p38 MAP kinase regulates BMP-4-stimulated VEGF synthesis via p70 S6 kinase in osteoblasts

Haruhiko Tokuda1,2, Daijiro Hatakeyama2,3, Toshiyuki Shibata3, Shigeru Akamatsu4, Yutaka Oiso5, and Osamu Kozawa2

1 Department of Internal Medicine, Chubu National Hospital, National Institute for Longevity Sciences, Obu, Aichi 474-8511; Departments of 2 Pharmacology, 3 Oral and Maxillofacial Surgery, and 4 Critical Care Medicine, Gifu University School of Medicine, Gifu 500-8705; and 5 First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466-8550, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously reported that p70 S6 kinase takes part in bone morphogenetic protein-4 (BMP-4)-stimulated vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. Recently, we showed that BMP-4-induced osteocalcin synthesis is regulated by p44/p42 MAP kinase and p38 MAP kinase in these cells. In the present study, we investigated whether the MAP kinases are involved in the BMP-4-stimulated synthesis of VEGF in MC3T3-E1 cells. PD-98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, failed to affect BMP-4-stimulated VEGF synthesis. SB-203580 and PD-169316, inhibitors of p38 MAP kinase, significantly reduced VEGF synthesis, whereas SB-202474, a negative control for p38 MAP kinase inhibitor, had little effect on VEGF synthesis. The BMP-4-stimulated phosphorylation of p38 MAP kinase was not affected by rapamycin, an inhibitor of p70 S6 kinase. On the contrary, SB-203580 and PD-169316 reduced the BMP-4-stimulated phosphorylation of p70 S6 kinase. In addition, anisomycin, an activator of p38 MAP kinase, phosphorylates p70 S6 kinase, and the phosphorylation was suppressed by SB-203580. LY-294002, an inhibitor of phosphatidylinositol 3-kinase, failed to suppress the phosphorylation of p38 MAP kinase induced by BMP-4. Not BMP-4 but anisomycin weakly induced the phosphorylation of phosphoinositide-dependent kinase-1. However, anisomycin had little effect on phosphorylation of either Akt or the mammalian target of rapamycin. Taken together, our results suggest that p38 MAP kinase functions in BMP-4-stimulated VEGF synthesis as a positive regulator at a point upstream from p70 S6 kinase in osteoblasts.

bone-morphogenetic protein; vascular endothelial growth factor; mitogen-activated protein kinase; osteoblast


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) has been characterized as a heparin-binding angiogenic factor that displays high specificity for endothelial cells (26). VEGF, which induces endothelial cell proliferation, angiogenesis, and capillary permeability, is produced and secreted from many cell types (26). It is well known that bone metabolism is regulated by osteoblasts and osteoclasts, responsible for bone formation and bone resorption, respectively (27). The formation of bone structures and bone remodeling result from the coupling process, bone resorption by osteoclasts, and subsequent deposition of new matrix by osteoblasts. During bone remodeling, capillary endothelial cells provide the microvasculature. It is currently recognized that the activity of osteoblasts, osteoclasts, and vascular endothelial cells are closely coordinated with one another to promote the bone-remodeling process (4). As for osteoblasts, it has been reported that prostaglandin (PG)E2, PGE1, and insulin-like growth factor I stimulate the synthesis of VEGF in osteoblasts (8, 9). Accumulating evidence suggests that VEGF secreted from osteoblasts plays important roles in bone remodeling. It has been reported that VEGF is an essential coordinator of extracellular matrix remodeling, angiogenesis, and bone formation in the growth plate (7). However, the exact mechanism underlying VEGF production in osteoblasts has not yet been precisely clarified.

