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Keywords:

  • basic fibroblast growth factor;
  • vascular endothelial growth factor;
  • p42/p44 mitogen-activated protein kinase;
  • p38 mitogen-activated protein kinase;
  • calcium; osteoblast

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O-aminophinoxy)-ethane-N,N,N,N-tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

IN THE PROCESSES of bone growth, repair, and remodeling, the formation of new capillaries is required.(1) Invasion of capillaries into the metaphyseal end of the growth plate is known to be an important step in endochondral bone formation, resulting in bone growth.(1) In the repair ofbone fracture, it is recognized that ossification by osteoblasts is associated with the sites of capillary penetration into the callus.(2) It also is known that bone remodeling carried out by osteoclasts and osteoblasts is accompanied with new capillaries extending.(3) During bone remodeling, capillary endothelial cells provide the microvasculature. Osteoblasts and osteoprogenitor cells, which locally proliferate and differentiate into osteoblasts, migrate into the resorption lacunae. Thus, currently, it is recognized that the activities of osteoblasts, osteoclasts, and capillary endothelial cells are closely coordinated and regulate bone metabolism.(4) These functional cells are considered to influence one another via humoral factors as well as by direct cell-to-cell contact.

Vascular endothelial growth factor (VEGF) is known as an essential mediator of angiogenesis.(5) VEGF binds to its respective tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), both of which are expressed on endothelial cells,(5) and promotes the mitogenic and chemotactic actions to endothelial cells.(5) In bone metabolism, it recently has been reported that inactivation of VEGF though systemic administration of a soluble receptor chimeric protein causes complete suppression of blood vessel invasion, which is concomitant with impaired trabecular bone formation and expansion of the hypertrophic chondrocyte zone in the mouse tibial epiphyseal growth plate.(6) VEGF is recognized to be synthesized and secreted by several types of cells including osteoblasts, (5,7–12) and the synthesis is regulated by various humoral factors. It has been shown that prostaglandin E1 and prostaglandin E2 increase messenger RNA (mRNA) for VEGF and protein in primary cultured rat calvarial cells and RCT-3 osteoblast-like cells.(9) In addition, 1,25-dihydroxyvitamin D3, insulin-like growth factor-I, and parathyroid hormone have been reported to induce the synthesis of VEGF in primary cultured human osteoblast-like cells and human osteosarcoma Saso-2 cells. (10–12) Thus, there is no longer any doubt that VEGF secreted from osteoblasts plays a crucial role in the regulation of bone metabolism. However, the mechanisms underlying VEGF synthesis in osteoblasts are still poorly understood.

Basic fibroblast growth factor (bFGF or FGF-2) is found in bone matrix, and cultured osteoblast-like cells reportedly produce bFGF. (13–15) In addition, bFGF expression in macrophages, osteoblasts, or chondrocytes is detected during fracture repair.(16) Thus, it is recognized that bFGF plays an important role in bone remodeling and fracture healing. There are four structurally related high affinity receptors (FGF receptors 1-4) that possess an intrinsic protein tyrosine kinase activity and induce tyrosine autophosphorylation of the receptor.(17,18) We have previously reported that FGF receptors 1 and 2 are autophosphorylated by bFGF in osteoblast-like MC3T3-E1 cells.(19) In a recent study, we have shown that p38 mitogen-activated protein (MAP) kinase, which belongs to the MAP kinase superfamily,(20) is activated by bFGF in a Ca2+-dependent manner, resulting in the synthesis of interleukin-6 (IL-6) in these cells.(21) However, the exact mechanism of bFGF signaling in osteoblasts has not been clarified fully yet.

In the present study, we examined the effect of bFGF on VEGF release in osteoblast-like MC3T3-E1 cells. Here, we show that bFGF stimulates VEGF release through p42/p44 MAP kinase activation via a Ca2+-dependent mechanism in osteoblasts and that the p38 MAP kinase activated by bFGF itself negatively regulates the VEGF release.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Materials

