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• W2076392488 abstract "Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10−12 to 10−11m) is sufficient for substantial increases in ERK activity. Treatment with PTH-(1–34) (10−8m), PTH-(1–31), or 8-bromo-cAMP failed to stimulate ERKs, whereas treatment with phorbol 12-myristate 13-acetate, serum, or PTH peptides lacking the N-terminal amino acids stimulated activity. Furthermore, the activation of ERKs was prevented by pretreatment of osteoblastic cells with inhibitors of protein kinase C (GF 109203X) and MEK (PD 98059). Treatment of UMR cells with epidermal growth factor (EGF), but not PTH, promoted tyrosine phosphorylation of the EGF receptor. Transient transfection of UMR cells with p21N17Ras did not block activation of ERKs following treatment with low concentrations of PTH. Thus, activation of ERKs and proliferation by PTH is protein kinase C-dependent, but stimulation occurs independently of the EGF receptor and Ras activation. Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10−12 to 10−11m) is sufficient for substantial increases in ERK activity. Treatment with PTH-(1–34) (10−8m), PTH-(1–31), or 8-bromo-cAMP failed to stimulate ERKs, whereas treatment with phorbol 12-myristate 13-acetate, serum, or PTH peptides lacking the N-terminal amino acids stimulated activity. Furthermore, the activation of ERKs was prevented by pretreatment of osteoblastic cells with inhibitors of protein kinase C (GF 109203X) and MEK (PD 98059). Treatment of UMR cells with epidermal growth factor (EGF), but not PTH, promoted tyrosine phosphorylation of the EGF receptor. Transient transfection of UMR cells with p21N17Ras did not block activation of ERKs following treatment with low concentrations of PTH. Thus, activation of ERKs and proliferation by PTH is protein kinase C-dependent, but stimulation occurs independently of the EGF receptor and Ras activation. parathyroid hormone PTH-related protein protein kinase A protein kinase C mitogen-activated protein kinase MAPK kinase kinase extracellular signal-regulated kinase MAPK/ERK kinase phorbol 12-myristate 13-acetate epidermal growth factor epidermal growth factor receptor myelin basic protein fetal bovine serum stress-activated protein kinase c-Jun N-terminal kinase Parathyroid hormone (PTH)1 is an essential regulator of calcium homeostasis (1Strewler G.J. N. Engl. J. Med. 2000; 342: 177-185Crossref PubMed Scopus (362) Google Scholar) and has both anabolic and catabolic effects in vivo on bone and in vitro on primary and clonal osteoblastic cells (2Dempster D.W. Cosman F. Parisien M. Shen V. Lindsay R. Endocr. Rev. 1993; 14: 690-709Crossref PubMed Scopus (671) Google Scholar, 3Partridge N.C. Bloch S.R. Pearman A.T. J. Cell. Biochem. 1994; 55: 321-327Crossref PubMed Scopus (116) Google Scholar). The dual functionality of PTH may stem from its ability to stimulate both adenylate cyclase (4Partridge N.C. Kemp B.E. Veroni M.C. Martin T.J. Endocrinology. 1981; 108: 220-225Crossref PubMed Scopus (143) Google Scholar) and phospholipase C (5Civitelli R. Reid I.R. Westbrook S. Avioli L.V. Hruska K.A. Am. J. Physiol. 1988; 255: E660-E667PubMed Google Scholar) as a result of ligand binding to the heterotrimeric G-protein-coupled PTH/PTHrP receptor (6Abou-Samra A.B. Juppner H. Force T. Freeman M.W. Kong X.F. Schipani E. Urena P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Crossref PubMed Scopus (1007) Google Scholar). The first 34 amino acids of PTH are necessary and sufficient for full biological activity of the intact hormone (7Habener J.F. Rosenblatt M. Potts Jr., J.T. Physiol. Rev. 1984; 64: 985-1053Crossref PubMed Scopus (262) Google Scholar). Furthermore, studies with N-terminal peptides of PTH demonstrate a requirement for the first 2 amino acids in the activation of adenylate cyclase (8Fujimori A. Cheng S.L. Avioli L.V. Civitelli R. Endocrinology. 