Sa1735

Proteasome Beta Subunits Interact With the PAI-2 and Reg1 Promoters andAre Required for Gastrin-Stimulated TranscriptionAdrian O'Hara, Alice E. Howarth, Andrea Varro, Rod Dimaline

The hormone gastrin helps maintain gastric mucosal integrity by regulating expression ofgenes such as plasminogen activator inhibitor 2 (PAI-2) and Regenerating protein 1 (Reg1) , butthe mechanisms involved are incompletely understood. We hypothesized that the proteasomebeta subunit PSMB1, recently implicated in the gastrin responsiveness of vesicular monoaminetransporter 2, may be required for gastrin-mediated upregulation of PAI-2 and Reg1. PSMB1was knocked down by siRNA in the gastric cancer cell line AGS-GR, which expresses thecholecystokinin 2 receptor, and the gastrin response to PAI-2 (1.6kb) and Reg1 (2.1kb)promoter-luciferase reporter constructs determined. In control cells (scrambled RNA) gastrin(2nM G17 for 6h) increased PAI-2 transcription by 9.2±1.5 fold (n=6), but in cells withPSMB1 knocked down (69.5±5.5% protein reduction), this was significantly reduced to2.1±0.2 fold (p,0.001, ANOVA). In control cells, gastrin increased Reg1 transcription by2.4±0.9 fold (n=9), but with PSMB1 knocked down, the response was abolished (1.2±0.1fold, p,0.03). To investigate PSMB1 binding to the promoters, ChIP assays were performedusing an anti-PSMB1 antibody for immunoprecipitation, or IgG control. In anti-PSMB1precipitated chromatin from cells treated with gastrin (2nM G17 for 2h), there was a 7.9±2.5(n=3) fold enrichment of DNA compared to control, determined by real time PCR usingprimers that generated a 250bp amplicon incorporating the region of the PAI-2 promoterknown to respond to gastrin. Using primers generating a 218bp amplicon including thegastrin responsive region of the Reg1 promoter we observed 10.0±4.6 fold enrichment.Immunocytochemistry of unstimulated AGS-GR cells revealed distribution of PSMB1throughout the cytoplasm and nucleus. After 2h stimulation with 2nM G17, cytoplasmicPSMB1 was barely detectable and nuclear staining was intensified; after 6h stimulation,PSMB1 was again visible in cytoplasm. Western blotting of nuclear and cytoplasmic extractsshowed that the nuclear:cytoplasmic ratio of PSMB1 changed from 1.7±0.4 in untreatedcells to 10.6±2.6 (n=7, p,0.01) after 2h stimulation with G17. This subcellular redistributionwas not seen with alpha (PSMA5) or regulatory (PSMC1) proteasome subunits. PSMB1distribution was unaffected by activation of other receptors expressed by AGS-GR cells,including histamine, epidermal growth factor, PGE2, or IL-8. The response to G17 washowever mimicked by phorbol 12-myristate 13-acetate (0.1μM), and prevented by theprotein kinase C (PKC) antagonist Ro-32-0432 (1μM). We conclude that proteasome betasubunits bind to the PAI-2 and Reg1 promoters in response to gastrin stimulation of AGS-GR cells and are required for transcription. Gastrin induces a PKC-dependent subcellularredistribution of beta proteasome subunits that may be linked to their transcriptional func-tions.

Sa1736

Protein Kinase D1 (PKD1) Promotes Migration of Intestinal Epithelial CellsSteven H. Young, James Sinnett-Smith, Enrique Rozengurt

