Biochemical and Biophysical Research Communications
Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion
Introduction
Bone homeostasis depends on the balance between the bone resorption by osteoclasts and formation by osteoblasts [1], [2]. Osteoclasts are multinucleated giant cells that have bone resorption activity. They also play a critical role in various pathological conditions associated with bone loss such as osteoporosis, rheumatoid arthritis and cancer metastasis [1], [2]. Therefore, a proper understanding of the regulatory mechanisms of osteoclastogenesis would provide a scientific basis for novel therapeutic strategies for such diseases.
Osteoclasts are differentiated from precursor cells of the monocyte/macrophage lineage in response to stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) [1], [2]. Nuclear factor of activated T cells (NFAT) c1, the master regulator of osteoclast differentiation, promotes mRNA expression of osteoclast related genes such as Ctsk (encoding Cathepsin K), Acp5 (tartrate-resistant acid phosphatase; TRAP), Dcstamp (dendritic cell specific transmembrane protein; DC-STAMP) and Ocstamp (osteoclast stimulatory transmembrane protein; OC-STAMP) in collaboration with other transcription factors, such as c-Fos and microphthalmia-associated transcription factor (MITF) [1], [2]. Osteoclast precursors become multinucleated cells by cell–cell fusion [1], [2], [3]. TRAP-positive mononucleated cells, but no multinucleated osteoclasts, were observed in DC-STAMP or OC-STAMP-deficient mice [4], [5]. Atp6v0d2-deficient mice exhibit an osteopetrotic phenotype due to impaired cell–cell fusion of osteoclast precursor cells [6]. These reports indicated that these molecules are important for cell–cell fusion during osteoclastogenesis, although the detailed molecular mechanisms at present remain unclear.
The level of gene expression is not only determined by the genomic sequence, but also by sequence-independent epigenetic regulation. Epigenetic regulatory mechanisms include histone modifications, such as methylation and acetylation, DNA methylation and microRNA-mediated regulation. Histone modifications are implicated in both gene activation and repression, depending on the specific lysine residue that is methylated or acetylated [7]. Histone H3 lysine 4 trimethylation (H3K4me3) activates transcription by recruiting nucleosome remodeling enzymes and histone acetylases [8]. In contrast, histone H3 lysine 27 trimethylation (H3K27me3) suppresses transcription by promoting chromatin compaction [8]. In osteoclastogenesis, it has been demonstrated that NFATc1 expression is regulated by a sequential change of histone modification from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(−) [9], suggesting the importance of this histone modification change in the regulation of osteoclast genes.
Cadherin is a calcium-dependent adhesion protein that is classified into the classical cadherins, desmosomal cadherins and protocadherins [10]. In osteoclasts, it has been reported that E-cadherin expression was involved in the fusion of osteoclast precursor cells into mature multinucleated osteoclasts in vitro [11]. However, the contribution of other cadherins in osteoclastogenesis is as yet unclear.
In this study, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis of H3K4me3 and H3K27me3 in RANKL-stimulated osteoclast precursor cells and screened for genes characterized by a histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(−). We also analyzed mRNA expression by RNA sequencing and selected the genes highly expressed and upregulated during osteoclastogenesis. Then we focused on the role of Pcdh7 (encoding protocadherin-7) in osteoclastogenesis. Here we report that Pcdh7, a non-clustered protocadherin, which is involved in cell–cell adhesion and signal transduction in neural and cancer cells [10], plays an important role in osteoclastogenesis by regulating cell–cell fusion.
Section snippets
In vitro osteoclastogenesis
In vitro osteoclastogenesis was performed as described previously with a minor modification [12], [13], [14]. Briefly, bone marrow cells were cultured with 10 ng/ml M-CSF (R&D Systems, Minneapolis, MN) to obtain bone marrow-derived monocyte/macrophage precursor cells (BMMs). These cells or infected cells described in the knockdown analysis were cultured with 12.5 or 25 ng/ml RANKL (PeproTech, Rocky Hill, NJ) and M-CSF for 3 or 4 days. Cyclosporin A (5 μg/ml; Sigma–Aldrich, St. Louis, MO) was
Selection of candidate osteoclast regulatory genes by ChIP sequencing of H3K4me3 and H3K27me3
To elucidate novel osteoclast regulatory genes that are epigenetically regulated during osteoclastogenesis, we performed ChIP sequencing of H3K4me3 and H3K27me3 in BMMs and mature osteoclasts generated by RANKL stimulation. Over 10% of the H3K4me3 peaks were located at transcription start sites (TSS) and more than half of them were located less than 5 kb from TSS. In contrast, H3K27me3 peaks were rarely located at TSS and less than 20% of them were located in regions less than 5 kb from TSS (Fig.
Discussion
Histone modification regulates gene expression in the course of the differentiation of various cell types [7]. NFATc1, a critical transcription factor, is regulated by H3K4me3 and H3K27me3 in osteoclastogenesis [9]. Using ChIP sequencing, we identified Pcdh7 to be under the control of a similar histone modification and it is one of the key osteoclast regulatory genes. Pcdh7, induced in an NFATc1-dependent manner, regulates osteoclast differentiation by promoting cell–cell fusion. A novel method
Acknowledgments
We thank T. Kitamura for providing Plat-E cell line. We also thank Y. Kadono, J. Hirose, and S. Fukuse for reagents, discussion and technical assistance. This work was supported in part by Grants for the ERATO Takayanagi Osteonetwork Project and PRESTO from Japan Science and Technology Agency; Grant-in-Aids for Scientific Research (B), Young Scientist (A) and Challenging Exploratory Research from the Japan Society for the Promotion of Science (JSPS); and Grants from Uehara Memorial Foundation,
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