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J. Biol. Chem., Vol. 276, Issue 34, 32204-32213, August 24, 2001

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Parathyroid Hormone-related Peptide Stimulates Osteogenic Cell Proliferation through Protein Kinase C Activation of the Ras/Mitogen-activated Protein Kinase Signaling Pathway^*

Dengshun Miao^§, Xin-Kang Tong^¶, George K. Chan, Dibyendu Panda, Peter S. McPherson^¶, and David Goltzman

From the Calcium Research Laboratory,  Department of Medicine and ^¶ Department of Neurology and Neurosurgery, McGill University Health Centre and McGill University, Montreal, Quebec H3A 1A1, Canada

Received for publication, February 5, 2001, and in revised form, May 31, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1). Several groups have also demonstrated that intermittent PTHrP administration increases bone mass in rats in vivo (2-8), and in humans, a 2-week course of PTHrP has been associated with activation of bone formation and suppression of bone resorption in postmenopausal women (9). Analogs of PTHrP have been developed in an attempt to improve its anabolic efficacy. One such analog, RS-66271, has received attention because of its pronounced bone anabolic activity when given intermittently to ovariectomized, osteopenic rats (10). A rapid increase in the number of osteoblasts on trabecular surfaces was observed following initiation of treatment (11). Additional evidence in support of an anabolic effect of PTHrP on bone comes from studies of heterozygous PTHrP "knockout" animals that exhibit haploinsufficiency and evidence of reduced trabecular bone volume (12). Despite these observations the cellular basis for the anabolic actions of PTHrP in vivo is still not well understood.

The osteogenic potential of bone marrow-derived mesenchymal stem cells has been very well characterized in vitro and in vivo (13-15). When cultured in the presence of dexamethasone, ascorbic acid, and -glycerophosphate, these cells proliferate and differentiate along the osteogenic lineage, producing bone-like nodules with a mineralized extracellular matrix. Treatment with 1,25-(OH)₂D₃, prostaglandin E₂, or PTH in vivo increases the number of osteogenic colonies in ex vivo bone marrow cultures suggesting that their effects may be mediated via bone marrow-derived mesenchymal stem cells (16-18). However, the mechanism of action of PTHrP on osteogenic cell proliferation and/or differentiation in vitro has not been reported.

Insight into the mechanism of action of PTH and PTHrP has been provided by the discovery of the type I PTH/PTHrP receptor (PTHR) (19, 20). This G protein-coupled receptor (GPCR) was shown to bind the NH₂-terminal regions of both PTH and PTHrP with almost equal affinity (21-23) and is associated with at least two signal transduction systems, the adenylyl cyclase/protein kinase A (PKA) pathway and the phospholipase C/protein kinase C pathway (21). Recently, Verheijen and Defize (24) have reported that PTH activates mitogen-activated protein kinase (MAPK) via a cAMP-mediated pathway independent of Ras in both Chinese hamster ovary R15 cells and parietal yolk sac carcinoma cells, and Swarthout et al. (25) have reported that PTH enhances proliferation in osteoblastic cells in vitro via a PKC-dependent activation of extracellular signal-regulated (ERK) kinase activity. In recent years, a number of GPCRs, operating through several subfamilies of heterotrimeric G proteins, have been shown to activate the MAP kinase cascade; this includes receptors for thrombin, bombesin, bradykinin, -adrenergic and nucleotide (P2Y) agonists (26-31). A variety of signaling effectors have been implicated in GPCR signaling to MAPK including Ras, receptor tyrosine kinases, Src family kinases, phosphatidylinositol 3-kinase (PI3K), and PKC isoenzymes (26, 27, 32). Both Ras-dependent and Ras-independent mechanisms of GPCR-mediated activation of MAPK have been described. Della Rocca et al. (33) 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 vitro by 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- 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, (34); 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).

Primary Bone Marrow Cell Cultures-- 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 -glycerophosphate, and 10⁸ M 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 (35). 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 0.01 M 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.

[³H]Thymidine Incorporation Assay-- 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.

Bromodeoxyuridine Incorporation Assay-- 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.

Cytochemical Staining for Alkaline Phosphatase (ALP) and von Kossa Staining for Calcified Colonies-- 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-- Computer-assisted image analysis was performed as described previously (36). 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.

Immunocytochemistry-- 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 (36). 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.

Double Staining with ALP and BrdUrd-- 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.

Western Blot Analysis-- 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).

RT-PCR-- 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'.

Protein Kinase A Assay-- 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 10⁷ M 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.

MAPK and Akt Assay-- 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 mM NaH₂PO₄, 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 (37) or Akt (Thr-308) (38). Membranes were stripped and re-probed with polyclonal antibodies against MAPK (ERK-1 and ERK-2) or Akt.

Ras Assays-- 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 -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 (37, 39-41). Following a 2-h incubation, the beads were washed 4 times with 1 ml of ice-cold Ras assay buffer, and material bound to the beads was eluted with SDS gel sample buffer and processed for SDS-polyacrylamide gel electrophoresis along with the starting material aliquot. The samples were processed for Western blots with the monoclonal antibody against Ras.

Statistical Analysis-- Data from image analysis of stained cell cultures are presented as means ± S.E. Statistical comparisons were made using one-way analysis of variance, with a probability of less than 0.05 being considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phenotypic Characterization of Cells from Bone Marrow Cell Cultures-- 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 (35). 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. 1B). Immunostaining for vimentin, a marker of mesenchymal cells (42, 43), showed that all adherent cells in the 6-day cultures of bone marrow cells were positive for vimentin (Fig. 1C). Cytochemical staining for ALP revealed that 83.7 ± 6.4% of cells were positive for ALP (Fig. 1D). Immunostaining for bone sialoprotein, type I collagen, and osteocalcin showed that all cells were positive for bone sialoprotein (Fig. 1E); all fibroblast-like cells but no small round cells were positive for type I collagen (Fig. 1F); and all cells were negative for osteocalcin (Fig. 1G). 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.

View larger version (88K): [in this window] [in a new window]   Fig. 1.   Phenotypic characterization of cells from bone marrow cell cultures. A, total RNA was extracted from 6-day BMC cultures, which had been incubated without PTHrP or with 107 M PTHrP for 6 days, and was subjected to RT-PCR as described under "Experimental Procedures." An RT-PCR product of the predicted size (264 base pairs) encoding PTHR was detected (lanes 2 and 3). Lane 1, molecular weight marker; lane 2, PTHrP; lane 3, +PTHrP; lane 4, water blank control for RT-PCR. B-G, cells from 6-day BMC cultures were fixed with PLP fixative and immunostained for PTHR (B), vimentin (C, Vim), bone sialoprotein (E, BSP), type I collagen (F, Col I), and osteocalcin (G, OCN) or stained histochemically for ALP (D) as described under "Experimental Procedures." Immunostaining for PTHR demonstrated that the receptor was localized mainly to the cell membrane (arrows) and cytoplasm (arrowheads). Histochemical staining for ALP and immunostaining for vimentin, BSP, collagen I, and osteocalcin on the 6-day cultures revealed that the majority of fibroblast-like cells was positive for ALP, and all of them were positive for vimentin, BSP, and collagen I but were negative for osteocalcin. The small round cells (C-G, arrows) were positive for vimentin and BSP but were negative for ALP, collagen I, and osteocalcin.

PTHrP Increases Alkaline Phosphatase-positive Colony Areas to a Greater Extent Than Mineralization-- 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. 2 and 3), i.e. mineralization was reduced.

View larger version (117K): [in this window] [in a new window]   Fig. 2.   Effect of PTHrP on ALP-positive colonies and calcified colonies in primary BMC cultures. Cells from 18-day primary BMC cultures incubated in the absence (Control) or presence of 107 M PTHrP (PTHrP) on day 4 were stained cytochemically for ALP and with the von Kossa method for calcified colonies as described under "Experimental Procedures." Upper panel, cytochemical staining for ALP. Bottom panel, double staining for ALP (red) and calcium (black).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Quantitative 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 107 M PTHrP (PTHrP) on day 4 were stained cytochemically for ALP and with the von Kossa method for calcified colonies as described under "Experimental Procedures." Left and 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.

PTHrP Enhances the Proliferation but Not Differentiation of Osteogenic Cells in Primary BMC Cultures-- 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 10⁷, 10⁸, and 10⁹ M 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 10⁷ M PTHrP treatment of cultures (76.5 ± 7.2%) than before (32.8 ± 2.2%) (Fig. 4, A and B). 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).

