Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways

Luisa Carpio, Julienne Gladu, David Goltzman, and Shafaat A. Rabbani

Department of Medicine, McGill University Health Center, Montreal, Quebec H3A 1A1, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Parathyroid hormone (PTH)-related peptide (PTHrP) can modulate the proliferation and differentiation of a number of cell types including osteoblasts. PTHrP can activate a G protein-coupled PTH/PTHrP receptor, which can interface with several second-messenger systems. In the current study, we have examined the signaling pathways involved in stimulated type I collagen and alkaline phosphatase expression in the human osteoblast-derived osteosarcoma cells, MG-63. By use of Northern blotting and histochemical analysis, maximum induction of these two markers of osteoblast differentiation occurred after 8 h of treatment with 100 nM PTHrP-(1-34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen and alkaline phosphatase stimulation in a dose-dependent manner. These effects of PTHrP could also be blocked by inhibiting the Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059. Transient transfection of MG-63 cells with a mutant form of Galpha , which can sequester beta gamma -subunits, showed significant downregulation of PTHrP-stimulated type I collagen expression, as did inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin. Consequently, the beta gamma -PI 3-kinase pathway may be involved in PTHrP stimulation of Ras. Collectively, these results demonstrate that, acting via its G protein-coupled receptor, PTHrP can induce indexes of osteoblast differentiation by utilizing multiple, perhaps parallel, signaling pathways.

parathyroid hormone-related peptide; osteoblast; cell differentiation; hypercalcemia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE SEARCH for the responsible pathogenetic factor in the development of hypercalcemia of malignancy culminated in the discovery of parathyroid hormone (PTH)-related peptide (PTHrP) a little more than a decade ago (6, 26, 32). Since then, PTHrP has been observed to be expressed by a wide variety of normal adult and fetal tissues (12, 30). Due to its wide tissue distribution and the degree to which it is conserved across evolution, it was proposed that PTHrP may well have a significant developmental role. It was subsequently discovered that PTHrP plays a role in the proliferation and differentiation of a variety of cell types, including chondrocytes, osteoblasts (18), and keratinocytes (16, 17). Despite the evidence that PTHrP plays an important role in cellular turnover and maturation, little is currently known of the molecular mechanisms involved in PTHrP-mediated cellular differentiation. Due to the NH2-terminal sequence homology between PTH and PTHrP, these two peptides can interact with a common receptor, PTH/PTHrP receptor (1). The presence of this receptor in bone is well established, and high receptor levels are seen in osteoblasts that are actively differentiating (24), suggesting a role for the receptor in osteoblast development. Active mineralization of bone matrix involves the production of type I collagen and alkaline phosphatase, which, among others, are established markers of osteoblast differentiation (4).

The common PTH/PTHrP receptor is a member of the family of seven transmembrane receptors that are coupled to heterotrimeric G proteins. PTH and PTHrP are known to activate several second-messenger pathways that are linked by distinct mediators to the PTH/PTHrP receptor (27). For example, ligand binding is known to stimulate both intracellular cAMP and inositol trisphosphate through Galpha s and Galpha q, respectively (1, 15). G protein-coupled receptors are also known to activate mitogen-activated protein kinase (MAPK) activity in a manner that is dependent on the profile of the involved G protein, the receptor to which it is coupled, and the cell type in which they are found (33). Previous studies have reported differential activation of protein kinase A (PKA) or PKC pathways, and this may be cell type specific and vary according to the differentiation stage and exposure time to the ligand. Activation of MAPK has been shown to be essential in the differentiation of several cell types, and it has been shown that PKA and/or PKC activation can influence MAPK activity. G protein activation will also involve the release of a beta gamma -dimer subunit (5, 21), and this subunit may regulate the phosphorylation of the protein Shc, which can then lead to the formation of a protein complex involving Shc-Grb2-Sos and the subsequent activation of the protooncogene Ras (11). Ras activation is known to result in activation of Raf and then in activation of the enzymes MAPK kinase (MEK) and MAPK (22). Receptors coupled to trimeric G proteins may therefore activate one or more of these possible pathways. Although it has been definitively established that PTH/PTHrP receptor signaling may occur via Galpha s and/or Galpha q, the question of beta gamma -mediated signaling by this receptor is not yet so clearly proven.

The present study was undertaken to further examine the possible role of multiple signal transduction pathways in the stimulation of osteoblast differentiation by PTHrP. Through the use of chemical inhibitors of signal transduction and transient transfection of a beta gamma -sequestering mutant form of Galpha s, and employing a human osteoblast-like osteosarcoma model, MG-63 cells, we show the importance of activation of PKA, PKC, and Ras by PTHrP in inducing osteoblast differentiation. Our results also demonstrate the involvement of MAPK, which may be a point of convergence of these activated signaling pathways in this system. These results, therefore, emphasize the involvement of multiple pathways in PTHrP-induced indexes of osteoblast differentiation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. MG-63 osteosarcoma cells were maintained in vitro in MEM [with Earle's Salts (ES)] supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin-streptomycin sulfate (GIBCO-BRL). For transient transfection assays, cells were plated at 1 × 105 cells/60-mm dish 24 h before transfection and growth in 5% CO2 in MEM-ES. Cells were incubated with 10 µg/ml lipofectin (GIBCO-BRL) and cultured overnight in serum-free MEM-ES culture medium with 0.1, 1, or 10 µg of plasmid DNA. After overnight incubation with lipofectin, fresh culture medium containing 10% FBS was added. PTHrP treatment assays were performed within 48 h after transfection.

PD-98059 (Biomol), wortmannin (Sigma Canada), B1086 (Eisai Research Institute, Andover MA), H-89 (Biomol), and chelerythrine chloride (Biomol) were dissolved in dimethyl sulfoxide and stored at appropriate stock concentrations and were diluted to the desired concentrations immediately before use.

The plasmid encoding the Galpha triple mutant was obtained from the laboratory of Dr. H. R. Bourne (University of California, San Francisco, CA) and has been previously described (13).

