Lipid deprivation increases surfactant phosphatidylcholine synthesis via a sterol-sensitive regulatory element within the CTP:phosphocholine cytidylyltransferase promoter.
Lipid-deprived mice increase alveolar surfactant disaturated phosphatidylcholine (DSPtdCho) synthesis compared with mice fed a standard diet by increasing expression of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme for DSPtdCho synthesis. We previously observed that lipid deprivation increases mRNA synthesis for CCT [Ryan, McCoy, Mathur, Field and Mallampalli (2000) J. Lipid Res. 41, 1268-1277]. To evaluate regulatory mechanisms for this gene, we cloned the proximal approximately 1900 bp of the 5' flanking sequence of the murine CCT gene, coupled this to a luciferase reporter, and examined transcriptional regulation in a murine alveolar epithelial type II cell line (MLE-12). The core promoter was localized to a region between -169 and +71 bp, which exhibited strong basal activity comparable with the simian virus 40 promoter. The full-length construct, from -1867 to +71, was induced 2-3-fold when cells were cultured in lipoprotein-deficient serum (LPDS), similar to the level of induction of the endogenous CCT gene. By deletional analysis the sterol regulatory element (SRE) was localized within a 240 bp region. LPDS activation of the CCT promoter was abolished by mutation of this SRE, and gel mobility-shift assays demonstrated specific binding of recombinant SRE-binding protein to this element within the CCT promoter. These observations indicate that sterol-regulated expression of CCT is mediated by an SRE within its 5' flanking region.
Mallampalli
Rama K
RK
The Veterans Administration Medical Center, Iowa City, IA 52246, U.S.A. rama-mallampalli@uiowa.edu
Ryan
Alan J
AJ
Carroll
James L
JL
Osborne
Timothy F
TF
Thomas
Christie P
CP
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics*; D017931:DNA Primers; D050356:Lipid Metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D013261:Sterols / Q000378:metabolism*; D013501:Surface-Active Agents / Q000378:metabolism*
D017931:DNA Primers; D010713:Phosphatidylcholines; D013261:Sterols; D013501:Surface-Active Agents; D019916:Choline-Phosphate Cytidylyltransferase