Patricia Ducy

Osf2/Cbfa1: A Transcriptional Activator of Osteoblast Differentiation

The osteoblast is the bone-forming cell. The molecular basis of osteoblast-specific gene expression and differentiation is unknown. We previously identified an osteoblast-specific cis-acting element, termed OSE2, in the Osteocalcin promoter. We have now cloned the cDNA encoding Osf2/Cbfa1, the protein that binds to OSE2. Osf2/Cbfa1 expression is initiated in the mesenchymal condensations of the developing skeleton, is strictly restricted to cells of the osteoblast lineage thereafter, and is regulated by BMP7 and vitamin D3. Osf2/Cbfa1 binds to and regulates the expression of multiple genes expressed in osteoblasts. Finally, forced expression of Osf2/Cbfa1 in nonosteoblastic cells induces the expression of the principal osteoblast-specific genes. This study identifies Osf2/Cbfa1 as an osteoblast-specific transcription factor and as a regulator of osteoblast differentiation.

A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation

The growth arrest and DNA damage-inducible 45β (GADD45β) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45β in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45β as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45β mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45β protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45β-/- mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45β in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45β stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45β plays an essential role during chondrocyte terminal differentiation.

STUDY OF OSTEOBLAST-SPECIFIC EXPRESSION OF ONE MOUSE OSTEOCALCIN GENE: CHARACTERIZATION OF THE FACTOR BINDING TO OSE2

In an attempt to understand the mechanisms of osteoblast-specific expression, we analyzed the promoter of the mouse osteocalcin gene 2, an osteoblast-specific gene. In this promoter, using a combination of DNA transfection experiments and DNA binding assays, we have identified two osteoblast-specific cis-acting elements called OSE1 and OSE2. Inspection of OSE2 DNA sequence and site-specific mutagenesis allowed us to define a core sequence for OSE2. This core sequence is identical to the DNA binding site of the PEBP2 alpha transcription factors, the mouse homologues of the Drosophila Runt protein. Here we show that OSF2, the factor present in osteoblast nuclear extracts and binding to OSE2, is immunologically related to the PEBP2 alpha transcription factors. In DNA cotransfection experiment, a human homologue of PEBP2 alpha transcription factor increases the activity of a short osteocalcin promoter through its binding to OSE2. Thus, this study presents evidence that OSF2 is a member of the PEBP2 alpha family of transcription factors.