SMYD2-Methylated PPARγ Facilitates Hypoxia-Induced Pulmonary Hypertension by Activating Mitophagy.

BACKGROUND: Hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs) and consequent pulmonary vascular remodeling are the crucial pathological features of pulmonary hypertension (PH). Protein methylation has been shown to be critically involved in PASMC proliferation and PH, but the underlying mechanism remains largely unknown. METHODS: PH animal models were generated by treating mice/rats with chronic hypoxia for 4 weeks. SMYD2-vTg mice (vascular smooth muscle cell-specific suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 (deformed epidural auto-regulatory factor-1) domain-containing protein 2 transgenic) or wild-type rats and mice treated with LLY-507 (3-cyano-5-{2-[4-[2-(3-methylindol-1-yl)ethyl]piperazin-1-yl]-phenyl}-N-[(3-pyrrolidin-1-yl)propyl]benzamide) were used to investigate the function of SMYD2 (suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 domain-containing protein 2) on PH development in vivo. Primary cultured rat PASMCs with SMYD2 knockdown or overexpression were used to explore the effects of SMYD2 on proliferation and to decipher the underlying mechanism. RESULTS: We demonstrated that the expression of the lysine methyltransferase SMYD2 was upregulated in the smooth muscle cells of pulmonary arteries from patients with PH and hypoxia-exposed rats/mice and in the cytoplasm of hypoxia-induced rat PASMCs. More importantly, targeted inhibition of SMYD2 by LLY-507 significantly attenuated hypoxia-induced pulmonary vascular remodeling and PH development in both male and female rats in vivo and reduced rat PASMC hyperproliferation in vitro. In contrast, SMYD2-vTg mice exhibited more severe PH phenotypes and related pathological changes than nontransgenic mice after 4 weeks of chronic hypoxia treatment. Furthermore, SMYD2 overexpression promoted, while SMYD2 knockdown suppressed, the proliferation of rat PASMCs by affecting the cell cycle checkpoint between S and G2 phases. Mechanistically, we revealed that SMYD2 directly interacted with and monomethylated PPARγ (peroxisome proliferator-activated receptor gamma) to inhibit the nuclear translocation and transcriptional activity of PPARγ, which further promoted mitophagy to facilitate PASMC proliferation and PH development. Furthermore, rosiglitazone, a PPARγ agonist, largely abolished the detrimental effects of SMYD2 overexpression on PASMC proliferation and PH. CONCLUSIONS: Our results demonstrated that SMYD2 monomethylates nonhistone PPARγ and inhibits its nuclear translocation and activation to accelerate PASMC proliferation and PH by triggering mitophagy, indicating that targeting SMYD2 or activating PPARγ are potential strategies for the prevention of PH.

Histone acetyltransferase P300 deficiency promotes ferroptosis of vascular smooth muscle cells by activating the HIF-1α/HMOX1 axis.

BACKGROUND: E1A-associated 300-kDa protein (P300), an endogenous histone acetyltransferase, contributes to modifications of the chromatin landscape of genes involved in multiple cardiovascular diseases. Ferroptosis of vascular smooth muscle cells (VSMCs) is a novel pathological mechanism of aortic dissection. However, whether P300 regulates VSMC ferroptosis remains unknown. METHODS: Cystine deprivation (CD) and imidazole ketone erastin (IKE) were used to induce VSMC ferroptosis. Two different knockdown plasmids targeting P300 and A-485 (a specific inhibitor of P300) were used to investigate the function of P300 in the ferroptosis of human aortic smooth muscle cells (HASMCs). Cell counting kit-8, lactate dehydrogenase and flow cytometry with propidium iodide staining were performed to assess the cell viability and death under the treatment of CD and IKE. BODIPY-C11 assay, immunofluorescence staining of 4-hydroxynonenal and malondialdehyde assay were conducted to detect the level of lipid peroxidation. Furthermore, co-immunoprecipitation was utilized to explore the interaction between P300 and HIF-1α, HIF-1α and P53. RESULTS: Compared with normal control, the protein level of P300 was significantly decreased in HASMCs treated with CD and IKE, which was largely nullified by the ferroptosis inhibitor ferrostatin-1 but not by the autophagy inhibitor or apoptosis inhibitor. Knockdown of P300 by short-hairpin RNA or inhibition of P300 activity by A-485 promoted CD- and IKE-induced HASMC ferroptosis, as evidenced by a reduction in cell viability and aggravation of lipid peroxidation of HASMCs. Furthermore, we found that hypoxia-inducible factor-1α (HIF-1α)/heme oxygenase 1 (HMOX1) pathway was responsible for the impacts of P300 on ferroptosis of HASMCs. The results of co-immunoprecipitation demonstrated that P300 and P53 competitively bound HIF-1α to regulate the expression of HMOX1. Under normal conditions, P300 interacted with HIF-1α to inhibit HMOX1 expression, while reduced expression of P300 induced by ferroptosis inducers would favor HIF-1α binding to P53 to trigger HMOX1 overexpression. Furthermore, the aggravated effects of P300 knockdown on HASMC ferroptosis were largely nullified by HIF-1α knockdown or the HIF-1α inhibitor BAY87-2243. CONCLUSION: Thus, our results revealed that P300 deficiency or inactivation facilitated CD- and IKE-induced VSMC ferroptosis by activating the HIF-1α/HMOX1 axis, which may contribute to the development of diseases related to VSMC ferroptosis.

