Anabolic effects of PTH in cyclooxygenase-2 knockout osteoblasts in vitro

PTH is a potent bone anabolic agent in vivo but anabolic effects on osteoblast differentiation in vitro are difficult to demonstrate. This study examined the role of cyclooxygenase (COX)-2 and prostaglandin (PG) production in the effects of PTH on osteoblast differentiation in vitro using marrow stromal cell (MSC) and calvarial osteoblast (COB) cultures from COX-2 knockout (KO) and wild type (WT) mice. Cells were treated with PTH (10 nM) or vehicle throughout culture. Alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA levels were measured at days 14 and 21, respectively, and mineralization at day 21. cAMP concentrations were measured in the presence of a phosphodiesterase inhibitor. PTH did not stimulate differentiation in cultures from WT mice but significantly increased ALP and OCN mRNA expression 6- to 7-fold in KO MSC cultures and 2- to 4-fold in KO COB cultures. PTH also increased mineralization in both KO MSC and COB cultures. Effects in KO cells were mimicked in WT MSC cultures treated with NS-398, an inhibitor of COX-2 activity. PTH increased cAMP concentrations similarly in WT and KO COBs. Differential gene responses to PTH in COX-2 KO COBs relative to WT COBs included greater fold-increases in the cAMP-mediated early response genes, c-fos and Nr4a2; increased IGF-1 mRNA expression; and decreased mRNA expression of MAP kinase phosphatase-1. PTH inhibited SOST mRNA expression 91% in COX-2 KO MSC cultures compared to 67% in WT cultures. We conclude that endogenous PGs inhibit the anabolic responses to PTH in vitro, possibly by desensitizing cAMP pathways.     

Decreased bone mineral density and reduced bone quality in H(+) /K(+) ATPase beta-subunit deficient mice

Proton pump inhibitors (PPIs) are widely used against gastroesophageal reflux disease. Recent epidemiological studies suggest that PPI users have an increased risk of fractures, but a causal relationship has been questioned. We have therefore investigated the skeletal phenotype in H⁺/K⁺ATPase beta‐subunit knockout (KO) female mice. Skeletal parameters were determined in 6‐ and 20‐month‐old KO mice and in wild‐type controls (WT). Whole body bone mineral density (BMD) and bone mineral content (BMC) were measured by dual energy X‐ray absorptiometry (DXA). Femurs were examined with µCT analyses and break force were examined by a three‐point bending test. Plasma levels of gastrin, RANKL, OPG, osteocalcin, leptin, and PTH were analyzed. KO mice had lower whole body BMC at 6 months (0.53 vs. 0.59 g, P = 0.035) and at 20 months (0.49 vs. 0.74 g, P < 0.01) compared to WT as well as lower BMD at 6 months (0.068 vs. 0.072 g/cm², P = 0.026) and 20 months (0.067 vs. 0.077 g/cm², P < 0.01). Mechanical strength was lower in KO mice at the age of 20 months (6.7 vs. 17.9 N, P < 0.01). Cortical thickness at 20 months and trabecular bone volume% at 6 months were significantly reduced in KO mice. Plasma OPG/RANKL ratio and PTH was increased in KO mice compared to controls. H⁺/K⁺ATPase beta subunit KO mice had decreased BMC and BMD, reduced cortical thickness and inferior mechanical bone strength. Whereas the mechanism is uncertain, these findings suggest a causal relationship between long‐term PPI use and an increased risk of fractures. J. Cell. Biochem. 113: 141–147, 2012. © 2011 Wiley Periodicals, Inc.     

Modulation of unloading-induced bone loss in mice with altered ERK signaling

Genetic variations mediate skeletal responsiveness to mechanical unloading, with individual space travelers exhibiting large variations in the extent of bone loss. We previously identified genomic regions harboring several hundred genes that can modulate the magnitude of skeletal adaptation to mechanical unloading. Here, bioinformatic filters aided in shortlisting 30 genes with bone-related and mechanoregulatory roles. The genes CD44, FGF2, NOD2, and Fas, all associated with ERK signaling, were then functionally tested in hindlimb-unloaded (HLU) knockout (KO) mice. Compared to their respective normally ambulating wildtype (WT) controls, all KO strains, except Fas mice, had lower trabecular bone volume, bone volume fraction, and/or trabecular number. For cortical bone and compared to ambulatory WT mice, CD44^?/? had impaired properties while FGF2^?/? showed enhanced indices. NOD2^?/? and Fas^?/? did not have a cortical phenotype. In all KO and WT groups, HLU resulted in impaired trabecular and cortical indices, primarily due to trabecular tissue loss and mitigation of cortical bone growth. The difference in trabecular separation between HLU and ambulatory controls was significantly greater in CD44^?/? and NOD2^?/? mice than in WT mice. In cortical bone, differences in cortical thickness, total pore volume, and cortical porosity between HLU and controls were aggravated in CD44^?/? mice. In contrast, deletion of NOD2 and Fas genes mitigated the differences in Po.V between HLU and control mice. Together, we narrowed a previous list of QTL-derived candidate genes from over 300 to 30, and showed that CD44, NOD2, and Fas have distinct functions in regulating changes in trabecular and cortical bone indices during unloading.     

