Estrogen receptor-alpha and endothelial nitric oxide synthase nuclear complex regulates transcription of human telomerase.
We report that in endothelial cells, the angiogenic effect of 17beta-estradiol (E2) is inhibited by the estrogen receptor (ER) antagonist ICI or the NO synthase (NOS) inhibitor 7-nitroindazole via downregulation of hTERT, the telomerase catalytic subunit, suggesting that E2 and NO are involved in controlling hTERT transcription. Quantitative Real-Time PCR and chromatin immunoprecipitations in E2-treated human umbilical vein endothelial cells, showed recruitment of ERs on the hTERT promoter and concomitant enrichment in histone 3 methylation at Lysine 79, a modification associated with transcription-competent chromatin. Confocal microscopy and re-chromatin immunoprecipitations revealed that on E2 induction, endothelial (e)NOS rapidly localized into the nucleus and associated with ERalpha on the hTERT promoter. Transfections of a constitutively active eNOS mutant (S1177D) strongly induced the hTERT promoter, indicating a direct role of the protein in hTERT transcriptional regulation. Mutation of the estrogen response element in the promoter abolished response to both ERs and active eNOS, demonstrating that the estrogen response element integrity is required for hTERT regulation by these factors. To investigate this novel regulation in a reduced NO environment, pulmonary endothelial cells were isolated from eNOS(-/-) mice and grown with/without E2. In wild-type cells, E2 significantly increased telomerase activity. In eNOS(-/-) cells, basal telomerase activity was rescued by exogenous eNOS or an NO donor, whereas responsiveness to E2 demanded the active protein. In conclusion, we document the novel findings of a combinatorial eNOS/ERalpha complex at the hTERT estrogen response element site and that active eNOS and ligand-activated ERs cooperate in regulating hTERT expression in the endothelium.
Department of Experimental Oncology, Regina Elena Cancer Institute, Rome, Italy.
Annalisa
A
Grasselli
Simona
S
Nanni
Claudia
C
Colussi
Aurora
A
Aiello
Valentina
V
Benvenuti
Gianluca
G
Ragone
Fabiola
F
Moretti
Ada
A
Sacchi
Silvia
S
Bacchetti
Carlo
C
Gaetano
Maurizio C
MC
Capogrossi
Alfredo
A
Pontecorvi
Antonella
A
Farsetti
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology*; D002478:Cells, Cultured; D019175:DNA Methylation; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D020125:Mutation, Missense; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000502:physiology; D019098:Telomerase / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology / Q000201:enzymology*
D004791:Enzyme Inhibitors; D047628:Estrogen Receptor alpha; D004967:Estrogens; D007191:Indazoles; D004958:Estradiol; C496314:NOS3 protein, human; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C509186:TERT protein, human; D019098:Telomerase; C080122:7-nitroindazole