Bone homeostasis is maintained by a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteoclast differentiation is induced by receptor activator of nuclear factor-kB ligand (RANKL), which activates the master transcription factor NFATc1; however, the mechanisms regulating NFATc1 at the protein level remain unclear. Ctdnep1 is a negative regulator of the BMP/TGF-beta and RANKL signaling pathways.

Knockdown of Ctdnep1 in RAW264.7 cells increases NFATc1 protein levels without altering its mRNA expression, suggesting post-translational regulation. This study aimed to elucidate the mechanisms by which Ctdnep1 regulates NFATc1 and osteoclast differentiation.

In myeloid-specific Ctdnep1-deficient mice, micro-computed tomography analysis revealed no significant changes in trabecular or cortical bone mass or osteoclast formation in vivo. In contrast, ex vivo osteoclast differentiation assays using bone marrow-derived macrophages showed increased formation of TRAP-positive multinucleated cells upon RANKL stimulation. Expression of osteoclast marker genes and NFATc1 protein levels were also elevated, indicating enhanced osteoclastogenesis.

Mechanistically, overexpression of Ctdnep1 in HEK293T cells reduced cytoplasmic NFATc1 protein levels. NFATc1 partially co-localized with Ctdnep1, and its signal intensity was decreased in regions with high Ctdnep1 expression, suggesting reduced protein stability. Furthermore, the transcriptional activity of constitutively active NFATc1 was suppressed by Ctdnep1.

The lack of an in vivo phenotype may reflect limited osteoclastogenic stimuli under physiological conditions, whereas under inflammatory or aging conditions, Ctdnep1 deficiency may further exacerbate bone resorption, highlighting its importance in pathological bone remodeling processes and diseases.