Parathyroid Hormone-related Peptide Stimulates Osteogenic Cell Proliferation through Protein Kinase C Activation of the Ras/Mitogen-activated Protein Kinase Signaling Pathway
2001; Elsevier BV; Volume: 276; Issue: 34 Linguagem: Inglês
10.1074/jbc.m101084200
ISSN1083-351X
AutoresDengshun Miao, Xin‐Kang Tong, George Chan, Dibyendu K. Panda, Peter S. McPherson, David Goltzman,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoWe investigated the mechanisms of parathyroid hormone-related peptide (PTHrP)-mediated effects on osteogenic cells in primary rat bone marrow cell (BMC) cultures. We first demonstrated by reverse transcriptase-polymerase chain reaction and immunocytochemistry that BMCs express the type I parathyroid hormone/PTHrP receptor. Treatment with PTHrP increased osteogenic cell proliferation as determined by [3H]thymidine and bromodeoxyuridine incorporation and augmented osteogenic colonies. Immunocytochemistry and Western blotting revealed no direct effect on expression of the osteoblast markers, type I collagen, bone sialoprotein, and osteocalcin, indicating that PTHrP did not directly stimulate differentiation in this system. PTHrP increased mitogen-activated protein kinase (MAPK) activity in BMC and MAPK activity, and PTHrP-induced osteogenic cell proliferation could be blocked by the MEK inhibitor PD-098059. PTHrP also increased Ras activity in BMC. Although wortmannin and H8, inhibitors of phosphoinositol 3-kinase and protein kinase A, respectively, did not block PTHrP-stimulated Ras or MAPK activity, chelerythrin chloride, a known protein kinase C inhibitor, did block these PTHrP actions as well as PTHrP-induced osteogenic cell proliferation. These results demonstrate that PTHrP stimulates osteogenic cell proliferation in rat marrow mesenchymal progenitor cells through protein kinase C-dependent activation of the Ras and MAPK signaling pathway. We investigated the mechanisms of parathyroid hormone-related peptide (PTHrP)-mediated effects on osteogenic cells in primary rat bone marrow cell (BMC) cultures. We first demonstrated by reverse transcriptase-polymerase chain reaction and immunocytochemistry that BMCs express the type I parathyroid hormone/PTHrP receptor. Treatment with PTHrP increased osteogenic cell proliferation as determined by [3H]thymidine and bromodeoxyuridine incorporation and augmented osteogenic colonies. Immunocytochemistry and Western blotting revealed no direct effect on expression of the osteoblast markers, type I collagen, bone sialoprotein, and osteocalcin, indicating that PTHrP did not directly stimulate differentiation in this system. PTHrP increased mitogen-activated protein kinase (MAPK) activity in BMC and MAPK activity, and PTHrP-induced osteogenic cell proliferation could be blocked by the MEK inhibitor PD-098059. PTHrP also increased Ras activity in BMC. Although wortmannin and H8, inhibitors of phosphoinositol 3-kinase and protein kinase A, respectively, did not block PTHrP-stimulated Ras or MAPK activity, chelerythrin chloride, a known protein kinase C inhibitor, did block these PTHrP actions as well as PTHrP-induced osteogenic cell proliferation. These results demonstrate that PTHrP stimulates osteogenic cell proliferation in rat marrow mesenchymal progenitor cells through protein kinase C-dependent activation of the Ras and MAPK signaling pathway. parathyroid hormone-related peptide malignancy-associated hypercalcemia parathyroid hormone bone marrow cell reverse transcriptase-polymerase chain reaction mitogen-activated protein kinase protein kinase C extracellular signal-regulated kinase G protein-coupled receptor phosphatidylinositol 3-kinase mitogen-activated protein protein kinase A tetradecanoylphorbol-13-acetate bromodeoxyuridine phosphate-buffered saline avidin-biotin-peroxidase complex alkaline phosphatase type I PTH/PTHrP receptor Parathyroid hormone-related peptide (PTHrP)1 was initially discovered as the pathogenetic mediator of malignancy-associated hypercalcemia (MAH). Originally, PTHrP was considered to be a skeletal catabolic agent as patients with MAH develop marked osteoclastic bone resorption. However, as is the case with PTH in hyperparathyroidism, this catabolic skeletal effect of PTHrP in MAH occurs in the context of continuous exposure of the skeleton to PTHrP. In contrast, administration of PTH to rodents on an intermittent basis increases bone mass. This anabolic effect of intermittent PTH administration in osteoporosis has been extensively explored (1Reeve J. J. Bone Miner. Res. 1996; 11: 440-445Crossref PubMed Scopus (93) Google Scholar). Several groups have also demonstrated that intermittent PTHrP administration increases bone mass in rats in vivo (2Everhart-Caye M. Inzucchi S.E. Guinness-Henry J. Mitnick M.A. Stewart A.F. J. Clin. Endocrinol. & Metab. 