Estrogen Modulates NFκB Signaling by Enhancing IκBα Levels and Blocking p65 Binding at the Promoters of Inflammatory Genes via Estrogen Receptor-β

Background: NF kappa B signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17 beta-estradiol, E2) inhibits expression of these genes in an estrogen receptor (ER)-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF)-alpha treated rat aortic smooth muscle cells (RASMCs). This study tested whether E2 inhibits NF kappa B signaling in RASMCs and defined the mechanisms. Methodology/Principal Findings: TNF-alpha treated RASMCs demonstrated rapid degradation of I kappa B alpha (10-30 min), followed by dramatic increases in I kappa B alpha mRNA and protein synthesis (40-60 min). E2 enhanced TNF-alpha induced I kappa B alpha synthesis without affecting I kappa B alpha degradation. Chromatin immunoprecipitation (ChIP) assays revealed that E2 pretreatment both enhanced TNF-alpha induced binding of NF kappa B p65 to the I kappa B alpha promoter and suppressed TNF-alpha induced binding of NF kappa B p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP)-1 and cytokine-induced neutrophil chemoattractant (CINC)-2 beta genes. ChIP analyses also demonstrated that ER beta can be recruited to the promoters of MCP-1 and CINC-2 beta during co-treatment with TNF-alpha and E2. Conclusions: These data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of I kappa B alpha, thus accelerating a negative feedback loop in NF kappa B signaling, and directly inhibiting binding of NF kappa B to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NF kappa B signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury.