Kyoichi Kishi

Skeletal muscle gene expression in space-flown rats

Skeletal muscles are vulnerable to marked atrophy under microgravity. This phenomenon is due to the transcriptional alteration of skeletal muscle cells to weightlessness. To further investigate this issue at a subcellular level, we examined the expression of ~26,000 gastrocnemius muscle genes in space‐flown rats by DNA microarray analysis. Comparison of the changes in gene expression among spaceflight, tail‐suspended, and denervated rats revealed that such changes were unique after spaceflight and not just an extension of simulated weightlessness. The microarray data showed two spaceflight‐specific gene expression patterns: 1) imbalanced expression of mitochondrial genes with disturbed expression of cytoskeletal molecules, including putative mitochondria‐anchoring proteins, A‐kinase anchoring protein, and cytoplasmic dynein, and 2) up‐regulated expression of ubiquitin ligase genes, MuRF‐1, Cbl‐b, and Siah‐1A, which are rate‐limiting enzymes of muscle protein degradation. Distorted expression of cytoskeletal genes during spaceflight resulted in dislocation of the mitochondria in the cell. Several oxidative stress‐inducible genes were highly expressed in the muscle of spaceflight rats. We postulate that mitochondrial dislocation during spaceflight has deleterious effects on muscle fibers, leading to atrophy in the form of insufficient energy provision for construction and leakage of reactive oxygen species from the mitochondria.

Space shuttle flight (STS-90) enhances degradation of rat myosin heavy chain in association with activation of ubiquitin–proteasome pathway

To elucidate the mechanisms of microgravity‐induced muscle atrophy, we focused on fast‐type myosin heavy chain (MHC) degradation and expression of proteases in atrophied gastrocnemius muscles of neonatal rats exposed to 16‐d spaceflight (STS‐90). The spaceflight stimulated ubiquitination of proteins, including a MHC molecule, and accumulation of MHC degradation fragments in the muscles. Semiquantitative reverse transcriptase‐polymerase chain reaction revealed that the spaceflight significantly increased mRNA levels of cathepsin L, proteasome components (RC2 and RC9), polyubiquitin, and ubiquitin‐conjugating enzyme in the muscles, compared with those of ground control rats. The levels of μ‐calpain, m‐calpain, cathepsin B, and cathepsin H mRNAs were not changed by the spaceflight. We also found that tail‐suspension of rats for 10 d or longer caused the ubiquitination and degradation of MHC in gastrocnemius muscle, as was observed in the spaceflight rats. In the muscle of suspended rats, these changes were closely associated with activation of proteasome and up‐regulation of expression of mRNA for the proteasome components and polyubiquitin. Administration of a cysteine protease inhibitor, E‐64, to the suspended rats did not prevent the MHC degradation. Our results suggest that spaceflight induces the degradation of muscle contractile proteins, including MHC, possibly through a ubiquitin‐dependent proteolytic pathway.

Type I Helicobacter pylori Lipopolysaccharide Stimulates Toll-Like Receptor 4 and Activates Mitogen Oxidase 1 in Gastric Pit Cells

ABSTRACT Guinea pig gastric pit cells express an isozyme of gp91-phox, mitogen oxidase 1 (Mox1), and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phox). Helicobacter pylori lipopolysaccharide (LPS) andEscherichia coli LPS have been shown to function as potent activators for the Mox1 oxidase. These cells spontaneously secreted about 10 nmol of superoxide anion (O2−)/mg of protein/h under LPS-free conditions. They expressed the mRNA and protein of Toll-like receptor 4 (TLR4) but not those of TLR2. LPS from type I H. pylori at 2.1 endotoxin units/ml or higher stimulated TLR4-mediated phosphorylations of transforming growth factor β-activated kinase 1 and its binding protein 1 induced TLR4 and p67-phox and up-regulated O2−production 10-fold. In contrast, none of these events occurred withH. pylori LPS from complete or partial deletion mutants of the cag pathogenicity island. Lipid A was confirmed to be a bioactive component for the priming effects, while removal of bisphosphates from lipid A completely eliminated the effects, suggesting the importance of the phosphorylation pattern besides the acylation pattern for the bioactivity. H. pylori LPS is generally accepted as having low toxicity; however, our results suggest that type I H. pylori lipid A may be a potent stimulator for innate immune responses of gastric mucosa by stimulating the TLR4 cascade and Mox1 oxidase in pit cells.

