Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by Nrf1 and Nrf2. tertButylhydroquinone (TBH) induces human GCLC via Nrf2-mediated trans activation of the antioxidantresponsive element (ARE). Interestingly, TBH also induces rat GCLC, but the rat GCLC promoter lacks ARE. This study examined the role of Nrf1 and Nrf2 in the transcriptional regulation of rat GCLC. The baseline and TBH-mediated increase in GCLC mRNA levels and rat GCLC promoter activity were lower in Nrf1 and Nrf2 null (F1 and F2) fibroblasts than in wild-type cells. The basal protein and mRNA levels and nuclear binding activities of c-Jun, c-Fos, p50, and p65 were lower in F1 and F2 cells and exhibited a blunted response to TBH. Lower c-Jun and p65 expression also occurs in Nrf2 null livers. Levels of other AP-1 and NF-B family members were either unaffected (i.e., JunB) or increased (i.e., Fra-1). Overexpression of Nrf1 and Nrf2 in respective cells restored the rat GCLC promoter activity and response to TBH but not if the AP-1 and NF-B binding sites were mutated. Fra-1 overexpression lowered endogenous GCLC expression and rat GCLC promoter activity, while Fra-1 antisense had the opposite effects. In conclusion, Nrf1 and Nrf2 regulate rat GCLC promoter by modulating the expression of key AP-1 and NF-B family members.Glutathione (GSH) is the main nonprotein thiol in mammalian cells that participates in many critical cellular functions, including antioxidant defense and cell growth (14,24,28). The synthesis of GSH from its constituent amino acids involves two ATP-requiring enzymatic steps: the formation of ␥-glutamylcysteine from glutamate and cysteine and the formation of GSH from ␥-glutamylcysteine and glycine. The first step of GSH biosynthesis is rate limiting and catalyzed by glutamatecysteine ligase (GCL, also known as ␥-glutamylcysteine synthetase), while the second step is catalyzed by GSH synthetase (14). The GCL enzyme is composed of a catalytic (GCLC, M r of ϳ73,000) and a modifier (GCLM, M r of ϳ30,000) subunit which are encoded by different genes and dissociate under reducing conditions (7,27,35). The catalytic subunit exhibits all of the catalytic activity of the isolated enzyme as well as feedback inhibition by GSH (27). The modifier subunit is enzymatically inactive but plays an important regulatory function by lowering the K m of GCL for glutamate and raising the K i for GSH (7,8). GCL is a major determinant of the overall GSH synthesis capacity, and changes in GCL activity can result from regulation at multiple levels affecting only the catalytic or modifier subunit or both (14). Both human GCLC and GCLM promoters have been cloned (4,5,16,18,34). Antioxidantresponse element (ARE, also known as electrophile response element, EpRE) and activator protein 1 (AP-1) are two cisacting elements present in the promoter of both human GCL subunits that have been implicated in their transcriptional regulation by oxidants and ␤-naphthoflavone (5, 14, 16, 18).Nrf1 and Nrf2, members of the cap 'n' collar-basic leu...

Rationale Foxp3+ T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. Objective Here, we address whether atherosclerosis impacts Treg plasticity and functionality in Apoe−/− mice, and what effect Treg plasticity might have on the pathology of atherosclerosis. Methods and Results We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3+ Tregs, and the accumulation of an intermediate Th1-like IFNγ+CCR5+ Treg subset (Th1/Tregs) within the aorta. Importantly, Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than T effector cells. We show that Th1/Tregs recovered from atherosclerotic mice are dysfunctional in suppression assays. Using an adoptive transfer system and plasticity-prone Mir146a−/− Tregs, we demonstrate that elevated IFNγ+ Mir146a−/− Th1/Tregs are unable to adequately reduce atherosclerosis, arterial Th1, or macrophage content within Apoe−/− mice, in comparison to Mir146a+/+ Tregs. Lastly, via single cell RNA-sequencing and RT-PCR we show that Th1/Tregs possess a unique transcriptional phenotype characterized by co-expression of Treg and Th1 lineage genes, and a down-regulation of Treg-related genes, including Ikzf2, Ikzf4, Tigit, Lilrb4, and Il10. Additionally, an ingenuity pathway analysis further implicates IFNγ, IFNα, IL-2, IL-7, CTLA4, T cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. Conclusions Atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ+ Th1/Tregs that may permit further arterial inflammation and atherogenesis.

