Biomedical subjects
S Nagata
Publications and source records attributed to S Nagata.
Dual control of neurite outgrowth by STAT3 and MAP kinase in PC12 cells stimulated with interleukin-6.
IL-6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). We explored the signals required for neurite outgrowth of PC12 cells by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte-colony stimulating factor (G-CSF) receptor and the cytoplasmic domain of gp130, a signal-transducing subunit of the IL-6 receptor. The mutants incapable of activating the MAP kinase cascade failed to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibited neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolished the ability to activate STAT3 did not inhibit, but rather stimulated neurite outgrowth. This mutant did not require NGF pretreatment for neurite outgrowth. Dominant-negative STAT3s mimicked NGF pretreatment, and NGF suppressed the IL-6-induced activation of STAT3, supporting the idea that STAT3 might regulate the differentiation of PC12 cells negatively. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF.
Enhancement of guanine-nucleotide exchange activity of C3G for Rap1 by the expression of Crk, CrkL, and Grb2.
Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.
Requirement of Fas for the development of autoimmune diabetes in nonobese diabetic mice.
Insulin-dependent diabetes mellitus (IDDM) is assumed to be a T cell-mediated autoimmune disease. To investigate the role of Fas-mediated cytotoxicity in pancreatic beta cell destruction, we established nonobese diabetic (NOD)-lymphoproliferation (lpr)/lpr mice lacking Fas. Out of three genotypes, female NOD-+/+ and NOD-+/lpr developed spontaneous diabetes by the age of 10 mo with the incidence of 68 and 62%, respectively. In contrast, NOD-lpr/lpr did not develop diabetes or insulitis. To further explore the role of Fas, adoptive transfer experiments were performed. When splenocytes were transferred from diabetic NOD, male NOD-+/+ and NOD-+/lpr developed diabetes with the incidence of 89 and 83%, respectively, whereas NOD-lpr/lpr did not show glycosuria by 12 wk after transfer. Severe mononuclear cell infiltration was revealed in islets of NOD-+/+ and NOD-+/lpr, whereas islet morphology remained intact in NOD-lpr/lpr. These results suggest that Fas-mediated cytotoxicity is required to initiate beta cell autoimmunity in NOD mice. Fas-Fas ligand system might be critical for autoimmune beta cell destruction leading to IDDM.
Jak1 plays an essential role for receptor phosphorylation and Stat activation in response to granulocyte colony-stimulating factor.
The proliferation and differentiation of neutrophils is regulated by granulocyte-specific colony-stimulating factor (G-CSF). G-CSF uses a receptor of the cytokine receptor superfamily and, in common with all members of the family, induces the tyrosine phosphorylation and activation of members of the Janus protein tyrosine kinase (Jak) family. In both myeloid cells and a human fibrosarcoma cell line expressing the G-CSF receptor, G-CSF induces the tyrosine phosphorylation and activation of Jak1, Jak2, and Tyk2. In addition, G-CSF induces the tyrosine phosphorylation of the receptor and members of the signal transducers and activators of transcription (Stat) family, including Stat3, as well as Stat1 and Stat5, depending on the cells involved. Using mutant cell lines lacking various Jaks, we show here that Jak1 is critical for G-CSF-mediated Stat activation, whereas Jak2 or Tyk2 are either not required or play redundant or ancillary roles. In the absence of Jak1, G-CSF induces activation of Jak2 and Tyk2, but fails to induce receptor tyrosine phosphorylation and induces dramatically reduced levels of Stat activation. A kinase-inactive Jak2, when overexpressed in cells lacking endogenous Jak2, can suppress Jak1 activation, receptor phosphorylation, and Stat activation, suggesting competition in the receptor complex either for Jak1 binding or substrates. Because the requirement for Jak1 is very similar to that previously shown for interleukin-6 signaling, the data support the concept that the G-CSF receptor and gp130 are both structurally and functionally similar.
Elevated serum level of Fas ligand correlates with the asymptomatic stage of human immunodeficiency virus infection.
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Evasion of cytotoxic T lymphocyte (CTL) responses by nef-dependent induction of Fas ligand (CD95L) expression on simian immunodeficiency virus-infected cells.