Role of nicotinamide adenine dinucleotide phosphate oxidase 1 in oxidative burst response to toll-like receptor 5 signaling in large intestinal epithelial cells

The NADPH oxidase 1 (Nox1) is a gp91phox homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O2−) of 160 nmol/mg protein/h and expressed the Nox1, p22phox, p67phox, and Rac1 mRNAs, but not the gp91phox, Nox4, p47phox, p40phox, and Rac2 mRNAs. They also expressed novel homologues of p47phox and p67phox (p41nox and p51nox, respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22phox, p51nox, and Rac1 mRNAs, but not the other NADPH component mRNAs, and secreted only small amounts of O2− (<2 nmol/mg protein/h). Cotransfection of p41nox and p51nox cDNAs in T84 cells enhanced PMA-stimulated O2− release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O2− release in association with the induction of Nox1 protein. The enhanced O2− production by cotransfection of p41nox and p51nox vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-β-activated kinase 1 and TGF-β-activated kinase 1-binding protein 1. A potent inhibitor for NF-κB (pyrrolidine dithiocarbamate) significantly blocked the rFliC-primed increase in O2− production and induction of Nox1 protein. These results suggest that p41nox and p51nox are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense.

Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest.

In animal models, several ubiquitin ligases play an important role in skeletal muscle atrophy caused by unloading. In this study we examined protein ubiquitination and ubiquitin ligase gene expression in quadriceps femoris muscle from healthy volunteers after 20‐day bedrest to clarify ubiquitin‐dependent proteolysis in human muscles after unloading. During bedrest, thickness and cross‐sectional area of the quadriceps femoris muscle decreased significantly by 4.6% and 3.7%, respectively. Ubiquitinated proteins accumulated in these atrophied human muscles. A real‐time reverse transcription–polymerase chain reaction system showed that bedrest significantly upregulated expression of two ubiquitin ligase genes, Cbl‐b and atrogin‐1. We also performed DNA microarray analysis to examine comprehensive gene expression in the atrophied muscle. Bedrest mainly suppressed the expression of muscle genes associated with control of gene expression in skeletal muscle. Our results suggest that, in humans, Cbl‐b– or atrogin‐1–mediated ubiquitination plays an important role in unloading‐induced muscle atrophy, and that unloading stress may preferentially inhibit transcriptional responses in skeletal muscle. Muscle Nerve, 2006

Deficiency of Cbl-b gene enhances infiltration and activation of macrophages in adipose tissue and causes peripheral insulin resistance in mice.

OBJECTIVE—c-Cbl plays an important role in whole-body fuel homeostasis by regulating insulin action. In the present study, we examined the role of Cbl-b, another member of the Cbl family, in insulin action. RESEARCH DESIGN AND METHODS—C57BL/6 (Cbl-b+/+) or Cbl-b-deficient (Cbl-b−/−) mice were subjected to insulin and glucose tolerance tests and a hyperinsulinemic-euglycemic clamp test. Infiltration of macrophages into white adipose tissue (WAT) was assessed by immunohistochemistry and flow cytometry. We examined macrophage activation using co-cultures of 3T3-L1 adipocytes and peritoneal macrophages. RESULTS—Elderly Cbl-b−/− mice developed glucose intolerance and peripheral insulin resistance; serum insulin concentrations after a glucose challenge were always higher in elderly Cbl-b−/− mice than age-matched Cbl-b+/+ mice. Deficiency of the Cbl-b gene significantly decreased the uptake of 2-deoxyglucose into WAT and glucose infusion rate, whereas fatty liver was apparent in elderly Cbl-b−/− mice. Cbl-b deficiency was associated with infiltration of macrophages into the WAT and expression of cytokines, such as tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein (MCP)-1. Co-culture of Cbl-b−/− macrophages with 3T3-L1 adipocytes induced leptin expression and dephosphorylation of insulin receptor substrate 1, leading to impaired glucose uptake in adipocytes. Furthermore, Vav1, a key factor in macrophage activation, was highly phosphorylated in peritoneal Cbl-b−/− macrophages compared with Cbl-b+/+ macrophages. Treatment with a neutralizing anti–MCP-1 antibody improved peripheral insulin resistance and macrophage infiltration into WAT in elderly Cbl-b−/− mice. CONCLUSIONS—Cbl-b is a negative regulator of macrophage infiltration and activation, and macrophage activation by Cbl-b deficiency contributes to the peripheral insulin resistance and glucose intolerance via cytokines secreted from macrophages.

