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• W2028188226 abstract "cAMP signaling, activated by extracellular stimuli such as parathyroid hormone, has cell type-specific effects important for cellular proliferation and differentiation in bone cells. Recent evidence of a second enzyme target for cAMP suggests divergent effects on extracellular-regulated kinase (ERK) activity depending on Epac/Rap1/B-Raf signaling. We investigated the molecular mechanism of the dual functionality of cAMP on cell proliferation in clonal bone cell types. MC3T3-E1 and ATDC5, but not MG63, express a 95-kDa isoform of B-Raf. cAMP stimulated Ras-independent and Rap1-dependent ERK phosphorylation and cell proliferation in B-Raf-expressing cells, but inhibited growth in B-Raf-lacking cells. The mitogenic action of cAMP was blocked by the ERK pathway inhibitor PD98059. In B-Raf-transduced MG63 cells, cAMP stimulated ERK activation and cell proliferation. Thus, B-Raf is the dominant molecular switch that permits differential cAMP-dependent regulation of ERK with important implications for cell proliferation in bone cells. These findings might explain the dual functionality of parathyroid hormone on osteoblastic cell proliferation. cAMP signaling, activated by extracellular stimuli such as parathyroid hormone, has cell type-specific effects important for cellular proliferation and differentiation in bone cells. Recent evidence of a second enzyme target for cAMP suggests divergent effects on extracellular-regulated kinase (ERK) activity depending on Epac/Rap1/B-Raf signaling. We investigated the molecular mechanism of the dual functionality of cAMP on cell proliferation in clonal bone cell types. MC3T3-E1 and ATDC5, but not MG63, express a 95-kDa isoform of B-Raf. cAMP stimulated Ras-independent and Rap1-dependent ERK phosphorylation and cell proliferation in B-Raf-expressing cells, but inhibited growth in B-Raf-lacking cells. The mitogenic action of cAMP was blocked by the ERK pathway inhibitor PD98059. In B-Raf-transduced MG63 cells, cAMP stimulated ERK activation and cell proliferation. Thus, B-Raf is the dominant molecular switch that permits differential cAMP-dependent regulation of ERK with important implications for cell proliferation in bone cells. These findings might explain the dual functionality of parathyroid hormone on osteoblastic cell proliferation. parathyroid hormone parathyroid hormone/parathyroid hormone-related protein PTHrP receptor cyclic adenosine monophosphate protein kinase A extracellular-regulated kinase guanine nucleotide exchange factor fetal calf serum Dulbecco's minimum essential medium 1-methyl-3-isobutylxanthine 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide bromodeoxyuridine dibutyryl-cAMP protein kinase C hemagglutinin cAMP-response element-binding protein In mammals, parathyroid hormone (PTH)1 is the most important hormone affecting bone growth and resorption. It shares the PTH/PTH-related protein (PTHrP) receptor (PPR) with PTHrP (1Habener J.F. Posenblatt M. Potts J.T., Jr. Physiol. Rev. 1984; 64: 985-1053Crossref PubMed Scopus (262) Google Scholar, 2Mannstadt M. Juppner H. Gardella T.J. Am. J. Physiol. 1999; 277: F665-F675Crossref PubMed Google Scholar, 3Juppner H. Abou-Samra A.B. Freeman M. Kong X.F. Schipani E. Richards J. Kolakowski L.F., Jr. Hock J. Potts J.T., Jr. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Crossref PubMed Scopus (1150) Google Scholar, 4Abou-Samra A.B. Juppner H. Force T. Freeman M.W. Kong X.F. Schipani E. Urena P. Richards J. Bonventre J.V. Potts J.T., Jr. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Crossref PubMed Scopus (1007) Google Scholar, 5Bringhurst F.R. Juppner H. Guo J. Urena P. Potts J.T., Jr. Kronenberg H.M. Abou-Samra A.B. Segre G.V. Endocrinology. 1993; 132: 2090-2098Crossref PubMed Google Scholar, 6Schipani E. Karga H. Karaplis A.C. Potts J.T., Jr. Kronenberg H.M. Segre G.V. Abou-Samra A.B. Juppner H. Endocrinology. 