Investigating the role of Fzd‐7 in liver donation and regeneration

Abstract

The Wnt‐Fzd‐LRP5/6‐β‐catenin signaling axis has important roles in liver homeostasis, regeneration and disease. Through use of liver‐ and cell‐type‐specific gene knockout mouse models, we and others have confirmed roles for β‐catenin and LRP5/6 in regulating metabolic zonation of the liver as well as initiation of liver regeneration after partial hepatectomy. Similarly, using cell‐type‐specific Wls gene knockout models we have identified endothelial cells and Kupffer cells as significant sources of Wnt proteins responsible for regulating β‐catenin signaling in liver regeneration. However, little is known about the actual frizzled (Fzd) receptor(s) that are critical to transmitting the Wnt signals that regulate metabolic zonation and liver regeneration. We do know that Fzd‐7 is highly expressed in some liver cancers. Here, we further investigate the role of Fzd‐7 in metabolic zonation and liver regeneration after partial hepatectomy. We used microdissection to isolate cells from 3 major zones of the liver (peri‐portal, mid‐zonal, pericentral) to assess the expression of Fzd receptors in each zone by qPCR. We observed ~3‐fold higher expression of Fzd‐7 in the pericentral zone, indicating a possible role in metabolic zonation. We also examined and found upregulation of Fzd‐7 gene expression at 12–24h after hepatectomy in mice. Therefore, we obtained Fzd‐7‐floxed mice to conditionally delete Fzd‐7 in the liver and investigate its role in metabolic zonation and liver regeneration. We injected Fzd‐7 floxed mice with AAV8‐TBG‐Cre to conditionally knockout Fzd‐7 from hepatocytes. Two weeks after administration of AAV8‐TBG‐Cre, we performed PCR analysis and confirmed successful knockout of the Fzd‐7 mRNA as well as detection of the floxed‐deletion in the liver DNA. However, analysis of GS and CYP2e1 showed no defect in metabolic zonation at 2 weeks post AAV injection. We also performed partial hepatectomy 14 days after AAV‐TBG‐Cre injection and analyzed the regenerating livers. Hepatocyte proliferation by BrdU staining was marginally lower in the AAV8‐Cre injected mice at 40h post hepatectomy indicating a possible delay in liver regeneration. Further characterization has shown compensatory increases in expression of other Fzd receptors in the AAV8‐Cre livers which may explain the lack of overt phenotype in these mice. Since Fzd‐7 protein may have longer half‐life, extended time points after AAV8‐TBG‐Cre injection to Fzd‐7‐floxed mice are currently under investigation. Additional characterization with Alb‐Cre‐Fzd‐7 liver knockouts is also being done. In conclusion, Fzd‐7 expression is highest in zone‐3 and is also upregulated early after hepatectomy. While acute deletion of Fzd‐7 did not reveal changes in zonation, a decrease in hepatocyte proliferation during regeneration was evident. Fzd‐7 may be an important mediator of Wnt‐β‐catenin signaling in the liver.Support or Funding InformationDepartment of Pathology and Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA