Inhibition Of FRNK On The Fibronectin-Induced HSC Migration And Molecular Mechanism

Actually, liver fibrosis is a repairing process of the body towards the injury. The moderate repair may reconstruct the liver normal structure, however, if not limited, the excess deposition of extracellular matrix (ECM) finally leads to a scar formation or liver fibrogenesis. The activation, proliferation, adhesion, migration of HSC and the production of ECM are the central events in the liver fibrogenesis. During liver fibrogenesis, the expression of integrin increases or is induced to express, just like a bridge connecting hepatic stellate cell (HSC) and ECM. Focal adhesion kinase (FAK) is the medium that connects integrin and the downstream signal molecules in integrin-signal transduction pathway, and is the convergence of many signal pathways. In the integrin-signal transduction pathway which FAK is central, except for the Ras-MAPK pathway, there is also phosphatidylinositol-3-kinase (PI3K) signal molecule. Through self-phosphorylation, FAK can recruit and activate the downstream signal molecules, thus modulate the cell framework and the expression of some genes,at last regulate cellular processes such as cell spreading, migration, proliferation, differentiation and cell survival etc. In our past study, FAK and extracellular signal-regulated kinase (ERK) have been found to change dynamically in liver fibrogenesis. Focal adhesion related non-kinase (FRNK) acts as an endogenous inhibitor of FAK phosphorylation. In recent years, study has focused on the integrin-FAK signal pathway and the role of it in cell adhesion and migration, but whether FAK takes part in the HSC migration process hasn't been proved. So with the help of cell culture in vitro, we set about our research from the interaction of HSC and ECM. We transfected HSC using FRNK expressing plasmid and studied the effects on the adhesion and migration of HSC after selectively breaking the phosphorylation of FAK and the role of some signal molecules in this process. The experiments contained three parts as below: Part 1: The Dynamic Expression of FAK,PI3K,ERK and AP-1 during Hepatic Fibrogenesis in Rats Objective:To investigate the role of FAK,PI3K,ERK and transcription factor AP-1(c-fos, c-jun), especially their phosphorylated forms in the hepatic fibrogenesis Methods:A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the formation of liver fibrosis. Dynamic expressions of FAK,p-FAK (Tyr³⁹⁷),PI3K,ERK,p-ERK and transcription factor AP-1 (c-fos,c-jun) were evaluated by RT-PCR at mRNA level and by Western blot at protein level, respectively. Results: â‘ Building of liver fibrosis model: The results of Hematoxylin and eosin staining and Masson's trichrome methods showed that in sham-operated group, the structure of liver lobule was integrin and the array of the liver flank was in order. But in model groups, the connective tissue hyperplasiaed broadly and the collagen fiber deposited around the central vein. The structure of the lobule was disturbed even the pseudo-lobules were formed. â‘¡Western blot analysis showed that in the sham-operated group, the actin was expressed a little, but with the development of liver fibrosis, the expression of it increased in model group. And the expression peak happened in the fourth week, 1.82 times of that of 1 wk and 14.53 times of that of sham-operated group; FAK, the molecular weight is 125 kD, expressed in normal rats, but with the period of making model prolonged, the expression increased 14.9,25.8,29.9 and 31.7 times than that of the sham-operated group. Phosphorylation of Tyr³⁹⁷ results in the activation of FAK. There was a little expression of p-FAK (Tyr³⁹⁷) in the sham-operated group, but with the development of liver fibrosis, the expression of p-FAK (Tyr³⁹⁷) increased and reached the peak value at the fourth week, which was 1.68 times of that of the first week and 6.16 times of that of sham-operated group, P<0.01; By RT-PCR analysis, FAK mRNAappeared in the liver of normal rat, and was upregulated on the 2nd day after bile duct ligation, and the upregulation continued during the liver fibrosis, the expression increased 4.7 times in the fourth week. â‘¢The PI3K, molecular weight 85 kD, was expressed in all groups. Compared with the sham-operated group, PI3K increased 1.20,1.54,1.90 and 7.41 times, and the difference was significant, P<0.05;â‘£With the development of liver fibrosis, the expression of ERK1/2 increased, and in the fourth week, ERK1/2 reached 1.89 times of that of sham-operated group. So that was p-ERK: compared with the sham-operated group, the p-ERK expression increased 1.01, 1.11, 1.18 and 1.46 times, respectively. According to our study, with the fibrosis developing, ERK mRNA increased simultaneously. ⑤Using the RT-PCR assay, c-fos and c-jun mRNA were expressed in sham-operated group. The levels of them were highest in the fourth week and 14.33 and 12.38 times of that of the sham-operated group. Conclusions: In liver fibrogenesis, the expression of FAK,PI3K,ERK and