RESUMO
Paneth cells are the primary source of C-type lysozyme, a ß-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1-/- hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.
Assuntos
Clostridiales/imunologia , Colite Ulcerativa/patologia , Muramidase/genética , Muramidase/metabolismo , Celulas de Paneth/metabolismo , Animais , Clostridiales/genética , Colite Ulcerativa/microbiologia , Doença de Crohn/patologia , Feminino , Microbioma Gastrointestinal/genética , Células Caliciformes/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT6/genéticaRESUMO
Paneth cells (PCs), a specialized secretory cell type in the small intestine, are increasingly recognized as having an essential role in host responses to microbiome and environmental stresses. Whether and how commensal and pathogenic microbes modify PC composition to modulate inflammation remain unclear. Using newly developed PC-reporter mice under conventional and gnotobiotic conditions, we determined PC transcriptomic heterogeneity in response to commensal and invasive microbes at single cell level. Infection expands the pool of CD74+ PCs, whose number correlates with auto or allogeneic inflammatory disease progressions in mice. Similar correlation was found in human inflammatory disease tissues. Infection-stimulated cytokines increase production of reactive oxygen species (ROS) and expression of a PC-specific mucosal pentraxin (Mptx2) in activated PCs. A PC-specific ablation of MyD88 reduced CD74+ PC population, thus ameliorating pathogen-induced systemic disease. A similar phenotype was also observed in mice lacking Mptx2. Thus, infection stimulates expansion of a PC subset that influences disease progression.
Assuntos
Microbiota , Celulas de Paneth , Humanos , Animais , Camundongos , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Intestino Delgado , Inflamação/patologia , Citocinas/metabolismoRESUMO
TFEB and TFE3 (TFEB/3), key regulators of lysosomal biogenesis and autophagy, play diverse roles depending on cell type. This study highlights a hitherto unrecognized role of TFEB/3 crucial for peripheral nerve repair. Specifically, they promote the generation of progenitor-like repair Schwann cells after axonal injury. In Schwann cell-specific TFEB/3 double knock-out mice of either sex, the TFEB/3 loss disrupts the transcriptomic reprogramming that is essential for the formation of repair Schwann cells. Consequently, mutant mice fail to populate the injured nerve with repair Schwann cells and exhibit defects in axon regrowth, target reinnervation, and functional recovery. TFEB/3 deficiency inhibits the expression of injury-responsive repair Schwann cell genes, despite the continued expression of c-jun, a previously identified regulator of repair Schwann cell function. TFEB/3 binding motifs are enriched in the enhancer regions of injury-responsive genes, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired despite TFEB/3 deficiency. These findings underscore a unique role of TFEB/3 in adult Schwann cells that is required for proper peripheral nerve regeneration.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Camundongos Knockout , Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Células de Schwann , Células de Schwann/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Camundongos , Traumatismos dos Nervos Periféricos/metabolismo , Regeneração Nervosa/fisiologia , Regeneração Nervosa/genética , Masculino , Feminino , Autofagia/fisiologia , Camundongos Endogâmicos C57BL , Nervo Isquiático/lesõesRESUMO
Ruminococcus gnavus is a mucolytic commensal bacterium whose increased gut colonization has been associated with chronic inflammatory and metabolic diseases in humans. Whether R. gnavus metabolites can modulate host intestinal physiology remains largely understudied. We performed untargeted metabolomic and bulk RNA-seq analyses using R. gnavus monocolonization in germ-free mice. Based on transcriptome-metabolome correlations, we tested the impact of specific arginine metabolites on intestinal epithelial production of nitric oxide (NO) and examined the effect of NO on the growth of various strains of R. gnavus in vitro and in nitric oxide synthase 2 (Nos2)-deficient mice. R. gnavus produces specific arginine, tryptophan, and tyrosine metabolites, some of which are regulated by the environmental richness of sialic acid and mucin. R. gnavus colonization promotes expression of amino acid transporters and enzymes involved in metabolic flux of arginine and associated metabolites into NO. R. gnavus induced elevated levels of NOS2, while Nos2 ablation resulted in R. gnavus expansion in vivo. The growth of various R. gnavus strains can be inhibited by NO. Specific R. gnavus metabolites modulate intestinal epithelial cell NOS2 abundance and reduce epithelial barrier function at higher concentrations. Intestinal colonization and interaction with R. gnavus are partially regulated by an arginine-NO metabolic pathway, whereby a balanced control by the gut epithelium may restrain R. gnavus growth in healthy individuals. Disruption in this arginine metabolic regulation will contribute to the expansion and blooming of R. gnavus.
