Osteoclast development in marrow cultured in calvaria-conditioned media

The precise signals responsible for recruitment and differentiation of osteoclasts (OCs) from their mononuclear precursors are poorly understood. Marrow mononuclear cells, a reputed source of OC precursors, fuse in culture, forming multinucleated cells. These cells, although similar to OCs, differ from osteoclasts in cell-surface morphology and are not recognized by an OC-specific monoclonal antibody. We have used the expression of an osteoclast-specific membrane epitope designated by monoclonal antibody 121F to delineate OCs from marrow-derived giant cells (MAGC). In this report we describe a series of experiments designed to better define the role of the bone environment in the osteoclast differentiation process. Periosteum-free calvariae from hatchling chicks or their conditioned media were combined with adherent Day 1 cultured marrow cells. The time course of OC marker expression was monitored by ELISA and the requirement for live bone and PTH was investigated. Freshly isolated marrow, MAGC, and calvariae were devoid of OC expression. Antigen expression developed in cultured MAGC after 4 days of coplating with either live bone or live bone-conditioned media. The presence of PTH in the cocultures or conditioned media from PTH-treated calvariae did not significantly alter the level of expression. These data indicate that live bone is, in part, responsible for the production of osteoclasts from mononuclear precursors.     

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.     

Impaired osteoclast formation in bone marrow cultures of Fgf2 null mice in response to parathyroid hormone

Fibroblast growth factor (FGF)-2 and parathyroid hormone (PTH) are potent inducers of osteoclast (OCL) formation, and PTH increases FGF-2 mRNA and protein expression in osteoblasts. To elucidate the role of endogenous FGF-2 in PTH responses, we examined PTH-induced OCL formation in bone marrow cultures from wild type and mice with a disruption of the Fgf2 gene. FGF-2-induced OCL formation was similar in marrow culture from both genotypes. In contrast, PTH-stimulated OCL formation in bone marrow cultures or co-cultures of osteoblast-spleen cells from Fgf2-/mice was significantly impaired. PTH increased RANKL mRNA expression in osteoblasts cultures from both genotypes. After 6 days of treatment, osteoprotegerin protein in cell supernatants was 40-fold higher in vehicle-treated and 30-fold higher in PTH-treated co-cultures of osteoblast and spleen cells from Fgf2-/mice compared with Fgf2+/+ mice. However, a neutralizing antibody to osteoprotegerin did not rescue reduced OCL formation in response to PTH. Injection of PTH caused hypercalcemia in Fgf2+/+ but not Fgf2-/mice. We conclude that PTH stimulates OCL formation and bone resorption in mice in part by endogenous FGF-2 synthesis by osteoblasts. Because RANKL- and interleukin-11-induced OCL formation was also reduced in bone marrow cultures from Fgf2-/mice, we further conclude that endogenous FGF-2 is necessary for maximal OCL formation by multiple bone resorbing factors.     

Amphiregulin-EGFR Signaling Mediates the Migration of Bone Marrow Mesenchymal Progenitors toward PTH-Stimulated Osteoblasts and Osteocytes

Intermittent administration of parathyroid hormone (PTH) dramatically increases bone mass and currently is one of the most effective treatments for osteoporosis. However, the detailed mechanisms are still largely unknown. Here we demonstrate that conditioned media from PTH-treated osteoblastic and osteocytic cells contain soluble chemotactic factors for bone marrow mesenchymal progenitors, which express a low amount of PTH receptor (PTH1R) and do not respond to PTH stimulation by increasing cAMP production or migrating toward PTH alone. Conditioned media from PTH-treated osteoblasts elevated phosphorylated Akt and p38MAPK amounts in mesenchymal progenitors and inhibition of these pathways blocked the migration of these progenitors toward conditioned media. Our previous and current studies revealed that PTH stimulates the expression of amphiregulin, an epidermal growth factor (EGF)-like ligand that signals through the EGF receptor (EGFR), in both osteoblasts and osteocytes. Interestingly, conditioned media from PTH-treated osteoblasts increased EGFR phosphorylation in mesenchymal progenitors. Using several different approaches, including inhibitor, neutralizing antibody, and siRNA, we demonstrate that PTH increases the release of amphiregulin from osteoblastic cells, which acts on the EGFRs expressed on mesenchymal progenitors to stimulate the Akt and p38MAPK pathways and subsequently promote their migration in vitro. Furthermore, inactivation of EGFR signaling specifically in osteoprogenitors/osteoblasts attenuated the anabolic actions of PTH on bone formation. Taken together, these results suggest a novel mechanism for the therapeutic effect of PTH on osteoporosis and an important role of EGFR signaling in mediating PTH''s anabolic actions on bone.     

