Temporal transcriptomic profiling uncovers multilevel suppression of RANKL-driven osteoclast differentiation by...

Receptor activator of nuclear factor κB ligand (RANKL) is the central driver of osteoclast differentiation and function, yet the regulatory checkpoints that restrain RANKL signaling remain incompletely defined. We previously demonstrated that 14-3-3ζ (Ywhaz) suppresses RANKL signaling by targeting TRAF6 at the post-translational level. Here, we identify a previously unrecognized transcriptional mechanism by which 14-3-3ζ constrains RANKL-induced osteoclastogenesis. Using an unbiased, time-resolved transcriptomic analysis (0-40 h) in primary rat bone marrow-derived preosteoclasts, we show that loss of 14-3-3ζ augments RANKL-induced expression of canonical osteoclast genes (Acp5, Ctsk, Car2), confirming its suppressive role in osteoclast differentiation. Beyond core osteoclast markers, 14-3-3ζ deficiency enhanced the induction of chemokines (Ccl7), focal adhesion (Rac2), and lysosomal markers (Lamp1, CTSZ), identifying its role in regulating the amplification of RANKL signaling. Mechanistically, we observed that 14-3-3ζ targets both positive and negative regulators of RANKL. While 14-3-3ζ promoted Lgr4, an inhibitor of RANKL signaling and NFATc1 expression, it concurrently suppressed SHP-2 (Ptpn11) expression, a phosphatase that promotes cell-cell fusion, a critical late step in osteoclast formation. Inhibition of SHP-2 activity with a specific pharmacological inhibitor abolished osteoclast differentiation in 14-3-3ζ-deficient cells, establishing SHP-2 as a functional effector downstream of 14-3-3ζ. Collectively, these findings reveal that 14-3-3ζ exerts dual transcriptional control over RANKL signaling, limiting both early osteoclastogenic gene induction and late-stage cell fusion. Loss of this regulatory brake increases susceptibility to RANKL-induced osteoclastogenesis, providing a mechanistic basis for enhanced bone loss, and highlighting 14-3-3ζ as a potential therapeutic target in inflammatory bone disorders.