Krishnamurthy Ramaswamy

Mechanism underlying inhibition of intestinal apical Cl–/OH– exchange following infection with enteropathogenic E. coli

Enteropathogenic E. coli (EPEC) is a major cause of infantile diarrhea, but the pathophysiology underlying associated diarrhea is poorly understood. We examined the role of the luminal membrane Cl(-)/OH(-) exchange process in EPEC pathogenesis using in vitro and in vivo models. Cl(-)/OH(-) exchange activity was measured as OH(-) gradient-driven (36)Cl(-) uptake. EPEC infection (60 minutes-3 hours) inhibited apical Cl(-)/OH(-) exchange activity in human intestinal Caco-2 and T84 cells. This effect was dependent upon the bacterial type III secretory system (TTSS) and involved secreted effector molecules EspG and EspG2, known to disrupt the host microtubular network. The microtubule-disrupting agent colchicine (100 muM, 3 hours) also inhibited (36)Cl(-) uptake. The plasma membrane expression of major apical anion exchanger DRA (SLC26A3) was considerably reduced in EPEC-infected cells, corresponding with decreased Cl(-)/OH(-) exchange activity. Confocal microscopic studies showed that EPEC infection caused a marked redistribution of DRA from the apical membrane to intracellular compartments. Interestingly, infection of cells with an EPEC mutant deficient in espG significantly attenuated the decrease in surface expression of DRA protein as compared with treatment with wild-type EPEC. EPEC infection in vivo (1 day) also caused marked redistribution of surface DRA protein in the mouse colon. Our data demonstrate that EspG and EspG2 play an important role in contributing to EPEC infection-associated inhibition of luminal membrane chloride transport via modulation of surface DRA expression.

Regulation of monocarboxylate transporter 1 (MCT1) promoter by butyrate in human intestinal epithelial cells: Involvement of NF-κB pathway

Butyrate, a short chain fatty acid (SCFA) produced by bacterial fermentation of undigested carbohydrates in the colon, constitutes the major fuel for colonocytes. We have earlier shown the role of apically localized monocarboxylate transporter isoform 1 (MCT1) in transport of butyrate into human colonic Caco‐2 cells. In an effort to study the regulation of MCT1 gene, we and others have cloned the promoter region of the MCT1 gene and identified cis elements for key transcription factors. A previous study has shown up‐regulation of MCT1 expression, and activity by butyrate in AA/C1 human colonic epithelial cells, however, the detailed mechanisms of this up‐regulation are not known. In this study, we demonstrate that butyrate, a substrate for MCT1, stimulates MCT1 promoter activity in Caco‐2 cells. This effect was dose dependent and specific to butyrate as other predominant SCFAs, acetate, and propionate, were ineffective. Utilizing progressive deletion constructs of the MCT1 promoter, we showed that the putative butyrate responsive elements are in the −229/+91 region of the promoter. Butyrate stimulation of the MCT1 promoter was found to be independent of PKC, PKA, and tyrosine kinases. However, specific inhibitors of the NF‐κB pathway, lactacystein (LC), and caffeic acid phenyl ester (CAPE) significantly reduced the MCT1 promoter stimulation by butyrate. Also, butyrate directly stimulated NF‐κB‐dependent luciferase reporter activity. Histone deacetylase (HDAC) inhibitor trichostatin A (TSA) also stimulated MCT1 promoter activity, however, unlike butyrate, this stimulation was unaltered by the NF‐κB inhibitors. Further, the combined effect of butyrate, and TSA on MCT1 promoter activity was additive, indicating that their mechanisms of action were independent. Our results demonstrate the involvement of NF‐κB pathway in the regulation of MCT1 promoter activity by butyrate. J. Cell. Biochem. 103: 1452–1463, 2008.

The enteropathogenic Escherichia coli effector protein EspF decreases sodium hydrogen exchanger 3 activity.

