Incadronate Amplifies Prostaglandin F2α-induced Vascular Endothelial Growth Factor Synthesis in Osteoblasts

We have previously reported that prostaglandin F2α (PGF2α) 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 PGF2α and the effect of incadronate on the VEGF synthesis in these cells. PGF2α significantly stimulated the VEGF synthesis in a dose-dependent manner between 1 pm and 10 μm. Cycloheximide reduced the PGF2α effect. PGF2α increased the levels of mRNA for VEGF. Cloprostenol, a PGF2α-sensitive receptor agonist, potently induced the VEGF synthesis. Indomethacin, an inhibitor of cyclooxygenase, significantly reduced the PGF2α-induced VEGF synthesis. Bisindolylmaleimide, an inhibitor of PKC, reduced the PGF2α-induced VEGF synthesis. The VEGF synthesis induced by PGF2α was significantly attenuated in the PKC down-regulated cells. PGF2α elicited the translocation of PKCβI from cytosol to membrane fraction. PD98059 or U0126, inhibitors of MEK, suppressed the VEGF synthesis induced by PGF2α. Farnesyltransferase inhibitor failed to affect the PGF2α-induced VEGF synthesis. Incadronate enhanced the synthesis of VEGF induced by PGF2α. NaF-induced VEGF synthesis was also amplified by incadronate. PD98059 suppressed the enhancement by incadronate of PGF2α-induced VEGF synthesis. Incadronate markedly enhanced the phosphorylation of Raf-1, MEK1/2, and p44/p42 MAPK induced by PGF2α 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 PGF2α 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. We have previously reported that prostaglandin F2α (PGF2α) 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 PGF2α and the effect of incadronate on the VEGF synthesis in these cells. PGF2α significantly stimulated the VEGF synthesis in a dose-dependent manner between 1 pm and 10 μm. Cycloheximide reduced the PGF2α effect. PGF2α increased the levels of mRNA for VEGF. Cloprostenol, a PGF2α-sensitive receptor agonist, potently induced the VEGF synthesis. Indomethacin, an inhibitor of cyclooxygenase, significantly reduced the PGF2α-induced VEGF synthesis. Bisindolylmaleimide, an inhibitor of PKC, reduced the PGF2α-induced VEGF synthesis. The VEGF synthesis induced by PGF2α was significantly attenuated in the PKC down-regulated cells. PGF2α elicited the translocation of PKCβI from cytosol to membrane fraction. PD98059 or U0126, inhibitors of MEK, suppressed the VEGF synthesis induced by PGF2α. Farnesyltransferase inhibitor failed to affect the PGF2α-induced VEGF synthesis. Incadronate enhanced the synthesis of VEGF induced by PGF2α. NaF-induced VEGF synthesis was also amplified by incadronate. PD98059 suppressed the enhancement by incadronate of PGF2α-induced VEGF synthesis. Incadronate markedly enhanced the phosphorylation of Raf-1, MEK1/2, and p44/p42 MAPK induced by PGF2α 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 PGF2α 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. 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 (1Nijweide P.J. Burger E.H. Feyen J.H.M. Physiol. Rev. 1986; 66: 855-886Crossref PubMed Scopus (397) Google Scholar). 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 κB) expression through their specific receptors on osteoblasts, suggesting that osteoblasts play pivotal roles in the regulation of bone resorption (2Suda T. Takahashi N. Udagawa N. Jimi E. Gillespie M.T. Martin T.J. Endocr. Rev. 1999; 20: 345-357Crossref PubMed Google Scholar). 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 (3Erlebacher A. Filvaroff E.H. Girelman S.E. Derynck R. Cell. 1995; 80: 371-378Abstract Full Text PDF PubMed Scopus (611) Google Scholar). Vascular endothelial growth factor (VEGF) 1The 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; α-MEM, α-minimum essential medium; FCS, fetal calf serum; ELISA, enzyme-linked immunosorbent assay; FP, PGF2α-sensitive receptor.1The 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; α-MEM, α-minimum essential medium; FCS, fetal calf serum; ELISA, enzyme-linked immunosorbent assay; FP, PGF2α-sensitive receptor. is a potent angiogenic factor that induces endothelial cell proliferation, angiogenesis, and capillary permeability (4Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). It is well recognized that VEGF is produced and secreted from a variety of cell types (4Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). 