Catabolic Effects of Human PTH (1-34) on Bone: Requirement of Monocyte Chemoattractant Protein-1 in Murine Model of Hyperparathyroidism.

The bone catabolic actions of parathyroid hormone (PTH) are seen in patients with hyperparathyroidism, or with infusion of PTH in rodents. We have previously shown that the chemokine, monocyte chemoattractant protein-1 (MCP-1), is a mediator of PTH's anabolic effects on bone. To determine its role in PTH's catabolic effects, we continuously infused female wild-type (WT) and MCP-1-/- mice with hPTH or vehicle. Microcomputed tomography (µCT) analysis of cortical bone showed that hPTH-infusion induced significant bone loss in WT mice. Further, µCT analysis of trabecular bone revealed that, compared with the vehicle-treated group, the PTH-treated WT mice had reduced trabecular thickness and trabecular number. Notably, MCP-1-/- mice were protected against PTH-induced cortical and trabecular bone loss as well as from increases in serum CTX (C-terminal crosslinking telopeptide of type I collagen) and TRACP-5b (tartrate-resistant acid phosphatase 5b). In vitro, bone marrow macrophages (BMMs) from MCP-1-/- and WT mice were cultured with M-CSF, RANKL and/or MCP-1. BMMs from MCP-1-/- mice showed decreased multinucleated osteoclast formation compared with WT mice. Taken together, our work demonstrates that MCP-1 has a role in PTH's catabolic effects on bone including monocyte and macrophage recruitment, osteoclast formation, bone resorption, and cortical and trabecular bone loss.

Monocyte chemoattractant protein-1 is a mediator of the anabolic action of parathyroid hormone on bone.

Parathyroid hormone (PTH) has a significant role as an anabolic hormone in bone when administered by intermittent injection. Previous microarray studies in our laboratory have shown that the most highly regulated gene, monocyte chemoattractant protein-1 (MCP-1), is rapidly and transiently induced when hPTH(1-34) is injected intermittently in rats. Through further in vivo studies, we found that rats treated with hPTH(1-34) showed a significant increase in serum MCP-1 levels 2 hours after PTH injection compared with basal levels. Using immunohistochemistry, increased MCP-1 expression in osteoblasts and osteocytes is evident after PTH treatment. PTH also increased the number of marrow macrophages. MCP-1 knockout mice injected daily with hPTH(1-34) showed less trabecular bone mineral density and bone volume compared with wild-type mice as measured by peripheral quantitative computed tomography (pQCT) and micro-computed tomography (µCT). Histomorphometric analysis revealed that the increase in osteoclast surface and osteoclast number observed with intermittent PTH treatment in the wild-type mice was completely eliminated in the MCP-1 null mice, as well as much lower numbers of macrophages. Consequently, the lack of osteoclast and macrophage activity in the MCP-1 null mice was paralleled by a reduction in bone formation. We conclude that osteoblast and osteocyte MCP-1 expression is an important mediator for the anabolic effects of PTH on bone.

Epidermal growth factor receptor plays an anabolic role in bone metabolism in vivo.

While the epidermal growth factor receptor (EGFR)-mediated signaling pathway has been shown to have vital roles in many developmental and pathologic processes, its functions in the development and homeostasis of the skeletal system has been poorly defined. To address its in vivo role, we constructed transgenic and pharmacologic mouse models and used peripheral quantitative computed tomography (pQCT), micro-computed tomography (µCT) and histomorphometry to analyze their trabecular and cortical bone phenotypes. We initially deleted the EGFR in preosteoblasts/osteoblasts using a Cre/loxP system (Col-Cre Egfr(f/f)), but no bone phenotype was observed because of incomplete deletion of the Egfr genomic locus. To further reduce the remaining osteoblastic EGFR activity, we introduced an EGFR dominant-negative allele, Wa5, and generated Col-Cre Egfr(Wa5/f) mice. At 3 and 7 months of age, both male and female mice exhibited a remarkable decrease in tibial trabecular bone mass with abnormalities in trabecular number and thickness. Histologic analyses revealed decreases in osteoblast number and mineralization activity and an increase in osteoclast number. Significant increases in trabecular pattern factor and structural model index indicate that trabecular microarchitecture was altered. The femurs of these mice were shorter and smaller with reduced cortical area and periosteal perimeter. Moreover, colony-forming unit-fibroblast (CFU-F) assay indicates that these mice had fewer bone marrow mesenchymal stem cells and committed progenitors. Similarly, administration of an EGFR inhibitor into wild-type mice caused a significant reduction in trabecular bone volume. In contrast, Egfr(Dsk5/+) mice with a constitutively active EGFR allele displayed increases in trabecular and cortical bone content. Taken together, these data demonstrate that the EGFR signaling pathway is an important bone regulator and that it primarily plays an anabolic role in bone metabolism.

