Article

CD4+ T-Cell Legumain Deficiency Attenuates Hypertensive Damage via Preservation of TRAF6

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Abstract

BACKGROUND T cells are central to the immune responses contributing to hypertension. LGMN (legumain) is highly expressed in T cells; however, its role in the pathogenesis of hypertension remains unclear. METHODS Peripheral blood samples were collected from patients with hypertension, and CD4+ T cells were sorted for gene expression and Western blotting analysis. TLGMNKO (T cell–specific LGMN-knockout) mice (Lgmn f/f /CD4 Cre ), regulatory T cell (Treg)–specific LGMN-knockout mice (Lgmn f/f /Foxp3 YFP Cre ), and RR-11a (LGMN inhibitor)–treated C57BL/6 mice were infused with Ang II (angiotensin II) or deoxycorticosterone acetate/salt to establish hypertensive animal models. Flow cytometry, 4-dimensional label-free proteomics, coimmunoprecipitation, Treg suppression, and in vivo Treg depletion or adoptive transfer were used to delineate the functional importance of T-cell LGMN in hypertension development. RESULTS LGMN mRNA expression was increased in CD4+ T cells isolated from hypertensive patients and mice, was positively correlated with both systolic and diastolic blood pressure, and was negatively correlated with serum IL (interleukin)-10 levels. TLGMNKO mice exhibited reduced Ang II–induced or deoxycorticosterone acetate/salt–induced hypertension and target organ damage relative to WT (wild-type) mice. Genetic and pharmacological inhibition of LGMN blocked Ang II–induced or deoxycorticosterone acetate/salt–induced immunoinhibitory Treg reduction in the kidneys and blood. Anti-CD25 antibody depletion of Tregs abolished the protective effects against Ang II–induced hypertension in TLGMNKO mice, and LGMN deletion in Tregs prevented Ang II–induced hypertension in mice. Mechanistically, endogenous LGMN impaired Treg differentiation and function by directly interacting with and facilitating the degradation of TRAF6 (tumor necrosis factor receptor–associated factor 6) via chaperone-mediated autophagy, thereby inhibiting NF-κB (nuclear factor kappa B) activation. Adoptive transfer of LGMN-deficient Tregs reversed Ang II–induced hypertension, whereas depletion of TRAF6 in LGMN-deficient Tregs blocked the protective effects. CONCLUSIONS LGMN deficiency in T cells prevents hypertension and its complications by promoting Treg differentiation and function. Specifically targeting LGMN in Tregs may be an innovative approach for hypertension treatment.

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... Hypertension is one of the most important risk factors for CVDs. Recent studies have shown that gene expression levels of LGMN is significantly increased in CD4 + T cells from hypertensive patients and mice [72]. LGMN directly interacts with and promotes the degradation of tumor necrosis factor receptor-associated factor 6 (TRAF6) through chaperonemediated autophagy, thereby inhibiting NF-κB activation and impairing regulatory T-cell (Treg) differentiation and function to prevent hypertension and its complications [72]. ...
... Recent studies have shown that gene expression levels of LGMN is significantly increased in CD4 + T cells from hypertensive patients and mice [72]. LGMN directly interacts with and promotes the degradation of tumor necrosis factor receptor-associated factor 6 (TRAF6) through chaperonemediated autophagy, thereby inhibiting NF-κB activation and impairing regulatory T-cell (Treg) differentiation and function to prevent hypertension and its complications [72]. ...
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Autocrine activation of the complement receptors C3aR and CD46 by complement activation components C3a and C3b produced through C3 cleavage by the protease cathepsin L (CTSL) during T cell stimulation is a requirement for IFN-γ production and Th1 induction in human CD4⁺ T cells. Thus, lack of autocrine CD46 activation, such as in CD46-deficient patients, is associated with defective Th1 responses and recurrent infections. We have identified LGMN [the gene coding for legumain, also known as asparaginyl endopeptidase (AEP)] as one of the key genes induced by CD46 co-stimulation during human CD4⁺ T cell activation. AEP processes and activates a range of proteins, among those α1-thymosin and CTSL, which both drive intrinsically Th1 activity—but has so far not been described to be functionally active in human T cells. Here we found that pharmacological inhibition of AEP during activation of human CD4⁺ T cells reduced CTSL activation and the CTSL-mediated generation of intracellular C3a. This translated into a specific reduction of IFN-γ production without affecting cell proliferation or survival. In line with these findings, CD4⁺ T cells isolated from Lgmn−/− mice also displayed a specific defect in IFN-γ secretion and Th1 induction. Furthermore, we did not observe a role for AEP-driven autocrine α1-thymosin activation in T cell-derived IFN-γ production. These data suggest that AEP is an “upstream” activator of the CTSL-C3-IFN-γ axis in human CD4⁺ T cells and hence an important supporter of human Th1 induction.
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Inflammatory processes have been shown to play an important role in the mechanisms involved in the pathogenesis of hypertension. Innate and adaptive immune responses participate in BP elevation and end‐organ damage. Here, we discuss recent studies focusing on novel inflammatory and immune mechanisms that play roles in BP elevation. Different subpopulations of cells involved in innate and adaptive immune responses, such as dendritic cells, monocytes/macrophages and NK cells, on the one hand, and B and T lymphocytes, on the other, contribute to the vascular and kidney injury in hypertension. Unconventional innate‐like T cells such as γδ T cells also participate in hypertensive mechanisms by priming both innate and adaptive immune cells, contributing to trigger vascular inflammation and BP elevation. These cells exert their effects in part via production of various cytokines including pro‐inflammatory IFN‐γ and IL‐17 and anti‐inflammatory IL‐10. The present review summarizes some of these immune mechanisms that participate in the pathophysiology of hypertension. Linked Articles This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc
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The superoxide-generating enzyme Nox2 contributes to hypertension and cardiovascular remodeling triggered by activation of the renin-angiotensin system. Multiple Nox2-expressing cells are implicated in angiotensin II (AngII)-induced pathophysiology, but the importance of Nox2 in leukocyte subsets is poorly understood. Here, we investigated the role of Nox2 in T cells, particularly Tregs. Mice globally deficient in Nox2 displayed increased numbers of Tregs in the heart at baseline whereas AngII-induced T-effector cell (Teffs) infiltration was inhibited. To investigate the role of Treg Nox2, we generated a mouse line with CD4-targeted Nox2 deficiency (Nox2fl/flCD4Cre+). These animals showed inhibition of AngII-induced hypertension and cardiac remodeling related to increased tissue-resident Tregs and reduction in infiltrating Teffs, including Th17 cells. The protection in Nox2fl/flCD4Cre+ mice was reversed by anti-CD25 Ab-depletion of Tregs. Mechanistically, Nox2-/y Tregs showed higher in vitro suppression of Teffs proliferation than WT Tregs, increased nuclear levels of FoxP3 and NF-κB, and enhanced transcription of CD25, CD39, and CD73. Adoptive transfer of Tregs confirmed that Nox2-deficient cells had greater inhibitory effects on AngII-induced heart remodeling than WT cells. These results identify a previously unrecognized role of Nox2 in modulating suppression of Tregs, which acts to enhance hypertension and cardiac remodeling.
