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Abraxas brother protein 1 (ABRO1) is a subunit of the deubiquitinating enzyme BRCC36-containing isopeptidase complex and plays important roles in cellular responses to stress by interacting with its binding partners, such as ubiquitin-specific peptidase 7, p53, activating transcription factor 4, THAP-domain containing 5, and serine hydroxymethyltransferase. However, the transcriptional regulation of ABRO1 remains unexplored. In this study, we identified and characterized the core regulatory elements of the human ABRO1 gene and mapped them to the ABRO1 promoter region. Additionally, 5′ rapid amplification of cDNA ends revealed that the transcriptional start site (TSS) was located −13 bp upstream from the start codon. Reporter gene, chromatin immunoprecipitation, and electrophoretic mobility shift assays demonstrated that ABRO1 transcription was regulated through cis-acting elements located in the region −89 to −59 bp upstream of the ABRO1 TSS and that these elements were targeted by yin yang 1 transcription factor (YY1). Moreover, YY1 overexpression increased human ABRO1 mRNA and protein expression, and small-interfering RNA-mediated downregulation of YY1 attenuated ABRO1 expression. These results suggested that YY1 positively regulated human ABRO1 expression by binding to cis-acting elements located in the ABRO1 TSS.

The nucleotide-binding domain and leucine-rich repeat-containing family pyrin domain containing 3 (NLRP3) inflammasome is involved in various acute and chronic liver diseases, however, it is not clear whether NLRP3 contributes to d -Galactosamine (D-GalN) plus lipopolysaccharide (LPS)-induced acute liver failure (ALF). This study aims to investigate the role of NLRP3 inflammasome in D-GalN/LPS-induced fatal hepatitis. We found that Nlrp3−/− and WT mice showed similar mortality against a lethal dose of D-GalN/LPS treatment. Serum ALT and AST levels, as well as liver necrosis area and hepatocyte apoptosis, were not significantly different between Nlrp3−/− and WT mice at 6 h after D-GalN/LPS injection. Moreover, the numbers of intrahepatic F4/80+ cells and Ly6G+ cells were comparable in two genotype mice following D-GalN/LPS treatment. Besides, Nlrp3−/− mice had reduced IL-1β levels but similar TNF-α, IL-6, and MCP-1 levels compared with WT mice upon D-GalN/LPS administration. Our findings revealed that NLRP3 ablation does not protect mice from D-GalN/LPS-induced fatal hepatitis and has a marginal effect on intrahepatic inflammatory response upon D-GalN/LPS treatment. This suggests that NLRP3 inflammasome does not appear to be a major contributor to D-GalN/LPS-induced ALF.

Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2−/− embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.

Hematopoietic Stem and Progenitor Cells In article number 2103838, Xiao‐Ming Yang, Chang‐Yan Li and co‐workers identify a critical regulator‐HEMGN, that is required for hematopoietic stem and progenitor cells (HSPCs) homing, survival, and reconstitution of blood cells during bone marrow transplantation (BMT). HEMGN acts as a protector which limits interferon‐γ (IFN‐γ) signaling by suppressing nuclear Stat1 tyrosine phosphorylation and assists HSPCs to combat the transplantation stress. Targeted Hemgn may be used to improve conditioning regimens and engraftment during bone marrow transplantation. [Image: see text]

Background Human hepatocellular carcinoma (HCC) lacks effective curative therapy and there is an urgent need to develop a novel molecular-targeted therapy for HCC. Selective tyrosine kinase inhibitors have shown promise in treating cancers including HCC. Tyrosine kinases c-Met and Trks are potential therapeutic targets of HCC and strategies to interrupt c-Met and Trks cross-signaling may result in increased effects on HCC inhibition. Methods The effects of Indo5 on c-Met and Trks activity were determined with in vitro kinase activity assay, cell-based signaling pathway activation, and kinases-driven cell transformation. The in vivo anti-tumor activity was determined with xenograft mice and liver orthotopic mice models. The co-expression of c-Met and TrkB in 180 pairs of HCC and adjacent normal tissues were detected using immunohistochemical staining. Results Indo5, a novel lead compound displayed biochemical potency against both c-Met and Trks with selectivity over 13 human kinases. Indo5 abrogated HGF-induced c-Met signaling activation and BDNF/NGF-induced Trks signal activation, c-Met or TrkB-mediated cell transformation and migration. Furthermore, Indo5 significantly decreased the growth of HCC cells in xenograft mice and improved the survival of mice with liver orthotopic tumors. In addition, co-expression of c-Met and TrkB in HCC patients was a predictor of poor prognosis, and combined inhibition of c-Met and TrkB exerted a synergistic suppressive effect on HCC. Conclusions These findings indicate that Indo5 is associated with marked suppression of c-Met and Trks co-expressing HCC, supporting its clinical development as an antitumor treatment for HCC patients with co-active c-Met and Trks signaling. Electronic supplementary material The online version of this article (10.1186/s13046-019-1104-4) contains supplementary material, which is available to authorized users.

Glycerol kinase (Gyk), consisting of 4 isoforms, plays a critical role in metabolism by converting glycerol to glycerol 3-phosphate in an ATP-dependent reaction. Only Gyk isoform b is present in whole cells, but its function in the nucleus remains elusive. Previous studies have shown that nuclear orphan receptor subfamily 4 group A member (NR4A)-1 is an important regulator of hepatic glucose homeostasis and lipid metabolism in adipose tissue. We aimed to elucidate the functional interaction between nuclear Gyk and NR4A1 during hepatic gluconeogenesis in the unfed state and diabetes. We identified nuclear Gyk as a novel corepressor of NR4A1 in the liver; moreover, this recruitment was dependent on the C-terminal ligand-binding domain instead of the N-terminal activation function 1 domain, which interacts with other NR4A1 coregulators. NR4A1 transcriptional activity was inhibited by Gyk via protein-protein interaction but not enzymatic activity. Moreover, Gyk overexpression suppressed NR4A1 ability to regulate the expression of target genes involved in hepatic gluconeogenesis in vitro and in vivo as well as blood glucose regulation, which was observed in both unfed and diabetic mice. These results highlight the moonlighting function of nuclear Gyk, which was found to act as a coregulator of NR4A1, participating in the regulation of hepatic glucose homeostasis in the unfed state and diabetes.-Miao, L., Yang, Y., Liu, Y., Lai, L., Wang, L., Zhan, Y., Yin, R., Yu, M., Li, C., Yang, X., Ge, C. Glycerol kinase interacts with nuclear receptor NR4A1 and regulates glucose metabolism in the liver.