Northern blot analysis. Total cellular RNA was extracted by TRIzol extraction from control and experimental cells after treatment with vehicle alone, PTHrP-(1-34) alone, or graded concentrations of the chemical inhibitors. Ten micrograms of total cellular RNA were electrophoresed on a 1.1% agarose-formaldehyde gel, transferred to a nylon membrane (Nytran; S&S, Keene, NH) by capillary blotting, and then fixed by drying and ultraviolet cross-linking for 10 min. The integrity of the RNA was assessed by ethidium bromide staining. Hybridization was carried out with a 32P-labeled type I collagen cDNA and with an 18S RNA probe with the use of a [32P]dCTP, as previously described (2). After a 24-h incubation at 65°C, filters were washed twice under low-stringency conditions [1× standard sodium citrate (SSC) and 1% SDS at 60°C for 2 × 20 min] and under high-stringency conditions (0.1× SSC and 0.1% SDS at 60°C for 2 × 20 min). Autoradiography of filters was carried out at -70°C using XAR film (Eastman Kodak, Rochester, NY). The levels of type I collagen expression were quantified by densitometric scanning using the MAC BAS v1.01 alias program.

Alkaline phosphatase detection. Alkaline phosphatase activity in control and PTHrP-treated MG-63 cells was detected by a histochemical reaction. Cells were fixed in citrate-buffered acetone. Slides were then immersed in alkaline-dye solution containing diazonium salts and incubated for 30 min. Cells were stained with Mayer's hematoxylin solution for 10 min to detect the insoluble pigments formed as a result of alkaline phosphatase activity. Slides were then evaluated as integrated densities of staining by use of Scion Image, where total staining intensity was measured (10).

Immune complex protein kinase assay. Cells were washed twice with ice-cold PBS. Ice-cold RIPA lysis buffer was added to cell monolayers and incubated on ice for 10 min. Cells were scraped and transferred to Eppendorf tubes and further disrupted by vortex. Cellular debris was pelletted, and the supernatant was retained. Upon Bio-Rad Protein Assay quantitation of total protein levels, 200-500 µg of total protein were coincubated with 0.2-2 µg of extracellular signal-regulated kinase (ERK)1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C. A 25-µl resuspended volume of protein G-agarose (Santa Cruz Biotechnology) was added and incubated at 4°C, rotating for 2-16 h. Immunoprecipitates were collected and rinsed with RIPA buffer.

Pellets were resuspended in 30 µl of appropriate kinase assay buffer containing 10-1,000 ng of peptide substrate myelin basic protein (MBP) and [gamma -32P]ATP (10 mCi/ml) and incubated at 30°C for 30 min. Kinase reaction was terminated by addition of an equal volume of 2× electrophoresis sample buffer and boiling for 2-3 min. Samples were analyzed by SDS-PAGE and autoradiography.

Statistical analysis. All data are shown as means ± SE. Statistical analysis of results was by Student's t-test or by analysis of variance. Significant values were taken at P < 0.05. The mean, SE, and P value measurements were performed using Excel software (Microsoft, Port Redmond, WA).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of PTHrP on indexes of osteoblast differentiation. MG-63 osteoblast-like osteosarcoma cells, which exhibit characteristics of early, immature osteoblasts, were incubated in the absence or presence of 100 nM PTHrP-(1-34) for 0, 1, 3, 6, 8, 12, and 16 h. As shown in Fig. 1A, treatment with PTHrP-(1-34) increased type I collagen mRNA transcript levels within 6 h, and peak levels were reached after 8 h of treatment. Type I collagen levels were augmented more than twofold at this time point compared with control, untreated cells. Type I collagen levels decreased to basal by ~16 h. A second marker of osteoblast differentiation, alkaline phosphatase, was detected by histochemical reaction. After treatment of cells with 100 nM PTHrP-(1-34) for 6, 12, and 24 h, cells were fixed, and alkaline phosphatase activity was detected by a histochemical reaction. As seen in Fig. 1, B and C, PTHrP induced alkaline phosphatase activity, with highest levels occurring at 24 h. Although staining density increased slightly in untreated cells, the overall increase in staining in treated cells was considerably greater, reaching levels as high as sevenfold after 12 h of treatment. These results indicate that PTHrP can induce indexes of differentiation in MG-63 osteoblastic cells.


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Fig. 1.   Effect of parathyroid hormone (PTH)-related peptide (PTHrP) on MG-63 cell differentiation. Human osteoblast-like osteosarcoma MG-63 cells were grown in 10% serum to 80% confluence and then incubated in serum-free conditions. Cells were then treated with vehicle or with 100 nM PTHrP-(1-34) for timed intervals. A: total cellular RNA was extracted from MG-63 cells. Fifteen micrograms of total cellular RNA for each time point (1-16 h) were electrophoresed onto a 1.1% agarose-formaldehyde gel. Filters were probed with type I collagen and 18S cDNA to determine the ratio of I collagen to 18S mRNA expression. B: alkaline phosphatase activity in control and PTHrP-treated MG-63 cells was detected by a histochemical reaction. Cells were fixed in citrate-buffered acetone. Slides were then immersed in alkaline-dye solution containing diazonium salts and incubated for 30 min. Cells were stained with Mayer's hematoxylin solution for 10 min to detect the insoluble pigments formed as a result of alkaline phosphate activity. Slides were then evaluated microscopically. C: alkaline phosphatase activity was quantified using the NIH Image-based Scion Image analysis program. Quantification is presented as staining density per field. Results represent means ± SE of 3 different experiments. *Significant differences from control (P < 0.05).

