title: Inhibition of lipopolysaccharide-induced osteoclast formation and bone resorption in vitro and in vivo by cysteine proteinase inhibitors.
abstract: Inflammation-induced bone destruction is a major treatment target in many inflammatory skeletal diseases. The aim of this study was to investigate if the cysteine proteinase inhibitors cystatin C, fungal cysteine proteinase inhibitor (E-64), andN-benzyloxycarbonyl-arginyl-leucyl-valyl-glycyl-diazomethane acetate (Z-RLVG-CHN2) can inhibit LPS-induced osteoclast formation. Mouse bone marrow macrophages (BMMs) were isolated and primed with receptor activator of NF-κB ligand (RANKL) for 24 h, followed by stimulation with LPS, with and without inhibitors. Adult mice were injected locally with LPS and then treated with E-64 and osteoclast formation assessed by the number of cathepsin K+multinucleated cells. Cystatin C inhibited LPS-induced osteoclast formation time and concentration dependently (IC50= 0.3 μM). The effect was associated with decreased mRNA and protein expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K and of the osteoclastogenic transcription factors c-Fos and NFATc1. LPS-induced osteoclast formation on bone slices was also inhibited by cystatin C, resulting in decreased pit formation and release of bone matrix proteins. Similar data were obtained with E-64 and Z-RLVG-CHN2Cystatin C was internalized in BMMs stimulated by LPS but not in unstimulated BMMs. Osteoclast formation induced by LPS was dependent on TNF-α, and the 3 inhibitors abolished LPS-induced TNF superfamily 2 (gene encoding TNF-α;Tnfsf2) mRNA expression without affectingIl1b,Il6, or oncostatin M (Osm) expression. Formation of osteoclasts in the skull bones after local LPS stimulation was inhibited by E-64. It is concluded that cysteine proteinase inhibitors effectively inhibit LPS-induced osteoclast formation in vivo and in vitro by inhibition of TNF-α expression. The targeting of cysteine proteinases might represent a novel treatment modality for prevention of inflammatory bone loss.
authors.affiliation: Department of Molecular Periodontology, Umeå University, Umeå, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Strålberg
authors.affiliation: Department of Molecular Periodontology, Umeå University, Umeå, Sweden.
authors.forename: Ali
authors.initials: A
authors.lastname: Kassem
authors.affiliation: Institute of Chemistry, University of Gdansk, Gdansk, Poland.
authors.forename: Franciszek
authors.initials: F
authors.lastname: Kasprzykowski
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Chemistry and Pharmacology, Lund University, Lund, Sweden.
authors.forename: Magnus
authors.initials: M
authors.lastname: Abrahamson
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Chemistry and Pharmacology, Lund University, Lund, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Grubb
authors.affiliation: Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; and.|Departments of Internal Medicine and Clinical Nutrition, Centre for Bone and Arthritis Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Catharina
authors.initials: C
authors.lastname: Lindholm
authors.affiliation: Department of Molecular Periodontology, Umeå University, Umeå, Sweden; ulf.lerner@gu.se.|Departments of Internal Medicine and Clinical Nutrition, Centre for Bone and Arthritis Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Ulf H
authors.initials: UH
authors.lastname: Lerner
mesh_terms: D000818:Animals; D050071:Bone Density Conservation Agents / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D001862:Bone Resorption / Q000139:chemically induced / Q000235:genetics / Q000276:immunology / Q000517:prevention & control*; D056657:Cathepsin K / Q000235:genetics / Q000276:immunology; D055316:Cystatin C / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004306:Dose-Response Relationship, Immunologic; D005786:Gene Expression Regulation; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D051379:Mice; D050778:NFATC Transcription Factors / Q000235:genetics / Q000276:immunology; D009842:Oligopeptides / Q000494:pharmacology*; D053683:Oncostatin M / Q000235:genetics / Q000276:immunology; D010010:Osteoclasts / Q000187:drug effects* / Q000276:immunology / Q000473:pathology; D061251:Primary Cell Culture; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000276:immunology; D053245:RANK Ligand / Q000037:antagonists & inhibitors / Q000494:pharmacology; D015398:Signal Transduction; D000071681:Tartrate-Resistant Acid Phosphatase / Q000235:genetics / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology
chemical_list: D050071:Bone Density Conservation Agents; D055316:Cystatin C; D015853:Cysteine Proteinase Inhibitors; D015850:Interleukin-6; D008070:Lipopolysaccharides; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D009842:Oligopeptides; D016760:Proto-Oncogene Proteins c-fos; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse; D014409:Tumor Necrosis Factor-alpha; C577686:interleukin-6, mouse; D053683:Oncostatin M; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D056657:Cathepsin K; C534132:Ctsk protein, mouse; D007930:Leucine; C024974:E 64
keywords: cystatin C; inflammation; macrophages; periodontitis; rheumatoid arthritis