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• W2068596181 abstract "The luteinizing hormone-releasing hormone (LHRH) receptor is a G protein-coupled receptor involved in the synthesis and release of pituitary gonadotropins and in the proliferation and apoptosis of pituitary cells. Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that has a mitogenic effect on pituitary cells. In this study, we used the αT3 gonadotrope cell line as a model to characterize the IGF-1R signaling pathways and to investigate whether this receptor interacts with the LHRH cascade. We found that IGF-1 activated the IGF-1R, insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase, and Akt in a time-dependent manner in αT3 cells. The MAPK (ERK1/2, p38, and JNK) pathways were only weakly activated by IGF-1. In contrast, LHRH strongly stimulated the MAPK pathways but had no effect on Akt activation. Cotreatment with IGF-1 and LHRH had various effects on these signaling pathways. 1) It strongly increased IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated phosphatidylinositol 3-kinase through activation of the epidermal growth factor receptor. 2) It had an additive effect on ERK1/2 activation without modifying the phosphorylation of p38 and JNK1/2. 3) It strongly reduced IGF-1 activation of Akt. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and cell cycle analysis revealed that, in addition to having an additive effect on ERK1/2 activation, cotreatment with IGF-1 and LHRH also had an additive effect on cell proliferation. The LHRH-induced inhibition of Akt stimulated by IGF-1 was completely blocked by Safingol, a protein kinase C (PKC) α-specific inhibitor, and by a dominant negative form of PKCα. Finally, we showed that the inhibitory effect of LHRH on IGF-1-induced PKCα-mediated Akt activation was associated with a marked reduction in Bad phosphorylation and a substantial decrease in the ability of IGF-1 to rescue αT3 cells from apoptosis induced by serum starvation. Our results demonstrate for the first time that several interactions take place between IGF-1 and LHRH receptors in gonadotrope cells. The luteinizing hormone-releasing hormone (LHRH) receptor is a G protein-coupled receptor involved in the synthesis and release of pituitary gonadotropins and in the proliferation and apoptosis of pituitary cells. Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that has a mitogenic effect on pituitary cells. In this study, we used the αT3 gonadotrope cell line as a model to characterize the IGF-1R signaling pathways and to investigate whether this receptor interacts with the LHRH cascade. We found that IGF-1 activated the IGF-1R, insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase, and Akt in a time-dependent manner in αT3 cells. The MAPK (ERK1/2, p38, and JNK) pathways were only weakly activated by IGF-1. In contrast, LHRH strongly stimulated the MAPK pathways but had no effect on Akt activation. Cotreatment with IGF-1 and LHRH had various effects on these signaling pathways. 1) It strongly increased IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated phosphatidylinositol 3-kinase through activation of the epidermal growth factor receptor. 2) It had an additive effect on ERK1/2 activation without modifying the phosphorylation of p38 and JNK1/2. 3) It strongly reduced IGF-1 activation of Akt. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and cell cycle analysis revealed that, in addition to having an additive effect on ERK1/2 activation, cotreatment with IGF-1 and LHRH also had an additive effect on cell proliferation. The LHRH-induced inhibition of Akt stimulated by IGF-1 was completely blocked by Safingol, a protein kinase C (PKC) α-specific inhibitor, and by a dominant negative form of PKCα. Finally, we showed that the inhibitory effect of LHRH on IGF-1-induced PKCα-mediated Akt activation was associated with a marked reduction in Bad phosphorylation and a substantial decrease in the ability of IGF-1 to rescue αT3 cells from apoptosis induced by serum starvation. Our results demonstrate for the first time that several interactions take place between IGF-1 and LHRH receptors in gonadotrope cells. Luteinizing hormone-releasing hormone (LHRH) 1The abbreviations used are: LHRH, luteinizing hormone-releasing hormone; LHRH-R, LHRH receptor; JNK, c-Jun N-terminal kinase; PI3K, phosphatidylinositol 3-kinase; IGF-1, insulin-like growth factor-1; IGF-1R, IGF-1 receptor; LH, Luteinizing hormone; FSH, follicle-stimulating hormone; EGF, epidermal growth factor; EGFR, EGF receptor; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PH, pleckstrin homology; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; IRS, insulin receptor substrate; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; FAK, focal adhesion kinase.1The abbreviations used are: LHRH, luteinizing hormone-releasing hormone; LHRH-R, LHRH receptor; JNK, c-Jun N-terminal kinase; PI3K, phosphatidylinositol 3-kinase; IGF-1, insulin-like growth factor-1; IGF-1R, IGF-1 receptor; LH, Luteinizing hormone; FSH, follicle-stimulating hormone; EGF, epidermal growth factor; EGFR, EGF receptor; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PH, pleckstrin homology; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; IRS, insulin receptor substrate; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; FAK, focal adhesion kinase. is a hypothalamic decapeptide, which plays a crucial role in normal reproductive function. In the pituitary, LHRH-I (mammalian LHRH) stimulates the synthesis and the release of the gonadotropins, LH and FSH, and promotes cell proliferation and apoptosis in pituitary cells (1Lewy H. Ashkenazi I.E. Naor Z. Mol. Cell. Endocrinol. 2003; 203: 25-32Crossref PubMed Scopus (12) Google Scholar, 2Imai A. Tamaya T. Vitam. Horm. 2000; 59: 1-33Crossref PubMed Google Scholar). The effects of LHRH-I are mediated by a cell surface receptor (LHRH-R) belonging to the G protein-coupled receptor superfamily (3Conn P.M. Crowley Jr., W.F. Annu. Rev. Med. 1994; 45: 391-405Crossref PubMed Scopus (327) Google Scholar, 4Sealfon S.C. Weinstein H. Millar R.P. Endocr. Rev. 1997; 18: 180-205Crossref PubMed Scopus (388) Google Scholar). LHRH-I binding to its cognate receptor leads to interaction of the receptor with heterotrimeric G proteins, including Gq/11. These G proteins in turn activate phospholipase C, leading to the production of diacylglycerol and the subsequent activation of protein kinase C (PKC) (5Naor Z. Harris D. Shacham S. Front. Neuroendocrinol. 1998; 19: 1-19Crossref PubMed Scopus (112) Google Scholar, 6Hawes B.E. Barnes S. Conn P.M. Endocrinology. 1993; 132: 2124-2130Crossref PubMed Google Scholar, 7Shah B.H. Milligan G. Mol. Pharmacol. 1994; 46: 1-7PubMed Google Scholar, 8Stanislaus D. Janovick J.A. Brothers S. Conn P.M. Mol. Endocrinol. 1997; 11: 738-746PubMed Google Scholar, 9Liu F. Usui I. Evans L.G. Austin D.A. Mellon P.L. Olefsky J.M. Webster J.G. J. Biol. Chem. 2002; 277: 32099-32108Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Activation of PKC then leads to activation of downstream protein kinases belonging to the MAPK family (9Liu F. Usui I. Evans L.G. Austin D.A. Mellon P.L. Olefsky J.M. Webster J.G. J. Biol. Chem. 2002; 277: 32099-32108Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 10Mitchell R. Sim P.J. Leslie T. Johnson M.S. Thomson F.J. J. Endocrinol. 