The esophageal lumen is lined by a stratified squamous epithelium comprised of proliferative basal cells that differentiate while migrating toward the luminal surface and eventually desquamate. Rapid epithelial renewal occurs, but the specific cell of origin that supports this high proliferative demand remains unknown. Herein, we have described a long-lived progenitor cell population in the mouse esophageal epithelium that is characterized by expression of keratin 15 (
Véronique Giroux, Ashley A. Lento, Mirazul Islam, Jason R. Pitarresi, Akriti Kharbanda, Kathryn E. Hamilton, Kelly A. Whelan, Apple Long, Ben Rhoades, Qiaosi Tang, Hiroshi Nakagawa, Christopher J. Lengner, Adam J. Bass, E. Paul Wileyto, Andres J. Klein-Szanto, Timothy C. Wang, Anil K. Rustgi
Cancer cells preferentially utilize glucose and glutamine, which provide macromolecules and antioxidants that sustain rapid cell division. Metabolic reprogramming in cancer drives an increased glycolytic rate that supports maximal production of these nutrients. The folate cycle, through transfer of a carbon unit between tetrahydrofolate and its derivatives in the cytoplasmic and mitochondrial compartments, produces other metabolites that are essential for cell growth, including nucleotides, methionine, and the antioxidant NADPH. Here, using hepatocellular carcinoma (HCC) as a cancer model, we have observed a reduction in growth rate upon withdrawal of folate. We found that an enzyme in the folate cycle, methylenetetrahydrofolate dehydrogenase 1–like (MTHFD1L), plays an essential role in support of cancer growth. We determined that MTHFD1L is transcriptionally activated by NRF2, a master regulator of redox homeostasis. Our observations further suggest that MTHFD1L contributes to the production and accumulation of NADPH to levels that are sufficient to combat oxidative stress in cancer cells. The elevation of oxidative stress through MTHFD1L knockdown or the use of methotrexate, an antifolate drug, sensitizes cancer cells to sorafenib, a targeted therapy for HCC. Taken together, our study identifies MTHFD1L in the folate cycle as an important metabolic pathway in cancer cells with the potential for therapeutic targeting.
Derek Lee, Iris Ming-Jing Xu, David Kung-Chun Chiu, Robin Kit-Ho Lai, Aki Pui-Wah Tse, Lynna Lan Li, Cheuk-Ting Law, Felice Ho-Ching Tsang, Larry Lai Wei, Cerise Yuen-Ki Chan, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong
Cholestatic liver fibrosis is caused by obstruction of the biliary tract and is associated with early activation of portal fibroblasts (PFs) that express Thy-1, fibulin 2, and the recently identified marker mesothelin (MSLN). Here, we have demonstrated that activated PFs (aPFs) and myofibroblasts play a critical role in the pathogenesis of liver fibrosis induced by bile duct ligation (BDL). Conditional ablation of MSLN+ aPFs in BDL-injured mice attenuated liver fibrosis by approximately 50%. Similar results were observed in MSLN-deficient mice (
Yukinori Koyama, Ping Wang, Shuang Liang, Keiko Iwaisako, Xiao Liu, Jun Xu, Mingjun Zhang, Mengxi Sun, Min Cong, Daniel Karin, Kojiro Taura, Chris Benner, Sven Heinz, Tapan Bera, David A. Brenner, Tatiana Kisseleva
Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the
Daniel O. Kechele, R. Eric Blue, Bailey Zwarycz, Scott T. Espenschied, Amanda T. Mah, Marni B. Siegel, Charles M. Perou, Shengli Ding, Scott T. Magness, P. Kay Lund, Kathleen M. Caron
Congenital tufting enteropathy (CTE) is a severe autosomal recessive human diarrheal disorder with characteristic intestinal epithelial dysplasia. CTE can be caused by mutations in genes encoding EpCAM, a putative adhesion molecule, and HAI-2, a cell surface protease inhibitor. A similar phenotype occurs in mice whose intestinal epithelial cells (IECs) fail to express the tight junction–associated protein claudin-7. EpCAM stabilizes claudin-7 in IECs, and HAI-2 regulates the cell surface serine protease matriptase, a known modifier of intestinal epithelial physiology. Therefore, we hypothesized that HAI-2, matriptase, EpCAM, and claudin-7 were functionally linked. Herein we have demonstrated that active matriptase cleaves EpCAM after Arg80 and that loss of HAI-2 in IECs led to unrestrained matriptase activity and efficient cleavage of EpCAM. Cleavage of EpCAM decreased its ability to associate with claudin-7 and targeted it for internalization and lysosomal degradation in conjunction with claudin-7. CTE-associated HAI-2 mutant proteins exhibited reduced ability to inhibit matriptase and also failed to efficiently stabilize claudin-7 in IECs. These results identify EpCAM as a substrate of matriptase and link HAI-2, matriptase, EpCAM, and claudin-7 in a functionally important pathway that causes disease when it is dysregulated.