Inoculation of macaques with live attenuated SIV strains has been shown to protect against subsequent challenge with wild-type SIV. The protective mechanism(s) remain obscure. To study the effect in more detail, we have investigated the role of virus-specific CTL responses in macaques infected with an attenuated SIV strain (pC8), which has a four-amino acid deletion in the nef gene, as compared with the wild-type SIVmac32H clone (pJ5). Cynomolgus macaques infected with pC8 were protected against subsequent challenge with pJ5 and did not develop any AIDS-like symptoms in the 12 months after infection. The pC8-induced protection was associated with high levels of virus-specific CTL responses to a variety of viral antigens. In contrast, pJ5-infected macaques had little, if any, detectable CTL response to the viral proteins after three months. The latter group of macaques also showed increased Fas expression and apoptotic cell death in both the CD4(+) and CD8(+) populations. In vitro, pJ5 but not pC8 leads to an increase in FasL expression on infected cells. Thus the expression of FasL may protect infected cells from CTL attack, killing viral-specific CTLs in the process, and providing a route for escaping the immune response, leading to the increased pathogenicity of pJ5. pC8, on the other hand does not induce FasL expression, allowing the development of a protective CTL response. Furthermore, interruption of the Fas-FasL interaction allows the regeneration of viral-specific CTL responses in pJ5-infected animals. This observation suggests an additional therapeutic approach to the treatment of AIDS.
Expression of a constitutively active phosphatidylinositol 3-kinase induces process formation in rat PC12 cells. Use of Cre/loxP recombination system.
It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogen-activated protein kinase or protein kinase B/Rac protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.
Decreased level of light-induced Fos expression in the suprachiasmatic nucleus of diabetic rats.
We assessed light-induced Fos-immunoreactive cells in the suprachiasmatic nucleus of diabetic rats. The number of Fos-immunoreactive cells significantly decreased in diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats as compared with control Long-Evans Tokushima Otsuka (LETO) rats. In contrast there was no decrease in the number of Fos-immunoreactive cells in young OLETF rats which have not yet developed diabetes. Two months after the administration of streptozotocin (STZ) to Wistar rats, the number of Fos-immunoreactive cells significantly decreased, although 1 week after the administration of STZ, the number had not yet changed in these STZ-induced diabetic rats. These results suggest that chronic diabetic (hyperglycemic) conditions may affect the light entraining responses in the suprachiasmatic nucleus (SCN).
The GLY1 gene of Saccharomyces cerevisiae encodes a low-specific L-threonine aldolase that catalyzes cleavage of L-allo-threonine and L-threonine to glycine--expression of the gene in Escherichia coli and purification and characterization of the enzyme.
The GLY1 gene of Saccharomyces cerevisiae is required for the biosynthesis of glycine for cell growth [McNeil, J. B., McIntosh, E. V., Taylor, B. V., Zhang, F-R., Tang, S. & Bognar, A. L. (1994) J. Biol. Chem. 269, 9155-9165], but its gene product has not been identified. We have found that the GLY1 protein is similar in primary structure to L-allo-threonine aldolase of Aeromonas jandiae DK-39, which stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The GLY1 gene was amplified by PCR, with a designed ribosome-binding site, cloned into pUC118, and expressed in Escherichia coli cells. The enzyme was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of about 170 kDa and consists of four subunits identical in molecular mass. The enzyme contains 2 mol pyridoxal 5'-phosphate/4 mol of subunit as a cofactor, and its absorption spectrum exhibits maxima at 280 nm and 420 nm. The enzyme catalyzes the cleavage of not only L-allo-threonine to glycine but also L-threonine. We have termed the enzyme a low-specific L-threonine aldolase to distinguish it from L-allo-threonine aldolase.
A target of phosphatidylinositol 3,4,5-trisphosphate with a zinc finger motif similar to that of the ADP-ribosylation-factor GTPase-activating protein and two pleckstrin homology domains.
We have purified a protein that binds phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] using beads bearing a PtdIns(3,4,5)P3 analogue. This protein, with a molecular mass of 43 kDa, was termed PtdIns(3,4,5)P3-binding protein. The partial amino acid sequences were determined and a full-length cDNA encoding the protein was isolated from bovine brain cDNA library. The clone harbored an open reading frame of 373 amino acids which contained one zinc finger motif similar to that of ADP-ribosylation-factor GTPase-activating protein and two pleckstrin homology domains. The entire sequence was 83% similar to centaurin alpha, another PtdIns(3,4,5)P3-binding protein. The protein bound PtdIns(3,4,5)P3 with a higher affinity than it did inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3-phosphate suggesting that the binding to PtdIns(3,4,5)P3 was specific. The binding activity was weaker in the mutants with a point mutation in the conserved sequences in each pleckstrin homology domain. Introduction of both mutations abolished the activity. These results suggest that this new binding protein binds PtdIns(3,4,5)P3 through two pleckstrin domains present in the molecule.
