ERRγ-inducible FGF23 promotes alcoholic liver injury through enhancing CYP2E1 mediated hepatic oxidative stress.

Fibroblast growth factor 23 (FGF23) is a member of endocrine FGF family, along with FGF15/19 and FGF21. Recent reports showed that under pathological conditions, liver produces FGF23, although the role of hepatic FGF23 remains nebulous. Here, we investigated the role of hepatic FGF23 in alcoholic liver disease (ALD) and delineated the underlying molecular mechanism. FGF23 expression was compared in livers from alcoholic hepatitis patients and healthy controls. The role of FGF23 was examined in hepatocyte-specific knock-out (LKO) mice of cannabinoid receptor type 1 (CB1R), estrogen related receptor γ (ERRγ), or FGF23. Animals were fed with an alcohol-containing liquid diet alone or in combination with ERRγ inverse agonist. FGF23 is mainly expressed in hepatocytes in the human liver, and it is upregulated in ALD patients. In mice, chronic alcohol feeding leads to liver damage and induced FGF23 in liver, but not in other organs. FGF23 is transcriptionally regulated by ERRγ in response to alcohol-mediated activation of the CB1R. Alcohol induced upregulation of hepatic FGF23 and plasma FGF23 levels is lost in ERRγ-LKO mice, and an inverse agonist mediated inhibition of ERRγ transactivation significantly improved alcoholic liver damage. Moreover, hepatic CYP2E1 induction in response to alcohol is FGF23 dependent. In line, FGF23-LKO mice display decreased hepatic CYP2E1 expression and improved ALD through reduced hepatocyte apoptosis and oxidative stress. We recognized CBIR-ERRγ-FGF23 axis in facilitating ALD pathology through hepatic CYP2E1 induction. Thus, we propose FGF23 as a potential therapeutic target to treat ALD.

Developmental roles of glomerular epithelial protein-1 in mice molar morphogenesis.

Glomerular epithelial protein-1 (Glepp1), a R3 subtype family of receptor-type protein tyrosine phosphatases, plays important role in the activation of Src family kinases and regulates cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, we firstly examined the functional evaluation of Glepp1 in tooth development and morphogenesis. The precise expression level and developmental function of Glepp1 were examined by RT-qPCR, in situ hybridization, and loss and gain of functional study using a range of in vitro organ cultivation methods. Expression of Glepp1 was detected in the developing tooth germs in cap and bell stage of tooth development. Knocking down Glepp1 at E13 for 2 days showed the altered expression levels of tooth development-related signaling molecules, including Bmps, Dspp, Fgf4, Lef1, and Shh. Moreover, transient knock down of Glepp1 revealed alterations in cellular physiology, examined by the localization patterns of Ki67 and E-cadherin. Similarly, knocking down of Glepp1 showed disrupted enamel rod and interrod formation in 3-week renal transplanted teeth. In addition, due to attrition of odontoblastic layers, the expression signals of Dspp and the localization of NESTIN were almost not detected after knock down of Glepp1; however, their expressions were increased after Glepp1 overexpression. Thus, our results suggested that Glepp1 plays modulating roles during odontogenesis by regulating the expression levels of signaling molecules and cellular events to achieve the proper structural formation of hard tissue matrices in mice molar development.

Transcriptomic profiling and the first spatial expression analysis of candidate genes in the salivary gland of the East Asian medicinal leech, Hirudo nipponia.

Hirudo nipponia, a blood-sucking leech native to East Asia, possesses a rich repertoire of active ingredients in its saliva, showcasing significant medical potential due to its anticoagulant, anti-inflammatory, and antibacterial effects against human diseases. Despite previous studies on the transcriptomic and proteomic characteristics of leech saliva, which have identified medicinal compounds, our knowledge of tissue-specific transcriptomes and their spatial expression patterns remains incomplete. In this study, we conducted an extensive transcriptomic profiling of the salivary gland tissue in H. nipponia based on de novo assemblies of tissue-specific transcriptomes from the salivary gland, teeth, and general head region. Through gene ontology (GO) analysis and hierarchical clustering, we discovered a novel set of anti-coagulant factors-i.e., Hni-Antistasin, Hni-Ghilanten, Hni-Bdellin, Hni-Hirudin-as well as a previously unrecognized immune-related gene, Hni-GLIPR1 and uncharacterized salivary gland specific transcripts. By employing in situ hybridization, we provided the first visualization of gene expression sites within the salivary gland of H. nipponia. Our findings expand on our understanding of transcripts specifically expressed in the salivary gland of blood-sucking leeches, offering valuable resources for the exploration of previously unidentified substances with medicinal applications.

