RESUMO
Creatine transporter (CrT1) mediates cellular uptake of creatine (Cr), a nutrient pivotal in maintaining energy homeostasis in various tissues including intestinal epithelial cells (IECs). The impact of CrT1 deficiency on the pathogenesis of various psychiatric and neurological disorders has been extensively investigated. However, there are no studies on its regulation in IECs in health and disease. Current studies have determined differential expression of CrT1 along the length of the mammalian intestine and its dysregulation in inflammatory bowel disease (IBD)-associated inflammation and Adherent Invasive E. coli (AIEC) infection. CrT1 mRNA and protein levels in normal intestines and their alterations in inflammation and following AIEC infection were determined in vitro in model IECs (Caco-2/IEC-6) and in vivo in SAMP1/YitFc mice, a model of spontaneous ileitis resembling human IBD. CrT1 is differentially expressed in different regions of mammalian intestines with its highest expression in jejunum. In vitro, CrT1 function (Na+-dependent 14C-Cr uptake), expression and promoter activity significantly decreased following TNFα/IL1ß treatments and AIEC infection. SAMP1 mice and ileal organoids generated from SAMP1 mice also showed decreased CrT1 mRNA and protein compared to AKR controls. Our studies suggest that Cr deficiency in IECs secondary to CrT1 dysregulation could be a key factor contributing to IBD pathogenesis.
Assuntos
Infecções por Escherichia coli , Mucosa Intestinal , Animais , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Camundongos , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Células CACO-2 , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Inflamação/metabolismo , Inflamação/genética , Inflamação/patologia , Escherichia coli , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Creatina/metabolismoRESUMO
Na+/H+ exchanger-3 (NHE-3) is the major apical membrane transporter involved in vectorial Na+ absorption in the intestine. Dysregulation of NHE-3 expression and/or function has been implicated in pathophysiology of diarrhea associated with gut inflammation and infections. Therefore, it is critical to understand the mechanisms involved in the regulation of NHE-3 expression. MicroRNAs (miRNAs) are highly conserved small RNAs that can regulate gene expression at the posttranscriptional level. To date, however, very little is known about the regulation of NHE-3 expression by microRNAs. Therefore, current studies were undertaken to examine the potential miRNA candidates that can regulate the expression of NHE-3 in intestinal epithelial cells. In silico analysis, using different algorithms, predicted several miRNAs that target NHE-3. MicroRNAs with highest context and target score, miR-326, miR-744-5p, and miR-330-5p, were selected for the current study. Human NHE-3 gene 3' untranslated region [3'UTR; 160 base pair (bp)] was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with mimics of miR-326, miR-744-5p, and miR-330-5p into Caco-2, HT-29, and SK-CO15 cells. Cotransfection of NHE-3 3' UTR with miR-326 and -miR-330-5p mimics resulted in a significant decrease in relative luciferase activity. Transfection of miR-326 and -330-5p mimics into SK-CO15 cells significantly decreased the NHE-3 protein expression, with no change in NHE-3 messenger ribonucleic acid (mRNA) levels. Our findings demonstrate a novel mechanism for posttranscriptional regulation of NHE-3 by miR-326 and -330-5p by translational repression. We speculate that miR-326 and -330-5p dependent pathways may be involved in modulating NHE-3 expression under physiological and pathophysiological conditions.
Assuntos
MicroRNAs , Trocador 3 de Sódio-Hidrogênio , Humanos , Células CACO-2 , Regulação para Baixo , Células Epiteliais/metabolismo , MicroRNAs/genética , Trocador 3 de Sódio-Hidrogênio/genéticaRESUMO
Autophagy, a process of degradation and recycling of macromolecules and organelles to maintain cellular homeostasis, has also been shown to help eliminate invading pathogens. Conversely, various pathogens including parasites have been shown to modulate/exploit host autophagy facilitating their intracellular infectious cycle. In this regard, Cryptosporidium parvum (CP), a protozoan parasite of small intestine is emerging as a major global health challenge. However, the pathophysiology of cryptosporidiosis is mostly unknown. We have recently demonstrated CP-induced epithelial barrier disruption via decreasing the expression of specific tight junction (TJ) and adherens junction (AJ) proteins such as occludin, claudin-4 and E-cadherin. Therefore, we utilised confluent Caco-2 cell monolayers as in vitro model of intestinal epithelial cells (IECs) to investigate the potential role of autophagy in the pathophysiology of cryptosporidiosis. Autophagy was assessed by increase in the ratio of LC3II (microtubule associated protein 1 light chain 3) to LC3I protein and decrease in p62/SQSTM1 protein levels. CP treatment of Caco-2 cells for 24 hr induced autophagy with a maximum effect observed with 0.5 × 106 oocyst/well. CP decreased mTOR (mammalian target of rapamycin, a suppressor of autophagy) phosphorylation, suggesting autophagy induction via mTOR inactivation. Measurement of autophagic flux utilizing the lysosomal inhibitor chloroquine (CQ) showed more pronounced increase in LC3II level in cells co-treated with CP + CQ as compared to CP or CQ alone, suggesting that CP-induced increase in LC3II was due to enhanced autophagosome formation rather than impaired lysosomal clearance. CP infection did not alter ATG7, a key autophagy protein. However, the decrease in occludin, claudin-4 and E-cadherin by CP was partially blocked following siRNA silencing of ATG7, suggesting the role of autophagy in CP-induced decrease in these TJ/AJ proteins. Our results provide novel evidence of autophagy induction by CP in host IECs that could alter important host cell processes contributing to the pathophysiology of cryptosporidiosis.
Assuntos
Autofagia , Cryptosporidium parvum/patogenicidade , Células Epiteliais/patologia , Células Epiteliais/parasitologia , Interações Hospedeiro-Parasita , Células CACO-2 , Humanos , Mucosa Intestinal/parasitologia , Proteínas de Junções Íntimas/metabolismoRESUMO
Dietary nutrients absorbed in the proximal small intestine and assimilated in different tissues have a profound effect on overall energy homeostasis, determined by a balance between body's energy intake and expenditure. In obesity, altered intestinal absorption and consequently tissue assimilation of nutrients may disturb the energy balance leading to metabolic abnormalities at the cellular level. The absorption of nutrients such as sugars, amino acids and fatty acids released from food digestion require high-capacity transporter proteins expressed in the intestinal epithelial absorptive cells. Furthermore, nutrient sensing by specific transporters/receptors expressed in the epithelial enteroendocrine cells triggers release of gut hormones involved in regulating energy homeostasis via their effects on appetite and food intake. Therefore, the intestinal epithelial cells play a pivotal role in the pathophysiology of obesity and associated complications. Over the past decade, gut microbiota has emerged as a key factor contributing to obesity via its effects on digestion and absorption of nutrients in the small intestine, and energy harvest from dietary fiber, undigested component of food, in the large intestine. Various mechanisms of microbiota effects on obesity have been implicated. However, the impact of obesity-associated microbiota on the intestinal nutrient transporters needs extensive investigation. This review marshals the limited studies addressing the altered structure and function of the gut epithelium in obesity with special emphasis on nutrient transporters and role of diet and microbiota. The review also discusses the thoughts and controversies and research gaps in this field.
Assuntos
Dieta , Mucosa Intestinal/fisiopatologia , Microbiota , Nutrientes/metabolismo , Obesidade/patologia , Animais , Humanos , Obesidade/etiologiaRESUMO
Short-chain fatty acids (SCFAs) produced by bacterial fermentation of dietary fiber exert myriad of beneficial effects including the amelioration of inflammation. SCFAs exist as anions at luminal pH; their entry into the cells depends on the expression and function of monocarboxylate transporters. In this regard, sodium-coupled monocarboxylate transporter-1 (SMCT-1) is one of the major proteins involved in the absorption of SCFA in the mammalian colon. However, very little is known about the mechanisms of regulation of SMCT-1 expression in health and disease. MicroRNAs (miRs) are known to play a key role in modulating gene expression. In silico analysis showed miR-29a, b, and c with highest context score and its binding region was conserved among mammals. The 3'-untranslated region (UTR) of human SMCT-1 gene was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with miR-29a, b, and c mimics into Caco-2 and/or T-84 cells. The presence of UTR of this gene significantly decreased luciferase activity compared with empty vector. Cotransfection with miR-29a, b, or c resulted in further decrease in 3'-UTR activity of SMCT-1 luciferase constructs. Mimic transfection significantly decreased SMCT-1 protein expression without altering mRNA expression. Furthermore, the expression of miR-29a and c were significantly lower in mouse colon compared with small intestine, consistent with higher levels of SMCT-1 protein in the colon. Our studies demonstrated a novel finding in which miR-29a, b, and c downregulate SMCT-1 expression in colonic epithelial cells and may partly explain the differential expression of these transporters along the length of the gastrointestinal (GI) tract.NEW & NOTEWORTHY Our study for the first time reports the posttranscriptional regulation of SMCT-1 by miR-29a, b, and c in colonic epithelial cells. We also demonstrate that the expression of these microRNAs is lower in the mouse proximal and distal colon which partially explains the higher expression level of SMCT-1 in the colon compared with small intestine.