RESUMO
Although Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Blocking the release of Wnt in distinct stromal cell types suggests obligatory functions of several stromal cell sources and yields different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affects colonic Reg4+ epithelial cell differentiation and impairs colonic epithelial regeneration after injury in mice. Single-cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin α2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that blockage of Wnt release from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated blockage of Wnt release from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.
Assuntos
Actinas/metabolismo , Colite Ulcerativa/metabolismo , Mucosa Intestinal/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Proteína Wnt3A/metabolismo , Cicatrização , Actinas/genética , Animais , Células Cultivadas , Colo/citologia , Colo/metabolismo , Colo/fisiologia , Mucosa Intestinal/citologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Proteínas Associadas a Pancreatite/genética , Proteínas Associadas a Pancreatite/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Proteína Wnt3A/genéticaRESUMO
RAB11 small GTPases and associated recycling endosome have been localized to mitotic spindles and implicated in regulating mitosis. However, the physiological significance of such regulation has not been observed in mammalian tissues. We have used newly engineered mouse models to investigate intestinal epithelial renewal in the absence of single or double isoforms of RAB11 family members: Rab11a and Rab11b. Comparing with single knockouts, mice with compound ablation demonstrate a defective cell cycle entry and robust mitotic arrest followed by apoptosis, leading to a total penetrance of lethality within 3 days of gene ablation. Upon Rab11 deletion ex vivo, enteroids show abnormal mitotic spindle formation and cell death. Untargeted proteomic profiling of Rab11a and Rab11b immunoprecipitates has uncovered a shared interactome containing mitotic spindle microtubule regulators. Disrupting Rab11 alters kinesin motor KIF11 function and impairs bipolar spindle formation and cell division. These data demonstrate that RAB11A and RAB11B redundantly control mitotic spindle function and intestinal progenitor cell division, a mechanism that may be utilized to govern the homeostasis and renewal of other mammalian tissues.
Assuntos
Proteômica , Proteínas rab de Ligação ao GTP , Animais , Camundongos , Mamíferos/metabolismo , Mitose , Proteínas rab de Ligação ao GTP/metabolismo , Fuso Acromático/metabolismo , Células-Tronco/metabolismoRESUMO
Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN-7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella-containing vacuoles after long-term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread.
Assuntos
Proteínas do Citoesqueleto , Salmonella typhimurium , Proteínas rho de Ligação ao GTP , Humanos , Actinas/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Citoesqueleto/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Salmonella typhimurium/patogenicidade , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismoRESUMO
BACKGROUND & AIMS: Excessive shedding of apoptotic enterocytes into the intestinal lumen is observed in inflammatory bowel disease and is correlated with disease relapse. Based on their cytolytic capacity and surveillance behavior, we investigated whether intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IELs) are actively involved in the shedding of enterocytes into the lumen. METHODS: Intravital microscopy was performed on GFP γδ T cell reporter mice treated with intraperitoneal lipopolysaccharide (10 mg/kg) for 90 minutes to induce tumor necrosis factor-mediated apoptosis. Cell shedding in various knockout or transgenic mice in the presence or absence of blocking antibody was quantified by immunostaining for ZO-1 funnels and cleaved caspase-3 (CC3). Granzyme A and granzyme B release from ex vivo-stimulated γδ IELs was quantified by enzyme-linked immunosorbent assay. Immunostaining for γδ T cell receptor and CC3 was performed on duodenal and ileal biopsies from controls and patients with Crohn's disease. RESULTS: Intravital microscopy of lipopolysaccharide-treated mice revealed that γδ IELs make extended contact with shedding enterocytes. These prolonged interactions require CD103 engagement by E-cadherin, and CD103 knockout or blockade significantly reduced lipopolysaccharide-induced shedding. Furthermore, we found that granzymes A and B, but not perforin, are required for cell shedding. These extracellular granzymes are released by γδ IELs both constitutively and after CD103/E-cadherin ligation. Moreover, we found that the frequency of γδ IEL localization to CC3-positive enterocytes is increased in Crohn's disease biopsies compared with healthy controls. CONCLUSIONS: Our results uncover a previously unrecognized role for γδ IELs in facilitating tumor necrosis factor-mediated shedding of apoptotic enterocytes via CD103-mediated extracellular granzyme release.
