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• W2017758314 abstract "Small differences in amplitude, duration, and temporal patterns of change in the concentration of free intracellular Ca2+ ([Ca2+]i) can profoundly affect cell physiology, altering programs of gene expression, cell proliferation, secretory activity, and cell survival. We report a novel mechanism for amplitude modulation of [Ca2+]i that involves mitogen-activated protein kinase (MAPK). We show that epidermal growth factor (EGF) potentiates gastrin-(1–17) (G17)-stimulated Ca2+ release from intracellular Ca2+ stores through a MAPK-dependent pathway. G17 activation of the cholecystokinin/gastrin receptor (CCK2R), a G protein-coupled receptor, stimulates release of Ca2+ from inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Pretreating rat intestinal epithelial cells expressing CCK2R with EGF increased the level of G17-stimulated Ca2+ release from intracellular stores. The stimulatory effect of EGF on CCK2R-mediated Ca2+ release requires activation of the MAPK kinase (MEK)1,2/extracellular signal-regulated kinase (ERK)1,2 pathway. Inhibition of the MEK1,2/ERK1,2 pathway by either serum starvation or treatment with selective MEK1,2 inhibitors PD98059 and U0126 or expression of a dominant-negative mutant form of MEK1 decreased the amplitude of the G17-stimulated Ca2+ release response. Activation of the MEK1,2/ERK1,2 pathway either by pretreating cells with EGF or by expression of constitutively active K-ras (K-rasV12G) or MEK1 (MEK1*) increased the amplitude of G17-stimulated Ca2+ release. Although EGF, MEK1*, and K-rasV12G activated the MEK1,2/ERK1,2 pathway, they did not increase [Ca2+]i in the absence of G17. These data demonstrate that the activation state of the MEK1,2/ERK1,2 pathway can modulate the amplitude of the CCK2R-mediated Ca2+ release response and identify a novel mechanism for cross-talk between EGF receptor- and CCK2R-regulated signaling pathways. Small differences in amplitude, duration, and temporal patterns of change in the concentration of free intracellular Ca2+ ([Ca2+]i) can profoundly affect cell physiology, altering programs of gene expression, cell proliferation, secretory activity, and cell survival. We report a novel mechanism for amplitude modulation of [Ca2+]i that involves mitogen-activated protein kinase (MAPK). We show that epidermal growth factor (EGF) potentiates gastrin-(1–17) (G17)-stimulated Ca2+ release from intracellular Ca2+ stores through a MAPK-dependent pathway. G17 activation of the cholecystokinin/gastrin receptor (CCK2R), a G protein-coupled receptor, stimulates release of Ca2+ from inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Pretreating rat intestinal epithelial cells expressing CCK2R with EGF increased the level of G17-stimulated Ca2+ release from intracellular stores. The stimulatory effect of EGF on CCK2R-mediated Ca2+ release requires activation of the MAPK kinase (MEK)1,2/extracellular signal-regulated kinase (ERK)1,2 pathway. Inhibition of the MEK1,2/ERK1,2 pathway by either serum starvation or treatment with selective MEK1,2 inhibitors PD98059 and U0126 or expression of a dominant-negative mutant form of MEK1 decreased the amplitude of the G17-stimulated Ca2+ release response. Activation of the MEK1,2/ERK1,2 pathway either by pretreating cells with EGF or by expression of constitutively active K-ras (K-rasV12G) or MEK1 (MEK1*) increased the amplitude of G17-stimulated Ca2+ release. Although EGF, MEK1*, and K-rasV12G activated the MEK1,2/ERK1,2 pathway, they did not increase [Ca2+]i in the absence of G17. These data demonstrate that the activation state of the MEK1,2/ERK1,2 pathway can modulate the amplitude of the CCK2R-mediated