Carbachol transactivation of the EGF receptor in T34 cells requires TGF-α release

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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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ammonium prepulse)was measuredas activity of Na+/H + exchange,and each sample was tested in the presenceand absenceof an inhibitor during the sameexperiment.In the presence of luminal 20/~M EIPA (isoform non-specific NHE inhibitor) the apical Na+/H + exchange rate was 0.033-+0.007 pH/min versus control 0.156-+0.008 pWmin (mean-+SEM, n=4, pO.05). Comparison of apical Na+/H÷ exchangeactivity in the presence of CI-versus absence of Cl-(substitution by either gluconate or NO3-) revealed no evidencefor a CI-dependenceof Na+/H+exchangein the colonic crypt. CONCLUSION:Even epithelial cells at the base of colonic crypts have potential to mediate electroneutral sodium absorption, and NHE-2 is likely the relevant apical NHE isoform of these epithelial cells.

calmodulin, Pyk-2 and Src kinases. EGFr phosphorylationcauses activation of ERK isoforms of the MAP kinase family and inhibition of carbachol-stimulatedchloride secretion. Studies in other systems have shown that carbachol causes EGFr ligand release. We investigated whether EGFr-mediatedlimitation of the chloride secretory response to carbachol involves release of an EGFr-hinding ligand. Methods: All experiments were conducted with confluent monolayersof T~ colonic epithelial cells grown on permeablesupports. Protein phosphorylation was measured by Western blotting and densitometry. Potassium channel conductance, which regulates calcium-mediatedchloride secretion, was measured by ~Rb efflux. TGF-a releasewas measuredby ELISA.Results:Pre-incubationof T~ cells with a neutralizingantibody (10 pg/ml) to the extracellular ligand binding domain of the EGFrdecreasedcarbachol (100 /~M) induced phosphorylationof the EGFrat 2 min by 70-+4% (p
2682 Ca2+ Activates Ci bat Not K+ Channels in NCM460 Ceils Heather M. Jones, State Unlv of New York, Buffalo, NY; Mary Pat Moyer, Incell Corp, San Antonio, TX; Peter Lance, Michael E. Duffey, State Univ of New York, Buffalo, NY

Intestinal secretion of water and electrolytes occurs as a result of stimulation of CI- and K÷ channels in the intestinal epithelium by agonists that act through the intracellular second messengers Ca2. and cAMP. We are studying activation of CI- and K+ channels by Ca2+ in the non-transformed immortalized colonic epithelial secretory cell line, NCM460. The Ca2÷ signaling response of NCM460 cells to the cholinergic agonist, carbachol, was investigated using fluorescencemicroscopy. The 340 nm to 380 nm fluorescenceratio of the Ca2+-sensitive dye, Fura-2, was used as an index of [Ca2+],. In resting cells this ratio averaged0.13_+0.3 (_+SEM, N=198), corresponding to a [Ca2+], of 35 nM. Exposure to carbachol (100 p.M) caused a rapid rise in the fluorescence ratio in 96% of cells to 0.41 _+0.08, correspondingto [Ca2+], of 210 nM. The response of CI and K+ channels to the carbachol-induced rise in [Ca2+],was measuredusing the whole-cell voltage-clamptechnique. Patch pipettes were filled with a simulated intracellular solution, and the cells were bathed in a standard NaCIsolution. When membrane potential was voltage-clamped to the K+ equilibrium potential (-80 mV), carbachol (100 p.M) induced an inwardly directed CI-current with a peak value of 66-+16 pA in 62% of cells tested. These results demonstrate that CI channels are activated by the carbachol-inducedrise in [Ca2+],.When membranepotentialwas clampedto the Cl'equilibrium potential (0 mV), carbacholinduced only insignificant K÷ current. This differs from the increase in K+ conductanceseen in other secretory epithelial cells where K+ channelsare co-activated with Cl-channelsto maintain membrane potential. To further characterizethe Ca2+-activated CI conductance, we measured whole-cell current during application of a rapid-step voltageclamp protocol in order to generatecurrent-voltage (IV) relationships. In 6 cells the conductance increasedduring carbachol application and the reversal potential shifted toward the CI equilibrium potential, consistent with activation of CI- channels but not K+ channels. The IV relationships were linear between -+120 mV. To examine the lack of a carbachoHnduced increase in K+ conductance,we designedoligonucleotideprimers to probe for the K+ channel, hlK1, a CaZ*-activatedchannel. RT-PCRusing NCM460cDNAshowedthat these cells express hlK1, suggesting that factors other than Ca2÷ regulate the K* conductance. Overall, these results show that NCM460 cells can be used to study the regulation of Cl-secretory channels by Ca2+.

