Identification of Glucagon-like Peptide-2 (GLP-2)-activated Signaling Pathways in Baby Hamster Kidney Fibroblasts Expressing the Rat GLP-2 Receptor
1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês
10.1074/jbc.274.43.30459
ISSN1083-351X
AutoresBernardo Yusta, Romel Somwar, Feng Wang, Donald G. Munroe, Sergio Grinstein, Amira Klip, Daniel J. Drucker,
Tópico(s)Ion Transport and Channel Regulation
ResumoGlucagon-like peptide-2 (GLP-2) promotes the expansion of the intestinal epithelium through stimulation of the GLP-2 receptor, a recently identified member of the glucagon-secretin G protein-coupled receptor superfamily. Although activation of G protein-coupled receptors may lead to stimulation of cell growth, the mechanisms transducing the GLP-2 signal to mitogenic proliferation remain unknown. We now report studies of GLP-2R signaling in baby hamster kidney (BHK) cells expressing a transfected rat GLP-2 receptor (BHK-GLP-2R cells). GLP-2, but not glucagon or GLP-1, increased the levels of cAMP and activated both cAMP-response element- and AP-1-dependent transcriptional activity in a dose-dependent manner. The activation of AP-1-luciferase activity was protein kinase A (PKA) -dependent and markedly diminished in the presence of a dominant negative inhibitor of PKA. Although GLP-2 stimulated the expression of c-fos, c-jun, junB, and zif268, and transiently increased p70 S6 kinase in quiescent BHK-GLP-2R cells, GLP-2 also inhibited extracellular signal-regulated kinase 1/2 and reduced serum-stimulated Elk-1 activity. Furthermore, no rise in intracellular calcium was observed following GLP-2 exposure in BHK-GLP-2R cells. Although GLP-2 stimulated both cAMP accumulation and cell proliferation, 8-bromo-cyclic AMP alone did not promote cell proliferation. These findings suggest that the GLP-2R may be coupled to activation of mitogenic signaling in heterologous cell types independent of PKA via as yet unidentified downstream mediators of GLP-2 action in vivo. Glucagon-like peptide-2 (GLP-2) promotes the expansion of the intestinal epithelium through stimulation of the GLP-2 receptor, a recently identified member of the glucagon-secretin G protein-coupled receptor superfamily. Although activation of G protein-coupled receptors may lead to stimulation of cell growth, the mechanisms transducing the GLP-2 signal to mitogenic proliferation remain unknown. We now report studies of GLP-2R signaling in baby hamster kidney (BHK) cells expressing a transfected rat GLP-2 receptor (BHK-GLP-2R cells). GLP-2, but not glucagon or GLP-1, increased the levels of cAMP and activated both cAMP-response element- and AP-1-dependent transcriptional activity in a dose-dependent manner. The activation of AP-1-luciferase activity was protein kinase A (PKA) -dependent and markedly diminished in the presence of a dominant negative inhibitor of PKA. Although GLP-2 stimulated the expression of c-fos, c-jun, junB, and zif268, and transiently increased p70 S6 kinase in quiescent BHK-GLP-2R cells, GLP-2 also inhibited extracellular signal-regulated kinase 1/2 and reduced serum-stimulated Elk-1 activity. Furthermore, no rise in intracellular calcium was observed following GLP-2 exposure in BHK-GLP-2R cells. Although GLP-2 stimulated both cAMP accumulation and cell proliferation, 8-bromo-cyclic AMP alone did not promote cell proliferation. These findings suggest that the GLP-2R may be coupled to activation of mitogenic signaling in heterologous cell types independent of PKA via as yet unidentified downstream mediators of GLP-2 action in vivo. glucagon-like peptide G protein-coupled receptor baby hamster kidney Dulbecco's modified Eagle's medium cAMP-response element mitogen-activated protein kinase protein kinase A immediate early 8-bromo-cyclic AMP extracellular signal-regulated kinase human The gastrointestinal mucosal epithelium contains a diverse number of specialized enteroendocrine cells that synthesize and secrete peptide hormones, frequently in a nutrient-dependent manner. Following secretion into circulation, gut-derived hormones may act in an endocrine manner by binding to receptors in tissues such as pancreas