Bone-morphogenetic proteins (BMPs) are multifunctional cytokines that were originally identified by their ability to form ectopic bone (14, 29). BMPs belong to the transforming growth factor-beta (TGF-beta ) superfamily (14, 29). BMPs are recognized as crucial regulatory factors in the early development of vertebrates (10). Osteoblasts reportedly synthesize BMP-2 and BMP-4, which stimulate alkaline phosphatase activity and the expression of osteocalcin, markers of mature osteoblast phenotype (2, 35, 36). The intracellular signaling of BMPs is mediated by Smad proteins such as Smad 1 and Smad 5, similar to TGF-beta (12, 14, 24). In addition to the Smad-signaling pathway, other signaling pathways have recently been shown to mediate TGF-beta superfamily signaling (13). We have previously reported (16) that BMP-4 stimulates the synthesis of VEGF in osteoblast-like MC3T3-E1 cells and that p70 S6 kinase is involved in the synthesis. Additionally, it has recently been shown that BMP-2 stimulates the phosphatidylinositol (PI) 3-kinase /p70 S6 kinase and p38 mitogen-activated protein (MAP) kinase cascades, which have a negative role in osteoblast differentiation (32). The MAP kinase superfamily plays a crucial role in the intracellular signaling of a variety of agonists (34). The three MAP kinases, p44/p42 MAP kinase, p38 MAP kinase, and stress-activated protein kinase/c-Jun NH2-terminal kinase, are known as central elements used by mammalian cells to transduce the diverse messages (34). It has been reported that p44/p42 MAP kinase is involved in BMP-2-induced osteoblast differentiation (23). We have recently shown (15) that BMP-4-stimulated osteocalcin synthesis is regulated by p44/p42 MAP kinase and p38 MAP kinase in MC3T3-E1 cells. In the present study, we investigated the possible involvement of the MAP kinases in BMP-4-stimulated VEGF synthesis in these cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. BMP-4 and mouse VEGF ELISA kits were purchased from R&D Systems (Tokyo, Japan). PD-98059, U-0126, SB-203580, PD-169316, SB-202474, rapamycin, anisomycin, and LY-294002 were obtained from Calbiochem-Novabiochem (La Jolla, CA). Phosphospecific p38 MAP kinase antibodies, p38 MAP kinase antibodies, phosphospecific p70 S6 kinase antibodies, p70 S6 kinase antibodies, phosphospecific phosphoinositide-dependent kinase (PDK)-1 antibodies, PDK-1 antibodies, phosphospecific Akt antibodies, and phosphospecific mammalian target of rapamycin (mTOR) antibodies were purchased from New England Biolabs (Beverly, MA). An ECL Western blotting system was obtained from Amersham Japan (Tokyo, Japan). Other materials and chemicals were obtained from Nacalai Tesque, (Kyoto, Japan). PD-98059, U-0126, SB-203580, PD-169316, SB-202474, rapamycin, and anisomycin were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect the assay for VEGF or Western blot analysis.

Cell culture. Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria (30) were maintained as previously described (17). In brief, the cells were cultured in alpha -minimum essential medium (alpha -MEM) containing 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% CO2-95% air. The cells were seeded into 35- or 90-mm-diameter dishes in alpha -MEM containing 10% FCS. After 5 days, the medium was exchanged for alpha -MEM containing 0.3% FCS. The cells were used for experiments after 24 h.

Assay for VEGF. The cultured cells were stimulated by BMP-4 in alpha -MEM containing 0.3% FCS for 48 h. The cells were pretreated with PD-98059, U-0126, SB-203580, PD-169316, or SB-202474 for 60 min as previously described (15). The reaction was terminated by collecting the medium, and VEGF in the medium was measured by a VEGF ELISA kit.

Western blot analysis. The cultured cells were stimulated by BMP-4 or anisomycin in alpha -MEM for the indicated periods. The cells were washed twice with phosphate-buffered saline (PBS) and then lysed, homogenized, and sonicated in a lysis buffer containing 62.5 mM Tris · HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol, and 10% glycerol. The cytosolic fraction was collected as the supernatant after centrifugation at 125,000 g for 10 min at 4°C. The soluble fraction containing 20 µg of protein was loaded, and SDS-polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli (20) in 10% polyacrylamide gel. Western blot analysis was performed as previously described (25) by using phosphospecific p38 MAP kinase antibodies, p38 MAP kinase antibodies, phosphospecific p70 S6 kinase antibodies, p70 S6 kinase antibodies, phosphospecific PDK-1 antibodies, PDK-1 antibodies, phosphospecific Akt antibodies, or phosphospecific mTOR antibodies, with peroxidase-labeled antibodies raised in goat against rabbit IgG being used as secondary antibodies. Peroxidase activity on the nitrocellulose sheet was visualized on X-ray film by means of the ECL Western blotting detection system.

