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Endocrinology Vol. 143, No. 5 1880-1888
Copyright © 2002 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

Parathyroid Hormone Inhibits c-Jun N-Terminal Kinase Activity in Rat Osteoblastic Cells by a Protein Kinase A-Dependent Pathway

Teresa A. Doggett, John T. Swarthout, Stephen C. Jefcoat, Jr., Dagmar Wilhelm1, Andreas Dieckmann, Peter Angel and Nicola C. Partridge

Cell and Molecular Biology Program (J.T.S.) and Department of Pharmacological and Physiological Science (T.A.D., J.T.S.), Saint Louis University School of Medicine, St. Louis, Missouri 63104; Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School (S.C.J., N.C.P.), Piscataway, New Jersey 08854; and German Cancer Research Center, Division of Signal Transduction and Growth Control (D.W., A.D., P.A.), D-69120 Heidelberg, Germany

Address all correspondence and requests for reprints to: Dr. Nicola C. Partridge, Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854. E-mail: . partrinc{at}umdnj.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment of osteoblastic cells with PTH initiates dual signaling cascades resulting in activation of both PKA and PKC. It has been shown that PTH either inhibits or stimulates ERKs depending on dose of the hormone; nevertheless, the ability of PTH to regulate other members of the MAPK family is unknown. Another member of this family, c-Jun-NH2-terminal kinase (JNK), is preferentially activated by cytokines and cellular stresses and plays a key role in regulating the activity of various transcription factors. We demonstrate that treatment of UMR 106-01 cells and rat calvarial osteoblasts with PTH (10-8 M), N-terminal peptides of PTH that selectively activate PKA, or 8-bromo-cAMP (activates PKA) results in the inhibition of JNK activity from high basal levels. Examination of the upstream members of the JNK cascade revealed that both stress-activated protein kinase/extracellular signal-related kinase kinase 1/MAPK kinase 4 and MAPK/extracellular signal-related kinase kinase kinase 1 activities were also inhibited after treatment with PTH (10-8 M). We conclude that treatment of osteoblastic cells with PTH is sufficient to inhibit high basal JNK activity by activation of the PKA signaling cascade.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH IS AN essential regulator of calcium homeostasis (1) and has both anabolic and catabolic effects in vivo on bone and in vitro on primary and clonal osteoblastic cells (1, 2). This dichotomy of function may result from PTH’s ability to activate 2 different signal transduction pathways in osteoblasts as a result of ligand binding to the heterotrimeric G protein-coupled PTH/PTHrP receptor (PTH1R) (3). Stimulation of Gs results in activation of adenylate cyclase and, consequently, cAMP-dependent PKA (4, 5), whereas stimulation of Gq leads to activation of PLC, culminating in increased intracellular levels of calcium and activation of PKC (6, 7). The first 34 amino acids of PTH are necessary and sufficient for full biological activity of the intact hormone (8). Furthermore, studies with N-terminal peptides of PTH demonstrate a requirement for the first 2 amino acids in the activation of adenylate cyclase (9, 10), whereas amino acids 29–32 are sufficient for PLC stimulation (11), and both N-terminal amino acids (3 and 13) and residues 25–34 of PTH-(1–34) encompass the principal region for receptor binding (12). Although these signaling pathways mediate the alterations of gene expression and proliferation in osteoblastic cells, the mechanism(s) involved is still poorly understood.

PTH mediates its biological effect in osteoblastic cells after binding to the PTH1R (3, 10, 13, 14). Although the immediate signaling pathways are known, the downstream mediators of these signaling cascades are unclear. Recent studies have shown that a variety of extracellular signals acting through G protein-coupled receptors culminate in regulation of the MAPK cascades (15, 16). Three subfamilies of MAPKs have been identified in mammalian cells; the ERK, the stress-activated protein kinase/c-Jun NH2-terminal kinases (JNK), and the p38 MAPKs. Recently, the ability of PTH to regulate ERKs has been established. Treatment of osteoblastic cells with high concentrations of PTH resulted in decreased ERK activity stimulated by basic fibroblast growth factor and platelet-derived growth factor-BB (17) or by epidermal growth factor (18). Conversely, although high concentrations of PTH activated ERKs in CHO-R15 (19), lower concentrations of PTH activated ERKs in rat osteoblastic cells (20) and OK cells (21). Although the regulation of ERKs is becoming increasingly clear, the ability of PTH to regulate other members of the MAPK family is unknown.

