RESUMO
BACKGROUND & AIMS: Diarrhea associated with inflammatory bowel diseases has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor (TNF). The intestinal mucosa of patients with inflammatory bowel diseases has reduced expression of solute carrier family 26 member 3 (SLC26A3, also called DRA). We investigated whether TNF directly affects expression of DRA in human intestinal epithelial cells (IECs) and in the intestines of mice, and studied the mechanisms of these effects. METHODS: We performed quantitative reverse transcription polymerase chain reaction, immunofluorescence, and immunoblot analyses in Caco-2, HT-29, and T-84 cells human IECs cultured in 2 or 3 dimensions with or without TNF (50 ng/mL for 6-24 hours). We purified nuclear extracts and quantified nuclear factor-κB (NF-κB) activation and DNA binding. We isolated intestinal crypts from C57BL/6 mice, cultured enteroids, incubated these with TNF (50 ng/mL, 24 hours), and quantified messenger RNAs. DRA-mediated exchange of Cl- for HCO3- was measured by uptake of 125I. Expression of the NF-κB inhibitor α (IkBa) was knocked down in Caco-2 cells with small interfering RNAs. Activation of NF-κB in response to TNF was measured by luciferase reporter assays; binding of the NF-κB subunit p65 in cells was analyzed in chromatin immunoprecipitation assays. DRA promoter activity was measured in a luciferase reporter assay. C57BL/6 mice were injected with TNF (5 µg/mouse for 3-6 hours) or vehicle (control); intestines were collected and analyzed by immunofluorescence, or RNA and protein were collected from the mucosa. RESULTS: Incubation of IECs with TNF reduced expression of DRA. Knockdown of NF-κB inhibitor α in IECs led to nuclear translocation of the NF-κB subunit p65 and reduced levels of DRA messenger RNA and protein. Expression of a transgene encoding p65 or p50 in IECs led to significant reductions in the promoter activity of DRA and its expression. In chromatin immunoprecipitation assays, p65 bound directly to the promoter of DRA, at the regions of -935 to -629 and -375 to -84. Injection of mice with TNF or incubation of crypt-derived enteroids with TNF reduced their expression of DRA messenger RNA and protein. CONCLUSIONS: In human IECs and intestinal tissues from mice, we found TNF to activate NF-κB, which reduced expression of the Cl- / HCO3- exchanger DRA (SLC26A3), via direct binding to the promoter of DRA. This pathway is an important therapeutic target for inflammatory bowel disease-associated diarrhea.
Assuntos
Antiporters/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Diarreia/etiologia , Células Epiteliais/efeitos dos fármacos , Doenças Inflamatórias Intestinais/complicações , Mucosa Intestinal/efeitos dos fármacos , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antiporters/genética , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/genética , Diarreia/genética , Diarreia/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células HT29 , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Regiões Promotoras Genéticas , Interferência de RNA , Transportadores de Sulfato , Fatores de Tempo , TransfecçãoRESUMO
The gut hormone, glucagon like peptide-1 (GLP-1) exerts anti-inflammatory effects. However, its clinical use is limited by its short half-life. Previously, we have shown that GLP-1 as a nanomedicine (GLP-1 in sterically stabilized phospholipid micelles, GLP-1-SSM) has increased in vivo stability. The current study was aimed at testing the efficacy of this GLP-1 nanomedicine in alleviating colonic inflammation and associated diarrhea in dextran sodium sulfate (DSS) induced mouse colitis model. Our results show that GLP-1-SSM treatment markedly alleviated the colitis phenotype by reducing the expression of pro-inflammatory cytokine IL-1ß, increasing goblet cells and preserving intestinal epithelial architecture in colitis model. Further, GLP-1-SSM alleviated diarrhea (as assessed by luminal fluid) by increasing protein expression of intestinal chloride transporter DRA (down regulated in adenoma). Our results indicate that GLP-1 nanomedicine may act as a novel therapeutic tool in alleviating gut inflammation and associated diarrhea in inflammatory bowel disease (IBD).
