| Recruitment and proliferation of Thy-1+ oval cells are a hallmark of liver regeneration following 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PHx) in rats. Oval cell activation, as part of a process of the progenitor dependent liver regeneration, is an orchestrated response induced by specific external stimuli and involves sequential changes in gene expression, growth factor production and morphological changes. To understand the molecular mechanism underlying oval cell response, we identified genes that are differentially expressed in Thy-1+ oval cells utilizing suppression subtractive hybridization methods. It turned out that connective tissue growth factor (CTGF), which is a heparan-binding, secretory protein and can promote the growth, migration, apoptosis, angiogenesis and differentiation in a cell type specific manner, is one of the candidates. The upregulation of CTGF was confirmed at both the RNA and protein levels in Northern and Western analyses. The induction pattern in CTGF coincides with that of oval cell activation. In addition, CTGF expression in Thy-1 + oval cells were verified by approaches including quantitative real time PCR, immunofluorescent staining and in situ hybridization. Furthermore, Northern analysis showed that CTGF upregulation was associated with those of TGF-beta1, procollagen type I and fibronectin gene expression. To inhibit CTGF synthesis, Iloprost, which is a stable prostacyclin derivative and has been shown to block TGF-beta induced CTGF synthesis, was administrated to rats treated with 2-AAF/PHx. Iloprost administration blocked CTGF induction in treated animals but did not affect TGF-beta1 expression. The inhibition of CTGF induction by Iloprost was associated with a significant decrease in oval cell proliferation and a lower level of alpha-fetoprotein (AFP) expression as compared to control animals. These results demonstrate that CTGF induction is important for robust oval cell response following 2-AAF/PHx treatment in rats. A yeast two-hybrid cDNA library specific for oval cell-aided liver regeneration was constructed and screened. Several interesting interactors including fibronectin, extracellular matrix protein 1 and histine rich glycoprotein were identified. Further characterization of these candidates in the future would help us to understand how CTGF regulates oval cell response during liver regeneration. |