transcription factor activator protein-1(c-fos,c-jun) increased obviously. So the increases of them play a pivotal role in the formation and development of liver fibrosis. Part 2: FRNK inhibits the HSC adhesion and migration Objective: FRNK plasmid was used to transfect HSCs, so as to evaluate the effects of breaking the phosphorylation of FAK on the HSC adhesion and migration. Methods: The adhesive inhibition of FRNK on HSCs was examined by toluidine blue colorimetric assay,and the inhibition of migration of FRNK on HSCs was evaluated by improved Boyden chamber. And the levels of FAK in HSCs were assayed by Western blot on protein level and RT-PCR on mRNA level. Results: â‘ Western blot analysis showed: the FAK expression increased obviously after cultivated with FN, compared with the control group, the expression increased 92.73%,P<0.01;But there was no significant difference among FN group, FN+liposome group and FN+liposome+empty plamid group,P>0.05. After transfection by FRNK gene for 48 h, the FAK expression of HSCs reduced significantly. Compared with the empty plasmid group, the expression of FAK(45448.7±3388.3)reduced 92.5%, there was statistic difference between them, P<0.01; The expression of p-FAK(Tyr397)protein (3213.6±1090.1) reduced after being transfected by FRNK, P<0.01;After HSCs transfected by plasmid FRNK for 8 h, FAK mRNA expression showed that there was no difference among FN group, FN+liposome group and FN+liposome+empty plamid group, but in the group transfected by FRNK, FAK mRNA reduced 27.4% than that in the empty-plasmid group.â‘¡The inhibition of proliferation by FRNK in HSCs: after being transfected by FRNK plasmid, the proliferation of HSCs was inhibited. The inhibition rates were 72.02%,82.04%,89.14% at 12 h,24 h,48 h, respectively. So FRNK plasmid could dramatically inhibit the proliferation of HSCs in time-dependent manner.â‘¢The inhibition of FRNK on HSC adhesion: compared with the control group, the adhesion rate increased in FN group, FN+ liposome group, FN+liposome+empty plamid group and non-FRNK group, but there was no statistic difference among them, P=0.767. After transfected by FRNK for 24 h,48 h the adhesion rate of HSCs reduced 37.56% and 38.85% than that of empty-plasmid group, there was significant difference (P=0.000) and was time-dependent. â‘£The inhibition of FRNK on HSCs migration: Compared with the control group, the cell number of migrating to the bottom chamber increased in FN group, FN+liposome group, FN+liposome+empty plamid group and non-FRNK empty plasmid group, but there was no statistic difference among them. After being transfected by FRNK, the number of crossing membrane of HSCs reduced 47.8% and 56.4% than that of empty-plasmid group, there was statistic difference and in time-dependent manner. Conclusions: The expression and phosphorylation of FAK in HSCs were inhibited after being transfected by FRNK. And FRNK can inhibit FAK both at protein and at mRNA levels, and inhibit the HSC proliferation, cell adhesion and migration in the same time course and in time-dependentmanner. Part 3:The molecular mechanism of the inhibiting migration of FRNK on HSCs Objective: To explore the molecular mechanism of FRNK's inhibiting HSCs migration induced by fibronectin. Methods: HSCs were cultured in vitro, and the expression of FAK, PI3K, ERK and transcription factor AP-1(c-fos,c-jun)were assessed using Western blot and RT-PCR assay. Results: â‘ Western blot showed: FAK expression increased obviously after cultivated with FN, compared with the control group, up to 92.73%,P<0.01;But there was no significant difference among FN group, FN+liposome group and FN+liposome +empty plamid group, P>0.05. After transfected by FRNK gene for 48 h, the expression of FAK reduced obviously in HSCs. Compared with the empty plasmid group, the expression of FAK (45448.7±3388.3) reduced 92.5%, there was statistic difference between them, P<0.01; The expression of p-FAK(Tyr³⁹⁷) protein (3213.6±1090.1) reduced after being transfected by FRNK, P<0.01;After transfected by FRNK for 8 h, we examined the level of FAK mRNA by RT-PCR. The results showed that there was no difference among FN group, FN+liposome group and FN+ liposome+empty plamid group, but in the group transfected by FRNK, FAK mRNA reduced 27.4% than that in the empty-plasmid group. â‘¡Western blot showed: Induced by FN, the expression of PI3K(189.9±8.0)increased much more than that in the control group(165.4±13.0). The increasing rate was 14.8%,P=0.013; But there was no significant difference among FN group, FN+liposome group and FN+liposome+empty plamid group, P>0.05. after being transfected by FRNK for 48 h, the expression of PI3K(86.7±6.4)of HSCs reduced obviously, compared with the empty plasmid group (189.4±11.3), it reduced 54.25%. there was statistic difference between them, P<0.01; The expression of PI3K protein reduced after being transfected by FRNK, P=0.000;After transfected by FRNK for 8 h, we examined the level of PI3K p85 mRNA by RT-PCR. The results showed that there is no difference...