ALBERT

Description: RNA-binding proteins (RBPs) play an important role in plant host-microbe interactions. In this study, we show that the plant RBP known as FPA, which regulates 3′-end mRNA polyadenylation, negatively regulates basal resistance to bacterial pathogen Pseudomonas syringae in Arabidopsis. A custom microarray analysis reveals that flg22, a peptide derived from bacterial flagellins, induces expression of alternatively polyadenylated isoforms of mRNA encoding the defence-related transcriptional repressor ETHYLENE RESPONSE FACTOR 4 (ERF4), which is regulated by FPA. Flg22 induces expression of a novel isoform of ERF4 that lacks the ERF-associated amphiphilic repression (EAR) motif, while FPA inhibits this induction. The EAR-lacking isoform of ERF4 acts as a transcriptional activator in vivo and suppresses the flg22-dependent reactive oxygen species burst. We propose that FPA controls use of proximal polyadenylation sites of ERF4, which quantitatively limit the defence response output. Scientific Reports 3 doi: 10.1038/srep02866

Description: Understanding lineage specific markers contributes to investigation into lineage commitment processes in hematopoiesis. Particularly in the human study, information about hematopoietic lineage divergence is essential to refine hematopoietic lineage tree. Lineage markers are also potentially useful for therapeutic target, such as CD20 in B-cell lymphoma, and CD33 in acute myeloid leukemia. We have recently reported that special AT-rich sequence-binding protein 1 (SATB1), a global chromatin organizer, promotes lymphocyte production from hematopoietic stem cells (HSCs) (Immunity 38;1105, 2013). Expression level of SATB1 increases with early lymphoid differentiation, whereas it is shut off in committed myeloid progenitors. To search a novel cell surface molecule that marks the point of branching lineage along early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSCs with mock-transduced HSCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (MS4A3). MS4A3, also called hematopoietic cell-specific transmembrane 4 (HTm4), is a member of the MS4A family. CD20, encoded by MS4A1 gene, belongs to the same family. We observed that expression level of MS4A3 in SATB1-overexpressed HSCs was decreased almost one tenth of that of mock HSCs. To confirm the relationship of SATB1 and MS4A3 in human hematopoietic cells, we first used chronic myeloid leukemia cell line K562, which was found to clearly express MS4A3 on their cell surface. While SATB1 expression was undetectable in original K562 cells, the exogenous expression of SATB1 significantly reduced their MS4A3 expression level, suggesting that SATB1 negatively regulates MS4A3 expression in human cells. Next, we analyzed MS4A3 expression pattern in primary human hematopoietic stem/progenitor cells. Bone marrow (BM) cells were obtained from healthy donors or patients with acute myeloid leukemia. The Institutional Review Board of Osaka University School of Medicine approved all of protocols, and written informed consents were obtained from all participants. Mononuclear cells were separated from the BM samples by density gradient centrifugation, and subsequently applied to cell sorting for Lineage marker-negative (Lin-) CD34+ CD38- HSCs, Lin- CD34+ CD38+ IL-3 receptor α (IL-3Rα)+ CD45RA- common myeloid progenitors (CMPs), Lin- CD34+ CD38+ IL3-Rα+ CD45RA+ granulocyte-macrophage progenitors (GMPs) and Lin- CD34+ CD38+ IL-3Rα- CD45RA-megakaryocyte-erythroid progenitors (MEPs). MS4A3 expression levels of the sorted cells were analyzed with real-time RT-PCR. We detected more than 10-fold amount of MS4A3 transcripts in CMPs than HSCs. Furthermore, its expression level continuously increased along myeloid lineage differentiation to GMP. On the other hand, megakaryocyte-erythroid lineage differentiation was not accompanied by MS4A3 expression and the amount of MS4A3 transcripts in MEPs was minimum as in HSCs. Flow cytometry analyses confirmed that HSCs and MEPs do not express MS4A3 on their cell surface whereas the MS4A3 expression on CMPs and GMPs is detectable. Further, the Lin- CD34+ CD38+ CD33+ cells could be fractionated according to the intensity of cell surface MS4A3 expression. To investigate the significance of cell surface MS4A3 expression for functional analyses of myeloid progenitor cells, we performed methylcellulose colony-forming assays. We found that MS4A3+ cells in Lin- CD34+ CD38+ CD33+ fraction only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. We also analyzed MS4A3 expression in BM cells obtained from patients with acute leukemia. Flow cytometry analyses revealed that leukemia cells of some patients expressed substantial amount of cell surface MS4A3. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis. In addition, our findings of MS4A3 expression on myeloid leukemia cells, while no expression on normal HSCs, imply that MS4A3 might be a therapeutic target molecule in myelogenous leukemia. Further studies would clarify the application of MS4A3 to anti-leukemia therapy. Disclosures No relevant conflicts of interest to declare.

