Proteomic and Phosphoproteomic Analyses Identify Novel Targets Involved in Beneficial Effect of Rolipram on Liver Fibrosis in Mice

Abstract

BackgroundChronic liver injury leads to the development of liver fibrosis for which there is no therapy. We have shown that phosphodiesterase 4 (PDE4) plays a pathogenic role in the development of hepatic fibrosis in a rat model of cholestatic liver injury. However, the involvement of PDE4 enzymes in hepatic fibrosis occurring in other forms of liver disease has not been examined. Here, we tested the effect of PDE4 inhibition on fibrogenesis in a mouse model of carbon tetrachloride (CCl4) induced fibrosis. Liver tissue proteomic and phosphoproteomic analysis was performed to identify the relevant novel proteins affected by PDE4 inhibition.MethodsHepatic fibrosis was established by repeated CCl4 injection for 4 weeks in C57Bl/6 mice. One group of mice received a PDE4 inhibitor, Rolipram (targeted to the liver), twice a week. Collagen deposition and expression of fibrogenic genes were assessed. Total liver proteins were digested and labeled with TMT‐10 plex reagents according to manufacturer's guidelines. Two TMT‐plexed sample flights were studied by 2DLC‐MS/MS using an Orbitrap ELITE (Thermo) mass spectrometer. Titanium dioxide phosphopeptide enrichment was performed on labeled peptides and data collected by 1D‐LCMS analysis. Spectra were matched to the mouse protein databank to identify and quantify the abundance. PEAKS Studio 8 software was used to quantitate the normalized phosphopeptide abundance. Quantitative cluster analysis, ANOVA, and Benjamini‐Hochberg corrected t‐test were performed to determine statistical significance.ResultsCCl4 injection led to a significant fibrosis as confirmed by Sirius red staining and hydroxyproline (HYP) content in mice. Importantly, PDE4 inhibition by Rolipram prevented HYP accumulation and the expression of genes involved in collagen synthesis (Hsp47) and crosslinking (Lox). Additionally, Mmp2 and Timp2 mRNA increases were markedly prevented by Rolipram. A total of 4,560 proteins in 3,841 clusters were observed at the protein (99.9% C) and peptide (99.9% CI at 2ppm mass accuracy). Quantitative cluster analysis identified 262 significantly regulated proteins. A total of 1,402 unique S/T/Y phosphopeptides were observed and derived from 606 unique protein accessions. Benjamini‐Hochberg corrected ANOVA p‐values identified 149 phosphopeptides regulated by either CCl4 or CCl4+Rolipram. Considering Log2FC, 22 unique peptides were increased by CCl4, and Rolipram reduced this effect. Ten phosphopeptides were observed where Rolipram appeared to have an additive effect with CCl4.ConclusionPDE4 inhibition modulated changes caused by CCl4 and attenuated the development of fibrosis by affecting several extracellular matrix proteins, profibrogenic and apoptotic signaling molecules. To our knowledge, this is the first hepatic proteomic analysis investigating the CCl4 and Rolipram‐responsive phosphoproteome in vivo.Support or Funding InformationR43AA021331, R44AA021331, P50AA024337, P20GM113226‐02This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.