2680 Guanylate Cyclase C (GC-C) Mediates Acid-Stimulated Duodenal Mucosal Bicarbonate Secretion (DMBS) Daniel L. Hogan, Univ of CA, San Diego, San Diego, CA; Diane L, Crumble, Univ of CA, San Diego, San Diego, CA; Stephen J. Keely, Univ of CA, San Diego, San Diego, CA; Elizabeth A. Mann, Meg SheiI-Puopoio, Ralph A. Gianella, Link, of Cincinnati, Cincinnati, OH; Kim E. Barrett, Univ of CA, San Diego, San Diego, CA; Jon L Isenberg, Univ of CA, San Diego, San Diego, CA; Vijaya S. K. Pratha, Univ of CA, San Diego, CA

Background:In GC-C knockout mice, DMBS quantitated in vitro, is impaired in response to agonists including: heat stable toxin of E. coil (STa, acting via cGMP), PGE2(viacAMP) and carbachol (via Ca2*). However, bicarbonatesecretory responsesare normal with direct intracellularstimulation by db-cAMP,8Br-cGMPand thapsigargin.The MAP-kinase/ERKphosphorylaUon signal-transduction pathway is involved in colonic epithelial chloride transport, and may be of importance in DMBS.Aim: To determineif GC-CmediatesDMBSin responseto a physiologic stimulus, luminal acidification,and to examineif cGUP and ERK phosphorylation participates in this response. Methods: Utilizing the GC-Cmurine model, GC-C(-/-) animals were compared to normal wildtype [WT; GC-C(+/+)] mice. Anesthesia was induced with hypnorm/midazolam (i.p.). The proximal duodenum (4-7ram) was cannulated, perfused with 154 mM NaCI (0.2 ml/min) and [HCO3-]measured by back titration. The duodenal segment was stimulated with HCI (10 mM, 5 min; 8.5/~moles). At least 6 animals were studied in each series. In addition, to determine levels of cGMP and phosphorylated ERK in response to acid stimulation, proximal duodenumwas excisedand exposedto 10 mM HCIfor 5 minutes. Samples were homogenizedin buffer and cGMP EIA or western blot analysis was performed. Results: Basal HCO3secretionwas diminished in GC-C(-/-) vs. WT, 2.6 -+ 0.5 vs. 5.0 -+ 0.8 p.mol/cm-h, respectively (P
2683 Ca*2-Oependent CI Secretion in T84 Epitholia: Rote ot SOts and Na*-K ÷ ATPase. Jaekyung Cecilia Song, Bath israel DeaconessMedical Ctr, Boston, MA; Patangi K. Rangachari, McMaster Univ, Hamilton Canada;Jeffrey B. Matthews, Beth Israel Deaconess Medical Ctr, Boston, MA

