Tetsuo Sudo

Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway

Acute-phase response factor (APRF) is a transcription factor that binds to the interleukin-6 (IL-6)-responsive elements identified in the promoters of various acute-phase protein genes. We report here the purification and cloning of APRF. APRF exhibits a 52.5% overall homology at the amino acid level with p91, a component of the interferon (IFN)-stimulated gene factor 3 complexes. The cloned APRF protein is tyrosine phosphorylated and translocated into the nucleus in response to IL-6, but not in response to IFN-gamma. Tyrosine phosphorylation was also observed in response to other cytokines, such as leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor, whose receptors share the IL-6 receptor signal transducer gp130. In contrast, we observed that p91 is not tyrosine phosphorylated in response to IL-6. These results suggest that this novel p91-related protein may play a major role in the gp130-mediated signaling pathway and that selective activation of p91-related factors may explain the diversity of cellular responses to different cytokines.

Calpain-induced Bax-cleavage product is a more potent inducer of apoptotic cell death than wild-type Bax.

Wild type (wt) p21 Bax was cleaved to generate p18 Bax during apoptotic processes by calpain, which was suggested to recognize a certain motif around amino acids 30-33 Phe-Ile-Gln-Asp (FIQD). In the present study, analysis of protein sequencing revealed that the cleavage site was between Gln28 and Gly29. The fragment lacking the NH(2)-terminal amino acids 1-28 (tBax(29)) was more apoptotic than wt Bax. The tBax(29)-induced apoptotic cell death was substantially resistant to Bcl-x(L)-mediated rescue, compared with wt Bax, in spite of the complex formation between these two molecules. Together, the tBax(29) would be valuable for the treatment of tumors with high levels of Bcl-x(L) as well as the understanding of Bax-mediated apoptotic processes.

dlk Inhibits Stem Cell Factor-Induced Colony Formation of Murine Hematopoietic Progenitors: Hes-1-Independent Effect

Delta‐like (dlk) is a family of transmembrane proteins containing epidermal growth factor‐like repeat motifs homologous to the notch/delta/serrate family. Recent studies suggest that dlk is a negative regulator of adipocyte differentiation, a promoting factor of cobblestone area colony formation, and a molecule which influences stromal cell‐pre‐B cell interactions and augments cellularity of developing thymocytes. However, the role of dlk in regulating the growth and differentiation of hematopoietic progenitors remains unclear. In the present study, we examined the effect of dlk on the proliferation of murine hematopoietic progenitors by hematopoietic growth factors. Soluble dlk‐IgG Fc chimeric protein completely inhibited the colony formation of lineage‐marker negative (Lin−) bone marrow cells by GM‐CSF, G‐CSF, or macrophage‐CSF (M‐CSF) in the presence of stem cell factor (SCF). However, dlk failed to inhibit the colony formation of Lin− bone marrow cells by CSF, as described above, or M‐CSF plus interleukin 3. Furthermore, dlk failed to inhibit the colony formation of Hes‐1‐null fetal liver cells by M‐CSF in the presence of SCF. These findings suggest that dlk is an important regulator of hematopoietic progenitor proliferation. Depending on the presence of SCF, dlk may act as a growth inhibitor, although dlk signaling does not mediate Hes‐1 transcription factor.

A germ cell-specific gene, Prmt5, works in somatic cell reprogramming.

Germ cells possess the unique ability to acquire totipotency during development in vivo as well as give rise to pluripotent stem cells under the appropriate conditions in vitro. Recent studies in which somatic cells were experimentally converted into pluripotent stem cells revealed that genes expressed in primordial germ cells (PGCs), such as Oct3/4, Sox2, and Lin28, are involved in this reprogramming. These findings suggest that PGCs may be useful for identifying factors that successfully and efficiently reprogram somatic cells into toti- and/or pluripotent stem cells. Here, we show that Blimp-1, Prdm14, and Prmt5, each of which is crucial for PGC development, have the potential to reprogram somatic cells into pluripotent stem cells. Among them, Prmt5 exhibited remarkable reprogramming of mouse embryonic fibroblasts into which Prmt5, Klf4, and Oct3/4 were introduced. The resulting cells exhibited pluripotent gene expression, teratoma formation, and germline transmission in chimeric mice, all of which were indistinguishable from those induced with embryonic stem cells. These data indicate that some of the factors that play essential roles in germ cell development are also active in somatic cell reprogramming.

