Deletion of WISP1 leads to higher sensitivity to carbon tetrachloride-induced liver damage

Abstract

WISP1 (Wnt-induced secreted protein-1) is a member of the CCN family of matricellular proteins. These highly conserved and secreted proteins can interact with integrins and modulate many biological processes like migration, proliferation, differentiation and apoptosis. CCN proteins have been show to play divergent roles in liver pathophysiology, in spite of having very similar structural domains. Importantly, little is known about WISP1 in this context. Gene array analysis of mouse liver after acute injury by intraperitoneal injection of CCl4 in C56BL/6N mice showed WISP1 mRNA upregulated during early injury phase, between 2h and 24h after CCl4 injection. These results were confirmed by real time PCR, showing a maximal induction of 40 fold over healthy liver at 24h. To establish the role of WISP1 in acute liver damage we used a knockout mouse model, in which WISP1 expression is disrupted by replacing Exon 2 with a resistance cassette and a stop codon. Heterozygous mice were bred to establish homozygous knockout and wild type lines which were validated by genotyping. Real time PCR analysis confirmed the absence of WISP1 mRNA expression in KO mice challenged with CCl4. Remarkably, deletion of WISP1 led to a significant increase of necrotic area at 24h. This was show by hematoxylin & eosin staining reaching 49% in KO vs. 27% in WT mice (p < 0.0001; n = 13 wild type, n = 11 KO) and serum transaminase activity (GPT: 8230 U/l in WT, 12300 U/l in KO). Importantly, immunofluorescence and real time PCR showed that WT and WISP1 KO mice have no significant differences in CYP2E1 expression, suggesting that the difference in tissue injury does not depend on alterations of CCl4 metabolism in KO mice. To further characterize this different phenotype, we performed a time course after CCl4 intoxication (460 mg/kg, i.p.) by collecting liver tissue at 30 min, 2h, 8h, 18h, and 24h. Interestingly, already at 18h KO mice showed a clear necrotic area around the central veins whereas WT mice showed ballooned hepatocytes with cytoplasmic translocation of high-mobility group protein B1 (HGMB1), indicating that WISP1 KO mice had a more advanced stage of liver injury at this time point than the WT counterparts. This phenotype was also observed upon intoxication with lower doses of CCl4 (132.4 mg/kg and 76 mg/kg). Analysis of cytokine expression by real time PCR showed a stronger induction of TNFα in WISP1 KO mice (7-fold v/s 10-fold at 18h, and 3-fold vs. 6-fold at 24h in WT vs. WISP1 KO respectively), and IL6 (5-fold vs. 10-fold at 18h, and no induction vs. 3-fold at 24h in WT v/s WISP1 KO respectively). Furthermore, western blot analysis showed marked alterations in signal transduction pathways in liver tissue of WISP1 KO mice, with stronger activations of p-JNK at 2h and p-STAT3 at 18h and 24h. Currently we are investigating the mechanism through which WISP1 exerts a protective role upon CCl4 stimulation in liver damage and in the subsequent regeneration process. In conclusion we have strong evidence that WISP1 plays an important role in acute liver damage.