1α,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-β activity via phospholipase A2-dependent production of lysophospholipid

1alpha,25(OH)(2)D-3 activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A(2) (PLA(2)). The purpose of this study was to determine if 1alpha,25(OH)(2)D-3 activates PI-PLC directly or through a PLA(2)-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by I alpha,25(OH)(2)D-3. Inhibitors and activators of PLA(2) were used to assess the inter-relation ship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1alpha,25(OH)(2)D-3, was increased within 30 s with peak activity at 1-3 min. PI-PLC activity in resting zone cells was unaffected by 1alpha,25(OH)(2)D-3. 1beta,25(OH)(2)D-3, 24R,25(OH)(2)D-3, actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, lalpha,25(OH)(2)D-3 regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA(2)-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as Ialpha,25(OH)(2)D-3; PLA(2)-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF(3)) reduced the effect of 1alpha,25(OH)(2)D-3. Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE > LPC). 1alpha,25(OH)(2)D-3 stimulated PI-PLC and PKC activities via Gq; GDPbetaS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-beta1a, PLC-beta1b, PLC-beta3 and PLC-gamma1 mRNA. Antibodies to PLC-beta1 and PLC-beta3 blocked the 1alpha,25(0H)(2)D-3 effect; antibodies to PLC-delta and PLC-gamma did not. Thus, 1alpha,25(OH)(2)D-3 regulates PLC-beta through PLA(2)-dependent production of lysophospholipid. (C) 2003 Elsevier Science Inc. All rights reserved.