Background Toll-like receptor 5 (TLR5)-mediated pathways play critical roles in regulating the hepatic immune response and show hepatoprotective effects in mouse models of hepatic diseases. However, the role of TLR5 in experimental models of liver regeneration has not been reported. This study aimed to investigate the role of TLR5 in partial hepatectomy (PHx)-induced liver regeneration. Methods We performed 2/3 PHx in wild-type (WT) mice, TLR5 knockout mice, or TLR5 agonist CBLB502 treated mice, as a model of liver regeneration. Bacterial flagellin content was measured with ELISA, and hepatic TLR5 expression was determined with quantitative PCR analyses and flow cytometry. To study the effects of TLR5 on hepatocyte proliferation, we analyzed bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression with immunohistochemistry (IHC) staining. The effects of TLR5 during the priming phase of liver regeneration were examined with quantitative PCR analyses of immediate early gene mRNA levels, and with Western blotting analysis of hepatic NF-κB and STAT3 activation. Cytokine and growth factor production after PHx were detected with real-time PCR and cytometric bead array (CBA) assays. Oil Red O staining and hepatic lipid concentrations were analyzed to examine the effect of TLR5 on hepatic lipid accumulation after PHx. Results The bacterial flagellin content in the serum and liver increased, and the hepatic TLR5 expression was significantly up-regulated in WT mice after PHx. TLR5-deficient mice exhibited diminished numbers of BrdU- and PCNA-positive cells, suppressed immediate early gene expression, and decreased cytokine and growth factor production. Moreover, PHx-induced hepatic NF-κB and STAT3 activation was inhibited in Tlr5−/− mice, as compared with WT mice. Consistently, the administration of CBLB502 significantly promoted PHx-mediated hepatocyte proliferation, which was correlated with enhanced production of proinflammatory cytokines and the recruitment of macrophages and neutrophils in the liver. Furthermore, Tlr5−/− mice displayed significantly lower hepatic lipid concentrations and smaller Oil Red O positive areas than those in control mice after PHx. Conclusion We reveal that TLR5 activation contributes to the initial events of liver regeneration after PHx. Our findings demonstrate that TLR5 signaling positively regulates liver regeneration and suggest the potential of TLR5 agonist to promote liver regeneration.

Background: The NOD-like receptor family protein 3 (NLRP3) inflammasome is involved in the progression of liver inflammation. NLRP3 inactivation has been shown to protect mice against multiple types of experimental liver injury. However, it is unclear whether NLRP3 contributes to D-Galactosamine (DGalN) plus lipopolysaccharide (LPS) induced fatal hepatitis. This study aims to examine the function of NLRP3 inflammasome in DGalN/LPS induced acute liver failure.Method: The expression of inflammasomes was detected by quantitative PCR. Wild type (WT) mice and NLRP3 knockout mice were given an intraperitoneal injection of DGalN plus LPS. Liver damage was examined at 6 h post DGalN/LPS administration by histological analysis and serum aminotransferases measurement. Hepatocyte apoptosis and pyroptosis was detected with TUNEL assay, immunohistochemistry (IHC) staining, and western blot. Hepatic macrophages (F4/80+) and neutrophils (Ly6G+) were analyzed by IHC staining. The proinflammatory cytokines levels in serum and liver were examined using cytometric bead array and quantitative PCR. Hepatic IL1β level was further detected by western blot.Result: The hepatic NLRP3 activity was upregulated in WT mice treated with DGalN/LPS. Nlrp3−/− and WT mice showed similar mortality against lethal dose of DGalN/LPS. Serum aminotransferases levels and liver necrosis area of Nlrp3−/− mice did not differ from that in WT mice after DGalN/LPS injection. The numbers of liver TUNEL positive cells and cleaved caspase3 positive hepatocytes were comparable between Nlrp3−/− and WT mice. The hepatic GSDMDNterm peptide production also showed no difference between Nlrp3−/− and WT mice. Mice treated with DGalN/LPS displayed significantly increased numbers of intrahepatic F4/80+ cells and Ly6G+ cells, but the cell numbers in Nlrp3−/− mice livers were similar to those in WT mice. Consistently, serum and hepatic TNFα, IL6, and MCP-1 levels were similar between Nlrp3−/− and WT mice upon DGalN/LPS administration, but serum IL1β and hepatic mature IL1β level in Nlrp3−/− mice was reduced.Conclusions: NLRP3 ablation does not protect mice from DGalN/LPS induced acute liver failure, nor affect hepatocyte apoptosis and pyroptosis. Deficiency of NLRP3 has a limited effect on intrahepatic inflammatory response induced by DGalN/LPS treatment. NLRP3 inflammasome does not appear to be a major contributor to DGalN/LPS induced fatal hepatitis.

