Expression And Effect Mechanism Of Activin Receptor-Interacting Protein 2 In Mouse Hepatocytes

Activin is a member of the transforming growth factor-beta (TGF-β) superfamily as growth and differentiation factors, not only can regulate secretion of follicle-stimulating hormone (FSH) and follistatin (FS) from anterior pituitary, but also can serve as an important regulator in the process of embryo formation, nerve differentiation, hematogenous cells proliferation, tissue repair and inflammation recovery. Activin contains three forms, activin A (pApA), activin B (pBpB) and activin AB (PApB). Activin A not only has the strongest effect, but also close related with liver disease. It is reported that activin can cause, liver fibrosis, and the inducer of liver fibrosis, TGF-β, can also depend on the effect of endogenetic activin partially.The actions of activin on target cells are tissue-specific, which associate with the difference of activin receptor signal transduction. Activin receptors are the members of Ser / Thr kinase receptors. Activin binds to receptor typeⅡto form a complex primarily. The complex interacts with the receptor typeⅠand makes it phosphorylated, then activates endocellular Smad protein binding to receptor typeⅠ. However, corresponding Smad protein can be expressed in target cell which interacts with activin. Therefore, it is difficult to explain tissue specificity of activin actions. ActRIPs are a new group of signal transduction regulators. The latest studies indicated activin receptor-interacting proteins (ActRIPs) were a group of signal transduction regulators. As Smad upstream signal regulators, ActRIPs can bind to ActRⅡand have an impact effect on endocellular signal transduction induced by Activin. The main characteristics of ActRIPs are the differences of its histological distribution and biological activity, which also are the crucial molecules to determinate histological specificity of activin effects. It has been demonstrated that ActRIP2 could mediate the endocytosis of ActRII A via RalBpl protein to suppress activin signal transduction. Therefore, it could infer that we can reverse the effect of activin on liver and achieve the goal of treatment of liver disease. In the present study, we explored the biological action of ActRIP2 in hepatocytes and its suppressive effect on hepatocytes pathological progress induced by activin.We analyzed the effect of ActRIP2 on mouse liver fibrosis in vivo by ActRIP 2 overexpression and gene silence using liver injury model mouse. We explored the mechanism of ActRIP2 mediating activin signal transduction in vitro and discussed the regulation factors of ActRIP2 expression in hepatocytes and effects of activin signal transduction mediated by ActRIP on Hepal-6 cells. The results indicated that ActRIP2 can suppress the biological activity mediated by activin in liver as activin negative regulator, and it may serve as an effective therapeutic target of liver injury and fibrosis.The biological signification of ActRIP2 in the process of mouse liver injury induced by Con AExperimental animal model is useful to explore the mechanism of various diseases and drug screenings. We obtained the model of human liver injury by injecting Con A (8mg/kg) into mouse C57BL/6 for 3 weeks and observed the interrelated changes at 24h, 72h, 120h, 168h after the last injection. The results showed that mouse liver injury is the most serious at 72h and it can be relieved depending on time by serum enzymological and pathological analysis. The pathological changes are negatively associated with the transcription level of ActRIP2 mRNA in liver tissues. The expression level of ActRⅡA mRNA peaked at 72h. However, activin A expression keeped high level all the time. The above data suggested that activin A-ActRⅡA pathway may be associated with the development of liver injury, whereas ActRIP2 can be a negative regulator in the process of mouse liver injury induced by Con A, so that it is useful for the repair of damage liver. Thus, upregulation of ActRIP 2 expression can suppress or reverse the effect of activin in liver diseases.The effects of ActRIP2 gene silence and overexpression on mouse liver injury induced by ConAWe injected pcDNA3-ActRIP2, ActRIP2 siRNA into mouse C57BL/6 via tail vein using liposome 24h before Con A stimulation for 9 weeks. The results showed that both serum ALT and AST increased, and all the liver indexes in ActRIP2 gene silence group are more raised than the group stimulated by ConA alone, with p＜0.01, which was coincident with the result by pathological slice examine. The hepatocytes were varicose, cytoplast showed balloon-like changes after dye, and the cells were cracked and necrotic. Whereas the indexes such as serum enzymology changes, liver changes, pathological changes were all close to normal in ActRIP2 overexpression group, which suggested that ActRIP expression changes can influence on the process of liver pathological injury. It may be that ActRIP2 expression suppresses the effects of activin A on hepatocyte death, ECM secretion increase and so on in liver tissue. ActRIP2 gene silence can inhibit feedback regulation of ActRIP2 in mouse to aggravate liver pathological damage. Thus, activin A may has effects on the development of liver fibrosis and block it's signal transduction in liver to control liver disease.The regulative effects of ActRIP2 on activin signal transduction in Hepal-6 cellsHepal-6 cells from mouse hepatoma cell line have activities of hepatocytes. It is showed that ActRIP2 can be expressed in Hepal-6 cytoplasm by immunohistochemical stain. The expression level of ActRIP2 mRNA can be increased in Hepal-6 cell stimulated by activin A(5ng/ml) after 12h and peak at 24h, and high levels can be kept. Whereas the expression level of ActRⅡA mRNA can be increased at 2h evidently, after which it can be reduced but keep in a certain level. However, the expression level of AcrRⅡB mRNA did not change obviously. The expression peak of ActRIP 2 mRNA is later than ActRIIA stimulated by activin A in Hepal-6 cells. Which indicated that ActRIP2 can participate in the negative feedback regulation of activin signal transduction in the late stage. We co-transfect pcDNA3-ActRIP2 and CAGA-lux plasmid into Hepal-6 cells and found that ActRIP2 can evidently suppress the transcription activity of gene induced by activin A. Expression level of ActRIIA on membrane surface was reduced after ActRIP2 overexpression in Hepal-6 cells, obviously. These findings suggest that ActRIP2 participates in the process of activin signal transduction in the late stage as a negative regulator.The regulation of ActRIP2 expressionActRIP2 can participate in activin-ActRⅡA signal transduction pathway in Hepal-6 cells as a negative regulator, and the expression level of which is associated with hepatocyte biological activity. It was found that activin A, endotoxin LPS, PMA and forskolin can promote the expression of ActRIP2 mRNA, whereas calcium ion vector A23187 can suppress ActRIP2 expression in hepal-6 cells. Thus, ActRIP2 expression can be regulated by various factors.The regulative effects of activin signal transduction mediated by ActRIP2 on Hepal-6 activityActivin can affect Hepal-6 cell biological activity evidently. For example, Activin A can promote synthesis of ECM and suppress its degradation by stimulating the mRNA expression level of FN and Timp2 and down regulating MMP2 expression. The mRNA expressions of FN, MMP2 and TIMP2 stimulated by activin A are reversed after ActRIP2 overexpression in Hepal-6 cells. Moreover, Activin A can inhibit Hepal-6 cell proliferation. The proliferous rate of Hepal-6 cells can arrive at 30% after stimulated by activin A (10ng/ml). The effect of activin A on Hepal-6 cell proliferation was reduced or disappear after transfected by pcDNA3-ActRIP2. The inhibition effect of activin A on Hepal-6 cell proliferation was evidently enhanced after ActRIP2 gene silence. It suggests that ActRIP2 can reverse the biological activity of activin as a negative regulator of activin signal transduction in hepatocytes.In conclusion, ActRIP2 can be expressed in hepatocytes and negatively regulate biological activity of activin in liver. The increase of ActRIP2 expression is useful for both the repair of damage liver and the suppression of formation and development of liver fibrosis. Thus, it may serve as an effective therapeutic target which can inhibit activin ontogenetic activity in liver.