title: Involvement of PU.1 in NFATc1 promoter function in osteoclast development.
abstract: BACKGROUND
  The transcription factors NFATc1 and PU.1 play important roles in osteoclast development. NFATc1 and PU.1 transactivate osteoclast-specific gene expression and a deficiency in NFATc1 or PU.1 genes causes osteopetrosis due to an insufficient development of osteoclasts. However, the existence of cross-regulation between NFATc1 and PU.1 is largely unknown. In the present study, the role of PU.1 in NFATc1 expression was investigated.
  
  
  METHODS
  Osteoclasts were generated from mouse bone marrow cells. PU.1 knockdown was performed with siRNA introduction. The mRNA levels in siRNA-introduced cells were determined by quantitative RT-PCR. The involvement of PU.1 in the NFATc1 promoter was analyzed by using a chromatin immunoprecipitation (ChIP) assay and a reporter assay. Retrovirus vector was used for enforced expression of PU.1.
  
  
  RESULTS
  Introduction of PU.1 siRNA into bone marrow-derived osteoclasts resulted in a decrease in NFATc1 mRNA level. A ChIP assay showed that PU.1 bound to the NFATc1 promoter in osteoclasts. NFATc1 promoter activity was reduced in PU.1 knockdown cells as assessed by a reporter assay. PU.1 siRNA introduction also downregulated the expression of osteoclast-specific genes and tartrate resistant acid phosphatase (TRAP) activity. Enforced expression of PU.1 using a retrovirus vector increased NFATc1 expression and TRAP activity. When NFATc1 expression was knocked down by using siRNA, the induction of osteoclast-specific genes and TRAP-positive cells was suppressed without affecting the expression level of PU.1.
  
  
  CONCLUSIONS
  These results indicate that PU.1 is involved in osteoclast development by transactivating NFATc1 expression via direct binding to the NFATc1 promoter.
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan; Department of Internal Medicine and Rheumatology, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Ishiyama
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan; Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Tokyo, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Yashiro
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Nakano
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan; Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Tokyo, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Kasakura
authors.affiliation: Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Tokyo, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Miura
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Mutsuko
authors.initials: M
authors.lastname: Hara
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan; Bay Bioscience Corporation, Hyogo, Japan.
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Kawai
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Maeda
authors.affiliation: Department of Internal Medicine and Rheumatology, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Tamura
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Ko
authors.initials: K
authors.lastname: Okumura
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Hideoki
authors.initials: H
authors.lastname: Ogawa
authors.affiliation: Department of Internal Medicine and Rheumatology, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Yoshinari
authors.initials: Y
authors.lastname: Takasaki
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan; Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Tokyo, Japan. Electronic address: chinishi@rs.tus.ac.jp.
authors.forename: Chiharu
authors.initials: C
authors.lastname: Nishiyama
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D051379:Mice; D008815:Mice, Inbred Strains; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D010010:Osteoclasts / Q000473:pathology*; D010024:Osteoporosis / Q000235:genetics / Q000276:immunology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D059467:Transcriptome; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D050778:NFATC Transcription Factors; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1
keywords: NFATc1; Osteoclasts; PU.1; Promoter; Transcription factor