PTEN-mediated AKT/β-catenin signaling enhances the proliferation and expansion of Lgr5+ hepatocytes

Abstract

Rationale: Compelling evidence suggests that Lgr5+ hepatocytes repair liver damage by promoting the regeneration of hepatocytes and ductal cells in the case of liver injury. The PTEN-mediated AKT/β-catenin signaling plays a key role in the regulation of innate immune regulation in the liver. However, the signaling pathways that control Lgr5+ hepatocyte proliferation in the liver remain unclear. Methods: In order to assess the involvement of PTEN-mediated AKT/β-catenin signaling in the expansion of Lgr5+ hepatocytes upon liver injuries, the Lgr5-CreER; Rosa-mTmG lineage tracing system was used to target Lgr5+ hepatocytes. Results: The tracing of Lgr5+ hepatocytes showed that PTEN deletion and β-catenin activation significantly promoted the proliferation of Lgr5+ hepatocytes. In converse, the simultaneous inhibition of PTEN and β-catenin limited Lgr5+ hepatocyte proliferation in the liver. Our findings provide an insight into understanding how PTEN-mediated AKT/β-catenin signaling regulates the proliferation of Lgr5+ hepatocytes. Conclusion: The outcomes can improve the application potential of Lgr5+ hepatocytes in the treatment of liver injury diseases and provide a new treatment option for liver cancer.

Keywords: AKT/β-catenin; Lgr5; hepatocyte; liver regeneration.; proliferation.

Figure 1

Both PTEN ablation and increased β-catenin signaling in Lgr5+ hepatocytes promoted the expansion of Lgr5+ hepatocytes. (A) Experimental scheme for tamoxifen injection and analysis of liver samples in Lgr5CreER; mTmG mice at 21 days post-injection. (B) Targeting of Lgr5+ hepatocytes and their progeny (green) in the liver with Lgr5CreER; mTmG reporter mice. Note the sporadic distribution of mGFP-labeled cells in the liver. (C) Representative images showing positive Lgr5+ hepatocytes and their progeny in Lgr5CreER; mTmG; PTEN^flox/flox mice. (D) A low-magnification image of a liver sample from Lgr5CreER; Ctnnb1^Δ(ex3); YFP mice (left panel). A magnified image of the left panel showed labeled Lgr5+ hepatocytes (right panel). Arrowheads indicate mGFP+ hepatocytes on day 21 after injection. (E) Phosphorylated β-catenin (white) immunostaining of liver tissue showing the specific colocalization of p-β-catenin and YFP (upper panel). Immunostaining of liver section from Lgr5CreER; Ctnnb1^Δ(ex3); YFP mice showed that mGFP-labeled cells colocalized with Albumin (white) (lower panel). (F) Immunostaining for Albumin (white) revealed colocalization of mGFP with Albumin-positive hepatocytes. (G-H) Immunostaining of liver tissues from Lgr5CreER; mTmG and Lgr5CreER; mTmG; PTEN^flox/flox mice for p-AKT antibody (white). Arrowheads indicate Lgr5+ hepatocytes with an activated AKT pathway. n = 3 mice for each genotype; CV: central vein; Scale bar: 50 µm.

Figure 2

PTEN-mediated AKT/β-catenin signaling controls Lgr5+ hepatocyte proliferation. (A) The number of labeled cells in the liver from Lgr5CreER; mTmG; Ctnnb1^flox/flox mice was significantly reduced upon β-catenin deletion. (B) Quantitation of mGFP+/YFP+ cells in liver samples from mice with different genotypes. (C) p-β-catenin immunostaining to show activation or inhibition of β-catenin signaling in the liver from different genotypes. (D) Sections of liver in Lgr5CreER; mTmG; PTEN^flox/flox; Ctnnb1^flox/flox mice showing that β-catenin deletion in PTEN-deficient Lgr5+ hepatocytes strongly blocked the proliferation of Lgr5+ hepatocytes (left panel). The high-magnification view of the left panel depicts sporadic mGFP+ cells (top right). Another higher magnification image showing Lgr5-labeled cells (bottom right). Arrowheads denote mGFP+ cells. (E) Quantitation of mGFP+ cells in liver samples from mice with different genotypes. ****, P<0.0001. Data shown are means ± SEM (n = 3 biological replicated experiments). Scale bar: 100 µm.

Figure 3

Schematic summary of the distribution of Lgr5+ hepatocytes and their progeny under alteration of PI3K/AKT and Wnt/β-catenin signaling. (A-E) Schematic depiction of the proliferative ability of Lgr5+ hepatocytes (green) during normal liver homeostasis (A) and under changes of PI3K/AKT and Wnt/β-catenin signaling (B-E). (F) Forced activation of β-catenin induced the expansion of Lgr5+ hepatocytes. (G) Schematic illustration of the crosstalk between Wnt/β-catenin and PI3K/AKT signaling pathways and their role in the proliferation of Lgr5+ hepatocytes.