beta}{sub 2} heterotetrameric state whereas Mn/MgATP induces a noncovalent association. Therefore, unlike the insulin receptor in which noncovalent association is sufficient for kinase activation, only the covalent assembly of the IGF-1 receptor {alpha}{beta} heterodimers into the {alpha}{sub 2}{beta}{sub 2} heterotetrameric holoreceptor complex is associated with ligand-stimulated protein kinase activation.« less

Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGF-beta signaling.

PubMed

Wu, Jia Wei; Krawitz, Ariel R; Chai, Jijie; Li, Wenyu; Zhang, Fangjiu; Luo, Kunxin; Shi, Yigong

2002-11-01

The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.

Epithelial and ectomesenchymal role of the type I TGF-β receptor ALK5 during facial morphogenesis and palatal fusion

PubMed Central

Dudas, Marek; Kim, Jieun; Li, Wai-Yee; Nagy, Andre; Larsson, Jonas; Karlsson, Stefan; Chai, Yang; Kaartinen, Vesa

2006-01-01

Transforming growth factor beta (TGF-β) proteins play important roles in morphogenesis of many craniofacial tissues; however, detailed biological mechanisms of TGF-β action, particularly in vivo, are still poorly understood. Here, we deleted the TGF-β type I receptor gene Alk5 specifically in the embryonic ectodermal and neural crest cell lineages. Failure in signaling via this receptor, either in the epithelium or in the mesenchyme, caused severe craniofacial defects including cleft palate. Moreover, the facial phenotypes of neural crest-specific Alk5 mutants included devastating facial cleft and appeared significantly more severe than the defects seen in corresponding mutants lacking the TGF-β type II receptor (TGFβRII), a prototypical binding partner of ALK5. Our data indicate that ALK5 plays unique, non-redundant cell-autonomous roles during facial development. Remarkable divergence between Tgfbr2 and Alk5 phenotypes, together with our biochemical in vitro data, imply that (1) ALK5 mediates signaling of a diverse set of ligands not limited to the three isoforms of TGF-β, and (2) ALK5 acts also in conjunction with type II receptors other than TGFβRII. PMID:16806156

Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

NASA Technical Reports Server (NTRS)

Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

1999-01-01

Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

Triiodothyronine, beta-adrenergic receptors, agonist responses, and exercise capacity.

PubMed

Martin, W H

1993-07-01

Although thyroid hormone excess results in increased beta-adrenergic receptor density or agonist responses in some cells of experimental animals, the role of these effects in contributing to clinical manifestations of hyperthyroidism in human subjects is unclear. To shed further light on this issue, we characterized the effect of 2 weeks of excess triiodothyronine administration on cardiac and metabolic responses to graded-dose isoproterenol infusion, skeletal muscle beta-adrenergic receptor density, and physiologic determinants of exercise capacity in young healthy subjects. The slope of the heart rate response to isoproterenol was 36% greater (p < 0.05) after triiodothyronine administration. In addition, beta-adrenergic receptor density was increased (p < 0.01) in all types of skeletal muscle fibers. Maximal oxygen uptake during treadmill exercise declined 5% (p < 0.001) after triiodothyronine administration because of a decrease in the arteriovenous oxygen difference (p < 0.05). The plasma lactate response to submaximal exercise was 25% greater (p < 0.01) in the hyperthyroid state. These effects were paralleled by a decrement in skeletal muscle oxidative capacity and a decrease in cross-sectional area of type 2A skeletal myocytes. Thus, thyroid hormone excess enhances cardiac beta-adrenergic sensitivity under in vivo conditions in human subjects. Nevertheless, exercise capacity is diminished in the hyperthyroid state, an effect that may be related to reduced skeletal muscle oxidative capacity and type 2A fiber atrophy.

Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture of colon tumour cell spheroids with normal cells after incubation with rhTGF- beta1 and/or CPT-11.