Protein determination. Protein concentrations in soluble extracts were determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA), with BSA as the standard protein. The absorbance of ELISA samples was measured at 450 nm with SLT-Labinstruments EAR 340 AT. Absorbance was correlated with concentration through a standard curve. The concentration of VEGF obtained (pg/ml) was adjusted against cell number at the end of incubation and presented as VEGF synthesis (pg/1 × 106 cells). A densitometric analysis was performed using Molecular Analyst/Macintosh (Bio-Rad Laboratories).

Statistical analysis. The data were analyzed by ANOVA followed by the Bonferroni method for multiple comparisons between pairs, and P < 0.05 was considered significant. All data are presented as means ± SD of triplicate determinations. Each experiment was repeated three times with similar results.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of PD-98059 or U-0126 on BMP-4-induced VEGF synthesis in MC3T3-E1 cells. We previously reported (15) that BMP-4 activates p44/p42 MAP kinase, resulting in negatively regulating the BMP-4-induced osteocalcin synthesis in MC3T3-E1 cells. To clarify whether p44/p42 MAP kinase is involved in the BMP-4-stimulated VEGF synthesis in these cells, we examined the effect of PD-98059, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (1), on the synthesis of VEGF. We have previously demonstrated (15) that PD-98059 (50 µM) significantly reduces the phosphorylation of p44/p42 MAP kinase induced by BMP-4 in MC3T3-E1 cells. In this study, PD-98059 at 30 µM markedly reduced the BMP-4-induced phosphorylation of p44/p42 MAP kinase (Fig. 1A). However, PD-98059 had little effect on BMP-4-induced VEGF synthesis in the range between 1 and 30 µM (Fig. 1B). We further examined the effect of U-0126, another specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (5), on the VEGF synthesis induced by BMP-4. U-0126 (3 µM) definitely reduced the BMP-4-induced phosphorylation of p44/p42 MAP kinase (Fig. 2A). However, U-0126 (3 µM), which by itself hardly affected VEGF synthesis, had little effect on the BMP-4-induced VEGF synthesis in these cells (Fig. 2B). We confirmed that the cell number changed little before and after these treatments (data not shown).


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Fig. 1.   Effects of PD-98059 on bone-morphogenetic protein (BMP)-4-induced p44/p42 MAP kinase phosphorylation and vascular endothelial growth factor (VEGF) synthesis in MC3T3-E1 cells. A: cultured cells were pretreated with 30 µM PD-98059 or vehicle for 60 min and then stimulated by 30 ng/ml BMP-4 or vehicle for 90 min. Extracts of cells were subjected to SDS-PAGE against phosphospecific p44/p42 MAP kinase or p44/p42 MAP kinase antibodies. Histogram shows quantitative representations of BMP-4-induced phosphorylation of p44/p42 MAP kinase obtained from laser densitometric analysis after normalization to the control of 3 different sample sets. B: cultured cells were pretreated with various doses of PD-98059 for 60 min and then stimulated by 30 ng/ml BMP-4 () or vehicle (open circle ) for 48 h. PD-98059 was present throughout the incubation. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations. *P < 0.05 compared with the value of BMP-4 alone.



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Fig. 2.   Effects of U-0126 on BMP-4-induced p44/p42 MAP kinase phosphorylation and VEGF synthesis in MC3T3-E1 cells. A: cultured cells were pretreated with 3 µM U-0126 or vehicle for 60 min and then stimulated by 30 ng/ml BMP-4 or vehicle for 90 min. Extracts of cells were subjected to SDS-PAGE against phosphospecific p44/p42 MAP kinase or p44/p42 MAP kinase antibodies. Histogram shows quantitative representations of BMP-4-induced phosphorylation of p44/p42 MAP kinase obtained from laser densitometric analysis after normalization to the control of 3 different sample sets. B: cultured cells were pretreated with 3 µM U-0126 or vehicle for 60 min and then stimulated by 30 ng/ml BMP-4 or vehicle for 48 h. U-0126 was present throughout the incubation. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations. *P < 0.05 compared with the value of BMP-4 alone.

