Apolipoprotein AI regulatory protein 1 (ARP-1) coordinates profibrogenic responses during liver fibrosis

Background: Liver fibrosis represents a general wound healing response elicited by different etiologic causes. The cells mainly in charge of this process are liver myofibroblasts, mesenchymal cells responsible for the synthesis of the extracellular matrix proteins. The main source of fibrogenic myofibroblasts are Hepatic Stellate Cells (HSC). During the last few years it has been given much value to the role that nuclear receptors play in impairing the HSC trans-differentiation process; indeed it’s well known that PPARg maintains HSC in the quiescent state, and inhibit collagene transcription. However, the detailed molecular mechanisms of this process are still unclear. ARP-1 is a orphan receptor capable of homo and heterodimerization with other nuclear receptors and is expressed during embriogenesis mainly in the mesenchimal, vascular and cardiac tissues. The aim of this study is to evaluate the role of ARP-1 nuclear receptor on liver fibrosis. Methods: ARP-1 expression has been investigated on CCl4 treated C57/Bl6 mice by immunofluorescence experiments. Murine and human HSC were isolated following standard procedures. To evaluate the role of ARP-1 in HSC activation and liver fibrosis, cultured HSC where transfected with an ARP-1 expression plasmid with specific reporter plasmids for ARP-1 and PPARg. Cell migration and proliferation as well as MMP2 activity and collagene synthesis assays, were performed. Proteomics profile of ARP-1 overexpressing or silenced HSC was performed with Fluorescence 2-D Difference Gel Electrophoresis (DIGE). The transcriptional effect of ARP-1 on collagene 1a2 (Col1A2) expression was evaluated on ARP-1 expressing-HSC transfected with various fragments or the full Col1A2 promoter; CHIP and EMSA experiments were also performed. Results: ARP-1 expression is increased during HSC activation both in vivo and in vitro. Increased expression is correlated with a specific inhibition of PPARg expression and transcriptional activity. In HSC cell culture, ARP-1 induces both MMP2 and Col1A2 expression and significantly increases cell invasiveness; these effects are reverted by dominant negative ARP-1 and by specific siRNA. Moreover ARP-1 increases the transcriptional activity of Col1A2 promoter. To further investigate this effect, we performed experiments with fragments of various length of Col1A2 promoter as well as CHIP and EMSA assays, identifying three regions containing putative ARP-1 responsive elements. Conclusions: ARP-1 expression is increased during HSC activation and regulates HSC invasiveness and collagene synthesis. These data indicate that ARP-1 is an important regulator of liver fibrogenesis.