Pharmacologically inhibiting nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome activation results in potent therapeutic effects in a wide variety of preclinical inflammatory disease models. NLRP3 deubiquitination is essential for efficient NLRP3 inflammasome activity, but it remains unclear whether this process can be harnessed for therapeutic benefit. Here, we show that thiolutin (THL), an inhibitor of the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease, blocks NLRP3 inflammasome activation by canonical, noncanonical, alternative, and transcription-independent pathways at nanomolar concentrations. In addition, THL potently inhibited the activation of multiple NLRP3 mutants linked with cryopyrin-associated periodic syndromes (CAPS). Treatment with THL alleviated NLRP3-related diseases in mouse models of lipopolysaccharide-induced sepsis, monosodium urate-induced peritonitis, experimental autoimmune encephalomyelitis, CAPS, and methionine-choline-deficient diet-induced nonalcoholic fatty liver disease. Mechanistic studies revealed that THL inhibits the BRCC3-containing isopeptidase complex (BRISC)-mediated NLRP3 deubiquitination and activation. In addition, we show that holomycin, a natural methyl derivative of THL, displays an even higher inhibitory activity against NLRP3 inflammasome than THL. Our study validates that posttranslational modification of NLRP3 can be pharmacologically targeted to prevent or treat NLRP3-associated inflammatory diseases. Future clinical development of derivatives of THL may provide new therapies for NLRP3-related diseases.

BRCA1/BRCA2-containing complex subunit 3 (BRCC3) is a lysine 63-specific deubiquitinase involved in multiple biological processes, such as DNA repair and immune responses. However, the regulation mechanism for BRCC3 protein stability is still unknown. Here, we demonstrate that BRCC3 is mainly degraded through the ubiquitin-proteasome pathway. The HECT-type E3 ubiquitin ligase WWP2 modulates BRCC3 ubiquitination and degradation. ABRO1, a subunit of the BRCC36 isopeptidase complex (BRISC), competes with WWP2 to bind to BRCC3, thereby preventing WWP2-mediated BRCC3 ubiquitination and enhancing BRCC3 stability. Functionally, we show that lentivirus-mediated overexpression of WWP2 in murine macrophages inhibits NLRP3 inflammasome activation by decreasing BRCC3 protein level. This study provides the first insights into the regulation of BRCC3 stability and expands our knowledge about the physiological function of WWP2.

Hematopoietic stem and progenitor cells (HSPCs) possess the remarkable ability to regenerate the whole blood system in response to ablated stress demands. Delineating the mechanisms that maintain HSPCs during regenerative stresses is increasingly important. Here, it is shown that Hemgn is significantly induced by hematopoietic stresses including irradiation and bone marrow transplantation (BMT). Hemgn deficiency does not disturb steady‐state hematopoiesis in young mice. Hemgn−/‐ HSPCs display defective engraftment activity during BMT with reduced homing and survival and increased apoptosis. Transcriptome profiling analysis reveals that upregulated genes in transplanted Hemgn−/− HSPCs are enriched for gene sets related to interferon gamma (IFN‐γ) signaling. Hemgn−/− HSPCs show enhanced responses to IFN‐γ treatment and increased aging over time. Blocking IFN‐γ signaling in irradiated recipients either pharmacologically or genetically rescues Hemgn−/‐ HSPCs engraftment defect. Mechanistical studies reveal that Hemgn deficiency sustain nuclear Stat1 tyrosine phosphorylation via suppressing T‐cell protein tyrosine phosphatase TC45 activity. Spermidine, a selective activator of TC45, rescues exacerbated phenotype of HSPCs in IFN‐γ‐treated Hemgn−/− mice. Collectively, these results identify that Hemgn is a critical regulator for successful engraftment and reconstitution of HSPCs in mice through negatively regulating IFN‐γ signaling. Targeted Hemgn may be used to improve conditioning regimens and engraftment during HSPCs transplantation.

// Chao Liu 1, 2, * , En-Dong Liu 2, * , Yun-Xiao Meng 3, * , Xiao-Ming Dong 4 , Ya-Lan Bi 3 , Huan-Wen Wu 3 , Yan-Chao Jin 2 , Ke Zhao 2 , Jian-Jie Li 5 , Miao Yu 2 , Yi-Qun Zhan 2 , Hui Chen 2 , Chang-Hui Ge 2 , Xiao-Ming Yang 1, 2, 4 and Chang-Yan Li 1, 2 1 An Hui Medical University, Hefei, 230032, China 2 State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 100850, China 3 Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tsinghua University, Beijing, 100730, China 4 Tianjin University, School of Chemical Engineering and Technology, Department of Pharmaceutical Engineering, Tianjin, 300072, China 5 Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Department of Thoracic Oncology, Peking University Cancer Hospital Institute, Beijing, 100871, China * These authors contributed equally to this work Correspondence to: Xiao-Ming Yang, email: xiaomingyang@sina.com Chang-Yan Li, email: fmmli@163.com Keywords: CK8, colitis, colitis-associated colorectal cancer, gut microbiota, TLR4 Received: February 21, 2017 Accepted: May 13, 2017 Published: May 27, 2017 ABSTRACT Keratin 8 (CK8) is the major component of the intermediate filaments of simple or single-layered epithelia. Gene targeting mice model suggest that CK8 is involved in colonic active ion transport, colorectal hyperplasia and inflammation. In the present study, we found that CK8 is downregulated in the colon during DSS-induced colitis and AOM/DSS-induced colitis-associated colorectal cancer (CAC) development. In human patients with colon cancer, CK8 is downregulated. Using CK8 heterozygous knockout mice (CK8 +/− ), we found that CK8 +/− mice are highly susceptible to DSS-induced colitis and more prone to AOM/DSS-induced CAC than wild type (WT) mice. The colonic permeability is increased with DSS or AOM/DSS treatment, leading to alteration of gut microbiota in CK8 +/− mice with CAC. Metagenomic analysis of fecal microbiota suggests Firmicutes and Proteobacteria are increased in CK8 +/− mice with CAC, while Bacteroidetes and Verrucomicrobia are decreased. Antibiotic treatment decreases the incidence of colorectal cancer tumorigenesis and TLR4 inhibitor attenuates the susceptibility of CK8 +/− mice to DSS-induced colitis. These data suggest CK8 protects mice from colitis and colitis-associated colorectal cancer by modulating colonic permeability and gut microbiota composition homeostasis.

