Faculty Bibliography

Receptor for advanced glycation end products (RAGE) is a multiligand member of the immunoglobulin superfamily of cell surface molecules whose repertoire of ligands includes advanced glycation end products (AGEs), amyloid fibrils, amphoterins and S100/calgranulins. The overlapping distribution of these ligands and cells overexpressing RAGE results in sustained receptor expression which is magnified via the apparent capacity of ligands to upregulate the receptor. We hypothesize that RAGE-ligand interaction is a propagation factor in a range of chronic disorders, based on the enhanced accumulation of the ligands in diseased tissues. For example, increased levels of AGEs in diabetes and renal insufficiency, amyloid fibrils in Alzheimer's disease brain, amphoterin in tumors and S100/calgranulins at sites of inflammation have been identified. The engagement of RAGE by its ligands can be considered the 'first hit' in a two-stage model, in which the second phase of cellular perturbation is mediated by superimposed accumulation of modified lipoproteins (in atherosclerosis), invading bacterial pathogens, ischemic stress and other factors. Taken together, these 'two hits' eventuate in a cellular response with a propensity towards tissue destruction rather than resolution of the offending pathogenic stimulus. Experimental data are cited regarding this hypothesis, though further studies will be required, especially with selective low molecular weight inhibitors of RAGE and RAGE knockout mice, to obtain additional proof in support of our concept

Activation of the zinc-finger transcription factor early growth response (Egr)-1, initially linked to developmental processes, is shown here to function as a master switch activated by ischemia to trigger expression of pivotal regulators of inflammation, coagulation and vascular hyperpermeability. Chemokine, adhesion receptor, procoagulant and permeability-related genes are coordinately upregulated by rapid ischemia-mediated activation of Egr-1. Deletion of the gene encoding Egr-1 strikingly diminished expression of these mediators of vascular injury in a murine model of lung ischemia/reperfusion, and enhanced animal survival and organ function. Rapid activation of Egr-1 in response to oxygen deprivation primes the vasculature for dysfunction manifest during reperfusion. These studies define a central and unifying role for Egr-1 activation in the pathogenesis of ischemic tissue damage

The transcription factor early growth response (Egr)-1 is an immediate-early gene product rapidly and transiently expressed after acute tissue injury. In contrast, in this report we demonstrate that lung tissue from patients undergoing lung reduction surgery for advanced emphysema, without clinical or anatomical evidence of acute infection, displays a selective and apparently sustained increase in Egr-1 transcripts and antigen, compared with a broad survey of other genes, including the transcription factor Sp1, whose levels were not significantly altered. Enhanced Egr-1 expression was especially evident in smooth muscle cells of bronchial and vascular walls, in alveolar macrophages, and some vascular endothelium. Gel shift analysis with (32)P-labeled Egr probe showed a band with nuclear extracts from emphysematous lung which was supershifted with antibody to Egr-1. Egr-1 has the capacity to regulate genes relevant to the pathophysiology of emphysema, namely those related to extracellular matrix formation and remodeling, thrombogenesis, and those encoding cytokines/chemokines and growth factors. Thus, we propose that further analysis of Egr-1, which appears to be up-regulated in a sustained fashion in patients with late stage emphysema, may provide insights into the pathogenesis of this destructive pulmonary disease, as well as a new facet in the biology of Egr-1

Hypoxia induces complex adaptive responses. In this report, induction of early growth response-1 (Egr-1) transcripts in lungs of mice subjected to hypoxia is shown to be dose and time dependent. Within 30 min of hypoxia, Egr-1 transcripts were approximately 20-fold elevated in 6% oxygen, approximately 5.2-fold increased by 10% oxygen, and returned to the normoxic baseline by 12% oxygen. Time course studies up to 48 h showed a biphasic profile with an initial steep rise in Egr-1 transcripts after 0.5 h of hypoxia and a second elevation beginning after 20-24 h. Hypoxic induction of Egr-1 was paralleled by enhanced expression of the downstream target gene tissue factor. Egr-1 and tissue factor antigen were visualized in bronchial and vascular smooth muscle and in alveolar macrophages. Egr-1 has the capacity to modulate expression of genes involved in the remodeling of the extracellular matrix and properties of smooth muscle, thus possibly contributing to the pulmonary response to chronic hypoxia

