Integrin α5β1 regulates PP2A complex assembly through PDE4D in atherosclerosis
Abstract
Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin α5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site, S651. Active PDE then hydrolyzes antiinflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site of PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in atherosclerosis-prone regions of arteries and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55α as the factor recruiting PP2A to PDE4D5. The B55α-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also, unexpectedly, stabilized the PP2A-B55α complex. The integrin-regulated, proatherosclerotic transcription factor Yap was also dephosphorylated and activated through this pathway. PDE4D5 therefore mediated matrix-specific regulation of endothelial cell phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex that has other targets. We believe these results may have widespread consequences for the control of cell function by integrins.
Introdution
Endothelial basement membranes in stable vessels, consisting primarily of collagen IV, laminins, and less abundant species, promote vessel maturation and stability (1, 2). By contrast, fibronectin (FN) is present at low levels in stable vessels in most tissues but is strongly upregulated in inflammation, developmental and postnatal angiogenesis, and atherosclerosis, as is its main receptor integrin α5β1 (3–6). FN has been most studied in atherogenesis, where it contributes to plaque formation (7). These effects are mediated in part by inhibition of the antiinflammatory cAMP/protein kinase A (cAMP/PKA) pathway (8). This occurs through binding of the α5 subunit cytoplasmic domain to the cAMP-specific phosphodiesterase PDE4D5, which results in dephosphorylation of PDE4D5 on an inhibitory site (9). The resultant increase in PDE catalytic activity suppresses cAMP/PKA signaling, thus priming endothelial cells (ECs) for inflammatory activation.
EC responses to fluid shear stress play major roles in vessel function, remodeling, and disease. Shear stress regulates artery remodeling to determine lumen diameter and during angiogenesis stabilizes vessel sprouts once flow is reestablished (10, 11). Disturbed flow patterns that arise in regions of arteries that curve sharply or branch induce local EC inflammatory activation. In the presence of systemic risk factors such as hyperlipidemia, hyperglycemia, hypertension, and elevated inflammatory mediators, these regions selectively develop atherosclerotic plaques. FN gene expression and matrix assembly are induced by disturbed flow (12, 13). FN is deposited at atherosclerosis-prone regions of arteries in WT mice and increases in atherosclerotic lesions in mice and humans (4, 14). Deletion of each of the individual isoforms of FN reduces plaque size in mice (15–17). However, in the 1 study in which it was examined, deletion of plasma FN also resulted in thinner fibrous caps with evidence of plaque rupture, suggesting reduced plaque stability (17).
Adherence of cells to FN promoted activation of NF-κB and other inflammatory pathways in response to disturbed flow, IL-1β, and oxidized LDL compared with adherence to collagens or laminin (4, 9, 18, 19). These effects were reversed by mutation of the integrin α5 cytoplasmic domain in vitro. To test in vivo, we introduced this mutation into mice, which suppressed atherosclerosis and improved recovery from hindlimb ischemia (9, 20). EC-specific deletion of integrin α5 also strongly reduced atherosclerosis in mouse models (21). However, other integrin α5 cytoplasmic domain effectors may also mediate these effects (22).
We set out to rigorously test the extent to which binding of PDE4D5 to integrin α5 mediates the role of α5 in vascular inflammation and atherosclerosis. Because complete deletion of PDE4D results in neonatal growth retardation and lethality (23), we mutated the integrin binding sequence in PDE4D5 to block this interaction without interfering with other functions. Positive results then prompted us to further examine the mechanism of PDE4D5 regulation. These studies revealed that PDE4D5 regulates a wider array of pathways via effects on phosphatase PP2A and other downstream targets.