1992; 130: 29-36Crossref PubMed Google Scholar, 9Azarani A. Goltzman D. Orlowski J. J. Biol. Chem. 1995; 270: 20004-20010Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), that amino acids 29–32 are sufficient for phospholipase C stimulation (10Jouishomme H. Whitfield J.F. Gagnon L. Maclean S. Isaacs R. Chakravarthy B. Durkin J. Neugebauer W. Willick G. Rixon R.H. J. Bone Miner. Res. 1994; 9: 943-949Crossref PubMed Scopus (124) Google Scholar), and that residues 25–34 are the principal receptor-binding region (11Gardella T.J. Wilson A.K. Keutmann H.T. Oberstein R. Potts Jr., J.T. Kronenberg M. Nussbaum S.R. Endocrinology. 1993; 132: 2024-2030Crossref PubMed Scopus (70) Google Scholar). The ability of PTH to activate adenylate cyclase and protein kinase A (PKA) is mediated via the stimulatory GTP-binding protein (Gs). PTH also directs the Gq-mediated activation of phospholipase C and ultimately PKC (5Civitelli R. Reid I.R. Westbrook S. Avioli L.V. Hruska K.A. Am. J. Physiol. 1988; 255: E660-E667PubMed Google Scholar), release of Ca2+ from intracellular stores (12Reid I.R. Civitelli R. Halstead L.R. Avioli L.V. Hruska K.A. Am. J. Physiol. 1987; 252: E45-E51Google Scholar), and activation of calcium channels (13Yamaguchi D.T. Kleeman C.R. Muallem S. J. Biol. Chem. 1987; 262: 14967-14973Abstract Full Text PDF PubMed Google Scholar, 14Yamaguchi D.T. Hahn T.J. Iida-Klein A. Kleeman C.R. Muallem S. J. Biol. Chem. 1987; 262: 7711-7718Abstract Full Text PDF PubMed Google Scholar). Although these signaling pathways mediate the alterations of gene expression and proliferation in osteoblastic cells, the mechanism(s) involved are still poorly understood. The MAPK signaling pathway is tightly coupled to the regulation of cell proliferation and viability. The PTH/PTHrP receptor couples to G-protein signaling pathways known to regulate MAPKs in many systems (15Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (612) Google Scholar). Furthermore, PTH stimulates proliferation in bone (2Dempster D.W. Cosman F. Parisien M. Shen V. Lindsay R. Endocr. Rev. 1993; 14: 690-709Crossref PubMed Scopus (671) Google Scholar) and kidney cells (16Garcia-Ocana A. Gomez-Casero E. Penaranda C. Esbrit P. Life Sci. 1998; 62: 2267-2273Crossref PubMed Scopus (18) Google Scholar). This combined evidence suggests that PTH may regulate MAPK in target tissues. Two of the best characterized MAPKs, ERK1/p42mapk and ERK2/p44mapk, are regulated by agonists interacting with growth factor receptor tyrosine kinases and G-protein-coupled receptors (17Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). ERK1 and ERK2 are activated by dual phosphorylation of threonine and tyrosine by the upstream MAPK kinase MEK1 or MEK2 (18Crews C.M. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8205-8209Crossref PubMed Scopus (152) Google Scholar, 19Crews C.M. Alessandrini A. Erikson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (740) Google Scholar), both of which are activated following phosphorylation by the MAPKKK Raf-1 (20Marshall M. Mol. Reprod. Dev. 1995; 42: 493-499Crossref PubMed Scopus (86) Google Scholar). It is known that activation of the MAPK pathway originates from several distinct classes of cell-surface receptors in a manner dependent on or independent of the small GTP-binding protein Ras (15Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (612) Google Scholar, 21Luttrell L. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). G-protein-coupled receptors have been shown to either activate (22Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 23Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (766) Google Scholar, 24Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar) or inhibit (25Burgering B.M. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (316) Google Scholar, 26Hordijk P.L. Verlaan I. Jalink K. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 3534-3538Abstract Full Text PDF PubMed Google Scholar, 27Verheijen M.H. Defize L.H. Endocrinology. 