RESUMO
Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.
Assuntos
Colite/enzimologia , Colo/enzimologia , Citotoxicidade Imunológica , Complexo II de Transporte de Elétrons/metabolismo , Células Epiteliais/enzimologia , Doença Enxerto-Hospedeiro/enzimologia , Mucosa Intestinal/enzimologia , Mitocôndrias/enzimologia , Linfócitos T/imunologia , Animais , Estudos de Casos e Controles , Comunicação Celular , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/patologia , Colo/imunologia , Colo/ultraestrutura , Modelos Animais de Doenças , Complexo II de Transporte de Elétrons/genética , Células Epiteliais/imunologia , Células Epiteliais/ultraestrutura , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Humanos , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Mucosa Intestinal/ultraestrutura , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/imunologia , Mitocôndrias/ultraestrutura , Fosforilação Oxidativa , Ácido Succínico/metabolismo , Linfócitos T/metabolismoRESUMO
Histone-modifying enzymes regulate transcription and are sensitive to availability of endogenous small-molecule metabolites, allowing chromatin to respond to changes in environment. The gut microbiota produces a myriad of metabolites that affect host physiology and susceptibility to disease; however, the underlying molecular events remain largely unknown. Here we demonstrate that microbial colonization regulates global histone acetylation and methylation in multiple host tissues in a diet-dependent manner: consumption of a "Western-type" diet prevents many of the microbiota-dependent chromatin changes that occur in a polysaccharide-rich diet. Finally, we demonstrate that supplementation of germ-free mice with short-chain fatty acids, major products of gut bacterial fermentation, is sufficient to recapitulate chromatin modification states and transcriptional responses associated with colonization. These findings have profound implications for understanding the complex functional interactions between diet, gut microbiota, and host health.
Assuntos
Dieta Ocidental , Epigênese Genética , Ácidos Graxos Voláteis/metabolismo , Microbioma Gastrointestinal/fisiologia , Tecido Adiposo/enzimologia , Tecido Adiposo/metabolismo , Animais , Colo/enzimologia , Colo/metabolismo , Metilação de DNA , Histonas/genética , Histonas/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de ÓrgãosRESUMO
BACKGROUND & AIMS: Because inflammatory bowel disease is increasing worldwide and can lead to colitis-associated carcinoma (CAC), new interventions are needed. We have shown that spermine oxidase (SMOX), which generates spermidine (Spd), regulates colitis. Here we determined whether Spd treatment reduces colitis and carcinogenesis. METHODS: SMOX was quantified in human colitis and associated dysplasia using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. We used wild-type (WT) and Smox-/- C57BL/6 mice treated with dextran sulfate sodium (DSS) or azoxymethane (AOM)-DSS as models of colitis and CAC, respectively. Mice with epithelial-specific deletion of Apc were used as a model of sporadic colon cancer. Animals were supplemented or not with Spd in the drinking water. Colonic polyamines, inflammation, tumorigenesis, transcriptomes, and microbiomes were assessed. RESULTS: SMOX messenger RNA levels were decreased in human ulcerative colitis tissues and inversely correlated with disease activity, and SMOX protein was reduced in colitis-associated dysplasia. DSS colitis and AOM-DSS-induced dysplasia and tumorigenesis were worsened in Smox-/- vs WT mice and improved in both genotypes with Spd. Tumor development caused by Apc deletion was also reduced by Spd. Smox deletion and AOM-DSS treatment were both strongly associated with increased expression of α-defensins, which was reduced by Spd. A shift in the microbiome, with reduced abundance of Prevotella and increased Proteobacteria and Deferribacteres, occurred in Smox-/- mice and was reversed with Spd. CONCLUSIONS: Loss of SMOX is associated with exacerbated colitis and CAC, increased α-defensin expression, and dysbiosis of the microbiome. Spd supplementation reverses these phenotypes, indicating that it has potential as an adjunctive treatment for colitis and chemopreventive for colon carcinogenesis.
