1α,25(OH)2D3 Regulates Chondrocyte Matrix Vesicle Protein Kinase C (PKC) Directly via G-protein-dependent Mechanisms and Indirectly via Incorporation of PKC during Matrix Vesicle Biogenesis

Abstract

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1␣,25 (OH) 2 D 3 . Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1␣,25(OH) 2 D 3 in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1␣,25(OH) 2 D 3 during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1␣,25 (OH) 2 D 3 , PKC in matrix vesicles was inhibited, while PKC␣ in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKC in matrix vesicles was increased, while PKC␣ in plasma membranes was unchanged. The effect of 1␣,25(OH) 2 D 3 was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1␣,25(OH) 2 D 3 membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1␣,25(OH) 2 D 3 -dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1␣,25(OH) 2 D 3 in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or G i /G s had no effect. However, inhibition of G q blocked the effect of 1␣,25(OH) 2 D 3 . The rapid effect of 1␣,25(OH) 2 D 3 also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1␣,25(OH) 2 D 3 at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.Costochondral growth plate chondrocytes metabolize 25 (OH)D 3 in a regulated manner, producing and secreting 1,25 (OH) 2 D 3 and 24,25(OH) 2 D 3 (1, 2). The physiological importance of this is not yet well understood. 1␣,25(OH) 2 D 3 exerts direct effects on matrix vesicles isolated from the extracellular matrix of growth plate chondrocytes (3), suggesting that the cells may use local production of the vitamin D metabolite as a mechanism for controlling events in the matrix. A number of observations support this hypothesis. Treatment of matrix vesicles with 1␣,25(OH) 2 D 3 causes increased alkaline phosphatase specific activity, which is associated with the onset of calcification (4). In addition, phospholipase A 2 (PLA 2 ) 1 specific activity is increased (5), which may lead to a loss of membrane integrity and the release of proteinases capable of remodeling the matrix (6, 7). One of the matrix metalloproteinases that are present in matrix vesicles, stromelysin-1 (MMP-3), has been shown to ...

Membrane Initiated Effects of 1α,25-Dihydroxyvitamin D3 in Prostate Cancer Cells: Effects on AP1 and CREB Mediated Transcription

“…Similar observations have been made in vitamin D responsive tissues, where membrane initiated signaling have been linked to PKA, PKC and MAPK signaling pathways (Schwartz et al, 2002;Dirks-Naylor and Lennon-Edwards, 2011). This study was aimed to further elucidate membrane initiated signaling by 1α,25(OH) 2 D 3 in LNCaP prostate cancer cells by evaluating the effects of 1α,25(OH) 2 D 3 on AP1 and CREB-dependent gene expression as well as testing the hypothesis that 1α,25(OH) 2 D 3 might evoke membrane initiated effects through TNF-α and TNF-α initiated signaling pathways.…”

“…There are no direct evidences that vitamin D receptors are coupled to a protein complex which includes adenylate cyclase. However, several studies have demonstrated that 1 ,25(OH) 2 D 3 evoke rapid increases in PKA activity, intracellular cAMP concentrations which have been found to be associated with G-protein-coupled signaling as well as regulation of Ca 2+ transport through Ca 2+ -channels (Massheimer et al, 1999;Schwartz et al, 2002;Dirks-Naylor & Lennon-Edwards, 2011).…”

Membrane actions of vitamin D metabolites 1α,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

“…Transfection of ROS 24/1 rat osteoblast-like cells, which lack a functional 1,25-nVDR, with annexin II restores transcaltachia in response to 1a,25(OH) 2 D 3 [Baran et al, 2000b]. Moreover, we have shown that when extracellular matrix vesicles are incubated directly with 1a,25(OH) 2 D 3 , PKC activity is regulated although no 1,25-nVDR is present and there is no possibility of gene transcription or protein synthesis [Schwartz et al, 2001a]. This is the first demonstration, however, that PKC is regulated by 1a,25(OH) 2 D 3 in intact chondrocytes that lack a functional 1,25-nVDR.…”

1α,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases

“…24R,25(OH) 2 D 3 stimulates PKCα in resting zone chondrocytes via a phospholipase D (PLD) dependent pathway which does not involve ERp60 [23]. This also results in production of DAG, but via a two-step process, and peak activation occurs later than is seen in 1α,25 (OH) 2 PKC activity in the extracellular organelles is increased due to the specific incorporation of PKC-zeta during production [24]. PKCζ is an atypical form of PKC, requiring neither lipid not Ca ++ as co-factors.…”