JNK is activated by dual phosphorylation on threonine and tyrosine residues by the dual specificity protein kinase stress-activated protein kinase/extracellular signal-related kinase kinase 1/MAPK kinase 4 (SEK1/MKK4) (22, 23) following phosphorylation and activation of SEK1 by MAPK/extracellular signal-related kinase kinase kinase 1 (MEKK1) (23). Activation of MEKK1 is believed to be mediated by various small G proteins, including Ras, Rac1, and Rho (24). JNKs are activated upon exposure of cells to environmental stresses and cytokines (25, 26) and are key regulators of transcription factors, including activating transcription factor-2, c-Jun, p53, and Elk-1, resulting in regulation of growth, differentiation, and cell apoptosis (27, 28). Phosphorylation and activation of c-Jun have been implicated in the initiation of apoptosis in response to various stresses, including removal of growth factors and the excitatory amino acid kainate (27, 29). Here we demonstrate that treatment of osteoblastic cells with PTH-(1–34) results in inhibition of JNK activity without affecting JNK protein levels. This inhibition of JNK activity by PTH is mediated by activation of the cAMP-dependent kinase PKA and is PKC independent. PTH treatment also results in inhibition of upstream members of the JNK cascade, including SEK1/MKK4 and MEKK1, but not p21-activated kinase-1 (PAK1).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), unless otherwise specified. Rat PTH [rPTH-(1–34)] and 8-bromo-cAMP (8Br-cAMP) were purchased from Sigma (St. Louis, MO). Isobutylmethylxanthine and phorbol myristate acetate (PMA) were obtained from Calbiochem (La Jolla, CA). Enhanced chemiluminescence reagents were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). NEN Life Science Products (Boston, MA) supplied [{gamma}-32P]ATP. Polyvinylidene difluoride membrane (Immobilon) was obtained from Millipore Corp. (Bedford, MA). Synthetic PTH peptides [-(1–31), -(3–34), -(13–34), and -(28–48)] were purchased from Bachem (King of Prussia, PA). Tissue culture media and reagents were obtained from the Washington University Tissue Culture Center (St. Louis, MO). FBS was a product of JRH Biosciences (Lenexa, KS). Protein levels in cell lysates were quantified using the Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay kit with BSA as standard and measuring the absorbance (595 nm) of samples with a spectrophotometer. All other chemicals were obtained from Sigma.

Cell culture
Osteoblastic cells were cultured as previously described (30) in Eagle’s MEM supplemented with nonessential amino acids, 25 mM HEPES (pH 7.3), penicillin (100 U/ml), and streptomycin (100 µg/ml), and 5% FBS for UMR 106-01 cells or 10% FBS for primary osteoblasts. For serum starvation, osteoblastic cells were allowed to grow to 70–80% confluence and then switched to Eagle’s MEM containing 0% FBS for 48 h unless stated otherwise. Primary osteoblasts were isolated from rat calvaria as previously described (31) and seeded at 6.24 x 103 cells/cm2.

Immunoblotting
Serum-starved osteoblastic cells were treated in the presence and absence of appropriate agents for the indicated time periods at 37 C. Medium was aspirated, and cells were washed twice in cold PBS and then lysed with lysis buffer A [10 mM Tris-HCl (pH 7.0), 30 mM sodium pyrophosphate, 0.1 mM sodium vanadate, 0.2 mM iodoacetic acid, 50 mM sodium chloride, 50 mM sodium fluoride, 5 mM zinc chloride, 1% Triton X-100, 10 mM dithiothreitol (DTT), 100 mM phenylmethylsulfonylfluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml pepstatin]. Monolayers were scraped into 1.5-ml Eppendorf tubes, and the lysates were cleared by centrifugation (12,000 x g, 10 min, 4 C). The protein contents of the supernatants were determined using the Bradford reagent from Bio-Rad Laboratories, Inc., at 595 nm.