Assuntos
Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Colite/genética , Neoplasias do Colo/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Espermidina/uso terapêutico , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Azoximetano , Colite/induzido quimicamente , Colite/enzimologia , Colite/prevenção & controle , Colite Ulcerativa/enzimologia , Colite Ulcerativa/genética , Colo/enzimologia , Colo/patologia , Neoplasias do Colo/prevenção & controle , Sulfato de Dextrana , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Lesões Pré-Cancerosas/enzimologia , Fatores de Proteção , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Espermidina/metabolismo , Espermidina/farmacologia , Redução de Peso/efeitos dos fármacos , alfa-Defensinas/genética , alfa-Defensinas/metabolismo , Poliamina OxidaseRESUMO
BACKGROUND & AIMS: Sulfation is a conjugation reaction essential for numerous biochemical and cellular functions in mammals. The 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the key enzyme to generate PAPS, which is the universal sulfonate donor for all sulfation reactions. The goal of this study was to determine whether and how PAPSS2 plays a role in colitis and colonic carcinogenesis. METHODS: Tissue arrays of human colon cancer specimens, gene expression data, and clinical features of cancer patients were analyzed. Intestinal-specific Papss2 knockout mice (Papss2ΔIE) were created and subjected to dextran sodium sulfate-induced colitis and colonic carcinogenesis induced by a combined treatment of azoxymethane and dextran sodium sulfate or azoxymethane alone. RESULTS: The expression of PAPSS2 is decreased in the colon cancers of mice and humans. The lower expression of PAPSS2 in colon cancer patients is correlated with worse survival. Papss2ΔIE mice showed heightened sensitivity to colitis and colon cancer by damaging the intestinal mucosal barrier, increasing intestinal permeability and bacteria infiltration, and worsening the intestinal tumor microenvironment. Mechanistically, the Papss2ΔIE mice exhibited reduced intestinal sulfomucin content. Metabolomic analyses revealed the accumulation of bile acids, including the Farnesoid X receptor antagonist bile acid tauro-ß-muricholic acid, and deficiency in the formation of bile acid sulfates in the colon of Papss2ΔIE mice. CONCLUSIONS: We have uncovered an important role of PAPSS2-mediated sulfation in colitis and colonic carcinogenesis. Intestinal sulfation may represent a potential diagnostic marker and PAPSS2 may serve as a potential therapeutic target for inflammatory bowel disease and colon cancer.
Assuntos
Neoplasias Associadas a Colite/prevenção & controle , Colite/prevenção & controle , Colo/enzimologia , Mucosa Intestinal/enzimologia , Mucinas/metabolismo , Complexos Multienzimáticos/metabolismo , Sulfato Adenililtransferase/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Colite/enzimologia , Colite/genética , Colite/patologia , Neoplasias Associadas a Colite/enzimologia , Neoplasias Associadas a Colite/genética , Neoplasias Associadas a Colite/patologia , Colo/patologia , Bases de Dados Genéticas , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/patologia , Metaboloma , Metabolômica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multienzimáticos/genética , Prognóstico , Receptores Citoplasmáticos e Nucleares/metabolismo , Sulfato Adenililtransferase/genéticaRESUMO
BACKGROUND & AIMS: The molecular checkpoints driving T cell activation and cytokine responses in ulcerative colitis (UC) are incompletely understood. Here, we studied the Tec kinase ITK in UC. METHODS: We analyzed patients with inflammatory bowel disease (n = 223) and evaluated ITK activity as well as the functional effects of cyclosporine-A (CsA). In addition, 3 independent murine colitis models were used to investigate the functional role of ITK. Finally, the activity of ITK was blocked via pharmacological inhibitors and genetically engineered mice. Readout parameters were mini-endoscopy, histopathology, mucosal T cell apoptosis, and cytokine production. RESULTS: We found an expansion of pITK-expressing mucosal CD4+ T cells in UC rather than Crohn's disease that correlated with disease severity. CsA suppressed activation of ITK in cultured CD4+ T cells and calcineurin-containing microclusters adjacent to the T cell receptor signaling complex. Functionally, the capacity of CsA to suppress activity of experimental colitis was critically dependent on ITK. Genetic inactivation of Itk via gene targeting or induction of allele-sensitive Itk mutants prevented experimental colitis in 3 colitis models, and treatment with pharmacological ITK blockers suppressed established colitis. In addition, ITK controlled apoptosis and activation of mucosal Th2 and Th17 lymphocytes via NFATc2 signaling pathways. CONCLUSIONS: ITK activation was detected in UC and could be down-regulated in cultured T cells by CsA administration. Selective targeting of ITK emerges as an attractive approach for treatment of chronic intestinal inflammation and potentially UC by driving resolution of mucosal inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Colite Ulcerativa/prevenção & controle , Colo/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Linfócitos Intraepiteliais/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Colite Ulcerativa/enzimologia , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Colo/enzimologia , Colo/imunologia , Colo/patologia , Ciclosporina/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Linfócitos Intraepiteliais/enzimologia , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/patologia , Camundongos Knockout , Terapia de Alvo Molecular , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