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Two distinct macrophage phenotypes contribute to kidney injury and repair during the progression of renal interstitial fibrosis; proinflammatory (M1) and antiinflammatory (M2) macrophages. Legumain, an asparaginyl endopeptidase of the cysteine protease family, is overexpressed in macrophages in some pathological conditions. However, the macrophage subtype and function of macrophage-derived legumain remains unclear. To resolve this we tested whether M2 macrophages contribute to the accumulation of legumain in the unilateral ureteral obstruction model. Legumain-null mice exhibited more severe fibrotic lesions after obstruction compared with wild-type control. In vitro, IL4-stimulated M2 polarization led to the overexpression and secretion of legumain. The levels of fibronectin and collagen I/III, major components of the extracellular matrix, were reduced in the conditioned medium of TGF-β1-stimulated tubular epithelial cells or fibroblasts after treatment with legumain or conditioned medium from IL4-stimulated macrophages. Administration of the legumain inhibitor RR-11a exacerbated fibrotic lesions following obstruction. Therapeutically, adoptive transfer of legumain-overexpressing macrophages or IL4-stimulated macrophages ameliorated the deposition of collagen and fibronectin induced by ureteral obstruction, either in the wild-type mice or in lgmn-/- mice. Thus, M2 macrophages overexpress and secret legumain and legumain mediates the anti-fibrotic effect of M2 macrophages in obstructive nephropathy.
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Rationale: Inflammation and immunity play crucial roles in the development of hypertension. Complement activation-mediated innate immune response is involved in the regulation of hypertension and target-organ damage. However, whether complement-mediated T-cell functions could regulate blood pressure elevation in hypertension is still unclear. Objective: We aim to determine whether C3aR (complement component 3a receptor) and C5aR (complement component 5a receptor) could regulate blood pressure via modulating regulatory T cells (Tregs). Methods and results: We showed that angiotensin II (Ang II)-induced hypertension resulted in an elevated expression of C3aR and C5aR in Foxp3 (forkhead box P3)+ Tregs. By using C3aR and C5aR DKO (double knockout) mice, we showed that C3aR and C5aR deficiency together strikingly decreased both systolic and diastolic blood pressure in response to Ang II compared with WT (wild type), single C3aR-deficient (C3aR-/-), or C5aR-deficient (C5aR-/-) mice. Flow cytometric analysis showed that Ang II-induced Treg reduction in the kidney and blood was also blocked in DKO mice. Histological analysis indicated that renal and vascular structure remodeling and damage after Ang II treatment were attenuated in DKO mice compared with WT mice. In vitro, Ang II was able to stimulate C3aR and C5aR expression in cultured CD4+CD25+ natural Tregs. CD3 and CD28 antibody stimuli downregulated Foxp3 expression in WT but not DKO Tregs. More important, depletion of Tregs with CD25 antibody abolished the protective effects against Ang II-induced hypertension and target-organ damage in DKO mice. Adoptive transfer of DKO Tregs showed much more profound protective effects against Ang II-induced hypertension than WT Treg transfer. Furthermore, we demonstrated that C5aR expression in Foxp3+ Tregs was higher in hypertensive patients compared with normotensive individuals. Conclusions: C3aR and C5aR DKO-mediated Treg function prevents Ang II-induced hypertension and target-organ damage. Targeting C3aR and C5aR in Tregs specifically may be an alternative novel approach for hypertension treatment.
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We previously showed that angiotensin II (Ang II) increases T cell production of IL-17A, and that mice deficient in IL-17A have blunted hypertension and attenuated renal and vascular dysfunction. It was recently shown that salt enhances IL-17A production from CD4+ T cells via a serum- and glucocorticoid-regulated kinase 1-dependent (SGK1-dependent) pathway. Thus, we tested the hypothesis that SGK1 signaling in T cells promotes hypertension and contributes to end-organ damage. We show that loss of T cell SGK1 results in a blunted hypertensive response to Ang II infusion by 25 mmHg. Importantly, renal and vascular inflammation is abrogated in these mice compared with control mice. Furthermore, mice lacking T cell SGK1 are protected from Ang II-induced endothelial dysfunction and renal injury. Loss of T cell SGK1 also blunts blood pressure and vascular inflammation in response to deoxycorticosterone acetate-salt (DOCA-salt) hypertension. Finally, we demonstrate that the Na+-K+-2Cl- cotransporter 1 (NKCC1) is upregulated in Th17 cells and is necessary for the salt-induced increase in SGK1 and the IL-23 receptor. These studies demonstrate that T cell SGK1 and NKCC1 may be novel therapeutic targets for the treatment of hypertension and identify a potentially new mechanism by which salt contributes to hypertension.
