24-25-dihydroxyvitamin-d-3 and lysophosphatidic-acid

Previously we showed that costochondral growth plate resting zone (RC) chondrocytes response primarily to 24R,25(OH)2D3 whereas prehypertrophic and hypertrophic (GC) cells respond to 1alpha,25(OH)2D3. 24R,25(OH)2D3 increases RC cell proliferation and inhibits activity of matrix processing enzymes, suggesting it stabilizes cells in the reserve zone, possibly by inhibiting the matrix degradation characteristic of apoptotic hypertrophic GC cells. To test this, apoptosis was induced in rat RC cells by treatment with exogenous inorganic phosphate (Pi). 24R,25(OH)2D3 blocked apoptotic effects in a dose-dependent manner. Similarly, apoptosis was induced in ATDC5 cell cultures and 24R,25(OH)2D3 blocked this effect. Further studies indicated that 24R,25(OH)2D3 acts via at least two independent pathways. 24R,25(OH)2D3 increases LPA receptor-1 (LPA R1) expression and production of lysophosphatidic acid (LPA), and subsequent LPA R1/3-dependent signaling, thereby decreasing p53 abundance. LPA also increases the Bcl-2/Bax ratio. In addition, 24R,25(OH)2D3 acts by increasing PKC activity. 24R,25(OH)2D3 stimulates 1-hydroxylase activity, resulting in increased levels of 1,25(OH)2D3, and it increases levels of phospholipase A2 activating protein, which is required for rapid 1alpha,25(OH)2D3-dependent activation of PKC in GC cells. These results suggest that 24R,25(OH)2D3 modulates growth plate development by controlling the rate and extent of RC chondrocyte transition to a GC chondrocyte phenotype.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Apoptosis; Calcitriol; Caspase 3; Cell Proliferation; Chondrocytes; Enzyme Activation; Growth Plate; Hypertrophy; Lysophospholipids; Male; Phospholipases A2; Rats; Rats, Sprague-Dawley; Tumor Suppressor Protein p53

Maintenance of the pool of chondrocytes in the resting zone of the growth plate in the presence of the physiological apoptogen inorganic phosphate (Pi) is crucial for skeletal development. Costochondral resting zone chondrocytes are regulated by the vitamin D metabolite 24R,25-dihydroxyvitamin D3 [24R,25(OH)(2)D(3)], with increased production of sulfated glycosaminoglycan-rich extracellular matrix, and reduced matrix metalloproteinase activity. The effects of 24R,25(OH)(2)D(3) are mediated by activation of phospholipase D (PLD), resulting in increased production of lysophosphatidic acid (LPA) and LPA-mediated proliferation, maturation, inhibition of Pi-induced apoptosis, and reduction of p53. However, the exact mechanism by which 24R,25(OH)(2)D(3) and LPA exert their effects is not fully understood. It was found that both 24R,25(OH)(2)D(3) and LPA attenuate Pi-induced caspase-3 activity. The actions of 24R,25(OH)(2)D(3) and LPA were dependent upon G(αi), LPA receptor(s) 1 and/or 3, PLD, phospholipase C (PLC), and intracellular calcium, phosphoinositide 3-kinase (PI(3)K) signaling, and nuclear export. 24R,25(OH)(2)D(3) decreased both p53 abundance and p53-medaited transcription and inhibited Pi-induced cytochrome c translocation. Moreover, LPA induced increased mdm2 phosphorylation, a negative regulator of p53. Taken together, these data show that 24R,25(OH)(2)D(3) inhibits Pi-induced apoptosis through Ca(2+), PLD, and PLC signaling and through LPA-LPA1/3-G(αi)-PI(3)K-mdm2-mediated p53 degradation, resulting in decreased cytochrome c translocation and caspase-3 activity.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Apoptosis; Calcium; Caspase 3; Cells, Cultured; Chondrocytes; Cytochromes c; Gene Expression Regulation; Lysophospholipids; Male; Phosphates; Phosphatidylinositol 3-Kinases; Phospholipase D; Proto-Oncogene Proteins c-mdm2; Rats; Rats, Sprague-Dawley; Receptors, Lysophosphatidic Acid; Signal Transduction; Tumor Suppressor Protein p53; Type C Phospholipases

Growth 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-gamma). 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.

Topics: 24,25-Dihydroxyvitamin D 3; Adolescent; Animals; Antineoplastic Agents; Apoptosis; Autocrine Communication; bcl-2-Associated X Protein; Benzophenanthridines; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Child; Chondrocytes; Cyclin-Dependent Kinase Inhibitor p21; Growth Plate; Humans; Lysophospholipids; Male; Osteogenesis; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptors, Lysophosphatidic Acid; Signal Transduction; Tumor Suppressor Protein p53