Effects of inhibiting PKC on indexes of osteoblast differentiation. To determine the involvement of PKC in PTHrP-mediated MG-63 cell differentiation, cells were pretreated with chelerythrine chloride (3, 7), a specific inhibitor of PKC, followed by coincubation with 100 nM PTHrP-(1-34) for 8 h, the time of maximal induction of type I collagen transcript levels by PTHrP treatment. As seen in Fig. 2, inhibition of PKC by chelerythrine chloride treatment resulted in a decrease in PTHrP-stimulated type I collagen levels in a dose-dependent manner. Cell morphology (Fig. 3), viability as determined by trypan blue dye exclusion (>95% viable), and basal levels of type I collagen mRNA were unaffected by treatment of MG-63 cells with chelerythrine chloride alone. These results were paralleled by a reduction in alkaline phosphatase levels. Pretreatment with 5.0 µM chelerythrine chloride followed by coincubation with 100 nM PTHrP-(1-34) for 24 h resulted in alkaline phosphatase levels that were comparable to levels in untreated cells cultured for the same time period (Fig. 3). The ability of this inhibitor to curtail the effects of PTHrP on MG-63 cell differentiation suggests that PKC activation is involved in this phenomenon.


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Fig. 2.   Effect of protein kinase C (PKC) inhibitor chelerythrine chloride (Ch Cl) on type I collagen gene expression in MG-63 cells. Top: MG-63 cells were grown in 10% serum to 80% confluence and then incubated overnight in serum-free conditions (Ctl). Cells were then pretreated with vehicle or Ch Cl for 1 h. Cells were then treated with 100 nM PTHrP-(1-34) for 8 h. Ctl represents the results of treatment with vehicle only for both the pretreatment and treatment periods. Levels of type I collagen and ratios of type I collagen to 18S mRNA were determined by Northern blot analysis, as described in MATERIALS AND METHODS. Bottom: results depict the ratios of type I collagen to 18S mRNA and are expressed as %change relative to Ctl, which was assigned a value of 100%. Bars represent means ± SE of 3 different experiments. *Significant differences in the ratios from Ctl cells (P < 0.05); **significant differences in the ratios from PTHrP-only-treated cells (P < 0.05).



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Fig. 3.   Effect of inhibitors of signal transduction on alkaline phosphatase levels. A: MG-63 cells were grown in 10% serum to 80% confluence on double-chamber slides and then incubated overnight in serum-free conditions. Cells were pretreated with vehicle or PKA inhibitor H-89 (30 µM) and PKC inhibitor Ch Cl (5.0 µM) for 1 h, phosphatidylinositol 3-kinase (PI 3K) inhibitor wortmannin (Wm; 100 nM) for 3 h, Ras inhibitor B1086 (5.0 µM), and mitogen-activated protein kinase (MAPK) inhibitor PD-98059 (100 µM) overnight. The cells were then treated with 100 nM PTHrP for 24 h. B: alkaline phosphatase activity was quantified using the NIH Image-based Scion Image analysis program. Quantification is presented as staining density per field. *Significant differences in the ratios from Ctl cells (P < 0.05); **significant differences in the ratios from PTHrP-only-treated cells (P < 0.05).

Effects of inhibiting PKA on indexes of osteoblast differentiation. In a number of cell types, PTHrP is known to be a strong activator of adenylate cyclase via its stimulation of Galpha s. The cAMP generated then leads to activation of PKA. To determine whether activation of PKA is involved in PTHrP-induced MG-63 cell differentiation, cells were pretreated with the PKA inhibitor H-89. After incubation in serum-free medium for ~16 h, cells were pretreated with H-89 at 15 and 30 µM concentrations for 1 h, followed by coincubation with 100 nM PTHrP-(1-34). Inhibition of PKA by H-89 treatment resulted in a dose-dependent decrease in PTHrP-stimulated type I collagen mRNA levels (Fig. 4). The specificity of this response was confirmed by the treatment of MG-63 cells with H-89 alone, which showed little to no effect on basal type I collagen mRNA levels. Cell viability by trypan blue dye exclusion (>94% viable) and morphology (Fig. 3) were unaffected by H-89 at the doses indicated. These results were paralleled by a reduction in alkaline phosphatase levels. Pretreatment with 30 µM H-89 for 1 h, followed by coincubation with 100 nM PTHrP-(1-34) for 24 h, resulted in levels of alkaline phosphatase that were comparable to those in untreated cells (Fig. 3). The ability of this inhibitor to curtail the effects of PTHrP on MG-63 cell differentiation suggests that PKA activation is involved in this phenomenon.


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Fig. 4.   Effect of the PKA inhibitor H-89 on type I collagen gene expression in MG-63 cells. Top: MG-63 cells were grown in 10% serum to 80% confluence and incubated overnight in serum-free conditions. Cells were then pretreated with vehicle or H-89 for 1 h and then incubated with 100 nM PTHrP-(1-34) for 8 h. Ctl represents results of treatment with vehicle only for both the pretreatment and treatment periods. Levels of type I collagen and ratios of type I collagen to 18S mRNA were determined by Northern blot analysis, as described in MATERIALS AND METHODS. Bottom: results depict the ratios of type I collagen to 18S mRNA and are expressed as %change relative to Ctl, which was assigned a value of 100%. Bars represent means ± SE of 3 different experiments. *Significant differences in the ratios from Ctl cells (P < 0.05); **significant differences in the ratios from PTHrP-only-treated cells (P < 0.05).

Effects of a Galpha s mutant on indexes of osteoblast differentiation. To confirm results obtained regarding PTHrP signaling via Galpha s in PTHrP-mediated osteoblast differentiation, MG-63 cells were transiently transfected with a Galpha s triple mutant. The Galpha s mutant used in our studies is designed to stabilize a receptor-Galpha s-beta gamma -complex, effectively blocking signaling from both Galpha s and beta gamma . After transient transfection, MG-63 cells were treated with 100 nM PTHrP-(1-34) for 8 h. As seen in Fig. 5, in cells transfected with the Galpha s mutant, PTHrP stimulation of type I collagen mRNA levels was significantly reduced, confirming the involvement of Galpha s in PTHrP-stimulated osteoblast differentiation and suggesting the possible involvement of beta gamma -subunits.


