Junji Shinoda

ETA receptor mediates the signaling of endothelin-1 in osteoblast-like cells

We previously reported that endothelin-1 (ET-1) stimulates phosphatidylcholine-hydrolyzing phospholipase D independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the characteristics of the receptors mediating ET-1-induced intracellular signaling pathway in MC3T3-E1 cells. Cyclo-D-Trp-D-Asp-Pro-D-Val-Leu (BQ123), a selective ETA receptor antagonist, significantly inhibited the ET-1-induced formation of inositol phosphates in a dose-dependent manner in the range between 22 nmol/L (IC50) and 2.2 mumol/L (IC50 x 100). On the contrary, N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma MeLeu-D-Trp(COOMe)-D-Nle-ONa (BQ788), a selective ETB receptor antagonist, had no effect on the ET-1-induced formation of inositol phosphates in the range between 1.2 nmol/L (IC50) and 120 nmol/L (IC50 x 100). BQ123 significantly suppressed the ET-1-induced formation of choline dose-dependently, however, BQ788 did not affect the choline formation. BQ123 also inhibited the ET-1-induced release of arachidonic acid, but BQ788 had little effect. The results strongly suggest that ETA receptor mediates the three intracellular signaling pathways of ET-1: (1) phosphoinositide hydrolysis by phospholipase C; (2) phosphatidylcholine hydrolysis by phospholipase D; (3) arachidonic acid release in osteoblast-like cells.

Thrombin induces proliferation of osteoblast-like cells through phosphatidylcholine hydrolysis.

We examined the effect of thrombin on phosphatidylcholine‐hydrolyzing phospholipase D activity in osteoblast‐like MC3T3‐E1 cells. Thrombin stimulated the formation of choline dose dependently in the range between 0.01 and 1 U/ml, but not the phosphocholine formation. Diisopropylfluorophosphate (DFP)‐inactivated thrombin had little effect on the choline formation. The combined effects of thrombin and 12‐O‐tetradecanoylphorbol‐13‐acetate, a protein kinase C‐activating phorbol ester, on the choline formation were additive. Staurosporine, an inhibitor of protein kinases, had little effect on the thrombin‐induced formation of choline. Combined addition of thrombin and NaF, an activator of heterotrimeric GTP‐binding protein, did not stimulate the formation of choline further. Pertussis toxin had little effect on the thrombin‐induced formation of choline. Thrombin stimulated Ca2+ influx from extracellular space time and dose dependently. The depletion of extracellular Ca2+ by EGTA exclusively reduced the thrombin‐induced choline formation. Thrombin had only a slight effect on phosphoinositide‐hydrolyzing phospholipase C activity. Thrombin induced diacylglycerol formation and DNA synthesis, and increased the number of MC3T3‐E1 cells, but DFP‐inactivated thrombin did not. Thrombin suppressed both basal and fetal calf serum‐induced alkaline phosphatase activity in these cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, inhibited both the thrombin‐induced diacylglycerol formation and DNA synthesis. These results suggest that thrombin stimulates phosphatidylcholine‐hydrolyzing phospholipase D due to self‐induced Ca2+ influx independently of protein kinase C activation in osteoblast‐like cells and that its proliferative effect depends on phospholipase D activation.

Basic fibroblast growth factor stimulates phosphatidylcholine‐hydrolyzing phospholipase D in osteoblast‐like cells

We examined the effect of basic fibroblast growth factor (bFGF) on the activation of phosphatidylcholine‐hydrolyzing phospholipase D in osteoblast‐like MC3T3‐E1 cells. bFGF stimulated both the formations of choline (EC50 was 30 ng/ml) and inositol phosphates (EC50 was 10 ng/ml). Calphostin C, an inhibitor of protein kinase C (PKC), had little effect on the bFGF‐induced formation of choline. bFGF stimulated the formation of choline also in PKC down regulated cells. Genistein and methyl 2,5‐dihydroxycinnamate, inhibitors of protein tyrosine kinases, significantly suppressed the bFGF‐induced formation of choline. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, enhanced the bFGF‐induced formation of choline. In vitro kinase assay for FGF receptors revealed that FGF receptor 1 and 2 were autophosphorylated after FGF stimulation. bFGF dose‐dependently stimulated DNA synthesis of these cells. These results strongly suggest that bFGF activates phosphatidylcholine‐hydrolyzing phospholipase D through the activation of tyrosine kinase, but independently of PKC activated by phosphoinositide hydrolysis in osteoblast‐like cells.

