Role of protein kinase C alpha, Arf, and cytoplasmic calcium transients in phospholipase D activation by sodium fluoride in osteoblast-like cells

The effect of fluoride on phospholipase D (PLD) activation was studied using in vitro culture of Saos-2, MG-63 osteosarcoma cells, and normal osteoblast-like cells derived from human bone explants. Millimolar concentrations of NaF induced a significant accumulation of phosphatidylethanol (PEG in Saos-2 cells but not in MG-63 and normal osteoblast-like cells, PLD activation,vas evident at 15 mM and concentration-dependent up to 50 mM. This stimulation was inhibited by deferoxamine, a chelator of Al3+, suggesting that PLD activation involves fluoride-sensitive G proteins, A good correlation was found between the levels of intracellular free Ca2+ and the activation of PLD, The time courses of the two responses were nearly identical, The ability of NaF to induce both responses was largely dependent on the presence of extracellular calcium, The calcium ionophore A23187 reproduced the effect of NaF, and this effect was antagonized by EGTA suggesting that PLD activation was, at least in part, a calcium-regulated event, Phorbol 12-myristate W-acetate (PMA) also stimulated PLD activity in human bone cells, Protein kinase C alpha (PKC alpha) and epsilon were expressed in Saos-2 cells. Acute pretreatment of cells with PLD reduced concomitantly the amounts of PKC alpha but not of PKC epsilon, and the subsequent activation of PLD elicited by PKC activators, The PLD response to NaF was not attenuated but rather enhanced by down-regulation of PKC alpha, Therefore, PKC alpha-induced PLD activation is unlikely to mediate the effect of NaF, Moreover, PMA and NaF showed a supraadditive effect on PLD activation in Saos-2 cells, This stimulation, in contrast to NaF alone, was not reduced by EGTA. Hence, mobilization of calcium by NaF cannot account for the enhanced PLD activation in response to PMA stimulation, Membrane Arf and RhoA contents were assessed by Western immunoblot analyses, Membranes derived from NaF-stimulated Saos-2 cells contained more Arf and RhoA when compared with membranes derived from control or PMA-stimulated cells, Translocation of the small GTPases was calcium-independent, We conclude that PLD activation by NaF in Saos-2 cells includes a fluoride-sensitive G protein, increases in the levels of intracellular calcium, and Arf/RhoA redistribution to membranes, The results also indicate that the NaF-induced ArF/RhoA translocation exerts in concert with PMA-activated PKC alpha a synergistic effect on the activation of PLD in Saos-2 cells.