Abstract 1396: Nitric Oxide is Required for Estrogen Regulation of hTERT Expression and Telomerase Activity in Human Endothelial Cells.
INTRODUCTION & BACKGROUND: We have previously reported that nitric oxide (NO) is required for the VEGF-dependent angiogenic response and telomerase activity in human umbilical endothelial cells (HUVEC). In this study, we investigated the role of NO as a potential regulator of the estrogen (E2) responsiveness of the catalytic subunit of human telomerase (hTERT), a non traditional target of the estrogen receptor signaling.
METHODS & RESULTS: Upon E2 stimulation, HUVEC showed a significant induction of telomerase activity evaluated by TRAP assay which was paralleled by a prompt stimulation of hTERT mRNA expression. This effect was prevented by the NOS inhibitor 7-nitroindazole (7N). In the presence of E2, gel retardation (EMSA) and chromatin immunoprecipitation (ChIP) assays clearly showed a specific recruitment of both ERa and ERb onto the hTERT promoter as the result of intrinsic estrogen-dependent chromatin remodeling. This effect was abrogated in the presence of the estrogen antagonist ICI. To determine the specific role of eNOS in the estrogen-dependent activation of hTERT promoter, bovine aortic endothelial cells (BAEC) were transiently transfected with the constitutively active eNOS mutant S1177D in the presence of E2 and/or 7N. Remarkably,, the eNOS inhibitor abolished E2 and NO responsiveness of hTERT promoter. To directly address the role of NO in the E2-dependent regulation of hTERT, pulmonary endothelial cells (PEC) were isolated from eNOS−/− mice and grown in absence or presence of E2. In this condition, E2 significantly increased telomerase activity in wild-type cells whereas eNOS−/− cells exhibited a barely detectable telomerase activity that was not rescued upon E2 treatment.
CONCLUSIONS: Our results provide novel evidences leading to a molecular dissection of the NO contribution to the estrogen-dependent regulation of hTERT and telomerase activity in endothelial cells.