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 (5Gerber H.-P. Vu T.H. Ryan A.M. Kowalski J. Werb Z. Ferrara N. Nat. Med. 1999; 5: 623-628Crossref PubMed Scopus (1673) Google Scholar). Moreover, evidence is accumulating that osteoblasts produce VEGF and secrete it in response to various humoral factors (4Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar, 6Harada S. Nagy J.A. Sullivan K.A. Thomas K.A. Endo N. Rodan G.A. J. Clin. Invest. 1994; 93: 2490-2496Crossref PubMed Scopus (361) Google Scholar, 7Goad D.L. Rubin J. Wang H. Tashijian Jr., A.H. Patterson C. Endocrinology. 1996; 137: 2262-2268Crossref PubMed Scopus (205) Google Scholar, 8Wang D.S. Yamazaki K. Nohtomi K. Shizume K. Ohsumi K. Shibuya M. Demura H. Sato K. J. Bone Miner. Res. 1996; 11: 472-479Crossref PubMed Scopus (123) Google Scholar, 9Schalaeppi J.M. Gutzwiller S. Finlenzeller G. Fournier B. Endocr. Res. 1997; 23: 213-229Crossref PubMed Scopus (79) Google Scholar). 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 (10Tokuda H. Kozawa T. J. Bone Miner. Res. PubMed Scopus Google Scholar, H. Kozawa M. T. J. PubMed Scopus Google Scholar). the mechanism VEGF synthesis in osteoblasts has been of have been as potent inhibitors of bone resorption and for the of various bone associated with increased bone resorption such as bone and H. A. Rodan G.A. M. Rodan G.A. of Bone Scholar). of to bone and is to the mechanism of the of H. A. Rodan G.A. M. Rodan G.A. of Bone Scholar). the at of the of on and are to through the to osteoblasts H. A. Rodan G.A. M. Rodan G.A. of Bone Scholar). It has been reported that and the synthesis of an inhibitor of bone resorption in cell C. H. Endocrinology. 1996; Scopus Google Scholar). and of osteoblasts through the activation of p44/p42 MAPK A.M. T. J. Clin. Invest. 1999; PubMed Scopus Google Scholar). and have recently been to the and activities of fetal osteoblasts B. M. Res. Google Scholar). It has also been reported that and the expression of mRNA for and RANKL in cells H. J. PubMed Scopus Google Scholar). Thus, it is currently that the of on bone metabolism are through osteoclasts also osteoblasts. are well to as of osteoblasts (1Nijweide P.J. Burger E.H. Feyen J.H.M. Physiol. Rev. 1986; 66: 855-886Crossref PubMed Scopus (397) Google Scholar, Rodan G.A. of Bone Scholar). PGF2α is recognized to a potent Rodan G.A. of Bone and stimulates the and the of osteoblasts Rodan G.A. of Bone Scholar). In M. H. K. J. Takahashi N. Kozawa Res. PubMed Scopus Google Scholar, A. J. H. J. Cell. 1994; PubMed Scopus Google we have that PGF2α stimulates phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase recognized to two of protein kinase C (PKC) activation PubMed Scopus Google Scholar, 1999; PubMed Scopus Google in osteoblast-like MC3T3-E1 cells. We have also reported that PGF2α activates p44/p42 MAPK through PKC in these cells H. Kozawa A. T. Cell. 1999; 11: PubMed Scopus Google Scholar). In the present study, we investigated PGF2α stimulates VEGF synthesis in MC3T3-E1 cells, and the mechanism of VEGF synthesis and the effect of incadronate on the VEGF synthesis. results strongly suggest that incadronate enhances the VEGF synthesis in osteoblasts and that the effect is at the point between PKC and Raf-1. and from was from and by and Bisindolylmaleimide, and inhibitor was from was from PKCβI p44/p42 MAPK p44/p42 MAPK and from mouse VEGF and a mouse VEGF mRNA from and a from and from and in PD98059 and in FTase inhibitor was in The concentration of or was which affect the for VEGF or the of osteoblast-like MC3T3-E1 cells from mouse M. H. S. S. J. PubMed Scopus Google as previously H. M. J. Res. PubMed Scopus Google Scholar). the cells in α-minimum essential fetal calf at in a of The cells into or in the was for The cells used for the cells with for as previously reported A. H. T. J. Physiol. 