Comprehensive analysis identified a reduction in ATP1A2 mediated by ARID3A in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is characterized by abdominal aorta dilatation and progressive structural impairment and is usually an asymptomatic and potentially lethal disease with a risk of rupture. To investigate the underlying mechanisms of AAA initiation and progression, seven AAA datasets related to human and mice were downloaded from the GEO database and reanalysed in the present study. After comprehensive bioinformatics analysis, we identified the enriched pathways associated with inflammation responses, vascular smooth muscle cell (VSMC) phenotype switching and cytokine secretion in AAA. Most importantly, we identified ATPase Na+ /K+ transporting subunit alpha 2 (ATP1A2) as a key gene that was significantly decreased in AAA samples of both human and mice; meanwhile, its reduction mainly occurred in VSMCs of the aorta; this finding was validated by immunostaining and Western blot in human and mouse AAA samples. Furthermore, we explored the potential upstream transcription factors (TFs) that regulate ATP1A2 expression. We found that the TF AT-rich interaction domain 3A (ARID3A) bound the promoter of ATP1A2 to suppress its expression. Our present study identified the ARID3A-ATP1A2 axis as a novel pathway in the pathological processes of AAA, further elucidating the molecular mechanism of AAA and providing potential therapeutic targets for AAA.

Targeting regulated cell death in aortic aneurysm and dissection therapy.

Regulated cell death (RCD) is a basic biological phenomenon associated with cell and tissue homeostasis. Recent studies have enriched our understanding of RCD, and many novel cell death types, such as ferroptosis and pyroptosis, have been discovered and defined. Aortic aneurysm and dissection (AAD) is a life-threatening condition, but the pathogenesis remains largely unclear. A series of studies have indicated that the death of smooth muscle cells, endothelial cells and inflammatory cells participates in the development of AAD and that corresponding interventions could alleviate disease progression. Many treatments against cell death have been used to impede the process of AAD in vitro and in vivo, which provides strategies to protect against this condition. In this review, we focus on various types of regulated cell death and provide a framework of their roles in AAD, and the information contributes to further exploration of the molecular mechanisms of AAD.

BRD4770 functions as a novel ferroptosis inhibitor to protect against aortic dissection.

