Cross-talk between Notch and Hedgehog Regulates Hepatic Stellate Cell Fate

Abstract

Liver repair involves phenotypic changes in hepatic stellate cells (HSC) and re-activation of morphogenic signaling pathways that modulate epithelial-to-mesenchymal/mesenchymal-to-epithelial transitions, such as Notch and Hedgehog (Hh). Hh stimulates HSC to become myofibroblasts (MF). Recent lineage tracing studies in adult mice with injured livers showed that some MF became multipotent progenitors to regenerate hepatocytes, cholangiocytes, and HSC. We studied primary HSC cultures and two different animal models of fibrosis to evaluate the hypothesis that activating the Notch pathway in HSC stimulates them to become (and remain) MF via a mechanism that involves an epithelial-to-mesenchymal-like transition, and requires cross-talk with the canonical Hh pathway. We found that when cultured HSC transitioned into MF, they activated Hh signaling, underwent an epithelial-to-mesenchymal-like transition, and increased Notch signaling. Blocking Notch signaling in MF-HSC 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 MF and Hh-responsive MF progeny (i.e., HSC and ductular cells). Conditionally disrupting Hh signaling in MF of bile duct-ligated (BDL) 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.

The outcome of liver injury is dictated by the efficiency of repair responses that replace damaged liver tissue with healthy hepatic parenchyma. Defective repair of chronic liver injury can result in cirrhosis, a scarring condition characterized by dramatic changes in the cellular composition of the liver. Outgrowth of progenitors and myofibroblasts (MF) is particularly prominent during scarring.(1) Since these cell types are critical for successful regeneration of damaged livers,(1, 2) their accumulation in cirrhotic liver suggests that scarring may occur because regenerative mechanisms become stalled prematurely. In order to restore healthy wound healing, therefore, it is necessary to characterize and prioritize the key signals that regulate the fate of cells that are required for liver repair.

Reconstruction of damaged adult liver utilizes several highly-conserved signaling pathways that orchestrate organogenesis during fetal development, including Wnt, Hedgehog (Hh), and Notch.(3) During embryogenesis, these pathways interact to modulate survival, proliferation, and differentiation of their target cells so that developing organs become appropriately populated with all of the cell types necessary for tissue-specific functions. For example, cross-talk between Hh and Notch controls the fate of embryonic stem cells,(4) zebrafish neural progenitors,(5) and Drosophila eye precursors.(6) In cancer biology, the importance of cell-autonomous cross-talk between Hh and Notch is also emerging. Overexpression of both the Notch and Hh signaling pathways occurs in a subpopulation of chemotherapy-resistant cancer stem cells and targeting Notch and Hh depleted this population.(7) However, whether similar cross-talk occurs when damaged adult livers are regenerated, which cell types are involved, and whether or not such signaling becomes deregulated during defective repair is not well-understood. Also uncertain is if/how these newly uncovered pathways in damaged adult liver fit into the classical paradigms for cirrhosis pathogenesis, and whether they are more or less important for that process than well-established regulators of adult liver growth, such as transforming growth factor-beta (TGF-β), which is generally credited for driving defective liver repair in adults.(1)

The aims of this study, therefore, were to investigate if and how Notch signaling regulates damage-related outgrowth of liver MF. We focused on MF derived from HSC because adult HSC are TGF-β-responsive cells that are also influenced by developmental morphogenic pathways, such as Wnt and Hh, which re-activate during adult liver repair. Adult HSC require Hh signaling to become and remain MF.(8) Recent lineage tracing studies in adult mice with injured livers demonstrated that some MF became multipotent progenitors that regenerated hepatocytes, cholangiocytes, and HSC. In parallel experiments, Cre recombinase-mediated knock down of canonical Hh signaling in cells expressing the MF gene, alpha-smooth muscle actin (ASMA), both blocked MF accumulation and inhibited outgrowth of ductular cells during cholestatic liver injury.(9) Both autocrine and paracrine signaling regulated by the Hh pathway might be involved. For example, Sonic hedgehog ligand is known to promote the transcription of Jagged-1,(10) and MF-derived Jagged-1 is thought to work in a paracrine fashion to promote ductular differentiation of Notch-responsive liver progenitors.(2) Previous work suggested that HSC themselves may also be capable of Notch signaling.(11) Most recently, Chen et al. reported that DAPT, a γ-secretase inhibitor that blocks Notch signaling, decreased expression of various MF genes in a rat HSC line (HSC-T6).(12) They also found that DAPT inhibited CCl₄-related fibrosis in rats and showed that this was accompanied by reduced hepatic expression of TGF-β, Snail, and various mesenchymal genes, but up-regulation of E-cadherin, suggesting that blocking Notch promoted mesenchymal-to-epithelial transitions.(13) An earlier study of cultured HSC, however, correlated induction of Notch-1 and Hes1 with suppression of ASMA expression and proliferation, and showed that knocking down expression of Notch-1 enhanced HSC growth.(14)

