Transcriptional repression of E-selectin and VCAM-1 gene expression by Oct-1 requires a specific co-repressor complex

The cell surface expression of the endothelial adhesion molecules, E-selectin and VCAM-1, plays a critical role in the recruitment of circulating leukocytes from the bloodstream to sites of inflammation in response to tissue injury and infection. Transcription of these genes, inactive in quiescent endothelial cells, is transiently induced by the inflammatory cytokines, TNFalpha and IL-1beta, through distinct signaling pathways that converge on the activation of the transcription factor, NF-kappaB. While the molecular mechanisms that control the cytokine-induced activation of NF-kappaB-dependent genes have been well-characterized, much less is known about the control mechanisms that mediate their selective repression. Although the role of IkappaB in preventing the nuclear localization of NF-kappaB has been described in detail as a model of repression for both E-selectin and VCAM-1, other mechanisms most likely exist for the active post-induction repression of these genes.; In Chapter II, I demonstrate through the employment of both transient transfection and single cell nuclear microinjection studies that TNFalpha-induced E-selectin and VCAM-1 gene expression is selectively repressed by the overexpression of the POU domain family transcription factor Oct-1, but not Oct-2, and that the mechanism of repression is DNA-independent. This regulation does not apply to all NF-kappaB target genes, as gene array studies indicate that Oct-1-mediated repression is promoter-specific. The results from these studies suggest that Oct-1 expression can be induced by the cytokine, IL-6. Experiments in Oct-1-deficient cells reveal that Oct-1 may also act constitutively in controlling the basal expression levels of certain genes.; In Chapter III, I show that Oct-1-mediated repression of TNFalpha-induced E-selectin and VCAM-1 gene expression depends on the engagement of a specific co-repressor complex as determined by both biochemical studies and single cell nuclear microinjection assays. Interestingly, Oct-1 does not repress IL-1-induced E-selectin and VCAM-1 gene expression, which is attributable to a nuclear translocation mechanism involving N-CoR.; In Chapter IV, I examine the effects of the transcription factor PPARgamma on TNFalpha-induced VCAM-1 expression in adipocytes. Biochemical and functional studies, which included macrophage binding assays, revealed that agonist bound PPARgamma downregulates VCAM-1 expression through interaction with an N-CoR/HDAC-containing co-repressor complex.