A. Rey, D. Manen, R. Rizzoli, S.L. Ferrari, J. Caverzasio⁎
Service of Bone Diseases, Department of Rehabilitation and Geriatrics, University Hospital of Geneva, CH-1211 Geneva 14, Switzerland Received 3 October 2006; revised 21 February 2007; accepted 28 February 2007
Available online 14 March 2007
Abstract
Increased bone formation by PTH mainly results from activation of osteoblasts, an effect largely mediated by the cAMP-PKA pathway. Other pathways, however, are likely to be involved in this process. In this study we investigated whether PTH can activate p38 MAPK and the role of this kinase in osteoblastic cells. Bovine PTH(1–34) and forskolin markedly increased alkaline phosphatase (ALP) activity and doubled osteocalcin (Oc) expression in early differentiating MC3T3-E1 cells. These effects were associated with increase in cellular cAMP and activation of the MAP kinases ERK and p38. Activation of these MAP kinases was detectable after 1 h incubation with 10−7M PTH and lasted 1–2 h. Activation of p38 was mimicked by 10μM forskolin and prevented by H89 suggesting a cAMP-PKA-dependent mechanism of p38 activation. Interestingly, PTH-induced ALP stimulation was dose-dependently inhibited by a specific p38 inhibitor with no change in the generation of cAMP and the production of osteocalcin. Similar inhibitory effect was obtained in cells stably expressing a dominant-negative p38 molecule. Finally, treatment of MC3T3-E1 cells with PTH for 3 weeks significantly enhanced matrix mineralization and this effect was markedly reduced by a selective p38 but not a specific MEK inhibitor.
In conclusion, data presented in this study indicate that PTH can activate p38 in early differentiating osteoblastic cells. Activation of p38 is cAMP-PKA-dependent and mediates PTH-induced stimulation of ALP which plays a critical role for the calcification of the bone matrix.
© 2007 Elsevier Inc. All rights reserved.
Keywords:PTH; Osteoblast; p38; Alkaline phosphatase; Mineralization
Introduction
Parathyroid hormone (PTH) is a major regulator of calcium/
phosphate and bone metabolism [25]. It is a unique bone anabolic agent that differentially enhances bone formation and bone resorption resulting in transient net bone gain in both human and rodents[1,5,17,21,33]. Daily administration of PTH increases the number of osteoblasts as well as bone formation [6]. Even though PTH can increase the proliferation of osteoblastic cells in vitro [9,11,34,35,39,44], there is little in vivo evidence that cell proliferation plays a major role in the anabolic effect of PTH[15]suggesting that this effect mainly results from either activation of pre-existing osteoblasts, increased differentiation of lining cells, reduced osteoblast
apoptosis or enhancement of the differentiation of pre-osteoblasts[3,7,22,27,34,36,40].
In osteoblasts, PTH binding to its membrane receptor PTH1R, a G-protein-coupled receptor, activates the protein kinase A (PKA) pathway through Gαs stimulation of cAMP production as well as the protein kinase C (PKC) and 1,4,5-inositol trisphosphate pathways through Gαq activation of phospholipase Cβ[24]. To date, the cAMP-PKA is considered as the main pathway in PTH signaling for the stimulation of osteoblasts. Indeed, in vivo injection of different PTH peptides that only activate this pathway exhibits anabolic effects on bone whereas this is not the case for those that activate the PKC pathway [2,14,38]. It has been documented that PKA directly phosphorylates transcription factors such as Cbfa1 and cAMP response element-binding proteins (CREB), thus regulating the transcription of several osteoblastic genes including alkaline phosphatase (ALP) and osteocalcin (Oc) [18,19,23,37,40].
Bone 41 (2007) 59–67
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⁎Corresponding author. Fax: +41 22 382 99 73.
E-mail address:Joseph.Caverzasio@medecine.unige.ch(J. Caverzasio).
8756-3282/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2007.02.031
Recent evidences, however, also implicate other pathways such as the mitogen-activated protein kinases (MAPKs). Effects of PTH on MAPKs in osteoblastic cells have been reported for ERK and JNK. Their stimulation or inhibition is largely dependent on cell type and culture conditions as well as the state of cell differentiation. Inhibition of ERK by PTH is PKA-dependent [45] whereas its activation is PKA- and PKC-dependent [11,30,44]. Changes in ERK activity by PTH are involved in alteration of both cell proliferation[11,30,44,45]and differentiation[20,26,48]. Recently, PKA-dependent inhibition of JNK activity by PTH has been described in UMR-106 osteosarcoma and rat calvarial cells[8]but its functional role in osteoblastic cells treated with this hormone remains to be investigated. Previous and recent studies from our laboratory indicate that JNK and p38 are important signaling pathways for cell differentiation induced by several osteotropic factors including α adrenergic agonists [43], serum growth factors [42]and BMP-2[12]. Whether the p38 MAP kinase pathway is regulated by PTH and plays any role in changes of osteoblastic cell activity induced by this hormone is not known. In the present study, we show that PTH can stimulate p38 in early dif-ferentiating osteoblastic cells, probably by a cAMP-PKA-dependent mechanism and that activation of p38 by PTH is important for the stimulation of ALP and matrix calcification induced by this peptide hormone.
Material and methods Reagents and antibodies
Fetal calf serum (FCS), glutamine, antibiotics and trypsin/EDTA were obtained from Gibco (Life Technologies, Basel, Switzerland). Alpha-modified essential medium (α-MEM) was purchased from Amimed (Bioconcept, Allschwill, Switzerland). U0126, SB202190 and Go6983 were obtained from Calbiochem-Novabiochem Corp. (San Diego, CA, USA). Bovine parathyroid hormone fragment 1–34 and forskolin (FSK) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The PKA inhibitor H89 was from Calbiochem (Merck Bioscience, Luzern, Switzerland). Polyclonal anti-JNK, anti-p38, anti-ERK and anti-p-cJun were obtained from Santa Cruz Biotechnol-ogy Inc (Santa Cruz, CA, USA). Polyclonal anti-pERK, anti-pp38 and monoclonal anti-HA-Tag were obtained from New England BioLabs (Cell Signaling Technology, MA, USA).
Cell culture
The MC4 subclone of mouse calvaria-derived MC3T3-E1 cells was obtained from ATCC American Type Culture Collection (Manassas, VA, USA). UMR-106 cells were kindly provided by Dr. T.J. Martin, University of Melbourne, Australia. Cells were cultured inα-MEM containing 10% FCS (vol/
vol), 0.5% nonessential amino acids (vol/vol), 100 IU/ml penicillin and 100μg/
ml streptomycin. They were seeded at 15,000 cells/cm2and cultured in a 37 °C humidified atmosphere of 5% CO2–95% air. Cells reached confluency after 48 h and culture medium was changed every 2–3 days. In experiments aimed at testing the effect of inhibitors or their vehicles, cells were preincubated for 1–2 h prior to and during the experiments.
Construction of the retroviral vector MSCVneo-dnp38 and infection of MC4 cells
Dominant-negative (dn) p38 cDNA tagged with an HA epitope in the 5′end was a generous gift of Dr. J. Han (The Scripp Research Institute, La Jolla, CA, USA). It was cloned into anXhoI site of the pMSCV neo plasmid (Clontech,
CA, USA). For the production of retroviral particles, PT67 packaging cells were transfected with either empty pMSCVneo or pMSCVneo-dnp38 with Metafecten (Bintex, Martinried/Planegg, Germany). Selection of pMSCV plasmids expressing cells was obtained after 1 week culture in 1 mg/ml G418 (Promega, Wallisen, Switzerland). Culture medium from packaging cells containing retroviral particles was filtered and directly added on MC4 cells in the presence of 8μg/ml Polybrene (Sigma-Aldrich, Basel, Switzerland) for 24 h.
Infected MC4 cells were then selected with 1 mg/ml G418 during 1 week before use for analysis of the PTH effect on cell differentiation.
Adenylyl cyclase activity
cAMP accumulation was determined as previously described[10]in the presence of 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Briefly, cells were first incubated for 2 h in medium containing 2μCi/ml of [3H]-adenine, then during 15 min with IBMX and subsequently exposed to either 100 nM bovine PTH(1–34) or 10μM forskolin for 15 min. The reaction was stopped with 1.2 M trichloroacetic acid. cAMP was isolated by a two-column chromatography methods as previously described [10]. Accumulation of [3H]-adenine was then determined by a standard liquid scintillation technique.
Western blotting analysis
MC4 cells treated with PTH(1–34) were rapidly frozen in liquid nitrogen and stored at−80 °C until used for analysis. Cells were lysed at 4 °C in buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10μg/ml aprotinin, 10μg/ml leupeptin, 2 mM Na3VO4, 0.01 μM calyculin A, 0.1 μM mycrocystin LR, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS for 10 min. Lysates were then cleared by centrifugation at 6000×gfor 30 min and a 75μl sample was diluted with an equal volume of 2× reducing sample buffer containing 125 mM Tris buffer (pH 6.8), 4%
SDS, 20% glycerol, 0.05% bromophenol blue and 200 mM dithiothreitol. The mixture was then heated at 70 °C for 30 min and subjected to gel electrophoresis on 6–15% gels. Following SDS–PAGE electrophoresis, proteins were transferred to Immobilon P membranes and immunoblotted with specific antibodies as previously described[4]. Detection was performed using peroxidase-coupled secondary antibody, enhanced chemiluminescence reaction and visualization by autoradiography (Amersham International plc, Little Chalfont, UK). Filters which were reprobed were stripped according to the manufacturer's protocol.
