Nuclear Redox-Sensitive Mechanisms in Endothelial Cells

Emory University, Atlanta GA

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Abstract

DESCRIPTION (provided by the applicant): Nuclear factor-kB (NF-kB) is a ubiquitous transcriptional control factor that is involved in diverse cellular processes including inflammation, apoptosis, and growth. Our objective is to understand the redox-sensitive mechanism by which NF-kB regulates VCAM-l gene expression in endothelial cells in response to various proinflammatory stimuli. We propose that the redox-sensitivity of the transactivation domain (TAD) of p65/NF-kB could be an important determinant factor in the redox regulation of VCAM-1 expression by NF-kB. Our studies suggest that redox-sensitive AP-1 (c-Fos/c-Jun) proteins can regulate the activity of the TAD pf p65, and that this effect is dependent on the stoichiometry of c-Fos/c-Jun. Previous studies have suggested that the redox-state of AP-1 can be modulated by the nuclear redox factor-1 (ref-1). Ref-1 catalyzes the reduction of highly conserved cysteine residue present in the DNA binding domain of AP-1 proteins suggesting that AP-1 redox regulation by Ref-1 could provide the basis for the redox-sensitive regulation of NF-kB. The transcriptional activity of p65 subunit of NF-kB is also regulated by various mechanisms such as its phosphorylation at distinct sites and its interaction with coactivators CBP/p300, which bind both c-Fos and c-Jun. We hypothesize that AP-1 (c-Fos/c-Jun) is an important component of the NF-kB complex that provides NF-kB the flexibility to regulate VCAM-1 gene expression through a redox-sensitive mechanism. We propose to gain insights into these mechanisms by modulating the redox state of AP-1 and the phosphorylation state of c-Jun through expressing antisense Ref-1 and dominant negative JNK into endothelial cells, respectively. To test the hypothesis we propose the following specific aims: 1) characterize the effect of stoichiometry of c-Fos/c-Jun on the activity of NF-kB and the endogenous VCAM-1 expression, 2) Characterize the effect of redox state of AP-1 proteins on the activity of NF-kB and the endogenous VCAM-1 expression, and 3) Characterize the effect of phosphorylation of NF-k-B associated c-Jun on the activity of NF-kB and the endogenous VCAM-1 gene expression. These studies will not only provide insights into the mechanism by which the redox-sensitive transcriptional activity of NF-kB is controlled but will also provide more general insights into the mechanisms by which redox state of cell can modulate protein-protein interaction and functional role of transcriptional factors.