PubMed

Paduch, Roman; Jakubowicz-Gil, Joanna; Kandefer-Szerszen, Martyna

2009-12-01

We studied the expression of inducible heat shock protein (HSP27, HSP72) and multidrug-resistance protein (MRP) in co-cultures of human colon carcinoma cell spheroids obtained from different grades of tumour with normal human colon epithelium, myofibroblast and endothelial cell monolayers. We also measured the influence of recombinant human transforming growth factor beta1 (rhTGF-beta1) and camptothecin (CPT-11), added as single agents or in combination, on the levels of the HSPs, MRP, interleukin (IL)-6 and nitric oxide (NO). An immunoblotting analysis with densitometry showed that rhTGF-beta1 and/or CPT-11 increased HSP27, HSP72 and MRP expression in tumour cells and myofibroblasts, as well as in co-cultures compared with appropriate controls. By contrast, in colonic epithelium, inhibition of HSPs and MRP was comparable with that of the control. In endothelial cells, HSP72 was undetectable. Direct interaction of colon tumour spheroids with normal myofibroblasts caused a significant, tumour-grade dependent increase in IL-6 production. Production of IL-6 was significantly lowered by rhTGF-beta1 and/or CPT-11. Tumour cell spheroids cultivated alone produced larger amounts of NO than normal cells. In co-culture, the level of the radical decreased compared with the sum of NO produced by the monocultures of the two types of cells. rhTGF-beta1 and/or CPT-11 decreased NO production both in tumour and normal cell monocultures and their co-cultures. In conclusion, direct interactions between tumour and normal cells influence the expression of HSP27, HSP72 and MRP, and alter IL-6 and NO production. rhTGF-beta1 and/or CPT-11 may potentate resistance to chemotherapy by increasing HSP and MRP expression but, on the other hand, they may limit tumour cell spread by decreasing the level of some soluble mediators of inflammation (IL-6 and NO).

Constitutive Smad signaling and Smad-dependent collagen gene expression in mouse embryonic fibroblasts lacking peroxisome proliferator-activated receptor-{gamma}

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

Ghosh, Asish K; Wei, Jun; Wu, Minghua

2008-09-19

Transforming growth factor-{beta} (TGF-{beta}), a potent inducer of collagen synthesis, is implicated in pathological fibrosis. Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a nuclear hormone receptor that regulates adipogenesis and numerous other biological processes. Here, we demonstrate that collagen gene expression was markedly elevated in mouse embryonic fibroblasts (MEFs) lacking PPAR-{gamma} compared to heterozygous control MEFs. Treatment with the PPAR-{gamma} ligand 15d-PGJ{sub 2} failed to down-regulate collagen gene expression in PPAR-{gamma} null MEFs, whereas reconstitution of these cells with ectopic PPAR-{gamma} resulted in their normalization. Compared to control MEFs, PPAR-{gamma} null MEFs displayed elevated levels of the Type I TGF-{beta} receptor (T{beta}RI),more » and secreted more TGF-{beta}1 into the media. Furthermore, PPAR-{gamma} null MEFs showed constitutive phosphorylation of cellular Smad2 and Smad3, even in the absence of exogenous TGF-{beta}, which was abrogated by the ALK5 inhibitor SB431542. Constitutive Smad2/3 phosphorylation in PPAR-{gamma} null MEFs was associated with Smad3 binding to its cognate DNA recognition sequences, and interaction with coactivator p300 previously implicated in TGF-{beta} responses. Taken together, these results indicate that loss of PPAR-{gamma} in MEFs is associated with upregulation of collagen synthesis, and activation of intracellular Smad signal transduction, due, at least in part, to autocrine TGF-{beta} stimulation.« less

Drug development targeting the glycogen synthase kinase-3beta (GSK-3beta)-mediated signal transduction pathway: the role of GSK-3beta in the maintenance of steady-state levels of insulin receptor signaling molecules and Na(v)1.7 sodium channel in adrenal chromaffin cells.

PubMed

Nemoto, Takayuki; Yanagita, Toshihiko; Kanai, Tasuku; Wada, Akihiko

2009-02-01

Glycogen synthase kinase-3 (GSK-3) is constitutively active in nonstimulated cells, where the majority of its substrates undergo inactivation/proteolysis by phosphorylation. Extracellular stimuli (e.g., insulin) catalyze inhibitory Ser(9)-phosphorylation of GSK-3beta, turning on signaling and causing other biological consequences otherwise constitutively suppressed by GSK-3beta. Regulated and dysregulated activities of GSK-3beta are pivotal to health, disease, and therapeutics (e.g., insulin resistance, neurodegeneration, tumorigenesis, inflammation); however, the underlying mechanisms of multifunctional GSK-3beta remain elusive. In cultured bovine adrenal chromaffin cells, 1) constitutive and negatively-regulated activities of GSK-3beta up- and down-regulated insulin receptor, insulin receptor substrate-1 (IRS-1), IRS-2, and Akt levels via controlling proteasomal degradation and protein synthesis; 2) nicotinic receptor/protein kinase C-alpha (PKC-alpha)/extracellular signal-regulated kinase (ERK) pathway up-regulated IRS-1 and IRS-2 levels, enhancing insulin-induced the phosphoinositide 3-kinase (PI3K)/Akt/GSK-3beta pathway; 3) inhibition of calcineurin by cyclosporin A or FK506 down-regulated IRS-2 level, attenuating insulin-like growth factor-I (IGF-I)-induced ERK and GSK-3beta pathways; and 4) insulin, IGF-I or therapeutics (e.g., lithium) up-regulated the voltage-dependent Na(v)1.7 sodium channel.