Interleukin-18 is regulated by parathyroid hormone and is required for its bone anabolic actions.

Interleukin-18 (IL-18) can regulate osteoblast and osteoclast function. We have identified, using cDNA microarray technology, that IL-18 expression is increased in UMR 106-01 rat osteoblastic cells in response to parathyroid hormone (PTH) treatment. Confirmation of these data using real-time reverse transcription-PCR showed that steady-state levels of IL-18 mRNA increased by 2 h (3-fold), peaked by 4 h (10-fold), and had diminished after 12 h (4.4-fold) and that this regulation was via the protein kinase A signaling pathway and did not involve activation of the PKC signal cascade. PTH regulation of IL-18 was confirmed at the protein level, and analysis of differentiating primary rat calvarial osteoblasts verified that both IL-18 mRNA and protein are regulated by PTH in primary rat osteoblasts. Promoter reporter assays revealed that PTH regulated the upstream IL-18 promoter and induced the exon 1 containing 1.1-kb IL-18 mRNA transcript in primary osteoblast cells. The in vivo physiological role of IL-18 in the anabolic actions of PTH on bone was then assessed using IL-18 knock-out mice. Female IL-18 null mice and wild-type littermate controls were injected with vehicle or 8 microg/100 g of human 1-38 PTH for 4 weeks. In IL-18 knock-out animals the anabolic effect of PTH (determined by bone mineral density changes in the proximal tibia) was abolished in trabecular bone but not in the cortical component. These data characterize the PTH regulation of IL-18 expression in osteoblastic cells and suggest that this cytokine is involved in the anabolic actions of PTH.

Determination of dual effects of parathyroid hormone on skeletal gene expression in vivo by microarray and network analysis.

Parathyroid hormone (PTH) stimulates bone formation when injected daily but causes severe bone loss with continuous infusion. The mechanism of its paradoxical effects is still elusive. In this study, we compared changes in the gene expression profile in bone induced by intermittent or continuous treatment with three different PTH peptides, PTH-(1-34), -(1-31), and -(3-34), in Sprague-Dawley female rats. PTH-(1-34) regulated numerous genes (approximately 1,000), but differentially, in both regimes. PTH-(1-31) regulated a similar number of genes in the intermittent regimen but fewer in the continuous regimen, consistent with its less potent catabolic effect. PTH-(3-34) regulated very few genes in both regimes, which suggests the protein kinase C pathway plays a limited role in mediating the dual effects of PTH, whereas the cAMP-dependent protein kinase A pathway appears to predominate. In the intermittent treatment, many genes encoding signaling mediators, transcription factors, cytokines, and proteases/protease inhibitors are regulated rapidly and cyclically with each PTH injection; genes associated with skeletal development show a slowly accruing pattern of expression. With continuous treatment, some genes are regulated from 6 h, and the mRNA levels are sustained with a longer infusion, whereas others show a kinetic decrease and then increase later. Significant up-regulation of genes stimulating osteoclastogenesis in the anabolic regime suggests a provocative and paradoxical theme for the anabolic effect of PTH that a full anabolic response requires a transient up-regulation of genes classically associated with a resorptive response. Ingenuity pathway analysis was performed on the microarray data. A novel signaling network was established that is differentially regulated in the two PTH treatment regimes. Key regulators are suggested to be AREG, CCL2, WNT4, and cAMP-responsive element modulator.

Discovery of a small molecule antagonist of the parathyroid hormone receptor by using an N-terminal parathyroid hormone peptide probe.

