Incadronate Amplifies Prostaglandin F2{alpha}-induced Vascular Endothelial Growth Factor Synthesis in Osteoblasts

ENHANCEMENT OF MAPK ACTIVITY*

Haruhiko Tokuda {ddagger} §, Atsushi Harada ¶, Kouseki Hirade §, Hiroyuki Matsuno §, Hidenori Ito ||, Kanefusa Kato ||, Yutaka Oiso ** and Osamu Kozawa § {ddagger}{ddagger}

From the {ddagger} Department of Internal Medicine, Chubu National Hospital, National Institute for Longevity Sciences, Obu, Aichi 474-8511, Japan, Department of Orthopaedics, Chubu National Hospital, National Institute for Longevity Sciences, Obu, Aichi 474-8511, Japan, § Department of Pharmacology, Gifu University School of Medicine, Gifu 500-8705, Japan, || Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0391, Japan, ** First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466-8550, Japan

Received for publication, September 6, 2002 , and in revised form, March 17, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously reported that prostaglandin F2{alpha} (PGF2{alpha}) activates p44/p42 mitogen-activated protein kinase (MAPK) through protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of vascular endothelial growth factor (VEGF) synthesis induced by PGF2{alpha} and the effect of incadronate on the VEGF synthesis in these cells. PGF2{alpha} significantly stimulated the VEGF synthesis in a dose-dependent manner between 1 pM and 10 µM. Cycloheximide reduced the PGF2{alpha} effect. PGF2{alpha} increased the levels of mRNA for VEGF. Cloprostenol, a PGF2{alpha}-sensitive receptor agonist, potently induced the VEGF synthesis. Indomethacin, an inhibitor of cyclooxygenase, significantly reduced the PGF2{alpha}-induced VEGF synthesis. Bisindolylmaleimide, an inhibitor of PKC, reduced the PGF2{alpha}-induced VEGF synthesis. The VEGF synthesis induced by PGF2{alpha} was significantly attenuated in the PKC down-regulated cells. PGF2{alpha} elicited the translocation of PKC{beta}I from cytosol to membrane fraction. PD98059 or U0126, inhibitors of MEK, suppressed the VEGF synthesis induced by PGF2{alpha}. Farnesyltransferase inhibitor failed to affect the PGF2{alpha}-induced VEGF synthesis. Incadronate enhanced the synthesis of VEGF induced by PGF2{alpha}. NaF-induced VEGF synthesis was also amplified by incadronate. PD98059 suppressed the enhancement by incadronate of PGF2{alpha}-induced VEGF synthesis. Incadronate markedly enhanced the phosphorylation of Raf-1, MEK1/2, and p44/p42 MAPK induced by PGF2{alpha} or 12-O-tetradecanoylphorbol-13-acetate, a PKC activator. Incadronate significantly enhanced the cloprostenol-increased level of VEGF concentration in mouse plasma in vivo. These results strongly suggest that PGF2{alpha} stimulates VEGF synthesis through the PKC-dependent activation of p44/p42 MAPK in osteoblasts and that the incadronate enhances the VEGF synthesis at the point between PKC and Raf-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone metabolism is mainly regulated by two functional cells, osteoblasts and osteoclasts; the former is responsible for bone formation, and the latter for bone resorption (1). Accumulating evidence indicates that bone-resorptive agents such as parathyroid hormone and 1,25-(OH)2 vitamin D3 up-regulate RANKL (receptor activator of nuclear factor {kappa}B) expression through their specific receptors on osteoblasts, suggesting that osteoblasts play pivotal roles in the regulation of bone resorption (2). The bone remodeling results from the coupling bone resorption by activated osteoclasts with subsequent deposition of new matrix by osteoblasts. During the process, capillary endothelial cells provide the microvasculature, and osteoblasts and osteoprogenitor cells, which proliferate locally and differentiate into osteoblasts, migrate into the resorption lacuna. Thus, it is currently recognized that the activities of osteoblasts, osteoclasts, and capillary endothelial cells are closely coordinated via humoral factors as well as by direct cell-to-cell contact and that these cells cooperatively regulate bone metabolism (3).

Vascular endothelial growth factor (VEGF)1 is a potent angiogenic factor that induces endothelial cell proliferation, angiogenesis, and capillary permeability (4). It is well recognized that VEGF is produced and secreted from a variety of cell types (4). It has been reported that inactivation of VEGF causes impaired trabecular bone formation and expansion of the hypertrophic chondrocyte zone in mouse tibial epiphyseal growth plate associated with the complete suppression of vascular invasion (5). Moreover, evidence is accumulating that osteoblasts produce VEGF and secrete it in response to various humoral factors (4, 6, 7, 8, 9). We have recently reported that basic fibroblast growth factor and prostaglandin (PG) E1 stimulate VEGF synthesis through the activation of p44/p42 mitogenactivated protein kinase (MAPK) and p38 MAPK, respectively, in osteoblast-like MC3T3-E1 cells (10, 11). However, the exact mechanism behind VEGF synthesis in osteoblasts has not yet been fully clarified.

Bisphosphonates, stable analogues of pyrophosphate, have been developed as potent inhibitors of bone resorption and effective tools for the treatment of various metabolic bone diseases associated with increased osteoclastic bone resorption such as Paget's disease, tumoral bone disease, and osteoporosis (12). Inhibition of osteoclast recruitment, osteoclastic adhesion to bone surface, and osteoclast activity is known to be the main mechanism of the anti-bone-resorptive actions of bisphosphonates (12). On the other hand, at least some of the effects of bisphosphonates on osteoclast development and function are proposed to be mediated through the actions to osteoblasts (12). It has been reported that ibandronate and alendronate induce the synthesis of an inhibitor of osteoclastic bone resorption in osteoblastic cell line CRP 10/30 (13). Etidronate, alendronate, pamidronate, and olpadronate reportedly prevent apoptosis of murine primary cultured osteoblasts through the activation of p44/p42 MAPK (14). Pamidronate and zoledronate have recently been shown to enhance the differentiation and bone-forming activities of primary cultured human fetal osteoblasts (15). It has also been reported that pamidronate and clodronate increase the expression of mRNA for osteopontin and RANKL in UMR 106-01 rat osteoblastic osteosarcoma cells (16). Thus, it is currently speculated that the effects of bisphosphonates on bone metabolism are exerted through not only osteoclasts but also osteoblasts.