Obesogenic diets (ODs) can affect AMPK activation in several sites as the colon, liver, and hypothalamus. OD intake can impair the hypothalamic AMPK regulation of energy homeostasis. Despite consuming ODs, not all subjects have the propensity to develop or progress to obesity. The obesity propensity is more associated with energy intake than expenditure dysregulations and may have a link with AMPK activity. While the effects of ODs are studied widely, few evaluate the short-term effects of terminating OD intake. Withdrawing from OD (WTD) is thought to improve or reverse the damages caused by the intake. Therefore, here we applied an OD intake and WTD protocol aiming to evaluate AMPK protein content and phosphorylation in the colon, liver, and hypothalamus and their relationship with obesity propensity. To this end, male Wistar rats (60 days) received control or high-sugar/high-fat (HSHF) OD for 30 days. Half of the animals were OD-withdrawn and fed the control diet for 48 h. After intake, we found a reduction in AMPK phosphorylation in the hypothalamus and colon, and after WTD, we found an increase in its hepatic and hypothalamic phosphorylation. The decrease in colon pAMPK/AMPK could be linked with hypothalamic pAMPK/AMPK after HSHF intake, while the increase in hepatic pAMPK/AMPK could have prevented the increase in hypothalamic pAMPK/AMPK. In the obesity-prone rats, we found higher levels of hypothalamic and colon pAMPK/AMPK despite the higher body mass gain. Our results highlight the relevance in multi-organ investigations and animal phenotype evaluation when studying the energy metabolism regulations.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Eixo Encéfalo-Intestino , Colo/enzimologia , Hipotálamo/enzimologia , Fígado/enzimologia , Obesidade/enzimologia , Animais , Dieta Hiperlipídica/efeitos adversos , Açúcares da Dieta/efeitos adversos , Masculino , Obesidade/etiologia , Ratos WistarRESUMO
Mouse and human data implicate the NOD1 and NOD2 sensors of the intestinal microbiome and the associated signal transduction via the receptor interacting protein kinase 2 (RIPK2) as a potential key signaling node for the development of inflammatory bowel disease (IBD) and an attractive target for pharmacological intervention. The TRUC mouse model of IBD was strongly indicated for evaluating RIPK2 antagonism for its effect on intestinal inflammation based on previous knockout studies with NOD1, NOD2, and RIPK2. We identified and profiled the BI 706039 molecule as a potent and specific functional inhibitor of both human and mouse RIPK2 and with favorable pharmacokinetic properties. We dosed BI 706039 in the spontaneous TRUC mouse model from age 28 to 56 days. Oral, daily administration of BI 706039 caused dose-responsive and significant improvement in colonic histopathological inflammation, colon weight, and terminal levels of protein-normalized fecal lipocalin (all P values <0.001). These observations correlated with dose responsively increasing systemic levels of the BI 706039 compound, splenic molecular target engagement of RIPK2, and modulation of inflammatory genes in the colon. This demonstrates that a relatively low oral dose of a potent and selective RIPK2 inhibitor can modulate signaling in the intestinal immune system and significantly improve disease associated intestinal inflammation.NEW & NOTEWORTHY The RIPK2 kinase at the apex of microbiome immunosensing is an attractive target for pharmacological intervention. A low oral dose of a RIPK2 inhibitor leads to significantly improved intestinal inflammation in the murine TRUC model of colitis. A selective and potent inhibitor of the RIPK2 kinase may represent a new class of therapeutics that target microbiome-driven signaling for the treatment of IBD.
Assuntos
Colite Ulcerativa/tratamento farmacológico , Colo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Animais , Disponibilidade Biológica , Células Cultivadas , Colite Ulcerativa/enzimologia , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Colo/enzimologia , Colo/patologia , Doença de Crohn/enzimologia , Doença de Crohn/patologia , Citocinas/genética , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Fezes/química , Humanos , Mediadores da Inflamação/metabolismo , Lipocalinas/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Biológicos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proteínas com Domínio T/genéticaRESUMO
miRNAs, a well-known group of noncoding RNAs, contribute to the pathogenesis of multiple diseases, including colitis-associated cancer (CAC). Our recent findings indicate that proinflammatory cytokines up-regulate c-MYC level, which subsequently activates cullin 4A and 4B (CUL4A/4B) and CRL4DCAF4 E3 ligases and promotes ubiquitination of suppression of tumorigenicity 7 in CAC. Herein, we identified and proved that miR-34b-5p can directly target c-MYC. In vitro oncogenic phenotype analyses and in vivo tumor formation assay indicated that miR-34b-5p overexpression could markedly decrease cell proliferation, colony formation, cell invasion, and tumor volumes. Overexpression of c-MYC in vitro could reverse the oncogenic phenotypes caused by miR-34b-5p up-regulation. In addition, the down-regulation of miR-34b-5p in CAC was dependent on the coregulation of the inflammatory microenvironment and DNA methylation. Collectively, our findings demonstrate that intracellular inflammation and DNA hypermethylation suppress miR-34b-5p expression, which limits its inhibitory effect on c-MYC and initiates the downstream events, including the induction of CRL4DCAF4 E3 ligase activity. The activated CRL4DCAF4 E3 ligase ubiquitinates suppression of tumorigenicity 7 and results in its degradation, eventually leading to the CAC tumorigenesis.