Ca2+ release response and identify a novel mechanism for cross-talk between EGF receptor- and CCK2R-regulated signaling pathways. The gastrointestinal peptide hormone gastrin-(1–17) (G17) 1The abbreviations used are: G17, gastrin-(1–17); CCK, cholecystokinin; CCK2R, CCK2/G17 receptor; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; EGFR, EGF receptor; RIE, rat intestinal epithelial; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase; MEK1*, constitutively active mutant of MEK1; dnMEK, dominant-negative (kinase-dead) mutant of MEK; EGFP, enhanced green fluorescent protein; GPCR, G protein-coupled receptor; GI, gastrointestinal.1The abbreviations used are: G17, gastrin-(1–17); CCK, cholecystokinin; CCK2R, CCK2/G17 receptor; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; EGFR, EGF receptor; RIE, rat intestinal epithelial; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase; MEK1*, constitutively active mutant of MEK1; dnMEK, dominant-negative (kinase-dead) mutant of MEK; EGFP, enhanced green fluorescent protein; GPCR, G protein-coupled receptor; GI, gastrointestinal. plays an essential role in the regulation of digestion by stimulating gastric acid secretion, histamine synthesis and release, and proliferation of the gastric epithelium and endocrine pancreas (1.Dockray G.J. J. Physiol. (Lond.). 1999; 518: 315-324Crossref Scopus (122) Google Scholar, 2.Wang R.N. Rehfeld J.F. Nielsen F.C. Kloppel G. Diabetologia. 1997; 40: 887-893Crossref PubMed Scopus (85) Google Scholar). In cancers of the stomach, pancreas, and colon, G17 promotes tumor cell proliferation, motility, and invasion (3.Rozengurt E. Walsh J.H. Annu. Rev. Physiol. 2001; 63: 49-76Crossref PubMed Scopus (178) Google Scholar, 4.Heasley L.E. Oncogene. 2001; 20: 1563-1569Crossref PubMed Scopus (175) Google Scholar, 5.Bierkamp C. Kowalski-Chauvel A. Dehez S. Fourmy D. Pradayrol L. Seva C. Oncogene. 2002; 21: 7656-7670Crossref PubMed Scopus (32) Google Scholar). The biological effects of G17 are mediated by the cholecystokinin-2 (CCK2)/G17 receptor (CCK2R) (previously named CCK-B receptor), a member of the G protein-coupled receptor superfamily. Three splice variants of the CCK2R have been identified (6.Kopin A.S. Lee Y.M. McBride E.W. Miller L.J. Lu M. Lin H.Y. Kolakowski Jr., L.F. Beinborn M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3605-3609Crossref PubMed Scopus (468) Google Scholar, 7.Hellmich M.R. Rui X.L. Hellmich H.L. Fleming R.Y. Evers B.M. Townsend Jr, C.M. J. Biol. Chem. 2000; 275: 32122-32128Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 8.Song I. Brown D.R. Wiltshire R.N. Gantz I. Trent J.M. Yamada T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9085-9089Crossref PubMed Scopus (113) Google Scholar) that bind the structurally related peptides cholecystokinin (CCK) and G17 with high affinities. An early event following agonist activation of CCK2R is the phospholipase Cβ-mediated elaboration of inositol 1,4,5-triphosphate (6.Kopin A.S. Lee Y.M. McBride E.W. Miller L.J. Lu M. Lin H.Y. Kolakowski Jr., L.F. Beinborn M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3605-3609Crossref PubMed Scopus (468) Google Scholar) from membrane phospholipids and the subsequent release of calcium (Ca2+) from inositol 1,4,5-triphosphate-sensitive intracellular Ca2+ stores. Calcium is an essential intracellular signal involved in many biological processes including fertilization, secretion, contraction, proliferation, differentiation, and apoptosis (9.Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4312) Google Scholar, 10.Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2250) Google Scholar). Small differences in the amplitude, duration, and/or temporal pattern of change in [Ca2+]i can have profound effects on cell physiology, altering programs of gene expression (11.Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1541) Google Scholar, 12.Dolmetsch R.E. Xu K. Lewis R.S. Nature. 1998; 392: 933-936Crossref PubMed Scopus (1659) Google Scholar), secretory activity (13.Hellmich M.R. Ives K.L. Udupi V. Soloff M.S. Greeley Jr., G.H. Christensen B.N. Townsend Jr, C.M. J. Biol. Chem. 1999; 274: 23901-23909Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), cell proliferation, and survival (14.Gallo V. Kingsbury A. Balazs R. Jorgensen O.S. J. Neurosci. 1987; 7: 2203-2213Crossref PubMed Google Scholar, 15.Koike T. Martin D.P. Johnson Jr, E.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6421-6425Crossref PubMed Scopus (371) Google Scholar, 16.Yano S. Tokumitsu H. Soderling T.R. Nature. 1998; 396: 584-587Crossref PubMed Scopus (528) Google Scholar). Calcium is an essential signaling molecule in G17-stimulated cell proliferation. In Chinese hamster ovary cells expressing recombinant CCK2R, an agonist-induced increase in mitogen-activated protein kinase (MAPK) activity and [3H]thymidine incorporation into DNA requires a slow oscillatory increase in [Ca2+]i (17.Akagi K. Nagao T. Urushidani T. Biochim. Biophys. Acta. 1999; 1452: 243-253Crossref PubMed Scopus (18) Google Scholar). In the rat pancreatic cancer cell line AR4-2J, a CCK2R-mediated increase in [Ca2+]i is required for formation of the Shc-Grb2-Sos protein complex and subsequent activation of MAPK (18.Daulhac L. Kowalski-Chauvel A. Pradayrol L. Vaysse N. Seva C. Biochem. J. 1997; 325: 383-389Crossref PubMed Scopus (47) Google Scholar). Defining the molecular mechanisms involved in CCK2R regulation of [Ca2+]i is necessary to understand the proliferative effects of G17 on normal and neoplastic cells. Like G17, epidermal growth factor (EGF) also stimulates the proliferation of gastric epithelial cells (19.Miyazaki Y. Shinomura Y. Higashiyama S. Kanayama S. Higashimoto Y. Tsutsui S. Zushi S. Taniguchi N. Matsuzawa Y. Biochem. Biophys. Res. Commun. 1996; 223: 36-41Crossref PubMed Scopus (24) Google Scholar, 20.Rutten M.J. Dempsey P.J. Solomon T.E. Coffey Jr, R.J. Am. J. Physiol. 1993; 265: G361-G369PubMed Google Scholar), and recently, the G17-related peptide CCK has been shown to synergize with EGF to stimulate DNA synthesis ([3H]thymidine incorporation), cyclin-D3 expression, and retinoblastoma protein phosphorylation in cells expressing both CCK2R and EGF receptors (21.Zhukova E. Sinnett-Smith J. Wong H. Chiu T. Rozengurt E. J. Cell. Physiol. 2001; 189: 291-305Crossref PubMed Scopus (29) Google Scholar). EGF and the related growth factors transforming growth factor-α and amphiregulin bind to a family of receptors known as type I receptor tyrosine kinases. This family is composed of four related receptors: the EGF receptor (EGFR/ErbB1/HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4) (22.Jorissen R.N. Walker F. Pouliot N. Garrett T.P. Ward C.W. Burgess A.W. Exp. Cell Res. 2003; 284: 31-53Crossref PubMed Scopus (1243) Google Scholar). The synergistic effect of CCK and EGF on regulation of cell cycle progression indicates cross-talk between CCK2R- and EGFR-regulated signaling pathways; however, the point at which their signal transduction pathways converge has not been identified. Because a G17-induced increase in [Ca2+]i is one of the initial events in CCK2R-regulated signal transduction, the aim of this study was to determine the effect of EGF treatment on CCK2R regulation of [Ca2+]i. We report that EGF affects this very early step in CCK2R-mediated signal transduction by potentiating G17-stimulated Ca2+ release from intracellular Ca2+ stores. The