The Effects Of High Mobility Group Box 1 Protein (HMGB1) On The Expression Of Tissue Factor (TF) And Its Mechanisms

Tissue factor (TF), a 263 amino acid membrane glycoprotein, is one major stimulator of the blood coagulation cascade. Recent studies revealed that TF plays an important role in the development and progression of acute coronary syndromes. TF protein levels and activity in atherosclerotic plaques are much higher than that of in the normal arterial wall. Moreover, the levels of TF in atherectomy specimens from patients with unstable angina or myocardial infarction are higher than those in patients with stable angina. However, the underlying causes for elevated TF expression in atherosclerotic plaques are not fully understood.High-mobility group box 1 protein (HMGB1) is a non-histone chromatin-binding protein. In normal condition, HMGB1 participates in stabilizing the chromatin structure and functioning as transcription factor which facilitates gene transcription. Recent studies showed that HMGB1 can be released and function as a proinflammatory cytokine, which plays an important role in many inflammatory diseases and critical conditions, such as endotoxemia, sepsis. A recent study demonstrated that extracellular HMGB1 levels are elevated atherosclerotic plaques, but not in normal arteries. However, extracellular HMGB1 in atherosclerotic plaques can stimulate TF expression is unclear.In present studies, we first determined the effects of HMGB1 on the expression of TF and Tissue factor pathway inhibitor (TFPI); then we determined the potential receptors that mediate HMGB1 stimulating the expression of TF; further we explored the molecular mechanism underlying the HMGB1 stimulating the expression of TF. The main results are as follows:1. The effects of HMGB1 on the expression of TF and TFPI: HMGB1 stimulated endothelial cells to express TF protein in a concentration-and time-dependent manner, but did not alter the expression of TFPI, as demonstrated by western-blot. HMGB1 also stimulated TF mRNA expression in endothelial cells and macrophage, as demonstrated by Real-time PCR, indicating that HMGB1 upregulates TF expression in transcription lever. Consistently, HMGB1 stimulation can upregulate TF activity in endothelial cells and macrophage as demonstrated by TF activity assay. In addition, our data showed that Endotoxin-neutralizing agent-polymyxin B (PMB) inhibited LPS-induced TF expression, but did not affect HMGB1-induced TF expression, as demonstrated by Real-time PCR. This result indicated that HMGB1-induced TF expression is not due to contamination of LPS.2. HMGB1-induced TF expression is mediated by the activation of all three receptors:To evaluate the potential involvement of receptors of HMGB1 in HMGB1-induced TF expression, endothelial cells were pre-treated with TLR4-, TLR2-, RAGE-specific neutralizing antibodies. Pre-incubation with each of the three antibodies uniformly attenuated HMGB1-induced TF mRNA expression and TF surface activity in endothelial cells, as demonstrated by Real-time PCR and TF activity assay respectively, indicating that HMGB1-induced TF expression is mediated by the activation of all three receptors.3. The molecular mechanism underlying the HMGB1 stimulating the expression of TF:HMGB1 stimulation increased expression of Egr-1 and nuclear translocation of NF-kB (c-Rel/p65) in endothelial cells, as demonstrated by western-blot and Real-time PCR. In addition, to further investigate the role of NF-kB and Egr-1 in the regulation of TF expression, we constructed plasmids containing the TF promoter region linked to a luciferase reporter. The reporter constructs contained either wild-type or mutated NF-kB and Egr-1 binding sites. Our data showed that HMGB1 stimulation can enhance the activity of wild-type TF gene promoter, whereas mutation of the NF-kB and Egr-1 binding sites inhibits HMGB1-mediated induction of reporter activity, supporting an important role for NF-B (c-Rel/p65) and Egr-1 in the regulation of HMGB1-mediated TF expression.In conclusion, our data suggest that HMGB1 induces TF expression in vascular endothelial cells via cell surface receptors (TLR4, TLR2, and RAGE), and through activation of transcription factors (NF-κB and Egr-1).