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Fig. 5.   Effect of expressing a dominant negative Galpha s triple mutant (Galpha TM) on PTHrP-induced type I collagen (1 collagen) gene expression in MG-63 cells. Top: MG-63 cells were grown in 10% serum to semiconfluence. Cells were then cultured in serum-free medium or transiently transfected for 12 h with the empty vector (pcDNA1) or with vector containing Galpha TM. Cells were then cultured for 24 h in serum-free medium. Untransfected and transfected cells were then treated with 100 nM PTHrP-(1-34) or vehicle for 8 h. After stimulation with 100 nM PTHrP-(1-34), total cellular RNA was extracted from untreated control and experimental cells. Twenty micrograms of total cellular RNA were electrophoresed onto a 1.1% agarose-formaldehyde gel. After transfer of RNA, filters were probed with a 32P-labeled type I collagen cDNA or with a 32P-labeled 18S RNA probe, as described in MATERIALS AND METHODS. Bottom: results depict the ratios of type I collagen to 18S mRNA and are expressed as %change relative to Ctl, which was assigned a value of 100%. Bars represent means ± SE of 3 different experiments. *Significant differences in ratios from vehicle-treated cells (Ctl) (P < 0.05); **significant differences in ratios from PTHrP-treated cells (P < 0.05).

Effects of inhibiting PI 3-kinase on indexes of osteoblast differentiation. To explore signal transduction components related to the beta gamma -complex that might be implicated in PTHrP-induced osteoblast differentiation, we examined the involvement of PI 3-kinase by using the chemical inhibitor wortmannin. In preliminary experiments, wortmannin was confirmed to have no effects on cellular viability by trypan blue dye exclusion (>97%) or morphology (Fig. 3) at the doses indicated. Cells were pretreated with wortmannin for 3 h, followed by coincubation with 100 nM PTHrP-(1-34). Type I collagen mRNA levels were minimally affected in cells treated with wortmannin alone. The ability of wortmannin to cause a dose-dependent decrease in PTHrP-stimulated type I collagen levels (Fig. 6) suggests that PI 3-kinase is a component of the PTHrP-induced signaling involved in MG-63 cell differentiation. This was confirmed by the ability of wortmannin to abrogate the PTHrP-mediated increase in alkaline phosphatase activity after 24 h of coincubation (Fig. 3). Alkaline phosphatase activity in the wortmannin-treated cells is comparable to basal, untreated cells (Fig. 4). Taken together, these results suggest the involvement of PI 3-kinase in PTHrP-mediated osteoblast differentiation.


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Fig. 6.   Effect of the PI 3K inhibitor Wm on type I collagen gene expression in MG-63 cells. Top: MG-63 cells were grown in 10% serum to 80% confluence and incubated overnight in serum-free conditions. Cells were then pretreated with vehicle or Wm (50 nM, 100 nM) for 3 h. Cells were then treated with 100 nM PTHrP-(1-34) for 8 h. Ctl represents results of treatment with vehicle only for both the pretreatment and treatment periods. Levels of type I collagen and ratios of type I collagen to 18S mRNA were determined by Northern blot analysis, as described in MATERIALS AND METHODS. Bottom: results depict the ratios of type I collagen to 18S mRNA and are expressed as %change relative to Ctl, which was assigned a value of 100%. Bars represent means ± SE of 3 different experiments. *Significant differences in the ratios from Ctl cells (P < 0.05). **significant differences in the ratios from PTHrP-only-treated cells (P < 0.05).

Effects of Ras inhibition on indexes of osteoblast differentiation. Ras proteins are a major point of convergence of numerous signal transduction pathways. Ras is required to be anchored to the plasma membrane to function and must undergo the posttranslational addition of a farnesyl group, which facilitates Ras insertion into the plasma membrane (28, 36). We therefore examined the effects of B1086, which is an inhibitor of the enzyme farnesyltransferase and therefore inhibits Ras activity. In preliminary experiments, B1086 was confirmed to have no effects of cellular viability as assessed by trypan blue dye exclusion (>97%) or morphology (Fig. 3) at the doses indicated. Overnight pretreatment of MG-63 cells with B1086 was followed by 8 h of coincubation with 100 nM PTHrP-(1-34). Total RNA was collected and subjected to Northern blot analysis. Type I collagen mRNA levels were significantly decreased in B1086-treated cells (Fig. 7), whereas type I collagen mRNA levels were little affected in cells treated with B1086 alone. Histochemical examination of alkaline phosphatase activity after identical treatment conditions also revealed a significant decrease in this parameter in B1086-treated cells (Fig. 3).


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Fig. 7.   Effect of the Ras inhibitor B1086 on type I collagen gene expression MG-63 cells. Top: MG-63 cells were grown in 10% serum to 80% confluence and then incubated overnight in serum-free conditions. Cells were pretreated with vehicle or Ras inhibitor B1086 (5 µM, 10 µM) for 16 h. The cells were then treated with 100 nM PTHrP for 8 h. Ctl represents results of treatment with vehicle only for both the pretreatment and treatment periods. Levels of type I collagen and ratios of type I collagen to 18S mRNA were determined by Northern blot analysis as described in MATERIALS AND METHODS. Bottom: results depict the ratios of type I collagen to 18S mRNA and are expressed as %change relative to Ctl, which was assigned a value of 100%. Bars represent means ± SE of 3 different experiments. *Significant differences in the ratios from Ctl cells (P < 0.05); **significant differences in the ratios from PTHrP-only-treated cells (P < 0.05).

