RhoA Mediates Angiotensin II–Induced Phospho-Ser536 Nuclear Factor κB/RelA Subunit Exchange on the Interleukin-6 Promoter in VSMCs

Abstract

Abstract-The vasoconstrictor angiotensin II (Ang II) accelerates atherosclerosis by inducing vascular gene expression programs, producing monocyte recruitment, and vascular remodeling. In vascular smooth muscle cells (VSMCs), Ang II signaling activates interleukin (IL)-6 expression, a cytokine producing acute-phase inflammation, mediated by the transcription factor nuclear factor B (NF-B). The classical NF-B activation pathway involves cytoplasmic-to-nuclear translocation of the potent RelA transactivating subunit; however, because nuclear RelA is present in VSMCs, the mechanism by which NF-B activity is controlled is incompletely understood. In this study, we focus on early activation steps controlling RelA activation. Although Ang II only weakly induces Ϸ1.5-fold RelA nuclear translocation, RelA is nevertheless required because short interfering RNA-mediated RelA knockdown inhibits inducible IL-6 expression. We find instead that Ang II stimulation rapidly induces RelA phosphorylation at serine residue 536, a critical regulatory site in its transactivating domain. Chromatin immunoprecipitation assays indicate no significant changes in total RelA binding to the native IL-6 promoter, but an apparent increase in fractional binding of phospho-Ser536 RelA. Inactivation of RhoA by treatment with Clostridium botulinum exoenzyme C3 exotoxin or expression of dominant negative RhoA blocks Ang II-inducible RelA Ser536 phosphorylation and IL-6 expression. Finally, enhanced phospho-Ser536 RelA formation in the aortae of rats chronically infused with Ang II was observed. Together, these data indicate a novel mechanism for Ang II-induced NF-B activation in VSMCs, mediated by RhoA-induced phospho-Ser536 RelA formation, IL-6 expression, and vascular inflammation.

Multifaceted Role of Angiotensin II in Vascular Inflammation and Aortic Aneurysmal Disease

“…These studies showed that Ang II induces NF-κB /RelA activation in VSMCs by increasing the relative abundance of phospho-Ser-536 RelA in the nucleoplasmic pool. We also confirmed Ang II-induced enhanced phospho-Ser-536 RelA formation in rat aortas treated with Ang II, establishing relevance to vascular signaling in vivo (Choudhary, et al, 2007;Cui, et al, 2006). Our group further showed that Ang II-induced RelA Ser-536 phosphorylation is mediated by NF-κB-inducing kinase (NIK), the major regulated step controlling non-canonical NF-κB signaling.…”

“…Interestingly, we also found that phospho-Ser-536 RelA formation was blocked by RhoA inhibition, suggesting that Ser-536 phosphorylation was mediated upstream by RhoA. In addition, RhoA inhibition also blocked Ang II-induced IL-6 expression, indicating that Ang II-inducible phospho-Ser-536 RelA was required for IL-6 activation (Cui, et al, 2006). Our studies in Ang II-stimulated VSMCs further showed that total RelA binding did not change on the native IL-6 promoter in response to Ang II, but fractional binding of phospho-Ser-536 RelA to the IL-6 promoter was increased (Choudhary, et al, 2007).…”

RelA Ser276 Phosphorylation Is Required for Activation of a Subset of NF-κB-Dependent Genes by Recruiting Cyclin-Dependent Kinase 9/Cyclin T1 Complexes

“…This phenomenon has been experimentally verified by us using Ang II, a ligand that activates phosphoSer 536 RelA formation without affecting total RelA nuclear abundance. In this case, an increase in the fraction of nuclear phospho-Ser 536 RelA can rapidly exchange with promoterbound hypophosphorylated RelA, producing a transition in gene expression from an inactive state to an activated one (14). Our ChIP analysis of phospho-Ser 276 RelA binding, however, indicates that this isoform does not rapidly exchange with the hypophosphorylated RelA bound to the Naf1 gene.…”

Mechanism of Ang II involvement in activation of NF-κB through phosphorylation of p65 during aging

“…It is known that several pro-inflammatory stimuli, such as TNFα, UV light, and LPS, induce phosphorylation of p65 at the Ser 536 residue, which is triggered by IKKαβ phosphorylation in the cytoplasm. Recent studies indicate that Ang II also activates NF-κB through the phosphorylation of p65 at Ser 536, which is shown to be mediated by IKKαβ phosphorylation in various cellular models (Cui et al 2006;Douillette et al 2006;Wei et al 2008). However, these findings are in contrast to earlier findings on TNFα showing Ser 276 phosphorylation by mitogenand stress-activated protein kinase-1 (MSK-1) in the nucleus (Viatour et al 2005;Zhong et al 2002).…”