Epigallocatechin-3-gallate suppresses transforming growth factor-beta signaling by interacting with the transforming growth factor-beta type ii receptor que es anoxia

AIM: to investigate the (-)-epigallocatechin-3-gallate (EGCG) binding to transforming growth factor-β (TGF-β) type II receptor (TGFRII).

METHODS: the expression of α-smooth muscle actin (α-SMA) was used as a marker for fibrotic change in human lung fibroblast MRC-5 cells. The α-SMA expression level was determined by western blotting and immunohistological analysis. We examined whether the anti-fibrotic effects of EGCG on MRC-5 cells was dependent on antioxidant mechanism by using edaravone and N-acetylcysteine (NAC). The suppression effects of EGCG on smad2/3 activation were studied by confocal fluorescence microscopy.


The binding of EGCG to recombinant TGFRII protein was analyzed by immunoprecipitation and affinity chromatography.Que es anoxia

RESULTS: when MRC-5 cells were treated with TGF-β, EGCG decreased the expression of α-SMA in a dose dependent manner, whereas catechin did not influence the α-SMA expression in the cells. Except for EGCG, antioxidant compounds (e.G., edaravone and NAC) had no effects on the TGF-β-induced α-SMA expression. Nuclear localization of phosphorylated smad2/3 was observed after TGF-β treatment; however, EGCG treatment attenuated the nuclear transportation of smad2/3 in the presence or absence of TGF-β. After a TGFRII expression vector was introduced into COS-7 cells, cell lysates were untreated or treated with EGCG or catechin. The immunoprecipitation experiments using the lysates showed that EGCG dose-dependently bound to tgfriiand that catechin did not at all.Que es anoxia affinity chromatography study indicated that EGCG would bind to TGFRII.

CONCLUSION: our results demonstrate that EGCG interacts with TGFRII and inhibits the expression of α-SMA via the TGF-β-smad2/3 pathway in human lung fibroblast MRC-5 cells.

INTRODUCTION

(-)-epigallocatechin-3-gallate (EGCG), the most biologically active constituent in green tea, has been recognized as a component that provides the beverage with potential benefits for human health[ 1]. The reported health-promoting properties of green tea include anti-cancer[ 1- 3], anti-obesity[ 4], anti-diabetic[ 5, 6], anti-atherosclerotic[ 7], anti-viral[ 8- 10], anti-bacterial[ 11- 13] and neuroprotective[ 14- 16] effects. The anti-fibrotic effects of green tea and its constituents, especially EGCG, on liver fibrosis[ 17- 19], pancreatic fibrosis[ 20] and pulmonary fibrosis[ 21] have been also reported.Que es anoxia

Activation of myofibroblasts is the one of the critical events during fibrosis development. Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that is pivotal in the regulation of myofibroblast activation, differentiation, migration, and extracellular matrix production; it also plays an important role in the initiation and progression of fibrosis[ 22]. However, the mechanisms by which EGCG influences TGF-β action on myofibroblast activation remain incompletely defined.

Tachibana et al[ 23] identified a catechin receptor for EGCG, and showed that this receptor partially mediates the function of EGCG. It is also known that EGCG shows its biological action by interacting with receptors other than the catechin receptor[ 24, 25].Que es anoxia in the present study, we investigated the possibility that EGCG might bind to the TGF-β type II receptor (TGFRII).

Western blotting

After washing with ice-cold phosphate buffer saline (PBS), cells were treated with 0.25% trypsin-EDTA solution (invitrogen, carlsbad, CA, united states), suspended in growth medium and collected by centrifugation at 700 g for 5 min. The pellets were washed with PBS, resuspended in lysis buffer (20 mmol/L tris-hcl, ph 7.4, 150 mmol/L nacl, 0.1% SDS, 1% triton X-100, 0.5% sodium deoxycholate), which contained a cocktail of protease inhibitors (roche molecular biochemicals, mannheim, germany) on ice, and centrifuged at 18000 g at 4 °C for 10 min.

Protein concentration was determined by a BCA protein assay kit (pierce, rockford, IL, united states).Que es anoxia cell lysates were suspended in SDS electrophoresis sample buffer and boiled for 5 min. Samples (2.5 μg of protein per lane) were separated on 10% polyacrylamide gels and then transferred to an immobilon P membrane (millipore, billerica, MA, united states). Antibody binding was detected by ECL plus (GE healthcare, buckinghamshire, united kingdom).

RESULTS

Effects of EGCG on the expression ofα -smooth muscle actin

The MRC-5 cell line, which is derived from human fetal lung fibroblasts, expresses α-SMA and is considered to be a myofibroblast cell line[ 26, 27]. Therefore, this cell line was used in this study.

MRC-5 cells were grown to 85% confluence, and then serum-starved (0.5% FBS) for 48 h. After serum starvation, cells were treated with TGF-β.Que es anoxia we and others usually use 1-2 ng/ml of TGF-β in culture media[ 27- 31]. A representative and frequently used marker of myofibroblast activation is α-SMA[ 32, 33]. Western blot analysis and immunohistological examination showed that expression of α-SMA was increased by TGF-β (figure 1). Whereas a catechin control showed no effects on α-SMA expression, EGCG dose-dependently abolished the increase in expression of α-SMA induced by TGF-β (figure 1B). The EGCG concentration used in this study was reasonable[ 34]. The expression of GAPDH also seemed to be decreased by a high dose of EGCG. The band densities of α-SMA and GAPDH were compared (figure 1B), and the result clearly showed the effects of EGCG on α-SMA.Que es anoxia

Figure 1 effects of (-)-epigallocatechin-3-gallate on expression of α-smooth muscle actin.

A: lysates of MRC-5 cells were obtained from cells treated with 0.5% FBS in DMEM alone, (-)-epigallocatechin-3-gallate (EGCG) (50 μmol/L), or catechin (50 μmol/L) for 24 h. After SDS-PAGE, proteins were blotted onto immobilon, and probed with anti-α-smooth muscle actin (α-SMA) antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control; B: MRC-5 cells were treated with the indicated amounts of EGCG. Α-SMA was detected in the same manner as in (A). The expression levels of α-SMA were normalized to those of GAPDH; C: expression of α-SMA in cells treated with EGCG (50 μmol/L) in the absence (-) or presence (+) of transforming growth factor-β (TGF-β) (2 ng/ml) were examined by confocal microscopy.Que es anoxia green: α-SMA (fluorescein isothiocyanate conjugated goat anti-mouse igg); red: actin stress fiber (rhodamine-labeled phalloidin); blue: nuclei (DAPI). FBS: fetal bovine serum.

Because EGCG is an antioxidant compound, we examined whether edaravone and NAC, two well-known antioxidant compounds, have similar effects. Neither treatment with edaravone (figure 2A) nor treatment with NAC (figure 2B) affected the increase in expression of α-SMA induced by TGF-β.

Figure 3 effects of (-)-epigallocatechin-3-gallate on activation and localization of smad2/3.

MRC-5 cells were treated with transforming growth factor-β (TGF-β) and/or (-)-epigallocatechin-3-gallate (EGCG). Cells were examined by confocal microscopy.Que es anoxia subcellular localization of smad2/3 (green) and actin stress fibers (red) are shown. Nuclei were stained by DAPI (blue). A: control; B: treated with TGF-β; C: treated with EGCG; D: treated with TGF-β and EGCG. DAPI: 4′,6-diamidino-2-phenylindole.

EGCG binds to TGFRII

Next, we examined the possibility that EGCG interferes with binding of TGF-β to the TGFRII. To this end, cells expressing large amounts of the receptor are preferable. Because COS-7 cells showed high transformation efficiency and marked expression of exogenous cdna, these cells were used for transformation experiments. A TGFRII expression vector was introduced into COS-7 cells. Cell lysates were untreated or treated with EGCG or catechin, and then subjected to immunoprecipitation with anti-TGFRII antibody.Que es anoxia in untreated lysate and lysate treated with catechin, TGFRII was precipitated by the antibody. When lysate was treated with EGCG, however, anti-TGFR did not precipitate TGFRII (figure 4).

