Exogenous PTH-Related Protein and PTH Improve Mineral and Skeletal Status in 25-Hydroxyvitamin D-1α-Hydroxylase and PTH Double Knockout Mice

Authors

  • Yingben Xue,

    1. Calcium Research Laboratory, McGill University Health Centre and Department of Medicine, McGill University, Montreal, Quebec, Canada
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  • Zengli Zhang,

    1. Calcium Research Laboratory, McGill University Health Centre and Department of Medicine, McGill University, Montreal, Quebec, Canada
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  • Andrew C Karaplis,

    1. Lady Davis Research Institute, Sir Mortimer B. Davis-Jewish General Hospital and Department of Medicine McGill University, Montreal, Quebec, Canada
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  • Geoffrey N Hendy,

    1. Calcium Research Laboratory, McGill University Health Centre and Department of Medicine, McGill University, Montreal, Quebec, Canada
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  • David Goltzman,

    1. Calcium Research Laboratory, McGill University Health Centre and Department of Medicine, McGill University, Montreal, Quebec, Canada
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  • Dengshun Miao MD, PhD

    Corresponding author
    1. Calcium Research Laboratory, McGill University Health Centre and Department of Medicine, McGill University, Montreal, Quebec, Canada
    Current affiliation:
    1. Center for Bone and Stem Cell Research, Nanjing Medical University, Nanjing, Jiangsu, China
    • Calcium Research Laboratory Room H4.67 Royal Victoria Hospital 687 Pine Avenue West Montreal, Quebec H3A 1A1, Canada
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  • The authors have no conflict of interest

Abstract

We examined the effect of NH2-terminal fragments of PTHrP and PTH in young mutant mice deficient in both PTH and 1,25-dihydroxyvitamin D. Both proteins prolonged murine survival by increasing serum calcium, apparently by enhancing renal calcium transporter expression. The dominant effect on the skeleton was an increase in both endochondral bone and appositional formation without increased bone resorption.

Introduction: PTH-related protein (PTHrP) was discovered as a hypercalcemic agent responsible for the syndrome of humeral hypercalcemia of malignancy, and PTH is the major protein hormone regulating calcium homeostasis. Both proteins have skeletal anabolic actions when administered intermittently. We examined effects of exogenous PTHrP(1-86) and PTH(1-34) in double null mutant mice deficient in both PTH and 25-hydroxyvitamin D-1α-hydroxylase {1α(OH)ase} to determine the action of these proteins in the absence of the two major regulators of calcium and skeletal homeostasis.

Materials and Methods: Mice heterozygous for the PTH null allele and for the 1α(OH)ase null allele were mated to generate pups homozygous for both null alleles. PTHrP(1-86) and PTH(1-34) were administered subcutaneously starting 4 days after birth. Serum biochemistry and skeletal radiology, histology, and histomorphometry were performed, and indices of bone formation, resorption, and renal calcium transport were determined by real time RT-PCR, Western blot, and immunohistochemical approaches.

Results: In the double mutant mice, which die within 3 weeks after birth with severe hypocalcemia, tetany, and skeletal defects, exogenous PTHrP and PTH enhanced survival of the animals by improving serum calcium. Both proteins increased renal calcium transporter expression and long bone length and augmented growth plate chondrocyte proliferation, differentiation, and cartilage matrix mineralization. Cortical and trabecular bone mass was increased with augmented osteoblast number and activity; however, bone resorption was not increased.

Conclusions: PTHrP and PTH reduced hypocalcemia by enhancing renal calcium reabsorption but not by increasing bone resorption. The major skeletal effects of exogenous PTHrP and PTH were to increase bone anabolism.

INTRODUCTION

PTH-RELATED PROTEIN (PTHrP) was discovered as a PTH-like factor responsible for humoral hypercalcemia of malignancy (HHM), (1–3) and PTH is known to be the major protein hormone defending against a reduction in extracellular fluid calcium. PTHrP and PTH can both bind to the type I PTH/PTHrP receptor (PTHR1)(4) and can thereby carry out similar actions. Thus, both peptides, through PTHR1, can increase 1,25 dihydroxyvitamin D3 {1,25(OH)2D3} by stimulating 25-hydroxyvitamin D 1α-hydroxylase {1α(OH)ase} and can enhance renal calcium reabsorption, although the relative potency of PTHrP and PTH in this respect has been uncertain. Like PTH, PTHrP can carry out either catabolic or anabolic actions in the skeleton depending on its secretory profile. As reported previously, HHM is primarily a skeletal catabolic disease, with uncoupled increase in bone resorption and suppression of bone formation. (5) In HHM and primary hyperparathyroidism, however, bone cells are presumably exposed to continuously elevated concentrations of PTHrP and PTH, respectively, that are secreted constitutively. (6, 7) In contrast to these catabolic skeletal effects of continuously elevated concentrations of PTHrP and PTH, there is very strong evidence that both proteins can have potent anabolic effects on the skeleton when administered intermittently. Daily injections of NH2-terminal forms of PTHrP and PTH increase skeletal mass in laboratory animals(6–8) and in postmenopausal women. (9, 10) Studies in mice lacking either PTHrP or PTH or both have shown that both peptides are required to achieve normal fetal skeletal morphogenesis. (11) However, adult Pth-null mice, when maintained on a normal calcium intake, exhibited reduced bone turnover, leading to increased rather than decreased trabecular and cortical bone volume. (12) We previously reported that PTHrP heterozygotes developed low trabecular bone mass by 3 months of age because of haploinsufficiency, thus showing that PTHrP is critical for maintaining trabecular bone. (13) To determine whether PTHrP plays a role in the accrual of increased bone mass in the PTH-deficient state, we compared PTH-null mice with mice homozygously null for PTH but heterozygously deleted for PTHrP, because PTHrP-null mice are unable to survive postpartum. PTHrP haploinsufficiency reduced trabecular bone of the PTH-null mice to levels below those in wildtype animals by decreasing osteoprogenitor cell recruitment, enhancing osteoblast apoptosis, and diminishing bone formation. (14) These results suggest that endogenous PTHrP is required for developing and maintaining bone mass in the adult. In support of these observations in vivo, we previously showed that PTHrP stimulates osteogenic cell proliferation in rat marrow mesenchymal progenitor cells through protein kinase C-dependent activation of the Ras and MAPK signaling pathway in vitro. (15)