Glycerol kinase (GYK) plays a critical role in hepatic metabolism by converting glycerol to glycerol 3-phosphate in an ATP-dependent reaction. GYK isoform b is the only glycerol kinase present in whole cells, and has a non-enzymatic moonlighting function in the nucleus. GYK isoform b acts as a co-regulator of nuclear receptor subfamily 4 group A1 (NR4A1) and participates in the regulation of hepatic glucose metabolism by protein–protein interaction with NR4A1. Herein, GYK expression was found to upregulate the expression of NR4A1-mediated lipid metabolism-related genes (SREBP1C, FASN, ACACA, and GPAM) in HEK293T and L02 cells, and in mouse in vivo studies. GYK expression increased blood levels of cholesterol, triglyceride, and high-density lipoprotein cholesterol, but not low-density lipoprotein cholesterol levels. It enhanced the transcriptional activity of Nr4a1 target genes by negatively cooperating with NR4A1 and its enzymatic activity or by other undefined moonlighting functions. This enhancement was observed in both normal and diabetic mice. We also found a feed-forward regulation loop between GYK and NR4A1, serving as part of a GYK-NR4A1 regulatory mechanism in hepatic metabolism. Thus, GYK regulates the effect of NR4A1 on hepatic lipid metabolism in normal and diabetic mice, partially through the cooperation of GYK and NR4A1.

During human erythroid maturation, Hsp70 translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. Failure of Hsp70 to localize to the nucleus was found in Myelodysplastic syndrome (MDS) erythroblasts and can induce dyserythropoiesis, with arrest of maturation and death of erythroblasts. However, the mechanism of the nuclear trafficking of Hsp70 in erythroblasts remains unknown. Here, we found the hematopoietic transcriptional regulator, EDAG, to be a novel binding partner of Hsp70 that forms a protein complex with Hsp70 and GATA-1 during human normal erythroid differentiation. EDAG overexpression blocked the cytoplasmic translocation of Hsp70 induced by EPO deprivation, inhibited GATA-1 degradation, thereby promoting erythroid maturation in an Hsp70-dependent manner. Furthermore, in myelodysplastic syndrome (MDS) patients with dyserythropoiesis, EDAG is dramatically down-regulated, and forced expression of EDAG has been found to restore the localization of Hsp70 in the nucleus and elevate the protein level of GATA-1 to a significant extent. In addition, EDAG rescued the dyserythropoiesis of MDS patients by increasing erythroid differentiation and decreasing cell apoptosis. This study demonstrates the molecular mechanism of Hsp70 nuclear sustaining during erythroid maturation and establishes that EDAG might be a suitable therapeutic target for dyserythropoiesis in MDS patients.

To investigate the effects of Listeria monocytogenes infection on hematopoietic stem and progenitor cell (HSPC) composition, cell cycle and cell colony-forming ability in mouse bone marrow.The C57BL/6J mice were divided into infected group and control group. The mice in injected group were infected intraperitoneally with 6.7×10Serum IFNγ levels peaked at 24 hours after infection with Listeria monocytogenes. After 24 h, the proportion of LSK, LSK in S phase, and short-term hematopoietic stem cells (ST-HSC) in the infected group were significantly higher than those in the control group (P＜0.001), long-term hematopoietic stem cells (LT-HSC) and the proportion of LT-HSC in S phase were significantly increased (P＜0.01), and the cell colony-forming ability of bone marrow significantly decreased (P＜0.01). [WTHZ]Conclusion: [WTB1]After infection with Listeria monocytogenes, bone marrow hematopoietic stem cells enter the proliferative state from rest, the cell colony-forming ability decreases, suggesting that Listeria monocytogenes infection can cause hematopoietic stem cell depletion.单核细胞增生性李斯特菌感染对小鼠骨髓造血干细胞的影响.探究单核细胞增生性李斯特菌感染对小鼠骨髓中造血干/祖细胞（HSPC）组成、细胞周期以及集落形成能力的影响.将C57BL/6J小鼠分为感染组和对照组，感染组小鼠经腹腔注射感染单核细胞增生性李斯特菌6.7×10感染单核细胞增生性李斯特菌24 h后，血清IFNγ水平达到高峰； 与对照组相比，小鼠骨髓细胞中LSK、处于S期的LSK以及短期造血干细胞（ST-HSC）的比例显著升高（P＜0.001），长期造血干细胞（LT-HSC）以及处于S期的LT-HSC的比例明显升高（P＜0.01），骨髓细胞集落形成能力明显降低（P＜0.01）.感染单核细胞增生性李斯特菌后，小鼠骨髓造血干细胞由静止进入增殖状态，细胞集落形成能力下降。这提示，单核细胞增生性李斯特菌感染可引起造血干细胞耗竭.

Bacterial flagellin is a pathogen-associated molecular pattern recognized by surface-localized Toll-like receptor 5 (TLR5) and cytosolic NOD-like receptor protein 4 (NLRC4). CBLB502, derived from Salmonella flagellin, exhibits high radioprotective efficacy in mice and primates by regulating TLR5 and the nuclear factor kappa B (NF-κB) signaling pathway. In this study, we examined the effects of CBLB502 and mutations in its NLRC4- and TLR5-binding domains on radioprotective efficacy and the immune inflammatory response. The results showed that CBLB502 mutation with I213A in the TLR5-binding domain significantly reduced NF-κB activity and radioprotective activity, whereas CBLB502 mutation with L292A in NLRC4-binding domain did not. Additionally, CBLB502 with both mutations greatly reduced NF-κB activity and eliminated radioprotection in mice. In contrast, NLRC4-binding domain mutation reduced the secretion of inflammatory interleukin-1β and interleukin-18. CBLB502 exerts its radioprotective effects through both the TLR5 and NLRC4 pathways. Additionally, deletion in the NLRC4-binding domain did not reduce radioprotective activity but reduced the inflammatory response.

