Akimichi Ohsaka

Granulocyte colony-stimulating factor promotes neovascularization by releasing vascular endothelial growth factor from neutrophils

The granulocyte colony‐stimulating factor (G‐CSF) promotes angiogenesis. However, the exact mechanism is not known. We demonstrate that vascular endothelial growth factor (VEGF) was released by Gr‐1+CD11b− neutrophils but not Gr‐1−CD11b+ monocytes prestimulated with G‐CSF in vitro and in vivo. Similarly, in vivo, concomitant with an increase in neutrophil numbers in circulation, G‐CSF augmented plasma VEGF level in vivo. Local G‐CSF administration into ischemic tissue increased capillary density and provided a functional vasculature and contributed to neovascularization of ischemic tissue. Blockade of the VEGF pathway abrogated G‐CSF‐induced angiogenesis. On the other hand, as we had shown previously, VEGF can induce endothelial progenitor cell (EPC) mobilization. Here, we show that G‐CSF also augmented the number of circulating VEGF receptor‐2 (VEGFR2) EPCs as compared with untreated controls. Blocking the VEGF/VEGFR1, but to a much lesser extent, the VEGF/VEGFR2 pathway in G‐CSF‐treated animals delayed tissue revascularization in a hindlimb model. These data clearly show that G‐CSF modulates angiogenesis by increasing myelomonocytic cells (VEGFR1+ neutrophils) and their release of VEGF. Our results indicated that administration of G‐CSF into ischemic tissue provides a novel and safe therapeutic strategy to improve neovascularization.

Activation of Integrin-Linked Kinase Is a Critical Prosurvival Pathway Induced in Leukemic Cells by Bone Marrow–Derived Stromal Cells

Integrin-linked kinase (ILK) directly interacts with beta integrins and phosphorylates Akt in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. In this study, we examined the functional role of ILK activation in leukemic and bone marrow stromal cells on their direct contact. Coculture of leukemic NB4 cells with bone marrow-derived stromal mesenchymal stem cells (MSC) resulted in robust activation of multiple signaling pathways, including ILK/Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducers and activators of transcription 3 (STAT3), and Notch1/Hes. Blockade of PI3K or ILK signaling with pharmacologic inhibitors LY294002 or QLT0267 specifically inhibited stroma-induced phosphorylation of Akt and glycogen synthase kinase 3beta, suppressed STAT3 and ERK1/2 activation, and decreased Notch1 and Hes1 expression in leukemic cells. This resulted in induction of apoptosis in both leukemic cell lines and in primary acute myelogenous leukemia samples that was not abrogated by MSC coculture. In turn, leukemic cells growing in direct contact with bone marrow stromal elements induce activation of Akt, ERK1/2, and STAT3 signaling in MSC, accompanied by significant increase in Hes1 and Bcl-2 proteins, which were all suppressed by QLT0267 and LY294002. In summary, our results indicate reciprocal activation of ILK/Akt in both leukemic and bone marrow stromal cells. We propose that ILK/Akt is a proximal signaling pathway critical for survival of leukemic cells within the bone marrow microenvironment. Hence, disruption of these interactions by ILK inhibitors represents a potential novel therapeutic strategy to eradicate leukemia in the bone marrow microenvironment by simultaneous targeting of both leukemic cells and activated bone marrow stromal cells.

Stimulation and priming of human neutrophils by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor: Qualitative and quantitative differences

Granulocyte colony-stimulating factor(G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) increased neutrophil C3bi-receptor expression and adherence and rapidly (less than 10 min) primed neutrophils to enhanced O2- release and membrane depolarization stimulated by chemotactic peptide. Direct triggering of O2- release in suspended neutrophils was also provoked by GM-CSF but not by G-CSF. GM-CSF-induced O2- release was inhibited by cyclic AMP agonists and cytochalasin B. The biological activity was greater in non-glycosylated GM-CSF than in glycosylated GM-CSF, whereas it was identical in glycosylated and non-glycosylated G-CSFs. Direct stimulation and priming by GM-CSF were consistently greater than those by G-CSF and the combined addition of the optimal concentrations of G-CSF and GM-CSF resulted in the effects of GM-CSF alone. These findings indicate that the effects of G-CSF and GM-CSF on neutrophil functions are qualitatively and quantitatively different from each other.

Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.

Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.