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Objective: To examine whether the long-term resting heart rate (RHR) pattern can predict the risk of having arterial stiffness in a large ongoing cohort study. Approach and results: This community-based cohort included 12 554 participants in the Kailun study, who were free of myocardial infarction, stroke, arrhythmia, and cancer. We used latent mixture modeling to identify RHR trajectories in 2006, 2008, and 2010. We used multivariate linear regression model to examine the association between RHR trajectory patterns and the risk of having arterial stiffness, which was assessed by brachial-ankle pulse wave velocity in 2010 to 2016. We adjusted for possible confounding factors, including socioeconomic status, lifestyle factors, use of medications, comorbidities, and serum concentrations of lipids, glucose, and high-sensitivity C-reactive proteins. We identified 5 distinct RHR trajectory patterns based on their 2006 status and on the pattern of change during 2006 to 2010 (low-stable, moderate-stable, moderate-increasing, elevated-decreasing, and elevated-stable). We found that individuals with elevated-stable RHR trajectory pattern had the highest brachial-ankle pulse wave velocity value and individuals with the low-stable RHR trajectory pattern had the lowest value (adjusted mean difference=157 cm/s; P<0.001). Adjusted odds ratio for risk of having arterial stiffness (brachial-ankle pulse wave velocity ≥1400 cm/s) was 4.14 (95% confidence interval, 2.61-6.57) relative to these 2 extreme categories. Consistently, a higher average RHR, a higher annual RHR increase rate, and a higher RHR variability were all associated with a higher risk of having arterial stiffness. Conclusions: Long-term RHR pattern is a strong predictor of having arterial stiffness.
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Background: -The recruitment of leukocytes to the vascular wall is a key step in hypertension development. Chemokine receptor CXCR2 mediates inflammatory cell chemotaxis in several diseases, however, the role of CXCR2 in hypertension development and the underlying mechanisms remain unknown. Methods: -Angiotensin II (490 ng·kg(-1)·min(-1)) or DOCA-salt-induced mouse hypertensive models in genetic ablation, pharmacological inhibition of CXCR2 and adoptive bone marrow transfer mice were used to determine the role of CXCR2 in hypertension (measured by radiotelemetry and tail-cuff system), inflammation (verified by flow cytometry and quantitative real-time PCR analysis), vascular remodeling (studied by haematoxylin and eosin (H&E) and Masson's trichrome staining), vascular dysfunction (assessed by aortic ring), and oxidative stress (indicated by NADPH oxidase activity, DHE staining and quantitative real-time PCR analysis). Moreover, the blood CXCR2(+) cells in normotensive controls and hypertension patients were analyzed by flow cytometry. Results: -Angiotensin II significantly up-regulated the expression of CXCR2 mRNA and protein, and increased the number of CD45(+) CXCR2(+) cells in mouse aorta (n=8 per group). Selective CXCR2 knockout (CXCR2(-/-)) or pharmacological inhibition of CXCR2 markedly reduced angiotensin II- or DOCA-salt-induced blood pressure elevation, aortic thickness and collagen deposition, accumulation of pro-inflammatory cells into the vascular wall, and the expression of cytokines (n=8 per group). CXCR2 inhibition also ameliorated angiotensin II-induced vascular dysfunction and reduced vascular superoxide formation, NADPH activity and the expression of NADPH oxidase subunits (n=6 per group). Bone marrow reconstitution of wild-type (WT) mice with CXCR2(-/-) bone marrow cells also significantly abolished angiotensin II-induced responses (n=6 per group). Importantly, CXCR2 blockade reversed established hypertension induced by angiotensin II or DOCA-salt challenge (n=10 per group). Furthermore, we demonstrated that CXCR2(+) pro-inflammatory cells were higher in hypertensive patients (n=30) compared with normotensive individuals (n=20). Conclusions: -Infiltration of CXCR2(+) cells plays a pathogenic role in arterial hypertension and vascular dysfunction. Inhibition of CXCR2 pathway may represent a novel therapeutic approach to treat hypertension.
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CD4(+)CD25(+)FoxP3(+) thymic-derived regulatory T cells (tTregs) are indispensable for maintaining immune system equilibrium. Adoptive transfer of tTregs is an effective means of suppressing graft-versus-host disease (GVHD) in murine models and in early human clinical trials. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an ubiquitin-conjugating enzyme that mediates nuclear factor κB (NF-κB) activation, plays an essential role in modulating regulatory T cell survival and function. MicroRNAs (miRNAs) are noncoding RNAs, which mediate RNA silencing and posttranscriptional gene repression. By performing comprehensive TaqMan Low Density Array miRNA assays, we identified 10 miRNAs differentially regulated in human tTreg compared with control T cells. One candidate, miR-146b, is preferentially and highly expressed in human naive tTregs compared with naive CD4 T cells. miRNA prediction software revealed that TRAF6 was the one of the top 10 scored mRNAs involved tTreg function with the highest probability as a potential miR-146b target. Antagomir-mediated knockdown of miRNA-146b, but not another miRNA-146 family member (miRNA-146a), enhanced TRAF6 expression. TRAF6, in turn, increases NF-κB activation, which is essential for tTreg function as well as Foxp3 protein and antiapoptotic gene expression, and downregulates proapoptotic gene expression. miR-146b knockdown increased the nuclear localization and expression of genes regulated by NF-κB, which was associated with enhanced tTreg survival, proliferation, and suppressive function measured in vitro and in vivo. TRAF6 inhibition had the opposite effects. We conclude that an miR-146b-TRAF6-NF-κB-FoxP3 signaling pathway restrains regulatory T cell survival, proliferation, and suppressor function. In vitro exposure of human tTregs to miR-146b antagomirs can be exploited to improve the clinical efficacy of human adoptive tTreg transfer in a GVHD setting.
