The Sclerostin-Independent Bone Anabolic Activity of Intermittent PTH Treatment Is Mediated by T-Cell–Produced Wnt10b

Authors

  • Jau-Yi Li,

    1. Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA
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  • Lindsey D Walker,

    1. Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA
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  • Abdul Malik Tyagi,

    1. Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA
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  • Jonathan Adams,

    1. Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA
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  • M Neale Weitzmann,

    1. Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA
    2. Atlanta VA Medical Center, Decatur, GA, USA
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  • Roberto Pacifici

    Corresponding author
    1. Division of Endocrinology, Metabolism and Lipids, Department of Medicine, Emory University, Atlanta, GA, USA
    2. Immunology and Molecular Pathogenesis Program, Emory University, Atlanta, GA, USA
    • Address correspondence to: Roberto Pacifici, MD, Division of Endocrinology, Metabolism and Lipids, Emory University School of Medicine, 101 Woodruff Circle, Room 1309, Atlanta, GA 30322, USA. E-mail: roberto.pacifici@emory.edu

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ABSTRACT

Both blunted osteocytic production of the Wnt inhibitor sclerostin (Scl) and increased T-cell production of the Wnt ligand Wnt10b contribute to the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. However, the relative contribution of these mechanisms is unknown. In this study, we modeled the repressive effects of iPTH on Scl production in mice by treatment with a neutralizing anti-Scl antibody (Scl-Ab) to determine the contribution of T-cell–produced Wnt10b to the Scl-independent modalities of action of iPTH. We report that combined treatment with Scl-Ab and iPTH was more potent than either iPTH or Scl-Ab alone in increasing stromal cell production of OPG, osteoblastogenesis, osteoblast life span, bone turnover, bone mineral density, and trabecular bone volume and structure in mice with T cells capable of producing Wnt10b. In T-cell–null mice and mice lacking T-cell production of Wnt10b, combined treatment increased bone turnover significantly more than iPTH or Scl-Ab alone. However, in these mice, combined treatment with Scl-Ab and iPTH was equally effective as Scl-Ab alone in increasing the osteoblastic pool, bone volume, density, and structure. These findings demonstrate that the Scl-independent activity of iPTH on osteoblasts and bone mass is mediated by T-cell–produced Wnt10b. The data provide a proof of concept of a more potent therapeutic effect of combined treatment with iPTH and Scl-Ab than either alone. © 2014 American Society for Bone and Mineral Research.

Introduction

Parathyroid hormone (PTH) is a major regulator of calcium metabolism that defends against hypocalcemia, in part by stimulating bone resorption and thereby the release of calcium from the skeleton. However, when injected daily, a regimen known as intermittent PTH (iPTH) treatment, the hormone markedly stimulates bone formation, leading to improved bone microarchitecture and increased strength.[1] As a result, intermittent treatment with the 1-34 fragment of PTH is an FDA-approved treatment modality for postmenopausal osteoporosis.

The effects of PTH on bone result from its binding to the PTH/PTH-related protein (PTHrP) receptor, expressed on bone marrow (BM) stromal cells (SCs), osteoblasts (OBs), and osteocytes.[2-4] iPTH stimulates bone formation by increasing the number of OBs,[5-7] a phenomenon achieved through activation of quiescent lining cells,[8] increased OB proliferation[9, 10] and differentiation,[9, 11, 12] attenuation of OB apoptosis,[13-16] and signaling in osteocytes.[17] The expansion of the osteoblastic pool induced by iPTH is initiated by the release from the bone matrix undergoing resorption of TGFβ, IGF-1, and other growth factors that recruit SCs to remodeling areas.[18-21] Subsequent events are driven primarily by the activation of Wnt signaling in osteoblastic cells.[22] Activation of Wnt signaling induces OB proliferation[23] and differentiation,[22, 24] prevents OB apoptosis,[15, 16, 25] and augments OB production of OPG, thus blunting bone resorption.[26] Wnt proteins initiate a canonical signaling cascade by binding to receptors of the Frizzled family together with the coreceptors LRP5-6, which results in the stabilization of cytosolic β-catenin. Wnt proteins also signal through noncanonical pathways that involve the Src/ERK and Pi3K/Akt cascades.[15]

iPTH activates Wnt signaling in OBs through multiple mechanisms that include Wnt ligand-independent activation of Wnt coreceptors,[27] increased production of Wnt ligands by bone and BM cells,[28, 29] and suppression of sclerostin (Scl) production.[30-32] Additional effects on the Wnt system have been described in models of hyperparathyroidism characterized by a continuous overproduction of PTH but not in mice treated with intermittent PTH. For example, continuous PTH treatment regulates the Wnt antagonist Dkk1[33, 34] and the Wnt receptor LRP6,[27] whereas iPTH does not.

The capacity of PTH to suppress Scl production,[30-32] the finding that serum levels of Scl are inversely correlated with PTH levels in healthy women,[35] and reports that women treated with teriparatide have decreased serum levels of Scl[36] have led to the hypothesis that repression of the SOST gene and the resulting inhibition of Scl production are a key mechanism of action of iPTH.[37] Studies in SOST transgenic and global SOST–/– mice revealed that Scl plays an important, yet nonexclusive, role in iPTH-induced anabolism.[38-40] The existence of a Scl-independent effect of iPTH was supported by the finding that iPTH equally elevates bone formation and resorption in WT and in SOST transgenic mice.[38] In addition, iPTH induced a significant increase in trabecular thickness and mineral apposition rate in Sost-null mice, thus demonstrating that iPTH stimulates bone formation also independently of Scl suppression.[38] However, the answer to the question of whether iPTH increases trabecular bone volume in the absence of Scl is still uncertain because no increase in bone volume or bone density was detected in the distal femur of both growing WT control and Sost-null mice in one study,[38] whereas a full anabolic activity of iPTH in the trabecular bone of skeletally mature Sost–/– mice was reported in another.[40] A cause of uncertainty concerning the relevance of Scl-independent modalities of action of iPTH is the confounding effect of the altered baseline bone density characteristic of Sost–/– and SOST BAC transgenic mice.[38-40] The fact that iPTH blunts but does not completely block Scl production further limits the usefulness of Sost–/– mice as a tool to investigate the mechanism of action of iPTH.

The mechanisms responsible for the Scl-independent activity of iPTH are largely unknown. Although SCs, OBs, and osteocytes represent the major targets of PTH in bone, reports from our laboratory have disclosed that T lymphocytes play a critical role in the mechanism of action of PTH.[29, 41-43] We have shown that treatment with iPTH increases the T-cell production of Wnt10b,[29, 43] a Wnt ligand that stimulates osteoblastogenesis by activating Wnt signaling in SCs and OBs. As a result, the bone anabolic activity of iPTH is markedly reduced in T-cell–deficient mice and in mice with a global or T-cell–specific disruption of Wnt10b production.[29, 43] Thus, stimulated T-cell production of Wnt10b is another critical mechanism of action of iPTH that, like Scl inhibition, contributes to the activation of Wnt signaling in osteoblastic cells.

The current study was designed to investigate the contribution of Scl inhibition and stimulated T-cell production of Wnt10 to the anabolic activity of iPTH in mice with normal baseline bone turnover and bone mass and capable of responding to iPTH with a partial suppression of Scl production.

To achieve these goals, mice were treated with iPTH and a neutralizing anti-Scl mAb[44] referred to hereafter as (Scl-Ab). We report that iPTH treatment potentiates the trabecular bone anabolism of Scl-Ab in WT mice, but not in T-cell–deficient mice, Wnt10b–/– mice, and mice lacking T-cell production of Wnt10b. These findings demonstrate that the anabolic activity of iPTH is not only the result of the repression of Scl production but also increased T-cell production of Wnt10b.

Materials and Methods

Animals

All of the animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University. Female C57BL6/J WT and TCRβ–/– mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). C57BL6/J Wnt10b–/– mice were generated as described[45] and provided by Dr TF Lane (University of California, Los Angeles, CA, USA). All mice were maintained under specific pathogen-free conditions and fed sterilized food and autoclaved water ad libitum.

Scl-Ab, irrelevant Ig, and iPTH treatment

The anti Scl-Ab was previously described[44, 46] and kindly provided by Novartis Pharmaceutical (Basel, Switzerland). Purified total mouse IgG2A (Lampire Biological Laboratories, Ottsville, PA, USA) was used as control irrelevant isotype matched Ig. Scl-Ab and irrelevant Ig were injected at 30 to 100 mg/kg/week iv for 4 weeks in 6-week-old mice. Mice were also injected with 80 µg/kg/day of human PTH (1-34) (Bachem California, Inc., Torrance, CA, USA) or vehicle daily subcutaneously for 4 weeks as described.[29, 43]

In vivo bone mineral density (BMD) measurements

Spinal BMD was measured in anesthetized mice using a PIXImus2 bone densitometer (GE Medical Systems, Lunar, Madison, WI, USA) as described.[47]

μCT measurements

Indices of trabecular and cortical bone volume and structure were measured in the spine (excised 5th lumbar spine body) and the femur, respectively, using a Scanco μCT-40 scanner (Scanco Medical, Bassersdorf, Switzerland). μCT scanning and analysis was performed as reported previously.[29, 42] Briefly, trabecular and cortical bone regions were evaluated using isotropic 12-µm voxels. For the vertebral trabecular region, we evaluated 250 transverse CT slices between the cranial and caudal end plates, excluding 100 µm near each end plate. For the femoral trabecular region, we analyzed 100 slices from the 50 slices under the distal growth plate. Femoral cortical bone was assessed using 50 continuous CT slides located at the femoral midshaft.

Quantitative bone histomorphometry

Vertebras were fixed in 10% neutral-buffered formalin for 48 hours, dehydrated and defattened at 4°C, and embedded in methyl methacrylate resin. In brief, 5-μm nonconsecutive longitudinal sections of L4 in the frontal midbody plane (RM2155 microtome, Leica Microsystems, Buffalo Grove, IL, USA) were cut and analyzed. Sections were stained with toluidine blue, Goldner's Trichrome, or tartrate-resistant acid phosphatase (TRAP) stains. Osteoblast and osteoclast number and surface were determined on stained sections, respectively, using a Merz grid (400 ×). The measurements, terminology, and units used for histomorphometric analysis were those recommended by the Nomenclature Committee of the American Society of Bone and Mineral Research.[48]

T-cell transfer

WT and Wnt10b–/– spleen T cells were purified by negative immunoselection using MACS Pan T cell isolation kit (Miltenyi Biotech, Auburn, CA, USA) and injected (5 × 106 cells per mouse) iv into TCRβ−/− recipient mice 3 weeks before treatment. Successful T-cell engraftment was confirmed by flow cytometry of the spleens of the recipient mice harvested at death.

