An Inverse Agonist GSK5182 Increases Protein Stability of the Orphan Nuclear Receptor ERRγ via Inhibition of Ubiquitination.

The orphan nuclear receptor, estrogen-related receptor γ (ERRγ) is a constitutively active transcription factor involved in mitochondrial metabolism and energy homeostasis. GSK5182, a specific inverse agonist of ERRγ that inhibits transcriptional activity, induces a conformational change in ERRγ, resulting in a loss of coactivator binding. However, the molecular mechanism underlying the stabilization of the ERRγ protein by its inverse agonist remains largely unknown. In this study, we found that GSK5182 inhibited ubiquitination of ERRγ, thereby stabilizing the ERRγ protein, using cell-based assays and confocal image analysis. Y326 of ERRγ was essential for stabilization by GSK5182, as ligand-induced stabilization of ERRγ was not observed with the ERRγ-Y326A mutant. GSK5182 suppressed ubiquitination of ERRγ by the E3 ligase Parkin and subsequent degradation. The inhibitory activity of GSK5182 was strong even when the ERRγ protein level was elevated, as ERRγ bound to GSK5182 recruited a corepressor, small heterodimer partner-interacting leucine zipper (SMILE), through the activation function 2 (AF-2) domain, without alteration of the nuclear localization or DNA-binding ability of ERRγ. In addition, the AF-2 domain of ERRγ was critical for the regulation of protein stability. Mutants in the AF-2 domain were present at higher levels than the wild type in the absence of GSK5182. Furthermore, the ERRγ-L449A/L451A mutant was no longer susceptible to GSK5182. Thus, the AF-2 domain of ERRγ is responsible for the regulation of transcriptional activity and protein stability by GSK5182. These findings suggest that GSK5182 regulates ERRγ by a unique molecular mechanism, increasing the inactive form of ERRγ via inhibition of ubiquitination.

Comparative Analysis of the Productivity and Immunogenicity of an Attenuated Classical Swine Fever Vaccine (LOM) and an Attenuated Live Marker Classical Swine Fever Vaccine (Flc-LOM-BErns) from Laboratory to Pig Farm.

Herein, we compared the productivity of pigs inoculated with one of two classical swine fever (CSF) vaccines (low virulent of Miyagi (LOM) or Flc-LOM-BErns) plus the swine erysipelothrix rhusiopathiae (SE) vaccine. The feed intake and weight increase of the pigs inoculated with Flc-LOM-BErns + SE were normal. However, the feed intake of the pigs inoculated with LOM + SE dropped sharply from four days post-vaccination (dpv). In addition, the slaughter date was an average of eight days later than that of the pigs inoculated with Flc-LOM-BErns + SE. All pigs inoculated with the Flc-LOM-BErns + SE vaccine were completely differentiated at 14 days against CSF Erns antibody and at approximately 45 days against the bovine viral diarrhea virus (BVDV) Erns antibody; the titers were maintained until slaughter. Leucopenia occurred temporarily in the LOM + SE group, but not in the Flc-LOM-BErns + SE group. Expression of tumor necrosis factor (TNF)-α and IFN-γ was significantly (p < 0.05) higher in the LOM + SE group than in the mock (no vaccine) group. When conducting the same experiment on a breeding farm, the results were similar to those of the laboratory experiments. In conclusion, the biggest advantage of replacing the CSF LOM vaccine with the Flc-LOM-BErns vaccine is improved productivity.

Frontline Science: Estrogen-related receptor γ increases poly(I:C)-mediated type I IFN expression in mouse macrophages.

Although type I IFNs (IFN-I) are important for the innate and adaptive immune responses to suppress viral replication, prolonged IFN-I signaling in macrophages suppresses the immune response. Nuclear receptor estrogen-related receptor γ (ERRγ) regulates the transcription of genes involved in endocrine and metabolic functions. However, the role of ERRγ in macrophage immune responses to viruses remains largely unknown. ERRγ expression was significantly induced in mouse bone marrow-derived macrophages (BMDMs) treated with polyinosinic-polycytidylic acid (poly(I:C)). Our results indicated that the induction of ERRγ expression by poly(I:C) is mediated through activation of the cytoplasmic dsRNA receptors, retinoic acid-inducible gene I and melanoma differentiation-associated protein 5. In BMDMs, overexpression of ERRγ significantly increased gene expression and secretion of the IFN-I genes, IFN-α and IFN-ß, whereas abolition of ERRγ significantly attenuated poly(I:C)-mediated IFN-I secretion. Chromatin immunoprecipitation assays and mutation analyses of the IFN-I promoters revealed that ERRγ regulates the transcription of IFN-α and IFN-ß by binding to a conserved ERR response element in each promoter region. Finally, GSK5182 significantly suppressed poly(I:C)-mediated induction of IFN-I gene expression and secretion in BMDMs. Taken together, these findings reveal a previously unrecognized role for ERRγ in the transcriptional control of innate and adaptive immune response to dsRNA virus replication.