PGs are well known to act as autocrine/paracrine modulators of osteoblasts (1, 17). Among them, PGF2{alpha} is recognized to be a potent bone-resorbing agent (17) and stimulates the proliferation and inhibits the differentiation of osteoblasts (17). In previous studies (18, 19), we have demonstrated that PGF2{alpha} stimulates both phosphoinositide-hydrolyzing phospholipase C (PIPLC) and phosphatidylcholine-hydrolyzing phospholipase D (PC-PLD), recognized to be two major pathways of physiological protein kinase C (PKC) activation (20, 21), in osteoblast-like MC3T3-E1 cells. We have also reported that PGF2{alpha} activates p44/p42 MAPK through PKC in these cells (22). In the present study, we investigated whether PGF2{alpha} stimulates VEGF synthesis in MC3T3-E1 cells, and if so, the mechanism of VEGF synthesis and the effect of incadronate on the VEGF synthesis. Our results strongly suggest that incadronate enhances the PGF2{alpha}-stimulated VEGF synthesis in osteoblasts and that the effect is exerted at the point between PKC and Raf-1.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—PGF2{alpha}, cycloheximide, PGE2, PGD2, indomethacin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma Chemical Co. (St. Louis, MO). Cloprostenol was purchased from BIOMOL Research Laboratories, Inc. (Plymouth, PA). Incadronate, alendronate, tiludronate, and etidronate were kindly provided by Yamanouchi Pharmaceutical Co. Ltd. (Tokyo, Japan), Teijin Ltd. (Tokyo, Japan), Meiji Seika Co. Ltd. (Kawasaki, Japan), and Sumitomo Pharmaceuticals Co. Ltd. (Osaka, Japan), respectively. Bisindolylmaleimide, 2'-amino-3'-methoxyflanone (PD98059) and H-Cys-Val-2-naphthylalanine-Met-OH (farnesyltransferase (FTase) inhibitor III) was obtained from Calbiochem-Novabiochem Co. (La Jolla, CA). 1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) was obtained from Promega Corp. (Madison, WI). PKC{beta}I antibodies, phospho-specific p44/p42 MAPK antibodies, p44/p42 MAPK antibodies, phospho-specific MEK1/2 antibodies, MEK1/2 antibodies, and phosphospecific Raf-1 antibodies were purchased from New England Bio-Labs, Inc. (Beverly, MA). A mouse VEGF ELISA kit and a mouse VEGF mRNA quantitation kit were purchased from R&D Systems, Inc. (Minneapolis, MN). An ECL Western blotting detection system and a QuickPrep total RNA extraction kit were purchased from Amersham Biosciences (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. PGF2{alpha}, cycloheximide, cloprostenol, PGE2, PGD2, and indomethacin were dissolved in ethanol. PD98059 and U0126 were dissolved in Me2SO. FTase inhibitor III was dissolved in acetic acid. The maximum concentration of ethanol, Me2SO, or acetic acid was 0.1%, which did not affect the assay for VEGF or the analysis of Western blot.

Cell Culture—Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria (23) were maintained as previously described (24). Briefly, 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-mm (5 x 104) or 90-mm (5 x 105) 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 48 h. When indicated, the cells were pretreated with 0.1 µM TPA for 24 h, as previously reported (25).

Assay for VEGF—The cells were stimulated by PGF2{alpha}, cloprostenol, PGE2, PGD2, or NaF in 1 ml of {alpha}-MEM containing 0.3% FCS for the indicated periods. In some experiments, the pretreatment with cycloheximide or indomethacin was performed for 30 min prior to the stimulation, and the pretreatment with bisindolylmaleimide, PD98059, or U0126 was performed for 60 min. The cells were pretreated with FTase inhibitor III for 4 h. The reaction was terminated by collecting the medium, and VEGF in the medium was measured by using a VEGF ELISA kit. When indicated, the cells were pretreated with incadronate, etidronate, tiludronate, or alendronate for 8 h.

Isolation of RNA and Quantification of mRNA for VEGF—The cultured cells were stimulated by PGF2{alpha} or PGE2 in 1 ml of {alpha}-MEN containing 0.3% FCS for 2 h. Total RNA was isolated with a QuickPrep total RNA extraction kit. Total RNA (3 µg) from each sample was subjected to the quantification of mRNA for VEGF using a mouse VEGF mRNA quantitation kit.

Analysis of PKC{beta}I, p44/p42 MAPK, MEK1/2, or Raf-1—The cultured cells were stimulated by PGF2{alpha} or TPA in 4 ml of {alpha}-MEM containing 0.3% FCS for the indicated periods. The cells were washed twice with phosphate-buffered saline and then 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 analysis of PKC{beta}I translocation to the plasma membrane was performed as previously described (26). The sample was centrifuged at 15,000 x g for 1 h to generate membrane and cytosolic fractions, and the pellet was resuspended in a lysis buffer. For the analysis of p44/p42 MAPK, MEK1/2, or Raf-1, the cytosolic fraction was collected as a supernatant after centrifugation at 125,000 x g for 10 min at 4 °C. SDS-PAGE was performed by Laemmli (27) in 10% polyacrylamide gel. Western blotting analysis was performed as described previously (28) by using PKC{beta}I antibodies, phospho-specific p44/p42 MAPK antibodies, p44/p42 MAPK antibodies, phospho-specific MEK1/2 antibodies, MEK1/2 antibodies, or phospho-specific Raf-1 antibodies, with peroxidaselabeled antibodies raised in goat against rabbit IgG being used as second antibodies. Peroxidase activity on the nitrocellulose sheet was visualized on x-ray film by means of the ECL Western blotting detection system. When indicated, the cells were pretreated with incadronate or vehicle for 8 h.

Assay for VEGF in Mouse Plasma in Vivo—C57B mice, obtained from SLC (Sizuoka, Japan), were divided into four groups, a control group (n = 3), a group treated with incadronate (n = 3), a group treated with cloprostenol (n = 3), and a group treated with both compounds (n = 6). Mice were placed in a supine position under anesthesia with an intraperitoneal injection of pentobarbital at a dose of 44 mg/kg. Catheters (Natume Co. Ltd., Tokyo, Japan) were connected to the left jugular vein for the infusion of either incadronate or buffered saline (0.15 M NaCl) and to the right jugular artery for monitoring blood pressure and pulse rate using a pressure transducer (AP601G Nihon Koden, Tokyo, Japan), respectively. Incadronate at a dose of 0.3 mg/kg (0.25 ml) was intravenously injected as a bolus (10% of total volume) and continuously infused for 2 h using an infusion pump via a right jugular vein. Cloprostenol at a dose of 0.2 µg per day was administered once a day by subcutaneous injection. In a group of combined treatments, the first administration of cloprostenol was performed at the end of infusion of incadronate, and the daily administration was continued for the indicated periods. Blood samples (0.3 ml) were collected via the jugular vein on sodium citrate (3.15%) under anesthesia with ether at the indicated time point. The plasma concentration of VEGF was measured by using a VEGF ELISA kit. The experiment was performed in accordance with the institutional guidelines.

Determination—The absorbance of ELISA samples was measured at 450 nm with an EL 340 Bio Kinetic Reader (Bio-Tek Instruments, Inc., Winooski, VT). The densitometric analysis was performed using Molecular Analyst/Macintosh (Bio-Rad Laboratories, Hercules, CA).

Statistical Analysis—The data were analyzed by analysis of variance followed by the Bonferroni method for multiple comparisons between pairs, and a value of p < 0.05 was considered significant. All data are presented as the mean ± S.E. of triplicate determinations. Each experiment was repeated three times with similar results.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of PGF2{alpha} on VEGF Synthesis and the Level of mRNA for VEGF in MC3T3-E1 Cells—PGF2{alpha} stimulated the VEGF synthesis time-dependently up to 48 h in osteoblast-like MC3T3-E1 cells (Fig. 1). The VEGF synthesis was significant after 3 h (Fig. 1). The stimulatory effect of PGF2{alpha} on VEGF synthesis was dose-dependent in the range between 1 pM and 10 µM (Fig. 2). The maximum effect of PGF2{alpha} was observed at 10 µM. Cycloheximide (10 µM), an inhibitor of protein synthesis (29), which by itself had no effect on the VEGF level, markedly inhibited the PGF2{alpha}-increased VEGF level (17 ± 2 pg/ml for control; 16 ± 2 pg/ml for 10 µM cycloheximide; 1921 ± 153 pg/ml for 10 µM PGF2{alpha} alone; and 759 ± 62 (p < 0.05, compared with the value of PGF2{alpha} alone) pg/ml for 10 µM PGF2{alpha} with 10 µM cycloheximide pretreatment, as measured during the stimulation for 48 h). PGF2{alpha} also increased the level of mRNA for VEGF (Table I). To clarify whether FP receptor is required for the PGF2{alpha}-induced VEGF synthesis or not in MC3T3-E1 cells, we examined the effect of cloprostenol, an FP agonist (30), on the VEGF synthesis. Cloprostenol significantly stimulated the VEGF synthesis (Table II). PGE2 is known to be abundantly produced by osteoblasts, including MC3T3-E1 cells, and act as a potent regulator of bone metabolism (17). Thus, we examined the effect of PGE2 on the VEGF level in these cells. PGE2 induced the VEGF synthesis (Table I) and increased the level of mRNA for VEGF (Table II). However, the effects were weaker than those of PGF2{alpha}. We have previously reported that PGE1 stimulates VEGF synthesis in MC3T3-E1 cells (11). In addition, we found that PGD2 induced VEGF synthesis in these cells (data not shown).



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FIG. 1.
Effect of PGF2{alpha} on VEGF synthesis in MC3T3-E1 cells. The cultured cells were stimulated by 10 µM PGF2{alpha} (closed circle) or vehicle (open circle) for the indicated periods. Each value represents the mean ± S.E. 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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FIG. 2.
Dose-dependent effect of PGF2{alpha} on VEGF synthesis in MC3T3-E1 cells. The cultured cells were stimulated by various doses of PGF2{alpha} for 48 h. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean ± S.E. 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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TABLE I
Effects of PGF2{alpha} or PGE2 on the level of mRNA for VEGF in MC3T3-E1 cells

 

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TABLE II
Effects of cloprostenol or PGE2 on VEGF synthesis in comparison with PGF2{alpha} in MC3T3-E1 cells

 

Effect of Indomethacin on the PGF2{alpha}-induced VEGF Synthesis in MC3T3-E1 Cells—It is well known that PGs act as autocrine/paracrine modulators of osteoblasts (1, 17). To clarify whether endogenous PG synthesis is involved in the VEGF synthesis induced by PGF2{alpha}, we investigated the effect of indomethacin, a cyclooxygenase inhibitor (31), on the PGF2{alpha}-induced VEGF synthesis in MC3T3-E1 cells. Indomethacin, which alone had little effect on the VEGF synthesis, reduced the VEGF synthesis stimulated by PGF2{alpha} in a dose-dependent manner in the range between 3 and 30 µM (Fig. 3).



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FIG. 3.
Effect of indomethacin on the PGF2{alpha}-induced VEGF synthesis in MC3T3-E1 cells. The cultured cells were pretreated with various doses of indomethacin for 30 min and then stimulated by 10 µM PGF2{alpha} (shaded bars) or vehicle (blank bars) for 48 h. Each value represents the mean ± S.E. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *, p < 0.05, compared with the value of PGF2{alpha} alone.