Assuntos
Colite/complicações , Neoplasias do Colo/patologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Colite/enzimologia , Colite/patologia , Colo/enzimologia , Colo/patologia , Neoplasias do Colo/complicações , Neoplasias do Colo/enzimologia , Proteínas Culina/genética , Proteínas Culina/metabolismo , Metilação de DNA , Regulação para Baixo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Inflamação , Proteínas Proto-Oncogênicas c-myc/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
In the present study, the effects of NLRP3 on radiation-induced tissue damage, including colon and skin damage in mice, and the possible mechanisms were explored in vivo and in vitro. The mice were subjected to whole abdomen radiation by timed exposure to X-ray at a cumulative dose of 14 Gy. The survival rate showed that NLRP3 deficiency increased the mortality rate in mice. Furthermore, colon damage, evaluated by H&E staining and barrier function analysis, were significantly aggravated by NLRP3 deficiency. Enhanced phosphorylation of p-TBK1 and p-IRF3 in colonic tissue as well as elevated IFN-ß levels in the serum indicated hyperactivation of cGAS-STING signaling. Moreover, radiation-induced expression of p-TBK1, p-IRF3, and IFN-ß in BMDMs increased in vitro after NLRP3 knockout. Thus, our study outcomes suggest that NLRP3 may protect mice from radiation-induced tissue damage via attenuating cGAS-STING signaling.
Assuntos
Colo/efeitos da radiação , Macrófagos/efeitos da radiação , Proteínas de Membrana/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleotidiltransferases/metabolismo , Lesões por Radiação/prevenção & controle , Úlcera Cutânea/prevenção & controle , Pele/efeitos da radiação , Animais , Células Cultivadas , Colo/enzimologia , Colo/patologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Macrófagos/enzimologia , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Lesões por Radiação/enzimologia , Lesões por Radiação/genética , Lesões por Radiação/patologia , Transdução de Sinais , Pele/enzimologia , Pele/patologia , Úlcera Cutânea/enzimologia , Úlcera Cutânea/genética , Úlcera Cutânea/patologiaRESUMO
Sialic acid and its associated metabolic enzymes have emerged as important components of the pathophysiology of type 2 diabetes (T2D). There is an elevation in the serum concentration of sialic acid in humans and animals with T2D. The present study investigated the modulation of mRNA expression level of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) and neuraminidase 1 (NEU1) genes in some organs of type 2 diabetic rats. T2D was induced using fructose-streptozotocin model and eight weeks after the induction of diabetes, sialic acid was assayed in the blood and organs (adipose tissue, brain, colon, kidney, liver, pancreas, skeletal muscle and spleen) followed by quantification of mRNA expression level of GNE and NEU1 genes by qPCR. The results showed a significant (P < 0.05) increase in sialic acid level in the serum and all the afore-mentioned organs investigated except in the adipose tissue and skeletal muscle of the diabetic rats compared the normal control. The expression GNE gene was only increased in the pancreas (1.8-fold) of the diabetic rats while there was a decrease in the expression of the gene in the colon. In contrast, the expression of NEU1 gene was increased in the spleen (3.5-fold), brain (2.2-fold), liver (1.9-fold), colon (1.5-fold) and kidney of the diabetic rats. It was concluded that the elevated level of sialic acid in the organs of diabetic rats, except the pancreas, might not be due to increased endogenous synthesis of sialic acid.
Assuntos
Diabetes Mellitus Tipo 2/enzimologia , Complexos Multienzimáticos/genética , Animais , Encéfalo/enzimologia , Colo/enzimologia , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/genética , Regulação Enzimológica da Expressão Gênica , Fígado/enzimologia , Ácido N-Acetilneuramínico/sangue , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/genética , Pâncreas/enzimologia , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Baço/enzimologiaRESUMO
Dendritic cells (DCs) play pivotal roles in maintaining intestinal homeostasis, but how the DCs regulate diverse immune networks on homeostasis breakdown remains largely unknown. Here, we report that, in response to epithelial barrier disruption, colonic DCs regulate the differentiation of type 1 regulatory T (Tr1) cells through p38α-dependent IL-27 production to initiate an effective immune response. Deletion of p38α in DCs, but not in T cells, led to increased Tr1 and protected mice from dextran sodium sulfate-induced acute colitis and chronic colitis-associated colorectal cancer. We show that higher levels of IL-27 in p38α-deficient colonic cDC1s, but not cDC2s, were responsible for the increase of Tr1 cells. Moreover, p38α-dependent IL-27 enhanced IL-22 secretion from intestinal group 3 innate lymphoid cells and protected epithelial barrier function. In p38α-deficient DCs, the TAK1-MKK4/7-JNK-c-Jun axis was hyperactivated, leading to high IL-27 levels, and inhibition of the JNK-c-Jun axis suppressed IL-27 expression. ChIP assay revealed direct binding of c-Jun to the promoter of Il27p28, which was further enhanced in p38α-deficient DCs. In summary, here we identify a key role for p38α signaling in DCs in regulating intestinal inflammatory response and tumorigenesis, and our finding may provide targets for the treatment of inflammatory intestinal diseases.
