Role Of SATB1on Hepatic Stellate Cell Activation And Liver Fibrosis

Background and aimLiver fibrosis, a major cause of high morbidity and mortality worldwide, is characterized by excessive and abnormal deposition of extracellular matrix, which leads to liver damage and seriously threatens human health. Hepatic stellate cells (HSCs) play a central role in the development of liver fibrosis. Previous studies have established the important role of TGF-β1and MAPK in driving the activated phenotype in HSCs. In the special AT-rich binding protein l(SATBl), a nuclear matrix attachment region binding protein, acted as a molecular switch that could modulate numerous gene expression. DNA microarray analysis revealed that SATB1modulates cell proliferation, apoptosis, adhesion, and migration by regulating genes involved in many key signaling pathways, many of which are closely related to liver fibrosis (e.g., the signaling pathways of TGF-β1, MAPK, Wnt, etc.). Thus far, the effect of SATB1expression on HSCs and liver fibrosis has remained unknown. Therefore, we infer that as a global gene regulator, SATB1may involve in the regulation of hepatic stellate cells activation and liver fibrosis.MethodIn order to investigate the relationship between SATB1and HSCs activation, a well-established rat model of liver fibrosis induced by thioacetamide (TAA) was used. HSCs isolated from both the normal and TAA rats were cultured for3days and then the expression of SATB1was measured. HSCs were freshly isolated and cultured for up to7days with or without TGF-β1treatment. The expression of SATB1was assayed by real-time PCR and Western blot. In order to investigate the role of SATB1on the activation of HSCs, chemically synthesized siRNAs directed against SATB1 were transfected into the HSCs (LX-2and rat HSCs) to establish SATB1-knockdown cells. In addition, LX-2were transfected with pcDNA3.1(-)-SATBl and rat HSCs were infected with AdSATB1to establish SATB1-overexpressed cells. The expression of SATB1, a-SMA, CTGF, COL1A1, COL3A1was determined by real-time PCR and Western blot. The effect of SATB1on cell proliferation and cyclin Dl/El expression were studied by CCK-8assay and real-time PCR. The effect of SATB1on cell migration and adhesion were investigated by transwell assay and cell adhesion. In order to investigate the special signal transduction pathway that SATB1regulated CTGF expression, Western blot was used to analyze the activation of Smad3, Smad4, Ras, Raf-1, MEK, ERK, P38, JNK, Ets-1in SATB1-knockdown cells and SATB1over-expression cells. In addition, we used the inhibitos of the Ras, Raf-1, MEK, ERK signaling molecules and Ets-1siRNA to treat SATB1over-expression cells. The rat liver fibrosis model were induced by TAA injection. The therapeutic effect and mechanism of SATB1on fibrotic rat liver were investigated by H&E staining, Masson’s trichrome staining, Sirius Red staining, the content of hydroxy-proline, IHC, real-time PCR and Western blot.ResultOur study showed that lower expression of SATB1was observed in activated rat HSCs isolated from TAA-induced fibrotic rats as compared with quiescent phenotypes from normal rats. In addition, a decline in SATB1expression was also observed in the two models of in-vitro activation of rat HSCs. SATB1silencing significantly increases expression of fibrosis-related genes (a-SMA, CTGF, COL1A1, and COL3A1) in LX-2and rat HSCs. Conversely, SATB1overexpression dramatically inhibits these genes expression, providing evidence that SATB1negatively regulates HSCs activation and collagen synthesis. Moreover, SATB1silencing significantly promotes LX-2cells proliferation by induction of cyclin E1; otherwise, upregulation of SATB1inhibits LX-2cells proliferation and cyclin E1 expression. Additionally, HSCs migration is induced or inhibited correspondingly after SATB1expression is silenced or enhanced. SATB1had no effect on the mRNA level of TGF-β1, Smad2, Smad3, Smad4and Smad7. At the same time, Smad3phosphorylation and Smad4nuclear translocation were also unchanged. After SATB1expression was silenced, the level of phospho-Ets-1(Thr38), phosphor-ERK, phosphor-MEK, Ras-GTP and the membrane translocation of Raf-1were significantly elevated,vice versa. The elevation of CTGF caused by the knock down of SATB1was noticeably reduced by treatment with Ets-1siRNA, U026, PD98059, GW5074and Manumycin A. Animal experiments were carried out to prove the effect of SATB1in vivo. Consistent with the in vitro data, adenovirus-mediated SATB1administration significantly attenuated ECM deposition and improved liver function compared with AdGFP or PBS injection. Additionally, the expression of α-SMA, CTGF, and COL1A1were also decreased after AdSATB1injection. These genes were mainly produced by activated HSCs, which indicated that SATB1may attenuate liver fibrosis through deactivating HSCs. Adenovirus-mediated SATB1administration significantly attenuate liver fibrosis.ConclusionsSATB1expression negatively correlated with HSCs activation and, in addition, inhibited HSCs activation, proliferation, and migration. Additionally, the CTGF expression was inhibited by SATB1through the Ras/Raf-1/MEK/ERK/Ets-1pathway in LX-2cells. Furthermore, the in vivo study showed that adenovirus-mediated SATB1injection can efficaciously inhibit HSCs activation and attenuate rat-liver fibrosis and improve liver function. Collectively, the evidence points to the important role of SATB1in HSCs activation and fibrosis.