Recently, we generated double knockout (KO) mice that are homozygous for both the 1α(OH)ase and PTH null alleles and compared them to mice null for each single gene and to wildtype mice at 2 weeks of age. (16) Double KO mice died 1–3 weeks postnatally, before the end of weaning, with severe tetany and hypocalcemia and skeletal defects that were more severe than in both types of single gene KO mice. In this study, we tested the hypothesis that the exogenous NH2-terminal domain of PTHrP {PTHrP(1-86)} and of PTH {PTH(1-34)} could improve mineral homeostasis in a hypocalcemic model and that this could occur in the absence of endogenous 1,25(OH)2D3 and endogenous PTH. We used the double KO mice to test this hypothesis. We also examined the mechanism of the effect of PTHrP and PTH in this setting in bone and kidney.

MATERIALS AND METHODS

Derivation of PTH and 1α(OH)ase double null mice

The derivation of the two parental strains of PTH−/− mice and 1α(OH)ase−/− mice by homologous recombination in embryonic stem cells was previously described by Miao et al. (11, 12) and Panda et al, (17) respectively. Briefly, a neomycin resistance gene was inserted into exon III of the mouse Pth gene, resulting in the replacement of the entire coding sequence of mature PTH. Lack of PTH expression in parathyroid glands was confirmed by immunostaining. (11) A neomycin resistance gene was inserted in place of exons VI, VII, and VIII of the mouse 1α(OH)ase gene, replacing both the ligand-binding and heme-binding domains. RT-PCR of renal RNA from homozygous 1α(OH)ase−/− mice confirmed that no 1α(OH)ase mRNA is expressed from this allele. (17) Mice heterozygous PTH null and mice heterozygous 1α(OH)ase null were fertile. PTH+/− mice and 1α(OH)ase+/− mice were mated and offspring heterozygous at both loci were mated to generate pups homozygous for both PTH and 1α(OH)ase null alleles (double KO). Lines were maintained by mating PTH+/−1α(OH)ase+/− males and PTH+/−1α(OH)ase+/− females. These mice were preserved on a mixed genetic background with contributions from C57BL/6J and BALB/c strains.

Genotyping of mice

Genomic DNA was isolated from tail fragments by standard phenol-chloroform extraction and isopropanol precipitation. To determine the genotype at both PTH and 1α(OH)ase loci, four PCRs were conducted for each animal. The presence of the wildtype Pth allele was detected using PTH forward primer 5′-GATGTCTGCAAACACCGTGGCTAA-3′ and PTH reverse primer 5′-TCCAAAGTTTCATTACAGTAGAAG-3′. The null Pth allele was detected using Neo forward primer 5′-TCTTGATTC CCACTTTGTGGTTCTA-3′ and PTH reverse primer. (18) To detect the wildtype 1α(OH)ase allele, DNA was amplified with forward primer 5′-AGACTGCACTCCACTCTGAG-3′ and reverse primer 5′-GTTTCCTACACGGATGTCTC-3′. For the neomycin gene, the primers were neo-F 5′-ACAACAGACAATCGGCTGCTC-3′ and neo-R 5′-CCATGGGTCACGACGAGATC-3′. (19)

In vivo experiments

All animal experiments were carried out in compliance with and approval by the Institutional Animal Care and Use Committee. Mutant mice and control littermates were maintained in a virus- and parasite-free barrier facility and exposed to a 12-h/12-h light/dark cycle. Ordinarily, double KO mice die with severe tetany 1–3 weeks postnatally, before the end of weaning. The wildtype and double mutant pups were injected subcutaneously with vehicle or 0.2 μg human PTHrP(1-86) or 0.2 μg rat PTH(1-34)/day starting at day 4 for 10 days or until 1 month of age. After PTHrP and PTH administration for 10 days, the wildtype mice and double KO mice were compared with the vehicle-treated wildtype and vehicle-treated double KO mice. One-month-old PTHrP-treated double KO mice that survived were also compared with vehicle-treated wildtype littermates.