Assuntos
Colite/enzimologia , Colo/imunologia , Neoplasias Colorretais/enzimologia , Células Dendríticas/enzimologia , Proteína Quinase 14 Ativada por Mitógeno/imunologia , Animais , Carcinogênese , Colite/genética , Colite/imunologia , Colite/patologia , Colo/enzimologia , Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Células Dendríticas/imunologia , Feminino , Humanos , Interleucina-27/genética , Interleucina-27/imunologia , Intestinos/imunologia , Intestinos/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 14 Ativada por Mitógeno/genética , Linfócitos T Reguladores/imunologiaRESUMO
Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.
Assuntos
Colo/enzimologia , Miocárdio/enzimologia , Próstata/enzimologia , Transglutaminases/metabolismo , Bexiga Urinária/enzimologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Clonagem Molecular , Estabilidade Enzimática , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Dodecilsulfato de Sódio/química , Especificidade por Substrato , Distribuição Tecidual , Transglutaminases/genéticaRESUMO
Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital disorder characterized by loss of smooth muscle contraction in the bladder and intestine. To date, three genes are known to be involved in MMIHS pathogenesis: ACTG2, MYH11, and LMOD1. However, for approximately 10% of affected individuals, the genetic cause of the disease is unknown, suggesting that other loci are most likely involved. Here, we report on three MMIHS-affected subjects from two consanguineous families with no variants in the known MMIHS-associated genes. By performing homozygosity mapping and whole-exome sequencing, we found homozygous variants in myosin light chain kinase (MYLK) in both families. We identified a 7 bp duplication (c.3838_3844dupGAAAGCG [p.Glu1282_Glyfs∗51]) in one family and a putative splice-site variant (c.3985+5C>A) in the other. Expression studies and splicing assays indicated that both variants affect normal MYLK expression. Because MYLK encodes an important kinase required for myosin activation and subsequent interaction with actin filaments, it is likely that in its absence, contraction of smooth muscle cells is impaired. The existence of a conditional-Mylk-knockout mouse model with severe gut dysmotility and abnormal function of the bladder supports the involvement of this gene in MMIHS pathogenesis. In aggregate, our findings implicate MYLK as a gene involved in the recessive form of MMIHS, confirming that this disease of the visceral organs is heterogeneous with a myopathic origin.
Assuntos
Anormalidades Múltiplas/enzimologia , Anormalidades Múltiplas/genética , Colo/anormalidades , Genes Recessivos , Pseudo-Obstrução Intestinal/enzimologia , Pseudo-Obstrução Intestinal/genética , Mutação/genética , Quinase de Cadeia Leve de Miosina/genética , Bexiga Urinária/anormalidades , Sequência de Bases , Colo/enzimologia , Feminino , Homozigoto , Humanos , Masculino , Linhagem , Bexiga Urinária/enzimologiaRESUMO
BACKGROUND & AIMS: Epithelial tight junctions are compromised in gastrointestinal disease. Processes that contribute to the resulting barrier loss include endocytic occludin removal from the tight junction and reduced occludin expression. Nevertheless, the relatively-normal basal phenotype of occludin knockout (KO) mice has been taken as evidence that occludin does not contribute to gastrointestinal barrier function. We asked whether stress could unmask occludin functions within intestinal epithelia. METHODS: Wildtype (WT), universal and intestinal epithelial-specific occludin KO, and villin-EGFP-occludin transgenic mice as well as WT and occludin knockdown (KD) Caco-2BBe cell monolayers were challenged with DSS, TNBS, staurosporine, 5-FU, or TNF. Occludin and caspase-3 expression were assessed in patient biopsies. RESULTS: Intestinal epithelial occludin loss limited severity of DSS- and TNBS-induced colitis due to epithelial resistance to apoptosis; activation of both intrinsic and extrinsic apoptotic pathways was blocked in occludin KO epithelia. Promoter analysis revealed that occludin enhances CASP3 transcription and, conversely, that occludin downregulation reduces caspase-3 expression. Analysis of biopsies from Crohn's disease and ulcerative colitis patients and normal controls demonstrated that disease-associated occludin downregulation was accompanied by and correlated with reduced caspase-3 expression. In vitro, cytokine-induced occludin downregulation resulted in reduced caspase-3 expression and resistance to intrinsic and extrinsic pathway apoptosis, demonstrating an overall protective effect of inflammation-induced occludin loss. CONCLUSIONS: The tight junction protein occludin regulates apoptosis by enhancing caspase-3 transcription. These data suggest that reduced epithelial caspase-3 expression downstream of occludin downregulation is a previously-unappreciated anti-apoptotic process that contributes to mucosal homeostasis in inflammatory conditions.
