The Cell-specific Expression of Endothelial Nitric-oxide Synthase

Abstract

The basis for the endothelial cell-restricted expression of endothelial nitric-oxide synthase (eNOS) is not known. While transgenic promoter/reporter mice demonstrated endothelium cell-specific eNOS expression, we found robust expression of episomal eNOS promoter/reporter constructs in cell types that do not express the native eNOS transcript. To explore the mechanism underlying this differential activity pattern of chromatin-versus episomebased eNOS promoters, we examined the methylation status of 5-regulatory sequences of the human eNOS gene. DNA methylation differed dramatically between endothelial and nonendothelial cell types, including vascular smooth muscle cells. This same cell type-specific methylation pattern was observed in vivo in endothelial and vascular smooth muscle cells of the mouse aorta at the native murine eNOS promoter. We addressed the functional consequences of methylation on eNOS transcription using transient transfection of in vitro methylated promoter/reporter constructs and found that methylated constructs exhibited a marked decrease in the synergistic action of Sp1, Sp3, and Ets1 on eNOS promoter activity. The addition of methyl-CpG-binding protein 2 further reduced the transcriptional activity of methylated eNOS constructs. Importantly, chromatin immunoprecipitation demonstrated the presence of Sp1, Sp3, and Ets1 at the native eNOS promoter in endothelial cells but not in vascular smooth muscle cells. Finally, robust expression of eNOS mRNA was induced in nonendothelial cell types following inhibition of DNA methyltransferase activity with 5-azacytidine, demonstrating the importance of DNA methylation-mediated repression. This report is the first to show that promoter DNA methylation plays an important role in the cell-specific expression of a constitutively expressed gene in the vascular endothelium.

Prospects for Epigenetic Epidemiology

“…To our knowledge, the dramatic difference in methylation levels observed in cancerous versus noncancerous tissue has not been found in other complex diseases, where methylation at any given CpG island or specific CpG sites in affected versus unaffected individuals may vary by less than 10% (83)(84)(85). Interpretation of functional consequences is therefore problematic.…”

Transcriptional regulation of endothelial nitric oxide synthase expression in uterine artery endothelial cells by c-Jun/AP-1

“…This is best exemplified by the fact that NOS3 mRNA and protein expression can be detected in various endothelial cells in vivo and in vitro under non-stimulated or stimulated conditions regardless of the origin of the cells. However, the NOS3 promoters of all species isolated so far, including sheep, also contain binding sites for multiple additional transcription factors participating in the complex regulation of NOS3 transcription in endothelial cells in a tissue/cell, and possibly species specific manner (Chan et al, 2004). Moreover, tissue and cell-specific transcriptional regulation of NOS3 expression is controlled by methylation (Chan et al, 2004) and histone acetylation (Fish et al, 2005;Gan et al, 2005) of the NOS3 promoter.…”

“…However, the NOS3 promoters of all species isolated so far, including sheep, also contain binding sites for multiple additional transcription factors participating in the complex regulation of NOS3 transcription in endothelial cells in a tissue/cell, and possibly species specific manner (Chan et al, 2004). Moreover, tissue and cell-specific transcriptional regulation of NOS3 expression is controlled by methylation (Chan et al, 2004) and histone acetylation (Fish et al, 2005;Gan et al, 2005) of the NOS3 promoter. In the present study, according to the sequence information of the 5′-flanking region of the sheep NOS3 gene, we hypothesized that the proximal consensus AP-1 (TGAGTCA) site positioned at the −682 to −676 upstream the ATG start codon in the sheep NOS3 gene plays an important role in the regulation of uterine artery endothelial NOS3 expression.…”

Implications of galactocerebrosidase and galactosylcerebroside metabolism in cancer cells

“…High-grade methylation leads to suppression, whereas low-grade methylation makes the DNA more susceptible for proteins that activate transcription. [24][25][26] DNA methylation has been described to downregulate the expression of the breast cancer-associated gene BRCA1. 25 In case of the GALC gene derived from HNSCC cells, transfection analysis of the promoter flanking the GALC coding region at the 5 0 end (base -176 to -24) has been conducted in a methylase-free setting that makes the presence of methylated DNA unlikely.…”