C/EBPα and PU.1 exhibit different responses to RANK signaling for osteoclastogenesis

The transcription factors C/EBPα and PU.1 are upregulated by RANKL through activation of its receptor RANK during osteoclastogenesis and are critical for osteoclast differentiation. Herein we investigated the mechanisms underlying how C/EBPα and PU.1 regulate osteoclast differentiation in response to RANK signaling. We showed that C/EBPα or PU.1 overexpression could initiate osteoclastogenesis and upregulate the expressions of the osteoclast genes encoding the nuclear factor of activated T-cells, C1, cathepsin K, and tartrate-resistant acid phosphatase independently of RANKL. However, while PU.1 upregulated C/EBPα, C/EBPα could not upregulate PU.1. RANK has a unique cytoplasmic domain, 535IVVY538 motif, which is crucial for osteoclast differentiation. We demonstrated that mutational inactivation of RANK IVVY motif blocked osteoclast differentiation and significantly attenuated C/EBPα, but not PU.1, expression, indicating that RANK-IVVY-induced signaling is dispensable to PU.1 upregulation during osteoclastogenesis. However, C/EBPα or PU.1 overexpression failed to promote osteoclastogenesis in cells expressing mutated RANK IVVY motif. We noted that RANK-IVVY-motif inactivation significantly repressed osteoclast genes as compared with a vector control, suggesting that IVVY motif might also negatively regulate osteoclast inhibitors during osteoclastogenesis. Consistently, IVVY-motif inactivation triggered upregulation of RBP-J, a potent osteoclast inhibitor, during osteoclastogenesis. Notably, C/EBPα or PU.1 overexpression in cells expressing mutated RANK IVVY motif failed to control the deregulated RBP-J expression, resulting in repression of osteoclast genes. Accordingly, RBP-J silencing in the mutant cells rescued osteoclastogenesis with C/EBPα or PU.1 overexpression. In conclusion, we revealed that while PU.1 and C/EBPα are critical for osteoclastogenesis, they respond differently to RANKL-induced activation of RANK IVVY motif.