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Endocrinology, doi:10.1210/en.2003-1695
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Endocrinology Vol. 145, No. 8 3554-3562
Copyright © 2004 by The Endocrine Society

Parathyroid Hormone-Related Peptide Is Required for Increased Trabecular Bone Volume in Parathyroid Hormone-Null Mice

Dengshun Miao, Jiarong Li, Yingben Xue, Hanyi Su, Andrew C. Karaplis and David Goltzman

Calcium Research Laboratory of the McGill University Health Center (D.M., J.L., Y.X., D.G.), Lady Davis Institute for Medical Research of the Sir Mortimer B. Davis-Jewish General Hospital (H.S., A.C.K.), and Department of Medicine, McGill University, Montréal, Québec, Canada

Address all correspondence and requests for reprints to: Dr. David Goltzman, Calcium Research Laboratory, Royal Victoria Hospital, 687 Pine Avenue W, Room H4.67, Montréal, Québec, Canada H3A 1A1. E-mail: david.goltzman{at}mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the relative contributions of PTH and PTHrP to the skeletal phenotype of mice deficient in PTH (PTH–/–). PTH–/– mice and PTH–/– mice lacking one allele encoding PTHrP (PTH–/–PTHrP+/–) were compared. Both mutants displayed similar biochemical abnormalities of hypoparathyroidism, but skeletal PTHrP mRNA and protein were decreased in PTH–/–PTHrP+/ – mice. PTH–/– mice had increased trabecular bone volume with diminished bone turnover. PTHrP haploinsufficiency reduced trabecular bone of the PTH–/– mice to levels below those in wild-type animals by decreasing osteoprogenitor cell recruitment, enhancing osteoblast apoptosis, and diminishing bone formation. The results show that the increased trabecular bone volume in PTH-deficient mice is due to diminished PTH-induced osteoclastic bone resorption and persistent PTHrP-stimulated osteoblastic bone formation. They also illustrate the changing role of PTHrP during bone development, demonstrate its bone- forming function in the postnatal state, and support its pharmacological potential as an anabolic agent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH BINDS TO cells of the osteoblastic lineage (1) and enhances both bone formation and bone resorption. PTH appears to exert discrete effects on trabecular bone in the intrauterine and postnatal environments. In fetal life, PTH is required to sustain a normal number of osteoblasts in the primary spongiosa, and the absence of PTH leads to diminished trabecular bone volume (2). In postnatal life, PTH contributes to maintaining normal extracellular fluid calcium levels, at least in part by enhancing trabecular bone resorption, and in the absence of PTH, trabecular bone mass is increased (3, 4). It is unclear, however, what mechanisms facilitate conversion of the PTH-deficient animal from a low trabecular bone mass state in utero to a high trabecular bone mass state in postnatal life.

PTHrP is an alternate ligand for the type I PTH/PTHrP receptor (PTHR) (5) and can mimic many of the actions of PTH. In PTHrP-haploinsufficient mice, trabecular bone volume is normal at birth, but is diminished by 3 months of age (6). Consequently, PTHrP appears to assume increasing importance in maintaining trabecular bone as the animal ages. To determine whether PTHrP plays a role in the accrual of increased bone mass in the PTH-deficient state, we compared mice with homozygous deletion of the gene encoding PTH (PTH–/– mice) with PTH–/– mice that were also heterozygous for PTHrP deletion (PTH–/–PTHrP+/– mice). We thus analyzed mineral and skeletal parameters in littermates that were wild-type, PTH–/–, or PTH–/–PTHrP+/–. The results indicate that PTHrP is required for the increased bone mass phenotype of hypoparathyroidism and offer new insights into the relative and changing roles of PTH and PTHrP during skeletal development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
We previously described the generation, by homologous recombination in embryonic stem cells, of mice carrying a disrupted PTH gene (2) and a disrupted PTHrP gene (7). To obtain wild-type, PTH–/–, PTHrP+/–, and PTH–/–PTHrP+/– littermates, male and female PTH+/– and PTHrP+/– mice on a C57BL/6J background were crossed. Genomic DNA was isolated from tail clips for genotyping by methods previously described (2, 7). Wild-type and mutant PTH alleles were detected using a 0.2-kb HindIII/XhoI genomic DNA fragment as a probe after digestion of tail tip DNA with BamHI. In detecting wild-type and mutant PTHrP alleles, a 0.62-kb SacI/XhoI genomic fragment was used after digestion of tail tip genomic DNA with PvuII. Mice were maintained in a virus- and parasite-free barrier facility and exposed to a 12-h light, 12-h dark cycle. After weaning, animals were fed normal mouse chow containing 0.97% calcium and 0.85% phosphorus (Charles River Laboratories, St. Constant, Canada) until death at 4 months of age. All animal experiments were carried out in compliance with and approval by the institutional animal care committee of McGill University.