Comparison of bone and parathyroid hormone as stimulators of osteoclast development and activity in calvarial cell cultures from normal and osteopetrotic (mi/mi) mice

Osteoclast development was studied in cell cultures prepared from calvaria of neonatal osteopetrotic (mi/mi) mice or their normal littermates, using tartrate-resistant acid phosphatase (TRAPase), as an osteoclast marker. In cultures from normal mice, treatment with 10 nM PTH for 4-5 days stimulated the formation of osteoclasts. However in cultures from mi/mi mice, this response was only 7% +/- 5% that of normal mice and they were significantly smaller than osteoclasts of normal mice. Mineralized bone particles elicited osteoclast development in cultures from both normal and mi/mi mice, and osteoclast size was identical for both genotypes. Seventy-eight to 96% of the TRAPase-positive cells bound 125I-CT, as demonstrated by autoradiography. 125I-CT binding characteristics were identical in cultures from both genotypes treated with bone particles, exhibiting a Kd of 3.3-3.6 x 10(-10) M. Addition of PTH stimulated 45Ca release from the added bone particles only in the case of cultures prepared from normal mice, and CT inhibited this response. Cells from normal mice were capable of excavating bone from the surface of smooth cortical bone wafers, but such excavations were rarely seen in the case of calvarial cells from mi/mi mice. Thus, PTH-driven differentiation of osteoclasts is arrested in calvarial cell cultures from mi/mi mice, but mi/mi preosteoclasts retain the ability to express certain osteoclast markers in response to bone derived signals. We hypothesize that the lack of activity of mi/mi osteoclasts is due to the failure of mi/mi preosteoclasts to respond appropriately to resorptive agents, or to cytokines elicited by these agents.     

Are Osteoclasts Needed for the Bone Anabolic Response to Parathyroid Hormone?: A STUDY OF INTERMITTENT PARATHYROID HORMONE WITH DENOSUMAB OR ALENDRONATE IN KNOCK-IN MICE EXPRESSING HUMANIZED RANKL*

PTH stimulates osteoblastic cells to form new bone and to produce osteoblast-osteoclast coupling factors such as RANKL. Whether osteoclasts or their activity are needed for PTH anabolism remains uncertain. We treated ovariectomized huRANKL knock-in mice with a human RANKL inhibitor denosumab (DMAb), alendronate (Aln), or vehicle for 4 weeks, followed by co-treatment with intermittent PTH for 4 weeks. Loss of bone mass and microarchitecture was prevented by Aln and further significantly improved by DMAb. PTH improved bone mass, microstructure, and strength, and was additive to Aln but not to DMAb. Aln inhibited biochemical and histomorphometrical indices of bone turnover, -i.e. osteocalcin and bone formation rate (BFR) on cancellous bone surfaces-, and Dmab inhibited them further. However Aln increased whereas Dmab suppressed osteoclast number and surfaces. PTH significantly increased osteocalcin and bone formation indices, in the absence or presence of either antiresorptive, although BFR remained lower in presence of Dmab. To further evaluate PTH effects in the complete absence of osteoclasts, high dose PTH was administered to RANK^−/− mice. PTH increased osteocalcin similarly in RANK^−/− and WT mice. It also increased BMD in RANK^−/− mice, although less than in WT. These results further indicate that osteoclasts are not strictly required for PTH anabolism, which presumably still occurs via stimulation of modeling-based bone formation. However the magnitude of PTH anabolic effects on the skeleton, in particular its additive effects with antiresorptives, depends on the extent of the remodeling space, as determined by the number and activity of osteoclasts on bone surfaces.     

