We previously demonstrated a correlation between wild-type p53 expression and appearance of osteoblastic-specific differentiation characteristics, as evidenced by basal osteocalcin gene expression in a mouse osteosarcoma tumor. The study reported here further explored the possibility of p53's having a distinct transcription-activating role in bone differentiation, in addition to its proposed role in G1 arrest and apoptosis. ROS17/2.3 osteoblastic osteosarcoma cells were stably transfected with a plasmid containing wild-type p53 binding sequences fused to the chloramphenicol acetyltransferase reporter gene. These cells were used to determine the transactivating role of p53 in regulation of osteocalcin gene expression. We chose two conditions under which osteocalcin expression is known to be upregulated: exposure of osteoblastic cells to differentiation-promoting medium and to vitamin D3. Exposure of the transfected cells to differentiation-promoting medium produced an increase in p53 transactivating activity correlating with the appearance of osteocalcin expression after about 1 wk. Vitamin D3 treatment resulted in upregulation of osteocalcin activity without a corresponding change in p53 transactivation activity or expression. In separate experiments, we tested whether changes in osteocalcin expression accompanied changes in p53 activity under conditions of downregulation of cell proliferation mediated by inhibition of DNA synthesis. Hydroxyurea treatment was used to inhibit DNA synthesis and produce growth arrest in osteoblastic cells. Inhibition of osteoblast cell proliferation was associated with a fourfold increase in p53 transactivating activity and a transient increase in osteocalcin steady-state expression. These results demonstrated a close relationship between p53 and osteocalcin and suggested a regulatory role for wild-type p53 in the control of basal osteocalcin gene expression in osteoblasts.

While estrogen's role in maintaining bone health relates to its action on osteoclasts, not much is presently known about the role of estrogen with respect to osteoblasts. Our laboratory is involved in studying the function of the p53 tumor suppressor gene in osteoblast differentiation. This study was therefore designed to understand the role of estrogen in osteoblast growth and differentiation and its effect on p53 function. ROS 17/2.8 cells, stably transfected with a construct containing multiple copies of a p53 response element fused to a chloramphenicol acetyl transferase (CAT) gene, were used to monitor wild-type p53 activity. Maximal p53 activity was observed when E2 was given at concentrations between 10(-12) and 10(-15) M. This increase in p53 activity was due to a change in transcription and peaked at about 16 hours after treatment. An increase in p53 activity was followed by an increase in expression of p53-regulated genes p21 and mdm2. This increase in p53 activity was partially inhibited by inclusion of estrogen antagonist ICI 182,780. Bone- specific markers osteocalcin and alkaline phophatase increased after treatment with E2, as did changes in estrogen receptors alpha and beta. Upregulation of osteocalcin was reduced when cycloheximide was added to E2, suggesting the presence of intermediates in the enhancement of osteocalcin gene transcription. These findings suggest that E2 can directly mediate an increase in p53 expression and function. The relevance of this to osteoblast differentiation is discussed.

Osteocalcin (OC) is a major noncollagenous bone matrix protein and an osteoblast marker whose expression is limited to mature osteoblasts during the late differentiation stage. In previous studies we have shown osteosarcomas to lose p53 function with a corresponding loss of osteocalcin gene expression. Introduction of wild type p53 resulted in re expression of the osteocalcin gene. Using gel shift and chromatin immunopreciptation assays, we have identified a putative p53 binding site within the rat OC promoter region and observed an increase in OC promoter activity when p53 accumulates using a CAT assay. The p53 inducible gene Mdm2 is a well-known downstream regulator of p53 levels. Our results showed a synergistic increase in the OC promoter activity when both p53 and MDM2 were transiently overexpressed. We further demonstrate that p53 is not degraded during overexpression of MDM2 protein. Increased OC expression was observed with concomitantly increased p53, VDR, and MDM2 levels in ROS17/2.8 cells during treatment with differentiation promoting (DP) media, but was significantly decreased when co-treated with DP media and the small molecule inhibitor of MDM2-p53 interaction, Nutlin-3. We have also observed a dramatic increase of the OC promoter activity in the presence of p53 and Mdm2 with inclusion of Cbfa-1 and p300 factors. Our results suggest that under some physiological conditions the oncoprotein MDM2 may cooperate with p53 to regulate the osteocalcin gene during osteoblastic differentiation.

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