Biochemical and hormone analyses
Serum calcium and phosphorus were determined by autoanalyzer (Synchron 67, Beckman Coulter, Fullerton, CA). Serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was measured by RIA (ImmunoDiagnostic Systems, Bolden UK). Two-site immunoradiometric assays were used to measure intact PTH (Immutopics, San Clemente, CA) and PTHrP (Nichols Institute Diagnostics, San Juan Capistrano, CA). The PTHrP assay recognized epitopes within the 1–40 and 60–72 regions of the molecule, and PTHrP-(1–86) was used as an assay standard.

RT-PCR
RNA was isolated from mouse tibiae and femora using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The forward and reverse primers used for amplification were: mouse PTHrP, 5'-AGACATTGCTATGGGAGCCAC-3' and 5'-TCTCCTGTTCTCTGCGTTTC-3'; receptor activator of nuclear factor-{kappa}B ligand (RANKL) mRNA, 5'-GGTCGGGCAATTCTGAATT-3' and 5'-GGGGAATTACAAAGTGCACCAG-3'; and osteoprotegerin (OPG), 5'-TCCTGGCACCTACCTAAAACAGCA-3' and 5'-CTACACTCTCGGCATTCACTTTGG-3'. The forward and reverse primers for GAPDH used as a loading control were 5'-CATGGAGAAGGCTGGGGCTC-3' and 5'-CACTGACACGTTGGCAGTGG-3'. The conditions for 32 cycles of PCRs were 94 C for 1 min, 61 C for 1 min, and 72 C for 1 min.

Quantitative real-time PCR
RT reactions were performed using the SuperScript First-Strand Synthesis System (Invitrogen). To determine the number of cDNA molecules in the reverse transcribed samples, real-time PCR analyses were performed using the LightCycler system (Roche, Indianapolis, IN). PCR was performed using 2 µl LightCycler DNA Master SYBR Green I (Roche), 0.25 µM of each 5' and 3' primer, and 2 µl samples or H2O to a final volume of 20 µl. The MgCl2 concentration was adjusted to 3 mM. Samples were denatured at 95 C for 10 sec, with a temperature transition rate of 20 C per sec. Amplification and fluorescence determination were carried out in four steps: denaturation at 95 C for 0 sec, with a temperature transition rate of 20 C/sec; annealing for 5 sec, with a temperature transition rate of 8 C/sec; extension at 72 C for 20 sec, with a temperature transition rate of 4 C/sec; and detection of SYBR Green fluorescence, which reflects the amount of double-stranded DNA, at 86 C for 3 sec. The amplification cycle number was 35. To discriminate specific from nonspecific cDNA products, a melting curve was obtained at the end of each run. Products were denatured at 95 C for 3 sec, and the temperature was then decreased to 58 C for 15 sec and raised slowly from 58 to 95 C using a temperature transition rate of 0.1 C/sec. To determine the number of copies of the targeted DNA in the samples, purified PCR fragments of known concentrations were serially diluted and served as external standards that were measured in each experiment. Data were normalized with GAPDH levels in the samples. The primer sequences used for the real-time PCR were the same as those used for routine PCR.

Western blot analysis
Proteins were extracted from long bones and quantitated by the protein assay (Bio-Rad, Mississauga, Canada). Protein samples (30 µg) were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Immunoblotting was carried out using antibodies against PTHrP-(1–34) peptide and tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Histology, histochemistry, and immunohistochemistry
Thyroparathyroidal tissue, femurs, tibiae, and lumber vertebral bodies were removed and fixed in 2% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate (PLP fixative) overnight at 4 C and processed histologically as previously described (8). The distal ends of femora, the proximal ends of the tibiae, and the first lumber vertebral bodies were decalcified in EDTA-glycerol solution for 5–7 d at 4 C. Decalcified tibiae and other tissues were dehydrated and embedded in paraffin, after which 5-µm sections were cut on a rotary microtome. The sections were stained with hematoxylin and eosin (HE) or histochemically for collagen (9), tartrate-resistant acid phosphatase (TRAP) activity (10), or alkaline phosphatase activity (11) as previously described.

Paraffin-embedded sections of decalcified skeleton and parathyroid glands were stained immunohistochemically for PTH, PTHrP, type I collagen, and RANKL (Santa Cruz Biotechnology, Inc.) using the avidin-biotin-peroxidase complex technique as described previously (2, 8).

Detection of apoptotic cells
Dewaxed paraffin sections were stained with an in situ cell death detection kit (Roche) to identify terminal deoxynucleotidyltransferase-mediated nick end-labeling of DNA strand breaks as previously described (2).

Microcomputed tomography (micro-CT)
Femora obtained from 4-month-old mice were dissected free of soft tissue, fixed overnight in 70% ethanol, and analyzed by micro-CT with a SkyScan 1072 scanner and associated analysis software (SkyScan, Antwerp, Belgium) (12). Image acquisition was performed at 100 kV and 98 µA, with a 0.9° rotation between frames. During scanning, the samples were enclosed in a tightly fitting plastic wrap to prevent movement and dehydration. Thresholding was applied to the images to segment the bone from the background. Two-dimensional images were used to generate three-dimensional renderings using the 3D Creator software supplied with the instrument. The resolution of the micro-CT images is 18.2 µm.

Double calcein labeling
Double calcein labeling was performed by ip injection of mice with 10 µg calcein/g body weight (C-0875, Sigma-Aldrich Corp., St. Louis, MO) at 10 and 3 d before death. Bones were harvested and embedded in LR White acrylic resin. Serial sections were cut, and the freshly cut surface of each section was viewed and imaged using fluorescence microscopy. The double calcein-labeled width of bone was measured using Northern Eclipse image analysis software version 6.0 (Empix Imaging, Inc., Mississauga, Canada), and the mineral apposition rate (MAR) was calculated as the interlabel width/labeling period.

Bone marrow cell cultures
Tibiae and femurs of 4-month-old wild-type, PTH–/– and PTH–/–PTHrP+/– mice were removed under aseptic conditions, and bone marrow cells were flushed out with DMEM containing 10% fetal calf serum, 50 µg/ml ascorbic acid, 10 mM ß-glycerophosphate, and 10–8 M dexamethasone. Cells were dispersed by repeated pipetting, and a single cell suspension was achieved by forcefully expelling the cells through a 22-gauge syringe needle. Total bone marrow cells (106) were cultured in 36-cm2 petri dishes in 5 ml of the above-described medium. Such cultures have previously been shown to contain osteogenic cell precursors, which in culture can produce mineralized bone nodules (13). The medium was changed on d 5, and cultures were maintained for 10 d. At the end of the culture period, cells were washed with PBS, fixed with PLP fixative, and stained with methylene blue or cytochemically for alkaline phosphatase (ALP) as described previously (8, 13, 14). After staining, total colony-forming unit-fibroblastic (CFU-f) number and ALP-positive CFU-f number were counted manually.