Description: Iron is essential for almost all organisms. However, free iron can be cytotoxic at high concentration and iron excess can have adverse effects on variety of cells, tissue, and organ functions. In immune systems, many reports have shown the effects of iron, some of which are complex and controversial. Iron deficiency has been reported to be associated with increased susceptibility to infection, but iron overload caused by dietary excess abnormal hemolysis or inherited disorders is also associated with heightened susceptibility to infections. On the other hand, elevated ferritin levels, primarily related to RBC transfusions, have been reported to increase the risk of acute and chronic GVHD in patients received hematopoietic cell transplantation. Although this iron-related risk of GVHD may reflect the organ damage, such as liver, kidney, and pancreas, it also may be caused by imbalance in immune systems. To exaggerate the effects of iron overload on immune system, we established iron-loaded models in mice. First, as acute iron-loaded model, 10mg of iron dextran, which is even equal to 200U of RCC transfusion in human (define as 200U of iron dextran), was intraperitoneally injected into mice once a day for 18 days. In peripheral blood, T cell and B cell populations were decreased but monocyte/macrophage and neutrophil populations were increased as compared in control mice administered with dextran only. And notably, regulatory T cell (CD4+Foxp3+; Treg) population was significantly reduced in iron loaded mice than control mice (1.40% vs 0.58%). Next, as chronic iron-loaded model, 2U of iron dextran was injected once a week 45 times. Although significant difference was not observed in Treg population in PB, that in spleen was significantly high in iron-loaded mice as compared with that in control mice (3.2% vs 1.7%, p

Description: Introduction: Anamorsin (AM, also called CIAPIN1) was originally isolated as a molecule that conferred resistance to apoptosis caused by growth factor deprivation. AM deficient (AM KO) mice die during late gestation; AM KO embryos are anemic and small compared to wild type (WT) embryos. It suggests that AM is indispensable for embryo growth and hematopoiesis. To determine which signaling pathways AM utilizes for these functions, we analyzed murine embryonic fibroblast (MEF) cells generated from E-14.5 AM KO or WT embryos. Proliferation of AM KO MEF cells was markedly retarded, and PKCθ, PKCδ, and p38MAPK were more highly phosphorylated in AM KO MEF cells. Expression of cyclinD1, the target molecule of p38MAPK, was down-regulated in AM KO MEF cells. P38MAPK inhibitor as well as PKC inhibitor restored expression of cyclinD1 and cell growth in AM KO MEF cells. These data suggested that PKCθ, PKCδ, and p38MAPK activation lead to cell cycle retardation in AM KO MEF cells. However, functions of AM still remain not fully understood. In order to elucidate functions of AM, we generated AM transgenic (Tg) mice under control of CAG promoter. Since our previous study showed AM overexpression in approximately 30% of B cell type malignant lymphoma (DLBCL and FL) cases, we focused on AM overexpressed B cells from AM Tg mice in this study. Methods and Results: At first, we compared the number of B cells in the peripheral blood and spleen size between AM Tg mice and WT mice and found no difference. Next, we analyzed the lipopolysaccharide (LPS) stimulated B cells. B cells were selected from spleen by using anti-B220 antibody. The purified B cells were exposed to LPS for three days and measured. Unexpectedly, LPS-stimulated proliferation of B cells from AM Tg mice was decreased compared to WT mice. Since we initially confirmed that AM overexpression did not affect TLR4 expression on B cells, we examined TLR4 signaling pathway activation status by detecting phosphorylation of the signal transduction molecules using Western-blotting, and found that phosphorylation of Erk1/2 and IKBα were decreased in LPS-stimulated B cells from AM Tg mice. Next, we performed cDNA microarray analysis to reveal the mechanisms of inhibition of the LPS signaling pathways by comparing the differential gene expression profiles in B cells of AM Tg and WT mice with or without LPS stimulation. We extracted 2375 genes from the data sets that met the following criteria: genes with 〉 2.0 fold change between LPS- vs LPS+ in AM Tg or WT mice, and secondly with 〉1.5 fold difference in the rate of gene expression change with LPS stimulation between AM Tg and WT mice, and performed upstream regulator analysis to predict the upstream regulators on the cascade of LPS signaling pathway with Ingenuity Pathway Analysis software. The data suggested that Ras activation was decreased in LPS-stimulated AM Tg B cells compared to WT B cells. Then, we confirmed the impairment of LPS induced Ras activation in AM Tg B cells using Ras