Indeed, the effects of Notch on MF differentiation and growth are complex and appear to vary according to the type of MF precursor. Notch signaling inhibits myofibroblastic differentiation of myoblast precursors and some types of fibroblasts.(15, 16) In contrast, it enhances MF differentiation of lung MF precursors,(17) airway epithelial cells,(18) and dermal fibroblasts.(19) Activating Notch also promotes epithelial-to-mesenchymal transition in kidney cells,(20) stimulates expansion of cardiac progenitors at the expense of MF,(21) and promotes an epithelial-to-mesenchymal transition process that enhances the stem-like properties of cancer stem cells.(22)

Notch signaling is critical for biliary morphogenesis during development.(23–25) As mentioned earlier, the fate of adult liver progenitors is also directed by Notch: increasing Notch signaling promotes differentiation along the biliary lineage, while suppressing the Notch pathway shifts progenitors towards an hepatocytic fate.(2) Deregulated Notch signaling has been implicated in the pathogenesis of hepatocellular carcinoma and cholangiocarcinoma.(26, 27) Despite growing evidence for Notch pathway involvement in liver cancer and fibrosis, it is unclear how Notch interfaces with other key signaling pathways that have been implicated in those disorders, or how Notch signaling in one type of liver cell (e.g., MF) might influence the accumulation of other types of liver cells (e.g., epithelial progenitors) that are required for adult liver repair.

In this study, we evaluate the hypothesis that Notch pathway activation in HSC stimulates them to become (and remain) MF via a mechanism that involves an epithelial-to-mesenchymal-like transition requiring cross-talk with canonical (i.e., TGF-β independent) Hedgehog signaling.

Materials and Methods

Full methods are available in Supplementary Materials/Methods.

Animals

Male C57BL/6 mice and Smo^tm2Amc/J (Smo/flox) mice were obtained from Jackson Laboratory (Bar Harbor, ME)(28). Smo/flox mice were crossed with ASMA-Cre-ER^T2 transgenic mice(29) to generate double-transgenic (DTG) mice in which treatment with tamoxifen induces conditional deletion of Smo in ASMA–positive cells.(9) 8–12 weeks old mice were subjected to bile duct ligation (BDL) or sham surgery for 14 days. Other 8–10 weeks old wild type mice were fed with a high fat diet and given intraperitoneal injection of either vehicle (olive oil) or CCl₄ (1 μL/g body weight, prediluted 1:3 in olive oil) twice per week for 2 weeks, and sacrificed 72 hours after last CCl₄ injection.(30) Animal experiments fulfilled National Institutes of Health and Duke University-Institutional Animal Care and Use Committee requirements for humane animal care.

Immunohistochemistry

Formalin-fixed, paraffin-embedded livers were prepared for immunohistochemistry.(9) Protocols and antibodies used are listed in Supplementary Materials/Methods.

Molecular Techniques

qRT-PCR and immunoblots were performed as described.(31)

Cell Isolation

Primary HSC were isolated from C57BL/6 mice using standard approaches. Purity of the preparations was rigorously analyzed as described.(9)

Pharmacological Manipulation of Notch and Hh Signaling

Day 4 primary HSC cultures were treated with γ-secretase inhibitor, DAPT (10 μM, Sigma-Aldrich, St Louis, MO) or Smoothened agonist, GDC-0449 (1 μM, Selleck Chemicals, Houston, TX), for 3 days. Controls were treated with DMSO. 603B cells were treated the same way for 2 days.