Intracellular Ca+2regulation of Cl-secretion (short-circuit current, I=) in T84 intestinal epithelia is incompletely understood.The time course and magnitude of the I= responsediffers among various Ca+2agonists.Carbachol(CCh)elicits a transient I= whereasthe responseto the Ca+2ATPase inhibitor thapsigargin {TO) is sustained. This is attributed to the generation of tipid inhibitors that terminate the CCh response. We wondered whether the sustained I,cevoked by Tg could reflect a role for store-operatedCa+2channels(SOCs) or the sustainedactivation of specific apical (AP) and basolateral (BL) transport pathways.Also, we examinedwhether SOCs are involved in the synergistic enhancement of the cAMP (forskolin) I,c response by Tg. Methods: Confluent T84 monolayers were examined by dual voltage-current clamp. BL K÷ conductancewas assessedby the current responseafter AP membranenystatin permeabilization and application of a K+ gradient (IK). BL Na-K ATPase and Na-K-2CI cotransporter (NKCC1) activity were measured as ouabain- and bumetanide*sensitiveeeRbuptake, respectively. All data had p3. Results: 1) Roleof SOCsin Tgresponse. A remove-and-replaceprotocol for buffer Ca+2revealed that the Tg-inducad Iscconsisted of two phases: an early transient phase that is unaffected by the presenceof BL Ca+2followed by a sustained phase that requires BL Ca÷2. BL addition of the SOCs inhibitor La+3dosedependently blocked the second but not the first phase of the I~. Removal of AP Ca+2did not affect Tg I=. 2) Influenceof SOCson transportpathways. Both the early SOCs-independent and late SOCs-dependentphases of the Tg response were blocked by AP DIDS, suggesting both phases depend on AP Ca+2-activatedCI channels. Tg evoked an early but transient increase in I~that was independent of 8L Ca+2and paralleled the early SOCs-independent phase of the I~. Tg evoked a later increase in =Rh uptake that was inhibited by ouabain but not bumetanide;this activation of Na-K ATPaseactivity required the presenceof BL Ca+2. 3) Requirementof SOCsfor enhancementof cAMP/~ Forskolin ~mulated an I~ independently of BL Ca+2, However, synergistic enhancementof this I~ by Tg was prevented by BL Ca*2 removal or by La÷3. Conclusion:Tg-induced CI- secretion consists of an initial SOCs-independent phase involving activation of BL K+ channels and a later SOCs-dependentphaseinvolving activation of Na-K ATPase.Sustained synergistic enhancementof cAMP-elicited secretion by Tg also involves BL SDCs.

2681 Apical Na*/H+Exchanga near the Base of Mouse Colonic Crypts Shaoyou Chu, Jingsong Chu, Marshall H. Montrose, Indiana Univ Sch of Medicine, Indianapolis, IN

BACKGROUND:It has been shown that colonic crypts can absorb fluid, but identity and focalization of the absorptive transporters remains speculative. Since intracellular pH (pHi) regulation and electroneutral NaCI absorption are closely related functions in mammalian colon, we askedwhether apical Na+/H+ exchangewas a featureof the relativelyundifferentiated cells near the base of colonic crypts, and sought to characterizeany such Na+/H+ function with respect to known NHE (Na+/H+ exchanger) isoforms or Cl-dependent Na÷/H+ exchange (CI-NHE). METHODS:Freshly isolated and muscle-stripped mouse distal colonic mucosa was mounted in a chamber, dye loaded with 5 p.lVl BCECF/AM,superfused from both luminal and basolateral sides and observed with a Zeiss LSM510 confocal microscope and a 40 x water immersion objective. Fluorescenceexcitation was alternated between 488 nm and 458 nm lines of an Ar-laser, with emission collected at 505 nm- 545nm. Ratio images of 488nm/ 458nm fluorescence were used to calculate intracellular pH (pHi) according to a calibration curve determined with isolated colonocytes loaded with BCECF/AM. Measurements of pH, were reportedfor the crypt epithelialcells approximatelylO~m from the crypt base. RESULTS: Both apical and basolateral Na÷/H+ exchange function are readily detected in these crypt epithelial cells. Rate of initial 4 min Na+ dependent pH, increase (from acidification by

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