Loss of the N-acetylgalactosamine side chain of the GPI-anchor impairs bone formation and brain functions and accelerates the prion disease pathology

Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrP^C, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)−galactose−sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrP^C GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases.     

Cytokine-independent Jak3 activation upon T cell receptor (TCR) stimulation through direct association of Jak3 and the TCR complex

Jak3 is responsible for growth signals by various cytokines such as interleukin (IL)-2, IL-4, and IL-7 through association with the common gamma chain (gammac) in lymphocytes. We found that T cells from Jak3-deficient mice exhibit impairment of not only cytokine signaling but also early activation signals and that Jak3 is phosphorylated upon T cell receptor (TCR) stimulation. TCR-mediated phosphorylation of Jak3 is independent of IL-2 receptor/gammac but is dependent on Lck and ZAP-70. Jak3 was found to be assembled with the TCR complex, particularly through direct association with CD3zeta via its JH4 region, which is a different region from that for gammac association. These results suggest that Jak3 plays a role not only in cell growth but also in T cell activation and represents cross-talk of a signaling molecule between TCR and growth signals.     

Site-specific phosphorylation of tau accompanied by activation of mitogen-activated protein kinase (MAPK) in brains of Niemann-Pick type C mice

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.     

Lecithinized Cu, Zn-superoxide dismutase limits the infarct size following ischemia-reperfusion injury in rat hearts in vivo

Ghrelin is a novel gut-brain peptide that binds to the growth hormone secretagogue receptor (GHS-R), thereby functioning in the regulation of growth hormone (GH) release and food intake. Ghrelin-producing cells are most abundant in the oxyntic glands of the stomach. The regulatory mechanism that governs the biosynthesis and secretion of ghrelin has not been clarified. We report that ghrelin mRNA expression in the gastric fundus was increased, but that ghrelin peptide content decreased after a 48-h fast. Both values returned to control levels after refeeding. The ghrelin plasma concentration in the gastric vein and systemic venous blood increased after 24- and 48-h fasts. Furthermore, des-octanoylated ghrelin and n-octanoylated ghrelin were found in rat stomach, with the ratio of des-octanoylated ghrelin to n-octanoylated ghrelin markedly increased after fasting. The ghrelin mRNA level in the stomach also increased after administration of insulin and leptin. Conversely, db/db mice, which are deficient in the leptin receptor, had lower ghrelin mRNA levels than control mice. These findings suggest that this novel gastrointestinal hormone plays a role in the regulation of energy balance.     

Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.     

Production of soluble granulocyte colony-stimulating factor receptors from myelomonocytic cells

It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b-CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b-CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.     

Inhibitory effect of a SALMFamide neuropeptide on secretion of gonad-stimulating substance from radial nerves in the starfish Asterina pectinifera

In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. The SALMFamide family is also known to an echinoderm neuropeptide. The present study examined effect of SALMFamide 1 (S1) on oocyte maturation of starfish Asterina pectinifera. Unlike GSS, S1 did not induce spawning in starfish ovary. In contrast, S1 was found to inhibit GSS secretion from radial nerves by treatment with high K+ concentration. Fifty percent inhibition was obtained by 0.1 mM S1. S1 did not have any effect on GSS- and 1-MeAde-induced oocyte maturation. Following incubation with a S1 antibody and subsequently with rhodamine-conjugated second antibody, neural networks were observed in ovaries. The networks were restricted mainly to their surface with little evidence of immunoreactivity inside the basement membranes. This indicates that neural networks are distributed in the ovarian wall. The result further suggests that S1 plays a role in oocyte maturation to regulate GSS secretion from the nervous system.