title: Arachidonic acid directly mediates the rapid effects of 24,25-dihydroxyvitamin D3 via protein kinase C and indirectly through prostaglandin production in resting zone chondrocytes.
abstract: Prior studies have shown that 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] plays a major role in resting zone chondrocyte differentiation and that this vitamin D metabolite regulates both phospholipase A2 and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipase A2 action and has been shown in other systems to affect a variety of cellular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH)2D3 on markers of proliferation, differentiation, and matrix production in resting zone chondrocytes and to characterize the mechanisms by which arachidonic acid regulates PKC, which was shown previously to mediate the rapid effects of 24,25-(OH)2D3 and arachidonic acid on these cells. Confluent, fourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [3H]thymidine incorporation and inhibited the activity of alkaline phosphatase and PKC, but had no effect on proteoglycan sulfation. In contrast, 24,25-(OH)2D3 inhibited [3H]thymidine incorporation and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC activity. In cultures treated with both agents, the effects of 24,25-(OH)2D3 were reversed by arachidonic acid. The PKC isoform affected by arachidonic acid was PKCalpha; cytosolic levels were decreased, but membrane levels were unaffected, indicating that translocation did not occur. Arachidonic acid had a direct effect on PKC in isolated plasma membranes and matrix vesicles, indicating a nongenomic mechanism. Plasma membrane PKCalpha was inhibited, and matrix vesicle PKCzeta was stimulated; these effects were blocked by 24,25-(OH)2D3. Studies using cyclooxygenase and lipoxygenase inhibitors indicate that the effects of arachidonic acid are due in part to PG production, but not to leukotriene production. This is supported by the fact that H8-dependent inhibition of protein kinase A, which mediates the effects of PGE2, had no effect on the direct action of arachidonic acid but did mediate the role of arachidonic acid in the cell response to 24,25-(OH)2D3. Diacylglycerol does not appear to be involved, indicating that phospholipase C and/or D do not play a role. Gamma-linolenic acid, an unsaturated precursor 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 regulator of 24,25-(OH)2D3 action in resting zone chondrocytes.
authors.affiliation: Department of Orthopedics, University of Texas Health Science Center, San Antonio 78284-7774, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Schwartz
authors.forename: V L
authors.initials: VL
authors.lastname: Sylvia
authors.forename: D
authors.initials: D
authors.lastname: Curry
authors.forename: M H
authors.initials: MH
authors.lastname: Luna
authors.forename: D D
authors.initials: DD
authors.lastname: Dean
authors.forename: B D
authors.initials: BD
authors.lastname: Boyan
mesh_terms: D015650:24,25-Dihydroxyvitamin D 3 / Q000494:pharmacology*; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D019902:Chondrocytes / Q000187:drug effects* / Q000502:physiology; D004075:Diglycerides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D008297:Male; D011453:Prostaglandins / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D013997:Time Factors; D017965:gamma-Linolenic Acid / Q000494:pharmacology
chemical_list: D004075:Diglycerides; D011453:Prostaglandins; C044295:1,2-dioctanoylglycerol; D016718:Arachidonic Acid; D015650:24,25-Dihydroxyvitamin D 3; D017965:gamma-Linolenic Acid; D011493:Protein Kinase C