Biochemical analysis of alkaline phosphatase and osteocalcin For the measurement of ALP activity, cells were harvested in 0.2% Nonidet P-40 and disrupted by sonication. The homogenate was centrifuged at 1500×gfor 5 min and ALP activity was determined in the supernatant by the method of Lowry et al.[29]. Oc released in the medium was measured by radioimmunoassay using a goat anti-mouse osteocalcin antibody and a donkey anti-goat secondary antibody (Biomedical Technologies, Inc., Stoughton, MA). Protein content was determined using the Pierce Coomassie Plus assay reagent (Pierce, Rockford, USA).
Matrix calcification
Calcium deposition in the matrix of MC3T3-E1 cells cultured for 3 weeks in the presence of 10 mMβ-glycerophosphate was quantified by Alizarin red staining as previously described[47]with minor modifications. Cells were washed twice with cold PBS, fixed in 70% ethanol for 10 min before staining for 30 s with 0.5%
Alizarin red in water (Sigma-Aldrich, Basel, Switzerland). Staining was followed by three washes with water and one with 70% ethanol. Matrix mineralization was quantified by extracting the Alizarin red stain with 100 nM cetylpyridinium chloride (Sigma-Aldrich, Basel, Switzerland) at room temperature during 4 h.
Absorbance of the extracted Alizarin red stain was measured at 570 nm.
Statistical analysis
All experiments were carried out independently at least 3 times. Results are expressed as the mean ± SEM. Comparative studies of means were
60 A. Rey et al. / Bone 41 (2007) 59–67
performed using one-way analysis of variance followed by a post hoc test (projected least significant difference Fisher) with a significance value of p<0.05.
Results
The cAMP pathway mediates PTH-induced stimulation of ALP and osteocalcin in early differentiating osteoblastic cells
In MC4 cells cultured for 8 days, which express high levels of parathyroid type I receptors (PTH1R) and a low basal ALP activity [46], bovine PTH(1–34) (PTH) induced a time and dose-dependent stimulation of this enzyme activity (Fig. 1).
This effect was detected with 10−9M and after 5 h incubation with 10−7M PTH. It was associated with a similar time–effect but a lower dose-dependent increase in the production of osteocalcin (Oc) (Fig. 2) and no change in total collagen content (data not shown). PTH effects on ALP and Oc were fully mimicked by 10 μM forskolin (FSK) (Fig. 3), a selective agonist of cAMP production. This observation suggests a role of the cAMP-PKA pathway in mediating activation of MC4 cells by PTH. The stimulation of ALP and Oc by PTH was also decreased by H89, an inhibitor of PKA, further supporting a role of the cAMP-PKA pathway (Fig. 4). In contrast, a selective
and large spectrum inhibitor of PKC had a weak effect (Fig. 4) suggesting a modest role of the PKC pathway in this hormonal response. As expected, cAMP stimulation by PTH was not affected by the presence of either PKA or PKC inhibitors (Table 1). Also, cAMP stimulation by 10μM FSK was nearly identical to the effect of 10−7M PTH (Table 1).
PTH activates ERK and p38 in MC4 cells
The role of MAP kinases in mediating changes in ALP activity and Oc production induced by PTH was then investigated by first measuring which MAP kinases are activated by PTH in early differentiating MC4 cells. As shown in Fig. 5, PTH increased the activity of ERK and p38.
Activation of both MAP kinases was delayed and transient. It was observed after 1 h exposure and lasted about 1–2 h. In this series of analysis, we also investigated whether PTH activates JNK. We could not find any direct increase in p-JNK by PTH but reproducibly found an increase in the phosphory-lation of a 50 kDa protein recognized by the anti-pcJun antibody (data not shown) suggesting either a weak activation of JNK in response to PTH that was not detected in our Western analysis or activation of a JNK-like kinase that remains to be investigated.
Fig. 1. Time course and dose effect of PTH on alkaline phosphatase activity in MC4 cells. Cells were cultured for 8 days in standard culture medium containing 10% FCS and exposed to either 10−7M PTH(1–34) for various incubation times (upper panel) or with different PTH concentrations for 24 h (lower panel).
Alkaline phosphatase activity (ALP) was then determined as described in Material and methods. Data are mean ± SEM of 3–4 determinations from a representative experiment. *p<0.01 compared with vehicle.
Fig. 2. Time course and dose effect of PTH on osteocalcin production in MC4 cells. Cells were cultured for 8 days in standard culture medium containing 10%
FCS and exposed to either 10−7M PTH(1–34) for various incubation times (upper panel) or with different PTH concentrations for 24 h (lower panel).
Osteocalcin content in the culture medium was then determined as described in Material and methods. Data are mean ± SEM of 3–4 determinations from a representative experiment. *p<0.01 compared with vehicle.
61 A. Rey et al. / Bone 41 (2007) 59–67
p38 activation by PTH is cAMP-PKA-dependent in osteoblastic cells
To determine whether activation of p38 by PTH is cAMP-dependent or -incAMP-dependent, we studied whether FSK also activates p38 in MC4 cells and investigated the influence of the PKA inhibitor H89 on PTH-induced activation of p38. As depicted inFig. 6(upper panel), PTH significantly increased the phosphorylation of p38 by 2.7 fold (p<0.01). FSK at 10μM, that had the same effect on ALP compared with PTH (Fig. 3), significantly activated p38 by 3.7 fold (p<0.01) thus fully mimicking the stimulation of p38 by PTH in these cells.
Evidences that cAMP might act through PKA for activation of p38 by PTH are supported by the complete inhibition of this signaling response by H89 at low and high concentrations (Fig. 5, lower panel).
Pharmacological inhibition of p38 MAP kinase or expression of dnp38 decreases PTH-induced ALP stimulation in
osteoblastic cells
Evaluation of the role of MAP kinases in PTH-induced MC4 osteoblastic differentiation was first performed using selective inhibitors of ERK and p38 pathways. As presented inFig. 7, the p38 inhibitor SB202190 significantly reduced the stimulation of ALP whereas it slightly enhanced the production of Oc induced by PTH. On the contrary, the MEK inhibitor U0126 markedly
reduced the PTH effect on Oc production but only weakly affected the change in ALP activity. These observations strongly suggested that p38 is involved in mediating the stimulation of ALP whereas ERK is rather implicated in the
Fig. 3. Effect of PTH(1–34) or forskolin on alkaline phosphatase activity and osteocalcin production in MC4 cells. Cells were cultured for 8 days in standard culture medium containing 10% FCS and exposed to either vehicles (Veh), 10−7M PTH(1–34) or 10μM forskolin (FSK) for 24 h. Alkaline phosphatase activity (ALP) and osteocalcin production were then determined as described in Material and methods. Data are mean ± SEM of 3–4 determinations from a
representative experiment. *p<0.01 compared with vehicle. Fig. 4. Effects of PKA and PKC inhibitors on PTH-induced alkaline phosphatase activity and osteocalcin production. MC4 cells were cultured for 8 days and preincubated either with 25μM H-89, a specific PKA inhibitor, 10μM Go6983, a selective PKC inhibitor, or vehicle for 1 h and then treated with or without 10−7M PTH(1–34) for 24 h. Alkaline phosphatase activity (ALP) and osteocalcin production were determined as described in Material and methods. Data are mean ± SEM of 4–6 determinations from a representative experiment. *p<0.01 compared with PTH-treated cells.
Table 1
Effects of PKA, PKC and MAP kinases inhibitors on PTH- and of forskolin-induced cAMP accumulation in MC4 cells
cAMP accumulation
MC4 cells were cultured for 8 days and preincubated for 2 h in medium containing 2μCi/ml [3H]-adenine and 1 mM IBMX during the last 15 min prior addition of either vehicle, H89 (PKA inhibitor), Gö6983 (PKC inhibitor), U0126 (MEK inhibitor) or SB202190 (p38 inhibitor) for 1 h and then with PTH or forskolin (FSK) for 15 min. cAMP was measured as described in Material and methods. Data are mean ± SEM of 3 determinations of a representative experiment. nd: not determined.
62 A. Rey et al. / Bone 41 (2007) 59–67
regulation of Oc by PTH in these cells. An effect of the p38 inhibitor on PTH-induced stimulation of ALP activity was also observed in UMR-106 cells. The increased ALP activity induced by PTH in these cells was very small (Veh 118.9 ± 2.1; PTH 134.6 ± 2.7 nmol/mg prot, p<0.05) compared with that in MC4 cells (Fig. 1) and was blunted by 10μM SB202190 (Veh-SB 99.7 ± 0.9; PTH-SB 104.0 ± 2.2 nmol/mg prot, n.s.). A significant role of p38 in PTH-induced ALP stimulation was further supported by the dose-dependent inhibition of
SB202190 on this bone marker in MC4 cells shown in Fig. 8 without any effect of this compound on basal ALP activity and cAMP production (Table 1). Data obtained with the p38 inhibitor were also supported by analysis of the PTH-induced ALP stimulation in three MC4 clones stably expressing HA-tagged dnp38 (Fig. 9A) in which a reduction of 35–40%
(p<0.01) of the PTH response was recorded compared with empty vector infected cells (Fig. 9B).
Impaired matrix mineralization induced by PTH in the presence of the p38 inhibitor
Finally, since ALP is a major regulator of matrix mineraliza-tion[31], we analyzed the influence of PTH on the calcification of bone matrix deposited by MC4 cells cultured in the presence or absence of MAP kinases inhibitors for 3 weeks. We reproducibly found that PTH induced a marked and significant increase in the deposition of calcium in the matrix of MC4 cells (Fig. 10).
Interestingly, the selective inhibitor of the ERK pathway had no significant effect whereas the p38 inhibitor markedly reduced matrix mineralization induced by PTH (Fig. 10). The lower
Fig. 5. Effect of PTH(1–34) on ERK and p38 MAP kinases. MC4 cells cultured for 8 days were exposed to 10−7M PTH(1–34) for various incubation times.
Cell lysates were then harvested and activation and expression levels of signaling molecules were investigated by Western blot analysis as described in Material and methods using specific antibodies against either phosphorylated or unphosphorylated molecules. Data are from two representative experiments.
Fig. 6. Effect of the cAMP-PKA pathway on p38 activation induced by PTH.
MC4 cells cultured for 8 days were either directly exposed to 10−7M PTH(1– 34) or 10μM forskolin for 2 h (A) or preincubated with 10 and 25μM H89 for 1 h before exposure to 10−7M PTH(1–34) for 2 h (B). Cell lysates were then harvested and activation and expression levels of p38 MAP kinase were investigated by Western blot analysis using specific antibodies against either phosphorylated or unphosphorylated p38 molecules. Data shown are mean ± SEM from 6 experiments for panel A and one representative experiment for panel B.p<0.01 compared with vehicle.
Fig. 7. Effect of MAP kinases inhibitors on PTH-induced stimulation of alkaline phosphatase activity and osteocalcin production. MC4 cells were cultured for 8 days and preincubated with either 10μM U0126, a specific MEK inhibitor, or 10μM SB202190, a specific p38 inhibitor, for 1 h prior incubation with or without 10−7M PTH(1–34) for 24 h. Alkaline phosphatase activity (ALP) and osteocalcin production were then determined as described in Material and methods. Data are mean ± SEM of 4–6 determinations from a representative experiment. *p<0.01 compared with vehicle. +p<0.01 compared with PTH-treated cells in the absence of inhibitors.
63 A. Rey et al. / Bone 41 (2007) 59–67
mineralization recorded in the presence of the p38 inhibitor was not due to loss of cells since the amount of proteins at the end of the 3 weeks study in this experimental group (5.89 ± 0.4 mg/well)
was similar to that of PTH-treated cells in the absence of this inhibitor (5.97 ± 0.2 mg/well).
Discussion
The molecular mechanism by which PTH increases osteo-blastic differentiation remains poorly understood. Very few studies have been published so far on the in vitro effect of PTH on activation of osteoblastic cell differentiation [3,40]. The reason for such a paucity of information on this topic is probably due to particular culture conditions that are required for inducing this hormonal response as suggested by reports indicating that PTH effects on osteoblast differentiation depend on either time exposure[18]or differentiation stages[19]. In the present study, we describe new in vitro experimental conditions in the MC4 subclone of MC3T3-E1 cells that leads to activation of the differentiation of osteoblastic cells by PTH and provide experimental evidences that MAP kinases ERK and p38 are probably involved in this cellular response. The MC4 subclone
The molecular mechanism by which PTH increases osteo-blastic differentiation remains poorly understood. Very few studies have been published so far on the in vitro effect of PTH on activation of osteoblastic cell differentiation [3,40]. The reason for such a paucity of information on this topic is probably due to particular culture conditions that are required for inducing this hormonal response as suggested by reports indicating that PTH effects on osteoblast differentiation depend on either time exposure[18]or differentiation stages[19]. In the present study, we describe new in vitro experimental conditions in the MC4 subclone of MC3T3-E1 cells that leads to activation of the differentiation of osteoblastic cells by PTH and provide experimental evidences that MAP kinases ERK and p38 are probably involved in this cellular response. The MC4 subclone