Enteropathogenic Escherichia coli (EPEC) have been previously shown to alter sodium hydrogen exchanger 3 (NHE3) activity in human intestinal epithelial cells. To further characterize these observations, PS120 fibroblasts transfected with NHE3 were studied. EPEC E2348/69 infection decreased NHE3 activity in PS120 fibroblasts. The effect on NHE3 was enhanced when PS120 cells were co‐transfected with the scaffolding/regulatory proteins NHERF1 or NHERF2 or EBP50 and E3KARP respectively. The decrease in NHE3 activity was dependent on an intact type III secretion system, although intimate attachment mediated by translocated intimin receptor was not required. Despite its ability to bind to NHERF proteins, the EPEC effector Map had no impact on the regulation of NHE activity. Instead, EspF was found to be responsible for decreased NHE3 activity. However, neither EspF‐induced apoptosis nor the interaction of EspF with sorting nexin‐9, an endocytic protein, were involved.

Expression of the Na+/H+ and Cl-/HCO-3 exchanger isoforms in proximal and distal human airways.

Recent studies have indicated the presence of Na+/H+and Cl-/[Formula: see text]exchange activities in lung alveolar and tracheal tissues of various species. To date, the identity of the Na+/H+(NHE) and Cl-/[Formula: see text](AE) exchanger isoforms and their regional distribution in human airways are not known. Molecular species of the NHE and AE gene families and their relative abundance in the human airway regions were assessed utilizing RT-PCR and the RNase protection assay, respectively. Organ donor lung epithelia from various bronchial regions (small, medium, and large bronchi and trachea) were harvested for RNA extraction. Gene-specific primers for the human NHE and AE isoforms were utilized for RT-PCR. Our results demonstrated that NHE1, AE2, and brain AE3 isoforms were expressed in all regions of the human airways, whereas NHE2, NHE3, AE1, and cardiac AE3 were not detected. RNase protection studies for NHE1 and AE2, utilizing glyceraldehyde-3-phosphate dehydrogenase as an internal standard, demonstrated that there were regional differences in the NHE1 mRNA levels in human airways. In contrast, the levels of AE2 mRNA remained unchanged. Differential expression of these isoforms in the human airways may have functional significance related to the airway absorption and secretion of electrolytes.

Molecular cloning, tissue distribution, and functional expression of the human Na+/H+exchanger NHE2

In the present report, we describe the cloning of a human colonic cDNA that describes the full-length Na+/H+exchanger (NHE) 2 coding region. The human NHE2 (hNHE2) cDNA encodes for a polypeptide of 812 amino acids with a 90% overall identity to both rabbit and rat NHE2 isoforms. In comparison with SLC9A2, recently reported as the human NHE2, the hNHE2 polypeptide is 115 amino acids longer in the NH2-terminal end and shows only an 84% DNA nucleotide sequence identity. Northern blot analysis revealed that hNHE2 message has an uneven tissue distribution, with high levels in the skeletal muscle, colon, and kidney and lower levels in the testis, prostate, ovary, and small intestine. Protein expression studies with hNHE2 clone showed that a 75-kDa protein was expressed. Stable expression of transfected full-length hNHE2 cDNA in Na+/H+exchange-deficient LAP1 cells exhibited Na+-dependent pH recovery after an acid prepulse that was inhibited by 0.1 mM amiloride. These data indicate that this cDNA is the true human NHE2 cDNA and that the encoded protein is capable of catalyzing Na+/H+exchange activity.In the present report, we describe the cloning of a human colonic cDNA that describes the full-length Na(+)/H(+) exchanger (NHE) 2 coding region. The human NHE2 (hNHE2) cDNA encodes for a polypeptide of 812 amino acids with a 90% overall identity to both rabbit and rat NHE2 isoforms. In comparison with SLC9A2, recently reported as the human NHE2, the hNHE2 polypeptide is 115 amino acids longer in the NH(2)-terminal end and shows only an 84% DNA nucleotide sequence identity. Northern blot analysis revealed that hNHE2 message has an uneven tissue distribution, with high levels in the skeletal muscle, colon, and kidney and lower levels in the testis, prostate, ovary, and small intestine. Protein expression studies with hNHE2 clone showed that a 75-kDa protein was expressed. Stable expression of transfected full-length hNHE2 cDNA in Na(+)/H(+) exchange-deficient LAP1 cells exhibited Na(+)-dependent pH recovery after an acid prepulse that was inhibited by 0.1 mM amiloride. These data indicate that this cDNA is the true human NHE2 cDNA and that the encoded protein is capable of catalyzing Na(+)/H(+) exchange activity.

