Stacey S. Huppert

Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1.

The Notch genes encode single-pass transmembrane receptors that transduce the extracellular signals responsible for cell fate determination during several steps of metazoan development. The mechanism by which extracellular signals affect gene transcription and ultimately cell fate decisions is beginning to emerge for the Notch signalling pathway. One paradigm is that ligand binding to Notch triggers a Presenilin1-dependent proteolytic release of the Notch intracellular domain from the membrane, resulting in low amounts of Notch intracellular domain which form a nuclear complex with CBF1/Su(H)/Lag1 to activate transcription of downstream targets. Not all observations clearly support this processing model, and the most rigorous test of it is to block processing in vivo and then determine the ability of unprocessed Notch to signal. Here we report that the phenotypes associated with a single point mutation at the intramembranous processing site of Notch1, Val1,744→Gly, resemble the null Notch1 phenotype. Our results show that efficient intramembranous processing of Notch1 is indispensable for embryonic viability and proper early embryonic development in vivo.

Liver‐specific deletion of histone deacetylase 3 disrupts metabolic transcriptional networks

Histone deacetylase 3 (Hdac3) is an enzymatic component of transcriptional repression complexes recruited by the nuclear hormone receptors. Inactivation of Hdac3 in cancer cell lines triggered apoptosis, and removal of Hdac3 in the germ line of mice caused embryonic lethality. Therefore, we deleted Hdac3 in the postnatal mouse liver. These mice developed hepatomegaly, which was the result of hepatocyte hypertrophy, and these morphological changes coincided with significant imbalances between carbohydrate and lipid metabolism. Loss of Hdac3 triggered changes in gene expression consistent with inactivation of repression mediated by nuclear hormone receptors. Loss of Hdac3 also increased the levels of Pparγ2, and treatment of these mice with a Pparγ antagonist partially reversed the lipid accumulation in the liver. In addition, gene expression analysis identified mammalian target of rapamycin signalling as being activated after deletion of Hdac3, and inhibition by rapamycin affected the accumulation of neutral lipids in Hdac3‐null livers. Thus, Hdac3 regulates metabolism through multiple signalling pathways in the liver, and deletion of Hdac3 disrupts normal metabolic homeostasis.

Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE

The four highly conserved Notch receptors receive short-range signals that control many biological processes during development and in adult vertebrate tissues. The involvement of Notch1 signaling in tissue self-renewal is less clear, however. We developed a novel genetic approach N1IP-CRE (Notch1 Intramembrane Proteolysis) to follow, at high resolution, the descendents of cells experiencing Notch1 activation in the mouse. By combining N1IP-CRE with loss-of-function analysis, Notch activation patterns were correlated with function during development, self-renewal and malignancy in selected tissues. Identification of many known functions of Notch1 throughout development validated the utility of this approach. Importantly, novel roles for Notch1 signaling were identified in heart, vasculature, retina and in the stem cell compartments of self-renewing epithelia. We find that the probability of Notch1 activation in different tissues does not always indicate a requirement for this receptor and that gradients of Notch1 activation are evident within one organ. These findings highlight an underappreciated layer of complexity of Notch signaling in vivo. Moreover, NIP-CRE represents a general strategy applicable for monitoring proteolysis-dependent signaling in vivo.

Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.

Alagille syndrome, a chronic hepatobiliary disease, is characterized by paucity of intrahepatic bile ducts (IHBDs). To determine the impact of Notch signaling specifically on IHBD arborization, we studied the influence of both chronic gain and loss of Notch function on the intact three‐dimensional IHBD structure using a series of mutant mouse models and a resin casting method. Impaired Notch signaling in bipotential hepatoblast progenitor cells (BHPCs) dose‐dependently decreased the density of peripheral IHBDs, whereas activation of Notch1 results in an increased density of peripheral IHBDs. Although Notch2 has a dominant role in IHBD formation, there is also a redundant role for other Notch receptors in determining the density of peripheral IHBDs. Because changes in IHBD density do not appear to be due to changes in cellular proliferation of bile duct progenitors, we suggest that Notch plays a permissive role in cooperation with other factors to influence lineage decisions of BHPCs and sustain peripheral IHBDs. Conclusion: There is a threshold requirement for Notch signaling at multiple steps, including IHBD tubulogenesis and maintenance, during hepatic development that determines the density of three‐dimensional peripheral IHBD architecture. (HEPATOLOGY 2010.)