Parathyroid hormone induces bone formation in phosphorylation-deficient PTHR1 knockin mice

Activation of G protein-coupled receptors by agonists leads to receptor phosphorylation, internalization of ligand receptor complexes, and desensitization of hormonal response. The role of parathyroid hormone (PTH) receptor 1, PTHR1, is well characterized and known to regulate cellular responsiveness in vitro. However, the role of PTHR1 phosphorylation in bone formation is yet to be investigated. We have previously demonstrated that impaired internalization and sustained cAMP stimulation of phosphorylation-deficient (PD) PTHR1 leads to exaggerated cAMP response to subcutaneous PTH infusion in a PD knockin mouse model. To understand the physiological role of receptor internalization on PTH bone anabolic action, we examined bone parameters of wild-type (WT) and PD knockin female and male mice following PTH treatment. We found a decrease in total and diaphyseal bone mineral density in female but not in male PD mice compared with WT controls at 3-6 mo of age. This effect was attenuated at older age groups. PTH administration displayed increased bone volume and trabecular thickness in the vertebrae and distal femora of both WT and PD animals. These results suggest that PTHR1 phosphorylation does not play a major role in the anabolic action of PTH.     

Deficiency of Adiponectin Protects against Ovariectomy-Induced Osteoporosis in Mice

Adipokine adiponectin (APN) has been recently reported to play a role in regulating bone mineral density (BMD). To explore the mechanism by which APN affects BMD, we investigated BMD and biomechanical strength properties of the femur and vertebra in sham-operated (Sham) and ovariectomized (OVX) APN knockout (KO) mice as compared to their operated wild-type (WT) littermates. The results show that APN deficiency has no effect on BMD but induces increased ALP activity and osteoclast cell number. While OVX indeed leads to significant bone loss in both femora and vertebras of WT mice with comparable osteogenic activity and a significant increase in osteoclast cell number when compared to that of sham control. However, no differences in BMD, ALP activity and osteoclast cell number were found between Sham and OVX mice deficient for APN. Further studies using bone marrow derived mesenchymal stem cells (MSCs) demonstrate an enhanced osteogenic differentiation and extracellular matrix calcification in APN KO mice. The possible mechanism for APN deletion induced acceleration of osteogenesis could involve increased proliferation of MSCs and higher expression of Runx2 and Osterix genes. These findings indicate that APN deficiency can protect against OVX-induced osteoporosis in mice, suggesting a potential role of APN in regulating the balance of bone formation and bone resorption, especially in the development of post-menopausal osteoporosis.     

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.     

Conditional disruption of IGF-I gene in type 1α collagen-expressing cells shows an essential role of IGF-I in skeletal anabolic response to loading

To establish a causal role for locally produced IGF-I in the mechanical strain response in the bone, we have generated mice with conditional disruption of the insulin-like growth factor (IGF) I gene in type 1α(2) collagen-expressing cells using the Cre-loxP approach. At 10 wk of age, loads adjusted to account for bone size difference were applied via four-point bending or axial loading (AL) in mice. Two wk of bending and AL produced significant increases in bone mineral density and bone size at the middiaphysis of wild-type (WT), but not knockout (KO), mice. In addition, AL produced an 8-25% increase in trabecular parameters (bone volume-tissue volume ratio, trabecular thickness, and trabecular bone mineral density) at the secondary spongiosa of WT, but not KO, mice. Histomorphometric analysis at the trabecular site revealed that AL increased osteoid width by 60% and decreased tartrate-resistance acidic phosphatase-labeled surface by 50% in the WT, but not KO, mice. Consistent with the in vivo data, blockade of IGF-I action with inhibitory IGF-binding protein (IGFBP4) in vitro completely abolished the fluid flow stress-induced MC3T3-E1 cell proliferation. One-way ANOVA revealed that expression levels of EFNB1, EFNB2, EFNA2, EphB2, and NR4a3 were different in the loaded bones of WT vs. KO mice and may, in part, be responsible for the increase in bone response to loading in the WT mice. In conclusion, IGF-I expressed in type 1 collagen-producing bone cells is critical for converting mechanical signal to anabolic signal in bone, and other growth factors cannot compensate for the loss of local IGF-I.     