SC purification

BM cells were collected at death, and SCs were purified as previously described.[41, 43] Briefly, BM was cultured for 7 days in α-MEM medium containing 10% fetal bovine serum (FBS) to allow the proliferation of SCs. After discarding nonadherent cells, adherent macrophages were eliminated by positive immunoselection by MACS Microbeads (Miltenyi Biotec) coupled to anti-CD11c antibody. This marker is expressed on nonadherent dendritic cells and adherent monocytes and macrophages. The remaining adherent cells were defined as SCs because they express ALP, type-I collagen, and Runx2 and have the capacity to form mineralization nodules when further cultured under mineralizing conditions.

Markers of bone turnover

Serum C-terminal telopeptide of collagen (CTX) was measured by a rodent-specific ELISA assay (Immunodiagnostic Systems, Scottsdale, AZ, USA). Serum P1NP was measured by ELISA (Immunodiagnostic Systems, Boldon, UK).

CFU-ALP assay

Colony-forming assays were carried out as described.[29, 41, 49]

Thymidine incorporation assay

The rate of SC proliferation was measured by [3H]-thymidine incorporation assay. SCs were purified and pulsed with [3H]-thymidine (0.5 μCi/10,000 cells) for 18 hours, and were harvested using a Cell Harvestor (Skatron, Inc., Sterling, VA, USA). [3H]-thymidine incorporation was determined by a LS 6000 IC Liquid Scintillation Counter (Beckman Coulter, Inc., Fullerton, CA, USA).

Apoptosis assay

The activity of caspase-3, the key protease in the induction of apoptosis, was measured in SCs using CaspACE Assay System (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol.

Statistical analysis

For each outcome, a two-way analysis-of-variance was applied that included the main effects for animal strain and treatment plus the statistical interaction between animal strain and treatment. When the statistical interaction between animal strain and treatment group was not statistically significant (p > 0.05) nor suggestive of an important interaction (p > 0.10), p values for the main effects tests were reported. When the statistical interaction was statistically significant or suggestive of an important interaction, then t tests were used to compare the differences between the treatment means for each animal strain, applying the Bonferroni correction for multiple comparisons.

Results

iPTH treatment promotes bone anabolism in mice treated with Scl-Ab

Based on a previous report that Scl-Ab at 12 mg/Kg/Wk increases bone volume as potently as iPTH treatment at 40 μg/kg/day,[46] we conducted a dose response study in six weeks old female C57BL6 WT mice to determine the dose of Scl-Ab required to model the partial repression of Scl production and the corresponding increase in the bone volume fraction (BV/TV) induced by iPTH. Analysis by μCT revealed (Supplemental Fig. S1) that Scl-Ab treatment at 30 mg/kg/week once weekly for 4 weeks induced a 43.5% increase in spinal trabecular bone volume (BV/TV). Treatment with Scl-Ab at 50 mg/kg/week increased BV/TV by the same amount as Scl-Ab at 30 mg/kg/week. A slightly higher (58.9%) increase in BV/TV was obtained with Scl-Ab at 100 mg/kg/week. Based on these findings, we selected 50 mg/kg/week as a dose capable of modeling the partial blunting effects of iPTH on Scl levels without inducing a complete Scl blockade. Therefore, all subsequent studies were conducted by treating 6-week-old female C57BL6 mice with Scl-Ab or control isotype matched irrelevant Ab (Irr.Ig) at the dose of 50 mg/kg iv once weekly for 4 weeks. In addition, mice were injected daily with vehicle or 80 μg/kg/day of hPTH 1-34 for 4 weeks, a treatment modality referred to hereafter as iPTH. The mice used for this investigation were WT mice, congenic TCRβ–/– mice, a strain completely devoid of αβ T cells, and global Wnt10b–/– mice. To control for strain-dependent confounders, the study also included TCRβ–/– mice subjected to adoptive transfer of T cells harvested from WT or Wnt10b–/– mice 3 weeks before the start of the 4-week-long treatment period. The adoptive transfer of WT and Wnt10b T cells is followed by the engraftment and homeostatic expansion of the donor T cells.[49-51] Flow cytometric analysis of splenocytes harvested at death from mice subjected to adoptive transfer of T cells confirmed the engraftment and the expansion of adoptively transferred T cells (Supplemental Fig. S2).

Dual X-ray absorptiometry (DXA) was utilized to measure the BMD of the spine in vivo. At baseline, WT and TCRβ–/– mice had similar BMD values. Because of age-related skeletal growth, BMD increased in WT vehicle-treated groups during the 4 weeks of the experiment (Fig. 1A). In WT mice, BMD increased significantly in response to treatment with either iPTH or Scl-Ab alone during the first 2 weeks of treatment. Attesting to persistent efficacy of the treatments, BMD continued to increase between week 2 and week 4. Combined treatment with Scl-Ab and iPTH was also effective in increasing BMD throughout the 4 weeks of the study. At week 4, combined treatment with Scl-Ab and iPTH resulted in a greater increase in spinal BMD than treatment with Scl-Ab alone or iPTH alone. The finding that iPTH had a significant effect on BMD in conditions of substantial Scl blockade confirms that the partial dampening of Scl production induced by iPTH does not account for the entire anabolic activity of iPTH.

Figure 1.

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on spine BMD in WT mice (A), TCRβ–/– mice (B), TCRβ–/– mice previously subjected to adoptive transfer of WT T cells (C) or Wnt10b T cells (D) and global Wnt10b–/– mice (E). In vivo spinal BMD was measured by DXA at 2 and 4 weeks of treatment (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle +Irr.Ig-treated group or the indicated group. #p < 0.05 compared with the corresponding group at 2 weeks.