Assuntos
Colite/tratamento farmacológico , Peptídeo 1 Semelhante ao Glucagon/administração & dosagem , Inflamação , Nanomedicina , Animais , Sulfato de Dextrana/uso terapêutico , Diarreia/tratamento farmacológico , Diarreia/etiologia , Modelos Animais de Doenças , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , CamundongosRESUMO
GPR120 (free fatty acid receptor-4) is a G protein-coupled receptor for medium- and long-chain unsaturated fatty acids, including ω-3 fatty acids. Recent studies have shown GPR120 to play cardinal roles in metabolic disorders via modulation of gut hormone secretion and insulin sensitivity and to exert anti-inflammatory effects in macrophages and adipose tissues. However, information on anti-inflammatory role of GPR120 at the level of intestinal epithelium is very limited. Current studies demonstrated differential levels of GPR120 mRNA and protein along the length of the human, mouse, and rat intestine and delineated distinct anti-inflammatory responses following GPR120 activation in model human intestinal epithelial Caco-2 cells, but not in model mouse intestinal epithelial endocrine cell line STC-1. In Caco-2 cells, GPR120 was internalized, bound to ß-arrestin-2, and attenuated NF-κB activation in response to 30-min exposure to the agonists GW9508, TUG-891, or docosahexaenoic acid. These effects were abrogated in response to small interfering RNA silencing of ß-arrestin-2. Treatment of STC-1 cells with these agonists did not induce receptor internalization and had no effects on NF-κB activation, although treatment with the agonists GW9508 or TUG-891 for 6 h augmented the synthesis and secretion of the gut hormone glucagon-like peptide-1 in this cell line. Our studies for the first time demonstrated a GPR120-mediated novel anti-inflammatory pathway in specific intestinal epithelial cell types that could be of therapeutic relevance to intestinal inflammatory disorders.
Assuntos
Inflamação/metabolismo , Mucosa Intestinal/imunologia , Receptores Acoplados a Proteínas G/imunologia , Animais , Células CACO-2 , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Imunoprecipitação , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/metabolismo , TransfecçãoRESUMO
All-trans-retinoic acid (ATRA) is an active vitamin A derivative known to modulate a number of physiological processes, including growth and development, differentiation, and gene transcription. The protective effect of ATRA in gut inflammation and diarrheal diseases has been documented. In this regard, down-regulated in adenoma (DRA, a key luminal membrane Cl(-) transporter involved in NaCl absorption) has been shown to be suppressed in intestinal inflammation. This suppression of DRA is associated with diarrheal phenotype. Therefore, current studies were undertaken to examine the effects of ATRA on DRA expression. DRA mRNA levels were significantly elevated (â¼4-fold) in response to ATRA with induction starting as early as 8 h of incubation. Similarly, ATRA increased DRA protein expression by â¼50%. Furthermore, DRA promoter activity was significantly increased in response to ATRA indicating transcriptional activation. ATRA effects on DRA expression appeared to be mediated via the RAR-ß receptor subtype, as ATRA remarkably induced RAR-ß mRNA levels, whereas RAR-ß knockdown substantially attenuated the ability of ATRA to increase DRA expression. Results obtained from agonist (CH-55) and antagonist (LE-135) studies further confirmed that ATRA exerts its effects through RAR-ß. Furthermore, ATRA treatment resulted in a significant increase in HNF-1ß mRNA levels. The ability of ATRA to induce DRA expression was inhibited in the presence of HNF-1ß siRNA indicative of its involvement in ATRA-induced effects on DRA expression. In conclusion, ATRA may act as an antidiarrheal agent by increasing DRA expression via the RAR-ß/HNF-1ß-dependent pathway.
Assuntos
Antiportadores de Cloreto-Bicarbonato/metabolismo , Fator 1-beta Nuclear de Hepatócito/genética , Mucosa Intestinal/efeitos dos fármacos , Tretinoína/farmacologia , Sequência de Bases , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/genética , Primers do DNA , Inativação Gênica , Fator 1-beta Nuclear de Hepatócito/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Transportadores de Sulfato , Regulação para Cima/genéticaRESUMO
Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl(-)) and sodium (Na(+)) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na(+)), CFTR (Cl(-)), and SLC26A3 (DRA) (Cl(-)/HCO3 (-)) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl(-) and Na(+) stool loss in MVID diarrhea.
