Shu-Min Zhang

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

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 (IKKβ/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 IKKβ in the cytoplasm and inhibits its downstream signaling. Moreover, deletion of the region of IRF3 responsible for the IRF3/IKKβ interaction inhibits the capacity of IRF3 to preserve glucose and lipid homeostasis. Conclusion: IRF3 interacts with IKKβ in the cytoplasm to inhibit IKKβ/NF‐κB signaling, thus alleviating hepatic inflammation, insulin resistance, and hepatic steatosis. (Hepatology 2014;59:870–885)

Interferon Regulatory Factor 9 Protects Against Cardiac Hypertrophy by Targeting Myocardin

Pathological cardiac hypertrophy is a major risk factor for heart failure. In this study, we identified interferon regulatory factor 9 (IRF9), a member of the IRF family, as a previously unidentified negative regulator of cardiac hypertrophy. The level of IRF9 expression was remarkably elevated in the hearts from animals with aortic banding–induced cardiac hypertrophy. IRF9-deficient mice exhibited pronounced cardiac hypertrophy after pressure overload, as demonstrated by increased cardiomyocyte size, extensive fibrosis, reduced cardiac function, and enhanced expression of hypertrophy markers, whereas transgenic mice with cardiac-specific overexpression of murine IRF9 exhibited a significant reduction in the hypertrophic response. Mechanistically, IRF9 competes with p300 for binding to the transcription activation domain of myocardin, a coactivator of serum response factor (SRF). This interaction markedly suppresses the transcriptional activity of myocardin because IRF9 overexpression strongly inhibits the ability of myocardin to activate CArG box–dependent reporters. These results provide compelling evidence that IRF9 inhibits the development of cardiac hypertrophy by suppressing the transcriptional activity of myocardin in the heart.

Interferon regulatory factor 9 protects against hepatic insulin resistance and steatosis in male mice

Obesity is a calorie‐excessive state associated with high risk of diabetes, atherosclerosis, and certain types of tumors. Obesity may induce inflammation and insulin resistance (IR). We found that the expression of interferon (IFN) regulatory factor 9 (IRF9), a major transcription factor mediating IFN responses, was lower in livers of obese mice than in those of their lean counterparts. Furthermore, whole‐body IRF9 knockout (KO) mice were more obese and had aggravated IR, hepatic steatosis, and inflammation after chronic high‐fat diet feeding. In contrast, adenoviral‐mediated hepatic IRF9 overexpression in both diet‐induced and genetically (ob/ob) obese mice showed markedly improved hepatic insulin sensitivity and attenuated hepatic steatosis and inflammation. We further employed a yeast two‐hybrid screening system to investigate the interactions between IRF9 and its cofactors. Importantly, we identified that IRF9 interacts with peroxisome proliferator‐activated receptor alpha (PPAR‐α), an important metabolism‐associated nuclear receptor, to activate PPAR‐α target genes. In addition, liver‐specific PPAR‐α overexpression rescued insulin sensitivity and ameliorated hepatic steatosis and inflammation in IRF9 KO mice. Conclusion: IRF9 attenuates hepatic IR, steatosis, and inflammation through interaction with PPAR‐α. (Hepatology 2013;58:603–616)

Interferon regulatory factor 7 deficiency prevents diet-induced obesity and insulin resistance

Obesity-related inflammation has been implicated in the pathogenesis of insulin resistance and type 2 diabetes. In this study, we addressed the potential role of interferon regulatory factor 7 (IRF7), a master regulator of type I interferon-dependent immune responses, in the regulation of energy metabolism. The expression levels of IRF7 were increased in white adipose tissue, liver tissue, and gastrocnemius muscle of both diet-induced obese mice and ob/ob mice compared with their lean counterparts. After feeding a high-fat diet (HFD) for 24 wk, IRF7 knockout (KO) mice showed less weight gain and adiposity than wild-type controls. KO of IRF7 improved glucose and lipid homeostasis and insulin sensitivity. Additionally, KO of IRF7 ameliorated diet-induced hepatic steatosis. Next, we assessed the inflammatory state of the IRF7 KO mice on the HFD. These mice showed less macrophage infiltration into multiple organs and were protected from local and systemic inflammation. This study demonstrates a role for IRF7 in diet-induced alterations in energy metabolism and insulin sensitivity. Our results also suggest that IRF7 is involved in the etiology of metabolic abnormalities, which suggests a new strategy for treating obesity and type 2 diabetes.