and liver, leading to the activation of signal transduction pathways and downstream physiological events. Consistent with their location in the intestinal mucosal epithelium, enteroendocrine peptides may function in part to regulate gastrointestinal motility and nutrient digestion and absorption. For example, gastrin promotes acid secretion, whereas secretin inhibits acid secretion and promotes pancreatic exocrine secretion. Peptide hormones structurally related to secretin, such as glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 (GLP-1),1 stimulate glucose-dependent insulin secretion from the pancreatic beta cells, and GLP-1, unlike the glucose-dependent insulinotropic polypeptide, also inhibits gastric emptying, glucagon secretion, and food intake in vivo (1Drucker D.J. Diabetes. 1998; 47: 159-169Crossref PubMed Google Scholar). The pleiotropic actions of the glucagon/secretin/glucose-dependent insulinotropic polypeptide peptide superfamily are mediated via binding to and activation of distinct G protein-coupled receptors (GPCRs). These GPCRs are encoded by unique genes, yet are structurally related, and often share common features with respect to utilization of signaling mechanisms following ligand activation. Glucagon-related peptides regulate metabolic events, hormone secretion, and intestinal growth. For example, glucagon regulates glycogenolysis and gluconeogenesis via activation of a hepatocyte glucagon receptor (2Jelinek L.J. Lok S. Rosenberg G.B. Smith R.A. Grant F.J. Biggs S. Bensch P.A. Kuijper J.L. Sheppard P.O. Sprecher C.A. O'Hara P.J. Foster D. Walker K.M. Chen L.H.J. McKernan P.A. Kindsvogel W. Science. 1993; 259: 1614-1616Crossref PubMed Scopus (368) Google Scholar), whereas GLP-1 stimulates glucose-dependent insulin secretion following activation of an islet beta cell GLP-1 receptor (3Thorens B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8641-8645Crossref PubMed Scopus (837) Google Scholar). Studies of glucagon and GLP-1 receptor signaling in cells expressing the endogenous receptor or in heterologous cells expressing transfected receptors demonstrate that both these peptides activate downstream signaling mechanisms coupled to the cAMP-dependent pathway (1Drucker D.J. Diabetes. 1998; 47: 159-169Crossref PubMed Google Scholar). In contrast to our understanding of the mechanisms underlying glucagon and GLP-1 action, much less is known about the biological activity of GLP-2, a 33-amino acid peptide located carboxyl-terminal to GLP-1 in the proglucagon precursor. GLP-2 administration to mice or rats promotes stimulation of crypt cell proliferation and inhibition of enterocyte apoptosis resulting in hyperplasia of the small bowel villous epithelium (4Drucker D.J. Ehrlich P. Asa S.L. Brubaker P.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7911-7916Crossref PubMed Scopus (715) Google Scholar, 5Tsai C.-H. Hill M. Asa S.L. Brubaker P.L. Drucker D.J. Am. J. Physiol. 1997; 273: E77-E84Crossref PubMed Google Scholar). GLP-2 also exerts trophic effects in animal models of both small and large bowel injury such as experimental small bowel resection or chemically induced colitis (6Scott R.B. Kirk D. MacNaughton W.K. Meddings J.B. Am. J. Physiol. 1998; 275: G911-G921Crossref PubMed Google Scholar, 7Drucker D.J. Yusta B. Boushey R.P. Deforest L. Brubaker P.L. Am. J. Physiol. 1999; 276: G79-G91PubMed Google Scholar). In addition to stimulation of epithelial proliferation, GLP-2 also acutely regulates gastric emptying (8Wojdemann M. Wettergren A. Hartmann B. Holst J.J. Scand. J. Gastroenterol. 1998; 33: 828-832Crossref PubMed Scopus (181) Google Scholar) and exerts rapid metabolic effects promoting stimulation of intestinal hexose transport within 30 min following intravenous GLP-2 infusion (9Cheeseman C.I. Tsang R. Am. J. Physiol. 1996; 271: G477-G482PubMed Google Scholar, 10Cheeseman C.I. Am. J. Physiol. 