Chuan-Jin Wu, Xu Feng, Michael Lu, Sohshi Morimura, Mark C. Udey
Interactions of diet, gut microbiota, and host genetics play important roles in the development of obesity and insulin resistance. Here, we have investigated the molecular links between gut microbiota, insulin resistance, and glucose metabolism in 3 inbred mouse strains with differing susceptibilities to metabolic syndrome using diet and antibiotic treatment. Antibiotic treatment altered intestinal microbiota, decreased tissue inflammation, improved insulin signaling in basal and stimulated states, and improved glucose metabolism in obesity- and diabetes-prone C57BL/6J mice on a high-fat diet (HFD). Many of these changes were reproduced by the transfer of gut microbiota from antibiotic-treated donors to germ-free or germ-depleted mice. These physiological changes closely correlated with changes in serum bile acids and levels of the antiinflammatory bile acid receptor Takeda G protein–coupled receptor 5 (TGR5) and were partially recapitulated by treatment with a TGR5 agonist. In contrast, antibiotic treatment of HFD-fed, obesity-resistant 129S1 and obesity-prone 129S6 mice did not improve metabolism, despite changes in microbiota and bile acids. These mice also failed to show a reduction in inflammatory gene expression in response to the TGR5 agonist. Thus, changes in bile acid and inflammatory signaling, insulin resistance, and glucose metabolism driven by an HFD can be modified by antibiotic-induced changes in gut microbiota; however, these effects depend on important interactions with the host’s genetic background and inflammatory potential.
Shiho Fujisaka, Siegfried Ussar, Clary Clish, Suzanne Devkota, Jonathan M. Dreyfuss, Masaji Sakaguchi, Marion Soto, Masahiro Konishi, Samir Softic, Emrah Altindis, Ning Li, Georg Gerber, Lynn Bry, C. Ronald Kahn
Radiotherapy causes dose-limiting toxicity and long-term complications in rapidly renewing tissues, including the gastrointestinal tract. Currently, there is no FDA-approved agent for the prevention or treatment of radiation-induced intestinal injury. In this study, we have shown that PD 0332991 (PD), an FDA-approved selective inhibitor of cyclin-dependent kinase 4/6 (CDK4/6), prevents radiation-induced lethal intestinal injury in mice. Treating mice with PD or a structurally distinct CDK4/6 inhibitor prior to radiation blocked proliferation and crypt apoptosis and improved crypt regeneration. PD treatment also enhanced LGR5+ stem cell survival and regeneration after radiation. PD was an on-target inhibitor of RB phosphorylation and blocked G1/S transition in the intestinal crypts. PD treatment strongly but reversibly inhibited radiation-induced p53 activation, which blocked p53-upregulated modulator of apoptosis–dependent (PUMA-dependent) apoptosis without affecting p21-dependent suppression of DNA damage accumulation, with a repair bias toward nonhomologous end joining. Further, deletion of
Liang Wei, Brian J. Leibowitz, Xinwei Wang, Michael Epperly, Joel Greenberger, Lin Zhang, Jian Yu
EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with
Dana M. Hardbower, Kshipra Singh, Mohammad Asim, Thomas G. Verriere, Danyvid Olivares-Villagómez, Daniel P. Barry, Margaret M. Allaman, M. Kay Washington, Richard M. Peek Jr., M. Blanca Piazuelo, Keith T. Wilson
Autism spectrum disorder (ASD) is an increasingly common behavioral condition that frequently presents with gastrointestinal (GI) disturbances. It is not clear, however, how gut dysfunction relates to core ASD features. Multiple, rare hyperfunctional coding variants of the serotonin (5-HT) transporter (SERT, encoded by
