Osteoclast Differentiation Factor Modulates Cell Cycle Machinery and Causes a Delay in S Phase Progression in RAW264 Cells

Meiyanto, Edy; Hoshijima, Mitsuhiro; Ogawa, Teiichiro; Ishida, Norihiro; Takeya, Tatsuo

doi:10.1006/bbrc.2001.4564

Cited by 38 publications

(32 citation statements)

References 27 publications

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“…Cell Culture and Osteoclastogenesis in Vitro-Soluble recombinant RANKL was constructed and expressed as a GST fusion protein as described previously (17) and finally purified free from LPS using the Detoxi-Gel TM Endotoxin Removing Gel (Pierce). GST was prepared using the same procedures.…”

Section: Methodsmentioning

confidence: 99%

“…GST was prepared using the same procedures. RAW264 mouse monocyte/macrophage line cells (18) were obtained from the RIKEN Cell Bank (Japan) and were maintained in Eagle medium supplemented with 10% fetal calf serum and 1% non-essential amino acids (Invitrogen) as described previously (17). For osteoclastogenesis in vitro, cells were plated at a density of 1.3 ϫ 10 4 /cm 2 unless otherwise noted with 10% fetal calf serum (HyClone) and incubated for 24 h, and then purified RANKL was added to the final concentration of 500 ng/ml (at 0 h) when needed.…”

Section: Methodsmentioning

confidence: 99%

“…RANKL binds the receptor called receptor activator of NFB (RANK) that is expressed in osteoclast precursors and induces osteoclast formation (14). 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 moreover, 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.…”

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confidence: 99%

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

Ishida

Hayashi

Hoshijima

et al. 2002

Journal of Biological Chemistry

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361

299

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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)...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Ishida

Hayashi

Hoshijima

et al. 2002

Journal of Biological Chemistry

Self Cite

361

299

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“…Cell types and stages of di erentiation are listed. Potential binding partners of p21 are proposed to di er at di erent stages, and include interactions postulated to mediate cell cycle inhibition (cyclin-cdk's, PCNA, E2F, TOK-1), apoptosis-inhibition (ASK, SAPK, procaspase-3, gadd45, myd118), proliferation (set, TK-1, cyclin-cdk4), subcellular localization (calmodulin, ciz-1), gene expression (E2F, CKII, p300) and di erentiation (C/EBPalpha, Stat3, p300, E2F) di erentiation stimulus is necessary for di erentiation commitment but not for growth arrest, whereas a later peak in p21 is associated with growth arrest (Casini and Pelicci, 1999;Matushansky et al, 2000b;Meiyanto et al, 2001).…”

Section: Cdki Cascades and DI Erentiationmentioning

confidence: 99%

Cell cycle regulators and hematopoiesis

Steinman

2002

Oncogene

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“…It has been reported that RANKL induced the dephosphorylation of pRb, and thereafter resulted in a delayed S-phase progression in RAW264 cells (Meiyanto et al, 2001). This suggests that RANKL exerts the anti-proliferative effects through Rb during osteoclast differentiation.…”

Section: Discussionmentioning

confidence: 92%

The Anti-proliferative Gene TIS21 Is Involved in Osteoclast Differentiation

Lee

Kwak²,

Lee³

et al. 2002

BMB Reports

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The remodeling process of bone is accompanied by complex changes in the expression levels of various genes. Several approaches have been employed to detect differentially-expressed genes in regard to osteoclast differentiation. In order to identify the genes that are involved in osteoclast differentiation, we used a cDNAarray-nylon membrane. Among 1,200 genes that showed a measurable signal, 19 genes were chosen for further study. Eleven genes were up-regulated; eight genes were downregulated. TIS21 was one of the up-regulated genes which were highly expressed in mature osteoclasts. To verify the cDNA microarray results, we carried out RT-PCR and real-time RT-PCR for the TIS21 gene. The TIS21 mRNA level was higher in differentiated-osteoclasts when compared to undifferentiated bone-marrow macrophages. Furthermore, the treatment with 1 µM of a TIS21 antisense oligonucleotide reduced the formation of osteoclasts from the bone-marrow-precursor cells bỹ 30%. These results provide evidence for the potential role of TIS21 in the differentiation of osteoclasts.

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Osteoclast Differentiation Factor Modulates Cell Cycle Machinery and Causes a Delay in S Phase Progression in RAW264 Cells

Cited by 38 publications

References 27 publications

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

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

Cell cycle regulators and hematopoiesis

The Anti-proliferative Gene TIS21 Is Involved in Osteoclast Differentiation

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