Alip Borthakur

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.

Siva-1 binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis

We previously cloned Siva-1 by using the cytoplasmic tail of CD27, a member of the tumor necrosis factor receptor family, as the bait in the yeast two-hybrid system. The Siva gene is organized into four exons that code for the predominant full-length Siva-1 transcript, whereas its alternate splice form, Siva-2, lacks exon 2 coding sequence. Various groups have demonstrated a role for Siva-1 in several apoptotic pathways. Interestingly, the proapoptotic properties of Siva-1 are lacking in Siva-2. The fact that Siva-1 is partly localized to mitochondria despite the absence of any mitochondrial targeting signal, it harbors a 20-aa-long putative amphipathic helical structure that is absent in Siva-2, and that its expression is restricted to double-positive (CD3+, CD4+, CD8+) thymocytes like BCL-XL, prompted us to test for a potential interaction between Siva-1 and BCL-XL. Here, we show that Siva-1 binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis. Indeed, the unique amphipathic helical region (SAH) present in Siva-1 is required for its binding to BCL-XL and sensitizing cells to UV radiation. Natural complexes of Siva-1/BCL-XL are detected in HUT78 and murine thymocyte, suggesting a potential role for Siva-1 in regulating T cell homeostasis.

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.

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.

A novel nutrient sensing mechanism underlies substrate-induced regulation of monocarboxylate transporter-1

Monocarboxylate transporter isoform-1 (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFAs) in the colon. Butyrate, a major SCFA, serves as the primary energy source for the colonic mucosa, maintains epithelial integrity, and ameliorates intestinal inflammation. Previous studies have shown substrate (butyrate)-induced upregulation of MCT1 expression and function via transcriptional mechanisms. The present studies provide evidence that short-term MCT1 regulation by substrates could be mediated via a novel nutrient sensing mechanism. Short-term regulation of MCT1 by butyrate was examined in vitro in human intestinal C2BBe1 and rat intestinal IEC-6 cells and ex vivo in rat intestinal mucosa. Effects of pectin feeding on MCT1, in vivo, were determined in rat model. Butyrate treatment (30-120 min) of C2BBe1 cells increased MCT1 function {p-(chloromercuri) benzene sulfonate (PCMBS)-sensitive [(14)C]butyrate uptake} in a pertussis toxin-sensitive manner. The effects were associated with decreased intracellular cAMP levels, increased V(max) of butyrate uptake, and GPR109A-dependent increase in apical membrane MCT1 level. Nicotinic acid, an agonist for the SCFA receptor GPR109A, also increased MCT1 function and decreased intracellular cAMP. Pectin feeding increased apical membrane MCT1 levels and nicotinate-induced transepithelial butyrate flux in rat colon. Our data provide strong evidence for substrate-induced enhancement of MCT1 surface expression and function via a novel nutrient sensing mechanism involving GPR109A as a SCFA sensor.

The Siva-1 putative amphipathic helical region (SAH) is sufficient to bind to BCL-XL and sensitize cells to UV radiation induced apoptosis

The human Siva gene is localized to chromosome 14q32-33 and gives rise to the full-length predominant form, Siva-1 and a minor alternate form, Siva-2 that appears to lack the proapoptotic properties of Siva-1. Our recent work has shown that the missing region in Siva-2 encodes a unique twenty amino acid putative amphipathic helical region (SAH, residues 36–55 in Siva-1). Despite the fact that Siva-1 does not belong to the BCL-2 family, it specifically interacts with the anti-apoptotic protein BCL-XL and sensitizes MCF7 breast cancer cells expressing BCL-XL to UV radiation induced apoptosis. Deletion mutagenesis has mapped the necessary region to the SAH in Siva-1. In this paper we demonstrate that the SAH region in Siva-1 is sufficient to specifically interact with the anti-apoptotic members of the BCL2 family such as BCL-XL and BCL-2 but not its apoptotic member BAX. Using transient transfections and direct microinjection of synthetic SAH peptides, we also demonstrate that the SAH region is sufficient to inhibit the BCL-XL mediated cell survival and render MDA-MB-231 and MCF7 breast cancer cells expressing BCL-XL highly susceptible to UV radiation induced apoptosis. The underlying mechanism of action of SAH mediated inhibition of BCL-XL (and/or BCL2) cell survival appears to be due to loss of mitochondrial integrity as reflected in enhanced cytochrome c release leading to the activation of caspase 9 and finally caspase 3.

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.

Enteropathogenic Escherichia coli Infection Inhibits Intestinal Serotonin Transporter Function and Expression

BACKGROUND & AIMS Serotonin transporter (SERT) plays a critical role in regulating serotonin (5-hydroxytryptamine [5-HT]) availability in the gut. Elevated 5-HT levels are associated with diarrheal conditions such as irritable bowel syndrome and enteric infections. Whether alteration in SERT activity contributes to the pathophysiology of diarrhea induced by the food-borne pathogen enteropathogenic Escherichia coli (EPEC) is not known. The present studies examined the effects of EPEC infection on SERT activity and expression in intestinal epithelial cells and elucidated the underlying mechanisms. METHODS Caco-2 cells as a model of human intestinal epithelia and EPEC-infected C57BL/6J mouse model of infection were utilized. SERT activity was measured as Na(+) and Cl(-) dependent (3)[H] 5-HT uptake. SERT expression was measured by real-time quantitative reverse-transcription polymerase chain reaction, Western blotting, and immunofluorescence studies. RESULTS Infection of Caco-2 cells with EPEC for 30-120 minutes decreased apical SERT activity (P < .001) in a type 3 secretion system dependent manner and via involvement of protein tyrosine phosphatases. EPEC infection decreased V(max) of the transporter; whereas cell surface biotinylation studies revealed no alteration in the cellular or plasma membrane content of SERT in Caco-2 cells. EPEC infection of mice (24 hours) reduced SERT immunostaining with a corresponding decrease in SERT messenger RNA levels, 5-HT uptake, and mucosal 5-HT content in the small intestine. CONCLUSIONS Our results demonstrate inhibition of SERT by EPEC and define the mechanisms underlying these effects. These data may aid in the development of a novel pharmacotherapy to modulate the serotonergic system in treatment of infectious diarrheal diseases.

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;)

B-cell CLL/Lymphoma 10 (BCL10) Is Required for NF-κB Production by Both Canonical and Noncanonical Pathways and for NF-κB-inducing Kinase (NIK) Phosphorylation

B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-κB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IκB-kinase (IKK)α−/− mouse embryonic fibroblasts, increases in phospho-IκBα, nuclear NF-κB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol. In IKKβ−/− cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-κB-inducing kinase (NIK), cytoplasmic NF-κB p100, and nuclear NF-κB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-κB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKβ−/− and IKKα−/− cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKγ, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-κB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.