Large Scale Gene Expression Analysis of Osteoclastogenesisin Vitro and Elucidation of NFAT2 as a Key Regulator

Norihiro Ishida,Koji Hayashi,Mitsuhiro Hoshijima,Takuya Ogawa,Shintaro Koga,Yuuki Miyatake,Masayoshi Kumegawa,Toru Kimura,Tatsuo Takeya

doi:10.1074/jbc.m205063200

Abstract

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.

Highlights

Specific factors/regulatory genes playing essential roles for cellular differentiation have been identified in various systems, and they have been shown to exert their effects eventually through the induction or repression of certain groups of genes [1,2,3,4]
The number of genes that appeared to be differentially expressed by a factor Ͼ2.0-fold between RANKL-treated and -untreated RAW264 cells and between RANKL- and GST-treated cells at each time point are shown in the top row (A) and the middle row (B), respectively
In our previous study using the same system described in the present study, we analyzed the expression profiles of cell cycle regulatory genes during the initial phase in committed cells to clarify the mechanisms controlling the coupling between withdrawal from the cell cycle and differentiation

Summary

Introduction

Specific factors/regulatory genes playing essential roles for cellular differentiation have been identified in various systems, and they have been shown to exert their effects eventually through the induction or repression of certain groups of genes [1,2,3,4]. Osteoclasts are multinucleated (MN) giant cells and present only in bone with the capacity to resorb mineralized tissues [9] They were reported to be formed by fusion of mononuclear precursor cells derived from colony-forming unit granulocyte macrophages (CFU-GM) and branch from the monocyte-macrophage lineage during the early stage of the differentiation process [9, 10]. On the basis of these findings, a system of in vitro osteoclastogenesis using the recombinant RANKL and mouse monocyte/macrophage-derived RAW264 cells was established [15,16,17] This system consists of a single cell type with a defined differentiation inducible factor, RANKL, and the entire process requires only 4 days to form mature osteoclasts under microscopic observation [17], making this an appropriate system to analyze a mammalian cell differentiation process. We further examined the behavior of NFAT2 and confirmed its crucial role in osteoclastogenesis in vitro

Methods

Results

Conclusion