Fibrin deposition is a salient feature of hypoxemic vasculature and results from induction of tissue factor. Such tissue factor expression in an oxygen deficient environment is driven by the transcription factor Early Growth Response (Egr)-1. Using homozygous null mice for the protein kinase C beta-isoform gene (PKCbeta null), PKCbeta is shown to be upstream of Egr-1 in this oxygen deprivation-mediated pathway for triggering procoagulant events. Whereas wild-type mice exposed to hypoxia (6%) displayed a robust increase in tissue factor transcripts and antigen, and vascular fibrin deposition, PKCbeta null animals showed a markedly blunted response. Consistent with a central role for Egr-1 in hypoxia-induced expression of tissue factor, PKCbeta null mice subjected to oxygen deprivation displayed at most a minor elevation in Egr-1 transcripts, antigen, and intensity of the gel shift band by electrophoretic mobility shift assay, compared with normoxic animals. These data firmly establish PKCbeta as a trigger for events leading to induction of Egr-1 and tissue factor under hypoxic conditions, and provide insight into a biologic cascade whereby oxygen deprivation recruits targets of PKCbeta and Egr-1, thereby amplifying the cellular response

Hypoxemia has long been associated with vascular fibrin formation leading to thrombosis. This review describes a pathway through which mononuclear phagocytes and vascular smooth muscle cells upregulate tissue factor under hypoxic conditions. Increased expression of tissue factor triggers events leading to vascular fibrin deposition, providing insight into a novel mechanism potentially underlying thrombosis in ischemic vasculature

Recent studies suggested that interruption of the interaction of advanced glycation end products (AGEs), with the signal-transducing receptor receptor for AGE (RAGE), by administration of the soluble, extracellular ligand-binding domain of RAGE, reversed vascular hyperpermeability and suppressed accelerated atherosclerosis in diabetic rodents. Since the precise molecular target of soluble RAGE in those settings was not elucidated, we tested the hypothesis that predominant specific AGEs within the tissues in disorders such as diabetes and renal failure, N(epsilon)-(carboxymethyl)lysine (CML) adducts, are ligands of RAGE. We demonstrate here that physiologically relevant CML modifications of proteins engage cellular RAGE, thereby activating key cell signaling pathways such as NF-kappaB and modulating gene expression. Thus, CML-RAGE interaction triggers processes intimately linked to accelerated vascular and inflammatory complications that typify disorders in which inflammation is an established component

Although oxygen deprivation has long been associated with triggering of the procoagulant pathway and venous thrombosis, blood hypoxemia and stasis by themselves do not lead to fibrin formation. A pathway is outlined through which diminished levels of oxygen activate the transcription factor early growth response-1 (Egr-1) leading to de novo transcription/translation of tissue factor in mononuclear phagocytes and smooth muscle cells, which eventuates in vascular fibrin deposition. The procoagulant response is magnified by concomitant suppression of fibrinolysis by hypoxia-mediated upregulation of plasminogen activator inhibitor-1. These data add a new facet to the biology of thrombosis associated with hypoxemia/stasis and imply that interference with mechanisms causing Egr-1 activation in response to oxygen deprivation might prevent vascular fibrin deposition occurring in ischemia without directly interfering with other pro/anticoagulant pathways

The paradigm for the response to hypoxia is erythropoietin gene expression; activation of hypoxia-inducible factor-1 (HIF-1) results in erythropoietin production. Previously, we found that oxygen deprivation induced tissue factor, especially in mononuclear phagocytes, by an early growth response (Egr-1)-dependent pathway without involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C., Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8298-8303). Now, we show that cultured monocytes subjected to hypoxia (pO2 approximately 12 torr) displayed increased Egr-1 expression because of de novo biosynthesis, with a approximately 10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase constructs localized elements responsible for hypoxia-enhanced expression to -424/-65, a region including EBS (ets binding site)-SRE (serum response element)-EBS and SRE-EBS-SRE sites. Further studies with each of these regions ligated to the basal thymidine kinase promoter and luciferase demonstrated that EBS sites in the element spanning -424/-375 were critical for hypoxia-enhanceable gene expression. These data suggested that an activated ets factor, such as Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced activation of Elk-1, and suppression of Elk-1 blocked up-regulation of Egr-1 transcription. The signaling cascade preceding Elk-1 activation in response to oxygen deprivation was traced to activation of protein kinase C-betaII, Raf, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase and mitogen-activated protein kinases. Comparable hypoxia-mediated Egr-1 induction and activation were observed in cultured hepatoma-derived cells deficient in HIF-1beta and wild-type hepatoma cells, indicating that the HIF-1 and Egr-1 pathways are initiated independently in response to oxygen deprivation. We propose that activation of Egr-1 in response to hypoxia induces a different facet of the adaptive response than HIF-1, one component of which causes expression of tissue factor, resulting in fibrin deposition