Effects of SB-203580 or PD-169316 on BMP-4-induced VEGF synthesis in MC3T3-E1 cells. We have shown (15) that BMP-4-activated p38 MAP kinase positively regulates the BMP-4-induced osteocalcin synthesis in MC3T3-E1 cells. To investigate the involvement of p38 MAP kinase in the synthesis of VEGF stimulated by BMP-4 in these cells, we next examined the effect of SB-203580, an inhibitor of p38 MAP kinase (3) on VEGF synthesis. SB-203580, which alone had little effect on the level of VEGF, significantly inhibited the VEGF synthesis stimulated by BMP-4 (Fig. 3A). The inhibitory effect of SB-203580 was dose dependent in the range between 1 and 30 µM. We confirmed that the cell number changed little by the treatment (8.1 ± 0.2 × 105 cells before incubation; 8.0 ± 0.3 × 105 cells after incubation with 30 µM SB-203580 and 30 ng/ml BMP-4; 7.9 ± 0.3 × 105 cells after incubation with 30 µM SB-203580 alone). In addition, PD-169316, another inhibitor of p38 MAP kinase (18), which by itself had little effect on VEGF level, markedly reduced the BMP-4-stimulated VEGF synthesis (Fig. 3B). The inhibitory effect of PD-169316 was dose dependent between 0.1 and 30 µM. The cell number was not affected by the treatment (data not shown).


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Fig. 3.   Effects of SB-203580 or PD-169316 on BMP-4-induced VEGF synthesis in MC3T3-E1 cells. Cultured cells were pretreated with various doses of SB-203580 (A), PD-169316 (B), or vehicle for 60 min and then stimulated by 30 ng/ml BMP-4 () or vehicle (open circle ) for 48 h. SB-203580 or PD-169316 was present throughout the incubation. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations. *P < 0.05 compared with the value of BMP-4 alone.

Effect of SB-202474 on BMP-4-induced VEGF synthesis in MC3T3-E1 cells. To clarify the involvement of p38 MAP kinase in the BMP-4-induced VEGF synthesis in MC3T3-E1 cells, we further examined the effect of SB-202474, a negative control for p38 MAP kinase inhibitor (22), on VEGF synthesis. SB-202474 (10 µM) failed to affect the VEGF synthesis induced by BMP-4, whereas SB-203580 (10 µM) significantly inhibited VEGF synthesis (Fig. 4). The cell number was not affected by the treatment (data not shown).


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Fig. 4.   Effects of SB-202474 or SB-203580 on BMP-4-induced VEGF synthesis in MC3T3-E1 cells. Cultured cells were pretreated with 10 µM SB-202474, 10 µM SB-203580, or vehicle for 60 min and then stimulated by 30 ng/ml BMP-4 (filled bars) or vehicle (open bars) for 48 h. SB-202474 or SB-203580 was present throughout the incubation. Each value represents the mean ± SD of triplicate determinations. *P < 0.05 compared with the value of BMP-4 alone or the value of BMP-4 with SB-202474 pretreatment. Similar results were obtained with 2 additional and different cell preparations.

Effects of rapamycin or LY-294002 on BMP-4-induced p38 MAP kinase phosphorylation in MC3T3-E1 cells. In a previous study (16), we reported that p70 S6 kinase and PI 3-kinase are involved in BMP-4-stimulated VEGF synthesis in osteoblast-like MC3T3-E1 cells. To clarify whether p70 S6 kinase affects the BMP-4-stimulated p38 MAP kinase activation in these cells, we examined the effect of rapamycin, a specific inhibitor of p70 S6 kinase (19, 28), on the phosphorylation of p38 MAP kinase induced by BMP-4. We previously demonstrated (16) that 30 ng/ml rapamycin significantly reduced the phosphorylation of p70 S6 kinase in these cells. However, rapamycin did not affect the BMP-4-induced p38 MAP kinase phosphorylation (Fig. 5). We next examined the effect of LY-294002, an inhibitor of PI 3-kinase (33), on the phosphorylation of p38 MAP kinase induced by BMP-4 in these cells. LY-294002 (10-50 µM) failed to suppress the BMP-4-induced phosphorylation of p38 MAP kinase (data not shown).


