Deficient p27 Phosphorylation at Serine 10 Increases Macrophage Foam Cell Formation and Aggravates Atherosclerosis Through a Proliferation-Independent Mechanism

Abstract

Objective— Genetic ablation of the growth suppressor p27 Kip1 (p27) in the mouse aggravates atherosclerosis coinciding with enhanced arterial cell proliferation. However, it is unknown whether molecular mechanisms that limit p27's protective function contribute to atherosclerosis development and whether p27 exerts proliferation-independent activities in the arterial wall. This study aims to provide insight into both questions by investigating the role in atherosclerosis of p27 phosphorylation at serine 10 (p27-phospho-Ser10), a major posttranslational modification of this protein. Methods and Results— Immunoblotting studies revealed a marked reduction in p27-phospho-Ser10 in atherosclerotic arteries from apolipoprotein E–null mice, and expression of the nonphosphorylatable mutant p27Ser10Ala, either global or restricted to bone marrow, accelerated atherosclerosis. p27Ser10Ala expression did not affect cell proliferation in early and advanced atheroma but activated RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) signaling and promoted macrophage foam cell formation in a ROCK-dependent manner. Supporting the clinical relevance of these findings, human atherosclerotic coronary arteries exhibited a prominent reduction in p27-phospho-Ser10 and increased ezrin/radixin/moesin protein phosphorylation, a marker of RhoA/ROCK activation. Conclusion— Scarce phosphorylation of p27 at Ser10 is a hallmark of human and mouse atherosclerosis and promotes disease progression in mice. This proatherogenic effect is mediated by a proliferation-independent mechanism that involves augmented foam cell formation owing to increased RhoA/ROCK activity. These findings unveil a new atheroprotective action of p27 and identify p27-phospho-Ser10 as an attractive target for the treatment of atherosclerosis.

Rho/ROCK pathway inhibition by CDK inhibitor p27kip1 participates in the onset of macrophage 3D-mesenchymal migration

“…The first type of mutant BMDMs, expresses p27 kip1 of which Ser10 is mutated to Ala, yielding mutant protein p27 kip1 Ser10Ala (Besson et al, 2006). In p27 kip1 Ser10Ala mice, total expression level of p27 kip1 Ser10Ala in macrophages is reduced compared to p27 kip1+/+ macrophages, but its nuclear localisation is increased (Fuster et al, 2011). The percentage of migration of p27 kip1 Ser10Ala BMDMs inside Matrigel was reduced by 43% 6 5 at 24 h compared to p27 kip1+/+ cells (Fig.…”

“…We next investigated Rho activity during macrophage 3D-mesenchymal migration and compared it to Rho activity during amoeboid migration, by performing western blot analysis of the phosphorylation states of two reliable markers of this pathway, cofilin and Ezrin-radixin-moesin (ERM) (Fuster et al, 2011;Takai et al, 2001). Migrating macrophages were lysed by boiling Laemmli buffer directly in matrices.…”

Impact of estrogens on atherosclerosis and bone in the apolipoprotein E–deficient mouse model

“…This methodology, which had been detailed previously, 10 is considered the gold standard in the field. Fixation was continued overnight at 4-C. After fixation, vessels were stained with Oil Red O solution (0.2% Oil Red O in 80% MetOH; Sigma) and opened longitudinally.…”

Age-dependent defective TGF-beta1 signaling in patients undergoing coronary artery bypass grafting

“…This atheroprotective effect is shared by its signaling protein p27, whose disruption has been shown to aggravate atherosclerotic lesions in animal models [7,8]. In human vascular samples, atherosclerotic lesions have been linked to reduced expression of p27 [9,10]. However, in advanced atherosclerosis TGF-β1 may behave as a proatherogenic substance by inducing enhanced fibrosis and increase of vascular tone, possibly by means of a defective signaling [2].…”