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Rationale: Accumulating evidence supports a role of adaptive immunity and particularly T cells in the pathogenesis of hypertension. Formation of memory T cells, which requires the co-stimulatory molecule CD70 on antigen presenting cells, is a cardinal feature of adaptive immunity. Objective: To test the hypothesis that CD70 and immunological memory contribute to the blood pressure elevation and renal dysfunction mediated by repeated hypertensive challenges. Methods and results: We imposed repeated hypertensive challenges using either L-NAME/high salt or repeated ang II stimulation in mice. During these challenges effector memory T (TEM) cells accumulated in the kidney and bone marrow. In the L-NAME/high salt model, memory T cells of the kidney were predominant sources of IFN-γ and IL-17A, known to contribute to hypertension. L-NAME/high salt increased macrophage and dendritic cell surface expression of CD70 by 3 to 5-fold. Mice lacking CD70 did not accumulate TEM cells and did not develop hypertension to either high salt or the second ang II challenge and were protected against renal damage. Bone marrow residing TEM cells proliferated and redistributed to the kidney in response to repeated salt feeding. Adoptively transferred TEM cells from hypertensive mice homed to the bone marrow and spleen and expanded upon salt feeding of the recipient mice. Conclusions: Our findings illustrate a previously undefined role of CD70 and long-lived TEM cells in the development of blood pressure elevation and end-organ damage that occur upon delayed exposure to mild hypertensive stimuli. Interventions to prevent repeated hypertensive surges could attenuate formation of hypertension-specific TEM cells.
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Background: Mimecan plays an important role in endothelial and vascular smooth muscle cell integrity and may be involved the pathology of arterial stiffness. However, the role of mimecan in arterial stiffness in patients with hypertension is not well defined. Methods and results: A total of 116 hypertension patients and 54 healthy controls were enrolled in the investigation. Hypertensive patients were divided into 2 groups: the with arterial stiffness group (brachial-ankle pulse wave velocity [baPWV] ≥1400 cm/s; n=83) and the without arterial stiffness group (baPWV <1400 cm/s; n=33). A noninvasive measure of vascular stiffness was performed using pulse wave velocity (PWV) measurement of baPWV. Hypertensive patients had higher baPWV, mimecan, and endothelin 1 (ET-1) than healthy controls. The arterial stiffness group had higher mimecan and endothelin 1 (ET-1) and lower ankle-brachial pressure index (ABI) than those without stiffness. In hypertensive patients, mimecan was inversely correlated with ABI (P<0.05) and positively correlated with baPWV, ET-1, and total cholesterol. On multivariable logistic regression analysis, diastolic blood pressure, mimecan, ET-1, and creatinine were independent predictors of arterial stiffness in hypertensive patients (P<0.05). Conclusions: Mimecan levels are higher in hypertensive patients than in healthy controls. Increased plasma mimecan levels are independently associated with increased arterial stiffness as assessed by baPWV.
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Background: Dysregulated BMP (bone morphogenetic protein) or TGF-β (transforming growth factor beta) signaling pathways are imperative in idiopathic and familial pulmonary arterial hypertension (PAH) as well as experimental pulmonary hypertension (PH) in rodent models. MED1 (mediator 1) is a key transcriptional co-activator and KLF4 (Krüppel-like factor 4) is a master transcription factor in endothelium. However, MED1 and KLF4 epigenetic and transcriptional regulations of the BMP/TGF-β axes in pulmonary endothelium and their dysregulations leading to PAH remain elusive. We investigate the MED1/KLF4 co-regulation of the BMP/TGF-β axes in endothelium by studying the epigenetic regulation of BMPR2 (BMP receptor type II), ETS-related gene (ERG), and TGFBR2 (TGF-β receptor 2) and their involvement in the PH. Methods: High-throughput screening involving data from RNA-seq, MED1 ChIP-seq, H3K27ac ChIP-seq, KLF4 ATAC-seq, and high-throughput chromosome conformation capture together with in silico computations were used to explore the epigenetic and transcriptional regulation of BMPR2, ERG, and TGFBR2 by MED1 and KLF4. In vitro experiments with cultured pulmonary arterial endothelial cells and bulk assays were used to validate results from these in silico analyses. Lung tissue from patients with idiopathic PAH, animals with experimental PH, and mice with endothelial ablation of MED1 (EC-MED1-/-) were used to study the PH-protective effect of MED1. Results: Levels of MED1 were decreased in lung tissue or pulmonary arterial endothelial cells from idiopathic PAH patients and rodent PH models. Mechanistically, MED1 acted synergistically with KLF4 to transactivate BMPR2, ERG, and TGFBR2 via chromatin remodeling and enhancer-promoter interactions. EC-MED1-/- mice showed PH susceptibility. In contrast, MED1 overexpression mitigated the PH phenotype in rodents. Conclusions: A homeostatic regulation of BMPR2, ERG, and TGFBR2 in ECs by MED1 synergistic with KLF4 is essential for the normal function of the pulmonary endothelium. Dysregulation of MED1 and the resulting impairment of the BMP/TGF-β signaling is implicated in the disease progression of PAH in humans and PH in rodent models.
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Mineralocorticoid receptor (MR) is a classic nuclear receptor and an effective drug target in the cardiovascular system. The function of MR in immune cells such as macrophages and T cells has been increasingly appreciated. The aim of this study was to investigate the function of Treg MR in the process of inflammatory bowel disease (IBD). We treated Treg MR‐deficient (MRflox/floxFoxp3YFP‐Cre, KO) mice and control (Foxp3YFP‐Cre, WT) mice with dextran sodium sulphate (DSS) to induce colitis and found that the severity of DSS‐induced colitis was markedly alleviated in Treg MR‐deficient mice, accompanied by reduced production of inflammatory cytokines, and relieved infiltration of monocytes, neutrophils and interferon γ+ T cells in colon lamina propria. Fecal microbiota of mice with colitis was analyzed by 16S rRNA gene sequencing and the composition of gut microbiota was vastly changed in Treg MR‐deficient mice. Furthermore, depletion of gut microbiota by antibiotics abolished the protective effects of Treg MR deficiency and resulted in similar severity of DSS‐induced colitis in WT and KO mice. Fecal microbiota transplantation from KO mice attenuated DSS‐induced colitis characterized by alleviated inflammatory infiltration compared to that from WT mice. Hence, our study demonstrates that Treg MR deficiency protects against DSS‐induced colitis by attenuation of colonic inflammatory infiltration. Gut microbiota is both sufficient and necessary for Treg MR deficiency to exert the beneficial effects.
