Negative Regulation by p70 S6 Kinase of FGF-2–Stimulated VEGF Release Through Stress-Activated Protein Kinase/c-Jun N-Terminal Kinase in Osteoblasts

Authors

  • Shinji Takai,

    1. Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu, Japan
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  • Haruhiko Tokuda,

    1. Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu, Japan
    2. Department of Clinical Laboratory, National Hospital for Geriatric Medicine, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan
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  • Yoshiteru Hanai,

    1. Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu, Japan
    2. Department of Clinical Laboratory, National Hospital for Geriatric Medicine, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan
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  • Atsushi Harada,

    1. Department of Functional Restoration, National Hospital for Geriatric Medicine, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan
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  • Eisuke Yasuda,

    1. Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu, Japan
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  • Rie Matsushima-Nishiwaki,

    1. Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu, Japan
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  • Hisaaki Kato,

    1. Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu, Japan
    2. Department of Emergency and Disaster Medicine, Gifu University Graduate School of Medicine, Gifu, Japan
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  • Shinji Ogura,

    1. Department of Emergency and Disaster Medicine, Gifu University Graduate School of Medicine, Gifu, Japan
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  • Toshiki Ohta,

    1. Department of Internal Medicine, National Hospital for Geriatric Medicine, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan
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  • Osamu Kozawa MD, PhD

    Corresponding author
    1. Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu, Japan
    • Yanagido1-1, Gifu 501-1194, Japan
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  • The authors state that they have no conflicts of interest.

Abstract

To clarify the mechanism of VEGF release in osteoblasts, we studied whether p70 S6 kinase is involved in basic FGF-2–stimulated VEGF release in osteoblast-like MC3T3-E1 cells. In this study, we show that p70 S6 kinase activated by FGF-2 negatively regulates VEGF release through SAPK/JNK in osteoblasts.

Introduction: Vascular endothelial growth factor (VEGF) plays an important role in bone metabolism. We have previously reported that fibroblast growth factor-2 (FGF-2) stimulates the release of VEGF through p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2–activated p38 MAP kinase negatively regulates VEGF release. However, the mechanism behind VEGF release in osteoblasts is not precisely known.

Materials and Methods: The levels of VEGF released from MC3T3-E1 cells were measured by enzyme immunoassay. The phosphorylation of each protein kinase was analyzed by Western blotting. To knock down p70 S6 kinase in MC3T3-E1 cells, the cells were transfected with siRNA to target p70 S6 kinase.

Results: FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase. Rapamycin significantly enhanced the FGF-2–stimulated VEGF release and VEGF mRNA expression. The FGF-2–induced phosphorylation of p70 S6 kinase was suppressed by rapamycin. Rapamycin markedly enhanced the FGF-2–induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. SP600125, a specific inhibitor of SAPK/JNK, suppressed the amplification by rapamycin of the FGF-2–stimulated VEGF release similar to the levels of FGF-2 with SP600125. Finally, downregulation of p70 S6 kinase by siRNA significantly enhanced the FGF-2–stimulated VEGF release and phosphorylation of SAPK/JNK.

Conclusions: These results strongly suggest that p70 S6 kinase limits FGF-2–stimulated VEGF release through self-regulation of SAPK/JNK, composing a negative feedback loop, in osteoblasts.

INTRODUCTION

It is well known that osteoblasts synthesize basic fibroblast growth factor (FGF)-2, and FGF-2 is embedded in bone matrix.(1,2) FGF-2 expression in osteoblasts is detected during fracture repair.(3) Bone metabolism is strictly regulated by osteoblasts and osteoclasts, which are responsible for bone formation and bone resorption, respectively.(4) Therefore, it is thought that FGF-2 plays a pivotal role in fracture healing, bone remodeling, and osteogenesis.(5) We have previously reported that FGF-2 autophosphorylates FGF receptors 1 and 2 among four structurally related high affinity receptors in osteoblast-like MC3T3-E1 cells.(6) In addition, we reported that FGF-2 stimulates induction of heat shock protein 27 in these cells.(7)