Statistics

Results are expressed as mean ± SEM. Analyses were performed using Student t test. P<.05 was considered significant.

Results

Activation of Notch Signaling in Desmin-expressing Cells during Hepatic Injury

We found up-regulation of mRNAs for Notch-2, Jagged-1, and several Notch-target genes (Hes1, Hey1, Hey2, and HeyL) in a mouse BDL model (Figure 1A), consistent with previous reports that adult liver injury activates Notch signaling.(2, 23) In addition to ductal cells (known Notch targets)(23), stromal cells expressed Notch-2, Jagged-1 and Hey2 post-BDL (Figure 1B, Supplementary Figure 1A). Some of these stromal cells co-stained with the HSC marker, Desmin, suggesting that activated Notch signaling occurs in MF-HSC during liver injury. Quantitative immunohistochemistry indicated that about 60% of the Desmin(+) cells co-expressed Notch-2 and/or Jagged-1, and 30% co-expressed Hey2. These findings were confirmed with FACS analysis of HSC isolated from BDL mice, which showed increased Notch-2, Jagged-1 and Hey2 compared to HSC harvested from sham controls (Figure 1C, Supplementary Figure 1B).

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.

We also examined mice treated with high fat (HF) diet ± CCl₄ for 2 weeks to provoke liver sinusoidal fibrosis. Compared to HF diet-fed controls, mice treated with HF diet/CCl₄ demonstrated increased mRNA expression of Notch-2, Jagged-1, Hes1, Hey1 and Hey2, as well a ductular marker, Keratin 19 (Krt19) (Figure 1D). As noted in BDL mice with portal-based fibrosis (Figure 1B–C), quantitative immunohistochemistry also demonstrated increased Notch-2, Jagged-1 and Hey2 expression in Desmin-positive cells of mice with CCl₄-induced sinusoidal fibrosis (Figure 1E, Supplementary Figure 1C).

Up-regulation of Notch Signaling during HSC Activation in vitro

Although it is established that cholangiocytes and their precursors are capable of Notch signaling,(24, 25, 27) it is uncertain if primary HSC and/or their progeny (e.g., MF-HSC) respond to Notch. Because immunohistochemistry and FACS revealed Notch signaling components in Desmin-expressing cells that accumulate in fibrotic livers (Figure 1B,C,E), we evaluated the expression of Notch pathway genes in primary mouse HSC (both freshly isolated HSC and 7-day, culture-activated MF-HSC) (Figure 2A–B). Results in the HSC were compared to those in a mouse ductular cell line (603B), which served as a positive control for Notch signaling (Figure 3). FACS showed that 603B express the cholangiocyte marker, Krt19, progenitor markers (Sox9, FN14, and CD24) and Notch pathway components (Notch-2 and Jagged-1) at very high levels, confirming that such cells are immature ductular-type cells with Notch signaling capability (Figure 3A). FACS similarly revealed that HSC express proteins that regulate Notch signaling, including the Notch ligand, Jagged-1, Notch-1 and Notch-2 receptors, and Numb, a Notch signaling repressor (Figure 2A, Supplementary Figure 2A). QRT-PCR analysis readily demonstrated mRNA for these factors (Figure 2B), while expression of another Notch ligand (Jagged-2) and other Notch receptors (Notch-3 and Notch-4) was detected at much lower levels (Supplementary Figure 2B).

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.