Background: Cell migration and proliferation in the GI mucosa play fundamental roles inhomeostatic turnover, restoration of tissue integrity after injury and pathogenesis of intestinaldisorders, including IBD and intestinal carcinogenesis. Despite its importance, the signalingmechanisms involved remain incompletely understood. Recently, we demonstrated thatprotein kinase D1 (PKD1), the foundingmember of a new family of protein kinases, stimulatesintestinal epithelial cell proliferation (J Biol Chem, 286, 511-520, 2011). Here, we examinedthe role of PKD1 in the regulation of migration of intestinal epithelial cells. Results: Woundingmonolayer cultures of intestinal epithelial cell line IEC-18 or IEC-6 induced rapid andselective PKD1 activation in the cells immediately adjacent to the wound edge, as judgedby immunofluorescence microscopy with an antibody that detects the phosphorylated stateof PKD1 at Ser916, an autophosphorylation site. An increase in PKD1 phosphorylation atSer916 was evident as early as 45 s after wounding, reached a maximum after 3 min, andpersisted for ≥15 min. PKD1 autophosphorylation at Ser916 in response to wounding wasprevented by the PKD family inhibitors kb NB 142-70 and CRT0066101. A rapid increasein PKD autophosphorylation was also elicited by wounding in IEC-6 cells. Using in vitrokinase assays after PKD1 immunoprecipitation, we corroborated that wounding IEC-18 cellsinduced rapid PKD1 catalytic activation. Further results indicate that PKD1 signaling isrequired to promote migration and polarization of intestinal epithelial cells into the denudedarea of the wound. Specifically, treatment with kb NB 142-70 or small interfering RNAstargeting PKD1 markedly reduced wound-induced migration in IEC-18 cells. PKD inhibitionalso prevented cell polarization of the cells at the wound, characterized by rearward position-ing of the nucleus and by prominent actin stress fibers at the posterior region of thelamellipodium. To test whether PKD1 promotes migration of intestinal epithelial cells invivo, we used transgenic mice that express elevated PKD1 protein in the small intestinalepithelium. Enterocyte migration, assayed by measuring the distance from the bottom ofthe crypt to the foremost BrdU-labeled enterocyte, was markedly increased in the PKD1transgenic mice. Conclusion: The results demonstrate that PKD1 activation is one of theearly events initiated by wounding a monolayer of intestinal epithelial cells and indicatethat PKD1 signaling promotes the migration of these cells in vitro and in vivo. We concludethat PKD1 plays a key role in mediating cell migration and proliferation in untransformedintestinal epithelial cells.

Sa1737

Intracellular CA2+ Oscillations Produced by the CA2+-Sensing Receptor AreMediated by Negative Feedback by Protein Kinase C At Thr888Steven H. Young, Osvaldo Rey, James Sinnett-Smith, Enrique Rozengurt

Background: The extracellular Ca2+-sensing receptor (CaR), a member of the C family ofheptahelical GPCRs, is expressed in many tissues and organs not directly involved in thecontrol of Ca2+ homeostasis, including intestinal epithelial cells. Our recent studies indicatethat CaR function negatively regulates the proliferation of colonic epithelial cells (J. Biol.Chem. 287,1158-1167, 2012). Consequently, the signaling mechanisms triggered via CaR

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activation are attracting intense attention. Studies in individual living cells have shown thatintracellular Ca2+ concentration ([Ca2+]i) oscillates upon stimulation of CaR by an elevationin extracellular Ca2+ concentration ([Ca2+]e). Here, we reexamined the notion that PKC-mediated phosphorylation of the CaR at Thr888 is necessary and sufficient for generating[Ca2+]i oscillations in response to an increase in [Ca2+]e. Results: To identify the mecha-nism(s) underlying [Ca2+]e-evoked [Ca2+]i oscillations via CaR, we analyzed the patternof [Ca2+]i response in 1335 individual HEK-293 cells transfected with the human CaR, anextensively used model system in studies of CaR regulation. An increase in [Ca2+]e from1.5 mM to 3 mM produced a rapid elevation in [Ca2+]i followed by striking oscillatoryfluctuations in [Ca2+]i that did not return to baseline values, a pattern known as sinusoidaloscillations. This pattern was observed in 70% of the cell population. Most other cells (27%)displayed a rapid peak and plateau response. To determine the role of PKC in the generationof [Ca2+]i oscillations, cells were exposed to increasing concentrations (0.5-5 μM) of thepreferential PKC inhibitor Ro-31-8220 prior to stimulation by extracellular Ca2+. Ro-31-8220at 3-5 μM completely eliminated the [Ca2+]e-evoked [Ca2+]i oscillations and transformed thepattern to a peak and sustained plateau response. Treatment with increasing concentrationsof other PKC inhibitors, including GFI or Go6983 produced an identical response. Similarly,treatment with GFI eliminated [Ca2+]e-evoked [Ca2+]i oscillations in colon-derived SW-480 cells expressing the CaR. To test further the role of PKC in the generation of oscillations,we expressed a CaR mutant in which the major PKC phosphorylation site, Thr888, wasconvereted to alanine (CaRT888A). None of the cells expressing CaRT888A (n= 225) showed[Ca2+]i oscillations after CaR activation. The [Ca2+]i response consisted of a rapid rise in[Ca2+]i followed by a sustained phase of elevated [Ca2+]i. Conclusion: Based on single cellanalysis, we substantiate the notion that sinusoidal [Ca2+]i oscillations induced by activationof the CaR result from periodic negative feedback involving PKC-mediated phosphorylationof the CaR at Thr888.

