H+ exchanger NHE-3 gene promoter

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NaPi cotransporter. The presence of putative vitamin D3 cis-elements in mPit-2 promoter further suggested the vitamin D3 regulation of intestinal mPit-2 may through transcriptional mechanism. This investigation was supported by NIDDK Grant 2RO1-DK33209 and M. W. Keck Foundation.

Analysis of the Lamina Prnpria Dendritic Cell Phenofypein Colon Tissue from Patients with Crohn's Disease Anje A. Velde, Acad Medical Ctr, Amsterdam Netherlands; Florry A. Vyth-Dreese, Netherlands Cancer Institute, Amsterdam Netherlands; Olle F. The, Acad Medical Ctr, Amsterdam Netherlands; Yvette Kooyk, Univ Medical Ctr St Radboud, Nijmegen Netherlands; Sander J. Deventer, Acad Medical Ctr, Amsterdam Netherlands

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BACKGROUND: Dendritic cells (DC) are professional antigen presenting cells that efficiently pick up antigen in the periphery and present this in the T call areas of draining lymph nodes resulting in T cell activation and differentiation. Antigen-dependent activation of Thl lymphocytes is believed to be a major pathogenic mechanism in Crohn's disease, but the phenotype of the antigen-presenting cells has been poorly defined. We have analyzedthe coexpression of activation and maturation markers on CD11c* DCs in the lamina propria (LP) of the colon of patients with Crohn's disease compared to normal colon. METHODS: 3 color confocal laser scanning microscopy (CLSM) analysis was performed of colonic resection specimens from patients with Crohn's disease and from patients with non-IBD related diSorders. The following markers were analyzed: CD4, CD68, CD80, CD83, DC-SIGN (Geijtenbeek et al, Cell 2000;100:575-585) and IL-3R. RESULTS:Counting 10 fields using 400x magnification yielded a mean of 29.9 + 2.9 CD11c- cells in the LP of patients with Crohn's disease compared to 13.8 + 1.9 in normal colon (p
normal Crohn's

CD4

CD68

CDSO

CD83

DC-SIGN

+/+/-

+/++

+ ++

+/. +

,/_ +

+/-:<25%, +:25-50%and ++:>50%cells showingco-expressio~with CD1lc

2675 Marked Difference of CD4*CD45RBh= Expression by Gut DertoN T Lymphnsytos in Crohn's Disease and Ulcerative Colitis; Differential Cytokine Synthesis of C045RB Subpopulations Tessa Hove, Olle F. The, Joost P. Bruggeman, Frederique Slors, Sander J.H. Deventer, Anje A. Velde, AMC, Amsterdam Netherlands Background: Evidence exists that T-lymphocytes are involved in the pathogenesis of IBD. In this study the phenotype of colonic mucosal lymphocytes, present in resection specimens from patients with active IBD and from controls was determined using FACScan analysis. Resu/ts: The percentage of CD4+CD45RB"~"-lymphocytes was significantly increased in patients with CD and UC (17.9 % and 40.9% resp.) compared to controls (2.5 %), whereas CD4÷CD45RO÷ and CD4÷CD45RA+ showed no significant differences. The number of cells expressing activation-markers CD4+CD69 +, CD4+CD134 *, HLA-DR* and CD44" was increasedin IBD. Furthermore, we investigatedfunctional differences betweenCD4+CD45RBw" and CD4* CD45RB~*-Iymphocytesby analysing the cytokine profile in peripheral blood. CD4*CD46RB'g"-lymphocytes produce significantly less IL-IO and IL4 and produce more TNFe than CD45RB~w-lymphocytes.In addition, CD4+CD45RB~* cells of IBD patients produce less IL-IO than CD4+CD45RB~°*-lymphocytesof controls. The IFN.y production of both subsets was decreased in IBD patients. There was a trend towards increased IL-4 production of CD4*CD45RB~°*-lymphocytesin CO patients, whereas in UC patients IL-4 production was significantly decreased.In conclusion, these data indicatethat both CDand UCare cbaracterised by an influx of CD4+CD45RB'0"-Iymphocytes.These CD4*CD45RB~-lymphocytes seem to be important in the pathogenesis of IBD as they produce more pro-inflammatory cytokines and less anti-inflammatory cytokines compared to CD4÷CD45RB~*-Iymphocytes.