 

Effects of PKC Down-regulation or Bisindolylmaleimide on PGF2{alpha}-induced VEGF Synthesis in MC3T3-E1 Cells—We have previously reported that PGF2{alpha} activates both PI-PLC and PC-PLD, known as major pathways of PKC activation (20, 21), in osteoblast-like MC3T3-E1 cells (18, 19). It has been reported that 24 h of pretreatment of TPA (0.1 µM), a PKC-activating phorbol ester (32), down-regulates PKC in these cells (33). We also found that the binding capacity of phorbol-12,13-dibutyrate, a PKC-activating phorbol ester (32), in MC3T3-E1 cells treated with TPA for 24 h is reduced to about 30% of the capacity in intact cells (25). To clarify the role of PKC in the PGF2{alpha}-stimulated VEGF synthesis in MC3T3-E1 cells, we examined the effect of TPA long term pretreatment on the VEGF. The effect of PGF2{alpha} on VEGF synthesis was significantly reduced in the PKC down-regulated cells compared with that in the cells without TPA pretreatment (Table III). In addition, we examined the effect of bisindolylmaleimide, an inhibitor of PKC (34), on the VEGF synthesis induced by PGF2{alpha} in MC3T3-E1 cells. Bisindolylmaleimide, which by itself had little effect on the level of VEGF, significantly suppressed the PGF2{alpha}-induced VEGF synthesis in a dose-dependent manner in the range between 1 and 10 µM (Fig. 4).


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TABLE III
Effect of PKC down-regulation on the PGF2{alpha}-induced VEGF synthesis in MC3T3-E1 cells

 


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FIG. 4.
Effect of bisindolylmaleimide on the PGF2{alpha}-induced VEGF synthesis in MC3T3-E1 cells. The cultured cells were pretreated with various doses of bisindolylmaleimide for 20 min and then stimulated by 10 µM PGF2{alpha} (shaded bars) or vehicle (blank bars) for 48 h. Each value represents the mean ± S.E. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *, p < 0.05, compared with the value of PGF2{alpha} alone.

 

Effect of PGF2{alpha} on the Translocation of PKC{beta}I to the Plasma Membrane of MC3T3-E1 Cells—It has been reported that, among the PKC family, PKC{alpha} and PKC{beta} mainly exist in osteoblast-like MC3T3-E1 cells, and short term treatment with TPA induces PKC{beta} translocation from cytosol to membrane (33). It is well recognized that the translocation of PKC from cytosol to plasma membrane occurs when PKC is activated (32). We examined the effect of PGF2{alpha} on the PKC{beta}I translocation to the plasma membrane of these cells. PGF2{alpha} elicited the PKC{beta}I translocation to the plasma membrane (Fig. 5). The maximum effect of PGF2{alpha} was observed at 5 min after the stimulation.



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FIG. 5.
Effect of PGF2{alpha} on the translocation of PKC{beta}I to the plasma membrane in MC3T3-E1 cells. The cultured cells were stimulated by 10 µM PGF2{alpha} for the indicated periods. The plasma membrane-rich extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against PKC{beta}I. The histogram shows quantitative representations of the levels of PKC{beta}I obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± S.E. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *, p < 0.05, compared with the value of control.

 

Effects of PD98059, U0126, or FTase Inhibitor III on the PGF2{alpha}-induced VEGF Synthesis in MC3T3-E1 Cells—We have previously reported that PGF2{alpha} activates p44/p42 MAPK in a PKC-dependent fashion in osteoblast-like MC3T3-E1 cells (22). To elucidate whether p44/p42 MAPK is involved in the PGF2{alpha}-induced VEGF synthesis in these cells, we examined the effect of PD98059, a specific inhibitor of MEK (35), on the VEGF synthesis induced by PGF2{alpha}. PD98059, which alone did not affect the level of VEGF, dose-dependently reduced the PGF2{alpha}-induced VEGF synthesis in the range between 0.1 and 50 µM (Fig. 6). The maximum inhibitory effect of PD98059 was observed at 50 µM, which caused about 85% reduction of the PGF2{alpha}-effect. U0126, another inhibitor of MEK (36), also suppressed the VEGF synthesis induced by PGF2{alpha} (data not shown).



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FIG. 6.
Effect of PD98059 on the PGF2{alpha}-induced VEGF synthesis in MC3T3-E1 cells. The cultured cells were pretreated with various doses of PD98059 for 60 min, and then stimulated by 10 µM PGF2{alpha} (closed circle) or vehicle (open circle) for 48 h. Each value represents the mean ± S.E. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *, p < 0.05, compared with the value of PGF2{alpha} alone.

 

On the other hand, it is well recognized that ligation of many receptors leads to the activation of p44/p42 MAPK through the activation of Ras (37). GTP-bound Ras interacts with Raf-1, and then Raf-1 activates MEK, the upstream kinase of p44/p42 MAPK (37). The farnesylation of C-terminal cysteine residue of Ras by FTase is known as the first step of post-translational modification of Ras, resulting in the translocalization of the Ras·Raf-1 complex to the membrane, an important process of Raf-1 activation (37, 38). To investigate whether Ras is involved in the VEGF synthesis induced by PGF2{alpha} in MC3T3-E1 cells, we examined the effect of FTase inhibitor III (39) on the VEGF synthesis. FTase inhibitor III, which by itself had little effect on VEGF synthesis, did not affect the VEGF synthesis stimulated by PGF2{alpha} (Table IV).


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TABLE IV
Effect of FTase inhibitor III on the PGF2{alpha}-induced VEGF synthesis in MC3T3-E1 cells

 

Effects of Incadronate on the PGF2{alpha}- or NaF-induced VEGF Synthesis in MC3T3-E1 Cells—Incadronate, which alone did not affect VEGF synthesis, significantly enhanced the VEGF synthesis induced by PGF2{alpha} in a dose-dependent manner in the range between 10 and 50 µM (Fig. 7). The maximum effect of incadronate on the PGF2{alpha}-induced VEGF synthesis was observed at 30 µM, which caused about 150% enhancement of the PGF2{alpha} effect. Other bisphosphonates such as etidronate, tiludronate, or alendronate failed to enhance the PGF2{alpha}-induced VEGF synthesis (data not shown). We found that PD98059 reduced the enhancement by incadronate of PGF2{alpha}-induced VEGF synthesis (Table V). FTase inhibitor III failed to affect the enhancement (data not shown).



