Akiko Sada

Matrix-embedded osteocytes regulate mobilization of hematopoietic stem/progenitor cells.

The bone marrow (BM) niche comprises multiple cell types that regulate hematopoietic stem/progenitor cell (HSPC) migration out of the niche and into the circulation. Here, we demonstrate that osteocytes, the major cellular component of mature bone, are regulators of HSPC egress. Granulocyte colony-stimulating factor (G-CSF), used clinically to mobilize HSPCs, induces changes in the morphology and gene expression of the osteocytic network that precedes changes in osteoblasts. This rapid response is likely under control of the sympathetic nervous system, since osteocytes express the β2-adrenergic receptor and surgical sympathectomy prevents it. Mice with targeted ablation of osteocytes or a disrupted osteocyte network have comparable numbers of HSPCs in the BM but fail to mobilize HSPCs in response to G-CSF. Taken together, these results indicate that the BM/bone niche interface is critically controlled from inside of the bone matrix and establish an important physiological role for skeletal tissues in hematopoietic function.

Osteocytes Regulate Primary Lymphoid Organs and Fat Metabolism

Osteocytes act as mechanosensors to control local bone volume. However, their roles in the homeostasis of remote organs are largely unknown. We show that ablation of osteocytes in mice (osteocyte-less [OL] mice) leads to severe lymphopenia, due to lack of lymphoid-supporting stroma in both the bone marrow and thymus, and complete loss of white adipose tissues. These effects were reversed when osteocytes were replenished within the bone. In contrast, neither in vivo supply of T cell progenitors and humoral factors via shared circulation with a normal parabiotic partner nor ablation of specific hypothalamic nuclei rescued thymic atrophy and fat loss in OL mice. Furthermore, ablation of the hypothalamus in OL mice led to hepatic steatosis, which was rescued by parabiosis with normal mice. Our results define a role for osteocytes as critical regulators of lymphopoiesis and fat metabolism and suggest that bone acts as a central regulator of multiple organs.

The role of transcriptional coactivator TRAP220 in myelomonocytic differentiation

The TRAP220 subunit of the thyroid hormone receptor‐associated polypeptide transcription coactivator complex (TRAP/Mediator complex), mammalian counterpart of the yeast Mediator complex, is proposed to act on a variety of major and specific biological events through physical interactions with nuclear receptors. The vitamin D receptor (VDR) and retinoic acid receptor (RAR), coupled with retinoid X receptor (RXR), are nuclear receptors which have important roles for monopoiesis and granulopoiesis, respectively. In this study, we present the functional role of TRAP220 in nuclear receptor‐mediated monopoiesis and granulopoiesis. The mouse Trap220−/– yolk sac hematopoietic progenitor cells were resistant to 1,25‐dihydroxyvitamin D3‐stimulated differentiation into monocytes/macrophages. Furthermore, flow cytometric analyses showed that HL‐60 cells, human promyelocytic leukemia cell line, wherein TRAP220 was down‐regulated, did not differentiate efficiently into monocytes and granulocytes by stimulation with 1,25‐dihydroxyvitamin D3 and all‐trans retinoic acid, correspondingly. The expression of direct target genes of VDR or RAR, as well as the differentiation marker genes, was low in the knockdown cells. These results indicated a crucial role of TRAP220 in the optimal VDR‐ and RAR‐mediated myelomonocytic differentiation processes in mammalian hematopoiesis.

Pharmacokinetics-based optimal dose prediction of donor source-dependent response to mycophenolate mofetil in unrelated hematopoietic cell transplantation

Mycophenolate mofetil (MMF) has been widely used for prophylaxis against graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (allo-SCT). However, no clear advantage over methotrexate has been reported, other than reduced incidence of mucositis. We speculated that the wide inter-individual variation of plasma mycophenolic acid (MPA) levels veiled the benefits of MMF. Data from 36 unrelated allogeneic bone marrow (allo-BMT) and cord blood transplantation (CBT) were analyzed retrospectively based on MPA area under the curve (AUC0–24h). In allo-BMT, high AUC0–24h (>30 μg h/ml) resulted in no incidence of grade II–IV acute/extensive chronic GVHD and tended to show higher overall and disease-free survival, lower relapse rates, and non-relapse mortality. In CBT, AUC0–24h less than 30 μg h/ml was sufficient for low incidence of acute/chronic GVHD and high survival. Strong correlation between AUC0–24h and C2h, plasma MPA concentration at 2 h after administration was observed. Single point assessment of C2h was shown to provide a useful surrogate of AUC0–24h to predict GVHD incidence. The results of this study suggest that individualized MMF dosing in a donor source-dependent fashion may be important for maximizing the benefit of MMF in allo-SCT.