Effects of MAPK inhibition on indexes of osteoblast differentiation. The MAPK family of serine/threonine kinases includes extracellular signal-regulated kinases (ERKs). Activation of the ERK group of MAP kinases may occur via Ras and may involve stimulation of the enzyme MEK, or MAPK kinase. MEK activates ERK MAPK directly and thus serves as a point of control in that selective inhibition by the chemical inhibitor of MEK, PD-98059 (2, 23), results in inhibition of MAPK. To determine the involvement of MAPK in the PTHrP-mediated induction of type I collagen expression, MG-63 cells were incubated overnight with PD-98059. PD-98059 was confirmed to have no effects of cellular viability by trypan blue dye exclusion (>97%) or morphology (Fig. 3) at the doses indicated. After pretreatment, cells were coincubated with 100 nM PTHrP for 8 h, and type I collagen levels were determined by Northern blot analysis. A dose-dependent decrease (Fig. 8) in PTHrP-stimulated type I collagen gene expression was observed, implicating MAPK in PTHrP-induced MG-63 differentiation. Cells treated with PD-98059 alone showed negligible effects on type I collagen mRNA levels, confirming the specificity of the response to PTHrP-induced increase of type I collagen mRNA. These results were confirmed by measurement of alkaline phosphatase activity in PD-98059-treated MG-63 cells (Fig. 3). Direct measurement of the effects of PTHrP on MAPK activity in MG-63 cells was also performed. PTHrP-(1-34) at 100 nM concentration induced an increase in MAPK activity as measured by in vitro kinase activity assay, as seen in Fig. 9. Peak levels of MBP phosphorylation by MAPK were observed at 15 min, with activity remaining significantly elevated at 20 and 25 min. By 30 min, levels of MBP phosphorylation had returned to basal, untreated levels.


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Fig. 8.   Effect of the MAPK kinase (MEK) inhibitor PD-98059 on type I collagen (a1 collagen) gene expression in MG-63 cells. Top: MG-63 cells were grown in 10% serum to 80% confluence and then incubated overnight in serum-free conditions. Cells were pretreated with vehicle or PD-98059 (50 µM, 100 µM) for 16 h, followed by PTHrP treatment for 8 h. Ctl represents results of treatment with vehicle only for both the pretreatment and treatment periods. Levels of type I collagen and ratios of type I collagen to 18S mRNA were determined by Northern blot analysis, as described in MATERIALS AND METHODS. Bottom: results depict the ratios of type I collagen to 18S mRNA and are expressed as %change relative to Ctl, which was assigned a value of 100%. Bars represent means ± SE of 3 different experiments. *Significant differences in the ratios from Ctl cells (P < 0.05); **significant differences in the ratios from PTHrP-only-treated cells (P < 0.05).



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Fig. 9.   Effect of the PTHrP-(1-34) on MAPK activity in MG-63 cells. Top: MG-63 cells were grown in 10% serum to 80% confluence and then incubated overnight in serum-free conditions. Cells were then treated with 100 nM PTHrP-(1-34) for timed intervals. After stimulation with PTHrP-(1-34), total cell lysates were collected from untreated control and from experimental cells. Between 200 and 500 µg of total cellular protein were immunoprecipitated with extracellular signal-regulated kinase-1 (ERK1) antibody. Immunoprecipitates were incubated in reconstituted kinase reaction buffer containing myelin basic protein (MBP) and [32P]ATP, as described in MATERIALS AND METHODS. Results represent means ± SE of 3 different experiments. *Significant differences from time 0 (P < 0.05).

We next investigated effects of the various inhibitors of upstream signaling on the ability of PTHrP to induce peak MAPK activity after 15 min of treatment (Fig. 10). All five inhibitors inhibited the ability of PTHrP to induce MAPK activity, thereby confirming the role of MAPK as a downstream target of these signaling pathways.


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Fig. 10.   Effect of inhibitors of signal transduction on MAPK activity levels. Top: MG-63 cells were grown in 10% serum to 80% confluence and then incubated overnight in serum-free conditions. Cells pretreated with vehicle or PKA inhibitor H-89 (30 µM) and PKC inhibitor Ch Cl (5.0 µM) for 1 h, PI 3K inhibitor Wm (100 nM) for 3 h, Ras inhibitor B1086 (5.0 µM), and MAPK inhibitor PD-98059 (100 µM) overnight. The cells were then treated with 100 nM PTHrP for 15 min. MAPK activity was then determined as described in MATERIALS AND METHODS. Bottom: results are expressed as %vehicle-only-treated cells (Ctl) and represent means ± SE of 3 different experiments. *Significant differences from Ctl (P < 0.05); **significant differences from PTHrP-only-treated cells (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies in our laboratory and others have demonstrated the capacity of PTH and PTHrP to affect the differentiation of a number of cell types, including osteoblasts. The current evidence indicates that PTHrP can act as an autocrine or paracrine growth and/or differentiation factor in a number of tissues (40-42). The existence of both PTHrP and the PTH/PTHrP receptor in bone and the ability of a variety of bone-derived cells to produce PTHrP both in culture and in vivo strongly suggest a local role in bone. Evidence for such a role was also provided by the effects of targeted disruption of the PTHrP gene in mice (19). Thus mice heterozygous for the null mutation displayed haploinsufficiency and decreased bone volume as the mice aged. Such observations, confirmed by targeted overexpression of the same gene (38), suggest that PTHrP may modulate the maturation and differentiation of osteoblasts.

Treatment of MG-63 cells with 100 nM PTHrP-(1-34) was seen to induce increased expression of the osteoblast differentiation markers type I collagen and alkaline phosphatase. Although production of type I collagen is not exclusive to the differentiating osteoblast but is also produced by fibroblastic cells, type I collagen is considered a useful osteoblast differentiation marker when expressed in an established sequence with other bone markers such as alkaline phosphatase (4). Both catabolic and anabolic effects of PTHrP and PTH may be observed in bone via the PTH/PTHrP receptor (8). Ishizuya et al. (14) have shown that these discrepancies appear to be a function of exposure time, such that intermittent exposure of cells to PTH-(1-34) of ~6 h will cause osteoblast differentiation. Treatment of MG-63 osteoblastic cells with PTHrP-(1-34) were consistent with these observations, in that levels of type I collagen mRNA were seen to rise at ~6 h, peak at 8 h, and begin to decline with extended exposure time. These results suggest that there may exist an inhibitory effect of prolonged exposure of osteoblasts to PTHrP. Subsequent experiments were therefore performed with 8-h incubation periods in keeping with these observations. The ability of the MG-63 cells to withstand prolonged exposure to each inhibitor was first verified by trypan blue dye exclusion viability experiments as well as by close observation for any adverse changes in cell morphology. It seems possible that receptor desensitization due to internalization did occur during this time interval, but this was not examined, and signaling events leading to eventual differentiation would appear to have been adequately initiated during the incubation times that were employed.

