INTRODUCTION & BACKGROUND: We previously reported that the hTERT promoter contains Estrogen Receptors (ER) Response Elements (EREs) active in human umbilical vein endothelial cells (HUVEC). Since Hypoxia Inducible Factors (HIF) too regulate hTERT via specific response elements (HREs), we queried whether estrogens and hypoxia cooperate in the control of hTERT expression. METHODS & RESULTS: HUVEC were cultured in normoxia or real hypoxia (<1% O2) with/without 17?-estradiol (E2, 10-7M) from 2-4 hours (h), and hTERT mRNA levels analysed by quantitative real-time PCR (qRT-PCR). E2 and hypoxia, when administered separately, significantly increased hTERT mRNA by 4h. Notably, upon combined stimuli the mRNA increase was advanced to 2h. To investigate the molecular mechanism underlying this process, we performed chromatin immune-precipitations (ChIP) on HUVEC grown in hypoxia or E2 for 30-90 and 45-90 minutes (min) respectively, using ER and HIF-1 specific antibodies followed by qRT-PCR of the hTERT promoter regions comprising the ERE and HRE. Specific ER recruitment on the ERE occurred upon E2 treatment with the expected 45/90 min attach/detach kinetics, while HIF-1 recruitment on the HRE upon hypoxia occurred at all time-points. Unexpectedly, ER occupancy of the ERE was seen also under hypoxia and was paralleled by an E2-inducible HIF-1 recruitment on both ERE and HRE. No recruitments were detected on the control cJun promoter. Given the synergy observed at the mRNA level, ChIP/qRT-PCR were performed with HUVEC grown in hypoxia plus E2. In these conditions, we detected a marked synergistic enhancement (5-fold over individual treatment) of HIF-1 recruitment onto the hTERT promoter. CONCLUSIONS: This work provides first evidence that a synergistic functional interaction between members of distinct transcription factor families contributes to the regulation of hTERT promoter activity and gene expression in human endothelial cells.
Regulation of hTERT Expression in Human Endothelial Cells Reveals A Functional Interaction between the Hypoxia Inducible Factor and the Estrogen Receptor
2007
Abstract
INTRODUCTION & BACKGROUND: We previously reported that the hTERT promoter contains Estrogen Receptors (ER) Response Elements (EREs) active in human umbilical vein endothelial cells (HUVEC). Since Hypoxia Inducible Factors (HIF) too regulate hTERT via specific response elements (HREs), we queried whether estrogens and hypoxia cooperate in the control of hTERT expression. METHODS & RESULTS: HUVEC were cultured in normoxia or real hypoxia (<1% O2) with/without 17?-estradiol (E2, 10-7M) from 2-4 hours (h), and hTERT mRNA levels analysed by quantitative real-time PCR (qRT-PCR). E2 and hypoxia, when administered separately, significantly increased hTERT mRNA by 4h. Notably, upon combined stimuli the mRNA increase was advanced to 2h. To investigate the molecular mechanism underlying this process, we performed chromatin immune-precipitations (ChIP) on HUVEC grown in hypoxia or E2 for 30-90 and 45-90 minutes (min) respectively, using ER and HIF-1 specific antibodies followed by qRT-PCR of the hTERT promoter regions comprising the ERE and HRE. Specific ER recruitment on the ERE occurred upon E2 treatment with the expected 45/90 min attach/detach kinetics, while HIF-1 recruitment on the HRE upon hypoxia occurred at all time-points. Unexpectedly, ER occupancy of the ERE was seen also under hypoxia and was paralleled by an E2-inducible HIF-1 recruitment on both ERE and HRE. No recruitments were detected on the control cJun promoter. Given the synergy observed at the mRNA level, ChIP/qRT-PCR were performed with HUVEC grown in hypoxia plus E2. In these conditions, we detected a marked synergistic enhancement (5-fold over individual treatment) of HIF-1 recruitment onto the hTERT promoter. CONCLUSIONS: This work provides first evidence that a synergistic functional interaction between members of distinct transcription factor families contributes to the regulation of hTERT promoter activity and gene expression in human endothelial cells.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
