The ATF site mediates downregulation of the cyclin A gene during contact inhibition in vascular endothelial cells.
Contact inhibition mediates monolayer formation and withdrawal from the cell cycle in vascular endothelial cells. In studying the cyclins--key regulators of the cell cycle--in bovine aortic endothelial cells (BAEC), we found that levels of cyclin A mRNA decreased in confluent BAEC despite the presence of 10% fetal calf serum. We then transfected into BAEC a series of plasmids containing various lengths of the human cyclin A 5' flanking sequence and the luciferase gene. Plasmids containing 3,200, 516, 406, 266, or 133 bp of the human cyclin A promoter directed high luciferase activity in growing but not confluent BAEC. In contrast, a plasmid containing 23 bp of the cyclin A promoter was associated with a 65-fold reduction in activity in growing BAEC, and the promoter activities of this plasmid were identical in both growing and confluent BAEC. Mutation of the activating transcription factor (ATF) consensus sequence at bp -80 to -73 of the cyclin A promoter decreased its activity, indicating the critical role of the ATF site. We identified by gel mobility shift analysis protein complexes that bound to the ATF site in nuclear extracts from growing but not confluent BAEC and identified (with antibodies) ATF-1 as a binding protein in nuclear extracts from growing cells. Also, ATF-1 mRNA levels decreased in confluent BAEC. Taken together, these data suggest that the ATF site and its cognate binding proteins play an important role in the downregulation of cyclin A gene expression during contact inhibition.
Yoshizumi
M
M
Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Hsieh
C M
CM
Zhou
F
F
Tsai
J C
JC
Patterson
C
C
Perrella
M A
MA
Lee
M E
ME
D051697:Activating Transcription Factor 1; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003260:Contact Inhibition; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D015533:Transcriptional Activation
D051697:Activating Transcription Factor 1; D016213:Cyclins; D004268:DNA-Binding Proteins; D014157:Transcription Factors