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Physiologic microbe-host interactions in the intestine require the maintenance of the microbiota in a luminal compartment through a complex interplay between epithelial and immune cells. However, the roles of mucosal myeloid cells in this process remain incompletely understood. In this study, we identified that decreased myeloid cell phagocytic activity promotes colon tumorigenesis. We show that this is due to bacterial accumulation in the lamina propria and present evidence that the underlying mechanism is bacterial induction of prostaglandin production by myeloid cells. Moreover, we show that similar events in the normal colonic mucosa lead to reductions in Tuft cells, goblet cells, and the mucus barrier of the colonic epithelium. These alterations are again linked to the induction of prostaglandin production in response to bacterial penetration of the mucosa. Altogether, our work highlights immune cell-epithelial cell interactions triggered by the microbiota that control intestinal immunity, epithelial differentiation, and carcinogenesis.

Singla A, Dwivedi A, Saksena S, Gill RK, Alrefai WA, Ramaswamy K, Dudeja PK. Mechanisms of lysophosphatidic acid (LPA) mediated stimulation of intestinal apical [C1.sup.-]/[OH.sup.-] exchange. Am J Physiol Gastrointest Liver Physio1298: G 182-G189, 2010. First published November 12, 2009; doi: 10.1152/ajpgi.00345.2009.--Lysophosphatidic acid (LPA), a potent bioactive phospholipid, is a natural component of food products like soy and egg yolk. LPA modulates a number of epithelial functions and has been shown to inhibit cholera toxin-induced diarrhea. Antidiarrheal effects of LPA are known to be mediated by inhibiting chloride secretion. However, the effects of LPA on chloride absorption in the mammalian intestine are not known. The present studies examined the effects of LPA on apical [Cl.sup.-]/[OH.sup.-] exchangers known to be involved in chloride absorption in intestinal epithelial cells. Caco-2 cells were treated with LPA, and [C1.sup.-]/[OH.sup.-] exchange activity was measured as DIDS-sensitive [sup.36][Cl.sup.-] uptake. Cell surface biotinylation studies were performed to evaluate the effect of LPA on cell surface levels of apical [Cl.sup.-]/[OH.sup.-] exchangers, downregulated in adenoma (DRA) (SLC26A3), and putative anion transporter-1 (SLC26A6). Treatment of Caco-2 cells with LPA (100 [micro]M) significantly stimulated [Cl.sup.-]/[OH.sup.-] exchange activity. Specific agonist for LPA2 receptor mimicked the effects of LPA. LPA-mediated stimulation of [C1.sup.-]/[OH.sup.-] exchange activity was dependent on activation of phosphatidylinositol 3-kinase/Akt signaling pathway. Consistent with the functional activity, LPA treatment resulted in increased levels of DRA on the apical membrane. Our results demonstrate that LPA stimulates apical [Cl.sup.-]/[OH.sup.-] exchange activity and surface levels of DRA in intestinal epithelial cells. This increase in [Cl.sup.-]/[OH.sup.-] exchange may contribute to the antidiarrheal effects of LPA. downregulated in adenoma; chloride absorption; human intestine; LPA receptor 2; phosphatidylinositol 3-kinase/Akt doi: 10.1152/ajpgi.00345.2009

Saksena S, Singla A, Goyal S, Katyal S, Bansal N, Gill RK, Alrefai WA, Ramaswamy K, Dudeja PK. Mechanisms of transcriptional modulation of the human anion exchanger SLC26A3 gene expression by IFN-[gamma]. Am J Physiol Gastrointest Liver Physiol 298: G159-G166, 2010. First published November 25, 2009; doi:10.1152/ajpgi.00374.2009.--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.sup.-]/[OH.sup.-] ([HCO.sub.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. DRA (downregulated in adenoma) promoter; STAT1 (signal transducer and activator of transcription 1); JAK (Janus kinases); chloride absorption doi: 10.1152/ajpgi.00374.2009