bFGF was kindly provided by Kaken Pharmaceutical Co., Ltd. (Tokyo, Japan). 2′-Amino-3′-methoxyflanone (PD98059) and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) were obtained from Calbiochem Novabiochem Co. (La Jolla, CA, U.S.A.). 1,2-bis-(O-Aminophinoxy)-ethane-N,N,N,N,-tetracetic acid tetracetoxymethyl ester (BAPTA/AM) was obtained from BIOMOL Research Laboratories, Inc. (Plymouth, PA, U.S.A.). A23187 and thapsigargin were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Phospho-specific p38 MAP kinase antibodies (rabbit polyclonal immunoglobulin G [IgG], affinity purified), p38 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified), phospho-specific p42/p44 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified), and p42/p44 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified) were purchased from New England Biolabs, Inc. (Beverly, MA, U.S.A.). Mouse VEGF enzyme immunoassay kit was purchased from R&D Systems, Inc. (Minneapolis, MN, U.S.A.). Enhanced chemiluminescence (ECL) Western blotting detection system was purchased from Amersham Japan (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. SB203580, PD98059, BAPTA/AM, A23187, and thapsigargin were dissolved in dimethyl sulfoxide (DMSO). The maximum concentration of DMSO was 0.1%, which did not affect the assay for VEGF or the analysis of MAP kinases.

Cell culture

Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria(22) were maintained as previously described.(23) Briefly, the cells were cultured in α-minimum essential medium (α-MEM) containing 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% CO2/95% air. The cells (5 × 104) were seeded into 35-mm-diameter dishes in 2 ml of α-MEM containing 10% FCS. After 5 days, the medium was exchanged for 2 ml of α-MEM containing 0.3% FCS. The cells were used for experiments after 48 h.

Assay for VEGF

The cultured cells were stimulated by bFGF, A23187, or thapsigargin in 1 ml of α-MEM containing 0.3% FCS for the indicated periods. When stimulated by A23187 or thapsigargin, the medium was exchanged for 1 ml of α-MEM containing 0.3% FCS after 1 h from the stimulation, and the cells were subsequently incubated for 24 h. When indicated, the cells were pretreated with SB203580 or PD98059 for 60 minutes. When we tested the effect of PD98059 on the VEGF release induced by bFGF and SB203580, the cells were pretreated with PD98059 for 60 minutes and the medium was subsequently exchanged for 1 ml of α-MEM containing 0.3% FCS. The cells were then pretreated with SB203580 for another 60 minutes. The pretreatment of BAPTA/AM was performed for 30 minutes. In addition, when indicated, extracellular Ca2+ was chelated with 2.4 mM [ethylenebis-(oxyethylenenitrilo)]tetracetic acid (EGTA). The reaction was terminated by collecting the medium, and VEGF in the medium was measured by a VEGF enzyme immunoassay kit.

Analysis of MAP kinases

The cultured cells were stimulated by bFGF or A23187 in 1 ml of α-MEM containing 0.3% FCS for the indicated periods. The cells were washed twice with 1 ml of phosphate-buffered saline 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 a supernatant after centrifugation at 125,000g for 10 minutes at 4°C. SDS-polyacrylamide gel electrophoresis (PAGE) was performed by Laemmli(24) in 10% polyacrylamide gel. Western blotting analysis was performed as described previously(25) by using phospho-specific p38 MAP kinase antibodies, p38 MAP kinase antibodies, phospho-specific p42/p44 MAP kinase antibodies, or p42/p44 MAP kinase antibodies and peroxidase-labeled antibodies raised in goat against rabbit IgG as second antibodies. Peroxidase activity on the nitrocellulose sheet was visualized on X-ray film by use of the ECL Western blotting detection system. When indicated, the cells were pretreated with SB203580 or PD98059 for 60 minutes.

Determination

The absorbance of enzyme immunoassay samples was measured at 450 nm with EL 340 Bio Kinetic Reader (Bio-Tek Instruments, Inc., Winooski, VT, U.S.A.).

Statistical analysis

The data were analyzed by analysis of variance (ANOVA) followed by the Bonferroni method for multiple comparison between pairs, and a value of p < 0.05 was considered significant. All data are presented as the mean ± SEM of triplicate determinations. Each experiment was repeated three times with similar results.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Effect of bFGF on VEGF release in MC3T3-E1 cells

bFGF stimulated the VEGF release time dependently up to 48 h in osteoblast-like MC3T3-E1 cells (Fig. 1). The VEGF release was significant after 3 h after the bFGF stimulation (Fig. 1). The stimulatory effect was dose dependent in the range between 10 and 100 ng/ml (Fig. 2). The maximum effect was observed at 100 ng/ml. The number of cells showed little change during the incubation (before the incubation, 62 ± 4.2 × 104 cells/dish; after the 48-h incubation with 50 ng/nl bFGF, 69 ± 3.5 × 104 cells/dish; after the 48-h incubation of control, 63 ± 4.0 × 104 cells/dish; each value represents the mean ± SEM of triplicate determinations; similar results were obtained with two additional and different cell preparations).

