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The transcription factor Sox4 is vital for fetal development, as Sox4-/- homozygotes die in utero. Sox4 mRNA is expressed in the early embryonic growth plate and is regulated by parathyroid hormone, but its function in bone modeling/remodeling is unknown. We report that Sox4+/- mice exhibit significantly lower bone mass (by dual-energy X-ray absorptiometry) from an early age, and fail to obtain the peak bone mass of wild-type (WT) animals. Microcomputed tomography (µCT), histomorphometry and biomechanical testing of Sox4+/- bones show reduced trabecular and cortical thickness, growth plate width, ultimate force and stiffness compared with WT. Bone formation rate (BFR) in 3-month-old Sox4+/- mice is 64% lower than in WT. Primary calvarial osteoblasts from Sox4+/- mice demonstrate markedly inhibited proliferation, differentiation and mineralization. In these cultures, osterix (Osx) and osteocalcin (OCN) mRNA expression was reduced, whereas Runx2 mRNA was unaffected. No functional defects were found in osteoclasts. Silencing of Sox4 by siRNA in WT osteoblasts replicated the defects observed in Sox4+/- cells. We demonstrate inhibited formation and altered microarchitecture of bone in Sox4+/-- mice versus WT, without apparent defects in bone resorption. Our results implicate the transcription factor Sox4 in regulation of bone formation, by acting upstream of Osx and independent of Runx2. [ABSTRACT FROM AUTHOR]

The human estrogen receptor alpha (ERalpha) gene is driven by multiple promoters, of which the F promoter alone is found to be active in primary osteoblasts. The study was aimed at identifying new regulatory pathways affecting transcription of the receptor in this cell lineage. We generated human osteoblast-like cells, Saos-2, stably transfected with a luciferase-reporter gene downstream of the human ERalpha F promoter (Saos F-Luc), and assayed the reporter response to differentiation-related signals. Over-confluence, shown to stimulate osteoblast differentiation, caused a time-dependent increase of F-promoter activity and correlated with an inactivation of protein kinase C alpha (PKCalpha ). PKC downregulation, obtained by long-term treatment with phorbol 12-myristate 13-acetate (PMA), resulted in promoter stimulation at similar levels in sub-confluent cells. The F promoter contains a putative PMA-responsive AP-1 site, but AP-1 activation was unremarkable in over-confluent cells. Treatment with PP1, a specific inhibitor of the non-receptor tyrosine-kinase c-Src, which is a negative regulator of osteoblast differentiation, showed that the activity of this kinase inhibits the F promoter. In PP1-treated cells, F-promoter activity was not further increased by PMA. Treatment with the generic kinase inhibitor 4-dimethylaminopyridine (DMAP) resulted in a dose-dependent induction of the promoter, which matched a parallel decrease of active c-Src. The effect was c-Src dependent, as DMAP caused no further promoter induction in PP1-treated cells. Overexpression of exogenous human ERalpha resulted in modest promoter stimulation, which required the ligand-independent activator function 1 of the receptor. In murine primary osteoblasts, additional ERalpha signal was observed upon induction of F promoter. In conclusion, we demonstrated a robust PKC/c-Src-dependent and estrogen-independent mechanism modulating transcription of ERalpha in osteoblasts, probably affecting estrogen responsiveness during cell differentiation.