Ets-1 protects vascular smooth muscle cells from undergoing apoptosis by activating p21WAF1/Cip1: ETS-1 regulates basal and and inducible p21WAF1/Cip: ETS-1 regulates basal and inducible p21WAF1/Cip1 transcription via distinct cis-acting elements in the p21WAF/Cip1 promoter.
The cyclin-dependent kinase inhibitor (CKI) p21WAF1/Cip1 is regulated at the level of transcription by nuclear factors such as the co-activator p300. It is presently unknown whether the Ets family of transcription factors control p21WAF1/Cip1 gene expression. Ets-1 inhibits apoptosis in vascular smooth muscle cells as determined by both fluorescein isothiocyanate-linked annexin V/propidium iodide staining of cells and fluorescence-activated cell sorting analysis and quantitative cytoplasmic histone-associated internucleosomal DNA fragmentation. p21WAF1/Cip1 can play a mitogenic and anti-apoptotic role in smooth muscle cells. Using transient transfection and Western blot analysis, we determined that Ets-1 activates p21WAF1/Cip1 transcription and protein expression. Electrophoretic mobility shift assays revealed that Ets-1 interacts selectively with the -1350GGAA-1347 Ets element in the p21WAF1/Cip1 promoter. Mutation of this element reduced basal and Ets1-inducible p21WAF1/Cip1 promoter-dependent expression. In contrast, the -1577GGAT-1574 motif mediates basal but not Ets-1 activation of the p21WAF1/Cip1 promoter. Co-immunoprecipitation and co-transfection analysis showed that Ets-1 binds p300 and cooperatively activates p21WAF1/Cip1 transcription. The phenotypic importance of Ets-1 regulation of p21WAF1/Cip1 was demonstrated by the capacity of antisense p21WAF1/Cip1 strategies to block Ets-1-inhibition of apoptosis and inhibit Ets-1-induction of proliferation.
Centre for Vascular Research, The University of New South Wales and Department of Haematology, The Prince of Wales Hospital, Sydney, New South Wales 2052, Australia.
Cuili
C
Zhang
Mary M
MM
Kavurma
Angela
A
Lai
Levon M
LM
Khachigian
D020816:Amino Acid Motifs; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002469:Cell Separation; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D053938:DNA Fragmentation; D004789:Enzyme Activation; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D009154:Mutation; D009707:Nucleosomes / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010641:Phenotype; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D050783:Proto-Oncogene Proteins c-ets; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; C494043:Ets1 protein, rat; D009707:Nucleosomes; D016376:Oligonucleotides, Antisense; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D008156:Luciferases