1994; 140: R15-R18Crossref PubMed Scopus (62) Google Scholar, 11Sim P.J. Wolbers W.B. Mitchell R. Mol. Cell. Endocrinol. 1995; 112: 257-263Crossref PubMed Scopus (43) Google Scholar, 12Reiss N. Llevi L.N. Shacham S. Harris D. Seger R. Naor Z. Endocrinology. 1997; 138: 1673-1682Crossref PubMed Scopus (114) Google Scholar, 13Roberson M.S. Misra-Press A. Laurance M.E. Stork P.J.S. Maurer R.A. Mol. Cell. Biol. 1995; 15: 3531-3539Crossref PubMed Google Scholar, 14Sundaresan S. Colin I.M. Pestell R.G. Jameson J.L. Endocrinology. 1996; 137: 304-311Crossref PubMed Scopus (142) Google Scholar, 15Naor Z. Benard O. Seger R. Trends Endocrinol. Metab. 2000; 11: 91-99Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Activation of LHRH-R also triggers calcium influx, resulting in an increase in intracellular calcium (16Conn P.M. Janovick J.A. Stanislaus D. Kuphal D. Jennes L. Vitam. Horm. 1995; 50: 151-214Crossref PubMed Scopus (60) Google Scholar) and cAMP levels (17Bourne G.A. Mol. Cell. Endocrinol. 1998; 58: 155-160Crossref Scopus (25) Google Scholar, 18Borgeat P. Chavancy G. Dupont A. Labrie F. Arimura A. Schally A.V. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2677-2681Crossref PubMed Scopus (159) Google Scholar) and leading to the activation of other protein kinases, such as the c-Jun N-terminal kinase (19Mulvaney J.M. Roberson M.S. J. Biol. Chem. 2000; 275: 14182-14189Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 20Johnson C.M. Hill C.S. Chawla S. Treisman R. Bading H. J. Neurosci. 1997; 17: 6189-6202Crossref PubMed Google Scholar). This LHRH-R-mediated pathway acts independently of the PKC and MAPK signaling pathways (21Roberson M.S. Zhang T. Ling H. Mulvaney J.M. Endocrinology. 1999; 140: 1310-1318Crossref PubMed Google Scholar). Thus, multiple signal transduction pathways may mediate LHRH-I action in the pituitary gland.Like LHRH-I, IGF-1 is a mitogen in pituitary cells (22Childs G.V. Unabia G. Endocrinology. 2001; 142: 847-853Crossref PubMed Scopus (26) Google Scholar, 23Doornbos R.P. Theelen M. Van der Hoeven P.C.J. Blitterswijk W.J.V. Verkleij A.J. Van Bergen en Henegouwen P.M.P. J. Biol. Chem. 1999; 274: 8589-8596Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 24Oomizu S. Takeuchi S. Takahashi S. J. Endocrinol. 1998; 157: 53-62Crossref PubMed Scopus (68) Google Scholar) and an anti-apoptotic factor in several cell lines (25Párrizas M. Saltiel A.R. LeRoith D. J. Biol. Chem. 1997; 272: 154-161Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 26Krause D. Lyons A. Fennelly C. O'Connor R. J. Biol. Chem. 2001; 276: 19244-19252Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). IGF-1 activates the intrinsic protein-tyrosine kinase activity of the IGF-1 receptor (IGF-1R) (27LeRoith D. Werner H. Beitner-Johnson D. Roberts Jr., C.T. Endocr. Rev. 1995; 16: 143-163Crossref PubMed Scopus (1241) Google Scholar). This activation results in auto-phosphorylation of the receptor and phosphorylation of various intracellular substrates, including several insulin receptor substrate (IRS) proteins (IRS-1 to -4) and the Src homology collagen protein (28Dupont J. Dunn S.E. Barrett J.C. LeRoith D. Recent Prog. Horm. Res. 2003; 58: 325-342Crossref PubMed Scopus (61) Google Scholar). Tyrosine-phosphorylated IRS-1 is a multisite docking protein for numerous Src homology 2 domain-containing proteins, including the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and the adapter protein, Grb2 (29Backer J.M. Myers M.G. Shoelson S.E. Chin D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (813) Google Scholar, 30Skolnick E.Y. Lee C.H. Batzer A. Vicentini L.M. Zhou M. Daly R. Myers M.G. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (604) Google Scholar). Downstream effectors of PI3K include the serine/threonine protein kinases, Akt/PKB and p70/S6 (31Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1180) Google Scholar). The binding of the IRS and Src homology collagen proteins to Grb2 and its associated guanine nucleotide exchange protein, mSos, leads to the activation of the Ras-Raf-MAPK pathway (27LeRoith D. Werner H. Beitner-Johnson D. Roberts Jr., C.T. Endocr. Rev. 1995; 16: 143-163Crossref PubMed Scopus (1241) Google Scholar). The role of the different pathways activated by IGF-1 during proliferative and anti-apoptotic responses is cell type-specific. For example, in human intestinal smooth muscle cells (32Kuemmerle J.F. Am. J. Physiol. 2003; 284: G411-G422Crossref PubMed Scopus (46) Google Scholar, 33Kuemmerle J.F. Bushman T.L. Am. J. Physiol. 1998; 275: G151-G158PubMed Google Scholar), IGF-1 induces growth by activating both the PI3K and MAPK-ERK1/2 pathways. In contrast, only the PI3K pathway is essential for IGF-1 signaling in proliferating MCF-7 cell lines (derived from human breast tumor) (34Dufourny B. Alblas J. van Teeffelen H.A. van Schaik F.M. van der Burg B. Steenbergh P.H. Sussenbach J.S. J. Biol. Chem. 1995; 272: 23589-23597Google Scholar).IGF-1 and IGF-1R are both expressed in pituitary cells (35Bach M.A. Bondy C.A. Endocrinology. 1992; 131: 2588-2594Crossref PubMed Scopus (84) Google Scholar). Like LHRH-I, IGF-1 treatment increases LH secretion by male rat pituitary cells (36Soldani R. Cagnacci A. Yen S.S.C. Eur. J. Endocrinol. 1994; 131: 641-645Crossref PubMed Scopus (95) Google Scholar). Moreover, IGF-1 enhances the LH response to LHRH-I in pituitary cells from male rats (37Xia Y.X. Weiss J.M. Polack S. Diedrich K. Ortmann O. Eur. J. Endocrinol. 2001; 144: 73-79Crossref PubMed Scopus (51) Google Scholar), pigs (38Whitley N.C. Barb C.R. Utley R.V. Popwell J.M. Kraeling R.R. Rampacek G.B. Biol. Reprod. 1995; 53: 1359-1364Crossref PubMed Scopus (28) Google Scholar), fish (39Weil C. Carre F. Blaise O. Breton B. Le Baril P.Y. Endocrinology. 1999; 140: 2054-2062Crossref PubMed Google Scholar), and sheep (40Hashizume T. Kumahara A. Fujino M. Okada K. Anim. Reprod. Sci. 2002; 70: 13-21Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). IGF-1 also regulates growth in pituitary gonadotrope cells (41Emons G. Muller V. Ortmann O. Schulz K.D. J. Steroid Biochem. Mol. Biol. 1998; 65: 199-206Crossref PubMed Scopus (125) Google Scholar). LHRH-I agonists inhibit cell proliferation in prostate cancer cells by interfering with some of the cellular mechanisms required for the mitogenic activity of IGF-1 (42Marelli M.M. Moretti R.M. Dondi D. Motta M. Limonta P. Endocrinology. 1999; 140: 329-334Crossref PubMed Scopus (60) Google Scholar). The degree of interaction between LHRH-I and IGF-1 signaling pathways in the pituitary gland remains unclear. In this study we have investigated these interactions by using αT3–1 gonadotrope cells derived from murine pituitary tumor cells. These cells produce the glycoprotein hormone α-subunit, the LHRH-R and SF-1. However, these cells do not produce LHβ, as they were derived from cells at an early stage of development during which LHβ was not produced. Although these cells do not express the α, LHβ, and FSHβ subunits (43Alarid E.T. Windle J.J. Whyte D.B. Mellon P.L. Development (Camb.). 1996; 122: 3319-3329PubMed Google Scholar, 44Windle J.J. Weiner R.I. Mellon P.L. Mol. Endocrinol. 