1996; 81: 199-208Crossref PubMed Scopus (0) Google Scholar, 3Fraher L.J. Caveney A.N. McFadden R.G. Am. J. Respir. Cell Mol. Biol. 1995; 12: 669-675Crossref PubMed Scopus (2) Google Scholar, 4Henry J.G. Mitnick M. Dann P.R. Stewart A.F. J. Clin. Endocrinol. & Metab. 1997; 82: 900-906PubMed Google Scholar, 5Hock J.M. 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Della Roccaet al. (33Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar) had reported that GPCRs could activate the MAP kinase cascade in a Ras-dependent manner. In the present studies, we investigated the effects of PTHrP on osteogenic cell proliferation and differentiation in vitroby employing primary rat bone marrow cell cultures. Our results demonstrate that PTHrP stimulates osteogenic cell proliferation through PKC-dependent activation of the Ras/MAPK signaling pathway. Human PTHrP-(1–34) was obtained from Peninsula Laboratories, Inc., CA. 12-O-Tetradecanoylphorbol-13-acetate (TPA), wortmannin, chelerythrin chloride, and H8 were purchased from Sigma. Antibodies were obtained as follows: rabbit anti-PTH receptor antibody (Babco, Berkeley, CA); mouse anti-vimentin monoclonal antibody (Medicorp, Montreal, Quebec, Canada); mouse anti-bromodeoxyuridine monoclonal antibody (Sigma); affinity-purified goat anti-human type I collagen antibody (Southern Biotechnology Associates, Inc., Birmingham, AL); goat anti-mouse osteocalcin (Biomedical Technologies, Inc., Stoughton, MA); rabbit anti-human bone sialoprotein LF-6, (34Bianco P. Fisher L.W. Young M.F. Termine J.D. Robey P.G. Calcif. Tissue Int. 1991; 49: 421-426Crossref PubMed Scopus (345) Google Scholar); rabbit anti-MAPK antibody (ERK-1 and ERK-2) (Santa Cruz Biotechnology Inc., Santa Cruz, CA); rabbit anti-activated, phosphorylated MAPK antibody (Promega, Madison, WI); rabbit anti-phospho-Akt (Thr-308) and Akt antibodies (New England Biolabs, Inc., Ontario, Canada); and mouse anti-Ras antibody (Transduction Laboratories, Lexington, KY). Tibiae and femurs of 200-g male Wistar rats were removed under aseptic conditions, and bone marrow cells (BMC) were flushed out with Dulbecco's modified Eagle's minimal essential medium containing 10% fetal calf serum, 50 µg/ml ascorbic acid, 10 mm β-glycerophosphate, and 10−8m dexamethasone. Cells were dispersed by repeated pipetting, and a single-cell suspension was achieved by forcefully expelling the cells through a 22-gauge syringe needle. 106 total bone marrow cells were cultured in 36-cm2 Petri dishes in 5 ml of the above-mentioned medium. The medium was changed every 4 days. The non-adherent cells containing hematopoietic elements were removed by pipetting gently when the medium was changed for the first time (35Conget P.A. Minguell J.J. J. Cell. Physiol. 1999; 181: 67-73Crossref PubMed Scopus (688) Google Scholar). Only monocytic cells remained. Cultures were maintained for 6–18 days. At the end of the culture period cells were washed with PBS, fixed with PLP fixative (2% paraformaldehyde containing 0.075 m lysine and 0.01m sodium periodate solution), and stained cytochemically or immunocytochemically. Proteins were extracted from these cells for Western blotting and MAPK and Ras assays, and RNA was extracted from these cells for RT-PCR, as described below. The proliferation of osteogenic cells was assessed by measuring the incorporation of [3H]thymidine into the trichloroacetic acid-insoluble fraction of cells. 4 × 105 BMC were cultured in 6-well plates under the conditions described in the figure legends and were pulsed with [3H]thymidine (1 µCi/ml of medium) for the last 6 h of incubation with test agents. After the incubation period, the medium was removed, and the cell monolayer was washed with ice-cold PBS and then detached by treatment with 1 ml of 0.25% trypsin for ∼5 min. Further tryptic activity was inhibited by addition of 1 ml of Dulbecco's modified Eagle's minimal essential medium containing 10% fetal calf serum. DNA containing incorporated [3H]thymidine was precipitated in cold 7.5% trichloroacetic acid, centrifuged, washed three times with 7.5% trichloroacetic acid, and then counted on an LKB Rackbeta scintillation counter. The proliferation of osteogenic cells was also assessed by bromodeoxyuridine (BrdUrd) incorporation assay. The cells from 6-day primary bone marrow cell cultures were pulsed with 5 µm BrdUrd for the last 6 h of incubation with test agents. After the culture period, cells were washed with PBS and fixed with PLP fixative solution. After air drying, the cells were stored at −20 °C until double staining for alkaline phosphatase and BrdUrd as described below. BrdUrd-positive cells were quantitated by image analysis. Cells from 18-day primary BMC cultures were incubated for 15 min at room temperature in 100 mm Tris maleate buffer containing 0.2 mg/ml naphthol AS-MX phosphate (Sigma) dissolved in ethylene glycol monomethyl ether (Sigma) as a substrate and fast red TR (0.4 mg/ml, Sigma) as a stain for the reaction product. After washing with distilled water and air drying, ALP-positive colony areas were measured by image analysis as described below. Following ALP staining, cells were stained with 3% silver nitrate for 20 min under ultraviolet light. After washing with distilled water and air drying, calcified positive colony areas were measured by image analysis. Computer-assisted image analysis was performed as described previously (36Miao D. Bai X. Panda D. McKee M.D. Karaplis A.C. Goltzman D. Endocrinology. 2001; 142: 926-939Crossref PubMed Scopus (139) Google Scholar). Briefly, images of stained culture dishes were photographed with transmitted light over a light box. All images were processed using Northern Eclipse image analysis software, version 5.0 (Empix Imaging Inc., Mississauga, Ontario, Canada). For determining the area of positive colonies in cultured cells, thresholds were set using green and red channels. The thresholds were determined interactively and empirically on the basis of three different images. Subsequently, this set threshold was used to analyze automatically all recorded images of all sections that were stained in the same staining session under identical conditions. Cultured cells in Petri dishes were stained immunocytochemically for type I PTH/PTHrP receptor (PTHR), vimentin, type I collagen, bone sialoprotein, and osteocalcin using the avidin-biotin-peroxidase complex (ABC) technique as described previously (36Miao D. Bai X. Panda D. McKee M.D. Karaplis A.C. Goltzman D. Endocrinology. 2001; 142: 926-939Crossref PubMed Scopus (139) Google Scholar). The cultured cells were first treated with 0.5% bovine testicular hyaluronidase (Sigma) for 30 min at 37 °C, to increase antibody penetration and access to epitopes. Primary antibody was applied to cells overnight at room temperature. As a negative control, the preimmune serum was substituted for the primary antibody. After washing with high salt buffer (50 mm Tris-HCl, 2.5% NaCl, 0.05% Tween 20, pH 7.6) for 10 min at room temperature followed by two 10-min washes with TBS (50 mm Tris-HCl, 150 mm NaCl, 0.01% Tween 20, pH 7.6), the cells were incubated with secondary antibody (biotinylated rabbit anti-goat IgG, biotinylated goat anti-rabbit IgG, biotinylated goat anti-mouse IgG, Fab special (Sigma)). Cells were then washed as before and incubated with the Vectastain ABC-AP kit (Vector Laboratories, Ontario, Canada) for 45 min. After washing as before, red pigmentation to demarcate regions of immunostaining was produced by a 10–15-min treatment with Fast Red TR/Naphthol AS-MX phosphate (Sigma, containing 1 mm levamisole as endogenous ALP inhibitor). After washing with distilled water, the sections were counter-stained with methyl green and mounted with Kaiser's glycerol jelly. Following ALP cytochemistry the cells cultured in Petri dishes were stained for BrdUrd reactivity by the ABC immunoperoxidase technique as described above, except for immunoreactivity detected by using the Vectastain Elite ABC kit and staining with Vector SG substrate (Vector Laboratories, Ontario, Canada). Antigenicity appeared as a gray coloration. Proteins were extracted from 18-day cultures and quantitated by the protein assay kit (Bio-Rad). Protein samples (30 µg) were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. Immunoblotting was carried out using antibodies as described above. Bands