Rab11a-FIP1 and Rab11a-FIP2 translocate to the secretory canaliculus of parietal cells upon stimulation with histamine

easily elevatedby pirenzepineand 4-DAMP methiodide, M1 and M3 antagonists, but not by famotidine or YM022, a gastrin antagonist.GastricpH of wild-type mice were partially sensitive to famotidine, the M1 and M3 antagonists,but insensitiveto YM022. This indicatesthat basal acid productions are mediated by the histaminergic and cholinergic pathways in wild-type mice and only by the cholinergic pathway in H2R null mice. Secratagogue-inducedacid secretions were measuredin mice maintainedon anesthesiawith ether. After thorough washes of gastric lumen with saline and pylorus ligation, saline, histamine (10 mg/kg), carbachol (0.05 mg/kg) or gastrin (1 mg/kg) were administered peritoneally.Gastricjuice was collected 30 min after the administration. Basal secretion was not different betweenwild-type (0.93 _+ 0.18 pEq/h 20) and H2R null mice (0.92 _+ 0.19 p,Eq/h). H2R null mice were unresponsive to histamine or gasttin, but responsiveto carbachol, with impaired ~iciency (4.67 _+ 0.39 vs 10.94 +_ 0.84/LEq/h). Thus, gastrin receptors on padatal cells seem not to participate directly in gastric acid production. We then examined immunohistochemically the effect of carbachol administration on subcellular localization of HK-ATPase. Carbachol induced translocation of HK-ATPaseand increase in parietal cell size in wild-type mice. In contrast, no apparent changes in sobcellular localization of HK-ATPase and parietal cell size was observed in H2R null mice. In conclusion, gastric acid secretion in the H2R null mice is only under the control of cholinergic stimulation. Consideringthe increasedgastrin levels and the impairment of HK-ATPasetranslocation,there may be a severedeficiencyin the acid production mechanism that cannot be balanced by cholinergic stimulation in H2R null mice.

545 Gut-Enriched Kruppei-Like Factor (GKLF) inhibits Cellular Proliferation by Blocking the G1/S Boundaryof the Cell Cycle Xinming Chen, David C. Johns, Deborah E. Geiman, Eduardo Marhen, Vincent W. Yang, Johns Hopldns Univ, Baltimore, MD BACKGROUND:GKLF(KLF4) belongs to the zinc finger-containing KLF family of transcription factors (Int J Biochem Cell Bin/32: 1103, 2000). Expression of GKLFis associated with growth arrest. Previousstudies indicatethat forced expressionof GKLFinhibits DNA synthesis. AIM: To determine the location in the celt cycle in which GKLF exerts its inhibitory effect. METHODS: An inducible system with regulated GKLF expression was established using a modified Ecdysone-lnducibleMammalianExpressionSystem(Inwtrogan). Stableclones(called EcR-RKO) were first established in a human colon cancer cell line, RKO, that expressed a transfected haterodimeric eedysoneand retinoid X receptor (EcR/RXR). An effector plssmid containing the enhanced green fluorescence protein (EGFP) and GKLF under the control of the ecdysone receptor element (EcRE) was then generatedand stably introduced into EcRRKO cells. GKLFwas highly induced upon addition of an ecdysone analogue, Ponasterone A, as evidenced by the appearance of EGFP and GKLF in the same cells using double immunofluorescence imaging, Western and Northern blot analyses.Cell cycle analyseswere performed foUowing various periods of induction by a Fluorescence Activated Celt Sorter (FACS).The sameeffector was also used as a shuttle vector to generatea replication-defective recombinant adenovirusthat harbors EGFPand GKLFunder the inducible promoter. Cell cycle analyses were similarly performed following infection and induction of EcR-RKO cells with the recombinant virus. RESULTS:In the absence of the inducer, GKLF-transfected EcR-RKO cells proliferated at a rate similar to that of untransfected EcR-RKOcells. However,the rate of proliferationof the transfected cells was significantly reducedin the presenceof Ponasterone A. FACSanalysesshowed that the proportion of transfected cells in the G1 phase of the cell cycle was significantly increased in the presence of PonasteroneA when compared to that in its absence. Conversely,the proportion of transfected cells in the S phase was significantly decreasedin cells treated with the inducer comparing to untreated cells. A similar G1/S block was observed in cells infected with the recombinantEGFP-GKLFadenovirusand induced with Ponasterone A, but not in infected but uninduced cells or in EGFPalone-containing virusinfected and induced cells. CONCLUSIONS:The negative effect of GKLF on cell proliferation is due to its ability to induce cell cycle arrest at the G1/S boundary.

