Differential regulation of the inducible nitric oxide synthase gene by estrogen receptors 1 and 2.

Estrogen has both rapid and longer term direct effects on cardiovascular tissues mediated by the two estrogen receptors, ESR1 and ESR2. Previous work identified that estrogen regulates the expression of inducible nitric oxide synthase (NOS2A) in vascular smooth muscle cells (VSMC). ESR2 knockout mice have vascular dysfunction due to dysregulation of NOS2A expression and these mice are hypertensive (Zhu et al. Science 2002 295 505-508). Here, we report studies to examine the differential regulation of NOS2A gene expression by ESR1 and 2. Immunoblotting and RT-PCR studies revealed that different VSMC lines expressed different levels of ESR1 and ESR2 protein and mRNA. VSMC from different vascular beds were studied, including aortic VSMC expressing ESR1 and radial (Rad) VSMC expressing ESR2. E(2) inhibited NO production and NOS2A protein expression in aortic VSMC. Human NOS2A promoter-reporter studies revealed suppression of NOS2A reporter activity by E(2) in aortic VSMC, and stimulation of NOS2A reporter activity by E(2) in Rad arterial VSMC. In heterologous expression studies of COS-7 cells lacking endogenous ER, E(2) treatment of COS-7 cells did not alter NOS2A reporter activity in the presence of ESR1, while reporter activity increased 2.3-fold in the presence of ESR2. Similar experiments in COS-7 cells using the selective estrogen receptor modulator raloxifene showed that raloxifene caused a reduction in NOS2A reporter activity with ESR1 coexpression and an increase with ESR2 coexpression. Rat VSMC expressing ESR2 but not ESR1 also showed increased NOS2A reporter activity with E(2) treatment, an effect lost when ESR1 was introduced into the cells. Taken together, these data support that hNOS2A transcription is regulated positively by ESR2 and negatively by ESR1 in VSMC, supporting differential actions of these two estrogen receptors on a physiologically relevant gene in VSMC.