Elimination of Wnt Secretion from Hepatic Stellate Cells Impairs Hepatocyte Proliferation After Partial Hepatectomy

Abstract

Wnt‐dependent β‐catenin activation contributes to hepatocyte proliferation following 2/3 partial hepatectomy (PH) through regulation of Cyclin‐D1 (Ccnd1) expression, as we have previously shown using β‐catenin or Wnt co‐receptors, low‐density lipoprotein receptor‐related protein 5–6, conditional knockouts (KO). However, given the redundancy between the 19 mammalian Wnt proteins and various cell sources for Wnt secretion, the precise mechanisms of Wnt signaling in liver regeneration (LR) are not completely understood. Recently, using endothelial cell (EC)‐specific deletion of Wntless (Wls), a cargo receptor required for Wnt secretion, we have reported that sinusoidal ECs secrete Wnt2 and Wnt9b to activate β‐catenin in hepatocytes and enhance Ccnd1 expression, which is indispensable for normal and optimal proliferation of hepatocytes during LR after PH. Alternatively, conditional deletion of Wls in hepatocytes or cholangiocytes did not impact LR, whereas Wls deletion in macrophages only marginally affected LR post‐PH. Here, to investigate the roles for hepatic stellate cell (HSC)‐secreted Wnts in LR, we characterize the HSC‐Wls‐KO, generated by interbreeding Wls‐floxed and Lecithin Retinol Acyl Transferase (LRAT)‐driven Cre (Lrat‐Cre) transgenic mice. HSC‐Wls‐KO mice showed no effect on liver weight or β‐catenin‐dependent pericentral gene expression at baseline. However, hepatocyte‐specific CCND1 protein normally evident in midzonal hepatocytes, was diminished in baseline KO liver as also evident by WB, whereas Ccnd1 expression was unaltered as seen by RNA‐seq and qPCR, suggesting posttranslational regulation. No difference in CCND1 protein was seen in the non‐parenchymal cells of the KO livers when compared to WT. After PH, HSC‐Wls‐KO showed modest decrease in gene expression of Ccnd1 at only 24 hour (h) post‐PH as compared to WT but comparable levels between the two groups were seen at 40h and 72h. While similiar PCNA+ staining in hepatocytes was observed at 40–72h post‐PH in HSC‐Wls‐KO and WT, HSC‐Wls‐KO displayed significantly diminished BrdU+ or pHH3+ hepatocytes at 40h, which rebounded by 72h, suggesting delayed regeneration due to the impaired hepatocyte replication.ConclusionAt baseline, Wnt ligands secreted by HSCs are essential for CCND1 protein expression in midzonal hepatocytes but not for pericentral zonation. Following PH, HSCs‐derived Wnts also contribute to normal Ccnd1 expression and hepatocyte mitosis.Support or Funding InformationThis work was supported by NIH grants 1R01DK62277, 1R01DK100287, R01CA204586 and Endowed Chair for Experimental Pathology to S.P.M., and by NIH grant 1P30DK120531‐01 to Pittsburgh Liver Research Center (PLRC) and by PLRC Pilot & Feasibility grant PF 2019‐05 to S.K.