AP-1 REGULATION OF THE RAT BONE SIALOPROTEIN GENE-TRANSCRIPTION IS MEDIATED THROUGH A TPA RESPONSE ELEMENT WITHIN A GLUCOCORTICOID RESPONSEUNIT IN THE GENE PROMOTER

Bone sialoprotein (BSP), a protein which has been implicated in the in itial mineralization of newly-formed bone, provides an early phenotypi c marker for differentiated osteoblasts. BSP expression is induced by glucocorticoids in association with osteoblast differentiation, and a glucocorticoid response element (GRE) overlapping a putative TRE (TPA, 12-O-tetradecanoylphorbol 13-acetate, response element) site has been identified in the rat BSP promoter (Ogata et al., 1995). Since AP-1 a nd the glucocorticoid receptor have a central role in regulating cell proliferation and differentiation, we have studied AP-1 activity, stim ulated by 100 ng/ml TPA in normal fetal rat calvarial cells and in tra nsformed rat osteosarcoma cells (ROS 17/2.8). A transient induction of both c-fos and c-jun mRNAs by TPA was observed in both cell populatio ns, together with an associated suppression of BSP mRNA in the fetal r at calvarial cells. Rat BSP promoter constructs, transiently transfect ed into ROS 17/2.8 cells, were used to show that TPA suppressed transc ription of a luciferase construct (-938/+60; pLUC6) that included the GRE/TRE, but not transcription of shorter contructs lacking this eleme nt. Notably, suppression of pLUC6 transcription by TPA was abrogated i n the presence of the synthetic glucocorticoid, dexamethasone. Gel mob ility shift analyses were performed using two double-stranded syntheti c oligonucleotides. These encompassed the TRE and either the distal pa ir of GRE half-sites (-936/-910; GRE 3) or the proximal pair of GRE ha lf-sites (-925/-899; GRE 4) that comprise the GRE/AP-1 element. The as say showed binding of both AP-1 complexes and recombinant c-Jun homodi mers. Additionally, either the c-Jun or glucocorticoid receptor could displace its counterpart from the GRE/TRE but not from consensus GRE a nd TRE oligonucleotides, indicating that the abrogation of AP-1-mediat ed gene suppression by glucocorticoids could involve competitive bindi ng. These studies, therefore, have identified a glucocorticoid respons e unit through which c-Fos and c-Jun can suppress the expression of BS P in proliferating pre-osteoblastic cells and through which glucocorti coids can ameliorate the effects of AP-1 and promote osteoblast differ entiation and the associated expression of BSP.