1990; 4: 597-603Crossref PubMed Scopus (440) Google Scholar), they proliferate and express the LHRH receptor. Thus, αT3 cells provide a good model system for determining whether interactions exist between the LHRH-I and IGF-1 signaling pathways.In this study, we characterized interactions taking place between the IGF-1R and LHRH-R signaling pathways during cell proliferation and apoptosis in αT3 cells. We demonstrate for the first time that LHRH-I treatment enhances IGF-1-induced, MAPK-ERK1/2 and PKC signaling pathway-mediated cell proliferation, whereas it strongly diminishes the anti-apoptotic effect of IGF-1 through inhibition of PKCα-mediated Akt activation.EXPERIMENTAL PROCEDURESMaterials—[α-32P]dCTP (6000 Ci/mmol) and [γ-32P]ATP (6000 Ci/mmol) were obtained from PerkinElmer Life Sciences. Recombinant human IGF-1 was purchased from R & D (Minneapolis, MN). Recombinant human LHRH-I, buserelin, and the LHRH antagonist, trifluoroacetate, were purchased from Sigma. Recombinant human EGF was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, and trypsin were purchased from Invitrogen. PI3K-specific inhibitor (LY294002) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. MEK1/2-specific inhibitor (U0126), p38 MAPK-specific inhibitor (SB202190), PKC-specific inhibitor (GF109203X), PKCδ-specific inhibitor (Rottlerin), PKCα-specific inhibitor (Safingol), EGF receptor tyrosine kinase-specific inhibitor (AG1470), and the calcium chelator, BAPTA, were obtained from Calbiochem. The JNK1/2 MAPK-specific inhibitor SP600125 was purchased from Biomol Research Laboratories (Plymouth Meeting, MA). All inhibitor stock solutions were prepared in MeSO. These stock solutions were prepared so that the concentration of MeSO added to the culture medium was below 0.1%.Rabbit polyclonal antibodies against phospho-Bad (phospho-Ser-136), Bad, phospho-Akt (phospho-Ser-473), Akt, phospho-ERK1/2 (phospho-Thr-202/Tyr-204), phospho-p38 (phospho-Thr-180/Tyr-182), phospho-JNK1/2 (phospho-Thr-183/Tyr-185), phospho-GSK3α/β (phospho-Ser-21/9), phospho-FKHR (phospho-Ser-256), phospho-Elk-1 (phospho-Ser-383), and cleaved caspase-3 were purchased from New England Biolabs (Beverly, MA). Rabbit polyclonal antibodies to EGFR (1005), phospho-EGFR (Tyr-1173), ERK2 (C14), p38 (C20), JNK2 (N18), IGF-1R (C20), and FAK (C20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to rabbit IRS-1, IRS-2, and the p85 regulatory subunit of mouse PI3K (αp85) were purchased from Upstate Biotechnology, Inc. Monoclonal anti-actin (clone AC) and monoclonal anti-phosphotyrosine (PY20) antibodies were obtained from Sigma and Transduction Laboratories (Lexington, KY), respectively. All antibodies were used at 1:1000 dilution.Preparation of Rat Anterior Pituitary Cells and Cell Cultures—Rat pituitary cells were prepared from 5-week-old female Wistar rats. Freshly removed anterior pituitary glands were washed twice in B1 buffer (0.25% bovine serum albumin, 0.01% deoxyribonuclease, 137 mm NaCl, 5 mm KCl, 25 mm HEPES, 0.6 mm NaH2PO4, 0.7 mm Na2HPO4 12H2O, and 6 mm glucose) before being incubated with type I collagenase (4 mg/ml) and grade II dispase (2 mg/ml) in buffer B1 at 37 °C for 30 min. Cells were centrifuged for 4 min at 200 × g and then incubated with 0.8% type V neuraminidase in B1 buffer for 10 min at 37 °C. After centrifugation (200 × g, 4 min), dispersed cells were cultured in DMEM without red phenol and supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 μg/ml), fungizone (1.25 μg/ml), and l-glutamine (200 mm).Mouse gonadotrope αT3 cell lines and LβT2 cells were provided by Dr. P. Mellon (La Jolla, CA). MCF-7, MDA-MB-231, OVCAR, and SKOV cells were obtained from the ATCC (Manassas, VA). All cell lines were routinely cultured in a humidified atmosphere of 95% air and 