543 Rablla-FIP1 And Rablla-FIP2 Translocate To The Secretory Canaliculus Of Parietal Cells Upon Stimulation With Histamine Chadwick M. Hales, Lynne A. Lapierre, Matthew Dorn, Medical Coil of Georgia and Augusta VAMC, Augusta, GA; Richard Gdner, Augusta State Univ, Augusta, GA; James R. Goldendng, Medical Coil of Georgia and Augusta VAMC, Augusta, GA Apical recycling is an important aspect of epithelial cell function. In pariata[calls, where apical recycling is critical for acid secretion, Rabl la is enrichedon tubulovesiclemembranes.Rab11a has been implicated in the regulation of apical recycling in a number of epithelial cells, including padetal cells, Until recently, two Rablla binding proteins had been discovered: Rabl 1a binding protein (Rabl laBP) and myosinVb.Current researchby our lab has discovered two other Rablla binding proteins called Rabl 1 family interacting protein 1 (FIPt) and Rabl 1a family interacting protein 2 (FIP2). Yeast two hybrid binding assays, immunocytochemistry and Rabtla overlays all demonstrate Rablla association with FIP1 and FIP2 through an amphipathic alpha helical motif. Since there is an enrichment of Rablla in gastric padetal cells and becauseRablla moves in concert with the H-K-ATPaseupon stimulation, we were interested in studying the location of FIP1 and FIP2 under stimulatsd and resting conditions. Both FIP1 and FIP2 codlstributed with Rablla and H-K-ATPasein preparations of enriched parietal cell tubulovesicles. In immunofluorescence studies of cultured rabbit parietal cells, FIP1 colocalizedwith Rablla and moved with Rablla after stimulation with histamine. FIP2 colocalized with the H-K-ATPase and moved with the H-K-ATPaseafter stimulation with histamine. These studies indicate that FIP1 and FIP2 are present on the tubulovesicles with Rablla and the H-K-ATPase,and that they may be involved in facilitating the recycling of the vesicles into and out of the canaliculus. These data may also indicate the formation of a multiprotein complex regulating tubulovesicle movement during stimulation.

546 Phosphorylation of the Cdx2 Activation Domain Is Mediated by Mitogen-Activated Protein Kinase Edmond H. H. M. Rings, Frangois Boudreau, Jennifer K. Taylor, Jennifer Moffett, Eun Ran Sub, Pater G. Traber, Univ of Pennsylvania,Philadelphia,PA Introduction: Cdx2 is an intestine-specifictranscription factor. Previously, we reported that the activation domain (AD) of Cdx2 resideswithin amino acids (AA) 15-180. The mechanistic basis for modulation of Cdx2 function in response to cellular signaling events is yet to be elucidated.Therefore,we hypothesizethat phosphorylationof the AD can modulatethe function of Cdx2. Methods:The AD of Cdx2 was further delineatedusing different portions of the Cdx2 protein fused to the Gal4-DNA binding domain (DBD) in transient transfections in NIH3T3 cells. Metabolic labeling of Ga[4-DBD Cdx2(15-180) fusion protein with ~P-orthophosphete in transiently transfected cells and subsequentimmunoprecipitationwere performed to study in vivo phosphorylation. Site directed mutagenesls was conducted to replace a potential phosphorylation site within the AD. Polyclonalantibodies were generatedto study a potential phosphorylation site: CNL was raised against AA 54-66 of mouse Cdx2 and P-Cdx2-S60was directed against the same epitope in which the serine 60 ($60) was phosphorylated.Cterm, raised against AA 263-278, was used as a control antibody. Results: Mapping of the AD indicatesthat a critical region for activation resides within AA 50-70. Substitution of a serine with alanine at AA 60 of Cdx2 (S60A) in Gal4-DBD Cdx2(15-180) reduces the incorporation of ~P-orthophosphate compared to the wild-type fusion protein. This indicates that this site is a major phosphoryiation site within the AD. Phosphoryt~on ol Cdx2 can be inhibited with the mitogen-activatedprotein kinase (MAPK) kinase (MEK)I inhibitor PD98059 or the MEKI/ 2 inhibitor U0126, resp. Thesedata support the notion that the $60 residue is phospherylated by a MAPK. P-Cdx2-S60,which exclusively recognizesthe phosphorylated $60 of the Cdx2 protein, detects Cdx2 mainly in the enterocytes of crypts and in the lower parts of the villi of the adult mouse small intestine, as revealedby immunohistochemistry. In contrast, CNL recognizes Cdx2 mainly in villous enterocytes, while Cterm recognizesCdx2 in both crypts and villi. In the colon, Cdx2 is phosphorylated in most colonocytes, similar to the presence of Cdx2as detectedby Cterm. Conclusions:Cdx2 is a phospho-proteinand its AD is phosphorylated by a MAPKat position $60. S60-phosphorylatedand S60-nonphosphorylatedCdx2 have different spatial expression patterns along the crypt-villus axis, suggesting distinct functions for these two forms of Cdx2 as they relate to intestinal differentiation.

