O-Glycosylation Regulates Ubiquitination and Degradation of the Anti-Inflammatory Protein A20 to Accelerate Atherosclerosis in Diabetic ApoE-Null Mice

Background: Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. Methodology/Principal Findings: High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis. Conclusions: High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes

A functional variant in the gene 3 ' untranslated region regulates HSP70 expression and is a potential candidate for insulin resistance-related abnormalities

[No abstract available

Tag polymorphisms at the A20 (TNFAIP3) locus are associated with lower gene expression and increased risk of coronary artery disease in type 2 diabetes

A20 or tumor necrosis factor (TNF)-induced protein 3 (TNFAIP3) is a negative regulator of nuclear factor-kappa B (NF-kappa B). We have investigated whether polymorphisms in this gene are associated with increased atherosclerosis in diabetic patients. Five tag single nucleotide polymorphisms (SNPs) were typed in 479 type 2 diabetic patients from Boston, including 239 coronary artery disease (CAD)positive case subjects and 240 CAD-negative control subjects. Two tag SNPs (rs5029930 and rs610604) were independently associated with CAD; adjusted odds ratios (ORs) for minor allele carriers were 2.3 (95% CI 1.4-3.8, P = 0.001) and 2.0 (1.3-2.9, P = 0.0008), respectively. The association with rs610604 was dependent on glycemic control, with ORs of 3.9 among subjects with A1C 7.0% (P for interaction = 0.015). A similar interaction pattern was found among 231 CAD-positive and 332 CAD-negative type 2 diabetic patients from Italy (OR 2.2, P = 0.05 vs. OR 0.9, P = 0.63 in the low vs. high A1C strata, P for interaction = 0.05). Quantitative RT-PCR in blood mononuclear cells from 83 nondiabetic subjects showed that rs610604 and rs5029930 minor allele homozygotes have 30-45% lower levels of A20 mRNA than major allele homozygotes, and heterozygotes have intermediate levels (P = 0.04 and 0.028, respectively). These findings point to variability in the A20/TNFAIP3 gene as a modulator of CAD risk in type 2 diabetes. This effect is mediated by allelic differences in A20 expression

Mutations at the BLK locus linked to maturity onset diabetes of the young and β-cell dysfunction

Maturity-onset diabetes of the young (MODY) is a subtype of diabetes defined by an autosomal pattern of inheritance and a young age at onset, often before age 25. MODY is genetically heterogeneous, with 8 distinct MODY genes identified to date and more believed to exist. We resequenced 732 kb of genomic sequence at 8p23 in 6 MODY families unlinked to known MODY genes that showed evidence of linkage at that location. Of the 410 sequence differences that we identified, 5 had a frequency <1% in the general population and segregated with diabetes in 3 of the families, including the 2 showing the strongest support for linkage at this location. The 5 mutations were all placed within 100 kb corresponding to the BLK gene. One resulted in an Ala71Thr substitution; the other 4 were noncoding and determined decreased in vitro promoter activity in reporter gene experiments. We found that BLK—a nonreceptor tyrosine-kinase of the src family of proto-oncogenes—is expressed in β-cells where it enhances insulin synthesis and secretion in response to glucose by up-regulating transcription factors Pdx1 and Nkx6.1. These actions are greatly attenuated by the Ala71Thr mutation. These findings point to BLK as a previously unrecognized modulator of β-cell function, the deficit of which may lead to the development of diabetes

Significant lethality following liver resection in A20 heterozygous knockout mice uncovers a key role for A20 in liver regeneration

Hepatic expression of A20, including in hepatocytes, increases in response to injury, inflammation and resection. This increase likely serves a hepatoprotective purpose. The characteristic unfettered liver inflammation and necrosis in A20 knockout mice established physiologic upregulation of A20 as integral to the anti-inflammatory and anti-apoptotic armamentarium of hepatocytes. However, the implication of physiologic upregulation of A20 in modulating hepatocytes' proliferative responses following liver resection remains controversial. To resolve the impact of A20 on hepatocyte proliferation and the liver's regenerative capacity, we examined whether decreased A20 expression, as in A20 heterozygous knockout mice, affects outcome following two-third partial hepatectomy. A20 heterozygous mice do not demonstrate a striking liver phenotype, indicating that their A20 expression levels are still sufficient to contain inflammation and cell death at baseline. However, usually benign partial hepatectomy provoked a staggering lethality (>40%) in these mice, uncovering an unsuspected phenotype. Heightened lethality in A20 heterozygous mice following partial hepatectomy resulted from impaired hepatocyte proliferation due to heightened levels of cyclin-dependent kinase inhibitor, p21, and deficient upregulation of cyclins D1, E and A, in the context of worsened liver steatosis. A20 heterozygous knockout minimally affected baseline liver transcriptome, mostly circadian rhythm genes. Nevertheless, this caused differential expression of >1000 genes post hepatectomy, hindering lipid metabolism, bile acid biosynthesis, insulin signaling and cell cycle, all critical cellular processes for liver regeneration. These results demonstrate that mere reduction of A20 levels causes worse outcome post hepatectomy than full knockout of bona fide liver pro-regenerative players such as IL-6, clearly ascertaining A20's primordial role in enabling liver regeneration. Clinical implications of these data are of utmost importance as they caution safety of extensive hepatectomy for donation or tumor in carriers of A20/TNFAIP3 single nucleotide polymorphisms alleles that decrease A20 expression or function, and prompt the development of A20-based liver pro-regenerative therapies

A20: linking a complex regulator of ubiquitylation to immunity and human disease

A20 (also known as TNFAIP3) is a potent anti-inflammatory signalling molecule that restricts multiple intracellular signalling cascades. Recent studies in three general areas have converged to highlight the clinical and biological importance of A20. First, human genetic studies have strongly linked polymorphisms and mutations in the gene encoding A20 to inflammatory, autoimmune and malignant diseases. Second, studies in gene-targeted mice have revealed that A20 regulates multiple immune cell functions and prevents experimental diseases that closely mimic human conditions. Third, biochemical studies have unveiled complex mechanisms by which A20 regulates ubiquitin-dependent nuclear factor-κB and cell-survival signals. Taken together, these studies are revealing the importance of A20-mediated regulation of ubiquitin-dependent signalling in human disease