Biochemistry

Serum concentrations of calcium and phosphorus and urine concentrations of calcium and creatinine were determined by routine methods using diagnostic reagents (Sigma). Calcium content in humeri was measured using atomic absorption analysis as described. (20)

RT-PCR and quantitative real-time PCR

RNA was isolated from mouse kidneys and long bones, using Trizol reagent (Invitrogen) according to the manufacturer's protocol. RT-PCR was performed by a one-step method using QIAGEN One-Step RT-PCR kit (QIAGEN, Mississauga, Canada) according to the manufacturer's instructions(15) with the primer sets shown in Table 1. RT reactions were performed using the SuperScript First-Strand Synthesis System (Invitrogen) as described. (14) To determine the number of cDNA molecules in the RT samples, real-time PCR was performed using the LightCycler system (Roche Molecular Biochemicals, Indianapolis, IN, USA). The conditions were 2 μl of LightCycler DNA master SYBR Green I (Roche), 0.25 μM of each 5′ and 3′ primer (see Table 1), and 2 μl of sample and/or H2O to a final volume of 20 μl. The MgCl2 concentration was adjusted to 3 mM. Samples were amplified for 35 cycles with a temperature transition rate of 20°C/s for all three steps, which were denaturation at 95°C for 10 s, annealing for 5 s, and extension at 72°C for 20 s. SYBR green fluorescence was measured to determine the amount of double-stranded DNA. 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 s; the temperature was decreased to 58°C for 15 s and raised slowly from 58°C to 95°C using a temperature transition rate of 0.1°C/s. To determine the number of copies of target DNA in the samples, purified PCR fragments of known concentrations were serially diluted and served as external standards for each experiment. Data were normalized to GAPDH levels.

Table Table 1.. RT-PCR Primers Used With Their Name, Orientation, Sense and Antisense Sequence, Annealing Temperature (Tm), and Length of Amplicon
original image

Western blot analysis

Proteins were extracted from kidneys and quantitated by a kit (Bio-Rad, Mississauga, Ontario, Canada). Thirty-microgram protein samples were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was carried out as described(15) using antibodies against TRPV5 (ECaC1), calbindin-D28K and calbindin-D9K, Na+/Ca2+ exchanger 1 (NCX1; Swant), and tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bands were visualized using enhanced chemiluminescence (ECL) (Amersham) and quantitated by Scion Image Beta 4.02 (Scion Corp.).

Skeletal radiography

Femurs were removed and dissected free of soft tissue. Contact radiographs were taken using a Faxitron model 805 radiographic inspection system (Faxitron Contact, Faxitron, Germany; 22-kV voltage and 4-minute exposure time). X-Omat TL film (Eastman Kodak, Rochester, NY, USA) was used and processed routinely.

μCT

Femurs obtained from 2-week-old or 1-month-old mice were dissected free of soft tissue, fixed overnight in 70% ethanol, and analyzed by μCT with a SkyScan 1072 scanner and associated analysis software (SkyScan, Antwerp, Belgium) as described. (14) Briefly, image acquisition was performed at 100 kV and 98 μA with a 0.9° rotation between frames. During scanning, the samples were enclosed in tightly fitting plastic wrap to prevent movement and dehydration. Thresholding was applied to the images to segment the bone from the background. 2D images were used to generate 3D renderings using the 3D Creator software supplied with the instrument. The resolution of the μCT images is 18.2 μm.

Histology

Femurs and tibias were removed and fixed in PLP fixative (2% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate) overnight at 4°C and processed histologically as described. (21) Proximal ends of tibias were decalcified in EDTA glycerol solution for 5–7 days at 4°C. Decalcified tibias 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 H&E or histochemically for alkaline phosphatase (ALP) activity or TRACP activity or immunohistochemically as described below. Alternatively, undecalcified tibias were embedded in LR white acrylic resin (London Resin Co., London, UK), and 1-μm sections were cut on an ultramicrotome. These sections were stained for mineral with the von Kossa staining procedure and counterstained with toluidine blue.

Immunohistochemical staining

Proliferating cell nuclear antigen (PCNA), types I and X collagens, and RANKL were determined by immunohistochemistry as described previously. (11, 12, 14, 21) Mouse monoclonal antibody against proliferating cell nuclear antigen (PCNA; Medicorp, Montreal, Canada), affinity-purified goat anti-human type I collagen (Col I) antibody (Southern Biotechnology Associates, Birmingham, AL, USA), rabbit anti-serum to type X collagen (a generous gift of Dr AR Poole, Shriners Hospital, Montreal, Canada), and affinity purified goat polyclonal antibody raised against a peptide mapping at the carboxy terminus of RANKL (C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied to dewaxed paraffin sections overnight at room temperature. As a negative control, preimmune serum was substituted for the primary antibody. After washing with high salt buffer (50 mM Tris-HCl, 2.5% NaCl, 0.05% Tween 20, pH 7.6) for 10 minutes at room temperature followed by two 10-minute washes with TBS, the sections were incubated with secondary antibody (biotinylated goat anti-rabbit IgG or biotinylated rabbit anti-goat IgG; Sigma), washed as before, and incubated with the Vectastain ABC-AP kit or the Vectastain Elite ABC kit (Vector Laboratories, Ontario, Canada) for 45 minutes. After washing as before, red pigmentation to demarcate regions of immunostaining was produced by a 10- to 15-minute treatment with Fast red TR/Naphthol AS-MX phosphate (Sigma; containing 1 mM levamisole as endogenous ALP inhibitor) or gray pigmentation was likewise produced using a Vector SG kit (Vector Laboratories). After washing with distilled water, the sections were counterstained with methyl green and mounted with Kaiser's glycerol jelly.