Macromolecular beta-adrenergic antagonists discriminating between receptor and antibody.

PubMed Central

Pitha, J; Zjawiony, J; Lefkowitz, R J; Caron, M G

1980-01-01

The beta-adrenergic antagonist, alprenolol, was attached in an irreversible manner to macromolecular dextran via side arms that differed in length. The ability of these macromolecules to bind to the beta-adrenergic receptor of frog erythrocytes and to catecholamine-binding antibodies raised against partially purified receptors was studied. Compared to the parent drug the potency of binding of macromolecular alprenolol to the receptor decreased about 1/10, 1/600, and 1/8000 when the length of the arm separating alprenolol from the dextran moiety was 13, 8, and 4 atoms, respectively. In contrast, the binding potencies of the parent drug and of all its macromolecular derivatives for the antibody were within the same order of magnitude. Thus, conversion of a drug to a macromolecular form may not only sustain its binding activity but may also lead in a higher selectivity. The macromolecular derivatives described here may be suitable probes for investigation of the location and of the molecular properties of the binding sites for beta-adrenergic drugs. PMID:6154947

Heterodimerization with beta2-adrenergic receptors promotes surface expression and functional activity of alpha1D-adrenergic receptors.

PubMed

Uberti, Michelle A; Hague, Chris; Oller, Heide; Minneman, Kenneth P; Hall, Randy A

2005-04-01

The alpha1D-adrenergic receptor (alpha1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote alpha1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of alpha1D-AR at the cell surface: alpha1B-AR and beta2-AR. Confocal imaging confirmed that coexpression with beta2-AR resulted in translocation of alpha1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that alpha1D-AR and beta2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total alpha1D-AR binding sites upon coexpression with beta2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with beta2-AR significantly enhanced the coupling of alpha1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of beta2-AR with alpha1D-AR also conferred the ability of alpha1D-AR to cointernalize upon beta2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of alpha1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between alpha1- and beta2-ARs that is mediated through heterodimerization and cross-internalization.

Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken.

PubMed

Cooley, James R; Yatskievych, Tatiana A; Antin, Parker B

2014-03-01

Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis. Copyright © 2013 Wiley Periodicals, Inc.

A critical role for transcription factor Smad4 in T cell function that is independent of transforming growth factor β receptor signaling.

PubMed

Gu, Ai-Di; Zhang, Song; Wang, Yunqi; Xiong, Hui; Curtis, Thomas A; Wan, Yisong Y

2015-01-20

Transforming growth factor-beta (TGF-β) suppresses T cell function to maintain self-tolerance and to promote tumor immune evasion. Yet how Smad4, a transcription factor component of TGF-β signaling, regulates T cell function remains unclear. Here we have demonstrated an essential role for Smad4 in promoting T cell function during autoimmunity and anti-tumor immunity. Smad4 deletion rescued the lethal autoimmunity resulting from transforming growth factor-beta receptor (TGF-βR) deletion and compromised T-cell-mediated tumor rejection. Although Smad4 was dispensable for T cell generation, homeostasis, and effector function, it was essential for T cell proliferation after activation in vitro and in vivo. The transcription factor Myc was identified to mediate Smad4-controlled T cell proliferation. This study thus reveals a requirement of Smad4 for T-cell-mediated autoimmunity and tumor rejection, which is beyond the current paradigm. It highlights a TGF-βR-independent role for Smad4 in promoting T cell function, autoimmunity, and anti-tumor immunity. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulation of pancreatic islet beta-cell mass by growth factor and hormone signaling.

PubMed

Huang, Yao; Chang, Yongchang

2014-01-01

Dysfunction and destruction of pancreatic islet beta cells is a hallmark of diabetes. Better understanding of cellular signals in beta cells will allow development of therapeutic strategies for diabetes, such as preservation and expansion of beta-cell mass and improvement of beta-cell function. During the past several decades, the number of studies analyzing the molecular mechanisms, including growth factor/hormone signaling pathways that impact islet beta-cell mass and function, has increased exponentially. Notably, somatolactogenic hormones including growth hormone (GH), prolactin (PRL), and insulin-like growth factor-1 (IGF-1) and their receptors (GHR, PRLR, and IGF-1R) are critically involved in beta-cell growth, survival, differentiation, and insulin secretion. In this chapter, we focus more narrowly on GH, PRL, and IGF-1 signaling, and GH-IGF-1 cross talk. We also discuss how these signaling aspects contribute to the regulation of beta-cell proliferation and apoptosis. In particular, our novel findings of GH-induced formation of GHR-JAK2-IGF-1R protein complex and synergistic effects of GH and IGF-1 on beta-cell signaling, proliferation, and antiapoptosis lead to a new concept that IGF-1R may serve as a proximal component of GH/GHR signaling. © 2014 Elsevier Inc. All rights reserved.