1993; 132: 2157-2165Crossref PubMed Scopus (153) Google Scholar, 7Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar, 8Gardella T.J. Juppner H. Trends Endocrinol. Metab. 2001; 12: 210-218Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), the only other known endogenous ligand for PPR. A series of gene disruption studies of PTHrP and/or PPR in mice (9Karaplis A.C. Luz A. Glowacki J. Bronson R.T. Tybulewicz V.L. Kronenberg H.M. Mulligan R.C. Genes Dev. 1994; 8: 277-289Crossref PubMed Scopus (1002) Google Scholar, 10Lanske B. Karaplis A.C. Lee K. Luz A. Vortkamp A. Pirro A. Karperien M. Defize L.H.K., Ho, C. Mulligan R.C. Abou-Samra A.B. Juppner H. Segre G.V. Kronenberg H.M. Science. 1996; 273: 663-666Crossref PubMed Scopus (1136) Google Scholar, 11Lanske B. Amling M. Neff L. Guiducci J. Baron R. Kronenberg H.M. J. Clin. Invest. 1999; 104: 399-407Crossref PubMed Scopus (204) Google Scholar, 12Calvi L.M. Sims N.A. Hunzelman J.L. Knight M.C. Giovannetti A. Saxton J.M. Kronenberg H.M. Baron R. Scipani E. J. Clin. Invest. 2001; 107: 277-286Crossref PubMed Scopus (310) Google Scholar) has indicated that PTH action on PPR can generate either bone-forming or bone-resorbing signaling, reflecting the complexity and heterogeneity of the osteoblast population and/or the regulatory microenvironment. PPR belongs to the class II G-protein-coupled receptor subfamily of receptors (7Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). PTH-(1–84), the natural hormone, and its congeners (e.g. PTH-(1–34)) strongly couple with PPR to activate adenylate cyclase and generate a cAMP signal (8Gardella T.J. Juppner H. Trends Endocrinol. Metab. 2001; 12: 210-218Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Several lines of evidence indicate that this signal activates ERKs (extracellular-regulated kinase) in the pathway that promotes bone growth in vivo and in vitro (13Ishizuya T. Yokose S. Hori M. Noda T. Suda T. Yoshiki S. Yamaguchi A. J. Clin. Invest. 1997; 99: 2961-2970Crossref PubMed Scopus (324) Google Scholar, 14Partridge N.C. Bloch S.R. Pearman A.T. J. Cell. Biochem. 1994; 55: 321-327Crossref PubMed Scopus (116) Google Scholar, 15Sabatini M. Lesur C. Pacherie M. Pastoureau P. Kucharczyk N. Fauchere J.L. Bonnet J. Bone. 1996; 18: 59-65Crossref PubMed Scopus (37) Google Scholar, 16Hilliker S. Wergedal J.E. Gruber H.E. Bettica P. Baylink D.J. Bone. 1996; 19: 469-477Crossref PubMed Scopus (51) Google Scholar, 17Schiller P.C. D'ippolito G. Roos B.A. Howard G.A. J. Bone Miner. Res. 1999; 14: 1504-1512Crossref PubMed Scopus (100) Google Scholar). However, how cAMP activates ERK is still unknown. The most important target of cAMP is protein kinase A (PKA), but activation of PKA is known to counteract the Ras/Raf-1/MEK signaling pathway (18Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1068Crossref PubMed Scopus (824) Google Scholar, 19Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar, 20Graves L.M. Bornfeldt K.E. Raines E.W. Potts B.C. Macdonald S.G. Ross R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10300-10304Crossref PubMed Scopus (404) Google Scholar) that is essential for activation of ERK and stimulation of cell proliferation (21Sevetson B.R. Kong X. Lawrence J.C., Jr. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10304-10309Crossref Scopus (345) Google Scholar, 22Whitmarsh A.J. Davis R.J. Nature. 2000; 403: 255-256Crossref PubMed Scopus (111) Google Scholar, 23Graves L.M. Guy H.I. Kozlowski P. Huang M. Lazarowski E. Pope R.M. Collins M.A. Dahlstrand E.N. Earp III, H.S. Evans D.R. Nature. 2000; 403: 328-332Crossref PubMed Scopus (177) Google Scholar). Thus, we have been in search of the pathway linking the cAMP signal to ERK activation. Recently, a second enzyme target of cAMP, cAMP-guanine nucleotide exchange factor (cAMP-GEF)/Epac, emerged as a Rap1-specific GEF (26Kawasaki H. Springett G.M. Mochizuki N. Toki S. Makaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1179) Google Scholar,27de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1634) Google Scholar), indicating that cAMP can modulate ERKs via the Epac/Rap1/B-Raf pathway in a PKA- and Ras-independent manner (24Vossler M.R. Yao H. York R.D. Pan M-G. Rim C.S. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 25York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J.S. Nature. 1998; 392: 622-626Crossref PubMed Scopus (762) Google Scholar, 26Kawasaki H. Springett G.M. Mochizuki N. Toki S. Makaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1179) Google Scholar, 27de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1634) Google Scholar). We now show that a variety of bone cell lines, clonal and primary, constitutively express the components of this pathway in a cell type-specific manner. Modulation of cell proliferation through cAMP signaling is regulated primarily by the expression pattern of B-Raf splice variants, and B-Raf appears to function as a molecular switch in this signaling system. The decisive role of B-Raf first identified in this study may explain the long-known dual functionality of PTH signaling in bone. MC3T3-E1, C3H10T1/2, C2C12, and ATDC5 were purchased from RIKEN Cell Bank (Ibaraki, Japan). ROS17/2.8 was obtained from Dr. Gideon Rodan (Merck), MG63 from Dr. Akifumi Togari (Aichi-Gakuin University, Nagoya, Japan), and MLO-Y4 and MLO-A5 from Dr. Lynda Bonewald (Texas Health Science Center, San Antonio, TX) (28Kato Y. Windle J.J. Koop B.A. Mundy G.R. Bonewald L.F. J. Bone Miner. Res. 1997; 12: 2014-2023Crossref PubMed Scopus (440) Google Scholar), MC3T3-E1 subclone4 (MC4) from Dr. Renny T. Franceschi (University of Michigan School of Dentistry, Ann Arbor, MI) (29Wang D. Christensen K. Chawla K. Xiao G. Krebsbach P.H. Franceschi R.T. J. Bone Miner. Res. 1999; 14: 893-903Crossref PubMed Scopus (546) Google Scholar), and PC12 cells from Dr. Akemichi Baba (Osaka University, Osaka, Japan). Human PTH-(1–34) was a gift from Suntory Ltd. (Osaka, Japan). HA-tagged N17Ras, V12Ras, N17Rap1, and V12Rap1 were from Dr. Daniel Altschuler of University of Pittsburgh (Pittsburgh, PA) (30Altschuler D. Ribeiro-Neto F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7475-7479Crossref PubMed Scopus (124) Google Scholar). HA-tagged Epac 1 and -2 cDNA were obtained from Dr. Johannnes L. Bos (University Medical Center Utrecht, Utrecht, The Netherlands) (31de Rooij J. Rehmann H. van Triest M. Cool R.H. Wittinghofer A. Bos J.L. J. Biol. Chem. 2000; 275: 20829-20836Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). FLAG-tagged B-Raf vector was kindly provided by Dr. Deborah Morrison (National Cancer Institute, Frederick, MD). Primary osteoblastic cells were cultured as described previously (32Fujita T. Fukuyama R. Izumo N. Hirai T. Meguro T. Nakamuta H. Koida M. Jpn. J. Pharmacol. 2001; 86: 405-416Crossref PubMed Scopus (21) Google Scholar). MC3T3-E1 was cultured in 10% fetal calf serum (FCS)/α-minimal essential minimum (α−MEM); C3H10T1/2 was cultured in 10% FCS/basal medium Eagle's; PC12, C2C12, and MG63 were cultured in 10% FCS/Dulbecco's modified Eagle's medium (DMEM), ROS17/2.8 and ATDC5 were in 10% FCS and 5% DMEM, F12, respectively. MLO-Y4 and MLO-A5 were cultured in 5% FBS, 5% FCS/α-MEM on collagen type I-coated plates as described previously (33Fujita T. Izumo N. Fukuyama R. Meguro T. Nakamuta H. Kohno T. Koida M. Biochem. Biophys. Res. Commun. 2001; 280: 348-352Crossref PubMed Scopus (66) Google Scholar). Total RNA was extracted using guanidinium thiocyanate/phenol/chloroform method as reported by Chomczynski and Sacchi (34Chomczynski P. Sacchi N. Anal. Biochem. 1987; 62: 156-159Crossref Scopus (63232) Google Scholar). Brain and calvaria were isolated from male ddY mice (Shimizu Experimental Supplies, Kyoto, Japan) and RNA samples were extracted. Northern blot analysis was performed under high stringency conditions as described previously (32Fujita T. Fukuyama R. Izumo N. Hirai T. Meguro T. Nakamuta H. Koida M. Jpn. J. Pharmacol. 