Phosphorylation of USP20 on Ser334 by IRAK1 promotes IL-1ß-evoked signaling in vascular smooth muscle cells and vascular inflammation.

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed ∼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1ß. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1ß induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1ß-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1ß-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.

Inhalation of an RNA aptamer that selectively binds extracellular histones protects from acute lung injury.

Acute lung injury (ALI) is a syndrome of acute inflammation, barrier disruption, and hypoxemic respiratory failure associated with high morbidity and mortality. Diverse conditions lead to ALI, including inhalation of toxic substances, aspiration of gastric contents, infection, and trauma. A shared mechanism of acute lung injury is cellular toxicity from damage-associated molecular patterns (DAMPs), including extracellular histones. We recently described the selection and efficacy of a histone-binding RNA aptamer (HBA7). The current study aimed to identify the effects of extracellular histones in the lung and determine if HBA7 protected mice from ALI. Histone proteins decreased metabolic activity, induced apoptosis, promoted proinflammatory cytokine production, and caused endothelial dysfunction and platelet activation in vitro. HBA7 prevented these effects. The oropharyngeal aspiration of histone proteins increased neutrophil and albumin levels in bronchoalveolar lavage fluid (BALF) and precipitated neutrophil infiltration, interstitial edema, and barrier disruption in alveoli in mice. Similarly, inhaling wood smoke particulate matter, as a clinically relevant model, increased lung inflammation and alveolar permeability. Treatment by HBA7 alleviated lung injury in both models of ALI. These findings demonstrate the pulmonary delivery of HBA7 as a nucleic acid-based therapeutic for ALI.

Drebrin attenuates atherosclerosis by limiting smooth muscle cell transdifferentiation.

AIMS: The F-actin-binding protein Drebrin inhibits smooth muscle cell (SMC) migration, proliferation, and pro-inflammatory signalling. Therefore, we tested the hypothesis that Drebrin constrains atherosclerosis. METHODS AND RESULTS: SM22-Cre+/Dbnflox/flox/Ldlr-/- (SMC-Dbn-/-/Ldlr-/-) and control mice (SM22-Cre+/Ldlr-/-, Dbnflox/flox/Ldlr-/-, and Ldlr-/-) were fed a western diet for 14-20 weeks. Brachiocephalic arteries of SMC-Dbn -/-/Ldlr-/- mice exhibited 1.5- or 1.8-fold greater cross-sectional lesion area than control mice at 14 or 20 weeks, respectively. Aortic atherosclerotic lesion surface area was 1.2-fold greater in SMC-Dbn-/-/Ldlr-/- mice. SMC-Dbn-/-/Ldlr-/- lesions comprised necrotic cores that were two-fold greater in size than those of control mice. Consistent with their bigger necrotic core size, lesions in SMC-Dbn-/- arteries also showed more transdifferentiation of SMCs to macrophage-like cells: 1.5- to 2.5-fold greater, assessed with BODIPY or with CD68, respectively. In vitro data were concordant: Dbn-/- SMCs had 1.7-fold higher levels of KLF4 and transdifferentiated to macrophage-like cells more readily than Dbnflox/flox SMCs upon cholesterol loading, as evidenced by greater up-regulation of CD68 and galectin-3. Adenovirally mediated Drebrin rescue produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs. During early atherogenesis, SMC-Dbn-/-/Ldlr-/- aortas demonstrated 1.6-fold higher levels of reactive oxygen species than control mouse aortas. The 1.8-fold higher levels of Nox1 in Dbn-/- SMCs were reduced to WT levels with KLF4 silencing. Inhibition of Nox1 chemically or with siRNA produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs. CONCLUSION: We conclude that SMC Drebrin limits atherosclerosis by constraining SMC Nox1 activity and SMC transdifferentiation to macrophage-like cells.

Influence of genetic background on albuminuria and kidney injury in Ins2(+/C96Y) (Akita) mice.

Previous studies have shown that Akita mice bearing the Ins2(+/C96Y) mutation have significant advantages as a type I diabetes platform for developing models of diabetic nephropathy (DN; Gurley SB, Clare SE, Snow KP, Hu A, Meyer TW, Coffman TM. Am J Physiol Renal Physiol 290: F214-F222, 2006). In view of the critical role for genetic factors in determining susceptibility to DN in humans, we investigated the role of genetic background on kidney injury in Akita mice. To generate a series of inbred Akita mouse lines, we back-crossed the Ins2(C96Y) mutation more than six generations onto the 129/SvEv and DBA/2 backgrounds and compared the extent of hyperglycemia and renal disease with the standard C57BL/6-Ins2(+/C96Y) line. Male mice from all three Akita strains developed marked and equivalent hyperglycemia. However, there were significant differences in the level of albuminuria among the lines with a hierarchy of DBA/2 > 129/SvEv > C57BL/6. Renal and glomerular hypertrophy was seen in all of the lines, but significant increases in mesangial matrix compared with baseline nondiabetic controls were observed only in the 129 and C57BL/6 backgrounds. In F1(DBA/2 x C57BL/6)-Ins2(+/C96Y) mice, the extent of albuminuria was similar to the parental DBA/2-Ins2(+/C96Y) line; they also developed marked hyperfiltration. These studies identify strong effects of genetic background to modify the renal phenotype associated with the Ins2(C96Y) mutation. Identification of these naturally occurring strain differences should prove useful for nephropathy modeling and may be exploited to allow identification of novel susceptibility alleles for albuminuria in diabetes.

Impact of genetic background on nephropathy in diabetic mice.

With the goal of identifying optimal platforms for developing better models of diabetic nephropathy in mice, we compared renal effects of streptozotocin (STZ)-induced diabetes among five common inbred mouse strains (C57BL/6, MRL/Mp, BALB/c, DBA/2, and 129/SvEv). We also evaluated the renal consequences of chemical and genetic diabetes on the same genetic background (C57BL/6). There was a hierarchical response of blood glucose level to the STZ regimen among the strains (DBA/2 > C57BL/6 > MRL/MP > 129/SvEv > BALB/c). In all five strains, males demonstrated much more robust hyperglycemia with STZ than females. STZ-induced diabetes was associated with modest levels of albuminuria in all of the strains but was greatest in the DBA/2 strain, which also had the most marked hyperglycemia. Renal structural changes on light microscopy were limited to the development of mesangial expansion, and, while there were some apparent differences among strains in susceptibility to renal pathological changes, there was a significant positive correlation between blood glucose and the degree of mesangial expansion, suggesting that most of the variability in renal pathological abnormalities was because of differences in hyperglycemia. Although the general character of renal involvement was similar between chemical and genetic diabetes, Akita mice developed more marked hyperglycemia, elevated blood pressures, and less variability in renal structural responses. Thus, among the strains and models tested, the DBA/2 genetic background and the Akita (Ins2(+/C96Y)) model may be the most useful platforms for model development.