Inhibition of G1 to S Phase Progression by a Novel Zinc Finger Protein P58TFL at P-bodies

We recently reported the translocation of the immunoglobulin (Ig) light chain κ locus gene with a possible tumor suppressor gene, TFL, in transformed follicular lymphoma. However, the functional significance in cell transformation remains to be elucidated. Here, we first identified two gene products, P58TFL and P36TFL, derived by alternative splicing. The expression was prominent in normal human lymphocytes but defective in some leukemia/lymphoma cell lines. Overexpression of either protein in a mouse pro-B cell line, Ba/F3, and a human leukemia cell line, Jurkat, inhibited G1 to S phase progression through suppression of retinoblastoma protein (Rb) phosphorylation. The dominant gene product, P58TFL, colocalized with mRNA-processing body markers, eukaryotic translation initiation factor 2C and DCP1 decapping-enzyme homolog A, but not with a stress granule maker, T-cell intracellular antigen 1, in the cytoplasm. Taken together with the unique CCCH-type zinc finger motif, the present study suggests that P58TFL could play an important role in the regulation of cell growth through posttranscriptional modification of cell cycle regulators, at least partially, upstream of Rb. (Mol Cancer Res 2009;7(6):880–9)

Posttranscriptional Modulation of Cytokine Production in T Cells for the Regulation of Excessive Inflammation by TFL

Posttranscriptional machinery regulates inflammation and is associated with autoimmunity as well as tumorigenesis in collaboration with transcription factors. We previously identified the tumor suppressor gene transformed follicular lymphoma (TFL) on 6q25 in a patient with follicular lymphoma, which transformed into diffuse large B cell lymphoma. TFL families have a common RNase domain that governs macrophage-mediated inflammation. In human peripheral blood, TFL is dominantly expressed at the glycine- and tryptophan-rich cytoplasmic processing bodies of T lymphocytes, and it is persistently upregulated in activated T cells. To address its physiological role, we established TFL−/− mice in which TFL−/− lymphocytes proliferated more rapidly than TFL+/+ upon stimulation with inappropriate cytokine secretion, including IL-2, IL-6, and IL-10. Moreover, TFL inhibited the synthesis of cytokines such as IL-2, IL-6, IL-10, TNF-α, and IL-17a by 3′ untranslated region RNA degradation. Experimental autoimmune encephalitis induced in TFL−/− mice demonstrated persistent severe paralysis. CNS-infiltrated CD4+ T cells in TFL−/− mice contained a higher proportion of Th17 cells than did those in TFL+/+ mice during the resolution phase, and IL-17a mRNA levels were markedly increased in TFL−/− cells. These results suggest that TFL may play an important role in attenuating local inflammation by suppressing the infiltration of Th17 cells in the CNS during the resolution phase of experimental autoimmune encephalitis. TFL is a novel gradual and persistent posttranscriptional regulator, and the TFL-driven attenuation of excessive inflammation could contribute to recovery from T cell–mediated autoimmune diseases.

Chronic eosinophilic Leukemia with the fip1l1-pdgfr± fusion gene in a patient with a history of combination chemotherapy