Article
Background: The development of thoracic aortic dissection (TAD) is closely related to the extracellular matrix (ECM) degradation and the vascular smooth muscle cell (VSMC) transformation from contractile to synthetic type. Legumain (Lgmn) degrades ECM components directly or by activating downstream signals. However, the role of Lgmn in the VSMC differentiation and the occurrence of TAD remains elusive. Methods: Microarray datasets concerning vascular dissection or aneurysm were downloaded from the Gene Expression Omnibus (GEO) database to screen differentially expressed genes (DEGs). Four-week-old male Lgmn knockout mice (Lgmn -/- ), macrophage-specific Lgmn knockout mice (Lgmn F/F ; LysM Cre ) and RR-11a treated C57BL/6 mice were given β-aminopropionitrile monofumarate (BAPN, 1g/kg/day) in drinking water for four weeks for TAD modeling. RNA-Seq analysis was performed to recapitulate transcriptome profile changes. Cell interaction was examined in macrophage and VSMC coculture system. The reciprocity of macrophage derived Lgmn with integrin αvβ3 in VSMCs was tested by co-immunoprecipitation assay and colocalization analyses. Results: Microarray datasets from the GEO database indicated up-regulated Lgmn in aorta from TAD patients and Ang II induced aortic abdominal aneurysm (AAA) mice. Elevated Lgmn was evidenced in the aortas and serums from TAD patients and BAPN-induced TAD mice in this study. BAPN induced TAD progression was significantly ameliorated in Lgmn deficient or inhibited mice. Macrophage specific deletion of Lgmn alleviated BAPN induced ECM degradation. Unbiased profiler PCR-array and GO analysis displayed that Lgmn regulated VSMC phenotype transformation. Macrophage specific deletion of Lgmn ameliorated VSMC phenotypic switch in BAPN treated mice. Macrophage derived Lgmn inhibited VSMC differentiation in vitro as assessed by macrophages and VSMCs coculture system. Mechanically, macrophage derived Lgmn bound to integrin αvβ3 in VSMCs and blocked integrin αvβ3, therefore attenuating Rho GTPase activation, down-regulating VSMCs differentiation markers and eventually exacerbating TAD development. Rho-kinase (ROCK) inhibitor Y-27632 reversed the protective role of Lgmn depletion in vascular dissection. Conclusions: These findings indicate that Lgmn signaling may be a novel target for the prevention and treatment of TAD.
Article
Shohei Hori recalls the early work that led to the identification of FOXP3 as the master regulator of regulatory T cells.
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High blood pressure is one of the most important risk factors for ischaemic heart disease, stroke, other cardiovascular diseases, chronic kidney disease and dementia. Mean blood pressure and the prevalence of raised blood pressure have declined substantially in high-income regions since at least the 1970s. By contrast, blood pressure has risen in East, South and Southeast Asia, Oceania and sub-Saharan Africa. Given these trends, the prevalence of hypertension is now higher in low-income and middle-income countries than in high-income countries. In 2015, an estimated 8.5 million deaths were attributable to systolic blood pressure >115 mmHg, 88% of which were in low-income and middle-income countries. Measures such as increasing the availability and affordability of fresh fruits and vegetables, lowering the sodium content of packaged and prepared food and staples such as bread, and improving the availability of dietary salt substitutes can help lower blood pressure in the entire population. The use and effectiveness of hypertension treatment vary substantially across countries. Factors influencing this variation include a country’s financial resources, the extent of health insurance and health facilities, how frequently people interact with physicians and non-physician health personnel, whether a clear and widely adopted clinical guideline exists and the availability of medicines. Scaling up treatment coverage and improving its community effectiveness can substantially reduce the health burden of hypertension.
Article
Background Hydrogen sulfide (H 2 S) has antihypertension and anti-inflammatory effects, and its endogenous-generation key enzyme cystathionine γ lyase (CSE) is expressed in CD4 ⁺ T cells. However, the role of CD4 ⁺ T-cell endogenous CSE/H 2 S in the development of hypertension is unclear. Methods Peripheral blood lymphocytes were isolated from hypertensive patients or spontaneously hypertensive rats, then H 2 S production and expression of its generation enzymes, cystathionine β synthase and CSE, were measured to determine the major H 2 S generation system changes in hypertension. Mice with CSE-specific knockout in T cells (conditional knockout, by CD4 cre mice hybridization) and CD4 null mice were generated for investigating the pathophysiological relevance of the CSE/H 2 S system. Results In lymphocytes, H 2 S from CSE, but not cystathionine β synthase, responded to blood pressure changes, supported by lymphocyte CSE protein changes and a negative correlation between H 2 S production with systolic blood pressure and diastolic blood pressure, but positive correlation with the serum level of interleukin 10 (an anti-inflammatory cytokine). Deletion of CSE in T cells elevated BP (5–8 mm Hg) under the physiological condition and exacerbated angiotensin II–induced hypertension. In keeping with hypertension, mesenteric artery dilation impaired association with arterial inflammation, an effect attributed to reduced immunoinhibitory T regulatory cell (Treg) numbers in the blood and kidney, thus causing excess CD4 ⁺ and CD8 ⁺ T cell infiltration in perivascular adipose tissues and kidney. CSE knockout CD4 ⁺ T cell transfer into CD4 null mice, also showed the similar phenotypes’ confirming the role of endogenous CSE/H 2 S action. Adoptive transfer of Tregs (to conditional knockout mice) reversed hypertension, vascular relaxation impairment, and immunocyte infiltration, which confirmed that conditional knockout–induced hypertension was attributable, in part, to the reduced Treg numbers. Mechanistically, endogenous CSE/H 2 S promoted Treg differentiation and proliferation by activating AMP-activated protein kinase. In part, it depended on activation of its upstream kinase, liver kinase B1, by sulfhydration to facilitate its substrate binding and phosphorylation. Conclusion The constitutive sulfhydration of liver kinase B1 by CSE-derived H 2 S activates its target kinase, AMP-activated protein kinase, and promotes Treg differentiation and proliferation, which attenuates the vascular and renal immune-inflammation, thereby preventing hypertension.
