Large scale gene expression analysis of osteoclastogenesis in vitro and elucidation of NFAT2 as a key regulator.
To understand the molecular events coupling between cell proliferation and differentiation by elucidating genes essential for the process, we conducted a large scale gene expression analysis of an in vitro osteoclastogenesis system consisting of recombinant RANKL and mouse RAW264 cells. The entire process leading to the formation of tartrate resistant acid phosphatase-positive multinucleated cells takes 3 days and plates become fully covered with multinucleated cells at 4 days. Microarray probing at eight time points revealed 635 genes that showed greater than 2-fold differential expression for at least one time point and they could be classified into six groups by the "k-means" clustering analysis. Among a group of 106 early inducible genes (within 2-5 h after RANKL stimulation), four genes including NFAT2 were identified as genes whose enhanced expressions were fairly correlated with an efficient induction of matured osteoclasts. Moreover, cyclosporin A significantly suppressed the multinucleated cell formation accompanying the reduction of the nuclear localization of NFAT2. When the expression of NFAT2 was suppressed by introducing antisense NFAT2, multinucleated cell formation was severely hampered. Functional analysis thus combined with gene analysis by microarray technology elucidated a key role of NFAT2 in osteoclastogenesis in vitro.
Ishida
Norihiro
N
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
Hayashi
Koji
K
Hoshijima
Mitsuhiro
M
Ogawa
Takuya
T
Koga
Shintaro
S
Miyatake
Yuuki
Y
Kumegawa
Masayoshi
M
Kimura
Toru
T
Takeya
Tatsuo
T
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000494:pharmacology; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D005982:Glutathione Transferase / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000494:pharmacology; D051379:Mice; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D020411:Oligonucleotide Array Sequence Analysis; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D013347:Subcellular Fractions / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*
D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D007093:Imidazoles; D008562:Membrane Glycoproteins; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D009687:Nuclear Proteins; D011725:Pyridines; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D014157:Transcription Factors; D016572:Cyclosporine; D005982:Glutathione Transferase; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole