title: CCR1 acts downstream of NFAT2 in osteoclastogenesis and enhances cell migration.
abstract: We found that a chemokine receptor gene, CCR1, acts downstream of NFAT2 in RANKL-stimulated RAW264 and bone marrow cells. The upstream regulatory region of CCR1 showed RANKL-dependent and CsA-suppressible promoter activity. Downregulation of the expression and function of CCR1 suppressed cell migration.
  
  
  BACKGROUND
  We previously reported that the expression of NFAT2 induced by RANKL is a key process for progression to multinucleated cells in an in vitro osteoclastogenesis system. Identifying the target genes of NFAT2 would thus be informative about the differentiation process. We focused here on chemokine and chemokine receptor genes that act downstream of NFAT2 in RAW264 cells as well as osteoclast precursors prepared from bone marrow cells.
  
  
  METHODS
  RAW264 mouse monocyte/macrophage line cells were cultured with or without cyclosporin A (CsA) in the presence of RANKL or glutathione S-transferase (GST). Osteoclast precursors were prepared from bone marrow cells. RANKL-inducible and CsA-suppressible genes were searched for by microarray analysis, and expression was confirmed by quantitative RT-PCR. Promoter activity was measured by luciferase gene reporter assay. Short interfering (si)RNA for CCR1 was introduced in RAW264 cells. Cell migration activity was examined using a Boyden chamber assay.
  
  
  CONCLUSIONS
  We identified the chemokine receptor gene CCR1 as a gene showing significant differential expression profiles in osteoclastogenesis in the presence versus the absence of CsA, an inhibitor of NFAT. This property was unique to CCR1 among the chemokine and chemokine receptor genes examined in both RAW264 and bone marrow cells. The upstream regulatory region was isolated from CCR1, and its RANKL-dependent and CsA-suppressible promoter activity was confirmed. The functional significance of CCR1 was assessed by monitoring the migration of cells in a transwell migration assay, and this activity was abolished when either CsA- or CCR1 siRNA-treated cells were used. Moreover, treatment with a Galpha inhibitor pertussis toxin (PTX) or methiolynated-regulated on activation, normal T cells expressed and secreted (Met-RANTES), an antagonist of CCR1, suppressed multinucleated cell formation in the bone marrow cell system. Together, these results suggest that the CCR1 signaling cascade is under the control of NFAT2 and seems to enhance the migration of differentiating osteoclasts.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Ishida
authors.forename: Koji
authors.initials: K
authors.lastname: Hayashi
authors.forename: Asuka
authors.initials: A
authors.lastname: Hattori
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Yogo
authors.forename: Toru
authors.initials: T
authors.lastname: Kimura
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Takeya
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D051379:Mice; D050778:NFATC Transcription Factors / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D054389:Receptors, CCR1; D019707:Receptors, Chemokine / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: C515301:Ccr1 protein, mouse; D004791:Enzyme Inhibitors; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D034741:RNA, Small Interfering; D054389:Receptors, CCR1; D019707:Receptors, Chemokine; D016572:Cyclosporine; D037342:Pertussis Toxin