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

1α,25(OH)₂D₃ activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A₂ (PLA₂). The purpose of this study was to determine if 1α,25(OH)₂D₃ activates PI-PLC directly or through a PLA₂-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 1α,25(OH)₂D₃. Inhibitors and activators of PLA₂ were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1α,25(OH)₂D₃, was increased within 30s with peak activity at 1-3min. PI-PLC activity in resting zone cells was unaffected by 1α,25(OH)₂D₃. 1β,25(OH)₂D₃, 24R,25(OH)₂D₃, actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1α,25(OH)₂D₃ regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA₂-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1α,25(OH)₂D₃; PLA₂-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF₃) reduced the effect of 1α,25(OH)₂D₃. Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1α,25(OH)₂D₃ stimulated PI-PLC and PKC activities via Gq; GDPβS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-β1a, PLC-β1b, PLC-β3 and PLC-γ1 mRNA. Antibodies to PLC-β1 and PLC-β3 blocked the 1α,25(OH)₂D₃ effect; antibodies to PLC-δ and PLC-γ did not. Thus, 1α,25(OH)₂D₃ regulates PLC-β through PLA₂-dependent production of lysophospholipid.