Transcription Factors Spl, Sp3 and AP-2 interact with the Haman Na*/H + Exchanger NHE-3 Gene Promoter Jateh M a l a Y , Refka Dahdal, Univ of Illinois, VA Chicago: Westslde, Chicago, IL; Pradeep K. Dudeja, Krishnamurthy Ramaswamy, Univ of Illinois at Chicago, Chicago, IL The Na'/H- exchanger isoforms, NHE-3 and NHE-2, are expressed on the apical membrane of the epithelial cells in the ileum and colon and implicated in the absorption of Na÷. These isoforms exhibit different responses to various stimuli. To investigate the molecular mechanisms involved in differential expression and regulation of these isoforms, we previously reported the cloning of a 3.3 kb fragment of the 5'-regulatory region of the human NHE-3 gone, and identified a number of potential transcription factor binding sites in the promoter region. In the current study, we generated progressive deletions of the 1.0 kb promoter region to define the cis-elemants and their cognate transcription factors. The deleted fragments were linked to a promoter-less luciferase reporter vector and transiently transfected into C2/bbe cells with pSV-bgal cotransfection serving as an internal control for transfection efficiency. A minimal promoter region (-95/+1) upstream from the transcription initiation site that contained the maximal promoter activity was defined. The regulatory response elements clustered within this region include two Spl response elements (-83 and - 68), two AP-2 motifs (-66 and - 18), an atypical TFIID binding site (-40), and a CACCCsequence (-31). The ability of recombinant AP-2 protein to bind to the putative AP-2 binding site (-66) was demonstrated by gel mobility shift assays (GMSA). Furthermore, DNA fontprinting experiments showed these interactions to occur not only within the proximal promoter-binding site, but also at a site further upstream. Two site specific mutations in the AP-2 (-66) binding site did not compete with the wild-typo NHE-3 AP-2 probe for binding to the AP-2 protein in a GMSA. The physiological significance of these mutations in the context of the NHE-3 promoter needs to be examined in transient transfecfion with the mutated promoter-reporter constructs. GMSA with the putative Spl binding site at (-83) exhibited at least three proteiNDNA complexes. The identities of two of the proteins were determined by supershift experiments as Spl and Sp3 transcdplton factors. Spl and Sp3, both bind to the Spl element, while AP-2 binds to the AP-2 element. In conclusion, our studies suggest that the transcriptional regulatory mechanism of the human NHE-3 gene is likely to be defined by a repertoire of transcription factors and their direct interactions with cis-elements in the promoter region as identified here for the Spl, Sp3 and AP-2 regulatory factors.