2001; 86: 405-416Crossref PubMed Scopus (21) Google Scholar). In brief, total RNA (20 μg) was electrophoresed in 1.2% agarose-formaldehyde gels, transferred on nylon membrane filters (Hybond N+, Amersham Biosciences, Buckinghamshire, UK), and hybridized with 32P-labeled cDNA probes. cDNAs encoding PPR and glyceraldehyde-3-phosphate dehydrogenase cloned by polymerase chain reaction (PCR) were used as probes (32Fujita T. Fukuyama R. Izumo N. Hirai T. Meguro T. Nakamuta H. Koida M. Jpn. J. Pharmacol. 2001; 86: 405-416Crossref PubMed Scopus (21) Google Scholar). After the final wash, the membrane was exposed to a BAS imaging plate (Fuji Film, Tokyo, Japan), and the relative signal intensity was estimated. For reverse transcription-PCR, a 0.5-μg RNA aliquot was reverse transcribed at 37 °C for 2 h in a 20-μl reaction volume containing 200 units of Superscript II (Invitrogen, Gaithersburg, MD), 4 μm random primers, 500 μm dNTPs, and 5 mm dithiothreitol. PCR was performed using one-tenth of the reverse transcription reaction volume and 30 pmol of following oligonucleotides. For Epac1, Epac1-F, 5′-GCTTCCTCCACAAACTCTCA-3′, Epac1-R, 5′-AACGCTGCCATCACCTCTCT-3′ (AN: NM_006105); for Epac2, Epac2-F, 5′-AGCCTTATCCCATCTTTCTA-3′, Epac2-R, 5′-CTGACTGTATTCGCCTCCAC-3′ (AN: NM_007023); for HPRT as an internal control, HPRT-F, 5′-GTTGAGAGATCATCTCCACC-3′, HPRT-R, 5′-AGCGATGATGAACCAGGTTA-3′. PCR was performed using Taq DNA polymerase on the following schedule: denaturation at 94 °C for 1 min, annealing at 64 °C for 1 min, and extension at 72 °C for 2 min on a TAKARA PCR thermal cycler MP system (Shiga, Japan); 30 cycles for the Epac and 20 cycles for the HPRT. Half of each reaction product was loaded onto a 2% agarose gel in 1 × Tris acetate-EDTA buffer. The number of cycles selected for each primer pair produced a linear relationship between the amount of input RNA and resulting PCR products. Cells were washed twice with incubation buffer (α-MEM containing 0.5 mm 1-methyl-3-isobutylxanthine (IBMX) and 1 mg/ml bovine serum albumin) and incubated for 30 min at 37 °C in the same buffer containing various concentrations of test agents. The reaction was terminated with trichloroacetic acid. cAMP was measured by radioimmunoassay (Amersham Biosciences) and the protein concentrations were estimated using a BCA protein assay kit (Pierce). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity was measured using a colorimetric assay (35Takuma K. Fujita T. Kimura Y. Tanabe M. Yamamuro A. Lee E. Mori K. Koyama Y. Baba A. Matsuda T. Eur. J. Pharmacol. 2000; 399: 1-8Crossref PubMed Scopus (33) Google Scholar). Cells were plated in 96-well plates at a density of 30,000 cells/well. Cells were treated with or without various agents for 4 days. The cells were then gently washed twice with 100 μl of phosphate-buffered saline and incubated at 37 °C for 2 h after the addition of 100 μl of 0.5 mg/ml MTT. Then, 50 μl of solubilizing solution containing 20% sodium dodecyl sulfate (SDS), 50% dimethylformamide, 2% acetic acid, and 2.5% of 1 m HCl, pH 4.7, was added to extract the dark blue crystals. After complete extraction, the absorbance was measured on a Bio-Rad Model 550 microplate reader (Bio-Rad, Hemel Hempstead, UK), using a test wavelength of 570 nm and a reference wavelength of 655 nm. Bromodeoxyuridine (BrdUrd) incorporation assay was performed using the colorimetric BrdUrd Cell proliferation Kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's protocol as described previously (36Fujita T. Meguro T. Izumo N. Yasutomi C. Fukuyama R. Nakamuta H. Koida M. Jpn. J. Pharmacol. 