Bone remodeling carried out by osteoclasts and osteoblasts is accompanied by angiogenesis and capillary outgrowth.(8,9) During bone remodeling, capillary endothelial cells provide the microvasculature. It is well recognized that the activities of osteoblasts, osteoclasts, and capillary endothelial cells are closely coordinated and regulate bone metabolism.(10) These functional cells are considered to influence one another through humoral factors and by direct cell-to-cell contact. Vascular endothelial growth factor (VEGF) is an angiogenic growth factor displaying high specificity for vascular endothelial cells.(11) VEGF, produced and secreted from a variety of cell types, increases capillary permeability and stimulates proliferation of endothelial cells.(11) As for bone metabolism, an inactivation of VEGF reportedly causes complete suppression of blood vessel invasion concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone in mouse tibial epiphyseal growth plate.(12) Evidence is accumulating that osteoblasts among bone cells produce and secrete VEGF in response to various physiological agents.(11,13–15) We have previously reported that FGF-2 stimulates VEGF release in MC3T3-E1 cells and that the release is positively regulated by p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK)(16,17) among the MAP kinase superfamily.(18) Based on these findings, it is currently recognized that VEGF secreted from osteoblasts may play an important role in the regulation of bone metabolism.(10,19) However, the mechanism behind VEGF synthesis in osteoblasts and its release from these cells is not fully understood.

p70 S6 kinase is a mitogen-activated serine/threonine kinase required for cell proliferation and G1 cell cycle progression.(20) As for osteoblasts, it has been shown that fluoroaluminate induces an increase in p70 S6 kinase phosphorylation.(21) In our previous study,(22) we reported that p70 S6 kinase plays as a positive regulator in bone morphogenetic protein-4–stimulated release of VEGF in osteoblast-like MC3T3-E1 cells. In addition, we recently showed that p38 MAP kinase, a member of the MAP kinase superfamily, functions at a point upstream from p70 S6 kinase in the release of VEGF in these cells.(23) However, the exact role of p70 S6 kinase in osteoblasts still remains unclear.

In this study, we investigated the involvement of p70 S6 kinase in the FGF-2–stimulated VEGF release in osteoblast-like MC3T3-E1 cells. We show that p70 S6 kinase activated by FGF-2 negatively regulates VEGF release through SAPK/JNK in these cells.

MATERIALS AND METHODS

Materials

FGF-2 and mouse VEGF enzyme immunoassay kits were purchased from R&D Systems (Minneapolis, MN, USA). Rapamycin and SP600125 were obtained from Calbiochem-Novabiochem Co. (La Jolla, CA, USA). Phospho-specific p70 S6 kinase antibodies, p70 S6 kinase antibodies, phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific p38 MAP kinase antibodies, p38 MAP kinase antibodies, phospho-specific SAPK/JNK antibodies, and SAPK/JNK antibodies were purchased from Cell Signaling (Beverly, MA, USA). ECL Western blotting detection system was purchased from Amersham Biosciences (Piscataway, NJ, USA). Control short interfering RNA (siRNA; Silencer Negative Control no. 1 siRNA) or p70 S6 kinase siRNA (Silencer Predesigned siRNA, 75849, 75755, and 75942) was purchased from Ambion (Austin, TX, USA). siLentFect was purchased from Bio-Rad (Hercules, CA, USA). Trizol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Omniscript Reverse Transcriptase Kit was purchased from QIAGEN (Hilden, Germany). Fast-Start DNA Master SYBR Green I was purchased from Roche Diagnostics (Mannheim, Germany). Other materials and chemicals were obtained from commercial sources. Rapamycin or SP600125 was dissolved in dimethyl sulfoxide (DMSO). The maximum concentration of DMSO 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(24) were maintained as previously described.(25) Briefly, the cells were cultured in α-MEM containing 10% FCS at 37°C in a humidified atmosphere of 5% CO2/95% air. The cells were seeded into 35- (5 × 104) or 90-mm-diameter (5 × 105) dishes in α-MEM containing 10% FCS. After 5 days, the medium was exchanged for α-MEM containing 0.3% FCS. The cells were used for experiments after 48 h.

