| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609
Address all correspondence and requests for reprints to: Dr. Baojie Li, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore 117609. E-mail: libj{at}imcb.nus.edu.sg.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Osteoblasts mature from osteoprogenitors that reside in the bone marrow (4, 5). During differentiation, osteoblast first produces an unmineralized extracellular matrix called osteoid (mainly type I collagen), which becomes mineralized naturally and encases the osteoblast, forming an osteocyte. Such mineralization involves hydroxyapatite formation and is one of the functions of osteoblasts. Other characteristics include up-regulation of alkaline phosphatase (ALP) and up-regulation of osteocalcin that occur at later stages of osteoblast differentiation (6, 7). For in vitro studies, osteoblasts can be isolated from calvaria of newborn animals or from bone marrow that contains osteoprogenitor cells (8). The calvarial osteoblast culture is a relatively pure population and is widely used for cell proliferation and differentiation studies. These primary cells undergo natural differentiation when cultured, and the differentiation process can be induced by factors such as bone morphogenetic proteins (BMPs), members of the TGFß superfamily, via the Smad signaling pathway (9).
MAPKs play important roles in cellular response to growth factors, cytokines, or environmental stress. They are classified into four classes: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase or stress-activated protein kinase, p38 MAPKs, and ERK5 (10). ERKs are involved in cell proliferation/transformation and survival, whereas c-Jun N-terminal kinase is involved in stress responses. p38 MAPKs are involved in many cellular processes, such as inflammatory responses, cell cycle control, apoptosis, and cell fate specification in imaginal discs of Drosophila (11). Recent evidence also suggests a role of p38 MAPK in cell differentiation of adipocytes, neurons, and myocytes (12, 13, 14). p38 MAPKs have four isoforms, p38
, p38ß, p38
, and p38
. Although p38 and p38ß are ubiquitously expressed, p38 is mainly expressed in muscle, and p38 is mainly expressed in kidney and lung. Some stimuli can activate all of the p38 MAPKs, whereas others can only activate a particular subset of p38MAPKs (12). p38 knockout mice are embryonic lethal due to defective placental organogenesis or failed erythropoiesis (15, 16). During differentiation of adipocytes, neurons, and cardiac myocytes, p38 MAPK was found activated in an MAPK kinase 6 (MKK6)-dependent manner. Blockade of p38 MAPKs with specific inhibitor or dominant negative forms of p38 MAPK or MKK6 was found to hinder the differentiation process of these cell types (17, 18, 19, 20). The activated MAPKs regulate the activity of transcription factors such as myocyte enhancer factor-2, CCAAT/enhancer-binding protein (C/EBP), and c-Jun to control gene expression and therefore differentiation (13).
The roles of ERKs and p38 MAPKs in the differentiation of osteoblast are disputable. Although one study of C2C12 indicates that p38 MAPK is required for BMP-2-induced expression of ALP and osteocalcin, and ERKs are necessary only for osteocalcin expression (21), a similar study gives opposite results (22). Studies using MC3T3-E1 cells suggest that activation of p38 is critical for ALP expression induced by fetal calf serum or epinephrine (23, 24, 25), but is not required for ALP expression stimulated with ascorbic acid (24). To clarify this matter, we used primary calvarial osteoblast and primary bone marrow osteoprogenitor cells to test the effect of p38 MAPK in osteoblast differentiation. We analyzed not only early markers such as ALP and osteocalcin, but also a later marker, mineral deposition. Using these primary cells, we compared natural and BMP-2-induced differentiation. Finally, we used MC3T3-E1 cells expressing dominant negative p38 MAPK to further confirm our studies using the inhibitor for p38 MAPKs. All of the experiments convincingly support a role for p38 MAPK, but not ERKs, in osteoblast differentiation. Furthermore, we found that p38 MAPK was activated at an early stage during primary calvarial osteoblast differentiation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
MEM supplemented with 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD).
Cultures were trypsinized upon confluence and then subcultured into 6- or 12-well plates or 100-mm petri dishes. Once confluence (d 0), cells were treated with different concentrations of SB203580 or PD98059 for 4 d in the differentiation medium (MEM containing 100 µg/ml ascorbic acid and 10 mM ß-glycerophosphate; Sigma-Aldrich Corp., St. Louis, MO). The inhibitors were then washed off, and the cells were cultured in the differentiation medium. To promote osteoblast differentiation, BMP-2 was added to the differentiation medium to a final concentration of 50 ng/ml from d 0.