Integrating spatiotemporal proteomics data with protein-protein interaction (PPI) data can help researchers make an in-depth exploration of their proteins of interest in a dynamic manner. However, there is still a lack of proper tools for the biologists who usually have few programming skills to construct a PPI network for a protein list, visualize active PPI subnetworks, and then select key nodes for further study. We propose a web-based platform named PPIExp that can automatically construct a PPI network, perform clustering analysis according to protein abundances, and perform functional enrichment analysis. More importantly, it provides multiple effective visualization interfaces, such as the interface to display the PPI network map, the interface to display a dendrogram and heatmap for the clustering result, and the interface to display the expression pattern of a selected protein. To visualize the active PPI subnetworks in specific space or time, it provides buttons to highlight the differentially expressed proteins under each condition on the network map. Additionally, to help researchers determine which proteins are worth further attention, PPIExp provides extensive one-click interactive operations to map node centrality measures to node size on the network and highlight three types of proteins, that is, the proteins in an enriched functional term, the coexpressed proteins selected from the dendgrogram and heatmap, and the proteins input by users. PPIExp is available at http://www.fgvis.com/expressvis/PPIExp .

Summary Effective visualization is important for knowledge discovery when analysing expression profile data. However, existing tools for visually integrating expression profile data with KEGG pathway maps lack extensive interactive visualization operations. KeggExp simultaneously presents the pathway map of one pathway, dendrogram and heatmap of the genes in the pathway and scatter map of one gene; and also provides interactive operations for highlighting specific genes on the pathway map, including differentially-expressed genes, co-expressed genes selected from the heatmap and user-input genes. With KeggExp, researchers, including those without programming skills, can take advantage of its interactive operations to determine key genes or pathways when analysing expression profile data. Availability and implementation Freely available at http://www.fgvis.com/expressvis/KeggExp/. Language: JavaScript, python; Libraries: D3.js, Rxjs, Angular, Django, Django rest frame work, Scipy. Supplementary information Supplementary data are available at Bioinformatics online.

CBLB502, a Toll-like receptor (TLR)5 agonist derived from Salmonella flagellin, was shown to protect mammalian hematopoietic and gastrointestinal systems from acute irradiation syndrome and to stimulate regeneration. To explore whether CBLB502 can improve testicular injuries caused by irradiation, mice were intraperitoneally injected with 0.2 mg/kg CBLB502 or vehicle control 30 min prior to applying 5.0 Gy ionizing radiation (IR). We observed these mice for the following 120 days and determined that CBLB502 pretreatment alleviated IR-induced oxidative stress, alleviated the distorted architecture of seminiferous tubules, reversed the decline of sperm quantity and quality, and helped recover male mouse fertility. Additionally, CBLB502 efficiently reduced DNA damage and chromosomal aberrations in IR-treated mice and their offspring. Due to the suppression of p53-dependent apoptosis, in IR-treated mice, CBLB502 was shown to significantly activate the nuclear factor kappa B (NFκB) pathway and reduce the apoptotic rate in association with an increase in anti-apoptotic B-cell lymphoma 2 levels and a decrease in the levels of DNA repair protein and proliferating cell nuclear antigen. Moreover, an IR-induced reduction in serum testosterone and superoxide dismutase levels and an increase in malondialdehyde levels were considerably reversed in CBLB502-pretreated mice. No significant reverse effects were found in Tlr5 knockout mice, suggesting that protection of the testis against IR by CBLB502 is primarily dependent on the TLR5 signaling pathway. Our results may help further investigations into potential CBLB502 applications for the protection of the male reproductive system during radiotherapy.

Background & aims Hepassocin (HPS) is a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage. In this paper, zebrafish were used to investigate the role of HPS in liver development. Methods and results During zebrafish development, HPS expression is enriched in liver throughout hepatogenesis. Knockdown of HPS using its specific morpholino leads to a smaller liver phenotype. Further results showed that the HPS knockdown has no effect on the expression of the early endoderm marker gata6 and early hepatic marker hhex. In addition, results showed that the smaller-liver phenotype in HPS morphants was caused by suppression of cell proliferation, not induction of cell apoptosis. Conclusions Current findings indicated that HPS is essential to the later stages of development in vertebrate liver organogenesis.

GATA-2, a member of zinc finger GATA transcription factor family, plays key role in the hematopoietic stem cells self-renewal and differentiation. The transforming growth factor-β (TGFβ) signaling pathway is a major signaling network that controls cell proliferation, differentiation and tumor suppression. Here we found that GATA-2 negatively regulated TGF-β signaling pathway in Smad4-dependent manner. GATA-2 specifically interacts with Smad4 with its N-terminal while the zinc finger domain of GATA-2 is essential for negative regulation of TGFβ. Although GATA-2 did not affect the phosphorylation of Smad2/3 and the complex Smad2/3/4 formation in response to TGFβ, the DNA binding activity of Smad4 was decreased significantly by GATA-2 overexpression. Overexpression of GATA-2 in K562 cells led to reduced TGFβ-induced erythroid differentiation while knockdown of GATA-2 enhanced TGFβ-induced erythroid differentiation. All these results suggest that GATA-2 is a novel negative regulator of TGFβ signal pathway.