Novel role of HDAC inhibitors in AML1/ETO AML cells: activation of apoptosis and phagocytosis through induction of annexin A1

The chimeric fusion protein AML1-ETO, created by the t(8;21) translocation, recruits histone deacetylase (HDAC) to AML1-dependent promoters, resulting in transcriptional repression of the target genes. We analyzed the transcriptional changes in t(8;21) Kasumi-1 AML cells in response to the HDAC inhibitors, depsipeptide (FK228) and suberoylanilide hydroxamic acid (SAHA), which induced marked growth inhibition and apoptosis. Using cDNA array, annexin A1 (ANXA1) was identified as one of the FK228-induced genes. Induction of ANXA1 mRNA was associated with histone acetylation in ANXA1 promoter and reversal of the HDAC-dependent suppression of C/EBPα by AML1-ETO with direct recruitment of C/EBPα to ANXA1 promoter. This led to increase in the N-terminal cleaved isoform of ANXA1 protein and accumulation of ANXA1 on cell membrane. Neutralization with anti-ANXA1 antibody or gene silencing with ANXA1 siRNA inhibited FK228-induced apoptosis, suggesting that the upregulation of endogenous ANXA1 promotes cell death. FK228-induced ANXA1 expression was associated with massive increase in cell attachment and engulfment of Kasumi-1 cells by human THP-1-derived macrophages, which was completely abrogated with ANXA1 knockdown via siRNA transfection or ANXA1 neutralization. These findings identify a novel mechanism of action of HDAC inhibitors, which induce the expression and externalization of ANXA1 in leukemic cells, which in turn mediates the phagocytic clearance of apoptotic cells by macrophages.

TGF‐β receptor kinase inhibitor LY2109761 reverses the anti‐apoptotic effects of TGF‐β1 in myelo‐monocytic leukaemic cells co‐cultured with stromal cells

Transforming growth factor β1 (TGF‐β1) is an essential regulator of cell proliferation, survival and apoptosis, depending on the cellular context. TGF‐β1 is also known to affect cell‐to‐cell interactions between tumour cells and stromal cells. We investigated the role of TGF‐β1 in the survival of myelo‐monocytic leukaemia cell lines co‐cultured with bone marrow (BM)‐derived mesenchymal stem cells (MSC). Treatment with recombinant human (rh)TGF‐β1 inhibited spontaneous and cytarabine‐induced apoptosis in U937 cells, most prominently in U937 cells directly attached to MSCs. Conversely, the pro‐survival effects of TGF‐β1 were inhibited by LY2109761 or TGF‐β1 neutralizing antibody. rhTGF‐β1 increased pro‐survival phosphorylation of Akt, which was inhibited by LY2109761. The combination of rhTGF‐β1 and MSC co‐culture induced significant upregulation of C/EBPβ gene (CEBPB) and protein expression along with increased C/EBPβ liver‐enriched activating protein: liver‐enriched inhibitory protein ratio, suggesting the novel role of C/EBPβ in TGF‐β1‐mediated U937 cell survival in the context of stromal cell support. In summary, these results indicate that TGF‐β1 produced by BM stromal cells promotes the survival and chemoresistance of leukaemia cells under the direct cell‐to‐cell interactions. The blockade of TGF‐β signalling by LY2109761, which effectively inhibited the pro‐survival signalling, may enhance the efficacy of chemotherapy against myelo‐monocytic leukaemic cells in the BM microenvironment.

PPARgamma-Active triterpenoid CDDO enhances ATRA-induced differentiation in APL

Acute promyelocytic leukemia (APL) is associated with oncogenic PML-RARα that acts as a dominant negative transcriptional repressor of retinoic acid (RA) receptor target genes by recruiting histone deacetylase (HDAC). The peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor family that forms heterodimers with retinoid X receptor (RXR). In addition to RAR targets, PML-RARα silence a wide range of nuclear receptor target genes including PPARγ targets. All-trans-retinoic acid (ATRA), a ligand for the RA receptor (RAR), restores normal retinoid signaling and induces terminal differentiation of APL cells; however, APL cells can develop resistance to ATRA. Using ATRA sensitive NB4 and ATRA-resistant derivative NB4MR2 cell lines, we demonstrate that PPARγ ligand 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) enhances pro-apoptotic and differentiating effects of ATRA in ATRA-sensitive NB4 cells and partially reverses ATRA resistance in NB4MR2 cells. The CDDO/ATRA combination synergistically induces RARβ2 expression both in ATRA-sensitive and -resistant APL cells. RARβ2 mRNA induction by CDDO/ATRA was mediated in part by enhanced H3-Lys9 acetylation in the RARβ2 promoter which in turn increased the affinity of RARβ for βRARE. PPARγ specific inhibitor T007 and silencing of PPARγ by siRNA diminished CDDO-induced maturation and RARβ2 mRNA along with PPARγ induction indicating that PPARγ activation is at least partially responsible for the RARβ2 transcription and maturation induction. In an in vivo mouse model of APL, CDDO derivative CDDO-methyl ester markedly enhanced ATRA-induced maturation and extended the survival of mice. In summary, these results provide rationale for the combined targeting of RAR and PPARγ nuclear receptors in the therapy of APL.

