BRISC is required for optimal activation of NF-κB in Kupffer cells induced by LPS and contributes to acute liver injury.

BRISC (BRCC3 isopeptidase complex) is a deubiquitinating enzyme that has been linked with inflammatory processes, but its role in liver diseases and the underlying mechanism are unknown. Here, we investigated the pathophysiological role of BRISC in acute liver failure using a mice model induced by D-galactosamine (D-GalN) plus lipopolysaccharide (LPS). We found that the expression of BRISC components was dramatically increased in kupffer cells (KCs) upon LPS treatment in vitro or by the injection of LPS in D-GalN-sensitized mice. D-GalN plus LPS-induced liver damage and mortality in global BRISC-null mice were markedly attenuated, which was accompanied by impaired hepatocyte death and hepatic inflammation response. Constantly, treatment with thiolutin, a potent BRISC inhibitor, remarkably alleviated D-GalN/LPS-induced liver injury in mice. By using bone marrow-reconstituted chimeric mice and cell-specific BRISC-deficient mice, we demonstrated that KCs are the key effector cells responsible for protection against D-GalN/LPS-induced liver injury in BRISC-deficient mice. Mechanistically, we found that hepatic and circulating levels of TNF-α, IL-6, MCP-1, and IL-1ß, as well as TNF-α- and MCP-1-producing KCs, in BRISC-deleted mice were dramatically decreased as early as 1 h after D-GalN/LPS challenge, which occurred prior to the elevation of the liver injury markers. Moreover, LPS-induced proinflammatory cytokines production in KCs was significantly diminished by BRISC deficiency in vitro, which was accompanied by potently attenuated NF-κB activation. Restoration of NF-κB activation by two small molecular activators of NF-κB p65 effectively reversed the suppression of cytokines production in ABRO1-deficient KCs by LPS. In conclusion, BRISC is required for optimal activation of NF-κB-mediated proinflammatory cytokines production in LPS-treated KCs and contributes to acute liver injury. This study opens the possibility to develop new strategies for the inhibition of KCs-driven inflammation in liver diseases.

VPS37C facilitates erythroid differentiation by promoting EKLF stability.

The process of erythroid differentiation is orchestrated at the molecular level by a complex network of transcription factors. Erythroid Krüppel-like factor (EKLF/KLF1) is a master erythroid gene regulator that directly regulates most aspects of terminal erythroid differentiation. However, the underlying regulatory mechanisms of EKLF protein stability are still largely unknown. In this study, we identified Vacuolar protein sorting 37 C (VPS37C), a core subunit of the Endosomal sorting complex required for transport-I (ESCRT-I) complex, as an essential regulator of EKLF stability. Our study showed that VPS37C interacts with EKLF and prevents K48-linked polyubiquitination of EKLF and proteasome-mediated EKLF degradation, thus enhancing EKLF protein stability and transcriptional activity. VPS37C overexpression in murine erythroleukemia (MEL) cells promotes hexamethylene bisacetamide (HMBA)-induced erythroid differentiation manifested by up-regulating erythroid-specific EKLF target genes and increasing benzidine-positive cells. In contrast, VPS37C knockdown inhibits HMBA-induced MEL cell erythroid differentiation. Particularly, the restoration of EKLF expression in VPS37C-knockdown MEL cells reverses erythroid-specific gene expression and hemoglobin production. Collectively, our study demonstrated VPS37C is a novel regulator of EKLF ubiquitination and degradation, which plays a positive role in erythroid differentiation of MEL cells by enhancing EKLF protein stability.

Investigation on Performances and Functions of Asphalt Mixtures Modified with Super Absorbent Polymer (SAP).

