Involvement of a Novel Rac/RhoA Guanosine Triphosphatase-Nuclear Factor-κB Inducing Kinase Signaling Pathway Mediating Angiotensin II-Induced RelA Transactivation

Abstract

Angiotensin II (Ang II) is the major effector peptide of the renin angiotensin system that induces inflammatory gene expression through the nuclear factor-kappaB (NF-kappaB) transcription factor. Activation of latent cytoplasmic NF-kappaB is controlled by distinct pathways, the best known being the canonical pathway controlling IkappaB kinase activation. Interestingly, Ang II only weakly activates the canonical pathway. Although basal nucleocytoplasmic RelA shuttling is required for Ang II stimulation, changes in RelA translocation do not account for its transcriptional effect. Instead, Ang II rapidly induced RelA phosphorylation at Ser residue 536, and complex formation with the Ser(536) kinase known as the NF-kappaB-inducing kinase (NIK)/MEKK14. The requirement of NIK in Ang II-inducible transcription was shown by expressing a dominant-negative NIK or small interfering RNA (siRNA)-mediated knockdown; both inhibited Ang II-induced transcription. Conversely, constitutively active NIK potently induced RelA transactivation activity. Consistent with its actions independent of the canonical pathway, NIK induces the activity of the RelA transactivation domains -1 and -2 in constitutively nuclear GAL4-RelA fusion proteins that do not bind IkappaBalpha. Ang II induces NIK activity, phosphorylation of its endogenous IkappaB kinase alpha substrate, and induction of nuclear NF-kappaB2 (p52) processing. NIK down-regulation prevents Ang II-induced phospho-Ser(536) RelA formation, indicating that it is essential for RelA activation. The Ang II pathway further involves the RhoA small GTP-binding protein because RhoA inhibition blocks Ang II-induced transcriptional activity and formation of phospho-Ser(536) RelA formation. Finally, we demonstrate that Ang II infusion in vivo rapidly induces phospho-Ser(536) RelA formation and activation of the NF-kappaB-dependent IL-6 gene. These data indicate that Ang II induces NF-kappaB-dependent transcription through an alternative pathway, being largely independent of IkappaB proteolysis, but mediated by the small GTPases Rac/RhoA, required for NIK.RelA complex formation and inducible Ser(536) RelA phosphorylation.

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.…”

“…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). 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.…”

“…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. NIK inhibition prevented Ang II-induced Ser-536 phosphorylation and NF-κB-dependent transcription (Choudhary, et al, 2007), indicating that it is essential for RelA activation. We also found that NIK induced the activity of the RelA transactivation domain-1 and -2 in constitutively nuclear RelA proteins and that RelA formed an inducible nuclear complex with NIK in response to Ang II stimulation.…”

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis

“…In these cells, substantial levels of RelA are found inactive in the nucleus under resting conditions. Ang II stimulation induces a pathway of RhoA and NIK activation, culminating in NIK-dependent phosphorylation of the nuclear RelA species on serine 536 (Choudhary et al, 2007;Cui et al, 2006). This phosphorylated pool of RelA is free from IB regulation and dynamically cycles through the nucleus, interacting with target genes (Bosisio et al, 2006;Sasaki et al, 2005).…”

Papel pró-inflamatório da angiotensina II na aorta de camundongos normotensos

“…No entanto, a Ang II induz a transcrição do NF-kB através de uma via independente da proteólise de IkB, mas mediada por pequenas GTPases Rac/Rho A, exigida pelo complexo NIK-Rel A e induzindo a fosforilação phospho-ser-536 Rel A (Han et al, 1999). Desse modo, a ativação NFkB/Rel A ocorre através do aumento nos níveis de phospho-ser-536 Rel A no núcleo, essa resposta já foi observada aorta de ratos tratados com Ang II (Choudhary, et al, 2007;Cui, et al, 2006). Quando ativado pela Ang II, o NF-kB estimula a expressão de IL-6 por fibroblastos da adventícia, monócitos recrutados e CMLVs (Han et al, 1999).…”