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1.
J Biol Chem ; 289(8): 4989-99, 2014 Feb 21.
Article in English | MEDLINE | ID: mdl-24375405

ABSTRACT

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein as well as a classic glycolytic enzyme, and its pleiotropic functions are achieved by various post-translational modifications and the resulting translocations to intracellular compartments. In the present study, GAPDH in the plasma membrane of BeWo choriocarcinoma cells displayed a striking acidic shift in two-dimensional electrophoresis after cell-cell fusion induction by forskolin. This post-translational modification was deamidation of multiple glutaminyl residues, as determined by molecular mass measurement and tandem mass spectrometry of acidic GAPDH isoforms. Transglutaminase (TG) inhibitors prevented this acidic shift and reduced cell fusion. Knockdown of the TG2 gene by short hairpin RNA reproduced these effects of TG inhibitors. Various GAPDH mutants with replacement of different numbers (one to seven) of Gln by Glu were expressed in BeWo cells. These deamidated mutants reversed the suppressive effect of wild-type GAPDH overexpression on cell fusion. Interestingly, the mutants accumulated in the plasma membrane, and this accumulation was increased according to the number of Gln/Glu substitutions. Considering that GAPDH binds F-actin via an electrostatic interaction and that the cytoskeleton is rearranged in trophoblastic cell fusion, TG2-dependent GAPDH deamidation was suggested to participate in actin cytoskeletal remodeling.


Subject(s)
Amides/metabolism , GTP-Binding Proteins/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Transglutaminases/metabolism , Trophoblasts/cytology , Trophoblasts/enzymology , Amino Acid Sequence , Cell Fusion , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , GTP-Binding Proteins/antagonists & inhibitors , Gene Knockdown Techniques , Giant Cells/cytology , Giant Cells/drug effects , Giant Cells/metabolism , Glutamic Acid/metabolism , Glutamine/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/isolation & purification , Humans , Molecular Sequence Data , Mutant Proteins/metabolism , Protein Glutamine gamma Glutamyltransferase 2 , Protein Processing, Post-Translational/drug effects , Transglutaminases/antagonists & inhibitors , Trophoblasts/drug effects
2.
Rheumatol Int ; 31(7): 967-9, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21113810

ABSTRACT

Therapeutics targeting sphingosine-1-phosphate (S1P), a kind of lipid mediator regulating immune cell trafficking, has been emerging rapidly as a novel line of regimen for autoimmune diseases, including rheumatoid arthritis (RA). Here, we propose that S1P-targeted therapy is beneficial not only for limiting inflammation but for preventing bone-resorptive disorders, such as osteoporosis, by controlling the migratory behavior of osteoclast precursors and therefore would be good for treating elderly female RA patients who suffer from postmenopausal osteoporosis and arthritis simultaneously.


Subject(s)
Arthritis, Rheumatoid/drug therapy , Lysophospholipids/agonists , Osteoclasts/drug effects , Osteoporosis, Postmenopausal/drug therapy , Receptors, Lysosphingolipid/agonists , Sphingosine/analogs & derivatives , Age Factors , Aged , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Arthritis, Rheumatoid/complications , Female , Humans , Lysophospholipids/physiology , Mice , Osteoclasts/physiology , Osteoporosis, Postmenopausal/immunology , Receptors, Lysosphingolipid/physiology , Sphingosine/agonists , Sphingosine/physiology , Treatment Outcome
3.
Rheumatol Int ; 28(3): 225-31, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17661048

ABSTRACT

Tetraspanin CD9 has been shown to be critically involved in multinucleation and cell fusion during osteoclastogenesis, however, its in vivo pathophysiological role in bone-resorbing disorders such as osteoporosis and rheumatoid arthritis, has not been elucidated. To investigate the involvement of tetraspanin CD9 in bone destruction in such diseases, we examined the expression and distribution of tetraspanin CD9 using murine experimental models of osteoporosis and arthritis. In results, CD9 protein is abundantly expressed on the activated osteoclasts in the bone tissues whose trabeculae are severely reduced in ovariectomy-induced osteoporosis. The expression of CD9 is also detected at the sites of bone erosion in arthritic lesions of collagen-induced arthritis (CIA), where tartate-resistant acid phosphatase (TRAP) staining-positive activated osteoclasts are present. These data suggest that tetraspanin CD9 play important roles in bone destructions in osteoporosis and arthritis, and therefore, functional alterations of tetraspanin CD9 may have therapeutic potential in such bone-resorptive disorders.


