PROTEIN-KINASE-A AND PROTEIN-KINASE-C POSITIVELY REGULATE G PROTEIN-DEPENDENT ACTIVATION OF PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C BY TUMOR-NECROSIS-FACTOR-ALPHA IN MC3T3-E1 OSTEOBLASTS

The role(s) of protein kinases in the regulation of G protein-dependen t activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha was investigated in the osteoblast cell line MC 3T3-E1. We have previously reported the stimulatory effects of tumor n ecrosis factor-alpha and A1F(4)(-), an activator of C proteins, on thi s phospholipase pathway documented by a decrease in mass of PI and rel ease of diacylglycerol. In this study, we further explored the mechani sm(s) by which the tumor necrosis factor or A1F(4)(-)-promoted breakdo wn of phosphatidylinositol and the polyphosphoinositides by phospholip ase C is regulated. Tumor necrosis factor-alpha was found to elicit a 4-5-fold increase in the formation of [H-3]inositol-l,4-phosphate and [H-3]inositol-l,4,5-phosphate; and a 36% increase in [H-3]inositol-1-p hosphate within 5 min in prelabeled cells. [H-3] inositol-4-phosphate, a metabolite of [H-3]inositol-1,4phosphate and [3H]inositol-1,4,5-pho sphate, was found to be the predominant phosphoinositol product of tum or necrosis factor-alpha and A1F(4)(-)-activated phospholipase C hydro lysis after 30 min. In addition, the preincubation of cells with pertu ssis toxin decreased the tumor necrosis factor-induced release of inos itol phosphates by 53%. Inhibitors of protein kinase C, including Et-1 8-OMe and H-7, dramatically decreased the formation of [H-3]inositol p hosphates stimulated by either tumor necrosis factor-alpha or A1F(4)(- ) by 90-100% but did not affect basal formation. The activation of cAM P-dependent protein kinase, or protein kinase A, by the treatment of c ells with forskolin or 8-BrcAMP augmented basal, tumor necrosis factor -alpha and A1F(4)(-)-induced [H-3]inositol phosphate formation. Theref ore, we report that protein kinases can regulate tumor necrosis factor -alpha-initiated signalling at the cell surface in osteoblasts through effects on the coupling between receptor, C-protein and phosphatidyli nositol-specific phospholipase. (C) 1997 Wiley-Liss, Inc.