DISCUSSION

In this study, we have demonstrated that EGCG both inhibits the signal transduction of TGF-β by binding to TGFRII and attenuates the expression of α-SMA in MRC-5 cells, which is a myofibroblast cell line, when it is stimulated by TGF-β. Myofibroblasts play crucial roles in the pathogenesis of tissue fibrosis[ 35]. Stimulation by TGF-β and other cytokines leads myofibroblasts to an activated state[ 36]. Activated myofibroblasts then secrete collagen and other components of the extracellular matrix, which can result in fibrosis[ 37].Que es anoxia

TGF-β is the most potent cytokine causing fibrosis. Both smad-dependent and smad-independent TGF-β signaling pathways are known. Initiation of both pathways takes place via binding of TGF-β to its receptor. TGF-β binds to a type II receptor, which then phosphorylates a TGF-β type I receptor. Subsequently, the type I receptor phosphorylates R-smads (receptor-regulated smads), and phosphorylated R-smads bind to co-smad (common-mediator smad). R-smad/co-smad complexes translocate into the nucleus, where they act as transcription factors[ 38]. In this manner, regulation of TGF-β target gene expression is carried out. Expression of many proteins in MRC-5 cells changes after stimulation by TGF-β[ 39].Que es anoxia A frequently used marker of the activation of myofibroblast is α-SMA; therefore, this protein was also used as a marker in this study. Expression changes after TGF-β stimulation in cells other than MRC-5 has been observed, for example, in IMR-90 human lung fibroblasts[ 33] and WI38-VA13 cells[ 40]. Besides α-SMA, upregulation of collagen I[ 41], fibronectin[ 27]and CTGF[ 42] has been reported when human lung fibroblasts are treated with TGF-β.

The expression of α-SMA is regulated by smad[ 43]. TGF-β increases the nuclear translocation of smad and expression of α-SMA. We examined the influence of EGCG treatment on smad2/3 appearance in MRC-5 cells. Immunohistological experiments indicated that EGCG inhibits the nuclear transportation of smad2/3.Que es anoxia

Moreover, we found that EGCG had suppressive effects on the expression of α-SMA in MRC-5 cells, whereas catechin did not. These data suggest that the effects are dependent on the gallate or pyrogallol moiety of EGCG.

Next, we investigated the mechanism of the inhibitory effect on the smad2/3 pathway. EGCG is a potent antioxidant and a lot of its health benefit effects are thought to be due to its antioxidative action[ 44- 46]. EGCG attenuated the increase in α-SMA expression brought about by TGF-β, whereas edaravone and NAC did not. These results indicate that the inhibitory effect of EGCG on α-SMA expression is independent of its antioxidative action.

We thought that part of the EGCG’s effects on α-SMA expression might arise through interference with receptor-ligand binding.Que es anoxia indeed, EGCG treated cell lysate containing TGFRII showed no immunoprecipitation with anti-TGFRII antibody. The interaction between EGCG and TGFRII was also confirmed by the affinity chromatography experiment. A likely explanation for this observation is that EGCG binds to TGFRII, thereby blocking the antibody from binding to TGFRII. Similarly, if EGCG binds to the TGF-β receptor, TGF-β would not be able to bind to its receptor and downstream signaling pathways would be ineffective.

In conclusion, we have shown that EGCG interacts with TGFRII and inhibits the expression of α-SMA via the TGF-β-smad2/3 pathway in MRC-5 cells, which are human lung fibroblasts. These results suggest that EGCG has anti-fibrotic effects that are crucial for the control of myofibroblast differentiation and extracellular matrix deposition, which are involved in fibrosis.Que es anoxia the evidence that EGCG is effective in the suppression of fibrosis may lead not only to better understanding of the biological roles of EGCG but also to clinical applications of this flavonoid.

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