[Abstract]

Fibronectin (FN) is deposited in subendothelial basement membranes in athero-prone regions of arteries with disturbed flow, where it promotes endothelial NFkB activation and expression of inflammatory mediators. We previously reported that fibronectin receptor integrin a5 directly binds phosphodiesterase (PDE) 4D and mutation of the integrin a5 cytoplasmic tail or the integrin binding site in PDE4D reduces atherosclerosis. Further analysis showed binding of PDE4D to a5 led to dephosphorylation of PDE4D by the phosphatase complex PP2A, which increased PDE and NFkB activity. These experiments unexpectedly revealed that the PDE4D promoted the PP2A-B55a holoenzyme assembly via direct interaction between B55a subunit of PP2A and N-terminal domain of PDE4D5. We therefore compared phosphoprotein profiles between (1) ECs on FN vs. matrigel (MG, basement membrane matrix) or (2) ECs expressing WT PDE4D vs. a mutant PDE4D with deletion of the PP2A-binding N-terminal domain. Among 205 hits showing >1.5-fold changes in phosphorylation from FN vs. MG analysis, 63 phosphorylation events were also changed in PDE4D WT vs. mutant analysis. Functional clustering analysis for the differentially phosphorylated hits showed that protein involved in cell-cell adhesion and RNA splicing are modulated by PDE4D-PP2A complex. This suggests that PP2A dephosphorylates FN-specific target proteins and promotes FN-dependent EC phenotypes.