The calcium-sensing receptor complements parathyroid hormone-induced bone turnover in discrete skeletal compartments in mice

Although the calcium-sensing receptor (CaSR) and parathyroid hormone (PTH) may each exert skeletal effects, it is uncertain how CaSR and PTH interact at the level of bone in primary hyperparathyroidism (PHPT). Therefore, we simulated PHPT with 2 wk of continuous PTH infusion in adult mice with deletion of the PTH gene (Pth(-/-) mice) and with deletion of both PTH and CaSR genes (Pth(-/-)-Casr (-/-) mice) and compared skeletal phenotypes. PTH infusion in Pth(-/-) mice increased cortical bone turnover, augmented cortical porosity, and reduced cortical bone volume, femoral bone mineral density (BMD), and bone mineral content (BMC); these effects were markedly attenuated in PTH-infused Pth(-/-)-Casr(-/-) mice. In the absence of CaSR, the PTH-stimulated expression of receptor activator of nuclear factor-κB ligand and tartrate-resistant acid phosphatase and PTH-stimulated osteoclastogenesis was also reduced. In trabecular bone, PTH-induced increases in bone turnover, trabecular bone volume, and trabecular number were lower in Pth(-/-)-Casr(-/-) mice than in Pth(-/-) mice. PTH-stimulated genetic markers of osteoblast activity were also lower. These results are consistent with a role for CaSR in modulating both PTH-induced bone resorption and PTH-induced bone formation in discrete skeletal compartments.     

Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including G_sα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of G_sα in the osteoblast lineage. Postnatal removal of G_sα in the osteoblast lineage (P-G_sα^OsxKO mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1–34) (80 μg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-G_sα^OsxKO mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-G_sα^OsxKO mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via G_sα but not phospholipase C via G_q/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of G_sα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond G_sα and G_q/11 act downstream of PTH on osteoblast differentiation.     

Effect of dietary calcium and 1,25-(OH)2D3 on the expression of calcium transport genes in calbindin-D9k and -D28k double knockout mice

The phenotypes of calbindin-D9k (CaBP-9k) and -28k (CaBP-28k) single knockout (KO) mice are similar to wild-type (WT) mice due to the compensatory action of other calcium transport proteins. In this study, we generated CaBP-9k/CaBP-28k double knockout (DKO) mice in order to investigate the importance of CaBP-9k and CaBP-28k in active calcium processing. Under normal dietary conditions, DKO mice did not exhibit any changes in phenotype or the expression of active calcium transport genes as compared to WT or CaBP-28k KO mice. Under calcium-deficient dietary conditions, the phenotype and expression of calcium transport genes in CaBP-28k KO mice were similar to WT, whereas in DKO mice, serum calcium levels and bone length were decreased. The intestinal and renal expression of transient receptor potential vanilloid member 6 (TRPV6) mRNA was significantly decreased in DKO mice fed a calcium-deficient diet as compared to CaBP-28k KO or WT mice, and DKO mice died after 4 weeks on a calcium-deficient diet. Body weight, bone mineral density (BMD) and bone length were significantly reduced in all mice fed a calcium and 1,25-(OH)₂D₃-deficient diet, as compared to a normal diet, and none of the mice survived more than 4 weeks. These results indicate that deletion of CaBP-28k alone does not affect body calcium homeostasis, but that deletion of CaBP-9k and CaBP-28k has a significant effect on calcium processing under calcium-deficient conditions, confirming the importance of dietary calcium and 1,25-(OH)₂D₃ during growth and development.     