IFN-beta1b augments glucocorticoid-induced suppression of tumor necrosis factor-alpha production by increasing the number of glucocorticoid receptors on a human monocytic cell line.

PubMed

Uitdehaag, B M; Hoekstra, K; Koper, J W; Polman, C H; Dijkstra, C D

2001-03-01

We studied the effect of recombinant interferon-beta1b (IFN-beta1b) on the sensitivity to glucocorticoids (GC) and on the number of GC receptors (GCR) in the human monocytic cell line THP-1. We found that IFN-beta1b augments the suppressive effect that dexamethasone has on the stimulated production of tumor necrosis factor-alpha (TNF-alpha), most likely related to the increased number of GCR observed after exposure to IFN-beta1b. This provides a possible clue to the mechanism of action of IFN-beta in multiple sclerosis.

[Density of beta-adrenergic receptors and left ventricular mass in patients with primary essential hypertension].

PubMed

Gajek, J; Zyśko, D; Spring, A

2000-08-01

Left ventricular hypertrophy (LVH) is one of the more important risk factors for sudden death. There are multiple factors for development of LVH in patients with hypertension. Sympathetic nervous system may play a key role causing afterload increase and neurohumoral mechanisms activation. The aim of the study was to determine beta-adrenergic receptors density and its relations to left ventricular mass in hypertensive subjects. The study was carried out in 63 patients (23 women and 40 men), mean age 43.3 +/- 11.6 yrs with primary hypertension: stage I--42 pts and stage II--21 pts. The control group consisted of 26 healthy persons matched for age and sex. We evaluated the density of beta-adrenergic receptors using 125I-cyanopindolol radioligand labeling method. Left ventricular dimensions were assessed by echocardiography (Hewlett-Packard 77010 CF) and left ventricular mass index (LVMI) was calculated. Systolic and diastolic blood pressure and LVMI was significantly higher in hypertension group 156.7 +/- 12.5 vs. 119.8 +/- 8.8 mmHg, p < 0.0001, 95.9/5.5 vs. 78.8 +/- 6.5 mmHg, p < 0.0001, 126.5 +/- 41.9 vs. 93.1 +/- 19.9 g/m2, p < 0.001 respectively. Beta-adrenergic receptors density was 40.7 +/- 29.9 fmol/ml in the hypertensive vs. 37.2 +/- 17.8 fmol/ml in control group (p = NS). There was no correlation between beta-adrenergic receptors density and LVMI. There was a statistically significant positive correlation between LVMI and systolic and diastolic blood pressure (r = 0.44, p < 0.05; r = 0.60, p < 0.01 respectively). 1. Beta-adrenergic receptors density was unchanged in patients with hypertension and did not correlate with LVMI. 2. A high positive correlation between blood pressure values and LVMI, but only in stage II hypertension was revealed.

Dominant expression of interleukin-10 and transforming growth factor-beta genes in activated T-cells of chronic active Epstein-Barr virus infection.

PubMed

Ohga, Shouichi; Nomura, Akihiko; Takada, Hidetoshi; Tanaka, Tamami; Furuno, Kenji; Takahata, Yasushi; Kinukawa, Naoko; Fukushima, Noriyasu; Imai, Shosuke; Hara, Toshiro

2004-11-01

Chronic active Epstein-Barr virus (EBV) infection is a chronic mononucleosis syndrome associated with clonal proliferation of EBV-carrying T-/natural killer (NK)-cells. High levels of circulating EBV and activated T-cells are sustained during the prolonged disease course, whereas it is not clear how ectopic EBV infection in T-/NK-cells has been established and maintained. To assess the biological role of activated T-cells in chronic active EBV infection (CAEBV), EBV DNA and cellular gene expressions in peripheral T-cells were quantified in CAEBV and infectious mononucleosis (IM) patients. In CAEBV, HLA-DR(+) T-cells had higher viral load and larger amounts of IFN gamma, IL-10, transforming growth factor-beta (TGF beta), and cytotoxic T lymphocyte antigen-4 (CTLA4) mRNA than HLA-DR(-)T-cells. HLA-DR(+) T cells of IM patients transcribed more IFN gamma and IL-10 than their HLA-DR(-)T cells. Expression levels of IFN gamma and forkhead box p3 (Foxp3) in CAEBV HLA-DR(+) T-cells were higher than in IM HLA-DR(+) T-cells. The effective variables to discriminate the positivity of HLA-DR were IL-10, IFN gamma, CTLA4, TGF beta, and IL-2 in the order of statistical weight. EBV load in CAEBV T-cells correlated with the expression levels of only IL-10 and TGF beta. These results suggest that CAEBV T-cells are activated to transcribe IFN gamma, IL-10, and TGF beta excessively, and the latter two genes are expressed preferentially in the EBV-infected subsets. The dominant expression of regulatory cytokines in T-cells may imply a viral evasion mechanism in the disease.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-05-15