Article
Rationale: Hypoxia promotes renal damage and progression of chronic kidney disease (CKD). The erythrocyte is the only cell type for oxygen (O2) delivery. Sphingosine 1-phosphate (S1P)-a highly enriched biolipid in erythrocytes-is recently reported to be induced under high altitude in normal humans to enhance O2 delivery. However, nothing is known about erythrocyte S1P in CKD. Objective: To investigate the function and metabolic basis of erythrocyte S1P in CKD with a goal to explore potential therapeutics. Methods and results: Using erythrocyte-specific SphK1 (sphingosine kinase 1; the only enzyme to produce S1P in erythrocytes) knockout mice (eSphK1 -/- ) in an experimental model of hypertensive CKD with Ang II (angiotensin II) infusion, we found severe renal hypoxia, hypertension, proteinuria, and fibrosis in Ang II-infused eSphk1 -/- mice compared with controls. Untargeted metabolomics profiling and in vivo U-13C6 isotopically labeled glucose flux analysis revealed that SphK1 is required for channeling glucose metabolism toward glycolysis versus pentose phosphate pathway, resulting in enhanced erythroid-specific Rapoport-Luebering shunt in Ang II-infused mice. Mechanistically, increased erythrocyte S1P functioning intracellularly activates AMPK (AMP-activated protein kinase) 1α and BPGM (bisphosphoglycerate mutase) by reducing ceramide/S1P ratio and inhibiting PP2A (protein phosphatase 2A), leading to increased 2,3-bisphosphoglycerate (an erythrocyte-specific metabolite negatively regulating Hb [hemoglobin]-O2-binding affinity) production and thus more O2 delivery to counteract kidney hypoxia and progression to CKD. Preclinical studies revealed that an AMPK agonist or a PP2A inhibitor rescued the severe CKD phenotype in Ang II-infused eSphK1 -/- mice and prevented development of CKD in the control mice by inducing 2,3-bisphosphoglycerate production and thus enhancing renal oxygenation. Translational research validated mouse findings in erythrocytes of hypertensive CKD patients and cultured human erythrocytes. Conclusions: Our study elucidates the beneficial role of eSphk1-S1P in hypertensive CKD by channeling glucose metabolism toward Rapoport-Luebering shunt and inducing 2,3-bisphosphoglycerate production and O2 delivery via a PP2A-AMPK1α signaling pathway. These findings reveal the metabolic and molecular basis of erythrocyte S1P in CKD and new therapeutic avenues.
Article
Background: Blood pressure (BP) often rises with aging, but exact mechanisms are still not completely understood. With aging, the level of pro-inflammatory cytokines increases in T lymphocyte. Prostaglandin (PG) D 2 , a pro-resolution mediator, suppresses Th1 cytokines through D-prostanoid receptor 1 (DP1). In this study, we aimed to investigate the role of PGD 2 /DP1 axis in T cells on age-related hypertension. Methods: To clarify the physiological and pathophysiological roles of DP1 in T cells with aging, peripheral blood samples were collected from young and older male participants and CD4 ⁺ T cells were sorted for gene expression, PG production and western bot assays. Mice BP was quantified by invasive telemetric monitor. Results: PGD 2 /DP1 axis was down-regulated in CD4 ⁺ T cells from older humans and aged mice, DP1 deletion in CD4+ T cells (TDP1KO) augmented age-related hypertension in aged male mice by enhancing Th1 cytokine secretion, vascular remodeling and CD4 ⁺ T cells infiltration, superoxide production in vasculature and kidneys. Conversely, forced expression of exogenous DP1 in T cells retarded age-associated hypertension in mice by reducing Th1 cytokine secretion. TNFα neutralization or IFNγ deletion ameliorated the age-related hypertension in TDP1KO mice. Mechanistically, DP1 inhibited Th1 activity via the Gαs/PKA/p-Sp1/NEDD4L pathway-mediated T-bet ubiquitination. T-bet deletion or forced NEDD4L expression in CD4 ⁺ T cell attenuated age-related hypertension in TDP1KO mice. DP1 receptor activation by BW245C prevented age-associated BP elevation and reduced vascular/renal superoxide production in male mice. Conclusions: PGD 2 /DP1 axis suppresses age-related Th1 activation and subsequent hypertensive response in male mice through increase of NEDD4L-mediated T-bet degradation by ubiquitination. Therefore, the T cell DP1 receptor may be an attractive therapeutic target for age-related hypertension in males.
Article
Background: The relationship between circulating regulatory T-cell (Tregs) subset distribution and hypertension severity in children with primary hypertension is not known. We aimed to find out if target organ damage (TOD) in children with primary hypertension is related to defects in Tregs distribution reflected by their phenotype characteristics. Methods: The study constituted 33 nontreated hypertensive children and 35 sex-matched and age-matched controls. Using multicolor flow cytometry technique, we assessed a distribution of the total Tregs (CD4CD25CD127) and their subsets (CD45RA-naive Tregs, CD45RA memory/activated Tregs, CD45RACD31 recent thymic emigrants Tregs and mature naive CD45RACD31 Tregs) in the whole blood. Results: Hypertensive children showed decreased percentage of the total Tregs, the CD45RA-naive Tregs, the total CD31 Tregs and the recent thymic emigrants Tregs but elevation of the CD45RA memory/activated Treg and mature naive CD45RACD31 Tregs. Decreased frequency of the total Tregs, naive Tregs and CD31-bearing Treg cell subsets (CD31 total Tregs, CD45RACD31 recent thymic emigrants Tregs) negatively correlated to TOD markers, arterial stiffness and blood pressure elevation. In contrast, increased percentage of memory Tregs and CD31 Tregs subsets positively correlated to organ damage markers, arterial stiffness and blood pressure values. These changes were independent of BMI, age, sex and hsCRP. Conclusion: Both diagnosis of hypertension, TOD and arterial stiffness in hypertensive children were associated with decreased population of total CD4 Tregs, limited output of recent thymic emigrants Tregs, and increased pool of activated/memory Tregs. Hypertension was an independent predictor of the circulating Treg subsets distribution irrespective of hsCRP.