Tyrosine kinase is involved in angiotensin II-stimulated phospholipase D activation in aortic smooth muscle cells: Function of Ca2+ influx

In the present study, we examined the effect of angiotensin II (Ang II) on phosphatidylcholine-hydrolyzing phospholipase D activity in subcultured rat aortic smooth muscle cells (SMC). Ang II dose-dependently stimulated the formation of choline and inositol phosphates. The effect of Ang II on the formation of inositol phosphates (EC50 was 0.249 +/- 0.091 nM) was more potent than that on the formation of choline (EC50 was 2.39 +/- 1.29 nM). A combination of Ang II and 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, additively stimulated the formation of choline. Staurosporine, an inhibitor of protein kinases, inhibited the TPA-induced formation of choline, but had little effect on the Ang II-induced choline formation. Ang II stimulated Ca2+ influx from extracellular space time- and dose-dependently. The depletion of extracellular Ca2+ by (ethylenebis(oxyethylenenitrilo)) tetraacetic acid (EGTA) significantly reduced the Ang II-induced formation of choline. Genistein and tyrphostin, protein tyrosine kinase inhibitors, significantly suppressed the Ang II-induced Ca2+ influx. Genistein and tyrphostin also suppressed the Ang II-induced formation of choline. These results suggest that Ang II stimulates phosphatidylcholine-hydrolyzing phospholipase D due to Ca2+ influx from the extracellular space in rat aortic SMC, and that protein tyrosine kinase is involved in the Ang II-induced Ca2+ influx, resulting in the promotion of phosphatidylcholine hydrolysis.

Arachidonic acid release induced by extracellular ATP in osteoblasts: role of phospholipase D

In a previous study, we have shown that extracellular ATP stimulates Ca2+ influx resulting in the release of arachidonic acid (AA) and prostaglandin E2 (PGE2) synthesis in osteoblast-like MC3T3-E1 cells. In addition, we have recently reported that extracellular ATP stimulates phosphatidylcholine hydrolysis by phospholipase D (PLD) independently from the activation of protein kinase C in these cells. It is well recognized that phosphatidylcholine is hydrolysed by PLD, generating phosphatidic acid, which can be further degraded by phosphatidic acid phosphohydrolase to diacylglycerol (DG). In the present study, we investigated the role of PLD activation in the extracellular ATP-induced AA release and PGE2 synthesis in osteoblast-like MC3T3-E1 cells. Extracellular ATP stimulated AA release dose-dependently in the range between 0.1 and 1 mM. Propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, significantly inhibited the AA release induced by extracellular ATP in a dose-dependent manner in the range between 100 and 300 microM. 1,6-Bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of DG lipase, significantly suppressed the AA release induced by extracellular ATP. Both the pretreatment of propranolol and RHC-80267 also inhibited the extracellular ATP-induced PGE2 synthesis. These results strongly suggest that the AA release induced by extracellular ATP is mediated at least in part by phosphatidylcholine hydrolysis by PLD in osteoblast-like cells.

Involvement of phospholipase D activation in endothelin-1-induced release of arachidonic acid in osteoblast-like cells

In a previous study, we have shown that endothelin‐1 (ET‐1) activates phospholipase D independently from protein kinase C in osteoblast‐like MC3T3‐E1 cells. It is well recognized that phosphatidylycholine hydrolysis by phospholipase D generates phosphatidic acid, which can be further degraded by phosphatidic acid phosphohydrolase to diacylglycerol. In the present study, we investigated the role of phospholipase D activation in ET‐1‐induced arachidonic acid release and prostaglandin E2 (PGE2) synthesis in osteoblast‐like MC3T3‐E1 cells. ET‐1 stimulated arachidonic acid release dose‐dependently in the range between 0.1 nM and 0.1 μM. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the ET‐1‐induced arachidonic acid release in a dose‐dependent manner as well as the ET‐1‐induced diacylglycerol formation. 1,6‐bis‐(cyclohexyloxyminocarbonylamino)‐hexane (RHC‐80267), an inhibitor of diacylglycerol lipase, significantly suppressed the ET‐1‐induced arachidonic acid release. The pretreatment with propranolol and RHC‐80267 also inhibited the ET‐1‐induced PGE2 synthesis. These results strongly suggest that phosphatidylcholine hydrolysis by phospholipase D is involved in the arachidonic acid release induced by ET‐1 in osteoblast‐like cells. J. Cell. Biochem. 64:376–381.