1997; 273: R1965-R1971PubMed Google Scholar). The actions of GLP-2 are transduced via a recently isolated novel member of the glucagon/secretin GPCR superfamily. The GLP-2 receptor, isolated by expression cloning, exhibits 50% homology to the glucagon and GLP-1 receptors, is expressed in the central nervous system and gut, and has been localized to human chromosome 17 (11Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar). Consistent with studies of glucagon and GLP-1 receptor signaling, the GLP-2 receptor is coupled to the adenylate cyclase pathway in transfected fibroblasts (11Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar). Although several studies suggest that both the glucagon and GLP-1 receptors may be coupled to multiple signal transduction pathways, little is known about the potential for GLP-2 to activate signaling via nonadenylate cyclase-dependent mechanisms. Furthermore, unlike glucagon and GLP-1, the major action of GLP-2 involves stimulation of cell growth, and the mechanisms coupling GLP-2 receptor activation, directly or indirectly, to cell proliferation have not been examined. As intestinal cells expressing the endogenous GLP-2R have not yet been identified, we have now analyzed the actions of GLP-2 on downstream signaling pathways and cell proliferation in baby hamster kidney (BHK) fibroblasts stably transfected with the rat GLP-2 receptor. Glucagon, GLP-1-(7–36)NH2, and rat GLP-2-(1–33) were from Bachem California Inc. (Torrance, CA). Recombinant human [Gly2]-GLP-2 was a kind gift from Allelix Biopharmaceuticals Inc. (Mississauga, ON). 3-Isobutyl-1-methylxanthine, forskolin, and 8-Br-cAMP were obtained from Sigma. The protein kinase A (PKA) inhibitor H-89 was from Calbiochem. The p3AP1-luciferase (12Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar) and pCRE/β-galactosidase (13Chen W. Shields T.S. Stork P.J. Cone R.D. Anal. Biochem. 1995; 226: 349-354Crossref PubMed Scopus (181) Google Scholar) reporter plasmids were gifts from C. A. Hauser (San Diego, CA) and R. D. Cone (Portland, OR), respectively. The expression plasmid MtR(AB) that encodes a dominant negative mutant of the PKA regulatory subunit (14Correll L.A. Woodford T.A. Corbin J.D. Mellon P.L. McKnight G.S. J. Biol. Chem. 1989; 264: 16672-16678Abstract Full Text PDF PubMed Google Scholar) was a gift from G. S. McKnight (Seattle, WA). The PathDetect Elk-1 trans-reporting system was purchased from Stratagene (La Jolla, CA). Anti-phospho-extracellular signal-regulated kinase (Erk) antibody was obtained from New England Biolabs (Beverly, MA). Polyclonal anti-Akt1 (C-20) and anti-p70 S6 kinase antibodies, p70 S6 kinase peptide substrate, and PKA and protein kinase C inhibitor peptides were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Whatman p81 filter paper was purchased from Whatman (Tewksbury, MA). Okadaic acid and microcystin were from Biomol (Plymouth Meeting, PA). Rapamycin was from Calbiochem. Polyclonal anti-Akt2 and Akt substrate peptide (Crosstide) were purchased from Upstate Biotechnology (Lake Placid, NY). [γ-32P]ATP (6000 Ci/mmol) and enhanced chemiluminescence (ECL) reagents were purchased from Amersham Pharmacia Biotech. Purified phosphatidylinositol was purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Oxalate-treated TLC Silica gel H plates (250 microns) were from Analtech (Newark, DE). Protein A- and protein G-Sepharose were purchased from Amersham Pharmacia Biotech. All electrophoresis and immunoblotting reagents were purchased from Bio-Rad. BHK fibroblast were grown in Dulbecco's modified Eagle's medium (DMEM, 4.5 g/l glucose) supplemented with 5% calf serum. Cells were transfected with cDNAs encoding the rat GLP-2 receptor (11Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar) or the rat GLP-1 receptor (15Wheeler M.B. Lu M. Dillon J.S. Leng X.-H. Chen C. Boyd III, A.E. Endocrinology. 