title: IL-6-accelerated calcification by induction of ROR2 in human adipose tissue-derived mesenchymal stem cells is STAT3 dependent.
abstract: OBJECTIVE
  The mechanisms of ectopic calcification in inflammatory diseases are poorly understood. We investigated the effects of inflammatory cytokines on the mechanisms of calcification in human adipose tissue-derived mesenchymal stem cells (hADSCs).
  
  
  METHODS
  The effects of inflammatory cytokines were evaluated using hADSCs cultured in osteoblast induction medium. mRNA expression was measured by real-time PCR and protein levels were measured by western blotting. Cell mineralization was evaluated by Alizarin Red S staining.
  
  
  RESULTS
  In hADSCs, administration of IL-6/soluble IL-6 receptor (sIL-6R), TNF or IL-1β accelerated calcification through enhanced expression of an osteoblast differentiation marker, runt-related transcription factor 2 (RUNX2). IL-6/sIL-6R had the greatest effect. The transcription of mRNA for receptor tyrosine kinase-like orphan receptor 2 (ROR2), involved in the non-canonical wingless-type (WNT) MMTV integration site pathway, was increased, while β-catenin expression, an essential factor in the canonical WNT signalling pathway for osteoblast differentiation, did not change. Suppression of signal transducer and activator of transcription 3 (STAT3), but not STAT1, by small interfering RNA (siRNA) exerted a strong inhibitory effect on RUNX2 and ROR2 expression, and inhibited accelerated calcification.
  
  
  CONCLUSIONS
  IL-6/sIL-6R stimulation accelerated the ROR2/WNT5A pathway in hADSCs in a STAT3-dependent manner, resulting in augmented calcification. These results suggest that the mechanisms of ectopic calcification accelerated by IL-6 in hADSCs may be involved in chronic inflammatory tissues and that IL-6 inhibitors may be beneficial in the treatment of ectopic calcification in inflammatory diseases.
authors.affiliation: First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Fukuyo
authors.affiliation: First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Yamaoka
authors.affiliation: First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Koshiro
authors.initials: K
authors.lastname: Sonomoto
authors.affiliation: First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan.First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Oshita
authors.affiliation: First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Okada
authors.affiliation: First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Saito
authors.affiliation: First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Yoshida
authors.affiliation: First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Kanazawa
authors.affiliation: First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Minami
authors.affiliation: First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu and Department of Physiology and Cell Biology, Graduate School of Medicine, School of Medicine, Kobe University, Kobe, Japan. tanaka@med.uoeh-u.ac.jp.
authors.forename: Yoshiya
authors.initials: Y
authors.lastname: Tanaka
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002113:Calcification, Physiologic / Q000187:drug effects* / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D053583:Interleukin-1beta / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism / Q000494:pharmacology*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D057050:Receptor Tyrosine Kinase-like Orphan Receptors / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D051153:Wnt Proteins / Q000378:metabolism; D000071818:Wnt-5a Protein; D051176:beta Catenin / Q000378:metabolism
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D053583:Interleukin-1beta; D015850:Interleukin-6; D011518:Proto-Oncogene Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D014409:Tumor Necrosis Factor-alpha; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D051176:beta Catenin; C078717:ROR2 protein, human; D057050:Receptor Tyrosine Kinase-like Orphan Receptors
keywords: ADSCs; IL-6; ROR2; STAT3; WNT5A; ectopic calcification