Inflammation has a critical role in the pathogenesis of diabetic complications, including diabetic nephropathy (DN). MicroRNAs have recently emerged as important regulators of DN. However, the role of microRNAs in the regulation of inflammation during DN is poorly understood. Here, we examined the in vivo role of microRNA-146a (miR-146a), a known anti-inflammatory microRNA, in the pathogenesis of DN. In a model of streptozotocin-induced diabetes, miR-146a 2/2 mice showed significantly exacerbated proteinuria, renal macrophage infiltration, glomerular hypertrophy, and fibrosis relative to the respective levels in control wild-type mice. Diabetes-induced upregulation of proinflammatory and profibrotic genes was significantly greater in the kidneys of miR-146a 2/2 than in the kidneys of wild-type mice. Notably, miR146a expression increased in both peritoneal and intrarenal macrophages in diabetic wild-type mice. Mechanistically, miR-146a deficiency during diabetes led to increased expression of M1 activation markers and suppression of M2 markers in macrophages. Concomitant with increased expression of proinflammatory cytokines, such as IL-1b and IL-18, markers of inflammasome activation also increased in the macrophages of diabetic miR-146a 2/2 mice. These studies suggest that in early DN, miR-146a upregulation exerts a protective effect by downregulating target inflammation-related genes, resulting in suppression of proinflammatory and inflammasome gene activation. Loss of this protective mechanism in miR-146a 2/2 mice leads to accelerated DN. Taken together, these results identify miR-146a as a novel anti-inflammatory noncoding RNA modulator of DN. 27: 227727: -228827: , 201627: . doi: 10.1681 Diabetic nephropathy (DN) is the leading cause of CKD and ESRD. 1-6 Development and progression of DN involve a complex interplay among metabolic, hemodynamic, growth, and inflammatory factors. 1,3,[7][8][9][10][11][12][13] The progressive decline in renal function during DN is a result of a multitude of pathologic changes in the kidneys, including glomerular and tubular hypertrophy, macrophage infiltration, extracellular matrix (ECM) accumulation in multiple renal cells, mesangial expansion, endothelial dysfunction, and podocyte injury. 1,3,[7][8][9][10][11][12][14][15][16] These pathologic changes clinically manifest as proteinuria and a steady deterioration in GFR. 3,4,16 Identification of molecular pathways that contribute to the pathophysiology of DN is imperative for the development of new therapeutic strategies. J Am Soc NephrolConversely, identification of factors that exert adaptive and protective roles in the early stages of DN can be exploited to prevent progression to ESRD.

Background & Aims-Methionine adenosyltransferase (MAT) catalyzes S-adenosylmethionine biosynthesis. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2β) encodes for a regulatory subunit that modulates the activity of MAT2A-encoded isoenzyme. We uncovered multiple splicing variants while characterizing its 5′-flanking region. The aims of our current study are to examine the expression pattern, regulation, and functions of the 2 major variants: V1 and V2.

GSH synthesis occurs via two enzymatic steps catalysed by GCL [glutamate-cysteine ligase, made up of GCLC (GCL catalytic subunit), and GCLM (GCL modifier subunit)] and GSS (GSH synthetase). Co-ordinated up-regulation of GCL and GSS further enhances GSH synthetic capacity. The present study examined whether TNFalpha (tumour necrosis factor alpha) influences the expression of rat GSH synthetic enzymes. To facilitate transcriptional studies of the rat GCLM, we cloned its 1.8 kb 5'-flanking region. TNFalpha induces the expression and recombinant promoter activities of GCLC, GCLM and GSS in H4IIE cells. TNFalpha induces NF-kappaB (nuclear factor kappaB) and AP-1 (activator protein 1) nuclear-binding activities. Blocking AP-1 with dominant negative c-Jun or NF-kappaB with IkappaBSR (IkappaB super-repressor, where IkappaB stands for inhibitory kappaB) lowered basal expression and inhibited the TNFalpha-mediated increase in mRNA levels of all three genes. While all three genes have multiple AP-1-binding sites, only GCLC has a NF-kappaB-binding site. Overexpression with p50 or p65 increased c-Jun mRNA levels, c-Jun-dependent promoter activity and the promoter activity of GCLM and GSS. Blocking NF-kappaB also lowered basal c-Jun expression and blunted the TNFalpha-mediated increase in c-Jun mRNA levels. TNFalpha treatment resulted in increased c-Jun and Nrf2 (nuclear factor erythroid 2-related factor 2) nuclear binding to the antioxidant response element of the rat GCLM and if this was prevented, TNFalpha no longer induced the GCLM promoter activity. In conclusion, both c-Jun and NF-kappaB are required for basal and TNFalpha-mediated induction of GSH synthetic enzymes in H4IIE cells. While NF-kappaB may exert a direct effect on the GCLC promoter, it induces the GCLM and GSS promoters indirectly via c-Jun.