5% CO2 in DMEM supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 300 μg/ml l-glutamine.Immunoprecipitation and Immunoblotting—Cell lysates were homogenized on ice in lysis buffer A (10 mm Tris (pH 7.4), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.5% Nonidet P-40) supplemented with various protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) and phosphatase inhibitors (100 mm sodium fluoride, 10 mm sodium pyrophosphate, and 2 mm sodium orthovanadate). Lysates were incubated on ice for 30 min and then centrifuged at 12,000 × g for 20 min at 4 °C. The protein concentration in the resulting supernatants was then determined using the BCA protein assay. Extracts containing 200 μg of protein were incubated with αIGF-1R, αIRS-1, or αIRS-2 (1:1000 dilution) for 16 h at 4 °C. Immune complexes were precipitated by incubation with protein G-agarose for 1 h at 4 °C as described previously (45Dupont J. Karas M. LeRoith D. J. Biol. Chem. 2000; 275: 35893-35901Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Immunoprecipitates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked by incubation for 1 h at room temperature with 5% nonfat milk dissolved in Tris-buffered saline supplemented with 0.1% Tween 20 and then probed with the appropriate antibodies. After extensive washing, immune complexes were detected using horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) and an enhanced chemiluminescence (ECL) detection system. The radiographs were scanned, and the optical density of each band was measured using the MacBas version 2.52 software (Fuji PhotoFilm). Western blotting was performed without immunoprecipitation using cell extracts containing 50 μg of protein.PI3K Assay—PI3K activity was determined as described previously (45Dupont J. Karas M. LeRoith D. J. Biol. Chem. 2000; 275: 35893-35901Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Cell lysates were prepared on ice in extraction buffer B (20 mm Tris (pH 7.5), 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 150 mm Na3VO4, 1% Nonidet P-40, 10% glycerol (v/v), 2 mm phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin in phosphate-buffered saline (PBS)). Cell lysates were cleared by centrifugation for 35 min at 40,000 × g at 4 °C. IRS-1, IRS-2, IGF-1R, or p85 were immunoprecipitated by incubating aliquots (containing 200 μg of total protein) of each of the supernatants with the appropriate antibodies (1:1000) overnight at 4 °C. Immunoprecipitates were collected using protein G-agarose beads and washed as follows: once in PBS supplemented with 1% Nonidet P-40 and 100 μm Na3VO4; twice in a buffer containing 100 mm Tris-HCl (pH 7.5), 500 mm LiCl2, 100 μm Na3VO4; and finally once in a buffer containing 10 mm Tris-HCl (pH 7.5), 100 mm NaCl, 1 mm EDTA, and 100 μm Na3VO4. Precipitates were then resuspended in 40 μl of a buffer containing 10 mm Tris-HCl (pH 7.5), 100 mm NaCl, and 1 mm EDTA. MnCl2 (10 μl of a 100 mm stock solution) and phosphatidylinositol (20 μg) were then added to each sample. PI3K assays were initiated by adding 10 μl of a 440 μm ATP solution containing 30 μCi of [γ-32P]ATP. Reactions were performed at room temperature and allowed to proceed for 10 min. Reactions were stopped by adding 20 μl of HCl (8N) and 160 μl of CHCl3/CH3OH (1:1). The reaction mixtures were then centrifuged at 3,000 × g for 4 min at 4 °C. The resulting organic phase was collected and subjected to silica gel TLC. TLC plates were developed by incubation in CHCl3/CH3OH/H2O/NH4OH (120:94:22.6:4) and then left to dry. Radioactivity was quantified using a PhosphorImager (Storm, Amersham Biosciences).Akt and ERK1/2 MAPK Activity Assays—Akt and