Cross-talk between Notch and Hedgehog regulates hepatic stellate cell fate in mice

Abstract

Liver repair involves phenotypic changes in hepatic stellate cells (HSCs) and reactivation of morphogenic signaling pathways that modulate epithelial-to-mesenchymal/mesenchymal-to-epithelial transitions, such as Notch and Hedgehog (Hh). Hh stimulates HSCs to become myofibroblasts (MFs). Recent lineage tracing studies in adult mice with injured livers showed that some MFs became multipotent progenitors to regenerate hepatocytes, cholangiocytes, and HSCs. We studied primary HSC cultures and two different animal models of fibrosis to evaluate the hypothesis that activating the Notch pathway in HSCs stimulates them to become (and remain) MFs through a mechanism that involves an epithelial-to-mesenchymal-like transition and requires cross-talk with the canonical Hh pathway. We found that when cultured HSCs transitioned into MFs, they activated Hh signaling, underwent an epithelial-to-mesenchymal-like transition, and increased Notch signaling. Blocking Notch signaling in MFs/HSCs suppressed Hh activity and caused a mesenchymal-to-epithelial-like transition. Inhibiting the Hh pathway suppressed Notch signaling and also induced a mesenchymal-to-epithelial-like transition. Manipulating Hh and Notch signaling in a mouse multipotent progenitor cell line evoked similar responses. In mice, liver injury increased Notch activity in MFs and Hh-responsive MF progeny (i.e., HSCs and ductular cells). Conditionally disrupting Hh signaling in MFs of bile-duct-ligated mice inhibited Notch signaling and blocked accumulation of both MF and ductular cells.

Conclusions: The Notch and Hedgehog pathways interact to control the fate of key cell types involved in adult liver repair by modulating epithelial-to-mesenchymal-like/mesenchymal-to-epithelial-like transitions.

Figure 1. Liver injuries increase Notch signaling in Desmin-expressing stromal cells

(A) qRT-PCR analysis of total liver mRNA from WT mice 14 days after sham or BDL surgery for expression of Notch pathway genes. *p<0.05 vs. sham, n=4. (B) Double immunohistochemistry for Notch-2, Jagged-1 or Hey2 (brown) with Desmin (green) in BDL mouse livers demonstrates co-localization (inset) of these markers. Percentages of double positive cells among Desmin+ cells were also quantified in 10 randomly selected fields. *p<0.001 vs. sham, n=3 mice/group. (C) FACS analysis of HSC isolated from WT mice 14 days after sham or BDL surgery for expression of ASMA, Notch-2, Jagged-1 or Hey2. Desmin was used as a marker for HSC. (D) qRT-PCR analysis of total mRNA from livers of WT mice treated for 14 days with High-fat diet/CCl₄. *p<0.05, **p<0.01 vs. High-fat diet controls, n=3. (E) Double immunohistochemistry for Notch-2, Jagged-1 or Hey2 (brown) and Desmin (green) in High-fat diet/CCl₄ mouse livers demonstrates co-localization (inset) of these markers. Magnification ×40. Percentages of double positive cells among Desmin+ cells were also quantified in 10 randomly selected fields. *p<0.001 vs. HF Ctrl, n=3 mice/group.

Figure 1. Liver injuries increase Notch signaling in Desmin-expressing stromal cells

(A) qRT-PCR analysis of total liver mRNA from WT mice 14 days after sham or BDL surgery for expression of Notch pathway genes. *p<0.05 vs. sham, n=4. (B) Double immunohistochemistry for Notch-2, Jagged-1 or Hey2 (brown) with Desmin (green) in BDL mouse livers demonstrates co-localization (inset) of these markers. Percentages of double positive cells among Desmin+ cells were also quantified in 10 randomly selected fields. *p<0.001 vs. sham, n=3 mice/group. (C) FACS analysis of HSC isolated from WT mice 14 days after sham or BDL surgery for expression of ASMA, Notch-2, Jagged-1 or Hey2. Desmin was used as a marker for HSC. (D) qRT-PCR analysis of total mRNA from livers of WT mice treated for 14 days with High-fat diet/CCl₄. *p<0.05, **p<0.01 vs. High-fat diet controls, n=3. (E) Double immunohistochemistry for Notch-2, Jagged-1 or Hey2 (brown) and Desmin (green) in High-fat diet/CCl₄ mouse livers demonstrates co-localization (inset) of these markers. Magnification ×40. Percentages of double positive cells among Desmin+ cells were also quantified in 10 randomly selected fields. *p<0.001 vs. HF Ctrl, n=3 mice/group.