Involvement of Phosphatidylcholine Hydrolysis by Phospholipase D in Extracellular ATP-Induced Arachidonic Acid Release in Aortic Smooth Muscle Cells

We investigated the effect of extracellular ATP on phosphatidylcholine-hydrolyzing phospholipase D activity and the role of phospholipase D activation in extracellular ATP-induced arachidonic acid release in cultured rat aortic smooth muscle cells. ATP significantly stimulated the formation of choline in a dose-dependent manner in the range between 0.01 and 0.5 mmol/L. However, ATP had no effect on the formation of phosphocholine. Staurosporine, an inhibitor of protein kinases, did not affect the ATP-induced formation of choline. ATP significantly stimulated arachidonic acid release in a dose-dependent manner in the range between 0.01 and 0.5 mmol/L. DL-Propranolol hydrochloride (propranolol), an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the ATP-induced release of arachidonic acid. 1,6-Bis(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a potent and selective inhibitor of diacylglycerol lipase, reduced ATP-induced arachidonic acid release. Quinacrine, a phospholipase A2 inhibitor, suppressed ATP-induced arachidonic acid release. Both propranolol and RHC-80267 markedly inhibited the ATP-induced synthesis of 6-ketoprostaglandin F1 alpha, a stable metabolite of prostacyclin. These results strongly suggest that extracellular ATP activates phosphatidylcholine-hydrolyzing phospholipase D independently of protein kinase C in aortic smooth muscle cells and that the arachidonic acid release induced by extracellular ATP is mediated, at least in part, through phosphatidylcholine hydrolysis by phospholipase D activation.

Function of Ca2+ influx in phospholipase D activation induced by prostaglandin F2α in osteoblast-like cells: Involvement of tyrosine kinase

We previously reported that prostaglandin F2 alpha (PGF2 alpha) induces Ca2+ influx from the extracellular space via protein tyrosine kinase in osteoblast-like MC3T3-E1 cells and that PGF2 alpha stimulates phosphatidylcholine-hydrolyzing phospholipase D in these cells (6, 12). In this study, we examined the relationship between the tyrosine kinase-regulated Ca2+ influx by PGF2 alpha and the activation of phospholipase D in MC3T3-E1 cells. The depletion of extracellular Ca2+ by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) markedly reduced the PGF2 alpha-induced formation of choline. Genistein, an inhibitor of protein tyrosine kinases, which by itself had little effect on choline formation, significantly suppressed the formation of choline induced by PGF2 alpha in a dose-dependent manner. Tyrphostin, an inhibitor of protein tyrosine kinases chemically distinct from genistein, also suppressed the PGF2 alpha-induced formation of choline. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, significantly enhanced the PGF2 alpha-induced formation of choline. These results strongly suggest that the phospholipase D activation by PGF2 alpha is dependent on extracellular Ca2+ in osteoblast-like cells and that protein tyrosine kinase is involved in the activation of phospholipase D.

Mechanism of thrombin-induced arachidonic acid release in osteoblast-like cells

In a previous study, we have reported that thrombin stimulates phosphatidylcholine hydrolysis by phospholipase (PL) D, but has little effect on phosphoinositide hydrolysis by PLC in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of the thrombin-induced arachidonic acid (AA) release in MC3T3-E1 cells. Thrombin stimulated AA release dose dependently in the range between 0.1 and 1 U/ml. Quinacrine, a PLA2 inhibitor, suppressed the thrombin-induced AA release. In addition, quinacrine also suppressed the thrombin-induced prostaglandin E2 synthesis in these cells. On the other hand, propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, did not affect the thrombin-induced AA release. 1(6-((17beta-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H- pyrrole-2,5-dione (U-73122), a PLC inhibitor, had no effect on the AA release by thrombin. In addition, 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of diacylglycerol lipase, had little effect on the thrombin-induced AA release. Neither propranolol, U-73122 nor RHC-80267 affect the thrombin-induced prostaglandin E2 synthesis. These results strongly suggest that thrombin induces AA release not by phosphatidylcholine hydrolysis by PLD nor phosphoinositide hydrolysis by PLC but mainly by PLA2 in osteoblast-like cells.

Function of Ca2' in Phosphatidylcholine-Hydrolyzing Phospholipase D Activation in Osteoblast-Like Cells

We investigated the function of Ca2+ in the activation of phosphatidylcholine (PC)-hydrolyzing phospholipase D (PLD) in osteoblast-like MC3T3-E1 cells. Fetal calf serum (FCS) stimulated the formation of choline in a dose-dependent manner in the range between 0.6% and 10%. The effect of a combination of FCS and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C (PKC) activator, on the formation of choline was additive. Staurosporine, an inhibitor of protein kinases, enhanced the formation of choline induced by FCS. BAPTA/AM, a chelator of intracellular Ca2+, inhibited the formation of choline induced by FCS. The depletion of extracellular Ca2+ by EGTA markedly reduced the FCS-induced formation of choline. SK&F 96365, an inhibitor of receptor-operated Ca2+ entry, significantly inhibited the choline formation induced by FCS. On the other hand, nifedipine, an inhibitor of L-type voltage-dependent Ca2+ channels, had little effect on the choline formation. TMB-8, an inhibitor of Ca2+ mobilization from intracellular Ca2+ store, significantly inhibited FCS-induced choline formation. These results strongly suggest that Ca2+ mobilization, through both the influx via receptor-operated Ca2+ channel and the release from intracellular Ca2+ store, plays an important role in the activation of PLD in osteoblast-like cells.