Assuntos
Apoptose , Caspase 3/metabolismo , Colite/enzimologia , Colo/enzimologia , Células Epiteliais/enzimologia , Mucosa Intestinal/enzimologia , Ocludina/metabolismo , Animais , Células CACO-2 , Estudos de Casos e Controles , Caspase 3/deficiência , Caspase 3/genética , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colite Ulcerativa/enzimologia , Colite Ulcerativa/patologia , Colo/patologia , Doença de Crohn/enzimologia , Doença de Crohn/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Células Epiteliais/patologia , Humanos , Mucosa Intestinal/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ocludina/deficiência , Ocludina/genética , Transdução de Sinais , Ácido Trinitrobenzenossulfônico , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
BACKGROUND & AIMS: It is not clear how regulation of T-cell function is altered during development of inflammatory bowel diseases (IBD). We studied the mechanisms by which geranylgeranyltransferase-mediated prenylation controls T-cell localization to the intestine and chronic inflammation. METHODS: We generated mice with T-cell-specific disruption of the geranylgeranyltransferase type I, beta subunit gene (Pggt1b), called Pggt1bΔCD4 mice, or the ras homolog family member A gene (Rhoa), called RhoaΔCD4 mice. We also studied mice with knockout of CDC42 or RAC1 and wild-type mice (controls). Intestinal tissues were analyzed by histology, multiphoton and confocal microscopy, and real-time polymerase chain reaction. Activation of CDC42, RAC1, and RHOA were measured with G-LISA, cell fractionation, and immunoblots. T cells and lamina propria mononuclear cells from mice were analyzed by flow cytometry or transferred to Rag1-/- mice. Mice were given injections of antibodies against integrin alpha4beta7 or gavaged with the RORC antagonist GSK805. We obtained peripheral blood and intestinal tissue samples from patients with and without IBD and analyzed them by flow cytometry. RESULTS: Pggt1bΔCD4 mice developed spontaneous colitis, characterized by thickening of the intestinal wall, edema, fibrosis, accumulation of T cells in the colon, and increased expression of inflammatory cytokines. Compared with control CD4+ T cells, PGGT1B-deficient CD4+ T cells expressed significantly higher levels of integrin alpha4beta7, which regulates their localization to the intestine. Inflammation induced by transfer of PGGT1B-deficient CD4+ T cells to Rag1-/- mice was blocked by injection of an antibody against integrin alpha4beta7. Lamina propria of Pggt1bΔCD4 mice had increased numbers of CD4+ T cells that expressed RORC and higher levels of cytokines produced by T-helper 17 cells (granulocyte-macrophage colony-stimulating factor, interleukin [IL]17A, IL17F, IL22, and tumor necrosis factor [TNF]). The RORC inverse agonist GSK805, but not antibodies against IL17A or IL17F, prevented colitis in Pggt1bΔCD4 mice. PGGT1B-deficient CD4+ T cells had decreased activation of RHOA. RhoAΔCD4 mice had a similar phenotype to Pggt1bΔCD4 mice, including development of colitis, increased numbers of CD4+ T cells in colon, increased expression of integrin alpha4beta7 by CD4+ T cells, and increased levels of IL17A and other inflammatory cytokines in lamina propria. T cells isolated from intestinal tissues from patients with IBD had significantly lower levels of PGGT1B than tissues from individuals without IBD. CONCLUSION: Loss of PGGT1B from T cells in mice impairs RHOA function, increasing CD4+ T-cell expression of integrin alpha4beta7 and localization to colon, resulting in increased expression of inflammatory cytokines and colitis. T cells isolated from gut tissues from patients with IBD have lower levels of PGGT1B than tissues from patients without IBD.