Osteoclast precursors in bone marrow and peritoneal cavity

Osteoclasts differentiate from cells that share some phenotypes with mature macrophages and monocytes, but early precursors for osteoclasts still remain obscure. To characterize osteoclast precursors, using monoclonal anti‐c‐Fms and anti‐c‐Kit antibodies, bone marrow cells were separated and the frequency of clonogenic progenitors were measured. Osteoclast precursors in the bone marrow mainly expressed c‐Kit and diminished in frequency when they expressed c‐Fms. In contrast to bone marrow, the precursors in the peritoneal cavity were enriched with a population of c‐Fms+. Injection of these antibodies into mice demonstrated that peritoneal osteoclast precursors were sensitive to anti‐c‐Fms but not to anti‐c‐Kit antibodies, whereas those in bone marrow only declined in the presence of both antibodies. Meanwhile, c‐Fms as opposed to c‐Kit played an essential role in the generation of osteoclasts in cultures. We also compared osteoclast precursors with colony forming cells (CFU‐M) by a macrophage colony stimulating factor. CFU‐M in bone marrow decreased when anti‐c‐Kit antibody was administered and no CFU‐M was detected in peritoneum. In this study, we show differences between proliferative potential osteoclast precursors maintained in bone marrow and peritoneum and between CFU‐M and osteoclast precursors. J. Cell. Physiol. 170:241–247, 1997.

Promotion of early osteoclastogenesis and B lymphopoiesis in the bone marrow of transgenic rats with the env-pX gene of human T-cell lymphotropic virus type I

Human T-cell lymphotropic virus type I (HTLV-I) is associated with various clinical disorders including adult T cell leukemia, myelopathy, arthropathy. Hypercalcemia resulting from osteoclast activation and a variety of hematopoietic abnormalities have been also observed in HTLV-I infected patients, however, precise mechanism about initial trigger(s) prior to presenting symptoms is still unknown. In this study, to assess effects of HTLV-I on hematopoiesis, we analysed characteristics of early hematopoietic precursors in HTLV-I env-pX transgenic rats. Progenitor cells for osteoclasts were significantly increased even in the marrow of asymptomatic env-pX rats. Progenitors for B cells were also highly enriched, while colony forming cells (CFC) elicited by GM-CSF(CFU-GM) and M-CSF(CFU-M) were comparable to normal littermates. Following arthritis in env-pX transgenic rats, osteoclastogenesis was further augmented and the CFCs were increased. Bone marrow cells carrying adjuvant-induced arthritis retained a constant number of progenitors for osteoclast and B lymphocytes, whereas the number of CFU-GM and CFU-M increased. These results indicate that the env-pX transgene affect early stages of osteoclast and B-cell lineages prior to developing diseases, in contrast, an increase of the CFCs was caused indirectly by arthritis. This study provides a novel standpoint for the mechanisms of pathogenesis by HTLV-I.

Tracing the Conversion Process from Primordial Germ Cells to Pluripotent Stem Cells in Mice