miR-146a inhibits inflammatory responses in human keratinocytes and in different mouse models of skin inflammation. Little is known about the role of miR-146b in the skin. In the present study, we confirmed the increased expression of miR-146a and miR-146b (miR-146a/b) in lesional skin of psoriasis patients. The expression of miR-146a was about 2-fold higher than that of miR-146b in healthy human skin and it was more strongly induced by stimulation of pro-inflammatory cytokines in keratinocytes and fibroblasts. miR-146a/b target genes regulating inflammatory responses or proliferation were altered in the skin of psoriasis patients, among which FERMT1 was verified as direct target of miR-146a. In silico analysis of genome-wide data from >4,000 psoriasis cases and >8,000 controls confirmed a moderate association between psoriasis and genetic variants in miR-146a gene. Transfection of miR-146a/b suppressed and inhibition enhanced keratinocyte proliferation and the expression of psoriasis-related target genes. Enhanced expression of miR-146a/b-influenced genes was detected in cultured keratinocytes from miR-146a−/− and skin fibroblasts from miR-146a−/− and miR-146b−/− mice stimulated with psoriasis-associated cytokines as compared to wild type mice. Our results indicate that besides miR-146a, miR-146b is expressed and might be capable of modulation of inflammatory responses and keratinocyte proliferation in psoriatic skin.

Aminoacylase 3 (AA3) deacetylates N-acetyl-aromatic amino acids and mercapturic acids including N-acetyl-1,2-dichlorovinyl-L-cysteine (Ac-DCVC), a metabolite of a xenobiotic trichloroethylene.Previous studies did not demonstrate metal-dependence of AA3 despite a high homology with a Zn 2+ -metalloenzyme aminoacylase 2 (AA2). A 3D model of mouse AA3 was created based on homology with AA2. The model showed a putative metal binding site formed by His21, Glu24 and His116, and Arg63, Asp68, Asn70, Arg71, Glu177 and Tyr287 potentially involved in catalysis/ substrate binding. The mutation of each of these residues to alanine inactivated AA3 except Asn70 and Arg71, therefore the corrected 3D model of mouse AA3 was created. Wild type (wt) mouse AA3 expressed in E. coli contained ~0.35 zinc atoms per monomer. Incubation with Co 2+ and Ni 2+ activated wt-AA3. In the cobalt-activated AA3 zinc was replaced with cobalt. Metal removal completely inactivated wt-AA3, whereas addition of Zn 2+ , Mn 2+ or Fe 2+ restored initial activity. Co 2+ and to a lesser extent Ni 2+ increased activity several times in comparison with intact wt-AA3. Co 2+ drastically increased the rate of deacetylation of Ac-DCVC and significantly increased the toxicity of Ac-DCVC in the HEK293T cells expressing wt-AA3. The results indicate that AA3 is a metalloenzyme significantly activated by Co 2+ and Ni 2+ .

microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake, preventing immune overreaction and malignant transformation by attenuating NF-κB signaling, putatively via repression of the Traf6 and Irak1 genes. The exact contribution of miR-146a–mediated silencing of these genes to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a–Traf6 signaling axis in the regulation of immune homeostasis using a genetic epistasis analysis in miR-146a−/− mice. We have uncovered a surprising separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued several significant miR-146a−/− phenotypes, such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the progressive bone marrow failure phenotype, as well as lymphomagenesis in miR-146a−/− mice, indicating that miR-146a controls these biological processes through different molecular mechanisms.