Mo1764

CXCL12 Oligomerization Dictates Divergent Signaling and MigratoryResponses in Intestinal Epithelial CellsLuke Drury, Joshua Ziarek, Brian F. Volkman, Michael B. Dwinell

Chemotactic migration classically exhibits a bell-shaped response over a narrow concentrationrange. Previous studies have shown the chemokine, CXCL12, exists in a monomer-dimerequilibrium with heightened concentrations resulting in preferential accumulation of homo-dimeric chemokine. Little is understood how CXCL12 oligomerization induces distinctcellular effects through the same cognent receptor, CXCR4. The objective of these studiesis to determine the effects of monomeric and dimeric CXCL12 on colorectal cancer cellmigration. HCT116 and HT29 human colonic carcinoma cells were utilized to examineboth chemotactic and restitutive migration respectively. Previously engineered preferentialmonomeric CXCL12(H25R), locked dimer CXCL12(2), and wild-type CXCL12(WT) wereutilized in our analyses. Transwell inserts and aspiration wounded monolayers demonstratedthat CXCL12(WT) and CXCL12(H25R) induced a strong chemotactic and restitutive migra-tion response. In contrast, CXCL12(2) dimer was unable to elicit cellular migration. Inagreement with those data, filamentous actin formation was induced by CXCL12(WT) andCXCL12(H25R) but not dimer. Intracellular calcium flux, a classical hallmark of G-proteincoupled receptor signaling, was elicited equally by each oligomeric variant. This responsewas blocked with addition of CXCR4-specific inhibitors indicating each of the variantsengaged the same cognate receptor. Similarly, CXCL12(2) dose-dependently inhibitedCXCR4, but not CXCR1, mediated chemotaxis. The lack of migration was subsequentlyshown using nuclear magnetic resonance analyses to reflect distinct interactions of themonomeric and dimeric ligand for CXCR4 homotypic receptor dimers. These latter datasupport the notion that double occupancy of dimeric receptor limits migration while singleoccupancy of the same receptors evokes strong migration responses. Together these dataunderlie the importance of CXCL12 oligomerization in dictating epithelial migration andsuggest the CXCL12 dimer may serve as a potential inhibitor of colorectal cancer metastasis.

Mo1765

Regulation of the Gene Encoding the Intestinal Bile Acid Transporter ASBT byCDX Transcription FactorsLi Ma, Gerd A. Kullak-Ublick, Jyrki J. Eloranta

Background and Aims: The apical sodium-dependent bile acid transporter (ASBT; SLC10A2)is expressed in the ileum, biliary tract, and kidney, and mediates bile acid absorption acrossthe apical membrane. CDX1 and CDX2 are intestine-specific transcription factors that regulatea broad range of genes involved in intestinal epithelial differentiation and proliferation.Aberrant esophageal expression of both ASBT and CDXs in Barrett's esophagus promptedus to study, whether the expression of the former gene is regulated by the latter twotranscription factors. Methods: Human intestine-derived epithelial cell lines Caco-2, T84,and LS174T were transfected with luciferase-linked promoter regions of the human ASBTgene, with or without expression plasmids harbouring transcription factor cDNAs, and dualluciferase activities were determined ~36 h later. Electrophoreticmobility shift assays (EMSAs)were performed using nuclear extracts from LS174T cells and In Vitro translated CDXproteins.Chromatin immunoprecipitations (ChIPs) were performed in T84 cells. Knockdowns ofCDXs with siRNAs were performed in T84 cells. Total RNAs were extracted from ileal biopsiesfrom 48 healthy patients, and analyzed by TaqMan real-time PCR. Results: Knockdown ofCDXs with siRNAs resulted in reduced endogenous ASBT mRNA expression in T84 cells.CDX1 and CDX2 strongly induced the activity of an ASBT promoter construct spanning nt-1688/+525 (numbering relative to transcriptional start). Several putative CDX binding siteswere predicted in silico within this region, and binding of CDXs to these sites was verifiedby EMSAs In Vitro and by ChIP assays within living cells. In cotransfection experiments,the known transactivator of theASBT gene, hepatocyte nuclear factor-1α (HNF-1α), increasedASBT promoter activity in a synergistic manner with CDXs. Significant correlation wasobserved between CDX1 and CDX2 mRNA expression levels in healthy ileal tissue, whereasno correlation was observed between either CDX1, CDX2, or HNF-1α and ASBT levels.Conclusions: The human ASBT promoter is activated transcriptionally by CDX1 and CDX2in intestinal cells. Our finding provides an explanation for the previous observation that ASBTis aberrantly expressed in esophageal metaplasia that also expresses CDX transcription factors.

