Screening for and validation of a hepatic fibrosis-related pathway induced by insulin-like growth factor-binding protein-related protein 1.

Abstract

Our previous studies characterized insulin-like growth factor-binding protein-related protein 1 (IGFBPrP1) as a molecule that promotes hepatic fibrogenesis, but its mechanism has not been fully elucidated. Here, we have investigated the effect of IGFBPrP1 on gene expression in the hepatic fibrosis-related pathway. Sprague-Dawley rats received injections of an adenovirus carrying IGFBPrP1 or EGFP cDNA into their tail veins. In hepatic preparations, hepatic stellate cell activation was determined by α-smooth muscle actin expression and hepatic fibrosis by Sirius red staining and hydroxyproline content analysis. IGFBPrP1-inducible genes of the hepatic fibrosis-related pathway were assessed by PCR array. Expression of IGFBPrP1 and transforming growth factor β1 (TGFβ1) and array results were evaluated by quantitative real-time PCR and western blotting. IGFBPrP1-overexpressing rats showed an increase in α-smooth muscle actin expression and collagen and hydroxyproline content in the liver. The PCR array results indicated that some genes were upregulated and some were downregulated in Ad-IGFBPrP1-infected rats. Among these, Egr1, MAP2K2 (MEK2) and MAPK3 (ERK1) expression increased, whereas PTEN and Hhip mRNA expression decreased. Egr1 protein levels increased and peaked 2 weeks after infection and then decreased gradually. PTEN protein decreased gradually in Ad-IGFBPrP1-infected rats with a concurrent increase in the degree of hepatic fibrosis. TGFβ1 levels increased during hepatic fibrosis development in liver tissues. Egr1, PTEN, Hhip, MAP2K2 (MEK2) and MAPK3 (ERK1) were identified as candidate genes of the IGFBPrP1-induced hepatic fibrosis-related pathway. IGFBPrP1 promoted hepatic fibrosis mainly by enhancing the TGFβ1 expression that it triggered.