drra (Down Regulated in Adenoma) Binds In Vitro to the Second PDZ Domain of NHERF and E31(ARP Georg LampreCht, Andreas Hell, Elena Lin Wu, Eberhard-Karls-University, Tuebingen Germany; Chris H. Yun, Johns Hopkins Univ Sch of Medicine, Baltimore, MD; Michael Gregor, Ursula Soldier, Eberhard-Kads-University, Tuebingen Germany Introduction: NaCI absorption in the gut is mediated by parallel Na/H- (NHE3) and CI/HCO3exchange (the gone product of dra). dra may also be involved together with CFTR in anion secretion in the duodenum. NHERF and E3KARP are two highly homologous PDZ adapter proteins. Their second PDZ domains have been shown to bind to NHE3 and CFTR. NHERF and E3KARP both bind independent of their PDZ domains to ezrin which mediates cAMP regulation of NHE3 and CFTR. Aim of the study: Because dra has a consensus sequence for PDZ binding (C-terminus ETKF), we studied whether dra also binds to one of the PDZ domains of NHERFand E3KARP. Binding to either of the PDZ domains would imply whether a common regulation of dra with NHE3or CFTRis mediated through binding to the different POZ domains or through binding of the adapter proteins to the cytosketeton (via ezrin). Methods and results: 1) NHERF, E3KARP and constructs of their PDZ domains and C-termini were expressed and purified as His-tag/S-tag-fusion proteins in Ecoli. The cytoplasmic tail of dra (C-dra) and a mutant lacking the PDZ interaction sequence (C-dra-ETKF-minus)were expressed as in vivo biotinylated fusion proteins in E.coli. NHERF, E3KARP and their constructs were bound to magnetic Nickel-agarose beads, which were then incubated with the bacterial lysates of the biotinylated C-dra and C-dra-ETKF-minus constructs. Bound biotinylated proteins were detected on western blots using streptavldin-HRP conjugate. The His-tag/S-tag constructs were purified to > 95% purity. C-dra but not C-dra-ETKF-minus bound to full length NHERF and E3KARP and to those constructs that contained the second PDZ domain. 2) Biotinylated 20 aa long peptldes of dra and CFTR and mutated peptides (C-terminal 4 aa changed to glycine) were bound to streptavidin beads and incubated with bacterial lysates of NHERF, E3KARP and constructs of their PDZ domains. Bound constructs were detected by S-tag wostem. NHERF, E3KARP and the constructs containing the second PDZ domain bound to the dm and CFTR peptide but not to the mutated peptides. Summary and conclusion: Like NHE3 and CFTRalso dra binds with its C-terminal ETKF-sequenceto the second PDZ domain of NHERFand E3KARP. The two PDZ domains of NHERFand E3KARPare therefore not used to form a direct physical link of dra with either NHE3 or CFTR. The suggested common localization and/or common regulation of dra with either NHE3 or CFTR may therefore be mediated through binding of NHERF or E3KARP to other proteins, most likely ezrin, which in turn binds to the actin cytoskeleton.

2676 Molecular Cloning and Functional Characterization of a Murine Type Ill Intestinal Sodium Dependent Phosphate Cotransporter (mPit-2) Promoter Liqun Bai, James F. Collins, Hua Xu, FayezK. Ghishan, Univ of Arizona, Tucson, AZ Intestinal sodium-dependent inorganic phosphate cotransport (NaPi) is critical for phosphate homeostasis in mammals. We previously cloned a type Ill NaPi cotransporter (mPit-2) from mouse intestine and demonstrated its expression in the enterocytes. The mRNA of intestinal mPit-2 was shown to be up-regulated by 1,25-dihydroxyvitamin D3 (Vitamin D3), a wellknown stimulator of small intestinal Na-Pi cotransport. This implies a potential transcriptional mechanism in the regulation of intestinal mPit-2 by vitamin D3.To elucidate the transcriptional mechanisms regulating the expression of mPit-2 gene, we have cloned and functionally characterized its 5'-flanking region. Screening a mouse Bac library using 5 -end specific cDNA of mPit-2 led to the isolation of a positive clone containing 2.6 kb of 5' flanking region. Primer extension analysis identified a major transcription initiation site at 375 bp upstream of the ATG start codon. Sequenceanalysis revealed that it contains a TATA box, several consensus binding motifs including CACCC, CREB, and NF-B. It also contains four putative vitamin D3responsive elements in a 1.2 kb upstream region of the published cDNA. Progressive 5' deletions of the 5' flanking region were fused to the luciferase reporter gene and transient expression measured following transfection into human colon carcinoma cell line (Caco-2). Transfection with these reporter constructs showed that these DNA fragments could drive luciferase reporter gene expression in this cell line, indicating a functional promoter. Analysis of luciterase activity revealed a minimal promoter region that lie between 225 bp upstream of the transcription initiation site and 61 bp into the first exon of the gene. Conclusion: We have cloned and functional characterized the promoter region of mouse intestinal type III

267g Cerbachol Transactivntion of the EGF Receptor in Tu Cells Requires TGF-a Release Declan F. McCole, Stephen J. Keely, Kim E. Barrett, UCSD Sch of Medicine, San Diego, CA Background/Aims: The epidermal growth factor receptor (EGFr) plays an important role in regulating calcium-mediated chloride secretion across intestinal epithelial cells. The calciumdependent secretagogue,carbachol, transactivates the EGFrvia an intracellular route involving

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