Assuntos
Alquil e Aril Transferases/deficiência , Quimiotaxia de Leucócito , Colite/enzimologia , Colo/enzimologia , Integrinas/metabolismo , Linfócitos T/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Imunidade Adaptativa , Alquil e Aril Transferases/genética , Animais , Estudos de Casos e Controles , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/patologia , Colo/imunologia , Colo/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/patologia , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTPRESUMO
BACKGROUND & AIMS: Many genetic and environmental factors, including family history, dietary fat, and inflammation, increase risk for colon cancer development. Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor that regulates systemic lipid homeostasis. We explored the role of intestinal PPARα in colon carcinogenesis. METHODS: Colon cancer was induced in mice with intestine-specific disruption of Ppara (PparaΔIE), Pparafl/fl (control), and mice with disruption of Ppara that express human PPARA (human PPARA transgenic mice), by administration of azoxymethane with or without dextran sulfate sodium (DSS). Colons were collected from mice and analyzed by immunoblots, quantitative polymerase chain reaction, and histopathology. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses were performed on urine and colons. We used molecular biology and biochemical approaches to study mechanisms in mouse colons, primary intestinal epithelial cells, and colon cancer cell lines. Gene expression data and clinical features of patients with colorectal tumors were obtained from Oncomine, and human colorectal-tumor specimens and adjacent normal tissues were collected and analyzed by immunohistochemistry. RESULTS: Levels of Ppara messenger RNA were reduced in colon tumors from mice. PparaΔIE mice developed more and larger colon tumors than control mice following administration of azoxymethane, with or without DSS. Metabolomic analyses revealed increases in methylation-related metabolites in urine and colons from PparaΔIE mice, compared with control mice, following administration of azoxymethane, with or without DSS. Levels of DNA methyltransferase 1 (DNMT1) and protein arginine methyltransferase 6 (PRMT6) were increased in colon tumors from PparaΔIE mice, compared with colon tumors from control mice. Depletion of PPARα reduced the expression of retinoblastoma protein, resulting in increased expression of DNMT1 and PRMT6. DNMT1 and PRMT6 decreased expression of the tumor suppressor genes Cdkn1a (P21) and Cdkn1b (p27) via DNA methylation and histone H3R2 dimethylation-mediated repression of transcription, respectively. Fenofibrate protected human PPARA transgenic mice from azoxymethane and DSS-induced colon cancer. Human colon adenocarcinoma specimens had lower levels of PPARA and retinoblastoma protein and higher levels of DNMT1 and PRMT6 than normal colon tissues. CONCLUSIONS: Loss of PPARα from the intestine promotes colon carcinogenesis by increasing DNMT1-mediated methylation of P21 and PRMT6-mediated methylation of p27 in mice. Human colorectal tumors have lower levels of PPARA messenger RNA and protein than nontumor tissues. Agents that activate PPARα might be developed for chemoprevention or treatment of colon cancer.
Assuntos
Adenocarcinoma/prevenção & controle , Colo/enzimologia , Neoplasias do Colo/prevenção & controle , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Proteínas Nucleares/metabolismo , PPAR alfa/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Anticarcinógenos/farmacologia , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colo/patologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/efeitos dos fármacos , Bases de Dados Genéticas , Modelos Animais de Doenças , Fenofibrato/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , PPAR alfa/agonistas , PPAR alfa/deficiência , PPAR alfa/genética , Proteína-Arginina N-Metiltransferases/genética , Transdução de SinaisRESUMO
BACKGROUND/AIMS: The colonic H+, K+ ATPase (HKA2) is a heterodimeric membrane protein that exchanges luminal K+ for intracellular H+ and is involved in maintaining potassium homeostasis. Under homeostatic conditions, the colonic HKA2 remains inactive, since most of the potassium is absorbed by the small intestine. In diarrheal states, potassium is secreted and compensatory potassium absorption becomes necessary. This study proposes a novel mechanism whereby the addition of penicillin G sodium salt (penG) to colonic crypts stimulates potassium uptake in the presence of intracellular nitric oxide (NO), under sodium-free (0-Na+) conditions. METHODS: Sprague Dawley rat colonic crypts were isolated and pHi changes were monitored through the ammonium prepulse technique. Increased proton extrusion in 0-Na+ conditions reflected heightened H+, K+ ATPase activity. Colonic crypts were exposed to penG, L-arginine (a NO precursor), and N-nitro l-arginine methyl ester (L-NAME, a NO synthase inhibitor). RESULTS: Isolated administration of penG significantly increased H+, K+ ATPase activity from baseline, p 0.0067. Co-administration of arginine and penG in 0-Na+ conditions further upregulated H+, K+ ATPase activity, p <0.0001. Crypt perfusion with L-NAME and penG demonstrated a significant reduction in H+, K+ ATPase activity, p 0.0058. CONCLUSION: Overall, acute exposure of colonic crypts to penG activates the H+, K+ ATPase in the presence of NO. This study provides new insights into colonic potassium homeostasis.