Smooth muscle cell (SMC) loss is the characteristic feature in the pathogenesis of aortic dissection (AD), and ferroptosis is a novel iron-dependent regulated cell death driven by the excessive lipid peroxidation accumulation. However, whether targeting ferroptosis is an effective approach for SMC loss and AD treatment remains unclear. Here, we found that the iron level, ferroptosis-related molecules TFR, HOMX1, ferritin and the lipid peroxidation product 4-hydroxynonenal were increased in the aorta of AD. Then, we screened several inhibitors of histone methyltransferases and found that BRD4770 had a protective effect on cystine deprivation-, imidazole ketone erastin- or RSL3-induced ferroptosis of SMCs. The classic ferroptosis pathways, System Xc--GPX4, FSP1-CoQ10 and GCH1-BH4 pathways which were inhibited by ferroptosis inducers, were re-activated by BRD4770 via inhibiting mono-, di- and tri- methylated histone H3 at lysine 9 (H3K9me1/2/3). RNA-sequencing analysis revealed that there was a positive feedback regulation between ferroptosis and inflammatory response, and BRD4770 can reverse the effects of inflammation activation on ferroptosis. More importantly, treatment with BRD4770 attenuated aortic dilation and decreased morbidity and mortality in a ß-Aminopropionitrile monofumarate-induced mouse AD model via inhibiting the inflammatory response, lipid peroxidation and ferroptosis. Taken together, our findings demonstrate that ferroptosis is a novel and critical pathological mechanism that is involved in SMC loss and AD development. BRD4770 is a novel ferroptosis inhibitor and has equivalent protective effect to Ferrostatin-1 at the optimal concentration. Translating insights into the anti-ferroptosis effects of BRD4770 may reveal a potential therapeutic approach for targeting SMC ferroptosis in AD.

Spliced X-box Binding Protein 1 Stimulates Adaptive Growth Through Activation of mTOR.

BACKGROUND: The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart. METHODS: We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner. RESULTS: We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth. CONCLUSIONS: Our results reveal a critical role of the XBP1s-FKBP11-mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.

Protein methylation functions as the posttranslational modification switch to regulate autophagy.

Studies over the past decades have elucidated the critical role of autophagy in human health and diseases. Although the processes of autophagy in the cytoplasm have been well studied, the posttranscriptional and epigenetic regulation mechanisms of autophagy are still poorly understood. Protein methylation, including histone methylation and non-histone protein methylation, is the most important type of posttranscriptional and epigenetic modification. Recent studies have shown that protein methylation is associated with effects on autophagosome formation, autophagy-related protein expression, and signaling pathway activation, but the details are still unclear. Thus, it is important to summarize the current status and discuss the future directions of research on protein methylation in the context of autophagy.

HDAC6 is associated with the formation of aortic dissection in human.

BACKGROUND: The pathological features of aortic dissection (AD) include vascular smooth muscle cell (VSMC) loss, elastic fiber fraction, and inflammatory responses in the aorta. However, little is known about the post-translational modification mechanisms responsible for these biological processes. METHODS: A total of 72 aorta samples, used for protein detection, were collected from 36 coronary artery disease (CAD, served as the control) patients and 36 type A AD (TAAD) patients. Chromatin immunoprecipitation (ChIP)-PCR was used to identify the genes regulated by H3K23ac, and tubastatin A, an inhibitor of HDAC6, was utilized to clarify the downstream mechanisms regulated by HDAC6. RESULTS: We found that the protein level of histone deacetylase HDAC6 was reduced in the aortas of patients suffering from TAAD and that the protein levels of H4K12ac, and H3K23ac significantly increased, while H3K18ac, H4K8ac, and H4K5ac dramatically decreased when compared with CAD patients. Although H3K23ac, H3K18ac, and H4K8ac increased in the human VSMCs after treatment with the HDAC6 inhibitor tubastatin A, only H3K23ac showed the same results in human tissues. Notably, the results of ChIP-PCR demonstrated that H3K23ac was enriched in extracellular matrix (ECM)-related genes, including Col1A2, Col3A1, CTGF, POSTN, MMP2, TIMP2, and ACTA2, in the aortic samples of TAAD patients. In addition, our results showed that HDAC6 regulates H4K20me2 and p-MEK1/2 in the pathological process of TAAD. CONCLUSIONS: These results indicate that HDAC6 is involved in human TAAD formation by regulating H3K23ac, H4K20me2 and p-MEK1/2, thus, providing a strategy for the treatment of TAAD by targeting protein post-translational modifications (PTMs), chiefly histone PTMs.

Disturbed energy and amino acid metabolism with their diagnostic potential in mitral valve disease revealed by untargeted plasma metabolic profiling.

