Hedgehog regulates yes-associated protein 1 in regenerating mouse liver

Abstract

Adult liver regeneration requires induction and suppression of proliferative activity in multiple types of liver cells. The mechanisms that orchestrate the global changes in gene expression that are required for proliferative activity to change within individual liver cells, and that coordinate proliferative activity among different types of liver cells, are not well understood. Morphogenic signaling pathways that are active during fetal development, including Hedgehog and Hippo/Yes-associated protein 1 (Yap1), regulate liver regeneration in adulthood. Cirrhosis and liver cancer result when these pathways become dysregulated, but relatively little is known about the mechanisms that coordinate and control morphogenic signaling during effective liver regeneration. We evaluated the hypothesis that the Hedgehog pathway controls Yap1 activation during liver regeneration by studying intact mice and cultured liver cells. In cultured hepatic stellate cells (HSCs), disrupting Hedgehog signaling blocked activation of Yap1, and knocking down Yap1 inhibited induction of both Yap1- and Hedgehog-regulated genes that enable HSC to become myofibroblasts (MFs). In mice, disrupting Hedgehog signaling in MFs inhibited liver regeneration after partial hepactectomy (PH). Reduced proliferative activity in the liver epithelial compartment resulted from loss of stroma-derived paracrine signals that activate Yap1 and the Hedgehog pathway in hepatocytes. This prevented hepatocytes from up-regulating Yap1- and Hedgehog-regulated transcription factors that normally promote their proliferation.

Conclusions: Morphogenic signaling in HSCs is necessary to reprogram hepatocytes to regenerate the liver epithelial compartment post-PH. This discovery identifies novel molecules that might be targeted to correct defective repair during cirrhosis and liver cancer. (Hepatology 2016;64:232-244).

Figure 1. Hedgehog signaling regulates Yap1 activity

HSC were isolated from Smo^flox/flox mice, and treated for 3 days with adenovirus bearing Cre recombinase (Ad-Cre) or adenovirus expressing green fluorescent protein (Ad-GFP, control). Cells were harvested for mRNA and protein analysis by qRT-PCR or western blot respectively. (A) Smo and Gli1 mRNA. (B) Yap1 mRNA. (C) Representative western blot showing pYap1 S112 Yap1, pLats1 T1079, Lats1, and beta-actin. (D) CTGF mRNA. (E) AREG mRNA. Results were expressed as fold change relative to control (Ad-GFP) treated HSC; mean ± SEM were graphed. *p<0.05 vs. control

Figure 2. Yap1 is a downstream effector of the Hedgehog pathway

HSC were treated with lentiviral vectors harboring distinct shRNA sequences for Yap1(YAP1D1, YAP1D2) or non-targeting control vectors (NT) and harvested for western blot and qRT-PCR analysis. Top: western blot showing Yap1 and Lamin B (nuclear protein loading control). Bottom: composite panel showing Desmin, αSMA, Snail, Collagen 1a1, Gli1 mRNA. Results were expressed as fold change relative to NT treated HSC; mean ± SEM were graphed; *p<0.05 vs. NT HSC.

Figure 3. Yap1 and Yap1-target genes are upregulated after PH

Wild type (WT) mice underwent PH. Whole livers were harvested for immunohistochemistry and qRT-PCR. (A) Representative Yap1 immunostaining in quiescent (Q) liver and at 48 and 96 h post-PH. Black arrows refer to Yap1 (+) cells. Inserts show YAP1 (+) ductular and hepatocytic cells. (B) Yap1 quantification. Results were expressed as mean ± SEM number of cells per high-powered field (HPF). (C) YAP1 mRNA, (D) CTGF mRNA, and (E) AREG mRNA; results were expressed as fold change relative to quiescent (time 0) livers; mean ± SEM were graphed. *p<0.05 vs. time 0.

Figure 4. Yap1 (+) cells are Hedgehog-responsive MF-HSC

WT livers were harvested as described in Figure legend 3. (A) Representative Yap1 (brown)/Desmin (green) double immunostaining in the quiescent (Q) liver (black arrows; 40x on right, 100x on left); Representative Yap1 (brown)/αSMA (green) double immunostaining 15 min after PH (black arrows; 40x on left, 100x on right). (B) Quantification of Yap1/αSMA double (+) cells. Results were expressed as mean ± SEM number of cells/high-powered field (HPF). (C) Yap1/Gli2 double (+) cells (solid columns) and whole liver Gli1 mRNA (red line) accumulate after PH. Results were expressed as fold change relative to quiescent livers; mean ± SEM. p<0.05 vs. quiescent livers or otherwise indicated.

Figure 5. Hedgehog-responsive MF-HSC regulate Yap1 expression in total liver

In separate experiments, αSMA-CreER^t2/SMO^flox/flox double transgenic (DTG) mice were treated with either vehicle (VEH) or TMX, and then sacrificed at 24, 48, 72, and 96h post-PH. Livers were harvested and analysed by immunohistochemistry. (A) Representative Yap1 immunostaining from VEH- and TMX-treated groups at time 0, and 48 h post-PH. (B) Yap1 quantification at time points after PH. Results were expressed as total number of Yap1 (+) cells per HPF; mean ± SEM; white columns represent VEH-treated DTG; black columns represent TMX-treated DTG. Liver RNA was analysed by qRT-PCR. (C) YAP1 mRNA. (D) CTGF mRNA. (E) AREG mRNA. Results were expressed as fold change relative to quiescent (time 0) livers; mean ± SEM were graphed; *p<0.05 vs time-matched VEH-treated DTG.

Figure 6. Hedgehog pathway activity in αSMA (+) cells promotes proliferation and de-differentiation in hepatocytes

αSMACreER^t2/Smo^flox/flox (DTG) mice were treated as described in Figure legend 5. Livers were analyzed by immunohistochemistry. (A) Yap1 (brown)/Gli2 (green) double immunostaining in VEH- and TMX-treated DTG 48 h post-PH (black arrows; 100x). In separate experiments, hepatocytes were isolated from VEH or TMX-treated DTG (n =10) and STG (n = 10) 48 h post-PH, and at time 0 (i.e. DO), and analyzed by qRT-PCR. (B) Gli1 mRNA. (C) YAP1 mRNA. (D – E) Proliferation markers: CCND1 (D) and FoxM1 (E) mRNA. (F) Hepatocyte marker, HNF4a mRNA. Results were expressed as fold change relative to DO hepatocytes; mean ± SEM were graphed; *p<0.05 versus DO or VEH-treated DTG hepatocytes.