1993; 133: 57-62Crossref PubMed Scopus (148) Google Scholar) cloned in the pcDNA3.1 eukaryotic expression vector (Invitrogen, San Diego, CA) or with pcDNA3.1 alone by calcium phosphate coprecipitation. Stably transfected cell populations were selected by growth in G418 (Life Technologies, Inc.) at 0.8 mg/ml for 2 weeks. Individual cell clones were obtained by limited dilution cloning, expanded for further characterization, and maintained in DMEM with 0.5 mg/ml G418. The BHK-GLP-2R clone utilized for the present studies was representative of several G418-resistant clones that expressed the GLP-2R and gave identical results in signal transduction studies. For transient transfection assays, BHK cells stably expressing the rat GLP-2 receptor (BHK-GLP-2R) or the empty vector pcDNA3.1 (BHK-pcDNA3) were plated in medium without G418. Cells were transfected at 60–70% confluency by calcium phosphate coprecipitation with either 10 μg of pCRE/β-galactosidase or 5 μg of p3AP1-luciferase reporter constructs plus pBluescript II (Stratagene) carrier DNA for a total of 20 μg of DNA. In the transfections involving the PathDetect Elk-1 trans-reporting system, the precipitate contained 9.5 μg of the GAL4-luciferase reporter plasmid, 0.5 μg of expression vector encoding the GAL4-Elk-1 chimeric trans-activator protein, and 10 μg of carrier DNA or MtR(AB) expression vector. This plasmid was also used in studies of GLP-2 activation of both CRE- and AP-1-dependent activity. Four hours after transfection, cells were glycerol-shocked and incubated for 18–20 h in DMEM + 0.2% calf serum. Indicated drugs or peptides were added for 6 h in DMEM supplemented with 0.1% calf serum and 10 μm 3-isobutyl-1-methylxanthine before harvesting cells for analysis of β-galactosidase and luciferase activities as described previously (13Chen W. Shields T.S. Stork P.J. Cone R.D. Anal. Biochem. 1995; 226: 349-354Crossref PubMed Scopus (181) Google Scholar, 16Kaestner K.H. Katz J. Liu Y. Drucker D.J. Schutz G. Genes Dev. 1999; 13: 495-504Crossref PubMed Scopus (211) Google Scholar, 17Jin T. Drucker D.J. Mol. Cell. Biol. 1996; 16: 19-28Crossref PubMed Scopus (114) Google Scholar). Reporter gene activities were normalized to the protein concentration in each cell extract. Protein content was determined using a Coomassie dye assay (Bio-Rad). Data are presented as the mean ± S.E. from a minimum of 3–4 independent transfections, each carried out in triplicate or quadruplicate. RNA was isolated using a modified acid-ethanol guanidinium thiocynate method as described previously (18Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar). For Northern blot analysis, RNA was size-fractionated in an agarose gel, transferred to a nylon membrane, and immobilized with ultraviolet light, and hybridization and washing were carried out as described previously (19Drucker D.J. Brubaker P.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3953-3957Crossref PubMed Scopus (108) Google Scholar). BHK-GLP-2R cells were grown in 24-well plates at 37° C and treated with 10 nmh[Gly2]-GLP-2 or 20 μm forskolin in DMEM supplemented with 0.1% calf serum and 10 μm3-isobutyl-1-methylxanthine. Incubations were terminated at the indicated times by the addition of chilled ethanol (65% final concentration). cAMP was measured in dried aliquots of ethanol extracts using a cAMP radioimmunoassay kit (Biomedical Technologies, Stoughton, MA), and cAMP data were normalized to the protein content/well. BHK-GLP-2R and BHK-pcDNA cells grown in 96-well plates were serum-starved for 24 h and then incubated for 48 h in serum-free medium in the absence or presence of h[Gly2]-GLP-2 at the indicated concentrations. Control cells were treated identically but were exposed to 5% calf serum for 48 h. Fresh medium and treatments were replaced every 24 h. At the end of the incubation period the number of viable cells in each condition was measured using the CellTiter 96 aqueous nonRadioactive cell proliferation assay kit (Promega, Madison, WI) according to the manufacturer's suggestions. Cytosolic-free calcium was measured as described previously (20Coppolino M.G. Woodside M.J. Demaurex N. Grinstein S. St-Arnaud R. Dedhar S. Nature. 