Assuntos
Antígenos CD/metabolismo , Doença de Crohn/metabolismo , Enterócitos/fisiologia , Granzimas/metabolismo , Cadeias alfa de Integrinas/metabolismo , Linfócitos Intraepiteliais/fisiologia , Adolescente , Adulto , Animais , Antígenos CD/genética , Apoptose , Caderinas/metabolismo , Caspase 3/metabolismo , Doença de Crohn/patologia , Duodeno/patologia , Enterócitos/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Íleo/patologia , Cadeias alfa de Integrinas/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Linfócitos Intraepiteliais/enzimologia , Linfócitos Intraepiteliais/patologia , Microscopia Intravital , Jejuno/imunologia , Jejuno/patologia , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
Within the intestinal epithelium, regulation of intracellular protein and vesicular trafficking is of utmost importance for barrier maintenance, immune responses, and tissue polarity. RAB11A is a small GTPase that mediates the anterograde transport of protein cargos to the plasma membrane. Loss of RAB11A-dependent trafficking in mature intestinal epithelial cells results in increased epithelial proliferation and nuclear accumulation of Yes-associated protein (YAP), a key Hippo-signaling transducer that senses cell-cell contacts and regulates tissue growth. However, it is unclear how RAB11A regulates YAP intracellular localizations. In this report, we examined the relationship of RAB11A to epithelial junctional complexes, YAP, and the associated consequences on colonic epithelial tissue repair. We found that RAB11A controls the biochemical associations of YAP with multiple components of adherens and tight junctions, including α-catenin, ß-catenin, and Merlin, a tumor suppressor. In the absence of RAB11A and Merlin, we observed enhanced YAP-ß-catenin complex formation and nuclear translocation. Upon chemical injury to the intestine, mice deficient in RAB11A were found to have reduced epithelial integrity, decreased YAP localization to adherens and tight junctions, and increased nuclear YAP accumulation in the colon epithelium. Thus, RAB11A-regulated trafficking regulates the Hippo-YAP signaling pathway for rapid reparative response after tissue injury.
Assuntos
Proteínas de Ciclo Celular/genética , Colite/genética , Neurofibromina 2/genética , Fatores de Transcrição/genética , beta Catenina/genética , Proteínas rab de Ligação ao GTP/genética , Junções Aderentes/genética , Animais , Células CACO-2 , Proliferação de Células/genética , Colite/induzido quimicamente , Colite/patologia , Colo/crescimento & desenvolvimento , Colo/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Epitélio/crescimento & desenvolvimento , Epitélio/patologia , Humanos , Camundongos , Junções Íntimas/genética , alfa Catenina/genéticaRESUMO
Differentiation of mesenchymal stem cells into adipocyte requires coordination of external stimuli and depends upon the functionality of the primary cilium. The Rab8 small GTPases are regulators of intracellular transport of membrane-bound structural and signaling cargo. However, the physiological contribution of the intrinsic trafficking network controlled by Rab8 to mesenchymal tissue differentiation has not been fully defined in vivo and in primary tissue cultures. Here, we show that mouse embryonic fibroblasts (MEFs) lacking Rab8 have severely impaired adipocyte differentiation in vivo and ex vivo. Immunofluorescent localization and biochemical analyses of Rab8a-deficient, Rab8b-deficient, and Rab8a and Rab8b double-deficient MEFs revealed that Rab8 controls the Lrp6 vesicular compartment, clearance of basal signalosome, traffic of frizzled two receptor, and thereby a proper attenuation of Wnt signaling in differentiating MEFs. Upon induction of adipogenesis program, Rab8a- and Rab8b-deficient MEFs exhibited severely defective lipid-droplet formation and abnormal cilia morphology, despite overall intact cilia growth and ciliary cargo transport. Our results suggest that intracellular Rab8 traffic regulates induction of adipogenesis via proper positioning of Wnt receptors for signaling control in mesenchymal cells.