Assuntos
Colo/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Óxido Nítrico/metabolismo , Penicilina G/farmacologia , Animais , Arginina/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
O-GlcNAcylation is a post-translational modification of thousands of intracellular proteins that dynamically regulates many fundamental cellular processes. Cellular O-GlcNAcylation levels are regulated by a unique couple of enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which adds and removes the GlcNAc residue, respectively. Maintenance of O-GlcNAc homeostasis is essential to ensure optimal cellular function and disruption of this homeostasis has been linked to the etiology of several human diseases including cancer. The mechanisms through which the cell maintains O-GlcNAc homeostasis are not fully understood but several studies have suggested that a reciprocal regulation of OGT and OGA expression could be one of them. In this study, we investigated the putative regulation of OGT and OGA expression in response to disruption in O-GlcNAc homeostasis in colon. We provide in vitro and in vivo evidences that in colon cells, modulation of O-GlcNAcylation levels leads to a compensatory regulation of OGT and OGA expression in an attempt to restore basal O-GlcNAcylation levels. Our results also suggests that the regulation of colonic OGA expression in response to changes in O-GlcNAc homeostasis occurs mostly at the transcriptional level whereas OGT regulation seems to rely mainly on post-transcriptional mechanisms.
Assuntos
Acetilglucosamina/metabolismo , Colo/enzimologia , Homeostase , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Colo/efeitos dos fármacos , Colo/patologia , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , Piranos/farmacologia , Tiazóis/farmacologia , Células Tumorais Cultivadas , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
The modifications of the hemodynamic system and hydromineral metabolism are physiological features characterizing a normal gestation. Thus, the ability to expand plasma volume without increasing the level of blood pressure is necessary for the correct perfusion of the placenta. The kidney is essential in this adaptation by reabsorbing avidly sodium and fluid. In this study, we observed that the H,K-ATPase type 2 (HKA2), an ion pump expressed in kidney and colon and already involved in the control of the K+ balance during gestation, is also required for the correct plasma volume expansion and to maintain normal blood pressure. Indeed, compared with WT pregnant mice that exhibit a 1.6-fold increase of their plasma volume, pregnant HKA2-null mice (HKA2KO) only modestly expand their extracellular volume (×1.2). The renal expression of the epithelial Na channel (ENaC) α- and γ-subunits and that of the pendrin are stimulated in gravid WT mice, whereas the Na/Cl- cotransporter (NCC) expression is downregulated. These modifications are all blunted in HKA2KO mice. This impeded renal adaptation to gestation is accompanied by the development of hypotension in the pregnant HKA2KO mice. Altogether, our results showed that the absence of the HKA2 during gestation leads to an "underfilled" situation and has established this transporter as a key player of the renal control of salt and potassium metabolism during gestation.
Assuntos
Pressão Sanguínea , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Rim/enzimologia , Volume Plasmático , Potássio/metabolismo , Sódio/metabolismo , Animais , Aquaporina 2/metabolismo , Colo/enzimologia , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Idade Gestacional , ATPase Trocadora de Hidrogênio-Potássio/deficiência , ATPase Trocadora de Hidrogênio-Potássio/genética , Homeostase , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismoRESUMO
Ulcerative colitis is a condition characterised by the infiltration of leukocytes into the gastrointestinal wall. Leukocyte-MPO catalyses hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) formation from chloride (Cl-) and thiocyanous (SCN-) anions, respectively. While HOCl indiscriminately oxidises biomolecules, HOSCN primarily targets low-molecular weight protein thiols. Oxidative damage mediated by HOSCN may be reversible, potentially decreasing MPO-associated host tissue destruction. This study investigated the effect of SCN- supplementation in a model of acute colitis. Female mice were supplemented dextran sodium sulphate (DSS, 3% w/v) in the presence of 10 mM Cl- or SCN- in drinking water ad libitum, or with salts (NaCl and NaSCN only) or water only (controls). Behavioural studies showed mice tolerated NaSCN and NaCl-treated water with water-seeking frequency. Ion-exchange chromatography showed increased fecal and plasma SCN- levels in thiocyanate supplemented mice; plasma SCN- reached similar fold-increase for smokers. Overall there was no difference in weight loss and clinical score, mucin levels, crypt integrity and extent of cellular infiltration between DSS/SCN- and DSS/Cl- groups. Neutrophil recruitment remained unchanged in DSS-treated mice, as assessed by fecal calprotectin levels. Total thiol and tyrosine phosphatase activity remained unchanged between DSS/Cl- and DSS/SCN- groups, however, colonic tissue showed a trend in decreased 3-chlorotyrosine (1.5-fold reduction, p < 0.051) and marked increase in colonic GCLC, the rate-limiting enzyme in glutathione synthesis. These data suggest that SCN- administration can modulate MPO activity towards a HOSCN-specific pathway, however, this does not alter the development of colitis within a DSS murine model.