Abstract 635: CD40-Filamin A Interactions Are Required for Translocation of CD40 to Lipid Rafts In Endothelial Cells and for Endothelial Cell Activation
Background and Aim: CD40 is a member of the co-stimulatory tumor necrosis factor receptor superfamily that is not only constitutively expressed on professional antigen presenting cells like macrophages, dendritic cells and B-cells, but also on endothelial cells. Interactions between CD40 and its ligand, CD40 ligand (CD40L) are crucial for proper immune cell activation. Activation of CD40 signaling plays a role in chronic diseases such as rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. However, since CD40-CD40L interactions modulate immune reactions as well as thrombosis, blocking CD40(L) can have adverse side-effects. Therefore, in search for new therapeutic targets, we aimed to identify new CD40-binding partners.
Methods and Results: We created a cDNA library of murine aortas containing atherosclerotic plaques at various stages and performed a yeast-two-hybrid with the C-terminal domain of CD40 as bait. We identified filamin A as a novel CD40 binding partner in atherosclerosis, which was confirmed by co-immunoprecipitation. Accordingly, using confocal microscopy we showed that, upon activation of CD40, filamin A was recruited to CD40 in endothelial cells. Site directed mutagenesis revealed that Filamin A binds to the intracellular domain of CD40, near the transmembrane domain, at a site distinct from the tumor necrosis receptor associated factor (TRAF) binding sites. In endothelial cells, CD40-signaling is dependent on intact lipid rafts. Interestingly, when we knocked-down filamin in endothelial cells using siRNAs, the translocation of CD40 to lipid rafts upon activation of CD40 signaling was inhibited, resulting in repression of CD40-mediated Akt signaling and subsequent inhibition of VCAM-1 and CCL-2, but not of CD40-mediated JNK signaling.
Conclusions: We show that CD40 interacts with filamin A in endothelial cells. This interaction is crucial for translocation of CD40 to the lipid rafts and CD40-mediated activation of the Akt pathway. The reduced upregulation of VCAM-1 and CCL-2 makes CD40-filamin interactions an interesting target for treatment of atherosclerosis.
Author Disclosures: C.M. van Tiel: None. P. Burger: None. N. Zelcer: None. P.L. Hordijk: None. J.D. van Buul: None. E. Lutgens: None.
- © 2015 by American Heart Association, Inc.