Analysis of TCRβ–/– mice revealed (Fig. 1B) that treatment with iPTH alone did not increase BMD in mice lacking T cells. By contrast, Scl-Ab similarly increased BMD at 2 and 4 weeks in WT and TCRβ–/– mice, thus demonstrating that the activity of Scl-Ab is T-cell independent. Moreover, we found that in mice lacking T cells, combined treatment with Scl-Ab and iPTH did not increase BMD over the levels induced by Scl-Ab alone at 2 and 4 weeks, thus indicating that the Scl-independent anabolic effect of iPTH is mediated by T cells.

To further investigate the role of T cells, we next analyzed the effects of iPTH and Scl-Ab in TCRβ–/– mice subjected to adoptive transfer of WT T cells 3 weeks before initiation of the treatments. Mimicking the results obtained in WT mice, we found (Fig. 1C) that BMD was increased significantly by iPTH alone and Scl-Ab alone and combined treatment. However, at 4 weeks, the increase in BMD observed in mice treated with iPTH plus Scl-Ab was significantly higher than that of mice treated with Scl-Ab only, confirming that T cells are required for iPTH to exert an anabolic effect superior to that achieved with Scl inhibition.

Because earlier studies had revealed that T cells potentiate the anabolic activity of iPTH through Wnt10b,[29, 43] we next analyzed the effects of iPTH and Scl-Ab in TCRβ–/– mice previously subjected to adoptive transfer of Wnt10b–/– T cells. These studies revealed (Fig. 1D) that treatment with iPTH alone did not significantly increase BMD in mice with T cells lacking Wnt10b production. In these mice, Scl-Ab caused a significant increase in spine BMD at 2 and 4 weeks. Importantly, combined treatment with Scl-Ab and iPTH did not increase BMD over the levels induced by Scl-Ab alone, thus demonstrating that the Scl-independent anabolic effect of iPTH is mediated by T-cell–produced Wnt10b.

To further investigate the relevance of Wnt10b, another study was conducted in global Wnt10b–/– mice. This strain is known to have a markedly lower femoral bone density and volume than WT littermates.[52] However, we found that at 6 weeks of age, Wnt10b–/– mice and WT controls had similar spinal bone density and volume (not shown). Treatment with iPTH alone did not significantly increase BMD compared with controls, whereas Scl-Ab induced a significant increase in BMD (Fig. 1E). Combined treatment with Scl-Ab and iPTH was not statistically superior to treatment with Scl-Ab alone, thus confirming the causal role of Wnt10b.

To assess the specific effects of the treatments in the trabecular and cortical compartments, bones were harvested at the end of the 4-week-long treatment period and analyzed by μCT. Measurements of spinal trabecular bone revealed (Fig. 2A) that BV/TV was increased 24% by iPTH alone, 59% by Scl-Ab alone, and 77% by combined treatment with iPTH plus Scl-Ab. The effect on BV/TV of combined treatment with Scl-Ab and iPTH was significantly higher than the effects of either iPTH alone or Scl-Ab alone. The finding that iPTH has a significant effect on BV/TV in conditions of Scl inhibition indicates that the inhibitory activity of iPTH on Scl production does not account for the entire anabolic activity of iPTH.

Figure 2.

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on spine trabecular bone volume (BV/TV) as measured by μCT scanning in WT mice (A), TCRβ−/− mice (B), TCRβ−/− mice previously subjected to adoptive transfer of WT T cells (C) or Wnt10b−/− T cells (D) and global Wnt10b−/− mice (E) (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the indicated group.

Analysis of samples from TCRβ–/– mice revealed (Fig. 2B) that treatment with iPTH alone did not increase BV/TV in mice lacking T cells. By contrast, Scl-Ab caused a large increase in BV/TV, thus demonstrating that the activity of Scl-Ab in trabecular bone is T-cell independent. Moreover, we found that combined treatment with Scl-Ab and iPTH did not increase BV/TV over the levels induced by Scl-Ab alone in mice lacking T cells, thus confirming that the Scl-independent anabolic effect of iPTH in trabecular bone is mediated by T cells.

Analysis of samples from TCRβ–/– mice reconstituted with WT T cells revealed (Fig. 2C) that trabecular BV/TV was increased 26% by iPTH alone, 59% by Scl-Ab alone, and 82% by combined treatment with iPTH plus Scl-Ab. Importantly, in these mice, the effect on BV/TV of combined treatment with Scl-Ab and iPTH was significantly higher than the effects of Scl-Ab alone, demonstrating that reconstitution of T-cell–deficient mice with WT T cells restores the capacity of iPTH to exert an anabolic effect in conditions of Scl inhibition.

Analysis of samples from TCRβ–/– mice reconstituted with Wnt10b–/– T cells revealed (Fig. 2D) that treatment with iPTH alone did not significantly increase BV/TV in mice with T cells lacking Wnt10b production. Scl-Ab increased BV/TV by 73%, a change higher than that induced by Scl-Ab in WT mice. Importantly, combined treatment with Scl-Ab and iPTH did not increase BV/TV over the levels induced by Scl-Ab alone, thus demonstrating that the Scl-independent anabolic effect of iPTH on trabecular bone is mediated by T-cell–produced Wnt10b.

