Tadashi Oshizawa

CD31 (PECAM-1)-bright cells derived from AC133-positive cells in human peripheral blood as endothelial-precursor cells

To clarify the process of endothelial differentiation, we isolated AC133+ cells and induced the in vitro differentiation of these cells into endothelial cells. AC133+ cells efficiently differentiated into endothelial cells when the cells were cultured on fibronectin‐coated dishes in the presence of vascular endothelial growth factor. Time‐course analysis of the alteration of endothelial markers on cultured AC133+ cells revealed that the expression of CD31 (PECAM‐1) on AC133+ cells was the earliest marker among all of the tested markers. Based on the hypothesis that CD31 is an early indicator during the endothelial differentiation, we examined the relationship between CD31 expression and the ability to differentiate into endothelial cells in cells derived from AC133+ cells. CD31‐bright cells, which were sorted from cultured AC133+ cells, differentiated more efficiently into endothelial cells than had CD31‐positive or CD31‐negative cells, suggesting that CD31‐bright cells may be precursor cells for endothelial cells. In the present study, we identified CD31+ cells derived from cultured AC133+ cells that are able to differentiate to endothelial cells as precursor cells.

Okadaic acid induces both augmentation and inhibition of opsonized zymosan-stimulated superoxide production by differentiated HL-60 cells. Possible involvement of dephosphorylation of a cytosolic 21K protein in respiratory burst

We found that okadaic acid (OA), a potent tumor promoter and a phosphatase inhibitor, has a unique opposing effect on opsonized zymosan (Op.-zym.)-elicited O2.- production by differentiated HL-60 cells in a narrow range of concentrations but does not induce any O2.- production by itself. Okadaic acid magnified the O2.- production 2.5-fold at 1.0 microM, while it inhibited it at 2.0 microM or higher concentrations. This effect of OA did not correspond to the changes in the expression of surface receptors (CD11b/CD18, CR3) for Op.-zym., because they were weakly down-regulated by OA at any concentration. Two-dimensional gel electrophoresis revealed that in the absence of OA, Op.-zym. induced rapid dephosphorylation of a cytosolic 21K protein with a very slight increase in phosphorylation of membranous p47phox, which is one of the cytosolic factors required for respiratory burst. In the presence of a stimulatory concentration (1.0 microM) of OA, the Op.-zym.-caused dephosphorylation of the 21K protein was still observed and the phosphorylation of p47phox was enhanced. In the presence of an inhibitory concentration (2.0 or 5.0 microM) of OA, the Op.-zym.-induced dephosphorylation of the 21K protein was strongly inhibited while p47phox was heavily phosphorylated. Acid hydrolysis of the 21K phosphoprotein yielded only phosphoserine as a phosphoamino acid. Furthermore, at least part of the 21K protein seemed to be associated with p67phox and p47phox, because it was co-immunoprecipitated with those cytosolic factors. These results suggest that a cytosolic 21K protein plays an important role in respiratory burst through dephosphorylation by a phosphoserine phosphatase, and that the dephosphorylated 21K protein may work synergistically with the phosphorylated p47phox on the pathway for activation of the respiratory burst oxidase.

Specific expression of annexin III in rat-small-hepatocytes ☆

Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.

HX531, a retinoid X receptor antagonist, inhibited the 9-cis retinoic acid-induced binding with steroid receptor coactivator-1 as detected by surface plasmon resonance

HX531 is a retinoid X receptor (RXR) antagonist that inhibits 9-cis retinoic acid-induced neutrophilic differentiation of HL-60 cells. In order to elucidate the inhibitory mechanism of HX531, we have developed a novel ligand sensor assay for RXR in which the receptor-coactivator interaction is directly monitored using surface plasmon resonance (SPR) biosensor technology. A 20-mer peptide from steroid receptor coactivator-1 (SRC-1), containing nuclear receptor interaction motif LXXLL was immobilized on the surface of a BIAcore sensor chip. Injection of human recombinant RXR with or without 9-cis retinoic acid resulted in ligand-dependent interaction with the SRC-1 peptide. Kinetic analysis revealed dissociation constants (KD) of 9-cis RA-preincubated RXR to SRC-1 was 5.92 x 10(-8)M. Using this technique, we found that 1 microM HX531 reduced the ka value of liganded-RXR with SRC-1, suggesting that HX531 reduced the affinity of RXR to SRC-1. This SPR assay system was applied to obtain quantitative kinetic data of RXR ligand binding to the SRC-1 peptide and the alteration of these data by antagonists.

Role of the p70 S6 kinase cascade in neutrophilic differentiation and proliferation of HL-60 cells—a study of transferrin receptor-positive and -negative cells obtained from dimethyl sulfoxide- or retinoic acid-treated HL-60 cells

Previously, we suggested that p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells; this conclusion was based on our analysis of transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells that appeared after treatment with dimethyl sulfoxide (Me(2)SO). In this study, we analyzed the upstream of p70 S6K in relation to the differentiation and proliferation of both cell types. The granulocyte colony-stimulating factor (G-CSF)-induced enhancement of phosphatidylinositol 3-kinase (PI3K) activity in Trf-R(+) cells was markedly higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited these activities. The wortmannin-dependent enhancement of neutrophilic differentiation was similar to that induced by rapamycin. From these results, we conclude that the PI3K/p70 S6K cascade may play an important role in negative regulation of neutrophilic differentiation in HL-60 cells. For the G-CSF-dependent proliferation, however, p70 S6K appears to be a highly important pathway through not only a PI3K-dependent but also possibly an independent cascade.

