Estrogen-related receptor alpha 1 up-regulates endothelial nitric oxide synthase expression

Abstract

The human estrogen-related receptor alpha1 (ERR alpha 1) is a member of an orphan receptor family closely related to the estrogen receptor. It has been demonstrated that estrogen modulates endothelial nitric oxide synthase (eNOS) expression through the estrogen receptor in endothelial cells. However, little is known about the relationship between ERR alpha 1 and eNOS. In this study, we show that ERR alpha 1 activates the estrogen response element (ERE) and eNOS promoter-dependent luciferase activity in COS-7 cells and bovine pulmonary artery endothelial cells. The endogenous ligand for ERR alpha 1 has not been identified, but we show that these actions are dependent on serum constituents because ERR alpha 1 fails to stimulate eNOS promoter-dependent luciferase activity in charcoal-treated serum. Furthermore, through the use of truncated eNOS promoter luciferase constructs, we demonstrate that the activation of eNOS transcription by ERR alpha 1 is mediated via three regions: base pairs -1001 to -743, base pairs -743 to -265, and downstream from base pair -265 on the eNOS promoter. In addition, ERR alpha 1 up-regulates eNOS mRNA and protein expression and stimulates eNOS activity in bovine pulmonary artery endothelial cells. These results suggest that ERR alpha 1 has a potential role in the regulation of eNOS expression and may stimulate NO production by endothelial cells, which may in turn result in a protective effect against atherosclerosis.

Fig. 1.

ERRα1-FLAG fusion protein expression. Twenty micrograms of total protein was extracted 24 and 48 h after transfection of COS-7 cells (A) and BPAEC (B) and subjected to SDS/10% PAGE. Western blots were performed with anti-FLAG monoclonal antibody (10 μg/ml). Lane 1, 0.5 μg of pCMV-Tag4A-transfected; lane 2, 1.0 μg of pCMV-Tag4A-transfected; lane 3, 0.5 μgofERRα1/pCMV-Tag4A-transfected; lane 4, 1.0 μgofERRα1/pCMV-Tag4A-transfected; lane 5, 0.2 μg of pCMV-Tag4A-transfected; lane 6, 0.4 μg of pCMV-Tag4A-transfected; lane 7, 0.2 μg of ERRα1/pCMV-Tag4A-transfected; lane 8, 0.4 μg of ERRα1/pCMV-Tag4A-transfected.

Fig. 2.

Transcriptional activity of ERRα1 in COS-7 cells. ERRα1/pcDNA3.1(+) or pcDNA3.1(+) cDNA were transiently cotransfected with 3×ERE-luciferase (open bar) or eNOS promoter (-1193/+109) luciferase (filled bar) cDNA into COS-7 cells. Cells were cultured for 48 h. ERE and eNOS promoter-dependent luciferase activities were determined and plotted as fold activation over the individual luciferase activity with vector alone (*, P < 0.05 vs. vector).

Fig. 3.

Transcriptional activity of ERRα1 in BPAEC. ERRα1/pcDNA3.1(+) or pcDNA3.1(+) cDNA were transiently cotransfected with 3×ERE-luciferase or eNOS promoter (-1193/+109) luciferase cDNA into BPAEC. Transfected cells were cultured for 24 h in medium containing normal (open bars) or charcoal-stripped (filled bars) serum. ERE and eNOS promoter-dependent luciferase activities were determined and plotted as fold activation over the individual luciferase activity with vector alone (*, P < 0.05 vs. vector).

Fig. 4.

ERRα1 transcriptional activity with truncated eNOS promoters in BPAEC. ERRα1/pcDNA3.1(+) cDNA was transiently cotransfected with four truncated eNOS promoter [-1193/+109, -1001/+109, -743/+109, or -265/+109] luciferase cDNAs into BPAEC. Cells were cultured for 24 h. eNOS promoter-dependent luciferase activities were determined and plotted as fold activation over the individual luciferase activity with vector alone (*, P < 0.05 vs. -1193/+109 eNOS promoter-dependent luciferase activity; †, P < 0.05 vs. -743/+109 eNOS promoter-dependent luciferase activity).

Fig. 5.

ERRα1 up-regulates endogenous eNOS mRNA expression in BPAEC. ERRα1/pcDNA3.1(+) (E) or pcDNA3.1(+) (V) cDNA were transiently transfected into BPAEC. Transfected cells were cultured for 12, 24, and 48 h. Ribonuclease protection assay was performed with radiolabeled human eNOS antisense RNA. Band intensities were quantified by using nih image software and were normalized to β-actin band intensity. The percent control of eNOS mRNA was then calculated and further normalized to pcDNA3.1(+) (vector only control) [*, P < 0.05 vs. before transfection (0 h)].

Fig. 6.

ERRα1 up-regulates endogenous eNOS protein expression in BPAEC. ERRα1/pcDNA3.1(+) (E) or pcDNA3.1(+) (V) cDNA were transiently transfected into BPAEC. Transfected cells were cultured for 12, 24, 48, and 72 h in medium containing normal serum (A and Right, open bars) or charcoal-stripped serum (B and Right, filled bars). Ten micrograms of total protein was extracted at each time point and separated by SDS/7.5% PAGE. Western blots were performed with anti-eNOS and anti-actin monoclonal antibodies. Each band intensity was quantified with nih image software and was normalized to the actin band intensity. The percent control of eNOS protein was then calculated and further normalized to pcDNA3.1(+) (vector only control) [*, P < 0.05 vs. before transfection (0 h)].

Fig. 7.

ERRα1 increases eNOS activity in BPAEC. ERRα1/pcDNA3.1(+) or pcDNA3.1(+) cDNA was transiently transfected into BPAEC. Transfected cells were cultured for 24, 48, and 72 h in medium containing normal serum (open bars) or charcoal-stripped serum (filled bars). eNOS activity was measured by conversion of [³H]arginine to [³H]citrulline. The percent control of eNOS activity was calculated and normalized to pcDNA3.1(+) transfections [*, P < 0.05 vs. before transfection (0 h)].