Lack of chronic immune activation in HIV-infected chimpanzees correlates with the resistance of T cells to Fas/Apo-1 (CD95)-induced apoptosis and preservation of a T helper 1 phenotype.
Chimpanzees are one of the few species, along with humans, susceptible to persistent HIV-1 infection. However, HIV-infected chimpanzees do not exhibit the marked immune system alterations seen in humans and remain relatively resistant to AIDS. In humans, HIV infection leads to unresponsiveness of T cells in response to TCR stimulation, associated with increased T cell death by apoptosis. In an effort to understand some of the mechanisms used to limit lentivirus infection in African nonhuman primates, we compared apoptosis in infected humans vs chimpanzees in CD4 and CD8 T cells in relation with the expression of Bcl-2 and Fas molecules. The intensity of apoptosis in CD4 and CD8 T cells from infected chimpanzees was very low, was not inducible by several TCR-dependent activators, and was comparable to that detected in noninfected chimpanzees. Moreover, CD45RO+ and HLA-DR+ subsets, which were shown to exhibit ex vivo a high propensity to undergo apoptosis in infected humans, were not modified in infected chimpanzees. Interestingly, in contrast to the situation found in infected humans, Fas ligation by agonistic Abs or recombinant human Fas ligand on CD4 and CD8 T cells from infected chimpanzees did not induce apoptosis in these subsets even when Bcl-2 was down-regulated. Finally, this resistance to apoptosis was associated with the predominance of CD3 T cells with a Th1 phenotype. Together these observations argue for a strong relationship among the absence of chronic immune stimulation in HIV-1-infected chimpanzees, the normal control of lymphocyte survival, and the resistance to disease progression.
Lethal effect of recombinant human Fas ligand in mice pretreated with Propionibacterium acnes.
Fas ligand (FasL) is a type II membrane protein. Binding of FasL to its receptor, Fas, induces apoptosis. Matrix metalloproteinase cleaves the membrane-bound human FasL to yield the active soluble form. Here, we have produced a large amount of human soluble rFasL using the yeast, Pichia pastoris. The purified rFasL was found to be glycosylated and to exist as a trimer. The rFasL was effective in inducing apoptosis in a Fas-expressing T cell or a fibroblast cell line. The ID50 of rFasL for mouse Fas-expressing T cells was about 0.5 ng/ml. The killing process with rFasL was quick. That is, >80% Fas-expressing mouse cells were killed within 1 h by a saturation concentration of human rFasL. Intravenous administration of 500 microg of human rFasL had a lethal effect in mice. When the mice were pretreated with Propionibacterium acnes, the subsequent injection of 30 microg of human rFasL induced hepatic failure and killed the mice within 24 h. These results indicated that the soluble human FasL is active in inducing apoptosis in vitro and in vivo, and its deleterious effect may be strengthened in patients who are suffering from bacterial infection.
Molecular cloning and characterization of the chromosomal gene for human lactoperoxidase.
Lactoperoxidase (LPO) is an oxidoreductase secreted into milk, and plays an important role in protecting the lactating mammary gland and the intestinal tract of the newborn infants against pathogenic microorganisms. In this study, the human LPO chromosomal gene was molecularly cloned, and its gene organization was determined. The human LPO gene was found to be arranged with the myeloperoxidase (MPO) gene in a tail-to-tail manner. Similar to the human MPO and eosinophil peroxidase (EPO) genes, the human LPO gene is split by 11 introns and spans 28 kb. Unlike most introns in mammalian gene, the 5' splice donor sequence of intron 11 starts with GC instead of GT. When the minigene comprised of exon 11, intron 11 and exon 12 of the human LPO gene was introduced into COS cells, the correct splicing of the intron was found, suggesting the intron 11 of the human LPO gene is functional. The coding sequence of human LPO consists of 2136 bp, and codes for a protein of 712 amino acids. The amino acid sequence of human LPO has 51% similarity with those of both human MPO and EPO, suggesting that these peroxidase genes have evolved from a common ancestral gene. On the other hand, the nucleotide sequences of the 5' promoter regions of these peroxidase genes exhibit no similarity among them, which agrees with their tissue-specific expression.