Kara Gross Margolis, Zhishan Li, Korey Stevanovic, Virginia Saurman, Narek Israelyan, George M. Anderson, Isaac Snyder, Jeremy Veenstra-VanderWeele, Randy D. Blakely, Michael D. Gershon
Although defects in intestinal barrier function are a key pathogenic factor in patients with inflammatory bowel diseases (IBDs), the molecular pathways driving disease-specific alterations of intestinal epithelial cells (IECs) are largely unknown. Here, we addressed this issue by characterizing the transcriptome of IECs from IBD patients using a genome-wide approach. We observed disease-specific alterations in IECs with markedly impaired Rho-A signaling in active IBD patients. Localization of epithelial Rho-A was shifted to the cytosol in IBDs, and inflammation was associated with suppressed Rho-A activation due to reduced expression of the Rho-A prenylation enzyme geranylgeranyltransferase-I (GGTase-I). Functionally, we found that mice with conditional loss of
Rocío López-Posadas, Christoph Becker, Claudia Günther, Stefan Tenzer, Kerstin Amann, Ulrike Billmeier, Raja Atreya, Gionata Fiorino, Stefania Vetrano, Silvio Danese, Arif B. Ekici, Stefan Wirtz, Veronika Thonn, Alastair J.M. Watson, Cord Brakebusch, Martin Bergö, Markus F. Neurath, Imke Atreya
The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (
Charlotte E. Egan, Chhinder P. Sodhi, Misty Good, Joyce Lin, Hongpeng Jia, Yukihiro Yamaguchi, Peng Lu, Congrong Ma, Maria F. Branca, Samantha Weyandt, William B. Fulton, Diego F. Niño, Thomas Prindle Jr., John A. Ozolek, David J. Hackam
Hirschsprung’s disease (HSCR) is a severe congenital anomaly of the enteric nervous system (ENS) characterized by functional intestinal obstruction due to a lack of intrinsic innervation in the distal bowel. Distal innervation deficiency results from incomplete colonization of the bowel by enteric neural crest cells (eNCCs), the ENS precursors. Here, we report the generation of a mouse model for HSCR — named Holstein — that contains an untargeted transgenic insertion upstream of the collagen-6α4 (
Rodolphe Soret, Mathilde Mennetrey, Karl F. Bergeron, Anne Dariel, Michel Neunlist, Franziska Grunder, Christophe Faure, David W. Silversides, Nicolas Pilon, for the Ente-Hirsch study group
Mesenchymal stem cell (MSC) therapy is an emerging field of regenerative medicine; however, it is often unclear how these cells mediate repair. Here, we investigated the use of MSCs in the treatment of intestinal disease and modeled abnormal repair by creating focal wounds in the colonic mucosa of prostaglandin-deficient mice. These wounds developed into ulcers that infiltrated the outer intestinal wall. We determined that penetrating ulcer formation in this model resulted from increased hypoxia and smooth muscle wall necrosis. Prostaglandin I2 (PGI2) stimulated VEGF-dependent angiogenesis to prevent penetrating ulcers. Treatment of mucosally injured WT mice with a VEGFR inhibitor resulted in the development of penetrating ulcers, further demonstrating that VEGF is critical for mucosal repair. We next used this model to address the role of transplanted colonic MSCs (cMSCs) in intestinal repair. Compared with intravenously injected cMSCs, mucosally injected cMSCs more effectively prevented the development of penetrating ulcers, as they were more efficiently recruited to colonic wounds. Importantly, mucosally injected cMSCs stimulated angiogenesis in a VEGF-dependent manner. Together, our results reveal that penetrating ulcer formation results from a reduction of local angiogenesis and targeted injection of MSCs can optimize transplantation therapy. Moreover, local MSC injection has potential for treating diseases with features of abnormal angiogenesis and repair.