Endogenous PTH deficiency impairs fracture healing and impedes the fracture-healing efficacy of exogenous PTH(1-34)

Background

Although the capacity of exogenous PTH1-34 to enhance the rate of bone repair is well established in animal models, our understanding of the mechanism(s) whereby PTH induces an anabolic response during skeletal repair remains limited. Furthermore it is unknown whether endogenous PTH is required for fracture healing and how the absence of endogenous PTH would influence the fracture-healing capacity of exogenous PTH.

Methodology/Principal Findings

Closed mid-diaphyseal femur fractures were created and stabilized with an intramedullary pin in 8-week-old wild-type and Pth null (Pth ^−/−) mice. Mice received daily injections of vehicle or of PTH1-34 (80 µg/kg) for 1–4 weeks post-fracture, and callus tissue properties were analyzed at 1, 2 and 4 weeks post-fracture. Cartilaginous callus areas were reduced at 1 week post-fracture, but were increased at 2 weeks post-fracture in vehicle-treated and PTH-treated Pth ^−/− mice compared to vehicle-treated and PTH-treated wild-type mice respectively. The mineralized callus areas, bony callus areas, osteoblast number and activity, osteoclast number and surface in callus tissues were all reduced in vehicle-treated and PTH-treated Pth ^−/− mice compared to vehicle-treated and PTH-treated wild-type mice, but were increased in PTH-treated wild-type and Pth ^−/− mice compared to vehicle-treated wild-type and Pth ^−/− mice.

Conclusions/Significance

Absence of endogenous PTH1-84 impedes bone fracture healing. Exogenous PTH1-34 can act in the absence of endogenous PTH but callus formation, including accelerated endochondral bone formation and callus remodeling as well as mechanical strength of the bone are greater when endogenous PTH is present. Results of this study suggest a complementary role for endogenous PTH1-84 and exogenous PTH1-34 in accelerating fracture healing.     

Amelioration of type I diabetes-induced osteoporosis by parathyroid hormone is associated with improved osteoblast survival

Type 1 diabetic osteoporosis results from impaired osteoblast activity and death. Therefore, anti-resorptive treatments may not effectively treat bone loss in this patient population. Intermittent parathyroid hormone (PTH) treatment stimulates bone remodeling and increases bone density in healthy subjects. However, PTH effects may be limited in patients with diseases that interfere with its signaling. Here, we examined the ability of 8 and 40 μg/kg intermittent PTH to counteract diabetic bone loss. PTH treatment reduced fat pad mass and blood glucose levels in non-diabetic PTH-treated mice, consistent with PTH-affecting glucose homeostasis. However, PTH treatment did not significantly affect general body parameters, including the blood glucose levels, of type 1 diabetic mice. We found that the high dose of PTH significantly increased tibial trabecular bone density parameters in control and diabetic mice, and the lower dose elevated trabecular bone parameters in diabetic mice. The increased bone density was due to increased mineral apposition and osteoblast surface, all of which are defective in type 1 diabetes. PTH treatment suppressed osteoblast apoptosis in diabetic bone, which could further contribute to the bone-enhancing effects. In addition, PTH treatment (40 μg/kg) reversed preexisting bone loss from diabetes. We conclude that intermittent PTH may increase type 1 diabetic trabecular bone volume through its anabolic effects on osteoblasts.     

Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin

Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.     