Compared to freshly isolated (day 0) HSC which were relatively enriched with cells expressing Notch-1 and Numb proteins, MF-HSC demonstrated much lower expression of Notch-1 and Numb but much higher expression of Jagged-1 and Notch-2 (Figure 2A, Supplementary Figure 2A), consistent with a previous report showing decreased Notch-1 expression during rat HSC culture activation.(11) Thus, expression of proteins regulating Notch signaling changed substantially during MF trans-differentiation. To determine if pathway activity also changed as quiescent (Q-)HSC transitioned into MF-HSC, qRT-PCR analysis was performed to assess expression of various Notch target genes (Hes1, Hey1, Hey2, and c-Myc) (Figure 2B). Hey2 and c-Myc mRNA expression increased significantly during HSC activation. This induction of Notch target genes occurred in conjunction with up-regulation of Jagged-1 and Notch-2 mRNAs and coincided with down-regulation of mRNAs for Notch-1 and Numb. The results suggest that HSC activate Notch signaling as they become MF. This possibility is supported by evidence that several Notch target genes (Hes1, Hey1, Hey2) mRNA levels in HSC are generally equal to or higher than their levels in ductular-type cells with acknowledged Notch signaling capability (Figure 2B).

Phenotypic and genotypic similarities in Notch-responsive liver cells

Notch regulates the fate of bipotent liver epithelial progenitors(2, 25) and lineage-tracing evidence in adult mice indicates that bipotent liver epithelial progenitors and HSC derive from a common multipotent progenitor that is controlled by the Hh pathway.(9, 32) Thus, it is conceivable that Notch interacts with Hh to direct the differentiation of adult progenitors during liver injury. We began to examine this issue by further characterizing 603B cells via FACS (Figure 3A–B), and using qRT PCR to compare gene expression in 603B cells, mature liver cells (primary mouse hepatocytes), and freshly-isolated or culture-activated primary HSC (Figure 3C).

FACS showed that although 97–99% of 603B cells express well-accepted markers of ductular progenitors (Krt19, Krt7, Sox9), only about a third express the biliary-associated transcription factor, HNF6. HNF4-alpha, a hepatocyte-associated transcription factor, is evident in ~50%, suggesting that 603B cells are capable of differentiating along both biliary and hepatocytic lineages. Consistent with that concept, virtually all of the cells (97–99%) express established markers of hepatoblasts (a.k.a. oval cells), such as CD24, FN14, and albumin. More than 80% of 603B also express a putative HSC marker, GFAP, suggesting that 603B cells may be multipotent (i.e., capable of differentiating into hepatocytes, cholangioctyes, and HSC). Indeed, about a third of 603B express Desmin and about 25% are ASMA-positive. Co-expression of ductular, hepatocytic, and HSC-markers occurs in Hh-responsive multipotent liver progenitors that are undergoing epithelial-mesenchymal transitions.(9) 99% of 603B co-express Krt7 (epithelial marker), vimentin (mesenchymal marker), and one or more Hh-target genes (Ptc, Gli1, Gli2), exhibiting the phenotype of multipotent liver progenitors that are in the midst of epithelial-mesenchymal transitions (Figure 3A–B).

QRT-PCR analysis provided additional evidence that 603B cells are transitioning multipotent liver progenitors. Compared to freshly-isolated primary hepatocytes from healthy adult mice, 603B express significantly higher mRNA levels of Hh-target genes (Ptc, Gli2), cholangiocyte-associated genes (e.g., Krt19 and HNF6) and HSC-associated genes (e.g., Desmin and GFAP), but significantly lower mRNA levels of the HNF4-α, a transcription factor that is strongly expressed by mature hepatocytes. As reported for transitional multipotent progenitors,(9) gene expression in 603B is more similar to HSC than hepatocytes. For example, primary HSC and 603B express comparable mRNA levels of Krt7, HNF6, AFP, Ptc and Gli2. mRNA levels of Desmin and GFAP are significantly lower in 603B than freshly-isolated HSC, however, and this discrepancy is magnified when HSC undergo culture activation to become MF (Figure 3C). Never-the-less, the aggregate data demonstrate genotypic and phenotypic similarities in Notch-responsive liver cells, and indicate that such cells are Hh-responsive and inherently plastic (i.e., capable of undergoing epithelial-mesenchymal transitions).