Computer-assisted image analysis
After HE staining or histochemical staining of sections from six mice of each genotype, images of fields were photographed with a Sony digital camera (Tokyo, Japan). Images of micrographs from single sections were digitally recorded using a rectangular template, and recordings were processed using Northern Eclipse image analysis software (2, 8). For determining the trabecular bone volume relative to the total volume in collagen-stained sections and the number and size of osteoclasts in TRAP histochemically stained sections, thresholds were set using green and red channels. The thresholds were determined as described previously (2). Trabecular volume was measured by quantitative histomorphometry (15) in the metaphyseal region from the distal (metaphyseal) side of the growth plate to 1 mm toward the diaphysis from this boundary. Cortical thickness was measured in the diaphysis region of femora. TRAP parameters were measured in the metaphyseal region at a 200-fold magnification with nonoverlapping sites spanning from the distal (metaphyseal) side of the growth plate to approximately 0.5 mm toward the diaphysis from this boundary. The number of osteoblasts was counted, and the surfaces of trabecular bone and osteoblasts were traced using Northern Eclipse image analysis software in the same region as TRAP parameters. Data from image analysis are presented as the mean ± SEM. Statistical comparisons were made using a two-way ANOVA, with P < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum biochemistry and parathyroid gland histology
Serum PTH was undetectable in PTH–/– mice that exhibited the typical biochemical features of hypoparathyroidism, i.e. hypocalcemia, hyperphosphatemia, and reduced serum concentrations of 1,25-(OH)2D3 (Fig. 1Go, A–D). These biochemical differences from wild-type animals persisted and were not significantly different in PTH–/–PTHrP+/– mice.



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  		FIG. 1. Serum chemistry and parathyroid glands. Serum PTH (A), calcium (B), phosphorus (C), and 1,25-(OH)2D3 (D) were determined in wild-type (WT), PTH–/–, and PTH–/–PTHrP+/ – mice as described in Materials and Methods. Values are the mean ± SEM of six determinations. *, P < 0.05, **, P < 0.001 (compared with wild-type mice). Representative micrographs of parathyroid glands and adjacent thyroid tissue stained with HE (E) and immunostained for PTH (F) in WT, PTH–/–, and PTH–/–PTHrP+/ – mice. Bars, 100 µm.

 
Parathyroid glands were enlarged in PTH–/– mice, compatible with the hypocalcemia and low serum 1,25-(OH)2D3 concentrations (16), but contained no immunoreactive PTH (Fig. 1Go, E and F). Similar findings were observed in the PTH–/–PTHrP+/– animals (Fig. 1Go, E and F).

Expression of PTHrP
Serum PTHrP concentrations were not significantly increased above wild-type levels in PTH–/– mice, but serum PTHrP was significantly below wild-type levels in PTHrP+/– and PTH–/–PTHrP+/– mice (Fig. 2AGo).



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  		FIG. 2. Serum PTHrP and expression of PTHrP protein and mRNA in skeletal tissues. A, Serum PTHrP was determined as described in Materials and Methods in wild-type (WT), PTH–/–, PTHrP+/–, and PTH–/–PTHrP+/– mice. Each value is the mean ± SEM of six determinations. #, P < 0.05; ##, P < 0.01 (compared with PTH–/– mice). B, Comparison of PTHrP mRNA expression, assessed by RT-PCR as described in Materials and Methods, in bone of WT, PTH–/–, PTHrP+/–, and PTH–/–PTHrP+/– mice. C, PTHrP mRNA expression as assessed by real-time PCR analysis was calculated as a ratio relative to the GAPDH mRNA level and expressed relative to levels in WT mice (100%). Each value is the mean ± SEM of triplicate determinations. **, P < 0.01 (compared with WT mice). ##, P < 0.01 (compared with PTH–/– mice). D, Western blots of long bone extracts for the expression of PTHrP. Tubulin was used as loading controls for Western blots. Comparison of PTHrP expression demonstrated by immunohistochemistry in the growth plate (E) and trabecular bone (F) of WT, PTH–/–, and PTH–/–PTHrP+/– mice. Bars, 25 µm.