View larger version (104K): [in this window] [in a new window]   Fig. 4.   Immunostaining for BrdUrd (BrdU), type I collagen, bone sialoprotein, and osteocalcin in cells from primary BMC cultures. Cells from 6-day primary BMC cultures incubated in the absence (Control) or presence of 107 M PTHrP (PTHrP) for the last 6 h were double-stained for ALP (red) and BrdUrd (gray) as described under "Experimental Procedures." Cells from 10-day primary BMC cultures treated without or with PTHrP on day 4 were stained immunocytochemically for type I collagen (Col I) and bone sialoprotein (BSP) as described under "Experimental Procedures." Cells from 18-day primary BMC cultures treated without or with PTHrP on day 4 were stained immunocytochemically for osteocalcin (OCN).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Western 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."

PTHrP-stimulated Osteogenic Cell Proliferation Occurs by Activation of MAPK-- 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 active, phosphorylated form of Erk-1/2 (PO₄ MAPK) (Fig. 6). MAPK activity was increased from 2 to 15 min after the stimulation by 10⁹ or 10⁷ M PTHrP, with a maximum at 5 min. MAPK activity returned to basal levels at 30 min. As a control, the levels of total MAPK were measured at various time points by stripping membranes and re-probing with an antibody that recognizes non-phosphorylated Erk-1/2 (MAPK). Total MAPK was constant over the 30-min interval (Fig. 6). To determine whether PTHrP activates MAPK in a MEK-dependent fashion, the cells were pretreated with 50 µM PD-98059, a known inhibitor of MEK, for 30 min and then were co-incubated with PTHrP for various time points. Treatment with PD98059 blocked basal and PTHrP-stimulated MAPK activity dramatically (Fig. 6). The levels of total MAPK remained constant at all these time points (Fig. 6).

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Effect of PTHrP and of PD98059 on MAPK activity. Cells from 6-day BMC cultures were transferred to serum-free media overnight and then incubated with 109 or 107 M PTHrP for various times (1st to 4th lines). In other experiments, cells were preincubated with 50 µM PD98059 for 30 min and then were incubated with 107 M PTHrP (5th and 6th lines) for various time points. After stimulation, cells were analyzed by PO4-MAPK or total MAPK as described under "Experimental Procedures."

To determine whether PTHrP stimulates osteogenic cell proliferation in a MAPK-dependent manner, the effect of PD98059 on osteogenic cell proliferation was investigated in 6-day BMC cultures using the [³H]thymidine incorporation assay. Treatment of cells with 10⁷ M PTHrP increased [³H]thymidine incorporation significantly by 95.1% of control cultures (Fig. 7). PD-98059 decreased [³H]thymidine incorporation of unstimulated cultures, and PTHrP stimulation above basal was eliminated (Fig. 7). Trypan blue exclusion demonstrated intact cell viability both before and after treatment with PD98059.

View larger version (8K): [in this window] [in a new window]   Fig. 7.   Effect of PD98059 on PTHrP-stimulated osteogenic cell proliferation in primary BMC cultures. Cells from 6-day BMC cultures were transferred to serum-free media overnight and then incubated for 6 h in the absence (0) or presence of 20 µM PD-98059 (PD), with 107 M PTHrP (PTHrP) or without PTHrP (Control). A [3H]thymidine incorporation assay was then performed as described under "Experimental Procedures." Each value represents the mean ± S.E. of three determinations. * denotes a statistically significant difference (p < 0.05) in the cultures with PTHrP relative to cultures without PTHrP.

PTHrP Activates MAPK via the Ras Pathway-- Several previous reports (33, 44) have shown that GPCR-mediated MAPK activation is Ras-dependent. Therefore, we examined whether Ras activation also occurred in our culture system. The levels of Ras-GTP in cells were determined by performing Ras pull-down assays with a GST-Raf1 fusion protein that has an increased affinity for Ras-GTP versus Ras-GDP (45, 46). Following PTHrP stimulation, there was a time-dependent increase in Ras activity. Ras activity was increased significantly from 1 to 5 min of PTHrP stimulation with maximal activation at 1 min. However, after 5 min there was a decline back toward basal levels (Fig. 8). The control levels of Ras in the crude cell lysates (starting material) at these time points were approximately the same (Fig. 8).

View larger version (10K): [in this window] [in a new window]   Fig. 8.   Effect of PTHrP on Ras. Cells from 6-day BMC cultures were transferred to serum-free media overnight and then challenged with 107 M PTHrP for various time points. After stimulation, cells were analyzed by Ras assay as described under "Experimental Procedures."