Phorbol myristate acetate potentiates superoxide release and membrane depolarization without affecting an increase in cytoplasmic free calcium in human granulocytes stimulated by the chemotactic peptide, lectins and the calcium ionophore

We investigated the inter-relationships of superoxide (O2-) release, membrane depolarization and an increase in cytoplasmic free Ca2+, [Ca2+]i, in human granulocytes stimulated by various agonists. When concanavalin A or the Ca2+ ionophore ionomycin was used as stimulus, an increase in [Ca2+]i clearly preceded the onset of membrane depolarization, which was followed by O2- release. On the other hand, when N-formylmethionylleucylphenylalanine or wheat-germ agglutinin was used as stimulus, no demonstrable lag was seen in any of the responses. O2- release and membrane depolarization stimulated by all these agonists were markedly potentiated in parallel by pretreatment of cells with a low concentration of phorbol myristate acetate (0.25 ng/ml), whereas an increase in [Ca2+]i was not affected or minimally potentiated. The lag time between addition of the stimulus (concanavalin A or ionomycin) and onset of membrane depolarization or O2- release was significantly reduced by pretreatment of cells with phorbol myristate acetate, whereas the lag time between addition of concanavalin A and onset of the increase in [Ca2+]i was not affected. The dose-response curves for triggering of O2- release and membrane depolarization by each of receptor-mediated agonists in phorbol myristate acetate-pretreated or control cells were identical. These findings suggest that; (a) an increase in [Ca2+]i stimulates membrane depolarization indirectly; (b) a low concentration of phorbol myristate acetate potentiates membrane depolarization and O2- release by acting primarily at the post-receptor level, in particular, at the level distal to an increase in [Ca2+]i, but not by augmenting an increase in [Ca2+]i; and (c) the system provoking membrane depolarization and the system activating NADPH oxidase share a common pathway, which may be susceptible to a low concentration of phorbol myristate acetate.

JAK2 , CALR , and MPL mutation spectrum in Japanese patients with myeloproliferative neoplasms

Recurrent somatic mutations in the JAK2 , MPL , and CALR genes have been described in patients diagnosed with Philadelphia-negative myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These mutations are generally

Multilineage expression of haemopoietic precursors with an abnormal clone in idiopathic myelofibrosis.

Summary. To clarify the lineage involvement of haemopoietic progenitor cells in idiopathic myelofibrosis (MF), simultaneous analysis of morphology and chromosomes were performed on single colonies from a 70‐year‐old Japanese woman with typical presentation of MF. The cytogenetic analysis of unstimu‐lated peripheral blood cells revealed three cell lines 47.XX, −6, +9, +der(6)t (1;6)(lqter→1q21::6p21→6qter), 46,XX, −6, +der(6) and 46.XX. Of 91 metaphases examined, the numbers of each cell line were 43, 41 and 7, respectively. Blood mononuclear cells were plated at 2 × 103 or 1 × 104/ml in methylcellulose medium containing phytohaemagglutinin‐stimulated leucocyte conditioned medium and erythropoietin. On days 10,12 and 14 of culture, 47 individual colonies were lifted and served for morphological and cytogenetic examination. Morphological examination revealed that the colonies contained neutrophils (n), macrophages (m), basophils (b), erythrocytes (E) and megakaryocytes (M). Twenty‐two of the 47 colonies had analysable metaphases, yielding totally 158 metaphases. Twelve colonies contained three or more metaphases. Eight colonies (3bE, IE, 2b, InEM, IbEM) were of the abnormal cell line 46,XX, − 6, + der(6), and four colonies (2E, IbE, ImE) were of the karyotypically normal cell line 46,XX. The other abnormal cell line 47.XX, −6, +9, +der(6) was not detected in any of the metaphases obtained from single colonies. These results provided direct evidence that the pluripotent stem cells were involved in MF.