1995; Scholar). for cells stimulated by or in 1 of for the In the with or was for to the and the with PD98059, or was for The cells with FTase inhibitor for The was by the and VEGF in the was by a VEGF the cells with or for of and of mRNA for cells stimulated by PGF2α or in 1 of for was with a from was to the of mRNA for VEGF a mouse VEGF mRNA of p44/p42 MAPK, MEK1/2, or cells stimulated by PGF2α or in of for the The cells with and and in a and The of PKCβI translocation to the plasma membrane was as previously J. J. Physiol. Google Scholar). The was at for 1 to membrane and and the was in a the of p44/p42 MAPK, MEK1/2, or Raf-1, the was as a at for 10 at was by PubMed Scopus Google in was as previously K. S. K. PubMed Scopus Google by PKCβI p44/p42 MAPK p44/p42 MAPK or with in used as on the was on by of the the cells with incadronate or for for VEGF in in from into a a with incadronate a with and a with in a with an of at a of to the for the of incadronate or and to the for and a Incadronate at a of was as a of and for an via a at a of was a by In a of the of was at the of of and the was for the via the on with at the The plasma concentration of VEGF was by a VEGF The was in with the of was at with an The was by of by the for between and a of was are as the S.E. of was with of VEGF and the of mRNA for VEGF in MC3T3-E1 stimulated the VEGF synthesis to in osteoblast-like MC3T3-E1 cells The VEGF synthesis was The effect of PGF2α on VEGF synthesis was dose-dependent in the between 1 pm and 10 The effect of PGF2α was at 10 μm. Cycloheximide an inhibitor of protein synthesis B. J. Full Text PDF PubMed Google which by effect on the VEGF markedly the VEGF level for for 10 for 10 PGF2α and with the of PGF2α for 10 PGF2α with 10 as the for PGF2α also increased the level of mRNA for VEGF receptor is for the PGF2α-induced VEGF synthesis or in MC3T3-E1 cells, we the effect of an M. PubMed Scopus Google on the VEGF synthesis. significantly stimulated the VEGF synthesis is to produced by osteoblasts, MC3T3-E1 cells, and as a potent of bone metabolism Rodan G.A. of Bone Scholar). Thus, we the effect of on the VEGF level in these cells. induced the VEGF synthesis and increased the level of mRNA for VEGF the of PGF2α. We have previously reported that stimulates VEGF synthesis in MC3T3-E1 cells H. Kozawa M. T. J. PubMed Scopus Google Scholar). In we that induced VEGF synthesis in these cells effect of PGF2α on VEGF synthesis in MC3T3-E1 cells. The cells stimulated by various of PGF2α for for cells have been from the S.E. of results with two and cell with the of of PGF2α or on the level of mRNA for VEGF in MC3T3-E1 cells stimulated by 10 10 or for The cells and was The from was for the of mRNA for VEGF. the S.E. of results with two and cell for with the of with the of with the of in a new of or on VEGF synthesis in with PGF2α in MC3T3-E1 cells stimulated by 10 10 10 or for the S.E. of results with two and cell with the of with the of with the of with the of in a new of on the PGF2α-induced VEGF in MC3T3-E1 is well that as of osteoblasts (1Nijweide P.J. Burger E.H. Feyen J.H.M. Physiol. Rev. 1986; 66: 855-886Crossref PubMed Scopus (397) Google Scholar, Rodan G.A. of Bone Scholar). synthesis is in the VEGF synthesis induced by we investigated the effect of a inhibitor J. PubMed Scopus Google on the PGF2α-induced VEGF synthesis in MC3T3-E1 cells. Indomethacin, which effect on the VEGF reduced the VEGF synthesis stimulated by PGF2α in a dose-dependent manner in the between and of PKC or on PGF2α-induced VEGF in MC3T3-E1 have previously reported that PGF2α activates and PC-PLD, as of PKC activation PubMed Scopus Google Scholar, 1999; PubMed Scopus Google in osteoblast-like MC3T3-E1 cells M. H. K. J. Takahashi N. Kozawa Res. PubMed Scopus Google Scholar, A. J. H. J. Cell. 1994; PubMed Scopus Google Scholar). It has been reported that of of a 1986; PubMed Scopus Google PKC in these cells T. N. PubMed Scopus Google Scholar). We also that the of a 1986; PubMed Scopus Google in MC3T3-E1 cells with for is reduced to of the in cells A. H. T. J. Physiol. 