siRNA transfection

To knock down p70 S6 kinse in MC3T3-E1 cells, the cells were transfected with control siRNA (Silencer Negative Control no. 1 siRNA) or p70 S6 kinase siRNA (Silencer Predesigned siRNA, 75849, 75755, and 75942; Ambion) using the siLentFect (Bio-Rad) according to the manufacturer's protocol. In brief, the cells were seeded in a 35-mm-diameter (1 × 105) dish in α-MEM containing 10% FCS and subcultured for 48 h. After that, the cells were incubated at 37°C for 48 h with 250 nM siRNA-siLentFect complexes.

VEGF assay

The cultured cells were stimulated by various doses of FGF-2 in 1 ml of α-MEM containing 0.3% FCS for the indicated periods. When indicated, the cells were pretreated with rapamycin or SP600125 for 60 minutes. The conditioned medium was collected at the end of the incubation, and the VEGF concentration was measured by ELISA kit.

Western blot analysis

The cultured cells were stimulated by FGF-2 in α-MEM containing 0.3% FCS for the indicated periods. The cells were washed twice with PBS, lysed, homogenized, and sonicated in a lysis buffer containing 62.5 mM Tris/HCl, pH 6.8, 2% 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-PAGE was performed according to Laemmli(26) in 10% polyacrylamide gel. Western blotting analysis was performed as described previously(27) using phospho-specific p70 S6 kinase antibodies, p70 S6 kinase antibodies, phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific p38 MAP kinase antibodies, p38 MAP kinase antibodies, phospho-specific SAPK/JNK antibodies, or SAPK/JNK antibodies, with peroxidase-labeled antibodies raised in goat against rabbit IgG being used as second antibodies. Peroxidase activity on the polyvinylidene difluoride (PVDF) sheet was visualized on X-ray film by means of the electrochemiluminescence (ECL) Western blotting detection system.

Determination

The absorbance of enzyme immunoassay samples was measured at 450 nm with EL 340 Bio Kinetic Reader (Bio-Tek Instruments, Winooski, VT, USA). The densitometric analysis was performed using Molecular Analyst/Macintosh (Bio-Rad Laboratories).

Real-time RT-PCR

The cultured cells were pretreated with rapamycin and/or SP600125 and stimulated by FGF-2 for the indicated time period. Total RNA was isolated and transcribed into cDNA using Trizol reagent and Omniscript Reverse Transcriptase Kit. Real-time PCR was performed using a Light Cycler system (Roche Diagnostics) in capillaries and Fast-Start DNA Master SYBR Green I provided with the kit. Sense and antisense primers were synthesized based on the report of Simpson et al.(28) for mouse VEGF mRNA and GAPDH mRNA. The amplified products were determined by melting curve analysis and agarose electrophoresis. VEGF mRNA levels were normalized with those of GAPDH mRNA.

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 the mean ± SE of triplicate determinations. Each experiment was repeated three times with similar results.

RESULTS

Effect of FGF-2 on the phosphorylation of p70 S6 kinase in MC3T3-E1 cells

To study whether FGF-2 activates p70 S6 kinase in osteoblast-like MC3T3-E1 cells, we examined the effect of FGF-2 on the phosphorylation of p70 S6 kinase. The stimulation of FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase (Fig. 1). The maximum effect was observed at 20 minutes after the stimulation of FGF-2.

Figure Figure 1.

