Study Of TSC1/TSC2 Involved In Proliferation And Differentiation Of Human Lung Embryo Fibroblasts

Pulmonary fibrosis (PF) comprises a group of interstitial disorders of the lung parenchyma that develop as a consequence of multiple causes, including radiotherapy and chemotherapy for lung neoplasms. Pulmonary fibrosis is regulated by a variety of cytokines. Pulmonary fibroblast plays an essential role in the formation and maintenance of lung structure and function. Recently the primary treatment of PF is using glucocorticoid, cytotoxic drug and so on, but the effects are not very ideal and there’re serious side effects for patients. However, the molecular mechanisms are still obscure. And finding the effective drugs for the pulmonary fibrosis has been the concern.The mTOR is serine-threonine kinase that receives input from various signaling pathways to activate translation and increases cell proliferation and growth. Inhibiting mTOR signaling pathway can reduce fibrosis. TSC1 and TSC2 gene products form a heterodimer which surpresses the mammalian target of rapamycin (mTOR).The TSC1 (on chromosome 9q34) or TSC2 (on chromosome 16p13.3) tumor suppressor genes encode the protein products TSC1 (130 kDa) and TSC2 (200 kDa) also known as hamartin and tuberin, respectively. Recent genetic studies demonstrated that high levels of tuberin had negative effects on cell proliferation, whereas downregulation or loss of tuberin upregulated proliferation rates, and hamartin also negatively affected the control of cellular proliferation. Recent studies demonstrated that active mTOR signaling pathway can promote kidney interstitial fibroblast activation and kidney fibrosis, and mice with fibroblast-specific Tscl deletion exhibit kidney interstitial fibroblast activation and kidney fibrosis. However, the function of TSC1 and TSC2 in the survival of human lung fibroblast is still unclear.In this study, we investigated the role of TSC1 and TSC2 in the TGF-β1-induced proliferation of human lung fibroblasts and their differentiation into myofibroblasts.1. The expression of TSC1 and TSC2 in HELF cellsWe sought to determine whether TGF-β1 regulated TSC1, TSC2 expression in activated HELF cells. As expected, results of Western blot demonstrated that expression of TSC1, TSC2 was suppressed in response to TGF-β1. Thus, our results suggested that down-regulation of TSC1, TSC2 may be involved in the differentiation and proliferation of HELF cells.2. Overexpression of TSC1 or TSC2 inhibits TGF-β1-induced HELF differentiation and proliferationWe firstly investigated the effect of pcDNA3-HA3-TSC1 on the expression of TSC1, pcDNA3-HA3-TSCl enhanced the expression of TSC1 and inhibited TGF-β1-induced differentiation. We found that induction of collagen I and a-SMA expressions were biomarkers of pulmonary fibrosis which were repressed in TSC1-overexpressed cells. Flow cytometry analysis showed that transfection of HELF cells with pcDNA3-HA3-TSC1 resulted in increased G0/G1 phase and decreased S and G2/M phases significantly. Secondly, we investigated the effect of pcDNA3-FLAG-TSC2 on the expression of TSC2, transfection of HELF cells with pcDNA3-FLAG-TSC2 significantly increased TSC2 expression and inhibited TGF-β1-induced differentiation while repressing the expression of Collagen I and a-SMA. Result of flow cytometry analysis demonstrated that transfection of HELF cells with pcDNA3-FLAG-TSC2 led to increased G0/G1 phase and decreased S and G2/M phases.3. Methylation of the TSC1 and the TSC2 promoter regionWe employed pyrosequencing to detect the methylation of TSC1 and TSC2 promoter region. The TGF-β1 treated group TSC1 promoter in Pos.1、Pos.2 and Pos.3 were found to be higher methylated than the control group. The TGF-β1-treated group TSC2 in Pos.2 、Pos.3、Pos.、Pos.5 and Pos.6 were found to be higher methylated than the control group. The effect of 5-azadC on the expression of TSC proteins was detected by Western blotting, and the result revealed that 2 μg/ml 5-azadC significantly up-regulated the protein levels of TSC 1 and TSC2.4. Effect of MeCP2 on TSC1 and TSC2The expression of MeCP2 was induced by TGF-β1 in a time-dependent manner. Results demonstrated that knockdown of MeCP2 with siRNA-MeCP2 significantly increased expression of TSC1 and TSC2 while inhibiting TGF-β1-induced cell differentiation and production of Collagen I and a-SMA. By contrast, overexpression of MeCP2, the effect of TSC proteins down-expression using pEGFP-C2-MeCP2 and up-expression of Collagen I and a-SMA was analyzed by Western blotting, respectively. Taken together, the results above suggested that MeCP2 had impacts on the expression of TSC1 and TSC2, and TGF-β1-induced proliferation of HELF cells were mediated by MeCP2.We provide novel evidence that TSC1 and TSC2, which may be affected by DNA methylation, play a vital role in modulating HELF cell proliferation and differentiation. Enhanced the expression of TSC1 and TSC2 may inhibit pulmonary fibrosis.