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Figure FIG. 1. Effect of bFGF on VEGF release in MC3T3-E1 cells. The cultured cells were stimulated by 50 ng/ml bFGF (closed circle) or vehicle (open circle) for the indicated periods. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. * p < 0.05, compared with the value of control.

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Figure FIG. 2. Dose-dependent effect of bFGF on VEGF release in MC3T3-E1 cells. The cultured cells were stimulated by various doses of bFGF for 24 h. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. * p < 0.05, compared with the value of control.

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Effect of SB203580 on bFGF-induced VEGF release in MC3T3-E1 cells

In a recent study,(21) we have shown that bFGF activates p38 MAP kinase in MC3T3-E1 cells, resulting in IL-6 synthesis. To clarify the role of p38 MAP kinase on the bFGF-induced VEGF release in MC3T3-E1 cells, we examined the effect of SB203580, a specific inhibitor of p38 MAP kinase,(26,27) on the VEGF release by bFGF. SB203580, which by itself did not affect VEGF release, significantly amplified the bFGF-induced VEGF release (Fig. 3). This effect of SB203580 was dose dependent in the range between 0.3 and 10 μM (Fig. 3). The maximum effect of SB203580 was observed at 10 μM, a dose that caused about 75% enhancement in the bFGF effect. We found that SB203580 markedly reduced the phosphorylation of p38 MAP kinase induced by bFGF (Fig. 4).

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Figure FIG. 3. Effect of SB203580 on the bFGF-induced VEGF release in MC3T3-E1 cells. The cultured cells were pretreated with various doses of SB203580 for 60 minutes, and then stimulated by 50 ng/ml bFGF (closed circle) or vehicle (open circle) for 24 h. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. * p < 0.05, compared with the value of bFGF alone.

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Figure FIG. 4. Effect of SB203580 on the phosphorylation of p38 MAP kinase in MC3T3-E1 cells. The cultured cells were pretreated with 50 μM SB203580 or vehicle for 60 minutes and then stimulated by 50 ng/ml bFGF or vehicle for 10 minutes. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p38 MAP kinase or p38 MAP kinase.

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Effect of bFGF on the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase in MC3T3-E1 cells

Next, we examined the effect of bFGF on the phosphorylation of p42/p44 MAP kinase, which belongs to the MAP kinase superfamily,(20) in order to investigate whether bFGF activates p42/p44 MAP kinase in MC3T3-E1 cells. bFGF time-dependently stimulated the phosphorylation of p42/p44 MAP kinase up to 90 minutes (Fig. 5). The maximum effect of bFGF on the phosphorylation of p42/p44 MAP kinase was observed at 90 minutes after the stimulation. As previously described,(21) bFGF stimulated the phosphorylation of p38 MAP kinase up to 90 minutes (Fig. 5). The maximum effect of bFGF on the p38 MAP kinase phosphorylation was observed at 10 minutes after the stimulation and decreased thereafter, which is consistent with our previous result.(21) The effect of bFGF on the phosphorylation of p42/p44 MAP kinase seems to be much slower than that on the p38 MAP kinase phosphorylation.

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Figure FIG. 5. Effects of bFGF on the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase in MC3T3-E1 cells. The cultured cells were stimulated by 50 ng/ml bFGF for 10 minutes (lane 2), 20 minutes (lane 3), 30 minutes (lane 4), 45 minutes (lane 5), 60 minutes (lane 6), 75 minutes (lane 7), and 90 minutes (lane 8). The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p42/p44 MAP kinase, p42/p44 MAP kinase, phospho-specific p38 MAP kinase, or p38 MAP kinase. Lane 1, control cells.

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Effect of PD98059 on the bFGF-induced VEGF release in MC3T3-E1 cells

To elucidate whether p42/p44 MAP kinase is involved in the release of VEGF induced by bFGF in MC3T3-E1 cells, we examined the effect of PD98059, an inhibitor of upstream kinase that activates p42/p44 MAP kinase,(28,29) on the bFGF-induced VEGF release. PD98059, which alone had little effect on VEGF release, markedly inhibited the bFGF-induced VEGF release in a dose-dependent manner in the range between 0.1 and 30 μM (Fig. 6). The maximum inhibitory effect of PD98059 was observed at 30 μM, a dose that led to about 85% reduction in the bFGF effect. We further investigated the effect of PD98059 on the enhancement by SB203580 of bFGF-induced VEGF release in these cells. PD98059 reduced the enhancement in parallel with the bFGF-induced VEGF release (Table 1).

