24,25-(OH)2D3 regulates protein kinase C through two distinct phospholipid-dependent mechanisms

Abstract

We have previously shown that 24,25-(OH)2D3 plays a major role in resting zone (RC) chondrocyte differentiation and that this vitamin D metabolite regulates protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 24,25-(OH)2D3 to stimulate PKC activation. Confluent, fourth passage RC cells from rat costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of RC cultures with 24,25-(OH)2D3 for 90 min produced a dose-dependent increase in diacylglycerol (DAG). Addition of R59022, a diacylglycerol kinase inhibitor, significantly increased PKC activity in cultures treated with 24,25-(OH)2D3. Addition of dioctanoylglycerol (DOG) to plasma membranes isolated from RC increased PKC activity 447-fold. Addition of pertussis toxin or cholera toxin to control cultures elevated basal PKC activity. When added together with 10(-9) M 24,25-(OH)2D3, there was an additive effect on PKC activity but in cultures treated with 10(-8) M 24,25-(OH)2D3, only the hormone-dependent stimulation of PKC was observed. The phospholipase C inhibitor, U73-122, had no effect on PKC activity, indicating that the DAG produced in response to 24,25-(OH)2D3 is not derived from phosphatidylinositol. Addition of the tyrosine kinase inhibitor, genistein, also had no effect on 24,25-(OH)2D3-stimulated PKC, further supporting the hypothesis that phospholipase C is not involved in the mechanism and that phospholipase D is responsible for the increase in DAG production. Phospholipase A2 inhibitors, quinacrine and AACOCF3, and the cyclooxygenase inhibitor indomethacin increased PKC activity in the RC cultures. Exogenous PGE2, one of the downstream products of phospholipase A2 action, inhibited PKC activity. These results suggest that 24,25-(OH)2D3 regulates PKC activity by two distinct phospholipid-dependent mechanisms: production of DAG via phospholipase D and inhibition of the production of PGE2 via inhibition of phospholipase A2 and cyclooxygenase.

24R,25-Dihydroxyvitamin D3 [24R,25(OH)2D3] controls growth plate development by inhibiting apoptosis in the reserve zone and stimulating response to 1α,25(OH)2D3 in hypertrophic cells

“…24R,25(OH) 2 D 3 increases the abundance of diacylglycerol (DAG) [6], an activator of many PKC isoforms. PKC activation by 24R,25(OH) 2 D 3 is maintained in the presence of chemical inhibitors targeted against either phosphatidylcholine (PC)-or phosphatidylinositol (PI)-specific phospholipase C (PLC) [7], indicating that the source of DAG is not due to the action of this enzyme.…”

“…PKC activation by 24R,25(OH) 2 D 3 is maintained in the presence of chemical inhibitors targeted against either phosphatidylcholine (PC)-or phosphatidylinositol (PI)-specific phospholipase C (PLC) [7], indicating that the source of DAG is not due to the action of this enzyme. Inhibition of tyrosine kinase signaling also does not attenuate rapid actions of 24R,25(OH) 2 D 3 , eliminating tyrosine kinases as a source of PKC activation [6]. Instead, activation of PKC by 24R,25(OH) 2 D 3 is dependent upon DAG derived from phosphatidic acid (PA) generated the actions of by phospholipase D (PLD), specifically PLD2 [8,9].…”

“…Following the initial decrease in PLA2, 24R,25(OH) 2 D 3 upregulates arachidonic acid turnover [14,15], altering fluidity of the plasma membrane [16], and increasing the production of prostaglandins E1 and E2 (PGE1, PGE2) to induce protein kinase A (PKA) activity [17]. Inhibition of PKA mitigated 24R,25(OH) 2 D 3 -induced rapid signaling and chondrocyte maturation, demonstrating the importance of this signaling pathway [6,17]. Together, activated PKC and PKA promote MEK and ERK1/2 signaling [1] in response 24R,25(OH) 2 D 3 to induce changes in gene transcription to promote chondrocyte maturation.…”

“…24R,25(OH) 2 D 3 -mediated effects are maintained in mice lacking the nuclear vitamin D receptor (nVDR) [4] and are not inhibited by Ab99, a blocking antibody targeted against the 1,25-dihydroxyvitamin D3 [1␣,25(OH) 2 [5]. Additionally, the effects of 24R,25(OH) 2 D 3 are rapid, inducing protein kinase C (PKC) activation in as little as 9 min [6]. These observations indicate that 24R,25(OH) 2 D 3 acts through a putative membrane-associated receptor (mVDR 24,25 ) that is distinct from the 1␣,25(OH) 2 D 3 -responsivie PDIA3 and functional nVDRs, but do not rule out the possibility that 24R,25(OH) 2 D 3 is acting via a VDR variant.…”

Physiological Importance of the 1,25(OH)2D3 Membrane Receptor and Evidence for a Membrane Receptor Specific for 24,25(OH)2D3

“…(1) It regulates membrane phospholipid metabolism through down-regulation of PLA 2 (8) and up-regulation of phospholipase D. (17) This results in changes in AA turnover and release and production of prostaglandin E 2 (9) and DAG. (18) It has been proposed that the rapid, membrane-mediated effects of 1,25(OH) 2 D 3 are mediated through the VDR after the latter is translocated to the cytoplasmic membrane. (19,20) Other reports suggested that specific membrane receptors exist for 1,25(OH) 2 …”