Tucker Collins

Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers.

Transcription of endothelial‐leukocyte adhesion molecule‐1 (E‐selectin or ELAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and intercellular adhesion molecule‐l (ICAM‐1) is induced by the inflammatory cytokines interleukin‐1 β (IL‐lβ) and tumor necrosis faetor‐α (TNFα). 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‐κB (NF‐κB) and a small group of other transcriptional activators. The organization of the cytokine‐inducible element in the E‐se‐ lectin promoter is remarkably similar to that of the virus‐indueible promoter of the human interferon‐β gene in that both promoters require NF‐κB, 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 inter‐ feron‐β 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 adhe‐sion molecules.—Collins, T., Read, M. A., Neisli, A. S., Whitley, M. Z., Thanos, D., Maniatis, T. Transcriptional regulation of endothelial cell adhesion molecules: NF‐κB and cytokine‐indueible enhancers. FASEBJ. 9, 899‐909 (1995)

Novel Inhibitors of Cytokine-induced IκBα Phosphorylation and Endothelial Cell Adhesion Molecule Expression Show Anti-inflammatory Effects in Vivo

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-α-induced phosphorylation of IκB-α, resulting in decreased nuclear factor-κB and decreased expression of adhesion molecules. The effects on both IκB-α phosphorylation and surface expression of E-selectin were irreversible and occurred at an IC50 of approximately 10 μm. These agents selectively and irreversibly inhibited the tumor necrosis factor-α-inducible phosphorylation of IκB-α without affecting the constitutive IκB-α 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 IκBα phosphorylation exert anti-inflammatory activity in vivo.

NF-κB: pivotal mediator or innocent bystander in atherogenesis?

Atherosclerosis and its clinical manifestations of heart attack, stroke, and peripheral vascular insufficiency are a major cause of morbidity and mortality among both men and women. Multiple risk factors including hypertension, diabetes mellitus, smoking, and lipoprotein disorders are involved in the pathogenesis of this chronic inflammatory disease of arteries. The development of early atherosclerotic lesions can be subdivided into initiation (formation of small fatty streaks), expansion (vertical and lateral growth and coalescence of fatty streaks), and progression to plaques (intimal smooth muscle cell recruitment, collagen deposition, and formation of a fibrous cap) (reviewed in ref.1). During the initiation and expansion of fatty streaks, circulating monocytes are recruited to the arterial intima where they are transformed into lipid-engorged macrophage foam cells. The arterial endothelium in these regions is activated and expresses inducible leukocyte adhesion molecules and chemokines. Production of cytokines and growth factors within lesions may amplify monocyte recruitment, stimulate macrophage proliferation, and induce migration of smooth muscle cells from the media to the intima of the vessel. Intimal smooth muscle cells deposit collagen and other ECM proteins, leading to the formation of a fibrous cap. Although clinically significant complications of atherosclerosis, such as plaque ulceration, rupture, and thrombosis, occur in established or advanced atherosclerotic plaques, understanding the mechanisms of early lesion formation offers the hope of intervening to delay or prevent lesion progression and complications. A select set of transcription factors may be critical in both the initiation and expansion of lesions, as well as in protecting the vessel wall from the formation of atherosclerotic lesions. In this overview, we will focus on one transcription factor, NF-κB, whose activation has been linked to the onset of atherosclerosis. NF-κB is composed of members of the Rel family that share a 300 amino acid region, known as the Rel homology domain, which mediates dimerization, nuclear translocation, DNA binding, and interaction with NF-κB inhibitors (reviewed in ref.2). Activation of NF-κB is controlled by a family of inhibitors, or IκBs, that bind to NF-κB dimers and mask the nuclear localization sequence of NF-κB, thus retaining the entire complex in the cytoplasm. Diverse stimuli activate NF-κB, through the phosphorylation and activation of the IκB kinase (IKK) complex. This complex consists of IKK-α and IKK-β heterodimers, a number of IKK-γ subunits, and possibly other components that have less certain significance. The activated IKK complex specifically phosphorylates the IκBs, which are then rapidly polyubiquitinated, targeting them for degradation by the proteosome. Following release from the inhibitor, NF-κB dimers translocate from the cytoplasm to the nucleus, where they bind target genes and stimulate transcription (Figure ​(Figure1).1). NF-κB activates a variety of target genes relevant to the pathophysiology of the vessel wall, including cytokines, chemokines, and leukocyte adhesion molecules, as well as genes that regulate cell proliferation and mediate cell survival. NF-κB also activates the IκBα gene, thus replenishing the cytoplasmic pool of its own inhibitor. Restored expression of IκB-α decreases NF-κB activation and diminishes expression of NF-κB–dependent genes. The NF-κB/IκB-α autoregulatory system ensures that the induction of NF-κB is transient and that the activated cell returns to a quiescent state. Physiological modulation and pathological activation of the NF-κB system may contribute to the changes in gene expression that occur during atherogenesis. Figure 1 Schematic representation of NF-κB as an integrator in atherogenesis. Many of the diverse agents associated with the onset of lesion formation interact with specific receptors. Angiotensin II (Ang II), cytokines, advanced glycosylation end products ...

