Abstract
1α,25(OH) 2D 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 1α,25(OH) 2D 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 1α,25(OH) 2D 3. Inhibitors and activators of PLA 2 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) 2D 3, was increased within 30 s with peak activity at 1–3 min. PI-PLC activity in resting zone cells was unaffected by 1α,25(OH) 2D 3. 1β,25(OH) 2D 3, 24 R,25(OH) 2D 3, actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1α,25(OH) 2D 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 1α,25(OH) 2D 3; PLA 2-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF 3) reduced the effect of 1α,25(OH) 2D 3. 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) 2D 3 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) 2D 3 effect; antibodies to PLC-δ and PLC-γ did not. Thus, 1α,25(OH) 2D 3 regulates PLC-β through PLA 2-dependent production of lysophospholipid.
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