1997; 386: 843-847Crossref PubMed Scopus (348) Google Scholar). Briefly, cells grown on 25-mm glass coverslips were loaded with Fura-2 by incubation with 2 μm of the precursor acetoxymethyl ester for 20 min at 37° C. Fura-2 fluorescence ratio measurements were made on a Nikon Diaphot TMD microscope equipped with a Fluor 4OX, 1.3 N. A. oil immersion objective and a high sensitivity photometer (d-104, Photon Technology Instruments), which was interfaced to a NEC computer with a 12 bit AID board (Labmaster). Illumination was provided by a 100 watt xenon lamp coupled to the microscope via a rotating mirror and fiber optic assembly (Ratiomaster, PTI). The cells were alternately excited at 340 and 380 nm while recording emission at 510 nm. Photometric data were acquired at 10 Hz using the Oscar software (PTI). Ionomycin and EGTA were used to calibrate the fluorescence ratio versus calcium concentration. Prior to all experimental manipulations for analysis of kinase activity, cells were deprived of serum overnight (12 h). MAPK phosphorylation was detected as described (21Somwar R. Sweeney G. Ramlal T. Klip A. Clin. Ther. 1998; 20: 125-140Abstract Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, BHK-GLP-2R cells were treated with 20 nm GLP-2 or 100 nm insulin for the indicated time periods. Cells were lysed in a solution containing 10% glycerol, 4% SDS, 115 mmTris/HCl (pH 6.8), 10 mm dithiothreitol, 0.25 mg/ml bromphenol blue, protease inhibitors (100 μmphenylmethylsulfonyl fluoride, 10 μm E-64, 1 μm pepstatin, 1 μm leupeptin), and phosphatase inhibitors (40 mm sodium fluoride, 7.5 mm sodium pyrophosphate, 1.5 mmNa3VO4). Lysates were passed five times through a 25-gauge syringe to sheer the DNA and boiled for 3 min. To detect MAPK phosphorylation, 30 μg of total cellular protein were resolved by 10% SDS-polyacrylamide gel electrophoresis, electrotransferred onto polyvinylidene difluoride membranes, and then immunoblotted with phospho-specific MAPK antibody (22Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1853) Google Scholar) (polyclonal, 1:1000 dilution). Protein was detected by the enhanced chemiluminescence method using goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5000 dilution) as the secondary antibody. p70 S6 kinase activity was determined as described previously (23Somwar R. Sumitani S. Taha C. Sweeney G. Klip A. Am. J. Physiol. 1998; 275: E618-E625PubMed Google Scholar). BHK-GLP-2R cells grown in 6-well plates were incubated with 20 nm h[Gly2]-GLP-2, washed twice with ice-cold phosphate-buffered saline, and lysed in 1 ml of lysis buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 20 mm β-glycerophosphate, 10 mm EDTA, 10 mm sodium pyrophosphate, 100 mm NaF, 1 mm Na3VO4, 1 mmdithiothreitol, 10 nm okadaic acid, and 1% (v/v) Nonidet P-40) containing a mixture of protease inhibitors (1 μmleupeptin, 1 μm pepstatin A, 10 μm E-64, and 200 μm phenylmethylsulfonyl fluoride). After 15 min of slow agitation and centrifugation (15,000 × g for 15 min), the supernatant was subjected to immunoprecipitation. p70 S6 kinase was immunoprecipitated using 250 μg of total protein and 1 μg of a rabbit polyclonal p70 S6 kinase antibody. The p70 S6 kinase immunocomplex was washed three times with wash buffer (50 mm Tris acetate, pH 8, 50 mm NaF, 5 mm sodium pyrophosphate, 5 mmβ-glycerophosphate, 1 mm Na3VO4, 1 mm EDTA, 1 mm EGTA, 10 nm okadaic acid, 0.1% (v/v) β-mercaptoethanol) including all the protease inhibitors used above and twice with kinase buffer (20 mm4-morpholinepropanesulfonic acid, pH 7.2, 25 mmβ-glycerophosphate, 5 mm EGTA, 2 mm EDTA, 20 mm MgCl2, 2 mmNa3VO4, and 1 mm dithiothreitol in a final volume of 50 μl of kinase buffer containing 1 μm protein kinase A and protein kinase C inhibitor peptides, 0.2 mm S6 peptide, and 0.25 mmMg-[γ-32P]ATP at 30 C for 10 min. Aliquots (30 μl) were transferred onto Whatman p81 filter papers and washed 3 times for 15 min with 175 mm phosphoric acid. 32P incorporated into the S6 peptide was measured by liquid scintillation counting. Immunoprecipitation of Akt and kinase assay was performed as described (23Somwar R. Sumitani S. Taha C. Sweeney G. Klip A. Am. J. Physiol. 