title: In vitro and in silico analysis of an inhibitory mechanism of osteoclastogenesis by salubrinal and guanabenz.
abstract: Inactivating bone-resorbing osteoclasts is a prime therapeutic strategy for the prevention of bone loss in patients with osteopenia and osteoporosis. Synthetic agents such as salubrinal and guanabenz, which attenuate stress to the endoplasmic reticulum, are reported to inhibit development of osteoclasts. However, the mechanism of their inhibitory action on osteoclasts is largely unknown. Using genome-wide expression profiles, we predicted key transcription factors that downregulated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis. Principal component analysis (PCA) predicted a list of transcription factors that were potentially responsible for reversing receptor activator of nuclear factor kappa-B ligand (RANKL)-driven stimulation of osteoclastogenesis. A partial silencing of NFATc1 allowed a selection of transcription factors that were likely to be located upstream of NFATc1. We validated the predicted transcription factors by focusing on two AP-1 transcription factors (c-Fos and JunB) using RAW264.7 pre-osteoclasts as well as primary bone marrow cells. As predicted, their mRNA and protein levels were elevated by RANKL, and the elevation was suppressed by salubrinal and guanabenz. A partial silencing of c-Fos or JunB by RNA interference decreased NFATc1 as well as tartrate-resistant acid phosphatase (TRAP) mRNA. Collectively, a systems-biology approach allows the prediction of a RANKL-salubrinal/guanabenz-NFATc1 regulatory axis, and in vitro assays validate an involvement of AP-1 transcription factors in suppression of osteoclastogenesis.
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA. Electronic address: hamamurk@iupui.edu.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Hamamura
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA.
authors.forename: Andy
authors.initials: A
authors.lastname: Chen
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA.
authors.forename: Nancy
authors.initials: N
authors.lastname: Tanjung
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA; Department of Orthopaedic Surgery, Mie University Graduate School of Medicine, Mie 514, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Takigawa
authors.affiliation: Department of Orthopaedic Surgery, Mie University Graduate School of Medicine, Mie 514, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Sudo
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA; Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yokota
mesh_terms: D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D002934:Cinnamates / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects; D006143:Guanabenz / Q000494:pharmacology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D050778:NFATC Transcription Factors / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D025341:Principal Component Analysis; D016760:Proto-Oncogene Proteins c-fos / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D002934:Cinnamates; D007527:Isoenzymes; C584097:JunB protein, mouse; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D016760:Proto-Oncogene Proteins c-fos; D053245:RANK Ligand; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C505689:Tnfsf11 protein, mouse; D018808:Transcription Factor AP-1; D014157:Transcription Factors; C496827:salubrinal; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D006143:Guanabenz; D013890:Thiourea
keywords: Guanabenz; JunB; NFATc1; Osteoclasts; Salubrinal; c-Fos