Activation of osteocalcin transcription involves interaction of protein kinase A- and protein kinase C-dependent pathways

Osteocalcin is a major noncollagenous protein component of bone extracellul ar matrix, synthesized and secreted exclusively by osteoblastic cells in th e late stage of maturation, and is considered indicator of osteoblast diffe rentiation. Osteocalcin expression is modulated by parathyroid hormone (PTH ) and a variety of other factors. The cAMP-dependent protein kinase pathway has been shown previously to have an essential role in PTH signaling and r egulation of osteocalcin expression. To determine the extent to which other pathways may also participate in osteocalcin expression, we used rat and h uman osteoblast-like cell lines to generate stably transfected clones in wh ich the osteocalcin promoter was fused to a luciferase reporter gene. These clones were examined for their responsiveness to agents known to activate or interfere with protein kinase A (PKA)- and protein kinase C (PKC)-depend ent pathways. We have found that forskolin, cAMP, and PTH, as well as insul in-like growth factor I (IGF-I) and basic fibroblast growth factor, all wer e effective in activating the osteocalcin promoter. Phorbol 12-myristate 13 -acetate (PIMA) was also a strong inducer of the promoter, indicating that PKC plays a role in expression of osteocalcin. In combination with PTH or f orskolin, the effect of PMA was additive to synergistic. Calphostin C, a se lective inhibitor of PRC, decreased the PMA-, PTH-, and IGF-I-induced lucif erase activity in a dose-dependent manner; a PKA inhibitor, H-89, also bloc ked the induction by PTH and IGF-I but not by PMA. We conclude that regulat ion of osteocalcin transcription is mediated by both PKA-dependent and PKC- dependent mechanisms and that the respective kinases reside on a linear or convergent pathway.