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

The bovine papillomavirus E5 protein requires a juxtamembrane negative charge for activation of the platelet-derived growth factor beta receptor and transformation of C127 cells.

PubMed

Klein, O; Kegler-Ebo, D; Su, J; Smith, S; DiMaio, D

1999-04-01

The bovine papillomavirus E5 gene encodes a 44-amino-acid, homodimeric transmembrane protein that is the smallest known transforming protein. The E5 protein transforms cultured fibroblasts by forming a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, leading to sustained receptor activation. Aspartic acid 33 in the extracellular juxtamembrane region of the E5 protein is important for cell transformation and interaction with the PDGF beta receptor. A. N. Meyer et al. (Proc. Natl. Acad. Sci USA 91:4634-4638, 1994) speculated that this residue interacted with lysine 499 on the receptor. We constructed E5 mutants containing all possible substitutions at position 33, as well as several double mutants containing substitutions at aspartic acid 33 and at glutamic acid 36, and we examined the ability of these mutants to transform C127 mouse fibroblasts and to bind to and induce activation of the PDGF beta receptor. There was an excellent correlation between the transformation activities of the various mutants and their ability to bind to and activate the PDGF beta receptor. Analysis of the mutants demonstrated that a juxtamembrane negative charge on the E5 protein was required for cell transformation and for productive interaction with the PDGF beta receptor and indicated that aspartic acid 33 was more important for these activities than was glutamic acid 36. These results are consistent with the existence of an essential juxtamembrane salt bridge between lysine 499 on the PDGF beta receptor and an acidic residue in the C terminus of the E5 protein and lend support to our proposed model for the complex between the E5 dimer and the PDGF beta receptor.

HIV-1 stimulates nuclear entry of amyloid beta via dynamin dependent EEA1 and TGF-β/Smad signaling

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

András, Ibolya E., E-mail: iandras@med.miami; Toborek, Michal, E-mail: mtoborek@med.miami.edu

Clinical evidence indicates increased amyloid deposition in HIV-1-infected brains, which contributes to neurocognitive dysfunction in infected patients. Here we show that HIV-1 exposure stimulates amyloid beta (Aβ) nuclear entry in human brain endothelial cells (HBMEC), the main component of the blood–brain barrier (BBB). Treatment with HIV-1 and/or Aβ resulted in concurrent increase in early endosomal antigen-1 (EEA1), Smad, and phosphorylated Smad (pSmad) in nuclear fraction of HBMEC. A series of inhibition and silencing studies indicated that Smad and EEA1 closely interact by influencing their own nuclear entry; the effect that was attenuated by dynasore, a blocker of GTP-ase activity ofmore » dynamin. Importantly, inhibition of dynamin, EEA1, or TGF-β/Smad effectively attenuated HIV-1-induced Aβ accumulation in the nuclei of HBMEC. The present study indicates that nuclear uptake of Aβ involves the dynamin-dependent EEA1 and TGF-β/Smad signaling pathways. These results identify potential novel targets to protect against HIV-1-associated dysregulation of amyloid processes at the BBB level. - Highlights: • HIV-1 induces nuclear accumulation of amyloid beta (Aβ) in brain endothelial cells. • EEA-1 and TGF-Β/Smad act in concert to regulate nuclear entry of Aβ. • Dynamin appropriates the EEA-1 and TGF-Β/Smad signaling. • Dynamin serves as a master regulator of HIV-1-induced nuclear accumulation of Aβ.« less

Acute Radiation-Induced Nocturia in Prostate Cancer Patients Is Associated With Pretreatment Symptoms, Radical Prostatectomy, and Genetic Markers in the TGF{beta}1 Gene