Histochemical staining for collagen, ALP, and TRACP

Enzyme histochemistry for ALP activity was performed as previously described. (22, 23) Briefly, after preincubation overnight in 1% magnesium chloride in 100 mM tris-maleate buffer (pH 9.2), dewaxed sections were incubated for 2 h at room temperature in 100 mM tris-maleate buffer containing naphthol AS-MX phosphate (0.2 mg/ml; Sigma) dissolved in ethylene glycol monomethyl ether (Sigma) as substrate, and Fast red TR (0.4 mg/ml; Sigma) as a stain for the reaction product. After washing with distilled water, the sections were counterstained with Vector methyl green nuclear counterstain (Vector Laboratories) and mounted with Kaiser's glycerol jelly.

Enzyme histochemistry for TRACP was performed as previously described. (24) Dewaxed sections were preincubated for 20 minutes in buffer containing 50 mM sodium acetate and 40 mM sodium tartrate at pH 5.0. Sections were incubated for 15 minutes at room temperature in the same buffer containing 2.5 mg/ml naphthol AS-MX phosphate (Sigma) in dimethylformamide as substrate and 0.5 mg/ml fast garnet GBC (Sigma) as a color indicator for the reaction product. After washing with distilled water, the sections were counterstained with methyl green and mounted in Kaiser's glycerol jelly.

Double calcein labeling

Double calcein labeling was performed by intraperitoneal injection of mice with 10 μg calcein/g body weight (C-0875; Sigma) at 4 days old and 3 days before death as described. (12) Bones were harvested and embedded in LR white acrylic resin as described above. Serial sections were cut, and the freshly cut surface of each section was viewed and imaged using a fluorescence microscopy. The double calcein labeled width of cortex and trabeculae was measured using Northern Eclipse image analysis software v6.0 (Empix Imaging, Mississauga, Canada), and the mineral apposition rate (MAR) was calculated as the interlabel width/labeling period.

Computer-assisted image analysis

After H&E staining or histochemical or immunohistochemical staining of sections from six mice of each genotype, images of fields were photographed with a Sony digital camera. Images of micrographs from single sections were digitally recorded using a rectangular template, and recordings were processed and analyzed using Northern Eclipse image analysis software as previously described. (12, 14, 17, 21)

Statistical analysis

Data from image analysis are presented as means ± SE. Statistical comparisons were made using a two-way ANOVA, with p < 0.05 being considered significant.

RESULTS

Effect of exogenous PTHrP and PTH on serum calcium and phosphorus, urine calcium, and calcium content of long bones

To determine whether exogenous PTHrP and PTH improved serum mineral ion levels in double KO mice, serum calcium and phosphorus and urine calcium/creatinine ratio were examined. The normal values of mouse serum calcium and phosphorus are 1.82-2.85 and 1.72-3.64 mM, respectively. Serum calcium levels rose from 48% of those of wildtype mice in vehicle-treated double KO mice to 70% and 76% of those of wildtype mice in PTHrP- and PTH-treated double KO mice, respectively, at 2 weeks of age (Fig. 1A). In contrast, serum calcium levels did not rise significantly in either PTHrP- or PTH-treated wildtype mice compared with the vehicle-treated mice (Fig. 1A). At 1 month of age, all vehicle-treated mice had died, but serum calcium was still 70% of wildtype mice in PTHrP-treated animals (Fig. 1A). The vehicle-treated double KO mice were hyperphosphatemic; however, serum phosphorus had decreased to normal levels in PTHrP- and PTH-treated double KO mice at 2 weeks of age, and these levels were maintained at 1 month of age in PTHrP-treated animals (Fig. 1B). Serum phosphorus levels were not altered in either PTHrP- or PTH-treated wildtype mice (Fig. 1B). The urine calcium/creatinine ratio was significantly elevated in vehicle-treated double KO mice compared with vehicle-treated wildtype mice. Urine calcium/creatinine ratio was significantly reduced in both PTHrP- and PTH-treated double KO mice relative to vehicle-treated double KO mice but was not normalized (Fig. 1C). No alteration of the urine calcium/creatinine ratio was observed in PTHrP- and PTH-treated wildtype mice (Fig. 1C). To determine whether the skeletal calcium content was altered in the double mutant animals and whether PTHrP and PTH administration changed skeletal calcium content, we measured the calcium content of humeri by atomic absorption analysis. The results revealed that the calcium content of humeri was decreased significantly in the double mutant animals and rose to normal levels after PTHrP and PTH administration (Fig. 1D). Only a slight, nonsignificant increase of the calcium content of humeri was found in the PTHrP- and PTH-treated wildtype mice (Fig. 1D).

Figure FIG. 1..

Biochemistry of serum, urine, and bone tissues. (A) Serum calcium and (B) phosphorus, (C) urine calcium/creatinine ratio, and (D) calcium content of humeri were determined in vehicle-treated wildtype (WT), PTHrP-treated wildtype (WT+PTHrP), PTH-treated wildtype (WT+PTH), vehicle-treated PTH−/−1α(OH)ase−/− mice (DKO), PTHrP-treated PTH−/− 1α(OH)ase−/− mice (DKO+PTHrP), and PTH-treated PTH−/− 1α(OH)ase−/− mice (DKO+PTH). The upper and lower levels of serum calcium are denoted by the upper and lower dotted lines, respectively, in A. Each value is the mean ± SE of determinations in six mice of each group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with vehicle-treated wildtype mice. ###p < 0.001 compared with vehicle-treated DKO mice.20

Effect of exogenous PTHrP on endogenous PTHrP expression and on renal calcium transporters in vivo

To examine expression of endogenous PTHrP in kidney, PTHrP was examined by RT-PCR and Western blot. Both mRNA and protein levels of renal endogenous PTHrP were increased in the vehicle-treated double KO mice compared with wildtype but were decreased in PTHrP-treated double KO mice at 2 weeks of age compared with their vehicle-treated wildtype littermates (Figs. 2A and 2B).

Figure FIG. 2..

Effect of exogenous PTHrP on endogenous PTHrP expression and on calcium transporters in vivo. (A) A specific PTHrP product was amplified from kidney RNA by RT-PCR. The GAPDH mRNA was amplified as a loading control. (B) Expression of kidney PTHrP protein was determined by Western blot with β-tubulin as loading control. Comparison of TRPV5, calbindin-D28K (CB28K), calbindin-D9K (CB9K), and NCX1 in kidney of vehicle-treated wildtype (WT), vehicle-treated PTH−/−1α(OH)ase−/− mice (DKO), and PTHrP-treated PTH−/−1α(OH)ase−/− mice (DKO+PTHrP). (C) Western blots (WB) of renal extracts for expression of TRPV5, CB28K, CB9K, and NCX1. β-tubulin was used as loading control for Western blots. (D) Specific TRPV5, CB28K, CB9K, and NCX1 products were amplified from the renal RNA by real-time RT-PCR. The GAPDH mRNA was used as control. mRNA expression assessed by real-time RT-PCR analysis is calculated as a ratio to the GAPDH mRNA level and expressed relative to levels of wildtype mice. *p < 0.05 compared with vehicle-treated wildtype mice. #p < 0.05 compared with vehicle-treated DKO mice.20

To determine whether the raised serum calcium levels induced by exogenous PTHrP and PTH were contributed to by increased renal reabsorption of calcium, we examined the mRNA and protein expression levels of the renal calcium transporters, TRPV5, calbindin-D28K, calbindin-D9K, and NCX1 by real time RT-PCR and Western blot. The results revealed that the mRNA and protein levels of these genes were downregulated in the kidney of double KO mice relative to wildtype but were upregulated by PTHrP administration (Figs. 2C and 2D). A similar upregulation of the renal calcium transporters by PTH administration was observed (data not shown).

Effect of exogenous PTHrP and PTH on skeletal phenotypes

To assess the effect of exogenous PTHrP and PTH administration on postnatal skeletal development, long bones were analyzed by radiography (Figs. 3A and 3B) and μCT (Figs. 3C and 3D). The results revealed that the length of femurs was shorter in vehicle-treated double KO mice than in wildtype mice, but the length approached that of their wildtype littermates at 2 weeks of age in PTHrP- and PTH-treated mice (Figs. 3A and 3E) and became almost normal at 1 month of age in PTHrP-treated double KO mice (Figs. 3B and 3E). No change in the length of femurs was found in the PTHrP- and PTH-treated wildtype mice relative to vehicle-treated controls (Figs. 3A and 3E). Radiolucency in epiphyses and metaphyses was increased in vehicle-treated double KO mice relative to wildtype mice; however, radiolucency decreased significantly at 2 weeks of age in PTHrP- and PTH-treated double KO mice (Fig. 3A) and was less than that of the vehicle-treated wildtype mice at 1 month of age in PTHrP-treated double KO mice (Fig. 3B). From 3D reconstructed longitudinal sections (Fig. 3C) and cross-sections (Fig. 3D) of the proximal ends of tibias, it can be seen that epiphyses were larger, unmineralized growth plate spaces were narrower, and trabecular and cortical bone volumes were greater in PTHrP-treated double KO mice compared with vehicle-treated double KO mice at 2 weeks of age. Similar results were seen with PTH (data not shown). By 1 month of age, all epiphyseal, trabecular, and cortical bone volumes were greater in PTHrP-treated double KO mice than in the vehicle-treated wildtype mice. Only a slight increase of the trabecular volume was found in the PTHrP- and PTH-treated wildtype mice compared with vehicle-treated wildtype mice (Fig. 3F). A significant increase of both trabecular and cortical bone volumes was observed in the PTHrP- and PTH-treated double KO mice (Figs. 3F and 3G).

Figure FIG. 3..

Effect of exogenous PTHrP and PTH on skeletal phenotypes. (A) Representative contact radiographs of the femurs from 2-week-old wildtype (WT) and PTH−/− 1α(OH)ase−/− mice (DKO) treated with vehicle (V) or PTHrP or PTH. (B) Representative contact radiographs of the femurs from 1-month-old vehicle-treated wildtype (WT) and PTHrP-treated PTH−/−1α(OH)ase−/− mice (DKO+PTHrP). Representative (C) longitudinal sections and (D) cross-sections of 3D reconstructed proximal ends of tibias. (E) Quantitation of femoral length. (F) Trabecular bone volume and (G) cortical volume were determined in vehicle-treated wildtype (WT), PTHrP-treated wildtype (WT+PTHrP), PTH-treated wildtype (WT+PTH), vehicle-treated PTH−/− 1α(OH)ase−/− mice (DKO), PTHrP-treated PTH−/−1α(OH)ase−/− mice (DKO+PTHrP), and PTH-treated PTH−/−1α(OH)ase−/− mice (DKO+PTH). Each value is the mean ± SE of determinations in six mice of each group. *p < 0.05 compared with vehicle-treated wildtype mice. #p < 0.05 compared with vehicle-treated DKO mice.20