2001; 85: 278-281Crossref PubMed Scopus (28) Google Scholar). There was no difference in either assay in the number of dead cells between the cell lines determined by a trypan blue exclusion assay. FCS was reduced to 1% for all treatment conditions. The day before transfection, cells were plated on 35-mm dishes at a density of 105 cells/ml, and then transfected by FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's protocol as described previously (37Fujita T. Izumo N. Fukuyama R. Meguro T. Yasutomi C. Nakamuta H. Koida M. Jpn. J. Pharmacol. 2001; 86: 86-96Crossref PubMed Scopus (24) Google Scholar). The vector containing both the green fluorescent protein and neomycin-resistant genes, pEGFP-N1 (CLONTECH, Valencia, CA), and the respective vectors were co-transfected in the cells. Subconfluent cells were trypsinized and plated at low density before selection. Subsequently, cells were selected by culturing them in the presence of 400 μg/ml G418 for 3 weeks. Three clones of each mutant from MC4 and MG63 were isolated. In this study, number 004, N17Rap1 MC4; number 005, V12 Rap1 MC4; number 001, N17 Ras MC4; number 021, N17Rap1 MG63; number 008, V12 Rap1 MG63; number 015, B-Raf MG63 were used. Cells were solubilized in Tris-HCl buffer, pH 6.8, containing 3% SDS and 10% glycerol and the protein concentrations were estimated using a BCA protein assay kit. The sample was mixed with 0.1% bromphenol blue and 0.05% 2-mercaptoethanol, boiled for 5 min, and then loaded (equal amounts of protein/lane) on 10% gels. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes for Western blotting, using antibodies against Epac1 (C-17), Epac2 (M-18), B-Raf (c19) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Rap1 (clone3; Transduction Labs, Lexington, KY), and phospho-ERK1/2, ERK1/2 (New England Biolabs, Beverly, MA), and horseradish peroxidase-conjugated antibody as described previously (37Fujita T. Izumo N. Fukuyama R. Meguro T. Yasutomi C. Nakamuta H. Koida M. Jpn. J. Pharmacol. 2001; 86: 86-96Crossref PubMed Scopus (24) Google Scholar). As a positive control, PC12 cell lysates were used. For selection of clones, anti-HA antibody (Y-11; Santa Cruz Biotechnology, Inc.) or anti-FLAG antibody (M2; Sigma) were used. Unless otherwise described, statistical analyses were performed using Student's t test. A p value of less than 0.05 was considered to be significant. Two-way analysis of variance was performed to determine statistical differences between cultures according to time in culture. B-Raf exists as a number of isoforms that are expressed primarily in neural and endocrine tissues (26Kawasaki H. Springett G.M. Mochizuki N. Toki S. Makaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1179) Google Scholar, 38Gonzalez-Robayna I.J. Falender A.E. Ochsner S. Firestone G.L. Richards J.S. Mol. Endocrinol. 2000; 14: 1283-1300Crossref PubMed Scopus (327) Google Scholar). Although the 95-kDa B-Raf isoform is found in both brain and spinal cord, its presence in bone cells has not been systematically explored. We measured B-Raf protein expression in cultures of fibroblast C3H10T1/2, osteoblast MC4, chondrocyte ATDC5, two osteocytic clones MLO-Y4 and MLO-A5, two osteosarcoma cell lines, ROS17/2.8 and MG63, and myoblast C2C12 cells by Western blotting using an antibody specific for B-Raf (Fig. 1 C,lower panel). ATDC5, MC4, C3H10T1/2, and the two types of MLO cells had both the 95- and 62-kDa B-Raf splice variants, and C2C12, MG63, and ROS17/2.8 cells expressed only the 62-kDa isoform. Additionally, Epac1 and Epac2, two isoforms of Epac, and Rap1 protein expression were investigated (Fig. 1). In vivo bone expressed Epac mRNA (Fig. 1 A). Similar Epac mRNA expression was observed in brain and PC12 cells. Epac2 expression was detected in all cells (Fig. 1 B). In contrast, Epac1 was not detected in ATDC5, C2C12, and ROS17/2.8, and Rap1 expression was limited and weak in C2C12 and ROS17/2.8 cells. Either expression pattern of 95-kDa B-Raf, Rap1, or Epac, however, failed to show any notable change during maturation (Fig. 1 D,right), while ERKs (p44 and p42) were gradually decreased (Fig. 1 D, left). Taken together, these results suggest that, among cell types tested, only primary calvaria cells predominantly express the 95-kDa B-Raf isoform while the other limited lines the 62-kDa isoform alone and this allows cell type-specific cAMP signal transduction and diverse proliferative reactions. Next, we performed Northern blot analysis using specific probes for PPR and examined cAMP accumulation levels triggered by PTH-(1–34) in some clonal cells (Fig.2). PPR mRNA expression was detected in ATDC5, MC3T3-E1, and MC4 cells. The MC4 cells expressed 10-fold more PPR mRNA compared with other lines (Fig. 2 A). As expected, PTH-(1–34) dose-dependently stimulated cAMP accumulation in these cells (Fig. 2 B). In MC4, cAMP accumulation levels were ∼100-fold compared with ATDC5 and MC3T3-E1. On the other hand, the increased cAMP induced by PTH-(1–34) was negligible and barely detectable in MG63, C3H10T1/2, and the two MLO cell lines (data not shown), although high enough to suppress ERK activity and cell proliferation (refer to Figs.3 B and 6 A). In development model using primary osteoblastic cells, Northern blot analysis showed that PPR mRNA expression levels were gradually increased up to 21 days and then gradually decreased (Fig.2 C). Similar results were reported in the MC3T3-E1 and ATDC5 developmental culture system (17Schiller P.C. D'ippolito G. Roos B.A. Howard G.A. J. Bone Miner. Res. 1999; 14: 1504-1512Crossref PubMed Scopus (100) Google Scholar, 39Shukunami C. Ishizeki K. Atsumi T. Ohta Y. Suzuki F. Hiraki Y. J. Bone Miner. Res. 1997; 12: 1174-1188Crossref PubMed Scopus (253) Google Scholar). In contrast, expression of ERK had a peak at the start of culture. Since the Epac pathway is expressed constantly, the ERK level peaked at the start may be an important variable to assure proliferative response toward cAMP signal that increases the cell number, as the main determinant of bone mass. These results suggest that increased intracellular cAMP is perhaps the major PTH-induced signaling mechanism in these osteoblastic cells during cell proliferation.Figure 3Regulation of ERK activity by cAMP in bone like cells. A, PTH-(1–34), Bt2cAMP (Db-cAMP), and forskolin (FK) stimulated ERK phosphorylation in ATDC5. Lysates (50 μg of proteins) from cells untreated or treated with 100 nm PTH were examined for the indicated times (left side). Indicated concentrations of PTH-(1–34) were prepared (right side). B, PTH-(1–34) stimulated ERK phosphorylation in ATDC5 and MC4, but suppressed phosphorylation in MG63 and ROS17/2.8. Cells were treated with the peptide for 10 min. C, MC4 cells were treated with agents at different concentrations for 10 min. D, effects of 1 μm H89 and 1 μm PD98059 on PTH-(1–34)- and forskolin-stimulated ERK phosphorylation in MC4. Cells were preincubated at 37 °C with either protein kinase inhibitors for 30 min before the addition of stimulants. Similar results were obtained from three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Regulation of ERK and growth by Rap1 and B-Raf in MG63 cells. A, PTH-(1–34), Bt2cAMP (Db-cAMP), and forskolin (FK) suppressed cell proliferation in MG63. B, effects of H89 and PD98059 on cell proliferation. Growth was inhibited by PD98059, while H89 did not affect either basal or stimulated cell growth.C, ERK activity in V12Rap1-, N17Rap1-, or B-Raf-transduced cells. D, there was increased BrdUrd incorporation in N17Rap1 and B-Raf clones, but BrdUrd incorporation was suppressed in V12Rap1 clones. The inhibition of BrdUrd incorporation by forskolin (10 nm) was diminished in N17Rap1. Vec, vector-transduced control line. #, versus B-Raf; *,versus control: *, p < 0.05; **,p < 0.01; and ***, p < 0.005; ##,p < 0.01. n = 6. Similar results were obtained from four additional experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the consequences of elevated intracellular cAMP on bone cellular ERK activity, we measured