EGFR Blockade Enriches for Lung Cancer Stem–like Cells through Notch3-Dependent Signaling

Mutations in the epidermal growth factor receptor (EGFR) are the most common actionable genetic abnormalities yet discovered in lung cancer. However, targeting these mutations with kinase inhibitors is not curative in advanced disease and has yet to demonstrate an impact on potentially curable, early-stage disease, with some data suggesting adverse outcomes. Here, we report that treatment of EGFR-mutated lung cancer cell lines with erlotinib, while showing robust cell death, enriches the ALDH(+) stem-like cells through EGFR-dependent activation of Notch3. In addition, we demonstrate that erlotinib treatment increases the clonogenicity of lung cancer cells in a sphere-forming assay, suggesting increased stem-like cell potential. We demonstrate that inhibition of EGFR kinase activity leads to activation of Notch transcriptional targets in a γ secretase inhibitor-sensitive manner and causes Notch activation, leading to an increase in ALDH high(+) cells. We also find a kinase-dependent physical association between the Notch3 and EGFR receptors and tyrosine phosphorylation of Notch3. This could explain the worsened survival observed in some studies of erlotinib treatment at early-stage disease, and suggests that specific dual targeting might overcome this adverse effect.

Analysis of transmembrane domain mutants is consistent with sequential cleavage of Notch by γ‐secretase

γ‐Secretase is a lipid‐embedded, intramembrane‐cleaving aspartyl protease that cleaves its substrates twice within their transmembrane domains (TMD): once near the cytosolic leaflet (at S3/ɛ) and again in the middle of the TMD (at S4/γ). To address whether this unusual process occurs in two independent or interdependent steps, we investigated how mutations at the S3/ɛ site in Notch1‐based substrates impact proteolysis. We demonstrate that such mutations greatly inhibit not only γ‐secretase‐mediated cleavage at S3 but also at S4, independent of their impact on NICD stability. These results, together with our previous observations, suggest that hydrolysis at the center of the Notch transmembrane domain (S4/γ) is dependent on the S3/ɛ cleavage. Notch (and perhaps all γ‐secretase substrates) may be cleaved by sequential proteolysis starting at S3.

Intrahepatic Bile Duct Regeneration in Mice Does Not Require Hnf6 or Notch Signaling through Rbpj

The potential for intrahepatic bile duct (IHBD) regeneration in patients with bile duct insufficiency diseases is poorly understood. Notch signaling and Hnf6 have each been shown to be important for the morphogenesis of IHBDs in mice. One congenital pediatric liver disease characterized by reduced numbers of IHBDs, Alagille syndrome, is associated with mutations in Notch signaling components. Therefore, we investigated whether liver cell plasticity could contribute to IHBD regeneration in mice with disruptions in Notch signaling and Hnf6. We studied a mouse model of bile duct insufficiency with liver epithelial cell-specific deficiencies in Hnf6 and Rbpj, a mediator of canonical Notch signaling. Albumin-Cre Hnf6(flox/flox)Rbpj(flox/flox) mice initially developed no peripheral bile ducts. The evolving postnatal liver phenotype was analyzed using IHBD resin casting, immunostaining, and serum chemistry. With age, Albumin-Cre Hnf6(flox/flox)Rbpj(flox/flox) mice mounted a ductular reaction extending through the hepatic tissue and then regenerated communicating peripheral IHBD branches. Rbpj and Hnf6 were determined to remain absent from biliary epithelial cells constituting the ductular reaction and the regenerated peripheral IHBDs. We report the expression of Sox9, a marker of biliary epithelial cells, in cells expressing hepatocyte markers. Tissue analysis indicates that reactive ductules did not arise directly from preexisting hilar IHBDs. We conclude that liver cell plasticity is competent for regeneration of IHBDs independent of Notch signaling via Rbpj and Hnf6.

Genetic interactions between hepatocyte nuclear factor-6 and Notch signaling regulate mouse intrahepatic bile duct development in vivo.