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

De Langhe, Sofie, E-mail: Sofie.DeLanghe@UGent.be; De Ruyck, Kim; Ost, Piet

2013-02-01

Purpose: After radiation therapy for prostate cancer, approximately 50% of the patients experience acute genitourinary symptoms, mostly nocturia. This may be highly bothersome with a major impact on the patient's quality of life. In the past, nocturia is seldom reported as a single, physiologically distinct endpoint, and little is known about its etiology. It is assumed that in addition to dose-volume parameters and patient- and therapy-related factors, a genetic component contributes to the development of radiation-induced damage. In this study, we investigated the association among dosimetric, clinical, and TGF{beta}1 polymorphisms and the development of acute radiation-induced nocturia in prostate cancermore » patients. Methods and Materials: Data were available for 322 prostate cancer patients treated with primary or postoperative intensity modulated radiation therapy (IMRT). Five genetic markers in the TGF{beta}1 gene (-800 G>A, -509 C>T, codon 10 T>C, codon 25 G>C, g.10780 T>G), and a high number of clinical and dosimetric parameters were considered. Toxicity was scored using an symptom scale developed in-house. Results: Radical prostatectomy (P<.001) and the presence of pretreatment nocturia (P<.001) are significantly associated with the occurrence of radiation-induced acute toxicity. The -509 CT/TT (P=.010) and codon 10 TC/CC (P=.005) genotypes are significantly associated with an increased risk for radiation-induced acute nocturia. Conclusions: Radical prostatectomy, the presence of pretreatment nocturia symptoms, and the variant alleles of TGF{beta}1 -509 C>T and codon 10 T>C are identified as factors involved in the development of acute radiation-induced nocturia. These findings may contribute to the research on prediction of late nocturia after IMRT for prostate cancer.« less

(1-->6)-beta-D-glucan as cell wall receptor for Pichia membranifaciens killer toxin.

PubMed

Santos, A; Marquina, D; Leal, J A; Peinado, J M

2000-05-01

The killer toxin from Pichia membranifaciens CYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1-->6)-beta-D-glucan of the cell wall of a sensitive yeast (Candida boidinii IGC 3430). The (1-->6)-beta-D-glucan was purified from cell walls of C. boidinii by alkali and hot-acetic acid extraction, a procedure which solubilizes glucans. The major fraction of receptor activity remained with the alkali-insoluble (1-->6)-beta- and (1-->3)-beta-D-glucans. The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and (1)H nuclear magnetic resonance) analyses of the fractions obtained showed that (1-->6)-beta-D-glucan was a receptor. Adsorption of most of the killer toxin to the (1-->6)-beta-D-glucan was complete within 2 min. Killer toxin adsorption to the linear (1-->6)-beta-D-glucan, pustulan, and a glucan from Penicillium allahabadense was observed. Other polysaccharides with different linkages failed to bind the killer toxin. The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines. This toxin shows its maximum killer activity in the presence of NaCl. This report is the first to identify the (1-->6)-beta-D-glucan as a receptor for this novel toxin.

Selectivity of beta-adrenergic stimulating and blocking agents.

PubMed

Löfdahl, C G; Svedmyr, N

1984-01-01

Studies have been performed to answer two questions: whether there are subgroups of beta 2-receptors separating effects in bronchial and skeletal muscle and whether beta 1-receptors in asthmatic airways mediate bronchoconstriction. Asthmatic patients have been studied in randomised cross-over trials. Effects on FEV1, heart rate and skeletal muscle tremor have been monitored. In some experimental studies, two new compounds, D2343 and QH-25, have shown a selectivity for beta 2-receptors in bronchial muscle compared to skeletal muscle. Studies in asthmatics did not confirm this. Thus, the beta 2-receptors in the two organs appear to be identical. The clinical effect of beta 1-receptors in the the airways was studied by giving selective beta 1-receptor blocking agents. It was shown that pafenolol , a beta-blocker more beta 1-selective than metoprolol, had less effect on FEV1 than metoprolol given in equipotent beta 1-blocking doses. Beta 1-receptor stimulation with a new selective beta 1-stimulating agent, prenalterol, did not give bronchodilation in doses which gave a significant increase of heart rate. Thus, beta 1-receptors do not contribute to bronchodilation in asthmatic patients.

[Preventing effect of TGF-beta1 antibody compounded with fibrin glue on postoperative adhesions of flexor tendon].