potentiating effect of EGF is mediated by the MEK1,2/ERK1,2 pathway. This study identifies a novel mechanism by which changes in the basal activation state of the MEK1,2/ERK1,2 pathway regulate the amplitude of the CCK2 R-mediated Ca2+ release response. Cell Lines—For most experiments, we used a nontransformed rat intestinal epithelial (RIE) cell line expressing recombinant human CCK2R called RIE/CCK2R. These cells were routinely cultured in DMEM supplemented with 400 μg/ml G418 and 10% heat-inactivated fetal bovine serum (FBS) at 37 °C. Other cell lines used included the rat pancreatic cancer cell line AR4-2J, the human prostate cancer cell line PC-3, and the human pancreatic carcinoid cell line BON. AR4-2J cells were cultured in DMEM supplemented with 10% FBS. PC-3 cells were cultured as recommended by the American Type Culture Collection (Manassas, VA). BON cells were cultured as described previously (23.Carraway R.E. Mitra S.P. Evers B.M. Townsend Jr, C.M. Regul. Pept. 1994; 53: 17-29Crossref PubMed Scopus (26) Google Scholar). Intracellular Ca2+ Imaging—RIE/CCK2R cells were cultured on 25-mm glass coverslips in DMEM supplemented with 10% FBS. To load the cells with the Ca2+ indicator dye Fura-2, they were first washed with a physiological medium (KRH) containing NaCl (125 mm), KCl (5 mm), KH2PO4 (1.2 mm), MgSO4 (1.2 mm), CaCl2 (2 mm), glucose (6 mm), HEPES (25 mm), pH 7.4, and then they were incubated with 2 μm Fura-2/AM (Molecular Probes, Eugene, OR) for 50 min at room temperature. Single cell changes in [Ca2+]i were recorded using a Nikon Diaphot inverted microscope (Garden City, NY) and a CCD camera (Dage-MTI, Inc., Michigan City, IN). Data points were collected every 1–8 s from ∼35 cells/coverslip and processed using ImageMaster software. Fluorescent ratios were converted into [Ca2+]i using the equation [Ca2+]i nm = Kd((R–Rmin)/(Rmax–R)) × β, as reported previously (48.Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Maximal (Rmax) and minimal (Rmin) values were determined following the addition of either 5 μm ionomycin or 5 mm EGTA, respectively. Analysis of statistical significance was performed using Student's unpaired t test. For multiple comparisons, one-way analysis of variance combined with the Tukey's post hoc test was used (GraphPad Software, San Diego, CA). Values are presented as the mean ± S.E. and are considered significant at p < 0.05. Competition Binding—RIE/CCK2R cells were plated into 24-well plates in DMEM supplemented with 10% FBS. After 2 days, cells were cultured in DMEM without FBS for an additional 24 h. Following a 5-min pretreatment with either EGF (1 ng/ml) or vehicle (H2O), the cells were washed with binding buffer (DMEM, 25 mm HEPES, 0.1% bovine serum albumin) and then incubated 1 h at room temperature in binding buffer containing 0.05 nm125I-labeled CCK-8 (specific activity = 2200 Ci/mmol, PerkinElmer Life Sciences) and various concentrations of unlabeled CCK-8 (1 pm–1 μm). The binding assay was terminated by rinsing the cells with an ice-cold solution of phosphate-buffered saline and 0.1% bovine serum albumin. After washing, the cells were lysed with 300 μl of 1 m NaOH and transferred to a glass tube. The amount of bound radioactivity was measured in a Cobra II gamma counter (Parkard Instrument Company, Downers Grove, IL). Total binding averaged ∼6% of the total counts added to the assays. Nonspecific binding was defined as the amount of radiolabeled CCK-8 bound to the cells in the presence of 1 μm unlabeled CCK-8. Each data point was determined in triplicate and is represented as the mean ± S.E. of three independent experiments. Nonlinear regression analysis was performed using Prism software (GraphPad Software). Western