Kyoko Tomizawa

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.

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.

Presence of activating transcription factor 4 (ATF4) in the porcine anterior pituitary

The two-hybrid system that identifies protein-protein interactions in a yeast expression system was used to investigate porcine anterior pituitary transcription factors. Four cDNA clones of a protein interacting with the leucine zipper domain of porcine cJun were obtained. Their nucleotide sequences revealed that they encode activating transcription factor 4 (ATF4). A full-length cDNA of porcine ATF4 was obtained by the polymerase chain reaction, and its deduced amino acid sequence showed 88 and 83% identity to human and mouse ATF4, respectively. Reverse transcription-polymerase chain reaction analysis of mRNAs prepared from 11 porcine tissues demonstrated that ATF4 is ubiquitous. Immunohistochemistry showed that ATF4 is present in the hormone producing cells of the anterior pituitary, but absent in some cells of the anterior pituitary. Further binding analysis revealed that ATF4 also interacts with itself and cFos. This evidence of ATF4 homodimerization, as well as heterodimerization with cJun and cFos in the anterior pituitary suggests a novel mechanism for the regulation of gene expression in this tissue.

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.

Multiple binding sites for nuclear proteins of the anterior pituitary are located in the 5′-flanking region of the porcine follicle-stimulating hormone (FSH) β-subunit gene

Gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), are synthesized specifically in the gonadotropes of the anterior pituitary. The aim of this study was to investigate nuclear factors that bind specifically to the porcine FSH beta-subunit gene. We examined nuclear protein binding to 2.75 kilobase pairs (kbp) of DNA adjacent to the porcine FSH beta-subunit gene: about 2.32 kbp of upstream DNA and 0.43 kbp of downstream DNA. The upstream region contains only TATA box, CACCC element, and some imperfect sequences of cAMP-responsive element, activator protein-1 binding site, and activator protein-2 binding site. Gel mobility shift assay using nuclear proteins extracted from the porcine anterior pituitary revealed that the proteins bound to a limited region of DNA, 107 bp long (designated as Fd2), located about -800 bp upstream from the transcription initiation site. Competitive binding assays demonstrated that the protein binding was sequence specific; the addition of excess amounts of several putative regulatory sequences and plasmid (non-homologous) DNA fragments did not reduce the binding. Furthermore, all five subfragments of Fd2 were also bound by the pituitary nuclear proteins, showing that the entire region of Fd2 is involved in this interaction. Southwestern blotting demonstrated that at least seven protein species of 110, 98, 78, 63, 52, 42, and 35 kDa recognize Fd2. Nuclear proteins from several other porcine tissues were also able to bind to the Fd2 fragment but the gel shift patterns were different and the bindings were weak, although only the cerebellum showed a pattern of binding that was similar to that of the anterior pituitary. These data suggest that multiple proteins of the anterior pituitary recognize a specific region of the porcine FSH beta-subunit gene.

Ovarian development induced in decapitated female Culex pipiens pallens mosquitoes by infusion of physiological quantities of 20-hydroxyecdysone together with amino acids.

Infusion of 20-hydroxyecdysone into the hemocoel of unfed decapitated female Culex pipiens pallens mosquitoes, at a very low rate of 500-2000pg per day, often stimulated oögenesis of this species, when the hormone was infused together with amino acids. The hormone alone or amino acids alone showed no such stimulatory effect. Previous reports that using an abdomen ligated immediately after a blood meal for hormone injection reduced the quantity of 20-hydroxyecdysone needed to activate unfed female Aedes aegypti by a few thousand times, are therefore due mainly to a sufficient supply of amino acids from the midgut in the isolated abdomen.

Baculovirus-insect cell production of bioactive porcine FSH

The in vitro and in vivo bioactivity of recombinant porcine FSH (rpFSH) produced from insect cells through use of a baculovirus expression system were studied and compared with those of natural FSH preparations. Determination of in vitro bioactivity, using the rat Sertoli cell aromatase bioassay, indicated that rpFSH is as active as purified pituitary FSH. Determination of in vivo bioactivity, using the mouse uterine weight bioassay, indicated that rpFSH is as active as purified pituitary FSH. Using the mouse Leydig cell testosterone bioassay, it was demonstrated that the intrinsic LH bioactivity of rpFSH is negligible. The increases in ovarian and uterine weight, and the stimulation in follicular growth in immature hypophysectomized rats induced by rpFSH supplemented with hCG were comparable to those induced by natural FSH preparations. Furthermore, rpFSH alone in hypophysectomized mice stimulated preantral follicular growth to preovulatory stages, and the subsequent injection of hCG caused ovulation. These results demonstrate that in vitro and in vivo biological characteristics of rpFSH produced from baculovirus-insect cells are indistinguishable from those of FSH isolated from natural sources.