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FIG. 7.
Effect of incadronate on the PGF2{alpha}-induced VEGF synthesis in MC3T3-E1 cells. The cultured cells were pretreated with various doses of incadronate for 8 h and then stimulated by 10 µM PGF2{alpha} (shaded bars) or vehicle (blank bars) for 48 h. Each value represents the mean ± S.E. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *, p < 0.05, compared with the value of PGF2{alpha} alone.

 

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TABLE V
Effect of PD98059 on the enhancement by incadronate on the PGF2{alpha}-induced VEGF synthesis in MC3T3-E1 cells

 

We have previously reported that heterotrimeric GTP-binding protein is involved in the PGF2{alpha}-induced activation of both PI-PLC and PC-PLD in osteoblast-like MC3T3-E1 cells (18, 19). Thus, we investigated the effect of incadronate on the VEGF synthesis stimulated by NaF, known as a direct activator of heterotrimeric GTP-binding proteins (40). NaF dose-dependently stimulated VEGF synthesis (data not shown). Incadronate significantly enhanced the NaF-induced VEGF synthesis (Table VI).


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TABLE VI
Effect of incadronate on the NaF-induced VEGF synthesis in MC3T3-E1 cells

 

Effects of Incadronate on the PGF2{alpha}- or TPA-induced Phosphorylation of p44/p42 MAPK in MC3T3-E1 Cells—We next examined the effect of incadronate on the phosphorylation of p44/p42 MAPK induced by PGF2{alpha} in MC3T3-E1 cells. Incadronate, which alone did not affect the p44/p42 MAPK phosphorylation, significantly enhanced the PGF2{alpha}-induced p44/p42 MAPK phosphorylation (Fig. 8A). To clarify whether the effect of incadronate is exerted at a point downstream of PKC or not, we examined the effect of incadronate on the TPA-induced phosphorylation of p44/p42 MAPK. We have already found that TPA phosphorylates p44/p42 MAPK in MC3T3-E1 cells (41). Incadronate markedly enhanced the TPA-induced phosphorylation of p44/p42 MAPK as well as the PGF2{alpha}-induced phosphorylation (Fig. 8B).



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FIG. 8.
Effects of incadronate on the PGF2{alpha}- or TPA-induced phosphorylation of p44/p42 MAPK in MC3T3-E1 cells. The cultured cells were pretreated with 50 µM incadronate or vehicle for 8 h and then stimulated by 10 µM PGF2{alpha} or vehicle for 30 min (A) or stimulated by 0.1 µM TPA or vehicle for 60 min (B). The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p44/p42 MAPK or p44/p42 MAPK. The histogram shows quantitative representations of the levels of PGF2{alpha}- or TPA-induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± S.E. of triplicate determinations. Similar results were obtained with two additional and different cell preparations.

 

Effects of Incadronate on the Phosphorylation of MEK1/2 and Raf-1 Induced by PGF2{alpha} or TPA in MC3T3-E1 Cells—It is well recognized that the activation of p44/p42 MAPK is regulated by MEK1/2, which is regulated by the upstream kinase known as Raf-1 (37). We found that PGF2{alpha} and TPA truly induced the MEK1/2 phosphorylation in osteoblast-like MC3T3-E1 cells (Fig. 9). Thus, we next examined the effect of incadronate on the phosphorylation of MEK1/2 induced by PGF2{alpha} in these cells. Incadronate, which by itself hardly affected the phosphorylation, significantly enhanced the phosphorylation of MEK1/2 induced by PGF2{alpha} (Fig. 9A). In addition, TPA-induced phosphorylation of MEK1/2 was markedly amplified by incadronate (Fig. 9B).



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FIG. 9.
Effects of incadronate on the PGF2{alpha}- or TPA-induced phosphorylation of MEK1/2 in MC3T3-E1 cells. The cultured cells were pretreated with 50 µM incadronate or vehicle for 8 h and then stimulated by 10 µM PGF2{alpha} or vehicle for 30 min (A) or stimulated by 0.1 µM TPA or vehicle for 60 min (B). The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific MEK1/2 or MEK1/2. The histogram shows quantitative representations of the levels of PGF2{alpha}- or TPA-induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± S.E. of triplicate determinations. Similar results were obtained with two additional and different cell preparations.

 

We found that PGF2{alpha} and TPA induced the phosphorylation of Raf-1 (Fig. 10). Furthermore, we investigated the effects of incadronate on the phosphorylation of Raf-1 induced by PGF2{alpha} or TPA. Incadronate alone had little effect on the phosphorylation of Raf-1 but markedly enhanced the PGF2{alpha}- or TPA -induced phosphorylation of Raf-1 (Fig. 10, A and B).



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FIG. 10.
Effects of incadronate on the PGF2{alpha}- or TPA-induced phosphorylation of Raf-1 in MC3T3-E1 cells. The cultured cells were pretreated with 50 µM incadronate or vehicle for 8 h and then stimulated by 10 µM PGF2{alpha} or vehicle for 15 min (A) or stimulated by 0.1 µM TPA or vehicle for 60 min (B). The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific Raf-1. The histogram shows quantitative representations of the levels of PGF2{alpha}- or TPA-induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± S.E. of triplicate determinations. Similar results were obtained with two additional and different cell preparations.

 

Effect of Cloprostenol or Incadronate on the Level of VEGF in Vivo—The level of VEGF in mouse plasma was significantly increased after the subcutaneous injection of cloprostenol (Table VII). The effect of cloprostenol was observed from 24 to 72 h (data not shown), and the maximum effect was observed at 48 h. Intravenous administration of incadronate, which by itself had little effect on the VEGF level in mouse plasma, significantly enhanced the cloprostenol-increased VEGF level (Table VII).