INTRODUCTION: Mitral valve disease (MVD), including mitral valve regurgitation (MR) and mitral valve stenosis (MS), is a chronic and progressive cardiac malady. However, the metabolic alterations in MVD is not well-understood till now. The current gold standard diagnostic test, transthoracic echocardiography, has limitations on high-throughput measurement and lacks molecular information for early diagnosis of the disease. OBJECTIVE: The present study aimed to investigate the biochemical alterations and to explore their diagnostic potential for MVD. METHODS: Plasma metabolic profile derangements and their diagnostic potential were non-invasively explored in 34 MR and 20 MS patients against their corresponding controls, using high-throughput NMR-based untargeted metabolomics. RESULTS: Eighteen differential metabolites were identified for MR and MS patients respectively, on the basis of multivariate and univariate data analysis, which were mainly involved in energy metabolism, amino acid metabolism, calcium metabolism and inflammation. These differential metabolites, notably the significantly down-regulated formate and lactate, showed high diagnostic potential for MVD by using Spearman's rank-order correlation analysis and ROC analysis. CONCLUSIONS: To the best of our knowledge, the present study is the first one that explores the metabolic derangements and their diagnostic values in MVD patients using metabolomics. The findings indicated that metabolic disturbance occurred in MVD patients, with plasma formate and lactate emerged as important candidate biomarkers for MVD.

Histone methyltransferase Setd2 is critical for the proliferation and differentiation of myoblasts.

Skeletal muscle cell proliferation and differentiation are tightly regulated. Epigenetic regulation is a major component of the regulatory mechanism governing these processes. Histone modification is part of the epigenetic code used for transcriptional regulation of chromatin through the establishment of an active or repressive state for genes involved in myogenesis in a temporal manner. Here, we uncovered the function of SET domain containing 2 (Setd2), an essential histone 3 lysine 36 trimethyltransferase, in regulating the proliferation and differentiation of myoblasts. Setd2 was silenced in the skeletal muscle myoblast cell line, C2C12, using the CRISPR/CAS9 system. The mutant cells exhibited defect in myotube formation. The myotube formation marker, myosin heavy chain (MHC), was downregulated earlier in Setd2 silenced cells compared to wild-type myoblasts during differentiation. The deficiency in Setd2 also resulted in repression of Myogenin (MyoG) expression, a key myogenic regulator during differentiation. In addition to the myoblast differentiation defect, decreased proliferation rate with significantly reduced levels of histone 3 phosphorylation, indicative of cell proliferation defect, were observed in the Setd2 silenced cells; suggesting an impaired proliferation phenotype. Furthermore, compromised G1/S- and G2/M-phase transition and decreased expression levels of major regulators of cell cycle G1/S checkpoints, cyclin D1, CDK4, CDK6, and cyclin E2 were detected in Setd2 silenced cells. Consistent with the cell cycle arrested phenotype, cyclin-dependent kinase inhibitor p21 was upregulated in Setd2 silenced cells. Together, this study demonstrates an essential role of Setd2 in myoblast proliferation and differentiation, and uncovers Setd2-mediated molecular mechanism through regulating MyoG and p21.

The Histone Methyltransferase Mixed Lineage Leukemia (MLL) 3 May Play a Potential Role on Clinical Dilated Cardiomyopathy.

Histone modifications play a critical role in the pathological processes of dilated cardiomyopathy (DCM). While the role and expression pattern of histone methyltransferases (HMTs), especially mixed lineage leukemia (MLL) families on DCM are unclear. To this end, twelve normal and fifteen DCM heart samples were included in the present study. A murine cardiac remodelling model was induced by transverse aortic constriction (TAC). Real-time PCR was performed to detect the expression levels of MLL families in the mouse and human left ventricles. The mRNA level of MLL3 was significantly increased in the mouse hearts treated by TAC surgery. Compared with normal hearts, higher mRNA and protein level of MLL3 was detected in the DCM hearts, and its expression level was closely associated with left ventricular end systolic diameter (LVEDD) and left ventricular ejection fraction (LVEF). However, the expression level of other MLL families (MLL, MLL2, MLL4, MLL5, SETD1A, and SETD1B) had no obvious change between control and DCM hearts or remodeled mouse hearts. Furthermore, the di-methylated histone H3 lysine 4 (H3K4me2) but not H3K4me3 was significantly increased in the DCM hearts. The protein levels of Smad3, GATA4, EGR1, which might regulate by MLL3, were remarkably elevated in the DCM hearts. Our hitherto unrecognized findings indicate that MLL3 has a potential role on pathological processes of DCM via regulating H3K4me2 and the expression of Smad3, GATA4, and EGR1.