DAPT Inhibits Notch Signaling in both Progenitors and HSCs in vitro

To investigate the functional significance of Notch signaling in HSC, the Notch pathway was suppressed by treating cultured primary MF-HSC with a γ-secretase inhibitor, DAPT. Results in HSC were compared to those in multipotent progenitor cells (603B), which served as a positive control for Notch signaling. As expected, studies in 603B showed that DAPT treatment significantly reduced expression of Jagged-1, Notch-2, and Notch target genes (Hes1, Hey1 and Hey2) (Figure 4). Inhibiting Notch signaling in 603B suppressed expression of cholangiocyte-associated genes (Krt7, Krt19, HNF1β, and HNF6), and permitted induction of hepatocyte lineage markers (alpha-fetoprotein (AFP), HNF1α, and HNF4α), consistent with previous reports that activation of Notch signaling drives liver progenitors towards the biliary lineage, while its suppression promotes differentiation along the hepatocytic lineage.(2, 24, 25) Blocking Notch signaling in 603B enhanced expression of glial fibrillary acidic protein (GFAP), a Q-HSC marker, but reduced ASMA, a MF-HSC marker, and TGF-β, a profibrogenic cytokine that promotes ductular differentiation of liver progenitors in developing embryos.(33) Blocking Notch also down-regulated key Hedgehog target genes, Gli1 and Patched (Ptc), in 603B. The aggregate findings suggest that Notch signaling interfaces with fibrogenic signals that are transduced by TGF-β and the Hh pathway in multipotent liver progenitor cells. This is particularly intriguing because both TGF-β and Hh signaling promote epithelial-to-mesenchymal transitions in developing embryos,(34) and Hh has been proven to stimulate epithelial-to-mesenchymal-like transitions in both adult HSC and progenitor cells.(8, 35)

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.

Having confirmed that DAPT performed as anticipated in Notch-responsive liver progenitor cells, we evaluated its actions in HSC. For these studies, primary murine HSC were cultured for 4 days to induce MF trans-differentiation and then treated with DAPT for an additional 3 days. As in 603B (Figure 4), MF-HSC showed DAPT-inhibited expression of Notch-2, Jagged-1, and several Notch target gene (Hey1, Hey2 and HeyL) mRNAs (Figure 5A). Immunocytochemistry confirmed that mRNA suppression was accompanied by decreased protein expression (Figure 5E). Blocking Notch signaling in MF-HSC also repressed typical MF-associated genes (ASMA, collagen and TGF-β) and Hh target genes that are known to be expressed by MF-HSC (Gli2, Ptc, and Sonic Hedgehog, Shh) (Figure 5B). In contrast, mRNA levels of various epithelial genes (bone morphogenic protein-7, desmoplakin, E-cadherin, AFP, HNF4α and Krt19) and Q-HSC markers (peroxisome proliferator activator receptor-γ, PPAR-γ, and GFAP) were up-regulated (Figure 5C). Immunocytochemistry confirmed the DAPT-induced reversion of MF-HSC to a more quiescent phenotype, showing decreased staining for ASMA, and Ki67 (proliferation marker) and increased oil red O staining indicative of neutral lipid accumulation (Figure 5F). Interestingly, when Notch signaling was inhibited and the MF-HSC reverted to a more quiescent phenotype, mRNA expression of Dlk1, a Notch-related gene that marks liver progenitors (36), and mRNAs encoding other progenitor cell markers (e.g., Nanog, Oct4, and FN14) were down-regulated (Figure 5D). Thus, Notch signaling is activated during culture-induced primary MF-HSC trans-differentiation, and this permits the cells to acquire a more mesenchymal phenotype with progenitor-like features. This process parallels activation-associated induction of Hh signaling and might be regulated by cross-talk between the Notch and Hh pathways because HSC require Hh signaling to become MF.(8, 31)

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.