1995; Scholar). the of PKC in the VEGF synthesis in MC3T3-E1 cells, we the effect of on the VEGF. The effect of PGF2α on VEGF synthesis was significantly reduced in the PKC down-regulated cells with that in the cells In we the effect of an inhibitor of PKC H. T. M. E. J. J. Full Text PDF PubMed Google on the VEGF synthesis induced by PGF2α in MC3T3-E1 cells. Bisindolylmaleimide, which by effect on the level of VEGF, significantly suppressed the PGF2α-induced VEGF synthesis in a dose-dependent manner in the between 1 and 10 of PKC on the PGF2α-induced VEGF synthesis in MC3T3-E1 cells with or for and stimulated by 10 PGF2α or for the S.E. of results with two and cell with the of PGF2α with the of PGF2α in a new of the of PKCβI to the of MC3T3-E1 has been reported the PKC and mainly in osteoblast-like MC3T3-E1 cells, and with induces translocation from cytosol to membrane T. N. PubMed Scopus Google Scholar). It is well recognized that the translocation of PKC from cytosol to plasma membrane PKC is activated 1986; PubMed Scopus Google Scholar). We the effect of PGF2α on the PKCβI translocation to the plasma membrane of these cells. PGF2α elicited the PKCβI translocation to the plasma membrane The effect of PGF2α was at the of PD98059, U0126, or FTase on the PGF2α-induced VEGF in MC3T3-E1 have previously reported that PGF2α activates p44/p42 MAPK in a PKC-dependent in osteoblast-like MC3T3-E1 cells H. Kozawa A. T. Cell. 1999; 11: PubMed Scopus Google Scholar). p44/p42 MAPK is in the PGF2α-induced VEGF synthesis in these cells, we the effect of PD98059, a specific inhibitor of A. J. 1995; Full Text Full Text PDF Scopus Google on the VEGF synthesis induced by PGF2α. PD98059, which affect the level of VEGF, reduced the PGF2α-induced VEGF synthesis in the between and The effect of PD98059 was at which of the U0126, inhibitor of J.M. J. Full Text Full Text PDF PubMed Scopus Google also suppressed the VEGF synthesis induced by PGF2α the it is well recognized that of receptors to the activation of p44/p42 MAPK through the activation of C. S. Physiol. Rev. 1999; PubMed Scopus Google Scholar). with Raf-1, and activates MEK, the kinase of p44/p42 MAPK C. S. Physiol. Rev. 1999; PubMed Scopus Google Scholar). The of of by FTase is as the of of in the of the to the an of activation C. S. Physiol. Rev. 1999; PubMed Scopus Google Scholar, A. J. 1994; Full Text PDF PubMed Google Scholar). is in the VEGF synthesis induced by PGF2α in MC3T3-E1 cells, we the effect of FTase inhibitor 1995; on the VEGF synthesis. FTase inhibitor which by effect on VEGF affect the VEGF synthesis stimulated by PGF2α of FTase inhibitor on the PGF2α-induced VEGF synthesis in MC3T3-E1 cells with FTase inhibitor or for and stimulated by 10 PGF2α or for the S.E. of results with two and cell inhibitor in a new of Incadronate on the or NaF-induced VEGF in MC3T3-E1 which affect VEGF significantly enhanced the VEGF synthesis induced by PGF2α in a dose-dependent manner in the between 10 and The effect of incadronate on the PGF2α-induced VEGF synthesis was at which enhancement of the PGF2α effect. such as or failed to the PGF2α-induced VEGF synthesis We that PD98059 reduced the enhancement by incadronate of PGF2α-induced VEGF synthesis FTase inhibitor failed to affect the enhancement of PD98059 on the enhancement by incadronate on the PGF2α-induced VEGF synthesis in MC3T3-E1 cells with incadronate or for the cells with PD98059 or for and stimulated by 10 PGF2α or for the S.E. of results with two and cell with the of PGF2α with the of PGF2α with the of the of incadronate and with the of PGF2α with the of the of incadronate and PGF2α. in a new We have previously reported that protein is in the PGF2α-induced activation of and in osteoblast-like MC3T3-E1 cells M. H. K. J. Takahashi N. Kozawa Res. PubMed Scopus Google Scholar, A. J. H. J. Cell. 1994; PubMed Scopus Google Scholar). Thus, we investigated the effect of incadronate on the VEGF synthesis stimulated by as a direct activator of Rev. PubMed Scopus Google Scholar). stimulated VEGF synthesis Incadronate significantly enhanced the NaF-induced VEGF synthesis of incadronate on the NaF-induced VEGF synthesis in MC3T3-E1 cells with various of incadronate for and stimulated by or for 1 an of the cells for the S.E. of results with two and cell with the of with the of in a new of Incadronate on the or of p44/p42 MAPK in MC3T3-E1 the effect of incadronate on the phosphorylation of p44/p42 MAPK