Interferon regulatory factor 8 modulates phenotypic switching of smooth muscle cells by regulating the activity of myocardin.

ABSTRACT Interferon regulatory factor 8 (IRF8), a member of the IRF transcription factor family, was recently implicated in vascular diseases. In the present study, using the mouse left carotid artery wire injury model, we unexpectedly observed that the expression of IRF8 was greatly enhanced in smooth muscle cells (SMCs) by injury. Compared with the wild-type controls, IRF8 global knockout mice exhibited reduced neointimal lesions and maintained SMC marker gene expression. We further generated SMC-specific IRF8 transgenic mice using an SM22α-driven IRF8 plasmid construct. In contrast to the knockout mice, mice with SMC-overexpressing IRF8 exhibited a synthetic phenotype and enhanced neointima formation. Mechanistically, IRF8 inhibited SMC marker gene expression through regulating serum response factor (SRF) transactivation in a myocardin-dependent manner. Furthermore, a coimmunoprecipitation assay indicated a direct interaction of IRF8 with myocardin, in which a specific region of myocardin was essential for recruiting acetyltransferase p300. Altogether, IRF8 is crucial in modulating SMC phenotype switching and neointima formation in response to vascular injury via direct interaction with the SRF/myocardin complex.

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.

Mindin regulates vascular smooth muscle cell phenotype and prevents neointima formation.

Mindin/spondin 2, an extracellular matrix (ECM) component that belongs to the thrombospondin type 1 (TSR) class of molecules, plays prominent roles in the regulation of inflammatory responses, angiogenesis and metabolic disorders. Our most recent studies indicated that mindin is largely involved in the initiation and development of cardiac and cerebrovascular diseases [Zhu et al. (2014) J. Hepatol. 60, 1046-1054; Bian et al. (2012) J. Mol. Med. 90, 895-910; Wang et al. (2013) Exp. Neurol. 247, 506-516; Yan et al. (2011) Cardiovasc. Res. 92, 85-94]. However, the regulatory functions of mindin in neointima formation remain unclear. In the present study, mindin expression was significantly down-regulated in platelet-derived growth factor-BB (PDGF-BB)-stimulated vascular smooth muscle cells (VSMCs) and wire injury-stimulated vascular tissue. Using a gain-of-function approach, overexpression of mindin in VSMCs exhibited strong anti-proliferative and anti-migratory effects on VSMCs, whereas significant suppression of intimal hyperplasia was observed in transgenic (TG) mice expressing mindin specifically in smooth muscle cells (SMCs). These mice exhibited blunted VSMC proliferation, migration and phenotypic switching. Conversely, deletion of mindin dramatically exacerbated neointima formation in a wire-injury mouse model, which was further confirmed in a balloon injury-induced vascular lesion model using a novel mindin-KO (knockout) rat strain. From a mechanistic standpoint, the AKT (Protein Kinase B)-GSK3β (glycogen synthase kinase 3β)/mTOR (mammalian target of rapamycin)-FOXO3A (forkhead box O)-FOXO1 signalling axis is responsible for the regulation of mindin during intimal thickening. Interestingly, an AKT inhibitor largely reversed mindin-KO-induced aggravated hyperplasia, suggesting that mindin-mediated neointima formation is AKT-dependent. Taken together, our findings demonstrate that mindin protects against vascular hyperplasia by suppression of abnormal VSMC proliferation, migration and phenotypic switching in an AKT-dependent manner. Up-regulation of mindin might represent an effective therapy for vascular-remodelling-related diseases.

Interferon Regulatory Factor 7 Protects Against Vascular Smooth Muscle Cell Proliferation and Neointima Formation