Subject(s)
Antigens, CD/metabolism , Arthritis, Experimental , Bone and Bones/pathology , Membrane Glycoproteins/metabolism , Osteoclasts/metabolism , Osteoporosis/etiology , Animals , Antigens, CD/genetics , Arthritis, Experimental/genetics , Arthritis, Experimental/pathology , Female , Gene Expression , Immunohistochemistry , Male , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Osteoporosis/physiopathology , Ovariectomy , Severity of Illness Index , Tetraspanin 29
4.
Allergol Int ; 56(4): 457-63, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17965585

ABSTRACT

BACKGROUND: Osteoclasts are bone-resorbing multinuclear polykaryons essential for bone remodeling, formed through cell fusion of mononuclear macrophage/monocyte lineage precursor cells upon stimulation by the RANK/RANKL system. Recent studies have revealed that a family of tetraspanin proteins, such as CD9, is critically involved in the cell fusion/polykaryon formation of these cell types. Until now, however, there is limited knowledge about the types of tetraspanins expressed in osteoclasts and their precursors. METHODS: The expression of different tetraspanin proteins in a monocyte/macrophage-lineage osteoclast precursor cell line, RAW264.7, was cyclopedically investigated using RT-PCR with specific primers and quantitative real-time RT-PCR. The function of two kinds of tetraspanins, Tspan-5 and NET-6, whose expression pattern was altered by RANKL stimulation, was examined by transfecting gene-specific short-interfering RNAs into these cell types. RESULTS: Of the 17 tetraspanins in mammalian hematopoietic cells, RAW264.7 cells express mRNA for 12 different kinds of tetraspanins, namely, CD9, CD37, CD53, CD63, CD81, CD82, CD151, NAG-2, NET-6, SAS, Tspan-3, and Tspan-5. Interestingly, during their maturation into osteoclasts upon RANKL stimulation, the transcript for Tspan-5 is up-regulated, whereas that for NET-6 is down-regulated. Targeted inhibition of Tspan-5 by using gene-specific RNA interference suppressed RANKL-induced cell fusion during osteoclastogenesis, whereas inhibition of NET-6 augmented the osteoclastogenesis itself. These results suggest that Tspan-5 and NET-6 have a reciprocal function during osteoclastogenesis, i.e., positive and negative regulation by Tspan-5 and NET-6, respectively. RANKL regulates osteoclastogenesis by altering the balances of these tetraspanin proteins. CONCLUSIONS: These data indicate that a diversity of tetraspanins is expressed in osteoclast precursors, and that cell fusion during osteoclastogenesis is regulated by cooperation of distinct tetraspanin family proteins such as Tspan-5 and NET-6. This study indicates that functional alterations of tetraspanin family proteins may have therapeutic potential in diseases where osteoclasts play a major role, such as rheumatoid arthritis and osteoporosis.


Subject(s)
Cell Differentiation/physiology , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Osteoclasts/cytology , Osteoclasts/metabolism , Animals , Cell Communication/genetics , Cell Line , Down-Regulation/genetics , Gene Expression Profiling , Membrane Proteins/physiology , Mice , Multigene Family , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/cytology , Stem Cells/metabolism , Tetraspanins , Up-Regulation/genetics
5.
J Bone Miner Res ; 22(10): 1612-20, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17576169