Assuntos
Enterócitos/metabolismo , Jejuno/metabolismo , Síndromes de Malabsorção/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Microvilosidades/patologia , Mucolipidoses/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Enterócitos/ultraestrutura , Regulação da Expressão Gênica , Humanos , Transporte de Íons , Jejuno/patologia , Jejuno/ultraestrutura , Síndromes de Malabsorção/genética , Síndromes de Malabsorção/patologia , Proteínas de Membrana Transportadoras/genética , Microvilosidades/genética , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Mucolipidoses/genética , Mucolipidoses/patologia , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Fenótipo , Fosfoproteínas/metabolismo , Interferência de RNA , Transdução de Sinais , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , Transportadores de Sulfato , Fatores de Transcrição , Transfecção , Proteínas de Sinalização YAPRESUMO
Impaired absorption of electrolytes is a hallmark of diarrhea associated with inflammation or enteric infections. Intestinal epithelial luminal membrane NHE3 (Na+/H+ exchanger 3) and DRA (Down-Regulated in Adenoma; Cl-/HCO3- exchanger) play key roles in mediating electroneutral NaCl absorption. We have previously shown decreased NHE3 and DRA function in response to short-term infection with enteropathogenic E coli (EPEC), a diarrheal pathogen. Recent studies have also shown substantial downregulation of DRA expression in a diarrheal model of infection with Citrobacter rodentium, the mouse counterpart of EPEC. Since our previous studies showed that the probiotic Lactobacillus acidophilus (LA) increased DRA and NHE3 function and expression and conferred protective effects in experimental colitis, we sought to evaluate the efficacy of LA in counteracting NHE3 and DRA inhibition and ameliorating diarrhea in a model of C rodentium infection. FVB/N mice challenged with C rodentium [1 × 109 colony-forming units (CFU)] with or without administration of live LA (3 × 109 CFU) were assessed for NHE3 and DRA mRNA and protein expression, mRNA levels of carbonic anhydrase, diarrheal phenotype (assessed by colonic weight-to-length ratio), myeloperoxidase activity, and proinflammatory cytokines. LA counteracted C rodentium-induced inhibition of colonic DRA, NHE3, and carbonic anhydrase I and IV expression and attenuated diarrheal phenotype and MPO activity. Furthermore, LA completely blocked C rodentium induction of IL-1ß, IFN-γ, and CXCL1 mRNA and C rodentium-induced STAT3 phosphorylation. In conclusion, our data provide mechanistic insights into antidiarrheal effects of LA in a model of infectious diarrhea and colitis.
Assuntos
Antiporters/metabolismo , Citrobacter rodentium , Diarreia/tratamento farmacológico , Infecções por Enterobacteriaceae/tratamento farmacológico , Lactobacillus acidophilus , Probióticos/uso terapêutico , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Colite/metabolismo , Colite/microbiologia , Colo/metabolismo , Colo/microbiologia , Citocinas/metabolismo , Diarreia/metabolismo , Diarreia/microbiologia , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Camundongos , Fosforilação , Trocador 3 de Sódio-Hidrogênio , Transportadores de Sulfato , Resultado do TratamentoRESUMO
Enteropathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl(-)/HCO3(-) exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl(-)/HCO3(-) exchange activity was measured as DIDS-sensitive (125)I(-) uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 µM) increased apical Cl(-)/HCO3(-) exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis.
Assuntos
Antiporters/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Escherichia coli Enteropatogênica , Infecções por Escherichia coli/metabolismo , Mucosa Intestinal/metabolismo , Animais , Western Blotting , Células CACO-2 , Clatrina/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de SulfatoRESUMO
Glycogen synthase 1 (GYS1), the rate-limiting enzyme in muscle glycogen synthesis, plays a central role in energy homeostasis and has been proposed as a therapeutic target in multiple glycogen storage diseases. Despite decades of investigation, there are no known potent, selective small-molecule inhibitors of this enzyme. Here, we report the preclinical characterization of MZ-101, a small molecule that potently inhibits GYS1 in vitro and in vivo without inhibiting GYS2, a related isoform essential for synthesizing liver glycogen. Chronic treatment with MZ-101 depleted muscle glycogen and was well tolerated in mice. Pompe disease, a glycogen storage disease caused by mutations in acid α glucosidase (GAA), results in pathological accumulation of glycogen and consequent autophagolysosomal abnormalities, metabolic dysregulation, and muscle atrophy. Enzyme replacement therapy (ERT) with recombinant GAA is the only approved treatment for Pompe disease, but it requires frequent infusions, and efficacy is limited by suboptimal skeletal muscle distribution. In a mouse model of Pompe disease, chronic oral administration of MZ-101 alone reduced glycogen buildup in skeletal muscle with comparable efficacy to ERT. In addition, treatment with MZ-101 in combination with ERT had an additive effect and could normalize muscle glycogen concentrations. Biochemical, metabolomic, and transcriptomic analyses of muscle tissue demonstrated that lowering of glycogen concentrations with MZ-101, alone or in combination with ERT, corrected the cellular pathology in this mouse model. These data suggest that substrate reduction therapy with GYS1 inhibition may be a promising therapeutic approach for Pompe disease and other glycogen storage diseases.