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TABLE VII
Effects of cloprostenol or incadronate on the level of VEGF in vivo

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we demonstrated that PGF2{alpha} induced VEGF synthesis in osteoblast-like MC3T3-E1 cells. We measured VEGF in the conditioned medium in this study. Although the VEGF was secreted from these cells, it is most likely that the changes in long term secretion represent changes in synthesis rather than a specific secretory process. We found the inhibitory effect of cycloheximide on the PGF2{alpha}-increased level of VEGF, suggesting that PGF2{alpha} stimulates de novo synthesis of VEGF protein. In addition, PGF2{alpha} stimulated the level of mRNA for VEGF in these cells. This is probably the first report showing the stimulatory effect of PGF2{alpha} on the VEGF synthesis in osteoblasts as far as we know. Cloprostenol (30) significantly induced the VEGF synthesis in MC3T3-E1 cells. Thus, it is probable that the effect of PGF2{alpha} on the VEGF synthesis is mediated through FP receptor. We also found that incadronate, which alone did not affect the VEGF level in mouse plasma, enhanced the cloprostenol-increased level of VEGF. Thus, it is probable that incadronate amplifies PGF2{alpha}-stimulated VEGF synthesis via FP receptor activation in vivo.

We have previously reported that PGE1 induces VEGF synthesis in these cells (11). Herein, we showed that PGE2 increased the level of mRNA for VEGF and stimulated the synthesis of VEGF. We also found that PGD2 induced VEGF synthesis in these cells. These findings indicate that several PGs induce the VEGF synthesis in osteoblasts. In the present study, indomethacin (31) reduced the PGF2{alpha}-stimulated VEGF synthesis, suggesting that the endogenous PGs are involved in the VEGF synthesis. PGE2 is known to be abundantly produced by osteoblasts, including MC3T3-E1 cells (17). We previously reported that PGF2{alpha} stimulates the synthesis of PGE2 in MC3T3-E1 cells (42). Based on these findings, it is possible that PGE2, at least in part, participates in the PGF2{alpha}-stimulated VEGF synthesis in these cells.

In our previous studies (18, 19, 22), we have shown that PGF2{alpha} activates PKC through both PI-PLC and PC-PLD via heterotrimeric GTP-binding protein and that p44/p42 MAPK is activated by PGF2{alpha} in a PKC-dependent manner. We here showed that bisindolylmaleimide (34) or the down-regulation of PKC suppressed the VEGF synthesis induced by PGF2{alpha}. It is well recognized that the translocation of PKC from cytosol to plasma membrane occurs when PKC is activated (32). We found that PGF2{alpha} truly elicited the PKC{beta}I translocation to the plasma membrane in MC3T3-E1 cells. Therefore, it is probable that PKC{beta}I is involved in the PGF2{alpha} signaling in these cells. In addition, PD98059 and U0126, inhibitors of MEK (35, 36), inhibited the PGF2{alpha}-induced VEGF synthesis, suggesting that the VEGF synthesis is mediated through the activation of p44/p42 MAPK. Therefore, our findings suggest that PGF2{alpha} stimulates VEGF synthesis through PKC- and probably PKC{beta}I-dependent activation of p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. It is well known that MEK1/2 is activated by the upstream kinase, Raf-1 (37). We previously demonstrated that TPA (32) induces the phosphorylation of p44/p42 MAPK in osteoblast-like MC3T3-E1 cells (41). Herein, we showed that TPA elicited the phosphorylation of Raf-1 and MEK1/2 in these cells, suggesting that PKC is an upstream regulator of Raf-1-MEK1/2-p44/p42 MAPK cascade. In addition, we here showed that FTase inhibitor III did not affect the PGF2{alpha}-induced VEGF synthesis. It is recognized that the farnesylation of C-terminal cysteine residue of Ras by FTase is the first step of the post-translational modification of Ras, which plays a role in Raf-1 activation (38). Therefore, it seems unlikely that Ras is involved in the PGF2{alpha}-induced VEGF synthesis in MC3T3-E1 cells.

In the present study, incadronate enhanced the PGF2{alpha}-induced VEGF synthesis in osteoblast-like MC3T3-E1 cells. We also showed that the VEGF synthesis induced by NaF (40) was augmented by incadronate. We have previously reported that heterotrimeric GTP-binding protein is involved in the PGF2{alpha} signaling in these cells (18, 19). Therefore, it is probable that the enhancement of PGF2{alpha}-induced VEGF synthesis by incadronate occurs at a point downstream from the heterotrimeric GTP-binding protein. We next demonstrated that incadronate amplified the phosphorylation of p44/p42 MAPK induced by PGF2{alpha} in MC3T3-E1 cells. In addition, PD98059 (35) suppressed the enhancement by incadronate of the PGF2{alpha}-induced VEGF synthesis in parallel with the VEGF synthesis stimulated by PGF2{alpha}. These findings strongly suggest that incadronate-induced amplification of p44/p42 MAPK activated by PGF2{alpha} is tightly coupled to the enhancement of the VEGF synthesis and that the effect is exerted at a point upstream of p44/p42 MAPK. We here presented that incadronate amplified the PGF2{alpha}-induced phosphorylation of MEK1/2 as well as that of Raf-1 in these cells, suggesting that the effect is exerted at a point upstream of Raf-1. Furthermore, the phosphorylation of p44/p42 MAPK, MEK1/2, and Raf-1 elicited by TPA (32) was enhanced by incadronate, indicating that the effect is exerted at a point downstream from PKC. We found that FTase inhibitor III did not affect the enhancement by incadronate of VEGF synthesis induced by PGF2{alpha} in these cells. Thus, it seems unlikely that the modulation of Ras farnesylation is involved in the enhancement by incadronate of PGF2{alpha}-stimulated VEGF synthesis. Taking our results into account, it is most likely that incadronate enhances PGF2{alpha}-induced VEGF synthesis, and the effect is exerted at the point between PKC and Raf-1 in osteoblast-like MC3T3-E1 cells. The potential mechanism of incadronate in PGF2{alpha}-induced VEGF synthesis in osteoblasts shown here is summarized in Fig. 11.



