The Regulative Effects Of FRNK On Collagen Metabolism In Hepatic Stellate Cells

Hepatic fibrosis, the final stage of which is cirrhosis, can result from several chronic liver diseases caused by many pathogenic factors, and the essence of this event is the pathological deposition of extracellular matrix (ECM). The activtion of hepatic stellate cells(HSC), which then synthesize a large quantity of collagen, especially Type[0]Ⅰcollagen, play a crucial role in the deposition of ECM . Therefore, the inhibition of collagen synthesis, particularly TypeⅠcollagen, is the central step to treat hepatic fibrosis.The synthesis and degradation of collagen in HSC are mainly modulated by matrix metalloproteinase (MMP). Recently, it has been documented that membrane-type matrix metalloproteinase-1(MT1-MMP) is the mainly membrane-type matrix metalloproteinase, which could not only degradate typeⅠcollagen but also regulate collagen degradation by activating matrix metalloproteinase-2(MMP2). MT1-MMP and MMP2 have the same inhibitive factor—tissue matrix metalloproteinasea-2 ( TIMP-2 ) , which promote collagen deposition by inhibiting their degradation effect on collagen.The collagen metabolism of HSC is modulated by many signal transduction pathways. Focal adhesion kinase (FAK) has been indicated as a point of convergence of integrin and growth factor signal pathways. The foundation research indicates that phosphorylated FAK has significant influence on collagen metabolism in many kinds of cells. Tyr397 is an autophosphorylation site in kinase region of FAK. phosphorylated Tyr397 can magnify the activity of FAK by activating the rest five phosphorylation sites of FAK in order. Therefore, we can conclude that Tyr397 is the trigger point of FAK in playing biology role.The C-terminal, non-catalytic domain of FAK, termed FRNK (FAK-related-non-kinase), is an endogenous inhibitor of FAK,and serve as a negative regulator of kinase activity. In many cells, overexpression of FRNK can inhibit cell collagen synthesis. But the effects of FRNK on collagen metabolism of HSC and their molecular mechanisms have not yet been reported.Therefore, using in vitro cell culture technique, the effects of selective blockade of FAK phosphoralation on the collagen metabolism in fibronectin (FN)-stimulated HSC were investigated by liposome-mediated transient transfection of FRNK into HSC, and its subsequent association with some signal molecules in the process also was explored.ObjectiveFRNK plasmid was used to transiently transfect FN-stimulated HSC in vitro, so as to evaluate the effects of inhibiting the phosphorylation of FAK by FRNK on collagen synthesis of HSC and the interaction of FAK signaling pathway with MT1-MMP,MMP2,TIMP-2 in hepatic fibrosis.MethodsHSC were cultured in vitro in RPMI 1640 medium supplemented with 2% Fetal Bovine Serum, 100 IU.mL-1 penicillin, 100μg·mL-1 streptomycin, 4 mmol·L-1 glutamine, and 1 mol·L-1 HEPES in a 37℃and 5% CO2 environment. The proliferation of HSC was stimulated by fibronectin (FN),and the experimental steps were supplied by Lipofectamine? Reagent. FRNK plasmids were transfected into HSCs mediated through liposomes. The cells were grouped into the following 5 groups:①control group;②FN group;③liposome group;④blank plasmid group;⑤FRNK plasmid group. 50μg·mL-1 FN was supplemented into culture medium from group②to⑤.The synthetic faculty of total and typeⅠcollagen of each group was examined by 3H-Pro incorporation assay, and the proliferation of HSC was evaluated by direct cell counting assay. The levels of FRNK, MT1-MMP, MMP2, TIMP-2 in HSC were assayed by Western blot on protein level and RT-PCR on mRNA level respectively.Results1 The FAK band of 125 kD was showed on Western Blot after FRNK transfection, and optical density value assay indicated that the expression of FAK protein was no significant difference at 0h, 24 h, 48 h, 72 h after FRNK plasmid has been transfected into HSC, P>0.05.At the same time, the FRNK band of 42 kD was showed, and with the prolongation of transfection time, the FRNK expression gradually increased. The FRNK protein was most strongly expressed at 48 h, and was decreased at 72h. There was a significant difference between FRNK and control groups (p<0.01).This showed that the FRNK expression was most strongly at 48h after successful FRNK plasmid transfection, while the FAK expression had no significant change before and after transfection.2 HSC total collagen synthesis was inhibited by FRNK:The 3H-Pro incorporation assay