Yuka Tanaka

Enhanced erythroid cell differentiation in hypoxic condition is in part contributed by miR-210.

Erythropoiesis, a process of erythroid production, is controlled by several factors including oxygen level. In this study, the effect of oxygen tension on erythropoiesis was investigated in K562 erythroleukemic cell line and erythroid progenitor cells derived from normal and β-thalassemia/hemoglobin (Hb) E individuals. The enhanced erythroid differentiation specific markers including increased levels of α-, β- and γ-globin gene expressions, numbers of HbF positive cells and the presence of glycophorin A surface marker were observed during cell culture under hypoxic atmosphere. The result also showed that miR-210, one of the hypoxia-induced miRNAs, was up-regulated in K562 and β-thalassemia/HbE progenitor cells cultured under hypoxic condition. Inhibition of miR-210 expression leads to reduction of the globin gene expression and delayed maturation in K562 and erythroid progenitor cells. This indicated that miR-210 contributes to hypoxia-induced erythroid differentiation in both K562 cells and β-thalassemia/HbE erythroid progenitor cells.

Imatinib induces demethylation of miR-203 gene : An epigenetic mechanism of anti-tumor effect of imatinib

MicroRNA (miRNA) is an important regulator of cellular proliferation, differentiation and death. Leukemia-specific signature of miRNAs suggests that epigenetic dysregulation of miRNAs is important for leukemogenesis. We focused on the role of DNA methylation of miR-203 which targets BCR-ABL1 mRNA. The microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment. Imatinib induced the demethylation of the miR-203 promoter region, resulting in low expression of targeted BCR-ABL1 gene, and loss of proliferation of leukemic cells. In conclusion, demethylation of miR-203 is one of the molecular mechanisms of imatinib-induced inhibition of BCR-ABL1-positive leukemic cells.

Induction of intrinsic apoptosis in leukaemia stem cells and in vivo zebrafish model by betulonic acid isolated from Walsura pinnata Hassk (Meliaceae)

BACKGROUND Leukaemia stem cells (LSC) have been associated with disease relapse and chemotherapy resistance. Betulonic acid (BA), a pentacyclic lupane-type triterpenoid, was reported to exhibit cytotoxicity toward various cancer cells and to be capable of inducing intrinsic apoptosis in solid tumours. However, the in vitro and in vivo apoptotic effects of BA against LSC remain unknown. HYPOTHESIS/PURPOSE We aimed to determine whether BA isolated from bark of Walsura pinnata Hassk (Meliaceae) has pro-apoptotic effects on LSC in in vitro and in vivo models. STUDY DESIGN/METHODS The population of high purity LSC was isolated from the Kasumi-1 cell line using magnetic sorting and characterised by flow cytometry. Cell viability was assessed using the MTS assay to examine dose- and time-dependent effects. The colony formation assay was performed in MethoCult® H4435 enriched media. Apoptosis was analysed using Annexin-V and propidium iodide staining, mitochondrial transmembrane potential was studied using JC-1 staining, and expression of apoptosis related genes (BAX, Bcl-2 and survivin) was evaluated by real time-polymerase chain reaction (RT-PCR). Caspase 3/7 and 9 activities were monitored through Promega Caspase-Glo® over a period of 24h. The in vivo antileukaemia activity was evaluated using LSC xenotransplanted zebrafish, observed for DNA fragmentation from apoptosis by TUNEL assay. RESULTS BA maintained its potency against the LSC population in comparison to parental Kasumi-1 cells (fold differences ≤ 1.94) over various treatment time points and significantly inhibited the formation of colonies by LSC. Apoptosis was triggered by BA through the upregulation of BAX and suppression of Bcl-2 and survivin genes with the loss of mitochondrial transmembrane potential, leading to the activation of caspase 9 followed by downstream caspase 3/7. BA was able to suppressed leukaemia formation and induced apoptosis in LSC xenotransplanted zebrafish. CONCLUSIONS The results demonstrate that BA inhibited the proliferative and colonogenic properties of LSC. BA induced apoptosis in LSC through the mitochondria pathway and was effective in the in vivo zebrafish model. Therefore, BA could be a lead compound for further development into a chemotherapy agent against LSC.

Localized SCF and IGF-1 secretion enhances erythropoiesis in the spleen of murine embryos

Fetal spleen is a major hematopoietic site prior to initiation of bone marrow hematopoiesis. Morphologic analysis suggested erythropoietic activity in fetal spleen, but it remained unclear how erythropoiesis was regulated. To address this question, we performed flow cytometric analysis and observed that the number of spleen erythroid cells increased 18.6-fold from 16.5 to 19.5 days post-coitum (dpc). Among erythropoietic cytokines, SCF and IGF-1 were primarily expressed in hematopoietic, endothelial and mesenchymal-like fetal spleen cells. Cultures treated with SCF and/or IGF-1R inhibitors showed significantly decreased CD45−c-Kit−CD71+/−Ter119+ erythroid cells and downregulated Gata1, Klf1 and &bgr;-major globin expression. Administration of these inhibitors to pregnant mice significantly decreased the number of CD45−c-Kit−CD71+/−Ter119+ cells and downregulated &bgr;-major globin gene expression in embryos derived from these mice. We conclude that fetal spleen is a major erythropoietic site where endothelial and mesenchymal-like cells primarily accelerate erythropoietic activity through SCF and IGF-1 secretion.