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FIG. 11.
Diagram of the potential mechanism of the enhancement by incadronate of the PGF2{alpha}-induced VEGF synthesis in MC3T3-E1 cells. +, positive modulation; GTP-binding protein, heterotrimeric GTP-binding protein; PI-PLC, phosphoinositide-hydrolyzing phospholipase C; PC-PLD, phosphatidylcholine-hydrolyzing phospholipase D; PA, phosphatidic acid; DAG, diacylglycerol; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; VEGF, vascular endothelial growth factor.

 

VEGF is established as a specific mitogen of vascular endothelial cells (4). Thus, it is speculated that the synthesis of VEGF by osteoblasts is an important intercellular mediator between the osteoblasts and the vascular endothelial cells. In addition, VEGF is reportedly involved in trabecular bone formation and expansion of the hypertrophic chondrocyte zone in the epiphyseal growth plate of mouse (5). The expansion of the capillary network providing microvasculature is thought to be essential for the process of bone remodeling. Thus, it is possible that PGF2{alpha}-induced VEGF synthesis by osteoblasts through an autocrine/paracrine mechanism promotes the development of vascular endothelial cells in the microenvironment, resulting in the modulation of bone remodeling. Interestingly, it has been reported that incadronate is able to produce intramembranous intramedullary bone formation in vivo. (43). We herein presented that incadronate truly up-regulated the cloprostenolincreased VEGF level in mouse plasma in vivo. It is well known that bisphosphonates mainly bind bone tissue (12), whereas cloprostenol is distributed to the whole body and acts through the activation of FP receptor. Therefore, our present findings strongly suggest that incadronate truly affects osteoblasts and enhances the PGF2{alpha}-induced VEGF synthesis in vivo. Taking our present findings into account, it is most likely that the enhancement of the VEGF synthesis by incadronate plays an important role in bone metabolism.

Nitrogen-containing bisphosphonates, including incadronate, are recognized to inhibit both farnesyl diphosphate synthase and squalene synthase in osteoclasts (12). These enzymes play pivotal roles in the mevalonate/cholesterol biosynthetic pathway. It has recently been reported that zoledronate, a nitrogen-containing bisphosphonate, induces differentiation of human fetal osteoblast cells via inhibition of the mevalonate pathway (44). Another recent report showed that connexin-43, a major gap junction protein, is an essential transducer of the p44/p42 MAPK-activating/anti-apoptotic effects of bisphosphonates in rat osteosarcoma ROS17/2.8 cells or rat calvarial osteoblasts (45). Herein, we found that other bisphosphonates, etidronate, tiludronate, or alendronate did not enhance the VEGF synthesis induced by PGF2{alpha}, suggesting that the enhancement by incadronate is not a common effect of bisphosphonates but a specific effect of incadronate. It has been shown that cycloheptylamino side chain is specific to incadronate (12). Therefore, it is likely that the unique effect of incadronate is attributed to the structural specificity. Further investigation would be required to clarify the details of the effects of bisphosphonates on bone-forming cells.

In conclusion, our present results strongly suggest that PGF2{alpha} stimulates VEGF synthesis through the activation of p44/p42 MAPK in osteoblasts and that incadronate enhances the VEGF synthesis at the point between PKC and Raf-1.


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

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 81-58-2672231; Fax: 81-58-267-2959; E-mail: okozawa{at}cc.gifu-u.ac.jp.

1 The abbreviations used are: VEGF, vascular endothelial growth factor; PG, prostaglandin; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; PI-PLC, phosphoinositide-hydrolyzing phospholipase C; PC-PLD, phosphatidylcholine-hydrolyzing phospholipase D; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; PD98059, 2'-amino-3'-methoxyflanone; FTase, farnesyltransferase; H-Cys-Val-2-naphthylalanine-Met-OH, FTase inhibitor III; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; {alpha}-MEM, {alpha}-minimum essential medium; FCS, fetal calf serum; ELISA, enzyme-linked immunosorbent assay; FP, PGF2{alpha}-sensitive receptor. Back