Resorption Controls Bone Anabolism Driven by Parathyroid Hormone (PTH) Receptor Signaling in Osteocytes

The contribution of remodeling-based bone formation coupled to osteoclast activity versus modeling-based bone formation that occurs independently of resorption, to the anabolic effect of PTH remains unclear. We addressed this question using transgenic mice with activated PTH receptor signaling in osteocytes that exhibit increased bone mass and remodeling, recognized skeletal effects of PTH elevation. Direct inhibition of bone formation was accomplished genetically by overexpressing the Wnt antagonist Sost/sclerostin; and resorption-dependent bone formation was inhibited pharmacologically with the bisphosphonate alendronate. We found that bone formation induced by osteocytic PTH receptor signaling on the periosteal surface depends on Wnt signaling but not on resorption. In contrast, bone formation on the endocortical surface results from a combination of Wnt-driven increased osteoblast number and resorption-dependent osteoblast activity. Moreover, elevated osteoclasts and intracortical/calvarial porosity is exacerbated by overexpressing Sost and reversed by blocking resorption. Furthermore, increased cancellous bone is abolished by Wnt inhibition but further increased by blocking resorption. Thus, resorption induced by PTH receptor signaling in osteocytes is critical for full anabolism in cortical bone, but tempers bone gain in cancellous bone. Dissecting underlying mechanisms of PTH receptor signaling would allow targeting actions in different bone compartments, enhancing the therapeutic potential of the pathway.     

Circulating levels of insulin-like growth factor-I and -II,and IGF-binding protein-3 in inflammation and after parathyroid hormone infusion

In order to assess if the anabolic action of PTH is related to changes in circulating levels of insulin-like growth factor-I and -II (IGF-I and -II), and IGF binding protein 3 (IGFBP-3), 24 h of PTH infusion was performed in healthy women and in patients with rheumatoid arthritis (RA), a state where both bone metabolism and PTH secretion is influenced by the inflammatory activity. The patients with RA had lower basal levels of both IGF-I and -II than the healthy controls (P < 0.05). In neither group did the IGFs change after 24 h of PTH administration, while IGFBP-3 was significantly increased in the healthy controls (4600 ± 1200 to 5750 ± 2200 μg/l, P < 0.05). IGFBP-3 was not affected by PTH infusion in patients with RA when the disease had high activity, but when inflammation had subsided they responded with a similar increase in IGFBP-3 as the control group and basal IGF-I and -II levels were normalised. Since IGFBP-3 can enhance the anabolic action of IGF-I, increased IGFBP-3 levels after PTH infusion may reflect a mechanism by which PTH is anabolic for bone. Inflammation may inhibit bone formation via decreased serum levels of IGFs and blocked IGFBP-3 response to PTH.     

Transgenic Overexpression of Ephrin B1 in Bone Cells Promotes Bone Formation and an Anabolic Response to Mechanical Loading in Mice

To test if ephrin B1 overexpression enhances bone mass, we generated transgenic mice overexpressing ephrin B1 under the control of a 3.6 kb rat collagen 1A1 promoter (Col3.6-Tg^efnb1). Col3.6-Tg^efnb1 mice express 6-, 12- and 14-fold greater levels of full-length ephrin B1 protein in bone marrow stromal cells, calvarial osteoblasts, and osteoclasts, respectively. The long bones of both genders of Col3.6-Tg^efnb1 mice have increased trabecular bone volume, trabecular number, and trabecular thickness and decreased trabecular separation. Enhanced bone formation and decreased bone resorption contributed to this increase in trabecular bone mass in Col3.6-Tg^efnb1 mice. Consistent with these findings, our in vitro studies showed that overexpression of ephrin B1 increased osteoblast differentiation and mineralization, osterix and collagen 1A1 expression in bone marrow stromal cells. Interaction of ephrin B1 with soluble clustered EphB2-Fc decreased osteoclast precursor differentiation into multinucleated cells. Furthermore, we demonstrated that the mechanical loading-induced increase in EphB2 expression and newly formed bone were significantly greater in the Col3.6-Tg^efnb1 mice than in WT littermate controls. Our findings that overexpression of ephrin B1 in bone cells enhances bone mass and promotes a skeletal anabolic response to mechanical loading suggest that manipulation of ephrin B1 actions in bone may provide a means to sensitize the skeleton to mechanical strain to stimulate new bone formation.     

Parathyroid Hormone (PTH)/PTH-related Peptide Type 1 Receptor (PPR) Signaling in Osteocytes Regulates Anabolic and Catabolic Skeletal Responses to PTH