The super absorbent polymer (SAP) has been attracting extensive concerns due to its strong capacity in water absorption and retention. The amorphous hydrogels formed by the post-absorbent SAP have the potential of clogging the micro-cracks in asphalt materials and refraining the rainwater from infiltrating. This provides the possibility of applying SAP in asphalt pavements to seal or fill the cracks and relieve the distresses caused by rainwater infiltration in the underlying layers. Before exploring the cracking sealing mechanism of SAPs in asphalt pavements, a series of experiments were performed to evaluate the feasibility and influences of SAPs in asphalt mastics and asphalt mixtures on their mechanical performances and functionalities. Firstly, the basic properties of SAPs were analyzed, and then the rheological properties of the asphalt mastics using SAP replacing mineral powder (10%, 20%, 30%, and 40% by volume) were explored. The water stability and infiltration reduction effect of the asphalt mixtures incorporated with SAP were evaluated by the Marshall stability test, immersion Marshall stability test, freeze-thaw splitting strength test, Cantabro test, and permeability test. The test results indicated that SAPs could be used in the asphalt mixtures to partially substitute mineral powder with desirable mechanical performances. When less than 10% of the mineral powder was replaced by the SAP, the high-temperature performance and fatigue life of the asphalt mastics could be improved to some extent, but both declined after the content of the SAP was larger than 10%. Due to the hydrogels formed by SAPs after water absorption, the water stability of the asphalt mixtures deteriorated with the increased content of SAPs. Moreover, the results from the permeability tests implied that the SAP hydrogels could fill the seepage channels in the material, thus improving the migration and infiltration resistances of the asphalt mixtures. With the increased contents of SAPs, the permeability coefficients of the asphalt mixtures could be reduced up to 55%. Based on the research findings in this study, when an appropriate amount of SAP was added in the asphalt materials, desirable temperature stability, water stability, and fatigue resistance could be achieved regarding actual requirements from applications. At the same time, the addition of SAPs could effectively refrain the infiltration and migration of rainwater in asphalt pavements, thus potentially mitigating the effect of water erosion on the underlying layers.

Protein phosphatase 2A catalytic subunit ß suppresses PMA/ionomycin-induced T-cell activation by negatively regulating PI3K/Akt signaling.

The precise regulation of the T-cell activation process is critical for overall immune homeostasis. Although protein phosphatase 2A (PP2A) is required for T-cell development and function, the role of PPP2CB, which is the catalytic subunit ß isoform of PP2A, remains unknown. In the present study, using a T cell-specific knockout mouse of PPP2CB (PPP2CBfl/fl Lck-Cre+ ), we demonstrated that PPP2CB was dispensable for T-cell development in the thymus and peripheral lymphoid organs. Furthermore, PPP2CB deletion did not affect T-cell receptor (TCR)-induced T-cell activation or cytokine-induced T-cell responses; however, it specifically enhanced phorbol myristate acetate (PMA) plus ionomycin-induced T-cell activation with increased cellular proliferation, elevated CD69 and CD25 expression, and enhanced cytokine production (inteferon-γ, interleukin-2 and tumor necrosis factor). Mechanistic analyses suggested that the PPP2CB deletion enhanced activation of the phosphoinositide 3-kinase/Akt signaling pathway and Ca2+ flux following stimulation with PMA plus ionomycin. Moreover, the specific PI3K inhibitor rescued the augmented cell activation in PPP2CB-deficient T cells. Using mass spectrometry-based phospho-peptide analysis, we identified potential substrates of PPP2CB during PMA plus ionomycin-induced T-cell activation. Collectively, our study provides evidence of the specific role of PPP2CB in controlling PMA plus ionomycin-induced T-cell activation.

Hemgn Protects Hematopoietic Stem and Progenitor Cells Against Transplantation Stress Through Negatively Regulating IFN-γ Signaling.

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.

Preclinical Pharmacokinetics, Tissue Distribution, and Primary Safety Evaluation of Indo5, a Novel Selective Inhibitor of c-Met and Trks.