Blotting—RIE/CCK2R cells were plated into 12-well plates at a density of ∼100,000 cells/well in DMEM supplemented with 10% FBS. After 2 days, cells were incubated with DMEM ± FBS for 24 h, treated as described in the figure legends, washed with ice-cold phosphate-buffered saline, and solubilized in lysis buffer containing 150 mm NaCl, 50 mm Tris, pH 7.4, 1 mm EGTA, 10 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100 at 4 °C. Protein concentrations of the supernatant were determined using the Bio-Rad DC protein assay kit. Protein (10 μg) from each sample was resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and probed with an antibody to the dual phosphorylated forms of ERK1 and ERK2 (Anti-ACTIVE® MAPK, Cat. No. V8031, Promega, Madison, WI). Immunoreactive proteins were visualized using the ECL Western blotting detection system (Amersham Biosciences). The total ERK content within the cell extracts was determined using an antibody that recognizes both active and inactive forms of ERK1 and ERK2 (Cat. No. SC-94, Santa Cruz Biotechnology Inc., Santa Cruz, CA). Transient Transfection—RIE/CCK2R cells were grown on coverslips in 6-well plates. After 24 h, cells were transiently co-transfected with 1 μg of one of several mutant expression constructs (constitutively active MEK1 (MEK1*), dominant-negative MEK1 (dnMEK1) (Upstate Biotech, Lake Placid, NY), and K-rasV12G (Dr. Aubrey Thompson, University of Texas Medical Branch, Galveston, TX)) and 0.1 μg of plasmid containing the cDNA for green fluorescence protein (pEGFP-C1, Clontech, Palo Alto, CA) using LipofectAMINE Plus reagent (Invitrogen). Twenty-four h post-transfection, the G17-stimulated Ca2+ response was measured in the EGFP-positive cells as described above. Transfection efficiency ranged from 10 to 20% of the cells and was assessed by counting the number of EGFP-positive cells/total cells in a microscopic field using a ×40 objective. Typically, 6–8 EGFP-positive cells were observed/field. Intracellular Ca2+ measurements were recorded from 32 to 48 EGFP-positive cells/experiment. EGF Potentiates G17-stimulated Increases in [Ca2+]i—The binding of G17 to CCK2R induced a rapid and transient increase in [Ca2+]i in RIE/CCK2R cells (Fig. 1A). To assess the effects of EGF on the G17-stimulated increase in [Ca2+]i, cells were cultured in medium without serum for 24 h. Under these conditions, the amplitude of the G17-induced Ca2+ response decreased by up to 50% (Fig. 1A). However, pretreating the serum-starved cells with EGF (1 ng/ml) reversed the inhibitory effects of serum starvation and increased the amplitude of the G17-stimulated Ca2+ response in a time-dependent manner (Fig. 1B). Simultaneous addition of EGF and G17 (10 nm) had no effect on [Ca2+]i when compared with cells treated with G17 alone; however, pretreating cells for 3, 4, or 5 min with EGF significantly increased the amplitude of the G17-induced Ca2+ response when compared with serum-starved cells (Fig. 1B). Five min of pretreatment with EGF was sufficient to increase the amplitude of the G17-stimulated increase in [Ca2+]i to the level observed in cells continuously cultured in 10% FBS (Fig. 1B). To assess whether the effect of EGF on G17-stimulated increases in [Ca2+]i was mediated by the EGF receptor, we pretreated cells with the EGFR tyrosine kinase inhibitor AG1478. Pretreatment with AG1478 (200 nm) had no effect on G17-stimulated increases in [Ca2+]i when used alone but completely blocked the potentiation effect of EGF on the G17-stimulated Ca2+ response (Fig. 1B). These data indicate that EGF potentiation of the G17-stimulated increases in [Ca2+]i required EGFR activation. Because EGF can activate phospholipase-γ, resulting in the production of inositol 1,4,5-triphosphate and the release of Ca2+ from intracellular stores (24.Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar), we assessed whether the potentiation effect of EGF on the G17-stimulated Ca2+ response was due to direct regulation of [Ca2+]i. Pretreating cells with EGF, up to a concentration of 25 ng/ml, had no effect on [Ca2+]i in RIE/CCK2R cells (Fig. 1C), indicating that its potentiation of the G17-stimulated Ca2+ response was not due to parallel regulation of [Ca2+]i. Pretreatment of RIE/CCK2R cells with EGF enhanced the efficacy of the G17-stimulated Ca2+ response but not the sensitivity. Pretreating serum-starved cells with EGF (1 ng/ml) for 5 min significantly increased the amplitude (efficacy) of the Ca2+ response induced by G17 over a broad range of concentrations from 0.01 to 100 nm (Fig. 2A). However, when the Ca2+ data was normalized to a percentage of maximum response there was no effect of EGF pretreatment on the EC50 value of the G17-stimulated Ca2+ response (Fig. 2B), suggesting that EGF was not altering the affinity of G17 for CCK2R. To examine this possibility, we performed a radiolabeled ligand binding analysis with and without pretreating the cells for 5 min with EGF (1 ng/ml). As expected EGF did not alter the IC50 value (affinity) of CCK binding to CCK2R (Fig. 2C), suggesting that the target of EGF action may be downstream of CCK2R. EGF Potentiation of the G17-stimulated Ca2+ Response Is Associated with an Increase in the Levels of Phosphorylated Extracellular Signal-regulated Kinase 1 and 2—An important intracellular signaling pathway regulated by the EGFR family is the mitogen-activated protein kinases. MAPKs are an evolutionarily conserved family of serine-threonine-directed kinases. Five subfamilies of MAPKs have been identified, which include ERK1 and ERK2, the c-Jun N-terminal kinases, the 38-kDa MAPKs, ERK5, and ERK-3s (25.Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2266) Google Scholar). The activities of MAPKs are regulated by upstream MAPK kinases, which are dual-specificity kinases that activate MAPKs by phosphorylating both the tyrosine and threonine residues present in the consensus sequence (TXY). Six MAPK kinase family members have been identified and are designated MEK1 through MEK5 and ERK-3 kinase. In many cell types, EGFR is coupled to the MEK1,2/ERK1,2 MAPK pathway through the Ras family of small GTP-binding proteins (26.Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1151) Google Scholar). Serum starvation reduces the activity of the MEK1,2/ERK1,2 pathway; therefore, we hypothesized that this pathway may mediate the stimulatory effects of EGF on G17-stimulated increases in [Ca2+]i. To test this hypothesis, we first assessed the time and dose dependence of EGF treatment on the level of phosphorylated (activated) ERK1 (pERK1) and ERK2 (pERK2) in serum-starved RIE/CCK2R cells. EGF (1 ng/ml) induced a time- and dose-dependent increase in the levels of pERK1 and pERK2 (Fig. 3). A long exposure film revealed an increase over base-line levels of pERK1 and pERK2 as early as 2 min after EGF treatment (Fig. 3A). A shorter exposure of an extended time course showed that the levels of pERK1 and pERK2 further increased at 10 and 20 min and began to decrease at 30 min (Fig. 3B). A dose-response analysis at 5 min showed a dose-dependent increase in the levels of pERK1 and pERK2 (Fig. 3C). When compared with untreated control cultures, an increase in the levels of pERK1 and pERK2 was detected in cells treated with as little as 0.1 ng/ml EGF for 5 min. Maximum levels of pERK1 and pERK2 were observed in cells treated with 5 ng/ml EGF for 5 min. An assessment of the effects of EGF on