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JAM-A Regulates Proliferation in the Intestinal Epithelium ThroughModulation of PI3K/AKT/β-Catenin SignalingPorfirio Nava Dominguez, Christopher Capaldo, Stefan Koch, Keli N. Kolegraff, CarolineR. Addis, Mattie Feasel, Charles A. Parkos, Asma Nusrat

The tight junction (TJ) localizes in the most apical region of the lateral membrane and isessential for regulating themovement of ions andmolecules through the paracellular pathway.Additionally, TJ proteins regulate several physiological process including differentiation andproliferation. TJs are formed by a core of transmembrane proteins that are anchored to theactin cytoskeleton via cytosolic plaque proteins. Previously we reported that the transmem-brane TJ protein Junctional Adhesion Molecule-A (JAM-A) regulates epithelial proliferation,but the signaling mechanisms underlying this process were unknown. Here, we report thatJAM-A controls intestinal epithelial cell proliferation by modulating PI3K/Akt/β-cateninsignaling. Using an siRNA approach to down-regulate JAM-A expression in a model intestinalepithelial cell line, we observed that decreased JAM-A expression leads to increased cellproliferation and enhanced transcriptional activity of β-catenin, a known regulator of cellproliferation. In addition, JAM-A deficient cells exhibit increased levels of activated Akt, aserine/threonine kinase that has been previously shown to regulate β-catenin-dependenttranscription. Consistent with a role for Akt/β-catenin signaling in our model, we foundthat Akt-mediated phosphorylation/inhibition of GSK3b (Ser9) was increased in cells lackingJAM-A. Furthermore, Akt inhibition using Triciribine significantly reduces both β-catenintranscriptional activity and cell proliferation in JAM-A deficient cells. In agreement with ourIn Vitro findings, JAM-A knockout mice display increased cell proliferation and enhancedAkt/β-catenin signaling in the intestinal epithelium. In conclusion these results offer amolecular basis for the link between JAM-A and regulation of proliferation in intestinalepithelial cells.

861a

TGF-β1 Upregulates Serotonin Transporter Function and Expression via PI3Kand SMAD 3-Mediated PathwaysRavinder K. Gill, Anoop Kumar, Arivarasu Natarajan Anbazhagan, Amika Singla, Saad F.Nazir, Shubha Priyamvada, Waddah A. Alrefai, Seema Saksena

The serotonin transporter (SERT) represents a critical mechanism for regulating the extracellu-lar availability of serotonin (5-HT). A decrease in SERT expression has been implicated invarious GI disorders. For example, transgenic mice with deletion of SERT exhibit diarrheainterspersed with transient constipation. Also, SERT expression is down-regulated in modelsof inflammation, ulcerative colitis patients, in response to enteric infections and proinflam-matory cytokines. However, therapeutic strategies harnessing the regulatory mechanismsthat increase SERT function and expression in intestinal epithelial cells have not beenexplored. In this regard, TGF-β1, an important multifunctional cytokine has protectiveeffects on intestinal mucosa. Given that the increase in SERT function may be of potentialtherapeutic value in alleviating GI symptoms, we hypothesized that TGF-β1 upregulatesintestinal SERT. Current studies were, therefore, designed to examine both acute and long-term effects of TGF-β1 on SERT utilizing Caco2 cells grown on Transwell inserts and C57/BL6 mice as an In Vivo model. SERT function was measured as NaCl dependent 3H-5-HTuptake from the apical side. Treatment of Caco-2 cells with TGF-β1 from basolateral side(10ng/ml, 60 min) stimulated SERT activity (~2 fold, P<0.005). This stimulation of SERTfunction was dependent upon activation of TGF-β1 receptor (TGFR) and PI3K dependentpathways as SB-431542, a specific TGFRI inhibitor and LY294002 (50 μM, PI3K inhibitor)blocked the stimulatory effect of TGF-β1, respectively. Further, TGF-β1 treatment of cellstransfected with SERT-GFP caused increased co-localization of SERT with fluorescent wheatgerm agglutinin on the apical surface concomitant with a decreased SERT expression in theintracellular compartments, indicating involvement of trafficking events. Additionally, TGF-β1 treatment (10ng/ml, 1h) ex vivo increased 3H-5-HT uptake in mouse ileum. Long-termtreatment of Caco-2 cells with TGF-β1 (10 ng/ml, 24h) also increased SERT function andmRNA levels. TGF-β1 mediated transcriptional modulation of responsive target genes occursvia phosphorylation and nuclear translocation of proteins called Smads (smad1-7). Interest-ingly, TGF-β1 stimulation of SERT function was abolished by SIS3, a Smad-3 inhibitor. InVivo administration of activated TGF-β1 (5 μg/mouse, 6h) by gavage increased SERT mRNAlevels in the colon (~ 3 fold) and increased mucosal 5-HT content. Our results demonstratethat TGF-β1 upregulates SERT by a dual mechanism involving participation of both post-translational (PI3K and trafficking) and transcriptional (smad3) mechanisms. These dataprovide novel insights into the molecular mechanisms involved in the regulation of SERTby TGF-β1 that may be relevant to the development of better therapeutics for gut disorders.