 
By both routine RT-PCR (Fig. 2BGo) and quantitative real-time RT-PCR (Fig. 2CGo), PTHrP mRNA levels in mRNA extracted from femoral bone were significantly increased in PTH–/– mice compared with wild-type mice and significantly reduced in PTHrP+/– and PTH–/–PTHrP+/– mice compared with PTH–/– mice. PTHrP protein levels in protein extracted from femora were increased in PTH–/– mice compared with wild-type mice and significantly reduced in PTHrP+/– and PTH–/–PTHrP+/– mice compared with wild-type and PTH–/– mice, as demonstrated by Western blots (Fig. 2DGo). PTHrP immunoreactivity in growth plate cartilage and osteoblastic cells was also increased in PTH–/– mice and decreased in PTH–/–PTHrP+/– animals (Fig. 2Go, E and F). The expression of PTH/PTHrP receptor protein was not different in bone of wild-type, PTH–/–, or PTH–/–PTHrP+/–animals (data not shown).

Trabecular bone volume
Trabecular bone volume was increased in PTH–/– mice compared with wild-type littermates, but was reduced not just to normal but to below wild-type levels in PTH–/–PTHrP+/– animals. This was observed by histological staining (Fig. 3AGo), immunostaining for type I collagen (Fig. 3BGo), and micro-CT (Fig. 3CGo) of long bones. Quantitation of bone volume by histomorphometry revealed significant increases in trabecular bone volume of tibiae, femora, and vertebrae of PTH–/– mice compared with wild-type mice and significant decreases in both PTHrP+/– and PTH–/–PTHrP+/– mice. These decreases were significant not only compared with PTH–/– mice, but also compared with wild-type mice (Fig. 3DGo). The cortical thickness in tibia and femur was increased in PTH–/– mice, but was no different than wild-type levels in PTHrP+/– mice. In PTH–/–PTHrP+/– mice, cortical thickness fell to the levels in wild-type mice (Fig. 3EGo).



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  		FIG. 3. Bone volume. Representative micrographs from wild-type (WT), PTH–/–, and PTH–/–PTHrP+/– mice of the distal ends of femora stained with HE (A) and immunostained with type I collagen (B). C, Two-dimensional images of the femora were obtained by micro-CT to generate three-dimensional reconstructions from 20 adjacent images as described in Materials and Methods. Trabecular bone volume relative to the tissue volume [BV/TV (%) D] and the cortical thickness (E) of long bones or vertebrae were determined by histomorphometric analysis as described in Materials and Methods. Each bar represents the mean ± SE of determinations in six animals of the same genotype. ***, P < 0.001 (compared with WT mice). ###, P < 0.001 (compared with PTH–/– mice).

 
Alterations in bone resorption
To determine whether changes in trabecular bone volume were due to alterations in bone resorption, osteoclast number and size were determined after staining for the marker TRAP (Fig. 4AGo). Both the number (Fig. 4BGo) and the size (Fig. 4CGo) of TRAP-positive osteoclasts were reduced in PTH–/– mice. Osteoclast size was also reduced in PTH–/–PTHrP+/– mice, and osteoclast numbers were even more markedly reduced in these animals (Fig. 4Go, B and C).



