Hisae Kobayashi

Role of the CD47-SHPS-1 system in regulation of cell migration.

SHPS‐1 is a transmembrane protein whose extracellular region interacts with CD47 and whose cytoplasmic region undergoes tyrosine phosphorylation and there by binds the protein tyrosine phosphatase SHP‐2. Formation of this complex is implicated in regulation of cell migration by an unknown mechanism. A CD47‐Fc fusion protein or antibodies to SHPS‐1 inhibited migration of human melanoma cells or of CHO cells overexpressing SHPS‐1. Overexpression of wild‐type SHPS‐1 promoted CHO cell migration, whereas expression of the SHPS‐1‐4F mutant, which lacks the phosphorylation sites required for SHP‐2 binding, had no effect. Antibodies to SHPS‐1 failed to inhibit migration of CHO cells expressing SHPS‐1‐4F. SHPS‐1 ligands induced the dephosphorylation of SHPS‐1 and dissociation of SHP‐2. Antibodies to SHPS‐1 also enhanced Rho activity and induced both formation of stress fibers and adoption of a less polarized morphology in melanoma cells. Our results suggest that engagement of SHPS‐1 by CD47 prevents the positive regulation of cell migration by this protein. The CD47–SHPS‐1 system and SHP‐2 might thus contribute to the inhibition of cell migration by cell–cell contact.

Regulation by SIRPα of dendritic cell homeostasis in lymphoid tissues

The molecular basis for regulation of dendritic cell (DC) development and homeostasis remains unclear. Signal regulatory protein α (SIRPα), an immunoglobulin superfamily protein that is predominantly expressed in DCs, mediates cell-cell signaling by interacting with CD47, another immunoglobulin superfamily protein. We now show that the number of CD11c(high) DCs (conventional DCs, or cDCs), in particular, that of CD8-CD4+ (CD4+) cDCs, is selectively reduced in secondary lymphoid tissues of mice expressing a mutant form of SIRPα that lacks the cytoplasmic region. We also found that SIRPα is required intrinsically within cDCs or DC precursors for the homeostasis of splenic CD4+ cDCs. Differentiation of bone marrow cells from SIRPα mutant mice into DCs induced by either macrophage-granulocyte colony-stimulating factor or Flt3 ligand in vitro was not impaired. Although the accumulation of the immediate precursors of cDCs in the spleen was also not impaired, the half-life of newly generated splenic CD4+ cDCs was markedly reduced in SIRPα mutant mice. Both hematopoietic and nonhematopoietic CD47 was found to be required for the homeostasis of CD4+ cDCs and CD8-CD4- (double negative) cDCs in the spleen. SIRPα as well as its ligand, CD47, are thus important for the homeostasis of CD4+ cDCs or double negative cDCs in lymphoid tissues.

Ectodomain Shedding of SHPS-1 and Its Role in Regulation of Cell Migration

SHPS-1 is a transmembrane protein whose cytoplasmic region undergoes tyrosine phosphorylation and then binds the protein-tyrosine phosphatase SHP-2. Formation of the SHPS-1-SHP-2 complex is implicated in regulation of cell migration. In addition, SHPS-1 and its ligand CD47 constitute an intercellular recognition system that contributes to inhibition of cell migration by cell-cell contact. The ectodomain of SHPS-1 has now been shown to be shed from cells in a reaction likely mediated by a metalloproteinase. This process was promoted by activation of protein kinase C or of Ras, and the released ectodomain exhibited minimal CD47-binding activity. Metalloproteinases catalyzed the cleavage of a recombinant SHPS-1-Fc fusion protein in vitro, and the primary cleavage site was localized to the juxtamembrane region of SHPS-1. Forced expression of an SHPS-1 mutant resistant to ectodomain shedding impaired cell migration, cell spreading, and reorganization of the actin cytoskeleton. It also increased the tyrosine phosphorylation of paxillin and FAK triggered by cell adhesion. These results suggest that shedding of the ectodomain of SHPS-1 plays an important role in regulation of cell migration and spreading by this protein.

Characterization of nucleotide pyrophosphatase-5 as an oligomannosidic glycoprotein in rat brain.

Membrane glycoproteins of neural cells play crucial roles in axon guidance, synaptogenesis, and neuronal transmission. We have here characterized membrane glycoproteins containing terminal alpha-mannose residues in rat brain membranes. Affinity purification using Galanthus nivalis agglutinin, that is highly specific for terminal alpha-mannose residues, revealed a 50-kDa protein as well as 80-kDa SHPS-1 and 45-kDa beta2 subunit of Na,K-ATPase in rat brain membranes. Combination of N-terminal peptide sequencing and mass spectrometry indicated that the 50-kDa protein was rat nucleotide pyrophosphatase-5 (NPP-5). In contrast to other NPPs, NPP-5 was a type-I transmembrane protein. Northern blot analysis showed that NPP-5 was highly expressed in brain, but also expressed in other peripheral tissues. However, we could not detect either the NPP activity or the lysophospholipase D activity in the immunoprecipitates with antibodies to NPP-5 from rat brain membranes. These data, therefore, suggest that NPP-5 is a neural oligomannosidic glycoprotein that may participate in neural cell communications.