PPARα Activators Inhibit Cytokine-Induced Vascular Cell Adhesion Molecule-1 Expression in Human Endothelial Cells

Background—Adhesion molecule expression on the endothelial cell (EC) surface is critical for leukocyte recruitment to atherosclerotic lesions. Better understanding of transcriptional regulation of adhesion molecules in ECs may provide important insight into plaque formation. Peroxisome proliferator–activated receptor-α (PPARα), a member of the nuclear receptor family, regulates gene expression in response to certain fatty acids and fibric acid derivatives. The present study investigated PPARα expression in human ECs and their regulation of vascular cell adhesion molecule-1 (VCAM-1). Methods and Results—Immunohistochemistry revealed that human carotid artery ECs express PPARα. Pretreatment of cultured human ECs with the PPARα activators fenofibrate or WY14643 inhibited TNF-α–induced VCAM-1 in a time- and concentration-dependent manner, an effect not seen with PPARγ activators. Both PPARα activators decreased cytokine-induced VCAM-1 mRNA expression without altering its mRNA half-life. Transient transfection...

Egr-1-Induced Endothelial Gene Expression: A Common Theme in Vascular Injury

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.

Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stress-responsive element.

The endothelial lining of blood vessels is constantly exposed to fluid mechanical forces generated by flowing blood. In vitro application of fluid shear stresses to cultured endothelial cells influences the expression of multiple genes, as reflected by changes in their steady-state mRNA levels. We have utilized the B chain of platelet-derived growth factor (PDGF-B) as a model to investigate the mechanisms of shear-stress-induced gene regulation in cultured bovine aortic endothelial cells (BAECs). Northern blot analysis revealed elevated endogenous PDGF-B transcript levels in BAECs, after exposure to a physiological level of laminar shear stress (10 dynes/cm2; 1 dyne = 100 mN) for 4 h. A transfected reporter gene, consisting of a 1.3-kb fragment of the human PDGF-B promoter coupled to chloramphenicol acetyltransferase (CAT), indicated a direct effect on transcriptional activity. Transfection of a series of PDGF-B-CAT deletion mutants led to the characterization of a cis-acting component within the PDGF-B promoter that was necessary for shear-stress responsiveness. In gel-shift assays, overlapping oligonucleotide probes of this region formed several protein-DNA complexes with nuclear extracts prepared from both static and shear-stressed BAECs. A 12-bp component (CTCTCAGAGACC) was identified that formed a distinct pattern of complexes with nuclear proteins extracted from shear-stressed BAECs. This shear-stress-responsive element does not encode binding sites for any known transcription factor but does contain a core binding sequence (GAGACC), as defined by deletion mutation in gel-shift assays. Interestingly, this putative transcription factor binding site is also present in the promoters of certain other endothelial genes, including tissue plasminogen activator, intercellular adhesion molecule 1, and transforming growth factor beta 1, that also are induced by shear stress. Thus, the expression of PDGF-B and other pathophysiologically relevant genes in vascular endothelium appears to be regulated, in part, by shear-stress-induced transcription factors interacting with a common promoter element.

Transcriptional Activation by NF-κB Requires Multiple Coactivators

ABSTRACT Nuclear factor-κB (NF-κB) plays a role in the transcriptional regulation of genes involved in inflammation and cell survival. In this report we demonstrate that NF-κB recruits a coactivator complex that has striking similarities to that recruited by nuclear receptors. Inactivation of either cyclic AMP response element binding protein (CREB)-binding protein (CBP), members of the p160 family of coactivators, or the CBP-associated factor (p/CAF) by nuclear antibody microinjection prevents NF-κB-dependent transactivation. Like nuclear receptor-dependent gene expression, NF-κB-dependent gene expression requires specific LXXLL motifs in one of the p160 family members, and enhancement of NF-κB activity requires the histone acetyltransferase (HAT) activity of p/CAF but not that of CBP. This coactivator complex is differentially recruited by members of the Rel family. The p50 homodimer fails to recruit coactivators, although the p50-p65 heterodimeric form of the transcription factor assembles the integrator complex. These findings provide new mechanistic insights into how this family of dimeric transcription factors has a differential effect on gene expression.

The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9.

The t(7;11)(p15;p15) translocation is a recurrent chromosomal abnormality associated primarily with acute myeloid leukaemia (FAB M2 and M4). We present here the molecular definition of this translocation. On chromosome 7 positional cloning revealed the consistent rearrangement of the HOXA9 gene, which encodes a class I homeodomain protein potentially involved in myeloid differentiation. On chromosome 11 the translocation targets the human homologue of NUPP98, a member of the GLFG nucleoporin family. Chimaeric messages spliced over the breakpoint fuse the GLFG repeat domains of NUP98 in-frame to the HOXA9 homeobox. The predicted NUP98–HOXA9 fusion protein may promote leukaemogenesis through inhibition of HOXA9-mediated terminal differentiation and/or aberrant nucleocytoplasmic transport.

Recruitment of CBP/p300 by the IFNβ Enhanceosome Is Required for Synergistic Activation of Transcription

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.

The proteasome pathway is required for cytokine-induced endothelial-leukocyte adhesion molecule expression.

Multiple cell adhesion proteins are up-regulated in vascular endothelial cells in response to TNF alpha and other inflammatory cytokines. This increase in cell adhesion gene expression is thought to require the transcription factor NF-kappa B. Here, we show that peptide aldehyde inhibitors of the proteasome, a multicatalytic protease recently shown to be required for the activation of NF-kappa B, block TNF alpha induction of the leukocyte adhesion molecules E-selectin, VCAM-1, and ICAM-1. Striking functional consequences of this inhibition were observed in analyses of leukocyte-endothelial interactions under defined flow conditions. Lymphocyte attachment to TNF alpha-treated endothelial monolayers was totally blocked, while neutrophil attachment was partially reduced but transmigration was essentially prevented.