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

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.

Fig. 1

Irf8 ^fl/fl ;Lyz2 ^cre/+ mice did not develop osteoporosis. a Representative μCT images of vertical (upper panels) and axial (lower panels) tibiae obtained from 8-week-old male mice of the indicated genotypes. b μCT analysis of bone volume per tissue volume (BV/TV) of tibia trabecular bone (wild-type, n = 8; Irf8 ^−/−, n = 6; control, n = 20; Irf8 ^fl/fl ;Lyz2 ^cre/+, n = 23). **p < 0.01; n.s. not significant

Fig. 2

Osteoclastogenesis induced in vitro by M-CSF and RANKL was enhanced in cultures of BMMs obtained from Irf8 ^fl/fl ;Lyz2 ^cre/+ mice. a Schema of conventional osteoclast culture system. b Representative TRAP staining of osteoclasts differentiated from BMMs cultured with M-CSF and RANKL (20 ng/mL) for the indicated genotypes (upper panels). Shown is TRAP activity of osteoclasts differentiated from BMMs cultured with M-CSF and the indicated doses of RANKL (lower graphs). c qPCR analysis of Irf8 mRNA expression in BMCs from control and Irf8 ^fl/fl ;Lyz2 ^cre/+ mice cultured with M-CSF for 0–4 days. d Western blot analysis of Irf8 and β-actin in BMMs differentiated from BMCs after 3 days of culture with M-CSF. *p < 0.05, **p < 0.01; n.s. not significant

Fig. 3

Lyz2 is up-regulated by M-CSF and down-regulated by RANKL in BMC and BMM cultures. a qPCR analysis of Ctsk, Irf8, and Lyz2 mRNA expressions in BMCs from wild-type mice cultured with M-CSF for days 0–3, then with M-CSF and RANKL (100 ng/mL) for days 4–6. b Schema of direct osteoclast culture system using BMCs. c Representative TRAP staining of osteoclasts differentiated from BMCs cultured with only M-CSF, or simultaneously with both M-CSF and RANKL (100 ng/mL) from the initiation of culture. d qPCR analysis of Ctsk, Irf8, and Lyz2 mRNA expressions in BMCs from wild-type mice cultured with only M-CSF, or simultaneously with both M-CSF and RANKL (100 ng/mL) for 4 days from the initiation of culture (lower panel). *p < 0.05, **p < 0.01

Fig. 4

Schematic representation of osteoclastogenesis of Irf8 ^fl/fl ;Lyz2 ^cre/+ mouse BMCs in vitro and in vivo. a Lyz2-cre was induced along with differentiation of BMCs from Irf8 ^fl/fl ;Lyz2 ^cre/+ mice into BMMs in cultures with M-CSF. Irf8 in the BMMs was then deleted by activation of the cre/loxP recombination system and osteoclastogenesis was accelerated. b Proposed novel in vivo differentiation lineage, in which osteoclast precursors among BMCs differentiate into osteoclasts by simultaneous exposure to M-CSF and RANKL, and do not show Lyz2/Lyz2-cre expression. This differentiation lineage may be dominant in vivo and could explain why Irf8 ^fl/fl ;Lyz2 ^cre/+ mice do not develop osteoporosis