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  		FIG. 4. Osteoclasts and RANKL expression. Representative micrographs of sections of the tibial metaphysis of wild-type (WT), PTH–/–, and PTH–/–PTHrP+/– mice stained histochemically for TRAP (A). Bar (horizontal), 50 µm. The number of TRAP-positive osteoclasts per square millimeters of bone surface (B) and the average size of TRAP-positive osteoclasts (C) were determined as described in Materials and Methods. Each bar represents the mean ± SE of determinations in six animals of the same genotype. ***, P < 0.001 (compared with WT mice). #, P < 0.05 (compared with PTH–/– mice). D, Representative micrographs of sections of the tibial metaphysis of WT, PTH–/–, and PTH–/–PTHrP+/– stained immunohistochemically for RANKL. Bar, 50 µm. E, Comparison of RANKL and OPG mRNA expression, assessed by RT-PCR, in bone of WT, PTH–/– and PTH–/–PTHrP+/– mice. F, RANKL and OPG mRNA expression, as assessed by real-time RT-PCR analysis, was calculated as a ratio relative to the GAPDH mRNA level and expressed relative to levels of wild-type mice (100%). Each value is the mean ± SEM of three determinations. ###, P < 0.001 (compared with PTH–/– mice). ***, P < 0.001 (compared with WT mice).

 
We also examined expression levels of RANKL and OPG, two requisite mediators of osteoclastogenesis (17, 18). Immunoreactive RANKL was detected in osteoblastic cells of wild-type mice, was reduced in PTH–/– mice, and was even more dramatically reduced in PTH–/–PTHrP+/– mice (Fig. 4DGo). Gene expression of RANKL mRNA extracted from bone was reduced in PTH–/– mice and especially in PTH–/–PTHrP+/– mice, as demonstrated by both routine RT-PCR (Fig. 4EGo) and quantitative real-time RT-PCR (Fig. 4FGo), while, simultaneously, gene expression of OPG mRNA was normal or elevated (Fig. 4Go, E and F). These reductions in the ratio of RANKL to OPG were therefore consistent with the decreases observed in the mutants with respect to osteoclast number and size, at least in the PTH–/–PTHrP+/– mice.

Alterations in bone formation
The MAR, determined by double calcein labeling, was decreased in PTH–/– mice, but was even more dramatically reduced in PTH–/–PTHrP+/– mice (Fig. 5Go, A and D). Osteoblast numbers were modestly reduced, and the osteoblast surface was very significantly reduced in PTH–/– mice (Fig. 5Go, B, E, and F). In PTH–/–PTHrP+/– mice, both the number and surface of osteoblasts were markedly decreased (Fig. 5Go, B, E, and F). Alkaline phosphatase staining confirmed the decreased osteoblastic activity in trabecular bone of PTH–/– mice and the even more dramatic reductions in trabecular bone of PTH–/–PTHrP+/– mice (Fig. 5Go, C and G).



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  		FIG. 5. Osteoblasts and mineral apposition rate. A, Micrographs of calcein double labeling in trabeculae were imaged from sections of the proximal ends of tibiae of wild-type (WT), PTH–/–, and PTH–/–PTHrP+/– mice as described in Materials and Methods. Bar, 50 µm. Representative micrographs of trabecular bone from WT, PTH–/–, and PTH–/– PTHrP+/– mice. Sections were stained with HE (B) or for ALP activity (C) as described in Materials and Methods. Bar, 100 µm. The MAR of trabeculae (D), osteoblast numbers presented as number per square millimeter of tissue area [N.Ob/T.Ar (#/mm2); E] osteoblast surface presented as a percent of bone surface [Ob.S/B.S (%); F], and ALP-positive area presented as a percentage of the tissue area are shown for each genotype. Each bar represents the mean ± SEM of determinations in six animals of the same genotype. ***, P < 0.001 (compared with wild-type mice). #, P < 0.05; ##, P < 0.01; ###, P < 0.001 (compared with PTH–/– mice).

 
To determine the mechanism of the reduction in osteoblastic indexes we examined apoptosis of osteoblasts and osteocytes. More apoptotic osteoblasts and osteocytes were present in PTH–/– mice than in wild-type mice (Fig. 6Go, A, B, and E). An even greater increase in apoptosis of these cells was observed in the PTH–/–PTHrP+/– animals (Fig. 6Go, A, B, and E).



