Diminished retinoic acid signaling in hepatic stellate cells in cholestatic liver fibrosis

Abstract

Hepatic stellate cells (HSC) participate in liver fibrogenesis via myofibroblastic activation, the extent of which appears to correlate with the loss of cellular vitamin A. The present study has tested a hypothesis that HSC activation is associated with diminished retinoic acid (RA) signaling. Pure HSC were isolated from rats with cholestatic liver fibrosis induced by bile duct ligation (BDL) and sham-operated animals (Sham). Northern blot analysis of HSC RNA from BDL confirmed fibrogenic activation of the cells with enhanced mRNA levels for procollagen-alpha1(I) and transforming growth factor-beta1 (TGF-beta1). Competitive polymerase chain reaction analysis showed selective reductions in the mRNA levels of RA receptor (RAR)-beta and retinoid X receptor (RXR)-alpha to 20 and 17% of the Sham HSC. The mRNA level for cellular retinol binding protein I, a gene with RA responsive element (RARE), was also suppressed by 78% in BDL. The concentrations of all-trans-RA and 9-cis-RA were decreased in HSC from BDL. Nuclear extracts of these cells showed diminished binding activity to the RARE, whereas activity of AP-1, a transcription factor known to be antagonized by RAR and RXR, was enhanced. These results demonstrate diminished RA signaling in HSC from cholestatic liver fibrosis, which appeared to have resulted from RA deficiency and suppressed expression of RAR-beta and RXR-alpha. Furthermore, the reciprocal enhancement of AP-1 activity and coordinately increased expression of an AP-1 responsive gene, TGF-beta1, suggest a permissive role of the diminished RA signaling in promoting AP-1 activity and TGF-beta1 expression.

PPARβ regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation

“…This alternative, however, seems unlikely since hepatic stellate cells in vitro rapidly loose their capacity to respond to retinoic acids, which is reflected by decreased retinoic acid and RAR levels (52,53). Here, we show that CRABP-I mRNA levels, after a transient rise between days 0 and 3, were reduced at later time-points.…”

“…Competition analysis showed that PPAR␤-RXR binding to a consensus DR1-like PPRE was actively competed for binding to the DR2-like RARE of the CRBP-I promoter. In contrast to PPAR␤, the expression of RARs becomes strongly reduced during hepatic stellate cell culturing (52,53). PPAR␤ might thus control the expression of CRBP-I by binding to responsive elements in the CRBP-I promoter.…”

Cellular Retinol-Binding Protein-1 Expression and Modulation during In Vivo and In Vitro Myofibroblastic Differentiation of Rat Hepatic Stellate Cells and Portal Fibroblasts

“…In this model, we did not detect clear modifications of CRBP-1 expression in lobular HSC. In contrast, Ohata et al (1997), using the same model, have shown that the mRNA expression of CRBP-1 was drastically reduced in isolated HSC. There are no ready explanations for this apparent discrepancy.…”

Peroxisome Proliferator-activated Receptor γ Induces a Phenotypic Switch from Activated to Quiescent Hepatic Stellate Cells

“…Electrophoretic Mobility Gel Shift Assay-HSC nuclear proteins (5-10 g) or PPAR␥, JunD, or RXR␣ in vitro translated using the coupled transcription/translation systems (Promega, Madison, WI) were incubated in a reaction mixture (20 mM HEPES, pH 7.6, 100 mM KCl, 0.2 mM EDTA, 2 mM dithiothreitol, 20% glycerol, 200 g/ml poly(dI-dC)) on ice for 15 min and further incubated with 1-2 ng of ␣-32 P-labeled doubled-stranded ARE-7 (the PPAR response element from the adipocyte fatty acid-binding protein gene) or AP-1 element (26) for an additional 30 min. The reaction mixture was then resolved on a 6% nondenaturing polyacrylamide gel in 4ϫ TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA).…”