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.
Highlights
The strength and health of bone tissues are regulated by a tight balance of bone resorption by osteoclasts and bone formation by osteoblasts
These results suggest that osteoclastogenesis in bone tissues of Irf8fl/fl;Lyz2cre/? mice is not enhanced in vivo
bone marrow-derived macrophages (BMMs) from the Irf8fl/fl;Lyz2cre/? mice with a low level of Interferon regulatory factor 8 (Irf8) expression induced by macrophage colony-stimulating factor (M-CSF) from bone marrow cells (BMCs) in vitro aggressively differentiated into osteoclasts by RANKL stimulation, similar to those from the Irf8-/- mice
Summary
The strength and health of bone tissues are regulated by a tight balance of bone resorption by osteoclasts and bone formation by osteoblasts. 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-jB 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. 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). Irf is expressed in osteoclast precursors and inhibits Nfatc function, and is downregulated by stimulation with RANKL, which leads to Nfatc auto-amplification and osteoclast differentiation.
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