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 extracellular matrix, synthesized and secreted exclusively by osteoblastic cells in the late stage of maturation, and is considered indicator of osteoblast differentiation. 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 regulation of osteocalcin expression. To determine the extent to which other pathways may also participate in osteocalcin expression, we used rat and human osteoblast-like cell lines to generate stably transfected clones in which 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)-dependent pathways. We have found that forskolin, cAMP, and PTH, as well as insulin-like growth factor I (IGF-I) and basic fibroblast growth factor, all were effective in activating the osteocalcin promoter. Phorbol 12-myristate 13-acetate (PMA) was also a strong inducer of the promoter, indicating that PKC plays a role in expression of osteocalcin. In combination with PTH or forskolin, the effect of PMA was additive to synergistic. Calphostin C, a selective inhibitor of PKC, decreased the PMA-, PTH-, and IGF-I-induced luciferase activity in a dose-dependent manner; a PKA inhibitor, H-89, also blocked the induction by PTH and IGF-I but not by PMA. We conclude that regulation 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.
Boguslawski
G
G
Endocrine Division, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA.
Hale
L V
LV
Yu
X P
XP
Miles
R R
RR
Onyia
J E
JE
Santerre
R F
RF
Chandrasekhar
S
S
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D015675:Osteocalcin / Q000235:genetics*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects
D009281:Naphthalenes; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D012333:RNA, Messenger; D015675:Osteocalcin; D005576:Colforsin; D003994:Bucladesine; D007334:Insulin-Like Growth Factor I; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C058819:calphostin C