G17-stimulated increases in [Ca2+]i revealed a good correspondence between both the time (Fig. 1B) and dose effects of EGF on ERK activation (Fig. 3C) and its potentiation of the G17-stimulated increase in [Ca2+]i (Fig. 3D). Together, these data suggest that EGF can increase the amplitude of the G17-stimulated Ca2+ response by increasing the activation state of the MEK1,2/ERK1,2 pathway. To further assess the role of the MEK1,2/ERK1,2 pathway in CCK2R regulation of [Ca2+]i, we next determined the effects of modulating the activities of MEK1 and MEK2 on G17-induced increases in [Ca2+]i. Inhibition of MEK1 and MEK2 Blocked the Potentiating Effects of Serum and EGF on G17-stimulated Ca2+ Release— Agonist binding to CCK2R induces an increase in [Ca2+]i that involves both the inositol 1,4,5-triphosphate-mediated release of Ca2+ from intracellular Ca2+ stores and the influx of extracellular Ca2+ across the plasma membrane. The total increase in [Ca2+]i induced by G17 is the sum of Ca2+ from these two sources. Because a G17-stimulated release of Ca2+ from intracellular stores is required for Ca2+ influx across the plasma membrane, we assessed the effects of MEK1,2 inhibitors on G17-stimulated Ca2+ release from internal stores. The amount of Ca2+ released from intracellular stores can be determined experimentally by bathing the cells in an extracellular solution without added Ca2+ and containing the Ca2+-chelating agent EGTA. Under these conditions, the change in [Ca2+]i induced by G17 stimulation is due to the release of Ca2+ from intracellular stores alone. To determine whether the activities of MEK1 and MEK2 were required for G17-stimulated Ca2+ release, RIE/CCK2R cells cultured in 10% FBS were pretreated for 5 min with different concentrations of the MEK inhibitor PD98059. Pretreatment of cells with PD98059 caused a dose-dependent decrease in the amplitude of the G17-stimulated Ca2+ release response (Fig. 4A). A concentration of 1 μm PD98059 reduced the amplitude of the change in [Ca2+]i by 28% (from 310 ± 12.1 nm to 221 ± 12.3 nm) (Fig. 4B). Treatment with 10 μm PD98059 resulted in a 93% reduction in [Ca2+]i. Western blot analysis showed detectable levels of pERK1 and pERK2 in cells cultured in 10% FBS but not in cells pretreated with 10 μm PD98059 for 5 min (Fig. 4B, inset). A similar dose-dependent inhibition of the G17-stimulated Ca2+ release was also observed in cells treated with another inhibitor of MEK1 and MEK2, U0126 (data not shown). The specificity of the chemical inhibitors for MEK1 was confirmed by transiently transfecting cells with a dominant-negative (kinase-dead) mutant of MEK1. To identify transfected cells, we co-transfected cells with an expression vector containing the cDNA for EGFP. G17-stimulated Ca2+ release was measured in EGFP-positive cells. When compared with EGFP-positive cells co-transfected with an empty expression vector, EGFP-positive cells co-transfected with dnMEK1 showed a significant decrease in the amplitude of G17-induced Ca2+ release (Fig. 4C). The peak change in [Ca2+]i decreased from 221 ± 24.4 nm (empty vector control) to 38.3 ± 12.2 nm (dnMEK1-transfected) (Fig. 4D). Although both the chemical inhibitors and forced expression of dnMEK1 reduced the peak levels of agonist-stimulated Ca2+ release, neither PD98059 treatment nor dnMEK expression affected base-line [Ca2+]i (Fig. 4, A and C), which is ∼100 nm. Together, these data demonstrate that inhibition of MEK activity is sufficient to reduce the amplitude of the G17-stimulated Ca2+ release from intracellular stores. To determine whether the MEK1,2/ERK1,2 pathway mediates the effect of EGF on CCK2 R-regulated [Ca2+]i, we assessed the effects of PD98059 treatment on EGF-induced