Assuntos
Mucosa Intestinal/metabolismo , MicroRNAs/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Células CACO-2 , Humanos , MicroRNAs/genética , Transportadores de Ácidos Monocarboxílicos/metabolismoRESUMO
Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na+/H+ and Cl-/HCO3- exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl-/HCO3- exchange, measured as DIDS-sensitive 36Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered Km with no effects on Vmax. Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl-/HCO3- exchangers, were unaltered. Thus, inhibition of villus cell Cl-/HCO3- exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl-, and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.
Assuntos
Ácidos Araquidônicos/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Enterócitos/metabolismo , Ileíte/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Ácidos Araquidônicos/farmacologia , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato/antagonistas & inibidores , Inibidores de Ciclo-Oxigenase/farmacologia , Enterócitos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ileíte/genética , Indóis/farmacologia , Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/farmacologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Piroxicam/farmacologiaRESUMO
The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl-/HCO3- exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl-/HCO3- exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.
Assuntos
Antiporters/genética , Antiportadores de Cloreto-Bicarbonato/genética , Criptosporidiose/genética , Cryptosporidium parvum/patogenicidade , Regulação da Expressão Gênica/genética , Mucosa Intestinal/metabolismo , Transportadores de Sulfato/genética , Animais , Anticorpos Neutralizantes/farmacologia , Antiporters/antagonistas & inibidores , Antiporters/metabolismo , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/antagonistas & inibidores , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloretos/metabolismo , Criptosporidiose/metabolismo , Criptosporidiose/parasitologia , Cryptosporidium parvum/fisiologia , Interações Hospedeiro-Parasita/genética , Humanos , Íleo/metabolismo , Íleo/parasitologia , Mucosa Intestinal/parasitologia , Transporte de Íons , Janus Quinases/genética , Janus Quinases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Organoides/metabolismo , Organoides/parasitologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transportadores de Sulfato/antagonistas & inibidores , Transportadores de Sulfato/metabolismoRESUMO
Putative anion transporter 1 (PAT1, SLC26A6), an intestinal epithelial Cl-/ HCO3- exchanger, also plays a key role in oxalate homeostasis via mediating intestinal oxalate secretion. Indeed, Slc26a6-null mice showed defect in intestinal oxalate secretion and high incidence of kidney stones. Recent emergence of PAT-1 as a novel therapeutic target for nephrolithiasis warrants detailed understanding of the mechanisms of PAT-1 regulation in health and disease. Therefore, we investigated the regulation of PAT-1 expression by microRNAs (miRNA), as they have been shown to play key role in modulating expression of other ion transporters. In silico analysis of PAT-1 3'-untranslated region (UTR) revealed potential binding sites for several miRNAs, suggesting the role of miRNAs in modulating PAT1 expression. miRNAs showing highest context scores (125a-5p, 339-5p, 423-5p, 485-5p, and 501-3p) were selected as candidates for their effects on the activity of a 263-bp PAT-1 3'-untranslated region (UTR) fragment cloned into pmirGLO vector upstream of luciferase. The 3'-UTR activity was measured by dual luciferase reporter assay in Caco-2, T-84, HT-29, and SK-CO15 cells. Transient transfection of PAT-1 3'-UTR significantly decreased the relative luciferase activity compared with the empty vector suggesting binding of potential miRNA(s) to the PAT-1 3'-UTR. Among all the selected candidates, cotransfection with miRNA mimics 125a-5p and 423-5p further decreased PAT-1 3'-UTR activity. Furthermore, increasing miR-125a-5p abundance via mimic transfection in Caco-2 cells decreased both mRNA and protein levels of PAT-1. Our results demonstrate a novel regulatory mechanism of intestinal PAT-1 expression via miR-125a-5p that could be of therapeutic importance in disorders associated with decreased PAT-1 expression and function.
Assuntos
Colo/metabolismo , Mucosa Intestinal/metabolismo , MicroRNAs/metabolismo , Ácido Oxálico/metabolismo , Transportadores de Sulfato/metabolismo , Regiões 3' não Traduzidas , Sítios de Ligação , Células CACO-2 , Regulação para Baixo , Células HT29 , Humanos , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transportadores de Sulfato/genéticaRESUMO
Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5 × 106 oocysts/well in Transwell inserts, 24 hr) increased paracellular permeability (FITC-dextran flux) in Caco-2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E-cadherin. Claudin 3, zonula occludens-1 (ZO1) and α-catenin were also significantly decreased, whereas claudins 1 and 2 and ß-catenin were not altered. Substantial downregulation of occludin, claudin 4, and E-cadherin was also observed in response to CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP-infected mouse ileum and jejunum but were unaltered in Caco-2 cells. Further, bafilomycin-A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco-2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection-induced diarrhoea.
Assuntos
Junções Aderentes/parasitologia , Moléculas de Adesão Celular/antagonistas & inibidores , Criptosporidiose/patologia , Cryptosporidium parvum/crescimento & desenvolvimento , Regulação para Baixo , Interações Hospedeiro-Patógeno , Junções Íntimas/parasitologia , Animais , Células CACO-2 , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Mucosa Intestinal/parasitologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , PermeabilidadeRESUMO
Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.
Assuntos
Fator 4 Nuclear de Hepatócito/metabolismo , Mucosa Intestinal/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Sítios de Ligação , Células CACO-2 , Regulação da Expressão Gênica , Células HCT116 , Fator 4 Nuclear de Hepatócito/genética , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Trocador 3 de Sódio-Hidrogênio/genéticaRESUMO
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
SLC26A3 [downregulated in adenoma (DRA)] plays a key role in mammalian intestinal NaCl absorption, in that it mediates apical membrane Cl-/[Formula: see text] exchange. DRA function and expression are significantly decreased in diarrhea associated with inflammatory bowel disease. DRA is also considered to be a marker of cellular differentiation and is predominantly expressed in differentiated epithelial cells. Caudal-type homeobox protein-2 (CDX2) is known to regulate genes involved in intestinal epithelial differentiation and proliferation. Reduced expression of both DRA and CDX2 in intestinal inflammation prompted us to study whether the DRA gene is directly regulated by CDX2. Our initial studies utilizing CDX2 knockout (CDX2fV/fV;Cre+) mice showed a marked reduction in DRA mRNA and protein levels in proximal and distal colon. In silico analysis of the DRA promoter showed two consensus sites for CDX2 binding. Therefore, we utilized Caco-2 cells as an in vitro model to examine if DRA is a direct target of CDX2 regulation. siRNA-mediated silencing of CDX2 in Caco-2 cells resulted in a marked (~50%) decrease in DRA mRNA and protein levels, whereas ectopic overexpression of CDX2 upregulated DRA expression and also stimulated DRA promoter activity, suggesting transcriptional regulation. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated direct binding of CDX2 to one of the two putative CDX2 binding sites in the DRA promoter (+645/+663). In summary, our studies, for the first time, demonstrate transcriptional regulation of DRA expression by CDX2, implying that reduced expression of DRA in inflammatory bowel disease-associated diarrhea may, in part, be due to downregulation of CDX2 in the inflamed mucosa.NEW & NOTEWORTHY SLC26A3 [downregulated in adenoma (DRA)] mediates intestinal luminal NaCl absorption and is downregulated in inflammatory bowel disease-associated diarrhea. Since both DRA and caudal-type homeobox protein-2 (CDX2) are reduced in intestinal inflammation and the DRA promoter harbors CDX2 binding sites, we examined whether the DRA gene is regulated by CDX2. Our studies, for the first time, demonstrate transcriptional regulation of DRA expression by CDX2 via direct binding to the DRA promoter, suggesting that reduced expression of DRA in inflammatory bowel disease-associated diarrhea could, in part, be attributed to downregulation of CDX2.