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Via de Sinalização Wnt , Proteínas rab de Ligação ao GTP/metabolismo , Adipogenia/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Cílios/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Proteínas rab de Ligação ao GTP/genéticaRESUMO
BACKGROUND & AIMS: Functions of intestinal stem cells (ISCs) are regulated by diet and metabolic pathways. Hepatocyte nuclear factor 4 (HNF4) family are transcription factors that bind fatty acids. We investigated how HNF4 transcription factors regulate metabolism and their functions in ISCs in mice. METHODS: We performed studies with Villin-CreERT2;Lgr5-EGFP-IRES-CreERT2;Hnf4αf/f;Hnf4γCrispr/Crispr mice, hereafter referred to Hnf4αγDKO. Mice were given tamoxifen to induce Cre recombinase. Mice transgenic with only Cre alleles (Villin-CreERT2, Lgr5-EGFP-IRES-CreERT2, Hnf4α+/+, and Hnf4γ+/+) or mice given vehicle were used as controls. Crypt and villus cells were isolated, incubated with fluorescently labeled fatty acids or glucose analog, and analyzed by confocal microscopy. Fatty acid oxidation activity and tricarboxylic acid (TCA) cycle metabolites were measured in cells collected from the proximal half of the small intestine of Hnf4αγDKO and control mice. We performed chromatin immunoprecipitation and gene expression profiling analyses to identify genes regulated by HNF4 factors. We established organoids from duodenal crypts, incubated them with labeled palmitate or acetate, and measured production of TCA cycle metabolites or fatty acids. Acetate, a precursor of acetyl coenzyme A (CoA) (a product of fatty acid ß-oxidation [FAO]), or dichloroacetate, a compound that promotes pyruvate oxidation and generation of mitochondrial acetyl-CoA, were used for metabolic intervention. RESULTS: Crypt cells rapidly absorbed labeled fatty acids, and messenger RNA levels of Lgr5+ stem cell markers (Lgr5, Olfm4, Smoc2, Msi1, and Ascl2) were down-regulated in organoids incubated with etomoxir, an inhibitor of FAO, indicating that FAO was required for renewal of ISCs. HNF4A and HNF4G were expressed in ISCs and throughout the intestinal epithelium. Single knockout of either HNF4A or HNF4G did not affect maintenance of ISCs, but double-knockout of HNF4A and HNF4G resulted in ISC loss; stem cells failed to renew. FAO supports ISC renewal, and HNF4 transcription factors directly activate FAO genes, including Acsl5 and Acsf2 (encode regulators of acyl-CoA synthesis), Slc27a2 (encodes a fatty acid transporter), Fabp2 (encodes fatty acid binding protein), and Hadh (encodes hydroxyacyl-CoA dehydrogenase). In the intestinal epithelium of Hnf4αγDKO mice, expression levels of FAO genes, FAO activity, and metabolites of TCA cycle were all significantly decreased, but fatty acid synthesis transcripts were increased, compared with control mice. The contribution of labeled palmitate or acetate to the TCA cycle was reduced in organoids derived from Hnf4αγDKO mice, compared with control mice. Incubation of organoids derived from double-knockout mice with acetate or dichloroacetate restored stem cells. CONCLUSIONS: In mice, the transcription factors HNF4A and HNF4G regulate the expression of genes required for FAO and are required for renewal of ISCs.