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Figure FIG. 6. Effect of PD98059 on the bFGF-induced VEGF release in MC3T3-E1 cells. The cultured cells were pretreated with various doses of PD98059 for 60 minutes and then stimulated by 50 ng/ml bFGF (closed circle) or vehicle (open circle) for 24 h. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05, compared with the value of bFGF alone.

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Table Table 1.. Effect of PD98059 on the Enhancement by SB203580 on bFGF-Induced VEGF Release in MC3T3-E1 Cells
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Effects of PD98059 or SB203580 on the p42/p44 MAP kinase phosphorylation induced by bFGF in MC3T3-E1 cells

We confirmed that PD98059 significantly inhibited the bFGF-stimulated phosphorylation of p42/p44 MAP kinase in MC3T3-E1 cells (Fig. 7A). To further investigate the role of p38 MAP kinase in the bFGF-induced VEGF release in MC3T3-E1 cells, we examined the effect of SB203580 on the phosphorylation of p42/p44 MAP kinase induced by bFGF. SB203580, which alone did not affect the basal levels of p42/p44 MAP kinase, markedly enhanced the phosphorylation of p42/p44 MAP kinase by bFGF (Fig. 7B).

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Figure FIG. 7. Effects of PD98059 or SB203580 on the bFGF-induced phosphorylation of p42/p44 MAP kinase in MC3T3-E1 cells. (A) The cultured cells were pretreated with 30 μM PD98059 or vehicle for 60 minutes and then stimulated by 50 ng/ml bFGF or vehicle for 90 minutes. (B) The cultured cells were pretreated with 10 μM SB203580 or vehicle for 60 minutes and then stimulated by 50 ng/ml bFGF or vehicle for 90 minutes. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p42/p44 MAP kinase or p42/p44 MAP kinase.

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Effects of depletion of extracellular Ca2+ by EGTA or BAPTA/AM on the bFGF-induced VEGF release in MC3T3-E1 cells

We previously reported that bFGF induces IL-6 synthesis via intracellular mobilization of Ca2+ in MC3T3-E1 cells.(21) To explore the role of Ca2+ in the release of VEGF induced by bFGF in MC3T3-E1 cells, we examined the effect of depletion of extracellular Ca2+ on the VEGF release. Chelating extracellular Ca2+ with 2.4 mM EGTA, which by itself had little effect on VEGF release, markedly suppressed the bFGF-induced VEGF release (Table 2). In addition, we examined the effect of BAPTA/AM, a chelator of intracellular Ca2+,(30) on the bFGF-induced VEGF release. BAPTA/AM (50 μM), which alone did not affect VEGF release, significantly reduced the VEGF release stimulated by bFGF (Table 2).

Table Table 2.. Effects of Extracellular Ca2+ Depletion by EGTA or BAPTA/AM on the bFGF-Induced VEGF Release in MC3T3-E1 Cells
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Effects of A23187 or thapsigargin on VEGF release in MC3T3-E1 cells

To further clarify the role of Ca2+ mobilization in the VEGF release stimulated by bFGF in MC3T3-E1 cells, we investigated the effect of A23187, known as a Ca ionophore, on the release of VEGF. VEGF release was significantly stimulated by A23187 alone (Table 3). In addition, thapsigargin, which is known to induce Ca2+ release from intracellular Ca2+ stores,(31) by itself also induced VEGF release (<11 ± 2 pg/ml for control and 246 ± 22 pg/ml for 0.1 μM of thapsigargin, after 1-h stimulation as measured during the incubation for 24 h).

Table Table 3.. Effects of PD98059 or SB203580 on the A23187-Induced VEGF Release in MC3T3-E1 Cells
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Effects of PD98059 or SB203580 on the VEGF release induced by A23187 in MC3T3-E1 cells

Next, we investigated the effect of PD98059 on the release of VEGF induced by A23187 in MC3T3-E1 cells. PD98059 (30 μM) significantly reduced the A23187-stimulated VEGF release (Table 3). This inhibitory effect was dose dependent in the range between 0.1 and 30 μM (data not shown). In addition, we examined the effect of SB203580 on the VEGF release by A23187. SB203580 (10 μM), which potently enhanced the bFGF-induced VEGF release as described previously, had little effect on the release of VEGF elicited by A23187 (Table 3).

