The suppression of the B cell population during senescence has been considered to be due to the suppression of interleukin-7 (IL-7) production and responsiveness to IL-7; however, the upregulation of transforming growth factor-beta (TGF-beta) was found to contribute to B cell suppression. To investigate the mechanism of this suppression based on the interrelationship between IL-7 and TGF-beta during senescence, senescence-accelerated mice (SAMs), the mouse model of aging, were used in this study to elucidate the mechanisms of B lymphopoietic suppression during aging. Similar to regular senescent mice, SAMs showed a decrease in the number of IL-7-responding B cell progenitors (i.e., colony-forming unit pre-B [CFU-pre-B] cells in the femoral bone marrow [BM]). A co-culture system of B lymphocytes and stromal cells that the authors established showed a significantly lower number of CFU-pre-B cells harvested when BM cells were co-cultured with senescent stromal cells than when they were co-cultured with young stromal cells. Interestingly, cells harvested from a senescent stroma and those from the control culture without stromal cells were higher in number than those harvested from a young stroma, thereby implying that an altered senescent stromal cell is unable to maintain self-renewal of the stem cell compartment. Because TGF-beta is supposed to suppress the proliferative capacity of pro-B/pre-B cells, we added a neutralizing anti-TGF-beta antibody to the co-culture system with a pro-B/pre-B cell-rich population to determine whether such suppression may be rescued. However, unexpectedly, any rescue was not observed and the number of CFU-pre-B cells remained unchanged when BM cells were co-cultured with senescent stromal cells compared with the co-culture with young stromal cells, which essentially showed an increase in the number of CFU-pre-B cells (P < 0.001 in 5 microg/ml). Furthermore, TGF-beta protein level in the supernatant of cultured senescent stroma cells was evaluated by enzyme-linked immunoabsorbent assay, but surprisingly, it was found that TGF-beta concentration was significantly lower than that of cultured young stromal cells. Thus, TGF-beta activity was assumed to decline particularly in a senescent stroma, which means a distinct difference between the senescent suppression of B lymphopoiesis and secondary B lymphocytopenia. Concerning proliferative signaling, on the other hand, the level of IL-7 gene expression in cells from freshly isolated BM decreased significantly with age. Therefore, the acceleration of proliferative signaling and the deceleration of suppressive signaling may both be altered and weakened in a senescent stroma (i.e., homeosuppression).

In the present study, we examined the role in hematopoiesis of cationic amino acid transporter 1 (CAT1) , IntroductionCytokines such as interleukin-3 (IL-3) and erythropoietin play an important role in the differentiation of hematopoietic stem cells. 1,2 However, the roles of small molecules such as vitamins, nucleic acids, and amino acids in cell differentiation have not been elucidated well, though it is likely that specific transporters are involved in importing such small hydrophilic molecules into the cells. The ABC transporter Bcrp1 (abcg2) is expressed mainly in hematopoietic stem cells, 3 and more recently, efflux transporter activities in murine hematopoietic stem cells (HSCs) were found to vary according to developmental and activation status. 4 Thiamine transporter Thtr-1 (slc19a2) gene knockout mice showed abnormalities of erythroid, myeloid, and megakaryocyte lineages in bone marrow when fed a thiamine-free diet. 5 The importance of CAT1 (cationic amino acid transporter)-mediated transport was recently underscored by the production of cat1 gene knockout mice, which exhibit anemia. 6 These reports indicate that substrates of transporters expressed on hematopoietic stem cells are physiologically essential in differentiation or proliferation of the cells, or both, and the expression levels and activities of these transporters are likely to be regulated stage and lineage specifically.Cationic amino acid transporter (CAT, slc7a), known as system y ϩ , transports cationic amino acids such as L-lysine, L-histidine, L-ornithine, and L-arginine. 7-9 System y ϩ is a facilitative process that is Na ϩ -independent and pH insensitive. CAT1 (slc7a1), whose mouse ortholog was first identified as a virus receptor, 10,11 is expressed ubiquitously, including fetal liver and bone marrow, though not adult liver. 12 It is known that fetal and embryonic liver plays a significant role in hematopoiesis. 13 Furthermore, CAT1 mRNA is induced in regenerating liver in a short-lived manner and has multiple sites for regulation of gene expression, indicating that system y ϩ is tightly regulated and essential for liver cells to enter mitosis. 14,15 These findings suggest that CAT1 could be involved in hematopoietic activity.One of the substrates of CAT1, L-arginine, is a precursor of nitric oxide (NO) and polyamines. NO can induce apoptosis in megakaryocytes and platelet formation. 16,17 L-ornithine is produced by arginase and further metabolized to polyamines, which regulate the cell cycle. 18 Recently, low concentrations of L-glutamine were reported to induce functional differentiation of U937 myelomonocytic cells. 19 These facts suggest that low-molecular-weight molecules such as amino acids may be involved in hematopoiesis, as well as large-molecular cytokines such as IL-3 and erythropoietin. In the present study, we investigated the effects of hCAT1 expression level and L-arginine on differentiation and proliferation of human cord blood cells and a cloned human erythroid model, K562 cells. Materials and methods Tissue cultur...

