Tumor Necrosis Factor Receptor-associated Factor-6 and Ribosomal S6 Kinase Intracellular Pathways Link the Angiotensin II AT1 Receptor to the Phosphorylation and Activation of the IκB Kinase Complex in Vascular Smooth Muscle Cells

Priscilla Doyon,Marc J Servant

doi:10.1074/jbc.m110.126433

Priscilla Doyon, Marc J Servant

Open Access

https://doi.org/10.1074/jbc.m110.126433

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Abstract

Activation of NF-κB transcription factors by locally produced angiotensin II (Ang II) is proposed to be involved in chronic inflammatory reactions leading to atherosclerosis development. However, a clear understanding of the signaling cascades coupling the Ang II AT1 receptors to the activation of NF-κB transcription factors is still lacking. Using primary cultured aortic vascular smooth muscle cells, we show that activation of the IKK complex and NF-κB transcription factors by Ang II is regulated by phosphorylation of the catalytic subunit IKKβ on serine residues 177 and 181 in the activation T-loop. The use of pharmacological inhibitors against conventional protein kinases C (PKCs), mitogen-activated/extracellular signal-regulated kinase (MEK) 1/2, ribosomal S6 kinase (RSK), and silencing RNA technology targeting PKCα, IKKβ subunit, tumor growth factor β-activating kinase-1 (TAK1), the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor-6 (TRAF6), and RSK isoforms, demonstrates the requirement of two distinct signaling pathway for the phosphorylation of IKKβ and the activation of the IKK complex by Ang II. Rapid phosphorylation of IKKβ requires a second messenger-dependent pathway composed of PKCα-TRAF6-TAK1, whereas sustained phosphorylation and activation of IKKβ requires the MEK1/2-ERK1/2-RSK pathway. Importantly, simultaneously targeting components of these two pathways completely blunts the phosphorylation of IKKβ and the proinflammatory effect of the octapeptide. This is the first report demonstrating activation of TAK1 by the AT1R. We propose a model whereby TRAF6-TAK1 and ERK-RSK intracellular pathways independently and sequentially converge to the T-loop phosphorylation for full activation of IKKβ, which is an essential step in the proinflammatory activity of Ang II.

Highlights

The NF-␬B family is represented by five members: p50, p65(RelA), c-Rel, p52, and RelB
tumor necrosis factor receptor-associated factor-6 (TRAF6) and ribosomal S6 kinase (RSK) Pathways Link the AT1R to the IKK Complex the classical pathway, the first phase of NF-␬B activation mainly consists of the regulated degradation of I␬B␣ and is triggered by prototypical activators such as tumor necrosis factor (TNF)-␣, lipopolysaccharide, IL-1␤, and phorbol 12-myristate 13-acetate
Essential Role of IKK␤ in the Proinflammatory Actions of angiotensin II (Ang II)—We and others have recently demonstrated AT1R-mediated activation of the IKK complex in primary vascular smooth muscle cells (VSMC) exposed to Ang II [5, 18]

Summary

The abbreviations used are

Ang II, angiotensin II; AT1R, Ang II AT1 receptor; RAS, renin angiotensin system; VSMC, vascular smooth muscle cells; IKK, I␬B kinase; RSK, ribosomal S6 kinase; TAK1, tumor growth factor ␤ activating kinase-1; TRAF6, TNF receptor-associated factor-6; PLC, phospholipase C; NIK, NF-␬B inducing kinase; LPA, lysophosphatidic acid; GPCR, G proteincoupled-receptors; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈtetraacetic acid; DMSO, dimethyl sulfoxide; DN, dominant-negative. TRAF6 and RSK Pathways Link the AT1R to the IKK Complex the classical pathway, the first phase of NF-␬B activation mainly consists of the regulated degradation of I␬B␣ and is triggered by prototypical activators such as tumor necrosis factor (TNF)-␣, lipopolysaccharide, IL-1␤, and phorbol 12-myristate 13-acetate These stimuli induce the phosphorylation of I␬B␣ at Ser and Ser in the N-terminal signal responsive domain by the canonical I␬B kinase (IKK) complex, which is composed of two catalytic subunits called IKK␣ and -␤, and one regulatory subunit called IKK␥. Genetic data about the molecular links between GPCR proximal events and activation of the IKK complex demonstrate that a complex formed by CARMA3, Bcl, and MALT1, better known as the CBM signalosome, constitute the missing link between G protein-coupled receptors (GPCRs) and IKK complex activation (20, 26 –28) In this complex, the ubiquitin E3 ligase MALT1 could act as the effector protein in AT1R signaling by inducing Lys63-linked IKK␥ polyubiquitination and IKK complex activation [26].

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION