[Show abstract][Hide abstract] ABSTRACT: GPR120 (free fatty acid receptor 4, FFAR4) is a G protein-coupled receptor for medium and long-chain unsaturated fatty acids (FA) including ω-3 FA. 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 β-arrestin-2, and attenuated NF-κB activation in response to 30 min exposure to the agonists GW9508, TUG-891 or docosahexaenoic acid (DHA). These effects were abrogated in response to siRNA silencing of β-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 hr augmented the synthesis and secretion of the gut hormone GLP-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.
Full-text · Article · Jan 2016 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: TGF-β1 is an important multifunctional cytokine with numerous protective effects on intestinal mucosa. The influence of TGF-β1 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-β1 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-β1 (10 ng/ml, 60 min) stimulated SERT activity (~2 fold, P<0.005). This stimulation of SERT function was dependent upon activation of TGF-β1 receptor (TGFRI) as SB-431542, a specific TGF-βRI inhibitor blocked the SERT stimulation. SERT activation in response to TGF-β1 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-β1-mediated increase in SERT function. TGF-β1 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-β1 showing increased luminal staining of SERT. Ussing chamber studies revealed increase in 3H-5-HT uptake in mouse ileum treated ex vivo with TGF-β1 (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-β1 offers a novel therapeutic strategy to treat GI disorders.
[Show abstract][Hide abstract] ABSTRACT: Celiac disease (CD) is an autoimmune disorder in individuals that carry DQ2 or DQ8 MHC class II haplotypes, triggered by the ingestion of gluten. There is no current treatment other than a gluten-free diet (GFD). We have previously shown that the BL-7010 copolymer poly(hydroxyethyl methacrylate-co-styrene sulfonate) (P(HEMA-co-SS)) binds with higher efficiency to gliadin than to other proteins present in the small intestine, ameliorating gliadin-induced pathology in the HLA-HCD4/DQ8 model of gluten sensitivity. The aim of this study was to investigate the efficiency of two batches of BL-7010 to interact with gliadin, essential vitamins and digestive enzymes not previously tested, and to assess the ability of the copolymer to reduce gluten-associated pathology using the NOD-DQ8 mouse model, which exhibits more significant small intestinal damage when challenged with gluten than HCD4/DQ8 mice. In addition, the safety and systemic exposure of BL-7010 was evaluated in vivo (in rats) and in vitro (genetic toxicity studies). In vitro binding data showed that BL-7010 interacted with high affinity with gliadin and that BL-7010 had no interaction with the tested vitamins and digestive enzymes. BL-7010 was effective at preventing gluten-induced decreases in villus-to-crypt ratios, intraepithelial lymphocytosis and alterations in paracellular permeability and putative anion transporter-1 mRNA expression in the small intestine. In rats, BL-7010 was well-tolerated and safe following 14 days of daily repeated administration of 3000 mg/kg. BL-7010 did not exhibit any mutagenic effect in the genetic toxicity studies. Using complementary animal models and chronic gluten exposure the results demonstrate that administration of BL-7010 is effective and safe and that it is able to decrease pathology associated with gliadin sensitization warranting the progression to Phase I trials in humans.
[Show abstract][Hide abstract] ABSTRACT: SLC26A3 or DRA (Down Regulated in Adenoma) 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 chloride diarrhea and gut inflammation. Our previous studies showed that increase in DRA function and expression by Lactobacillus acidophilus and its culture supernatant (CS) might underlie anti-diarrheal effects of this probiotic strain. However the effects of Bifidobacterium species, important inhabitants of human colon, on intestinal Cl-/HCO3- exchange activity are not known. Our current results demonstrated that CS derived from B. breve, B. infantis or B. bifidum increased the anion exchange activity (~ 1.8-2.4 fold) in Caco-2 cells. Consistent with the function, CS also increased the protein as well as mRNA levels of DRA (but not PAT-1). CS of all three species of Bifidobacteria increased DRA promoter activity (-1183/+114bp) in Caco-2 cells (1.5-1.8 fold). Further, the increase in DRA mRNA expression by CS of B. breve and B.infantis was blocked in the presence of transcription inhibitor, Actinomycin D (5μM) and ERK1/2 MAPK pathway inhibitor (U0126, 10μM). Administration of live B. breve, B. infantis and B. bifidum by oral gavage to mice for 24 h increased the DRA mRNA and protein levels in the colon. These data demonstrate an upregulation of DRA via activation of ERK1/2 pathway that may underlie potential anti-diarrheal effects of Bifidobacterium species.
