Abstract
Binding of receptor activator of NF-κB ligand (RANKL) to its receptor RANK on osteoclast (OC) precursors up-regulates c-Fos and CCAAT/enhancer-binding protein-α (C/EBPα), two critical OC transcription factors. However, the effects of c-Fos and C/EBPα on osteoclastogenesis have not been compared. Herein, we demonstrate that overexpression of c-Fos or C/EBPα in OC precursors up-regulates OC genes and initiates osteoclastogenesis independently of RANKL. However, although C/EBPα up-regulated c-Fos, c-Fos failed to up-regulate C/EBPα in OC precursors. Consistently, C/EBPα overexpression more strongly promoted OC differentiation than did c-Fos overexpression. RANK has a cytoplasmic 535IVVY538 (IVVY) motif that is essential for osteoclastogenesis, and we found that mutation of the IVVY motif blocked OC differentiation by partly inhibiting expression of C/EBPα but not expression of c-Fos. We therefore hypothesized that C/EBPα overexpression might rescue osteoclastogenesis in cells expressing the mutated IVVY motif. However, overexpression of C/EBPα or c-Fos failed to stimulate osteoclastogenesis in the mutant cells. Notably, the IVVY motif mutation abrogated OC gene expression compared with a vector control, suggesting that the IVVY motif might counteract OC inhibitors during osteoclastogenesis. Consistently, the IVVY motif mutant triggered up-regulation of recombinant recognition sequence-binding protein at the Jκ site (RBP-J) protein, a potent OC inhibitor. Mechanistically, C/EBPα or c-Fos overexpression in the mutant cells failed to control the up-regulated RBP-J expression, leading to suppression of OC genes. Accordingly, RBP-J silencing in the mutant cells rescued osteoclastogenesis with C/EBPα or c-Fos overexpression with C/EBPα exhibiting a stronger osteoclastogenic effect. Collectively, our findings indicate that C/EBPα is a stronger inducer of OC differentiation than c-Fos, partly via C/EBPα regulation by the RANK 535IVVY538 motif.
Highlights
B–D, mouse bone marrow (MBM) cells expressing a GFP control, FLAG-NFATc1, FLAG-C/EBP␣, or FLAG-c-Fos were treated with macrophage colony-stimulating factor (M-CSF) (10 ng/ml) for 4 days for Western blot analysis using -actin as a loading control from at least three independent experiments (B) or tartrate-resistant acid phosphatase (TRAP) staining for osteoclastogenesis (C)
Transient stimulation of mouse bone marrow (MBM) cells, widely used as primary OC precursors, with M-CSF and RANKL was shown to up-regulate NFATc1, c-Fos, and C/EBP␣ during osteoclastogenesis (14 –16, 25). We recapitulated this finding and confirmed that stimulation of MBM cells by M-CSF and RANKL for 12 h could up-regulate the expressions of NFATc1, C/EBP␣, and c-Fos as compared with M-CSF alone (Fig. 1A), supporting their involvement in early stages of osteoclastogenesis
In addressing the molecular basis of this finding, we showed that overexpression of C/EBP␣, c-Fos, or NFATc1 could significantly up-regulate the OC genes encoding cathepsin K (Ctsk) (Fig. 1E) and TRAP (Fig. 1F) as compared with a GFP control in the absence of RANKL [27]
Summary
C/EBP␣ overexpression shows a stronger osteoclastogenic effect than c-Fos overexpression c-Fos is a critical transcription factor for osteoclastogenesis [12, 13]. Stronger osteoclastogenic effect than c-Fos. we found that MBM cells doubly expressing Fas-mIVVY and C/EBP␣, c-Fos, or NFATc1 failed to generate OCs with 10 (Fig. 4, C and D) or even 100 ng/ml (Fig. 4, C and E) ␣-Fas as compared with cells expressing the chimeric receptor with normal RANK, indicating that gene overexpression could not rescue osteoclastogenesis from inactivation of the RANK IVVY motif. We found that, to the RANKL-induced osteoclastogenesis assays (Fig. 2, E and F), C/EBP␣ or c-Fos overexpression showed no overt effect on OC size from the ␣-Fas-mediated osteoclastogenesis assays (Fig. 4, F and G) In confirming this finding, we demonstrated that RAW264.7 cells doubly expressing Fas-RANK and C/EBP␣, c-Fos, or NFATc1 could promote OC differentiation with both permissive (10 ng/ml) and optimum (100 ng/ml) ␣-Fas stimulation as compared with the GFP control (Fig. S3, A–E). The results indicated that RBP-J silencing could initiate osteoclastogenesis in cells expressing the mutated RANK IVVY motif, and overexpression of C/EBP␣, c-Fos, or NFATc1 in the mutant cells could further enhance OC differentiation
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