Total protein (50 µg) for each sample was boiled for 5 min in SDS sample buffer [63 mM Tris buffer (pH 6.8), 1% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 0.1% bromophenol blue], and SDS-PAGE was performed with 6% and 12% stacking and resolving gels, respectively. Proteins were transferred electrophoretically to polyvinylidene difluoride membrane at 100 V for 1 h. Membranes were placed in blocking buffer [5% powdered milk in 20 mM Tris-HCl-buffered saline with 0.01% Tween 20 (TBST), pH 7.2] for 1 h. Membranes were then incubated with the appropriate antibody diluted with 5% milk in TBST for 16 h at 4 C, washed three times with Tris-HCl-buffered saline, and incubated for 2 h with goat antirabbit peroxidase-labeled antibody diluted 1:5000 in 5% milk in TBST. Labeled bands were visualized with enhanced chemiluminescence reagent (Amersham Pharmacia Biotech), and membranes were exposed to Kodak film (Rochester, NY). In some instances 25–50 µg whole cell lysate were used and transferred directly to polyvinylidene difluoride membranes after SDS-PAGE for Western blot analysis.

In vitro kinase assays
JNK activities were assayed using an in vitro JNK assay (32). JNKs were immunoprecipitated from cell lysates (200 µg) with 3 µg each of anti-JNK1 and anti-JNK2 in lysis buffer A for 1 h at 4 C on a rotating platform, protein A-agarose beads were added, and the samples were rocked for an additional hour. Immune complexes were washed twice with lysis buffer A and once with buffer B [20 mM Tris-HCl (pH 7.5), 10 mM DTT, 0.1 M NaCl, and 12 mM MgCl2], then incubated with 2 µg glutathione-S-transferase (GST)-c-Jun-(1–79) and 4 µCi [{gamma}-32P]ATP in buffer B (final volume, 25 µl) for 20 min at 30 C. The activity of MEKK1 was assayed using GST-SEK1 bound to agarose beads (PharMingen, San Diego, CA). MEKK1 was immunoprecipitated as described above, using 3 µg anti-MEKK1 antibody. Immune complexes were combined with 2 µg GST-SEK1 agarose and 4 µCi [{gamma}-32P]ATP in buffer B and incubated for 15 min at 30 C. SEK1 was immunoprecipitated, and activity was measured using 2 µg GST-JNK1 as substrate with 4 µCi [{gamma}-32P]ATP in buffer B and incubated for 20 min at 30 C. PAK1 activity was assayed by 32P incorporation into kinase-deficient MEKK1-(1–301) (Upstate Biotechnology, Inc., Lake Placid, NY) after immunoprecipitation of PAK1 (using 3.0 µg each of both C-20 and N-19 antibodies), as described above, and immune complexes were washed twice with buffer A and once with buffer C [50 mM HEPES (pH 7.5), 10 mM MnCl2, 10 mM MgCl2, 1 mM DTT, and 50 µM ATP]. Immunoprecipitates were incubated in buffer C containing 4 µg MEKK1-(1–301) and 4 µCi [{gamma}-32P]ATP for 20 min at 30 C. All reactions were terminated by the addition of 7.5 µl of 3x SDS sample buffer and boiling for 5 min. After centrifugation for 10 min at 4 C, each reaction was resolved on a 12.75% SDS-PAGE gel, fixed, dried, and exposed to x-ray film. Quantitation was performed with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Statistical analysis
Experimental significance was assessed using one-way ANOVA with post hoc analysis of multiple comparisons using the pairwise Dunnett’s method. A t test analysis was also performed to assess the significance of differences between individual treatment groups. All data shown are representative of at least three separate experiments with similar results. Asterisks were used on graphs to indicate significant differences in mean values (P <= 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time-course inhibition of JNK activity in osteoblastic cells
A complete time course was performed in both UMR 106-01 cells and normal rat osteoblasts to determine the optimal time for inhibition of JNK activity after treatment with PTH-(1–34). After 48 h of serum deprivation, cells were treated with 10-8 M PTH-(1–34), and cell lysates were collected every 15 min over a 60-min period. Western blot analysis measuring the relative levels of JNK1/JNK2 revealed that the relative levels of JNK protein are equal at all time points and that JNK1 protein levels are greater than the level of JNK2 protein (Fig. 1Go, A and B). Equal amounts of total protein from whole cell lysates were used to assess total JNK activity by immunoprecipitation of JNK and incubation with c-Jun in the presence of radiolabeled [{gamma}-32P]ATP. PTH treatment resulted in significant (P < 0.05) inhibition of JNK activity by 45 min in UMR 106-01 cells and by 30 min in primary osteoblasts (Fig. 1Go, A and B). Maximal inhibition (>50%) occurred at 45 min for UMR 106-01 cells and at 60 min for primary rat osteoblasts (Fig. 1Go, A and B), with JNK activity returning to basal levels 90 min after PTH treatment (data not shown).