PTHrP Activates Ras/MAPK via PKC but Not via the PKA or PI3K Pathways-- To determine possible upstream mediators of Ras/MAPK activation by PTHrP, we examined the PKA, PI3K, and PKC signaling pathways. The cells were first pretreated with 10 µM H8 or 10 µM wortmannin, inhibitors of PKA and PI3K, respectively. Treatment with H8 inhibited both basal and PTHrP-stimulated PKA (Fig. 9A). PTHrP did not stimulate Akt, a known downstream target of PI3K, (data not shown). However, wortmannin did markedly reduce Akt activity (Fig. 9B, 5th and 6th lines). Despite the effectiveness of H8 and wortmannin as kinase inhibitors in these cells, PTHrP treatment in the presence of these inhibitors led to the previously observed increases in MAPK activity (Fig. 9B, 1st and 3rd lines, respectively). As a control, the levels of total MAPK in H8- or wortmannin-pretreated cultures were assessed, and these remained essentially constant (Fig. 9B, 2nd and 4th lines, respectively). In contrast, stimulation of MAPK activity by PTHrP was blocked by pretreatment with 5 µM chelerythrin chloride, a known PKC inhibitor (Fig. 10, 1st line). The levels of total MAPK in chelerythrin chloride pretreated cultures at these time points also remained constant (Fig. 10, 2nd line).

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Effect of inhibitors of PKA and PI3K on PTHrP-stimulated MAPK. A, cells from 6-day BMC cultures were transferred to serum-free media overnight and then preincubated without or with 10 µM H8 for 30 min and then incubated without or with 107 M PTHrP for 10 min. PKA assay was then performed as described under "Experimental Procedures." Each result is the mean ± S.E. of 6 replicates. The asterisk represents a significant difference at p < 0.05 in the cultures with PTHrP relative to cultures without PTHrP. # denotes a statistically significant difference (p < 0.05) in the cultures with H8 relative to cultures without H8. B, cells from 6-day BMC cultures were transferred to serum-free media overnight and preincubated with 10 µM H8 (1st and 2nd lines) or 10 µM wortmannin (3rd and 4th lines) for 30 min and then incubated with 107 M PTHrP or treated with 10 µM wortmannin alone for various time points. After stimulation, cells were analyzed by MAPK assay (1st to 4th lines) or Akt assay (5th and 6th lines) as described under "Experimental Procedures."

View larger version (33K): [in this window] [in a new window]   Fig. 10.   Effects of an inhibitor or activator of PKC on MAPK and Ras. Cells from 6-day BMC cultures were transferred to serum-free media overnight and preincubated with 5 µM chelerythrine chloride (1st to 4th lines) for 30 min and then incubated with 107 M PTHrP or treated with 1 µM TPA alone (5th to 8th lines) for various time points. After stimulation, cells were analyzed by MAPK assay (1st and 2nd lines and 5th and 6th lines) or Ras assay (lines 3 and 4 and lines 7 and 8) as described under "Experimental Procedures."

To pursue this finding, we next examined the effect of the PKC inhibitor on PTHrP-stimulated Ras activity. Ras stimulation by PTHrP was also blocked by chelerythrin chloride treatment (Fig. 10, 3rd line). The control levels of total Ras in the crude cell lysates (starting material) at all these time points were essentially constant (Fig. 10, 4th line). These results suggest that PTHrP activates Ras/MAPK via PKC but not via the PKA or PI3K pathways.

To substantiate further that PTHrP activates Ras/MAPK via PKC signaling, we examined whether TPA, a PKC activator, can activate both MAPK and Ras in our cell culture system. TPA caused a progressive increase in MAPK activity from 1 to 30 min (Fig. 10, 5th line) and a significant increase in Ras activity at these same time points (Fig. 10, 7th line). The control levels of total MAPK and Ras in the crude cell lysates (starting material) remained constant during these periods (Fig. 10, 6th and 8th lines, respectively).

To determine whether PTHrP stimulates osteogenic cell proliferation in a PKC-dependent manner, the effect of the PKC inhibitor, chelerythrin chloride, on osteogenic cell proliferation was investigated in 6-day BMC cultures using the [³H]thymidine incorporation assay. Treatment of cells with 10⁷ M PTHrP increased [³H]thymidine incorporation significantly by 97.0% of control cultures, but this stimulation was blocked in the presence of 1.5 µM chelerythrin chloride (Fig. 11). Chelerythrin chloride decreased [³H]thymidine incorporation in unstimulated cultures and completely inhibited PTHrP-stimulated [³H]thymidine incorporation (Fig. 11). Trypan blue exclusion demonstrated intact cell viability both before and after treatment with chelerythrin chloride.