1995; 136: 3331-3337Crossref PubMed Google Scholar) MAPKs. MAPK activation by Gq-coupled agonists is PKC-dependent, but pertussis toxin-insensitive, and because PKC can directly phosphorylate Raf (28Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1161) Google Scholar), ERK activation may or may not involve Ras (29Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (409) Google Scholar, 30Ueda Y. Hirai S. Osada S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 31Della Rocca G.J. Maudsley S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 1999; 274: 13978-13984Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Gs-coupled receptors activate or inhibit MAPK in a cell type-specific manner (26Hordijk P.L. Verlaan I. Jalink K. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 3534-3538Abstract Full Text PDF PubMed Google Scholar, 32Frodin M. Peraldi P. Van Obberghen E. J. Biol. Chem. 1994; 269: 6207-6214Abstract Full Text PDF PubMed Google Scholar, 33Crespo P. Cachero T.G. Xu N. Gutkind J.S. J. Biol. Chem. 1995; 270: 25259-25265Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Previously, it was shown that PTH-(1–34) could inhibit MAPK in UMR 106-01 and ROS 17/2.8 cells (27Verheijen M.H. Defize L.H. Endocrinology. 1995; 136: 3331-3337Crossref PubMed Google Scholar) and inhibits cellular proliferation and DNA synthesis in UMR 106-01 cells (4Partridge N.C. Kemp B.E. Veroni M.C. Martin T.J. Endocrinology. 1981; 108: 220-225Crossref PubMed Scopus (143) Google Scholar, 34Partridge N.C. Opie A.L. Opie R.T. Martin T.J. Calcif. Tissue Int. 1985; 37: 519-525Crossref PubMed Scopus (63) Google Scholar) via a PKA-dependent pathway. In MC3T3-E1 osteoblasts, PMA was shown to stimulate both proliferation and phosphorylation of ERKs, and these effects were blocked by pretreatment with forskolin (35Siddhanti S.R. Hartle J.E., II Quarles L.D. Endocrinology. 1995; 136: 4834-4841Crossref PubMed Google Scholar). Additionally, low concentrations of PTH were shown to activate ERKs and proliferation in opossum kidney cells (36Cole J.A. Endocrinology. 1999; 140: 5771-5779Crossref PubMed Google Scholar). Since PKC is known to activate MAPK, it is possible that the ability of PTH to activate PKC and ERKs is a potential mechanism for the anabolic effects of PTH. Here, we demonstrate that activation of ERKs and proliferation in osteoblastic cells occurs following treatment with low concentrations of PTH. This activation is dependent upon PKC, but not PKA, and does not require activation of the small G-protein Ras or phosphorylation of the EGFR. This study supports PTH as a mitogen and demonstrates that the proliferative effects of PTH are dependent upon selective PKC activation, resulting in ERK activation. Synthetic rat PTH-(1–34), protein A-acrylic beads, myelin basic protein (MBP), and 8-bromo-cAMP were purchased from Sigma. Isobutylmethylxanthine, ionomycin, PMA, PD 98059, H-89, and GF 109203X were purchased from Calbiochem. Enhanced chemiluminescence (ECL) reagents were obtained from Amersham Pharmacia Biotech. PerkinElmer Life Sciences supplied [γ-32P]ATP and [3H]thymidine. Polyvinylidene difluoride membrane (Immobilon) was purchased from Millipore Corp. (Bedford, MA). Synthetic PTH peptides (PTH-(1–31), -(3–34), -(13–34), and -(28–48)) were purchased from Bachem (King of Prussia, PA). Tissue culture media and reagents were obtained from the Washington University Tissue Culture Center (St. Louis, MO). Fetal bovine serum was a product of JRH Biosciences (Lenexa, KS). All other chemicals were obtained from Sigma. Polyclonal anti-ERK1 (p44mapk) and anti-ERK2 (p42mapk), polyclonal anti-EGFR, and monoclonal anti-phosphotyrosine (PY20) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies were purchased from Sigma. The pcDNA3 plasmid alone or containing dominant-negative mutant p21N17Ras under the control of the cytomegalovirus promoter was graciously provided by Dr. Joseph Baldassare (Saint Louis University School of Medicine). Osteoblastic cells were cultured as previously described (37Puccinelli J.M. Omura T.H. Strege D.W. Jeffrey J.J. Partridge N.C. J. Cell. Physiol. 