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.

Regulation of NF-B occurs through phosphorylation-dependent ubiquitination of IB␣, which is degradedNF-B is a transcription factor required for inducible expression of a number of proinflammatory mediators including cytokines, chemokines, and leukocyte adhesion molecules (6). In addition, NF-B regulates the expression of survival genes which prevent cell death in response to tumor necrosis factor alpha (TNF-␣) (7,37,59,62). NF-B is a member of the Rel family of proteins and is typically a heterodimer composed of p50 and p65 subunits. In quiescent cells, NF-B is retained in the cytosol bound to IB, a family of inhibitory proteins which mask the nuclear localization and DNA binding sequences on NF-B (5, 22). Stimulation of these cells with various cytokines, lipopolysaccharide, viruses, antigens, or oxidants triggers signaling events that ultimately lead to the phosphorylation and degradation of IB, allowing NF-B to translocate into the nucleus and activate target genes (3,21,38,54).Phosphorylation of Ser 32 and Ser 36 has been shown to target IB for ubiquitination and subsequent proteolysis by the ubiquitin-proteasome pathway (UPP) of protein degradation (2,8,45,49). The UPP is the principal pathway for intracellular protein turnover, including regulatory proteins (9). Protein substrates that enter the UPP are first marked by the covalent ligation of polyubiquitin chains mediated by a cascade of enzymes called E1 (ubiquitin activation enzyme), E2 (ubiquitinconjugating enzyme), and E3 (ubiquitin ligase) (9). In a reaction requiring ATP, ubiquitin is activated by E1 and charged onto an E2 through a thioester formed between the active-site cysteine residue in the E2 and the C-terminal glycine of ubiquitin. The E3 then directs the transfer of ubiquitin from the E2 onto lysine residues within specific substrate proteins, ultimately resulting in the formation of a ubiquitin-protein conjugate. Polyubiquitinated proteins are then recognized and degraded by the 26S proteasome complex to yield small peptides and monomeric ubiquitin.Recently, the receptor component of the IB E3 was identified as a member of the ␤TrCP (beta-transducin repeatcontaining protein) family of proteins called E3RS

SummaryStructural analysis of the promoters of several endothelial genes induced at sites of inflammatory or immune responses reveals binding sites for the transcription factor nuclear factor KB (NF-KB). Endothelial cells express transcripts encoding the pS0/p105 and p65 components of NF-IcB and the tel-related proto-oncogene c-re/; steady state levels of these transcripts are transiently increased by tumor necrosis factor ce (TNF-ce). Western blotting revealed that stimulation of endothelial calls with TNF-ce resulted in nuclear accumulation of the p50 and p65 components of NF-KB. Ultraviolet crosslinking and immunoprecipitation demonstrated binding of the p50 and p65 components of NF-KB to the E-selectin xB site. Endothelial cells express an inhibitor of NF-gB activation, IgB-ce (MAD-3). Protein levels of this inhibitor fall rapidly after TNF-cx stimulation. In parallel, p50 and p65 accumulate in the nucleus and RNA transcript levels for IgB-o~ are dramatically upreguhted. Recombinant p65 stimulates expression of E-sdectin promoter-reporter constructs. IKB-ce inhibits p65 or TNF-ce-stimulated E-sdectin promoter-reporter gene expression in transfected endothelial cells. The NF-gB and IKB-ce system may be an inducible regulatory mechanism in endothelial activation.

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