1998; 275: E618-E625PubMed Google Scholar) with modifications. Anti-Akt antibodies were pre-coupled to a mixture of protein A- and protein G-Sepharose beads by incubating 2 μg of antibody/condition with 20 μl of protein A-Sepharose (100 mg/ml) and 20 μl of protein G-Sepharose (100 mg/ml) for a minimum of 2 h. The anti-Akt-bead complexes were washed twice with ice-cold phosphate-buffered saline and once with ice-cold lysis buffer. Akt was immunoprecipitated by incubating 200 μg of total cellular protein with the anti-Akt-bead complex for 2–3 h under constant rotation (4° C). The Akt1 immunocomplex was isolated and washed 4 times with 1 ml of wash buffer (25 mmHEPES, pH 7.8, 10% glycerol (v/v), 1% Triton X-100 (v/v), 0.1% bovine serum albumin (v/v), 1 m NaCl, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 μm microcystin, 100 nm okadaic acid) and twice with 1 ml of kinase buffer (50 mm Tris/HCl, pH 7.5, 10 mm MgCl2 and 1 mmdithiothreitol). This was then incubated under constant agitation for 30 min at 30° C with 30 μl of reaction mixture (kinase buffer containing 5 μm ATP, 2 μCi of [γ-32P]ATP, and 100 μm Crosstide). Following the reaction, 30 μl of the supernatant were transferred onto Whatman p81 filter paper and treated as described above for p70 S6 kinase assay. To stain F-actin, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 20 min at room temperature and then permeabilized using 0.1% Triton X-100 in phosphate-buffered saline. Permeabilization was followed by incubation with a 1:1000 dilution of rhodamine phalloidin (Molecular Probes) for 45 min at room temperature. The samples were then washed extensively and mounted using Dako mounting medium. Samples were visualized by epifluorescence on a Leica DM-IRB microscope, and images were acquired with a MicroMax 2 cooled charge-coupled device camera (Princeton Instruments) using WinView software and a PC compatible computer. The initial characterization of GLP-2R signaling was carried out in COS cells transiently transfected with the GLP-2R (11Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar). As cell lines that express an endogenous GLP-2R have not yet been identified, we chose to establish in vitro models for reproducible analysis of GLP-2 action by generating BHK fibroblast clones that stably expressed the rat GLP-2 receptor. BHK cells were transfected with an expression vector containing the full-length rat GLP-2R coding sequence under the control of the cytomegalovirus promoter in the pcDNA3.1 expression vector. Following selection with the antibiotic G418, surviving clones were expanded and characterized for GLP-2R expression. Several BHK-GLP-2R cell lines were identified that expressed the GLP-2R and responded identically to GLP-2. A representative clone, hereafter referred to as BHK-GLP-2R, was chosen for more detailed analysis of GLP-2-dependent signal transduction. Studies were carried out with either native rat GLP-2 or h[Gly2]-GLP-2, a protease-resistant GLP-2 analogue recently shown to exhibit greater in vitro and in vivo stability compared with the native peptide (24Drucker D.J. Shi Q. Crivici A. Sumner-Smith M. Tavares W. Hill M. Deforest L. Cooper S. Brubaker P.L. Nature Biotechnol. 1997; 15: 673-677Crossref PubMed Scopus (225) Google Scholar). As analysis of GLP-2R signaling in transiently transfected COS cells suggested GLP-2 activated the adenylate cyclase pathway (11Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar), we initially analyzed the GLP-2-dependent activation of a cAMP-dependent reporter gene, CRE-β-galactosidase, in transfected BHK-GLP-2R cells. The activity of this reporter gene has been shown to correlate, in a linear manner, with accumulation of intracellular cAMP (13Chen W. Shields T.S. Stork P.J. Cone R.D. Anal. Biochem. 1995; 226: 349-354Crossref PubMed Scopus (181) Google Scholar). Both rGLP-2 and h[Gly2]-GLP-2, from 0.01 to 20 nm, increased β-galactosidase activity in BHK-GLP-2R cells (Fig. 1 A). Furthermore, the level of β-galactosidase induction following transfection of CRE-β-galactosidase and incubation with GLP-2 was similar in magnitude to that obtained by treating the cells with either forskolin or 8-bromo-cyclic AMP, two well characterized activators of the adenylate cyclase pathway (Fig. 1 B). In contrast, the structurally related peptides glucagon and GLP-1 did not stimulate β-galactosidase activity in BHK-GLP-2R cells (Fig. 1 A). Furthermore, GLP-2 had no effect on the activity of a cotransfected CRE-β-galactosidase reporter gene in control cells stably expressing the parental expression vector pcDNA3.1 (Fig. 