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  		Figure 1. Inhibition of JNK activity by PTH is time dependent in rat osteoblastic cells. Osteoblastic cells were serum-starved for 48 h before treatment with 10-8 M PTH-(1–34) or control medium, and lysates were collected at the times shown during the 60-min treatment. JNKs were immunoprecipitated, and activity was assessed with in vitro kinase assays using GST-c-Jun as the substrate. One representative in vitro kinase assay is shown for each cell type. Western blots are shown of whole cell lysates probed with anti-JNK1 and anti-JNK2 antibodies to assess the relative abundance of either kinase as the result of PTH treatment. The graphed data show the mean and SEM of three individual experiments, expressed as the percent JNK activity in PTH-treated lysates in relation to the activity in vehicle-treated lysates for each time point. The asterisks indicate that the PTH-treated sample is significantly different (P < 0.05) from the vehicle-treated control at that time point.

 
Inhibition of JNK activity by PTH in osteoblastic cells is dose dependent
Having determined the maximal time of inhibition of JNK by PTH, we tested whether this inhibition was dose dependent. Osteoblastic cells were treated for 45 min with increasing concentrations of PTH ranging from 10-12–10-8 M. Maximal inhibition of JNK activity occurred at concentrations of PTH higher than 10-10 M (Fig. 2Go, B and D). Interestingly, treatment with lower concentrations of PTH (10-12 M in primary osteoblasts, 10-115 M in UMR cells) resulted in a modest, yet significant, increase in JNK activity (Fig. 2Go, B and D). The difference in JNK activity was not due to alteration in the level of JNK expression as determined by Western blot analysis (Fig. 2Go, A and C).



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  		Figure 2. Dose-dependent inhibition of JNK activity in UMR 106-01 and normal rat osteoblastic cell lysates. Cells were serum-deprived for 48 h and treated with increasing concentrations of PTH-(1–34) for 45 min. A and C, Representative results of the in vitro kinase assay using GST-c-Jun as substrate. Western blots of whole cell lysates probed with anti-JNK1 and JNK2 antibodies were performed to show relative levels of JNK protein present in each lysate. B and D, Inhibition of JNK activity in lysates from UMR 106-01 cells and primary rat osteoblasts after treatment with increasing concentrations of PTH. Values are presented as the fold difference in JNK activity from the zero control and represent data from three separate experiments ± SEM. Asterisks indicate a significant difference (P < 0.05) from the control samples in each experiment.

 
Inhibition of JNKs in osteoblastic cells is PKA dependent
PTH leads to activation of both PKA and PKC (2); therefore, to determine whether inhibition of JNK activity by PTH in UMR 106-01 cells and normal rat osteoblastic cells was due to PKA or PKC activation, osteoblastic cells were treated for 45 min with PTH peptides that selectively activate PKA or PKC. Treatment of osteoblastic cells with either PTH-(1–34) or PTH-(1–31) resulted in inhibition of JNK activity (Fig. 3Go, A–C). Treatment with PTH-(1–34) (10-8 M) resulted in significant (P = 0.001) inhibition of JNKs (Fig. 3DGo). However, peptides lacking the first two N-terminal amino acids failed to inhibit JNK activity in either UMR 106-01 cells or normal rat osteoblastic cells (Fig. 3Go, A–C). Western blot analysis showed that inhibition was not due to differences in levels of JNK expression in PTH peptide-treated cells (Fig. 3Go, A and B).



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  		Figure 3. Inhibition of JNK activity in UMR 106-01 and normal rat osteoblastic cells is PKA dependent. Cells were serum-deprived for 48 h, then treated for 45 min with PTH peptides (10-8 M), and JNK assays were performed. A and B, Representative results obtained from in vitro kinase assays. Western blots of whole cell lysates probed with anti-JNK1 and anti-JNK2 antibodies were performed to assess levels of JNK protein in each lysate. C, Inhibition of JNK activity in lysates from UMR 106-01 cells after treatment with N-terminal PTH peptides. The results are presented as the fold inhibition of JNK activity from control and represent data from three separate experiments ± SEM. Asterisks indicate activities that were significantly different (P < 0.05) from the control in each experiment. D, Cells were serum-deprived for 48 h, then treated with 10-8 M PTH-(1–34), 8Br-cAMP (5 mM), PMA (10-6 M) for 45 min, and JNK assays were performed.

 
To confirm our observations that PKA activation was important for inhibition of JNK activity by PTH, we compared the inhibition elicited by treatment with 10-8 M PTH-(1–34), 8Br-cAMP (5 mM), and PMA (10-6 M). In UMR 106-01 cells, inhibition of JNK activity was observed after treatment with PTH-(1–34) and the cAMP analog 8Br-cAMP (Fig. 3DGo) with similar results observed in normal rat osteoblastic cells (data not shown). PMA treatment of either cell type did not result in inhibition of JNK activity. Similarly, PTH-(3–34), which has been shown to raise intracellular calcium levels (33), did not inhibit JNK activity. This together with the results of treatment with other PTH peptides indicate that PTH inhibition of JNK activity is probably dependent upon cAMP.

PTH inhibition of upstream members of the JNK cascade
PTH inhibits JNKs in a dose- and time-dependent manner. Furthermore, this inhibition occurs after activation of PKA by PTH. However, it is unknown whether inhibition of JNK by PTH is a direct effect of PKA, or whether PTH regulates the activity of upstream members of the JNK cascade. To address the effects of PTH on upstream activating kinases, we performed in vitro kinase assays for SEK1 and MEKK1 activity in lysates from UMR 106-01 cells treated with 10-8 M PTH-(1–34) over a 60-min time course. Treatment of UMR 106-01 cells with 10-8 M PTH-(1–34) resulted in an inhibition of SEK1 and MEKK1 activity, as demonstrated by phosphorylation of GST-JNK1 and GST-SEK1, respectively (Fig. 4Go). The inhibition of SEK1 and MEKK1 followed a time course similar to that observed with the in vitro JNK assay (Fig. 1Go), with significant inhibition (~40%) occurring 45 min after PTH treatment for both SEK1 (P = 0.003) and MEKK1 (P = 0.02). The inhibition was only significant for SEK1 activities (10% decrease) after 30 min of PTH treatment, possibly reflecting the amplification of the effect from one level to the next. Western blots of UMR whole cell lysates revealed that the overall levels of SEK1 and MEKK1 did not change over the 60-min time course.



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  		Figure 4. PTH-(1–34) treatment of UMR 106-01 cells results in the inhibition of the upstream kinases SEK1 and MEKK1. UMR cells were treated with 10-8 M PTH-(1–34) or vehicle, and cell lysates were collected every 15 min over a 60-min period. A, Representative kinase assays with phosphorylated substrates shown [GST-JNK1 for SEK1, GST-SEK1 for MEKK1, MEKK1-(1–301) for PAK1]. Western blots for the enzymes are also shown. B, Graphs showing percent difference in SEK1 and MEKK1 activities in PTH-treated lysates compared with the untreated control lysates. The results are shown as the percent change in enzyme activity from the control value at each time point after PTH treatment and represent data from three separate experiments ± SEM. Asterisks indicate activities significantly different (P < 0.05) from their respective untreated controls at each time point.