Nicholas A. Manieri, Madison R. Mack, Molly D. Himmelrich, Daniel L. Worthley, Elaine M. Hanson, Lars Eckmann, Timothy C. Wang, Thaddeus S. Stappenbeck
Increasing evidence indicates that the gut microbiota can be altered to ameliorate or prevent disease states, and engineering the gut microbiota to therapeutically modulate host metabolism is an emerging goal of microbiome research. In the intestine, bacterial urease converts host-derived urea to ammonia and carbon dioxide, contributing to hyperammonemia-associated neurotoxicity and encephalopathy in patients with liver disease. Here, we engineered murine gut microbiota to reduce urease activity. Animals were depleted of their preexisting gut microbiota and then inoculated with altered Schaedler flora (ASF), a defined consortium of 8 bacteria with minimal urease gene content. This protocol resulted in establishment of a persistent new community that promoted a long-term reduction in fecal urease activity and ammonia production. Moreover, in a murine model of hepatic injury, ASF transplantation was associated with decreased morbidity and mortality. These results provide proof of concept that inoculation of a prepared host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility.
Ting-Chin David Shen, Lindsey Albenberg, Kyle Bittinger, Christel Chehoud, Ying-Yu Chen, Colleen A. Judge, Lillian Chau, Josephine Ni, Michael Sheng, Andrew Lin, Benjamin J. Wilkins, Elizabeth L. Buza, James D. Lewis, Yevgeny Daikhin, Ilana Nissim, Marc Yudkoff, Frederic D. Bushman, Gary D. Wu
Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells, especially in tissues that are constantly replenished like the esophagus. Disruption of this balance is associated with pathological conditions, including eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic upon proinflammatory stimulation. However, how basal cells respond to the inflammatory environment at the molecular level remains undetermined. We previously reported that the bone morphogenetic protein (BMP) signaling pathway is critical for epithelial morphogenesis in the embryonic esophagus. Here, we address how this pathway regulates tissue homeostasis and EoE development in the adult esophagus. BMP signaling was specifically activated in differentiated squamous epithelium, but not in basal progenitor cells, which express the BMP antagonist follistatin. Previous reports indicate that increased BMP activity promotes Barrett’s intestinal differentiation; however, in mice, basal progenitor cell–specific expression of constitutively active BMP promoted squamous differentiation. Moreover, BMP activation increased intracellular ROS levels, initiating an NRF2-mediated oxidative response during basal progenitor cell differentiation. In both a mouse EoE model and human biopsies, reduced squamous differentiation was associated with high levels of follistatin and disrupted BMP/NRF2 pathways. We therefore propose a model in which normal squamous differentiation of basal progenitor cells is mediated by BMP-driven NRF2 activation and basal cell hyperplasia is promoted by disruption of BMP signaling in EoE.
Ming Jiang, Wei-Yao Ku, Zhongren Zhou, Evan S. Dellon, Gary W. Falk, Hiroshi Nakagawa, Mei-Lun Wang, Kuancan Liu, Jun Wang, David A. Katzka, Xiaopeng Lan, Jeffrey H. Peters, Jianwen Que
Epithelial restitution is an essential process that is required to repair barrier function at mucosal surfaces following injury. Prolonged breaches in epithelial barrier function result in inflammation and further damage; therefore, a better understanding of the epithelial restitution process has potential for improving the development of therapeutics. In this work, we demonstrate that endogenous annexin A1 (ANXA1) is released as a component of extracellular vesicles (EVs) derived from intestinal epithelial cells, and these ANXA1-containing EVs activate wound repair circuits. Compared with healthy controls, patients with active inflammatory bowel disease had elevated levels of secreted ANXA1-containing EVs in sera, indicating that ANXA1-containing EVs are systemically distributed in response to the inflammatory process and could potentially serve as a biomarker of intestinal mucosal inflammation. Local intestinal delivery of an exogenous ANXA1 mimetic peptide (Ac2-26) encapsulated within targeted polymeric nanoparticles (Ac2-26 Col IV NPs) accelerated healing of murine colonic wounds after biopsy-induced injury. Moreover, one-time systemic administration of Ac2-26 Col IV NPs accelerated recovery following experimentally induced colitis. Together, our results suggest that local delivery of proresolving peptides encapsulated within nanoparticles may represent a potential therapeutic strategy for clinical situations characterized by chronic mucosal injury, such as is seen in patients with IBD.