Notch signaling and hepatocyte nuclear factor‐6 (HNF‐6) are two genetic factors known to affect lineage commitment in the bipotential hepatoblast progenitor cell (BHPC) population. A genetic interaction involving Notch signaling and HNF‐6 in mice has been inferred through separate experiments showing that both affect BHPC specification and bile duct morphogenesis. To define the genetic interaction between HNF‐6 and Notch signaling in an in vivo mouse model, we examined the effects of BHPC‐specific loss of HNF‐6 alone and within the background of BHPC‐specific loss of recombination signal binding protein immunoglobulin kappa J (RBP‐J), the common DNA‐binding partner of all Notch receptors. Isolated loss of HNF‐6 in this mouse model fails to demonstrate a phenotypic variance in bile duct development compared to control. However, when HNF‐6 loss is combined with RBP‐J loss, a phenotype consisting of cholestasis, hepatic necrosis, and fibrosis is observed that is more severe than the phenotype seen with Notch signaling loss alone. This phenotype is associated with significant intrahepatic biliary system abnormalities, including an early decrease in biliary epithelial cells, evolving to ductular proliferation and a decrease in the density of communicating peripheral bile duct branches. In this in vivo model, simultaneous loss of both HNF‐6 and RBP‐J results in down‐regulation of both HNF‐1β and Sox9 (sex determining region Y–related HMG box transcription factor 9). Conclusion: HNF‐6 and Notch signaling interact in vivo to control expression of downstream mediators essential to the normal development of the intrahepatic biliary system. This study provides a model to investigate genetic interactions of factors important to intrahepatic bile duct development and their effect on cholestatic liver disease phenotypes. (HEPATOLOGY 2012;55:232–242)

Notch1 Mutation Leads to Valvular Calcification Through Enhanced Myofibroblast Mechanotransduction

Objective— Calcific aortic valve disease (CAVD) is a significant cardiovascular disorder, and controversy exists as to whether it is primarily a dystrophic or osteogenic process in vivo. In this study, we sought to clarify the mechanism of CAVD by assessing a genetic mutation, Notch1 heterozygosity, which leads to CAVD with 100% penetrance in humans. Approach and Results— Murine immortalized Notch1+/− aortic valve interstitial cells (AVICs) were isolated and expanded in vitro. Molecular signaling of wild-type and Notch1+/− AVICs were compared to identify changes in pathways that have been linked to CAVD—transforming growth factor-&bgr;1/bone morphogenetic protein, mitogen-activated protein kinase, and phosphoinositide 3-kinase/protein kinase B—and assessed for calcification potential. Additionally, AVIC mechanobiology was studied in a physiologically relevant, dynamic mechanical environment (10% cyclic strain) to investigate differences in responses between the cell types. We found that Notch1+/− AVICs resembled a myofibroblast-like phenotype expressing higher amounts of cadherin-11, a known mediator of dystrophic calcification, and decreased Runx2, a known osteogenic marker. We determined that cadherin-11 expression is regulated by Akt activity, and inhibition of Akt phosphorylation significantly reduced cadherin-11 expression. Moreover, in the presence of cyclic strain, Notch1+/− AVICs exhibited significantly upregulated phosphorylation of Akt at Ser473 and smooth muscle &agr;-actin expression, indicative of a fully activated myofibroblast. Finally, these Notch1-mediated alterations led to enhanced dystrophic calcific nodule formation. Conclusions— This study presents novel insights in our understanding of Notch1-mediated CAVD by demonstrating that the mutation leads to AVICs that are fully activated myofibroblasts, resulting in dystrophic, but not osteogenic, calcification.

Defects in hepatic Notch signaling result in disruption of the communicating intrahepatic bile duct network in mice

SUMMARY Abnormal Notch signaling in humans results in Alagille syndrome, a pleiotropic disease characterized by a paucity of intrahepatic bile ducts (IHBDs). It is not clear how IHBD paucity develops as a consequence of atypical Notch signaling, whether by a developmental lack of bile duct formation, a post-natal lack of branching and elongation or an inability to maintain formed ducts. Previous studies have focused on the role of Notch in IHBD development, and demonstrated a dosage requirement of Notch signaling for proper IHBD formation. In this study, we use resin casting and X-ray microtomography (microCT) analysis to address the role of Notch signaling in the maintenance of formed IHBDs upon chronic loss or gain of Notch function. Our data show that constitutive expression of the Notch1 intracellular domain in bi-potential hepatoblast progenitor cells (BHPCs) results in increased IHBD branches at post-natal day 60 (P60), which are maintained at P90 and P120. By contrast, loss of Notch signaling via BHPC-specific deletion of RBP-J (RBP KO), the DNA-binding partner for all Notch receptors, results in progressive loss of intact IHBD branches with age. Interestingly, in RBP KO mice, we observed a reduction in bile ducts per portal vein at P60; no further reduction had occurred at P120. Thus, bile duct structures are not lost with age; instead, we propose a model in which BHPC-specific loss of Notch signaling results in an initial developmental defect resulting in fewer bile ducts being formed, and in an acquired post-natal defect in the maintenance of intact IHBD architecture as a result of irresolvable cholestasis. Our studies reveal a previously unappreciated role for Notch signaling in the post-natal maintenance of an intact communicating IHBD structure, and suggest that liver defects observed in Alagille syndrome patients might be more complex than bile duct paucity.