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In IBD patients, integration between a hyper-activated immune system and epithelial cell plasticity underlies colon cancer development. However, molecular regulation of such a circuity remains undefined. Claudin-1 (Cld-1), a tight-junction integral protein deregulation alters colonic epithelial cell (CEC) differentiation, and promotes colitis severity while impairing colitis-associated injury/repair. Tumorigenesis is a product of an unregulated wound-healing process and therefore we postulated that upregulated Cld-1 levels render IBD patients susceptible to the colitis-associated cancer (CAC). Villin Cld-1 mice are used to carryout overexpressed studies in mice. The role of deregulated Cld-1 expression in CAC and the underlying mechanism was determined using a well-constructed study scheme and mouse models of DSS colitis/recovery and CAC. Using an inclusive investigative scheme, we here report that upregulated Cld-1 expression promotes susceptibility to the CAC and its malignancy. Increased mucosal inflammation and defective epithelial homeostasis accompanied the increased CAC in Villin-Cld-1-Tg mice. We further found significantly increased levels of protumorigenic M2 macrophages and β-cateninSer552 (β-CatSer552) expression in the CAC in Cld-1Tg vs. WT mice. Mechanistic studies identified the role of PI3K/Akt signaling in Cld-1-dependent activation of the β-CatSer552, which, in turn, was dependent on proinflammatory signals. Our studies identify a critical role of Cld-1 in promoting susceptibility to CAC. Importantly, these effects of deregulated Cld-1 were not associated with altered tight junction integrity, but on its noncanonical role in regulating Notch/PI3K/Wnt/ β-CatSer552 signaling. Overall, outcome from our current studies identifies Cld-1 as potential prognostic biomarker for IBD severity and CAC, and a novel therapeutic target.
The mechanism of how organ shape emerges and specifies cell fate is not understood. Pancreatic duct and endocrine lineages arise in a spatially distinct domain from the acinar lineage. Whether these lineages are pre-determined or settle once these niches have been established remains unknown. Here, we reconcile these two apparently opposing models, demonstrating that pancreatic progenitors re-localize to establish the niche that will determine their ultimate fate. We identify a p120ctn-regulated mechanism for coordination of organ architecture and cellular fate mediated by differential E-cadherin based cell sorting. Reduced p120ctn expression is necessary and sufficient to re-localize a subset of progenitors to the peripheral tip domain, where they acquire an acinar fate. The same mechanism is used re-iteratively during endocrine specification, where it balances the choice between the alpha and beta cell fates. In conclusion, organ patterning is regulated by p120ctn-mediated cellular positioning, which precedes and determines pancreatic progenitor fate.
Copyright © 2019 Elsevier Inc. All rights reserved.
During mouse pancreas organogenesis, endocrine cells are born from progenitors residing in an epithelial plexus niche. After a period in a lineage-primed state, progenitors become endocrine committed via upregulation of We find that the to transition is associated with distinct stages of an epithelial egression process: narrowing the apical surface of the cell, basalward cell movement and eventual cell-rear detachment from the apical lumen surface to allow clustering as nascent islets under the basement membrane. Apical narrowing, basalward movement and transcriptional upregulation still occur without Neurog3 protein, suggesting that morphogenetic cues deployed within the plexus initiate endocrine commitment upstream or independently of Neurog3. Neurog3 is required for cell-rear detachment and complete endocrine-cell birth. The ROCK-nmMyoII pathway coordinates epithelial-cell morphogenesis and the progression through -expressing states. NmMyoII is necessary for apical narrowing, basalward cell displacement and upregulation, but all three are limited by ROCK activity. We propose that ROCK-nmMyoII activity, gene-dose and Notch signaling integrate endocrine fate allocation with epithelial plexus growth and morphogenesis, representing a feedback control circuit that coordinates morphogenesis with lineage diversification in the endocrine-birth niche.
© 2018. Published by The Company of Biologists Ltd.
Von Hippel-Lindau (VHL) gene mutations induce neural tissue hemangioblastomas, as well as highly vascularized clear cell renal cell carcinomas (ccRCCs). Pathological vessel remodeling arises from misregulation of HIFs and VEGF, among other genes. Variation in disease penetrance has long been recognized in relation to genotype. We show Vhl mutations also disrupt Notch signaling, causing mutation-specific vascular abnormalities, e.g., type 1 (null) vs. type 2B (murine G518A representing human R167Q). In conditional mutation retina vasculature, Vhl-null mutation (i.e., UBCCreER/+Vhlfl/fl) had little effect on initial vessel branching, but it severely reduced arterial and venous branching at later stages. Interestingly, this mutation accelerated arterial maturation, as observed in retina vessel morphology and aberrant α-smooth muscle actin localization, particularly in vascular pericytes. RNA sequencing analysis identified gene expression changes within several key pathways, including Notch and smooth muscle cell contractility. Notch inhibition failed to reverse later-stage branching defects but rescued the accelerated arterialization. Retinal vessels harboring the type 2B Vhl mutation (i.e., UBCCreER/+Vhlfl/2B) displayed stage-specific changes in vessel branching and an advanced progression toward an arterial phenotype. Disrupting Notch signaling in type 2B mutants increased both artery and vein branching and restored arterial maturation toward nonmutant levels. By revealing differential effects of the null and type 2B Vhl mutations on vessel branching and maturation, these data may provide insight into the variability of VHL-associated vascular changes - particularly the heterogeneity and aggressiveness in ccRCC vessel growth - and also suggest Notch pathway targets for treating VHL syndrome.
γ-Secretase cleavage of the Notch receptor transmembrane domain is a critical signaling event for various cellular processes. Efforts to develop inhibitors of γ-secretase cleavage of the amyloid-β precursor C99 protein as potential Alzheimer's disease therapeutics have been confounded by toxicity resulting from the inhibition of normal cleavage of Notch. We present biochemical and structural data for the combined transmembrane and juxtamembrane Notch domains (Notch-TMD) that illuminate Notch signaling and that can be compared and contrasted with the corresponding traits of C99. The Notch-TMD and C99 have very different conformations, adapt differently to changes in model membrane hydrophobic span, and exhibit different cholesterol-binding properties. These differences may be exploited in the design of agents that inhibit cleavage of C99 while allowing Notch cleavage.
Citrobacter rodentium uses a type III secretion system (T3SS) to induce colonic crypt hyperplasia in mice, thereby gaining an edge during its competition with the gut microbiota through an unknown mechanism. Here, we show that by triggering colonic crypt hyperplasia, the C. rodentium T3SS induced an excessive expansion of undifferentiated Ki67-positive epithelial cells, which increased oxygenation of the mucosal surface and drove an aerobic C. rodentium expansion in the colon. Treatment of mice with the γ-secretase inhibitor dibenzazepine to diminish Notch-driven colonic crypt hyperplasia curtailed the fitness advantage conferred by aerobic respiration during C. rodentium infection. We conclude that C. rodentium uses its T3SS to induce histopathological lesions that generate an intestinal microenvironment in which growth of the pathogen is fueled by aerobic respiration.
Copyright © 2016, American Association for the Advancement of Science.
In many cancers, aberrant Notch activity has been demonstrated to play a role in the initiation and maintenance of the neoplastic phenotype and in cancer stem cells, which may allude to its additional involvement in metastasis and resistance to therapy. Therefore, Notch is an exceedingly attractive therapeutic target in cancer, but the full range of potential targets within the pathway has been underexplored. To date, there are no small-molecule inhibitors that directly target the intracellular Notch pathway or the assembly of the transcriptional activation complex. Here, we describe an in vitro assay that quantitatively measures the assembly of the Notch transcriptional complex on DNA. Integrating this approach with computer-aided drug design, we explored potential ligand-binding sites and screened for compounds that could disrupt the assembly of the Notch transcriptional activation complex. We identified a small-molecule inhibitor, termed Inhibitor of Mastermind Recruitment-1 (IMR-1), that disrupted the recruitment of Mastermind-like 1 to the Notch transcriptional activation complex on chromatin, thereby attenuating Notch target gene transcription. Furthermore, IMR-1 inhibited the growth of Notch-dependent cell lines and significantly abrogated the growth of patient-derived tumor xenografts. Taken together, our findings suggest that a novel class of Notch inhibitors targeting the transcriptional activation complex may represent a new paradigm for Notch-based anticancer therapeutics, warranting further preclinical characterization. Cancer Res; 76(12); 3593-603. ©2016 AACR.