Mo1766

Increased Enhancer Activity of the 14010*C Variant Associated With LactasePersistenceTine G. Jensen, Anke Liebert, Rikke H. Lewinsky, Dallas M. Swallow, Jørgen Olsen, JesperT. Troelsen

Background and Aims In most people worldwide, intestinal lactase expression declines inchildhood. In many others, particularly in Europeans, lactase expression persists into adultlife. The lactase persistence phenotype is in Europe associated with the 13910*T singlenucleotide variant located 13910 bp upstream the lactase gene in an enhancer region thataffects lactase promoter activity. This variant falls in an Oct 1 binding site and shows greaterOct 1 binding than the ancestral variant and increases enhancer activity. Several othervariants have been identified very close to the 13910 position, which are associated withlactase persistence in the Middle East and Africa. One of them, the 14010*C, is associatedwith lactase persistence in East Africa. The aim of the project is to investigate the generegulatory ability of the 14010*C variant. Methods The 450 bp LCT enhancer containingthe ancestral non-persistent variant, the European ( 13910*T) variant or East African (14010*C) variants was subjected to a functional reporter gene analysis measuring theenhancer activity of the variants. DNA-protein interactions were investigated by gel shift,supershift and DNase footprinting assays. Results Here we show that the region containingthe 14010*C variant generates a stronger enhancer effect than the ancestral 14010*G variant.Deletion analysis reveals that the 14010 position is located in a 144 bp region that repressesthe enhancer activity. The 14010*C variant shows reduced repression compared with the

S-645 AGA Abstracts

14010*G variant, explaining the stronger enhancer effect of the East African lactase persist-ence variant. Binding sites for Oct 1 and HNF1α were identified surrounding the 14010position. The higher enhancer activity is associated with an increased Oct 1 binding to theregion containing the 14010*C variant. Conclusions Lactase persistent variants influenceLCT enhancer activity by modulating the activity of the enhancer. The East African 14010*Cvariant is located between an Oct-1 site and an HNF1α binding site and is correlated withan increased Oct 1 binding which increases the enhancer activity.

Mo1767

CFTR is Necessary for Vacuolar ATPase Proton Pump (VATPase) Localizationand Function in Polarized Intestinal CaCo2BBe CellsAnne Collaco, Peter J. Geibel, John P. Geibel, Nadia Ameen