The murine cationic amino acid transporter 1 (mCAT1) protein mediates membrane transports of L-arginine, L-lysine, L-histidine, and L-ornithine. The importance of mCAT1-mediated transport was underscored by the phenotype of mCAT1 gene-knockout mice, which exhibit anemia. 1) In addition, we reported that L-arginine that is a substrate of CAT1 is essential for the differentiation to erythrocyte by in vitro cell culture study.2) It has been also observed that L-arginine deficiency affects maturation of early B cells in the bone marrow, but not development of the T cells in the thymus. 3)However, the detailed biological role of the L-arginine in proliferation and differentiation of blood cells and K562 cells that are human leukemia cell line is not clear. L-Arginine is the precursor of polyamines, nitric oxide (NO), and agmatine that are associated with cell proliferation and differentiation.4) While NO can induce an apoptosis of megakaryocytes and a formation of platelet, 5,6) it was reported that NO inhibited differentiation of K562 cells. 7) L-Ornithine is produced by arginase and further metabolized into polyamines such as putrescine, spermidine and spermine, which regulate the cell cycle.8) Moreover, there is some evidence that agmatine acts as an antiproliferative molecule through induction of the protein antizyme.9) Antizyme inhibits ornithine decarboxylase (ODC), hence polyamine biosynthesis. At the same time, antizyme suppresses polyamine uptake. Based on these reports, it was considered that polyamines might be metabolic products of L-arginine that can facilitate differentiation of hematopoietic precursors to erythrocytes.Polyamines (putrescine, spermidine and spermine) play a crucial role in regulating gene expression, signal transduction, ion channel function, DNA and protein synthesis, as well as cell proliferation and differentiation.10) The intracellular polyamine concentration in human erythrocytes is reported to be relatively high; putrescine 0.6 mM (males), and 0.9 mM (females); spermidine 18 and 24 mM; spermine 19 and 15 mM. 11) Polyamines are also scavengers of reactive oxygen species, thereby protecting DNA, protein, and lipids from oxidative damage.12) Available evidence shows that polyamines are key regulators of angiogenesis, early mammalian embryogenesis, placental trophoblast growth, and embryonic development.13) The cellular polyamines are thought to be supplied through de novo synthesis from L-arginine and transport, while the mammalian polyamine transporter gene has not been identified, 14,15) unlike bacterial and/or fungal polyamine transporter. 10,16) De novo polyamine synthesis is governed in part by the activity of ornithine decarboxylase (ODC), which catalyzes the conversion of the amino acid, L-ornithine to the diamine and putrescine. When the ODC activity was inhibited by alpha-difluoromethylornithine (DFMO) in K562 cells, proliferation of K562 cells was inhibited through abolishment of intracellular polyamines.17) It was also reported that spermine induced hemoglobin production i...

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Hematopoiesis occurs in the bone marrow, where primitive hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment composed of stromal cells. We examined the hematopoietic supportive ability of stromal cells in a 3D culture system using polymer particles with grafted epoxy polymer chains. Umbilical cord blood-derived CD34(+) cells were co-cultivated with MS-5 stromal cells. They formed a 3D structure in the culture dish in the presence of particles, and the total numbers of cells and the numbers of hematopoietic progenitor cells, including colony-forming unit (CFU)-Mix, CFU-granulocyte-macrophage, CFU-megakaryocyte and burst-forming unit-erythroid, were measured every seven days. The hematopoietic supportive activity of the 3D culture containing polymer particles and stromal cells was superior to that of 2D culture, and allowed the expansion and maintenance of hematopoietic progenitor cells for more than 12 weeks. Various types of hematopoietic cells, including granulocytes, macrophages and megakaryocytes at different maturation stages, appeared in the 3D culture, suggesting that the CD34(+) cells were able to differentiate into a range of blood cell types. Morphological examination showed that MS-5 stromal cells grew on the surface of the particles and bridged the gaps between them to form a 3D structure. Hematopoietic cells slipped into the 3D layer and proliferated within it, relying on the presence of the MS-5 cells. These results suggest that this 3D culture system using polymer particles reproduced the hematopoietic phenomenon in vitro, and might thus provide a new tool for investigating hematopoietic stem cell-stromal cell interactions.

In this study, we examined the age-associated defect of stromal cells, which support B cell development, treated with 5-fluorouracil (5-FU) to induce severe perturbation of hematopoiesis, including B lymphocyte development, using SAMP1 mice exhibiting senescence-mimicking stromal-cell impairment after 30 weeks of age. Significant findings of this study are as follows: first, a marked and prolonged decrease in number of CFU-preB cells in non-SCI mice (58% of the steady-state level) associated with more markedly depressed number of CFU-preB cells in SCI mice (20% of the steady-state level), despite the absence of difference in the number of CFU-GMs during the period; second, in the non-SCI mice, a significant and prolonged up-regulation of GM-CSF and IL-6, positive regulators of myelopoiesis and suppressive factors of B lymphopoiesis, was observed. In SCI mice, greater and prolonged suppression of B lymphopoiesis was clearly demonstrated by the significant up-regulation of the negative regulator TNF-alpha associated with the concomitant marked down-regulation of the positive regulator SDF-1, although the increases of GM-CSF and IL-6 were limited. That is, 'negative complementation' makes preB recovery after 5-FU treatment impaired and prolonged. Principal component analysis clearly showed differences in the cytokine expression patterns in both early and later phases and the time course of the expression pattern of each cytokine between SCI and non-SCI mice without supervising information. An impaired regulation of the expressions of not only positive but also negative regulators after 5-FU treatment was, in part, the cause of the impaired regeneration of CFU-preB cells in SCI mice.