 
Although both SEK1 and MEKK1 had been reported to be upstream members of the JNK cascade, there were few reports on the nature of the kinases located upstream of MEKK1. One possible kinase is PAK1, which is known to be able to directly phosphorylate MEKK1 (34). We therefore examined whether PTH-(1–34) treatment of UMR 106-01 cells resulted in inhibition of PAK1 activity. Interestingly, the activity of PAK1 in PTH-treated UMR 106-01 cells was not repressed but, rather, was increased over the 60-min time course and was maximal 45 min (2.5-fold increase) after PTH treatment (Fig. 4Go), ruling out the possibility that PKA-induced repression of JNK is mediated by down-modulation of PAK1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PTH has been shown to induce changes in osteoblastic gene expression, including decreased mRNA levels for both collagen and osteopontin, with a concomitant increase in collagenase-3 expression (2). Changes in gene expression are a direct result of PTH’s ability to activate two different signal transduction pathways in the osteoblast as a result of ligand binding to the heterotrimeric G protein-coupled PTH1R (3). Stimulation of Gs results in activation of adenylate cyclase and, consequently, PKA (4, 5), whereas stimulation of Gq leads to activation of PLC, culminating in increased intracellular levels of calcium and activation of PKC (6, 7). The activation of PKA by PTH is a prerequisite for the expression of members of the activator protein-1 (AP-1) family (c-Jun and c-Fos) of transcription factors (2). The presence of AP-1-binding sites in a multitude of osteoblastic genes, including collagenase-3, suggests that Fos/Jun heterodimers may play an important role in mediating the effects of osteogenic stimuli (35, 36, 37). However, little is known of PTH’s ability to mediate the activation of AP-1 transcription factors. Although JNKs are known to phosphorylate the amino-terminal trans-activation domain of c-Jun (38), the consequences of PTH-induced signaling on JNK activity is unknown. A related subclass of the MAPK family, the ERKs, was found to be activated by PTH in osteoblastic and kidney cells (17, 18, 19, 20, 21). This regulation is a consequence of both PKA and PKC activation (20, 21, 39), and in UMR 106-01 cells low concentrations of PTH result in a PKC-dependent increase in ERK activation and cellular proliferation (20). This combined evidence lead to the present study, in which we examined the ability of PTH to regulate the stress-activated protein kinase/JNK pathway.

Although activation of JNKs was anticipated, we provide evidence that treatment of both UMR 106-01 and normal rat osteoblastic cells with high doses of PTH-(1–34) results in the marked inhibition of JNK activity (Fig. 2Go). These same conditions were previously shown to activate transcription of c-Jun and collagenase-3 (35, 36, 40) and may indicate that although PTH increases the transcription of c-Jun, it may not be involved in the activation of this transcription factor. This is supported by the very inefficient hyperphosphorylation of c-Jun in PTH-treated cells (37). The ability of N-terminal PTH peptides PTH-(1–34) and PTH-(1–31) (activates PKA) or the cAMP analog 8Br-cAMP to inhibit JNK activity demonstrated PKA activation to be essential for this effect (Fig. 3Go). Furthermore, activation of PKA as a prerequisite to JNK inhibition was supported by the time of maximal inhibition (Fig. 1Go). Although activation of PKA by PTH in osteoblastic cells occurs in minutes (4, 5), inhibition of JNK activities occurs around 30 min. JNK is activated by dual phosphorylation on threonine and tyrosine residues by the dual specificity protein kinase SEK1/MKK4 (22, 23) after phosphorylation and activation of SEK1 by MEKK1 (23). Addition of PTH resulted in significant inhibition of SEK1 as early as 30 min and of MEKK1 by 45 min after treatment (Fig. 4Go) consistent with SEK1 and MEKK1 being upstream mediators of JNK activation. Additionally, activation of MEKK1 is mediated by various small G proteins, including Ras, Rac1, and Rho (41); however, transfection of osteoblastic cells with dominant negative RasN17 or Rho (19) had no effect on JNK basal levels or the ability of PTH to regulate JNK activity (data not shown). Two members of this family, Cdc42 and Rac1, are particularly important in the regulation of new protrusions and adhesions at the periphery of the cell. Both of these small G proteins have been implicated in the JNK cascade, acting through PAKs (42). Although we can rule out Rho and Ras, we have not investigated the role of Cdc42 or Rac in JNK inhibition by PTH. However, we did examine the effector of these small G proteins, the serine/threonine kinase PAK1 (42). It has been demonstrated that PAK1 is able to phosphorylate the N-terminal of MEKK1-(1–301) (43), and it has been proposed that the direct interaction of MEKK1 with PAK1 and Rac/Cdc42 is necessary for phosphorylation and the subsequent activation of the JNK pathway (43). There is a disparity in the activation of each of these kinases. PTH treatment of UMR 106-01 cells resulted in an increase in PAK1 activity and a transient inhibition of MEKK1 and SEK1 activity (Fig. 4Go). The easiest explanation is that although PAK1 is a substrate for Rac, it is only one of more than a dozen (34), and its role as a physiological regulator of JNK is unresolved (34, 44). Therefore, although JNK activity is elevated as a consequence of MEKK1 activation, the events upstream of MEKK1 are unknown.

