Mesenchyme-specific overexpression of nucleolar protein 66 in mice inhibits skeletal growth and bone formation.

Previous studies showed that nucleolar protein 66 (NO66), the Jumonji C-domain-containing histone demethylase for methylated histone H3K4 and H3K36 (H3K36me), negatively regulates osteoblast differentiation in vitro by inhibiting the activity of transcription factor osterix (Osx). However, whether NO66 affects mammalian skeletogenesis in vivo is not yet known. Here, we generated transgenic (TG) mice overexpressing a flag-tagged NO66 transgene driven by the Prx1 (paired related homeobox 1) promoter. We found that NO66 overexpression in Prx1-expressing mesenchymal cells inhibited skeletal growth and bone formation. The inhibitory phenotype was associated with >50% decreases in chondrocyte/osteoblast proliferation and differentiation. Moreover, we found that in bones of NO66-TG mice, expression of Igf1, Igf1 receptor (Igf1r), runt-related transcription factor 2, and Osx was significantly down-regulated (P < 0.05). Consistent with these results, we observed >50% reduction in levels of phosphorylated protein kinase B (Akt) and H3K36me3 in bones of NO66-TG mice, suggesting an inverse correlation between NO66 histone demethylase and the activity of IGF1R/Akt signaling. This correlation was further confirmed by in vitro assays of C2C12 cells with NO66 overexpression. We propose that the decrease in the IGF1R/Akt signaling pathway in mice with mesenchymal overexpression of NO66 may contribute in part to the inhibition of skeletal growth and bone formation.