Regulation of endothelial thrombomodulin expression by inflammatory cytokines is mediated by activation of nuclear factor-kappa B

Abstract

Inflammation and thrombosis are increasingly recognized as interrelated biologic processes. Endothelial cell expression of thrombomodulin (TM), a key component of the anticoagulant protein C pathway, is potently inhibited by inflammatory cytokines. Because the mechanism underlying this effect is largely unknown, we investigated a potential role for the inflammatory transcription factor nuclear factor-kappa B (NF-B). Blocking NF-B activation effectively prevented cytokineinduced down-regulation of TM, both in vitro and in a mouse model of tumor necrosis factor-␣ (TNF-␣)-mediated lung injury. Although the TM promoter lacks a classic NF-B consensus site, it does contain tandem Ets transcription factor binding sites previously shown to be important for both constitutive TM gene expression and cytokine-induced repression. Using electrophoretic mobility shift assay and chromatin immunoprecipitation, we found that multiple Ets species bind to the TNF-␣ response element within the TM promoter. Although cytokine exposure did not alter Ets factor binding, it did reduce binding of p300, a coactivator required by Ets for full transcriptional activity. Overexpression of p300 also prevented TM repression by cytokines. We conclude that NF-B is a critical mediator of TM repression by cytokines. Further evidence suggests a mechanism involving competition by NF-B for limited pools of the transcriptional coactivator p300 necessary for TM gene expression. IntroductionSystemic inflammatory conditions, such as bacterial sepsis and vasculitis, are frequently complicated by the development of pathologic thrombosis. Inflammatory processes can shift the hemostatic balance toward thrombus formation not only by stimulating tissue factor-dependent coagulation but also by inhibiting anticoagulant and fibrinolytic pathways. 1 Thrombomodulin (TM), a 100-kDa transmembrane protein expressed in abundance by vascular endothelial cells, is a critical component of the anticoagulant protein C pathway. 2 TM binds thrombin and alters its active site specificity to facilitate proteolytic activation of circulating protein C. In concert with its cofactor protein S, activated protein C (APC) enzymatically degrades factors Va and VIIIa of the clotting cascade, thereby suppressing further thrombin generation. Growing clinical evidence suggests that dysfunction of the TM-APC pathway caused by inflammation compromises vascular thromboresistance. For example, TM expression is markedly reduced in skin biopsy specimens taken from patients with severe bacterial sepsis complicated by microvascular thrombosis and associated with abnormally low circulating levels of APC. 3 Impaired TM activity provides the rationale for administering recombinant APC to patients with severe sepsis, a therapy that has been shown to reduce the risk of death in such patients by nearly 20%. 4,5 In vitro studies have demonstrated that endothelial TM expression is potently inhibited by inflammatory mediators such as bacterial endotoxin and several inflammatory cytokines. [6][7][8] Of these...

Sirt1 protects against thrombomodulin down-regulation and lung coagulation after particulate matter exposure

“…31,32,44 TNF-␣ has been reported to down-regulate TM and KLF2 expression by activating NF-B. 49 An IL-6-mediated pathway has been demonstrated to play a critical role in PM-induced thrombotic events.…”

“…45,46 NF-B also physically interacts with p300, a transcriptional coactivator required for many transcription factors including those controlling TM expression. 31,32,49,50 NF-B has been reported to down-regulate TM expression indirectly by competing for the limited pool of p300 in the nucleus. 31,32,49,50 Acetylation has recently been demonstrated as a key mechanism for NF-B activation.…”

“…[27][28][29][30] It has been reported that Sirt1 deacetylates NF-B, thereby inhibiting its activity in several in vitro and in vivo systems. [27][28][29][30] NF-B activation has been reported to suppress TM expression, 31,32 implying that Sirt1 may be involved in protection against both inflammation and coagulation.…”

Thrombosis in Multiple Myeloma

“…Some authors have suggested an inhibition of platelet aggregation, 26 and others a prevention of the up-regulation of prothrombotic molecules such as thrombomodulin. 27 Further research is needed to explore the mechanisms for the treatment-related effects on the risk of VTE with these agents.…”

Peripheral Nerve Protein Expression and Carbonyl Content in N,N-Diethlydithiocarbamate Myelinopathy

“…The use of disulfiram for alcohol aversion therapy is probably the best recognized pharmaceutical application of a dithiocarbamate but additional new therapeutic applications of dithiocarbamates are currently being investigated for cancer (1), AIDS (2), inflammatory diseases, and atherosclerosis (3,4). In addition, dithiocarbamates have been proposed as biological tools to modulate gene transcription (5,6) and as antiviral agents (7)(8)(9). Delineating the hazards and mechanisms of dithiocarbamates is important for determining the risks associated with environmental exposures to dithiocarbamates and for developing safer and more efficacious dithiocarbamates for therapeutic purposes.…”