INTRODUCTION & BACKGROUND: The role of Nitric Oxide (NO) in estrogen (E2) dependent genomic regulation of gene expression is poorly characterized. We investigated NO contribution to E2 regulation of the human telomerase catalytic subunit (hTERT) expression and activity. METHODS & RESULTS: In human umbilical vein endothelial cells (HUVEC) E2 treatment had strong pro-angiogenic effect which was abrogated by the estrogen receptor antagonist ICI, the hTERT inhibitor AZT or the NOS inhibitor 7-nitroindazole (7N). Specifically, 7N prevented hTERT induction and activity in the presence of E2, revealing a role of NO in E2-dependent genomic regulation of hTERT expression. Quantitative Real-Time PCR coupled with Chromatin Immune-precipitations, performed on the hTERT promoter in the presence or absence of E2, showed recruitment of estrogen receptor alpha (ER-?) and beta (ER-?) and enriched histone 3 methylation on Lysine 79, a modification associated with actively transcribed chromatin. This effect was abolished by ICI or 7N. Transient transfection of a constitutively active eNOS mutant (eNOSmut) resulted in strong induction of a hTERT promoter reporter construct, which was again abrogated by 7N. Intriguingly, transfection of a hTERT promoter with a mutated estrogen responsive element (ERE) abolished E2- or eNOSmut-dependent transcription, indicating that the integrity of binding to ERE is important for hTERT response to NO. To further investigate the role of NO, pulmonary endothelial cells were isolated from eNOS-KO mice and grown with/without E2. In wild type cells, E2 significantly increased telomerase activity whereas eNOS-KO cells were insensitive to this treatment. Addition of a NO donor substantially rescued E2-dependent telomerase activity. CONCLUSIONS: These results provide evidence for a novel role of NO in the E2-dependent genomic regulation of hTERT expression and telomerase activity in human endothelial cells.
Novel Role For Nitric Oxide In The Estrogen-Dependent Genomic Regulation Of The Human Catalytic Telomerase Subunit Expression And Telomerase Activity.
2007
Abstract
INTRODUCTION & BACKGROUND: The role of Nitric Oxide (NO) in estrogen (E2) dependent genomic regulation of gene expression is poorly characterized. We investigated NO contribution to E2 regulation of the human telomerase catalytic subunit (hTERT) expression and activity. METHODS & RESULTS: In human umbilical vein endothelial cells (HUVEC) E2 treatment had strong pro-angiogenic effect which was abrogated by the estrogen receptor antagonist ICI, the hTERT inhibitor AZT or the NOS inhibitor 7-nitroindazole (7N). Specifically, 7N prevented hTERT induction and activity in the presence of E2, revealing a role of NO in E2-dependent genomic regulation of hTERT expression. Quantitative Real-Time PCR coupled with Chromatin Immune-precipitations, performed on the hTERT promoter in the presence or absence of E2, showed recruitment of estrogen receptor alpha (ER-?) and beta (ER-?) and enriched histone 3 methylation on Lysine 79, a modification associated with actively transcribed chromatin. This effect was abolished by ICI or 7N. Transient transfection of a constitutively active eNOS mutant (eNOSmut) resulted in strong induction of a hTERT promoter reporter construct, which was again abrogated by 7N. Intriguingly, transfection of a hTERT promoter with a mutated estrogen responsive element (ERE) abolished E2- or eNOSmut-dependent transcription, indicating that the integrity of binding to ERE is important for hTERT response to NO. To further investigate the role of NO, pulmonary endothelial cells were isolated from eNOS-KO mice and grown with/without E2. In wild type cells, E2 significantly increased telomerase activity whereas eNOS-KO cells were insensitive to this treatment. Addition of a NO donor substantially rescued E2-dependent telomerase activity. CONCLUSIONS: These results provide evidence for a novel role of NO in the E2-dependent genomic regulation of hTERT expression and telomerase activity in human endothelial cells.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