Glial TIM-3 Modulates Immune Responses in the Brain Tumor Microenvironment.

T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3), a potential immunotherapeutic target for cancer, has been shown to display diverse characteristics in a context-dependent manner. Thus, it would be useful to delineate the precise functional features of TIM-3 in a given situation. Here, we report that glial TIM-3 shows distinctive properties in the brain tumor microenvironment. TIM-3 was expressed on both growing tumor cells and their surrounding cells including glia and T cells in an orthotopic mouse glioma model. The expression pattern of TIM-3 was distinct from those of other immune checkpoint molecules in tumor-exposed and tumor-infiltrating glia. Comparison of cells from tumor-bearing and contralateral hemispheres of a glioma model showed that TIM-3 expression was lower in tumor-infiltrating CD11b+CD45mid glial cells but higher in tumor-infiltrating CD8+ T cells. In TIM-3 mutant mice with intracellular signaling defects and Cre-inducible TIM-3 mice, TIM-3 affected the expression of several immune-associated molecules including iNOS and PD-L1 in primary glia-exposed conditioned media (CM) from brain tumors. Further, TIM-3 was cross-regulated by TLR2, but not by TLR4, in brain tumor CM- or Pam3CSK4-exposed glia. In addition, following exposure to tumor CM, IFNγ production was lower in T cells cocultured with TIM-3-defective glia than with normal glia. Collectively, these findings suggest that glial TIM-3 actively and distinctively responds to brain tumor, and plays specific intracellular and intercellular immunoregulatory roles that might be different from TIM-3 on T cells in the brain tumor microenvironment. SIGNIFICANCE: TIM-3 is typically thought of as a T-cell checkpoint receptor. This study demonstrates a role for TIM-3 in mediating myeloid cell responses in glioblastoma.

Hepatocyte toll-like receptor 4 mediates lipopolysaccharide-induced hepcidin expression.

Hepcidin expression is induced by inflammatory molecules such as lipopolysaccharide (LPS) via a macrophage-mediated pathway. Although hepatocytes directly respond to LPS, the molecular mechanism underlying toll-like receptor (TLR)-dependent hepcidin expression by hepatocytes is mostly unknown. Here we show that LPS can directly induce the mRNA expression and secretion of hepcidin by hepatocytes via TLR4 activation. Using hepatocytes deficient in TLR4, myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing interferon-ß (TRIF), we demonstrated that LPS-induced hepcidin expression by hepatocytes is regulated by its specific receptor, TLR4, via a MyD88-dependent signaling pathway. Hepcidin promoter activity was significantly increased by MyD88-dependent downstream signaling molecules (interleukin-1 receptor-associated kinase (IRAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6), which activate c-Jun N-terminal kinase (JNK) and activator protein-1 (AP-1). We then confirmed that LPS stimulation induced the phosphorylation of JNK and c-Jun, and observed strong occupancy of the hepcidin promoter by c-Jun. Promoter mutation analysis also identified the AP-1-binding site on the hepcidin promoter. Finally, bone marrow transplantation between wild-type and TLR4 knockout mice revealed that hepatic TLR4-dependent hepcidin expression was comparable to macrophage TLR4-dependent hepcidin expression induced by LPS. Taken together, these results suggest that TLR4 expressed by hepatocytes regulates hepcidin expression via the IRAK-TRAF6-JNK-AP-1 axis.

Orphan nuclear receptor SHP regulates iron metabolism through inhibition of BMP6-mediated hepcidin expression.