Effect of FGF-2 on the phosphorylation of p70 S6 kinase in MC3T3-E1 cells. The cultured cells were stimulated by 70 ng/ml FGF-2 for the indicated periods. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p70 S6 kinase or p70 S6 kinase. The histogram shows quantitative representations of the levels of FGF-2–induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SE of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05 compared with the value of control.

Effect of rapamycin on the FGF-2–stimulated VEGF release in MC3T3-E1 cells

To clarify whether p70 S6 kinase is involved in the FGF-2–induced release of VEGF in MC3T3-E1 cells, we examined the effect of rapamycin, a specific inhibitor of p70 S6 kinase,(29,30) on the FGF-2–induced release of VEGF. Rapamycin, which alone did not affect the basal levels of VEGF, significantly enhanced the FGF-2–induced release of VEGF in a time-dependent manner (Fig. 2A). The amplifying effect of rapamycin was dose dependent in a range between 1 and 50 ng/ml (Fig. 2B). Rapamycin at 50 ng/ml caused ∼200% enhancement in the FGF-2 effect.

Figure Figure 2.

Effect of rapamycin on the FGF-2–stimulated VEGF release in MC3T3-E1 cells. (A) The cultured cells were pretreated with 10 ng/ml rapamycin (circle symbols) or vehicle (square symbols) for 60 minutes and stimulated by 70 ng/ml FGF-2 (solid symbols) or vehicle (open symbols) for the indicated periods. *p < 0.05 compared with the control. **p < 0.05 compared with the value of FGF-2 alone. (B) The cultured cells were pretreated with various doses of rapamycin for 60 minutes and stimulated by 70 ng/ml FGF-2 (•) or vehicle (○) for 24 h. *p < 0.05 compared with the value of FGF-2 alone. Each value represents the mean ± SE of triplicate determinations. Similar results were obtained with two additional and different cell preparations.

Effect of rapamycin on the FGF-2–stimulated VEGF mRNA expression in MC3T3-E1 cells

To clarify whether the enhancement of FGF-2–stimulated VEGF release by rapamycin is mediated through transcriptional events, we examined the effect of rapamycin on the FGF-2–induced VEGF mRNA expression by real-time PCR. We found that rapamycin significantly upregulated the FGF-2–induced VEGF mRNA expression 6 h after stimulation (3.5 ± 0.3-fold for 70 ng/ml FGF-2 alone compared with the control; 5.2 ± 0.3-fold for 70 ng/ml FGF-2 with 50 ng/ml rapamycin compared the control). These results suggest that the amplifying effect of rapamycin is mediated, at least in part, by upregulation of VEGF mRNA expression in osteoblast-like MC3T3-E1 cells.

Effects of rapamycin on the FGF-2–induced phosphorylation of p70 S6 kinase in MC3T3-E1 cells

We next examined the effect of rapamycin on the FGF-2–induced phosphorylation of p70 S6 kinase. Rapamycin almost completely attenuated the FGF-2–induced phosphorylation of p70 S6 kinase in a range between 10 and 50 ng/ml (Fig. 3).

Figure Figure 3.

Effect of rapamycin on the FGF-2–induced phosphorylation of p70 S6 kinase in MC3T3-E1 cells. The cultured cells were pretreated with 10 ng/ml rapamycin or vehicle for 60 minutes and stimulated by 30 ng/ml FGF-2 or vehicle for 10 minutes. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p70 S6 kinase or p70 S6 kinase. The histogram shows quantitative representations of the levels of FGF-2–induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SE of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05 compared with the control. **p < 0.05 compared with the value of FGF-2 alone.

Effects of rapamycin on the FGF-2–induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, or SAPK/JNK in MC3T3-E1 cells

To study whether the amplifying effect of rapamycin on the FGF-2–stimulated VEGF release was through activation of p44/p42 MAP kinase, p38 MAP kinase, or SAPK/JNK in MC3T3-E1 cells, we examined the effect of rapamycin on the phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, or SAPK/JNK induced by FGF-2. Rapamycin failed to affect the FGF-2–induced phosphorylation of p44/p42 MAP kinase (Fig. 4A) or p38 MAP kinase (Fig. 4B). However, the FGF-2–induced phosphorylation of SAPK/JNK was markedly enhanced by rapamycin (Fig. 5). Rapamycin at 10 ng/ml caused ∼100% enhancement in the FGF-2 effect.

