Identification of signal transduction pathways and promoter sequences thatmediate parathyroid hormone 1-38 inhibition of osteoprotegerin gene expression

Osteoprotegerin (OPG), a secreted member of the tumor necrosis receptor sup erfamily, is a potent inhibitor of osteoclast formation and bone resorption . Parathyroid hormone (PTH), a potent inducer of osteoclast formation, supp resses OPG mRNA expression in vitro and in vivo. To determine the molecular basis of this inhibition, we analyzed the effects of PTH on the human OPG promoter (-5917 to +19) fused with beta -galactosidase reporter gene in sta ble and transient transfections into rat osteoblast-like UMR106 cells. The effect of PTH on OPG promoter expression was biphasic and concentration-dep endent. PTH (1-100 nM) induced the transcriptional activity of the OPG prom oter (1.7-fold) at 8 h followed by a gradual decrease with maximal inhibiti on (6.6-fold) at 24-48 h. To ascertain the signal transduction pathways med iating PTH (1-38) effects on OPG gene expression, we compared the effects o f PTH with PTH analogs, parathyroid hormone-related protein 1-34 (PTHrP 1-3 4), forskolin, 3-isobutyl-1-methylxanthine (IBMX), dibutyryl cAMP, phorbol- 12-myristate-13-acetate (PMA), thapsigargin and calcium ionophore A23187. P TH 1-31 and PTHrP 1-34, which stimulate the cAMP/PKA pathway, and other act ivators of cAMP/PKA, forskolin, IBMX, N-6, O-2'- dibityryl adenosine 3',5-c yclic monophosphate (dibutyryl cAMP), all elicited a similar biphasic respo nse on OPG promoter expression. PTH analogs PTH 3-34 and PTH 7-34, that do not stimulate cAMP production, had no effect on OPG expression. In contrast , phorbol-12-myristate-13-acetate (PMA), an activator of PKC, stimulated OP G promoter expression, while thapsigargin and calcium ionophore A23187, whi ch increase intracellular Ca2+, showed a close-dependent inhibition of OPG promoter expression. To delineate the promoter sequences that mediate the i nhibitory effects of PTH on OPG transcription, we analyzed systematic delet ions of the OPG promoter for responsiveness in transient transfection assay s. The major inhibitory effects of PTH were localized to 391 bp (-372 to +1 9) of the proximal promoter. Deletions of the promoter region led to a comp lete loss of responsiveness. Taken together, these results demonstrate that the inhibitory effects of PTH on OPG are mediated at the transcriptional l evel through cis elements in the proximal promoter. The similar biphasic re sponse of OPG to PTH, PTH 1-31, PTHrP 1-34, forskolin, IBMX and dibutyryl c AMP suggests that PTH regulates OPG transcription via activation of the cAM P/PKA signal transduction pathway. (C) 2001 Wiley-Liss, Inc.