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