Figure Figure 4.

Effects of rapamycin on the FGF-2–induced phosphorylation of p44/p42 MAP kinase or p38 MAP kinase in MC3T3-E1 cells. (A) The cultured cells were pretreated with various doses of rapamycin for 60 minutes and stimulated by 30 ng/ml FGF-2 or vehicle for 20 minutes. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p44/p42 MAP kinase or p44/p42 MAP kinase. (B) The cultured cells were pretreated with various doses of rapamycin for 60 minutes and stimulated by 30 ng/ml FGF-2 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. The histogram shows quantitative representations of the levels of FGF-2–induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SE of triplicate determinations. Similar results were obtained with two additional and different cell preparations.

Figure Figure 5.

Effect of rapamycin on the FGF-2–induced phosphorylation of SAPK/JNK in MC3T3-E1 cells. The cultured cells were pretreated with various doses of rapamycin for 60 minutes and stimulated by 30 ng/ml FGF-2 or vehicle for 20 minutes. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific SAPK/JNK or SAPK/JNK. The histogram shows quantitative representations of the levels of FGF-2-induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SE of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05 compared with the control. **p < 0.05 compared with the value of FGF-2 alone.

Effects of SP600125 on the amplification by rapamycin of the FGF-2–induced VEGF release and phosphorylation of SAPK/JNK in MC3T3-E1 cells

SP600125, a specific SAPK/JNK inhibitor,(31) which by itself did not affect the basal levels of VEGF, significantly reduced the enhancement by rapamycin of FGF-2–induced VEGF release (Table 1). The enhanced levels by rapamycin of FGF-2–induced VEGF release were reduced by SP600125 similar to the levels by FGF-2 with SP600125 treatment. In addition, SP600125 almost completely suppressed both the FGF-2–induced phosphorylation of SAPK/JNK and its enhancement by rapamycin (Fig. 6).

Table Table 1.. Effect of SP600125 on the Enhancement by Rapamycin of the FGF-2–Stimulated VEGF Release in MC3T3-E1 Cells
original image
Figure Figure 6.

Effect of SP600125 on the enhancement by rapamycin of the FGF-2–induced phosphorylation of SAPK/JNK in MC3T3-E1 cells. The cultured cells were pretreated with 50 μM SP600125 or vehicle for 60 minutes and incubated by 30 ng/ml rapamycin or vehicle for 60 minutes. The cells were stimulated by 30 ng/ml FGF-2 or vehicle for 20 minutes. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific SAPK/JNK or SAPK/JNK. The histogram shows quantitative representations of the levels of FGF-2–induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SE of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05 compared with the control. **p < 0.05 compared with the value of FGF-2 alone. ***p < 0.05 compared with the value of FGF-2 with rapamycin pretreatment.

Effects of SP600125 on the upregulation by rapamycin of the FGF-2–stimulated VEGF mRNA expression in MC3T3-E1 cells

To clarify whether the suppressive effect by SP600125 of FGF-2–stimulated VEGF release is mediated through transcriptional events, we examined the effect of SP600125 in the presence or absence of rapamycin on the FGF-2–stimulated VEGF mRNA expression by real-time PCR. We found that SP600125 significantly downregulated the FGF-2–induced VEGF mRNA expression 2 h after stimulation (5.0 ± 0.9-fold for 70 ng/ml FGF-2 alone compared with the control; 3.2 ± 0.4-fold for 70 ng/ml FGF-2 with 10 μM SP600125 compared with the control; 3.0 ± 0.3-fold for 70 ng/ml FGF-2 with 10 μM SP600125 and 50 ng/ml rapamycin compared with the control). These results suggest that the suppressive effect of SP600125 is mediated, at least in part, by downregulation of VEGF mRNA expression in osteoblast-like MC3T3-E1 cells.