Article
Traumatic brain injury (TBI) is associated in some studies with clinical dementia, and neuropathological features, including amyloid plaque deposition and Tau neurofibrillary degeneration commonly identified in Alzheimer's disease (AD). However, the molecular mechanisms linking TBI to AD remain unclear. Here we show that TBI activates transcription factor CCAAT/Enhancer Binding Protein Beta (C/EBPβ), increasing delta-secretase (AEP) expression. Activated AEP cleaves both APP and Tau at APP N585 and Tau N368 sites, respectively, which mediate AD pathogenesis by promoting Aβ production and Tau hyperphosphorylation and inducing neuroinflammation and neurotoxicity. Knockout of AEP or C/EBPβ diminishes TBI-induced AD-like pathology and cognitive impairment in the 3xTg AD mouse model. Remarkably, viral expression of AEP-resistant Tau N368A in the hippocampus of 3xTg mice also ameliorates the pathological and cognitive consequences of TBI. Finally, clinical TBI activates C/EBPβ and escalates AEP expression, leading to APP N585 and Tau N368 proteolytic cleavage in TBI patient brains. Hence, our findings support a potential role for AEP in linking TBI exposure with AD pathogenesis.
Article
Immune mechanisms have been recognized to have a role in the pathogenesis of hypertension, vascular disease and kidney damage in humans and animals for many decades. Contemporary advances in experimentation have permitted a deeper understanding of the mechanisms by which inflammation and immunity participate in cardiovascular disease, and multiple observations have demonstrated strong correlations between the discoveries made in animals and those made in patients with hypertension. Of note, striking phenotypic similarities have been observed in the infiltration of immune cells in the kidney and the development of end-organ damage in patients and animal models with sodium-sensitive hypertension. The available data suggest that an initial salt-induced increase in renal perfusion pressure, which is likely independent of immune mechanisms, induces the infiltration of immune cells into the kidney. The mechanisms mediating immune cell infiltration in the kidney are not well understood but likely involve tissue damage, the direct influence of salt to stimulate immune cell activation, sympathetic nerve stimulation or other factors. The infiltrating cells then release cytokines, free radicals and other factors that contribute to renal damage as well as increased retention of sodium and water and vascular resistance, which lead to the further development of hypertension.
Article
Life-threatening cardiovascular events occur despite control of conventional risk factors. Inflammation, as measured by high-sensitivity C-reactive protein (hsCRP) concentration, is associated with future vascular events in both primary and secondary prevention, independent of usual risk markers. Statins are powerful lipid-lowering agents with clinically relevant anti-inflammatory effects. Recent data support targeting the interleukin (IL)-1-to-IL-6-to-CRP signaling pathway as an adjunctive method for atheroprotection. The CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcomes Study) trial showed that reducing inflammation through IL-1β inhibition significantly reduced vascular risk, beyond that achievable with lipid lowering. CANTOS further demonstrated a 31% reduction in cardiovascular mortality and all-cause mortality among patients treated with canakinumab who achieved the largest reductions in hsCRP, as well as efficacy in high-risk patients with chronic kidney disease and diabetes. This review outlines the clinical implications of CANTOS for patients with “residual inflammatory risk,” the potential benefits and risks associated with anti-inflammatory therapy, and the importance of CANTOS for future drug development.
Article
CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.
Article
Rationale: Microvascular inflammation and endothelial dysfunction secondary to unchecked activation of endothelium plays a critical role in the pathophysiology of sepsis and organ failure. The intrinsic signaling mechanisms responsible for dampening excessive activation of endothelial cells are not completely understood. Objective: To determine the central role of Yes-associated protein (YAP), the major transcriptional co-activator of the Hippo pathway, in modulating the strength and magnitude of endothelial activation and vascular inflammation. Methods and Results: Endothelial specific YAP knockout mice showed increased basal expression of E-selectin and intercellular adhesion molecule-1 in endothelial cells, a greater number of adherent neutrophils in post-capillary venules, and increased neutrophil counts in bronchoalveolar lavage fluid. Lipopolysaccharide challenge of these mice augmented nuclear factor-кB (NF-кB) activation, expression of endothelial adhesion proteins, neutrophil and monocyte adhesion to cremaster muscle venules, transendothelial neutrophil migration, and lung inflammatory injury. Deletion of YAP in endothelial cells also markedly augmented the inflammatory response and cardiovascular dysfunction in a polymicrobial sepsis model induced by cecal ligation and puncture. YAP functioned by interacting with the E3 ubiquitin-protein ligase Toll-like receptor signaling adaptor tumor necrosis factor receptor-associated factor 6 (TRAF6) to ubiquitinate TRAF6, and thus promoted TRAF6 degradation and modification resulting in inhibition of NF-кB activation. TRAF6 depletion in endothelial cells rescued the augmented inflammatory phenotype in mice with endothelial cell specific deletion of YAP. Conclusions: YAP modulates the activation of endothelial cells and suppresses vascular inflammation through preventing TRAF6-mediated NF-кB activation, and is hence essential for limiting the severity of sepsis-induced inflammation and organ failure.
Article
Chaperone-mediated autophagy (CMA) was the first studied process that indicated that degradation of intracellular components by the lysosome can be selective - a concept that is now well accepted for other forms of autophagy. Lysosomes can degrade cellular cytosol in a nonspecific manner but can also discriminate what to target for degradation with the involvement of a degradation tag, a chaperone and a sophisticated mechanism to make the selected proteins cross the lysosomal membrane through a dedicated translocation complex. Recent studies modulating CMA activity in vivo using transgenic mouse models have demonstrated that selectivity confers on CMA the ability to participate in the regulation of multiple cellular functions. Timely degradation of specific cellular proteins by CMA modulates, for example, glucose and lipid metabolism, DNA repair, cellular reprograming and the cellular response to stress. These findings expand the physiological relevance of CMA beyond its originally identified role in protein quality control and reveal that CMA failure with age may aggravate diseases, such as ageing-associated neurodegeneration and cancer.