Assuntos
Antiporters/metabolismo , Fator de Transcrição CDX2/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Animais , Antiporters/genética , Fator de Transcrição CDX2/genética , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Interferência de RNA , RNA Interferente Pequeno , Transportadores de SulfatoRESUMO
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
SLC26A3 or Downregulated in adenoma (DRA) is the major Cl(-)/HCO3 (-) exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Alterations in DRA function and expression have been implicated in diarrheal diseases associated with inflammation or infection. Therefore, agents that upregulate DRA activity may serve as potential antidiarrheals. In this regard, sphingosine-1-phosphate (S1P), a member of the bioactive sphingolipid family, has been shown to modulate various cellular processes including improvement of intestinal barrier function. However, the role of S1P in modulating intestinal chloride absorption by regulating DRA is not known. Therefore, the present studies were designed to examine the direct effects of S1P on apical Cl(-)/HCO3 (-) exchange activity and DRA expression. S1P significantly increased Cl(-)/HCO3 (-) exchange activity and also significantly increased DRA mRNA and protein expression. Increased DRA mRNA by S1P was accompanied by enhanced DRA promoter activity, indicating involvement of transcriptional mechanisms. The specific S1P receptor subtype-2 (S1PR2) antagonist JTE-013 blocked the stimulatory effects of S1P on DRA promoter activity, indicating the involvement of S1PR2 S1P-mediated increase in DRA promoter activity involved PI3K/Akt pathway. Progressive deletions of the DRA promoter indicated that the putative S1P-responsive elements are present in the -790/-398 region of the DRA promoter. Furthermore, results obtained from electrophoretic mobility shift assay showed that S1P stimulated DRA promoter activity via increased binding of Ying-Yang1 (YY1) in the S1P-responsive region. In conclusion, transcriptional modulation of DRA expression and function in response to S1P through a PI3/Akt pathway represents a novel role of S1P as a potential proabsorptive agent.
Assuntos
Antiportadores de Cloreto-Bicarbonato/metabolismo , Lisofosfolipídeos/farmacologia , Regiões Promotoras Genéticas , Esfingosina/análogos & derivados , Bicarbonatos/metabolismo , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/genética , Cloretos/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/farmacologia , Transportadores de Sulfato , Fator de Transcrição YY1/metabolismoRESUMO
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
Butyrate, a key short-chain fatty acid metabolite of colonic luminal bacterial action on dietary fiber, serves as a primary fuel for the colonocytes, ameliorates mucosal inflammation, and stimulates NaCl absorption. Absorption of butyrate into the colonocytes is essential for these intracellular effects. Monocarboxylate transporter 1 (MCT1) plays a major role in colonic luminal butyrate absorption. Previous studies (Tan J, McKenzie C, Potamitis M, Thorburn AN, Mackay CR, Macia L. Adv Immunol 121: 91-119, 2014.) showed decreased MCT1 expression and function in intestinal inflammation. We have previously shown (Borthakur A, Gill RK, Hodges K, Ramaswamy K, Hecht G, Dudeja PK. Am J Physiol Gastrointest Liver Physiol 290: G30-G35, 2006.) impaired butyrate absorption in human intestinal epithelial Caco-2 cells due to decreased MCT1 level at the apical cell surface following enteropathogenic E. coli (EPEC) infection. Current studies, therefore, examined the potential role of probiotic Lactobacilli in stimulating MCT1-mediated butyrate uptake and counteracting EPEC inhibition of MCT1 function. Of the five species of Lactobacilli, short-term (3 h) treatment with L. acidophilus (LA) significantly increased MCT1-mediated butyrate uptake in Caco-2 cells. Heat-killed LA was ineffective, whereas the conditioned culture supernatant of LA (LA-CS) was equally effective in stimulating MCT1 function, indicating that the effects are mediated by LA-secreted soluble factor(s). Furthermore, LA-CS increased apical membrane levels of MCT1 protein via decreasing its basal endocytosis, suggesting that LA-CS stimulation of butyrate uptake could be secondary to increased levels of MCT1 on the apical cell surface. LA-CS also attenuated EPEC inhibition of butyrate uptake and EPEC-mediated endocytosis of MCT1. Our studies highlight distinct role of specific LA-secreted molecules in modulating colonic butyrate absorption.
Assuntos
Ácido Butírico/metabolismo , Escherichia coli Enteropatogênica/fisiologia , Mucosa Intestinal/metabolismo , Lactobacillus acidophilus/fisiologia , Transporte Biológico , Biotinilação , Células CACO-2 , Técnicas de Cultura de Células , Humanos , Immunoblotting , Mucosa Intestinal/microbiologia , Intestinos/microbiologiaRESUMO
SLC26A3, or downregulated in adenoma (DRA), plays a major role in mediating Cl(-) absorption in the mammalian intestine. Disturbances in DRA function and expression have been implicated in intestinal disorders such as congenital Cl(-) diarrhea and gut inflammation. We previously showed that an increase in DRA function and expression by Lactobacillus acidophilus and its culture supernatant (CS) might underlie antidiarrheal effects of this probiotic strain. However, the effects of Bifidobacterium species, important inhabitants of the human colon, on intestinal Cl(-)/HCO3 (-) exchange activity are not known. Our current results demonstrate that CS derived from Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum increased anion exchange activity in Caco-2 cells (â¼1.8- to 2.4-fold). Consistent with the increase in DRA function, CS also increased the protein, as well as the mRNA, level of DRA (but not putative anion transporter 1). CS of all three Bifidobacterium sp. increased DRA promoter activity (-1,183/+114 bp) in Caco-2 cells (1.5- to 1.8-fold). Furthermore, the increase in DRA mRNA expression by CS of B. breve and B. infantis was blocked in the presence of the transcription inhibitor actinomycin D (5 µM) and the ERK1/2 MAPK pathway inhibitor U0126 (10 µM). Administration of live B. breve, B. infantis, and B. bifidum by oral gavage to mice for 24 h increased DRA mRNA and protein levels in the colon. These data demonstrate an upregulation of DRA via activation of the ERK1/2 pathway that may underlie potential antidiarrheal effects of Bifidobacterium sp.