    ACKNOWLEDGMENTS
 
We are very grateful to Daijiro Hatakeyama and Takahiro Kumagai for their skillful technical assistance.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nijweide, P. J., Burger, E. H., and Feyen, J. H. M. (1986) Physiol. Rev. 66, 855–886[Free Full Text]
  2. Suda, T., Takahashi, N., Udagawa, N., Jimi, E., Gillespie, M. T., and Martin, T. J. (1999) Endocr. Rev. 20, 345–357[Abstract/Free Full Text]
  3. Erlebacher, A., Filvaroff, E. H., Girelman, S. E., and Derynck, R. (1995) Cell 80, 371–378[Medline] [Order article via Infotrieve]
  4. Ferrara, N., and Davis-Smyth, T. (1997) Endocr. Rev. 18, 4–25[Abstract/Free Full Text]
  5. Gerber, H.-P., Vu, T. H., Ryan, A. M., Kowalski, J., Werb, Z., and Ferrara, N. (1999) Nat. Med. 5, 623–628[CrossRef][Medline] [Order article via Infotrieve]
  6. Harada, S., Nagy, J. A., Sullivan, K. A., Thomas, K. A., Endo, N., and Rodan, G. A. (1994) J. Clin. Invest. 93, 2490–2496[Medline] [Order article via Infotrieve]
  7. Goad, D. L., Rubin, J., Wang, H., Tashijian, A. H., Jr., and Patterson, C. (1996) Endocrinology 137, 2262–2268[Abstract]
  8. Wang, D. S., Yamazaki, K., Nohtomi, K., Shizume, K., Ohsumi, K., Shibuya, M., Demura, H., and Sato, K. (1996) J. Bone Miner. Res. 11, 472–479[Medline] [Order article via Infotrieve]
  9. Schalaeppi, J. M., Gutzwiller, S., Finlenzeller, G., and Fournier, B. (1997) Endocr. Res. 23, 213–229[Medline] [Order article via Infotrieve]
  10. Tokuda, H., Kozawa, O., and Uematsu, T. (2000) J. Bone Miner. Res. 15, 2371–2379[Medline] [Order article via Infotrieve]
  11. Tokuda, H., Kozawa, O., Miwa, M., and Uematsu, T. (2001) J. Endocrinol. 170, 629–638[Abstract/Free Full Text]
  12. Fleisch, H., Reszka, A., Rodan, G. A., and Rogers, M. (2002) in Principles of Bone Biology, 2nd. Ed. (Raisz, L. G., and Rodan, G. A., eds) pp. 1361–1385, Academic Press, San Diego, CA
  13. Vitte, C., Fleisch, H., and Guenther, H. L. (1996) Endocrinology 136, 2324–2333
  14. Plotkin, L. I., Weinstein, R. S., Parfitt, A. M., Roberson, P. K., Manolagas, S. C., and Bellido, T. (1999) J. Clin. Invest. 104, 1363–1374[Abstract/Free Full Text]
  15. Reinholz, G. C., Getz, B., Pederson, L., Sanders, E. S., Subramaniam, M., Ingle, J. N., and Spelsberg, T. C. (2000) Cancer Res. 60, 6001–6007[Abstract/Free Full Text]
  16. Mackie, P. S., Fisher, J. L., Zhou, H., and Choong, P. F. (2001) Br. J. Cancer 84, 951–958[CrossRef][Medline] [Order article via Infotrieve]
  17. Pilbeam, C. C., Harrison, J. R., and Raisz, L. G. (1996) in Principles of Bone Biology (Raisz, L. G., and Rodan, G. A., eds) pp. 715–728, Academic Press, San Diego, CA
  18. Miwa, M., Tokuda, H., Tsushita, K., Kotoyori, J., Takahashi, Y., Ozaki, N., Kozawa, O., and Oiso, Y. (1990) Biochem. Biophys. Res. Commun. 171, 1229–1235[Medline] [Order article via Infotrieve]
  19. Kozawa, O., Suzuki, A., Kotoyori, J., Tokuda, H., Watanabe, Y., Ito, Y., and Oiso, Y. (1994) J. Cell. Biochem. 55, 373–379[Medline] [Order article via Infotrieve]
  20. Nishizuka, Y. (1992) Science 258, 607–614[Medline] [Order article via Infotrieve]
  21. Exton, J. H. (1999) Biochim. Biophys. Acta 1439, 121–133[Medline] [Order article via Infotrieve]
  22. Tokuda, H., Kozawa, O., Harada, A., and Uematsu, T. (1999) Cell. Signal. 11, 325–330[CrossRef][Medline] [Order article via Infotrieve]
  23. Sudo, M., Kodama, H., Amagai, Y., Yamamoto, S., and Kasai, S. (1983) J. Cell Biol. 96, 191–198[Abstract]
  24. Kozawa, O., Tokuda, H., Miwa, M., Kotoyori, J., and Oiso, Y. (1992) Exp. Cell Res. 198, 130–134[Medline] [Order article via Infotrieve]
  25. Kozawa, O., Suzuki, A., Tokuda, H., and Uematsu, T. (1995) Am. J. Physiol. 272, E208–E211
  26. Miao, L., Dai, Y., and Zhang, J. (2002) Am. J. Physiol. 283, H983–H989
  27. Laemmli, U. K. (1970) Nature 227, 680–685[Medline] [Order article via Infotrieve]
  28. Kato, K., Goto, S., Hasegawa, K., and Inaguma, Y. (1993) J Biochem. (Tokyo) 114, 640–647[Abstract]
  29. Obrig, T. G., Culp, W. J., Mckeehan, W. L., and Hardesty, B. (1971) J. Biol. Chem. 246, 174–181[Abstract/Free Full Text]
  30. Dukes, M., Russell, W., and Walpole, A. L. (1974) Nature 250, 330–331[Medline] [Order article via Infotrieve]
  31. Smith, W. L. (1989) Biochem. J. 259, 315–324[Medline] [Order article via Infotrieve]
  32. Nishizuka, Y. (1986) Science 233, 305–312[Medline] [Order article via Infotrieve]
  33. Sakai, T., Okano, Y., Nozawa, Y., and Oka, N. (1992) Cell Calcium 13, 329–340[Medline] [Order article via Infotrieve]
  34. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., and Kirlowsky, J. (1991) J. Biol. Chem. 266, 15771–15781[Abstract/Free Full Text]
  35. Alessi, D. R., Cuenda, A., Cohen, P., Dulley, D. T., and Sartiel, A. R. (1995) J. Biol. Chem. 270, 27484–27494
  36. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. (1998) J. Biol. Chem. 273, 18623–18632[Abstract/Free Full Text]
  37. Widmann, C., Gibson, S., Jarpe, M. B., and Johnson, G. L. (1999) Physiol. Rev. 79, 143–180[Abstract/Free Full Text]
  38. Kikuchi, A., and Williams, L. T. (1994) J. Biol. Chem. 269, 20054–20059[Abstract/Free Full Text]
  39. Hamilton, A. D., and Sebti, S. M. (1995) Drug News Perspect. 8, 138
  40. Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615–649[CrossRef][Medline] [Order article via Infotrieve]
  41. Hatakeyama, D., Kozawa, O., Otsuka, T., Shibata, T., and Uematsu, T. (2002) J. Cell. Biochem. 85, 621–628[CrossRef][Medline] [Order article via Infotrieve]
  42. Tokuda, H., Oiso, Y., and Kozawa, O. (1992) J. Cell. Biochem. 48, 262–268[Medline] [Order article via Infotrieve]
  43. Nii, A., Fujimoto, R., Okazaki, A., Narita, K., and Miki, H. (1994) Toxicol. Pathol. 22, 536–544[Medline] [Order article via Infotrieve]
  44. Reinholz, G. G., Getz, B., Sanders, E. S., Karpeisky, M. Y., Padyukova, N. S., Mikhailov, S. N., Ingle, J. N., and Spelsberg, T. C. (2002) Breast Cancer Res. Treat. 71, 257–268[CrossRef][Medline] [Order article via Infotrieve]
  45. Plotkin, L. I., Manolagas, S. C., and Bellido, T. (2002) J. Biol. Chem. 277, 8648–8657[Abstract/Free Full Text]