[Show abstract][Hide abstract] ABSTRACT: Alveolar type II lung epithelia produce surfactant, an essential surface-active material highly enriched with disaturated phosphatidylcholine (DSPC), which requires a key regulatory enzyme, CTP:phosphocholine cytidylyltransferase alpha (CCTalpha), for its synthesis before its export apically into the alveolus. In this study, we examined whether surfactant phosphatidylcholine (PC) synthesis and export are physiologically linked. Stable overexpression of CCTalpha in lung epithelial cell lines increased rates of PC synthesis and cellular DSPC mass without altering total cellular PC content. Overexpression of CCTalpha was associated with i) increased basolateral, rather than apical, PC export catalyzed by ABCA1; ii) basolateral export of significant levels of unsaturated (nonsurfactant) PC; and iii) transcriptional activation of the ABCA1 gene via a liver X receptor/retinoic acid receptor-independent pathway. Cells exposed to PC vesicles exhibited a dose-dependent increase in ABCA1 transcriptional activity. These data provide the first evidence that surfactant PC synthesis is linked to its export via a basolateral lipid efflux pathway. This pathway is mediated, in part, by a phospholipid sensor, ABCA1, that appears to partake in the autoregulation of both cellular content and composition of PC, thereby providing a potentially novel exit route for a newly synthesized pool of PC distinct from surfactant.
Preview · Article · Oct 2004 · The Journal of Lipid Research
[Show abstract][Hide abstract] ABSTRACT: Gene therapy requires the presence of a robust and yet small promoter to drive high-level expression of desired proteins. In comparative analysis, we investigated the promoter strength of the CTP:phosphocholine cytidylyltransferase promoter (CCT alpha) with other commonly used promoters, which were all cloned into a similar background vector (PGL3 basic). Transient promoter-reporter assays in murine lung epithelial (MLE-12) cells revealed that the core CCT alpha promoter (240 bp) was observed to exhibit a 40-fold, 8-fold, and 3-fold higher level of activity compared with the simian virus 40, human cytomegalovirus, and Rous sarcoma virus promoters, respectively. The CCT alpha promoter was significantly more active than the Clara cell 10, thymidine kinase, and phosphoglycerate kinase promoters. This pattern of high-level expression for CCT alpha was detected primarily in cell lines of distal lung epithelial origin (MLE-12, RLE, H441) and was reduced in other cell lines (A549, CHO, HepG 2). CCT alpha promoter-reporter activity, CCT alpha transcript levels, and immunoreactive protein levels increased significantly in the presence of all-trans retinoic acid. The CCT alpha promoter, in a retinoic acid-inducible manner, efficiently directed expression of murine erythropoietin in MLE-12 cells. Collectively, these observations suggest that the CCT alpha construct might be useful to drive high-level, regulatable expression of heterologous proteins in alveolar epithelia.
Preview · Article · Feb 2004 · American Journal of Respiratory Cell and Molecular Biology