Essential roles of SHPS-1 in induction of contact hypersensitivity of skin.

SHPS-1 is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 and is abundant on the surface of CD11c(+) dendritic cells (DCs). We recently showed that SHPS-1 is essential for priming by DCs of CD4(+) T cells and for development of Th17 cell-mediated experimental autoimmunity. We have now further evaluated the importance of SHPS-1 and that of its ligand CD47 in contact hypersensitivity (CHS) to 2,4-dinitro-1-fluorobenzene (DNFB). Whereas the DNFB-induced CHS response was impaired in mice that express a mutant form of SHPS-1 lacking most of the cytoplasmic region, it was unaffected in CD47-deficient mice. Moreover, treatment of wild-type mice with mAbs to SHPS-1 that either block or do not block the binding of SHPS-1 to CD47 inhibited the CHS response. A mAb to CD47 had no such effect. The 2,4-dinitro-benzenesulfonic acid-induced proliferation of, and production of IFN-gamma or IL-17 by, T cells from DNFB-sensitized wild-type mice were inhibited by either mAb to SHPS-1 but not by that to CD47. In contrast, the blocking mAbs to SHPS-1, but not that to CD47, inhibited an allogeneic mixed leukocyte reaction. Both mAbs to SHPS-1, but not that to CD47, also inhibited the lipopolysaccharide- or polyinosinic-polycytidylic acid-induced production of TNF-alpha by DCs. These results suggest that SHPS-1 is essential for development of CHS, likely as a result of its positive regulation of the priming by DCs of CD4(+) T cells. However, such regulation by SHPS-1 does not appear to require its interaction with CD47.

Inhibitory Effects of WR-2721 and Cysteamine on Tumor Initiation in Mammary Glands of Pregnant Rats by Radiation

Abstract Inano, H., Onoda, M., Suzuki, K., Kobayashi, H. and Wakabayashi, K. Inhibitory Effects of WR-2721 and Cysteamine on Tumor Initiation in Mammary Glands of Pregnant Rats by Radiation. We evaluated the effect of WR-2721 [S-2-(3-aminopropylamino)-ethylphosphorothioic acid] and cysteamine (2-mercaptoethylamine) on the development of radiation-induced mammary tumors in rats. Pregnant rats were treated with WR-2721 or cysteamine 30 min prior to whole-body irradiation with γ rays from a 60Co source at a dose of 1.5 or 2.6 Gy. Additional pregnant rats were given saline and then exposed to γ rays at a dose of 0, 1.5 or 2.6 Gy as a control. All rats were implanted with pellets of diethylstilbestrol, a tumor promoter, 1 month after termination of nursing and were observed for 1 year to detect palpable mammary tumors. No mammary tumors developed in the saline-injected nonirradiated rats. However, when rats were irradiated with 1.5 or 2.6 Gy after saline treatment, the incidence of mammary tumors was high (71.4 and 92.3%, respectively). Administration of WR-2721 or cysteamine prior to irradiation with 1.5 Gy significantly decreased the tumor incidence (23.8 and 20.8%, respectively). Tumor prevention by either agent was less effective at the higher dose. The appearance of the first mammary tumor occurred later in rats treated with WR-2721 or cysteamine than in the control rats. An increasing rate of adenocarcinoma in the control group was observed with increasing dose from 1.5 Gy up to 2.6 Gy. However, the development of adenocarcinoma did not increase after pretreatment with WR-2721 or cysteamine in rats irradiated with 2.6 Gy. Many of the mammary tumors that developed in the control rats were of the ER+PgR+ type. Administration of WR-2721 produced no tumors of the ER+PgR+ type. Cysteamine treatment increased the development of ER-negative tumors. The serum concentration of progesterone was significantly higher in rats treated with WR-2721 or cysteamine than in the control rats. On the other hand, the estradiol-17β concentration was reduced by treatment with WR-2721, but not significantly compared to the control. WR-2721 and cysteamine had no effect on the prolactin concentration of the irradiated rats. The results suggest that administration of WR-2721 or cysteamine prior to the irradiation has a potent preventive effect on the initiation phase during mammary tumorigenesis.

Comparative effect of chlormadinone acetate and diethylstilbestrol as promoters in mammary tumorigenesis of rats irradiated with γ-rays during lactation

The purpose of this study was to determine the promotional role of estrogen and progestin in the development of radiation-induced mammary tumors. To eliminate the effects of endogenous ovarian hormones on tumor promotion, all rats were ovariectomized immediately after the initiation by irradiation with 2.6 Gy γ-rays at day 21 of lactation, and were divided into 3 groups. For the control experiment, rats were implanted with a cholesterol pellet 1 month after the irradiation. Only one rat developed a fibroadenoma (4.3% mammary tumor incidence) during the 1 year period of the implantation. In the other two groups, chlormadinone acetate (CMA) to increase progestin level or diethylstilbestrol (DES) to increase estrogenic activity were administered, respectively, as tumor promoters for 1 year. Treatment with CMA did not significantly increase the incidence of mammary tumors as compared with the controls. However, administration of DES resulted in a significantly higher mammary tumor incidence (79.3%) than control treatment. Compared with cholesterol administration, DES treatment caused an increase in prolactin concentration in serum (5-fold), and reduction of estradiol-17β concentration (22% of control). These results suggest that DES ia a potent effective promoter for tumorigenesis of radiation-initiated mammary cells, but CMA is not. DES may act directly on the irradiated mammary cells by binding to ER, and indirectly by stimulating prolactin secretion from the pituitary glands.