Effect of exogenous PTHrP and PTH on chondrocyte proliferation and differentiation and on cartilage mineralization

To assess the effect of exogenous PTHrP and PTH administration on endochondral bone formation, we examined the histology of the cartilaginous growth plates, the proliferation and differentiation of chondrocytes, and the mineralization of the hypertrophic zone in 2-week-old mice. The growth plate width (Fig. 4A) was narrower, PCNA+ chondrocytes (Figs. 4B and 4F) were decreased, and the type X collagen-positive hypertrophic zone (Figs. 4C and 4G) was dramatically narrowed and less mineralized (Figs. 4D and 4H) in vehicle-treated double KO mice compared with wildtype littermates. After 10 days of PTHrP or PTH administration, all these parameters were improved in the double KO mice, although they were still below wildtype levels. At 1 month of age, the growth plate width was almost normal with an increased mineralized hypertrophic zone in PTHrP-treated double KO mice compared with vehicle-treated wildtype mice (data not shown).

Figure FIG. 4..

Effect of exogenous PTHrP and PTH on chondrocyte proliferation and differentiation and on cartilage mineralization. Paraffin-embedded sections of tibias from vehicle-treated wildtype (WT), vehicle-treated PTH−/−1α(OH)ase−/− mice (DKO), PTHrP-treated PTH−/−1α(OH)ase−/− mice (DKO+PTHrP), and PTH-treated PTH−/− 1α(OH)ase−/− mice (DKO+PTH) at 2 weeks of age were (A) stained with H&E and immunostained for (B) PCNA or (C) type X collagen. (D) Undecalcified sections of tibias were stained with the von Kossa procedure. Scale bars in A-D represent 100, 25, 25, and 50 μm, respectively. (E) Width of the hypertrophic zone of growth plates was determined. (F) Numbers of PCNA+ chondrocytes of total chondrocytes per field were determined by image analysis, and the PCNA+ percentages of total chondrocytes were calculated. (G) Type X collagen immunopositive area as a percentage of the growth plate field was determined. (H) Mineralized area as a percent of the cartilage matrix per field was determined by image analysis. Each value is the mean ± SE of determinations in six animals of each group. *p < 0.05; **p < 0.01; ***p < 0.001 in the mutant mice relative to the vehicle-treated wildtype mice. ##p < 0.005; ###p < 0.001 compared with vehicle-treated DKO mice.20

Effect of exogenous PTHrP and PTH on osteoblastic bone formation parameters

To determine whether the alteration of bone volume was associated with an alteration of osteoblastic bone formation, MAR, histomorphometric analysis for osteoid volume, osteoblast number, and Col I deposition in bone matrix were performed at 2 weeks of age. MAR and mineralization in both trabecular and cortical bone (Figs. 5A-5D), ALP+ osteoblasts, and matrix deposition of Col I (Figs. 6A-6D) were increased in PTHrP- and PTH-treated double mutants compared with vehicle-treated double mutants, although they did not reach the levels observed in vehicle-treated wildtype mice. Consistent with these observations, osteoblastic gene expression levels of Cbfa1 (Runx2), ALP, Col I, and osteocalcin (OCN) were all upregulated after 10-day PTHrP administration to double KO mice as shown by real-time RT-PCR (Fig. 6F). These results indicate that stimulation of osteoblastic bone formation is a major factor contributing to the bone anabolic actions of exogenous PTHrP and PTH.

Figure FIG. 5..

Effect of exogenous PTHrP and PTH on mineral apposition rate and osteoid volume. (A) Representative micrographs of calcein double labeling and (B) sections stained with the von Kossa procedure in the trabeculae and cortex were imaged from ethanol fixed and undecalcified LR white resin embedded sections of the proximal ends of tibias of the vehicle-treated wildtype (WT), vehicle-treated PTH−/−1α(OH)ase−/− mice (DKO), PTHrP-treated PTH−/− 1α(OH)ase−/− mice (DKO+PTHrP), and PTH-treated PTH−/−1α(OH)ase−/− mice (DKO+PTH). (C) MAR of trabeculae and cortex of the same treated animals was determined. (D) Osteoid volume was determined in undecalcified von Kossa-stained sections and is presented as a percent of bone volume (OV/BV, %) of trabeculae and cortex. Each value is the mean ± SE of determinations in six animals of each group. *p < 0.05 relative to vehicle-treated wildtype mice. #p < 0.05 compared with vehicle-treated DKO mice.20

Figure FIG. 6..

Effect of exogenous PTHrP and PTH on osteoblastic bone formation parameters. Representative micrographs of tibial sections, stained (A) histochemically for ALP activity and (B) immunostained for type I collagen (Col I) from the vehicle-treated wildtype (WT), vehicle-treated PTH−/−1α(OH)ase−/− mice (DKO), PTHrP-treated PTH−/−1α(OH)ase−/− mice (DKO+PTHrP), and PTH-treated PTH−/− 1α(OH)ase−/− mice (DKO+PTH). Scale bar = 25 μm. (C) Number of osteoblasts per mm2 were counted in the primary spongiosa of H&E-stained tibias of the mice and presented as mean ± SE. The (D) ALP+ and (E) Col I+ areas as a percent of the tissue area were determined in the metaphyseal regions for each group. (F) Real-time RT-PCR of long bone extracts for expression of Cbfa I, ALP, Col I, and OCN. GAPDH mRNA was used as loading controls. mRNA expression assessed by real-time RT-PCR is calculated as a ratio to the GAPDH mRNA level and expressed relative to levels in wildtype mice. Each value is the mean ± SE of determinations in six animals of each group. *p < 0.05, **p < 0.01; ***p < 0.001 in the mutant mice relative to the vehicle-treated wildtype mice. #p < 0.05; ###p < 0.001 compared with the vehicle-treated DKO mice.20

