Regulation of apoptosis, induced by phosphatidylcholine synthesis inhibition

Abstract

PC is the most abundant phospholipid in cellular membranes of mammalian tissues. In addition to its structural role in membranes and lipoproteins, PC functions as a major source of intracellular signalling molecules. All eukaryotic cell types and tissues display unique and stable profiles of PC and perturbation of PC homeostasis, by pharmacological agents or by genetic engineering, leads to cell death via a process called apoptosis. Although several studies describe a relation between inhibition of PC synthesis and apoptosis, the underlying signalling pathways that mediate this cell death have not been identified. Therefore, the aim of this study was to identify signalling pathways that mediate apoptosis during the inhibition of PC. With the help of a genetic model, a temperature sensitive mutant MT58, we investigated the effect of PC depletion on several potential stress pathways. Furthermore we tried to elucidate which cellular event might be the trigger for the induction of apoptotic processes during prolonged inhibition of PC synthesis. We first analysed the effect of prolonged inhibition of PC synthesis on viable MT58 cells (24 h). Despite the fact that PC synthesis is severely decreased after 24h at 40 °C, PC breakdown is even increased in MT58 cells. This results in a decrease of PC and especially in PC species, which are mono-saturated at the sn-1 position. Overall, cellular membranes in MT58 cells have the tendency to maintain a more saturated composition during incubation at the non-permissive temperature. After 24 h of PC depletion, membranes of the secretory pathway that are in a dynamic equilibrium were affected the most, such as the ER and the Golgi. These structures were highly disrupted in MT58 cells, whilst the plasma membrane, mitochondriae and the membrane of the nucleus seem to be intact. Furthermore a large accumulation of lipid droplets in the cell was occurring during PC depletion. Because of the dramatic impact of PC depletion on ER and Golgi structures, the role of the ER stress response in the apoptotic process of PC depleted cells was investigated. Inhibition of PC synthesis leads to the induction of the ER stress-related, pro-apoptotic transcription factor CHOP/GADD153, but does not influence canonical ER stress events, like induction of the ER chaperone Bip, inhibition of protein translation or caspase 12 activation. To identify the role of CHOP induction in PC depletion we performed studies using the promoter region of CHOP. Mutants of all putative regulatory elements in the CHOP promoter were made and we observed that CHOP expression during PC depletion is mediated via activation of a conserved region, the C/EBP-ATF site. Activation of this site seems to depend on the binding of transcription factor ATF2. Phosphorylation of ATF2 is essential for binding to the C/EBP site, but during PC depletion ATF2 phosphorylation is not mediated by JNK kinase. However, this kinase plays a role in the apoptotic process of PC depletion, because treatment of MT58 cells with the JNK inhibitor SP600125 can rescue the cells from apoptosis. Chemical agents that inhibit PC synthesis are often used to investigate the role of PC depletion in apoptosis and furthermore they are used as neoplastic agents during anti-cancer therapy. Therefore we compared the effects of a familiar used pharmacological drug of PC synthesis inhibition, HePC with the results we found in our genetic model. Although both systems lead to an inhibition of PC synthesis and the induction of apoptosis, fundamental differences between them were observed. Treatment of HePC did not result in depletion of PC, apoptotic processes were not preceded by an induction of CHOP and HePC treated cells can be rescued by both LysoPC and LysoPE. Therefore HePC treated cells induce apoptosis probably via other pathways than the inhibition of PC. Considering the underlying regulatory pathway of PC depletion-induced apoptosis, we have identified CHOP/GADD153, ATF2 and JNK as key players in cellular stress responses to inhibition of PC synthesis. Internal structures as Golgi and ER are disrupted in PC depleted cells and this event could be a trigger for the observed cellular stress pathways that eventually result in apoptosis.