Assuntos
Ácidos Graxos/metabolismo , Fator 4 Nuclear de Hepatócito/fisiologia , Intestino Delgado/citologia , Células-Tronco/metabolismo , Animais , Duodeno/citologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Mucosa Intestinal/citologia , Camundongos , Camundongos Knockout , Organoides/metabolismo , OxirreduçãoRESUMO
During development, the embryo transitions from a metabolism favoring glycolysis to a metabolism favoring mitochondrial respiration. How metabolic shifts regulate developmental processes, or how developmental processes regulate metabolic shifts, remains unclear. To test the requirement of mitochondrial function in developing endoderm-derived tissues, we genetically inactivated the mitochondrial transcription factor, Tfam, using the Shh-Cre driver. Tfam mutants did not survive postnatally, exhibiting defects in lung development. In the developing intestine, TFAM-loss was tolerated until late fetal development, during which the process of villus elongation was compromised. While progenitor cell populations appeared unperturbed, markers of enterocyte maturation were diminished and villi were blunted. Loss of TFAM was also tested in the adult intestinal epithelium, where enterocyte maturation was similarly dependent upon the mitochondrial transcription factor. While progenitor cells in the transit amplifying zone of the adult intestine remained proliferative, intestinal stem cell renewal was dependent upon TFAM, as indicated by molecular profiling and intestinal organoid formation assays. Taken together, these studies point to critical roles for the mitochondrial regulator TFAM for multiple aspects of intestinal development and maturation, and highlight the importance of mitochondrial regulators in tissue development and homeostasis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/fisiologia , Mucosa Intestinal/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Autorrenovação Celular/fisiologia , Proteínas de Ligação a DNA/genética , Feto/metabolismo , Regulação da Expressão Gênica/genética , Glicólise/genética , Glicólise/fisiologia , Proteínas de Grupo de Alta Mobilidade/genética , Mucosa Intestinal/embriologia , Mucosa Intestinal/crescimento & desenvolvimento , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Organogênese/genética , Organogênese/fisiologia , Organoides/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Misplaced formation of microvilli to basolateral domains and intracellular inclusions in enterocytes are pathognomonic features in congenital enteropathy associated with mutation of the apical plasma membrane receptor syntaxin 3 (STX3). Although the demonstrated binding of Myo5b to the Rab8a and Rab11a small GTPases in vitro implicates cytoskeleton-dependent membrane sorting, the mechanisms underlying the microvillar location defect remain unclear. By selective or combinatory disruption of Rab8a and Rab11a membrane traffic in vivo, we demonstrate that transport of distinct cargo to the apical brush border rely on either individual or both Rab regulators, whereas certain basolateral cargos are redundantly transported by both factors. Enterocyte-specific Rab8a and Rab11a double-knockout mouse neonates showed immediate postnatal lethality and more severe enteropathy than single knockouts, with extensive formation of microvilli along basolateral surfaces. Notably, following an inducible Rab11a deletion from neonatal enterocytes, basolateral microvilli were induced within 3â days. These data identify a potentially important and distinct mechanism for a characteristic microvillus defect exhibited by enterocytes of patients with neonatal enteropathy.
Assuntos
Enterócitos/metabolismo , Doenças Genéticas Inatas/metabolismo , Enteropatias/metabolismo , Microvilosidades/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Animais , Enterócitos/patologia , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Enteropatias/congênito , Enteropatias/patologia , Camundongos , Camundongos Knockout , Microvilosidades/genética , Microvilosidades/patologia , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Wntless (Wls) transports Wnt molecules for secretion; however, the cellular mechanism underlying the initial assembly of Wnt secretory vesicles is still not fully defined. Here, we performed proteomic and mutagenic analyses of mammalian Wls, and report a mechanism for formation of early Wnt secretory vesicles on ER membrane. Wls forms a complex with SEC12 (also known as PREB), an ER membrane-localized guanine nucleotide-exchange factor (GEF) activator of the SAR1 (the SAR1A isoform) small GTPase. Compared to palmitoylation-deficient Wnt molecules, binding of mature Wnt to Wls increases Wls-SEC12 interaction and promotes association of Wls with SAR1, the key activator of the COPII machinery. Incorporation of Wls into this exporting ER compartment is affected by Wnt ligand binding and SEC12 binding to Wls, as well as the structural integrity and, potentially, the folding of the cytosolic tail of Wls. In contrast, Wls-SEC12 binding is stable, with the interacting interface biochemically mapped to cytosolic segments of individual proteins. Mutant Wls that fails to communicate with the COPII machinery cannot effectively support Wnt secretion. These data suggest that formation of early Wnt secretory vesicles is carefully regulated to ensure proper export of functional ligands.