Once-daily s.c. administration of either human parathyroid hormone (PTH)-(1-84) or recombinant human PTH-(1-34) provides for dramatic increases in bone mass in women with postmenopausal osteoporosis. We initiated a program to discover orally bioavailable small molecule equivalents of these peptides. A traditional high-throughput screening approach using cAMP activation of the PTH/PTH-related peptide receptor (PPR) as a readout failed to provide any lead compounds. Accordingly, we designed a new screen for this receptor that used a modified N-terminal fragment of PTH as a probe for small molecule binding to the transmembrane region of the PPR, driven by the assumption that the pharmacological properties (agonist/antagonist) of compounds that bound to this putative signaling domain of the PPR could be altered by chemical modification. We developed DPC-AJ1951, a 14 amino acid peptide that acts as a potent agonist of the PPR, and characterized its activity in ex vivo and in vivo assays of bone resorption. In addition, we studied its ability to initiate gene transcription by using microarray technology. Together, these experiments indicated that the highly modified 14 amino acid peptide induces qualitatively similar biological responses to those produced by PTH-(1-34), albeit with lower potency relative to the parent peptide. Encouraged by these data, we performed a screen of a small compound collection by using DPC-AJ1951 as the ligand. These studies led to the identification of the benzoxazepinone SW106, a previously unrecognized small molecule antagonist for the PPR. The binding of SW106 to the PPR was rationalized by using a homology receptor model.

Is exogenous tissue plasminogen activator necessary for antithrombotic efficacy of an inhibitor of thrombin activatable fibrinolysis inhibitor (TAFI) in rats?

INTRODUCTION: TAFI indirectly reduces the action of tPA on plasminogen. Whether exogenous tPA is necessary for TAFI inhibitor efficacy is unclear. Potato carboxypeptidase inhibitor (PCI), a TAFI inhibitor, has shown variable tPA dependence in rat models of arteriovenous shunt thrombosis (required) and microthrombosis (not required). This study was designed to further explore the importance of exogenous tPA in revealing PCI activity in rat models of venous and arterial thrombosis and provoked bleeding. METHODS: PCI was given as a bolus (5, 10 mg/kg) +/- infusion (5, 10 mg/kg/h) and with or without low dose tPA (5, 10, 25 microg/kg/min). In each instance tPA was adjusted to produce subthreshold thrombus reduction. Arterial thrombosis was induced by FeCl2; venous thrombosis by tissue factor or FeCl2. Bleeding was induced by kidney incision with PCI given (5 mg + 5 mg/kg/h) in the presence or absence of tPA (10, 150, 200 microg/kg/min). RESULTS: PCI was ineffective without exogenous tPA in all tested thrombosis models. With exogenous tPA, PCI decreased thrombus weight 85% in tissue factor thrombosis, 59% in FeCl2 thrombosis, and 46% in arterial thrombosis. PCI prolonged bleeding only when combined with a relatively high tPA dose (200 microg/kg/min) that increased bleeding alone. CONCLUSIONS: If the current results predict clinical efficacy, the need for exogenous tPA in combination with TAFI inhibition is a potential problem. However, in acute settings where intravenous fibrinolytics are administered, or indications in which tPA production increases, TAFI inhibitors may prove to be safe and moderately effective profibrinolytic agents.

Deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI) protected mice from ferric chloride-induced vena cava thrombosis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. Since the role of TAFI in thrombus formation is still controversial in mice, our present study was designed to evaluate mice deficient in TAFI (TAFI(-/-)) on FeCl(3)-induced vena cava and carotid artery thrombosis. Parallel studies were carried out in wild-type mice using a potato carboxypeptidase inhibitor (PCI), a selective inhibitor of activated TAFI (TAFIa). Significant reduction in thrombus formation was observed in TAFI(-/-) mice (n = 8, P < 0.05 compared to wild-type littermates) but not in heterozygous (TAFI(+/-)) mice in 3.5% FeCl(3)-induced vena cava thrombosis. A similar effect was observed following treatment with 5 mg/kg bolus plus 5 mg/kg/h PCI in the same venous thrombosis model in C57BL/6 mice (n = 8, P < 0.01 compared to vehicle). No compositional difference was observed for the venous thrombi in TAFI(-/-) and wild-type littermates with or without PCI treatment using histological assessment. In contrast, neither TAFI deficiency nor treatment with PCI showed antithrombotic efficacy in the 3.5% FeCl(3)-induced carotid artery thrombosis model. In a tail transection bleeding time model, both TAFI deficiency and PCI treatment increased bleeding time up to 4.5 and 3.5 times, respectively, over controls (P < 0.05, n = 8). Similar ex vivo fibrinolytic activities were demonstrated for both TAFI deficiency and PCI treatment as enhanced lysis of thrombin-induced plasma clots and lysis of whole blood clot in a thrombelastograph. These data provide direct evidence for the role of TAFIa in vena cava thrombosis without the addition of exogenous thrombolytic in mice. The strong ex vivo fibrinolytic activity of TAFI deficiency or TAFIa inhibition by PCI provides a biomarker of TAFIa inhibition that tracks in vivo antithrombotic efficacy.