Analysis of samples from Wnt10b–/– mice revealed (Fig. 2E) that treatment with iPTH alone did not significantly increase BV/TV compared with controls, wherea Scl-Ab induced a 34% increase in BV/TV. Combined treatment with Scl-Ab and iPTH was not statistically superior to treatment with Scl-Ab alone, thus confirming the causal role of Wnt10b.

Parameters of trabecular structure were also differentially affected by combined treatment with iPTH plus Scl-Ab versus treatment with either iPTH or Scl-Ab alone. In fact, trabecular thickness (Tb.Th), trabecular number (Tb.N), and connectivity density (Conn.D) were more potently improved by combined treatment than by treatment with a single agent in WT mice and in T-cell–deficient mice reconstituted with WT T cells (Supplemental Figs. S3 to S5). By contrast, combined treatment was as effective as treatment with Scl-Ab alone in TCRβ−/− mice, TCRβ−/− mice previously reconstituted with Wnt10b−/− T cells, and global Wnt10b−/− mice.

Although T-cell–produced Wnt10b is required for iPTH to induce trabecular anabolism, iPTH induces cortical bone accretion by a T-cell–independent mechanism.[29] Accordingly, analysis of femoral cortical bone revealed that iPTH increased cortical volume (Fig. 3) and cortical thickness (Supplemental Fig. S6) in all groups of mice except for Wnt10b–/– mice. Treatment with Scl-Ab also induced a significant increase in cortical volume (Fig. 3) and cortical thickness (Supplemental Fig. S6) in all groups of mice. Attesting to the capacity of iPTH to exert Scl-independent and T-cell–independent effects on cortical bone, we found that combined treatment with iPTH and Scl-Ab increased cortical volume (Fig. 3) and cortical thickness (Supplemental Fig. S6) more than treatment with Scl-Ab alone or iPTH alone in all groups except for Wnt10b–/– mice.

Figure 3.

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on femoral cortical volume (Co.Vo) as measured by μCT scanning in WT mice (A), TCRβ−/− mice (B), TCRβ−/− mice previously subjected to adoptive transfer of WT T cells (C) or Wnt10b−/− T cells (D) and global Wnt10b−/− mice (E) (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the indicated group.

Analysis of vertebral cancellous bone by bone histomorphometry (Fig. 4) revealed that two static indices of bone formation, the number of OBs per bone surface (N.Ob/BS) and the percentage of surfaces covered by OBs (Ob.S/BS), increased significantly in response to treatment with iPTH alone in WT mice and in TCRβ–/– mice previously reconstituted with WT T cells. These indices of bone formation did not increase in response to iPTH in TCRβ–/– mice, TCRβ–/– mice previously reconstituted with Wnt10b–/– T cells, and global Wnt10b–/– mice. Treatment with Scl-Ab did not increase histomorphometric indices of bone formation in all groups of mice. This finding is consistent with recent studies in animals and humans demonstrating that Scl-Ab causes long-term increases in BMD but only a transitory, short-lasting increase in bone formation.[53-55] Combined treatment with iPTH and Scl-Ab increased N.Ob/BS and Ob.S/BS above those of Scl-Ab–treated mice in all groups of mice except Wnt10b-null mice. The findings demonstrate that iPTH has the capacity to regulate trabecular bone formation via a Scl-independent, Wnt10b-dependent mechanism.

Figure 4.

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on histomorphometric indices of bone formation and resorption. (A) Number of osteoblasts per mm bone surface (N.Ob/BS). (B) Percentage of bone surface covered by osteoblasts (Ob.S/BS). (C) Number of osteoclasts per mm bone surface (N.Oc/BS). (D) Percentage of bone surface covered by osteoclasts (Oc.S/BS). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle + Irr.Ig-treated group. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the Scl-Ab–treated group.

Measurements of serum type 1 procollagen amino-terminal propeptide (P1NP), a marker of bone formation that reflects primarily changes in cortical bone, revealed that P1NP levels increased significantly in response to iPTH treatment in all groups of mice except global Wnt10b–/– mice (Fig. 5A–E). Treatment with Scl-Ab did not induce an increase in P1NP levels in all groups of mice. Combined treatment with iPTH and Scl-Ab increased P1NP levels above those of Scl-Ab–treated mice in all groups of mice, indicating that iPTH has the capacity to regulate global bone formation by a Scl-independent mechanism.

Figure 5.

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on serum P1NP levels in WT mice (A), TCRβ−/− mice (B), TCRβ–/– mice previously subjected to adoptive transfer of WT T cells (C) or Wnt10b−/− T cells (D) and global Wnt10b−/− mice (E) (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the indicated group.

Histomorphometric analysis showed no significant effects of iPTH alone on two indices of trabecular bone resorption, the number of OCs per bone surface (N.Oc/BS), and the percentage of surfaces covered by OCs (Oc.S/BS) in all groups of mice (Fig. 4). Scl-Ab alone had no effects in all mice except T-cell–null mice, whereas combined treatment was more potent than Scl-Ab in WT mice.

Consistent with the lower sensitivity of biochemical markers compared with bone histomorphometry, serum CTX levels, an index of bone resorption, were not increased by iPTH, Scl-Ab, or combined treatment in all groups of mice (Fig. 6).

Figure 6.

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on serum CTX levels in WT mice (A), TCRβ−/− mice (B), TCRβ−/− mice previously subjected to adoptive transfer of WT T cells (C) or Wnt10b−/− T cells (D) and global Wnt10b−/− mice (E) (n = 10 mice per group). *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the indicated group.

