SUMMARY Parathyroid hormone (PTH) suppresses Dickkopf 1 (Dkk1) expression in osteoblasts. To determine whether this suppression is essential for PTH-mediated Wnt signaling and bone formation, we examined mice that overexpress Dkk1 in osteoblasts (Dkk1 mice). Dkk1 mice were osteopenic due to abnormal osteoblast and osteoclast activity. When fed a low calcium diet, and in two other models of hyperparathyroidism, these mice failed to develop the peritrabecular stromal cell response (“osteitis fibrosis”) and new bone formation seen in wild type mice. Despite these effects of Dkk1 overexpression, PTH still activated Wnt signaling in Dkk1 mice and in osteoblastic cells cultured from these mice. In cultured MC3T3E1 preosteoblastic cells, PTH dramatically suppressed Dkk1 expression, induced PKA-mediated phosphorylation of β-catenin and significantly enhanced Lef1 expression. Our findings indicate that the full actions of PTH require intact Wnt signaling but that PTH can activate the Wnt pathway despite overexpression of Dkk1.

The Wnt signaling pathway has recently been demonstrated to play an important role in bone cell function. In previous studies using DNA microarray analyses, we observed a change in some of the molecular components of the canonical Wnt pathway namely, frizzled-1 (FZD-1) and axil, in response to continuous parathyroid hormone (PTH) treatment in rats. In the present study, we further explored other components of the Wnt signaling pathway in rat distal metaphyseal bone in vivo, and rat osteoblastic osteosarcoma cells (UMR 106) in culture. Several Wnt pathway components, including low-density lipoprotein-receptor-related protein 5 (LRP5), LRP6, FZD-1, Dickkopf-1 (Dkk-1), and Kremen-1 (KRM-1), were expressed in bone in vivo and in osteoblasts in vitro. Continuous exposure to PTH (1-38) both in vivo and in vitro upregulated the mRNA expression of LRP6 and FZD-1 and decreased LRP5 and Dkk-1. These effects in UMR 106 cells were associated with an increase in beta-catenin as measured by Western blots and resulted in functional activation (three to six-fold) of a downstream Wnt responsive TBE6-luciferase (TCF/LEF-binding element) reporter gene. Activation of the TBE6-luciferase reporter gene by PTH (1-38) in UMR 106 cells was inhibited by the protein kinase A (PKA) inhibitor, H89. Activation was mimicked by PTH (1-31), PTH-related protein (1-34), and forskolin, but both PTH (3-34) and (7-34) had no effect. These findings suggest that the effect of PTH on the canonical Wnt signaling pathway occurs at least in part via the cAMP-PKA pathway through the differential regulation of the receptor complex proteins (FZD-1/LRP5 or LRP6) and the antagonist (Dkk-1). Taken together, these results reveal a possible role for the Wnt signaling pathway in PTH actions in bone.

To better understand the complex roles of transforming growth factor-beta (TGF-β) in bone metabolism, we examined the impact of a range of TGF-β concentrations on osteoclast differentiation. In co-cultures of support cells and spleen or marrow osteoclast precursors, low TGF-β concentrations stimulated while high concentrations inhibited differentiation. We investigated the influences of TGF-β on macrophage colony stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) expression and found a dose dependent inhibition of M-CSF expression. RANKL expression was elevated at low TGF-β concentrations with a less dramatic increase in OPG. Addition of OPG blocked differentiation at the stimulatory TGF-β dose. Thus, low TGF-β concentrations elevated the RANKL/OPG ratio while high concentrations did not, supporting that, at low TGF-β concentrations, there is sufficient M-CSF and a high RANKL/OPG ratio to stimulate differentiation. At high TGF-β concentrations, the RANKL/OPG ratio and M-CSF expression were both repressed and there was no differentiation. We examined whether TGF-β-mediated repression of osteoclasts differentiation is due to these changes by adding M-CSF and/or RANKL and did not observe any impact on differentiation repression. We studied direct TGF-βimpacts on osteoclast precursors by culturing spleen or marrow cells with M-CSF and RANKL. TGF-β treatment dose-dependently stimulated osteoclast differentiation. These data indicate that low TGF-β levels stimulate osteoclast differentiation by impacting the RANKL/OPG ratio while high TGF-β levels repress osteoclast differentiation by multiple avenues including mechanisms independent of the RANKL/OPG ratio or M-CSF expression regulation.Transforming growth factor-beta (TGF-β) is a ubiquitous multifunctional cytokine that has a spectrum of influences. The variety of reported responses to TGF-β depends, at least in part, on experimental conditions as well as the cell type under study. Within the bone environment, TGF-β is a key regulator of bone metabolism. Although all TGF-β isoforms bind to the same receptor complex, there have been some reports of different cellular responses to the different isoforms (Jennings et al

Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblastsecreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE 2 )), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the ␤-galactosidase reporter gene (pOPG5.9␤gal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9␤gal reporter construct led to 39-, 7-, and 16-fold increases in ␤-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE 2 elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE 2 element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequencespecific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.Bone growth, development, and maintenance in mammals is a highly regulated process. The level of bone mass is dependent on the balance between bone formation and resorption. At the cellular level, this balance involves the coordinate regulation and interaction of the component cell types: bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts are derived from mesenchymal stem cells, and their formation and function are under the control of an osteoblast-specific transcription factor known as core binding factor a1 (Cbfa1) or osteoblast specific factor 2 (Osf2) 1 (1-3). Cbfa1 has been shown to regulate the expression of genes that characterize the osteoblast phenotype, including osteocalcin, osteopontin, type I collagen, bone sialoprotein, and collagenase-3, by binding to DNA sequence elements called OSE 2 that are present in the control regions of these genes (1, 4 -6). Cbfa1 knock out animals completely lack bones because of a maturational arrest of osteoblasts and die soon after delivery (2, 3). Further, haploinsufficiency of the Cbfa1 gene product in mice and humans heterozygous for the Cbfa...

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