Inhibiting Hedgehog Signaling Blocks Notch Signaling in vitro

To further examine possible cross-talk between Notch and Hh signaling, the two Notch-responsive cell types (603B and primary MF-HSC) were treated with a Hh signaling antagonist, GDC-0449. GDC-0449 directly interacts with and inhibits the Hh co-receptor, Smoothened.(37) Earlier work has proven that GDC-0449 recapitulates the effect of Smoothened gene knock-down in MF-HSC, with both approaches inhibiting canonical Hh signaling, thereby blocking the nuclear localization and transcriptional activation of Gli DNA-binding proteins.(31) In both cell types, antagonizing Smoothened caused suppression of Notch-2, Jagged-1 and Notch target genes (Figures 6A–B), demonstrating that canonical Hh pathway activity promotes the expression of Notch signaling pathway genes. Given that DAPT, a γ-secretase inhibitor that specifically blocks Notch signaling, suppressed expression of Shh ligand, Gli2 (Hh-regulated transcription factor), and Ptc (a direct transcriptional target of Gli) (Figure 5), the Notch pathway seems to stimulate Hh pathway activity. Hence, the results identify a previously unsuspected Hh-Notch positive feedback loop that regulates cell fate decisions in immature ductular-type cells and MF-HSC. In certain types of adult liver injury, these two cell types accumulate and intermingle within fibrotic septae that extend outward from portal tracts to cause bridging fibrosis, an antecedent to cirrhosis.(38) This suggests that Notch-Hh interactions might regulate cirrhosis pathogenesis by controlling the fate of two key cell types that are involved in liver repair.

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.

Blocking Hh Signaling in MF Inhibits Notch Signaling in vivo

To verify that Hh signaling regulates Notch signaling in vivo, as observed in vitro, and to evaluate the functional implications of this interaction for liver repair, we used a genetic approach to conditionally delete Smoothened in MF-HSC. Double transgenic (DTG) mice were created by crossing Smo^flox/flox mice with ASMA-Cre-ER^T2 mice. Treating such DTG mice with tamoxifen (TMX) induced selective deletion of the floxed Smo gene, but only in ASMA-expressing cells,(31) providing a useful tool for examining the effects of Hh signaling in MF-HSC and their progeny.(9) DTG mice underwent BDL to provoke liver injury and compensatory repair responses. Four days later, treatment with either vehicle or TMX was initiated and given every other day through day 10; mice were sacrificed on day 14 post-BDL for liver tissue analysis. In an earlier study, we showed that this approach knocked down expression of Smo in the liver, reduced the hepatic content of ASMA(+) cells by >85%, and significantly decreased collagen gene expression, hepatic hydroxyproline content, and Sirius red staining, as well as the accumulation of Krt19(+) ductular cells.(9) In this study, we confirmed that TMX reduced both Smo and ASMA expression (Figure 6C), and showed that decreasing Hh-responsive MF dramatically decreased numbers of Notch-2(+) and Hey2(+) cells, both along liver sinusoids (co-localized with Desmin(+) cells) and in residual ductular structures (Figure 6D). QRT-PCR analysis of whole liver RNA demonstrated that the loss of Notch-2-expressing cells in TMX-treated DTG mice was accompanied by significantly reduced whole liver expression of Notch target genes compared to the vehicle-treated controls (Figure 6C). Immunoblot analysis of whole liver lysates confirmed that suppression of Notch signaling was accompanied by the expected loss of proteins that mark ductular-type cells and their progenitors (e.g., Krt19 and HNF6), with concomitant induction of the hepatocyte-enriched transcription factor, HNF4α (Supplementary figure 3C). Interestingly, however, we were unable to detect differences in expression of Jagged-1 mRNA (Figure 6C) or protein (Supplementary figure 3A) in our BDL-mice despite significant reductions in αMSA-expressing cells at the time point we examined. Immunohistochemistry demonstrated co-localization of Jagged-1 in Desmin(+) stromal cells that persisted after Smo deletion, suggesting that unlike culture-activated MF-HSC (Figure 5A), in vivo-activated HSC maintain Jagged-1 expression for at least a while after they revert from a myofibroblastic state to a more quiescent HSC phenotype. To determine whether or not Jagged-1 is able to activate Notch signaling after Smo knock-down, we tested responses to recombinant Jagged-1 ligand in primary HSC from Smo^flox/flox mice after the HSC were culture-activated to MF and treated with Cre-recombinase adenoviral vectors to delete Smo. Results were compared to Smo^flox/flox HSC treated with control adenoviral vectors (Ad-GFP). Jagged-1 significantly increased expression of Notch 2 and Notch target genes in control HSC, but had no effect in Smo-depleted HSC (Supplementary Figure 3B). Thus, the aggregate in vivo and in vitro data suggest that the Hh pathway modulates Notch signaling down-stream of Jagged-1 in liver cells, at least in part by promoting expression of Notch-2. Abrogating canonical Hh signaling prevents Jagged-1 from inducing Notch-2 and is sufficient to cause liver cells to become relatively resistant to Jagged-1, thereby inhibiting Jagged-Notch signaling and blocking induction of Notch target genes. This blocked the outgrowth of both myofibroblastic and ductular cells, and reduced fibrosis during cholestatic liver injury (present data and (9)). Given that blocking Notch inhibited Hh in cultured MF (Figure 5B), and inhibiting Notch signaling also decreased liver fibrosis in rats treated with CCl₄,(13) it seems likely that the Hh and Notch pathways interact to control HSC fate in vivo, as they do in vitro. Future experiments that conditionally disrupt Notch signaling in MF are needed to resolve that issue.

Discussion

This study demonstrates for the first time that primary HSC use the Notch signaling pathway to regulate their trans-differentiation. We found that as HSC become MF in culture, they up-regulate their expression of the Notch ligand, Jagged-1, as well as the Notch-2 receptor, while down-regulating their expression of Notch-1 receptor and Numb, a Notch signaling inhibitor. Our findings in primary mouse HSC differ somewhat from those that were reported recently in a T antigen-transformed rat HSC line which was shown to express mainly Notch-3.(12) As was noted in that immortalized rat HSC line, however, we also found that primary MF-HSC reverted to a less myofibroblastic phenotype when treated with DAPT, a specific Notch signaling inhibitor. Moreover, we showed that inhibiting Notch permitted the primary MF-HSC to reacquire markers of Q-HSC (e.g., GFAP and PPAR-γ), re-accumulate lipid, become less proliferative, and to express several genes that typify epithelial cells (e.g., E-cadherin and Desmoplakin). Evidence that blocking Notch signaling permits a mesenchymal-to-epithelial-like transition in primary MF-HSC is novel, but consistent with the known ability of Notch to promote epithelial-to-mesenchymal transitions.(39) Indeed, we observed that DAPT also decreased Notch signaling and mesenchymal gene expression in an immature ductular cell line (603B) with multipotent liver epithelial progenitor features. During this process, we observed that 603B exhibited not only the expected down-regulation of ductular progenitor markers (e.g., HNF1, HNF6, Krt19) and reciprocal up-regulation of hepatocytic progenitor markers (e.g., HNF4α and AFP), but also showed increased expression of the Q-HSC gene, GFAP.

Evidence that a Notch-regulated progenitor for hepatocytes and cholangiocytes can also differentiate into Notch-sensitive cells that express markers of HSC is consistent with an earlier lineage tracing study in adult mice which suggested a common lineage for such bipotent liver epithelial progenitors and HSC,(32) as well as a more recent lineage tracing study which proved that ASMA- and GFAP-expressing cells give rise to hepatocytes and ductular cells during adult liver injury.(9) MF derived from HSC express several markers of multi-potent progenitors, including Oct4.(40) Other adult epithelial tissues are known to harbor subpopulations of differentiated (non-stem) cells that are capable of de-differentiating into stem-like cells;(41) passage of such non-stem cells through epithelial-to-mesenchymal transitions has been closely connected to their entrance into the stem cell state.(42) These findings have prompted speculation that stem cell compartments in adult tissues might be replenished by contextual signals within the microenvironment that reactivate pluripotency factors, such as Oct4, in subpopulations of mature cells with intrinsic phenotypic plasticity.(41)