Monocarboxylate transporter (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFA) such as butyrate in the human colon. Previous studies from our laboratory have demonstrated that phorbol ester, PMA (1 [micro]M, 24 h), upregulates butyrate transport and MCT1 protein expression in human intestinal Caco-2 cells. However, the molecular mechanisms involved in the transcriptional regulation of MCT1 gene expression by PMA in the intestine are not known. In the present study, we showed that PMA (0.1 [micro]M, 24 h) increased the MCT1 promoter activity (-871/+ 91) by approximately fourfold. A corresponding increase in MCT1 mRNA abundance in response to PMA was also observed. PMA-induced stimulation of MCT1 promoter activity was observed as early as 1 h and persisted until 24 h, suggesting that the effects of PMA are attributable to initial PKC activation. Kinase inhibitor and phosphorylation studies indicated that these effects may be mediated through activation of the atypical PKC-[xi] isoform. 5'-deletion studies demonstrated that the MCT1 core promoter region (-229/+91) is the PMA-responsive region. Site-directed mutagenesis studies showed the predominant involvement of potential activator protein 2 (AP2) binding site in the activation of MCT1 promoter activity by PMA. In addition, overexpression of AP2 in Caco-2 cells significantly increased MCT1 promoter activity in a dose-dependent manner. These findings showing the regulation of MCT1 promoter by PKC and AP2 are of significant importance for an understanding of the molecular regulation of SCFA absorption in the human intestine. short-chain fatty acid absorption; transcriptional regulation; human intestine; protein kinase C-[xi]; activator protein 2

The enteric serotonin transporter (SERT) plays a critical role in modulating serotonin availability and thus has been implicated in the pathogenesis of various intestinal disorders. To date, SERT expression and function in the human intestine have not been investigated. Current studies were designed to characterize the function, expression, distribution, and membrane localization of SERT in the native human intestine. Real-time PCR studies showed relatively higher SERT mRNA expression in the human small intestine compared with colon (ileum [much greater than] duodenum [much greater than] jejunum). Northern blot analysis revealed three mRNA hybridizing species encoding SERT (3.0, 4.9, and 6.8 kb) in the hurhan ileum. Consistent with SERT mRNA expression, SERT immunostaining was mainly detected in the epithelial cells of human duodenal and ileal resected tissues. Notably, SERT expression was localized predominantly to the apical and intracellular compartments and was distributed throughout the crypt-villus axis. Immunoblotting studies detected a prominent protein band (~70 kDa) in the ileal apical plasma membrane vesicles (AMVs) isolated from mucosa obtained from organ-donor intestine. Functional studies showed that uptake of [[sup.3]H]serotonin (150 nM) in human ileal AMVs was 1) significantly increased in the presence of both [Na.sup.+] and C[I.sup.-]; 2) inhibited (~50%) by the neuronal SERT inhibitor, fluoxetine (10 [micro]M) and by unlabeled 5-HT; and 3) exhibited saturation kinetics indicating the presence of a carrier-mediated process. Our studies demonstrated differential expression of SERT across various regions of the human intestine and provide evidence for the existence of a functional SERT capable of removing intraluminal serotonin in human ileal epithelial cells. serotonin uptake; serotonin transporter expression

NiemannPick C1-like 1 (NPC1L1) is an essential intestinal component of cholesterol absorption. However, little is known about the molecular regulation of intestinal NPC1L1 expression and promoter activity. We demonstrated that human NPC1L1 mRNA expression was significantly decreased by 25-hydroxycholesterol but increased in response to cellular cholesterol depletion achieved by incubation with Mevinolin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) in human intestinal Caco-2 cells. We also showed that a -1741/+56 fragment of the NPC1L1 gene demonstrated high promoter activity in Caco-2 cells that was reduced by 25-hydroxycholesterol and stimulated by cholesterol depletion. Interestingly, we showed that the NPC1L1 promoter is remarkably transactivated by the overexpression of sterol regulatory element (SRE) binding protein (SREBP)-2, suggesting its involvement in the sterol-induced alteration in NPC1L1 promoter activity. Finally, we identified two putative SREs in the human NPC1L1 promoter and established their essential roles in mediating the effects of cholesterol on promoter activity. Our study demonstrated the modulation of human NPC1L1 expression and promoter activity by cholesterol in a SREBP-2-dependent mechanism. oxysterols; intestinal cholesterol absorption