Polyglutamine diseases are a group of neurodegenerative disorders caused by expansion of a CAG repeat that encodes polyglutamine in each respective disease gene. The transcription factor THAP11, a member of THAP family, is involved in cell growth, ES cell pluripotency and embryogenesis. Previous studies suggest that THAP11 protein contains a 29-residue repeat polyglutamine motif and the number of polyglutamine ranges from 20 to 41 in Indian population. We have investigated the CAG numbers at the THAP11 locus in normal individuals and neurodegenerative disease patients of Chinese Han population and a 38Q expansion (THAP11(38Q)) was found in patients. Using fluorescence confocal-based cell imaging, THAP11(38Q) protein formed intranuclear inclusions easier than THAP11(29Q) in PC12 cells. Enhanced toxicity was investigated in THAP11(38Q)-expressing cells by growth inhibition and G0/G1 arrest. CREB-mediated transcription activity was inhibited by THAP11(38Q). The transcription factor, TBP, coactivator CBP, and chaperon protein, HSP70, could be recruited to THAP11(38Q). These results indicate that expansion of the polyglutamine in THAP11 forms intracellular aggregation and is toxic in PC12 cells, suggesting a putative role of THAP11 in polyglutamine disease.

Toll-like receptor-5 (TLR5) signaling regulates the immune privileged status of the liver and is involved in hepatic immune disorders. However, the role of TLR5 has not yet been investigated in experimental models of concanavalin A (Con A)-mediated liver injury. Here, we show that TLR5 is highly up-regulated in the hepatic mononuclear cells of mice during Con A-induced hepatitis. Increased mortality and liver histopathology of TLR5-deficient mice correlated with excessive production of proinflammatory cytokines, suggesting that TLR5 knockout mice were more susceptible to Con A-induced hepatitis. We also report that administration of CBLB502, an exogenous TLR5 agonist, substantially alleviated Con A-mediated hepatitis in wild-type mice as shown by increased survival rates, reduced aminotransferase and proinflammatory cytokine production, impaired lymphocyte infiltration, and ameliorated hepatocyte necrosis and/or apoptosis. Mechanistic studies revealed that CBLB502 acts as a negative regulator in limiting T-cell/natural killer T-cell activity and cytokine production in the Con A-hepatitis model. Bone marrow transplantation experiments showed that TLR5 in bone marrow-derived cells contributed to the hepatoprotective efficacy of CBLB502 against Con A-induced liver injury. Moreover, interleukin-6 elevation induced by CBLB502 is an important protective factor against Con A-induced liver injury. In addition, we demonstrate that CBLB502 suppresses α-galactosylceramide-induced natural killer T cell-dependent inflammatory liver injury. Conclusion The TLR5 signaling pathway plays an important role in T cell-mediated hepatic injury and may be exploited for therapeutic treatment of inflammatory liver diseases. (Hepatology 2017;65:2059-2073).

EWSR1, participating in transcription and splicing, has been identified as a translocation partner for various transcription factors, resulting in translocation, which in turn plays crucial roles in tumorigenesis. Recent studies have investigated the role of EWSR1 in mitosis. However, the effect of EWSR1 on mitosis is poorly understood. Here, we observed that depletion of EWSR1 resulted in cell cycle arrest in the mitotic phase, mainly due to an increase in the time from nuclear envelope breakdown to metaphase, resulting in a high percentage of unaligned chromosomes and multipolar spindles. We also demonstrated that EWSR1 is a spindle-associated protein that interacts with α-tubulin during mitosis. EWSR1 depletion increased the cold-sensitivity of spindle microtubules, and decreased the rate of spindle assembly. EWSR1 regulated the level of microtubule acetylation in the mitotic spindle; microtubule acetylation was rescued in EWSR1-depleted mitotic cells following suppression of HDAC6 activity by its specific inhibitor or siRNA treatment. In summary, these results suggest that EWSR1 regulates the acetylation of microtubules in a cell cycle-dependent manner through its dynamic location on spindle MTs, and may be a novel regulator for mitosis progress independent of its translocation.

Toll-like receptors (TLRs) have critical roles in innate immunity and inflammation and the detailed mechanisms by which TLR signaling is fine tuned remain unclear. Keratin 8 (CK8) belongs to the type II keratin family and is the major compontent of the intermediate filaments of simple or single-layered epithelia. Here we report that down-regulation of CK8 in mice enhanced TLR-mediated responses, rendering mice more susceptible to lipopolysaccharide (LPS)-induced endotoxin shock and Escherichia coli–caused septic peritonitis with reduced survival, elevated levels of inflammation cytokines and more severe tissue damage. We found that CK8 suppressed TLR-induced nuclear factor (NF)-κB activation and interacted with the adaptor tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to prevent its polyubiquitination. Our findings demonstrate a novel role of CK8 in negative regulation of TLR/NF-κB signaling and highlight a previously unidentified nonclassical function for CK8 in limiting inflammatory responses.

Erythroid differentiation-associated gene (EDAG) is a haematopoietic tissue-specific transcription regulator that plays a key role in maintaining the homeostasis of haematopoietic lineage commitment. In acute myeloid leukaemia (AML) patients, the high expression level of EDAG is associated with poor prognosis. NPM1 (nucleophosmin/B23), a ubiquitous nucleolar phosphoprotein, comprises a multifunctional protein that is involved in several cellular processes, including ribosome biogenesis, centrosome duplication, cell cycle progression, cell growth and transformation. Various studies have implicated NPM1 overexpression in promoting tumour cell proliferation, blocking the differentiation of leukaemia cells and resisting apoptosis. In the present study, using co-immunoprecipitation, we characterized EDAG as a physiological binding partner of NPM1; The N-terminal (amino acids 1-124) region of EDAG interacts with the N-terminal (amino acids 118-187) of NPM1. Under cycloheximide treatment, the stability of NPM1 protein was enhanced by EDAG overexpression, whereas knockdown of EDAG by lentivirus-mediated small interfering RNA resulted in an increased degradation rate of NPM1 in K562 cells. During 4β-phorbol l2-myristate 13-acetate-induced K562 megakaryocytic differentiation, overexpression of EDAG prevented the down-regulation of NPM1 proteins, whereas knockdown of EDAG accelerated the down-regulation of NPM1. EDAG deletion mutant lacking the binding domain with NPM1 lost the ability to stabilize NPM1 protein. Furthermore, knockdown of EDAG in K562 cells led to increased cell apoptosis induced by imatinib, and re-expression of NPM1 attenuated the increased apoptosis. These results suggest that EDAG enhances the protein stability of NPM1 via binding to NPM1, which plays a critical role in the anti-apoptosis of leukaemia cells.

THAP11 is an essential factor involved in ES cell pluripotency and cell growth. Here, we identified THAP11 as a novel physiological binding partner of PCBP1. In HepG2 cells, THAP11 overexpression inhibited CD44 v6 expression and cell invasion. However, when deleting the binding domain with PCBP1 or endogenous PCBP1 was knocked down, THAP11 failed to inhibit CD44 v6 expression, indicating that THAP11 regulates CD44 v6 expression through interacting with PCBP1. In HCC patients, the expression of THAP11 mRNA significantly correlated with PCBP1 mRNA expression. Our results suggest a novel role of THAP11 in CD44 alternative splicing and hepatoma invasion. Structured summary of protein interactions: THAP11 physically interacts with PCBP1 by anti bait coimmunoprecipitation ( View interaction ) THAP11 physically interacts with PCBP1 by anti tag coimmunoprecipitation (View Interaction: 1 , 2 ) THAP11 and PCBP1 colocalize by fluorescence microscopy ( View interaction )

Human augmenter of liver regeneration (hALR) is a sulfhydryl oxidase that is highly expressed in spermatogonia and early spermatocytes. To investigate the physiological effects of hALR in spermatogenesis, we generated a hALR transgenic mouse model driven by the human TSPY (testis-specific protein, Y-encoded) promoter that allows the transgene to be specifically activated in the testes. hALR content was found to be increased in both germ cells. The histological and TUNEL analysis of transgenic testes revealed a number of spermatogenetic defects including primary spermatocyte overpopulation followed by depletion through apoptosis, degenerating and detached nucleated germ cells, haploid cell loss and intraepithelial vacuoles of varying sizes. In line with these features, adult transgenic male mice also displayed a reduction in fertility. Our data suggest that regulated spatial and temporal expression of hALR is required for normal testicular development and spermatogenesis, and overexpression of hALR results in influencing the sperm morphology and quantity and the eventual reduction in male fertility. Present findings in the mouse may be of interest to human male fertility.

EDAG is multifunctional transcriptional regulator primarily expressed in the linloc-kit+Sca-1+ hematopoietic stem cells (HSC) and CD34+ progenitor cells. Previous studies indicate that EDAG is required for maintaining hematopoietic lineage commitment balance. Here using ex vivo culture and HSC transplantation models, we report that EDAG enhances the proliferative potential of human cord blood CD34+ cells, increases survival, prevents cell apoptosis and promotes their repopulating capacity. Moreover, EDAG overexpression induces rapid entry of CD34+ cells into the cell cycle. Gene expression profile analysis indicate that EDAG knockdown leads to down-regulation of various positive cell cycle regulators including cyclin A, B, D, and E. Together these data provides novel insights into EDAG in regulation of expansion and survival of human hematopoietic stem/progenitor cells.

Human hepassocin (HPS) was originally detected by subtractive and differential cDNA cloning as a liver-specific gene that was markedly upregulated during liver regeneration. Previous studies suggested that HPS showed mitogenic activity on isolated hepatocytes in vitro. However, its in vivo functions remained largely unknown. Therefore, the function of recombinant human HPS during liver regeneration and chemically induced liver injury was investigated.The proliferation of primary hepatocytes was examined by [(3)H]thymidine incorporation and immunohistological staining of proliferating cell nuclear antigen (PCNA). RNA interference was performed to knock down the endogenous expression of HPS. The proliferation of L02 cells was examined by MTS assay. The phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2) was investigated by western blotting analysis. Assessment of liver injury (histology, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels) and of apoptosis, by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, was performed.Purified recombinant human HPS showed specific mitogenic activity on primary hepatocytes and normal liver cell lines in a mitogen-activated protein kinase (MAPK)-dependent manner and stimulated the proliferation of hepatocytes in rats with 70% partial hepatectomy. Administration of HPS to rats after d-galactose and carbon tetrachloride (CCl(4)) treatment protected against liver injury (minimal liver necrosis, depressed ALT and AST levels, and decreased lethality), reduced apoptosis and enhanced proliferation. Knock-down of endogenous HPS in vivo enhanced the liver injury induced by d-galactose by increasing the apoptosis and elevating ALT and AST levels.HPS is a hepatic growth factor which can accelerate hepatocyte proliferation in vivo and protect against liver injury. These data point to the potential interest of HPS in the treatment of fulminant hepatic failure.

Hepassocin (HPS), is a liver-specific gene with mitogenic activity on isolated hepatocytes. It is up-regulated following partial hepatectomy and down-regulated frequently in heptocellular carcinoma (HCC). However, very little is known about the HPS transcription regulation mechanism. In this study, we identified HNF1alpha (hepatocyte nuclear factor-1alpha) as an important liver-specific cis-acting element for HPS using in vivo luciferase assays. Deletion of the HNF1 binding site not only led to a complete loss of HPS promoter activity in vivo but also abolished the induction of the HPS promoter by HNF1alpha. An electrophoretic mobility shift assay demonstrated that HNF1alpha interacted with the HPS gene promoter in vitro. Chromatin immunoprecipitation showed that HNF1alpha interacted with HMGB1 and CREB-binding protein, and all of them were recruited to the HPS promoter in vivo. Moreover, HNF1alpha expression was lower in HCC cell lines and tissues and correlated significantly with the down-regulation of HPS expression. Re-expression of HNF1alpha in human hepatoma HepG2 cells reinduced HPS expression. In contrast, knockdown of endogenous HNF1alpha expression by small interfering RNA resulted in a significant reduction of HPS expression. Furthermore, we found that partial hepatectomy and IL-6 significantly induced promoter activity of HPS, depending on STAT3 and HNF1 binding sites in the HPS promoter. These results demonstrate that the HNF1 binding site and HNF1alpha are critical to liver-specific expression of HPS, and down-regulation or loss of HNF1alpha causes, at least in part, the transcriptional down-regulation of HPS in HCC.

