Thrombin-dependent NF- B Activation and Monocyte/Endothelial Adhesion Are Mediated by the CARMA3{middle dot}Bcl10{middle dot}MALT1 Signalosome

Cellular and Molecular Biology Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2010; 285(53):41432-42. DOI: 10.1074/jbc.M110.158949
Source: PubMed


Thrombin is a potent modulator of endothelial function and, through stimulation of NF-κB, induces endothelial expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These cell surface adhesion molecules recruit inflammatory cells to the vessel wall and thereby participate in the development of atherosclerosis, which is increasingly recognized as an inflammatory condition. The principal receptor for thrombin on endothelial cells is protease-activated receptor-1 (PAR-1), a member of the G protein-coupled receptor superfamily. Although it is known that PAR-1 signaling to NF-κB depends on initial PKC activation, the subsequent steps leading to stimulation of the canonical NF-κB machinery have remained unclear. Here, we demonstrate that a complex of proteins containing CARMA3, Bcl10, and MALT1 links PAR-1 activation to stimulation of the IκB kinase complex. IκB kinase in turn phosphorylates IκB, leading to its degradation and the release of active NF-κB. Further, we find that although this CARMA3·Bcl10·MALT1 signalosome shares features with a CARMA1-containing signalosome found in lymphocytes, there are significant differences in how the signalosomes communicate with their cognate receptors. Specifically, whereas the CARMA1-containing lymphocyte complex relies on 3-phosphoinositide-dependent protein kinase 1 for assembly and activation, the CARMA3-containing endothelial signalosome functions completely independent of 3-phosphoinositide-dependent protein kinase 1 and instead relies on β-arrestin 2 for assembly. Finally, we show that thrombin-dependent adhesion of monocytes to endothelial cells requires an intact endothelial CARMA3·Bcl10·MALT1 signalosome, underscoring the importance of the signalosome in mediating one of the most significant pro-atherogenic effects of thrombin.

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    • "Therefore, it is plausible to postulate that thrombin stimulates MSCs to express and secrete FN via PAR-1- and PAR-2-mediated ERK pathway. Interestingly, blockage to PAR-1 and PAR-2 had little effect on the phosphorylation of NFκB, though PAR-1- and PAR-2-mediated NFκB activation by thrombin has been reported in epithelial and endothelial cells [56,57]. The discrepancy should be due to different cell types used in the experiments, and further investigations are needed to clarify the observation that thrombin resulted in NFκB activation in human bone marrow MSCs. "
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    ABSTRACT: Fibronectin (FN) is commonly used in the development of serum-free media for the expansion of mesenchymal stem cells (MSCs). This study was aimed to observe if thrombin could stimulate FN secretion by human bone marrow MSCs and investigate the potential underlying mechanisms. PCR was performed to detect the expression of the protease-activated receptors (PARs) in MSCs. After thrombin treatment, the expression level and secretion of FN were observed by RT-PCR, immunofluorescence staining and ELISA respectively, and the activation of ERK1/2 and NF kappa B pathways was revealed by western blotting, with or without pre-treatment of small-molecule blockers specific for PAR-1 and -2. The phenotypic and functional activities of thrombin-treated MSCs were also observed. PCR analysis showed that human bone marrow MSCs expressed two subtypes of PARs, PAR-1 and PAR-2. Thrombin treatment enhanced MSCs to express FN at mRNA and protein levels and promoted FN secretion by MSCs, accompanied by potent adherence to the culture plastic. Thrombin induced prompt phosphorylation of ERK 1/2 and NF kappa B p65 and the stimulatory effects of thrombin on FN secretion were blunted by specific inhibitors of these signalling molecules. Blockage to PAR-1 and PAR-2 partially abrogated thrombin-elicited FN secretion by MSCs and ERK 1/2 phosphorylation, whereas that of NF kappa B p65 was unaffected. Moreover, thrombin-treated MSCs maintained the phenotypic features, in vitro osteogenesis and adipogenesis capacities, and inhibitory activity on PHA-induced allogeneic lymphocyte proliferation. Thrombin could promote FN secretion by MSCs via PAR- mediated ERK 1/2 activation, while NF kappa B might be also involved in an undefined manner.
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    • "Therefore, protein C deficiency in DIC may be predictive of aggravated inflammatory processes as well as coagulopathy. Similarly, antithrombin also inhibits inflammatory processes by downregulating thrombin, which induces various inflammatory responses through stimulation of the nuclear factor kappa-light-chain-enhancer of activated B cells [18]. Hence, antithrombin deficiency in DIC also aggravates the inflammatory response. "
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    • "More recently, ligation of ICAM-1 has also been shown to induce endothelial permeability [18], suggesting dual function of ICAM-1 in mediating PMN transmigration and EC permeability associated with lung inflammation. We and others have shown that up-regulation of ICAM-1 expression by thrombin depends primarily on activation of the transcription factor NF-κB (predominantly RelA/p65 homodimer) [19] and that this response is mediated through activation of the GTP-binding protein (G-protein) coupled receptor, protease-activated receptor-1 (PAR-1) [19], [20]. In most cases, the initiating event in NF-κB activation involves stimulation of IκBβ kinase (IKKβ) activity which phosphorylates two specific serine residues (Ser32 and Ser36) of IκBα, an inhibitory protein that retains NF-κB in the cytoplasm. "
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