Activation of Osteocalcin Transcription Involves Interaction of Protein Kinase A- and Protein Kinase C-dependent Pathways

Abstract

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 insulinlike 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 PKAdependent and PKC-dependent mechanisms and that the respective kinases reside on a linear or convergent pathway.

Parathyroid Hormone Induction of the Osteocalcin Gene

“…In vitro, PTH inhibits bone marker gene expression and mineralization in differentiated osteoblasts and cementoblasts (49,56). In contrast, PTH rapidly induces osteoblastspecific gene expression in undifferentiated, proliferating osteoblasts (8,16,57,58). Thus, PTH may preferentially stimulate osteoblast differentiation in immature cells while inhibiting it in more mature cells.…”

“…For example, Boguslawski et al (16) showed that PKA and PKC pathways cooperatively interact in PTH-mediated OCN gene expression in rat and human osteoblast cell lines. Consistent with these reports, the present study shows that these two pathways are also required for PTH-induced mouse OCN gene expression in MC3T3-E1 preosteoblast cells.…”

“…Although the functions of OCN are poorly understood, gene deletion studies suggest possible functions in bone remodeling (10). The expression of OCN is regulated by a number of calcitropic hormones and growth factors including 1,25-dihydroxy vitamin D 3 (11)(12)(13), glucocorticoids (14,15), PTH (16), bone morphogenetic proteins (17), basic fibroblast growth factor 2 (18), tumor necrosis factor-␣ (19), and transforming growth factor ␤ (20). A number of transcription factors that bind to specific regions of the osteocalcin gene promoter have also been identified.…”

Diverse Effects of Cyclic AMP Variants on Osteogenic and Adipogenic Differentiation of Human Mesenchymal Stromal Cells

“…via cAMP and forskolin [23]. In contrast, numerous studies have implicated this pathway in adipogenic differentiation.…”

A Tryptamine Derivative, SST-VEDI-1, Inhibits Apoptosis and Stimulates Mineralization in Osteoblasts

“…some amines derived from thyroid hormone decarboxylation and deiodination, known as thyronamines, have been shown to be endogenous compounds that stimulate caMP production via activation of Taar1 [36]. Cyclic aMP signaling regulates both osteogenic and apoptotic events [37,38]. VEdi-1 and thyronamines have a common basic chemical structure, thus we set out to determine whether VEdi-1 affects caMP production in osteoblasts.…”