BM cells harvested at death were utilized to assess the formation of alkaline phosphatase (ALP)-positive colony-forming unit-fibroblast (CFU-F), herein defined as CFU-ALP, an index of SC commitment to the osteoblastic lineage. iPTH treatment increased CFU-ALP formation in the BM of T-cell–replete mice by approximately twofold, whereas it had no effect in that of mice lacking T cells, T-cell–produced Wnt10b, or global Wnt10b production (Fig. 7A). Treatment with Scl-Ab did not increase the number of CFU-ALP in any group. Combined treatment was more effective than treatment with Scl-Ab alone in WT mice and T-cell–null mice reconstituted with WT T cells, but not in mice lacking T cells, T-cell production of Wnt10b, or global Wnt10b production.

Figure 7.

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on CFU-ALP formation, SC number, SC proliferation, and SC apoptosis in WT mice, TCRβ−/− mice, TCRβ−/− mice previously subjected to adoptive transfer of WT T cells or Wnt10b−/− T cells, and global Wnt10b−/− mice. (A) Whole BM was cultured for 7 days to assess the formation CFU-ALP. (B) BM harvested at death was cultured for 1 week and SCs purified and counted. (C) SCs were purified from BM cultured for 1 week, seeded in equal number and pulsed with 3H-thymidine for 18 hours to assess their proliferation. Data are expressed in CPM. (D) SCs were purified from BM cultured for 1 week and the rate of apoptosis quantified by determinations of caspase3 activity (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle or to the indicated group. #p < 0.05 compared with Scl-Ab treatment.

To determine the effects of iPTH and Scl-Ab on osteoblastogenesis, BM was cultured for 1 week to allow SCs to proliferate. SCs were then purified and counted. This analysis revealed that in vivo iPTH treatment increases the number of SCs in samples from mice with WT T cells, whereas it had no effects in SCs from mice lacking T cells, T-cell–produced Wnt10b, or global Wnt10b production (Fig. 7B). Treatment with Scl-Ab increased SC number in WT mice but not in the remaining groups. Combined treatment with iPTH and Scl-Ab was more potent than Scl-Ab alone but only in mice with WT T cells. To investigate the mechanism involved, BM was cultured for 1 week and SCs purified and used to determine their rate of proliferation and apoptosis. These experiments revealed that iPTH increases significantly the proliferation of SCs from mice with WT T cells, whereas it had no effect on the proliferation of SCs from mice lacking T cells, T-cell–produced Wnt10b, or global Wnt10b production (Fig. 7C). Treatment with Scl-Ab did not increase SC proliferation in any group. Combined treatment with iPTH and Scl-Ab was significantly more potent than Scl-Ab alone in mice with WT T cells but not in the groups lacking T cells, T-cell production of Wnt10b, or global Wnt10b production.

iPTH decreased the rate of SC apoptosis in WT mice and T-cell–null mice reconstituted with WT and Wnt10b−/− T cells, whereas it had no effects in SCs from mice lacking T cells or global Wnt10b production (Fig. 7D). Treatment with Scl-Ab did not affect SC apoptosis in all groups. Combined treatment with iPTH and Scl-Ab was significantly more potent than Scl-Ab alone in diminishing SC apoptosis but only in mice with WT T cells.

Analysis of the expression levels of osteoblastic genes in SCs revealed that iPTH treatment increased the expression of type 1 collagen, Runx2, osterix, bone sialoprotein, and osteocalcin mRNAs by approximately two- to fourfold in SCs from mice with WT T cells, whereas it had no effects in BM from mice lacking T cells, T-cell–produced Wnt10b, or global Wnt10b production (Fig. 8A–E). Treatment with Scl-Ab did not affect SC differentiation in all groups. Combined treatment with iPTH and Scl-Ab revealed that iPTH was still capable of promoting SC differentiation in conditions of Scl blockade but only in mice with WT T cells. These findings demonstrate that iPTH regulates some aspects of OB differentiation and life span through a Wnt10b-dependent, Scl-independent mechanism.

Figure 8.

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on OB differentiation in WT mice (A), TCRβ−/− mice (B), TCRβ−/− mice previously subjected to adoptive transfer of WT T cells (C), or Wnt10b−/− T cells (D) and global Wnt10b−/− mice (E). SCs were purified from BM cultured for 1 week and the level of OB marker gene mRNAs, bone sialoprotein (BSP), type I collagen (Col1), osteocalcin (Ocn), osterix (Osx), and runt related transcription factor 2 (Runx2) analyzed by RT-PCR (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle. #p < 0.05 compared with Scl-Ab treatment.

Activation of Wnt signaling augments the production of OPG,[26] a soluble decoy receptor for the osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL). Therefore, we measured the SC mRNA levels of OPG and RANKL to investigate whether changes in these cytokine levels contribute to the changes in bone turnover induced by iPTH, Scl-Ab, and combined treatment. This analysis revealed that iPTH treatment increased the expression of OPG mRNAs by approximately threefold in SCs from mice with WT T cells, whereas it had no effects in BM from mice lacking T cells, T-cell–produced Wnt10b, or global Wnt10b production (Fig. 9A). Treatment with Scl-Ab increased OPG expression in WT mice but not in the remaining groups. Combined treatment with iPTH and Scl-Ab stimulated OPG expression more potently than iPTH alone or Scl-Ab alone in mice with WT T cells but not in the other groups. By contrast, in all groups of mice, neither iPTH, Scl-Ab, or combined treatment induced a significant increase in RANKL (Fig. 9B). These findings suggest that iPTH regulates OPG production through a T-cell/Wnt10b–dependent mechanism.