EDAG, a hematopoietic tissue-specific protein, is involved in the regulation of proliferation, differentiation and apoptosis of hematopoietic cells. In this study, a dose-dependent inhibition of EDAG expression by PMA was observed in K562 cells. The responsive element for the PMA-induced inhibition was contained in the region between -211 and +32bp of the EDAG gene promoter. By oligonucleotide-directed mutagenesis, EMSA, ChIP and transient transfection assays, we found that two tandem repeat GATA-1 sites in the promoter of EDAG gene played an important role in the PMA-mediated down-regulation of the EDAG gene expression in K562 cells. The kinetics of EDAG expression during PMA induction showed that the levels of EDAG expression were down-regulated concomitantly with GATA-1 down-expression. Decreased GATA-1 expression by siRNA reduced expression of EDAG in K562 cells, and restored expression of GATA-1 significantly rescued EDAG expression from PMA-mediated suppression. Overexpression of EDAG in K562 cells inhibited the megakaryocytic differentiation induced by PMA which raised the interesting possibility that PMA induced K562 cells differentiation toward megakaryocytic phenotype through, at least in part, the inhibition of EDAG expression. In vivo analysis confirmed that EDAG was highly expressed in primitive progenitor cells and down-regulated in megakaryocytes which was consistent with the expression pattern of GATA-1. Furthermore, PKC and MAPK specific inhibitors treatment attenuated the down-regulation of EDAG induced by PMA. Taken together, these results suggest that the inhibition of the EDAG gene by PMA is mediated through down-regulation of transcription factor GATA-1 and involved the PKC/MAPK signaling pathway.

Hepatocyte nuclear factor (HNF)-1alpha is one of the liver-enriched transcription factors involved in many tissue-specific expressions of hepatic genes. The molecular mechanisms for determining HNF1alpha-mediated transactivation have not been explained fully. To identify unknown proteins that interact with HNF1alpha, we developed a co-IP-MS strategy to search HNF1alpha interactions, and high mobility group protein-B1 (HMGB1), a chromosomal protein, was identified as a novel HNF1alpha-interacting protein. In vitro glutathione S-transferase pull-down and in vivo co-immunoprecipitation studies confirmed an interaction between HMGB1 and HNF1alpha. The protein-protein interaction was mediated through the HMG box domains of HMGB1 and the homeodomain of HNF1alpha. Furthermore, electrophoretic mobility shift assay and chromatin-immunoprecipitation assay demonstrated that HMGB1 was recruited to endogenous HNF1alpha-responsive promoters and enhanced HNF1alpha binding to its cognate DNA sequences. Moreover, luciferase reporter analyses showed that HMGB1 potentiated the transcriptional activities of HNF1alpha in cultured cells, and downregulation of HMGB1 by RNA interference specifically affected the HNF1alpha-dependent gene expression in HepG2 cell. Taken together, these findings raise the intriguing possibility that HMGB1 is a new cofactor of HNF1alpha and participates in HNF1alpha-mediated transcription regulation through protein-protein interaction.

Nrf2 is the key transcription factor regulating the antioxidant response which is crucial for cytoprotection against extracellular stresses. Numerousin vivostudies indicate that Nrf2 plays a protective role in anti-inflammatory response. 3-(3-Pyridylmethylidene)-2-indolinone (PMID) is a synthesized derivative of 2-indolinone compounds. Our previous study suggested that PMID induces the activation of Nrf2/ARE pathway, then protecting against oxidative stress-mediated cell death. However, little is known regarding the anti-inflammatory properties of PMID in severe inflammatory phenotypes. In the present study we determined if PMID treatment protects mice from dextran sodium sulphate- (DSS-) induced colitis. The result suggests that treatment with PMID prior to colitis induction significantly reduced body weight loss, shortened colon length, and decreased disease activity index compared to control mice. Histopathological analysis of the colon revealed attenuated inflammation in PMID pretreated animals. The levels of inflammatory markers in colon tissue and serum were reduced associated with inhibition of NF-κB activation. The expression levels of Nrf2-dependent genes such as HO-1, NQO1, and Nrf2 were increased in PMID pretreated mice. However, PMID pretreatment did not prevent DSS-induced colitis in Nrf2 knockout mice. These data indicate that PMID pretreatment in mice confers protection against DSS-induced colitis in Nrf2-dependent manner, suggesting a potential role of PMID in anti-inflammatory response.

Highlights • GIT2 depletion attenuates Con A-induced immunological hepatic injuries. • GIT2 depletion suppressed the activation and function of mouse CD4+ T cells. • GIT2 depletion suppressed liver infiltration by lymphoid cells after Con A treatment. • There were lower levels of proinflammatory cytokines in Git2−/− mice after Con A injection., G protein-coupled receptor kinase interactor 2 (GIT2) is a signaling scaffold protein involved in regulation of cytoskeletal dynamics and the internalization of G protein-coupled receptors (GPCRs). The short-splice form of GIT2 is expressed in peripheral T cells and thymocytes. However, the functions of GIT2 in T cells have not yet been determined. We show that treatment with Con A in a model of polyclonal T-lymphocyte activation resulted in marked inhibitions in the intrahepatic infiltration of inflammatory cells, cytokine response and acute liver failure in Git2−/− mice. CD4+ T cells from Git2−/− mice showed significant impairment in proliferation, cytokine production and signal transduction upon TCR-stimulated activation. Our results suggested that GIT2 plays an important role in T-cell function in vivo and in vitro.