potentiation of G17-stimulated Ca2+ release from intracellular stores. First, we determined the effects of PD98059 on the levels of pERK1 and pERK2 in serum-starved RIE/CCK2R cells. A long exposure film showed low levels of pERK1 and pERK2 in serum-starved cells compared with cells cultured in 10% FBS (Fig. 4E, lanes 2 and 1, respectively). Pretreating the cells with PD98059 (10 μm for 5 min) reduced pERK1 and pERK2 to undetectable levels (Fig. 4E, lane 3). Treatment of serum-starved cells with EGF (1 ng/ml for 5 min) stimulated an increase in the levels of pERK1 and pERK2, which was completely blocked by PD98059 (10 μm) (Fig. 4E, lanes 5 and 4, respectively). Analysis of G17-stimulated Ca2+ responses from cells treated the same way showed a good correspondence between the relative levels of pERK1 and pERK2 and the amplitude of the G17-stimulated Ca2+ release response. PD98059 treatment decreased both basal and EGF-stimulated pERK levels in serum-starved cells and also reduced basal and EGF-enhanced G17-stimulated Ca2+ release (Fig. 4F). Neither G17-stimulated Activation of the MEK1,2/ERK1,2 Pathway nor EGF Activation of the Phosphatidylinositol 3-Kinase Pathway Is Involved in the Potentiation of CCK2R-mediated Ca2+ Response—It is well established that in addition to regulation of [Ca2+]i, CCK2R is coupled to the MEK1,2/ERK1,2 pathway. We reported previously G17-stimulated increases in ERK activation in RIE/CCK2R cells (27.Guo Y.S. Cheng J.Z. Jin G.F. Gutkind J.S. Hellmich M.R. Townsend Jr, C.M. J. Biol. Chem. 2002; 277: 48755-48763Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). To determine whether G17 stimulation of the MEK1,2/ERK1,2 pathway plays a role in CCK2 R-mediated Ca2+ release, we compared the time courses of G17-stimulated Ca2+ release and ERK activation (Fig. 5). We found that the G17-stimulated increase in Ca2+ release from intracellular stores preceded a detectable increase in the levels of G17-stimulated ERK activation, suggesting that CCK2R-mediated activation of the MEK1,2/ERK1,2 pathway is not responsible for the potentiation of the G17-induced Ca2+ release response. EGF can activate other pathways in addition to the MEK1,2/ERK1,2 pathway, including the phosphatidylinositol 3-kinase pathway (22.Jorissen R.N. Walker F. Pouliot N. Garrett T.P. Ward C.W. Burgess A.W. Exp. Cell Res. 2003; 284: 31-53Crossref PubMed Scopus (1243) Google Scholar). To assess the possible involvement of the phosphatidylinositol 3-kinase pathway in EGF potentiation of G17-stimulated Ca2+ release, we pretreated cells cultured in 10% FBS with two commonly used inhibitors of the phosphatidylinositol 3-kinase pathway, wortmannin and LY294002. Unlike the MEK inhibitors, which blocked the potentiation effect of EGF on G17-stimulated Ca2+ release, neither wortmannin (100 nm) nor LY294002 (10 μm) effected EGF potentiation of G17-stimulated Ca2+ release (Fig. 6). Wortmannin and LY294002 also had no effect on G17-stimulated Ca2+ release in the absence of EGF (Fig. 6). Together, the data support the conclusion that EGF potentiates CCK2 R-mediated Ca2+ release from intracellular stores by increasing the activation state of the MEK1,2/ERK1,2 pathway. Furthermore, the inhibitory effect of PD98059 on G17-stimulated Ca2+ release in the absence of EGF suggests that altering the basal activation state of the MEK1,2/ERK1,2 pathway may be sufficient to modulate the efficacy of the G17-stimula" @default.
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• W2017758314 title "Epidermal Growth Factor Potentiates Cholecystokinin/Gastrin Receptor-mediated Ca2+ Release by Activation of Mitogen-activated Protein Kinases" @default.
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