Effect of p70 S6 kinase-siRNA on the FGF-2–stimulated VEGF release in MC3T3-E1 cells

To confirm the results from rapamycin, we examined the effects of p70 S6 kinase downregulation by p70 S6 kinase siRNA on the VEGF release and the phosphorylation of SAPK/JNK induced by FGF-2 in osteoblast-like MC3T3-E1 cells. We found that p70 S6 kinase siRNA (250 nM) successfully reduced the p70 S6 kinase levels compared with those of control siRNA determined by the band intensity of Western blot analysis (Fig. 7A). The FGF-2–induced levels of VEGF release in p70 S6 kinase–downregulated cells were truly enhanced compared with those in control siRNA-transfected cells. Downregulation of p70 S6 kinase caused ∼180% enhancement in the FGF-2 effect (Table 2). FGF-2 significantly enhanced the phosphorylation levels of SAPK/JNK in the p70 S6 kinase–downregulated cells (Fig. 7B).

Table Table 2.. Effect of p70 S6 Kinase (S6K) siRNA on the FGF-2–Stimulated VEGF Release in MC3T3-E1 Cells
original image
Figure Figure 7.

Effect of p70 S6 kinase-siRNA in MC3T3-E1 cells and effect of p70 S6 kinase downregulation on the FGF-2–inducedphosphorylation of SAPK/JNK in MC3T3-E1 cells. The cells were transfected with 250 nM of control siRNA or p70 S6 kinase siRNA by using the siLentFect. (A) Cells were incubated at 37°C for 48 h with siRNA-siLentFect complexes and subsequently harvested for preparation of Western blot analysis. (B) The cultured cells transfected with control siRNA or p70 S6 kinase siRNA were stimulated by 70 ng/ml FGF-2 or vehicle for 20 minutes and subsequently harvested for preparation of Western blot analysis. The histogram shows quantitative representations of the levels of FGF-2–induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SE of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05 compared with the value of vehicle. **p < 0.05 compared with the value of FGF-2 with control siRNA transfection.

DISCUSSION

In this study, we found that FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase in osteoblast-like MC3T3-E1 cells, using phospho-specific p70 S6 kinase (Thr389) antibodies. It is generally recognized that the activity of p70 S6 kinase is regulated by multiple phosphorylation events.(20) It has been shown that phosphorylation at Thr389 among the phosphorylation most strongly correlates with p70 S6 kinase activity.(20) Thus, it is probable that FGF-2 induces the activation of p70 S6 kinase in osteoblast-like MC3T3-E1 cells. To the best of our knowledge, this is probably the first report showing the FGF-2–induced p70 S6 kinase activation in osteoblasts.

We have previously reported that FGF-2 stimulates the release of VEGF in osteoblast-like MC3T3-E1 cells.(16) Therefore, we studied whether p70 S6 kinase functions in FGF-2–stimulated VEGF release in osteoblast-like MC3T3-E1 cells. Rapamycin, a specific inhibitor of p70 S6 kinase,(29,30) markedly enhanced the FGF-2–stimulated release of VEGF and expression of VEGF mRNA in MC3T3-E1 cells. We confirmed that the FGF-2–induced phosphorylation of p70 S6 kinase was truly attenuated by rapamycin. In addition, downregulation of p70 S6 kinase by siRNA markedly enhanced FGF-2–stimulated VEGF release in these cells. These results strongly suggest that FGF-2–stimulated VEGF release is reduced by the activation of p70 S6 kinase. Therefore, it is probable that FGF-2 activates the p70 S6 kinase pathway, resulting in negatively regulating the release of VEGF. We speculate that p70 S6 kinase signaling activated by FGF-2 limits the FGF-2–induced over-release of VEGF in osteoblast-like MC3T3-E1 cells. As far as we know, this finding is the first report to show that the activation of p70 S6 kinase leads to negative feedback regulation of VEGF release in osteoblasts.