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  		FIG. 6. Apoptotic osteoblasts and ex vivo bone marrow cell cultures. Apoptotic osteoblasts and osteocytes whose nuclei stained red were detected by terminal deoxynucleotidyltransferase- mediated nick end-labeling of DNA strand break assay in the trabeculae from 2-month-old (A) and 4-month-old (B) mice as described in Materials and Methods. Bars, 25 µm. Ex vivo primary bone marrow cultures stained with methylene blue to show total CFU-f (C) or cytochemically to show CFU-fap (D) as described in Materials and Methods. The percentages of apoptotic osteoblasts and osteocytes are shown as the mean ± SEM of determinations in six animals of the same genotype (E), and CFU-f and CFU-fap numbers (F) are the mean ± SEM of triplicate determinations from three replicate experiments, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with wild-type mice). #, P < 0.05; ##, P < 0.01 (compared with PTH–/– mice).

 
To assess the extent of osteoblast recruitment from bone marrow precursors, bone marrow cells were cultured and then stained to determine total CFU-f (Fig. 6CGo) and ALP-positive CFU-f, (CFU-fap; Fig. 6DGo). Total CFU-f and CFU-fap numbers were decreased in PTH–/– mice, but were even more substantially reduced in PTH–/–PTHrP+/– mice (Fig. 6Go, C, D, and F).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several studies in both animals (3, 4) and adult humans (19, 20, 21, 22) have confirmed the presence of increased bone mass in hypoparathyroidism. Our studies in postnatal hypoparathyroid (PTH–/–) animals demonstrate a reduction in bone turnover associated with an increase in trabecular bone volume. We previously reported, however, that fetal mice at term that are deficient in PTH have reduced trabecular bone compared with wild-type littermates (2). This reduction was not due to increased osteoclastic activity, which was actually diminished. The reduction in trabecular bone mass was, in fact, due to decreased osteoblasts. In our current studies in the postnatal PTH–/– mouse, when trabecular bone volume was increased, the reduced osteoclastic activity due to PTH deficiency was still apparent. In addition, however, osteoblastic bone formation was decreased below wild-type levels. Nevertheless, the presence of augmented trabecular bone volume indicated that, on balance, formation was exceeding resorption. Consequently, we sought to determine the mechanism that might be responsible for this relative increase in bone formation relative to resorption in the absence of PTH.

PTHrP shares limited, but important, amino acid sequence homology with PTH (23, 24). This homology, which appears within the biologically important NH2-terminal domain, permits the NH2-terminal sequence of the two peptides to bind to a common receptor, termed the type I PTHR (5). This interaction is known to be the molecular basis of the analogous physiological actions of PTHrP and PTH; for example, in circumstances when they are oversecreted, such as tumor-induced hypercalcemia (23, 24) and primary hyperparathyroidism, respectively. PTH and PTHrP appear to bind to receptors on both osteoblastic stromal cells and osteoblasts (1, 6), through which they stimulate new bone formation and, after activating the RANKL signaling system, also stimulate osteoclastic bone resorption. We previously demonstrated that mice heterozygous for PTHrP gene deletion (PTHrP+/– mice) exhibit haploinsufficiency (6). Although these animals appear normal at birth, they develop progressive osteopenia with age, associated with reduced skeletal PTHrP levels. Consequently, PTHrP seems essential for the maintenance of normal trabecular bone mass in postnatal growing animals. In the present study we confirmed the reduction in trabecular bone volume in PTHrP+/– mice in which PTH levels are normal. Furthermore, we investigated whether PTHrP-mediated activation of osteoblastic bone formation might contribute to the postnatal increases in bone mass observed in hypoparathyroid mice in the face of reduced PTH-induced bone resorption.