1991; 147: 505-513Crossref PubMed Scopus (7) Google Scholar) in Eagle's minimal essential medium supplemented with nonessential amino acids, 25 mm HEPES (pH 7.3), 100 units/ml penicillin, 100 μg/ml streptomycin, and 5% fetal bovine serum (FBS) for UMR 106-01 cells or 10% FBS for primary osteoblasts. For serum starvation, osteoblastic cells were allowed to grow to 70–80% confluence and then switched to Eagle's minimal essential medium without FBS for 48 h unless stated otherwise. Primary osteoblasts were isolated from rat calvariae as described (38Shalhoub V. Gerstenfeld L.C. Collart D. Lian J.B. Stein G.S. Biochemistry. 1989; 28: 5318-5322Crossref PubMed Scopus (60) Google Scholar) and seeded at 6.24 × 103 cells/cm2. UMR 106-01 cells were seeded at 1 × 106 cells/100-mm Petri dish and transiently transfected with pcDNA3 plasmids containing dominant-negative mutant p21N17Ras. Transfections were performed as described (39Tyson D.R. Swarthout J.T. Partridge N.C. Endocrinology. 1999; 140: 1255-1261Crossref PubMed Google Scholar) using Effectene (QIAGEN Inc.). Briefly, cells were seeded, allowed to adhere overnight, and transfected with 1.0 μg of total DNA. 18 h after transfection, UMR cells were serum-starved for 48 h and treated as described below for the immune complex assay. Osteoblastic cells were seeded at 3 × 104/cm2 (UMR 106-01) or 6.24 × 103/cm2 (primary rat osteoblastic cells), allowed to adhere overnight, serum-starved for 24 h, and treated continuously in the presence or absence of appropriate agents for 24 h. Osteoblastic cells were pulsed-labeled with 1 μCi/ml [3H]thymidine for 2 h just prior to the end of the incubation period and harvested by trypsinization, followed by centrifugation. The cell pellet was resuspended in cold 10% trichloroacetic acid and vortexed vigorously to lyse the cells. The lysate was incubated on ice for 20 min, passed through a 0.45-μm HA filter (Millipore Corp.), washed once with cold 5% trichloroacetic acid and once with cold 70% ethanol, dried, and placed in 10 ml of scintillation fluid. Radioactivity was measured in a liquid scintillation counter (Beckman LS 3801). PKC activity was measured using a Pierce colorimetric PKC assay kit, SpinZymeTM format (product no. 29542). Cell lysis and membrane preparation were performed as previously described (40Chakravarthy B.R. Bussey A. Whitfield J.F. Sikorska M. Williams R.E. Durkin J.P. Anal. Biochem. 1991; 196: 144-150Crossref PubMed Scopus (113) Google Scholar). PKC activity was measured in lysates prepared from cytosolic and membrane fractions by incubation with fluorescently labeled MBP substrate. The reaction mixture was applied to a separation unit with an affinity membrane that specifically binds phosphorylated substrate. Quantitation of the phosphorylated product was performed by measuring absorbance at 570 nm, with the amount of peptide obtained being directly proportional to the amount of specific kinase activity present in each sample. Extrapolation of activity was performed from a linear regression generated using known amounts of PKC active enzyme. Serum-starved osteoblastic cells were treated in the presence and absence of appropriate agents for the indicated time periods at 37 °C. The medium was aspirated, and cells were washed twice with cold phosphate-buffered saline and then lysed in ice-cold lysis buffer (20 mm Tris-HCl (pH 8.0), 10% glycerol, 1% Triton X-100, 2 mm EDTA, 50 mmβ-glycerophosphate, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin). Monolayers were scraped into 1.5-ml Eppendorf tubes and incubated on ice for 30 min. The lysates were cleared by centrifugation (12,000 rpm, 10 min, 4 °C). Protein contents of the supernatants were determined using the Bradford reagent (Bio-Rad) at 595 nm. For each