Aim: Hepatic stellate cells (HSCs) play an important role in the pathogenesis of liver fibrosis and cirrhosis. Raf kinase inhibitor protein (RKIP), an inhibitor of extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway, has been proved to suppress tumor metastasis. Interestingly, RKIP promotes cell migration in Madin–Darby canine kidney epithelial cells. However, the effects of RKIP on HSC behaviours are unknown. The purpose of the present study is to investigate the role of RKIP in HSC proliferation, apoptosis and migration.
Methods: Two types of cells, freshly isolated HSC and HSC-T6 cell line, were used in this study. The amount of RKIP, the phosphorylation of RKIP, Raf and ERK (pRKIP, pRaf and pERK) were analysed in quiescent and activated HSCs by Western blots. HSC-T6 cells were transfected with RKIP-expressing plasmid or treated with locostatin, a RKIP inhibitor. HSC proliferation, apoptosis and migration were evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) staining and Transwell cell migration assay respectively.
Results: In activated HSCs, RKIP protein expression was downregulated whereas pRKIP, pRaf and pERK were upregulated. RKIP overexpression significantly mitigated the phosphorylation of RKIP, Raf and ERK. This in turn inhibited HSC proliferation. Locostatin not only inhibited RKIP protein expression but also, to some extent, reversed the RKIP-inhibited phosphorylation of RKIP, Raf and ERK. RKIP augmented HSC migration and enhanced wound closure. Locostatin reversed the effects of RKIP.
Conclusion: Raf kinase inhibitor protein inhibits ERK/MAPK signalling and this inhibition impedes HSC proliferation. RKIP promotes HSC migration and wound closure.