Effect of exogenous PTHrP and PTH on osteoclastic parameters

To determine whether PTHrP and PTH can also exert a catabolic effect on bone in PTH- and 1,25(OH)2D3-deficient mice, histochemical staining for TRACP (Fig. 7A) and immunostaining for RANKL (Fig. 7B) were performed. The results revealed that PTHrP and PTH administered to double KO mice did not increase TRACP+ osteoclasts compared with vehicle-treated double KO mice (Figs. 7A and 7C), although RANKL (Figs. 7B and 7D) immunoreactivity in osteoblastic cells was increased. When the ratio of RANKL/OPG mRNA levels was determined by real-time RT-PCR, the ratio of RANKL/OPG mRNA levels was reduced in vehicle-treated double KO mice and in PTHrP- and PTH-treated double KO mice compared with wildtype mice (Fig. 7E). The ratio alteration after PTHrP and PTH administration was consistent with that of a reduced TRACP+ osteoclast number. Consequently, PTHrP/PTH-induced osteoclastic stimulation was not observed.

Figure FIG. 7..

Effect of exogenous PTHrP and PTH on osteoclastic parameters. Representative micrographs of sections of the tibial metaphysis (A) stained histochemically for TRACP activity and (B) immunostained for RANKL in the vehicle-treated wildtype (WT), vehicle-treated PTH−/−1α(OH)ase−/− mice (DKO), PTHrP-treated PTH−/− 1α(OH)ase−/− mice (DKO+PTHrP), and PTH-treated PTH−/−1α(OH)ase−/− mice (DKO+PTH). Scale bars in A and B represent 25 μm. (C) Number of TRACP+ osteoclasts per field of tissue and (D) RANKL immunopositive area relative to tissue area. Each value is the mean ± SE of determinations in three animals of each group. (E) Real-time RT-PCR was performed on bone extracts for RANKL and OPG mRNA with GAPDH mRNA as control. mRNA expression assessed by real-time RT-PCR analysis is calculated as a ratio to the GAPDH mRNA level and expressed relative to levels of wildtype mice. RANKL/OPG mRNA levels are presented as the mean ± SE of determinations in six animals of each group. *p < 0.05; **p < 0.01; ***p < 0.001 relative to vehicle-treated wildtype mice. ###p < 0.001 compared with the vehicle-treated DKO mice.20

DISCUSSION

To better define the direct action of PTHrP on mineral ion homeostasis and bone metabolism, this study used a double KO animal model that is homozygous for both 1α(OH)ase and PTH null alleles. These double KO mice died 1–3 weeks postnatally, before the end of weaning, with severe hypocalcemia and skeletal defects. (16) Use of this animal model can avoid interference from 1,25(OH)2D3 and PTH to study the function of the NH2-terminal domains of PTHrP and PTH per se in vivo. This study shows that exogenous PTHrP(1-86) and PTH(1-34) administration improved serum calcium levels and survival and increased the length of long bones and augmented bone mass.

Interestingly, the same doses of PTHrP(1-86) and PTH (1-34) administered to wildtype mice as to double mutants showed little effect on serum or urine calcium, serum phosphorus, or calcium content of long bones. Consequently, the potency of the two proteins seemed to be enhanced in the double mutant mice. The similarity of actions of the two administered proteins strongly indicates that they both acted through the PTHR1. One possible explanation for the relatively increased skeletal and renal actions in the mutant mice may be that the absence of endogenous PTH resulted in reduced competition for receptor occupancy in target cells of bone and kidney accompanied by upregulation of PTHR1. We cannot, of course, exclude that alterations in blood calcium and/or phosphorus per se modulated the relatively increased efficacy of PTHrP and PTH in the mutant mice and further studies will be required to distinguish among these possibilities.

Levels of endogenous PTHrP in the kidney were increased in the vehicle-treated double KO mice, but were decreased in PTHrP-treated double KO mice at 2 weeks of age compared with their vehicle-treated wildtype littermates. These findings suggest that endogenous PTHrP mRNA and protein levels may be regulated directly or indirectly by calcium and/or phosphate because levels of these ions were markedly different between wildtype and double mutants and between double mutants and PTHrP-treated animals. Indeed, previous studies have also shown calcium-mediated modulation of PTHrP in lactating murine breast tissue. (25) A direct downregulating effect of PTHrP per se, however, also seems possible, in view of the fact that the levels of endogenous PTHrP, after treatment with exogenous peptide, were reduced below wildtype levels. These results also indicate that the increased endogenous PTHrP was not capable of maintaining serum calcium at levels sufficient for the survival of the double KO mice and that, under physiological conditions, endogenous PTHrP can only play a minor role in mineral ion homeostasis, even though, when oversecreted by tumors, it can cause hypercalcemia.

Although there is general agreement that hypercalcemia occurs among patients with HHM as a result of osteoclastic bone resorption, our results have shown that PTHrP administered daily, which did increase serum calcium levels, did not enhance osteoclastic bone resorption in the double mutant mice. Consequently the skeletal catabolic effects of PTHrP in HHM as well as of PTH in primary hyperparathyroidism may be caused by persistent rather than intermittent secretion of PTHrP but significant increases in calcemia may occur even in the absence of bone resorption. It is possible that deficient levels of 1,25(OH)2D3 contributed to reduced PTH-induced osteoclastogenesis because inappropriately low PTH-induced bone resorption has previously been noted in vitamin D deficiency with secondary hyperparathyroidism. (19) There is overwhelming evidence that interaction with osteoblastic stromal cell-derived RANKL induces osteoclast differentiation. (26) Osteoprotegerin (OPG) is a secreted stromal cell-derived decoy receptor that specifically binds RANKL and inhibits osteoclast differentiation. (26) The balance of RANKL relative to OPG expression modulates the rate of osteoclast differentiation and many factors that influence osteoclast differentiation do so by regulating OPG and RANKL expression in stromal support cells. (27) Therefore, we examined the ratio of RANKL/OPG mRNA expression using real time RT-PCR. Our results showed a consistent reduction of the TRACP+ osteoclast number and the ratio of RANKL/OPG mRNA expression in the double KO mice and the PTHrP/PTH-treated double KO mice. Our results are therefore consistent with previous reports that PTH/PTHrP-dependent bone resorption and hypercalcemia are blocked by OPG. (28, 29) Overall, these results show that the major effect of daily administration of exogenous NH2-terminal PTHrP/PTH is to increase bone anabolism and not bone resorption.

Although the pathophysiology responsible for HHM has become increasingly clear over the past two decades, the role of PTHrP in stimulating renal calcium reabsorption in subjects with HHM is still controversial, and the molecular mechanism of PTHrP stimulation of renal calcium reabsorption is unclear. The transcellular pathway of calcium transport through the renal tubular epithelial cell involves the entry of calcium through the calcium channel, TRPV5, located on the apical surface of distal tubule epithelial cells. Subsequently, the cation binds to calbindin-D9K or to calbindin-D28K and diffuses through the cytosol to the basolateral membrane. Ca2+ ions are then extruded into the blood by a Na+/Ca2+ exchanger (NCX1). (30, 31) This study examined the effect of PTHrP and of PTH on these components of the renal transcellular calcium transport system by real-time RT-PCR and Western blot. Our results showed that TRPV5, calbindin-D28K, calbindin-D9K, and NCX1 mRNA and protein levels in kidney were all increased in parallel with serum calcium levels in PTHrP- and PTH-treated double KO mice in vivo and that renal calcium levels declined. In previous studies, 1,25(OH)2D3 and the vitamin D receptor have been reported to modulate calcium transporters in vivo(30, 31) and PTH has previously been shown to modulate calcium transporters in vitro. (32) Our current studies are, however, the first to report modulation by PTHrP. In view of the fact that enhanced intestinal calcium transport mediated by 1,25(OH)2D3 could not have occurred in these 1α(OH)ase-deficient mutants and because bone resorption was not enhanced by PTHrP or PTH, the major mechanism whereby exogenous PTHrP and PTH increased serum calcium seemed to be by stimulating renal calcium absorption.

Existing evidence indicates that PTHrP and PTH can have potent anabolic effects on the skeleton when administered intermittently. However, the exact mechanism of the skeletal anabolic action of these proteins is unclear. A striking finding from this study is that exogenous PTHrP, similar to PTH, exerts a very strong skeletal anabolic action. In part, this is by stimulating endochondral bone formation. Our results show that many endochondral bone formation parameters were improved after 10 days of exogenous PTHrP or PTH administration to the double KO mice. These include increased long bone length, increased epiphyseal volume, upregulated chondrocyte proliferation, and differentiation as well as increased cartilaginous matrix mineralization. Long bone length, epiphyseal volume, and cartilaginous growth plates were virtually normalized in 1-month-old PTHrP-treated double KO mice. Our results are consistent with the functions of PTHrP in regulating endochondral bone development as shown in both the PTHrP and the PTHR1 null mice. (33, 34) That is, PTHrP accelerates growth of cartilage by promoting chondrocyte proliferation. However, a number of the positive effects on the growth plate of both PTHrP and PTH including increased chondrocyte differentiation and cartilaginous matrix mineralization and perhaps even to an extent increased chondrocyte proliferation(35) may have been indirect and mediated by the raised concentrations of extracellular calcium. It is also possible that exogenously administered PTHrP(1-86) differs in its effects on the growth plate from endogenous PTHrP, which expresses both the NH2-terminal domain that binds the common PTHR1, and more carboxyl regions of the molecule, which may differ in their effects on skeletal function.

In addition to stimulating endochondral bone formation, exogenous PTHrP and PTH administration also stimulated appositional osteoblastic bone formation at both trabeculae and cortices. Our results show that PTHrP and PTH administration increased MAR, ALP+ osteoblast number, and Col I deposition in bone matrix. Consistent with these observations, osteoblastic gene expression levels of Cbfa1, ALP, Col I, and OCN were all upregulated after 10 days of PTHrP or PTH administration to the double KO mice.

Consequently, both proteins, administered daily, produced beneficial skeletal effects. Such effects may, furthermore, be enhanced by strategies to reduce endogenous PTH production and to limit the bone-resorbing actions of 1,25(OH)2D3.

Acknowledgements

This work was supported by operating grants to DM, ACK, GNH, and DG from the Canadian Institutes of Health Research and to DG from the National Cancer Institute of Canada.

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