Interferon regulatory factors: at the crossroads of immunity, metabolism, and disease.

The interferon-regulatory factor (IRF) family comprises nine members in mammals. Although this transcription factor family was originally thought to function primarily in the immune system, contributing to both the innate immune response and the development of immune cells, recent advances have revealed that IRFs plays critical roles in other biological processes, such as metabolism. Accordingly, abnormalities in the expression and/or function of IRFs have increasingly been linked to disease. Herein, we provide an update on the recent progress regarding the regulation of immune responses and immune cell development associated with IRFs. Additionally, we discuss the relationships between IRFs and immunity, metabolism, and disease, with a particular focus on the role of IRFs as stress sensors. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

A critical role for interferon regulatory factor 9 in cerebral ischemic stroke.

The failure of past efforts to develop effective stroke treatments is at least partially because these treatments often interfered with essential physiological functions, even though they are targeted toward pathophysiological events, such as inflammation, excitotoxicity, and oxidative stress. Thus, the direct targeting of endogenous neuroprotective or destructive elements holds promise as a potential new approach to treating this devastating condition. Interferon regulatory factor 9 (IRF9), a transcription factor that regulates innate immune responses, has been implicated in neurological pathology. Here, we provide new evidence that IRF9 directly mediates neuronal death in male mice. In response to ischemia/reperfusion (I/R), IRF9 accumulated in neurons. IRF9 deficiency markedly mitigated both poststroke neuronal death and neurological deficits, whereas the neuron-specific overexpression of IRF9 sensitized neurons to death. The histone deacetylase Sirt1 was identified as a novel negative transcriptional target of IRF9 both in vivo and in vitro. IRF9 inhibits Sirt1 deacetylase activity, culminating in the acetylation and activation of p53-mediated cell death signaling. Importantly, both the genetic and pharmacological manipulation of Sirt1 effectively counteracted the pathophysiological effects of IRF9 on stroke outcome. These findings indicate that, rather than activating a delayed innate immune response, IRF9 directly activates neuronal death signaling pathways through the downregulation of Sirt1 deacetylase in response to acute I/R stress.

Growth/differentiation factor 1 alleviates pressure overload-induced cardiac hypertrophy and dysfunction.

Pathological cardiac hypertrophy is a major risk factor for developing heart failure, the leading cause of death in the world. Growth/differentiation factor 1 (GDF1), a transforming growth factor-ß family member, is a regulator of cell growth and differentiation in both embryonic and adult tissues. Evidence from human and animal studies suggests that GDF1 may play an important role in cardiac physiology and pathology. However, a critical role for GDF1 in cardiac remodelling has not been investigated. Here, we performed gain-of-function and loss-of-function studies using cardiac-specific GDF1 knockout mice and transgenic mice to determine the role of GDF1 in pathological cardiac hypertrophy, which was induced by aortic banding (AB). The extent of cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. Our results demonstrated that cardiac specific GDF1 overexpression in the heart markedly attenuated cardiac hypertrophy, fibrosis, and cardiac dysfunction, whereas loss of GDF1 in cardiomyocytes exaggerated the pathological cardiac hypertrophy and dysfunction in response to pressure overload. Mechanistically, we revealed that the cardioprotective effect of GDF1 on cardiac remodeling was associated with the inhibition of the MEK-ERK1/2 and Smad signaling cascades. Collectively, our data suggest that GDF1 plays a protective role in cardiac remodeling via the negative regulation of the MEK-ERK1/2 and Smad signaling pathways.

Interferon regulatory factor 9 is a key mediator of hepatic ischemia/reperfusion injury.