Serum proteome profiles identifies parathyroid hormone physiologic response

Parathyroid hormone (amino acids 1-34) (PTH) regulates bone and calcium homeostasis. The magnitude of the effects of PTH on bone varies in osteoporosis patients. We employed ProteinChip technology to generate protein profiles from sera of mice treated once daily with PTH or vehicle for 3 or 11 days. Data analyses on selected arrays indicated significant increases in serum proteins or peptides in PTH-treated groups, compared to vehicle-controls. The magnitude of change increased with duration of treatment. Anion-exchange fractionation of sera prior to profiling on array surfaces increased the number of proteins detected that were regulated by PTH. The optimized purification conditions developed "on-chip" for subsets of proteins, reflected corresponding behavior with process-compatible chromatographic resins under elution chromatography. We have identified and evaluated subsets of serum proteins regulated by PTH treatment, using a combination of ProteinChip technology, column chromatography, PAGE and LC-MS/MS. Our data demonstrate the feasibility of using a panel of serum proteins to detect PTH responsiveness in humans.     

Consequences of daily administered parathyroid hormone on myeloma growth, bone disease, and molecular profiling of whole myelomatous bone

Background

Induction of osteolytic bone lesions in multiple myeloma is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Current management of myeloma bone disease is limited to the use of antiresorptive agents such as bisphosphonates.

Methodology/Principal Findings

We tested the effects of daily administered parathyroid hormone (PTH) on bone disease and myeloma growth, and we investigated molecular mechanisms by analyzing gene expression profiles of unique myeloma cell lines and primary myeloma cells engrafted in SCID-rab and SCID-hu mouse models. PTH resulted in increased bone mineral density of myelomatous bones and reduced tumor burden, which reflected the dependence of primary myeloma cells on the bone marrow microenvironment. Treatment with PTH also increased bone mineral density of uninvolved murine bones in myelomatous hosts and bone mineral density of implanted human bones in nonmyelomatous hosts. In myelomatous bone, PTH markedly increased the number of osteoblasts and bone-formation parameters, and the number of osteoclasts was unaffected or moderately reduced. Pretreatment with PTH before injecting myeloma cells increased bone mineral density of the implanted bone and delayed tumor progression. Human global gene expression profiling of myelomatous bones from SCID-hu mice treated with PTH or saline revealed activation of multiple distinct pathways involved in bone formation and coupling; involvement of Wnt signaling was prominent. Treatment with PTH also downregulated markers typically expressed by osteoclasts and myeloma cells, and altered expression of genes that control oxidative stress and inflammation. PTH receptors were not expressed by myeloma cells, and PTH had no effect on myeloma cell growth in vitro.

Conclusions/Significance

We conclude that PTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis and attenuated bone resorption and myeloma growth; mechanisms involve increased osteoblast production of anti-myeloma factors and minimized myeloma induction of inflammatory conditions.     

Impaired bone anabolic response to parathyroid hormone in Fgf2-/- and Fgf2+/- mice

Since parathyroid hormone (PTH) increased FGF2 mRNA and protein expression in osteoblasts, and serum FGF-2 was increased in osteoporotic patients treated with PTH, we assessed whether the anabolic effect of PTH was impaired in Fgf2-/- mice. Eight-week-old Fgf2+/+ and Fgf2-/- male mice were treated with rhPTH 1-34 (80mug/kg) for 4 weeks. Micro-CT and histomorphometry demonstrated that PTH significantly increased parameters of bone formation in femurs from Fgf2+/+ mice but the changes were smaller and not significant in Fgf2-/- mice. IGF-1 was significantly reduced in serum from PTH-treated Fgf2-/- mice. DEXA analysis of femurs from Fgf2+/+, Fgf2+/-, and Fgf2-/- mice treated with rhPTH (160mug/kg) for 10 days showed that PTH significantly increased femoral BMD in Fgf2+/+ by 18%; by only 3% in Fgf2+/- mice and reduced by 3% in Fgf2-/- mice. We conclude that endogenous Fgf2 is important for maximum bone anabolic effect of PTH in mice.     

