Diss. (sammanfattning) Stockholm : Karol. Inst., 2002. / Härtill 6 uppsatser.

Lysophosphatidic acid (LPA) is the simplest of the glycerol lipids and regulates a number of cellular processes such as morphological changes, migration, proliferation, and inhibition of apoptosis. LPA exerts these effects through activation of the G-protein coupled receptors (GPCRs) LPA1-6 and the intracellular fatty acid receptor peroxisome proliferator-activated receptor-gamma (PPARγ). The overall goal of this thesis was to determine 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. The LPA effect depends upon increased proteasomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription. The result of LPA-mediated inhibition of p53 in A549 cells is enhanced resistance to chemotherapeutic-induced apoptosis. 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)[subscript2]D [subscript 3]] via a phospholipase D-dependent pathway, suggesting downstream phospholipid metabolites are involved. In this study, we showed that 24R,25(OH)[subscript 2]D[subscript 3] stimulates rat costochondral RC cells to release LPA. Additionally, we demonstrated that RC cells respond to LPA with increased proliferation, maturation, and inhibition of apoptosis. In the final study, the mechanism of LPA and 24R,25(OH)[subscript 2]D[subscript 3]-mediated inhibition of chondrocyte apoptosis was further investigated. Our data show that 24R,25(OH)[subscript 2]D[subscript 3] inhibits apoptosis through Ca⁺⁺, PLD, and PLC signaling and through LPA/Gαi/PI[subscript 3]K/mdm2-mediated degradation of p53, resulting in decreased caspase-3 activity. Collectively, our data establish LPA, vitamin D, and p53 as an anti-apoptotic signaling axis.

The growth plate resting zone consists of hyaline-like chondrocytes disbursed in a proteoglycan rich extracellular matrix. These cells give rise to the columns of the growth zone, consisting of progressively hypertrophic cells. Proliferation of resting zone chondrocytes induced by systemic and local stimuli is the driving force of longitudinal growth of long bones. Therefore, homeostasis of this cell population has great importance. Although the regulation of proliferation and differentiation of these cells has been well studied, little is known about the regulation of their apoptosis. We have previously shown that chelerythrine and tamoxifen induce apoptosis in resting zone chondrocytes in a nitric oxide (NO)-dependent pathway. In this study we explored two physiological apoptogens: inorganic phosphate (Pi) and 17β-estradiol (E₂). We found NO production is necessary in Pi-induced apoptosis. We also found that NO donors induced chondrocyte apoptosis by up-regulating p53 expression, Bax/Bcl-2 expression ratio and cytochrome C release from mitochondria, as well as caspase-3 activity, indicating that NO induces chondrocyte apoptosis in a mitochondrial pathway. Mitogen activated protein kinase (MAPK) activity was involved. A c-Jun N-terminal kinase (JNK) inhibitor, but not inhibitors of p38 or extracellular signal-regulated kinase (ERK1/2), was able to block NO-induced apoptosis, indicating that JNK is necessary in this pathway. Taken together, Pi elevates NO production, which leads to a mitochondrial apoptotic pathway dependent on JNK. On the other hand, although E₂caused apoptosis in resting zone chondrocytes in a dose-dependent manner, up-regulated p53 and Bax, and induced release of cytochrome C from the mitochondria, which indicated a mitochondrial apoptotic pathway, the apoptosis did not involve elevated nitric oxide production or MAPK as was found in Pi-induced apoptosis. This study elucidates the signaling pathway underlying Pi and E₂-induced chondrocyte apoptosis. It has important implications on understanding the development of mammalian growth plate. It also provides further information about the physiological functions of estrogen on longitudinal bone growth.