1 C). To verify that the activation of CRE-dependent β-galactosidase activity reflected the accumulation of intracellular cAMP following GLP-2 stimulation, we compared the levels of cAMP in BHK-GLP-2R cells at various time points after incubation of cells with either 10 nm h[Gly2]-GLP-2 or 20 μmforskolin. The relative magnitude and kinetics of intracellular cAMP accumulation were comparable from 10 to 360 min following exposure of cells to either reagent (Fig. 1 D). Furthermore, the EC50 for stimulation of CRE-dependent β-galactosidase activity was ∼0.06 nm, identical to the value reported for GLP-2-stimulation of cAMP accumulation in 293-EBNA cells expressing the rat GLP-2 receptor (11Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar). As the structurally related peptides glucagon and GLP-1 stimulate AP-1-dependent signaling pathways (25Susini S. Roche E. Prentki M. Schlegel W. FASEB J. 1998; 12: 1173-1182Crossref PubMed Scopus (124) Google Scholar, 26Hansen L.H. Gromada J. Bouchelouche P. Whitmore T. Jelinek L. Kindsvogel W. Nishimura E. Am. J. Physiol. 1998; 274: C1552-C1562Crossref PubMed Google Scholar), we next ascertained whether activation of the GLP-2 receptor was also coupled to AP-1-dependent transcriptional activation. BHK-GLP-2R cells were transfected with a reporter gene containing three tandemly linked AP-1 sites adjacent to a luciferase reporter gene. h[Gly2]-GLP-2, at concentrations of 0.01–20 nm, stimulated a 3–4-fold induction of AP-1-dependent luciferase activity in BHK-GLP-2R cells (Fig.2 A) but not in BHK cells stably transfected with the expression vector alone (BHK-pcDNA3.1, Fig. 2 B). Induction of AP-1-directed luciferase activity was also observed with activators of the adenylate cyclase pathway such as forskolin and 8-bromo-cyclic AMP (Fig. 2 C); however, the relative magnitude of induction with these PKA activators was less than that observed for h[Gly2]-GLP-2 alone (Fig. 2 C,p < 0.05). Similarly, exposure of BHK-GLP-2R cells to 10% fetal calf serum significantly activated AP-1-directed luciferase activity (Fig. 2 C). Taken together, these findings establish the sensitivity and specificity of GLP-2-induction of adenylate cyclase and AP-1-dependent pathways in BHK-GLP-2R cells in vitro. The induction of AP-1-luciferase activity by both forskolin and 8-Br-cAMP suggested that GLP-2 might activate AP-1 activity via PKA-dependent mechanisms. To examine this possibility, the CRE-β-galactosidase and AP1-luciferase reporter genes were transfected into BHK-GLP-2R cells in the presence or absence of a cDNA encoding a dominant negative inhibitor of PKA, MtR(AB) (14Correll L.A. Woodford T.A. Corbin J.D. Mellon P.L. McKnight G.S. J. Biol. Chem. 1989; 264: 16672-16678Abstract Full Text PDF PubMed Google Scholar). The GLP-2-dependent induction of β-galactosidase activity was reduced by 80% in the presence of the cotransfected PKA inhibitor (Fig. 3). Similarly, the forskolin induction of CRE-β-galactosidase was reduced by ∼80% in similar experiments, consistent with the results of previous studies (14Correll L.A. Woodford T.A. Corbin J.D. Mellon P.L. McKnight G.S. J. Biol. Chem. 1989; 264: 16672-16678Abstract Full Text PDF PubMed Google Scholar). Furthermore both the GLP-2- and forskolin-dependent activation of AP-1-luciferase activity were also significantly reduced in the presence of the PKA inhibitor MtR(AB) (Fig. 3, p< 0.001–0.005). Similar results were also obtained with the PKA inhibitor H89 (data not shown). However, whereas the forskolin induction of AP-1 activity was eliminated in the presence of PKA inhibition, a small but detectable GLP-2-induction of AP-1 luciferase was still observed in the presence of MtR(AB). These findings suggest the existence of alternate pathways independent of PKA for induction of AP-1 activity. Consistent with the existence of these alternate pathways, the serum induction of AP-1-dependent luciferase activity was not diminished by co-transfection with the PKA inhibitor plasmid (Fig. 3). As activation of the AP-1 pathway is frequently associated with stimulation of cell proliferation, the finding that GLP-2 activate
Referência(s)