View larger version (9K): [in this window] [in a new window]   Fig. 11.   Effect of PKC inhibition on PTHrP-stimulated osteogenic cell proliferation in primary BMC cultures. Cells from 6-day BMC cultures were transferred to serum-free media overnight and then incubated for 6 h in the absence (0) or presence of 1.5 µM chelerythrine chloride (chelerythrin), with 107 M PTHrP (PTHrP) or without PTHrP (Control). A [3H]thymidine incorporation assay was performed as described under "Experimental Procedures." Each value represents the mean ± S.E. of three determinations. * denotes a statistically significant difference (p < 0.05) in the cultures with PTHrP relative to cultures without PTHrP.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The anabolic effects of PTHrP have been demonstrated in rodents and in humans in vivo (2-9); however, the molecular and cellular basis for these actions of PTHrP are unclear. Treatment with 1,25-(OH)₂D₃, prostaglandin E₂, or PTH in vivo increases the number of osteogenic colonies in ex vivo bone marrow cultures suggesting that their effects may be mediated by bone marrow-derived mesenchymal stem cells (16-18). These investigations have not as yet been performed with PTHrP either in vivo or in vitro. In the present study, we employed a primary BMC culture system to investigate the anabolic effects of PTHrP on osteogenic cell proliferation and differentiation in vitro. Under the conditions we used, these cultures appeared, early on, to consist mainly of mesenchymal stromal cells or pre-osteoblastic cells and expressed both PTHR mRNA and PTHR protein. Subsequently, their osteogenic potential was observed in view of the fact that cells displayed an osteoblastic phenotype expressing ALP, type I collagen, bone sialoprotein, and osteocalcin and formed calcified nodules. The increase in differentiation markers was, however, only proportional to the increase in cells induced by PTHrP, and mineralization of the cultures was also not increased directly by PTHrP. Consequently, treatment with PTHrP primarily stimulated the proliferation of the osteogenic cells, which then differentiated into an osteoblastic phenotype.

Previous studies have indicated that development of osteogenic cells proceeds through a complex series of maturation events leading from a mesenchymal stem cell through a committed progenitor cell or cells to a differentiated osteoblast and therefore to an osteocyte (47, 48). Consequently, PTHrP may act at a number of loci. The capacity of PTHrP to influence commitment to the osteogenic lineage was not tested and may represent an important function. Similarly, the capacity to inhibit apoptosis of cells of the osteoblast lineage would also appear to be of importance but was not tested (49). Nevertheless, from our current studies, a major role for PTHrP appears to be the capacity to stimulate proliferation of an osteogenic progenitor cell, which can then differentiate into a more mature osteoblastic cell. Transient administration of PTHrP as performed in this study may therefore be desirable for the anabolic effects of this molecule in order to expand the progenitor population which, in the absence of sustained PTHrP exposure, can then differentiate. Indeed, in the presence of sustained PTHrP, it is possible that the capacity to differentiate and mineralize may in fact be impeded. This may therefore be reflected in vivo in the observation that in malignancy-associated hypercalcemia, where very high sustained ambient PTHrP levels occur, bone formation may be impaired resulting in uncoupling of the resorption-formation paradigm (50).

To gain further insight into the molecular mechanism of PTHrP action in stimulating proliferation of these progenitor cells, we examined whether PTHrP induces activation of the MAPK signaling pathway. Our results demonstrated that PTHrP induces activation of MAPK in a time-dependent and an MEK-dependent fashion in these bone marrow cells. Furthermore, PTHrP-induced stimulation of osteogenic cell proliferation occurred in a MAPK-dependent manner. This is therefore the first report of a role for PTHrP in stimulating MAPK-dependent mitogenesis in osteogenic cells. These results are consistent with previous studies, which reported that PTH causes MAPK activation in CHO-R15 cells (24) leads to MAPK activation and DNA synthesis in the renal proximal tubule-like OK cell line (51) and increases extracellular signal-regulated kinase activity and proliferation in an osteosarcoma cell line and in calvarial osteoblasts (25).

We next examined activators of MAPK in our system. The mechanism of activation of the MAPK signaling pathway by GPCRs is poorly understood, although it is becoming evident that signal transduction by certain GPCRs utilizes many of the same intermediates as those activated by receptor tyrosine kinases (33, 52). Our results show that PTHrP induced a time-dependent increase in Ras activity. These results differ from studies showing that PTH activates MAPK by a Ras-independent manner in Chinese hamster ovary R15 cells and parietal yolk sac carcinoma cells (24) and in osteosarcoma cells and calvarial osteoblasts (25). These differences may reflect the fact that significant degrees of heterogeneity of downstream signaling via GPCRs exist between cell types (53) or may reflect differences in the use of pharmacologic inhibitors versus transfected dominant negative inhibitors (25) to probe the Ras pathway.