ERK activity using antibodies specific for the active phosphorylated ERK. For these experiments, a variety of agents that can increase intracellular levels of cAMP or act as a cAMP analogue, forskolin, dibutyryl-cAMP (Bt2cAMP), and 8-(4-chlorphenylthio)-cyclopencyladenosine, were used. Only clonal cells expressing 95-kDa B-Raf, PTH-(1–34), forskolin, Bt2cAMP, and 8-(4-chlorphenylthio)-cyclopencyladenosine (8-CPT) increased the activity of ERK 2–12-fold more than in unstimulated cells (Fig. 3, B and C, and data not shown). Maximal activation by 1 nm PTH-(1–34) occurred rapidly, within 5–30 min and its activation was reduced by a higher concentration of the peptide (Fig. 3 A). IBMX increases cAMP levels by inhibiting its degradation by cAMP phosphodiesterase. IBMX also potentiated ERK activity (Fig. 3 C), indicating that the potentiation was due to increased intracellular cAMP. PTH-(1–34)-induced ERK phosphorylation was completely inhibited by the ERK pathway inhibitor PD98059. When cells were treated with H89, low concentrations of PTH-(1–34)-induced ERK activation were not affected, while increased ERK activation was observed with high concentrations of the peptide (Fig. 3 D). These results suggest that PTH stimulates intracellular cAMP accumulation, cAMP then activates Rap1 via GEF molecules, both Epac dependently and PKA independently, which in turn leads to B-Raf activation and results in ERK activation. Therefore, PKA might inhibit cAMP-mediated ERK activation when excess intracellular cAMP accumulates. In our preliminary experiments, calcitonin also stimulated cAMP-ERK signaling mechanisms in two MLO cell lines that express functional calcitonin receptors. 2T. Fujita, T. Meguro, R. Fukuyama, H. Nakamuta, and M. Koida, unpublished data. Therefore, cAMP-induced ERK activation by other hormones and factors was thought to be a common regulatory pathway in bone cells. In neuronal cells, cAMP signaling has an important role in cell differentiation and survival through a Rap1-B-Raf expression-dependent mechanism (24Vossler M.R. Yao H. York R.D. Pan M-G. Rim C.S. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). Activated ERK provides a mitogenic and a differentiating signal in many cell types (22Whitmarsh A.J. Davis R.J. Nature. 2000; 403: 255-256Crossref PubMed Scopus (111) Google Scholar, 23Graves L.M. Guy H.I. Kozlowski P. Huang M. Lazarowski E. Pope R.M. Collins M.A. Dahlstrand E.N. Earp III, H.S. Evans D.R. Nature. 2000; 403: 328-332Crossref PubMed Scopus (177) Google Scholar). To determine whether cAMP stimulates cell proliferation, we examined cell proliferation of bone cells assessed by MTT and BrdUrd incorporation assays. MTT activity and BrdUrd incorporation were directly correlated with the counted cell number of MC4 and MG63 cells in our preliminary experiments, thus growth was estimated using the MTT method. There was no correlation, however, between MTT activity and BrdUrd incorporation in ATDC5 and gene-transduced cells. Therefore, the BrdUrd method was used to measure cell proliferation (data not shown). PTH-(1–34), Bt2cAMP, and forskolin stimulated BrdUrd incorporation in two B-Raf-expressing bone cell lines in a low concentration range (ATDC5 and MC4; Fig.4, A and B). With increased ERK activity, IBMX also potentiated cell proliferation in basal and cAMP-stimulated conditions (Fig. 4 C), indicating that growth potentiation was due to the increase in intracellular cAMP. The ERK pathway inhibitor PD98059 inhibited cAMP-triggered ERK activation and cell proliferation (Figs. 3 D and4 D). Basal cell proliferation of MC4 was not affected by 1 μm PD98059. The PKA inhibitor H89 (1 μm) did not affect low concentrations cAMP-induced cell growth (Fig.4 D), indicating that if the PKA signaling pathway was inhibited, the mitogenic action of cAMP was not blocked because the signaling pathway of the Epac remained active. In contrast to the low concentration effects