The compound [3-(1H-benzimidazol-2-methylene)-5-(2-methylphenylaminosulfo)-2-indolone], known as Indo5, is a novel selective inhibitor of c-Met and Trks, and it is a promising anticancer candidate against hepatocellular carcinoma (HCC). Assessing the pharmacokinetic properties, tissue distribution, and toxicity of Indo5 is critical for its medicinal evaluation. A series of sensitive and specific liquid chromatography-tandem mass spectrometry methods were developed and validated to determine the concentration of Indo5 in rat plasma and tissue homogenates. These methods were then applied to investigate the pharmacokinetics and tissue distribution of Indo5 in rats. After intravenous injection of Indo5, the maximum concentration (Cmax) and the time at which Cmax was reached (Tmax) were 1,565.3 ± 286.2 ng/ml and 1 min, respectively. After oral administration, Cmax and Tmax were 54.7 ± 10.4 ng/ml and 2.0 ± 0.48 h, respectively. We calculated the absolute oral bioavailability of Indo5 in rats to be 1.59%. Following intravenous injection, the concentrations of Indo5 in various tissues showed the following order: liver > kidney ≈ heart > lung ≈ large intestine ≈ small intestine ≈ stomach > spleen > brain ≈ testes; hence, Indo5 distributed highest in the liver and could not cross the blood-brain or blood-testes barriers. Continuous injection of Indo5 for 21 days did not lead to liver injury, considering unchanged ALT and AST levels, normal histological architecture of the liver, and normal number and frequencies of immune cells in the liver, indicating a very low toxicity of Indo5 in vivo. Collectively, our findings provide a comprehensive understanding of the biological actions of Indo5 in vivo and further support its development as an antitumor treatment for HCC patients.

Pharmacological targeting of NLRP3 deubiquitination for treatment of NLRP3-associated inflammatory diseases.

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.

Toll-like receptor 5-mediated signaling enhances liver regeneration in mice.

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.

Transcriptional regulation of human abraxas brother protein 1 expression by yin yang 1.

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.

ABRO1 stabilizes the deubiquitinase BRCC3 through inhibiting its degradation mediated by the E3 ubiquitin ligase WWP2.

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.

NLRP3 is dispensable for d-galactosamine/lipopolysaccharide-induced acute liver failure.

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-1ß 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.

EDAG mediates Hsp70 nuclear localization in erythroblasts and rescues dyserythropoiesis in myelodysplastic syndrome.

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.

GPS2 promotes erythroid differentiation by control of the stability of EKLF protein.

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.

Glycerol kinase enhances hepatic lipid metabolism by repressing nuclear receptor subfamily 4 group A1 in the nucleus.

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.

NLRC4 Mutation in flagellin-derived peptide CBLB502 ligand-binding domain reduces the inflammatory response but not radioprotective activity.

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-1ß 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.

[Dynamics of Proliferation and Differentiation after Transplantation of Hematopoietic Cells in Mouse].

OBJECTIVE: 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. METHODS: The C57BL/6J mice were divided into infected group and control group. The mice in injected group were infected intraperitoneally with 6.7×106 CFU Listeria monocytogenes,while the mice in control group were injecfed with PBS of same volume.The serum levels of IFNγ were detected at different time points. After 24 hours, the HS/PC composition, cell cycle and cell colony-forming ability in bone marrow of mice were measured, and the difference between the control group and the infected group was statistically analyzed. RESULTS: Serum 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.

A selective c-Met and Trks inhibitor Indo5 suppresses hepatocellular carcinoma growth.

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.

Glycerol kinase interacts with nuclear receptor NR4A1 and regulates glucose metabolism in the liver.

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.

ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination.

Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that ABRO1, a subunit of the BRISC deubiquitinase complex, is necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1ß and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1. Moreover, we report that ABRO1 deficiency results in a remarkable attenuation in the syndrome severity of NLRP3-associated inflammatory diseases, including MSU- and Alum-induced peritonitis and LPS-induced sepsis in mice. Mechanistic studies reveal that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, subsequently recruiting the BRISC to remove K63-linked ubiquitin chains of NLRP3 upon stimulation with activators. Furthermore, deficiency of BRCC3, the catalytically active component of BRISC, displays similar phenotypes to ABRO1 knockout mice. Our findings reveal an ABRO1-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome and provide novel potential targets for treating NLRP3-associated inflammatory diseases.

CBLB502, a Toll-like receptor 5 agonist, offers protection against radiation-induced male reproductive system damage in mice.

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.