During liver injury, the hepatic microenvironment changes dramatically and factors that are not expressed in healthy adult livers, such as Jagged and Hh ligands, accumulate. Many of the cell types required for liver repair are Hh-responsive, including HSC and bipotent liver progenitors. Activating Hh signaling in such cells globally impacts their fate, provoking epithelial-to-mesenchymal-like transitions, stimulating proliferation, and enhancing survival.(43) Here we demonstrate for the first time that Hh interacts with Notch to orchestrate these cell fate changes in primary HSC. We showed that blocking Notch signaling with DAPT inhibited expression of Hh target genes, such as Ptc, while GDC-0449, a direct antagonist of Smoothened, reduced expression of Notch-2, Hes1, Hey2, and HeyL. MF-HSC require cross-talk between the Notch and Hh pathways to retain their myofibroblastic phenotype because blocking either pathway suppressed expression of typical MF markers (e.g., ASMA and collagen), while inducing re-expression of quiescent markers (e.g., PPAR-γ and GFAP). Parallel studies in 603B confirm that similar Hh-Notch interactions regulate cell fate decisions in multipotent liver progenitors. In addition, cross-talk with other key repair-related signaling pathways is likely to be involved because we found that DAPT suppressed expression of TGF-β mRNA in both MF-HSC and the progenitor cell line, and GDC-0449 has been reported to inhibit TGF-β expression in MF-HSC.(44) TGF-β interacts with its receptors to initiate signals that activate Gli-family factors independently of Smoothened,(45) suggesting that Notch-Hh cross-talk might promote activation of other signaling pathways that re-enforce their actions on down-stream targets.

To clarify the ultimate biological relevance of Hh-Notch interactions in adult liver repair, therefore, we used a Cre recombinase-driven approach to target ASMA-expressing cells, and deleted Smoothened to abrogate canonical (i.e., TGFβ-independent) Hh signaling in mice with ongoing cholestatic liver injury induced by BDL. We found that knocking down Hh signaling in MF significantly inhibited Notch signaling, decreasing whole liver expression of various Notch target genes by 40–60%. This inhibited accumulation of cells that express ductular markers, such as Krt19 and HNF6 (p<0.05 and 0.005 vs. respective vehicle-treated controls). As expected by data generated here and in our earlier work,(9, 31) blocking Hh signaling in MF significantly decreased accumulation of collagen-producing cells and decreased liver fibrosis post-BDL. Contrary to our prediction, however, the depletion of MF did not appreciably reduce hepatic expression of Jagged-1. Immunohistochemistry localized Jagged-1 to Desmin(+) stromal cells that persisted after Smo depletion, suggesting that MF-HSC that revert to quiescence when Hh signaling is abrogated in vivo retain Jagged-1. Hh-deficient cells are relatively resistant to Jagged-Notch signaling, however, because treating Smo-depleted cells with recombinant Jagged-1 failed to evoke induction of Notch-2 or increase expression of Notch-regulated genes. Given present and published evidence for the inherent plasticity of HSC and HSC-derived MF,(40) additional research will be necessary to determine if the outcomes observed after Smo knockdown in MF of BDL mice reflect disruption of Hh-Notch interactions that control epithelial-to-mesenchymal-like/mesenchymal-to-epithelial-like transitions in these wound-healing cells. In any case, the new evidence that Hh signaling influences Notch pathway activity in the injured adult mouse livers complements data that demonstrate mutually re-enforcing cross-talk between these two signaling pathways in cultured adult liver cells. Stated another way, both in vitro and in vivo, activating the Hh pathway stimulates Notch signaling, and the latter further enhances pro-fibrogenic Hh signaling. The newly-identified positive feed-back loop provides a previously unsuspected mechanism that helps to explain why a recent study found that treating rats with a Notch inhibitor reduced CCl₄-induced liver fibrosis.(13)

In summary, our latest discoveries complement work by other groups and together, extend growing evidence that adult liver repair is controlled by reactivated morphogenic signaling pathways that orchestrate organogenesis during development, such as Notch and Hedgehog. These pathways clearly act in concert during adult organ repair, and likely coordinate during development as well. In the adult liver, these mechanisms appear to involve modulation of fundamental fate decisions in subpopulations of adult liver cells that retain high levels of inherent plasticity. Although additional research is needed to clarify the nuances of this insight, it has already identified a myriad of novel diagnostic and therapeutic targets that might be exploited to improve the outcomes of adult liver injury.

Abbreviations

ASMA

α-smooth muscle actin

BDL

bile duct ligation

DTG

double transgenic

Hes

hairy and enhancer of split

Hey

hairy/enhancer-of-split related with YRPW

Hh

Hedgehog

HSC

Hepatic stellate cell

Krt 7

keratin 7

Krt19

keratin 19

MF

myofibroblast

Smo

smoothened