Multiple toll-like receptors (TLRs) display differential bacterial and ligand specificity in the earthworm, Eisenia andrei.

Toll-like receptors (TLRs), an ancient and well-conserved group of pattern recognition receptors (PRRs), recognize conserved pathogen-associated molecular patterns. TLRs consist of three domains: the extracellular N-terminal domain, containing one or more leucine-rich repeats (LRRs), responsible for the recognizing and binding of antigens; the type-I transmembrane domain; and the intracellular domain known as the Toll/Interleukin-1 receptor (TIR) domain required for the downstream signaling pathway. We identified six new full-length complementary DNA (cDNA) sequences, Ean-TLR1/2/3/4/5/6. The deduced amino acid sequences indicate that Ean-TLRs consist of one signal peptide, one LRR N-terminal domain (Ean-TLR4/5), varying numbers of LRRs, one (Ean-TLR1/2/3/4/5) or two (Ean-TLR6) LRR C-terminal domains, one type-I transmembrane domain, and a TIR domain. In addition, a TIR domain alignment revealed that three conserved motifs, designated as Box 1, Box 2, and Box 3, contain essential amino acid residues for downstream signaling activity. Phylogenetic analysis of earthworm TLRs generated two separate evolutionary branches representing single (sccTLR) and multiple (mccTLR) cysteine cluster TLRs. Ean-TLR1/2/3/4 (sccTLR type) and Ean-TLR6 (mccTLR type) were clustered with corresponding types of previously reported earthworm TLRs as well as TLRs from Clitellata and Polychaete. As PRRs, earthworm TLRs should be capable of sensing a diverse range of pathogens. Except for Ean-TLR3, which was not responsive to any bacteria, earthworm TLR expression was significantly induced by Gram-positive but not Gram-negative bacteria. Moreover, it is likely that earthworms can differentiate between different species of Gram-positive bacteria via their TLR responses. The ligand specificity of earthworm TLRs suggests that their pathogenic ligand recognition is likely to be as specific and diverse as the mammalian TLR pathogen-sensing system.

TMBIM6 deficiency leads to bone loss by accelerating osteoclastogenesis.

TMBIM6 is an endoplasmic reticulum (ER) protein that modulates various physiological and pathological processes, including metabolism and cancer. However, its involvement in bone remodeling has not been investigated. In this study, we demonstrate that TMBIM6 serves as a crucial negative regulator of osteoclast differentiation, a process essential for bone remodeling. Our investigation of Tmbim6-knockout mice revealed an osteoporotic phenotype, and knockdown of Tmbim6 inhibited the formation of multinucleated tartrate-resistant acid phosphatase-positive cells, which are characteristic of osteoclasts. Transcriptome and immunoblot analyses uncovered that TMBIM6 exerts its inhibitory effect on osteoclastogenesis by scavenging reactive oxygen species and preventing p65 nuclear localization. Additionally, TMBIM6 depletion was found to promote p65 localization to osteoclast-related gene promoters. Notably, treatment with N-acetyl cysteine, an antioxidant, impeded the osteoclastogenesis induced by TMBIM6-depleted cells, supporting the role of TMBIM6 in redox regulation. Furthermore, we discovered that TMBIM6 controls redox regulation via NRF2 signaling pathways. Our findings establish TMBIM6 as a critical regulator of osteoclastogenesis and suggest its potential as a therapeutic target for the treatment of osteoporosis.

Evaluation of deoxythymidine-based cationic amphiphiles as antimicrobial, antibiofilm, and anti-inflammatory agents.

OBJECTIVES: We recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. METHODS: Minimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. RESULTS: ADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. CONCLUSION: Our findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.

