Thrombin Down-Regulates Tissue Factor Pathway Inhibitor Expression in a PI3K/Nuclear Factor-κB–Dependent Manner in Human Pleural Mesothelial Cells

Abstract

Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of the extrinsic coagulation cascade, and its expression is reported to be relatively stable. Various pathophysiologic agents have been shown to influence TFPI activity by regulating its expression or by modifying the protein. It is not clear how TFPI activity is regulated in normal physiology or in injury. Because thrombin and TFPI are locally elaborated in pleural injury, we sought to determine if thrombin could regulate TFPI in human pleural mesothelial cells (HPMCs). Thrombin significantly decreased TFPI mRNA and protein levels by . 70%. Thrombin-mediated down-regulation of TFPI promoted factor X activation by HPMCs. The ability of thrombin to significantly decrease TFPI mRNA and protein levels was maintained at nanomolar concentrations. Protease-activated receptor (PAR)-1, a mediator of thrombin signaling, is detectable in the mesothelium in human and murine pleural injury. PAR-1 silencing blocked thrombin-mediated decrements of TFPI in HPMCs. Thrombin activates PI3K/Akt and nuclear factor kB (NF-kB) signaling in HPMCs. Inhibition of PI3K (by PX-866) and NF-kB (by SN50) prevented thrombin-mediated TFPI mRNA and protein downregulation. These are the first studies to demonstrate that thrombin decreases TFPI expression in HPMCs. Our findings demonstrate a novel mechanism by which thrombin regulates TFPI expression in HPMCs and promotes an unrestricted procoagulant response, and suggest that interactions between PI3K and NF-kB signaling pathways are linked in HPMCs and control TFPI expression. These findings raise the possibility that targeting this pathway could limit the ability of the mesothelium to support extravascular fibrin deposition and organization associated with pleural injury.

Mesomesenchymal transition of pleural mesothelial cells is PI3K and NF-κB dependent

“…Cells were treated with TGF-␤ (5 ng/ml, R&D Systems, Minneapolis, MN), plasmin (6 nM, Sekisui, Lexington, MA), and thrombin (7 nM, Enzyme Research Laboratories, South Bend, IN) as previously described (8,19). Briefly, cells were serum starved for 16 -24 h prior to treatment.…”

“…Briefly, cells were serum starved for 16 -24 h prior to treatment. Serum-starved cells were treated with TGF-␤, plasmin, and thrombin for 48 h. Cells were then lysed and cleared, and protein concentration was determined (8,19). Lysates were then probed for changes in ␣-SMA (cat.…”

“…Cells were treated with varying doses of PI3K/ Akt and NF-B inhibitors prior to treatment with inducers of MesoMT. PI3K/Akt signaling was blocked with the PI3K inhibitor PX866 (1-0.05 M, a kind gift from Scott Peterson, Oncothyreon) and the pan-Akt inhibitor Akt VIII (AKT8, 2-0.25 M, Calbiochem, Billerica, MA) as previously described (8). NF-B signaling was blocked by use of SN50 (20-1 M, Calbiochem), as previously described (8), and Bay-11-7085 (10 M, Calbiochem) according to manufacturer's instructions.…”

“…PI3K/Akt signaling was blocked with the PI3K inhibitor PX866 (1-0.05 M, a kind gift from Scott Peterson, Oncothyreon) and the pan-Akt inhibitor Akt VIII (AKT8, 2-0.25 M, Calbiochem, Billerica, MA) as previously described (8). NF-B signaling was blocked by use of SN50 (20-1 M, Calbiochem), as previously described (8), and Bay-11-7085 (10 M, Calbiochem) according to manufacturer's instructions. Briefly cells were serum starved in the presence of the inhibitor for 16 -24 h. Cells were washed and then treated with PBS, TGF-␤, plasmin, or thrombin in the presence of inhibitors and allowed to incubate for another 48 h to assess their impact on the progression of MesoMT.…”

“…HPMCs were adenovirally transduced as previously described (8). Briefly, cells were transduced with ␤-galactosidase (LacZ) or myristoylated Akt (Myr-Akt), a constitutively active isoform of Akt, at a concentration of 2 multiplicities of infection for 24 h. Cells were then placed in serum-free media for 48 h and then assayed for markers of MesoMT.…”