PARATHYROID-HORMONE (PTH-1-34) REGULATION OF RAT OSTEOCALCIN GENE-TRANSCRIPTION

Osteocalcin (OC) is a bone-specific extracellular matrix protein expre ssed by mature osteoblasts during late stages of differentiation. Prev ious studies have shown that forskolin, an activator of adenylate cycl ase, stimulated OC production. Because PTH has been shown to activate several intracellular signal transduction pathways including cAMP, ino sitol phosphate and intracellular calcium mobilization, we investigate d whether PTH action on cAMP accumulation leads to OC promoter activat ion. The rat OC promoter (1095 bp) was cloned into the promoterless lu ciferase gene reporter vector. The transcriptional activity of the rat OC promoter was evaluated after transfection of SaOS-2, an osteosarco ma cell line, with the OC promoter followed by treatment with PTH. Max imal OC promoter activity was observed within 4-8 h after the addition of 10(-8) M PTH, whereas very Little induction was seen after 24 and 48 h of treatment. The induction of OC promoter activity by PTH was co ncentration dependent. PTH analogs (PTH 1-84, PTH 1-34, and PTH 1-31) that stimulate intracellular cAMP accumulation, induced OC promoter ac tivity, whereas other PTH: analogs (PTH 3-34, PTH 7-34, PTH 13-34, and PTH 53-84) that do not stimulate cAMP production had no effect on OC promoter activation. Furthermore, PTH activation of the OC promoter wa s significantly enhanced in the presence of 3-isobutyl-1-methylxanthin e (IBMX), a phosphodiesterase inhibitor. Inactivation of cAMP-dependen t protein kinase A activity by either a selective protein kinase A inh ibitor, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5 isoquinolinesulfonam ide), or antisense oligonucleotide directed against the regulatory sub unit of cAMP-dependent protein kinase A, led to a corresponding loss o f OC promoter activation by PTH. 5' deletion analysis of the OC promot er demonstrated that the promoter (1095 bp) exhibited the greatest res ponse to PTH, whereas the -198 bp construct of the OC promoter, contai ning only one cAMP response element and OC box, was no longer responsi ve. The constructs with further deletions (-120, -92, and -74) retaine d PTH responsiveness, but to a lesser extent. In summary, our results indicate that PTH activation of the OC promoter is a rapid event and m ediated by the cAMP-dependent protein kinase A pathway. Although the n ovel cAMP response region overlapping the OC box is required for activ ation, full activation may require several cis-acting cAMP response el ements or other response elements.