Transcription of endothelial-leukocyte adhesion molecule-1 (E-selectin or ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) is induced by the inflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha). The positive regulatory domains required for maximal levels of cytokine induction have been defined in the promoters of all three genes. DNA binding studies reveal a requirement for nuclear factor-kappa B (NF-kappa B) and a small group of other transcriptional activators. The organization of the cytokine-inducible element in the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human interferon-beta gene in that both promoters require NF-kappa B, activating transcription factor-2 (ATF-2), and high mobility group protein I(Y) for induction. Based on this structural similarity, a model has been proposed for the cytokine-induced E-selectin enhancer that is similar to the stereospecific complex proposed for the interferon-beta gene promoter. In these models, multiple DNA bending proteins facilitate the assembly of higher order complexes of transcriptional activators that interact as a unit with the basal transcriptional machinery. The assembly of unique enhancer complexes from similar sets of transcriptional factors may provide the specificity required to regulate complex patterns of gene expression and correlate with the distinct patterns of expression of the leukocyte adhesion molecules.

We have identified two compounds that inhibit the expression of endothelial-leukocyte adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. These compounds act by inhibiting tumor necrosis factor-␣-induced phosphorylation of IB-␣, resulting in decreased nuclear factor-B and decreased expression of adhesion molecules. The effects on both IB-␣ phosphorylation and surface expression of E-selectin were irreversible and occurred at an IC 50 of approximately 10 M. These agents selectively and irreversibly inhibited the tumor necrosis factor-␣-inducible phosphorylation of IB-␣ without affecting the constitutive IB-␣ phosphorylation. Although these compounds exhibited other activities, including stimulation of the stress-activated protein kinases, p38 and JNK-1, and activation of tyrosine phosphorylation of a 130 -140-kDa protein, these effects are probably distinct from the effects on adhesion molecule expression since they were reversible. One compound was evaluated in vivo and shown to be a potent anti-inflammatory drug in two animal models of inflammation. The compound reduced edema formation in a dose-dependent manner in the rat carrageenan paw edema assay and reduced paw swelling in a rat adjuvant arthritis model. These studies suggest that inhibitors of cytokine-inducible IB␣ phosphorylation exert anti-inflammatory activity in vivo.The adhesion of circulating leukocytes to vascular endothelium is critical to inflammatory responses (reviewed in Refs. 1-3). Interaction of the selectin family of adhesion proteins and lectin counter-receptors is the predominant mechanism mediating initial adhesion between leukocytes and the vessel wall. The expression of endothelial-leukocyte adhesion molecule-1 (E-selectin, CD62E), vascular cell adhesion molecule-1 (VCAM-1, 1 CD106), and intercellular adhesion molecule-1 (ICAM-1, CD54) on the surface of endothelial cells is elevated at sites of inflammation (2, 4). Induction of these molecules by tumor necrosis factor-␣ (TNF␣) and other inflammatory cytokines is regulated at the level of gene transcription and requires binding of the transcription factor nuclear factor-B (NF-B) to the regulatory regions within the promoters of each of these genes (5-12).The NF-B/Rel transcription factor family plays an important role in cytokine-induced gene activation (13-15). The Rel family includes p50 (NFKB1), p52 (NFKB2), p65 (RelA), RelB, v-Rel, and c-Rel. In endothelial cells, the p50⅐p65 heterodimer is the predominant species that binds to B consensus sequences in the VCAM-1, ICAM-1, and E-selectin genes and activates gene transcription. NF-B is located in the cytoplasm of cells in an inactive form in association with the inhibitor IB-␣. In response to TNF␣ stimulation, IB-␣ is phosphorylated on 2 serine residues (Ser-32 and Ser-36), ubiquitinated, and degraded by a proteosome-dependent pathway allowing active NF-B to translocate to the nucleus where it can activate gene expression (16 -23). Many NF-B-dependent genes including the adhesion m...