The role of c-Myc on granulocyte colony-stimulating factor-dependent neutrophilic proliferation and differentiation of HL-60 cells

We have previously suggested that phosphatidylinositol 3-kinase (PI3K)/p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells on the basis of analysis of transferrin receptor (Trf-R)-positive (Trf-R(+)) and -negative (Trf-R(-)) cells that appear after treatment with dimethyl sulfoxide (DMSO). In the present study, we analyzed the downstream events of p70 S6K in differentiation and proliferation of both cell types, with a particular focus on c-Myc. Similar to p70 S6K, we found that the expression of c-Myc in Trf-R(+) cells is also higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity, c-Myc expression, and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited p70 S6K activity, c-Myc expression, and G-CSF-dependent proliferation, indicating that the extent of c-Myc inhibition by these inhibitors correlates with a reduction in proliferation, and that c-Myc is downstream from PI3K/p70 S6K. We also determined phosphorylation of the 4E-binding protein 1 (4E-BP1), which is regulated downstream of the mammalian target of rapamycin. The addition of G-CSF failed to enhance the phosphorylation state of 4E-BP1 of HL-60 cells 2 days after DMSO differentiation. An antisense oligonucleotide for c-myc inhibited both G-CSF-dependent enhancement of c-Myc expression and proliferation in Trf-R(+) cells, but did not enhance the differentiation in terms of O(2)(-)-generating ability or fMLP-R expression. In contrast, antisense oligonucleotide for c-myc promoted fMLP-R on non-treated HL-60 cells. We therefore conclude that the PI3K/p70 S6K/c-Myc cascade plays an important role in neutrophilic proliferation in HL-60 cells. Unlike that of rapamycin, however, the antisense oligonucleotide for c-myc could not promote differentiation of Trf-R(+) cells cultured with G-CSF, indicating that another target downstream of p70 S6K may control the differentiation of HL-60 cells in terms of the signal transduction of G-CSF.

A new role of thrombopoietin enhancing EX vivo expansion of endothelial precursor cells derived from AC133 positive cells

We previously reported that CD31bright cells, which were sorted from cultured AC133+ cells of adult peripheral blood cells, differentiated more efficiently into endothelial cells than CD31+ cells or CD31- cells, suggesting that CD31bright cells may be endothelial precursor cells. In this study, we found that CD31bright cells have a strong ability to release cytokines. The mixture of vascular endothelial growth factor (VEGF), thrombopoietin (TPO), and stem cell factor stimulated ex vivo expansion of the total cell number from cultured AC133+ cells of adult peripheral blood cells and cord blood cells, resulting in incrementation of the adhesion cells, in which endothelial nitric oxide synthase and kinase insert domain-containing receptor were positive. Moreover, the mixture of VEGF and TPO increased the CD31bright cell population when compared with VEGF alone or the mixture of VEGF and stem cell factor. These data suggest that TPO is an important growth factor that can promote endothelial precursor cells expansion ex vivo.

Granulocyte colony-stimulating factor promotes the translocation of protein kinase Cι in neutrophilic differentiation cells

Previously, we suggested that the phosphatidylinositol 3‐kinase (PI3K)‐p70 S6 kinase (p70 S6K) pathway plays an important role in granulocyte colony‐stimulating factor (G‐CSF)‐dependent enhancement of the neutrophilic differentiation and proliferation of HL‐60 cells. While atypical protein kinase C (PKC) has been reported to be a regulator of p70 S6K, abundant expression of PKCι was observed in myeloid and lymphoid cells. Therefore, we analyzed the participation of PKCι in G‐CSF‐dependent proliferation. The maximum stimulation of PKCι was observed from 15 to 30 min after the addition of G‐CSF. From 5 to 15 min into this lag time, PKCι was found to translocate from the nucleus to the membrane. At 30 min it re‐translocated to the cytosol. This dynamic translocation of PKCι was also observed in G‐CSF‐stimulated myeloperoxidase‐positive cells differentiated from cord blood cells. Small interfering RNA for PKCι inhibited G‐CSF‐induced proliferation and the promotion of neutrophilic differentiation of HL‐60 cells. These data indicate that the G‐CSF‐induced dynamic translocation and activation processes of PKCι are important to neutrophilic proliferation. J. Cell. Physiol. 211: 189–196, 2007.

Granulocyte colony‐stimulating factor‐induced dephosphorylation of a 45 kD cytosolic protein in HL‐60 cells differentiating into neutrophils

Granulocyte colony‐stimulating factor (G‐CSF)‐induced alteration of phosphoprotein during differentiation of HL‐60 cells was studied. From the two‐dimensional gel electrophoresis analysis of phosphoproteins, a 45 kD phosphoprotein in the cytosolic fraction of DMSO‐pretreated HL‐60 cells was rapidly dephosphorylated by the addition of G‐CSF. This 45 kD phosphoprotein migrated into four or five spots between 4.5 and 5.5 pI. The dephosphorylation of 45 kD protein was observed within at least 10 min and reached a maximum at 60 min. Phosphoamino acid analysis showed that only serine residue of 45 kD phosphoprotein was phosphorylated, suggesting that G‐CSF induced an activation of serine phosphatase. Furthermore, Staurosporine and calphostin C inhibited the phosphorylation of 45 kD protein, suggesting that protein kinase C or its downstream kinase(s) is involved in the phosphorylation of 45 kD protein. These results indicate that G‐CSF causes dephosphorylation of a 45 kD cytosolic phosphoprotein which may play a role in signal transduction of G‐CSF.