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CAM Kinase IIγ Inhibits Activity and Directly Binds the C-Terminal Domainof the Intestinal Brush Border Na/H Exchanger NHE3 in a Dymanic, Ca2+Dependent MannerMirza Zizak, Rafiquel Sarker, Tian-e Chen, Boyoung Cha, Marjan Gucek, Robert N. Cole,Ming C. Tse, Mark Donowitz

Background: NHE3, the epithelial brush border Na/H exchanger, is acutely regulated bychanges in BB trafficking. A multiprotein complex which links NHE3 to the cytoskeletonassociates with the NHE3 C-terminal domain between A.a. 586 and 605. Ca2+/calmodulindependent protein kinase II (CaM KII) is one of the proteins which binds in this area,helping set basal NHE3 by inhibiting its activity. The goal of these studies was to identifywhich CaMKII isoform(s) binds NHE3, localize its binding site, and characterize how thisbinding regulates Na+/H+ exchange. Methods: PS120 fibroblasts stably transfected withNHE3 or Caco-2/BBe cells transiently infected with adenovirus-rabbit NHE3 were studied.NHE3 activity was measured by BCECF/fluorescence. IP studies were performed with HA-or VSVG-antibodies coupled to beads to precipitate NHE3 or anti-CaMKIIγ. LC-MS/MS andRT-PCR were used to identify the CaMKII isoforms. GST-fusion proteins were used for pulldown studies. Results: CaMKII inhibits basal NHE3 activity in Caco-2 and PS120 cells since

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inhibiting NHE3 (KN-62 or KN-93) stimulated basal NHE3 activity. This process wasNHERF2 dependent based on knocking down NHERF1 and NHERF2 separately in Caco-2 cells (lentivirus-shRNA-puromycin). The Caco-2 cells expressed only the γ and δ CaMKIIisoforms, as detected by RT-PCR. LC-MS/MS demonstrated that NHE3 binds to the γ isoformof CaMKII . CaMKII2γ bound to a His-fusion protein corresponding to aa 582-667 of NHE3.This occurred in the absence of calmodulin or autophosphorylation of CaMKII. Binding wasfurther localized to aa 586-605 using NHE3 truncations. CaMK2γ co-precipitated with NHE3in a Ca2+ dependent manner, with more binding in the absence of Ca2+ . Site directedmutagenesis of NHE3 showed that Leu596 and Met598 were critical for binding since doublemutant (L596Q,M598A) completely abolished binding of CaMKIIγ to NHE3 in In Vivo co-immunoprecipitation studies. Conclusions: 1) The CaMKII isoforms present in Caco-2 cellsare γ and δ, while PS120 cells expressed only the γ isoform. 2) CaMKII inhibits NHE3 inPS120 and Caco-2 cells under basal conditions by a NHERF2 dependent process. 3) NHE3is a CaMKIIγ binding protein. Binding occurs between aa 586-605. 4) This binding occursunder basal Ca2+ conditions , involves aa 596 and 598, and is reduced by elevated Ca2+.5) CaMKIIγ is a dynamic regulator of NHE3, manipulation of which should be consideredfor treatment of diarrheal diseases.

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K+ Efflux Mediated via Serosal KCNN4 (Kcnn4b), but Not Mucosal Kcnma1Channels Provide the Driving Force for cAMP-Induced Anion Secretion in RatDistal ColonVazhaikkurichi M. Rajendran, Geoffrey I. Sandle