It is known that PTH/PTHrP receptor activation can lead to activation of multiple G proteins, namely Galpha q and Galpha s, with subsequent activation of phospholipase C (PLC) and adenylate cyclase (25), respectively. Stimulation of PLC will result in subsequent production of inositol 1,4,5-trisphosphate and diacylglycerol (1, 2, 39), leading to mobilization of calcium and PKC, respectively. Although there are varying reports regarding the involvement of PLC in osteoblast differentiation, we have seen that inhibition of PKC by treatment with chelerythrine chloride can block induction of type I collagen and alkaline phosphatase. Stimulation of the PTH/PTHrP receptor also results in activation of adenylate cyclase followed by a rapid increase in intracellular cAMP and subsequent activation of PKA. We show that the inhibition of adenylate cyclase, by treatment with H-89, results in an abrogation of the PTHrP-(1-34)-stimulated increase in type I collagen mRNA expression and also interrupts the production of the later marker of MG-63 cell differentiation, alkaline phosphatase.

Although activation of PKA and PKC is known to occur in minutes, the ability of these short-term signals to influence such a late-developing cellular phenomenon as differentiation is well documented in the literature. Previous studies by Tsai et al. (35) show that 100 nM PTHrP induces cAMP in UMR106 osteosarcoma cells in minutes, with subsequent occurrence of differentiation at later time points. In several studies investigating the effects of PTH on osteoblast activity, the PKC and PKA signaling pathways appear to be activated simultaneously and seem to cooperate in the anabolic effects of the shared receptor in bone cells. Cross talk between these two signal transduction systems may therefore occur in osteoblasts after PTH stimulation. Further studies to elucidate this possibility were also undertaken by combined inhibitor treatment of MG-63 cells, but these show additive, and not synergistic, effects (data not shown).

Although it has been well established that the PTH/PTHrP receptor can signal through both Galpha s and Galpha q (1), the question of PTHrP signal transduction via beta gamma -subunit-dependent pathways remains to be definitively answered. The ability of the transient transfection of a Galpha s mutant (29), which sequesters beta gamma -subunits, to abrogate the effects of PTHrP treatment on osteoblast differentiation markers may implicate beta gamma -signaling. However, because the dominant negative Galpha s (8) mutant utilized also inhibits signaling via the endogenous Galpha s subunit, the results obtained from our experiment may have simply confirmed results obtained by inhibition of adenylate cyclase by H-89. Nevertheless, data obtained by chemical inhibition of PI 3-kinase are consistent with a role for the beta gamma -subunits in mediating some of the effects of PTHrP. Further studies in which beta gamma -subunits are selectively inhibited will be required, but such studies are currently technically challenging.

PI 3-kinase is a heterodimeric cytosolic protein composed of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (9). PI 3-kinase is stimulated by both G protein-coupled receptors and receptor tyrosine kinases. Stephens et al. (31) first discovered that PI 3-kinase is specifically activated by purified beta gamma -subunits. The ability of the chemical inhibitor of PI 3-kinase, wortmannin, to cause a significant reduction in the expression of both osteoblast differentiation markers in our studies suggests that PI 3-kinase is involved in PTHrP-mediated MG-63 cell differentiation. Because PI 3-kinase inhibition is seen to decrease beta gamma -dependent MAPK activation, it is commonly presumed that beta gamma -subunits activate PI 3-kinase and thus initiate a cascade of events that leads to the phosphorylation of the protein Shc. Whether Rac or other GTP-exchange proteins are involved in PI 3-kinase-mediated pathways was not tested. Nevertheless, the interaction of beta gamma -subunits with SH2-containing proteins can be mediated by PI 3-kinase, such that Shc complexes to the SH2-containing adaptor protein Grb2. Grb2 can then stably associate with the Ras guanine nucleotide exchange factor Sos. Ras is anchored to the plasma membrane, where Sos can stimulate the exchange of GDP for GTP, thereby leading to Ras activation. We have found that chemical inhibition of Ras by treatment with B1086, a farnesyltransferase inhibitor, also diminished the effect of PTHrP-(1-34) on induction of both type I collagen levels and alkaline phosphatase levels.

One of the best-characterized downstream targets of Ras is the family of MAPKs (20). MAPKs are a family of serine/threonine kinases involved in the transduction of cellular signals to the nucleus. These proteins regulate a vast range of cellular processes including proliferation, differentiation, transformation, inflammation, apoptosis, and cytoskeletal rearrangement. Previous data also support the pivotal role of MAPKs in the induction of phenotypic indexes of osteoblast differentiation (34, 43). Upstream of MAPK, the activation of Ras will lead to the sequential activation of Raf1, a serine/threonine kinase, and MEK, whose substrates are the ERK1 and ERK2 of the MAPK family. It has also been shown that activation of MAPK can occur independently of Ras activation, presumably via direct activation of Raf or MEK by PKA (37) or PKC. Treatment of MG-63 cells with the MAPK inhibitor PD-98059 interrupted PTHrP stimulation of indexes of osteoblast differentiation, thereby suggesting that MAPK is involved in this process. The ability of PTHrP to induce MAPK activity in MG-63 osteoblastic cells complements the results of the studies on the effect of PD-98059 on PTHrP induction of markers of differentiation. Furthermore, the capacity of multiple inhibitors of upstream signaling to diminish MAPK activity assay seems to suggest that these multiple signaling pathways converge to some extent at MAPK. Whether other members of the MAPK family, such as p38K or c-Jun NH2-terminal protein kinase, are also involved in PTHrP signaling remains to be determined.

