Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro

Emi Saito; Dai Suzuki; Daisuke Kurotaki; Ayako Mochizuki; Yoko Manome; Tetsuo Suzawa; Yoichi Toyoshima; Takahiro Ichikawa; Takahiro Funatsu; Tomio Inoue; Masamichi Takami; Tomohiko Tamura; Katsunori Inagaki; Ryutaro Kamijo

doi:10.1007/s10616-016-0013-z

Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro

Cytotechnology. 2017 Jun;69(3):443-450. doi: 10.1007/s10616-016-0013-z. Epub 2016 Aug 8.

Authors

Emi Saito^{1

2}, Dai Suzuki³, Daisuke Kurotaki⁴, Ayako Mochizuki⁵, Yoko Manome^{1

6}, Tetsuo Suzawa¹, Yoichi Toyoshima², Takahiro Ichikawa¹, Takahiro Funatsu⁶, Tomio Inoue⁵, Masamichi Takami⁷, Tomohiko Tamura⁴, Katsunori Inagaki², Ryutaro Kamijo¹

Affiliations

¹ Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
² Department of Orthopedic Surgery, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
³ Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. dai.suzuki@dent.showa-u.ac.jp.
⁴ Department of Immunology, Graduate School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004, Japan.
⁵ Department of Oral Physiology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
⁶ 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.
⁷ Department of Pharmacology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.

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 (Irf8 ^fl/fl ;Lyz2 ^cre/+) mice. We found that trabecular bone volume in the Irf8 ^fl/fl ;Lyz2 ^cre/+ 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 Irf8 ^fl/fl ;Lyz2 ^cre/+ 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 Irf8 ^fl/fl ;Lyz2 ^cre/+ 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.

Keywords: BMMs; Irf8; Lysozyme M; Lyz2; Osteoclasts.