David L. Halladay

Catabolic Effects of Continuous Human PTH (1–38) in Vivo Is Associated with Sustained Stimulation of RANKL and Inhibition of Osteoprotegerin and Gene-Associated Bone Formation

Continuous infusion of PTH in vivo results in active bone resorption. To investigate the molecular basis of the catabolic effect of PTH in vivo, we evaluated the role of OPG and RANKL, which are known to influence osteoclast formation and function. Weanling rats fed a calcium-free diet were parathyroidectomized and infused with PTH via an Alzet pump to examine: 1) the changes of serum-ionized calcium and osteoclast number, 2) the expression of OPG/RANKL mRNA and protein, and 3) the expression of osteoblast phenotype bone formation-associated genes such as osteoblast specific transcription factor, osteocalcin, bone sialoprotein, and type I collagen. PTH (1–38) (0.01–20 μg/100 g) continuous infusion for 1–24 h resulted in a dose-dependent increase in serum-ionized calcium in parathyroidectomized rats and a corresponding dose-dependent increase in osteoclast number, indicating an increased bone resorption. At 20 μg/100 g PTH dose level, serum-ionized calcium was 2.1-fold of the vehicle control and not differ...

Effects of parathyroid hormone on Wnt signaling pathway in bone

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 β‐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.

The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function.

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 osteoblast-secreted 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 (OSE2)), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the β-galactosidase reporter gene (pOPG5.9βgal) 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 pOPG5.9βgal reporter construct led to 39-, 7-, and 16-fold increases in β-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE2 elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE2 element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequence-specific 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.

Orally bioavailable GSK-3α/β dual inhibitor increases markers of cellular differentiation in vitro and bone mass in vivo

GSK‐3, a component of the canonical Wnt signaling pathway, is implicated in regulation of bone mass. The effect of a small molecule GSK‐3 inhibitor was evaluated in pre‐osteoblasts and in osteopenic rats. GSK‐3 inhibitor induced osteoblast differentiation in vitro and increased markers of bone formation in vitro and in vivo with concomitant increased bone mass and strength in rats.

Changes in Osteoblast, Chondrocyte, and Adipocyte Lineages Mediate the Bone Anabolic Actions of PTH and Small Molecule GSK‐3 Inhibitor

Parathyroid hormone (PTH) and glycogen synthase kinase‐3 (GSK‐3) inhibitor 603281‐31‐8, administered once daily increased bone formation in vivo. We investigated the molecular mechanisms of the anabolic responses of PTH and 603281‐31‐8 in rat osteopenia model. Female 6‐month‐old rats were ovariectomized (Ovx) and permitted to lose bone for 1 month, followed by treatment with PTH (1–38) at 10 µg/kg/day s.c. or 603281‐31‐8 at 3 mg/kg/day p.o. for 60 days. Twenty‐four hours after the last treatment, RNA from distal femur metaphysis was subjected to gene expression analysis. Differentially expressed genes (P < 0.05) were subjected to pathway analysis to delineate relevant bio‐processes involved in skeletal biology. Genes involved in morphogenesis, cell growth/differentiation, and apoptosis were significantly altered by Ovx and the treatments. Analysis of morphogenesis genes showed an overrepresentation of genes involved in osteogenesis, chondrogenesis, and adipogenesis. A striking finding was that Ovx decreased several markers of osteogenesis/chondrogenesis and increased markers of adipogenesis/lipid metabolism. Treatment with either PTH or the GSK‐3 inhibitor reversed these effects, albeit at different levels. Histological analysis confirmed that osteopenia in Ovx animals was associated with three‐fold increase in marrow adiposity. PTH and GSK‐3 inhibitor restored bone volume, and reversed or normalized marrow adiposity. Ex vivo studies showed that PTH and GSK‐3 inhibitor increased the ratio of colony forming marrow stromal progenitors (CFU‐fs) that were alkaline phosphatase positive (putative osteoblasts). Our results suggest that the bone anabolic actions of PTH and GSK‐3 inhibitor in vivo involve concerted effects on mesenchymal lineages; osteoblasts, chondrocytes, and adipocytes. J. Cell. Biochem. 102: 1504–1518, 2007.

Analysis of regulator of G‐protein signaling‐2 (RGS‐2) expression and function in osteoblastic cells

Regulator of G‐protein signaling‐2 (RGS‐2) belongs to a novel family of GTPase‐activating proteins that rapidly turn‐off G‐protein coupled receptor signaling. RGS proteins contain a characteristic RGS domain by which they interact with the α‐subunit of G‐proteins and drive them into their inactive GDP‐bound forms. Previously, we have reported that RGS‐2 mRNA is rapidly and transiently increased by PTH in rat bone and in osteoblast cultures in vitro. In this study, we further explored the molecular basis for the regulation of RGS‐2 by cloning and functionally characterizing the RGS‐2 gene promoter. We cloned 2.3‐ and 2.8‐kb fragments of the 5′‐flanking regions of the rat and mouse RGS‐2 genes, respectively, and generated a stable clone of UMR106 osteoblastic cells containing the rat RGS‐2 promoter driving the β‐gal reporter gene (p2.3RGS‐2‐β‐gal). Treatment of the stable clone with PTH resulted in a maximal 2.2‐ to 3.6‐fold increase in promoter activity at 8 h, reminiscent of the early response observed with endogenous RGS‐2 mRNA regulation. Further, PTH (1–38), (1–31), PTHrP (1–34), and forskolin, which elevate cAMP levels, stimulated the promoter, while PTH (3–34) and (7–34), which do not readily stimulate cAMP accumulation, and PMA that directly activates protein kinase C, had no effect on promoter activity. Taken together, these results implicate the involvement of the Gαs‐adenylate cyclase‐protein kinase A pathway in stimulating RGS‐2 expression. Maintenance of a hyperphosphorylated state via the inhibition of type 2A protein phosphatases by okadaic acid, resulted in a strong dose‐dependent increase in transcriptional activity of the RGS‐2 promoter as well as that of the endogenous RGS‐2 gene. Furthermore, overexpression of the osteoblast‐specific transcription factor Runx2 also led to a stimulation of RGS‐2 promoter activity. Functional analysis using RGS‐2 overexpression suggests the potential negative regulatory effects of RGS‐2 on PTH‐ and forskolin‐induced cAMP production in osteoblastic cells. In summary, our data suggest that PTH treatment results in a direct transcriptional stimulation of RGS‐2 that in turn may play a role in modulating the duration/intensity of PTH receptor signaling. J. Cell. Biochem. 85: 837–850, 2002.

A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers

The non‐mineral component of bone matrix consists of 90% collagenous, 10% non‐collagenous proteins. These proteins regulate mineralization, growth, cell signaling and differentiation, and provide bone with its tensile strength. Expression of bone matrix proteins have historically been studied individually or in small numbers owing to limitations in analytical technologies. Current mass‐spectrometric and separations technologies allow a global view of protein expression patterns in complex samples. To our knowledge, no proteome profile of bone matrix has yet been reported. Therefore, we have used mass spectrometry as a tool to generate a profile of proteins present in the extracellular matrix of adult rat bone. Overall, 108 and 25 proteins were identified with high confidence in the metaphysis and diaphysis, respectively, using a bottom up proteomic technique. Twenty‐one of these proteins were present in both the metaphysis and diaphysis including the bone specific proteins, osteocalcin, type I collagen, osteopontin, osteoregulin, and bone sialoprotein. Interestingly, type II collagen, a protein thought to be exclusively expressed in cartilage, was identified in both the metaphysis and diaphysis. This observation was validated by Western blot. Additionally, the presence of aggrecan, another protein expressed in cartilage was identified in the bone matrix extracts by Western blot. The proteome profile generated using this technology represents an initial survey of the acid soluble proteins of bone matrix which provides a reference for the analysis of deviations from the normal composition due to perturbations or disease states. J. Cell. Biochem. 101: 466–476, 2007.

Identification of signal transduction pathways and promoter sequences that mediate parathyroid hormone 1–38 inhibition of osteoprotegerin gene expression

Osteoprotegerin (OPG), a secreted member of the tumor necrosis receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Parathyroid hormone (PTH), a potent inducer of osteoclast formation, suppresses OPG mRNA expression in vitro and in vivo. To determine the molecular basis of this inhibition, we analyzed the effects of PTH on the human OPG promoter (−5917 to +19) fused with β‐galactosidase reporter gene in stable and transient transfections into rat osteoblast‐like UMR106 cells. The effect of PTH on OPG promoter expression was biphasic and concentration‐dependent. PTH (1–100 nM) induced the transcriptional activity of the OPG promoter (1.7‐fold) at 8 h followed by a gradual decrease with maximal inhibition (6.6‐fold) at 24–48 h. To ascertain the signal transduction pathways mediating PTH (1–38) effects on OPG gene expression, we compared the effects of PTH with PTH analogs, parathyroid hormone‐related protein 1–34 (PTHrP 1–34), forskolin, 3‐isobutyl‐1‐methylxanthine (IBMX), dibutyryl cAMP, phorbol‐12‐myristate‐13‐acetate (PMA), thapsigargin and calcium ionophore A23187. PTH 1–31 and PTHrP 1–34, which stimulate the cAMP/PKA pathway, and other activators of cAMP/PKA, forskolin, IBMX, N6, O2′‐dibityryl adenosine 3′,5′‐cyclic monophosphate (dibutyryl cAMP), all elicited a similar biphasic response on OPG promoter expression. PTH analogs PTH 3–34 and PTH 7–34, that do not stimulate cAMP production, had no effect on OPG expression. In contrast, phorbol‐12‐myristate‐13‐acetate (PMA), an activator of PKC, stimulated OPG promoter expression, while thapsigargin and calcium ionophore A23187, which increase intracellular Ca2+, showed a dose‐dependent inhibition of OPG promoter expression. To delineate the promoter sequences that mediate the inhibitory effects of PTH on OPG transcription, we analyzed systematic deletions of the OPG promoter for responsiveness in transient transfection assays. The major inhibitory effects of PTH were localized to 391 bp (−372 to +19) of the proximal promoter. Deletions of the promoter region led to a complete loss of responsiveness. Taken together, these results demonstrate that the inhibitory effects of PTH on OPG are mediated at the transcriptional level through cis elements in the proximal promoter. The similar biphasic response of OPG to PTH, PTH 1–31, PTHrP 1–34, forskolin, IBMX and dibutyryl cAMP suggests that PTH regulates OPG transcription via activation of the cAMP/PKA signal transduction pathway. J. Cell. Biochem. 84: 1–11, 2002.

Parathyroid hormone (hPTH 1–38) stimulates the expression of UBP41, an ubiquitin‐specific protease, in bone

Parathyroid hormone (PTH) stimulates bone formation in both animals and humans, and the expression of a number of genes has been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon, we used differential display reverse transcription polymerase chain reaction (DDRT‐PCR) and screened for genes, which are differentially expressed in osteoblast‐enriched femoral metaphyseal primary spongiosa of young male rats after a single subcutaneous (s.c.) injection of hPTH (1–38) (8 μg/100 g). We found and cloned one full‐length cDNA, which encodes a putative 348 amino acid protein. Sequence analysis of this protein demonstrates a 98, 93.7, and 82.5% identity with mouse, human, and chicken ubiquitin‐specific protease UBP41, respectively. Northern blot analysis confirmed that a 3.8–4 kb UBP41 mRNA transcript was rapidly increased 1 h after acute hPTH (1–38) exposure in both metaphyseal (6‐ to 8‐fold) and diaphyseal (3‐fold) bone, but returned to control levels by 24 h after exposure. In contrast, continuous exposure to hPTH (1–38), resulted in a rapid and sustained elevation of UBP41 mRNA. PTH (1–31), which stimulates intracellular cAMP, and PTHrP (1–34) both induced UBP41 mRNA expression; whereas PTH analogs (3–34) and (7–34), that do not stimulate cAMP, had no effect on UBP41 expression. UBP41 mRNA expression was also rapidly induced 1 h after injection of PGE2, but returned to the control level by 6 to 24 h. In vitro, UBP41 mRNA is expressed in primary osteoblasts (metaphyseal and diaphyseal derived) and in the osteoblast‐like cell lines UMR106, ROS17/2.8, and BALC. PTH (1–38) treatment induced UPB41 expression (3.6‐ to 13‐fold) in both primary cultures of osteoblasts and in UMR106 cells. Further analysis in UMR 106 cells demonstrated that PGE2, forskolin and dibutyryl cAMP increased UBP41 mRNA expression 4‐, 4.5‐, and 2.4‐fold, respectively. Tissue distribution analysis of UBP41 mRNA detected transcripts in brain, heart, skeletal muscle, kidney, liver, and testis. Together, these results demonstrate that UBP41, an ubiquitin‐specific protease, is selectively upregulated in bone by the osteotropic agents PTH, PTHrP, and PGE2, possibly via the PKA/cAMP pathway. We speculate that the rapid induction of UBP41 in response to these physiological regulators contributes to the mechanism by which either the structure, activity, half‐life or localization of essential proteins are modified to maintain bone homeostasis. J. Cell. Biochem. 85: 229–242, 2002.