was used to determine HSC total collagen synthesis. The result showed that,compared with control, FN increase HSC total collagen synthesis,P<0.05; but there were no significant difference among FN group, liposome group and non-FRNK plamid group, P>0.05;while compared with non-FRNK plasmid group, HSC total collagen synthesis significantly decreased at 48h after FRNK transfection,P<0.01. This showed that HSC total collagen synthesis was increased by FN stimulation, and effectively inhibited by using FRNK transfection.3 HSC type I collagen synthesis was inhibited by FRNK: HSC type I collagen synthesis was detected by using 3H-Pro incorporation assay. The result showed that,compared with control, FN increase HSC type I collagen synthesis,P<0.01; but there were no significant difference among FN group, liposome group and non-FRNK plamid group, P>0.05;while compared with non-FRNK plasmid group, HSC type I collagen synthesis significantly decreased at 48h after FRNK transfection,P<0.01. This showed that HSC type I collagen synthesis was increased by FN stimulation, and significantly inhibited by using FRNK transfection.4 The expression of MT1-MMP was up-regulated by FRNK : Western Blot showed that the expression of MT1-MMP protein was obviously lower in FN group than that in control group (1.02±0.18 vs 1.55±0.27), reduced by 34.19 %, p<0.01; but there were no significant difference among FN group, liposome group and non-FRNK plamid group,P>0.05; the expression of MT1-MMP protein was obviously higher in FRNK plamid group at 48h after transfection than that in non-FRNK plamid group (2.25±0.54 vs 0.99±0.88), increased by 51.77%, the difference was significant, P<0.01. Ten hours after FRNK transfection, RT-PCR was used to detect the mRNA of MT1-MMP andβ-actin. The results showed that there were no significant difference among FN group, liposome group and non-FRNK plamid group, P>0.05. The expression of MT1-MMP mRNA was significantly higher in FRNK plamid group than that in non-FRNK plamid group (1.58±0.18 vs 1.00±0.10), there was a significant difference, P<0.01.5 The expression of MMP2 was up-regulated by FRNK: Western Blot showed that the expression of MMP2 protein was significantly lower in FN group than that in control group (1.13±0.17 vs 1.46±0.20), reduced by 22.60 %, P<0.01; but there were no significant difference among FN group, liposome group and non-FRNK plamid group,P>0.05; the expression of MMP2 protein was significantly higher in FRNK plamid group at 48h after transfection than that in non-FRNK plamid group (2.26±0.14 vs 1.09±0.15), increased by 51.77%, there was a significant difference, P<0.01. RT-PCR was used to detect the mRNA of MMP2 andβ-actin at 10h after FRNK transfection, the results showed that there were no significant difference among FN group, liposome group and non-FRNK plamid group, P>0.05. The expression of MMP2 mRNA was obviously higher in FRNK plamid group than that in non-FRNK plamid group (1.65±0.04 vs 0.99±0.03), there was a significant difference, P<0.01.6 The expression of TIMP-2 was down-regulated by FRNK: Western Blot showed that the level of TIMP-2 protein was significantly higher in FN group than that in control group (2.26±0.13 vs 1.44±0.09), increased by 36.28 %, P<0.01; but there were no significant difference among FN group, liposome group and non-FRNK plamid group,P>0.05; the expression of TIMP-2 protein was significantly lower in FRNK plamid group at 48h after transfection than that in non-FRNK plamid group (1.14±0.10 vs 2.23±0.15), decreased by 48.88%, there was a significant difference, P<0.01. RT-PCR was used to detect the mRNA of TIMP-2 andβ-actin at 10h after FRNK transfection, and the scan assay showed that there were no significant difference among FN group, liposome group and non-FRNK plamid group, P>0.05. The expression of TIMP-2 mRNA was obviously lower in FRNK plamid group than that in non-FRNK plamid group (0.97±0.04 vs 1.69±0.04), there was a significant difference, P<0.01.7 The value of MMP2/TIMP-2 was enhanced: The expression of MMP2 and TIMP-2 at both transcriptional and translational levels among all groups were made a ratio, and the results showed that the ratios of MMP2/TIMP-2 at both levels were enhanced when the FRNK was overexpressed in HSC.ConclusionsFRNK transfection mediated through liposome induced the overexpression of exogenous FRNK in HSC, and the synthesis of total and typeⅠcollagen in HSCs was inhibited, this can be related to the up-regulation of MT1-MMP and MMP2/TIMP-2 by FRNK.