The Role Of Rho-Rock On Myofibroblast Transdifferentiation In Lung Fibroblasts

Interstitial lung disease / pulmonary fibrosis is a serious risk to human health, the incidence rate is in a gradually rising trend. At present there is no effective treatment for the unknown etiology as well as unclear pathogenesis. Although it's difficult to eliminate formation of pulmonary fibrosis, pulmonary fibrosis is a chronic, progressive pathological process. The strategies through exploring the molecular mechanisms of the pathogenesis of pulmonary fibrosis in delaying or blocking the process of fibrosis development still has important practical significance and has always been a focus throughout the pneumo-science circle internationally.In the process of fibrosis, lung fibroblasts into myofibroblasts play a key role. Myofibroblasts are fibroblast-like cells with the characteristics of smooth muscle cells, particularly expression ofα-smooth muscle actin (α-SMA). Fibroblasts can transdifferentiate to myofibroblasts following the exposure to a variety of cytokine, among which TGF-βis a potent stimulus for myofibroblast differentiation. Myofibroblasts differentiation promoted by TGF-β/Smad is a definite mechanism in the process of pulmonary fibrosis. It is proved that TGF-β/Smad is a main but not only pathway of myofibroblasts differentiation. Research has found Rho-Rock signal pathway regulates the expression of myofibroblast phenotype marker—α-SMA, however, the specific mechanism is not clear yet. It is reasonable to hypothesize that Rho-Rock and TGF-β/Smad signal transduction pathways are commonly involved in the fibroblast-myofibroblasts transdifferentiation , and the two transduction pathways may have cross-talk. Therefore, Rho-Rock and TGF-β/Smad play an important role in the occurrence and development of pulmonary fibrosis. If our assumptions can be tested, it may not only improve Rho-Rock system, Rho-Rock signaling pathway in lung fibrosis gives a more objective assessment of the role, but also further improves the TGF-β/Smad signal transduction pathway.Objective: To identify the contribution of Rho-Rock signal pathway to the transdifferentiation of lung fibroblast-myofibroblast under the stimulation of TGF-β1 and to elucidate the role of Rho-Rock signaling in the progression of lung fibrosis, the cross-talking between Rho-Rock and TGF-β/Smad in lung fibrosis was also detected so as to provide a new theoretical basis for the prevention of pulmonary fibrosis.Method: The human embryonic lung fibroblast were cultured in vitro,fibroblasts were treated with either TGF-β1 alone or with one of the following blocking agents : Y-27632 ( a inhibitor of Rho-Rock signaling ) and Staurosporine ( inhibitor of Serine and Threonine kinases , a blocking agent of TGF-β/Smad signaling ), flow cytometry and Cytochemical immunofluorescence were performed to detect the expression ofα-SMA , flow cytometry was used to observe cell circle , ELISA were performed to test the content of extracellular matrix in cell culture supernatants , RT-PCR was performed to detect the levels of RhoA,RhoC,Rock1 and Smad2 mRNA.Result:(1)TGF-β1 can significantly promote lung fibroblastα-SMA expression which indicates myofibroblast differentiation has increased, whileα-SMA expression is potently inhibited with the addition of the Y-27632 or Staurosporine.(2)TGF-β1 decreased the cells of G0/G1 phase and increased the percentage of cells in S phase . Fibroblasts were treated with TGF-β1 and Y-27632 or Staurosporine, the percentages of cells in G0/G1 phase increased, in S and G2/M phase decreased.(3)TGF-β1 increased FN,LN,type I collagen production in supernatants of lung fibroblasts. In contrast, Y-27632 or Staurosporine significantly reduced,TGF-β1 stimulated FN,LN,type I collagen production in supernatants of lung fibroblasts.(4) TGF-β1 up-regulated the mRNA level of RhoA,RhoC,Rock1,Smad2 in lung fibroblasts. In contrast , Y-27632 or Staurosporine reduced the RhoA,Rock1,Smad2 mRNA expression stimulated by TGF-β1, Y-27632 also attenuated RhoC mRNA expression driven by TGF-β1.Conclusion:( 1 ) Rho-Rock pathway participated in lung fibroblast-myofibroblast differentiation, blocked the pathway which may inhibit fibroblast proliferation and extracellular matrix synthesis.(2)Rho-Rock pathway and TGF-β/Smad pathway cross-talks between each other.