“TF-kB”-Dependent Suppression of Agonist-Mediated Tissue Factor Induction by Histone Deacetylase Inhibitors in Human Endothelial Cells and Monocytes.

Histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) and Sodium Butyrate (NaB) can regulate gene expression by acetylating histones and transcription factors. HDACi attenuate expression of certain NF-kB-regulated cytokines, including TNF-α, IL-1β, IL-6, and IFN-γ in some inflammatory disease models. Human tissue fatctor (TF) expression is, in part, governed by a unique, NF-kB-related “TF-kB” promoter binding site. We hypothesized that HDACi would inhibit induction of TF expression by physiologic agonists. We assessed the effect of TSA and NaB upon TF expression in human umbilical vein endothelial cells (HUVEC) and peripheral blood mononuclear cells (PBM) in response to 1 ng/ml TNF-α, 1 ng/ml IL-1β, 1 μg/ml LPS, and 100 μM HOSCN, a potent phagocyte peroxidase-derived oxidant. HUVEC monlayers and PBM were pretreated with TSA (0.01–1 μM) or NaB (1–10 mM) for 4 h prior to agonist addition and 4 h later whole cell lysates were assayed for TF activity by 1-stage clotting assay and western blots probed for TF. Both TSA and NaB inhibit HUVEC TF activity and protein expression up to 90% with respective IC50’s of 30–50 nM and 2 mM. TSA decreases PBM TF activity induced by LPS and IL-1β by 50–65% with similar potency. In striking contrast, western blots show no effect of TSA on agonist-induced expression of the NF-kB-regulated adhesion molecules ICAM-1 and E-selectin. RT-PCR analysis shows TNF-α stimulation induces a 22-fold increase of TF mRNA that is attenuated up to 70% by TSA treatment without affecting the 10-fold ICAM-1 mRNA increase. TSA did not influence TF translational efficiency as detected by polyribosomal TF mRNA distribution and TSA TF inhibition was unaffected by the ubiquitin-dependent protein degradation inhibitor MG-132. EMSA analysis of HUVEC nuclear extracts using probes for the SP1, AP-1, Egr-1 and TF-kB transcription factor binding sites in the TF gene promoter shows that SP1, AP-1 and Egr-1 are constitutively activated in HUVEC and are unaffected by LPS or TNF-α irrespective of whether TSA is present. However, both TNF-α and LPS induce abundant p65/p50 and p65/c-Rel heterodimer binding to both the NF-kB and TF-kB probes. 0.3 μM TSA nearly completely abolishes TF-kB activation without affecting NF-kB activation. TSA has no effect on TNF-α-induced cytoplasmic IkB-α degradation or nuclear translocation of c-Rel, p65, or p50. Chromatin immunoprecipitation assay shows that TSA strongly inhibits p65 binding to the authentic endogenous HUVEC TF-kB site in the TF promoter induced by TNF-α without affecting p65 binding to the ICAM-1 NF-kB site. A HUVEC promoter-luciferase reporter system confirms the complete specificity of TSA’s inhibitory effect on LPS- or TNF-α- induced TF expression for TF-kB rather than NF-kB. We conclude that HDACi transcriptionally inhibit agonist-induced TF expression in human endothelial cells and monocytes by specific blockade of TF-kB activation. We propose that histone deacetylases play a previously unsuspected role in regulating TF expression and HDACi might be a novel therapy for thrombotic diseases.