No preview · Article · Aug 2014 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: Intestinal NPC1L1 transporter is essential for cholesterol absorption and the maintenance of cholesterol homeostasis in the body. NPC1L1 is differentially expressed along the gastrointestinal tract with very low levels in the colon as compared to the small intestine. Current studies were undertaken to examine whether DNA methylation was responsible for segment specific expression of NPC1L1. Treatment of mice with 5-azacytidine (i.p) resulted in a significant dose-dependent increase in NPC1L1 mRNA expression in the colon. The lack of expression of NPC1L1 in the normal colon was associated with high levels of methylation in the area flanking 3 Kb fragment upstream of the initiation site of mouse NPC1L1 gene in mouse colon as analyzed by EpiTyper Massarray. The high level of methylation in the colon was observed in specific CpG dinucleotides and was significantly decreased in response to 5-azacytidine. Similar to mouse NPC1L1, 5-azacytidine treatment also increased the level of human NPC1L1 mRNA expression in intestinal HuTu-80 cell line in a dose and time-dependent manner. Silencing the expression of DNA methyltransferase DNMT1, 2, 3A and 3B alone by siRNA did not affect NPC1L1 expression in HuTu-80 cells. However, the simultaneous attenuation of DNMT1 and 3B expression caused a significant increase in NPC1L1 mRNA expression as compared to control. Also, in vitro methylation of human NPC1L1 promoter significantly decreased NPC1L1 promoter activity in human intestinal Caco2 cells. In conclusion, our data demonstrated for the first time, that DNA methylation in the promoter region of NPC1L1 gene appears to be a major mechanism underlying differential expression of NPC1L1 along the length of the GI tract.
Full-text · Article · Jun 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: SLC26A3 (Down-regulated 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. In order to investigate potential modulation of DRA expression by microRNA, 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 to 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 as compared to empty vector. Co-transfection of the DRA 3'UTR luciferase vector with a miR-494 mimic further decreased luciferase activity as compared to cells transfected with negative control. The transfection of a miR-494 mimic into Caco2 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.
No preview · Article · Oct 2013 · AJP Gastrointestinal and Liver Physiology
[Show abstract][Hide abstract] ABSTRACT: Probiotics have been used as alternative prevention and therapy modalities in intestinal inflammatory disorders including inflammatory bowel diseases (IBD) and necrotizing enterocolitis (NEC). Pathophysiology of IBD and NEC includes the production of diverse lipid mediators, including platelet-activating factor (PAF) that mediate inflammatory responses in the disease. PAF is known to activate NF-κB, however, the mechanisms of PAF-induced inflammation are not fully defined. We have recently described a novel PAF-triggered pathway of NF-κB activation and IL-8 production in intestinal epithelial cells (IECs), requiring the pivotal role of the adaptor protein Bcl10 and its interactions with CARMA3 and MALT1. The current studies examined the potential role of the probiotic Lactobacillus acidophilus in reversing the PAF-induced, Bcl10-dependent NF-κB activation and IL-8 production in IECs. PAF treatment (5 µM×24 h) of NCM460 and Caco-2 cells significantly increased nuclear p65 NF-κB levels and IL-8 secretion (2-3-fold, P<0.05), compared to control, which were blocked by pretreatment of the cells for 6 h with L. acidophilus (LA) or its culture supernatant (CS), followed by continued treatments with PAF for 24 h. LA-CS also attenuated PAF-induced increase in Bcl10 mRNA and protein levels and Bcl10 promoter activity. LA-CS did not alter PAF-induced interaction of Bcl10 with CARMA3, but attenuated Bcl10 interaction with MALT1 and also PAF-induced ubiquitination of IKKγ. Efficacy of bacteria-free CS of LA in counteracting PAF-induced inflammatory cascade suggests that soluble factor(s) in the CS of LA mediate these effects. These results define a novel mechanism by which probiotics counteract PAF-induced inflammation in IECs.
[Show abstract][Hide abstract] ABSTRACT: Intestinal P-glycoprotein (P-gp/MDR1), encoded by the ATP-binding cassette B1 (ABCB1) gene, is primarily involved in the transepithelial efflux of toxic metabolites and xenobiotics from the mucosa into the gut lumen. Reduced Pgp function and expression has been shown to be associated with intestinal inflammatory disorders. Keratinocyte growth factor-2 (KGF2) has emerged as a potential target for modulation of intestinal inflammation and maintenance of gut mucosal integrity. Whether KGF2 directly regulates Pgp in the human intestine is not known. Therefore, the present studies were undertaken to determine the modulation of Pgp by KGF2 utilizing Caco-2 cells. Short-term treatment of Caco2 cells with KGF2 (10 ng/ml, 1h) increased Pgp activity (~2 fold, P<0.05) as measured by verapamil-sensitive (3)H-Digoxin flux. This increase in Pgp function was associated with an increase in surface Pgp levels. Specific fibroblast growth factor receptor (FGFR) antagonist, PD161570 blocked KGF2-mediated increase in Pgp activity. Inhibition of MAP kinase pathway by PD98059 attenuated the stimulatory effects of KGF2 on Pgp activity. siRNA knockdown of Erk1/2 MAPK blocked the increase in surface Pgp levels by KGF2. Long-term treatment with KGF2 (10 ng/ml, 24h) also significantly increased PgP activity, mRNA, protein expression and promoter activity. The long-term effects of KGF2 on Pgp promoter activity were also blocked by the FGFR antagonist and mediated by Erk1/2 MAPK pathway. In conclusion, our findings define the post-translational and transcriptional mechanisms underlying stimulation of Pgp function and expression by KGF2 that may contribute to the beneficial effects of KGF2 in intestinal inflammatory disorders.
Full-text · Article · Jan 2013 · AJP Gastrointestinal and Liver Physiology