Giovanna Leoni, Philipp-Alexander Neumann, Nazila Kamaly, Miguel Quiros, Hikaru Nishio, Hefin R. Jones, Ronen Sumagin, Roland S. Hilgarth, Ashfaqul Alam, Gabrielle Fredman, Ioannis Argyris, Emile Rijcken, Dennis Kusters, Chris Reutelingsperger, Mauro Perretti, Charles A. Parkos, Omid C. Farokhzad, Andrew S. Neish, Asma Nusrat
Cystic fibrosis (CF) intestinal disease is associated with the pathological manifestation mucoviscidosis, which is the secretion of tenacious, viscid mucus that plugs ducts and glands of epithelial-lined organs. Goblet cells are the principal cell type involved in exocytosis of mucin granules; however, little is known about the exocytotic process of goblet cells in the CF intestine. Using intestinal organoids from a CF mouse model, we determined that CF goblet cells have altered exocytotic dynamics, which involved intrathecal granule swelling that was abruptly followed by incomplete release of partially decondensated mucus. Some CF goblet cells exhibited an ectopic granule location and distorted cellular morphology, a phenotype that is consistent with retrograde intracellular granule movement during exocytosis. Increasing the luminal concentration of bicarbonate, which mimics CF transmembrane conductance regulator–mediated anion secretion, increased spontaneous degranulation in WT goblet cells and improved exocytotic dynamics in CF goblet cells; however, there was still an apparent incoordination between granule decondensation and exocytosis in the CF goblet cells. Compared with those within WT goblet cells, mucin granules within CF goblet cells had an alkaline pH, which may adversely affect the polyionic composition of the mucins. Together, these findings indicate that goblet cell dysfunction is an epithelial-autonomous defect in the CF intestine that likely contributes to the pathology of mucoviscidosis and the intestinal manifestations of obstruction and inflammation.
Jinghua Liu, Nancy M. Walker, Akifumi Ootani, Ashlee M. Strubberg, Lane L. Clarke
The intracellular protein HMGB1 is released from cells and acts as a damage-associated molecular pattern molecule during many diseases, including inflammatory bowel disease (IBD); however, the intracellular function of HMGB1 during inflammation is poorly understood. Here, we demonstrated that cytosolic HMGB1 regulates apoptosis by protecting the autophagy proteins beclin 1 and ATG5 from calpain-mediated cleavage during inflammation. Colitis in mice with an intestinal epithelial cell–specific
Xiaorong Zhu, Jeannette S. Messer, Yunwei Wang, Fanfei Lin, Candace M. Cham, Jonathan Chang, Timothy R. Billiar, Michael T. Lotze, David L. Boone, Eugene B. Chang
Satiety and other core physiological functions are modulated by sensory signals arising from the surface of the gut. Luminal nutrients and bacteria stimulate epithelial biosensors called enteroendocrine cells. Despite being electrically excitable, enteroendocrine cells are generally thought to communicate indirectly with nerves through hormone secretion and not through direct cell-nerve contact. However, we recently uncovered in intestinal enteroendocrine cells a cytoplasmic process that we named neuropod. Here, we determined that neuropods provide a direct connection between enteroendocrine cells and neurons innervating the small intestine and colon. Using cell-specific transgenic mice to study neural circuits, we found that enteroendocrine cells have the necessary elements for neurotransmission, including expression of genes that encode pre-, post-, and transsynaptic proteins. This neuroepithelial circuit was reconstituted in vitro by coculturing single enteroendocrine cells with sensory neurons. We used a monosynaptic rabies virus to define the circuit’s functional connectivity in vivo and determined that delivery of this neurotropic virus into the colon lumen resulted in the infection of mucosal nerves through enteroendocrine cells. This neuroepithelial circuit can serve as both a sensory conduit for food and gut microbes to interact with the nervous system and a portal for viruses to enter the enteric and central nervous systems.
Diego V. Bohórquez, Rafiq A. Shahid, Alan Erdmann, Alex M. Kreger, Yu Wang, Nicole Calakos, Fan Wang, Rodger A. Liddle
Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barrett’s esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barrett’s metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barrett’s pathogenesis. By microarray analysis, we found that the transcription factor
David H. Wang, Anjana Tiwari, Monica E. Kim, Nicholas J. Clemons, Nanda L. Regmi, William A. Hodges, David M. Berman, Elizabeth A. Montgomery, D. Neil Watkins, Xi Zhang, Qiuyang Zhang, Chunfa Jie, Stuart J. Spechler, Rhonda F. Souza