Chronic inflammation is an independent risk factor for ischemic heart disease. Persistent elevations in circulating cytokines from the vascular wall and hepatic acute phase reactants affect the progression of atherosclerotic lesions. Angiotensin II (AID, the effector of the renin angiotensin system, potentiates atherogenesis and has pro-inflammatory actions through its ability to induce Nuclear Factor kappaB (NF-kappaB), a critical signaling molecule that controls chemokine expression by vascular smooth muscle cells (VSMCs) and hepatic acute phase reactant synthesis by hepatocytes. In the previous granting period, we have demonstrated that AII induces NF-KappaB activation through a distinct intracellular signaling pathway than that induced by cytokines. In this renewal, we will pursue the hypothesis that AH induces Nuclear Factor kappaB (NF-kappaB) by the MAP3Ks TGF Beta-activated kinase (TAK1), NF-kappaB inducing kinase (NIK), and I kappaB Kinase alpha (IKKalpha) that participate in a novel alternative signaling pathway directly controlling nuclear NF-tall activity. We propose four aims: 1. To determine the molecular mechanism(s) for NIK dependent Rel A activation. We will determine whether AII induces NIK kinase activity and if this occurs by nuclear translocation. We will identify domains of the transactivating Rel A subunit of NF-kappaB that are activated by NIK. 2. To determine the relationship between the IKK and AII signaling pathways. The effect of AII on IKKbeta kinase activity will be measured in IP-kinase assays and the requirement for catalytic IKKbeta determined by transfection experiments using a dominant negative (DN) inhibitor. We will determine the role of the regulatory IKKgamma subunit in AII signaling and identify which regulatory isoform mediates the AII and NIK pathways. 3. To determine the relationship of the TAKI-IKKa signaling pathway on AII-and NIK-induced NF-kappaB activation.
This aim will determine the effect of AII on TAK1 kinase activity, and whether All- and NIK signaling is sensitive to TAK1 DN expression. We will determine the membrane proteins associated with the TAK1-IKKgamma-delta-assembled membrane preactivation complex using matrix-assisted laser desorption/ionization (MALDI) analyses. 4. To determine the requirement of NIK and TAK1 in AII induced chemokine production by VSMCs. Nylon based eDNA macroarrays will identify the spectrum of AII-induced chemokines. Short interfering RNA (siRNA) mediated """"""""knockdowns"""""""" will be performed to identify the spectrum of NIK- and TAK1-dependent chemokines induced by AII. The mechanism(s) how AII mediates vascular inflammation is significant in cardiovascular biology. These studies will identify new therapeutic targets that can disrupt the vicious cycle of inflammation and atherosclerosis.