Perillaldehyde Inhibits AHR Signaling and Activates NRF2 Antioxidant Pathway in Human Keratinocytes

The skin covers the outer surface of the body, so the epidermal keratinocytes within it are susceptible to reactive oxygen species (ROS) generated by environmental pollutants such as benzo(a)pyrene (BaP), a potent activator of aryl hydrocarbon receptor (AHR). Antioxidant activity is generally mediated by the nuclear factor-erythroid 2-related factor-2 (NRF2) and heme oxygenase-1 (HO1) axis in human keratinocytes. Perillaldehyde is the main component of Perilla frutescens, which is a medicinal antioxidant herb traditionally consumed in East Asia. However, the effect of perillaldehyde on the AHR/ROS and/or NRF2/HO1 pathways remains unknown. In human keratinocytes, we found that perillaldehyde (1) inhibited BaP-induced AHR activation and ROS production, (2) inhibited BaP/AHR-mediated release of the CCL2 chemokine, and (3) activated the NRF2/HO1 antioxidant pathway. Perillaldehyde is thus potentially useful for managing inflammatory skin diseases or disorders related to oxidative stress.

Embryonic Intra-Aortic Clusters Undergo Myeloid Differentiation Mediated by Mesonephros-Derived CSF1 in Mouse

The aorta-gonad-mesonephros (AGM) region contains intra-aortic clusters (IACs) thought to have acquired hematopoietic stem cell (HSC) potential in vertebrate embryos. To assess extrinsic regulation of IACs in the AGM region, we employed mouse embryos harboring a Sall1-GFP reporter gene, which allows identification of mesonephros cells based on GFP expression. Analysis of AGM region tissue sections confirmed mesonephros GFP expression. Mesonephric cells sorted at E10.5 expressed mRNA encoding Csf1, a hematopoietic cytokine, and corresponding protein, based on real-time PCR and immunocytochemistry, respectively. Further analysis indicated that some IACs express the CSF1 receptor, CSF1R. Expression of Cebpa and Irf8 mRNAs was higher in CSF1R-positive IACs, whereas that of Cebpε and Gfi1 mRNAs was lower relative to CSF1R-negative IACs, suggesting that CSF1/CSF1R signaling functions in IAC myeloid differentiation by modulating expression of these transcription factors. Colony formation assays using CSF1R-positive IACs revealed increased numbers of myeloid colonies in the presence of CSF1. Analysis using an intra-cellular signaling array indicated the greatest fold increase of Cleaved Caspase-3 in AGM cells in the presence of CSF1. Immunohistochemistry revealed that Cleaved Caspase-3 is primarily expressed in IACs in the AGM region, and incubation of IACs with CSF1 up-regulated Cleaved Caspase-3. Overall, our findings suggest that CSF1 secreted from mesonephros accelerates IAC myeloid differentiation in the AGM region, possibly via Caspase-3 cleavage.

Embryonic hematopoietic progenitor cells reside in muscle before bone marrow hematopoiesis

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.

Corrigendum to “Perillaldehyde Inhibits AHR Signaling and Activates NRF2 Antioxidant Pathway in Human Keratinocytes”

[This corrects the article DOI: 10.1155/2018/9524657.].

Glucagon-like peptide-1 analogue liraglutide facilitates wound healing by activating PI3K/Akt pathway in keratinocytes

AIMS Diabetes induces various skin troubles including foot ulcer. This type of skin ulcer is refractory but the pathogenesis is not so certain. Recent study show that glucagon-like peptide-1 (GLP-1) analogues reduce foot complications with diabetes (Pérez et al., 2015), however, the role of GLP-1/GLP-1R axis is not fully understood, and clear evidence of GLP-1 to facilitate wound closure is still lacking. In this study, we investigated whether a potent GLP-1R agonist liraglutide affects wound healing process. METHODS The expression of GLP-1R in HaCaT cells were indentified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunoblotting analysis. To assess the effect on wound closure in keratinocytes, we performed in vitro scratch assay using the IncuCyte system (Essen BioSciences, Ann Arborm MI). We applied ointment containing liraglutide on full-thickness wounds in the dorsum of female balb/c mice (n = 6) until healing. To investigate the effect on PI3K/Akt pathway, we used IncuCyte system in HaCaT treated with PI3K inhibitor and Akt inhibitor. RESULTS Keratinocytes expressed GLP-1R and liraglutide induced their migration. Liraglutide facilitated the wound healing in mice. Liraglutide upregulated keratinocyte migration via PI3K/Akt activation. CONCLUSIONS Our study suggests that liraglutide may be a potential target drug to improve skin ulcer with diabetes through its specific receptor GLP-1.

Plasma microRNA-451 as a novel hemolytic marker for β0-thalassemia/HbE disease

In Southeast Asia, particularly in Thailand, β0-thalassemia/hemoglobin E (HbE) disease is a common hereditary hematological disease. It is associated with pathophysiological processes, such as the intramedullary destruction of immature erythroid cells and peripheral hemolysis of mature red blood cells. MicroRNA (miR) sequences, which are short non-coding RNA that regulate gene expression in a suppressive manner, serve a crucial role in human erythropoiesis. In the present study, the plasma levels of the erythroid-expressed miRNAs, miR-451 and miR-155, were analyzed in 23 patients with β0-thalassemia/HbE and 16 control subjects. Reverse transcription-quantitative polymerase chain reaction analysis revealed significantly higher levels of plasma miR-451 and miR-155 in β0-thalassemia/HbE patients when compared to the control subjects. Notably, among the β0-thalassemia/HbE patients, a significant increase in miR-451 levels was detected in severe cases when compared with mild cases. The levels of plasma miR-451 correlated with reticulocyte and platelet counts. The results suggest that increased plasma miR-451 levels may be associated with the degree of hemolysis and accelerated erythropoiesis in β0-thalassemia/HbE patients. In conclusion, miR-451 may represent a relevant biomarker for pathological erythropoiesis associated with β0-thalassemia/HbE.