Background:The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. Results: Cnr2 activation down-regulates 5-lipoxygenase (Alox5) expression by suppressing the JNK/c-Jun activation. Conclusion: The Cnr2-JNK-Alox5 axis modulates leukocyte inflammatory migration. Significance: Linking two important regulators in leukocyte inflammatory migration and providing a potential therapeutic strategy for treating human inflammation-associated diseases.
BackgroundDNA methyltransferase 1 (Dnmt1) regulates expression of many critical genes through maintaining parental DNA methylation patterns on daughter DNA strands during mitosis. It is essential for embryonic development and diverse biological processes, including maintenance of hematopoietic stem and progenitor cells (HSPCs). However, the precise molecular mechanism of how Dnmt1 is involved in HSPC maintenance remains unexplored.MethodsAn N-ethyl-N-nitrosourea (ENU)-based genetic screening was performed to identify putative mutants with defects in definitive HSPCs during hematopoiesis in zebrafish. The expression of hematopoietic markers was analyzed via whole mount in situ hybridization assay (WISH). Positional cloning approach was carried out to identify the gene responsible for the defective definitive hematopoiesis in the mutants. Analyses of the mechanism were conducted by morpholino-mediated gene knockdown, mRNA injection rescue assays, anti-phosphorylated histone H3 (pH3) immunostaining and TUNEL assay, quantitative real-time PCR, and bisulfite sequencing analysis.ResultsA heritable mutant line with impaired HSPCs of definitive hematopoiesis was identified. Positional cloning demonstrated that a stop codon mutation was introduced in dnmt1 which resulted in a predicted truncated Dnmt1 lacking the DNA methylation catalytic domain. Molecular analysis revealed that expression of CCAAT/enhancer-binding protein alpha (C/ebpa) was upregulated, which correlated with hypomethylation of CpG islands in the regulation regions of cebpa gene in Dnmt1 deficient HSPCs. Overexpression of a transcriptional repressive SUMO-C/ebpa fusion protein could rescue hematological defects in the dnmt1 mutants. Finally, dnmt1 and cebpa double null embryos exhibited no obvious abnormal hematopoiesis indicated that the HSPC defects triggered by dnmt1 mutation were C/ebpa dependent.ConclusionsDnmt1 is required for HSPC maintenance via cebpa regulation during definitive hematopoiesis in zebrafish.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0115-7) contains supplementary material, which is available to authorized users.
In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.
The interrelationship between genetic susceptibility and carcinogenic exposure is important in cancer development. Polymorphisms in detoxification enzymes of the glutathione-S-transferases (GST) family are associated with an increased incidence of lymphoma. Here we investigated the molecular connection of the genetic polymorphism of GSTT1 to the response of lymphocytes to polycyclic aromatic hydrocarbons (PAH). In neoplastic situation, GSTT1 deletions were more frequently observed in lymphoma patients (54.9%) than in normal controls (42.0%, P = 0.009), resulting in an increased risk for lymphoma in individuals with GSTT1-null genotype (Odds ratio = 1.698, 95% confidence interval = 1.145–2.518). GSTT1 gene and protein expression were accordingly decreased in GSTT1-deleting patients, consistent with activated profile of cell cycle regulation genes. Mimicking environmental exposure using long-term repeat culture with low-dose PAH metabolite Hydroquinone, malignant B- and T-lymphocytes presented increased DNA damage, pCHK1/MYC expression and cell proliferation, which were counteracted by ectopic expression of GSTT1. Moreover, GSTT1 expression retarded xenograft tumor formation of Hydroquinone-treated lymphoma cells in nude mice. In non-neoplastic situation, when zebrafish was exposed to PAH Benzo(a)pyrene, molecular silencing of gstt1 enhanced the proliferation of normal lymphocytes and upregulated myca expression. Collectively, these findings suggested that GSTT1 deletion is related to genetic predisposition to lymphoma, particularly interacting with environmental pollutants containing PAH.
Epigenetic processes such as DNA methylation are essential for processes of gene expression in normal mammalian development. DNA methyltransferases (DNMT) are responsible for initiating and maintaining DNA methylation. It is known that 5-Aza-CdR, an inhibitor of DNMT induces cytotoxicity by reducing DNMT activity in various tumor cell lines. However, disturbances in neuronal DNA methylation may also play a role in altered brain functions. Thus, it was of interest to determine whether alterations in DNA methylation might be associated with neuronal functions by using 5-Aza-CdR, on mouse hippocampus-derived neuronal HT22 cell line. In particular, the aim of this study was to investigate the effects of 5-Aza-CdR on cell growth inhibition, cell cycle arrest, apoptosis as well as the expression levels of DNMT in HT22 cells. HT22 cells were incubated with 5 or 20 μmol/L 5-Aza-CdR for 24 h. Data showed that 5-Aza-CdR at both concentrations significantly inhibited proliferation of HT22 cells and exacerbated cytoplasmic vacuolization. Flow cytometry analysis demonstrated that 5-Aza-CdR treatment at both concentrations decreased early apoptosis but enhanced late apoptosis. Cell cycle analysis illustrated that 5-Aza-CdR treatment induced S phase arrest. Further, incubation with 5-Aza-CdR produced a down-regulation in expression of mRNA and protein DNMT1 and 3A but no marked changes were noted in DNMT 3B and p21 expression. In addition, DNMT1 activity was significantly decreased at both 5-Aza-CdR concentrations. Evidence indicates that 5-Aza-CdR induced cytotoxicity was associated with altered mRNA and protein expression of DNMT 1 and 3A associated with reduced DNMT1 activity in HT22 cells which might affect brain functions.
The regulation of hematopoiesis is generally evolutionarily conserved from zebrafish to mammals, including hematopoietic stem cell formation and blood cell lineage differentiation. In zebrafish, primitive granulocytes originate at two distinct regions, the anterior lateral plate mesoderm (A-LPM) and the intermediate cell mass (ICM). Few studies in the zebrafish have examined genes specifically required for the granulocytic lineage. In this study, we identified the responsible gene for a zebrafish mutant that has relatively normal hematopoiesis, except decreased expression of the granulocyte-specific gene mpx. Positional cloning revealed that phospholipase C gamma-1 (plcg1) was mutated. Deficiency of plcg1 function specifically affected development of granulocytes, especially the maturation process. These results suggested that plcg1 functioned specifically in zebrafish ICM granulopoiesis for the first time. Our studies suggest that specific pathways regulate the differentiation of the hematopoietic lineages.
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