Lactobacillus acidophilus stimulates the expression of SLC26A3 via a transcriptional mechanism

Clinical efficacy of probiotics in treating various forms of diarrhea has been clearly established. However, mechanisms underlying antidiarrheal effects of probiotics are not completely defined. Diarrhea is caused either by decreased absorption or increased secretion of electrolytes and solutes in the intestine. In this regard, the electroneutral absorption of two major electrolytes, Na(+) and Cl(-), occurs mainly through the coupled operation of Na(+)/H(+) exchangers and Cl(-)/OH(-) exchangers. Previous studies from our laboratory have shown that Lactobacillus acidophilus (LA) acutely stimulated Cl(-)/OH(-) exchange activity via an increase in the surface levels of the apical anion exchanger SLC26A3 (DRA). However, whether probiotics influence SLC26A3 expression and promoter activity has not been examined. The present studies were, therefore, undertaken to investigate the long-term effects of LA on SLC26A3 expression and promoter activity. Treatment of Caco-2 cells with LA for 6-24 h resulted in a significant increase in Cl(-)/OH(-) exchange activity. DRA mRNA levels were also significantly elevated in response to LA treatment starting as early as 8 h. Additionally, the promoter activity of DRA was increased by more than twofold following 8 h LA treatment of Caco-2 cells. Similar to the in vitro studies, in vivo studies using mice gavaged with LA also showed significantly increased DRA mRNA ( approximately 4-fold) and protein expression in the colonic regions as assessed by Western blot analysis and immunofluorescence. In conclusion, increase in DRA promoter activity and expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of LA.

Taurodeoxycholate Modulates Apical Cl−/OH− Exchange Activity in Caco2 Cells

Bile acid malabsorption has been shown to be associated with diarrhea in cases such as ileal resection Crohn’s disease of the ileum, and radiation enteritis. The mechanisms of bile acid-induced diarrhea are not fully understood. Although the induction of colonic chloride secretion in response to bile acids has been extensively investigated, to date the direct effect of bile acids on intestinal chloride absorption has not been well defined. Therefore, the current studies were undertaken to investigate the effect of bile acids on the apical Cl−/OH− exchange process utilizing Caco2 monolayers as an in vitro cellular model. Cl−/OH− exchange activity was measured as DIDS-sensitive pH gradient-driven 36Cl uptake. The results are summarized as follows: (i) short-term exposure (20 min) of Caco2 cells to taurodeoxycholate (TDC; 200 μM) and glycochenodeoxycholate (GCDC; 200 μM) acids significantly inhibited apical Cl−/OH− exchange (by ∼60–70%); (ii) the Ca2+ chelator BAPTA-AM blocked the inhibition by TDC; (iii) the reduction in Cl−/OH− exchange by TDC was reversed by the PKC inhibitor, chelerythrine chloride; (iv) functional and inhibitor studies indicated that TDC induced inhibition of Cl−/OH− exchange was mediated via the activation of the PKCβI isoform; (v) the effect of TDC on apical Cl−/OH− exchange was completely blocked by the PI3 kinase inhibitor LY294002 (5 μM); and (vi) the PKA inhibitor, RpcAMP, had no effect on TDC induced inhibition of Cl−/OH− exchange. In conclusion, our studies provide direct evidence for inhibition of human intestinal apical Cl−/OH− exchange activity by bile acids via Ca2+-, PI3 kinase-, and PKCβI-dependent pathways in Caco2 cells.

