Thrombin-Dependent NF-κB Activation and Monocyte/Endothelial Adhesion Is Mediated by a ß-Arrestin/CARMA3-Bcl10-MALT1 Signaling Complex; Implications for Atherogenesis.
Phillip C Delekta, Ingrid J Apel, Shufang Gu, Katy Siu, Yoshiyuki Hattori, Linda M McAllister-Lucas, Peter C Lucas. University of Michigan Medical School, Ann Arbor; Dokkyo University School of Medicine, Mibu, Tochigi, Japan
Background: Thrombin is a potent modulator of endothelial function, and through stimulation of NF-κB, induces endothelial expression of adhesion molecules (eg, ICAM-1 and VCAM-1). These cell-surface 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 PAR-1, a member of the GPCR superfamily. While it is known that PAR-1 signaling to NF-κB is a key step in endothelial dysfunction, the steps leading to stimulation of NF-κB have remained unclear.
Design: Endothelial cell culture models based on the mouse SVEC4-10 and human EA.hy923 lines were used to test the role of a signaling complex composed of three principal proteins, CARMA3, Bcl10, and MALT1, in mediating NF-κB activation downstream of PAR-1. RNA interference was used to abrogate expression of each protein and the affect on NF-κB activation was assessed through detection of IκB phosphorylation, NF-κB nuclear translocation, and expression of the NF-κB gene targets, VCAM-1 and ICAM-1. In addition, the impact of Bcl10 knock-down on thrombin-responsive endothelial/monocyte adhesion was evaluated, as was recruitment of monocytes to the aortic intima of Bcl10-/- mice. Finally, the role of ß-arrestin2 was evaluated through RNA interference and through the use of MEFs derived from ß-arrestin2-/- mice.
Results: We demonstrate that a complex of proteins including CARMA3, Bcl10, and MALT1 link PAR-1 activation to stimulation of the canonical NF-κB signaling pathway. Further, we provide evidence that ß-arrestin2 serves as a scaffold to link the PAR-1 receptor to the CARMA3/Bcl10/MALT1 signaling complex. Disruption of the complex impairs thrombin-dependent induction of adhesion molecules, thereby reducing attachment of circulating monocytes.
Conclusions: The CARMA3/Bcl10/MALT1 complex shares features with an analogous CARMA1-containing complex found in lymphocytes. We now show that the CARMA3-containing complex functions in cells outside the immune system, contributing to endothelial dysfunction and recruitment of inflammatory cells. Since atherogenesis is an inflammatory condition, we are exploring the use of MALT1 inhibitors as a therapeutic approach to thrombin-dependent vascular disease.
Monday, February 28, 2011 11:00 AM
Platform Session: Section G 2, Monday Morning