were visualized using the ECL chemiluminescence detection method (Amersham Pharmacia Biotech). Total RNA for RT-PCR was extracted from 6-day BMC cultures by a single-step method, using Trizol reagent, and reversed-transcribed and amplified by PCR using Qiagen one-step TR-PCR kit (Qiagen Inc., Mississauga, Ontario, Canada) according to the manufacturer's instructions. We used the following primer sets to amplify PTHR: forward 5′-TGCTTGCCACTAAGCTTCG-3′ and reverse 5′-TCCTAATCTCTGCCTGCACC-3′. Cells from 6-day BMC cultures were transferred to serum-free media overnight and then preincubated with or without 10 µm H8 for 30 min and then incubated with or without 10−7m PTHrP for 10 min. Protein kinase A (PKA) activity was assessed with PKA Assay Kit (Upstate Biotechnology, Inc.) according to the manufacturer's instructions. Six readings were taken for each treatment. Cells from 6-day BMC cultures were transferred into serum-free media overnight and then challenged with various reagents as described in the figure legends. After stimulation, cells were washed in phosphate-buffered saline (PBS, 20 mmNaH2PO4, 0.9% NaCl, pH 7.4) and lysed for 20 min with lysis buffer (20 mm Tris-Cl, pH 7.4, 150 mm NaCl, 0.1% Nonidet P-40, 1% glycerol, 0.2 mm sodium vanadate, and a protease mixture tablet/10 ml of buffer). The samples were collected and microcentrifuged at 14,000 rpm for 5 min, and the supernatants were collected, assayed for protein, and prepared for Western blot analysis with antibody against the active, phosphorylated form of MAPK (37Tong X.K. Hussain N.K. Adams A.G. O'Bryan J.P. McPherson P.S. J. Biol. Chem. 2000; 275: 29894-29899Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) or Akt (Thr-308) (38Huang H.M. Huang C.J. Yen J.J. Blood. 2000; 96: 1764-1771Crossref PubMed Google Scholar). Membranes were stripped and re-probed with polyclonal antibodies against MAPK (ERK-1 and ERK-2) or Akt. Cells, treated as described for the MAPK assay, were washed in PBS and then scraped from the dish with 0.3 ml of ice-cold Ras assay buffer (20 mm Tris, pH 7.5, containing 1 mm EDTA, 10% glycerol, 1% Triton X-100, 100 mm KCl, 5 mm sodium fluoride, 0.2 mm sodium vanadate, 5 mm MgCl2, 0.05% mm β-mercaptoethanol, and a protease mixture tablet/10 ml of buffer). The extracts were microcentrifuged at 14,000 rpm for 3 min, and a 0.03-ml aliquot of the supernatant was retained as a starting material, and the remainder was applied to ∼25 µg of a glutathione S-transferase fusion protein encoding the Ras-GTP binding domain of Raf1 coupled to glutathione-Sepharose beads (37Tong X.K. Hussain N.K. Adams A.G. O'Bryan J.P. McPherson P.S. J. Biol. Chem. 2000; 275: 29894-29899Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 39de Rooij J. Bos J.L. 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Cells were characterized in early (6 day) cultures of bone marrow cells. Following removal of the non-adherent cells at 4 days, only two kind of adherent cells were observed morphologically. Over 90% of cells were large fibroblast-like stromal cells, and less than 10% were small round cells of the monocyte/macrophage lineage (35Conget P.A. Minguell J.J. J. Cell. Physiol. 1999; 181: 67-73Crossref PubMed Scopus (688) Google Scholar). Oil red O staining was negative for the presence of adipocytes. Expression of the PTH/PTHrP receptor (PTHR) was assessed by RT-PCR and immunocytochemical staining. PTHR mRNA was expressed in cultures and was not apparently up- or down-regulated by treatment of the cells with PTHrP for 6 days (Fig.1A). PTHR protein was evident in all fibroblast-like stomal cells and was localized mainly to the membrane and cytoplasm of these cells (Fig. 1 B). Immunostaining for vimentin, a marker of mesenchymal cells (42Altmannsberger M. Osborn M. Schauer A. Weber K. Lab. Invest. 