Figure 2. Notch signaling is activated during transdifferentiation of primary HSC

(A) FACS analysis of quiescent (freshly isolated, day 0) and myofibroblastic (culture day 7) HSC. Desmin and α-smooth muscle actin (ASMA) were used as markers for quiescent or myofibroblastic HSC, respectively. (B) qRT-PCR analysis of Notch inhibitor (Numb), receptors (Notch-1 and Notch-2), ligand (Jagged-1) and target genes (Hes1, Hey1, Hey2 and c-Myc) in quiescent and myofibroblastic HSC. Results were compared to gene expression in ductular progenitor cells (603B), *p<0.05, **p<0.01, ***p<0.001, n=3.

Figure 3. Notch-responsive liver progenitors (603B) co-express ductular, hepatocytic, HSC, and mesenchymal markers

(A) FACS analysis confirmed that 603B are mouse ductular progenitors with active Notch signaling. Gray lines indicate isotype controls. (B) FACS analysis of 603B demonstrated expression of other ductular markers (Krt7 and HNF6), but also hepatocytic markers (HNF4α, AFP and Albumin), Hh signaling factors/target genes (Ptc, Gli1, and Gli2), mesenchymal markers (Vimentin and ASMA) and HSC-associated markers (Desmin and GFAP) (C) Comparison of gene expression in 603B with primary mouse hepatocytes (mHep) and freshly-isolated or culture-activated primary mouse HSC (d0 mHSC and d7 mHSC, respectively) by qRT-PCR analysis, n=3/group. # signifies non-detectable signal.

Figure 4. Inhibiting Notch signaling suppresses Hedgehog signaling and promotes a mesenchymal-to-epithelial-like transition and hepatocytic differentiation in ductular-type progenitor cells

qRT-PCR analysis of 603B treated with DAPT (a γ-secretase inhibitor) for 48 hours for changes in (A) Notch pathway genes, (B) Epithelial/quiescence genes, and (C) Myofibroblast (MF)/Hedgehog (Hh) genes. *p<0.05 vs. DMSO control, n=3.

Figure 5. Notch inhibition suppresses Hedgehog signaling and promotes a mesenchymal-to-epithelial-like transition in primary HSC

qRT-PCR analysis of primary MF-HSC treated with DAPT for 3 days for changes in (A) Notch genes, (B) MF/Hh target genes, (C) Epithelial/quiescence genes, and (D) Progenitor genes, *p<0.05 vs. DMSO control, n=3. (E) DAPT-treated MF-HSC were stained for cleaved Notch-2, Jagged-1 and Hey2 protein. Scale bar: 150μM. (F) Effect of DAPT on HSC expression of ASMA, proliferation (Ki67) and lipid content (Oil Red O) was examined.

Figure 6. Blocking Hedgehog signaling in myofibroblastic liver cells inhibits Notch signaling

(A) qRT-PCR analysis of 603B treated with an Hh inhibitor, GDC-0449 or DMSO for 48 hours for changes in Hh target genes (Ptc and Gli1), Notch genes (Notch-2, Jagged-1, Hey1 and Hey2), and epithelial genes (AFP and HNF4α). *p<0.05 vs. DMSO control, n=3. (B) qRT-PCR analysis of primary MF-HSC treated with GDC-0449 for 3 days. *p<0.05, **p<0.01 vs. DMSO control. (C–E) ASMA-Cre-ER^T2–Smo-flox (double transgenic, DTG) mice were subjected to BDL and treated with vehicle (VEH, olive oil, n=3) or tamoxifen (TMX, n=4) every other day from day 4–10 post-BDL. (C) qRT-PCR analysis of total liver mRNA, *p<0.05. (D) Representative immunohistochemistry and quantification for Notch-2 and Hey2. Scale bar: 100μm. *p<0.05, **p<0.01. (E) Double staining of Notch-2 or Hey2 (brown) with Desmin (green) in liver sections described in Figure 5D. The percentages of Notch-2/Desmin or Hey2/Desmin double-positive cells among Desmin+ cells were also quantified. At least 10 fields were counted per mouse. *p<0.05, n=3.