Sclerostin is expressed in osteoclasts from aged mice and reduces osteoclast‐mediated stimulation of mineralization

Osteoclast‐mediated bone resorption precedes osteoblast‐mediated bone formation through early adulthood, but formation fails to keep pace with resorption during aging. We previously identified several factors produced by osteoclasts that promote bone formation. In this study, we determined if osteoclast‐produced factors contribute to the impaired bone formation with aging. We previously found that mice between the ages of 18 and 22 months develop age‐related bone loss. Bone marrow‐derived pre‐osteoclasts were isolated from 6‐week, 12‐month, and 18‐ to 24‐month‐old mice and differentiated into osteoclasts in vitro. Conditioned media were collected and compared for osteoblast mineralization support. Conditioned medium from osteoclasts from all ages was able to support mineralization of bone marrow stromal cells. Concentrating the conditioned medium from 6‐week‐old and 12‐month‐old mouse marrow cells‐derived osteoclasts enhanced mineralization support whereas concentrated conditioned medium from 18‐ to 24‐month‐old mouse marrow‐derived osteoclasts repressed mineralization compared to base medium. This observation suggests that an inhibitor of mineralization was secreted by aged murine osteoclasts. Gene and protein analysis revealed that the Wnt antagonist sclerostin was significantly elevated in the conditioned media from 24‐month‐old mouse cells compared to 6‐week‐old mouse cells. Antibodies directed to sclerostin neutralized the influences of the aged mouse cell concentrated conditioned media on mineralization. Sclerostin is primarily produced by osteocytes in young animals. This study demonstrates that osteoclasts from aged mice also produce sclerostin in quantities that may contribute to the age‐related impairment in bone formation. J. Cell. Biochem. 114: 1901–1907, 2013. © 2013 Wiley Periodicals, Inc.     

Rapid activation of the medullary bone osteoclast cell surface by parathyroid hormone

Quantitative transmission electron microscope methods were used to determine the response of functionally inactive avian medullary bone osteoclasts to parathyroid hormone (PTH). Egg-lying Japanese quail were used during a period of the egg cycle when medullary bone was not being resorbed for egg shell calcification and when medullary bone osteoclasts were functionally inactive. Ruffled borders adjacent to bone surfaces were rarely, if ever, found on these cells. 20 min after the administration of PTH, over 70% of the osteoclast profiles had ruffled borders adjacent to bone surfaces. These ruffled borders were bounded by filamentous-rich "clear zones" and resembled ruffled borders found on functionally active cells. There was also a marked increase in plasma calcium levels after PTH administration. This study demonstrates that PTH stimulates the de novo generation of ruffled borders on osteoclasts in vivo and suggests that osteoclasts may be involved in the acute regulation of calcium metabolism by exogenous PTH.     

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.     

Parathyroid hormone (PTH) and PTH-like protein (PLP) stimulate interleukin-6 production by osteogenic cells: a possible role of interleukin-6 in osteoclastogenesis

Osteogenic cells mediate PTH-stimulated osteoclastic bone resorption by a yet unidentified mechanism. We show that primairy rat osteoblast-like cells and the clonal osteogenic sarcoma cell line UMR-106 produce interleukin-6 (IL-6) and that bPTH(1-84) and synthetic hPLP(1-34) stimulate this production dose-dependently. With both peptides a close relation between IL-6 and cyclic-AMP production was found, though for PTH concentrations higher than 2.10(-8) M a clear dissociation was observed. Significant IL-6 activity was also detected in media of cultures of 17-day-old fetal mouse radii and metacarpals which was clearly stimulated by PTH. The source of IL-6 in these bone explants seems to be the osteogenic (cartilage) cells. Treatment of bone explants with IL-6 induced osteoclastic resorption which, however, depended on the bone resorption system used. This bone resorbing action of IL-6 is exerted probably through an effect on the formation of osteoclasts (osteoclastogenesis) rather than on the activation of already existing mature osteoclasts. We suggest that IL-6 produced by osteogenic cells may be a mediator in PTH-stimulated osteoclastic bone resorption.     