PubMed

Zhang, Zhimin; Liu, Jian; Meng, Guolin; Wu, Yaoping

2008-03-01

To explore the preventing effects of TGF-beta1 antibody (TGF-beta1Ab) compounded with fibrin glue (FG) on postoperative adhesions of flexor tendon. Seventy-two Leghorn chickens were randomly divided into 4 groups (groups A, B, C and D), 18 chickens for each group, and the long flexor tendons of the 3rd and 4th toes in zone II of all chickens were transversed and sutured with the 4-strand cruciate repair technique to make defect models. In group A, 0.2 mL TGF-beta1 Ab was applied at repair site. In group B, 0.2 mL FG was applied at repair site. In group C, 0.2 mL TGF-beta1Ab and FG was applied at repair site. In group D, 0.2 mL normal sodium was applied at repair site. At 1, 3 and 8 weeks after operation, the tendons of 6 chickens in each group were harvested for morphological and histological evaluation. Six specimens of each group were obtained for biomechanical test at 3 and 8 weeks. The gross-observation showed that the differences in grading of tendon adhesion were not significant among 4 groups at 1 week after operation (P > 0.05), but the differences were significant between groups A, B, D and group C at 3 and 8 weeks after operation (P < 0.05). Histological observation showed that collagen fibers arranged irregularly in groups A, B and D, but arranged regularly in group C at 3 and 8 weeks' after operation. At 3 weeks after operation the gliding excursion ratio of the tendon in groups A, B, C and D were 0.45 +/- 0.05, 0.40 +/- 0.10, 0.79 +/- 0.09 and 0.25 +/- 0.07 respectively; the simulated active flexion ratio were 0.61 +/- 0.02, 0.67 +/- 0.03, 0.91 +/- 0.03 and 0.53 +/- 0.04 respectively; the work of flexion were (18.00 +/- 0.77), (17.80 +/- 1.13), (27.60 +/- 1.73) and (15.60 +/- 1.27) degrees/N respectively. There were significant differences between group C and other three groups (P < 0.05). The tendon anastomosis breaking strength were (14.2 +/- 1.9), (15.2 +/- 2.2), (16.0 +/- 2.2) and (14.7 +/- 2.7) N, showing no significant differences among 4 groups

Down-regulation of transforming growth factor beta-2 expression is associated with the reduction of cyclosporin induced gingival overgrowth in rats treated with roxithromycin: an experimental study

PubMed Central

2009-01-01

Background Gingival overgrowth (GO) is a common side effect of the chronic use of cyclosporine (CsA), an immunosuppressant widely used to prevent rejection in transplant patients. Recent studies have reported elevated levels of specific cytokines in gingival overgrowth tissue, particularly TGF-beta, suggesting that this growth factor plays a role in the accumulation of extracellular matrix materials. The effectiveness of azithromycin, a macrolide antibiotic, in the regression of this undesirable side effect has also been demonstrated. Methods In this study, we created an experimental model for assessing the therapeutic effect of roxithromycin in GO and the expression of transforming growth factor beta (TGF-beta2) through immunohistochemistry. We used four groups of rats totaling 32 individuals. GO was induced during five weeks and drug treatment was given on the 6th week as follows: group 1 received saline; group 2 received CsA and was treated with saline on the 6th week; group 3 received CsA and, on the 6th week, ampicilin; and group 4 received CsA during 5 weeks and, on the 6th week, was treated with roxithromycin. Results The results demonstrated that roxithromycin treatment was effective in reducing cyclosporine-induced GO in rats. Both epithelial and connective tissue showed a decrease in thickness and a significant reduction in TGF-beta2 expression, with a lower number of fibroblasts, reduction in fibrotic areas and decrease in inflammatory infiltrate. Conclusion The present data suggest that the down-regulation of TGF-beta2 expression may be an important mechanism of action by which roxithromycin inhibits GO. PMID:19995419

Effects of sequential artificial tear and cyclosporine emulsion therapy on conjunctival goblet cell density and transforming growth factor-beta2 production.

PubMed

Pflugfelder, Stephen C; De Paiva, Cintia S; Villarreal, Arturo L; Stern, Michael E

2008-01-01

To evaluate the effects of sequential treatment with artificial tears and cyclosporine emulsion on conjunctival goblet cell density and production of transforming growth factor (TGF)-beta2 in patients with dry eye disease. Patients with dry eye disease (N = 6) defined by an Ocular Surface Disease Index symptom score >or=25, Schirmer test 1 <10 mm, and corneal fluorescein and conjunctival lissamine green staining scores >or=3 were treated with artificial tears (Refresh Plus; Allergan, Irvine, CA) 4 times a day for 4 weeks, followed by 0.05% cyclosporine emulsion (Restasis; Allergan) twice a day for 12 weeks. Impression cytology was performed on the bulbar conjunctiva of both eyes at baseline, after artificial tear therapy, and after 6 and 12 weeks of cyclosporine therapy. Goblet cells were counted in 5 representative microscopic fields per membrane in those taken from the temporal and inferior bulbar conjunctiva of the worse eye, and membranes taken from the fellow eye were immunostained for TGF-beta2. There were no differences in mean goblet cell density between baseline and 4 weeks of artificial tears in the temporal and inferior bulbar specimens. After 6 weeks of cyclosporine emulsion, goblet cell density was significantly greater than baseline and artificial tears in the inferior bulbar conjunctiva (P < 0.01). After 12 weeks of cyclosporine emulsion, goblet cell density was significantly greater than baseline and artificial tears in both temporal and inferior bulbar sites (P < 0.01). The number of TGF-beta2-positive goblet cells was also noted to increase after 6 and 12 weeks of cyclosporine therapy (P < 0.001). Cyclosporine emulsion, but not artificial tears, increases goblet cell density and production of the immunoregulatory factor TGF-beta2 in the bulbar conjunctiva in patients with dry eye.