Upregulation of the ERRγ-VDAC1 axis underlies the molecular pathogenesis of pancreatitis.

Emerging evidence suggest that transcription factors play multiple roles in the development of pancreatitis, a necroinflammatory condition lacking specific therapy. Estrogen-related receptor γ (ERRγ), a pleiotropic transcription factor, has been reported to play a vital role in pancreatic acinar cell (PAC) homeostasis. However, the role of ERRγ in PAC dysfunction remains hitherto unknown. Here, we demonstrated in both mice models and human cohorts that pancreatitis is associated with an increase in ERRγ gene expression via activation of STAT3. Acinar-specific ERRγ haploinsufficiency or pharmacological inhibition of ERRγ significantly impaired the progression of pancreatitis both in vitro and in vivo. Using systematic transcriptomic analysis, we identified that voltage-dependent anion channel 1 (VDAC1) acts as a molecular mediator of ERRγ. Mechanistically, we showed that induction of ERRγ in cultured acinar cells and mouse pancreata enhanced VDAC1 expression by directly binding to specific site of the Vdac1 gene promoter and resulted in VDAC1 oligomerization. Notably, VDAC1, whose expression and oligomerization were dependent on ERRγ, modulates mitochondrial Ca2+ and ROS levels. Inhibition of the ERRγ-VDAC1 axis could alleviate mitochondrial Ca2+ accumulation, ROS formation and inhibit progression of pancreatitis. Using two different mouse models of pancreatitis, we showed that pharmacological blockade of ERRγ-VDAC1 pathway has therapeutic benefits in mitigating progression of pancreatitis. Likewise, using PRSS1R122H-Tg mice to mimic human hereditary pancreatitis, we demonstrated that ERRγ inhibitor also alleviated pancreatitis. Our findings highlight the importance of ERRγ in pancreatitis progression and suggests its therapeutic intervention for prevention and treatment of pancreatitis.

DN200434, an orally available inverse agonist of estrogen-related receptor γ, induces ferroptosis in sorafenib-resistant hepatocellular carcinoma.

Sorafenib, originally identified as an inhibitor of multiple oncogenic kinases, induces ferroptosis in hepatocellular carcinoma (HCC) cells. Several pathways that mitigate sorafenib-induced ferroptosis confer drug resistance; thus strategies that enhance ferroptosis increase sorafenib efficacy. Orphan nuclear receptor estrogen-related receptor γ (ERRγ) is upregulated in human HCC tissues and plays a role in cancer cell proliferation. The aim of this study was to determine whether inhibition of ERRγ with DN200434, an orally available inverse agonist, can overcome resistance to sorafenib through induction of ferroptosis. Sorafenib-resistant HCC cells were less sensitive to sorafenibinduced ferroptosis and showed significantly higher ERRγ levels than sorafenib-sensitive HCC cells. DN200434 induced lipid peroxidation and ferroptosis in sorafenib-resistant HCC cells. Mechanistically, DN200434 increased mitochondrial ROS generation by reducing glutathione/glutathione disulfide levels, which subsequently reduced mTOR activity and GPX4 levels. DN200434 induced amplification of the antitumor effects of sorafenib was confirmed in a tumor xenograft model. The present results indicate that DN200434 may be a novel therapeutic strategy to re-sensitize HCC cells to sorafenib. [BMB Reports 2022; 55(11): 547-552].

Estrogen-Related Receptor γ Maintains Pancreatic Acinar Cell Function and Identity by Regulating Cellular Metabolism.

