PMID- 27502007 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20191120 IS - 0920-9069 (Print) IS - 1573-0778 (Electronic) IS - 0920-9069 (Linking) VI - 69 IP - 3 DP - 2017 Jun TI - Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro. PG - 443-450 LID - 10.1007/s10616-016-0013-z [doi] AB - 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. FAU - Saito, Emi AU - Saito E AD - Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. AD - Department of Orthopedic Surgery, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. FAU - Suzuki, Dai AU - Suzuki D AUID- ORCID: 0000-0002-9905-0774 AD - Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. dai.suzuki@dent.showa-u.ac.jp. FAU - Kurotaki, Daisuke AU - Kurotaki D AD - Department of Immunology, Graduate School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004, Japan. FAU - Mochizuki, Ayako AU - Mochizuki A AD - Department of Oral Physiology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. FAU - Manome, Yoko AU - Manome Y AD - Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. AD - 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. FAU - Suzawa, Tetsuo AU - Suzawa T AD - Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. FAU - Toyoshima, Yoichi AU - Toyoshima Y AD - Department of Orthopedic Surgery, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. FAU - Ichikawa, Takahiro AU - Ichikawa T AD - Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. FAU - Funatsu, Takahiro AU - Funatsu T AD - 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. FAU - Inoue, Tomio AU - Inoue T AD - Department of Oral Physiology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. FAU - Takami, Masamichi AU - Takami M AD - Department of Pharmacology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. FAU - Tamura, Tomohiko AU - Tamura T AD - Department of Immunology, Graduate School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004, Japan. FAU - Inagaki, Katsunori AU - Inagaki K AD - Department of Orthopedic Surgery, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. FAU - Kamijo, Ryutaro AU - Kamijo R AD - Departments of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan. LA - eng PT - Journal Article DEP - 20160808 PL - United States TA - Cytotechnology JT - Cytotechnology JID - 8807027 PMC - PMC5461233 OTO - NOTNLM OT - BMMs OT - Irf8 OT - Lysozyme M OT - Lyz2 OT - Osteoclasts COIS- The authors have no conflicts of interest to declare in regard to this study. EDAT- 2016/08/10 06:00 MHDA- 2016/08/10 06:01 PMCR- 2016/08/08 CRDT- 2016/08/10 06:00 PHST- 2016/07/04 00:00 [received] PHST- 2016/07/20 00:00 [accepted] PHST- 2016/08/10 06:00 [pubmed] PHST- 2016/08/10 06:01 [medline] PHST- 2016/08/10 06:00 [entrez] PHST- 2016/08/08 00:00 [pmc-release] AID - 10.1007/s10616-016-0013-z [pii] AID - 13 [pii] AID - 10.1007/s10616-016-0013-z [doi] PST - ppublish SO - Cytotechnology. 2017 Jun;69(3):443-450. doi: 10.1007/s10616-016-0013-z. Epub 2016 Aug 8.