Background Interferon regulatory factor 7 (IRF7), a member of the interferon regulatory factor family, plays important roles in innate immunity and immune cell differentiation. However, the role of IRF7 in neointima formation is currently unknown. Methods and Results Significant decreases in IRF7 expression were observed in vascular smooth muscle cells (VSMCs) following carotid artery injury in vivo and platelet‐derived growth factor‐BB (PDGF‐BB) stimulation in vitro. Compared with non‐transgenic (NTG) controls, SMC‐specific IRF7 transgenic (IRF7‐TG) mice displayed reduced neointima formation and VSMC proliferation in response to carotid injury, whereas a global knockout of IRF7 (IRF7‐KO) resulted in the opposite effect. Notably, a novel IRF7‐KO rat strain was successfully generated and used to further confirm the effects of IRF7 deletion on the acceleration of intimal hyperplasia based on a balloon injury‐induced vascular lesion model. Mechanistically, IRF7s inhibition of carotid thickening and the expression of VSMC proliferation markers was dependent on the interaction of IRF7 with activating transcription factor 3 (ATF3) and its downstream target, proliferating cell nuclear antigen (PCNA). The evidence that IRF7/ATF3‐double‐TG (DTG) and IRF7/ATF3‐double‐KO (DKO) mice abolished the regulatory effects exhibited by the IRF7‐TG and IRF7‐KO mice, respectively, validated the underlying molecular events of IRF7‐ATF3 interaction. Conclusions These findings demonstrated that IRF7 modulated VSMC proliferation and neointima formation by interacting with ATF3, thereby inhibiting the ATF3‐mediated induction of PCNA transcription. The results of this study indicate that IRF7 is a novel modulator of neointima formation and VSMC proliferation and may represent a promising target for vascular disease therapy.

Interferon regulatory factor 3 protects against adverse neo-intima formation

AIMS Vascular smooth muscle cell (VSMC) proliferation is central to the pathophysiology of neo-intima formation. Interferon regulatory factor 3 (IRF3) inhibits the growth of cancer cells and fibroblasts. However, the role of IRF3 in vascular neo-intima formation is unknown. We evaluated the protective role of IRF3 against neo-intima formation in mice and the underlying mechanisms. METHODS AND RESULTS IRF3 expression was down-regulated in VSMCs after carotid wire injury in vivo, and in SMCs after platelet-derived growth factor (PDGF)-BB challenge in vitro. Global knockout of IRF3 (IRF3-KO) led to accelerated neo-intima formation and proliferation of VSMCs, whereas the opposite was seen in SMC-specific IRF3 transgenic mice. Mechanistically, we identified IRF3 as a novel regulator of peroxisome proliferator-activated receptor γ (PPARγ), a negative regulator of SMC proliferation after vascular injury. Binding of IRF3 to the AB domain of PPARγ in the nucleus of SMCs facilitated PPARγ transactivation, resulting in decreased proliferation cell nuclear antigen expression and suppressed proliferation. Overexpression of wild-type, but not truncated, IRF3 with a mutated IRF association domain (IAD) retained the ability to exert anti-proliferative effect. CONCLUSIONS IRF3 inhibits VSMC proliferation and neo-intima formation after vascular injury through PPARγ activation.

CARD3 Deficiency Exacerbates Diet-Induced Obesity, Hepatosteatosis, and Insulin Resistance in Male Mice

Caspase activation and recruitment domain 3 (CARD3) is a 61-kDa protein kinase with an N-terminal serine/threonine kinase domain and a C-terminal CARD. Previous research on the function of CARD3 has focused on its role in the immune response and inflammatory diseases. Obesity is now a worldwide health problem and is generally recognized as an inflammatory disease. Unexpectedly, we found that CARD3 expression was lower during obesity. In this study, we explored the biological and genetic bases of obesity using CARD3-knockout (KO) and wild-type (WT) mice fed a high-fat diet (HFD) for 24 weeks. We demonstrate that KO mice were more obese than their WT littermates, and KO mice exhibited obvious visceral fat accumulation and liver weight gains after 24 weeks of HFD feeding. We also observed more severe hepatosteatosis in KO mice compared with the WT controls. Hepatic steatosis in the HFD-fed KO mice was linked to a significant increase in the expression of key lipogenic and cholesterol synthesis enzymes, whereas the expression of the enzymes involves in β-oxidation was dramatically reduced. Furthermore, we confirmed the repression of AMP-activated protein kinase signaling and activation of the endoplasmic reticulum stress response. Fatty liver impaired the global glucose and lipid metabolism, which further exacerbated the insulin resistance associated with the repression of Akt signaling and up-regulated systemic inflammation through the M1/M2 (pro- and anti-inflammation) type switch and the activation of the nuclear factor-κB pathway. Our studies demonstrate the crucial role of CARD3 in metabolism and indicate that CARD3 deficiency promotes the diet-induced phenotype of type 2 diabetes.