BACKGROUND & AIMS: Mitochondrial dysfunction disrupts the synthesis and secretion of digestive enzymes in pancreatic acinar cells and plays a primary role in the etiology of exocrine pancreas disorders. However, the transcriptional mechanisms that regulate mitochondrial function to support acinar cell physiology are poorly understood. Here, we aim to elucidate the function of estrogen-related receptor γ (ERRγ) in pancreatic acinar cell mitochondrial homeostasis and energy production. METHODS: Two models of ERRγ inhibition, GSK5182-treated wild-type mice and ERRγ conditional knock-out (cKO) mice, were established to investigate ERRγ function in the exocrine pancreas. To identify the functional role of ERRγ in pancreatic acinar cells, we performed histologic and transcriptome analysis with the pancreas isolated from ERRγ cKO mice. To determine the relevance of these findings for human disease, we analyzed transcriptome data from multiple independent human cohorts and conducted genetic association studies for ESRRG variants in 2 distinct human pancreatitis cohorts. RESULTS: Blocking ERRγ function in mice by genetic deletion or inverse agonist treatment results in striking pancreatitis-like phenotypes accompanied by inflammation, fibrosis, and cell death. Mechanistically, loss of ERRγ in primary acini abrogates messenger RNA expression and protein levels of mitochondrial oxidative phosphorylation complex genes, resulting in defective acinar cell energetics. Mitochondrial dysfunction due to ERRγ deletion further triggers autophagy dysfunction, endoplasmic reticulum stress, and production of reactive oxygen species, ultimately leading to cell death. Interestingly, ERRγ-deficient acinar cells that escape cell death acquire ductal cell characteristics, indicating a role for ERRγ in acinar-to-ductal metaplasia. Consistent with our findings in ERRγ cKO mice, ERRγ expression was significantly reduced in patients with chronic pancreatitis compared with normal subjects. Furthermore, candidate locus region genetic association studies revealed multiple single nucleotide variants for ERRγ that are associated with chronic pancreatitis. CONCLUSIONS: Collectively, our findings highlight an essential role for ERRγ in maintaining the transcriptional program that supports acinar cell mitochondrial function and organellar homeostasis and provide a novel molecular link between ERRγ and exocrine pancreas disorders.

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.

Discovery of novel phenaleno isoquinolinium-based fluorescence imaging agents for sentinel lymph node mapping.

Fluorescence imaging agents have recently received huge attention due to their important role in disease diagnostics. However, the intrinsic problems of these probes, such as complex synthetic routes and high molecular weight, remain challenging. Here, we developed novel phenaleno isoquinolinium-based fluorescent agents, Medical Fluorophores 37-41 (MF37-41), applicable to the quantitative and sensitive detection of sentinel lymph nodes (SLNs). These imaging agents showed no adverse effects on the proliferation of immune and normal cells and did not induce in vivo toxicity. In vivo fluorescence lifetime imaging demonstrated the accumulation of phenaleno isoquinolinium salts in the SLNs of nude mice within 15 min postinjection, consistent with our biodistribution findings. These results suggest that phenaleno isoquinolinium salts are feasible fluorescence imaging agents that can be used as potential lymphatic tracers.

Transcriptional upregulation of multiple earthworm chitinase genes following bacterial challenge suggests their implications in innate immunity.

BACKGROUND: Chitinase is a multi-functional enzyme that catalyzes the hydrolysis of ß-1,4-linkages between N-acetylglucosamines (GlcNAc) in chitin. Recent studies imply that earthworm chitinase is implicated in self-defense immunity against chitin-containing pathogens. However, a direct relationship of earthworm chitinase with innate immunity has not yet been established. OBJECTIVE: In this study, earthworm (Eisenia andrei) chitinase expression was examined following bacterial challenge by Bacillus subtilis. METHODS: RNA sequencing (RNA-seq) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to quantitatively evaluate mRNA expression changes in response to bacterial stimulation. RESULTS: Multiple chitinase-related mRNAs were found to be upregulated, among which EaChi3, EaChi4, and EaChi2 were upregulated by approximately eightfold, eightfold, and 2.5-fold, respectively. This strongly suggested that earthworm chitinases may act as inducible humoral effectors in earthworm innate immunity. The primary structures of all three chitinases contained an N-terminal glycol_18 domain with two chitin-binding and chitin-catalyzing domains, and a C-terminal proline, glycine, serine, threonine (PGST)-rich domain. In addition, EaChi2 had a chitin-binding peritrophin-A domain at the end of the C-terminus with 5 cysteine residues possibly contributing two intradomain disulfide bonds. Multiple sequence alignment of the catalytic domain centers of glycol_18 domain displayed highly conserved chitin-binding and chitin-catalyzing domains in which three essential amino acid residues (D, D, E) for catalyzing activity are well conserved except EaChi4. The critical glutamic acid (E) residue was substituted for glutamine (Q) in EaChi4 indicating that it is devoid of catalytic activity. CONCLUSIONS: To our knowledge, this is the first report providing direct evidence that multiple earthworm chitinases are bacteria-responsive, strongly suggesting that earthworm chitinases are inducible humoral effectors in earthworm innate immunity. In addition, our results possibly suggest that earthworm EaChi4 may function as a pattern recognition molecule modulating the downstream immune pathway.