Effects of PD98059 or SB203580 on the A23187-induced phosphorylation of MAP kinases in MC3T3-E1 cells

A23187 (1 μM) time-dependently induced the phosphorylation of p42/p44 MAP kinase (Fig. 8A). The maximum effect of A23187 on the phosphorylation was observed at 10 minutes after the stimulation and decreased thereafter. We found that PD98059 (30 μM) markedly suppressed the phosphorylation of p42/p44 MAP kinase induced by A23187 (Fig. 8B). On the other hand, SB203580 did not affect the A23187-induced phosphorylation of p42/p44 MAP kinase (Fig. 8C). The phosphorylation of p38 MAP kinase induced by A23187 was reduced by SB203580 (Fig. 8D).

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Figure FIG. 8. Effects of PD98059 or SB203580 on the A23187-induced phosphorylation of p42/p44 MAP kinase or p38 MAP kinase in MC3T3-E1 cells. (A) The cultured cells were stimulated by 3 μM A23187 for 1 minute (lane 2), 3 minutes (lane 3), 5 minutes (lane 4), 10 minutes (lane 5), 20 minutes (lane 6), and 30 minutes (lane 7). Lane 1, control cells. (B) The cultured cells were pretreated with 30 μM PD98059 or vehicle for 60 minutes and then stimulated by 3 μM A23187 or vehicle for 10 minutes. (C) The cultured cells were pretreated with 10 μM SB203580 or vehicle for 60 minutes and then stimulated by 3 μM A23187 or vehicle for 10 minutes. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p42/p44 MAP kinase or p42/p44 MAP kinase. (D) The cultured cells were pretreated with 50 μM SB203580 or vehicle for 60 minutes and then stimulated by 3 μM A23187 or vehicle for 10 minutes. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p38 MAP kinase or p38 MAP kinase.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

In the present study, we showed that bFGF stimulated VEGF release in osteoblast-like MC3T3-E1 cells. We measured VEGF in the conditioned medium in this study, and the levels of VEGF were undetectable up to 3 h after the bFGF stimulation. Although the VEGF was secreted from these cells, it is most likely that the changes in long-term secretion present changes in synthesis rather than a specific secretory process. To the best of our knowledge, this is probably the first report showing the stimulatory effect of bFGF on VEGF release in osteoblasts.

Herein, we showed that the depletion of extracellular Ca2+ by EGTA reduced the release of VEGF stimulated by bFGF in MC3T3-E1 cells. We also showed that BAPTA/AM decreased the VEGF release. Thus, these results suggest that Ca2+ mobilization is involved in the bFGF-induced VEGF release in MC3T3-E1 cells. In addition, we showed that A23187 or thapsigargin alone elicited the VEGF release. Therefore, most likely, intracellular Ca2+ mobilization is required for the bFGF-induced VEGF release in osteoblast-like MC3T3-E1 cells. However, the inhibition of bFGF-induced VEGF release by Ca2+ depletion was partial. It is possible that bFGF-induced VEGF release is mediated by a Ca2+-dependent pathway and a Ca2+-independent pathway.

MAP kinases play important roles in intracellular signaling of a variety of agonists.(20) Previously, we have shown that bFGF activates p38 MAP kinase via Ca2+ mobilization, resulting in IL-6 synthesis in MC3T3-E1 cells.(21) Thus, next we investigated whether or not p38 MAP kinase is involved in bFGF-induced VEGF release. SB203580 markedly enhanced the bFGF-induced VEGF release. We confirmed that SB203580 inhibited the phosphorylation of p38 MAP kinase induced by bFGF. Thus, it seems that p38 MAP kinase activation inhibits the VEGF release by bFGF. Therefore, these results suggest that the VEGF release induced by bFGF is negatively regulated by p38 MAP kinase activated by bFGF itself in osteoblast-like MC3T3-E1 cells.