ABSTRACT

UNLABELLED: We showed that RGS18, a myeloid lineage-specific RGS protein that is inhibited after activation of the RANK/RANKL system, is a negative regulator of osteoclastogenesis. RGS18 acts through an external acidosis-sensing osteoclastogenic mechanism through the OGR1/NFAT pathway. INTRODUCTION: Osteoclasts are bone-resorbing multinuclear giant cells that are differentiated from mononuclear macrophage/monocyte lineage precursors stimulated by the RANK/RANKL system. The regulators of G-protein signaling (RGS) family is a diverse group of proteins that accelerate intrinsic GTP hydrolysis on heterotrimeric G-protein alpha subunits and play crucial roles in physiological regulation of G-protein-mediated cell signaling in various tissues and organs. We examined the expression and function of RGS18, a myeloid lineage-specific RGS protein, during osteoclastogenesis. MATERIALS AND METHODS: A macrophage/monocyte lineage cell line, RAW264.7, and primary osteoclast precursor monocytes derived from mouse bone marrow cultured with macrophage-colony stimulating factor (M-CSF) (bone marrow-derived monocytes [BMMs]) were used in this study. Both cell types differentiate into osteoclast-like cells on activation by RANKL. Expression of different RGS proteins, including RGS18, was assessed by gene-specific RT-PCR. The subcellular distribution of RGS18 on native osteoclasts in bone tissues, as well as in RAW264.7 cells, was examined by immunohistochemistry using a specific polyclonal antibody. Short interfering RNA against RGS18 was used to inhibit the function endogenous RGS18 in these cell types. Activation of NFATc1, an osteoclastogenic transcription factor, on external acidosis was assessed by visualizing the nuclear localization of NFATc1 visualized with anti-NFATc1 antibody. RESULTS: RAW264.7 and BMM cells both expressed mRNA for 10 different mammalian RGS proteins, including RGS18. Expression of RGS18 is significantly inhibited by RANKL both cell types, and inhibition of RGS18 function using RNA interference prominently enhanced osteoclastogenesis on stimulation with RANKL. The effect of RGS18 inhibition was reversed by blocking of proton-sensing OGR1 signaling, and overexpression of exogenous RGS18 inhibited extracellular acidosis-mediated NFATc1 activation. Immunohistochemical studies of mouse bone tissues revealed expression of RGS18 in osteoclasts in vivo. CONCLUSIONS: RGS18 acts as a negative regulator of the acidosis-induced osteoclastogenic OGR1/NFAT signaling pathway, and RANKL stimulates osteoclastogenesis by inhibiting expression of RGS18. Therefore, the results suggest a novel control mechanism of osteoclastogenesis by RGS proteins.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , NFATC Transcription Factors/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , Osteogenesis , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Acids/pharmacology , Animals , Bone and Bones/metabolism , Cell Nucleus/metabolism , Cells, Cultured , GTP-Binding Proteins/genetics , Gene Expression Regulation , Hydrogen-Ion Concentration , Intracellular Signaling Peptides and Proteins/genetics , Mice , Osteoclasts/drug effects , Osteogenesis/drug effects , Protein Subunits/genetics , RANK Ligand/metabolism , RGS Proteins , RNA, Small Interfering/genetics , Signal Transduction/drug effects
6.
J Bone Miner Res ; 21(6): 965-76, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16753027

ABSTRACT

UNLABELLED: We showed that CD9, a member of tetraspanin superfamily proteins, is expressed in a specific membrane microdomain, called "lipid raft," and is crucial for cell fusion during osteoclastogenesis after activation of the RANK/RANKL system. INTRODUCTION: Osteoclasts are bone-resorbing multinuclear polykaryons that are essential for bone remodeling and are formed through cell fusion of mononuclear macrophage/monocyte lineage precursors. Although osteoclastogenesis has been shown to be critically regulated by the RANK/RANKL system, the mechanism how precursor cells fuse with each other remains unclear. We examined the function of CD9, a member of tetraspanin superfamily, which has previously been shown to form macromolecular membrane microdomains and to regulate cell-cell fusion in various cell types. MATERIALS AND METHODS: We used RAW264.7, a macrophage/monocyte lineage cell line, which can differentiate into osteoclast-like polykaryons on the application of RANKL. Expression and distribution of CD9 was assessed by Western blotting, fluorescence-assorted cell sorting (FACS) and immunohistochemistry with light and electron microscopy. A specific neutralizing antibody and RNA interference were used to inhibit the function of CD9, and green fluorescent protein (GFP)-CD9 was exogenously expressed to enhance the effect of CD9. The distribution of CD9 in lipid microdomain was examined by biochemical (sucrose density gradient) isolation and imaging technique. RESULTS: CD9 is expressed on cell surfaces of RAW264.7, which is enhanced by RANKL. Targeted inhibition of CD9 decreases the number of osteoclast-like cells. On the other hand, overexpression of CD9 promotes spontaneous cell fusion even in the absence of RANKL. CD9 is localized in detergent-insoluble "lipid raft" microdomain in RANKL stimulation, and disruption of lipid rafts markedly reduces the formation of osteoclast-like polykaryons. Immunohistochemical studies of bone tissues revealed the expression of CD9 in osteoclasts in vivo. CONCLUSIONS: These data suggest that function of tetraspanin CD9 and its expression in lipid rafts are crucial for cell fusion during osteoclastogenesis.