Central Sarcopenia, Frailty and Comorbidity as Predictor of Surgical Outcome in Elderly Patients with Degenerative Spine Disease.

OBJECTIVE: People are living longer and the elderly population continues to increase. The incidence of degenerative spinal diseases (DSDs) in the elderly population is quite high. Therefore, we are facing more cases of DSD and offering more surgical solutions in geriatric patients. Understanding the significance and association of frailty and central sarcopenia as risk factors for spinal surgery in elderly patients will be helpful in improving surgical outcomes. We conducted a retrospective cohort analysis of prospectively collected data to assess the impact of preoperative central sarcopenia, frailty, and comorbidity on surgical outcome in elderly patients with DSD. METHODS: We conducted a retrospective analysis of patients who underwent elective spinal surgery performed from January 1, 2019 to September 30, 2020 at our hospital. We included patients aged 65 and over who underwent surgery on the thoracic or lumbar spine and were diagnosed as DSD. Central sarcopenia was measured by the 50th percentile of psoas : L4 vertebral index (PLVI) using the cross-sectional area of the psoas muscle. We used the Korean version of the fatigue, resistance, ambulation, illnesses, and loss of weight (K-FRAIL) scale to measure frailty. Comorbidity was confirmed and scored using the Charlson Comorbidity Index (CCI). As a tool for measuring surgical outcome, we used the Clavien-Dindo (CD) classification for postoperative complications and the length of stay (LOS). RESULTS: This study included 85 patients (35 males and 50 females). The mean age was 74.05±6.47 years. Using the K-FRAIL scale, four patients were scored as robust, 44 patients were pre-frail and 37 patients were frail. The mean PLVI was 0.61±0.19. According to the CD classification, 50 patients were classified as grade 1, 19 as grade 2, and four as grade 4. The mean LOS was 12.35±8.17 days. Multivariate stepwise regression analysis showed that postoperative complication was significantly associated with surgical invasiveness and K-FRAIL scale. LOS was significantly associated with surgical invasiveness and CCI. K-FRAIL scale showed a significant correlation with CCI and PLVI. CONCLUSION: The present study demonstrates that frailty, comorbidity, and surgical invasiveness are important risk factors for postoperative complications and LOS in elderly patients with DSD. Preoperative recognition of these factors may be useful for perioperative optimization, risk stratification, and patient counseling.

Targeting the Nuclear Receptor-Binding SET Domain Family of Histone Lysine Methyltransferases for Cancer Therapy: Recent Progress and Perspectives.

Nuclear receptor-binding SET domain (NSD) proteins are a class of histone lysine methyltransferases (HKMTases) that are amplified, mutated, translocated, or overexpressed in various types of cancers. Several campaigns to develop NSD inhibitors for cancer treatment have begun following recent advances in knowledge of NSD1, NSD2, and NSD3 structures and functions as well as the U.S. FDA approval of the first HKMTase inhibitor (tazemetostat, an EZH2 inhibitor) to treat follicular lymphoma and epithelioid sarcoma. This perspective highlights recent findings on the structures of catalytic su(var), enhancer-of-zeste, trithorax (SET) domains and other functional domains of NSD methyltransferases. In addition, recent progress and efforts to discover NSD-specific small molecule inhibitors against cancer-targeting catalytic SET domains, plant homeodomains, and proline-tryptophan-tryptophan-proline domains are summarized.

Nontargeted Metabolomics by High-Resolution Mass Spectrometry to Study the In Vitro Metabolism of a Dual Inverse Agonist of Estrogen-Related Receptors ß and γ, DN203368.