We have shown that the IL-6 synthesis induced by prostaglandin F, tumor necrosis factor α, or endothelin-1 is mediated through p42/p44 MAP kinase activation in MC3T3-E1 cells. (32–34) Here, we showed that bFGF elicited the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase in MC3T3-E1 cells. It is well recognized that MAP kinases are activated by phosphorylation of threonine and tyrosine residues by dual-specificity MAP kinase kinase.(20,35) Therefore, our finding suggests that bFGF activates p42/p44 MAP kinase in these cells in addition to p38 MAP kinase as previously reported.(21) Thus, we tried to test the effect of PD98059 on the bFGF-induced VEGF release and showed that PD98059 significantly inhibited the VEGF release. PD98059 is a well-known inhibitor of upstream kinase that activates p42/p44 MAP kinase,(28,29) and here we confirmed that PD98059 truly suppressed the phosphorylation of p42/p44 MAP kinase induced by bFGF in these cells. These results suggest that bFGF stimulates VEGF release via activation of p42/p44 MAP kinase in osteoblast-like MC3T3-E1 cells. Furthermore, we showed that A23187 stimulated the phosphorylation of p42/p44 MAP kinase and that PD98059 inhibited the A23187-induced VEGF release in these cells. Based on our findings, most likely bFGF activates p42/p44 MAP kinase via mobilization of intracellular Ca2+, resulting in VEGF release in osteoblast-like MC3T3-E1 cells. It is probable that the p38 MAP kinase activated by bFGF limits the bFGF-induced VEGF release in these cells.

In addition, we showed that SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF and that PD98059 reduced the enhancement of bFGF-induced VEGF release by SB203580 in MC3T3-E1 cells. Therefore, p38 MAP kinase seems to act at a point upstream from p42/p44 MAP kinase and negatively regulates the VEGF release induced by bFGF in osteoblast-like MC3T3-E1 cells. Here, we showed that the bFGF-induced phosphorylation of p42/p44 MAP kinase was later than that of p38 MAP kinase. It is possible that the later phosphorylation of p42/p44 MAP kinase is caused by the inhibition by p38 MAP kinase pathway at earlier time points. Moreover, SB203580 hardly affected either VEGF release or p42/p44 MAP kinase phosphorylation induced by A23187. Thus, it seems unlikely that the effect of p38 MAP kinase is exerted at a point downstream from Ca2+ mobilization. Taking our results into account as a whole, most likely bFGF divergently stimulates p42/p44 MAP kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and the former induces the VEGF release and the latter negatively acts on bFGF stimulation of p42/p44 MAP kinase activity as well as of subsequent VEGF release. In addition, the A23187-induced VEGF release was partially inhibited by PD98059. Therefore, it is possible that p38 MAP kinase as well as p42/p44 MAP kinase might be involved in the Ca2+ mobilization-independent VEGF release induced by bFGF in these cells.

The physiological significance of the divergent activation of p42/p44 MAP kinase and p38 MAP kinase by bFGF remains unclear. However, it is probable that the VEGF synthesis induced by bFGF is under the strict control of these MAP kinases in osteoblasts. Bone remodeling is accompanied with new capillaries extending.(4) Because VEGF is a specific mitogen of vascular endothelial cells,(5) our present results lead us to speculate that bFGF-stimulated p42/p44 MAP kinase acts as a stimulative regulator of bone microvasculature development and bFGF-activated p38 MAP kinase acts as a suppressive regulator of it, resulting in promoting bone remodeling. Further investigation would be required to clarify the details.

The concentration of bFGF stimulating the VEGF release observed in the present study was much higher than the physiological concentrations reported by previous in vivo reports.(36,37) bFGF produced by osteoblast is accumulated in extracellular matrix of bone, (13–15) suggesting that the osteoblasts that make contact with bone matrix are possibly exposed to relatively high doses of bFGF. Thus, it is probable that under the physiological conditions, bFGF stimulates the synthesis of VEGF through the mechanism shown here. bFGF reportedly induces VEGF expression through an autocrine/paracrine mechanism in the endothelial cells of forming capillaries.(38) Therefore, it is possible that the bFGF-induced VEGF synthesis in osteoblasts and endothelial cells plays an important role in bone growth, repair, and remodeling through regulating capillary endothelial cells.

In conclusion, our present results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by p38 MAP kinase activated by bFGF itself.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The authors are very grateful to Masaichi Miwa, Daijiro Hatakeyama, and Hidenori Kawamura for their skillful technical assistance. This investigation was supported in part by the Research Grant for Longevity Science (10C-03), the Research Grant of Cooperative Studies for Longevity Sciences for National Sanatoria, the Health Science Research Grant for Comprehensive Research on Aging and Health from the Ministry of Health and Welfare of Japan, and a Grant-in-Aid for Scientific Research (09671041) from the Ministry of Education, Science, Sports, and Culture of Japan.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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