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Endocrinology Vol. 148, No. 4 1629-1637
Copyright © 2007 by The Endocrine Society

p38 Mitogen-Activated Protein Kinase Regulates Osteoblast Differentiation through Osterix

Xueying Wang, Choon Hong Goh and Baojie Li

The Institute of Molecular and Cell Biology, Singapore 138673, Republic of Singapore

Address all correspondence and requests for reprints to: Baojie Li, The Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Republic of Singapore. E-mail: libj{at}imcb.a-star.edu.sg.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
p38 MAPK has been shown to regulate osteoblast differentiation. Inhibition of this kinase with inhibitors or dominant-negative mutant impedes osteoblast differentiation. Yet the molecular mechanism behind this regulation is not well understood. Here we provide evidence that the effect of p38 MAPK on osteoblast differentiation can be mediated by osterix (Osx), a transcription factor necessary and sufficient for osteoblast differentiation. Inhibition of p38 MAPK had minimal effects on differentiation of p53–/– osteoblasts, which had sustained Osx expression. Inhibition of p38 MAPK down-regulated the expression of Osx at both protein and mRNA levels, but not other transcription factors involved in osteoblast differentiation. More importantly, this inhibitory effect could be significantly relieved in osteoblasts overexpressing Osx. Further experiments support that Osx expression is mainly controlled by bone morphogenetic proteins existing in the culture medium, secreted by osteoblasts or provided by serum, and p38 MAPK plays a positive role in bone morphogenetic proteins-induced Osx expression. These findings identify a novel mechanism by which p38 MAPK regulates osteoblast differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BONE IS A DYNAMIC organ that is remodeled throughout the lifetime, with old bone being replaced by newly formed bone. Bone formation is carried out by the osteoblast (OB), and bone resorption is carried out by the osteoclast. Coordinated action of OBs and osteoclasts is essential for maintaining an optimal bone density. Disruption of the coordination leads to development of bone diseases such as osteoporosis and osteosclerosis (1). Osteoporosis is estimated to affect more than 200 million people worldwide and causes morbidity and mortality in the aged population (2).

Bone formation is a function of OBs that are derived from bone marrow mesenchymal stem cells (MSC). The maturation of MSC to functional osteocytes is a multiple step process that involves cell expansion and differentiation. Many extracellular signals affect OB differentiation. Among the best studied are bone morphogenetic proteins (BMPs) and the Wnt molecules (3, 4, 5). BMPs activate the Smad1/5/8 signaling pathway, and Wnt activates the ß-catenin pathway to regulate OB differentiation. In addition, it has been shown that MAPK pathways are also involved in OB differentiation, which can be activated by BMPs as well as other growth factors. Although inhibition of ERKs was found to have a minimal effect on OB differentiation, inhibition of p38 MAPK by inhibitors or by dominant-negative p38 MAPK impedes OB differentiation in primary OB and some OB cell lines (6, 7, 8, 9, 10, 11). Yet the molecular mechanisms by which p38 MAPK regulates OB differentiation are not well understood.

Downstream of these signaling pathways are transcription factors that control OB differentiation, some of which are regulated by BMPs and/or Wnt via the aforementioned signaling pathways (12, 13). Among them, Runx2 and osterix (Osx) are expressed specifically in OBs and chondrocytes. Biochemical and genetic evidence supports that Runx2 and Osx are each sufficient and essential for OB differentiation and bone calcification. Ectopic expression of either Runx2 or Osx turns on OB-specific genes in fibroblasts (14, 15). Deficiency in either one results in the absence of mature OBs and a lack of calcified bones in mouse (15, 16, 17). In addition, some transcription factors, which are expressed in many other tissues as well, were reported to regulate OB differentiation and bone formation, e.g. Atf4 and Dlx5 (18, 19, 20). Recently, p53, a tumor suppressor, has been reported to negatively regulate OB differentiation, bone formation, and OB-dependent osteoclastogenesis (21). p53 deficiency could rescue the inadequate bone development of Mdm2 knockout mice (22). p53 functions as a transcription activator or repressor depending on the target genes. p53 might execute its negative role in OB differentiation by repressing the expression of Osx (23).