©2016 American Association for Cancer Research.
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with Notch pathway mutations. While both normal activated and leukemic T cells can utilize aerobic glycolysis to support proliferation, it is unclear to what extent these cell populations are metabolically similar and if differences reveal T-ALL vulnerabilities. Here we show that aerobic glycolysis is surprisingly less active in T-ALL cells than proliferating normal T cells and that T-ALL cells are metabolically distinct. Oncogenic Notch promoted glycolysis but also induced metabolic stress that activated 5' AMP-activated kinase (AMPK). Unlike stimulated T cells, AMPK actively restrained aerobic glycolysis in T-ALL cells through inhibition of mTORC1 while promoting oxidative metabolism and mitochondrial Complex I activity. Importantly, AMPK deficiency or inhibition of Complex I led to T-ALL cell death and reduced disease burden. Thus, AMPK simultaneously inhibits anabolic growth signaling and is essential to promote mitochondrial pathways that mitigate metabolic stress and apoptosis in T-ALL.
Copyright © 2016 Elsevier Inc. All rights reserved.
Transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) play key roles in regulating the response to renal injury but are thought to mediate divergent effects on cell behavior. However, how TGF-β signaling alters the response to HGF in epithelia, the key site of HGF signaling in the injured kidney, is not well studied. Contrary to our expectation, we showed that deletion of the TGF-β type II receptor in conditionally immortalized proximal tubule (PT) cells impaired HGF-dependent signaling. This reduced signaling was due to decreased transcription of c-Met, the HGF receptor, and the TGF-β-dependent c-Met transcription and increased response to HGF in PT cells were mediated by the Notch pathway. The interactions of TGF-β, HGF, and Notch pathways had biologically significant effects on branching morphogenesis, cell morphology, migration, and proliferation. In conclusion, epithelial TGF-β signaling promotes HGF signaling in a Notch-dependent pathway. These findings suggest that TGF-β modulates PT responses not only by direct effects, but also by affecting other growth factor signaling pathways.
In the mammalian pancreas, endocrine cells undergo lineage allocation upon emergence from a bipotent duct/endocrine progenitor pool, which resides in the "trunk epithelium." Major questions remain regarding how niche environments are organized within this epithelium to coordinate endocrine differentiation with programs of epithelial growth, maturation, and morphogenesis. We used EdU pulse-chase and tissue-reconstruction approaches to analyze how endocrine progenitors and their differentiating progeny are assembled within the trunk as it undergoes remodeling from an irregular plexus of tubules to form the eventual mature, branched ductal arbor. The bulk of endocrine progenitors is maintained in an epithelial "plexus state," which is a transient intermediate during epithelial maturation within which endocrine cell differentiation is continually robust and surprisingly long-lived. Within the plexus, local feedback effects derived from the differentiating and delaminating endocrine cells nonautonomously regulate the flux of endocrine cell birth as well as proliferative growth of the bipotent cell population using Notch-dependent and Notch-independent influences, respectively. These feedback effects in turn maintain the plexus state to ensure prolonged allocation of endocrine cells late into gestation. These findings begin to define a niche-like environment guiding the genesis of the endocrine pancreas and advance current models for how differentiation is coordinated with the growth and morphogenesis of the developing pancreatic epithelium.
© 2015 Bankaitis et al.; Published by Cold Spring Harbor Laboratory Press.