Activating transcription factor 4 regulates osteoclast differentiation in mice
Huiling Cao, Shibing Yu, Zhi Yao, Deborah L. Galson, Yu Jiang, Xiaoyan Zhang, Jie Fan, Binfeng Lu, Youfei Guan, Min Luo, Yumei Lai, Yibei Zhu, Noriyoshi Kurihara, Kenneth Patrene, G. David Roodman, Guozhi Xiao
Huiling Cao, Shibing Yu, Zhi Yao, Deborah L. Galson, Yu Jiang, Xiaoyan Zhang, Jie Fan, Binfeng Lu, Youfei Guan, Min Luo, Yumei Lai, Yibei Zhu, Noriyoshi Kurihara, Kenneth Patrene, G. David Roodman, Guozhi Xiao
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Research Article Bone biology

Activating transcription factor 4 regulates osteoclast differentiation in mice

Abstract

Activating transcription factor 4 (ATF4) is a critical transcription factor for osteoblast (OBL) function and bone formation; however, a direct role in osteoclasts (OCLs) has not been established. Here, we targeted expression of ATF4 to the OCL lineage using the Trap promoter or through deletion of Atf4 in mice. OCL differentiation was drastically decreased in Atf4–/– bone marrow monocyte (BMM) cultures and bones. Coculture of Atf4–/– BMMs with WT OBLs or a high concentration of RANKL failed to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity.

Authors

Huiling Cao, Shibing Yu, Zhi Yao, Deborah L. Galson, Yu Jiang, Xiaoyan Zhang, Jie Fan, Binfeng Lu, Youfei Guan, Min Luo, Yumei Lai, Yibei Zhu, Noriyoshi Kurihara, Kenneth Patrene, G. David Roodman, Guozhi Xiao

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Figure 6

ATF4 is upregulated by M-CSF and PI3K/AKT and is required for M-CSF induction of RANK expression in BMMs.

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ATF4 is upregulated by M-CSF and PI3K/AKT and is required for M-CSF indu...
(A and B) Effects of M-CSF on ATF4 in BMMs. Cells were cultured with or without 30 ng/ml M-CSF for the indicated times, followed by Western blot (A) or real-time RT-PCR (B) for ATF4. (C) Effects of various inhibitors or activators on the level of ATF4 in BMMs. Cells were cultured in M-CSF–containing medium with and without the indicated inhibitors or activators (10 μM) for 24 hours. LY, LY294002; SB, SB209580; GF, GF109203X. (D) Effect of PI3K/AKT inhibition on OCL differentiation. BMMs were seeded in proliferation medium for 3 days and treated with increasing concentrations of LY294002 for 24 hours. Inhibitor was then removed by switching cells to differentiation medium for 5 days, followed by TRAP staining. *P < 0.01 versus 0 μm. (E and F) COS-7 cells were transfected with 1.0 μg pCMV/ATF4 expression plasmid. After 24 hours, cells were treated with or without 10 μM LY294002 as well as with or without 10 μg/ml CHX (E) or 10 μM MG115 (F) for another 24 hours. (G) IHC. Purified CD11b+ BMMs were seeded in proliferation medium for 72 hours, followed by IHC with an anti-RANK antibody or control IgG. (H) Western blot. Primary BMMs were seeded in 35-mm dishes in proliferation medium for 72 hours. (I) Real-time RT-PCR. WT and Atf4–/– BMMs were cultured in proliferation medium for 3 days and switched to 2% FBS α-MEM without M-CSF overnight. Cells were then treated with 10 ng/ml M-CSF for the indicated times. *P < 0.01, WT versus KO. Original magnification, ×200.