The probiotic Lactobacillus plantarum counteracts TNF-α-induced downregulation of SMCT1 expression and function

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.

Mechanisms of transcriptional modulation of the human anion exchanger SLC26A3 gene expression by IFN-γ

Two members of the SLC26 gene family, SLC26A3 or DRA (downregulated in adenoma) and SLC26A6 (putative anion transporter 1, PAT1), are known to play a major role in apical Cl(-)/OH(-) (HCO(3)(-)) exchange process in the human intestine. We have previously shown the inhibitory effects of IFN-gamma (30 ng/ml, 24 h) on both SLC26A3 and A6 expression and promoter activity. We also demonstrated that the effects of IFN-gamma on SLC26A6 gene expression were mediated via IRF-1 transcription factor. However, the molecular mechanisms underlying the transcriptional modulation of SLC26A3 gene expression by IFN-gamma in the intestine are not known. The present studies were, therefore, designed to elucidate the signaling mechanisms and transcription factor(s) involved in mediating the inhibitory effects of IFN-gamma on DRA promoter (p--1183/+114) activity. Deletion analysis indicated that the IFN-gamma response element is located within the -1183 to -790 region, and sequence analysis of this region revealed the presence of potential gamma-activated site (GAS), a binding site (-933/-925 bp) for signal transducer and activator of transcription factor 1 (STAT1). Mutations in the potential GAS element abrogated the inhibitory effects of IFN-gamma. These studies provide evidence for the involvement of STAT1 in the inhibition of SLC26A3 gene expression by IFN-gamma in the human intestine.

Platelet‐activating factor‐induced NF‐κB activation and IL‐8 production in intestinal epithelial cells are Bcl10‐dependent

Background: Platelet‐activating factor (PAF), a potent proinflammatory phospholipid mediator, has been implicated in inducing intestinal inflammation in diseases such as inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC). However, its mechanisms of inducing inflammatory responses are not fully understood. Therefore, studies were designed to explore the mechanisms of PAF‐induced inflammatory cascade in intestinal epithelial cells. Methods: Nuclear factor kappa B (NF‐&kgr;B) activation was measured by luciferase assay and enzyme‐linked immunosorbent assay (ELISA), and interleukin 8 (IL‐8) production was determined by ELISA. B‐cell lymphoma 10 (Bcl10), caspase recruitment domain‐containing membrane‐associated guanylate kinase protein 3 (CARMA3), and mucosa‐associated lymphoid tissue lymphoma translocation protein 1 (MALT1) mRNA and protein levels were assessed by real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) and Western blot, respectively. siRNA silencing of Bcl10 was used to examine its role in PAF‐induced NF‐&kgr;B activation and IL‐8 production. The promoter region of the Bcl10 gene was cloned with the PCR method and promoter activity measured by luciferase assay. Results: The adaptor protein Bcl10 appeared to play an important role in the PAF‐induced inflammatory pathway in human intestinal epithelial cells. Bcl10 was required for PAF‐induced I&kgr;B&agr; phosphorylation, NF‐&kgr;B activation, and IL‐8 production in NCM460, a cell line derived from normal human colon, and Caco‐2, a transformed human intestinal cell line. PAF also stimulated Bcl10 interactions with CARMA3 and MALT1, and upregulated Bcl10 expression in these cells via transcriptional regulation. Conclusions: These findings highlight a novel PAF‐induced inflammatory pathway in intestinal epithelial cells, requiring Bcl10 as a critical mediator and involving CARMA3/Bcl10/MALT1 interactions. The proinflammatory effects of PAF play prominent roles in the pathogenesis of IBD and this pathway may present important targets for intervention in chronic inflammatory diseases of the intestine. (Inflamm Bowel Dis 2009;)