Assuntos
Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptores Acoplados a Proteínas G/genética , Vesículas Secretórias/genética , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Lipoilação/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/genética , Proteômica , Receptores Acoplados a Proteínas G/metabolismo , Vesículas Secretórias/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
During late gestation, villi extend into the intestinal lumen to dramatically increase the surface area of the intestinal epithelium, preparing the gut for the neonatal diet. Incomplete development of the intestine is the most common gastrointestinal complication in neonates, but the causes are unclear. We provide evidence in mice that Yin Yang 1 (Yy1) is crucial for intestinal villus development. YY1 loss in the developing endoderm had no apparent consequences until late gestation, after which the intestine differentiated poorly and exhibited severely stunted villi. Transcriptome analysis revealed that YY1 is required for mitochondrial gene expression, and ultrastructural analysis confirmed compromised mitochondrial integrity in the mutant intestine. We found increased oxidative phosphorylation gene expression at the onset of villus elongation, suggesting that aerobic respiration might function as a regulator of villus growth. Mitochondrial inhibitors blocked villus growth in a fashion similar to Yy1 loss, thus further linking oxidative phosphorylation with late-gestation intestinal development. Interestingly, we find that necrotizing enterocolitis patients also exhibit decreased expression of oxidative phosphorylation genes. Our study highlights the still unappreciated role of metabolic regulation during organogenesis, and suggests that it might contribute to neonatal gastrointestinal disorders.
Assuntos
Mucosa Intestinal/metabolismo , Intestinos/citologia , Organogênese/fisiologia , Fator de Transcrição YY1/metabolismo , Aerobiose/genética , Aerobiose/fisiologia , Animais , Western Blotting , Genótipo , Imuno-Histoquímica , Masculino , Camundongos , Organogênese/genética , Fosforilação Oxidativa , Transcriptoma/genética , Fator de Transcrição YY1/genéticaRESUMO
Compartmentalization of Toll-like receptors (TLRs) in intestinal epithelial cells (IECs) regulates distinct immune responses to microbes; however, the specific cellular machinery that controls this mechanism has not been fully identified. Here we provide genetic evidences that the recycling endosomal compartment in enterocytes maintains a homeostatic TLR9 intracellular distribution, supporting mucosal tolerance to normal microbiota. Genetic ablation of a recycling endosome resident small GTPase, Rab11a, a gene adjacent to a Crohn's disease risk locus, in mouse IECs and in Drosophila midgut caused epithelial cell-intrinsic cytokine production, inflammatory bowel phenotype, and early mortality. Unlike wild-type controls, germ-free Rab11a-deficient mouse intestines failed to tolerate the intraluminal stimulation of microbial agonists. Thus, Rab11a endosome controls intestinal host-microbial homeostasis at least partially via sorting TLRs.
Assuntos
Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Enterócitos/imunologia , Enterócitos/microbiologia , Microbiota/imunologia , Receptor Toll-Like 9/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Endossomos/imunologia , Deleção de Genes , Homeostase , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Camundongos , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Receptor Toll-Like 9/imunologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/imunologiaRESUMO
Communication between stem and niche supporting cells maintains the homeostasis of adult tissues. Wnt signaling is a crucial regulator of the stem cell niche, but the mechanism that governs Wnt ligand delivery in this compartment has not been fully investigated. We identified that Wnt secretion is partly dependent on Rab8a-mediated anterograde transport of Gpr177 (wntless), a Wnt-specific transmembrane transporter. Gpr177 binds to Rab8a, depletion of which compromises Gpr177 traffic, thereby weakening the secretion of multiple Wnts. Analyses of generic Wnt/ß-catenin targets in Rab8a knockout mouse intestinal crypts indicate reduced signaling activities; maturation of Paneth cells - a Wnt-dependent cell type - is severely affected. Rab8a knockout crypts show an expansion of Lgr5(+) and Hopx(+) cells in vivo. However, in vitro, the knockout enteroids exhibit significantly weakened growth that can be partly restored by exogenous Wnts or Gsk3ß inhibitors. Immunogold labeling and surface protein isolation identified decreased plasma membrane localization of Gpr177 in Rab8a knockout Paneth cells and fibroblasts. Upon stimulation by exogenous Wnts, Rab8a-deficient cells show ligand-induced Lrp6 phosphorylation and transcriptional reporter activation. Rab8a thus controls Wnt delivery in producing cells and is crucial for Paneth cell maturation. Our data highlight the profound tissue plasticity that occurs in response to stress induced by depletion of a stem cell niche signal.
