Signal Transduction Pathways Mediating Parathyroid Hormone Stimulation of Bone Sialoprotein Gene Expression in Osteoblasts

Abstract

Bone sialoprotein is a major noncollagenous protein of bone. Parathyroid hormone (PTH) was shown to cause a 2-4-fold increase in the steady-state levels of bone sialoprotein mRNAs within primary cultures of embryonic osteoblasts. The induction could be mimicked by both forskolin and phorbol 12-myristate 13-acetate and was not inhibited by cycloheximide. Transient expression of a ϳ1200-base pair avian 5 bsp promoter/reporter construct demonstrated similar inductions as mRNA levels. Co-transfection of an expression plasmid encoding heat-stable inhibitor of cAMP-dependent protein kinase, a peptide inhibitor of PKA, decreased both the basal and PTH-induced bsp transcription, while co-expression of the catalytic subunit of PKA-induced bsp expression 3-fold. Protein kinase C activation, on the other hand, did not appear to work through its activation of c-fos, since co-transfection of an expression clone for c-fos had no effect. Interestingly, heat-stable inhibitor of cAMP-dependent protein kinase also inhibited the phorbol 12-myristate 13-acetate induction, suggesting that the protein kinase C acts through some form of interaction with the cAMP/PKA pathway. A half-cAMP response element site in the bsp promoter was identified as the cis-acting element that mediated the PTH response by the transient transfections with reporter constructs containing nested deletions of the promoter or a heterologous promoter containing the cAMP response element. In conclusion, these data indicate that PTH stimulation of bsp gene expression is specific to osteoblasts and mediated by changing cellular cAMP/PKA levels. They further suggest that although protein kinase C is capable of stimulating the gene by itself, it plays a minimal role in mediating the PTH induction of bone sialoprotein. PTH,1 like a number of other peptide hormones, mediates its effects through interaction with its receptor, which modulates its activities through specific G proteins that activate or inhibit adenylate cyclase production of cAMP. The levels of cAMP then control the activity of protein kinase A (PKA), which serves as the cAMP intracellular second signal transducer (1-3). There now is considerable evidence that the interaction of PTH with its receptor also leads to the activation of phospholipase C, which in turn causes intracellular calcium ion and diacylglycerol release and further activates members of the protein kinase C (PKC) family (4 -7). Thus, both PKA and PKC may be activated by PTH, and at the nuclear level the actions of both PKA and PKC families of kinases through the phosphorylation of specific members of the leucine zipper family of transcription factors affect the transcriptional activity of specific genes. These transcription factors fall primarily into two broad classes, the cAMP response element binding protein family or CREBs, which includes a variety of members of the CREB, CREM, and ATF classes of factors, and members of the AP1 family, whose primary members include fos, jun, and fra (8 -11). In general, the action of PKA is mediated t...

Human Osteocalcin and Bone Sialoprotein Mediating Osteomimicry of Prostate Cancer Cells: Role of cAMP-Dependent Protein Kinase A Signaling Pathway

“…Boguslawski et al (30) observed that rat and human osteoblast-like cell lines stably transfected with osteocalcin promoter reporter construct responded to parathyroid hormone and growth factors ( fibroblast growth factor-2 and insulin-like growth factor-I) via enhanced osteocalcin transcription mediated by a PKA-dependent pathway. A similar activation of bone sialoprotein promoter activity was documented for fibroblast growth factor-2 (54, 56), parathyroid hormone (33,53), and prostaglandin E 2 (34) in which bone sialoprotein transcription was stimulated through a PKA-dependent pathway, although a protein kinase C-mediated pathway may play a minor role (33). The present study differs from previous reports in two important aspects.…”

“…The cAMP-responsive element (CRE), a cis-element, interacts with a basic domain/leucine zipper motif contained within a transcription factor termed CRE-binding protein (CREB) through the cAMP-dependent PKA pathway (32). Studies in which both osteocalcin (30) and bone sialoprotein (33,34) promoter activities were stimulated by the cAMPdependent PKA pathway suggest the presence of the putative CRE site within these promoter regions that may interact with CREB to initiate osteocalcin and bone sialoprotein expression.…”

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.…”

runt Homology Domain Transcription Factors (Runx, Cbfa, and AML) Mediate Repression of the Bone Sialoprotein Promoter: Evidence for Promoter Context-Dependent Activity of Cbfa Proteins

“…The plasmid construct containing bp Ϫ1,239 to ϩ25 of the BSP promoter in the sense direction relative to the CAT coding sequence in the pCAT basic vector (Promega Corp.) was previously described (59). Nested deletions from the 5Ј end of the BSP promoter were generated by PCR amplification of selected segments of the promoter from bp Ϫ620, Ϫ524, or Ϫ131 to ϩ25 and have been previously described (58). Site-directed mutagenesis was performed to incorporate 2-nucleotide substitutions into the core binding motif (TGG) of each individual Cbfa site (Fig.…”