Lysophosphatidic acid signaling promotes proliferation, differentiation, and cell survival in rat growth plate chondrocytes

AbstractGrowth plate cartilage is responsible for long bone growth in children and adolescents and is regulated by vitamin D metabolites in a cell zone-specific manner. Resting zone chondrocytes (RC cells) are regulated by 24,25-dihydroxyvitamin D3 via a phospholipase D-dependent pathway, suggesting downstream phospholipid metabolites are involved. In this study, we showed that 24R,25(OH)2D3 stimulates rat costochondral RC chondrocytes to release lysophosphatidic acid (LPA) and, therefore sought to determine the role of LPA signaling in these cells. RC cells expressed the G-protein coupled receptors LPA1–5 and peroxisome proliferator-activated receptor gamma (PPAR-γ). LPA and the LPA1/3 selective agonist OMPT increased proliferation and two maturation markers, alkaline phosphatase activity and [35S]-sulfate incorporation. LPA and 24R,25(OH)2D3's effects were inhibited by the LPA1/3 selective antagonist VPC32183(S). Furthermore, apoptosis induced by either inorganic phosphate or chelerythrine was attenuated by LPA, based on DNA fragmentation, TUNEL staining, caspase-3 activity, and Bcl-2:Bax protein ratio. LPA prevented apoptotic signaling by decreasing the abundance, nuclear localization, and transcriptional activity of the tumor-suppressor p53. LPA treatment also regulated the expression of the p53-target genes Bcl-2 and Bax to enhance cell survival. Collectively, these data suggest that LPA promotes differentiation and survival in RC chondrocytes, demonstrating a novel physiological function of LPA-signaling

1,25-Dihydroxy Vitamin D3 Is an Autocrine Regulator of Extracellular Matrix Turnover and Growth Factor Release via ERp60-Activated Matrix Vesicle Matrix Metalloproteinases

As growth plate chondrocytes mature and hypertrophy, they reorganize their proteoglycan-rich type II collagen extracellular matrix (ECM), involving 1,25(OH)2D3-dependent regulation of matrix metalloproteinases (MMPs). Stromelysin-1 (MMP-3) and 72-kD gelatinase (MMP-2) are found in extracellular matrix vesicles (MVs) and release and activate ECM-bound latent TGF-β1 and TGF-β2, respectively. 1,25(OH)2D3 regulates incorporation of MMP-2 and MMP-3 into MVs and release of these enzymes in the ECM. Plasma membranes (PMs) and MVs contain the 1α,25(OH)2D3 membrane receptor ERp60 (protein disulfide isomerase A3), phospholipase A2 (PLA2), PLA2-activating protein, the nuclear vitamin D receptor and caveolin-1. 1,25(OH)2D3 secreted by chondrocytes binds MV ERp60, activating PLA2. Resulting lysophospholipids destabilize MV membranes, releasing active MMPs. We examined 1,25(OH)2D3-dependent activation of latent TGF-β1 stored in cartilage ECM. Interestingly, TGF-β1 regulates 1,25(OH)2D3 production. 1α,25(OH)2D3 activates PM protein kinase C (PKC)-α via ERp60-dependent PLA2-signaling, lysophospholipid production and phospholipase C-γ. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching MVs in PKC-ζ. Direct activation of MV MMP-3 requires ERp60 based on blocking antibodies and PKC based on inhibitor studies. However, treatment of MVs with 1,25(OH)2D3 decreases MV PKC-ζ activity, suggesting more complex feedback mechanisms, potentially involving MV lipid signaling. Our observations indicate that one role of MVs is to provide MMPs at sites distant from the cells. Chondrocytes secrete 1,25(OH)2D3, which acts directly on MV-membranes via ERp60, releasing MMPs. MMP-specific ECM components are hydrolyzed, resulting in release and activation of growth factors that can act back on the cells