Paracrine interactions in bone-secreted products of osteoblasts permit osteoclasts to respond to parathyroid hormone

During bone remodeling, activation of resorption is followed by a cycle of formation and this ordered sequence of events has long suggested that local interactions between osteoclasts and osteoblasts are an important regulatory mechanism in bone metabolism. To study this phenomenon, we have prepared bone cells containing primarily osteoclasts by brief digestion of mice calvariae in collagenase, overnight attachment to polystyrene tissue culture flasks in serumless medium supplemented with OB (osteoblast) cell conditioned medium and subsequent growth in low serum. These OC (osteoclast) cells were found to be highly enriched in acid phosphatase activity and expressed cAMP responses to PTH (parathyroid hormone) and prostaglandin E2 but exhibited no PTH-stimulated hyaluronate synthesis in contrast to prostaglandin E2. PTH effects on hyaluronate, however, could be restored upon coculture of OC cells with OB cells (noncontact) or with OB cell conditioned medium, thereby suggesting that OB cells regulate OC cell PTH responsiveness and/or differentiation by soluble cell products secreted into the medium.     

The role of strain in the response of rapidly growing young male rat bones to parathyroid hormone

Human parathyroid hormone (hPTH 1-34) stimulates an anabolic response in human and animal skeletons; however, it is unclear if the effect is strain dependent. To determine if the anabolic response to hPTH (1-34) was dependent upon strain in rats we used 2 outbred strains (Sprague Dawley, Wistar), 2 inbred strains (Fischer 344, Wistar spontaneously hypertensive:SHR), and 2 mutant strains (Zucker obese, Zucker lean) of rats. Male rats, 5 weeks of age, from each strain were treated subcutaneously with 80 microg/kg body weight hPTH (1-34) or vehicle for 12 days. The response to PTH was similar in all strains whereby PTH exerted an anabolic effect on femoral bone mass and cancellous bone histology that was independent of strain differences. Histomorphometric indices of bone volume, mineralized surface and bone formation in lumbar vertebrae increased in all PTH-treated rats. Additionally, femur bone mineral content and bone mineral density measured by dual energy X-ray absorptiometry (DEXA), and ash weight increased in all PTH-treated rats. These increases occurred regardless of strain. In summary, PTH exerted comparable anabolic effects on bone mass, bone mineral density and bone formation in all rat models tested demonstrating that the skeletal responsiveness to PTH was not dependent upon strain.     

Parathyroid hormone stimulates osteoblastic expression of MCP-1 to recruit and increase the fusion of pre/osteoclasts

The clinical findings that alendronate blunted the anabolic effect of human parathyroid hormone (PTH) on bone formation suggest that active resorption is involved and enhances the anabolic effect. PTH signals via its receptor on the osteoblast membrane, and osteoclasts are impacted indirectly via the products of osteoblasts. Microarray with RNA from rats injected with human PTH or vehicle showed a strong association between the stimulation of monocyte chemoattractant protein-1 (MCP-1) and the anabolic effects of PTH. PTH rapidly and dramatically stimulated MCP-1 mRNA in the femora of rats receiving daily injections of PTH or in primary osteoblastic and UMR 106-01 cells. The stimulation of MCP-1 mRNA was dose-dependent and a primary response to PTH signaling via the cAMP-dependent protein kinase pathway in vitro. Studies with the mouse monocyte cell line RAW 264.7 and mouse bone marrow proved that osteoblastic MCP-1 can potently recruit osteoclast monocyte precursors and facilitate receptor activator of NF-kappaB ligand-induced osteoclastogenesis and, in particular, enhanced fusion. Our model suggests that PTH-induced osteoblastic expression of MCP-1 is involved in recruitment and differentiation at the stage of multinucleation of osteoclast precursors. This information provides a rationale for increased osteoclast activity in the anabolic effects of PTH in addition to receptor activator of NF-kappaB ligand stimulation to initiate greater bone remodeling.