Arachidonic Acid Directly Mediates the Rapid Effects of 24,25-Dihydroxyvitamin D₃Via Protein Kinase C and Indirectly through Prostaglandin Production in Resting Zone Chondrocytes¹

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.

Activation of the Phosphatidylinositol 3-Kinase-Akt/Protein Kinase B Signaling Pathway in Arachidonic Acid-stimulated Human Myeloid and Endothelial Cells

“…The actions of 20:46 include the stimulation of neutrophil superoxide production and degranulation (2,3), insulin secretion by isolated rat islets of Langerhans (4), induction of the MAP kinase phosphatase-1 gene in vascular smooth muscle cells (5), differentiation of HL60 cells (6) and zone chondrocytes (7), adherence of neutrophils and human breast cancer cells to various substrata (3,8), and proliferation of certain tumor and normal cell types (9,10). On the other hand, 20:46 has been reported to inhibit cell-cell communication via gap junctions (11,12), cytokineinduced adhesion molecule expression in endothelial cells (13), and proliferation of HL60 cells in conjunction with the induction of differentiation (6).…”

Effect of a vitamin D3-based nutritional supplement (`Videchol') on carbohydrate metabolism of rats following chronic low dose-rate irradiation

“…Consequently, the biosynthesis of new mRNA molecules and the corresponding protein translation are activated, completing the hormonal response [5,6]. As well as this traditional receptor-mediated mechanism of action of vitamin D 3 hydroxymetabolites a new non-classical mechanism of action has been established-rapid membrane-mediated events, some of which do not require new gene expression or protein synthesis [7,8]. It has been found that vitamin D 3 hormonal active metabolites can regulate protein kinase C activity through two distinct phospholipid-dependent mechanisms [9][10][11].…”