Compared with other types of cells that are commonly used for the analysis of signaling pathways, such as HeLa, HEK-293, and fibroblasts, the level of JNK activity present in serum-starved osteoblastic cells was relatively high. Moreover, JNK activity is high in both transformed and normal osteoblastic cells, suggesting that this is a mechanism common to both cell types and not simply a consequence of being transformed. It is known that the JNK subclass of MAPKs is activated in response to UV irradiation, cytokines, and certain mitogens (38); however, none of these was employed in our system. Previous reports demonstrated removal of tropic factors was sufficient to activate JNKs and to induce apoptosis in numerous cells (45, 46, 47). As our experiments were performed after removal of serum, we tested this as a mechanism of JNK activation. JNK activity was elevated in cells regardless of culture with or without serum, and PTH could inhibit JNKs even in the presence of serum (data not shown). JNKs are also activated after stretching of cardiac fibroblasts as a consequence of growth on extracellular matrix proteins (48, 49) or by imparted mechanical stress, as observed in osteoblastic periodontal ligament cells (50). Cultured osteoblastic cells are elongated and stellate in appearance with multiple processes when grown at low density (51). Furthermore, there is a strong correlation between cell density and polymerization of cytoskeletal actin and myosin microfilaments in osteoblastic cells (52). As our cells are plated at low densities, JNK activities may be elevated basally by signals originating from adhesion sites. Increases in cAMP lead to a decrease in cytoskeletal actin and myosin filaments (52). Therefore, inhibition of JNKs by PTH may be a consequence of enhanced cAMP levels.

In summary, we have demonstrated that treatment of osteoblastic cells with high doses of PTH results in the inhibition of JNK activity, that it is cAMP dependent, and that the activity of upstream members of the JNK pathway, namely MEKK1 and SEK1, are also inhibited, indicating that PKA negatively regulates these upstream members of the JNK pathway (Fig. 5Go). Whether PTH inhibition of JNK is a consequence of cAMP-mediated cytoskeletal rearrangements or is a direct effect on regulation of the activation of Rho family guanosine triphosphatases and/or Rac guanidine exchange factors is at present unclear and may be worth further pursuit.



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  		Figure 5. Model of the regulatory levels in the JNK cascade affected by PTH/PKA signaling in the osteoblast. PTH (10-8 M) activation of its receptor and release of the {alpha}-subunit of the heterotrimeric G protein ({alpha}s) lead to the activation of adenylyl cyclase (AC) and an increase in intracellular cAMP concentrations. The increase in cAMP causes release of the regulatory subunits (R) from the catalytic subunits (C) of PKA, allowing it to carry out functions that lead to inhibition of the JAK signaling cascade at the MEKK1 level, resulting in an amplified decrease in SEK1 and JNK activities.

 


   Footnotes
 
This work was supported in part by NIH Grant DK-48109.

1 Present address: Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, D-76021 Karlsruhe, Germany. Back

Abbreviations: AP-1, Activator protein-1; 8Br-cAMP, 8-bromo-cAMP; DTT, dithiothreitol; GST, glutathione-S-transferase; JNK, c-Jun N-terminal kinase; MEKK1, MAPK/extracellular signal-related kinase kinase kinase 1; PAK1, p21-activated kinase-1; PMA, phorbol 12-myristate 13-acetate; PTH1R, PTH/PTHrP receptor; SEK1/MKK4, stress-activated protein kinase/extracellular signal-related kinase kinase 1/MAPK kinase 4; TBST, Tris-buffered saline/Tween 20.

Received August 3, 2001.

Accepted for publication December 29, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dempster DW, Cosman F, Parisien M, Shen V, Lindsay R 1993 Anabolic actions of parathyroid hormone on bone. Endocr Rev 14:690–709[Abstract/Free Full Text]
  2. Partridge NC, Bloch SR, Pearman AT 1994 Signal transduction pathways mediating parathyroid hormone regulation of osteoblastic gene expression. J Cell Biochem 55:321–327[CrossRef][Medline]
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