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

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.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 (1-4). Therefore, gene expression profiling based on fine statistical analysis in addition to an elucidation of key factors/ genes might be essential to understand the molecular mechanisms underlying the differentiation process of a certain cell type. Fortunately, recent advances in the technology for assaying RNA in a highly parallel fashion (5-7), coupled with the completion/progress of several mammalian genome projects, make the approach feasible if a refined system is available. Here, we describe the broad outlines of gene expression during osteoclastogenesis in vitro, in particular during the initial stage, and explain the identification and characterization of genes essential for osteoclastogenesis on the basis of profiling characteristics. A similar approach using a different cell system was reported recently (8).Osteoclasts are multinucleated (MN) 1 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). Recently, a key factor responsible for initiating this differentiation process was identified and named receptor activator of NFB ligand (RANKL) (or osteoclast differentiation factor (ODF)/TNF-related activation-induced cytokine TRANCE) (11-13)...

Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and NF-κB transactivation by RANKL

“…9,17 The expression of this transcription factor was shown to be induced by RANKL during osteoclastogenesis. 16,17 As the TAK1-MKK6-p38 pathway positively regulated Oc differentiation, we investigated whether this MAPK signalling pathway is linked to NFATc1 induction by RANKL. BMMs were infected with TAK1-DN or the control viruses and cultured for 24 h in the presence of RANKL and NFATc1 expression was assessed by Western analyses.…”

“…Recent studies have shown that the expression of NFATc1 is induced by RANKL treatment 16,17 and NFATc1-deficient embryonic stem cell lines failed to differentiate to Ocs. 17 It has been indicated in other cell types that MAPKs are involved in the regulation of NFAT family proteins.…”

Global gene expression analysis in liver of obese diabetic db/db mice treated with metformin

“…DNA microarray analysis DNA microarray analysis using Affymetrix GeneChip technology was performed as described previously [28]. Briefly, 10 μg of total RNA was used as a template for cDNA synthesis.…”

The RANK/RANKL/osteoprotegerin system in rheumatoid arthritis: New insights from animal models

“…The TRAF-binding domains of RANK are important for the RANK-dependent induction of NF-B and JNK (21,23). In addition, the signaling pathways via the transcription factors c-Fos and nuclear factor of activated T cells c1 (NF-ATc1) seem to be important for RANKL-induced osteoclastogenesis, as outlined in recent studies (24)(25)(26). RANKL also activates the antiapoptotic serine/ threonine kinase protein kinase B/Akt through a signaling complex involving c-Src and TRAF6, which interact with one another and with RANK after receptor engagement (27).…”