Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.
Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.
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DOI：
10.1007/s10616-016-0013-z
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发表时间：
2017-06
期刊：
影响因子：
2.2
通讯作者：
Kamijo R
中科院分区：
文献类型：
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作者：
Saito E;Suzuki D;Kurotaki D;Mochizuki A;Manome Y;Suzawa T;Toyoshima Y;Ichikawa T;Funatsu T;Inoue T;Takami M;Tamura T;Inagaki K;Kamijo R
干扰素调节因子8（Irf 8）是一种负调节破骨细胞分化的转录因子，Irf 8全基因敲除（Irf 8 −/−）小鼠已被证明具有因破骨细胞数量增加而导致的骨体积减少。然而，详细分析Irf 8在具有单核细胞/巨噬细胞谱系的破骨细胞前体中的功能是困难的，因为Irf 8 −/−小鼠中造血细胞的数量和性质发生了严重改变。因此，为了清楚地阐明Irf 8在破骨细胞生成过程中的功能，我们建立了骨髓细胞特异性Irf 8条件性敲除（Irf 8 fl/fl ; Lyz 2 cre/+）小鼠。我们发现Irf 8 fl/fl ; Lyz 2 cre/+小鼠的骨小梁体积未受到显著影响，而暴露于M-CSF和RANKL显著增加了体外破骨细胞中的TRAP活性，这些破骨细胞通过添加M-CSF从这些小鼠的骨髓细胞（BMC）诱导的骨髓源性巨噬细胞（BMSCs）进行破骨细胞生成。我们的结果还表明，从Irf 8 fl/fl ; Lyz 2 cre/+小鼠中获得并与M-CSF一起培养的BMMs中Irf 8 mRNA和蛋白的表达减少。这些发现预测当BMCs在具有M-CSF的培养物中分化为BMCs时，Lyz 2/Lyz 2-cre表达被诱导。在破骨细胞分化培养物中，在培养的前3天，M-CSF逐渐增加Lyz 2，然后在接下来的3天内加入RANKL和M-CSF迅速降低。此外，当从培养开始与M-CSF和RANKL同时培养时，BMCs分化成破骨细胞，同时保持低水平的Lyz 2表达。这些发现表明，Lyz 2-cre表达沿着由从Irf 8 fl/fl ; Lyz 2 cre/+小鼠获得并与M-CSF一起培养的BMCs分化为BMCs。此外，Irf 8被下调的cre/loxP重组系统的激活，在BERGOS和破骨细胞生成加速。基于我们的研究结果，我们提出了在体内存在一个新的谱系的破骨细胞前体之间的BMC，分化成破骨细胞没有上调Lyz 2的表达。本文的在线版本（doi：10.1007/s10616-016-0013-z）包含补充材料，可供授权用户使用。
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. The online version of this article (doi:10.1007/s10616-016-0013-z) contains supplementary material, which is available to authorized users.