induced by PGF2α in MC3T3-E1 cells. which affect the p44/p42 MAPK significantly enhanced the PGF2α-induced p44/p42 MAPK phosphorylation the effect of incadronate is at a point of PKC or we the effect of incadronate on the phosphorylation of p44/p42 We have that p44/p42 MAPK in MC3T3-E1 cells Kozawa T. T. T. J. Cell. PubMed Scopus Google Scholar). Incadronate markedly enhanced the phosphorylation of p44/p42 MAPK as well as the PGF2α-induced phosphorylation of Incadronate on the of and by in MC3T3-E1 is well recognized that the activation of p44/p42 MAPK is regulated by MEK1/2, which is regulated by the kinase as C. S. Physiol. Rev. 1999; PubMed Scopus Google Scholar). We that PGF2α and induced the phosphorylation in osteoblast-like MC3T3-E1 cells Thus, we the effect of incadronate on the phosphorylation of induced by PGF2α in these cells. which by the significantly enhanced the phosphorylation of induced by PGF2α In phosphorylation of was markedly amplified by incadronate We that PGF2α and induced the phosphorylation of we investigated the of incadronate on the phosphorylation of induced by PGF2α or Incadronate effect on the phosphorylation of markedly enhanced the or phosphorylation of and of or Incadronate on the of VEGF in level of VEGF in mouse plasma was significantly increased the of The effect of was from to and the effect was at of which by effect on the VEGF level in mouse significantly enhanced the cloprostenol-increased VEGF level of or incadronate on the level of VEGF in or was as a and for or was for by from the of the S.E. of results with two and with the of with the of with the of with the of in a new In the present study, we that PGF2α induced VEGF synthesis in osteoblast-like MC3T3-E1 cells. We VEGF in the in the VEGF was secreted from these cells, it is that the in in synthesis a specific We the effect of on the level of VEGF, suggesting that PGF2α stimulates synthesis of VEGF In PGF2α stimulated the level of mRNA for VEGF in these cells. is the the effect of PGF2α on the VEGF synthesis in osteoblasts as as we M. PubMed Scopus Google significantly induced the VEGF synthesis in MC3T3-E1 cells. Thus, it is that the effect of PGF2α on the VEGF synthesis is through receptor. We also that which affect the VEGF level in mouse enhanced the cloprostenol-increased level of VEGF. Thus, it is that incadronate VEGF synthesis via receptor activation in vivo. We have previously reported that induces VEGF synthesis in these cells H. Kozawa M. T. J. PubMed Scopus Google Scholar). we that increased the level of mRNA for VEGF and stimulated the synthesis of VEGF. We also that induced VEGF synthesis in these cells. These that the VEGF synthesis in osteoblasts. In the present study, J. PubMed Scopus Google reduced the VEGF suggesting that the are in the VEGF synthesis. is to produced by osteoblasts, MC3T3-E1 cells Rodan G.A. of Bone Scholar). We previously reported that PGF2α stimulates the synthesis of in MC3T3-E1 cells H. Kozawa J. Cell. PubMed Scopus Google Scholar). on these it is that at in in the VEGF synthesis in these cells. In M. H. K. J. Takahashi N. Kozawa Res. PubMed Scopus Google Scholar, A. J. H. J. Cell. 1994; PubMed Scopus Google Scholar, H. Kozawa A. T. Cell. 1999; 11: PubMed Scopus Google we have that PGF2α activates PKC through and via protein and that p44/p42 MAPK is activated by PGF2α in a PKC-dependent We that H. T. M. E. J. J. Full Text PDF PubMed Google or the of PKC suppressed the VEGF synthesis induced by PGF2α. It is well recognized that the translocation of PKC from cytosol to plasma membrane PKC is activated 1986; PubMed Scopus Google Scholar). We that PGF2α elicited the PKCβI translocation to the plasma membrane in MC3T3-E1 cells. it is that PKCβI is in the PGF2α in these cells. In PD98059 and U0126, inhibitors of A. J. 1995; Full Text Full Text PDF Scopus Google Scholar, J.M. J. Full Text Full Text PDF PubMed Scopus Google the PGF2α-induced VEGF suggesting that the VEGF synthesis is through the activation of p44/p42 suggest that PGF2α stimulates VEGF synthesis through and activation of p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. It is well that is activated by the C. S. Physiol. Rev. 