Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro
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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.
KeywordsBMMs Irf8 Lysozyme M Lyz2 Osteoclasts
The strength and health of bone tissues are regulated by a tight balance of bone resorption by osteoclasts and bone formation by osteoblasts. When that balance is destructed along with enhanced osteoclast and/or failed osteoblast function, bone develops osteopenia or osteoporosis, which is associated with a high risk of fracture. Osteoclasts are differentiated from osteoclast precursors with a monocyte/macrophage lineage following stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL), which are secreted from osteoblasts and osteocytes (Kobayashi et al. 2009; Nakashima et al. 2012). Osteoclast differentiation and activation are strictly controlled by various transcription factors, including AP-1 (Fos/Jun) and nuclear factor of activated T cells c1 (NFATc1), which are activated by RANKL signaling. Differentiated osteoclasts express several specific markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, a cysteine protease.
We previously reported that interferon regulatory factor 8 (IRF8), a transcription factor otherwise known as interferon consensus sequence binding protein (ICSBP), negatively regulates osteoclast differentiation (Zhao et al. 2009). Irf8 is expressed in osteoclast precursors and inhibits Nfatc1 function, and is down-regulated by stimulation with RANKL, which leads to Nfatc1 auto-amplification and osteoclast differentiation. Furthermore, Irf8 global knockout (Irf8 −/−) mice show reduced bone volume as a result of increased numbers of osteoclasts.
The population and properties of hematopoietic cells in Irf8 −/− mouse bone marrow and spleen tissues are dramatically changed, along with development of chronic myelogenous leukemia and splenomegaly (Holtschke et al. 1996; Tamura et al. 2015; Tamura and Ozato 2002), thus it is difficult to clearly analyze the functions of Irf8 in monocytes/macrophages in the osteoclast precursor stage. In the present study, to investigate the detailed functions of Irf8 in monocytes/macrophages during osoteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout (Irf8 fl/fl ;Lyz2 cre/+) mice by crossing Irf8-flox with Lyz2-cre mice, and analyzed bone phenotype and osteoclast differentiation.
Materials and methods
Generation of mice
All animal experiments were conducted in accordance with the guidelines of Showa University. The Irf8 knockout (Irf8 −/−) mice (C57BL/6) used in this study have been described (Holtschke et al. 1996). Myeloid cell-specific Irf8 conditional knockout (Irf8 fl/fl ;Lyz2 cre/+) mice (C57BL/6) were generated by mating Irf8-flox with Lyz2-cre knock-in mice (Clausen et al. 1999; Feng et al. 2011). The primers used for genotyping are shown in Table S1.
Following euthanasia, tibiae were dissected and subjected to three-dimensional micro-computed tomography (μCT) with a ScanXmate-L090H (Comscan Tecno, Yokohama, Japan). Three-dimensional microstructural image data thus obtained were reconstructed using TRI/3D-BON software (Ratoc System Engineering, Tokyo, Japan).
Mouse bone marrow cells (BMCs) were collected from the femora and tibiae of 6- to 8-week-old male mice. Bone marrow-derived macrophages (BMMs) were formed from BMCs cultured in α-MEM supplemented with 10 % fetal bovine serum and M-CSF (50 ng/mL) for 3–4 days at 37 °C in a CO2 incubator (5 % CO2, 95 % air). Osteoclasts were formed from BMM (or BMC in Fig. 3c, d) cultures with M-CSF and various concentrations of RANKL after 3–4 days. Human M-CSF (Leucoprol) and RANKL was purchased from Kyowa Hakko Kogyo (Tokyo, Japan) and R&D Systems (Minneapolis, MN, USA), respectively.
Detection of osteoclasts and measurement of TRAP activity
After culturing, cells were fixed with formalin and stained for tartrate-resistant acid phosphatase (TRAP; osteoclast marker) using a conventional method with naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis, MO, USA) and fast red violet LB salt (Sigma-Aldrich) dissolved in 0.1 M acetic buffer (pH 5.0) containing 1 % tartrate acid (Suda et al. 1997). To evaluate the generation of osteoclasts, we used a TRAP activity assay, as previously described (Mochizuki et al. 2006).
Western blot analysis
Cells were lysed in RIPA buffer and quantified using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). SDS-polyacrylamide gel electrophoresis sample buffer was added to protein samples (30 μg), followed by boiling for 5 min. Next, the samples were loaded onto pre-cast gradient Mini-PROTEAN TGX Gels (Bio-Rad Laboratories, Hercules, CA, USA), then separated and transferred to PVDF Immobilon-P membranes (Merck Millipore, Billerica, MA, USA) using a Mini Trans-Blot Cell system (Bio-Rad Laboratories). The membranes were blocked in 5 % BSA Tris-buffered saline-Tween 20, incubated with primary antibodies according to the supplier’s instructions, and incubated with appropriate HRP-conjugated secondary antibodies prior to signal detection with SuperSignal West Substrate (Thermo Scientific) using a VersaDoc Imaging System (Bio-Rad Laboratories). The antibodies used in the present experiment were as follows: anti-Irf8 antibody (Icsbp) #sc6058 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β-actin antibody #A5060 (Sigma-Aldrich), anti-goat IgG-HRP #sc2768 (Santa Cruz Biotechnology), and anti-rabbit IgG-HRP #7074 (Cell Signaling Technology, Danvers, MA, USA).
Quantitative real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), then reverse-transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Quantitative real-time PCR (qPCR) was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO) and the StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primer sequences were as follows: Actb, 5′-AGATGACCCAGATCATGTTTGAGA-3′ and 5′-CACAGCCTGGATGGCTACGT-3′; Irf8, 5′-GGTGGATGCTTCCATCTTCAA-3′ and 5′-GTGGCTGGTTCAGCTTTGTCT-3′; Ctsk, 5′-CGACTATCGAAAGAAAGGATACGTT-3′ and 5′-AGCCCAACAGGAACCACACT-3′; and Lyz2, 5′-AATGGCTGGCTACTATGGAGTCA-3′ and 5′-TGCTCTCGTGCTGAGCTAAACA-3′. Expressions were normalized to that of Actb.
The results are expressed as the mean ± SD for each experiment. Student’s two-tailed t test was used, with p < 0.05 considered to indicate significance.
On the other hand, this is the first study to show that osteoclast precursors among BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when simultaneously exposed to both M-CSF and RANKL from the initiation of culture. That finding raises the possibility of another osteoclast lineage that differentiates from osteoclast precursors among BMCs and does not express Lyz2/Lyz2-cre, which may be dominant in vivo as compared to osteoclasts with BMM lineage (Fig. 4b). Irf8 mRNA expression was not reduced in BMCs from Irf8 fl/fl ;Lyz2 cre/+ mice and those mice did not demonstrate osteoporosis. Thus, if most osteoclasts differentiate from BMMs in vivo, then Irf8 fl/fl ;Lyz2 cre/+ mice should develop osteoporosis caused by enhancement of excessive bone resorption induced by osteoclastogenesis, the same as seen in Irf8 −/− mice. However, that does not occur in Irf8 fl/fl ;Lyz2 cre/+ mice. A previous study showed that mice obtained by crossing Nfatc1 conditional knockout mice with Lyz2-cre mice had no alterations in bone density (Aliprantis et al. 2008). Nfatc1 is a master regulator of osteoclast differentiation and activated by RANKL, and its transcriptional activity and expression are inhibited by Irf8 in osteoclast precursors (Zhao et al. 2009). In other words, the main portion of osteoclasts in vivo is the results of differentiation from osteoclast precursors under regulations of Irf8 and Nfatc1, without Lyz2/Lyz2-cre expression. However, Lyz2-cre mice have been used in osteoclast differentiation studies, and conditional knockout mice have been found to have such bone phenotypes as osteoporosis (Albers et al. 2013; Martin-Millan et al. 2010) and osteopetrosis (Kenner et al. 2004; Wang et al. 2008). Thus, the process of osteoclast differentiation may be dependent on a combination of between expression level and/or function of each regulator, and two types of osteoclast precursors, those with and without Lyz2/Lyz2-cre expression. Additional studies are needed to elucidate the mechanisms involved.
We express our gratitude to M. Benton for his help with our work. We thank H. Morse III (National Institute of Allergy and Infectious Diseases, Rockville, MD, USA) for providing Irf8-flox mice. This study was supported in part by Grants-in-Aid for Scientific Research (B) (MT: 26293398) and (C) (AM: 15K11052) from the Japan Society for the Promotion of Science.
Compliance with ethical standards
Conflicts of interest
The authors have no conflicts of interest to declare in regard to this study.
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