title: Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.
abstract: Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout (Irf8-/-) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8-/-mice are severely altered. Therefore, to clearly elucidate the functions of Irf8 during osteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout (Irf8fl/fl;Lyz2cre/+) mice. We found that trabecular bone volume in the Irf8fl/fl;Lyz2cre/+mice was not significantly affected, while exposure to M-CSF and RANKL significantly increased TRAP activity in vitro in osteoclasts that underwent osteoclastogenesis from bone marrow-derived macrophages (BMMs) induced from bone marrow cells (BMCs) of those mice by addition of M-CSF. Our results also showed that expression of Irf8 mRNA and protein in BMMs obtained from Irf8fl/fl;Lyz2cre/+mice and cultured with M-CSF was reduced. These findings predicted that Lyz2/Lyz2-cre expression is induced when BMCs differentiate into BMMs in cultures with M-CSF. In osteoclast differentiation cultures, Lyz2 was gradually increased by M-CSF during the first 3 days of culture, then rapidly decreased by the addition of RANKL with M-CSF during the next 3 days. Furthermore, BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when cultured simultaneously with both M-CSF and RANKL from the initiation of culture. These findings suggest that Lyz2-cre expression is induced along with differentiation to BMMs by BMCs obtained from Irf8fl/fl;Lyz2cre/+mice and cultured with M-CSF. In addition, Irf8 was down-regulated by activation of the cre/loxP recombination system in BMMs and osteoclastogenesis was accelerated. Based on our results, we propose the existence in vivo of a new lineage of osteoclast precursors among BMCs, which differentiate into osteoclasts without up-regulation of Lyz2 expression.
authors: Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.|Department of Orthopedic Surgery, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
authors: Emi
authors: E
authors: Saito
authors: Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. dai.suzuki@dent.showa-u.ac.jp.
authors: Dai
authors: http://orcid.org/0000-0002-9905-0774
authors: D
authors: Suzuki
authors: Department of Immunology, Graduate School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004, Japan.
authors: Daisuke
authors: D
authors: Kurotaki
authors: Department of Oral Physiology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
authors: Ayako
authors: A
authors: Mochizuki
authors: Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.|Division of Dentistry for Persons with Disabilities, Department of Special Needs Dentistry, Showa University Dental Hospital, 2-1-1 Kitasenzoku, Ota, Tokyo, 145-8515, Japan.
authors: Yoko
authors: Y
authors: Manome
authors: Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
authors: Tetsuo
authors: T
authors: Suzawa
authors: Department of Orthopedic Surgery, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
authors: Yoichi
authors: Y
authors: Toyoshima
authors: Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
authors: Takahiro
authors: T
authors: Ichikawa
authors: Division of Dentistry for Persons with Disabilities, Department of Special Needs Dentistry, Showa University Dental Hospital, 2-1-1 Kitasenzoku, Ota, Tokyo, 145-8515, Japan.
authors: Takahiro
authors: T
authors: Funatsu
authors: Department of Oral Physiology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
authors: Tomio
authors: T
authors: Inoue
authors: Department of Pharmacology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
authors: Masamichi
authors: M
authors: Takami
authors: Department of Immunology, Graduate School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004, Japan.
authors: Tomohiko
authors: T
authors: Tamura
authors: Department of Orthopedic Surgery, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
authors: Katsunori
authors: K
authors: Inagaki
authors: Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
authors: Ryutaro
authors: R
authors: Kamijo
keywords: BMMs; Irf8; Lysozyme M; Lyz2; Osteoclasts