activation assay kit. Furthermore, we found that AM mRNA level was increased (1.8-4.0 fold) in WT B cells at 4 hours after LPS stimulation. From these data, it was shown that AM overexpression in AM Tg B cells inhibited Ras signaling pathways and retarded the cell proliferation of LPS-stimulated B cells. Conclusion and Discussion: In our previous study, we showed that AM negatively regulates novel PKCs and p38MAPK by using MEFcells generated from AM KO mice, while in this study it was shown that overexpressed AM negatively regulates Ras signaling pathways by using AM Tg B cells. Although there are no reports other than ours that show the relationships between the signaling molecules and AM, our present data showed the novel role of AM that regulates the signaling molecules in the different manner. LPS increased AM expression via Ras activation in B cells, and AM overexpression inhibited Ras, which suggested AM might induce a negative feedback loop that attenuates Ras activation in LPS stimulated B cells. Our previous data showed that low international prognostic index (IPI) DLBCL patients with AM overexpression had a poor prognosis, especially in the patients who received chemotherapy without rituximab. In those patients, AM overexpression might contribute at least in part to chemotherapy resistance possibly through negative regulation of Ras. In summary, we showed that overexpressed AM is one of negative regulators of LPS induced signaling pathways in B cells. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 610 Introduction: Iron has crucial roles in many cellular biological processes. Cellular iron uptake and export must be tightly regulated. Insufficient iron concentrations impair the function of numerous iron proteins, whereas excess free iron can oxidize and damage the contents of cells. Anamorsin (AM, also called CIAPIN-1) is an anti-apoptotic factor, which we originally isolated as a molecule that confers factor-independent survival of hematopoietic cells. AM-deficient mice are embryonic lethal at late gestation due to the defect of definitive hematopoiesis. It is thought that AM plays a crucial role in hematopoiesis, however its precise biological mechanisms remain unclear. Recently, it was reported that the yeast AM homolog, Dre2, was implicated in cytosolic iron-sulfur (Fe/S) cluster assembly (Zhang Y., et al. Mol.Cell.Biol. 28:5569–5582, 2008). The AM carries conserved cysteine motifs (CX2CXC and twin CX2C) at its C termini, which may form iron binding sites. In this study, we have focused on the possibility that AM may be involved in the maturation of Fe/S cluster and the cellular iron homeostasis, especially, the regulation of labile iron pool (LIP) and that AM may affect the accumulation of reactive oxygen species (ROS), leading to impaired erythropoiesis. Methods and Results: To analyze the function of Fe/S protein, we established wild-type cell lines (AMWT) and AM-deficient cell lines (AMKO) from wild-type and AM-deficient fetal liver (14.5dpc) respectively by using SV40 large T antigen. Iron regulatory protein 1 (IRP1) is a well-known Fe/S protein with dual functions. In the presence of Fe/S cluster, IRP1 functions as a cytosolic aconitase. While, in the absence of Fe/S cluster, IRP1 stabilizes the transferrin receptor (TfR) mRNA by binding to the iron responsive element (IRE). We compared the aconitase activity and the IRE binding activity of IRP1 between AMWT and AMKO. The results showed that the cytosolic aconitase activity in AMKO decreased approximately 30% compared to AMWT and the IRE binding activity of IRP1 in AMKO increased 3-fold compared to AMWT. Furthermore, we compared the iron homeostasis. In the presence of iron chelator, desferrioxamine, the expression of TfR in AMWT was markedly elevated, while it was hardly elevated in AMKO. The LIP is a pool of chelatable and redox-active iron, which serves as a crossroad of cell iron metabolism. The measurement of LIP with the metal-sensitive sensor calcein acetoxymethyl ester showed that AMKO had 5-fold higher cellular LIP than AMWT. Moreover we evaluated the accumulation of ROS and the induction of apoptosis by extracellular iron uptake between AMWT and AMKO. The results showed the accumulation of ROS and the induction of apoptosis in AMKO were enhanced about twice as much as in AMWT. These enhancements could be restored by transduction of AM expressing retrovirus vector to AMKO. We also evaluated the effects of AM-deficiency on erythroid differentiation. Fetal liver cells from wild-type or AM-deficient embryos (14.5dpc) were divided into primitive and more matured erythroid populations based on their expression of CD71 and Ter119 by FACS analysis. AM-deficient fetal liver cells had a significant increase in the CD71low TER119low population, containing primitive erythroid progenitors, compared to wild-type (9.4±2.1% vs. 5.2±1.1%, P