thromboplastin and prolinedithiocarbamate

Our previous data demonstrated that nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are involved in the process of anti-β2GPI/β2GPI-induced tissue factor (TF) expression in monocytes. However, the role of NF-κB and AP-1 in pathogenic mechanisms of antiphospholipid syndrome (APS) in vivo has been rarely studied. This study aimed to investigate whether NF-κB and c-Jun/AP-1 are involved in anti-β2GPI-induced expression of prothrombotic and proinflammatory molecules in mouse. IgG-APS or anti-β2GPI antibodies were injected into BALB/c mice in the presence or absence of PDTC (a specific inhibitor of NF-κB) and Curcumin (a potent inhibitor of AP-1) treatment. Our data showed that both IgG-APS and anti-β2GPI could induce the activation of NF-κB and c-Jun/AP-1 in mouse peritoneal macrophages. The anti-β2GPI-induced TF activity in homogenates of carotid arteries and peritoneal macrophages from mice could significantly decrease after PDTC and/or Curcumin treatment, in which PDTC showed the strongest inhibitory effect, but combination of two inhibitors had no synergistic effect. Furthermore, anti-β2GPI-induced expression of TF, VCAM-1, ICAM-1 and E-selectin in the aorta and expression of TF, IL-1β, IL-6 and TNF-α in peritoneal macrophages of mice were also significantly attenuated by PDTC and/or Curcumin treatment. These results indicate that both NF-κB and c-Jun/AP-1 are involved in regulating anti-β2GPI-induced expression of prothrombotic and proinflammatory molecules in vivo. Inhibition of NF-κB and c-Jun/AP-1 pathways may be beneficial for the prevention and treatment of thrombosis and inflammation in patients with APS.

Topics: Animals; Antibodies; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Curcumin; E-Selectin; Immunoglobulin G; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-1beta; Interleukin-6; Macrophages, Peritoneal; Male; Mice, Inbred BALB C; NF-kappa B; Phosphorylation; Proline; Real-Time Polymerase Chain Reaction; Thiocarbamates; Thromboplastin; Thrombosis; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

To explore the roles of NF-κB in factor VIIa-induced proliferation and migration of a colon cancer cell line (SW620) in vitro and its possible mechanism.. The expression levels of NF-κB (p65), inhibitory protein of NF-κB (IκB-α), caspase-7, interleukin 8 (IL-8) and tissue factor (TF) in SW620 cells treated with factor VIIa, PDTC (an inhibitor of NF-κB) and other factors were measured by Western-blotting and real-time PCR. Proliferation and migration of the cells were analyzed by flow cytometry and Transwell assay, respectively.. Factor VIIa down-regulated the IκB-α level in SW620 cells and increased the intranuclear level of NF-κB. Those effects of factor VIIa were blocked by anti-TF or anti-PAR2 antibodies. The effects of factor VIIa on proliferation and migration of SW620 cells, expression of IL-8, TF as well as caspase-7, were interfered by PDTC (the inhibitor of NF-κB).. TF/VIIa complex activates NF-κB pathway via PAR2, thereby up-regulates IL-8 and down-regulates caspase-7 expression in SW620 cells, finally promotes proliferation and migration of colon cancer cells. In addition, TF/VIIa/PAR2/NF-κB pathway also upregulates TF expression, thus to create a positive feedback loop of TF/VIIa/PAR2/NF-κB/TF.

Topics: Antineoplastic Agents; Caspase 7; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colonic Neoplasms; Factor VIIa; Humans; I-kappa B Proteins; Interleukin-8; NF-KappaB Inhibitor alpha; Proline; Receptor, PAR-2; RNA, Messenger; Thiocarbamates; Thromboplastin; Transcription Factor RelA

The vascular endothelial cell (EC) is a primary target of infection with Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever. Changes in gene transcription elicited by intracellular infection, including EC expression of the coagulation pathway initiator known as tissue factor (TF), may contribute to the vascular pathology observed during disease. Nuclear run-on analysis of uninfected and infected, cultured human endothelial cells revealed that the rate of TF mRNA transcription is enhanced more than twofold at 3 h following infection, thus coinciding with increased steady-state levels of TF mRNA. TF mRNA remained relatively unstable during infection, with a half-life of 1.6 h. The eukaryotic protein synthesis inhibitor cycloheximide did not block R. rickettsii-induced increase in TF mRNA levels and actually resulted in its superinduction, thus revealing that de novo synthesis of host cell protein was not prerequisite to this transcriptional response. Involvement of the transcription factor NF-kappaB in R. rickettsii-induced TF expression was demonstrated by using two unrelated inhibitors of NF-kappaB activation. The antioxidant pyrrolidinedithiocarbamate and the proteasome inhibitor N-tosyl-L-phenylalanine chloromethyl ketone blocked expression of TF mRNA and activity during infection. This study demonstrates that R. rickettsii infection results in transcriptional activation of the TF gene and that this response involves activation of the transcription factor NF-kappaB.

Topics: Cells, Cultured; Cycloheximide; Endothelium, Vascular; Gene Expression Regulation; Humans; NF-kappa B; Proline; RNA, Messenger; Rocky Mountain Spotted Fever; Thiocarbamates; Thromboplastin; Tosylphenylalanyl Chloromethyl Ketone; Transcription, Genetic