Overexpression of Runx2 directed by the matrix metalloproteinase-13 promoter containing the AP-1 and Runx/RD/Cbfa sites alters bone remodeling in vivo.

The activator protein-1 (AP-1) and runt domain binding (Runx/RD/Cbfa) sites and their respective binding proteins, c-Fos/c-Jun and Runx2 (Cbfa1), regulate the rat matrix metalloproteinase-13 (MMP-13) promoter in both parathyroid hormone (PTH)-treated and differentiating osteoblastic cells in culture. To determine the importance of these regulatory sites in the expression of MMP-13 in vivo, transgenic mice containing either wild-type (-456 or -148) or AP-1 and Runx/RD/Cbfa sites mutated (-148A3R3) MMP-13 promoters fused with the E. coli lacZ reporter were generated. The wild-type transgenic lines expressed higher levels of bacterial beta-galactosidase in bone, teeth, and skin compared to the mutant and non-transgenic lines. Next, we investigated if overexpression of Runx2 directed by the MMP-13 promoter regulated expression of bone specific genes in vivo, and whether this causes morphological changes in these animals. Real time RT-PCR experiments identified increased mRNA expression of bone forming genes and decreased MMP-13 in the tibiae of transgenic mice (14 days and 6 weeks old). Histomorphometric analyses of the proximal tibiae showed increased bone mineralization surface, mineral apposition rate, and bone formation rate in the transgenic mice which appears to be due to decreased osteoclast number. Since MMP-13 is likely to play a role in recruiting osteoclasts to the bone surface, decreased expression of MMP-13 may cause reduced osteoclast-mediated bone resorption, resulting in greater bone formation in transgenic mice. In summary, we show here that the 148 bp upstream of the MMP-13 transcriptional start site is sufficient and necessary for gene expression in bone, teeth, and skin in vivo and the AP-1 and Runx/RD/Cbfa sites are likely to regulate this. Overexpression of Runx2 by these regulatory elements appears to alter the balance between the bone formation-bone resorption processes in vivo.

Amphiregulin is a novel growth factor involved in normal bone development and in the cellular response to parathyroid hormone stimulation.

Parathyroid hormone (PTH) is the major mediator of calcium homeostasis and bone remodeling and is now known to be an effective drug for osteoporosis treatment. Yet the mechanisms responsible for its functions in bone are largely unknown. Here we report that the expression of amphiregulin (AR), a member of the epidermal growth factor (EGF) family, is rapidly and highly up-regulated by PTH in several osteoblastic cell lines and bone tissues. Other osteotropic hormones (1alpha,25-dihydroxyvitamin D3 and prostaglandin E2) also strongly stimulate AR expression. We found all EGF-like ligands and their receptors are expressed in osteoblasts, but AR is the only member that is highly regulated by PTH. Functional studies demonstrated that although AR is a potent growth factor for preosteoblasts, it completely inhibits further differentiation. AR also strongly and quickly stimulated Akt and ERK phosphorylation and c-fos and c-jun expression in an EGF receptor-dependent manner. Moreover, AR null mice displayed significantly less tibial trabecular bone than wild-type mice. Taken together, we have identified a novel growth factor that is PTH-regulated and appears to have an important role in bone metabolism.

Characterization of a new class of selective nonsteroidal progesterone receptor agonists.

The identification of a new series of selective nonsteroidal progesterone receptor (PR) agonists is reported. Using a high-throughput screening assay based on the measurement of transactivation of a mouse mammary tumor virus promoter-driven luciferase reporter (MMTV-Luc) in human breast cancer T47D cells, a benzimidazole-2-thione analog was identified. Compound 1 showed an apparent EC50 of 53 nM and efficacy of 93% with respect to progesterone. It binds to PR with high affinity (Ki nM), but had no or very low affinity for other steroid hormone receptors. Structure-activity relationship studies of a series of benzimidazole-2-thione analogs revealed critical positions for high PR binding affinity and transactivation potency as well as receptor selectivity, as exemplified by 25. Compound 25 binds to human PR with high affinity (Ki nM) and had at least > 1000-fold selectivity for PR versus other steroid receptors. Molecular modeling studies suggested that these agonists overlap favorably with progesterone in the ligand-binding domain of PR. In T47D cells, compound 25 acted as a full agonist in the MMTV-Luc reporter assay, as well as in the induction of endogenous alkaline phosphatase activity with apparent EC50 values of 4 and 9 nM, respectively. In the immature rat model, compound 25 provided a significant suppression of estrogen-induced endometrium hypertrophy as measured by luminal epithelial height. In contrast, compound 25 was inactive in the luteinizing hormone release assay in young ovariectomized rats. These benzimidazole-2-thione analogs constitute a new series of nonsteroidal PR agonists with an excellent steroid receptor selectivity profile. The differential activities observed in the in vivo progestogenic assays in rat models suggest that these analogs can act as selective PR modulators.

Characterization of bone structure in leptin receptor-deficient Zucker (fa/fa) rats.

UNLABELLED: To investigate the role of leptin in bone formation, the skeleton of the obese female leptin receptor-deficient Zucker rat was examined using pQCT, microCT, and histomorphometry. A trend toward decreasing structural and bone formation parameters in these rats as they age suggest that leptin has a small positive effect on bone. INTRODUCTION: Evidence in the literature has suggested the possible role of leptin in bone formation. Leptin deficiency or leptin receptor deficiency results in higher bone mass. In an attempt to further investigate leptin's role in bone formation, we examined the skeleton of obese leptin receptor-deficient Zucker rats. METHODS: Female leptin receptor-deficient Zucker (fa/fa) rats and their homozygous (Fa/Fa) and heterozygous (Fa/fa) lean controls were used at 9 and 15 weeks of age (n = 5). Bone mineral density of the proximal tibia was measured by peripheral quantitative computed tomography (pQCT). Microcomputed tomography (microCT) was used for the analysis of trabecular architecture in the proximal tibia metaphysis and cortical bone at the tibia-fibula junction. Static and dynamic parameters of bone resorption and formation were quantitated by histomorphometry. Statistical analysis was performed by Dunnett's one-way ANOVA. RESULTS: Analysis of the proximal tibia by pQCT show no significant differences in the bone mineral density of obese rats compared with their corresponding lean controls in either age group. Trabecular architecture measured by microCT indicate a trends toward decreasing bone volume (BV/TV) in the obese animals, evident by a decrease in trabecular number and thickness with an increase in trabecular separation. Histomorphometric evaluation further shows significant increases in osteoclast surface in the obese rats at both 9 and 15 weeks without a change in osteoclast number. Osteoid surface in the obese animals was also found to be decreased by 15 weeks of age. Fluorescent-based measurements of bone formation were not significantly different. Differences in the cortical compartment were not observed at either age. CONCLUSION: Based on the observed skeletal phenotype of the Zucker (fa/fa) rat, it is suggested that leptin exerts a positive effect on bone.

The ability of statins to inhibit bone resorption is directly related to their inhibitory effect on HMG-CoA reductase activity.

Statins, which are inhibitors of 3-hydroxy-3-glutaryl-coenzyme A (HMG-CoA) reductase, decrease the hepatic biosynthesis of cholesterol by blocking the mevalonate pathway. Nitrogen-containing bisphosphonate drugs also inhibit the mevalonate pathway, preventing the production of the isoprenoids, which consequently results in the inhibition of osteoclast formation and osteoclast function. Therefore, we hypothesized that statins could affect bone metabolism in vivo through effects on osteoclastic bone resorption. In vitro, cerivastatin inhibited the parathyroid hormone (PTH)-stimulated bone resorption. Using a panel of 40 statin analogs, which showed variable effects on HMG-CoA reductase activity, we found that the ability of compounds to inhibit bone resorption is directly related to HMG-CoA reductase activity. However, in the thyro-parathyrodectomy (TPTX) model for bone resorption in the rat in vivo, cerivastatin did not prevent experimentally induced increases in bone resorption. The lack of effect of cerivastatin in this model is not related to a limited penetration of the target tissue (bone marrow), because a significant effect on HMG-CoA reductase activity was demonstrated in the total rat bone marrow cell extracts of rats posttreatment in vivo. Furthermore, cerivastatin inhibited protein prenylation in osteoclasts isolated from the rabbit bone marrow of rabbits after treatment in vivo. In contrast to other studies, none of the statins tested showed anabolic effects in parietal bone explant cultures. Taken together, we conclude that statins inhibit bone resorption in vitro, which correlates directly with the potency of the compounds for inhibition of HMG-CoA reductase activity. However, cerivastatin does not affect bone resorption in the rat TPTX model in vivo.