ERK1/2 MAPK kinase activities were measured using the appropriate assay kits (Cell Signaling Technology, Beverly, MA). αT3 cells were stimulated as described in the figure legends and lysed using cell lysis buffer. Cell lysates containing 200 μg of total protein were immunoprecipitated by incubation with immobilized Akt or phospho-ERK1/2 monoclonal antibodies, with gentle rocking for 3 h at 4 °C. After two washes with cell lysis buffer followed by two washes with kinase buffer (25 mm Tris (pH 7.5), 5 mm β-glycerophosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4,10 mm MgCl2), the immunoprecipitates were resuspended in 40 μl of kinase buffer supplemented with 200 μm ATP and 1 μg of GSK-3 or Elk-1 fusion proteins. Samples were incubated for 30 min at 30 °C. The reaction was terminated by adding 20 μl of 3× SDS loading buffer. Samples were analyzed by probing with the phospho-GSK-3α/β (Ser-21/9) or phospho-Elk-1 (Ser-383) antibodies.Cell Proliferation Assay—For growth studies, αT3 cells were seeded in 96-well plates (10,000 cells per well) in DMEM supplemented with 10% of FBS. After 1 day, cells were transferred to serum-free medium and allowed to grow for 24 h before being incubated with various stimulants (see figure legends for details). Growth was analyzed using the MTT assay as described previously (45Dupont J. Karas M. LeRoith D. J. Biol. Chem. 2000; 275: 35893-35901Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar).Flow Cytometry and Cell Cycle Analysis—αT3 cultures, in which ∼75% of the cells were confluent, were washed and transferred to serum-free DMEM and allowed to grow for 24 h. Cells were subjected to various treatments (see figure legends for details). After treatment with IGF-1 (10 nm) and/or LHRH (10 nm) for 18 h, cells were trypsinized, centrifuged, and washed twice with PBS. Cell pellets were resuspended in citrate buffer (250 mm sucrose, 40 mm trisodium citrate (pH 7.6), 5% Me2SO) and stored at -70 °C. Nuclei were obtained by incubating the cells first in 300 μl of solution A (3.4 mm trisodium citrate (pH 7.6), 1 mm Tris, 3 mm spermine tetrahydrochloride, 0.2% Nonidet P-40, 100 μg/ml trypsin) for 5 min at room temperature and then in 300 μl of solution B (3.4 mm trisodium citrate (pH 7.6), 1 mm Tris, 3 mm spermine tetrahydrochloride, 0.2% Nonidet P-40, 100 μg/ml ribonuclease A, and 500 μg/ml trypsin inhibitor) for 5 min at room temperature. Nuclei were stained with 30 μl of propidium iodide solution (1 mg/ml), and DNA staining was analyzed by flow cytometry using a FACSCalibur system with the CellQuest software. Cell cycle analysis was performed using the ModfitLT™ software (version 2).Cell Death Assays—Apoptotic cells were detected by using terminal dUTP nick-end labeling assay with ApopTag Plus Peroxidase, according to the manufacturer's instructions (Intergen Co., Purchase, NY). One thousand cells were counted, and the values obtained were used to calculate the percentage of labeled cells.Transient Transfection of MCF-7 and αT3 Cells—MCF-7 and αT3 cells were plated out the day before transfection. Cells were transiently transfected using the LipofectAMINE transfection reagent (Invitrogen) according to the manufacturer's instructions. Cells were transfected with 1 μg of each DNA construct (mouse LHRH-R construct for MCF-7 cells and PKCα DN or focal adhesion kinase dominant negative for αT3 cells) or 1 μg of empty pcDNA3.1 vector (negative control). Twenty four hours after transfection, cells were transferred to serum-free DMEM and allowed to grow for 16 h before being stimulated with various ligands, see figure legends for details. Cells were then washed with ice-cold PBS, and