of cAMP, high concentration cAMP-induced cell proliferation was normalized to control levels. H89 did not affect the low concentration cAMP stimulation, whereas it potentiated the high concentration cAMP-mediated activation of cell growth. Thus, PKA might function to inhibit cell proliferation via ERK signal-induced mechanisms only with high concentrations of intracellular cAMP. Because Rap1 is a transducer of cAMP-mediated regulation of ERK, we next tested the hypothesis that the cAMP effect on ERK activity and MC4 proliferation is the result of Rap1 activation. We established MC4 overexpressing dominant negative N17Rap1 or the constitutively active V12Rap1 mutants, and mutants of Ras were generated and several independently isolated clones analyzed for Rap1 expression by Western blotting using HA antibody. All clones demonstrated elevated Rap1 or Ras expression compared with vector–transfected control lines (data not shown). ERK activation by PTH-(1–34), Bt2cAMP, and forskolin was observed in Ras mutant clones, whereas it was completely blocked in N17Rap1-transduced MC4 cells, and accelerated in V12Rap1-transduced cells (Fig.5 A). Thus, cAMP actions on ERK were mediated via Rap1 activation and activated Rap1 induced ERK activation. We next tested" @default.
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• W2028188226 title "New Signaling Pathway for Parathyroid Hormone and Cyclic AMP Action on Extracellular-regulated Kinase and Cell Proliferation in Bone Cells" @default.
• W2028188226 cites W1619161113 @default.
• W2028188226 cites W1667204010 @default.
• W2028188226 cites W1885709862 @default.
• W2028188226 cites W1965963242 @default.
• W2028188226 cites W1968093456 @default.
• W2028188226 cites W1970199559 @default.
• W2028188226 cites W1973755084 @default.
• W2028188226 cites W1976849281 @default.
• W2028188226 cites W1991732341 @default.
• W2028188226 cites W1993876642 @default.
• W2028188226 cites W1994816216 @default.
• W2028188226 cites W2000367276 @default.
• W2028188226 cites W2007478852 @default.
• W2028188226 cites W2010637356 @default.
• W2028188226 cites W2012415301 @default.
• W2028188226 cites W2016164334 @default.
• W2028188226 cites W2017351769 @default.
• W2028188226 cites W2022713347 @default.
• W2028188226 cites W2032991286 @default.
• W2028188226 cites W2036258710 @default.
• W2028188226 cites W2039629780 @default.
• W2028188226 cites W2045876058 @default.
• W2028188226 cites W2046862403 @default.
• W2028188226 cites W2047057752 @default.
• W2028188226 cites W2051926344 @default.
• W2028188226 cites W2054571309 @default.
• W2028188226 cites W2058966826 @default.
• W2028188226 cites W2059320983 @default.
• W2028188226 cites W2062273595 @default.
• W2028188226 cites W2063578899 @default.
• W2028188226 cites W2064157894 @default.
• W2028188226 cites W2066553743 @default.
• W2028188226 cites W2068901208 @default.
• W2028188226 cites W2076392488 @default.
• W2028188226 cites W2078037416 @default.
• W2028188226 cites W2079246839 @default.
• W2028188226 cites W2079541312 @default.
• W2028188226 cites W2084488354 @default.
• W2028188226 cites W2085530184 @default.
• W2028188226 cites W2090327416 @default.
• W2028188226 cites W2091800872 @default.
• W2028188226 cites W2100503761 @default.
• W2028188226 cites W2106405240 @default.
• W2028188226 cites W2127765989 @default.
• W2028188226 cites W2135087298 @default.
• W2028188226 cites W2141263471 @default.
• W2028188226 cites W2151887581 @default.
• W2028188226 cites W2158938925 @default.
• W2028188226 cites W2163047938 @default.
• W2028188226 cites W2180631471 @default.
• W2028188226 cites W2266367605 @default.
• W2028188226 cites W230923890 @default.
• W2028188226 cites W4231134694 @default.
• W2028188226 cites W4294216491 @default.
• W2028188226 cites W4377079766 @default.
• W2028188226 doi "https://doi.org/10.1074/jbc.m110364200" @default.
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