assay, 100 μg of total protein were used; the volume was adjusted to 200 μl; 3 μg of anti-ERK1 and anti-ERK2 antibodies were added; and the samples were rotated for 4 h at 4 °C. At this time, 30 μl of protein A-acrylic beads were added, and the samples were rotated from 2 h to overnight at 4 °C. The samples were then centrifuged (12,000 rpm, 10 min, 4 °C). The immune complex was washed three times with cold lysis buffer, three times with 0.5 m LiCl and 100 mmTris (pH 7.6), and once with assay buffer (20 mm Tris-HCl (pH 7.2), 1.5 mm EGTA, 0.03% Brij 35, 50 mmβ-glycerophosphate, and 1 mm dithiothreitol) and then resuspended in 20 μl of reaction buffer (20 mm Tris-HCl (pH 7.3), 50 μm ATP, 5 μCi of [γ-32P]ATP, and 10 mm MgCl2) containing 10 μg of MBP/assay for 30 min at 30 °C. Reactions were terminated via the addition of 7.5 μl of SDS sample buffer (0.063m Tris (pH 6.8), 1% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.1% bromphenol blue) and boiling for 5 min. Following centrifugation for 10 min at 4 °C, 10 μl of each reaction were resolved on a 12.75% SDS-polyacrylamide gel; and after drying, the gel was exposed to x-ray film. Quantitation was performed by scanning the gel with a PhosphorImager (Molecular Dynamics, Inc.). Cell lysates containing 50 μg of total protein in lysis buffer were boiled for 5 min with 2× SDS sample buffer, centrifuged (12,000 rpm, 3 min), and placed on ice. SDS-polyacrylamide gel electrophoresis was performed with 6 and 12% stacking and resolving gels, respectively. The proteins were transferred electrophoretically to polyvinylidene difluoride membrane at 100 V for 1 h. After blocking the membrane in 0.1% Tween 20, 138 mm NaCl, 5 mm KCl, and 25 mm Tris-HCl (pH 8.0) containing 5% (w/v) nonfat dry milk, the membrane was probed with anti-ERK1/ERK2 antibodies (diluted 1:1000), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (diluted 1:10,000). The antigen-antibody complexes were detected by ECL following the manufacturer's protocol. UMR 106-01 cells grown in 100-mm dishes were treated as indicated. Lysates were prepared as described for the ERK immune complex assay, and equal amounts of protein (0.2 mg) from each sample were incubated overnight at 4 °C with 3 μg of polyclonal anti-EGFR or monoclonal anti-phosphotyrosine (PY20) antibody. Samples were then incubated for an additional 2 h with protein A/G-agarose at 4 °C, and the antigen-antibody complexes were collected by centrifugation. Pellets were boiled for 5 min in 2× SDS sample buffer and resolved on 4–15% gradient gels (Bio-Rad). After transfer to polyvinylidene difluoride membranes, samples were immunoblotted with PY20 to assess EGFR phosphorylation content or with anti-EGFR antibody to identify the EGFR. Experimental significance was assessed using one-way analysis of variance with post hoc analysis of multiple comparison using Dunnett's pairwise method. All other data shown are representative of at least three separate experiments with similar results. Although PTH (10−8m) activates PKA and is inhibitory to cellular proliferation and DNA synthesis in UMR 106-01 cells (4Partridge N.C. Kemp B.E. Veroni M.C. Martin T.J. Endocrinology. 1981; 108: 220-225Crossref PubMed Scopus (143) Google Scholar, 34Partridge N.C. Opie A.L. Opie R.T. Martin T.J. Calcif. Tissue Int. 1985; 37: 519-525Crossref PubMed Scopus (63) Google Scholar), lower doses result in significant increases (p < 0.05) in cell number (34Partridge N.C. Opie A.L. Opie R.T. Martin T.J. Calcif. Tissue Int. 1985; 37: 519-525Crossref PubMed Scopus (63) Google Scholar). Here, we assayed for changes in DNA synthesis following treatment of UMR 106-01 cells with different doses of PTH. Furthermore, we used N-terminal PTH peptides to determine any involvement of PKC or PKA. Following serum starvation for 24 h, UMR 106-01 cells were treated with PTH-(1–34) (10−8m) alone or with the selective inhibitor PD 98059 (MEK), GF 109203X (PKC), or H-89 (PKA). As