It is probable, then, that PTH/PTHrP activation in response to PTHrP initiates parallel signaling via Galpha s leading to PKA activation, via Galpha q leading to activation of PKC, and via beta gamma leading to activation of PI 3-kinase and Ras (Fig. 11). Convergence of these signals may then occur via activation of MAPK. Further study is now required to extend these observations to other osteoblastic cell models and to determine whether other kinases, such as the stress-activated kinases, play a role in PTHrP-mediated osteoblast differentiation. The elucidation of the signaling pathways involved in the effects of PTHrP on enhancing differentiation of osteoblastic cells clearly has important implications with regard to our understanding of the actions of this hormone in normal physiology and particularly with respect to its anabolic actions in bone.


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Fig. 11.   Schematic diagram of PTHrP receptor signaling involved in regulating differentiation in MG-63 osteoblastic cells. PLC, phospholipase C; IP3, inositol triphosphate; DAG, diacylglycerol; PIP2, phosphatidylinosotol phosphate-2.


    FOOTNOTES

Address for reprint requests and other correspondence: S. A. Rabbani, Calcium Research Laboratory, Royal Victoria Hospital, 687 Pine Ave. West, Rm. H4.72, Montreal, QC H3A 1A1 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.

Received 29 January 2001; accepted in final form 26 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abou-Samra, AB, Juppner H, Force T, Freeman MW, Kong XF, Schipani E, Urena P, Richards J, Bonventre JV, Potts JT, Jr, Kronenberg HM, and Segre GV. Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related-peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium. Proc Natl Acad Sci USA 89: 2732-2736, 1992[Abstract].

2.   Aklilu, F, Gladu J, Goltzman D, and Rabbani SA. Role of mitogen-activated protein kinases in the induction of parathyroid hormone-related protein. Cancer Res 60: 1753-1760, 2000[Abstract/Free Full Text].

3.   Araujo dos Santos, A, and Giestal de Araujo E. The effect of PKC activation on the survival of rat retinal ganglion cells in culture. Brain Res 853: 338-343, 2000[ISI][Medline].

4.   Aubin, JE, Liu F, Malaval L, and Gupta AK. Osteoblast and chondroblast differentiation. Bone 17: 77-83, 1995.

5.   Birnbaumer, L. Which G protein subunits are the active mediators of signal transduction? Trends Pharmacol Sci 8: 209-211, 1987[ISI].

6.   Burtis, WJ, Brady TG, Orloff JJ, Ersbak JB, Warrell RP, Jr, Olson BR, Wu TL, Mittnick ME, Broadus AE, and Stewart AF. Immunochemical characterization of circulating parathyroid hormone-related protein in patients with humoral hypercalcemia. N Engl J Med 322: 1106-1112, 1990[Abstract].

7.   Cai, YC, Ma L, Fan GH, Zhao J, Jiang LZ, and Pei G. Activation of N-methyl-D-aspartate receptor attenuates acute responsiveness of delta -opioid receptors. Mol Pharmacol 51: 583-587, 1997[Abstract/Free Full Text].

8.   Canalis, E, McCarthy TL, and Centrella M. Differential effects of continuous and transient treatment with parathyroid hormone-related peptide (PTHrP) on bone collagen synthesis. Endocrinology 126: 1806-1812, 1990[Abstract].

9.   Carter, AN, and Downes CP. Phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 88: 7908-7912, 1992[Abstract].

10.   Choi, HJ, Hyun MS, Jung GJ, Kim SS, and Hong SH. Tumor angiogenesis as a prognostic predictor in colorectal carcinoma with special reference to mode of metastasis and recurrence. Oncology 55: 575-581, 1998[ISI][Medline].

11.   Clapham, DEG Protein beta gamma subunits. Annu Rev Pharmacol Toxicol 37: 167-203, 1997[ISI][Medline].

12.   Guise, TA, Yoneda T, Yates AJ, and Mundy GR. The combined effect of tumor-produced parathyroid hormone-related protein in patients with humoral hypercalcemia of cancer. N Engl J Med 322: 1106-1112, 1993[Abstract].

13.   Iiri, T, Bell SM, Baranski TJ, Fujita T, and Bourne HR. A Gsa mutant designed to inhibit receptor signaling through Gs. Proc Natl Acad Sci USA 96: 499-504, 1999[Abstract/Free Full Text].

14.   Ishizuya, T, Yokose S, Hori M, Noda T, Suda T, Yoshiki S, and Yamaguchi A. Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells. J Clin Invest 99: 2961-2970, 1997[Abstract/Free Full Text].

15.   Juppner, H, Abou-Samra AB, Freeman M, Kong XF, Schipani E, Richards J, Kolakowski LF, Jr, Hock J, Potts JT, Jr, Kronenberg HM, and Segre GV. A G protein-linked receptor for parathyroid hormone, and parathyroid hormone-related peptide. Science 254: 1024-1026, 1991[ISI][Medline].

16.   Kaiser, SM, Laneuville P, Bernier SM, Rhim JS, Kremer R, and Goltzman D. Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide. J Biol Chem 267: 13623-13628, 1992[Abstract/Free Full Text].

17.   Kaiser, SM, Sebag M, Rhim JS, Kremer R, and Goltzman D. Antisense-mediated inhibition of parathyroid hormone-related peptide production in a keratinocyte cell line impedes differentiation. Mol Endocrinol 8: 139-147, 1994[Abstract].

18.   Kano, J, Sugimoto T, Fukase M, and Chihara K. The direct involvement of cAMP-dependent protein kinase in the regulation of collagen synthesis by parathyroid hormone (PTH) and parathyroid hormone-related peptide in osteoblast-like osteosarcoma cells (UMR-106). Biochem Biophys Res Commun 184: 525-529, 1992[ISI][Medline].

19.   Karaplis, AC, Luz A, Glowacki J, Bronson RT, Tybulewicz VL, Kronenberg HM, and Mulligan RC. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev 8: 277-289, 1994[Abstract].