The vacuolar ATPase (VATPase) proton pump serves an important role in organellar acid-ification and regulation of luminal pH in epithelial tissues. Movement of chloride is necessaryfor pump function in organelle or luminal acidification. Polarized intestinal CaCo2BBe cellsexpress endogenous CFTR and VATPase in apical endocytic and recycling vesicles andundergo cAMP-regulated traffic to the apical domain. Furthermore, cAMP-activated apicaltraffic of VATPase is markedly reduced in the intestines of CF knock out mice comparedto WT, indicating a physiologic role for CFTR in VATPase traffic (Collaco et.al; DDW, AbsT1837, 2009). We hypothesized that CFTR chloride channels are functionally linked toVATPase in intestinal epithelial cells. Here we used polarized CaCo2BBe cells and examinedVATPase function in the presence and absence of pharmacologic inhibition of CFTR andVATPase. Methods: CaCo2BBe were grown to confluence on glass coverslips and transferredto the stage of fluorescence microscopy that was connected to a video imaging system. Cellswere then loaded with the pH sensitive dye BCECF to monitor intracellular pH. The cellswere then excited with 490nm/440nm light while monitoring the emission at 535nm. Toconfirm functional VATPase activity, CaCo2BBe cells were exposed to a 20mM NH4Clsolution (NH4Cl) to provide a proton load and then washed into a Na-free (0Na) Hepesbuffer solution. Rates of recovery in the absence of Na were attributed to VATPase activity.In a separate series of studies cells were exposed to (50nM) concanamycin, a specific inhibitorof the VATPase. To evaluate the functional link between CFTR and VATPase, cells wereexposed to the specific CFTR inhibitor CFinh172 (20μM) to block CFTR activity prior toexposure to NH4Cl and 0Na. The rate of recovery was compared to cells exposed to vehiclealone, following NH4Cl and 0Na. Results: In 5 separate dishes with measurements derivedfrom 20 cells per dish (n=100) we found that all cells have a robust Na-independent recoveryindicative of VATPase. In a separate series of studies in 5 dishes, exposure to concanamycineffectively blocked the pH recovery in the 0Na solution. In an additional 5 dishes, cellspretreated with CFTRinh172 had no pH recovery following an acid load and exposure toNa-free solution. Matched vehicle controls (n=60) showed a robust recovery. Conclusions:In the present study we have now confirmed the presence of an active VATPase that canpump excess protons out of the cytosol. Furthermore, we also demonstrated that inhibitionof CFTR inactivates the VATPase in CaCo2BBe cells. These studies now provide functionalevidence supporting our previously presented immunohistochemical data. This is the firstdemonstration that CFTR directly modulates VATPase function in intestinal CaCo2BBe cells.

Mo1768

Epcam Deficient Mice Show Significant Morbidity and Pathology ResemblingCongenital Tufting EnteropathyMamata Sivagnanam, James L. Mueller, Matthew D. Mcgeough, Carla Pena, Hal M.Hoffman

Congenital tufting enteropathy (CTE) is an inherited intractable diarrhea of infancy presentingwith chronic diarrhea, electrolyte imbalances, and impaired growth. The diagnosis of CTEis made with the recognition of villous changes of the small intestinal epithelium includingvillous atrophy, crypt hyperplasia and focal epithelial tufts. Most cases of CTE lead tointestinal failure and require prolonged parenteral nutrition or small bowel transplant. Wepreviously identified mutations in the gene for Epithelial Cell Adhesion molecule (EpCAM)in our CTE patients. mRNA from a subset of these patients has show splicing of exon 4.Additionally, significant decreases in protein levels of EpCAM have been seen by westernblot and immunohistochemistry in intestinal tissue from CTE patients. In normal adulttissues EpCAM is expressed on the basolateral surface of simple, pseudo-stratified, andtransitional epithelia. EpCAM has been implicated to have a role in organogenesis and celladhesion, though its role in the normal gut development and function remains unclear. Theobjective of this project is to elucidate the role of EpCAM in intestinal development andfunction using In Vivo model of intestinal epithelial cell function. We achieved this goalwith a mutant EpCAM mouse model. Previous groups have shown EpCAM over-expressingmice or mice with absent expression. We have developed a novel mouse model with adeletion of exon 4 of EpCAM (as seen in some of our CTE patients). This is the first modelto have decreased, but not absent, EpCAM expression. To confirm successful attenuationof EpCAM we have performed IHC utilizing the well-characterized and readily availableG8.8 antibody to confirm decreased protein expression (Fig.1) in mutant vs. wild-type mice.The mutant mice have shown significant morbidity and mortality. In most instances survivalof the mutant mice is limited to 7 days or less. These mice are also noted to have significantdecreased weights as compared to their WT and HET littermates. Microscopic imaging ofthe small intestine and colon shows significant pathologic changes throughout the duodenum,jejunum and ileum in MUT mice. H&E staining reveals blunting of the intestinal villi,edema, crowding of enterocytes and tufted villi. Abnormal invaginations between enterocyteswere also appreciated. Some mice also show significant necrosis and cell death of the mucosa.HET and WT mice show normal histology. These findings are very similar to those inpatients with CTE (Fig. 2). This is the first murine model mimicking changes in EpCAMseen in CTE patients. This model will serve to further elucidate mechanism of CTE andpotentially lead to improved therapeutic options.

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