1999; PubMed Scopus Google Scholar). We previously that 1986; PubMed Scopus Google induces the phosphorylation of p44/p42 MAPK in osteoblast-like MC3T3-E1 cells Kozawa T. T. T. J. Cell. PubMed Scopus Google Scholar). we that elicited the phosphorylation of and in these cells, suggesting that PKC is an of MAPK In we that FTase inhibitor affect the PGF2α-induced VEGF synthesis. It is recognized that the of of by FTase is the of the of which a in activation A. J. 1994; Full Text PDF PubMed Google Scholar). it that is in the PGF2α-induced VEGF synthesis in MC3T3-E1 cells. In the present study, incadronate enhanced the PGF2α-induced VEGF synthesis in osteoblast-like MC3T3-E1 cells. We also that the VEGF synthesis induced by Rev. PubMed Scopus Google was by incadronate. We have previously reported that protein is in the PGF2α in these cells M. H. K. J. Takahashi N. Kozawa Res. PubMed Scopus Google Scholar, A. J. H. J. Cell. 1994; PubMed Scopus Google Scholar). it is that the enhancement of PGF2α-induced VEGF synthesis by incadronate at a point from the We that incadronate amplified the phosphorylation of p44/p42 MAPK induced by PGF2α in MC3T3-E1 cells. In PD98059 A. J. 1995; Full Text Full Text PDF Scopus Google suppressed the enhancement by incadronate of the PGF2α-induced VEGF synthesis in with the VEGF synthesis stimulated by PGF2α. These strongly suggest that of p44/p42 MAPK activated by PGF2α is to the enhancement of the VEGF synthesis and that the effect is at a point of p44/p42 We that incadronate amplified the PGF2α-induced phosphorylation of as well as that of in these cells, suggesting that the effect is at a point of Raf-1. the phosphorylation of p44/p42 MAPK, MEK1/2, and elicited by 1986; PubMed Scopus Google was enhanced by that the effect is at a point from We that FTase inhibitor affect the enhancement by incadronate of VEGF synthesis induced by PGF2α in these cells. Thus, it that the of is in the enhancement by incadronate of VEGF synthesis. results into it is that incadronate enhances PGF2α-induced VEGF and the effect is at the point between PKC and in osteoblast-like MC3T3-E1 cells. The mechanism of incadronate in PGF2α-induced VEGF synthesis in osteoblasts is in VEGF is as a specific of vascular endothelial cells (4Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). Thus, it is that the synthesis of VEGF by osteoblasts is an between the osteoblasts and the vascular endothelial cells. In VEGF is in trabecular bone formation and expansion of the hypertrophic chondrocyte zone in the epiphyseal growth plate of mouse (5Gerber H.-P. Vu T.H. Ryan A.M. Kowalski J. Werb Z. Ferrara N. Nat. Med. 1999; 5: 623-628Crossref PubMed Scopus (1673) Google Scholar). The expansion of the capillary is to essential for the of bone Thus, it is that PGF2α-induced VEGF synthesis by osteoblasts through an mechanism the of vascular endothelial cells in the in the of bone it has been reported that incadronate is to produce bone formation in vivo. A. R. A. K. H. 1994; PubMed Scopus Google Scholar). We that incadronate the VEGF level in mouse plasma in vivo. It is well that mainly bone H. A. Rodan G.A. M. Rodan G.A. of Bone is to the and through the activation of receptor. present strongly suggest that incadronate osteoblasts and enhances the PGF2α-induced VEGF synthesis in vivo. present into it is that the enhancement of the VEGF synthesis by incadronate an in bone are recognized to and in osteoclasts H. A. Rodan G.A. M. Rodan G.A. of Bone Scholar). These play pivotal roles in the It has recently been reported that a induces of fetal cells via of the B. Res. PubMed Scopus Google Scholar). that a is an essential of the p44/p42 of in cells or osteoblasts T. J. Full Text Full Text PDF PubMed Scopus Google Scholar). we that or the VEGF synthesis induced by suggesting that the enhancement by incadronate is a effect of a specific effect of incadronate. It has been that is specific to incadronate H. A. Rodan G.A. M. Rodan G.A. of Bone Scholar). it is that the effect of incadronate is to the to the of the of on cells. In present results strongly suggest that PGF2α 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. We are to and for their