DN203368 ((E)-3-[1-(4-[4-isopropylpiperazine-1-yl]phenyl) 3-methyl-2-phenylbut-1-en-1-yl] phenol) is a 4-hydroxy tamoxifen analog that is a dual inverse agonist of estrogen-related receptor ß/γ (ERRß/γ). ERRγ is an orphan nuclear receptor that plays an important role in development and homeostasis and holds potential as a novel therapeutic target in metabolic diseases such as diabetes mellitus, obesity, and cancer. ERRß is also one of the orphan nuclear receptors critical for many biological processes, such as development. We investigated the in vitro metabolism of DN203368 by conventional and metabolomic approaches using high-resolution mass spectrometry. The compound (100 µM) was incubated with rat and human liver microsomes in the presence of NADPH. In the metabolomic approach, the m/z value and retention time information obtained from the sample and heat-inactivated control group were statistically evaluated using principal component analysis and orthogonal partial least-squares discriminant analysis. Significant features responsible for group separation were then identified using tandem mass spectra. Seven metabolites of DN203368 were identified in rat liver microsomes and the metabolic pathways include hydroxylation (M1-3), N-oxidation (M4), N-deisopropylation (M5), N,N-dealkylation (M6), and oxidation and dehydrogenation (M7). Only five metabolites (M2, M3, and M5-M7) were detected in human liver microsomes. In the conventional approach using extracted ion monitoring for values of mass increase or decrease by known metabolic reactions, only five metabolites (M1-M5) were found in rat liver microsomes, whereas three metabolites (M2, M3, and M5) were found in human liver microsomes. This study revealed that nontargeted metabolomics combined with high-resolution mass spectrometry and multivariate analysis could be a more efficient tool for drug metabolite identification than the conventional approach. These results might also be useful for understanding the pharmacokinetics and metabolism of DN203368 in animals and humans.

Orphan nuclear receptor ERRγ regulates hepatic TGF-ß2 expression and fibrogenic response in CCl4-induced acute liver injury.

Acute liver injury results from the complex interactions of various pathological processes. The TGF-ß superfamily plays a crucial role in orchestrating fibrogenic response. In contrast to TGF-ß1, a role of TGF-ß2 in hepatic fibrogenic response has not been fully investigated. In this study, we showed that TGF-ß2 gene expression and secretion are induced in the liver of CCl4 (1 ml/kg)-treated WT mice. Studies with hepatocyte specific ERRγ knockout mice or treatment with an ERRγ-specific inverse agonist, GSK5182 (40 mg/kg), indicated that CCl4-induced hepatic TGF-ß2 production is ERRγ dependent. Moreover, IL6 was found as upstream signal to induce hepatic ERRγ and TGF-ß2 gene expression in CCl4-mediated acute toxicity model. Over-expression of ERRγ was sufficient to induce hepatic TGF-ß2 expression, whereas ERRγ depletion markedly reduces IL6-induced TGF-ß2 gene expression and secretion in vitro and in vivo. Promoter assays showed that ERRγ directly binds to an ERR response element in the TGF-ß2 promoter to induce TGF-ß2 transcription. Finally, GSK5182 diminished CCl4-induced fibrogenic response through inhibition of ERRγ-mediated TGF-ß2 production. Taken together, these results firstly demonstrate that ERRγ can regulate the TGF-ß2-mediated fibrogenic response in a mouse model of CC14-induced acute liver injury.

Novel quinoline-based fluorescent bioimaging probe, KSNP117, for sentinel lymph node mapping.

Fluorescent imaging agents with biocompatibility and high sensitivity are urgently required for the accurate detection of sentinel lymph nodes (SLNs). Herein, we report the design of a novel quinoline-based fluorescent probe, designated KSNP117, which can be applied as a biomedical imaging agent in the sensitive and quantitative detection of SLNs. KSNP117 exerted no adverse effects on the proliferation of ovary and immune cells and also showed excellent serum stability with photo-brightening effects. In vivo fluorescent imaging revealed the accumulation of KSNP117 in the SLNs of nude mice within 10 min post injection, without in vivo toxicity, which was consistent with the findings of ex vivo imaging. These results support the potential of KSNP117 as a promising lymphatic tracer for biomedical imaging applications.