Assuntos
Celulas de Paneth/citologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/citologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Ativação Transcricional , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The molecular mechanisms leading to and responsible for age-related, sporadic Alzheimer's disease (AD) remain largely unknown. It is well documented that aging patients with elevated levels of the amino acid metabolite homocysteine (Hcy) are at high risk of developing AD. We investigated the impact of Hcy on molecular clearance pathways in mammalian cells, including in vitro cultured induced pluripotent stem cell-derived forebrain neurons and in vivo neurons in mouse brains. Exposure to Hcy resulted in up-regulation of the mechanistic target of rapamycin complex 1 (mTORC1) activity, one of the major kinases in cells that is tightly linked to anabolic and catabolic pathways. Hcy is sensed by a constitutive protein complex composed of leucyl-tRNA-synthetase and folliculin, which regulates mTOR tethering to lysosomal membranes. In hyperhomocysteinemic human cells and cystathionine ß-synthase-deficient mouse brains, we find an acute and chronic inhibition of the molecular clearance of protein products resulting in a buildup of abnormal proteins, including ß-amyloid and phospho-Tau. Formation of these protein aggregates leads to AD-like neurodegeneration. This pathology can be prevented by inhibition of mTORC1 or by induction of autophagy. We conclude that an increase of intracellular Hcy levels predisposes neurons to develop abnormal protein aggregates, which are hallmarks of AD and its associated onset and pathophysiology with age.-Khayati, K., Antikainen, H., Bonder, E. M., Weber, G. F., Kruger, W. D., Jakubowski, H., Dobrowolski, R. The amino acid metabolite homocysteine activates mTORC1 to inhibit autophagy and form abnormal proteins in human neurons and mice.
Assuntos
Autofagia/fisiologia , Regulação da Expressão Gênica/fisiologia , Homocisteína/metabolismo , Complexos Multiproteicos/metabolismo , Neurônios/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
The intestinal stem cell fuels the highest rate of tissue turnover in the body and has been implicated in intestinal disease and cancer; understanding the regulatory mechanisms controlling intestinal stem cell physiology is of great importance. Here, we provide evidence that the transcription factor YY1 is essential for intestinal stem cell renewal. We observe that YY1 loss skews normal homeostatic cell turnover, with an increase in proliferating crypt cells and a decrease in their differentiated villous progeny. Increased crypt cell numbers come at the expense of Lgr5(+) stem cells. On YY1 deletion, Lgr5(+) cells accelerate their commitment to the differentiated population, exhibit increased levels of apoptosis, and fail to maintain stem cell renewal. Loss of Yy1 in the intestine is ultimately fatal. Mechanistically, YY1 seems to play a role in stem cell energy metabolism, with mitochondrial complex I genes bound directly by YY1 and their transcript levels decreasing on YY1 loss. These unappreciated YY1 functions broaden our understanding of metabolic regulation in intestinal stem cell homeostasis.
Assuntos
Divisão Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Intestinos/citologia , Mitocôndrias/metabolismo , Células-Tronco/fisiologia , Fator de Transcrição YY1/metabolismo , Animais , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Camundongos , Camundongos Knockout , Análise em Microsséries , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Fator de Transcrição YY1/genéticaRESUMO
Rab11a has been conceived as a prominent regulatory component of the recycling endosome, which acts as a nexus in the endo- and exocytotic networks. The precise in vivo role of Rab11a in mouse embryonic development is unknown. We globally ablated Rab11a and examined the phenotypic and molecular outcomes in Rab11a(null) blastocysts and mouse embryonic fibroblasts. Using multiple trafficking assays and complementation analyses, we determined, among multiple important membrane-associated and soluble cargos, the critical contribution of Rab11a vesicular traffic to the secretion of multiple soluble MMPs. Rab11a(null) embryos were able to properly form normal blastocysts but died at peri-implantation stages. Our data suggest that Rab11a critically controls mouse blastocyst development and soluble matrix metalloproteinase secretion.