Article
Both conventional T (Tconv) cells and regulatory T (Treg) cells are activated through ligation of the T cell receptor (TCR) complex, leading to the induction of the transcription factor NF-κB. In Tconv cells, NF-κB regulates expression of genes essential for T cell activation, proliferation, and function. However the role of NF-κB in Treg function remains unclear. We conditionally deleted canonical NF-κB members p65 and c-Rel in developing and mature Treg cells and found they have unique but partially redundant roles. c-Rel was critical for thymic Treg development while p65 was essential for mature Treg identity and maintenance of immune tolerance. Transcriptome and NF-κB p65 binding analyses demonstrated a lineage specific, NF-κB-dependent transcriptional program, enabled by enhanced chromatin accessibility. These dual roles of canonical NF-κB in Tconv and Treg cells highlight the functional plasticity of the NF-κB signaling pathway and underscores the need for more selective strategies to therapeutically target NF-κB.
Article
The immunosuppressive calcineurin inhibitors cyclosporine A and tacrolimus alter T-cell subsets and can cause hypertension, vascular dysfunction, and renal toxicity. We and others have reported that cyclosporine A and tacrolimus decrease anti-inflammatory regulatory T cells and increase proinflammatory interleukin-17-producing T cells; therefore, we hypothesized that inhibition of these effects using noncellular therapies would prevent the hypertension, endothelial dysfunction, and renal glomerular injury induced by calcineurin inhibitor therapy. Daily treatment of mice with cyclosporine A or tacrolimus for 1 week significantly decreased CD4(+)/FoxP3(+) regulatory T cells in the spleen and lymph nodes, as well as induced hypertension, vascular injury and dysfunction, and glomerular mesangial expansion in mice. Daily cotreatment with all-trans retinoic acid reported to increase regulatory T cells and decrease interleukin-17-producing T cells, prevented all of the detrimental effects of cyclosporine A and tacrolimus. All-trans retinoic acid also increased regulatory T cells and prevented the hypertension, endothelial dysfunction, and glomerular injury in genetically modified mice that phenocopy calcineurin inhibitor-treated mice (FKBP12-Tie2 knockout). Treatment with an interleukin-17-neutralizing antibody also increased regulatory T-cell levels and prevented the hypertension, endothelial dysfunction, and glomerular injury in cyclosporine A-treated and tacrolimus-treated mice and FKBP12-Tie2 knockout mice, whereas an isotype control had no effect. Augmenting regulatory T cells and inhibiting interleukin-17 signaling using noncellular therapies prevents the cardiovascular and renal toxicity of calcineurin inhibitors in mice.
Article
Rationale: Hypertension remains to be a global public health burden and demands novel intervention strategies such as targeting T cells and T-cell-derived cytokines. Mineralocorticoid receptor (MR) antagonists have been clinically used to treat hypertension. However, the function of T-cell MR in blood pressure (BP) regulation has not been elucidated. Objective: We aim to determine the role of T-cell MR in BP regulation and to explore the mechanism. Methods and results: Using T-cell MR knockout mouse in combination with angiotensin II-induced hypertensive mouse model, we demonstrated that MR deficiency in T cells strikingly decreased both systolic and diastolic BP and attenuated renal and vascular damage. Flow cytometric analysis showed that T-cell MR knockout mitigated angiotensin II-induced accumulation of interferon-gamma (IFN-γ)-producing T cells, particularly CD8(+) population, in both kidneys and aortas. Similarly, eplerenone attenuated angiotensin II-induced elevation of BP and accumulation of IFN-γ-producing T cells in wild-type mice. In cultured CD8(+) T cells, T-cell MR knockout suppressed IFN-γ expression whereas T-cell MR overexpression and aldosterone both enhanced IFN-γ expression. At the molecular level, MR interacted with NFAT1 (nuclear factor of activated T-cells 1) and activator protein-1 in T cells. Finally, T-cell MR overexpressing mice manifested more elevated BP compared with control mice after angiotensin II infusion and such difference was abolished by IFN-γ-neutralizing antibodies. Conclusions: MR may interact with NFAT1 and activator protein-1 to control IFN-γ in T cells and to regulate target organ damage and ultimately BP. Targeting MR in T cells specifically may be an effective novel approach for hypertension treatment.
Article
Inflammation is essential in the initial development and progression of many cardiovascular diseases involving innate and adaptive immune responses. The role of CD4(+)CD25(+)FOXP3(+) regulatory T (TREG) cells in the modulation of inflammation and immunity has received increasing attention. Given the important role of TREG cells in the induction and maintenance of immune homeostasis and tolerance, dysregulation in the generation or function of TREG cells can trigger abnormal immune responses and lead to pathology. A wealth of evidence from experimental and clinical studies has indicated that TREG cells might have an important role in protecting against cardiovascular disease, in particular atherosclerosis and abdominal aortic aneurysm. In this Review, we provide an overview of the roles of TREG cells in the pathogenesis of a number of cardiovascular diseases, including atherosclerosis, hypertension, ischaemic stroke, abdominal aortic aneurysm, Kawasaki disease, pulmonary arterial hypertension, myocardial infarction and remodelling, postischaemic neovascularization, myocarditis and dilated cardiomyopathy, and heart failure. Although the exact molecular mechanisms underlying the cardioprotective effects of TREG cells are still to be elucidated, targeted therapies with TREG cells might provide a promising and novel future approach to the prevention and treatment of cardiovascular diseases.
Article
Legumain (EC 3.4.22.34) is an asparaginyl endopeptidase. Strong legumain activity was observed in the mouse kidney, and legumain was highly expressed in tumors. We previously reported that bovine kidney annexin A2 was co-purified with legumain and that legumain cleaved the N-terminal region of annexin A2 at an Asn residue in vitro and in vivo. Recently, we found that transcription of the legumain gene is regulated by the p53 tumor suppressor in HCT116 cells. We and others reported that DJ-1/PARK7, a cancer- and Parkinson's disease-associated protein, works as a coactivator to various transcription factors, including the androgen receptor, p53, PSF, Nrf2, SREBP and RREB1. In this study, we found that expression levels of legumain mRNA and protein and legumain activity were increased in DJ-1-knockout cells. Furthermore, we found that DJ-1 binds to the p53-binding site on intron 1 of the mouse legumain gene in wild-type cells and that cleavage of annexin A2 was increased in DJ-1-knockout cells. These results suggest that legumain expression and activation and cleavage of annexin A2 are regulated by DJ-1 through p53.