Lysophosphatidylcholine phosphorylates CREB and activates the jun2TRE site of c-jun promoter in vascular endothelial cells.
Lysophosphatidylcholine (lyso-PC), a polar phospholipid increased in atherogenic lipoproteins and atherosclerotic lesions, has been shown to induce transcription of a variety of endothelial genes relevant to atherogenesis. Lyso-PC has been shown to activate c-jun N-terminal kinase (JNK) and activator protein 1 (AP-1) and thereby stimulate transcription of the c-jun gene. Here we provide evidence that lyso-PC can phosphorylate cyclic AMP responsive element binding protein (CREB) and thereby activate the jun2 12-O-tetradecanoylphorbol 13-acetate response element (jun2TRE) site of the c-jun promoter, which appears to be the major molecular mechanism involved in lyso-PC-induced c-jun gene expression in cultured bovine aortic endothelial cells (BAEC). Transient transfection of BAEC with a 1.6-kbp c-jun promoter and luciferase reporter fusion gene resulted in a 12.9-fold increase in luciferase activity by lyso-PC treatment. Serial deletion mutation in c-jun promoter and luciferase reporter gene assay revealed that the 5' promoter region between nucleotide numbers -268 and -127, which contains a jun2TRE binding sequence, was most crucial for lyso-PC-induced transcription. The 5' promoter region between -76 and -27, which contains an AP-1 site, also affected lyso-PC-induced transcription of the c-jun gene. Point mutation in the jun2TRE site reduced lyso-PC-induced transcription of the c-jun promoter-luciferase fusion gene by a 70.3% decrease in c-jun promoter activity. Electrophoretic mobility shift assays showed increased binding of (32)P-labeled oligonucleotides with jun2TRE in nuclear extracts isolated from lyso-PC-treated BAEC, which was abolished or supershifted by anti-CREB antibody. Immunoblotting with anti-phosphorylated CREB antibody showed rapid phosphorylation of this protein after lyso-PC treatment. These results indicate that lyso-PC phosphorylates CREB, which was then bound to the jun2TRE site of the c-jun promoter and activated transcription. Activation of jun2TRE may play a key role in the transcriptional activation of c-jun as well as other endothelial genes depending upon these transcription factors.
Ueno
Y
Y
Department of Neurosurgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Kume
N
N
Miyamoto
S
S
Morimoto
M
M
Kataoka
H
H
Ochi
H
H
Nishi
E
E
Moriwaki
H
H
Minami
M
M
Hashimoto
N
N
Kita
T
T
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D016758:Genes, jun; D008244:Lysophosphatidylcholines / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects
D017362:Cyclic AMP Response Element-Binding Protein; D008244:Lysophosphatidylcholines