Beta-catenin signaling in murine liver zonation and regeneration: A Wnt-Wnt situation!

Abstract

Liver-specific β-catenin knockout (β-Catenin-LKO) mice have revealed an essential role of β-catenin in metabolic zonation where it regulates pericentral gene expression and in initiating liver regeneration (LR) after partial hepatectomy (PH), by regulating expression of Cyclin-D1. However what regulates β-catenin activity in these events remains an enigma. Here, we investigate to what extent β-catenin activation is Wnt-signaling dependent and the potential cell source of Wnts. We studied liver-specific Lrp5/6 KO (Lrp-LKO) mice where Wnt-signaling was abolished in hepatocytes while the β-catenin gene remained intact. Intriguingly, like β-catenin-LKO mice, Lrp-LKO exhibited a defect in metabolic zonation observed as lack of glutamine synthetase (GS), Cyp1a2 and Cyp2e1. Lrp-LKO also displayed a significant delay in initiation of LR due to absence of β-catenin-TCF4 association and lack of Cyclin-D1. To address the source of Wnt proteins in liver, we investigated conditional Wntless (Wls) KO mice, which lacked ability to secrete Wnts from either liver epithelial cells (Wls-LKO), or macrophages including Kupffer cells (Wls-MKO), or endothelial cells (Wls-EKO). While Wls-EKO was embryonic lethal precluding further analysis in adult hepatic homeostasis and growth, Wls-LKO and Wls-MKO were viable but did not show any defect in hepatic zonation. Wls-LKO showed normal initiation of LR, however Wls-MKO showed a significant but temporal deficit in LR that was associated with decreased β-catenin-TCF4 association and diminished Cyclin-D1 expression. Conclusion Wnt-signaling is the major upstream effector of β-catenin activity in pericentral hepatocytes and during LR. Hepatocytes, cholangiocytes or macrophages are not the source of Wnts in regulating hepatic zonation. However, Kupffer cells are a major contributing source of Wnt secretion necessary for β-catenin activation during LR.

Endothelial Wnts regulate β‐catenin signaling in murine liver zonation and regeneration: A sequel to the Wnt–Wnt situation

“…Similar to β‐catenin‐LKO and LRP5‐6‐LDKO, we observed a significantly lower liver weight to body weight ratio in EC‐Wls‐KO6, 7, 14 (Fig. 2A).…”

“…Mice were fed regular chow in standard caging and kept under a 12‐hour light–dark cycle with no enrichment. Lyve1‐cre mice were purchased from Jackson Laboratories15 and bred with Wls flox/flox mice 7, 14. Lyve1‐cre +/– ;Wls flox/flox are hereafter referred to as EC‐Wls‐KO.…”

“…Whole‐cell protein lysate preparation was performed using radio immunoprecipitation assay buffer7, and 30 μg of protein was used for gel electrophoresis for western blots (WBs). Immunoprecipitation (IP) was performed using 750 μg of precleared protein and A/G agarose beads, as described 18.…”

“…For quantification, hepatocytes with mitotic figures were counted in three to five random 200 × fields from three livers from each group at 24, 40, and 72 hours. For immunohistochemistry (IHC), paraffin sections were processed as described 6, 7. Antibody dilutions are listed in http://onlinelibrary.wiley.com/doi/10.1002/hep4.1196/full.…”

“…To address cell source and identification of Wnts and because there are 19 Wnts in the mammalian genome with several expressed in various hepatic cells,13 we used mice that allowed cell‐specific deletion of Wntless (Wls), a cargo receptor essential for all Wnt secretion 14. Conditional deletion of Wls from liver ECs showed no effect on zonation or LR, while its loss from macrophages did not impact zonation but marginally affected hepatocyte proliferation at 40 hours after PH 7. Conditional loss of Wls from all ECs (tyrosine kinase 2 [Tie2]‐Cre) was embryonic lethal and precluded analysis for zonation and LR 7…”