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Fig. 5.   Effect of rapamycin on BMP-4-induced p38 MAP kinase phosphorylation in MC3T3-E1 cells. Cultured cells were pretreated with 30 ng/ml rapamycin or vehicle for 20 min and then stimulated by 30 ng/ml BMP-4 or vehicle for 90 min. Extracts of cells were subjected to SDS-PAGE against phosphospecific p38 MAP kinase or p38 MAP kinase antibodies. Histogram shows quantitative representations of BMP-4-induced phosphorylation of p38 MAP kinase obtained from laser densitometric analysis after normalization to the control of 3 different sample sets. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations.

Effect of SB-203580 on BMP-4-induced p70 S6 kinase phosphorylation in MC3T3-E1 cells. To investigate whether p38 MAP kinase affects the phosphorylation of p70 S6 kinase stimulated by BMP-4 in MC3T3-E1 cells, we examined the effect of SB-203580 on p70 S6 kinase phosphorylation. SB-203580 markedly reduced the BMP-4-induced p70 S6 kinase phosphorylation (Fig. 6). According to the densitometric analysis, SB-203580 caused an almost complete reduction in the BMP-4 effect.


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Fig. 6.   Effect of SB-203580 on BMP-4-induced p70 S6 kinase phosphorylation in MC3T3-E1 cells. Cultured cells were pretreated with 30 µM SB-203580 or vehicle for 60 min and then stimulated by 30 ng/ml BMP-4 or vehicle for 3 h. Extracts of cells were subjected to SDS-PAGE against phosphospecific p70 S6 kinase or p70 S6 kinase antibodies. Histogram shows quantitative representations of BMP-4-induced phosphorylation of p70 S6 kinase obtained from laser densitometric analysis after normalization to the control of 3 different sample sets. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations. *P < 0.05 compared with the value of BMP-4 alone.

Effect of anisomycin on phosphorylation of p70 S6 kinase in MC3T3-E1 cells. To clarify the relationship between p38 MAP kinase and p70 S6 kinase in MC3T3-E1 cells, we examined the effect of anisomycin, an activator of p38 MAP kinase (11), on the phosphorylation of p70 S6 kinase. We confirmed that anisomycin phosphorylates p38 MAP kinase in these cells (Fig. 7). p38 MAP kinase was time dependently phosphorylated by anisomycin. The maximum effect of anisomycin on the phosphorylation of p38 MAP kinase was observed at 20 min after the stimulation. In addition, anisomycin phosphorylates p70 S6 kinase in MC3T3-E1 cells (Fig. 7). The maximum effect on the p70 S6 kinase phosphorylation was observed at 60 min after the stimulation.


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Fig. 7.   Effect of anisomycin on the phosphorylation of p38 MAP kinase or p70 S6 kinase in MC3T3-E1 cells. Cultured cells were stimulated by 0.1 µM anisomycin for the indicated periods. Extracts of cells were subjected to SDS-PAGE against phosphospecific p38 MAP kinase, p38 MAP kinase, phosphospecific p70 S6 kinase, or p70 S6 kinase antibodies.

Effect of SB-203580 on anisomycin-induced phosphorylation of p70 S6 kinase in MC3T3-E1 cells. To elucidate the relationship between p38 MAP kinase and p70 S6 kinase in MC3T3-E1 cells, we further examined the effect of SB-203580 on the anisomycin-induced phosphorylation of p70 S6 kinase. SB-203580 significantly reduced the anisomycin-stimulated phosphorylation of p70 S6 kinase (Fig. 8).