shown in Fig. 1 A, PTH at this concentration was unable on its own to stimulate proliferation. However, inhibition of PKA by the addition of H-89 resulted in a significant ∼2-fold increase (p = 0.001) in [3H]thymidine incorporation, whereas GF 109203X and PD 98059 had no effect. We next examined the ability of N-terminal PTH deletion peptides to stimulate proliferation (Fig. 1 B). Following serum starvation, UMR 106-01 cells were incubated with PTH-(1–31) (activates PKA, but not PKC) or with PTH-(3–34) (unable to activate PKA). Since PTH-(3–34) also promotes release of intracellular calcium, UMR 106-01 cells were also treated with PTH-(13–34) and PTH-(28–48), peptides that selectively activate PKC. We observed significant increases in [3H]thymidine incorporation following treatment with PTH-(3–34) (p = 0.003), PTH-(13–34) (p = 0.0007), and PTH-(28–48) (p = 0.01), but not with PTH-(1–31). Finally, we determined if the significant increases in proliferation we previously observed with low doses of PTH (34Partridge N.C. Opie A.L. Opie R.T. Martin T.J. Calcif. Tissue Int. 1985; 37: 519-525Crossref PubMed Scopus (63) Google Scholar) and with PTH-(3–34) were PKC-dependent. Following serum starvation, UMR 106-01 cells were incubated with either PTH-(1–34) (10−11m) or PTH-(3–34) (10−8m) with or without the PKC inhibitor GF 109203X at 5 μm (Fig. 1 C). We observed an ∼3-fold increase in thymidine incorporation following treatment with 10−11m PTH-(1–34) (p = 0.01) or PTH-(3–34) (p = 0.01), and both were significantly inhibited by the addition of GF 109203X (p = 0.01 and 0.03, respectively). To correlate enhanced proliferation with an increase in cell number, a complete dose response experiment with PTH-(1–34) followed by cell counting was performed. We observed a significant increase (p = 0.016) in cell number from 3.7 × 105/well with controls to 5.2 × 105/well following treatment with 10−11m PTH for 72 h. Since we observed significant increases in proliferation of UMR 106-01 cells at low doses of PTH (10−11m) and these increases appear to be PKC-dependent, we performed a dose response experiment with PTH to determine the minimal concentration required for activation of PKC (Fig. 2). Following 24 h of serum starvation, cells were treated with increasing concentrations of PTH for 2 min, and PKC activity was assayed in both the cytoplasmic and membrane fractions. PKC activity was measured by a colorimetric assay and is defined as picomoles of phosphate incorporated into substrate at 37 °C for 30 min. We observed a significant increase (p < 0.001) in translocation to the membrane and associated PKC activity at 10−11m PTH. Although 10−11m PTH was sufficient to activate PKC, higher concentrations were necessary for increased PKA activity. We have previously shown PKA to be activated to 30% of the maximum at 10−9m PTH in UMR 106-01 cells, with half-maximal activation at 5 × 10−8m PTH (41Partridge N.C. Kemp B.E. Livesey A. Martin T.J. Endocrinology. 1982; 111: 178-183Crossref PubMed Scopus (64) Google Scholar). These results are similar to those of Fujimori et al. (42Fujimori A. Cheng S.L. Avioli L.V. Civitelli R. Endocrinology. 1991; 128: 3032-3039Crossref PubMed Scopus (86) Google Scholar). The ERK subclass (ERK1/p42mapk and ERK2/p44mapk) of MAPKs is activated by major signaling pathways regulating cell proliferation (43Janknecht R. Cahill M.A. Nordheim A. Carcinogenesis. 1995; 16: 443-450Crossref PubMed Scopus (112) Google Scholar, 44Janknecht R. Hunter T. EMBO J. 1997; 16: 1620-1627Crossref PubMed Scopus (204) Google Scholar, 45Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1136) Google Scholar). It is known that high concentrations of PTH, which result in elevated levels of cAMP and activation of PKA (4Partridge N.C. Kemp B.E. Veroni M.C. Martin T.J. Endocrinology. 