total protein was extracted.Statistics—Data were analyzed by one-way analysis of variance using the Statview 5.0 software. Values are expressed as means ± S.E. Values of p < 0.05 were considered statistically significant.RESULTSIGF-1R Signaling Pathways in αT3 Gonadotrope Cells—We first determined whether αT3 cells expressed the IGF-1 receptor (Fig. 1). Immunoblot analysis using an antibody recognizing the β-subunit of the IGF-1R revealed that αT3 cells did express the IGF-1R. NWTb3 (NIH-3T3 overexpressing the human IGF-1R) (46Blakesley V. Kato H. Roberts C.T. LeRoith D. J. Biol. Chem. 1995; 270: 2764-2769Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and the human breast cancer cell line, MCF-7, were used as positive controls (Fig. 1). We then investigated the functionality of the IGF-1 receptor in αT3 cells. Serum-starved cells were exposed to 10 nm IGF-1 for different times (Fig. 2). We found that IGF-1 induced a rapid and substantial increase in tyrosine phosphorylation of the IGF-1R β-subunit (15-fold increase after 1 min of treatment) and of IRS-1 (5-fold increase after 1 min of treatment). Tyrosine phosphorylation levels then declined over the next 30 min. IGF-1R is involved in two main signaling pathways: the PI3K/Akt pathway and the MAPK (ERK1/2, p38, and JNK1/2) pathway (28Dupont J. Dunn S.E. Barrett J.C. LeRoith D. Recent Prog. Horm. Res. 2003; 58: 325-342Crossref PubMed Scopus (61) Google Scholar). Thus, we tested whether these kinases were activated in response to treatment of αT3 cells with IGF-1. IGF-1 stimulated PI3K activity (data not shown) and resulted in a 5-fold increase in the levels of phosphorylated Ser-473 and Thr-308 Akt after 1 min of treatment. The level of phosphorylated Akt increased with the length of the treatment, reaching a maximum of 20-fold after 10 min (Fig. 2C). IGF-1 also stimulated phosphorylation of two downstream targets of Akt: the transcription factor FKHR and the kinase GSK3 α/β (Fig. 2D). Although the MAPKs, ERK1/2, p38, and JNK1/2, were present in the αT3 extracts, we detected only a small increase in the level of phosphorylation of these kinases in response to IGF-1 (Fig. 2E). Thus, our results indicate that αT3 cells are equipped with a functional IGF-1 receptor.Fig. 2IGF-1 signaling pathways in αT3 cells. Time course of IGF-1-stimulated IGF-1R (A), IRS-1 (B), Akt (C), GSK3α/β and FKHR (D), and MAPKs (ERK1/2, p38, and JNK1/2) (E) activation in αT3 cells. αT3 cells were incubated in serum-free DMEM overnight before treatment with 10 nm IGF-1 for 0, 0.5, 1, 10, 30, or 60 min. Whole cell lysates were separated by SDS-PAGE and immunoblotted with different antibodies (phosphotyrosine, phospho-MAPK (ERK1/2, p38, and JNK1/2), phospho-Akt Ser and Thr, phospho-GSK3α/β, and phospho-FKHR). The blots were then stripped and reblotted with appropriate antibodies to determine total proteins loading. The blots are typical examples of an experiment that was replicated three to four times.View Large Image Figure ViewerDownload (PPT)LHRH-I Signaling Pathways in αT3 Gonadotrope Cells— Previous studies (48Perrin M.H. Bilez" @default.
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• W2068596181 date "2004-12-01" @default.
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• W2068596181 title "The Luteinizing Hormone-releasing Hormone Inhibits the Anti-apoptotic Activity of Insulin-like Growth Factor-1 in Pituitary αT3 Cells by Protein Kinase Cα-mediated Negative Regulation of Akt" @default.
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• W2068596181 doi "https://doi.org/10.1074/jbc.m404571200" @default.
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