Background: High anion (Cl- and HCO3-) and K+ concentrations are present in cholera-

induced diarrheal stools, as cholera toxin (cAMP) activates both mucosal anion (CFTR) andK+ channels. Both Kcnn4c (IK, KCa3.1) and Kcnma1 (BK, KCa1.1) channels that mediateelectrogenic K+ secretion are present on the apical membranes of colon. In recent studies,we have shown that activation of mucosal Kcnn4c by DC-EBIO (a Kcnn4 channel opener,which also activates CFTR), provided the driving force for anion secretion. cAMP (choleratoxin) has also been shown to induce active K+ secretion. Aim: Present study was initiatedto identify whether mucosal Kcnn4 (Kcnn4c) and/or Kcnma1 channels mediate the cAMP-induced electrogenic K+ secretion and that cAMP-induced K+ secretion also provided thedriving force for anion secretion. Methods: Mucosal to serosal (m-s) and serosal to mucosal(s-m) 86Rb (K+ surrogate) fluxes were measured under voltage clamp conditions in rat distalcolonic mucosa mounted In Vitro. Net K+ fluxes were calculated by subtracting s-m fromm-s. Positive and negative net fluxes represent absorption and secretion, respectively. Short-circuit current (Isc) reflected anion secretion. Intracellular cAMPwas increased using forskolin(FSK, 10 μM). Results: Mucosal addition of VO4 (a P-type ATPase inhibitor) inhibited netK+ absorption present in normal distal colon. In the presence of mucosal VO4, serosaladdition of FSK induced both anion (2.1 ± 0.3 vs 7.2 ± 0.8 μEq/h ● cm2) and net K+

secretion (-0.63 ± 0.06 μEq/h ● cm2). Mucosal addition of iberiotoxin (IbTX; a Kcnma1channel blocker) and charybdotoxin (CTX; a common inhibitor of Kcnn4 and Kcnma1)inhibited FSK-induced K+ secretion by 92 and 96%, respectively. In contrast, neither IbTXnor CTX significantly affected the FSK-induced Isc. Serosal addition of chromanol 293B (acAMP-activated K+ channel blocker) and TRAM-34 (a Kcnn4 channel blocker) inhibited theFSK-induced Isc by 12 and 78%, respectively. Simultaneous addition of chromanol 293Band TRAM-34 inhibited the FSK-induced anion secretion by 86%. Neither chromanol 293Bnor TRAM-34 affected the FSK-induced K+ secretion. Conclusions: 1) K+ efflux mediatedvia serosal Kcnn4 channels, but not mucosal Kcnma1 channels that mediate K+ secretionprovide the driving force for cAMP-induced anion secretion; 2) cAMP-induced K+ secretionmediated via Kcnma1 channels contribute to the high concentration of K+ present in secretorydiarrhea; and 3) cAMP-induced Cl- secretion and K+ secretion occur independently ofone another.

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Activation of AMPK Inhibits Guanylin Induced Chloride Secretion in RatColonSascha Kopic, Peter J. Geibel, John P. Geibel

INTRODUCTION: Acute Diarrheal Illness (ADI) is the second leading cause of mortality inchildren under five, resulting in over 2 million deaths each year due to dehydration andloss of vital salts. In children, ADI and malnutrition are inexorably linked, creating a tragiccombination often resulting in long-term disabilities that adversely affect the productivitypotential of these individuals if or when reaching adulthood. Secretory diarrhea occurs asa result of increased intestinal ion secretion through chloride channels and a concomitantimpairment of electrolyte absorption. Our preliminary findings and observations from othergroups indicate that the “cellular energy sensor” AMP-activated protein kinase (AMPK) caninteract with a variety of these ion transport mechanisms, including the cystic fibrosistransmembrane conductance regulator (CFTR), which represents the main chloride conduct-ance pathway during secretory diarrhea. The aim of this investigation is to exploit thisinteraction by inhibiting toxin induced chloride hypersecretion through activation of AMPK.METHODS: Single isolated rat colonic crypts were placed in a perfusion chamber and loadedwith the fluorescent chloride indicator dye N-(ethoxycarbonylmethyl)-6-methoxyquinolin-ium bromide (MQAE). Changes in intracellular chloride concentrations were monitored inreal time using a digital imaging setup. Following perfusion with a standard HEPES bufferedRinger's solution; isolated crypts were exposed to a chloride-free solution to observe thekinetics and amount of chloride efflux. Subsequently, the rate of chloride efflux (Δ(F/F0)/sec) was calculated and used for statistical comparison. Chloride efflux was stimulated bythe cyclic nucleotide activators forskolin (cAMP), or guanylin (cGMP), an analogue of theheat-stable enterotoxin (STa) of E.coli. AMPK was activated by the cell-permeable AMPanalogue 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR). RESULTS: Bothforskolin and guanylin increased chloride efflux in comparison to unstimulated conditions.Forskolin and guanylin induced chloride secretion could be abrogated by activating AMPKwith AICAR. CONCLUSION: Activation of AMPKmay thus effectively ameliorate pathologicalion handling during secretory diarrhea and may represent a novel supplemental therapeuticapproach in the treatment of ADI.

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