To further understand how p38 MAPK regulates OB differentiation, we studied the differentiation of p53–/– OB and found that these OBs were refractory to the negative effect of p38 MAPK inhibition. p38 MAPK inhibition in wild-type OBs led to reduction of Osx but not Runx2, Atf4, or Dlx5. More importantly, p53–/– OBs show elevated Osx, which was sustained even in the presence of p38 MAPK inhibitors, suggesting that Osx might mediate the effect of p38 MAPK on OB differentiation. To prove this, we generated calvarial OBs that overexpressed Osx with a retroviral vector. It was found that these cells were significantly resistant to the inhibition of p38 MAPK. These results indicate that inhibition of p38 MAPK leads to a decrease in Osx levels, which in turn impedes OB differentiation. Moreover, the driving force for OB differentiation in the culture medium was confirmed to be BMPs, which controls Osx expression in a dosage-dependent manner. Further studies suggest that p38 MAPK acts on Osx promoter and plays a positive role in BMP-induced Osx expression and OB differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Calvarial OB isolation and culture
Calvarial OBs were isolated from new-born pups of p53–/– and control littermates (23). All mice were bred and used following the guidelines of the Institute of Molecular and Cell Biology, with protocols approved by Animal Care and Use Committee, Singapore. Briefly, calvaria were extracted from newborn pups, washed with PBS, and digested in {alpha}-MEM with dispase and collagenase IV for 20 min at 37 C for four times. Cells from the last three digestions were collected and cultured in {alpha}-MEM supplemented with 10% fetal calf serum, glutamine, and pen/strep, at 37 C in the presence of 5% CO2. They were passaged a couple of times before being used for different experiments. Cell proliferation was determined with water-soluble tetrazolium-1 assay that measures the metabolic activity of cells (Roche Applied Science, Penzberg, Germany).

Differentiation assays
For OB differentiation, calvarial OBs were cultured in {alpha}-MEM with 50 µg/ml of ascorbic acid and 10 mM of ß-glycerolphosphate. To make sure that the difference in OB differentiation is not caused by the differential in cell growth rates, we plated sufficient cells (5 x 105 cells per well in a 24-well plate) so that the cultures were confluent the second day. The medium was changed every 3 d. To test the effect of different inhibitors, they were included in the culture for 3 d or as indicated in the text and washed off. Afterward, culture was continued in differentiation medium without the inhibitors, until the cultures were used for different assays. This was to prevent possible toxic effects of the inhibitors on OB cultures. SB203580 and PD98059 were obtained from Calbiochem (San Diego, CA).

Alkaline phosphatase (ALP) staining, ALP quantitation assays, and Von Kossa staining
OBs were cultured in differentiation medium for different periods of time, fixed, and stained for ALP with a kit (Sigma, St. Louis, MO). To quantify the enzymatic activities of ALP, cells were collected, lysed, and used for quantitation assay (Sigma). The protein concentration of these lysates was determined by Bio-Rad method and was used to normalize ALP activity. The relative ALP activity is defined as millimoles of p-nitrophenol phosphate hydrolyzed per minute per milligram of total protein (units). For bone mineralization assay, OBs were cultured in differentiation medium for different periods of time, washed, and bone mineralization was assessed by Von Kossa staining (23).

Retroviral infection
To express Osx in calvarial OBs, a retroviral vector was used. The coding sequence of Osx (the longest version) was cloned into retroviral vector pMSCVpuro (Clontech, Mountain View, CA). The vector and Osx constructs were transfected into Plat E cells to produce the retrovirus (24). Calvarial OBs were infected with control viruses and viruses expressing Osx, and then selected against puromycin for 3 d before splitting for differentiation assays.

Luciferase assay
The Osx promoter (a 2.0-kb fragment from the start of transcription) was cloned into pGL3 (luciferase basic vector) (Promega, Madison, WI) (23). p38 MAPK or dominant-negative p38 MAPK expression constructs, the promoter plasmid (pGL3-Osx-Luc), and renilla plasmid (internal control) was cotransfected into C2C12 or MC3T3-E1 cells. Cells were harvested 48 h later, washed with PBS, and lysed with reporter lysis buffer (Promega). The luciferase activities were measured following the manufacturer’s procedures and were normalized against the renilla activity. All transient expressions in this assay were carried out in triplicates.

RT-PCR
Total mRNA was isolated from OBs growing on 60-mm dishes using TRIzol reagent (Invitrogen, Carlsbad, CA) and used for RT-PCR assays. Total RNA was subjected to DNase treatment (Ambion, Austin, TX) and quantitated. Five micrograms of total mRNA was reverse transcribed into cDNA using AMV (Promega) reverse transcriptase. The total reaction was used in the PCR with the following primers: Osx (197 bp): forward, 5'-TGA GGA AGA AGC CCA TTC AC, reverse, 5'-ACT TCT TCT CCC GGG TGT G; Runx2 (113 bp): forward, 5'-TGG CAG CAC GCT ATT AAA TC, reverse, 5'-TCT GCC GCT AGA ATT CAA AA; Atf4: forward, 5'-TTC CAC TCC AGA GCA TTC CT, reverse, 5'-CAG GTG GGT CAT AAG GTT TG; RankL: forward, 5'-CAG AAG ACA GCA CTC ACT GC, reverse, 5'-GAA CCC GAT GGG ATG C; Opg: forward, 5'-CTG CCT GGG AAG AAG ATC AG, reverse, 5'-TTG TGA AGC TGT GCA GGA AC; M-Csf: forward, 5'-CTG GAA GGA GGA TCA GCA AG, reverse, 5'-ATG TCT GAG GGT CTC GAT GG; ß-actin (104 bp): forward, 5'-AGA TGT GGA TCA GCA AGC AG, reverse, 5'-GCG CAA GTT AGG TTT TGT CA. PCR was carried out for 30 cycles of denaturation (94 C for 30 sec), annealing (57 C for 30 sec), and extension (72 C for 1 min), and one cycle of final extension (72 C for 10 min), which was just enough to detect the PCR products.

The detection and quantification of target mRNA were performed with semiquantitative RT-PCR. The amplification for each mRNA was performed in the linear range for RT-PCR by optimizing the template concentration and limiting the amplification cycles to less than 30 to ensure exponential amplification.

Western blot analysis
Cells were washed with PBS and lysed in a buffer containing 50 mM Tris (pH 7.5), 100 mM KCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, 1 mM sodium orthorvanadate, 10 mM sodium fluoride, 1 mM ß-glycerolphosphate, and 10 µg/ml each of aprotonin, pepstatin, and leupeptin. Protein concentrations were determined by the Bio-Rad protein quantitation assay. The same amounts of protein (20 µg) were fractionated by electrophoresis on a 10% SDS-PAGE gel, transferred to a Immobilon-P polyvinylidene difluoride membrane (Millipore, Billerica, MA), probed with polyclonal anti-Osx antibodies (developed by X. Wang et al.), and visualized using an ECL kit (Amersham, Piscataway, NJ). Anti-p53, anti-phospho-Smad1/5, anti-Smad1, anti-phospho-p38, and anti-p38 antibodies were purchased from Cell Signaling (Danvers, MA), anti-actin antibodies were from Sigma, and anti-Runx2 antibodies were from Abcam (Cambridge, UK).

Statistical analysis
Each experiment was repeated three times. Statistical analysis was performed using an unpaired t test (STATISTICA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of p38 MAPK impeded differentiation of p53+/+ but not p53–/– OBs
To understand how p38 MAPK regulates OB differentiation, we tested the responses of p53–/– OBs to p38 MAPK inhibition as these mutant OBs show enhanced differentiation (23). p53–/– and control OBs were pretreated for 3 d with different concentrations of SB203580, a commonly used inhibitor for p38{alpha} and p38ß MAPKs, cultured in differentiation medium for 4 more days, and stained for ALP expression. Previous studies showed that 5–20 µM SB203580 inhibited p38 MAPK and OB differentiation in a dosage-dependent manner (6, 25). This is also a range used in many other cell types. SB203580, at the concentration of 5–10 µM, is sufficient to markedly repress OB differentiation, manifested by a reduction in ALP expression and mineral deposition (6). Moreover, 10 µM of SB203580 is sufficient to diminish oxidative stress-induced up-regulation of antioxidant protein peroxiredoxin in OBs (25). However, SB203580, at this range of concentrations, did not have a significant effect on cell proliferation (6). To minimize possible interference of cell growth on differentiation, an excess number of OBs were plated in the present study, leaving little room for cell proliferation. Furthermore, inhibitors were present in the culture only for 3 d. Under these conditions, we did not observe a significant difference in cell death rates between p53–/– and control OBs with or without SB203580 (<10% dead cells in all cultures justified by trypan blue staining), nor a significant difference in cell numbers (justified by water-soluble tetrazolium-1 assay and the protein levels of these cell lysates) (data not shown). Yet, SB203580, at the same concentrations, inhibited ALP staining in a dosage-dependent manner in wild-type OBs, but not much in p53–/– OBs (Fig. 1AGo). Quantitation of ALP (normalized to the total protein levels) confirmed this observation (Fig. 1BGo). In wild-type cells, SB203580 at 5 µM was able to inhibit 70% of ALP activity in wild-type cells, whereas the inhibition was less than 10% in p53–/– cells. At 10 µM, the inhibition was 90% in wild-type cells and less than 20% in p53–/– cells. At higher concentrations like 20 µM, SB203580 was able to inhibit ALP activity in p53–/– cells, but it may reflect a nonspecific effect because 5–10 µM is sufficient to inhibit p38 to more than 50%. Nevertheless, in the presence of 20 µM SB203580, p53–/– cells still showed ALP levels equivalent to that of wild-type cells in the absence of SB203580 (Fig. 1BGo). Moreover, inhibition of p38 MAPK impeded expression of osteocalcin, another OB-specific marker, and bone mineralization of wild-type OB cultures but not much in p53–/– OB cultures (Fig. 1Go, C and D). Hence, SB203580 showed little effect on osteocalcin expression or mineral deposition in p53–/– cells. The results on all three OB differentiation markers support the notion that p53 negatively regulates OB differentiation and that cells with a deficiency of p53 could at least partially overcome the negative effects of p38 MAPK inhibition.


Figure 1
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  		FIG. 1. p53–/– OBs were resistant to the negative effects of p38 MAPK inhibition. A, p53–/– OBs were resistant to the negative effects of p38 MAPK inhibition on ALP staining. p53–/– and control OBs were plated onto 24-well plates. Different dosages (5, 10, 15, and 20 µM) of SB203580 (SB) or 20 µM of PD98059 (PD) were added to the cultures for 3 d in differentiation medium, washed off, and cultured to d 7 for ALP staining. B, Quantitation data from A that were normalized to the total protein levels. C, RT-PCR assays showed that inhibition of p38 MAPK reduced the mRNA levels of osteocalcin. The basal level of osteocalcin in wild-type cells was set at 1.0. D, p53–/– OBs were resistant to the negative effects of p38 MAPK inhibition on bone mineral deposition. The experiments were carried out as in A. The inhibitor was washed off and the cells were cultured until d 21 for Von Kossa assay. *, P < 0.05 when compared with unstimulated controls. **, P < 0.05 when compared with the counterparts of wild-type cells.

 
In contrast, inhibition of ERKs with PD98059 showed minimal effect on ALP levels in wild-type OBs, but an enhancement of ALP expression in p53–/– OBs (Fig. 1Go, A and B). These results indicated that ERKs and p38 MAPK played distinct roles in OB differentiation and that p53–/– OBs provide a model to dissect how p38 MAPK regulates OB differentiation. Further studies will be needed to understand why inhibition of ERKs made such a difference in OB differentiation in the presence or absence of p53.

Inhibition of p38 MAPK down-regulated Osx in wild-type OBs but not in p53–/– OBs
The differential responses of p53–/– and control OBs to p38 MAPK inhibition suggest that p53 target gene(s) might be responsible for the effect of p38 MAPK. One legitimate candidate is Osx because p53–/– OBs acquired enhanced differentiation potential due to elevated expression of Osx (23). If this is true, inhibition of p38 MAPK would alter the levels of Osx in OBs. Indeed, we found that SB203580 was able to reduce the levels of Osx mRNA in wild-type cells, but not much in p53–/– OBs (Fig. 2AGo). Western blot analysis confirmed this observation (Fig. 2BGo). Interestingly, mRNA levels for Runx2, Atf4, and Dlx5 were not affected by either inhibition of p38 MAPK or by p53 deficiency (Fig. 2AGo). Nor was there a difference in Smad1 or Smad5 mRNA levels (Fig. 2AGo). Western blot analysis confirmed that Runx2 was not affected by inhibition of p38 MAPK (Fig. 2CGo). The fact that expression of Runx2, Atf4, and Dlx5 was not affected by p38 MAPK inhibition or by p53 deficiency suggests that regulation of Osx is a relatively specific event. The positive correlation between Osx and OB differentiation in the presence of p38 MAPK inhibition suggests that the effect of p38 MAPK on OB differentiation is likely to be mediated by Osx. A similar role for Osx in mediating the effect of Atm and c-Abl on OB differentiation has been reported (23, 26). The results also support the notion that modulation of Osx expression plays an important role in OB differentiation.


Figure 2
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  		FIG. 2. p38 MAPK inhibition down-regulated expression of Osx in normal OBs, but not much in p53–/– OBs. A, RT-PCR assays showed that the mRNA levels of Osx, but not Atf4, Runx2, Smad1/5, or Dlx5, were repressed by inhibition of p38 MAPK in wild-type OBs. B, Western blot analysis showed that Osx protein levels were down-regulated by p38 MAPK inhibition in wild-type OBs. The basal level of Osx mRNA or protein in wild-type cells was set at 1.0. C, p38 MAPK inhibition did not alter the protein levels of Runx2. Normal OBs were cultured in differentiation medium with or without SB203580 (SB) for different periods of time. The protein levels of Runx2 were determined by Western blot analysis.

 
Overexpression of Osx rescued the defect in OB differentiation caused by p38 MAPK inhibition
Having shown that inhibition of p38 MAPK led to reduced Osx expression as well as compromised OB differentiation, and that p53–/– OBs failed to show a significant down-regulation of Osx or defective differentiation in response to p38 MAPK inhibition, we wanted to prove that altered expression of Osx is mediating the effect of p38 MAPK. Osx has been demonstrated to be sufficient and essential for OB differentiation. We reasoned that cells overexpressing Osx would be resistant to the negative effect of p38 MAPK inhibition. We then generated primary OB that ectopically expressed Osx by a retroviral vector. Primary calvarial OBs were infected with control virus or virus expressing Osx, selected for 3 d, and then split for experiments. RT-PCR and Western blot analysis showed that Osx was much higher in cells infected with the retroviral-Osx, which could not be markedly down-regulated by inhibition of p38 MAPK (Fig. 3AGo and data not shown). At d 1 after plating, OBs infected with control virus or virus expressing Osx showed little ALP staining (data not shown). At d 4, inhibition of p38 MAPK obstructed ALP expression in control OBs, whereas OB expressing Osx showed sustained ALP expression (Fig. 3Go, B and C). A similar observation was made for bone mineral deposition (Fig. 3DGo). Although we observed that SB203580, especially at higher concentrations, inhibited ALP expression even in the presence of overexpressed Osx, we think that this could be explained by the fact that retroviral driven Osx expression was not exactly the same as that of the endogenous gene, in terms of the strength and the duration of expression. Alternatively, it is possible that Osx is not the only downstream target of the p38 MAPK pathway that regulates OB differentiation. These results suggest that the effect of p38 MAPK on OB differentiation is, at least in part, mediated by Osx.


Figure 3
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  		FIG. 3. OBs overexpressing Osx were resistant to the negative effect of the p38 MAPK inhibition. A, Western blot analysis showed that the protein levels of Osx were higher in cells infected with virus expressing Osx. Primary OBs infected with retroviruses expressing Osx or just empty vector were cultured in the presence of SB203580 (SB) (5, 10, 15 µM), PD98059 (PD) (20 µM), or noggin plus chordin (N+C) for 3 d and collected. The protein levels of Osx were determined by Western blot analysis. B, ALP expression sustained in the presence of p38 MAPK inhibition in OBs overexpressing Osx. Primary OBs infected with retroviruses expressing Osx or just empty vector were cultured in differentiation medium for different periods of time, and were then stained for ALP. C, Quantitation data from B. D, Bone mineral deposition sustained in the presence of p38 MAPK inhibition in OBs overexpressing Osx. *, P < 0.05 when compared with unstimulated controls. **, P < 0.05 when compared with the counterparts of wild-type cells.

 
BMPs were required and sufficient for OB differentiation and Osx expression
It has been reported that BMP molecules can stimulate OB differentiation. BMPs can be synthesized and secreted into the medium by OBs (27). In addition, serum might contain certain amounts of BMPs as well. To test whether BMPs in the medium are required for OB differentiation, we used noggin and chordin, BMPs binding proteins, to block their action (4). It was found that noggin (0.25 µg/ml) and chordin (0.5 µg/ml) reduced the expression of Osx at mRNA and protein levels and inhibited OB differentiation with a decrease in ALP and mineralization (Fig. 4Go, A–C). The combination of the two BMP binding proteins had a much better effect than a single protein (data not shown). These results indicate that BMPs in the cultures are required for Osx expression and for OB differentiation.


Figure 4
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  		FIG. 4. Blockade of BMP action inhibited Osx expression and OB differentiation. A, OBs showed reduced expression of Osx in the presence of noggin and chordin (N+C). Normal OBs were incubated with noggin and chordin for 4 d and collected for total RNA isolation and for assessment of Osx mRNA (left panel) or protein levels (right panel). The basal level of Osx mRNA or protein in wild-type cells was set at 1.0. B, OBs expressed minimal levels of ALP in the presence of noggin and chordin. C, Von Kossa staining showing that noggin and chordin also repressed OB mineralization.

 
To determine whether BMPs were sufficient for OB differentiation and for up-regulation of Osx, we added different amounts of BMP2 into the culture of wild-type and p53–/– OB. We found that BMP2 promoted OB differentiation, manifested by an increase in ALP (Fig. 5Go, A and B). This was associated with increased expression of Osx in a BMP2 dosage-dependent manner (Fig. 5Go, C and D). We found that p53–/– cells still showed a more advanced differentiation than control OBs, and this was accompanied by an enhanced induction of Osx (Fig. 5Go). This was confirmed by studies using serum-starved cells with lower dosages of BMP2 (Fig. 5EGo). Lower dosage of BMP2 showed a strong induction of Osx in serum-starved cells but not in nonstarved cells, probably due to the presence of BMPs in the serum. These results confirmed that BMP2 is sufficient in induction of Osx and in promotion of OB differentiation. They also indicate that BMPs, which are provided by the serum or secreted by OBs themselves, are the major driving force for OB differentiation under our experimental settings (Fig. 5Go, A–C). It is likely that the effects of BMP2 on OB differentiation are mediated by Osx.


Figure 5
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  		FIG. 5. BMP2 up-regulates Osx expression and OB differentiation. A, p53–/– and control wild-type OBs were incubated with different doses of BMP2 in differentiation medium for different periods of time and then used for ALP staining. B, Quantitation data for A. The experiment was repeated three times. C, BMP2 induced the expression of Osx at the mRNA levels. p53–/– and control wild-type OBs were incubated with different doses of BMP2 (nanograms per milliliter) in differentiation medium and then used for RT-PCR assays to assess the mRNA levels of Osx. D, Quantitation data for Osx from C. E, BMP2 (lower dosages, nanograms per milliliter) induced the expression of Osx at the mRNA levels in the absence of serum. The experiments were carried out as C except that cells were serum starved. *, P < 0.05 when compared with unstimulated controls. **, P < 0.05 when compared with the counterparts of wild-type cells.

 
Osx expression was regulated by p38 MAPK pathway in a p53-independent manner
Having shown that Osx might mediate the effect of p38 MAPK on OB differentiation and that BMPs in the medium are the main driving force of OB differentiation, it is conceivable that p38 MAPK should affect BMPs-induced Osx expression and subsequently OB differentiation. It has been previously reported that BMP2 can activate p38 MAPK (7, 27). Consistently we found that, in serum-starved OBs, as low as 10 ng/ml of BMP2 was able to activate p38 MAPK (Fig. 6AGo). Yet, in the presence of serum, noggin and chordin could not block the activation of p38 MAPK (Fig. 6BGo), suggesting that other factors in the serum might be responsible for this activation. The fact that noggin and chordin could block OB differentiation even in the presence of active p38 MAPK (Fig. 4Go) suggests that Smad1/5/8 pathway itself is also crucial for Osx expression and OB differentiation.


Figure 6
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  		FIG. 6. Involvement of p38 MAPK in BMP-induced Osx transcription. A, BMP2 activated p38 MAPK in the absence of serum. OBs were starved from serum for overnight and then stimulated with 10 or 50 ng/ml BMP2 for 1 h. p38 MAPK activation was determined by Western blot using antibodies that specifically recognize activated p38 MAPK. B, Noggin and chordin (N+C) did not block p38 MAPK activation in the presence of serum. Cells were cultured for different periods of time in the presence or absence of noggin and chordin. p38 MAPK activation was determined by Western blot as described in A. C, RT-PCR shows that inhibition of p38 MAPK compromised BMP2-induced Osx up-regulation. Wild-type OBs were serum starved for 12 h, pretreated with 15 µM SB203580 (SB) for 1 h, and then further treated with BMP2 (10 or 50 ng/ml) for 12 h. Total RNA was isolated and used for RT-PCR. The basal level of Osx mRNA in wild-type cells was set at 1.0. D, Western blot shows that inhibition of p38 MAPK compromised BMP2-induced Osx up-regulation at the protein level. OBs were starved from serum overnight, pretreated with 10 µM SB203580, and then stimulated with 10 ng/ml of BMP2 for 6 or 12 h. The basal level of Osx mRNA or protein in wild-type cells was set at 1.0. E, Inhibition of p38 MAPK compromised the Osx promoter activity in a reporter assay. MC3T3 cells were transfected with the reporter construct (1 µg per 35-mm plate), cultured for 48 h in the presence of different concentration of SB203580, PD98059 (PD), or BMP2, and then collected for luciferase assay. F, Coexpression of dominant-negative (DN) p38 MAPK inhibited the Osx promoter activity. Each of these constructs (1 µg per 35-mm plate) was cotransfected into MC3T3 cells with the reporter gene, and luciferase activity was measured 48 h after transfection. G, Inhibition of p38 MAPK decreased the protein levels of p53. OBs were cultured in differentiation medium for different periods of time in the presence or absence of 10 µM SB203580. The protein levels of p53 were determined by Western blot analysis. *, P < 0.05 when compared with untreated controls.

 
We then tested whether inhibition of p38 MAPK, which is activated either by BMPs or by other factors in the serum, directly affect BMP2-induced Osx up-regulation in our experimental settings. OBs were pretreated with 10 or 15 µM of SB203580 and then different amounts of BMP2 were added to the culture. The cells were collected and the mRNA levels of Osx were determined by RT-PCR. It was found that inhibition of p38 MAPK compromised the induction of Osx at mRNA and protein levels by BMP2 (Fig. 6Go, C and D, and data not shown). These results suggest that BMP2-activated p38 MAPK is required for maximal induction of Osx. Similar results have been reported in human MSC (28, 29). To further understand at what level p38 MAPK regulates expression of Osx, we tried a reporter assay in which luciferase is driven by Osx promoter, which could be activated by BMP2. It was found that inhibition of p38 MAPK could reduce the promoter activity of the Osx, whereas inhibition of ERKs even showed a slight enhancement (Fig. 6EGo). Coexpression of dominant-negative p38{alpha} MAPK gave rise to the same results, whereas wild-type p38{alpha} MAPK showed a minimal effect (Fig. 6FGo). These results confirmed that p38 MAPK regulates the expression of Osx through transcription. Taken together, these results suggest that BMPR-Smad1/5/8 pathway and the p38 MAPK pathway might cooperate in induction of Osx. Blockade of either pathway negatively affects Osx expression and OB differentiation.

We have shown that both p53 and p38 MAPK regulate the expression of Osx (23). Is it possible that p53 mediates the effect of p38 MAPK on OB differentiation? If it is true, we would see that inhibition of p38 MAPK up-regulates p53, leading to inhibition of Osx expression. We harvested OBs cultured in differentiation medium for different periods of time in the presence or absence of 10 µM SB203580. The p53 protein levels were determined by Western blot analysis. It appeared that inhibition of p38 MAPK led to a slight reduction in the p53 levels (Fig. 6GGo), which theoretically would result in an up-regulation of Osx. These results suggest that p53 is unlikely to mediate the effect of p38 MAPK on OB differentiation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
p38 MAPK is known to play an important role in stress response. It is also involved in cell differentiation (30). Two of the best studied examples are osteoclasts and myocytes (31, 32, 33). p38 MAPK can phosphorylate MEF2 and E47 (a MyoD partner) to regulate the expression of myoblast-specific genes and thereafter myoblast differentiation (34). The mechanisms whereby p38 MAPK regulates osteoclast differentiation are not well understood. Previous studies have shown that p38 MAPK activation plays important roles in differentiation of primary calvarial OBs, bone marrow osteoprogenitor cells, and some immortalized OB or stromal cell lines. In this study, we provided evidence that one mechanism by which p38 MAPK (mainly {alpha} and ß isoforms) regulates OB differentiation is through up-regulating the expression of Osx. This conclusion is supported by the following observations: 1) inhibition of p38 MAPK leads to reduction of Osx at both mRNA and protein levels, correlated with diminished OB differentiation; 2) a knockout cell culture (p53–/– OBs), which has elevated Osx levels, is refractory to the negative effect of p38 MAPK inhibition; 3) more importantly, normal OBs with elevated expression of Osx with a retroviral vector show resistance to the negative effect of p38 MAPK inhibition; and 4) BMP2 induces differentiation of OBs and the expression of Osx, and this can be compromised by p38 MAPK inhibition. Collectively, these results strongly suggest that Osx channels the effect of p38 MAPK on OB differentiation. Although previous reports have studied the role for p38 MAPK in the expression of ALP and osteocalcin (8, 28), our studies identified a link between p38 MAPK and a master transcription factor Osx, which controls not only the expression of many OB markers but also bone mineralization (Fig. 7Go). Moreover, these findings also suggest that other intracellular stimuli, which regulate p38 MAPK pathway, might modulate the expression of Osx to control OB differentiation.


Figure 7
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  		FIG. 7. A schematic diagram showing participation of p38 MAPK and BMPR-Smad1/5/8 in regulation of Osx expression. Dotted lines indicate that confirmation is needed. X, An unknown protein that is induced by BMPs and activates Osx transcription.