Subject(s)
Antigens, CD/metabolism , Carrier Proteins/metabolism , Membrane Glycoproteins/metabolism , Membrane Microdomains/metabolism , Osteoclasts/metabolism , Animals , Antigens, CD/drug effects , Antigens, CD/ultrastructure , Bone and Bones/cytology , Bone and Bones/metabolism , Carrier Proteins/genetics , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Cell Fusion , Cell Line, Tumor , Cells, Cultured , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/genetics , Membrane Glycoproteins/pharmacology , Membrane Glycoproteins/ultrastructure , Membrane Microdomains/chemistry , Mice , Osteoclasts/cytology , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Tetraspanin 29 , Up-Regulation
7.
Water Res ; 39(19): 4693-704, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16257033

ABSTRACT

We conducted a comparison of the characteristics of dissolved organic matter (DOM) taken from the bio-toilet and other sources. A characterisation of DOM was carried out to assess the stability of the compost generated during the thermophilic and aerobic biodegradation of faeces. In addition, levels of soluble microbial products generated in the bio-toilet composting reactor were compared with those taken from other sources. The results showed that (i) the main component of DOM from the bio-toilet are solutes with molecular weight (MW)>30,000 Da (40%), whereas micromolecules (MW< 1000 Da) constituted more than 60% of the DOM from other solid samples, while liquid samples reached even more than 90%; (ii) the DOM stabilisation level in the composting reactor of the bio-toilet system was greater than that shown by DOM from other sources; (iii) stabilisation of DOM in the bio-toilet system was characterised by an increasing amount of macromolecules (MW>30,000 Da) after a decreasing trend was observed in the early stages of the biodegradation process; and (iv) net production of lipopolysaccharide (LPS) in wastewater treatment plants is greater than in the bio-toilet.


Subject(s)
Bacteria, Aerobic/metabolism , Conservation of Natural Resources , Feces/microbiology , Organic Chemicals/metabolism , Refuse Disposal/methods , Biodegradation, Environmental , Molecular Weight , Time Factors
8.
J Biol Chem ; 279(42): 44065-73, 2004 Oct 15.
Article in English | MEDLINE | ID: mdl-15310750

ABSTRACT

The inwardly rectifying K+ channel subunit Kir5.1 is expressed abundantly in the brain, but its precise distribution and function are still largely unknown. Because Kir5.1 is co-expressed with Kir4.1 in retinal glial Muller cells, we have compared the biochemical and immunological properties of Kir5.1 and Kir4.1 in the mouse brain. Immunoprecipitation experiments suggested that brain expressed at least two subsets of Kir channels, heteromeric Kir4.1/5.1 and homomeric Kir4.1. Immunolabeling using specific antibodies showed that channels comprising Kir4.1 and Kir5.1 subunits were assembled in a region-specific fashion. Heteromeric Kir4.1/5.1 was identified in the neocortex and in the glomeruli of the olfactory bulb. Homomeric Kir4.1 was confined to the hippocampus and the thalamus. Homomeric Kir5.1 was not identified. Kir4.1/5.1 and Kir4.1 expression appeared to occur only in astrocytes, specifically in the membrane domains facing the pia mater and blood vessels or in the processes surrounding synapses. Both Kir4.1/5.1 and Kir4.1 could be associated with PDZ domain-containing syntrophins, which might be involved in the subcellular targeting of these astrocyte Kir channels. Because heteromeric Kir4.1/5.1 and homomeric Kir4.1 have distinct ion channel properties (Tanemoto, M., Kittaka, N., Inanobe, A., and Kurachi, Y. (2000) J. Physiol. (Lond.) 525, 587-592 and Tucker, S. J., Imbrici, P., Salvatore, L., D'Adamo, M. C., and Pessia, M. (2000) J. Biol. Chem. 275, 16404-16407), it is plausible that these channels play differential physiological roles in the K+ -buffering action of brain astrocytes in a region-specific manner.