Prominent Expression of Spinocerebellar Ataxia Type-1 (SCA1) Gene Encoding Ataxin-1 in LH-Producing Cells, LβT2

The LH-producing cell line, LbetaT2, and non LH-producing cell line, alphaT3-1 cells, established from a pituitary tumor, were employed for cDNA subtraction cloning to identify genes with expression unique to LH producing cells. Several cDNAs that code for known as well as for many unidentified clones were discovered. Most clones were the spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1, the abnormality of which causes neurodegeneration and loss of cerebellar Purkinje cells. We examined whether the expression of SCA1 gene in LbetaT2 cells is related to hormone production. We also compared the expression of SCA1 with that in various other pituitary tumor derived cell lines, and confirmed the prominent expression of SCA1 in LbetaT2 cells. The effect of gonadal factor(s) for SCA1 gene expression was examined. The expression level in female rats was low and did not change during the estrus cycle, but increased significantly after ovariectomy and did not return to the normal level under low and high doses of estrogen. In the male pituitary SCA1 gene expression increased markedly after castration and was not decreased by estrogen or testosterone. The Ontogeny of SCA1 gene expression was investigated in porcine fetal and postnatal pituitaries and revealed biphasic and sexually dimorphic expression. Transient expression of SCA1 gene was observed at fetal day 50 and 65 in males and day 40 in females, followed by a decline and increased expression before birth in both genders. Thus the expression of SCA1 gene is prominent in LH-producing cells and is not under direct control of gonadal factor(s) in both genders. In addition to the variable expression of SCA1 gene during the fetus period, the present results provide a novel aspect to the understanding of Boucher-Neuhauser syndrome (Ataxia Hypogonadism Choroidal Dystrophy).

Recombinant porcine follicle stimulating hormone produced in baculovirus-insect cells induces rat ovulation in vivo and gene expression of tissue plasminogen activator in vitro.

Superovulatory responses in cattle are known to be highly variable. In the present study, a recombinant porcine follicle stimulating hormone (rpFSH) produced in baculovirus-insect cells was utilised to evaluate the role of this recombinant FSH in control of the ovulatory process. Immature hypophysectomised rats were implanted with oestrogen pellet (10 mg diethylstilbestrol) and then primed with pregnant mare serum gonadotropin (PMSG, 17.5 IU, sc). Fifty-two hours later, 100 microg rpFSH or saline was injected (sc) to induce ovulation. All rats that received rpFSH ovulated with about eight ova rat(-1), whereas none of the control animals did. Ovulation induced by rpFSH was associated with an increase in the ovarian activity and message levels of tissue-type plasminogen activator (tPA), a protease important in the preovulatory degradation of the follicle wall. Furthermore, addition of rpFSH to the cultured rat granulosa cells resulted in a significant increase in tPA enzyme activity. These results demonstrate that rpFSH produced in baculovirus-insect cells has biological potency in ovulation as well as gene expression of tPA, providing a large advantage of this massive expression system in the reproduction of domestic animals.

Increased Thermo-Stability of Rat Prolactin after Replacing Glutamic Acid at Position 118 by Lysine

Abstract We examined the structural stabilities after heat treatment of 22 mutants of rat prolactin (rPRL) with amino acid replacements at 15 different positions and recombinant wild-type rPRL (WT-PRL) as part of our series of studies on site-directed mutagenesis of rPRL. When WT-PRL at low concentrations (0.1 ∼ 10 ng/ ml) was heated at 100°C for 20 min, it lost its Nb2 proliferation activity, whereas at high concentrations (above 1 fig/ml), its activity remained. Temperature-dependent loss of the proliferation activity of 10 ng/ml WT-PRL after heat treatment for 5 min was observed. Next, we examined the proliferation activities of the 22 mutants heated at 60 and 70°C for 5 min. After treatment at 60°C, all the mutants retained their initial proliferation activities, whereas treatment at 70°C reduced their activities to about 63%, except for one in which glutamic acid at position 118 was replaced by lysine (E118K), suggesting that the mutations did not induce structural instability. The mutant E118K retained 84% of its initial activity after treatment at 70°C, significantly (P < 0.01) higher than the WT-PRL value. The temperature-dependency profile of the Nb2 proliferation activity of E118K also showed it had significantly increased thermo-stability. Meanwhile another mutant (E118Q) at the same residue showed no increased thermo-stability, suggesting that changing a negative charge (E) to a positive one (K) at position 118 induces ionic bond formation with a neighboring negative charge, resulting in thermostabilization of the structure of PRL.