ABSTRACT To understand mechanisms underlying acquisition of pluripotency, it is critical to identify cells that can be converted to pluripotent stem cells. For this purpose, we focused on unipotent primordial germ cells (PGCs), which can be reprogrammed into pluripotent embryonic germ (EG) cells under defined conditions. Treatment of PGCs with combinations of signaling inhibitors, including inhibitors of MAP2K (MEK), GSK3B (GSK-3beta), and TGFB (TGFbeta) type 1 receptors, induced cells to enter a pluripotent state at a high frequency (12.1%) by Day 10 of culture. When we employed fluorescence-activated cell sorting to monitor conversion of candidate cells to a pluripotent state, we observed a cell cycle shift to S phase, indicating enrichment of pluripotent cells, during the early phase of EG formation. Transcriptome analysis revealed that PGCs retained expression of some pluripotent stem cell-associated genes, such as Pou5f1 and Sox2, during EG cell formation. On the other hand, PGCs lost their germ lineage characteristics and acquired expression of pluripotent stem cell markers, such as Klf4 and Eras. The overall gene expression profiles revealed by this system provide novel insight into how pluripotency is acquired in germ-committed cells.

Detection of Granulocyte Colony‐stimulating Factor Produced by a Newly Established Human Hepatoma Cell Line Using a Simple Bioassay System

A colony‐stimulating factor(CSF)‐producing tumor cell line (KX‐87) was established from a patient with hepatocellular carcinoma and marked granuloeytosis. This cell line formed tumors on nude mice in high frequency and the mice revealed marked granuloeytosis. In clonogenic assays of human bone marrow cells, KX‐87 conditioned medium (CM) supported the formation of colonies mainly consisting of neutrophilic granulocytes but had no burst‐promoting activity. The molecular weight of the colony‐stimulating activity (CSA) in KX‐87CM was estimated about 25,000 daltons by gel filtration and a new bioassay system. In principle, a subline of murine hemopoietic cell line NFS‐60 was cloned which was dependent on KX‐87CM. Then the growth of this subline was examined by a rapid and sensitive colorimetric tetrazolium assay. From these results, it was concluded that the CSA which KX‐87 cell line produced was G‐CSF.

Granulocyte-macrophage colony-stimulating factor enhances macrophage accessory function in Con A-stimulated T-cell proliferation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to augment various macrophage (M phi) functions, including antigen presentation in the antibody-producing response. We investigated the augmentative effect of GM-CSF on M phi A-cell activity in concanavalin A-stimulated T-cell proliferation. Pretreatment with GM-CSF of peritoneal M phi enhanced the T-cell proliferative response. This effect of GM-CSF was dose dependent and GM-CSF supplementation was needed at the beginning of M phi culture. We observed that GM-CSF induced M phi spreading and firm attachment accompanied with enlargement of the cytoplasm, but could not induce de novo expression of Ia antigen. GM-CSF treatment enabled M phi to produce more interleukin (IL)-1 and IL-6 upon stimulation with lipopolysaccharides or polyinosinic-polycytidylic acid, but was unable to stimulate M phi directly. This was confirmed by Northern blot analysis. These results indicate that GM-CSF augments M phi A-cell activity through the enhancement of the capacity of M phi to produce IL-1 and IL-6.

Establishment of megakaryoblastic cell lines by coinfection of Abelson murine leukemia virus and recombinant SV40-retrovirus.

Murine embryonic cells including yolk sac prepared from 8‐day embryos were co‐infected with Abelson murine leukemia virus (A‐MuLV) and/or a recombinant retrovirus containing large T and small t antigens, and early region of simian virus 40 (M‐SV40). By coinfection with A‐MuLV and M‐SV40, megakaryoblastic cells were obtained in addition to mast cells and fibroblastic cells. However, following infection with A‐MuLV or M‐SV40 alone, no megakaryoblastic cells were detected, although mast cells and/or fibroblastic cells developed. The same results were obtained in several experiments. By single‐cell transfer, 6 acetyl‐cholinesterase (AchE)‐positive clonal cell lines were established. Characteristics of megakaryocytes, such as AchE, glycoproteins lIb and IlIa, and platelet peroxidase were detected in two representative cells (C1 and C8). More significant changes expressing differentiation were observed following treatment with phorbol myristate acetate or pokeweed mitogen‐stimulated murine spleen cell conditioned medium, although release of platelets was not observed. This is the first report showing development of megakaryocytic cells as the result of coinfection with retroviruses.