Rer1 and calnexin regulate endoplasmic reticulum retention of a peripheral myelin protein 22 mutant that causes type 1A Charcot-Marie-Tooth disease.

Peripheral myelin protein 22 (PMP22) resides in the plasma membrane and is required for myelin formation in the peripheral nervous system. Many PMP22 mutants accumulate in excess in the endoplasmic reticulum (ER) and lead to the inherited neuropathies of Charcot-Marie-Tooth (CMT) disease. However, the mechanism through which PMP22 mutants accumulate in the ER is unknown. Here, we studied the quality control mechanisms for the PMP22 mutants L16P and G150D, which were originally identified in mice and patients with CMT. We found that the ER-localised ubiquitin ligase Hrd1/SYVN1 mediates ER-associated degradation (ERAD) of PMP22(L16P) and PMP22(G150D), and another ubiquitin ligase, gp78/AMFR, mediates ERAD of PMP22(G150D) as well. We also found that PMP22(L16P), but not PMP22(G150D), is partly released from the ER by loss of Rer1, which is a Golgi-localised sorting receptor for ER retrieval. Rer1 interacts with the wild-type and mutant forms of PMP22. Interestingly, release of PMP22(L16P) from the ER was more prominent with simultaneous knockdown of Rer1 and the ER-localised chaperone calnexin than with the knockdown of each gene. These results suggest that CMT disease-related PMP22(L16P) is trapped in the ER by calnexin-dependent ER retention and Rer1-mediated early Golgi retrieval systems and partly degraded by the Hrd1-mediated ERAD system.

Expression of Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 in Pancreatic β-Cells and Its Role in Promotion of Insulin Secretion and Protection against Diabetes

Insulin secretion by beta-cells of pancreatic islets is regulated by various soluble factors including glucose and hormones. The importance of direct cell-cell communication among beta-cells or between beta-cells and other cell types for such regulation has remained unclear, however. Transmembrane proteins Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) and its ligand CD47 interact through their extracellular regions and contribute to intercellular communication. We now show that both SHPS-1 and CD47 are prominently expressed in beta-cells of the pancreas. The plasma insulin level in the randomly fed state was markedly reduced in mice that express a mutant form of SHPS-1 lacking most of the cytoplasmic region compared with that in wild-type (WT) mice, although the blood glucose concentrations of the two types of mice were similar. This reduction in the plasma insulin level of SHPS-1 mutant mice was even more pronounced in animals maintained on a high-fat diet. Glucose tolerance was also markedly impaired in SHPS-1 mutant mice on a high-fat diet, whereas both peripheral insulin sensitivity and the insulin content of the pancreas in the mutant animals were similar to those of WT mice. Glucose-stimulated insulin secretion was similar for islets isolated from WT or SHPS-1 mutant mice. The impaired glucose tolerance of SHPS-1 mutant mice was ameliorated by treatment with the alpha2-adrenergic antagonist yohimbine. These results suggest that SHPS-1 promotes insulin secretion from beta-cells and thereby protects against diabetes. Preventing of alpha2-adrenergic receptor-mediated inhibition of insulin secretion may partly participate in such a function of SHPS-1.

DIF-1, an anti-tumor substance found in Dictyostelium discoideum, inhibits progesterone-induced oocyte maturation in Xenopus laevis

Differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) is a putative morphogen that induces stalk-cell formation in the cellular slime mold Dictyostelium discoideum. DIF-1 has previously been shown to suppress cell growth in mammalian cells. In this study, we examined the effects of DIF-1 on the progesterone-induced germinal vesicle breakdown in Xenopus laevis, which is thought to be mediated by a decrease in intracellular cAMP and the subsequent activation of mitogen-activated protein kinase (MAPK) and maturation-promoting factor, a complex of cdc2 and cyclin B, which regulates germinal vesicle breakdown. DIF-1 at 10-40 microM inhibited progesterone-induced germinal vesicle breakdown in de-folliculated oocytes in a dose-dependent manner. Progesterone-induced cdc2 activation, MAPK activation, and c-Mos accumulation were inhibited by DIF-1. Furthermore, DIF-1 was found to inhibit the progesterone-induced cAMP decrease in the oocytes. These results indicate that DIF-1 inhibits progesterone-induced germinal vesicle breakdown possibly by blocking the progesterone-induced decrease in [cAMP](i) and the subsequent events in Xenopus oocytes.