Epigallocatechin-3-gallate suppresses transforming growth factor-beta signaling by interacting with the transforming growth factor-beta typeⅡreceptor

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 inhuman 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.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 TGFRⅡ and that catechin did not at all. 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.

Effect of mango seed kernel extract on the adipogenesis in 3T3-L1 adipocytes and in rats fed a high fat diet

Mangoes (Mangifera indica L.) are one of the most important tropical foods. The seed is one of the main by-products of mango processing. Therefore, it is important to find an economically viable use for this waste (e.g., as a food additive or supplement with high nutraceutical value). We investigated the anti-obesity effects of mango seed kernel extract with hot water (MSKE-W) in 3T3-L1 adipocytes and in a high fat diet (HFD)-induced obesity rat model. MSKE-W caused a significant decrease in the activity of glycerol 2-phosphate dehydrogenase in 3T3-L1 adipocytes without eliciting cell cytotoxicity and inhibited cellular lipid accumulation through down-regulation of transcription factors such as PPARγ and C/EBPα. In the animal model, rats fed an HFD containing 1% MSKE-W gained less weight than rats fed an HFD alone. The visceral fat mass in rats fed an HFD containing 1% MSKE-W tended to be lower than that in rats fed an HFD alone. Furthermore, histological examination of rat livers from an HFD showed steatohepatitis. However, rats on an HFD containning 1% MSKE-W showed no histopathological changes in liver tissue. Our results indicate that MSKE-W influences anti-obesity effects, both in vitro and in vivo, and suggest that MSKE-W provides a novel preventive potential against obesity.

Interaction of calcium- and integrin-binding protein 1 with integrin <i>α</i>11 and its possible involvement in pulmonary fibrosis

Integrin α11 (ITGA11) is one of the collagen-binding integrin α chains;however, its biological significance remains unknown. To determine the functions of ITGA11, we performed a yeast two-hybrid screen using the cytoplasmic domain of ITGA11 as bait and transformed an EGY48 yeast strain with the bait-containing plasmid using the plasmid from a human lung fibroblast cDNA library. This screen identified calcium- and integrin-binding protein 1 (CIB1) as prey. Recombinant ITGA11 and CIB1 were expressed in mammalian cells and used in coimmunoprecipitation experiments, which showed that full-length ITGA11 and CIB1 are also associated in vivo. Over-expression of CIB1 in the human lung myofibroblast MRC-5 cells decreased the expression of α-smooth muscle actin and fibronectin. Using a mouse model of pulmonary fibrosis (bleomycin-treatment), we detected elevated expression of CIB1 in lung tissues compared with controls. These data suggest that CIB1 may regulate pulmonary fibrosis in concert with IT-GA11.