Abstract
Many of the effects of 1α,25-(OH) 2D 3 and 24 R,25-(OH) 2D 3 on costochondral chondrocytes are mediated by the protein kinase C (PKC) signal transduction pathway. 1α,25-(OH) 2D 3 activates PKC in costochondral growth zone chondrocytes through a specific membrane receptor (1α,25-mVDR), involving rapid increases in diacylglycerol via a phospholipase C (PLC)-dependent mechanism. 24 R,25-(OH) 2D 3 activates PKC in resting zone chondrocytes. Although diacylglycerol is increased by 24 R,25-(OH) 2D 3, PLC is not involved, suggesting a phospholipase D (PLD)-dependent mechanism. Here, we show that resting zone and growth zone cells express mRNAs for PLD1a, PLD1b, and PLD2. Both cell types have PLD activity, but levels are higher in resting zone cells. 24 R,25-(OH) 2D 3, but not 24 S,25-(OH) 2D 3 or 1α,25-(OH) 2D 3, stimulates PLD activity in resting zone cells within 3 min via nongenomic mechanisms. Neither 1α,25-(OH) 2D 3 nor 24 R,25-(OH) 2D 3 affected PLD in growth zone cells. Basal and 24 R,25-(OH) 2D 3-stimulated PLD were inhibited by the PLD inhibitors wortmannin and EDS. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase), PKC, phosphatidylinositol-specific PLC (PI-PLC), and phosphatidylcholine-specific PLC (PC-PLC) had no effect on PLD activity. Thus, 24 R,25-(OH) 2D 3 stimulates PLD, and PI 3-kinase, PI-PLC and PKC are not involved, whereas PLD is required for stimulation of PKC by 24 R,25-(OH) 2D 3. Pertussis toxin, GDPβS, and GTPγS had no effect on 24 R,25-(OH) 2D 3-dependent PLD when added to cell cultures, indicating that G-proteins are not involved. These data show that PKC activation in resting zone cells is mediated by PLD and suggest that a functional 24 R,25-(OH) 2D 3-mVDR is required. The results also support the conclusion that the 24 R,25-(OH) 2D 3-responsive PLD is PLD2, since this PLD isoform is G-protein-independent.
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