Abstract 20088: Ubiquitin-Specific Protease 20 Attenuates Atherosclerosis through Activity in Smooth Muscle Cells
Integral to atherosclerosis (athero) is smooth muscle cell (SMC) NFκB-activating signaling, which involves reversible ubiquitination that enhances protein activity downstream of pro-atherogenic tumor necrosis factor receptor-1 (TNFR1) and TLR4. De-ubiquitination of pro-inflammatory signaling proteins is mediated, in part, by USP20 (ubiquitin-specific protease 20). We therefore tested the hypothesis that USP20 activity in SMCs attenuates athero. We used C57BL/6-congenic Ldlr-/- male mice without (control) or with SMC-specific (SM22α promoter-driven) expression of murine USP20 (SMC-USP20-Tg) or its dominant-negative (C154S/H643Q) mutant (SMC-DN-USP20-Tg). Transgenes expressed 3-fold above endogenous SMC USP20 by IB; all mice had equivalent weights, BPs and heart rates. After 14 wk on a Western diet, control mice had 13±1% aortic involvement by Sudan IV-positive lesions (en face), which was 18±2% less than SMC-DN-USP20-Tg and 15±1% more than SMC-USP20-Tg mice (p<0.01, n≥13/group). Congruent data derived from Tg and WT carotid arteries orthotopically transplanted into congenic Apoe-/- mice: SMC-DN-USP20-Tg arteries had 1.9-fold greater athero lesion area (p<0.01) and 2-fold greater lesion macrophage prevalence (by CD68 staining, p<0.01). In vitro, siRNA-induced USP20 silencing in WT SMCs augmented TNF-induced activation of NFκB, judged by IκB kinase-β-mediated phosphorylation (phos) of p65-NFκB on Ser536 (↑60%) and IκB degradation (↑65%) (p<0.05). TLR4 activation in SMCs induced USP20 phos on Ser333, as judged by IB with a phospho-specific IgG. Phospho-Ser333 USP20 is catalytically inactive, as judged by heterologous expression of S333D- and S333A-USP20 mutants in HEK cells. Using purified USP20, we found a 2-fold increase in Ser333 phos upon incubation with purified IRAK1, a kinase activated by TLR4. In human peroneal arteries from amputated legs, SMC levels of phospho-Ser333-USP20 were 3.8±0.7-fold higher in segments with athero as compared with no athero, despite equivalent levels of total USP20 and SMC-actin (quantitative immunofluorescence, p<0.05). We conclude that USP20 activity in SMCs attenuates athero by impeding NFκB activation, and that USP20 activity itself is attenuated by IRAK1-mediated phosphorylation.
Author Disclosures: J. Wu: None. L. Zhang: Research Grant; Modest; AHA. P. Jean-Charles: None. A. Davidson: None. L. Brian: None. R. Qi: None. S.K. Shenoy: Research Grant; Significant; NIH. N.J. Freedman: Research Grant; Significant; NIH.
- © 2016 by American Heart Association, Inc.