Sa1738

Crosstalk Between PKD1 and β-Catenin Signaling Pathways in IntestinalEpithelial CellsYang Ni, James Sinnett-Smith, Steven H. Young, Enrique Rozengurt

Rationale: β-catenin signaling plays a critical role in promoting normal and abnormal intes-tinal epithelial cell proliferation in response to Wnt ligands, pro-inflammatory cytokinesand growth factors. Our recent results demonstrated that protein kinase D1 (PKD1) signalingstimulates intestinal epithelial cell proliferation, as revealed by using epithelial cells inculture and a novel PKD1 transgenic mouse model (J Biol Chem, 286, 511-520, 2011). Wehypothesize that crosstalk between PKD1 and β-catenin signaling provides a novelmechanismleading to intestinal epithelial cell proliferation. Results: To identify a model system toexplore PKD1/β-catenin crosstalk, we determined the redistribution of endogenous β-cateninin response to the Gq-coupled receptor agonist angiotensin II (ANGII), a potent inducer ofPKD1 activation in intestinal IEC-18 cells. Stimulation of these cells with ANG II induceda dramatic increase in β-catenin nuclear localization. The redistribution of β-catenin inresponse to ANGII was prevented by cell treatment with the PKD family inhibitors kb NB142-70 and CRT006610. Similar results were obtained with IEC-6 cells. These resultsindicated that PKD1 activation mediates GPCR-induced β-catenin nuclear localization inintestinal epithelial cells. Furthermore, we demonstrated that Gq-coupled agonists inducecomplex formation between PKD1 and β-catenin in intestinal epithelial IEC-18 cells asdetected by co-immunoprecipitation. The association of PKD1 with β-catenin was prominentas early as 10 min after cell stimulation and remained nearly at the maximal level for atleast 4 h. Exposure of the cells to kb NB 142-70 blocked the association of PKD1 with β-catenin, implying that PKD1/β-catenin complex formation depends on PKD1 activation.Given that β-catenin phosphorylation at Ser552 promotes its dissociation from cell-cellcontacts, induces its nuclear translocation and stimulates its transcriptional activity, we alsoexamined whether GPCR/PKD1 stimulates phosphorylation of β-catenin at this importantsite. Stimulation of IEC-18 cells with ANG II induced striking β-catenin phosphorylationat Ser552, an effect prevented by either inhibitors of PKD family activity (kb NB 142-70 orCRT006610) or by siRNA-mediated knockdown of PKD1 expression. PKD1/ β-catenin cros-stalk also occurs in intestinal epithelial cell in vivo, since an increase in β-catenin nuclearlocalization was demonstrated by inmunohistochemistry in transgenic mice that overexpressPKD1 in intestinal epithelial cells. Conclusion: Several lines of evidence indicate that GPCRactivation induces crosstalk between PKD1 and β-catenin signaling, thus providing a novelmechanism in the regulation of intestinal epithelial cell proliferation.

Sa1739

Beta-Nicotinamide Adenine Dinucleotide (Beta-NAD) Is a Ligand for theAdenosine A1 Receptor Expressed by Neurons in the Guinea Pig EntericNervous System (ENS)Guo-Du Wang, Xiyu Wang, Fei Zou, Jackie D. Wood

Background and Aims: Adenosine is formed from the breakdown of ATP by ectonucleotidasesin themilieu surrounding the ENS. It acts at A1 type P1 receptors to suppress neurotransmitterrelease. A1-mediated presynaptic inhibition suppresses multiple forms of neurotransmissionin the ENS. Beta-NAD is a putative inhibitory neurotransmitter at P2Y1 receptors at intestinalneuromuscular junctions. Excitatory P2Y1 receptors are expressed also by secretomotorneurons that stimulate mucosal chloride and bicarbonate secretion. We examined its actionson ENS synaptic transmission and neuronal excitability. Methods: Standard intracellular"sharp" microelectrodes recorded synaptic events and membrane potentials in neurons ofthe small and large intestine. RT-PCR examined purinergic receptor expression. Results:Bath application of beta-NAD (10-30μM) mimicked the actions of adenosine, which wasmembrane hyperpolarization and suppression of spike discharge evoked by intracellularinjection of depolarizing current. Fast nicotinic EPSPs and slow EPSPs were reduced inamplitude by beta-NAD. This was the case for S-type neurons in 17 preparations from theileum or colon and for AH-type neurons in 25 myenteric plexus whole-mount preparationsfrom the same regions. Application of 20μM adenosine, the adenosine A1 receptor agonist,2'-me CCPA (10μM), 100-300μMATP or 100-300μMADPmimicked the action of adenosine.Bath application of 10-30μM beta-NAD mimicked the actions of each of the purinergic

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