Figure 9.

Analysis of the effects (mean ± SEM) of iPTH, Scl-Ab, and combined treatment on SC expression of OPG (A) and RANKL (B) mRNA in WT mice, TCRβ−/− mice, TCRβ−/− mice previously subjected to adoptive transfer of WT T cells or Wnt10b−/− T cells and global Wnt10b−/− mice. SCs were purified from BM cultured for 1 week and the level of OPG and RANKL mRNA analyzed by RT-PCR (n = 10 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle or to the indicated group. #p < 0.05 compared with Scl-Ab treatment.

Discussion

We report that iPTH stimulates OPG production, osteoblastogenesis, and increases OB life span bone volume and bone density in mice concomitantly treated with a dose of Scl-Ab that models the partial repression of Scl production induced by iPTH. These findings demonstrate that iPTH exerts part of its anabolic activity through a mechanism independent of its capacity to blunt Scl production. We also show that the Scl-independent anabolic activity of iPTH in trabecular bone is mediated by T-cell–produced Wnt10b. The data provide a proof of concept of a more potent effect of combined treatment with iPTH and Scl-Ab than either alone that may be useful in the treatment of osteoporosis.

After the discovery that iPTH blunts the osteocytic production of Scl,[32] studies have been conducted in global Sost−/− mice and SOST BAC transgenic mice to determine the relevance of Scl in the mechanism of action of iPTH.[38-40, 56] These studies suggested that iPTH promotes bone anabolism through Scl-dependent and -independent mechanisms. However, the conclusions of these earlier reports are weakened by major methodological limitations. First, iPTH decreases but does not completely suppress the production of Scl, whereas the global genetic models used in earlier studies are characterized by either the complete absence of Scl or the constitutive production of elevated amounts of Scl. More importantly, the presence of high bone formation and bone mass in the Sost–/– mice and of low bone mass in the SOST BAC transgenic mice prevents a conclusive assessment of the strength of the Scl-independent effects of iPTH.[37, 39]

To investigate this matter further, we made use of a Scl-Ab known to neutralize the biological activity of Scl.[44, 46] This Scl-Ab was previously found to have mild anabolic effects in C57BL/6 mice at 12 mg/kg/week.[44] In another study, the Scl-Ab was injected at 12 mg/kg/week for 5 weeks and found to increase bone density and volume in a manner similar to iPTH at 40 μg/kg/day.[46] In this study, we used Scl-Ab at 50 mg/kg/week to generate anabolic conditions exceeding those generated by the partial blockade of Scl production induced by iPTH, without completely neutralizing circulating Scl. This approach was selected to determine with a high degree of confidence whether the anabolic activity of iPTH is partially owing to mechanisms other than inhibition of sclerostin production. As expected, at this dose, Scl-Ab increased bone volume more than iPTH, demonstrating that iPTH blunts circulating Scl levels to a smaller extent than Scl-Ab at 50 mg/kg/week. Therefore, in physiologic conditions, the Scl-independent effect of iPTH is likely to be greater than observed in this study.

We found that both PTH and Scl-Ab markedly increased bone density and indices of trabecular and cortical volume and structure. In vivo bone density measurements showed that Scl-Ab increased BMD in the first 2 weeks as well as in the last 2 weeks of treatment, indicating that the Scl-Ab maintained its capacity to neutralize Scl for the entire study. Selective measurements of trabecular and cortical bone were obtained by in vitro μCT utilizing samples harvested at death. Trabecular measurements were obtained in the spine but not in the femur because preliminary studies (not shown) revealed that combined treatment with Scl-Ab and iPTH induces massive formation of woven bone in the femur, a phenomenon that prevented the accurate quantification of the additive effect of the treatments. Cortical indices were instead measured in the distal femur because an accurate demarcation of cortical areas in the spine is not feasible with the Scanco μCT scanner utilized for our studies.

Measurements of bone density volume and structure clearly demonstrated an additive effect of Scl-Ab and iPTH treatment, a finding that confirms that the inhibitory effect of iPTH on Scl production does not account for the full anabolic activity of iPTH. Importantly, the additive effect of Scl-Ab and iPTH in trabecular bone was observed only in mice with WT T cells, but not in those lacking T cells, T-cell production of Wnt10b, or global Wnt10b production. By contrast, the additive effect of Scl-Ab and iPTH in cortical bone was observed in mice with or without T-cell production of Wnt10b, although it was abolished in mice lacking global production of Wnt10b. These findings confirm earlier reports from our laboratory that T-cell–produced Wnt10b specifically potentiates the anabolic activity of iPTH in trabecular but not cortical bone.[29, 43] Moreover, the data demonstrate that T-cell production of Wnt10b accounts for the Scl-independent activity of iPTH in trabecular bone. The mechanism responsible for the Scl-independent activity of iPTH in cortical bone remains to be determined. In the BM, iPTH specifically stimulates Wnt10b production by CD8+ cells without affecting Wnt10b production by other lineages.[29] However, it is possible that increased production of Wnt10b by osteocytes or osteoblasts may contribute to the T-cell–independent effects of iPTH on cortical bone.