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Fig. 8.   Effect of SB-203580 on anisomycin-induced p70 S6 kinase phosphorylation in MC3T3-E1 cells. Cultured cells were pretreated with 20 µM SB-203580 or vehicle for 60 min and then stimulated by 0.1 µM anisomycin or vehicle for 3 h. Extracts of cells were subjected to SDS-PAGE against phosphospecific p70 S6 kinase or p70 S6 kinase antibodies. Histogram shows quantitative representations of anisomycin-induced phosphorylation of p70 S6 kinase obtained from laser densitometric analysis after normalization to the control of 3 different sample sets. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations. *P < 0.05 compared with the value of anisomycin alone.

Effects of BMP-4 or anisomycin on phosphorylation of PDK-1 in MC3T3-E1 cells. It is generally recognized that PDK-1 and mTOR are involved in the activation of p70 S6 kinase (31). We examined the effects of anisomycin or BMP-4 on the phosphorylation of PDK-1 in MC3T3-E1 cells. Anisomycin (0.1 µM) induced the phosphorylation of PDK-1 in parallel with that of p38 MAP kinase (Fig. 9). However, BMP-4 (30 ng/ml) had little effect on the phosphorylation of PDK-1 (Fig. 9). Furthermore, anisomycin affected neither mTOR nor Akt in these cells (data not shown).


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Fig. 9.   Effect of BMP-4 or anisomycin on the phosphorylation of PDK-1 in MC3T3-E1 cells. Cultured cells were stimulated by 30 ng/ml BMP-4 for the indicated periods or 0.1 µM anisomycin or vehicle for 120 min. Extracts of cells were subjected to SDS-PAGE against phosphospecific p38 MAP kinase, p38 MAP kinase, phosphospecific phosphoinositide-dependent kinase (PDK)-1, or PDK-1 antibodies.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In a previous study (16), we showed that BMP-4 stimulates VEGF synthesis in osteoblast-like MC3T3-E1 cells. We recently reported (15) that p44/p42 MAP kinase acts as a negative regulator in the BMP-4-stimulated synthesis of osteocalcin, whereas p38 MAP kinase takes part in the osteocalcin synthesis as a positive regulator in these cells. In the present study, we investigated whether there is a causal relationship between these MAP kinases and the BMP-4-stimulated VEGF synthesis in MC3T3-E1 cells. PD-98059, a specific inhibitor of mitogen-activated and extracellular signal-regulated kinase kinase (1), failed to affect the BMP-4-induced VEGF synthesis. Therefore, it seems unlikely that p44/p42 MAP kinase is involved in the synthesis of VEGF stimulated by BMP-4 in osteoblast-like MC3T3-E1 cells. Next, we showed herein that SB-203580, an inhibitor of p38 MAP kinase (3), suppressed the BMP-4-induced VEGF synthesis. Because SB-203580 reportedly blocks PDK activity and Akt activation in addition to p38 MAP kinase (21), we additionally examined the effect of PD-169316, another inhibitor of p38 MAP kinase (18), on VEGF synthesis. We found that PD-169316 reduced the synthesis of VEGF induced by BMP-4. Thus our findings suggest that p38 MAP kinase is involved in the BMP-4-stimulated VEGF synthesis in MC3T3-E1 cells. Furthermore, SB-202474, a negative control of p38 MAP kinase inhibitor (22), had little effect on VEGF synthesis. Taking these results into account, it is most likely that p38 MAP kinase takes part in BMP-4-induced VEGF synthesis in osteoblast-like MC3T3-E1 cells.