BACKGROUND & AIMS: Hepatic ischemia/reperfusion (I/R) injury is characterized by anoxic cell injury and the generation of inflammatory mediators, leading to hepatic parenchymal cell death. The activation of interferon regulatory factors (IRFs) has been implicated in hepatic I/R injury, but the role of IRF9 in this progression is unclear. METHODS: We investigated the function and molecular mechanisms of IRF9 in transgene and knockout mice subjected to warm I/R of the liver. Isolated hepatocytes from IRF9 transgene and knockout mice were subjected to hypoxia/reoxygenation (H/R) injury to determine the in vitro effects of IRF9. RESULTS: The injuries were augmented in IRF9-overexpressing mice that were subjected to warm I/R of the liver. In contrast, a deficiency in IRF9 markedly reduced the necrotic area, serum alanine amino transferase/aspartate amino transferase (ALT/AST), immune cell infiltration, inflammatory cytokine levels, and hepatocyte apoptosis after liver I/R. Sirtuin (SIRT) 1 levels were significantly higher and the acetylation of p53 was decreased in the IRF9 knockout mice. Notably, IRF9 suppressed the activity of the SIRT1 promoter luciferase reporter and deacetylase activity. Liver injuries were significantly more severe in the IRF9/SIRT1 double knockout (DKO) mice in the I/R model, eliminating the protective effects observed in the IRF9 knockout mice. CONCLUSIONS: IRF9 has a novel function of inducing hepatocyte apoptosis after I/R injury by decreasing SIRT1 expression and increasing acetyl-p53 levels. Targeting IRF9 may be a potential strategy for ameliorating ischemic liver injury after liver surgery.

Regulatory role of CARD3 in left ventricular remodelling and dysfunction after myocardial infarction.

Caspase activation and recruitment domain 3 (CARD3) is a caspase recruitment domain (CARD)-containing serine/threonine kinase and plays a pivotal role in apoptosis, immunity, tissue development and proliferation. To date, the causal relationship between CARD3 and myocardial infarction (MI) remains largely unexplored. This study aimed to identify the functional significance of CARD3 in the regulation of cardiac remodelling after MI and the underlying mechanisms of its effects. The levels of CARD3 expression were up-regulated in failing human and mouse post-infarction hearts. In addition, CARD3-knockout (KO) mice and transgenic mice overexpressing CARD3 in the heart were then generated and subjected to MI. Compared with wild-type (WT) control mice, CARD3-KO mice developed smaller infarct sizes, improved survival rates, and preserved left ventricle (LV) function after MI. Significantly, CARD3-KO hearts had less cardiomyocyte apoptosis and inflammatory cell infiltration in the infarct border zone. Attenuated LV remodelling was also observed in the KO hearts following MI, with reduced cardiac hypertrophy and fibrosis. Conversely, CARD3 overexpression resulted in the opposite MI-induced phenotype. Similar results were observed in ex vivo-cultured neonatal rat cardiomyocytes exposed to hypoxia. Mechanistically, we discovered that the CARD3-mediated detrimental effects of MI were associated with the activation of the NF-κB and p38 signalling cascades. Taken together, these data demonstrate that CARD3 serves as a novel positive modulator of ventricular remodelling after MI via the regulation of the NF-κB and p38 signalling. Thus, CARD3 may be a promising therapeutic target for the treatment of heart failure after MI.

Interferon regulatory factor 3 constrains IKKß/NF-κB signaling to alleviate hepatic steatosis and insulin resistance.

UNLABELLED: Obesity and related metabolic diseases associated with chronic low-grade inflammation greatly compromise human health. Previous observations on the roles of interferon regulatory factors (IRFs) in the regulation of metabolism prompted investigation of the involvement of a key family member, IRF3, in metabolic disorders. IRF3 expression in the liver is decreased in animals with diet-induced and genetic obesity. The global knockout (KO) of IRF3 significantly promotes chronic high-fat diet (HFD)-induced hepatic insulin resistance and steatosis; in contrast, adenoviral-mediated hepatic IRF3 overexpression preserves glucose and lipid homeostasis. Furthermore, systemic and hepatic inflammation, which is increased in IRF3 KO mice, is attenuated by the overexpression of hepatic IRF3. Importantly, inhibitor of nuclear factor kappa B kinase beta subunit / nuclear factor kappa B (IKKß/NF-κB) signaling is repressed by IRF3, and hepatic overexpression of the inhibitor of κB-α (IκBα) reverses HFD-induced insulin resistance and steatosis in IRF3 KO mice. Mechanistically, IRF3 interacts with the kinase domain of IKKß in the cytoplasm and inhibits its downstream signaling. Moreover, deletion of the region of IRF3 responsible for the IRF3/IKKß interaction inhibits the capacity of IRF3 to preserve glucose and lipid homeostasis. CONCLUSION: IRF3 interacts with IKKß in the cytoplasm to inhibit IKKß/NF-κB signaling, thus alleviating hepatic inflammation, insulin resistance, and hepatic steatosis.