Activation of Wnt signaling in SCs and OBs induces OB proliferation[23] and differentiation,[22, 24] promotes OB survival,[15, 16, 25] and decreases bone resorption by increasing the osteoblastic production of OPG.[26] In keeping with the capacity of iPTH to activate canonical Wnt signaling in osteoblastic cells, we found that treatment of WT mice with iPTH resulted in a significant increase in histomorphometric and biochemical indices of bone formation. We also found that iPTH increased the recruitment of SCs toward the osteoblastic lineage and promoted OB proliferation, life span, and differentiation. A trend toward an increase in histomorphometric and biochemical indices of bone resorption was also observed; however, these changes did not reach statistical significance. The observed increase in OPG production by BM SCs might be a reason why iPTH did not increase bone resorption in a significant manner.

Surprisingly, treatment with Scl-Ab was not associated with an increase in histomorphometric and biochemical indices of bone formation and resorption measured at the end of the study, or with changes in indices of osteoblastogenesis and OB life span. However, Scl-Ab did increase SC OPG production, albeit only in WT mice. The lack of a stimulation of bone formation in response to Scl-Ab is in contrast with the results of earlier short-term animal studies with Scl-Ab.[57-60] However, our findings are in agreement with the result of recent long-term studies in animals and humans.[53-55] For example, in a study in ovx rats, it was found that serum osteocalcin, a marker of bone formation, increased acutely after initiation of Scl-Ab treatment but returned to baseline by week 9, whereas histomorphometric indices of bone formation peaked at week 6.[53] In another study in monkeys, osteocalcin and histomorphometric indices of bone formation peaked at 3 months and returned to baseline at 6 months.[54] In both studies, bone density continued to increase when bone formation had already returned to baseline levels. Moreover, a phase-2 human trial with Scl-Ab demonstrated that although Scl-Ab strikingly increased bone density and effectively neutralized circulating Scl for the entire 12 months of the study, Scl-Ab caused only a transitory short-term increase in the serum levels of P1NP and osteocalcin.[55] Markers of bone resorption were also affected by Scl-Ab for the first few weeks of the study only. The dissociation between long-term effects of Scl-Ab on bone mass and short-term effects on bone turnover opens the possibility that the long-term anabolic activity of Scl may occur through effects unrelated to typical changes in bone turnover. Taken together, the available data suggest that in our study Scl-Ab was no longer stimulating bone formation at the time of sacrifice in spite of a persistent effect on bone density on the second half of the study.

Importantly, in WT mice, histomorphometric indices of bone formation and resorption were increased by combined treatment with Scl-Ab and iPTH. These findings demonstrate that iPTH stimulates bone formation independently of Scl inhibition. The data also indicate that although Scl-Ab alone does not affect bone turnover, a synergistic interaction of Scl-Ab and iPTH induces complex changes in bone turnover that result in an increase in bone volume and bone density.

Serum P1NP levels are proportional to the rate of bone formation as well as the size of the bone compartment producing P1NP. Therefore, serum P1NP levels reflect primarily the rate of cortical bone formation. Accordingly, combined treatment resulted in higher serum P1NP levels than treatment with Scl-Ab alone in all groups of mice, indicating that iPTH increases cortical bone formation by a T-cell/Wnt10b–independent mechanism. By contrast, combined treatment resulted in higher CFU-ALP formation, number of SCs, SC proliferation and life span, and OB differentiation than treatment with Scl-Ab alone but only in mice with WT T cells. In mice lacking global or T-cell–specific production of Wnt10b, all of these indices were similar in mice treated with Scl-Ab only or Scl-Ab and iPTH. Thus, the osteoblastogenic activity of iPTH is Scl-independent and specifically driven by T-cell–produced Wnt10b.

In summary, our data are consistent with a complex modality of action of iPTH that include suppression of Scl production and increased T-cell production of Wnt10b (Fig. 10). In conditions of normal baseline bone turnover and bone mass and partial Scl blockade that mimic the repressive activity of the hormone on Scl production, iPTH stimulates osteoblastogenesis, OB life span bone density, and trabecular bone volume independently of Scl through a Wnt10b-mediated mechanism.

Figure 10.

Schematic representation of the mechanism of action of iPTH. In baseline conditions, T cells produce low levels of Wnt10b, whereas osteocytes secrete high levels of Scl. The presence of a low Wnt10b/Scl ration prevents the activation of Wnt signaling in OBs leading to low osteoblastogenesis and high OB apoptosis. Treatment with iPTH increases the T-cell production of Wnt10b and blunts the osteocytic secretion of Scl, thus increasing the Wnt10b/Scl ration. Wnt10b is now capable of activating Wnt signaling in OBs leading to increased osteoblastogenesis and decreased OB apoptosis.

Disclosures

All authors state that they have no conflicts of interest.

Acknowledgments

This study was supported by grants from the National Institutes of Health (AR54625, AR49659, and AR061453). MNW gratefully acknowledges financial support by the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development (5I01BX000105), NIAMS (AR059364, AR056090, and AR053607), NIA (AG040013), and by the Georgia Research Alliance.

We thank Dr Michaela Kneissel from the Novartis Institutes for Biomedical Research (NIBR), Basel, Switzerland, for processing the samples for bone histomorphometry for the partial analysis of the histomorphometric indices and for the kind donation of the Scl antibody, which stems from a collaboration between NIBR and MorhoSys (Martinsried, Germany).

We also thank the Histomorphometry and Molecular Analysis Core Laboratory at the University of Alabama at Birmingham, Center for Metabolic Bone Disease NIH, P30 AR 04603, for part of the histomorphometric analysis.

Authors' roles: JUL performed the research, analyzed the data, and wrote the paper. LDW performed the research. AMT performed the research. JA performed the research. MNW interpreted the data and reviewed the manuscript. RP designed the study and wrote the manuscript.

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