1981; 108: 220-225Crossref PubMed Scopus (143) Google Scholar, 46Chase L.R. Aurbach G.D. J. Biol. Chem. 1970; 245: 1520-1526Abstract Full Text PDF PubMed Google Scholar), inhibit proliferation (34Partridge N.C. Opie A.L. Opie R.T. Martin T.J. Calcif. Tissue Int. 1985; 37: 519-525Crossref PubMed Scopus (63) Google Scholar) and growth factor-stimulated MAPK activity (25Burgering B.M. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (316) Google Scholar, 27Verheijen M.H. Defize L.H. Endocrinology. 1995; 136: 3331-3337Crossref PubMed Google Scholar) in UMR 106-01 and fibroblast cells. However, since we had demonstrated activation of PKC by N-terminal PTH peptides or low doses of PTH-(1–34) that were also able to stimulate proliferation, we investigated the effect of PTH on MAPK activity in UMR 106-01 osteoblastic cells (Fig. 3). To quantify the effect of PTH-(1–34) on ERK activity, we performed an in vitro kinase assay with immunoprecipitated ERK1/ERK2 in which MBP was a substrate. ERK activity was measured in osteoblastic cells 15 min after the addition of PTH, which we had found was the time of maximal stimulation (data not shown). Treatment of UMR cells with 10−12 and 10−11m PTH resulted in significant increases (p< 0.05) in the -fold stimulation of ERK activity (2.9- and 2.8-fold, respectively) above basal levels (Fig. 3). Additionally, UMR cells were treated with 5% FBS as a positive control for ERK activation. Treatment for 15 min with 5% FBS resulted in a 2.8-fold increase in ERK activity above basal levels (Fig. 3). Next, a complete dose response was performed with increasing concentrations of PTH (Fig.4 A). Treatment of UMR cells with 10−12, 10−11, 10−10, 10−9, or 10−8m PTH or 5% FBS resulted in 3.2-, 2.6-, 2.9-, 1.5-, 1.6-, and 3.3-fold increases above basal activation. The addition of medium containing 5% fetal bovine serum was performed as a positive control for ERK activation. In primary osteoblasts (Fig. 4 B), the results were similar, with 3.9-, 5-, 4.7-, 1.2-, 1.4-, and 10-fold increases above basal activation. These results clearly demonstrate that activation of MAPK by PTH is dose-dependent. Treatment of osteoblastic cells with concentrations of PTH in the lower range stimulated ERKs, whereas higher concentrations, which activated PKA maximally, were unable to stimulate ERKs. Interestingly, although treatment with 10−12m PTH was unable to significantly (p = 0.15) stimulate PKC above basal levels (Fig. 2), the level of activation was sufficient to stimulate ERK activity. The ERK assays were performed with equal amounts of total protein from whole cell lysates. Therefore, Western blotting was performed to examine the relative levels of ERK1/ERK2 protein present for each assay.Figure 4Effect of various concentrations of PTH on ERK activities in UMR 106-01 and primary osteoblasts. ERK activation was assayed for a wide range of PTH concentrations in osteoblastic cells following serum starvation. Cells were serum-starved for 48 h and treated with Eagle's minimal essential medium without (control (C)) or with 10−12, 10−11, 10−10, 10−9, or 10−8m PTH or 5% FBS for 15 min. ERK activity in cell lysates from osteoblastic cells (A, UMR 106-01 cells; B, primary rat osteoblasts) was analyzed by immune complex kinase assays with polyclonal anti-ERK1 and anti-ERK2 antibodies and MBP as substrate (upper panels). Western blot analysis was performed to examine the relative levels of ERK1/ERK2 protein present for each assay (lower panels).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Experiments were performed using the cAMP analog 8-bromo-cAMP (10−3m), the calcium ionophore ionomycin (10−7m), or the phorbol ester PMA (10−6m) to determine which signaling cascade leads to activation of ERKs by PTH (Fig. 5). Following 48 h of" @default.
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• W2076392488 title "Stimulation of Extracellular Signal-regulated Kinases and Proliferation in Rat Osteoblastic Cells by Parathyroid Hormone Is Protein Kinase C-dependent" @default.
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