CBP (CREB-binding protein) and p300 are versatile coactivators that link transcriptional activators to the basal transcriptional apparatus. In the present study, we identify CBP and p300 as coactivators of the nuclear factor-B (NF-B) component p65 (RelA). Consistent with their role as coactivators, both CBP and p300 potentiated p65-activated transcription of E-selectin and VCAM-1-CAT reporter constructs. The N-and C-terminal domains of both CBP͞p300 functionally interact with a region of p65 containing the transcriptional activation domain as demonstrated by mammalian two-hybrid assays. Direct physical interactions of CBP͞p300 with p65 were demonstrated by glutathione Stransferase fusion protein binding, and coimmunoprecipitation͞Western blot studies. The adenovirus E1A 12S protein, which complexes with CBP and p300, inhibited p65-dependent gene expression. Reporter gene expression could be rescued from E1A inhibition by overexpression of CBP or p300. CBP and p300 act as coactivators of p65-driven gene activation and may play an important role in the cytokine-induced expression of various immune and inf lammatory genes.

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Atherosclerotic lesions form at distinct sites in the arterial tree, suggesting that hemodynamic forces influence the initiation of atherogenesis. If NF-B plays a role in atherogenesis, then the activation of this signal transduction pathway in arterial endothelium should show topographic variation. The expression of NF-B͞ IB components and NF-B activation was evaluated by specific antibody staining, en face confocal microscopy, and image analysis of endothelium in regions of mouse proximal aorta with high and low probability (HP and LP) for atherosclerotic lesion development. In control C57BL͞6 mice, expression levels of p65, IB␣, and IB␤ were 5-to 18-fold higher in the HP region, yet NF-B was activated in a minority of endothelial cells. This suggested that NF-B signal transduction was primed for activation in HP regions on encountering an activation stimulus. Lipopolysaccharide treatment or feeding low-density lipoprotein receptor knockout mice an atherogenic diet resulted in NF-B activation and up-regulated expression of NF-B-inducible genes predominantly in HP region endothelium. Preferential regional activation of endothelial NF-B by systemic stimuli, including hypercholesterolemia, may contribute to the localization of atherosclerotic lesions at sites with high steadystate expression levels of NF-B͞IB components.atherosclerosis ͉ p65 ͉ IB␣ ͉ VCAM-1 ͉ E-selectin

A number of pathophysiologically relevant genes, including platelet-derived growth factor B-chain (PDGF-B), are induced in the vasculature after acute mechanical injury. In rat aorta, the activated expression of these genes was preceded by a marked increase in the amount of the early-growth-response gene product Egr-1 at the endothelial wound edge. Egr-1 interacts with a novel element in the proximal PDGF-B promoter, as well as with consensus elements in the promoters of other genes induced by endothelial injury. This interaction is crucial for injury-induced PDGF-B promoter-dependent expression. Sp1, whose binding site in the PDGF-B promoter overlaps that of Egr-1, occupies this element in unstimulated cells and is displaced by increasing amounts of Egr-1. These findings implicate Egr-1 in the up-regulated expression of PDGF-B and other potent mediators in mechanically injured arterial endothelial cells.

Human ECs express PPARalpha, a potentially important regulator of atherogenesis through its transcriptional control of VCAM-1 gene expression. Such findings also have implications regarding the clinical use of lipid-lowering agents, like fibric acids, which can activate PPARalpha.

Transcriptional activation of the IFN beta gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-kappa B, IRF1, ATF2/c-Jun, and the architectural protein HMG I(Y). The level of transcription generated by all of these activators is greater than the sum of the levels generated by individual factors, a phenomenon designated transcriptional synergy. We demonstrate that this synergy, in the context of the enhanceosome, requires a new protein-protein interaction domain in the p65 subunit of NF-kappa B. Transcriptional synergy requires recruitment of the CBP/p300 coactivator to the enhanceosome, via a new activating surface assembled from the novel p65 domain and the activation domains of all of the activators. Deletion, substitution, or rearrangement of any one of the activation domains in the context of the enhanceosome decreases both recruitment of CBP and transcriptional synergy.