We first showed that in PTH–/– mice skeletal levels of PTHrP mRNA and protein are elevated and therefore could contribute to the increased bone mass of PTH–/– animals. We previously reported that 1,25-(OH)2D3 is a potent inhibitor of PTHrP gene transcription (25). It is therefore possible that the reduced 1,25-(OH)2D3 concentrations in PTH–/– mice contributed to the observed increase in skeletal PTHrP. We next showed that the double mutants, lacking one PTHrP allele, do indeed exhibit skeletal PTHrP deficiency. Furthermore the presence of reduced PTHrP not only reversed the high bone mass phenotype of the hypoparathyroid mouse, but converted it to a low bone mass phenotype, with trabecular bone volume below wild-type levels. This was not due to accelerated bone resorption, but, in fact, was associated with an even further reduction in RANKL production and osteoclastic activity. The reduction was due mechanistically to both a decreased capacity to generate osteoblast progenitors, as demonstrated by the ex vivo bone marrow cultures, and an increase in osteoblast and osteocyte apoptosis.

The reduction of trabecular bone volume in the double mutants demonstrates that PTHrP is essential for maintenance of the high bone mass phenotype in hypoparathyroid mice. Consequently, increased trabecular bone volume in hypoparathyroid mice results from diminished PTH- induced bone resorption coupled with persistent PTHrP- induced bone formation. The reduction of trabecular bone volume below wild-type levels in PTH–/–PTHrP+/– mice confirms that PTHrP is required for the maintenance of normal bone mass in the postnatal growing animal and is not a phenomenon induced to compensate for a lack of PTH- induced osteoblastic activity in the hypoparathyroid state.

Different results were observed in cortical bone. Cortical bone thickness was elevated in PTH–/– mice, but was normal in PTHrP+/– mice when circulating PTH was normal. Consequently, endogenous circulating PTH appears to be the major regulator of cortical bone turnover. However, in PTH–/– PTHrP+/– mice, cortical bone volume was reduced to wild-type levels. Consequently, PTHrP deficiency does affect cortical bone turnover, but predominantly in the absence of PTH. The mechanism of this interaction will require further study.

Our overall results, therefore, point to discrete and changing physiological roles for PTH and PTHrP in the fetal and postnatal environments. In the fetal environment, where ambient calcium is maintained through the maternal circulation, PTH appears to play a critical physiological anabolic role in forming trabecular bone. In contrast, the major fetal function of PTHrP appears to be in directing the sequential controlled proliferation and differentiation of chondrocytes for normal cartilaginous growth plate development (7, 26). In the postnatal situation, where environmental stresses to maintain calcium homeostasis differ, PTH release is signaled by a reduction in extracellular fluid calcium, and PTH acts in bone primarily to cause resorption to contribute to the restoration of normal calcium homeostasis. In contrast, PTHrP appears to be a developmental switch that now functions in the postnatal state as the major PTHR ligand to stimulate new trabecular bone formation.

These studies therefore demonstrate a physiological anabolic role for both PTH and PTHrP and indicate that they vary developmentally and with changing environments. These physiological roles may underpin the pharmacological efficacy of both hormones (27, 28) on inducing bone accrual.


   Acknowledgments
 
We acknowledge the staff of the Center for Bone and Periodontal Research (www.bonecentre.ca) for their excellent technical assistance.


   Footnotes
 
This work was supported by operating grants from the Canadian Institutes of Health Research (to D.M., A.C.K., and D.G.) and from the National Cancer Institute of Canada (to A.C.K. and D.G.).

Abbreviations: ALP, Alkaline phosphatase; CFU-f, colony-forming unit-fibroblastic; CFU-fap, alkaline phosphatase-positive colony- forming unit-fibroblastic; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; HE, hematoxylin and eosin; MAR, mineral apposition rate; micro-CT, microcomputed tomography; OPG, osteoprotegerin; PLP fixative, 2% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate; PTHR, PTH/PTH-related peptide receptor; RANKL, receptor activator of nuclear factor-{kappa}B ligand; TRAP, tartrate-resistant acid phosphatase.

Received December 14, 2003.

Accepted for publication April 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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