20.   Khosravi-Far, R, White MA, Westwick JK, Solski PA, Chrzanowska-Wodnicka M, Van Aelst L, Wigler MH, and Der CK. Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation. Mol Cell Biol 16: 3923-3933, 1996[Abstract].

21.   Logothetis, DE, Kurachi Y, Galper J, Neer EJ, and Clapman DE. The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature 325: 321-326, 1987[ISI][Medline].

22.   Lopez-Ilasca, M. Signaling from G-protein-coupled receptors to mitogen-activated protein (MAP)-kinase cascades. Biochem Pharmacol 56: 269-277, 1998[ISI][Medline].

23.   Mayer, AM, Brenic S, and Glaser KB. Pharmacological targeting of signaling pathways in protein kinase C-stimulated superoxide generation in neutrophil-like HL-60 cells: effect of phorbol ester, arachidonic acid and inhibitors of kinase(s), phosphatase(s) and phospholipase A2. J Pharmacol Exp Ther 279: 633-644, 1996[Abstract].

24.   McCauley, LK, Koh AJ, Beecher CA, Cui Y, Rosol TJ, and Franceschi RT. PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis. J Cell Biochem 61: 638-647, 1996[ISI][Medline].

25.   Morris, AJ, and Malbon CC. Physiological regulation of G protein-linked signaling. Physiol Rev 79: 1373-1430, 1999[Abstract/Free Full Text].

26.   Moseley, JM, Kubota M, Diefenbach-Jagger H, Wettenhall RE, Kemp BE, Suva LJ, Rodda CP, Ebeling PR, Hudson PJ, Zajac JD, and Martin TJ. Parathyroid hormone-related protein purified from a lung cancer cell line. Proc Natl Acad Sci USA 84: 5048-5052, 1987[Abstract].

27.   Partridge, NC, Bloch SR, and Pearman AT. Signal transduction pathways mediating parathyroid hormone regulation of osteoblast gene expression. J Cell Biochem 55: 321-327, 1994[ISI][Medline].

28.   Schlessinger, J, and Ullrich A. Growth factor signaling by receptor tyrosine kinases. Neuron 9: 383-391, 1992[ISI][Medline].

29.   Skoglund, G, Mehboob HA, and Holz GG. Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin gene cAMP response element. Diabetes 49: 1156-1164, 2000[Abstract].

30.   Soifer, NE, Van Why SK, Ganz MB, Kashgarian M, Siegel NJ, and Stewart AF. Expression of parathyroid hormone-related protein in the rat glomerulus and tubule during recovery from renal ischemia. J Clin Invest 92: 2850-2857, 1993[ISI][Medline].

31.   Stephens, L, Smrcka A, Cooke FT, Jackson TR, Sternweis PC, and Hawkins PT. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits. Cell 77: 83-93, 1994[ISI][Medline].

32.   Strewler, GJ, Stern PH, Jacobs JW, Eveloff J, Klein RF, Leung SC, Rosenblatt M, and Nissenson RA. Parathyroid hormone-like protein from human renal carcinoma cells: structural and functional homology with parathyroid hormone. J Clin Invest 80: 1803-1807, 1987[ISI][Medline].

33.   Sugden, PH, and Clerk A. Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors. Cell Signal 9: 337-351, 1997[ISI][Medline].

34.   Takeuchi, Y, Suzawa M, Kikuchi T, Nishida E, Fujita T, and Matsumoto T. Differentiation and transforming growth factor-1beta receptor down-regulation by collagen 11alpha 211beta integrin interaction is mediated by focal adhesion kinase and its downstream murine osteoblastic cells. J Biol Chem 272: 29309-29316, 1997[Abstract/Free Full Text].

35.   Tsai, JA, Bucht E, Stark A, Sjostedt U, and Torring O. Parathyroid hormone-related protein (1-37) induces cAMP response in human osteoblast-like cells. Calcif Tissue Int 62: 250-254, 1998[ISI][Medline].

36.   Ullrich, A, and Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell 61: 203-212, 1990[ISI][Medline].

37.   Verheijen, MHG, and Defize LHK Parathyroid hormone activates mitogen-activated protein kinase via a cAMP-mediated pathway independent of Ras. J Biol Chem 272: 3423-3429, 1997[Abstract/Free Full Text].

38.   Weir, EC, Philbrick WM, Amling M, Neff LA, Baron R, and Broadus AE. Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation. Proc Natl Acad Sci USA 93: 10240-10245, 1996[Abstract/Free Full Text].

39.   Wu, S, Pirola CJ, Green J, Yamaguchi DT, Okano K, Juppner H, Forrester JS, Fagin JA, and Clemons TL. Effects of N-terminal, midregion, and C-terminal parathyroid hormone-related peptides on adenosine 3',5'-monophosphate and cytoplasmic free calcium in rat aortic smooth muscle cells and UMR-106 osteoblast-like cells. Endocrinology 133: 2437-2444, 1993[Abstract].

40.   Wysolmerski, JJ, Broadus AE, Zhou J, Fuchs E, Milstone LM, and Philbrick WM. Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development. Proc Natl Acad Sci USA 91: 1133-1137, 1994[Abstract].

41.   Wysolmerski, JJ, McCaughern-Carucci JF, Daifotis AG, Broadus AE, and Philbrick WM. Overexpression of parathyroid hormone-related protein or parathyroid hormone in transgenic mice impairs branching morphogenesis during mammary gland development. Development 121: 3539-3547, 1995[Abstract/Free Full Text].

42.   Wysolmerski, JJ, and Stewart AF. The physiology of parathyroid hormone-related protein: an emerging role as a developmental factor. Annu Rev Physiol 60: 431-460, 1998[ISI][Medline].

43.   Xiao, G, Jiang D, Thomas P, Benson MD, Guan K, Karsenty G, and Franceschi RT. MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. J Biol Chem 275: 4453-4459, 2000[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 281(3):E489-E499
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