Small heterodimer partner (SHP) is a transcriptional corepressor regulating diverse metabolic processes. Here, we show that SHP acts as an intrinsic negative regulator of iron homeostasis. SHP-deficient mice maintained on a high-iron diet showed increased serum hepcidin levels, decreased expression of the iron exporter ferroportin as well as iron accumulation compared to WT mice. Conversely, overexpression of either SHP or AMP-activated protein kinase (AMPK), a metabolic sensor inducing SHP expression, suppressed BMP6-induced hepcidin expression. In addition, an inhibitory effect of AMPK activators metformin and AICAR on BMP6-mediated hepcidin gene expression was significantly attenuated by ablation of SHP expression. Interestingly, SHP physically interacted with SMAD1 and suppressed BMP6-mediated recruitment of the SMAD complex to the hepcidin gene promoter by inhibiting the formation of SMAD1 and SMAD4 complex. Finally, overexpression of SHP and metformin treatment of BMP6 stimulated mice substantially restored hepcidin expression and serum iron to baseline levels. These results reveal a previously unrecognized role for SHP in the transcriptional control of iron homeostasis.

The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression.

BACKGROUND: Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. RESULTS: Activation of the hepatic CB1 receptor by arachidonyl-2'-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRß, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. CONCLUSION: Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion.

Attenuation of Cigarette Smoke-Induced Emphysema in Mice by Apolipoprotein A-1 Overexpression.

Chronic inflammation, oxidative stress, and proteolysis participate primarily in the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. COPD is a highly prevalent smoking-related disease for which no effective therapy exists to improve the disease course. Although apolipoprotein A-1 (ApoA1) has antiinflammatory and antioxidant properties as well as cholesterol efflux potential, its role in cigarette smoke (CS)-induced emphysema has not been determined. Therefore, we investigated whether human ApoA1 transgenic (TG) mice, with conditionally induced alveolar epithelium to overexpress ApoA1, are protected against the CS-induced lung inflammatory response and development of emphysema. In this study, ApoA1 levels were significantly decreased in the lungs of patients with COPD and in the lungs of mice exposed to CS. ApoA1 TG mice did not develop emphysema when chronically exposed to CS. Compared with the control TG mice, ApoA1 overexpression attenuated lung inflammation, oxidative stress, metalloprotease activation, and apoptosis in CS-exposed mouse lungs. To explore a plausible mechanism of antiapoptotic activity of ApoA1, alveolar epithelial cells (A549) were treated with CS extract (CSE). ApoA1 prevented CSE-induced translocation of Fas and downstream death-inducing signaling complex into lipid rafts, thereby inhibiting Fas-mediated apoptosis. Taken together, the data showed that ApoA1 overexpression attenuated CS-induced lung inflammation and emphysema in mice. Augmentation of ApoA1 in the lung may have therapeutic potential in preventing smoking-related COPD/emphysema.

In vitro cytotoxicity on human ovarian cancer cells by T-type calcium channel blockers.

The growth inhibition of human cancer cells via T-type Ca(2+) channel blockade has been well known. Herein, a series of new 3,4-dihydroquinazoline derivatives were synthesized via a brief SAR study on KYS05090 template and evaluated for both T-type Ca(2+) channel (Cav3.1) blockade and cytotoxicity on three human ovarian cancer cells (SK-OV-3, A2780 and A2780-T). Most of compounds except 6i generally exhibited more potent cytotoxicity on SK-OV-3 than mibefradil as a positive control regardless of the degree of T-type channel blockade. In particular, eight compounds (KYS05090, 6a and 6c-6h) showing strong channel blockade exhibited almost equal and more potent cytotoxicity on A2780 when compared to mibefradil. On A2780-T paclitaxel-resistant human ovarian carcinoma, two compounds (KYS05090 and 6d) were 20-fold more active than mibefradil. With respect to cell cycle arrest effect on A2780 and A2780-T cells, KYS05090 induced large proportion of sub-G1 phase in the cell cycle progression of A2780 and A2780-T, meaning the induction of cancer cell death instead of cell cycle arrest via blocking T-type Ca(2+) channel. Among new analogues, compounds 6g and 6h induced cell cycle arrest at G1 phase of A2780 and A2780-T cells in dose-dependent manner and exhibited strong anti-proliferation effects of ovarian cancer cells by blocking T-type Ca(2+) channel. Furthermore, 6g and 6h possessing strong cytotoxic effects could induce apoptosis of A2780 cells, which was detected by confocal micrographs using DAPI staining.

WTAP regulates migration and invasion of cholangiocarcinoma cells.

