| Lysophosphatidic acid (LPA) is a bioactive acid lysophospholipid that regulates a wide array of cellular processes such as migration, proliferation, inhibition of apoptosis, wound healing, and morphological changes through activation of G-protein coupled receptors. The overall goal of this thesis was to understand the mechanisms by which LPA enhances cell survival by inhibiting apoptosis. The project was divided into three studies: (1) to determine the mechanism of LPA-mediated inhibition of p53 in A549 lung carcinoma cells, (2) to investigate the regulation of growth plate chondrocytes by LPA, and (3) to determine the mechanisms of LPA-mediated effects in the growth plate.;In the first study, evidence is provided that LPA reduces the cellular abundance of the tumor suppressor p53 in A549 lung carcinoma cells, which express endogenous LPA receptors. The LPA effect depends upon increased proteosomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription. Inhibition of phosphatidylinositide 3-kinase (PI3K) protected cells from the LPA-induced reduction of p53, which implicates this signaling pathway in the mechanism of LPA-induced loss of p53. LPA partially protected A549 cells from actinomycin D induction of both apoptosis and increased p53 abundance. These results identify p53 as a target of LPA action and provide a new dimension for understanding how LPA stimulates cancer cell division, protects against apoptosis, and thereby promotes tumor progression.;In the second study, the role of LPA in resting zone chondrocytes (RC cells) was investigated. RC cells are regulated by 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] 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 cells to release LPA. RC cells also expressed LPA receptors 1-5 (LPA1-5) and the LPA-responsive intracellular fatty acid receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma). LPA and the LPA1/3-selective agonist OMPT increased proliferation and maturation markers. The anti-apoptotic effects of LPA and 24R,25(OH)2D3 were inhibited by the LPA1/3-selective antagonist VPC32183(S). Furthermore, apoptosis induced by either inorganic phosphate (Pi) or chelerythrine was attenuated by LPA. LPA prevented apoptotic signaling by decreasing the abundance, nuclear localization, and transcriptional activity of 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.;In the final study, the mechanism of LPA and 24R,25(OH)2D 3-mediated inhibition of chondrocyte apoptosis was further investigated. We found that both 24R,25(OH)2D3 and LPA rescue of Pi-induced caspase-3 activity. The actions of 24R,25(OH)2D3 were dependent upon Galphai, LPA1/3, phospholipase D (PLD), phospholipase C (PLC), and calcium (Ca++); whereas, those of LPA were dependent on Galphai and PI 3K signaling and nuclear export. 24R,25(OH)2D3 decreased both p53 abundance and p53-medaited transcription and inhibited Pi-induced cytochrome c translocation. Moreover, LPA induced an increase mdm2 phosphorylation, a negative regulator of p53. Taken together, these data show that 24R,25(OH)2D3 inhibits Pi-induced apoptosis through Ca++, PLD, and PLC signaling and through LPA/LPA1/Galpha i/PI3K/mdm2-mediated degradation of p53, resulting in decreased caspase-3 activity. |