 
How does p38 MAPK regulate Osx expression? It is known that Osx expression is induced by BMPs (35). Here we confirmed that BMPs, provided by the serum or secreted by OBs themselves, are required for in vitro OB differentiation. Blockade of BMP actions by noggin and chordin down-regulated the levels of Osx and inhibited OB differentiation. We also confirmed that BMPs are sufficient for Osx up-regulation. Addition of BMP2 up-regulated mRNA levels of Osx and promoted OB differentiation. Yet the available data suggest that Osx is not a direct target gene of BMP-Smad1/5/8, based on two facts. First, induction of Osx mRNA by BMP2 needs 6–8 h, whereas induction of Id1, a known direct target gene, only requires 30 min. Second, blockade of protein synthesis by cycloheximide appeared to inhibit Osx induction by BMP2, suggesting that an unidentified BMP direct target gene controls Osx expression (data not shown). How does p38 MAPK fit in this scenario? It is well known that BMPs activate the p38 MAPK pathway. Here we show that activation of p38 MAPK is required for maximal Osx up-regulation induced by BMP2 in primary calvarial OBs and that p38 MAPK works on the promoter of Osx. These observations suggest that p38 MAPK might be involved in either the expression or the activation of the unidentified transcription factor that is responsible for Osx transactivation (Fig. 7Go). Future efforts will focus on identification of the cis-elements and transcription factors that control Osx transcription and a possible link of p38 MAPK to these transcription factors. Although our and others’ studies indicate that p38 MAPK plays a positive role in Osx expression during OB differentiation, it does not play a significant role in the transcriptional regulation of Osx in response to tumor necrosis factor (36). Neither does it play a significant role in Osx expression controlled by p53 (23). These results indicate that multiple pathways exist that regulate Osx expression.

p53–/– OBs are insensitive to the negative effect of p38 MAPK inhibition on OB differentiation. This is likely caused by elevated expression of Osx in p53–/– OB. Studies in tumorigenesis suggest that p38 acts upstream of p53 and functions as a tumor suppressor. p38 MAPK is reported to phosphorylate p53 to activate p53, leading to cell cycle arrest or apoptosis (37). Thus p38 MAPK and p53 have a similar function in tumor suppression. However, p38 MAPK and p53 appear to have opposite functions in OB differentiation. Although p38 MAPK is required for OB differentiation, p53 plays a negative role (23). Indeed, we found that inhibition of p38 MAPK down-regulated the protein levels of p53, which theoretically would augment Osx expression. Therefore, it is unlikely that p38 MAPK regulates Osx expression and OB differentiation via p53. Osx is essential for OB maturation and bone calcification. Ectopic expression of Osx enhances OB differentiation. Recent studies suggest that Osx might be a major mediator of OB differentiation in response to extracellular stimulations. p53–/– mice show osteosclerotic phenotypes and enhanced OB differentiation, accompanied by elevated levels of Osx, but not Runx2 or other transcription factors (23). Atm–/– and c-Abl–/– mice show osteoporotic phenotypes and defective OB differentiation (23, 26, 38). Deficiency of c-Abl or Atm also leads to down-regulation of Osx but not Runx2 or other transcription factors. Similarly, inhibition of p38 MAPK impedes OB differentiation, accompanied with a decrease in Osx but not Runx2. More importantly, manipulation of Osx levels by small interfering RNA or retroviral expression influences the differentiation process of normal or p53–/– OBs (23). A positive correlation between the levels of Osx and the advancement of differentiation was observed in all those cases. Another implication of these studies is that Atf4 and Dlx5 are probably not regulated by Osx and likely act upstream of Osx, as demonstrated for Runx2 (15).

In summary, this study contributes to our understanding of OB differentiation in several aspects. First, studies on normal OBs, p53–/– OBs, and OBs overexpressing Osx established a positive correlation between the levels of Osx and the advancement of OB differentiation, reinforcing the concept that Osx might be an important mediator from extracellular stimuli to OB differentiation. Second, p38 MAPK signaling pathway regulates OB differentiation via modulating the expression of Osx in response to BMPs and probably other signaling molecules (Fig. 7Go). Because blocking Smad1/5/8 activation impeded OB differentiation even in the presence of activated p38 MAPK (Figs. 4Go and 6BGo), cooperation of BMPR-Smad pathway and the p38 MAPK pathway may play crucial roles in controlling Osx expression. Third, this study showed that BMPs are important regulators of OB differentiation and their effects are likely mediated by Osx.


   Acknowledgments
 
We thank Drs. Wai Fook Leong and Fung Ling Jenny Chau for helpful discussions, and Hang In Ian, Wan Qing Leow, Linna Soh, Wai Fook Leong, and Kelvin Yong for technical support.


   Footnotes
 
This work was supported by the Agency for Science, Technology and Research of the Republic of Singapore.

X.W. and B.L. are adjunct members of the Department of Medicine of the National University of Singapore.

Disclosure Summary: The authors have nothing to declare.

First Published Online December 21, 2006

Abbreviations: ALP, Alkaline phosphatase; BMP, bone morphogenetic protein; MSC, mesenchymal stem cell; OB, osteoblast; Osx, osterix.

Received July 25, 2006.

Accepted for publication December 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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