Arachidonic acid directly mediates the rapid effects of 24,25-dihydroxyvitamin D-3 via protein kinase C and indirectly through prostaglandin production in resting zone chondrocytes

Prior studies have shown that 24,25-dihydroxyvitamin D-3 [24,25(OH)(2)D-3] plays a major role in resting zone chondrocyte differentiation and that thi s vitamin D metabolite regulates both phospholipase A, and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipas e A, action and has been shown in other systems to affect a variety of cell ular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH)(2)D- 3 on markers of proliferation, differentiation, and matrix production in re sting zone chondrocytes and to characterize the mechanisms by which arachid onic acid regulates PKC, which was shown previously to mediate the rapid ef fects of 24,25-(OH)(2)D-3 and arachidonic acid on these cells. Confluent, f ourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [H-3]thymidine incorporation and inhibited the act ivity of alkaline phosphatase and PKC, but had no effect on proteoglycan su lfation. In contrast, 24,25(OH)(2)D-3 inhibited [H-3]thymidine incorporatio n and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC acti vity. In cultures treated with both agents, the effects of 24,25-(OH)(2)D-3 were reversed by arachidonic acid. The PKC isoform affected by arachidonic acid was PKC alpha; cytosolic levels were decreased, but membrane levels w ere unaffected, indicating that translocation did not occur. Arachidonic ac id had a direct effect on PKC in isolated plasma membranes and matrix vesic les, indicating a nongenomic mechanism. Plasma membrane PKCa was inhibited, and matrix vesicle PKC zeta was stimulated; these effects were blocked by 24,25-(OH)(2)D-3. Studies using cyclooxygenase and lipoxygenase inhibitors indicate that the effects of arachidonic acid are due in part to PG product ion, but not to leukotriene; production. This is supported by the fact that H8-dependent inhibition of protein kinase A, which mediates the effects of PGE,, had no effect on the direct action of arachidonic acid but did media te the role of arachidonic acid in the cell response to 24,25(OH)(2)D-3. Di acylglycerol does not appear to be involved, indicating that phospholipase C and/or D do not play a role, gamma-Linolenic acid, an unsaturated precurs or of arachidonic acid, elicited a similar response in matrix vesicles but not plasma membranes, whereas palmitic acid, a saturated fatty acid, had no effect. These data suggest that arachidonic acid may act as a negative reg ulator of 24,25-(OH)(2)D-3 action in resting zone chondrocytes.