Cysteine supplementation prevents unweighting-induced ubiquitination in association with redox regulation in rat skeletal muscle

Abstract We have previously reported that spaceflight and tail suspension enhanced degradation of rat myosin heavy chain (MHC) in association with activation of a ubiquitindependent proteolytic pathway [Ikemoto et al., FASEB J. 15 (2001), 1279 1281]. To elucidate whether the ubiquitination is accompanied by oxidative stress, we measured markers for oxidative stress, such as thiobarbituric acidreactive substance (TBARS) and glutathione disulfide (GSSG), in gastrocnemius muscle of tailsuspended rats. Glutathione (GSH) concentration in the muscle significantly decreased from day 5 and reached a minimum value on day 10. Tail suspension reciprocally increased concentrations of TBARS and GSSG in parallel with enhancement of protein ubiquitination, suggesting that oxidative stress may play an important role in protein ubiquitination caused by tail suspension. To prevent ubiquitination associated with oxidative stress, we also administered an antioxidative nutrient, cysteine, to tailsuspended rats. Intragastric supplementation of 140 mg/rat of cysteine for 2 weeks or longer normalized the ratio of GSH to GSSG in the muscle and suppressed protein ubiquitination and MHC fragmentation, compared with supplementation of the equimolar amount of alanine. The cysteine supplementation significantly suppressed the loss of hindlimb muscle mass. Our results suggest that supplementation of antioxidative nutrients, such as cysteine, may be beneficial for preventing ubiquitination of muscle proteins caused by unweighting.

Osteoactivin fragments produced by ectodomain shedding induce MMP-3 expression via ERK pathway in mouse NIH-3T3 fibroblasts

Intact osteoactivin, a novel type I membrane glycoprotein, were shed at a dibasic motif in the juxtamembrane region in C2C12 myoblasts. Extracellular fragments were secreted into the culture media by a putative metalloprotease. Extracellular fragments of osteoactivin, but not control protein, induced matrix metalloprotease‐3 (MMP‐3) expression in NIH‐3T3 fibroblasts. Epidermal growth factor (ERK) kinase inhibitors inhibited the osteoactivin‐mediated MMP‐3 expression, whereas the extracellular fragment of osteoactivin activated ERK1/2 and p38 in the mitogen‐activated protein kinase pathway. Our results suggest that the extracellular fragments of osteoactivin produced by shedding act as a growth factor to induce MMP‐3 expression via the ERK pathway in fibroblasts.

Glutathione depletion impairs transcriptional activation of heat shock genes in primary cultures of guinea pig gastric mucosal cells.

When primary cultures of guinea pig gastric mucosal cells were exposed to heat (43 degree C), ethanol, hydrogen peroxide (H2O2), or diamide, heat shock proteins (HSP90, HSP70, HSP60, and HSC73) were rapidly synthesized. The extent of each HSP induction varied with the type of stress. Ethanol, H2O2, and diamide increased the syntheses of several other undefined proteins besides the HSPs. However, none of these proteins were induced by exposure to heat or the reagents, when intracellular glutathione was depleted to <10% of the control level by pretreatment with DL-buthionine-[S,R]-sulfoximine. Gel mobility shift assay using a synthetic oligonucleotide coding HSP70 heat shock element showed that glutathione depletion inhibited the heat- and the reagent-initiated activation of the heat shock factor 1 (HSF1) and did not promote the expression of HSP70 mRNA. Immunoblot analysis with antiserum against HSF1 demonstrated that the steady-state level of HSF1 was not changed in glutathione-depleted cells, but glutathione depletion inhibited the nuclear translocation of HSF1 after exposure to heat stress. These results suggest that intracellular glutathione may support early and important biochemical events in the acquisition by gastric mucosal cells of an adaptive response to irritants.

Geranylgeranylacetone stimulates mucin synthesis in cultured guinea pig gastric pit cells by inducing a neuronal nitric oxide synthase

Abstract: Nitric oxide (NO) has been considered to play an important role in the regulation of blood flow, mucosal integrity, and mucus production in the stomach. We investigated the stimulatory actions of epidermal growth factor (EGF) and a cytoprotective compound, geranylgeranylacetone (GGA), on mucin synthesis in guinea pig gastric pre-pit cells, maintained in a serum-free culture system. GGA increased [3H]glucosamine uptake and the accumulation of mucus granules positive for galactose oxidase-Schiff reaction in the cells. This stimulatory action of GGA was equivalent to that of EGF, but GGA did not stimulate the cell growth. Both EGF and GGA increased the release of NO degeneration products, NO2− and NO3−. The [3H]glucosamine uptake was completely inhibited by the non-selective NO synthase (NOS) inhibitors, NG-nitro-l-arginine and NG-monomethyl-l-arginine, and it was only partially inhibited by a more selective inhibitor for inducible NOS isoform (iNOS), aminoguanidine. Northern blotting with a cDNA probe for rat iNOS, and Western blotting with a polyclonal antibody against iNOS, demonstrated that GGA did not up-regulate the iNOS mRNA expression nor induce its protein. In contrast, GGA and EGF induced neuronal NOS, but not endothelial NOS, which was confirmed by immunoblot analyses with antibodies against these constitutive NOS isoforms. Thus, the present experiments suggests that GGA, as well as EGF, stimulates mucin synthesis at least in part through an NO-dependent pathway, leading to an increase in the integrity of the gastric mucosa.