[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.
PLoS ONE 11/2014; 9(11):e109972. · 3.53 Impact Factor
[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.
[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.
Journal of Biological Chemistry 06/2014; · 4.60 Impact Factor
[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.
AJP Gastrointestinal and Liver Physiology 10/2013; · 3.65 Impact Factor
[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.
PLoS ONE 10/2013; 8(10):e75664. · 3.53 Impact Factor
[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.
AJP Gastrointestinal and Liver Physiology 01/2013; · 3.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carrageenan, a sulfated polysaccharide that is widely used as a food additive, induces inflammatory responses in animal models and human cells. The carrageenan-induced inflammatory cascades involve toll-like receptor (TLR)4- and B-cell leukemia/lymphoma (BCL)10-dependent activation of NF-κB, leading to increased IL-8 production. Translocations involving BCL10 in the mucosa-associated lymphoid tissue (MALT) lymphomas are associated with constitutive activation of NF-κB. This report presents a mechanism by which carrageenan exposure leads to prolonged activation of both BCL10 and NF-κB in human colonic epithelial cells. Study findings demonstrate that nuclear RelA and RelB bind to an NF-κB binding motif in the BCL10 promoter in human colonic epithelial NCM460 and HT-29 cells. In vitro oligonucleotide binding assay, non-radioactive gel shift assay, and chromatin immunoprecipitation (ChIP) indicate binding of RelA and RelB to the BCL10 promoter. Prolonged inflammation follows activation of the BCL10-NFκB inflammatory loop in response to carrageenan, shown by increased BCL10, RelA, and IL-8 for 36 to 48h and increased RelB for 24h following withdrawal of carrageenan after 12h. In contrast, exposure to dextran sulfate sodium, which does not cause inflammation through TLR4 and BCL10 in the colonic epithelial cells, did not provoke prolonged activation of inflammation. The carrageenan-enhanced BCL10 promoter activity was blocked by caffeic acid phenethyl ester (CAPE) and MB-132 which inhibit NF-κB activation. These results indicate that NF-κB binding to the BCL10 promoter can lead to prolonged activation of the carrageenan-induced inflammatory cascade by a transcriptional mechanism involving an NF-κB-BCL10 loop.
Biochimica et Biophysica Acta 05/2012; 1822(8):1300-7. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the impact of B cell leukemia/lymphoma (BCL) 10 on the phosphorylation of crucial mediators in NF-κB-mediated inflammatory pathways, human colonic epithelial cells were exposed to carrageenan (CGN), a sulfated polysaccharide commonly used as a food additive and known to induce NF-κB nuclear translocation by both canonical and noncanonical pathways. Phosphorylations of intermediates in inflammatory cascades, including NF-κB-inducing kinase (NIK) at Thr(559), transforming growth factor-β-activating kinase (TAK) 1 at Thr(184), Thr(187), and Ser(192), and inhibitory factor κBα (IκBα) at Ser(32), were examined following mutation of BCL10 at Ser(138) and at Ser(218). Specific phosphoantibodies were used for detection by enzyme-linked immunosorbent assay, immunoblot, and confocal microscopy of differences in phosphorylation following transfection by mutated BCL10. Both mutations demonstrated dominant-negative effects, with inhibition of phospho(Ser(32))-IκBα to less than control levels. Both of the BCL10 mutations reduced the CGN-induced increases in nuclear RelA and p50, but only the Ser(138) mutation inhibited the CGN-induced increases in nuclear RelB and p52 and in NIK Thr(559) phosphorylation. Hence, the phosphorylation of BCL10 Ser(138), but not Ser(218), emerged as a critical event in activation of the noncanonical pathway of NF-κB activation. Either BCL10 Ser(138) or Ser(218) mutation inhibited the phosphorylation of TAK1 at Thr(184) and at Thr(187), but not at Ser(192). These findings indicate that BCL10 phosphorylations act upstream of phosphorylations of NIK, TAK1, and IκBα and differentially affect the canonical and noncanonical pathways of NF-κB activation.
[Show abstract][Hide abstract] ABSTRACT: Serotonin transporter (SERT) regulates extracellular availability of serotonin and is a potential pharmacological target for gastrointestinal disorders. A decrease in SERT has been implicated in intestinal inflammatory and diarrheal disorders. However, little is known regarding regulation of SERT in the intestine. Epidermal growth factor (EGF) is known to influence intestinal electrolyte and nutrient transport processes and has protective effects on intestinal mucosa. Whether EGF regulates SERT in the human intestine is not known. The present studies examined the regulation of SERT by EGF, utilizing Caco-2 cells grown on Transwell inserts as an in vitro model. Treatment with EGF from the basolateral side (10 ng/ml, 24 h) significantly stimulated SERT activity (∼2-fold, P < 0.01) and mRNA levels compared with control. EGF increased the activities of the two alternate promoter constructs for human SERT gene: SERT promoter 1 (hSERTp1, upstream of exon 1a) and SERT promoter 2 (hSERTp2, upstream of exon 2). Inhibition of EGF receptor (EGFR) tyrosine kinase activity by PD168393 (1 nM) blocked the stimulatory effects of EGF on SERT promoters. Progressive deletions of the SERT promoter indicated that the putative EGF-responsive elements are present in the -672/-472 region of the hSERTp1 and regions spanning -1195/-738 and -152/+123 of hSERTp2. EGF markedly increased the binding of Caco-2 nuclear proteins to the potential AP-1 cis-elements present in EGF-responsive regions of hSERTp1 and p2. Overexpression of c-jun but not c-fos specifically transactivated hSERTp2, with no effects on hSERTp1. Our findings define novel mechanisms of transcriptional regulation of SERT by EGF via EGFR at the promoter level that may contribute to the beneficial effects of EGF in gut disorders.
[Show abstract][Hide abstract] ABSTRACT: The major short-chain fatty acid (SCFA) butyrate is produced in the colonic lumen by bacterial fermentation of dietary fiber. Butyrate serves as primary fuel for the colonocytes and also ameliorates mucosal inflammation. Disturbed energy homeostasis seen in inflamed mucosa of inflammatory bowel disease patients has been attributed to impaired absorption of butyrate. Since sodium-coupled monocarboxylate transporter 1 (SMCT1, SLC5A8) has recently been shown to play a role in Na(+)-coupled transport of monocarboxylates, including SCFA, such as luminal butyrate, we examined the effects of proinflammatory TNF-α on SMCT1 expression and function and potential anti-inflammatory role of probiotic Lactobacillus species in counteracting the TNF-α effects. Rat intestinal epithelial cell (IEC)-6 or human intestinal Caco-2 cells were treated with TNF-α in the presence or absence of Lactobacilli culture supernatants (CS). TNF-α treatments for 24 h dose-dependently inhibited SMCT1-mediated, Na(+)-dependent butyrate uptake and SMCT1 mRNA expression in IEC-6 cells and SMCT1 promoter activity in Caco-2 cells. CS of L. plantarum (LP) stimulated Na(+)-dependent butyrate uptake (2.5-fold, P < 0.05), SMCT1 mRNA expression, and promoter activity. Furthermore, preincubating the cells with LP-CS followed by coincubation with TNF-α significantly attenuated the inhibitory effects of TNF-α on SMCT1 function, expression, and promoter activity. In vivo, oral administration of live LP enhanced SMCT1 mRNA expression in the colonic and ileal tissues of C57BL/6 mice after 24 h. Efficacy of LP or their secreted soluble factors to stimulate SMCT1 expression and function and to counteract the inhibitory effects of TNF-α on butyrate absorption could have potential therapeutic value.