Subject(s)
Astrocytes/physiology , Potassium Channels, Inwardly Rectifying/genetics , Animals , Astrocytes/cytology , Brain/cytology , Brain/physiology , Female , Kidney/physiology , Mice , Mice, Inbred C57BL , Organ Specificity , Potassium Channels, Inwardly Rectifying/analysis , Protein Subunits/analysis , Protein Subunits/genetics
9.
Eur J Neurosci ; 19(1): 76-84, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14750965

ABSTRACT

Cochlear endolymph contains 150 mm K+ and has a highly positive potential of approximately +80 mV. The specialized ionic composition and high potential in endolymph are essential for hearing and maintained by circulation of K+ from perilymph to endolymph through the cochlear lateral wall. Various types of K+ channel such as Kir4.1 and KCNQ1/KCNE1 are expressed in stria vascularis of the lateral wall and play essential roles in K+ circulation. In this study, we examined a distribution of another K+ channel, Kir5.1, and found it specifically expressed in the spiral ligament of the cochlear lateral wall. Specific immunoreactivity for Kir5.1 was detected in type II, IV and V fibrocytes of the ligament and spiral limbus, all of which are directly involved in K+ circulation. Kir5.1 was not found in either type I or III fibrocytes. Although Kir5.1 assembles with Kir4.1 to form a functional Kir channel in renal epithelia and retinal Müller cells, double-immunolabelling revealed that they were expressed in distinct regions in the cochlea lateral wall, i.e. Kir4.1 only in stria vascularis vs. Kir5.1 in spiral ligament. During development, the expression of Kir5.1 subunits started significantly later than Kir4.1 and was correlated with the 'rapid' phase of the elevation of endocochlear potential (EP). Kir5.1 and Kir4.1 channel-subunits may therefore play distinct functional roles in K+ circulation in the cochlear lateral wall.


Subject(s)
Cochlea/metabolism , Endolymph/metabolism , Fibroblasts/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Potassium/metabolism , Stria Vascularis/metabolism , Animals , Animals, Newborn , Cell Differentiation/physiology , Cochlea/growth & development , Cochlea/ultrastructure , Fibroblasts/ultrastructure , Gene Expression Regulation, Developmental/physiology , Hearing/physiology , Immunohistochemistry , Male , Mechanotransduction, Cellular/physiology , Membrane Potentials/physiology , Microscopy, Electron , Potassium Channels, Inwardly Rectifying/biosynthesis , Rats , Rats, Wistar , Stria Vascularis/ultrastructure
10.
Am J Physiol Cell Physiol ; 285(2): C260-7, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12686518

ABSTRACT

Kir5.1 is an inwardly rectifying K+ channel subunit whose functional role has not been fully elucidated. Expression and distribution of Kir5.1 in retina were examined with a specific polyclonal antibody. Kir5.1 immunoreactivity was detected in glial Müller cells and in some retinal neurons. In the Kir5.1-positive neurons the expression of glutamic acid decarboxylase (GAD65) was detected, suggesting that they may be GABAergic-amacrine cells. In Müller cells, spots of Kir5.1 immunoreactivity distributed diffusely at the cell body and in the distal portions, where Kir4.1 immunoreactivity largely overlapped. In addition, Kir4.1 immunoreactivity without Kir5.1 was strongly concentrated at the endfoot of Müller cells facing the vitreous surface or in the processes surrounding vessels. The immunoprecipitant obtained from retina with anti-Kir4.1 antibody contained Kir5.1. These results suggest that heterotetrameric Kir4.1/Kir5.1 channels may exist in the cell body and distal portion of Müller cells, whereas homomeric Kir4.1 channels are clustered in the endfeet and surrounding vessels. It is possible that homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels play different functional roles in the K+-buffering action of Müller cells.


Subject(s)
Cell Membrane/metabolism , Neuroglia/metabolism , Neurons/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Retina/metabolism , Amacrine Cells/cytology , Amacrine Cells/metabolism , Animals , Capillaries/cytology , Capillaries/metabolism , Cells, Cultured , Glutamate Decarboxylase/metabolism , Immunohistochemistry , Isoenzymes/metabolism , Male , Membrane Potentials/physiology , Neuroglia/cytology , Neurons/cytology , Potassium/metabolism , Rats , Rats, Inbred WKY , Retina/cytology , gamma-Aminobutyric Acid/metabolism
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