The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

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

Watanabe, H.; Grubb, J.H.; Sly, W.S.

1990-10-01

The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functionalmore » receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.« less

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed Central

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-01-01

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

[The mechanism of vasculogenesis: the critical role of transforming growth factor-beta 1 in the formation of vessel-like structures during the differentiation in vitro of murine embryonic stem cells].

PubMed

Tsung, H C; Yao, Z

1996-09-01

When ES-5 cells were transfected with an exogenous porcine TGF-beta 1 gene, one can obtain clones of genetically modified ES cells with over-expression of the transfected gene. We called the genetically modified ES-5 cells as ES-T cells. When ES-T cells were used to study their differentiation in vitro by all trans-retinoic acid (RA), it was soon noticed that embryoid bodies of ES-T cells can exclusively differentiate into endothelial cells and vessel-like structures, but not in their parent ES-5 cells. The above result is the first indication that the differentiation of tubular structures in embryoid bodies of ES-T cells may somehow be related to TGF-beta 1. To demonstrate further the role of TGF-beta 1 in the formation of vessel-like structures, the cultured ES-5 cells in the presence of added rhTGF-beta 1 were closely followed in the course of their differentiation. We have, thus, demonstrated the promoting effects of exogenous rhTGF-beta 1 in the formation of vessel-like structures, morphologically similar to those structures derived from ES-T6 cells, during the differentiation of ES-5 cells, both in monolayer culture, in three dimensional collagen gel and in embryoid bodies cultured on gelatin-coated tissue culture wells. Addition of suitable amount of anti-TGF-beta 1 monoclonal antibody IgG (TB21) to the culture medium of embryoid bodies of ES-T6 cells could effectively abolish the formation of vessel-like structures induced by retinoic acid. The percentage of the inhibition was very high, giving a figure comparable to that of atypical vessel-like structures formed in the control embryoid bodies from their parent ES-5 cells. The flat epithelial-like cells and round cells differentiated from embryoid bodies of ES-T6 cells were stained rather strongly for laminin and type IV collagen by immunofluorescent procedure. The above results indicate clearly that TGF-beta 1 is a crucial factor in organizing the differentiated derivatives (endothelial-like cells and

beta-Endorphin-induced analgesia is inhibited by synthetic analogs of beta-endorphin.

PubMed

Nicolas, P; Hammonds, R G; Li, C H

1984-05-01

Competitive antagonism of human beta-endorphin (beta h-EP)-induced analgesia by synthetic beta h-EP analogs with high in vitro opiate receptor binding to in vivo analgesic potency ratio has been demonstrated. A parallel shift of the dose-response curve for analgesia to the right was observed when either beta h-EP or [ Trp27 ] -beta h-EP was coinjected with various doses of [Gln8, Gly31 ]-beta h-EP-Gly-Gly-NH2, [Arg9,19,24,28,29]-beta h-EP, or [ Cys11 ,26, Phe27 , Gly31 ]-beta h-EP. It was estimated that the most potent antagonist, [Gln8, Gly31 ]-beta h-EP-Gly-NH2, is at least 200 times more potent than naloxone.

beta-Endorphin-induced analgesia is inhibited by synthetic analogs of beta-endorphin.

PubMed Central

Nicolas, P; Hammonds, R G; Li, C H

1984-01-01

Competitive antagonism of human beta-endorphin (beta h-EP)-induced analgesia by synthetic beta h-EP analogs with high in vitro opiate receptor binding to in vivo analgesic potency ratio has been demonstrated. A parallel shift of the dose-response curve for analgesia to the right was observed when either beta h-EP or [ Trp27 ] -beta h-EP was coinjected with various doses of [Gln8, Gly31 ]-beta h-EP-Gly-Gly-NH2, [Arg9,19,24,28,29]-beta h-EP, or [ Cys11 ,26, Phe27 , Gly31 ]-beta h-EP. It was estimated that the most potent antagonist, [Gln8, Gly31 ]-beta h-EP-Gly-NH2, is at least 200 times more potent than naloxone. PMID:6328494