We have previously shown that FGF-2 induces the phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, and SAPK/JNK in MC3T3-E1 cells.(16,17) It is generally recognized that the MAP kinase superfamily mediates intracellular signaling of extracellular agonists and plays a crucial role in cellular functions including proliferation, differentiation, and apoptosis in a variety of cells.(18) Three major MAP kinases, p44/p42 MAP kinase, p38 MAP kinase, and SAPK/JNK, are known as central elements used by mammalian cells to transducer the diverse messages.(18) In our previous studies,(16,17) we showed that p44/p42 MAP kinase and SAPK/JNK act as positive regulators in FGF-2–induced VEGF release. On the other hand, FGF-2–activated p38 MAP kinase negatively regulates VEGF release. These findings lead us to speculate that there is cross-talk regulation between p70 S6 kinase and these MAP kinases in FGF-2–stimulated VEGF release in these cells. However, rapamycin(29,30) failed to affect the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. Therefore, it seems unlikely that the p70 S6 kinase signaling pathway affects FGF-2–stimulated release of VEGF through the amplification of activities of p44/p42 MAP kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. On the contrary, we showed that the phosphorylation levels of FGF-2–induced SAPK/JNK were strengthened by rapamycin and p70 S6 kinase siRNA transfection. In addition, the amplification by rapamycin of the FGF-2–stimulated VEGF release and mRNA expression was suppressed by SP600125, a specific inhibitor of SAPK/JNK,(31) similar to the levels of FGF-2 with SP600125. Furthermore, we showed that SP600125 almost completely reduced the rapamycin-enhanced phosphorylation of SAPK/JNK and FGF-2–induced phosphorylation of SAPK/JNK. Taking our findings into account, it is probable that the p70 S6 kinase signal pathway affects the FGF-2–stimulated VEGF release through upregulation of SAPK/JNK in osteoblast-like MC3T3-E1 cells.

The p70 S6 kinase pathway is recognized to play a crucial role in various cellular functions, especially cell cycle progression.(20) These results indicate that the p70 S6 kinase pathway in osteoblasts has an important role in the control of the production of VEGF, one of the key regulators of bone metabolism. Bone remodeling carried out by osteoclasts and osteoblasts is accompanied by angiogenesis and capillary outgrowth.(10) Because VEGF is a specific mitogen of vascular endothelial cells,(11) these results lead us to speculate that FGF-2–activated p70 S6 kinase signaling acts as a negative regulator of the microvasculature development in bone. Thus, the p70 S6 kinase pathway in osteoblasts might be considered to be a new candidate as a molecular target of bone resorption concurrent with various bone diseases. On the contrary, we have previously shown that p70 S6 kinase acts as a positive regulator in bone morphogenetic protein-4–stimulated release of VEGF in MC3T3-E1 cells.(22) The physiological significance of a regulatory mechanism by p70 S6 kinase in osteoblasts still remains unclear. Further study is required to clarify the exact roles of p70 S6 kinase in osteoblasts and bone metabolism.

In conclusion, these results strongly suggest that p70 S6 kinase plays an important role in the regulation of FGF-2–stimulated VEGF release in osteoblasts and may serve as a negative feedback mechanism to prevent oversynthesizing of VEGF through SAPK/JNK in these cells.

Acknowledgements

We thank Yoko Kawamura and Seiko Sakakibara for skillful technical assistance. This study was supported in part by Grant-in-Aid for Scientific Research (16590873 and 16591482) for the Ministry of Education, Science, Sports and Culture of Japan, Research Grants for Longevity Sciences (15A-1 and 15C-2), and Research on Proteomics and Research on Longevity Sciences from the Ministry of Health, Labour and Welfare of Japan.

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