1981; 45: 427-434PubMed Google Scholar, 43Gown A.M. Vogel A.M. J. Cell Biol. 1982; 95: 414-424Crossref PubMed Scopus (211) Google Scholar), showed that all adherent cells in the 6-day cultures of bone marrow cells were positive for vimentin (Fig. 1 C). Cytochemical staining for ALP revealed that 83.7 ± 6.4% of cells were positive for ALP (Fig. 1 D). Immunostaining for bone sialoprotein, type I collagen, and osteocalcin showed that all cells were positive for bone sialoprotein (Fig. 1 E); all fibroblast-like cells but no small round cells were positive for type I collagen (Fig. 1 F); and all cells were negative for osteocalcin (Fig. 1 G). Consequently all cells at 6 days of culture expressed vimentin and bone sialoprotein, and the majority of cells was positive for ALP and type I collagen, and none expressed osteocalcin. The majority of the cells (over 90%) at this stage was therefore mesenchymal stromal cells (pre-osteoblastic cells) but not mature osteoblasts. We next explored the potential action of PTHrP on BMCs. Following 18 days in culture, cells were stained cytochemically for ALP to identify potential osteogenic areas and with von Kossa to identify calcified colonies. Positive ALP and calcified areas were quantitated by image analysis. Treatment with PTHrP at day 4 resulted in a significant increase in ALP-positive colony area compared with control cultures. However, calcified colony area was not altered significantly between control and PTHrP-treated cultures. Consequently, the ratio of ALP-positive colony area to calcified colony area was significantly increased in PTHrP-treated cultures compared with control cultures (Figs.2and3), i.e. mineralization was reduced.Figure 3Quantitative data from cytochemical staining for ALP and from von Kossa staining for calcium. Cells from 18-day primary BMC cultures incubated in the absence (Control) or presence of 10−7m PTHrP (PTHrP) on day 4 were stained cytochemically for ALP and with the von Kossa method for calcified colonies as described under "Experimental Procedures." Leftand middle bars, resulting stained cultures were quantitated by image analysis and expressed as a percent positive area for ALP and CA, respectively, per dish. Each value is the mean ± S.E. of three determinations.Right bars, results are also expressed as the ratio of Ca/ALP area × 100%. * denotes a statistically significant difference (p < 0.05) in the PTHrP-treated cultures relative to the control cultures.View Large Image Figure ViewerDownload (PPT) To assess further whether the increase of ALP-positive colony area in PTHrP-treated cultures resulted from osteogenic cell proliferation or differentiation, we performed [3H]thymidine incorporation and BrdUrd assays and analyzed cultured cells for phenotypic indices of osteoblasts by immunocytochemical staining and Western blotting. Treatment of cultures with 10−7, 10−8, and 10−9m PTHrP increased [3H]thymidine incorporation by 149.2 ± 12.8, 79.8 ± 6.5, and 53.2 ± 4.2%, respectively, in 6-day cultures, as compared with control cultures. This stimulation was also confirmed by BrdUrd incorporation assay, which demonstrated more BrdUrd-positive cells after 10−7m PTHrP treatment of cultures (76.5 ± 7.2%) than before (32.8 ± 2.2%) (Fig. 4, A andB). Consequently, both methods confirmed the proliferative effect of PTHrP in these cells. We then explored the effect of PTHrP treatment on the expression of a variety of phenotypic markers of osteoblast differentiation in cells within the colonies. When these cells were cultured for 10 days or more, almost all cells expressed type I collagen, bone sialoprotein, and osteocalcin. Consequently, these cells expressed several indices consistent with an osteoblast phenotype. After PTHrP treatment increased numbers of cells staining for type I collagen, bone sialoprotein, and osteocalcin in colonies were observed (Fig. 4, C–H). However, no difference was found in the levels of vimentin, type I collagen, bone sialoprotein, and osteocalcin per unit of cell protein following PTHrP treatment (Fig. 5).Figure 5Western blot analysis of vimentin, type I collagen, bone sialoprotein, and osteocalcin in cells from primary BMC cultures. BMC were cultured as in Fig. 4 for 10 days, after which protein was extracted for Western blotting for vimentin (VIM), type I collagen (Col I), and bone sialoprotein (BSP) or for 18 days, after which protein was extracted for osteocalcin (OCN). Extracts were analyzed by Western blot employing 30 µg of extracted protein as described under "Experimental Procedures."View Large Image Figure ViewerDownload (PPT) To explore the possible mechanism by which PTHrP stimulates osteogenic cell proliferation, we first examined whether PTHrP activates endogenous MAPK in osteogenic cells from 6-day BMC cultures. PTHrP treatment resulted in a time-dependent increase in MAPK activity, as determined by Western blots with an antibody against the
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