Effects of 1?,25-(OH)2D3 on rat growth zone chondrocytes are mediated via cyclooxygenase-1 and phospholipase A2

Abstract

Abstract1a,25-(OH) 2 D 3 mediates its effects on growth zone chondrocytes via rapid membrane-associated events as well as through traditional nuclear receptor mechanisms. The membrane-associated signaling pathways include rapid production of diacylglycerol and activation of protein kinase C (PKC), as well as activation of phospholipase A 2 (PLA 2 ), increased production of arachidonic acid, and increased production of prostaglandins. This study examined the roles of PLA 2 and cyclooxygenase (Cox) in the mechanism of action of 1a,25-(OH) 2 D 3 in these cells to determine whether one or both enzymes catalyze the rate limiting step and whether constitutive or inducible Cox is involved. Cultures were incubated with 1a,25-(OH) 2 D 3 for 9 min to measure PKC or for 24 h to measure physiological responses ([ 3 H]-thymidine incorporation, alkaline phosphatase speci®c activity, [35 S]-sulfate incorporation). Based on RT-PCR and Northern blot analysis, growth zone chondrocytes expressed mRNAs for both Cox-1 and Cox-2 and neither Cox was modulated by 1a,25-(OH) 2 D 3 . To examine the role of Cox, the cultures were also treated with resveratrol (a speci®c inhibitor of Cox-1), NS-398 (a speci®c inhibitor of Cox-2), or indomethacin (a general Cox inhibitor). The results showed that Cox-1 inhibition reduced the 1a,25-(OH) 2 D 3 -dependent effects on proliferation, differentiation, and matrix production, whereas inhibition of Cox-2 only had an effect on proliferation. The effects of Cox inhibition were not rate limiting, based on experiments in which PLA 2 was activated with melittin or inhibited with quinacrine. However, at least part of the action of 1a,25-(OH) 2 D 3 was regulated by metabolism of arachidonic acid to prostaglandins. This supports the hypothesis that 1a,25-(OH) 2 D 3 exerts its effects via more than one signaling pathway and that these pathways are interrelated via the modulation of PLA 2 as a rate-limiting step. PKC regulation may occur at multiple stages in the signal transduction cascade.

Phospholipase A2 activating protein is required for 1α,25-dihydroxyvitamin D3 dependent rapid activation of protein kinase C via Pdia3

“…Taken together with previously published studies [10], the results suggest the following pathway (Fig. 8): 1␣,25(OH) 2 D 3 binds the Pdia3-Cav-1 membrane receptor complex in caveolae, triggering binding of PLAA to Pdia3.…”

“…PLA 2 action generates arachidonic acid (AA) and lysophospholipid (LPL) [7]. AA can either increase PKC␣ activity directly [9], or it is processed further to PGE 2 , which acts via its EP1 receptor to increase cyclic AMP [10]. Phosphatidylinositol-specific PLC␤ is activated via G␣q and lysophospholipid, generating diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) [7,11].…”

ESWT Enhances Expression of Pdia-3 which is a Key Factor of 1α,25-Dihydroxyvitamin D3 Rapid Membrane Signaling Pathway in Treatment of Early Osteoarthritis Knee

“…Our current study provides the first evidence from immunohistochemistry that shockwave therapy can induce articular cartilages expression of Pdia-3, the critical transcription factor responsible for the matrix formation of chondrocyte. Recent studies reported that 1α,25(OH)2D3 rapidly stimulated membrane signaling via Pdia-3 dependent activation in growth zone chondrocytes and promotes the production of matrix protein [11,13,20,22,42,43]. The present study showed the decrease of cartilage matrix loss and increased aggrecan and collagen II expression in shockwave group.…”

“…1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) is essential in calcium homeostasis to regulation of endochondral ossification [11]. In vitamin D deficiency, bone matrix synthesis and cartilage growth are inhibited.…”

Mechanisms regulating differential activation of membrane-mediated signaling by 1α,25(OH)2D3 and 24R,25(OH)2D3

“…Not surprisingly, inhibition of arachidonic acid metabolism with the general cyclooxygenase inhibitor indomethacin causes growth zone cells and resting zone cells to respond to 1␣,25(OH) 2 D 3 and 24R,25(OH) 2 D 3 in a manner like their response to exogenous arachidonic acid. Inhibition of inducible cyclooxygenase 2 does not alter that response, whereas inhibition of constitutive cyclooxygenase 1 reduces the arachidonic acid-like effect [22]. This observation shows that PLA 2 is rate limiting because it changes substrate concencentration for the consistutive form of the enzyme, and it shows that metabolites of arachidonic acid are also involved in the mechanism.…”