544 CUGGP2, a 54-kDa RNA-bindingProtein Modulates Apolipoprotein B mRNA Editing by Sequectering apobec-1 and ACF Shrikant Anant, Jeffrey O. Henderson,Debnath Mukhopadhyay,Susan Kennedy,Nicholas O. Davidson,Washington Univ Sch of Medicine, St. Louis, MO Apolipoprotein B (apoB) circulates in two distinct forms, apoBlO0 and apoB48. ApoBlO0, synthesized exclusively in the human liver, is present as a major protein component of low density lipoprotein particles whereasapoB48,synthesizedin the intestine, is found in association with chylomicron and chylomicron remnant particles. Both proteins are the product of a singe APOB gene but a single C to U editing event resu~ in the conversion of a CAA (giutamine) codon to a UAA stop codon and the translation of the smaller apoB48 protein. The minimal editing complex consists of two proteins, apohec-1 and ACF. Apobec-1 is the catalytic subunit of the editing enzyme and encodes an RNA-specific cytidine deaminase activity, required for the editing reaction. ACF, the apobec-1 complemantationfactor is the structural componentof the editing enzymethat is requiredfor RNA editing in vitro. However, the composition of the holoenzymein vivo is currently unknown. We have now identified, by yeast two-hybrid screening, a 54-kDa protein, CUGBP2, using apobec-1 as bait. CUGBP2 binds to apobec-1 and ACF, both in vivo and in vitro. Contoca[ microscopy demonstrated that CUGBP2interacts and sequestersapobec-1 in the cytoplasm. On the other hand, CUGBP2 interacts and colocalizeswith ACF in the nucleus. Immunodepletionof CUGBP2from bovine liver $100 extracts resulted in loss of complementationactivity, which was reconstitutedwith addition of exogenousACF but not CUGBP2.These data suggest that in cells, CUGBP2binds to apobec-1 and to ACF. Knockout of CUGBP2 expression in McArdle cells using antisense oligonucleotides resulted in a two-fold increase in editing activity suggesting that CUGBP2 is a negative regulator of apoB RNA editing. We determinedthat CUGBP2 is an RNA binding protein, binding to AU-dch sequences.Usinga seriesof apoBRNAmutants we havedetermined that CUGBP2binds to a sequence5' to the edited base. Immunoprecpitationof CUGBP2from $100 extracts of McArdle cells co-precipitated apoB RNA suggesting that it is bound to the RNA in vivo. Analysis of the CUGBP2bound RNA demonstrated preferential binding to edited RNA. Taken together, these data suggest that CUGBP2 is an integral member of the apoB mRNA editing enzyme which plays an important role in modulating RNA editing activity.

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Susrase-lsomaltase Gene Transcription Requires eoth Cdx and HNF-1 Regulatory Sites and Is Modulated by the in vivo Interaction Between Cdx2 and HNF-I~ Proteins. Francois Boudreau, Edmond H H M Rings, Jennifer Moffatt, Gary P. Swain, Peter G. Traher, Univ of Pennsylvania,Philadelphia, PA INTRODUCTION:Sucrase-isomaitase(SI) gene is an entemcyte-specificgone expressedin a complex developmentalpattern. A short evolutionarily conserved SI gone promoter recapitulates developmentalexpression of SI in the mouse small intestine. This short gene contains two regulatory sites, SIF3 and SIF1, that interact with HNF-1 and Cdx proteins, respectively. We previously reported that HNF-1 regulatory site is essential to support SI transcription in mouse transganic mice as well as during enterocytedifferentiation. This observation prompted us to investigate the importance of the Cdx site to regulate SI transcription. METHODS: Transganic mice were generated using constructs harboring point mutations in the SIF1 elementthat linked nucleotides-8500 to + 54 of the rest goneto the human growth hormone

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