We have reported (16) that p70 S6 kinase participates in BMP-4-stimulated VEGF synthesis as a positive regulator in osteoblast-like MC3T3-E1 cells. We have demonstrated (19, 28) that rapamycin did not affect the BMP-4-induced p38 MAP kinase phosphorylation. Thus it seems unlikely that p70 S6 kinase functions at a point upstream from p38 MAP kinase. On the other hand, SB-203580 inhibited the BMP-4-stimulated phosphorylation of p70 S6 kinase. Therefore, our findings suggest that p38 MAP kinase affects the p70 S6 kinase phosphorylation induced by BMP-4 in MC3T3-E1 cells. It has been shown that BMP-2 stimulates the PI 3-kinase/p70 S6 kinase and p38 MAP kinase cascades in C2C12 cells (32). We have reported (16) that wortmannin or LY-294002, inhibitors of PI 3-kinase, significantly reduced BMP-4-induced VEGF synthesis in MC3T3-E1 cells, suggesting that PI 3-kinase is involved in BMP-4-induced VEGF synthesis. However, LY-294002 failed to suppress the phosphorylation of p38 MAP kinase induced by BMP-4. Next, we compared the time course of phosphorylation of p38 MAP kinase with that of phosphorylation of p70 S6 kinase. The time course of the phosphorylation of p38 MAP kinase stimulated by anisomycin, an activator of p38 MAP kinase (11), was faster than that of p70 S6 kinase in osteoblast-like MC3T3-E1 cells. We previously reported (15) that BMP-4 phosphorylates p38 MAP kinase and that the maximum effect of BMP-4 is observed at 90 min after the stimulation. On the other hand, p70 S6 kinase phosphorylation induced by BMP-4 is observed at 3 h in these cells (16). Thus these results suggest that p38 MAP kinase acts at a point upstream from p70 S6 kinase in MC3T3-E1 cells. Moreover, we showed that SB-203580 inhibited the phosphorylation of p70 S6 kinase stimulated by anisomycin. These results strongly suggest that p38 MAP kinase is an upstream regulator of p70 S6 kinase in osteoblast-like MC3T3-E1 cells.

It is recognized that PDK-1, Akt, and mTOR, possible downstream effectors of PI 3-kinase, are involved in the activation of p70 S6 kinase (31). Herein, we showed that the phosphorylation of PDK-1 was induced by anisomycin in MC3T3-E1 cells. Thus it is possible that activation of PDK-1 is regulated by p38 MAP kinase in these cells. However, BMP-4 hardly affected the phosphorylation of PDK-1 in these cells. It is unlikely that the physiological activation of p38 MAP kinase induced by BMP-4 potently elicits the PDK-1 activation in osteoblasts. We previously reported (16) that BMP-4 did not affect the phosphorylation of Akt. In addition, we confirmed here that not only BMP-4 but also anisomycin failed to induce the phosphorylation of mTOR or Akt in these cells. Taken together, it is likely that PI 3-kinase and p38 MAP kinase act at points upstream from p70 S6 kinase in BMP-4-stimulated VEGF synthesis without involvement of PDK-1, Akt, or mTOR in osteoblast-like MC3T3-E1 cells.

VEGF is well known to be an angiogenic growth factor displaying high specificity for endothelial cells and promoting angiogenesis, providing the microvasculature (26) that is essential for bone remodeling. In our recent study (15), we reported that p38 MAP kinase is a positive regulator of the BMP-4-induced synthesis of osteocalcin, an osteoblast-specific phenotype marker. It is generally recognized that osteocalcin normally functions to limit, without impairing, bone resorption and mineralization. With these findings taken into account, it is probable that BMP-4-activated p38 MAP kinase regulates bone remodeling via VEGF and osteocalcin synthesis. On the other hand, BMP-2-mediated p38 MAP kinase reportedly acts as a negative regulator of osteoblast differentiation in C2C12 cells (32). C2C12 cells are murine myoblasts, whereas MC3T3-E1 cells are murine preosteoblastic cells. Therefore, the discrepancy in the role of p38 MAP kinase between C2C12 cells and osteoblasts-like MC3T3-E1 cells might be due to the difference of cell differentiation stage.

In conclusion, our present findings strongly suggest that p38 MAP kinase regulates BMP-4-stimulated VEGF synthesis via p70 S6 kinase in osteoblasts.


    FOOTNOTES

Address for reprint requests and other correspondence: O. Kozawa, Dept. of Pharmacology, Gifu Univ. School of Medicine, Gifu 500-8705, Japan (E-mail: okozawa{at}cc.gifu-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published March 11, 2003;10.1152/ajpendo.00300.2002

Received 8 August 2002; accepted in final form 19 February 2003.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 284(6):E1202-E1209
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