Acute oral toxicity of microcystin-LR, a cyanobacterial hepatotoxin, in mice.
Microcystin-LR (MCLR) is a hepatotoxic peptide produced by Microcystis aeruginosa, an alga found worldwide in reservoirs for drinking supply; however, acute oral toxicity of purified MCLR remains unknown. Therefore, a single dose of MCLR (more than 95% purity) ranging from 8.0 to 20.0 mg/kg body weight was orally given to female 6-week old BALB/c mice, and lethality and pathological changes were observed. Median lethal dose (LD50) of the orally given MCLR estimated by the up and down method was 10.9 mg/kg, which was 167 times higher than the i.p. LD50 value (65.4 microgram/kg by moving average method). Orally administrated toxin caused primarily hepatocellular injuries with characteristics of hemorrhage and necrosis. In situ end-labeling as well as electron microscopic observation revealed an induction of apoptotic cell death to hepatocytes. These results indicate the lethality of MCLR was much lower in oral dosage than by i.p. administration, but toxic effects are similar. In addition, apoptosis is considered one of major components in MCLR-induced hepatotoxicity.
Fas involvement in human NK cell apoptosis: lack of a requirement for CD16-mediated events.
Propriocidal regulation of T cells refers to apoptosis induced by interleukin-2 (IL-2) activation with subsequent antigen receptor stimulation. We previously reported that natural killer (NK) cells also exhibit propriocidal death. Cell death can be induced following occupancy of the Fc gamma RIII (CD16) receptor when NK cells were pretreated with IL-2, IL-12, or IL-15. Here we show other triggering receptors on NK cells such as CD44, anti-NK-receptor antibodies, and pharmacological activation can result in the cell death signal. Requirement for cell interactions indicated that cell contact was required; however, unlike cell-mediated lysis, extracellular calcium was not required. Like T cells, the process of cell death for NK cells was receptor-induced apoptosis. Activation-induced apoptosis of T cells is mediated by members of the tumor necrosis factor (TNF) cytokine superfamily. We examined the involvement of TNF receptor family members or Fas in this rapid cell death. Antibody directed against Fas, TNFR60, TNFR80, LTBR, and LT alpha failed to inhibit receptor-induced death. Therefore, NK cells appear to demonstrate a rapid apoptotic episode when CD16 is cross-linked, but the mechanism of this apoptosis is quite different than was observed in T cells with CD3. The direct examination of the Fas pathway on activated NK cells revealed that susceptibility required longer treatment times and IL-2 activation. This susceptibility was paralleled by increased Fas-ligand expression. Therefore, NK cells can demonstrate an apoptotic response to CD16, CD44, NK receptors, and Fas. The enumeration of ligands capable of eliciting NK cell death and the in vivo relevance of this observation require further study.
Electron microscopic immunohistochemistry of ocular and extraocular pseudoexfoliative material.
To investigate the nature of extraocular pseudoexfoliative (PSX) material, and to validate the hypothesis that the PSX syndrome is a systemic disorder, we studied the immunoreactivity of intraocular and extraocular PSX material from patients with the PSX syndrome using antibodies against proteins of extracellular matrices. Surgical specimens of four different tissues were obtained from human eyes with the PSX syndrome: nine trabecular tissues, three cataractous lenses, six bulbar conjunctivas, and seven lid skins. These tissues were processed for electron microscopic immunohistochemistry, and stained with antibodies against vitronectin, fibronectin, laminin, and elastin, by an indirect immunogold procedure. Density of the gold particles located on the PSX material was estimated by video image analysis. Statistical analysis of the data revealed that the PSX material from trabecular tissues, lenses, bulbar conjunctivas, and lid skins had almost identical immunoreactivity to the antibodies studied. PSX materials of trabecular tissues, lenses, bulbar conjunctivas, and lid skins were found to have not only a similar ultrastructure, but also almost identical immunohistochemical characteristics. These results support the hypothesis that the PSX syndrome is a systemic disorder.