Parathyroid hormone (PTH) is the only Food and Drug Administration-approved anabolic agent to treat osteoporosis; however, the cellular targets of PTH action in bone remain controversial. PTH modulates bone turnover by binding to the PTH/PTH-related peptide (PTHrP) type 1 receptor (PPR), a G-protein-coupled receptor highly expressed in bone and kidneys. Osteocytes, the most abundant cells in adult bone, also express PPR. However, the physiological relevance of PPR signaling in osteocytes remains to be elucidated. Toward this goal, we generated mice with PPR deletion in osteocytes (Ocy-PPRKO). Skeletal analysis of these mice revealed a significant increase in bone mineral density and trabecular and cortical bone parameters. Osteoblast activities were reduced in these animals, as demonstrated by decreased collagen type I α1 mRNA and receptor activator of NF-κB ligand (RANKL) expression. Importantly, when subjected to an anabolic or catabolic PTH regimen, Ocy-PPRKO animals demonstrated blunted skeletal responses. PTH failed to suppress SOST/Sclerostin or induce RANKL expression in Ocy-PPRKO animals compared with controls. In vitro, osteoclastogenesis was significantly impaired in Ocy-PPRKO upon PTH administration, indicating that osteocytes control osteoclast formation through a PPR-mediated mechanism. Taken together, these data indicate that PPR signaling in osteocytes is required for bone remodeling, and receptor signaling in osteocytes is needed for anabolic and catabolic skeletal responses.     

Bone anabolic effects of parathyroid hormone are blunted by deletion of the Wnt antagonist secreted frizzled-related protein-1

Secreted frizzled-related protein (sFRP)-1 is a Wnt antagonist that when deleted in mice leads to increased trabecular bone formation in adult animals after 13 weeks of age. Treatment of mice with parathyroid hormone (PTH) also increases trabecular bone formation, and some of the anabolic actions of this hormone may result from altered expression of Wnt pathway components. To test this hypothesis, we treated +/+ and -/- female sFRP-1 mice with PTH 1-34 for 30 days and measured distal femur trabecular bone parameters by peripheral quantitative computed tomography (pQCT) and high-resolution micro-computed tomography. During the course of the 32-week study, volumetric bone mineral density (vBMD) declined 41% in vehicle-treated +/+ mice, but increased 24% in vehicle-treated -/- animals. At 8 weeks of age when vBMD was not altered by deletion of sFRP-1, treatment of +/+ and -/- mice with PTH increased vBMD by 147 and 163%, respectively. In contrast, at 24 weeks of age when vBMD was 75% higher in -/- mice than in +/+ controls, treatment with PTH increased vBMD 164% in +/+ animals, but only 58% in -/- mice. Furthermore, at 36 weeks of age when vBMD was 117% higher in -/- mice than in +/+ controls, treatment with PTH increased vBMD 74% in +/+ animals, while no increase was observed in -/- mice. At each of these time points, PTH treatment increased vBMD to a similar level in +/+ and -/- mice, and this level declined with age. In addition, at 36 weeks of age, the vBMD level reached by PTH treatment of +/+ mice was the same as that achieved solely by deletion of sFRP-1. These results indicate that loss of sFRP-1 and PTH treatment increase vBMD to a similar extent. Moreover, as the effects of sFRP-1 deletion on vBMD increase, the ability of PTH to enhance vBMD declines suggesting that there are overlapping mechanisms of action.     

Biglycan and chondroitin sulfate play pivotal roles in bone toughness via retaining bound water in bone mineral matrix

Recent in vitro evidence shows that glycosaminoglycans (GAGs) and proteoglycans (PGs) in bone matrix may functionally be involved in the tissue-level toughness of bone. In this study, we showed the effect of biglycan (Bgn), a small leucine-rich proteoglycan enriched in extracellular matrix of bone and the associated GAG subtype, chondroitin sulfate (CS), on the toughness of bone in vivo, using wild-type (WT) and Bgn deficient mice. The amount of total GAGs and CS in the mineralized compartment of Bgn KO mouse bone matrix decreased significantly, associated with the reduction of the toughness of bone, in comparison with those of WT mice. However, such differences between WT and Bgn KO mice diminished once the bound water was removed from bone matrix. In addition, CS was identified as the major subtype in bone matrix. We then supplemented CS to both WT and Bgn KO mice to test whether supplemental GAGs could improve the tissue-level toughness of bone. After intradermal administration of CS, the toughness of WT bone was greatly improved, with the GAGs and bound water amount in the bone matrix increased, while such improvement was not observed in Bgn KO mice or with supplementation of dermatan sulfate (DS). Moreover, CS supplemented WT mice exhibited higher bone mineral density and reduced osteoclastogenesis. Interestingly, Bgn KO bone did not show such differences irrespective of the intradermal administration of CS. In summary, the results of this study suggest that Bgn and CS in bone matrix play a pivotal role in imparting the toughness to bone most likely via retaining bound water in bone matrix. Moreover, supplementation of CS improves the toughness of bone in mouse models.