BACKGROUND: Wilms' tumor 1-associating protein (WTAP) is a nuclear protein that has been associated with the regulation of proliferation and apoptosis. Although its dynamic expression and physiological functions in vascular cells have been reported, its expression and roles in cholangiocarcinoma cells are poorly characterized. METHODS: To examine the expression of WTAP in patient tissues, we performed immunohistochemistry. To examine motility of cholangiocarcinoma cells, we employed Boyden chamber, wound healing and Matrigel invasion assays, and a liver xenograft model. RESULTS: Immunohistochemistry in patient tissues showed WTAP overexpression in cholangiocarcinoma tissues and correlation of WTAP expression with metastasis of cholangiocarcinoma cells. Overexpression or knockdown of WTAP significantly increased or decreased the motility of cholangiocarcinoma cells. Moreover, WTAP overexpression or knockdown significantly increased or decreased tumorigenicity of cholangiocarcinoma cells in an orthotopic xenograft model. Furthermore, microarray study showed that WTAP induce the expressions of MMP7, MMP28, cathepsin H and Muc1. CONCLUSION: WTAP is overexpressed in cholangiocarcinoma and regulates motility of cholangiocarcinoma cells.

Expression and roles of NUPR1 in cholangiocarcinoma cells.

Nuclear protein-1 (NUPR1) is a small nuclear protein that is responsive to various stress stimuli. Although NUPR1 has been associated with cancer development, its expression and roles in cholangiocarcinoma have not yet been described. In the present study, we found that NUPR1 was over-expressed in human cholangiocarcinoma tissues, using immunohistochemistry. The role of NUPR1 in cholangiocarcinoma was examined by its specific siRNA. NUPR1 siRNA decreased proliferation, migration and invasion of human cholangiocarcinoma cell lines (HuCCT1 and SNU1196 cells). From these results, we conclude that NUPR1 is over-expressed in cholangiocarcinoma and regulates the proliferation and motility of cancer cells.

Functional delivery of DNAzyme with iron oxide nanoparticles for hepatitis C virus gene knockdown.

DNAzyme is an attractive therapeutic oligonucleotide which enables cleavage of mRNA in a sequence-specific manner and thus, silencing target gene. A particularly important challenge in achieving the successful down-regulation of gene expression is to efficiently deliver DNAzymes to disease sites and cells. Here, we report the nanoparticle-assisted functional delivery of therapeutic DNAzyme for the treatment of hepatitis C by inducing knockdown of hepatitis C virus (HCV) gene, NS3. HCV NS3 gene encodes helicase and protease which are essential for the virus replication. The nanocomplex showed efficient NS3 knockdown while not evoking undesired immune responses or notable cytotoxicity. We also demonstrated the DNAzyme conjugated nanoparticle system could be applicable in vivo by showing the accumulation of the nanoparticles in liver, and more specifically, in hepatocytes. We believe that the present work is a successful demonstration of effective, functional, non-immunostimulatory DNAzyme delivery system based on inorganic nanoparticles with high potential for further therapeutic application of DNAzyme in the treatment of hepatitis C.

SNAI1 is involved in the proliferation and migration of glioblastoma cells.

Glioblastoma is the most common type of astrocytoma in the brain. Due to its high invasiveness and chemoresistance, patients with advanced stage of glioblastoma have a poor prognosis. SNAI1, an important regulator of epithelial-mesenchymal transition, has been associated with metastasis in various carcinoma cells. However, its roles in glioblastoma cells have been poorly characterized. To examine roles of SNAI1 in glioblastoma cells, we knockdowned SNAI1 expression using siRNA. SNAI1 siRNA increased the expression level of E-cadherin and decreased that of vimentin. In the water-soluble tetrazolium salt (WST-1) assay, SNAI1 siRNA inhibited the proliferation of U87-MG and GBM05 glioblastoma cells. Moreover, in the Boyden chamber assay and Matrigel invasion assay, SNAI1 siRNA inhibited serum-induced migration and invasion of glioblastoma cells. These results suggested that SNAI1 is involved in the proliferation and migration of glioblastoma cells.

Growth inhibition of human cancer cells in vitro by T-type calcium channel blockers.

This paper describes the preliminary biological results that novel T-type calcium channel blockers inhibit the growth of human cancer cells by blocking calcium influx into the cell, based on unknown mechanism on the cell cycle responsible for cellular proliferation. Among the selected compounds from compound library, compound 9c (KYS05041) was identified to be nearly equipotent with Cisplatin against some human cancers in the micromolar range.