Assuntos
Doença de Depósito de Glicogênio Tipo II , Camundongos , Animais , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Glicogênio Sintase/metabolismo , Glicogênio Sintase/farmacologia , Camundongos Knockout , Glicogênio/metabolismo , Músculo Esquelético/metabolismo , Terapia de Reposição de Enzimas/métodosRESUMO
AIM: P-glycoprotein (Pgp/MDR1) plays a major role in intestinal homeostasis. Decrease in Pgp function and expression has been implicated in the pathogenesis of IBD. However, inhibitory mechanisms involved in the decrease of Pgp in inflammation are not fully understood. Angiotensin II (Ang II), a peptide hormone predominantly expressed in the epithelial cells of the crypt-villus junction of the intestine, has been shown to exert pro-inflammatory effects in the gut. It is increased in IBD patients and animals with experimental colitis. Whether Ang II directly influences Pgp is not known. METHODS: Pgp activity was measured as verapamil-sensitive 3 H-digoxin flux. Pgp surface expression and exocytosis were measured by cell surface biotinylation studies. Signalling pathways were elucidated by Western blot analysis and pharmacological approaches. RESULTS: Ang II (10 nM) significantly inhibited Pgp activity at 60 minutes. Ang II-mediated effects on Pgp function were receptor-mediated as the Ang II receptor 1 (ATR1) antagonist, losartan, blocked Pgp inhibition. Ang II effects on Pgp activity appeared to be mediated via PI3 kinase, p38 MAPK and Akt signalling. Ang II-mediated inhibition of Pgp activity was associated with a decrease in the surface membrane expression of Pgp protein via decreased exocytosis and was found to be dependent on the Akt pathway. Short-term treatment of Ang II (2 mg/kg b.wt., 2 hours) to mice also decreased the membrane expression of Pgp protein levels in ileum and colon. CONCLUSION: Our findings provide novel insights into the role of Ang II and ATR1 in decreasing Pgp expression in intestinal inflammation.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Angiotensina II/farmacologia , Células Epiteliais/metabolismo , Mucosa Intestinal/citologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Angiotensina II/administração & dosagem , Animais , Células CACO-2 , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
TGF-ß1 is an important multifunctional cytokine with numerous protective effects on intestinal mucosa. The influence of TGF-ß1 on serotonin transporter (SERT) activity, the critical mechanism regulating the extracellular availability of serotonin (5-HT), is not known. Current studies were designed to examine acute effects of TGF-ß1 on SERT. Model human intestinal Caco-2 cells grown as monolayer's or as cysts in 3D culture and ex vivo mouse model were utilized. Treatment of Caco-2 cells with TGF-ß1 (10 ng/ml, 60 min) stimulated SERT activity (~2 fold, P<0.005). This stimulation of SERT function was dependent upon activation of TGF-ß1 receptor (TGFRI) as SB-431542, a specific TGF-ßRI inhibitor blocked the SERT stimulation. SERT activation in response to TGF-ß1 was attenuated by inhibition of PI3K and occurred via enhanced recruitment of SERT-GFP to apical surface in a PI3K dependent manner. The exocytosis inhibitor brefeldin A (2.5 µM) attenuated the TGF-ß1-mediated increase in SERT function. TGF-ß1 increased the association of SERT with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3) and promoted exocytosis of SERT. Caco-2 cells grown as cysts in 3D culture recapitulated the effects of TGF-ß1 showing increased luminal staining of SERT. Ussing chamber studies revealed increase in 3H-5-HT uptake in mouse ileum treated ex vivo with TGF-ß1 (10 ng/ml, 1h). These data demonstrate a novel mechanism rapidly regulating intestinal SERT via PI3K and STX3. Since decreased SERT is implicated in various gastro-intestinal disorders e.g IBD, IBS and diarrhea, understanding mechanisms stimulating SERT function by TGF-ß1 offers a novel therapeutic strategy to treat GI disorders.