Assuntos
Bifidobacterium/fisiologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Células Epiteliais/microbiologia , Mucosa Intestinal/microbiologia , Probióticos , Animais , Antiporters/genética , Antiporters/metabolismo , Bicarbonatos/metabolismo , Bifidobacterium/classificação , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/genética , Cloretos/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HT29 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos Endogâmicos C57BL , Inibidores da Síntese de Ácido Nucleico/farmacologia , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais , Transportadores de Sulfato , Fatores de Tempo , Transcrição Gênica , Transfecção , Regulação para CimaRESUMO
SLC26A3 [downregulated in adenoma (DRA)] is a Cl(-)/HCO3(-) exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs (miRNAs) are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. To investigate potential modulation of DRA expression by miRNA, five different in silico algorithms were used to predict the miRNAs that target DRA. Of these miRNAs, miR-494 was shown to have a highly conserved putative binding site in the DRA 3'-untranslated region (3'-UTR) compared with other DRA-targeting miRNAs in vertebrates. Transfection with pmirGLO dual luciferase vector containing DRA 3'-UTR (pmirGLO-3'-UTR DRA) resulted in a significant decrease in relative luciferase activity compared with empty vector. Cotransfection of the DRA 3'-UTR luciferase vector with a miR-494 mimic further decreased luciferase activity compared with cells transfected with negative control. The transfection of a miR-494 mimic into Caco-2 and T-84 cells significantly increased the expression of miR-494 and concomitantly decreased the DRA protein expression. Mutation of the seed sequences for miR-494 in 3'-UTR of DRA abrogated the effect of miR-494 on 3'-UTR. These data demonstrate a novel regulatory mechanism of DRA expression via miR-494 and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for diarrheal diseases.
Assuntos
Antiportadores de Cloreto-Bicarbonato/antagonistas & inibidores , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , MicroRNAs/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Regiões 3' não Traduzidas/genética , Western Blotting , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/biossíntese , Antiportadores de Cloreto-Bicarbonato/genética , Clonagem Molecular , Biologia Computacional , Diarreia/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transportadores de SulfatoRESUMO
Probiotics, including Lactobacilli, are commensal bacteria that have been used in clinical trials and experimental models for the prevention and treatment of diarrheal disorders. Our previous studies have shown that Lactobacillus acidophilus (LA) and its culture supernatant (CS) stimulated Cl(-)/HCO3 (-) exchange activity, acutely via an increase in the surface levels of downregulated in adenoma (DRA, SLC26A3) and in long-term treatments via increasing its expression involving transcriptional mechanisms. However, the role of LA in modulating DRA activity under inflammatory conditions is not known. Current in vitro studies using human intestinal epithelial Caco-2 cells examined the efficacy of LA or its CS in counteracting the inhibitory effects of interferon-γ (IFN-γ) on Cl(-)/HCO3 (-) exchange activity. Pretreatment of cells with LA or LA-CS for 1 h followed by coincubation with IFN-γ significantly alleviated the inhibitory effects of IFN-γ on Cl(-)/HCO3 (-) exchange activity. In the in vivo model of dextran sulfate sodium-induced experimental colitis (3% in drinking water for 7 days) in C57BL/6J mice, administration of live LA (3 × 10(9) colony-forming units) via oral gavage attenuated colonic inflammation. LA administration also counteracted the colitis-induced decrease in DRA mRNA and protein levels. Efficacy of LA or its secreted soluble factors in alleviating inflammation and inflammation-associated dysregulation of DRA activity could justify their therapeutic potential in inflammatory diarrheal diseases.