Hypereosinophilic syndrome (HES) was diagnosed in December 2000 in a 43-year-old man on the basis of persistent eosinophilia (11.7 x 109/L) and a normal karyotype of the bone marrow cells. He had developed intra-abdominal non-Hodgkin’s lymphoma and in 1992 had received 3 courses of combination chemotherapy with doxorubicin (Adriamycin), cyclophosphamide, vincristine, methotrexate, bleomycin, and prednisolone. The patient was orally given prednisolone (10 mg/day) and cyclophosphamide (50 mg/day) as HES treatment without a subsequent improvement of the eosinophilia. In May 2003, anemia (hemoglobin, 7.9 g/dL) and thrombocytopenia (65 x 109/L) manifested with progressive eosinophilia (21.0 ×109/L) and a small number of blasts. The patient became febrile and was admitted in July 2003. Cytogenetic reexamination of the bone marrow cells disclosed the deletion of 4q12, indicating the presence of a fusion of the Fip1-like 1 (FIP1L1) gene to the plateletderived growth factor receptor α (PDGFRα) gene and consequently the clonal nature of his hematopoietic cells. DNA sequence analysis demonstrated that the breakpoints of the FIP1L1 and PDGFRα genes were present in exon 9 and exon 12, respectively. Treatment with imatinib mesylate (300 mg/day) promptly brought about complete remission. Although a number of similar eosinophilic cases have been reported, our patient may be the first such patient with a history of chemotherapy.

Deregulation of a possible tumour suppressor gene, ZC3H12D, by translocation of IGK@ in transformed follicular lymphoma with t(2;6)(p12;q25).

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A multicenter analysis of the FIP1L1-αPDGFR fusion gene in Japanese idiopathic hypereosinophilic syndrome: an aberrant splicing skipping the αPDGFR exon 12

To study the clinical characteristics of hypereosionophilic syndrome and chronic eosinophilic leukemia (HES/CEL) in Japan, the clinical data of 29 HES/CEL patients throughout the country were surveyed. Moreover, the involvement of the FIP1L1-αPDGFR fusion gene resulting from a cryptic del (4)(q12q12) was examined in 24 cases. The FIP1L1-αPDGFR messenger RNA (mRNA) was detected in three patients (13% of patients fulfilled WHO criteria and 17% of Chusid criteria). One had a novel fusion transcript, which skipped the exon 12 of αPDGFR. The transcript appears to be generated by a splicing mechanism that is different from the previously reported splicing patterns. In silico analysis, the exon skipping was not related to a disruption of the exonic splicing enhancers within the exon but strongly associated with the loss of the vast majority of the FIP1L intron 8a where intronic splicing enhancers were accumulated. Unexpectedly, pseudo-chimera DNA fragments with some shared characteristic features were occasionally generated from healthy control samples by reverse transcriptase polymerase chain reaction (RT-PCR). Considering the relatively low incidence of the FIP1L1-αPDGFR transcript positive case, extreme care must therefore be taken when making a diagnosis using RT-PCR before imatinib therapy.

G-CSF-induced sympathetic tone provokes fever and primes antimobilizing functions of neutrophils via PGE2

Granulocyte colony-stimulating factor (G-CSF) is widely used for peripheral blood stem/progenitor mobilization. G-CSF causes low-grade fever that is ameliorated by nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting the activation of arachidonic acid (AA) cascade. How G-CSF regulated this reaction was assessed. G-CSF treatment in mice resulted in fever, which was canceled in prostaglandin E synthase (mPGES-1)-deficient mice. Mobilization efficiency was twice as high in chimeric mice lacking mPGES-1, specifically in hematopoietic cells, suggesting that prostaglandin E2 (PGE2) from hematopoietic cells modulated the bone marrow (BM) microenvironment. Neutrophils from steady-state BM constitutively expressed mPGES-1 and significantly enhanced PGE2 production in vitro by β-adrenergic stimulation, but not by G-CSF, which was inhibited by an NSAID. Although neutrophils expressed all β-adrenergic receptors, only β3-agonist induced this phenomenon. Liquid chromatography-tandem mass spectrometry traced β-agonist-induced PGE2 synthesis from exogenous deuterium-labeled AA. Spontaneous PGE2 production was highly efficient in Gr-1high neutrophils among BM cells from G-CSF-treated mice. In addition to these in vitro data, the in vivo depletion of Gr-1high neutrophils disrupted G-CSF-induced fever. Furthermore, sympathetic denervation eliminated both neutrophil priming for PGE2 production and fever during G-CSF treatment. Thus, sympathetic tone-primed BM neutrophils were identified as one of the major PGE2 producers. PGE2 upregulated osteopontin, specifically in preosteoblasts, to retain progenitors in the BM via EP4 receptor. Thus, the sympathetic nervous system regulated neutrophils as an indispensable PGE2 source to modulate BM microenvironment and body temperature. This study provided a novel mechanistic insight into the communication of the nervous system, BM niche components, and hematopoietic cells.