Orphan nuclear receptor ERR-γ regulates hepatic FGF23 production in acute kidney injury.

Fibroblast growth factor 23 (FGF23), a hormone generally derived from bone, is important in phosphate and vitamin D homeostasis. In acute kidney injury (AKI) patients, high-circulating FGF23 levels are associated with disease progression and mortality. However, the organ and cell type of FGF23 production in AKI and the molecular mechanism of its excessive production are still unidentified. For insight, we investigated folic acid (FA)-induced AKI in mice. Interestingly, simultaneous with FGF23, orphan nuclear receptor ERR-γ expression is increased in the liver of FA-treated mice, and ectopic overexpression of ERR-γ was sufficient to induce hepatic FGF23 production. In patients and in mice, AKI is accompanied by up-regulated systemic IL-6, which was previously identified as an upstream regulator of ERR-γ expression in the liver. Administration of IL-6 neutralizing antibody to FA-treated mice or of recombinant IL-6 to healthy mice confirms IL-6 as an upstream regulator of hepatic ERR-γ-mediated FGF23 production. A significant (P < 0.001) interconnection between high IL-6 and FGF23 levels as a predictor of AKI in patients that underwent cardiac surgery was also found, suggesting the clinical relevance of the finding. Finally, liver-specific depletion of ERR-γ or treatment with an inverse ERR-γ agonist decreased hepatic FGF23 expression and plasma FGF23 levels in mice with FA-induced AKI. Thus, inverse agonist of ERR-γ may represent a therapeutic strategy to reduce adverse plasma FGF23 levels in AKI.

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.

Identification and expression of adenosine deaminases acting on tRNA (ADAT) during early tail regeneration of the earthworm.

BACKGROUND: RNA editing is a widespread phenomenon in all metazoans. One of the common RNA editing event is the chemical conversion of adenosine to inosine (A-to-I) catalyzed by adenosine deaminases acting on tRNA (ADAT). During D. melanogaster development, the ADAT1 transcript was found to localize mainly to the central nervous system including brain and ventral nerve cord during brain development. Although an earthworm adenosine deaminases acting on mRNA (ADAR) has been identified and its possible implication in earthworm regeneration has been investigated, there is little accumulated information on ADAT and tRNA editing in the annelid including terrestrial earthworms. OBJECTIVE: This study aimed to investigate the molecular characteristics and the expression pattern of earthworm ADAT during tail regeneration to understand its physiological significance. METHODS: Nucleotide sequence of Ean-ADAT was retrieved from the genome assembly of Eisenia andrei via Basic Local Alignment Search Tool (BLAST). The genome assembly of Eisenia andrei was downloaded from National Genomics Data Center ( http://bigd.big.ac.cn/gwh/ ). The alignment and phylogenetic relationship of the core deaminase domains of ADATs and ADARs were analyzed. Its temporal expression during early tail regeneration was measured using real-time PCR. RESULTS: The open reading frame of Ean-ADAT consists of 1719 nucleotides encoding 573 amino acids. Domain analysis indicates that Ean-ADAT has a deaminase domain composed of 498 amino acids and a predicted nuclear localization signal at the N-terminal. Its subcellular localization was predicted to be nuclear. The core deaminase region of Ean-ADAT encompasses the three active-site motifs, including zinc-chelating residues and a glutamate residue for catalytic activity. In addition, Ean-ADAT shares highly conserved RNA recognition region flanking the third cysteine of the deaminase motif with other ADAT1s even from the yeast. Multiple sequence alignment and phylogenetic analysis indicate that Ean-ADAT shows greater similarity to vertebrate ADARs than to yeast Tad1p. Ean-ADAT mRNA expression began to remarkably decrease before 12 h post-amputation, showing a tendency to gradual decrease until 7 dpa and then it slightly rebounded at 10 dpa. CONCLUSIONS: Our results demonstrate that Ean-ADAT belongs to a class of ADAT1s and support the hypothesis of a common evolutionary origin for ADARs and ADATs. The temporal expression of Ean-ADAT could suggest that its activity is unrelated to the molecular mechanisms of dedifferentiation.