Alkaline phosphatase assay
Osteoblast cultures were stained for ALP activity using the ALP kit (Sigma-Aldrich Corp.) on d 4 unless otherwise indicated. To quantitate ALP activities, a semiquantitative method using -naphthyl phosphate as the substrate and Fast Blue salt (Sigma-Aldrich Corp.) as the diazonium salt was used. Briefly, cells were washed three times with ice-cold Tris-buffered saline, pH 7.4, and scraped immediately upon addition of ice-cold 50 mM Tris-buffered saline, and the collected lysates were sonicated for 20 sec at 4 C. The kinase assay was performed in assay buffer (10 mM MgCl2 and 0.1 M alkaline buffer, pH 10.3) containing 10 mM p-nitrophenylphosphate in alkaline buffer (3.71 mg/ml assay buffer) as the substrate. Tubes were incubated in a 37 C water bath and timed. The reaction was stopped by the addition of 0.3 N NaOH. Reaction mixtures were transferred into cuvettes, and absorbance was read at OD405. The relative ALP activity is defined as millimoles of p-nitrophenol phosphate hydrolyzed per minute per milligram of total protein (units).
von Kossa staining
After 2–4 wk in culture, cells were washed three times with PBS, fixed with neutral formalin solution for 5 min, and rinsed with deionized water. After the addition of 5% silver nitrate solution, the wells were exposed to UV light for 1 h. The reaction was stopped by treatment with sodium thiosulfate.
Western blot analysis
Cells were washed with cold PBS and lysed in cold 50 mM Tris (pH 7.5), 100 mM KCl, 1 mM EDTA, 0.5% Nonidet P-40, and 1 mM phenylmethylsulfonylfluoride) and phosphatase inhibitors. Lysates were clarified by centrifugation (10 min, 13,000 x g, 4 C). Protein concentrations were determined by the method developed by Bio-Rad Laboratories, Inc. (Hercules, CA). Twenty micrograms of total protein were fractionated onto a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). The membrane was blocked in 5% nonfat dry milk in Tris-buffered saline/Tween 20 buffer; probed with antiphosphorylated p38 MAPK, antiphosphorylated ERKs (Cell Signaling), anti-p38 MAPK (Santa Cruz Biotechnology, Inc.), or anti-ERK antibodies (Cell Signaling, Beverly, MA), followed by incubation with antirabbit antibodies conjugated with horseradish peroxidase; and visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL).
Northern blot analysis of osteocalcin mRNA
After a time-course treatment with or without inhibitors, cells were collected, and total RNA was isolated using TRIzol (Life Technologies, Inc.). RNA was quantitated at OD260 using a spectrophotometer. Ten micrograms of total RNA were fractionated onto a 1.5% formaldehyde agarose gel, transferred to Nytran membrane (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). Membrane filters were rinsed with 2x standard saline citrate and cross-linked under UV light. The membrane was then hybridized with RNA probes prepared from osteocalcin cDNA, and osteocalcin mRNA was detected after exposure to x-ray films.
Statistical analysis
Each experiment was repeated three times. Statistical analysis was performed using an unpaired t test (STATISTICA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, upper panel). We found that SB203580 inhibited ALP activity. On plates in the presence of 10 µM SB203580, ALP intensity was significantly weaker than that in controls, suggesting that inhibition of p38 MAPK reduced the expression of the early marker of osteoblast differentiation. PD98059, a specific inhibitor for ERKs, was used to determine the role of ERK in osteoblast differentiation. We found that ALP levels in the presence of the ERK inhibitor were similar to the control values even at 20 µM PD98059, a concentration demonstrated to inhibit ERK activity in osteoblasts (26), suggesting that inhibition of ERK had no effect on ALP expression. Furthermore, we used a quantitative ALP assay to confirm the results obtained from ALP staining. Similarly, cells were treated with different inhibitors for 4 d, cells were lysed, and ALP activities were determined with a reaction that turns the substrate p-nitrophenylphosphate to p-nitrophenol, which can be measured by absorbance reading at OD405. Protein levels were measured by the Bio-Rad Laboratories, Inc., method and were used to normalize ALP activity. We did not detect any significant change in protein levels in the presence of SB203580 or PD98059 (data not shown). Consistent with histochemical data, inhibition of p38 MAPK significantly reduced the expression of ALP (Fig. 1B), whereas ERK inhibitor had no significant effect. Our results are consistent with previous studies showing that p38 is required for ALP expression in MC3T3-E1 cells (23).
|
, A and B). Inhibition of p38 MAPK significantly suppressed BMP-2-induced expression of ALP, whereas inhibition of ERKs enhanced the the BMP-2 effect on ALP expression (Fig. 1, A and B). These results suggest that p38 activation is necessary for BMP-2-induced ALP expression and that ERK activation may play a negative role in BMP-2-induced ALP expression.
Inhibition of p38 MAPK suppressed osteocalcin expression and mineral deposition in calvarial osteoblasts
To determine whether activation of p38 MAPK is required for later stages of osteoblast differentiation, osteocalcin expression in the presence of SB203580 was compared with that in control cultures. Upon confluence (d 0), primary osteoblasts were treated with 10 µM SB203580 for 4 d in the differentiation medium and then continuously cultured without the inhibitor for up to 7, 14, or 21 d. Cells were then collected, and total RNA was isolated. Northern blot analysis was carried out to assess the expression levels of osteocalcin that were normalized to 28S rRNA. The experiments were repeated three times, and the results from a representative experiment are shown (Fig. 2A). We found that in the control cultures, osteocalcin mRNA started to increase from d 7 onward. However, in the presence of SB203580, the levels of osteocalcin were compromised on d 14 and 21. Quantitation analysis with densitometry revealed an average 40% inhibition of osteocalcin up-regulation (data not shown). These results suggest that activation of p38 MAPK is required for maximal induction of osteocalcin, yet to a much lesser extent than that of ALP. The reason for the difference is not clear.
|
), very little mineral deposition was observed. In the presence of BMP-2, mineral deposition was significantly increased, and SB203580 was found to inhibit mineralization in a dosage-dependent manner. On d 28, control cells showed increased mineral deposition that was dramatically suppressed by SB203580 (Fig. 2B). The presence of BMP-2 partially relieved the inhibition, suggesting that BMP-2 could partially overcome the inhibitory effect of SB203580 in osteoblast differentiation. In the absence of BMP-2, PD98059 treatment did not affect mineral deposition, but in the presence of BMP-2, PD98059 exhibited a stimulatory effect on mineral deposition, consistent with the finding that PD98059 enhanced ALP expression in the presence of BMP-2 (Fig. 1B).
Activation of p38 MAPK during calvarial osteoblast differentiation
We have demonstrated that inhibition of p38 MAPK interfered with the differentiation process of calvarial osteoblasts. We next investigated whether p38 MAPK was activated during osteoblast differentiation. Cell lysates were collected from primary calvarial osteoblasts at different stages of differentiation, and the same amount of total proteins was analyzed by Western blot using antibodies specifically recognizing the active form of p38 MAPK (Fig. 3). Compared with controls (d 0), the activity of p38 MAPK was increased from d 3 and was kept in the activated form until d 9 (Fig. 3), consistent with a role for p38 MAPK in early stages of osteoblast differentiation. The same blot was stripped and reprobed with anti-p38 MAPK antibody that recognizes total p38 MAPK. We found that the p38 MAPK level decreased slightly from d 3–9 (Fig. 3), suggesting that p38 MAPK activation was even underestimated. We conclude that p38 MAPK was activated during osteoblast differentiation at an early step.
|
), with a maximum on d 2 and 3, followed by decrease from d 4 onward. We conclude that ERK was activated during osteoblast differentiation, although they are not required for differentiation.
ALP expression and mineral deposition decreased in cell lines expressing the dominant negative p38
Although the pharmacological inhibitors are very useful for signal transduction studies, they could have side-effects. Other strategies include using the dominant negative version of p38 MAPK (kinase-deficient mutation), which inhibits endogenous p38 kinase activity. To test whether a dominant negative p38 MAPK has a similar effect as the inhibitor, cell lines stably expressing dominant negative p38 MAPK were established from MC3T3-E1, a murine calvarial osteoblast line that is widely used in the field. A construct expressing dominant negative p38 MAPK [tagged with hemagglutinin (HA)], along with a Neo-resistant gene was used to transfect MC3T3-E1 cells by the calcium phosphate method, and Neor clones were selected. The expression of p38 MAPK was confirmed by Western blot using anti-HA antibodies (Fig. 4A). Dominant negative p38 MAPK was stably expressed in clones 7, 14, and 18. Western blot analysis using anti-p38 MAPK antibodies that recognize both endogenous p38 and the dominant negative p38 MAPK did not reveal a significant increase in the level of p38 MAPK in the three stable cell lines, suggesting that dominant negative p38 MAPK is expressed at a level much lower than that of endogenous p38 MAPK. This could be due to the fact that interference with p38 MAPK activity by a dominant negative p38 is deleterious to cells. Therefore, only the cells expressing low levels of dominant negative p38 MAPK were selected.
|
). Figure 4C shows a microscopic view of the ALP staining. Although the parental strain showed some ALP-positive cells, dominant negative clone 7 showed much less and dominant negative clones 14 and 18 did not show any.
Mineral deposition in these stable lines and the parental line was assayed by von Kossa staining. The staining was extremely weak, and only microscopic views of 5-wk plates were shown (Fig. 4D). Mineralization in dominant negative clone 7 clone was reduced compared with that in the parental cells, and mineralization in dominant negative clones 14 and 18 cultures was missing. The more severe defect in differentiation observed in clones 14 and 18 correlates with the higher levels of dominant negative p38 MAPK expressed in these cells.
Inhibition of p38 MAPK suppressed bone marrow osteoprogenitor cell differentiation
We used primary and immortalized calvarial osteoblast cultures to demonstrate that inhibition of p38 MAPK interfered with the differentiation process of osteoblasts, manifested by the lower expression of ALP and lesser mineral deposition. We then studied the effect of inhibition of p38 MAPK on the differentiation of osteoprecursor cells isolated from the bone marrow to further support our conclusion drawn from studies using calvarial osteoblasts and MC3T3-E1. Bone marrow stromal cultures contain the precursors for osteoblasts in addition to adipocytes and fibroblasts. The precursor cells can spontaneously differentiate into osteoblasts, with the expression of ALP and mineral deposition. Bone marrow stromal cultures were prepared from 2-month-old mice and propagated to passage 2 before being plated for experiments. Similarly, bone marrow stromal cultures were treated with inhibitors for p38 MAPK or ERK for 4 d, and then ALP was stained on plates (Fig. 5A). SB203580 inhibited the expression of ALP at concentrations of 2.5 and 10 µM, such that staining intensity was weaker than that in controls. Quantitative assay confirmed these results (Fig. 5B). However, PD98059 did not have much effect on ALP activity, as the quantitative assays based on three separate experiments did not reveal a significant difference between control cell cultures and cell cultures in presence of 20 µM PD98059.
|
). Staining of d 7 plates was negative (data not shown). We detected a significant loss of mineralization in the presence of SB203580 on d 14, 21, and 28 plates, whereas PD98059 did not exhibit any dramatic effect, as the mineralization level was similar to that in the controls. Taken together, these data suggest that p38 MAPK activation is necessary for the differentiation of bone marrow osteoprogenitor cells. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We provided evidence to support a role for p38 MAPK in the differentiation of another cell type, the osteoblast. To date, activation of p38 MAPK has been implicated in the differentiation of a variety of cell types, including neurons, myocytes, adipocytes, chondrocytes, osteoclasts, intestinal epithelia, and thymocytes (reviewed in Refs.27 and28). These studies suggest that p38 MAPK may play a general role in the cell differentiation process, probably through regulating different transcription factors, such as activating transcription factor-2, p53, growth arrest and DNA damage inducible 153 (GADD153), C/EBP, and myocyte enhancer factor-2. In most of these studies, a p38 MAPK-specific inhibitor, SB230580, was used. Based on the facts that SB203580 mainly targets p38 and p38ß and that we used the dominant negative p38 in stable line studies, we believe that it is the p38 isoform that is important for osteoblast differentiation.
We also demonstrated that ERKs did not play a necessary role in osteoblast differentiation. Blockage of ERKs with a specific drug had no significant effect on either calvarial or bone marrow stromal osteoprogenitor cell differentiation, although ERK activation was observed at an early stage during osteoblast differentiation. The ERK signaling pathway has been known to be involved in cell proliferation and cell survival. Our results are not in agreement with findings made by Lai et al. (28), who found that expression of dominant negative ERK1 not only inhibited human osteoblast differentiation, but also proliferation, cell spreading, and migration. One explanation for the discrepancy is the cell source. Osteoblasts isolated from human bone chips were used in these experiments. We used primary calvarial osteoblasts and bone marrow stromal cultures of mouse origin, and these cells are probably at an earlier stage of maturation. Another possibility is the difference between the inhibitor and the dominant negative form of ERK1. Dominant negative ERK1 also affects cell growth, whereas PD98059 does not. It is likely that the growth condition affects differentiation. There is another report showing that ERKs may phosphorylate core binding factor 1 (Cbfa1) and therefore affect osteoblast differentiation; they found that the concentration of PD98059 required to maximally inhibit osteocalcin up-regulation is 100 µM. In our experiments the concentration is 20 µM, which is sufficient to inhibit the ERKs in calvarial osteoblasts (26). The inhibitor for ERKs was present for only 4 d and was washed away. Higher concentrations of PD98059 may inhibit the activation of other kinases. Based upon our results, ERK activation may not play a necessary role in osteoblast differentiation. On the contrary, our results indicate that ERKs may play a negative role in BMP-2-induced osteoblast differentiation.
Osteoblast differentiation is stimulated by local factors such as BMPs. Inhibition of p38 MAPK caused a delay in osteoblast differentiation. This delay was partially released by the presence of BMP-2, suggesting that BMP-2 is able to overcome the loss of signals from p38 MAPKs during osteoblast differentiation. Activation of p38 MAPKs by BMP-2 in osteoblast has been documented, although to a lesser extent (25, 29). BMPs control osteoblast differentiation through Smad proteins that are inhibited by Tob (30). Activation of the BMP-Smad pathway may somehow compensate for the loss of p38 MAPK. Further experiments will be needed to investigate the mechanism.
How does p38 MAPK participate in osteoblast differentiation? As a serine/threonine kinase, it is likely that p38 MAPK transmits signals via phosphorylation of transcription factors directly or indirectly by activating other kinases that subsequently phosphorylate its downstream targets. When phosphorylated by p38 MAPK directly or its downstream kinases, these transcription factors either activate their trans-activation ability or increase their DNA binding affinity. The ultimate outcome is specific up-regulation of a set of genes that are involved in initiating or maintaining differentiation. In adipocytes, p38 MAPKs were known to phosphorylate C/EBPß and regulate adipocyte-specific gene expression (17). C/EBP proteins have also been demonstrated to synergize with Runx2/Cbfa1 to control osteoblast-specific genes such as osteocalcin (31). Cbfa1 is a master transcription factor for the osteoblast lineage. A mouse deficient in Runx2/Cbfa1 completely lacks bones (32, 33). Another possible candidate is serum response factor, which is known to be regulated by p38 MAPK and plays an important role in osteoblast differentiation (34). Recently, a novel zinc finger transcription factor, osterix, has been found to be required for osteoblast differentiation and bone formation and to function downstream of Runx2/Cbfa1 (35). Whether osterix is a target of p38 MAPK needs further investigation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: ALP, Alkaline phosphatase; BMP, bone morphogenetic protein; Cbfa1, core binding factor 1; C/EBP, CCAAT/enhancer-binding protein; ERK, extracellular signal-regulated kinase; HA, hemagglutinin.
Received August 19, 2002.
Accepted for publication January 28, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
MAP kinase in placental but not embryonic cardiovascular development. Mol Cell 6:109–116[CrossRef][Medline]
in erythropoietin expression: a role for stress kinases in erythropoiesis. Cell 102:221–231[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  E. Grenier, F. S. Maupas, J.-F. Beaulieu, E. Seidman, E. Delvin, A. Sane, E. Tremblay, C. Garofalo, and E. Levy Effect of retinoic acid on cell proliferation and differentiation as well as on lipid synthesis, lipoprotein secretion, and apolipoprotein biogenesis Am J Physiol Gastrointest Liver Physiol, December 1, 2007; 293(6): G1178 - G1189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Caverzasio and D. Manen Essential Role of Wnt3a-Mediated Activation of Mitogen-Activated Protein Kinase p38 for the Stimulation of Alkaline Phosphatase Activity and Matrix Mineralization in C3H10T1/2 Mesenchymal Cells Endocrinology, November 1, 2007; 148(11): 5323 - 5330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, C. H. Goh, and B. Li p38 Mitogen-Activated Protein Kinase Regulates Osteoblast Differentiation through Osterix Endocrinology, April 1, 2007; 148(4): 1629 - 1637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Pino, J. M. Rodriguez, S. Rios, P. Astudillo, L. Leiva, G. Seitz, M. Fernandez, and J P. Rodriguez Aromatase activity of human mesenchymal stem cells is stimulated by early differentiation, vitamin D and leptin J. Endocrinol., December 1, 2006; 191(3): 715 - 725. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Lu, L. Gilbert, X. He, J. Rubin, and M. S. Nanes Transcriptional Regulation of the Osterix (Osx, Sp7) Promoter by Tumor Necrosis Factor Identifies Disparate Effects of Mitogen-activated Protein Kinase and NF{kappa}B Pathways J. Biol. Chem., March 10, 2006; 281(10): 6297 - 6306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-L. Kuo, Y.-L. Hsu, C.-H. Chang, and J.-K. Chang Osthole-Mediated Cell Differentiation through Bone Morphogenetic Protein-2/p38 and Extracellular Signal-Regulated Kinase 1/2 Pathway in Human Osteoblast Cells J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1290 - 1299. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