Interferon regulatory factor 8 protects against cerebral ischaemic-reperfusion injury.

Interferon regulatory factor 8 (IRF8), a transcriptional regulator in the IRF family, has been implicated in innate immunity, immune cell differentiation and tumour cell apoptosis. In the present study, we found that IRF8 is constitutively expressed in the brain and suppressed after cerebral ischaemia in a time-dependent manner. IRF8 knockout (IRF8-KO) mice, wild type (WT) mice, neuron-specific IRF8 transgenic (TG) mice and non-transgenic mice were used in a transient cerebral ischaemic model. The IRF8 knockout mice exhibited aggravated apoptosis, inflammation and oxidative injury in the ischaemic brain, eventually leading to poorer stroke outcomes, whereas neuron-specific IRF8 transgenic mice showed a marked inhibition of apoptosis and improved stroke outcomes. To model ischaemia/reperfusion conditions in vitro, primary cortical neurons were cultured and subjected to transient oxygen and glucose deprivation for 60 min. Similar to the in vivo study, IRF8 knockdown by Ad-shIRF8 resulted in increased apoptosis, whereas IRF8 over-expression by Ad-IRF8 significantly decreased neuronal apoptosis. These data indicate that IRF8 is strongly protective in ischaemic stroke by regulating neuronal apoptosis, the inflammatory response and oxidative stress. In the present study, we found that the transcriptional factor IRF8 plays a protective role in the cerebral ischaemic-reperfusion injury by attenuating neuronal apoptosis, oxidative stress and inflammation. Besides the known function of IRF8 in regulating the inflammatory gene expression, we first demonstrated that IRF8 can directly modulate apoptosis and oxidative stress by controlling the relative genes expression.

Mindin/Spondin 2 inhibits hepatic steatosis, insulin resistance, and obesity via interaction with peroxisome proliferator-activated receptor α in mice.

BACKGROUND & AIMS: Obesity and its related pathologies, such as hepatic steatosis, are associated with chronic inflammation and insulin resistance (IR), which contribute to cardiovascular disease. Our previous studies indicated that Spondin 2 has a protective role in the context of cardiovascular and cerebrovascular diseases. Whether Spondin 2 is also associated with the development of hepatic steatosis and IR remains unclear. METHODS: Wild-type mice, Spondin 2-knockout (KO) mice, hepatic-specific Spondin 2 transgenic (Spondin 2-TG) mice, high fat diet (HFD)-induced obese mice injected with an adenovirus expressing Spondin 2-specific shRNA or a Spondin 2 mutant and genetically obese (ob/ob) mice injected with an adenovirus expressing Spondin 2 were fed normal chow (NC) or HFD for indicated time to induce obesity, hepatic steatosis, inflammation, and IR. Biomedical, histological, and metabolic analyses were conducted to identify pathologic alterations in these mice. The molecular mechanisms of Spondin 2 functions were explored in mice and in hepatocytes or cell lines. RESULTS: Consistent with Spondin 2 repression in the livers of HFD-induced and ob/ob mice, the Spondin 2-KO or hepatic-specific Spondin 2 knockdown mice exhibited more severe obesity, hepatic steatosis, inflammation, and IR upon HFD. Conversely, these pathological conditions were significantly improved in the Spondin 2-TG mice or Spondin 2-overexpressing ob/ob mice. Spondin 2 interacts with PPARα to regulate PPARα-target genes, thereby improving the pathological phenotypes. In contrast, the hepatic overexpression of mutant Spondin 2 without the PPARα-interacting domain failed to improve the aggravated phenotypes observed in the Spondin 2-KO mice. CONCLUSION: Spondin 2 regulates hepatic lipid metabolism and alleviates hepatic steatosis, obesity, inflammation, and IR in mice via its interaction with PPARα.