We also examined possible activators of Ras. Receptor tyrosine kinases induce MAP kinase activation through the sequential interaction of the signaling proteins Grb2, Sos, Ras, Raf, and MEK. However, GPCRs stimulate MAPK through G and G subunits. The molecules that subsequently intervene are still poorly defined. Based on previous studies examining PTHrP signaling, two major signal transduction systems have been shown (54). One is a G_s-mediated increase in cAMP, leading to activation of PKA. The other is a G_q-mediated activation of phospholipase C, leading to increases in intracellular inositol triphosphate and diacylglycerol and activation of PKC. Linkage of GPCRs to the MAPK signaling pathway through PI3K has been demonstrated in COS-7 cells overexpressing PI3K (32), and linkage through PKC has been demonstrated in osteosarcoma cells and in calvarial osteoblasts (25). In the present study, we examined three possible pathways that might stimulate Ras: PI3K, PKA, and PKC. Our results showed that in our system wortmannin, an inhibitor of the PI3K pathway, could not block MAPK activity stimulated by PTHrP. Moreover, we also observed that H8, an inhibitor of the PKA pathway, could also not block MAPK activity stimulated by PTHrP. We did demonstrate, however, that chelerythrin chloride, a known PKC inhibitor, blocked PTHrP-induced Ras/MAPK activation and osteogenic cell proliferation. Furthermore, TPA, a PKC activator, was able to activate both Ras and MAP kinase activity in the cell population we examined. Our results differ from a previous report that demonstrated that PKC activates Raf in a Ras-independent fashion (55). However, other groups (40) have shown that stimulation of PKC in COS cells led to activation of Ras and formation of Ras-Raf-1 complexes that activate Raf-1. Sato et al. (56) also reported that TPA induces activation of Ras and ERK in MCF-7 cells. Consequently, we conclude that PKC is an upstream component of the Ras/MAPK signaling pathway in primary BMC cultures.

In summary, our results demonstrate that primary osteogenic cells of rat bone marrow are direct targets of PTHrP. The amino-terminal domain of PTHrP can, as shown previously, bind to PTHR, enhance phospholipase C activation via G_q, increase intracellular inositol triphosphate and diacylglycerol, and cause activation of PKC. Our results demonstrate that this PKC activation can lead to stimulation of the RAS > Raf > MEK > MAPK cascade in marrow progenitor cells and contribute to PTHrP enhancement of osteogenic cell proliferation (Fig. 12).

View larger version (19K): [in this window] [in a new window]   Fig. 12.   Schematic summary of PTHrP signaling through the MAPK cascade. The abbreviations used are: DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; IP3, inositol triphosphate; MEK, MAPK/ERK kinase; and PLC, phospholipase C.

	ACKNOWLEDGEMENT

We thank Isabel Bolivar for the work on the PKA assay.

	FOOTNOTES

^* This work was supported in part by Grant MT-5775 (to D. G.) from the Canadian Institutes for Health Research of Canada, by Grant 00731 (to D. G.) from the National Cancer Institute of Canada, and by a research contract (to P. S. M.) from Biochem Pharma (Laval, Quebec, Canada).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipient of a fellowship from the Canadian Institutes for Health Research of Canada.

To whom correspondence should be addressed: Calcium Research Laboratory, Dept. of Medicine, Royal Victoria Hospital, H4.67, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada. Tel.: 514-843-1632; Fax: 514-843-1712; E-mail: david.goltzman@muhc.mcgill.ca.

Published, JBC Papers in Press, June 11, 2001, DOI 10.1074/jbc.M101084200

	ABBREVIATIONS

The abbreviations used are: PTHrP, parathyroid hormone-related peptide; MAH, malignancy-associated hypercalcemia; PTH, parathyroid hormone; BMC, bone marrow cell; RT-PCR, reverse transcriptase-polymerase chain reaction; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; PI3K, phosphatidylinositol 3-kinase; MAP, mitogen-activated protein; PKA, protein kinase A; TPA, tetradecanoylphorbol-13-acetate; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline; ABC, avidin-biotin-peroxidase complex; ALP, alkaline phosphatase; PTHR, type I PTH/PTHrP receptor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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