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Endocrinology Vol. 139, No. 12 5194-5204
Copyright © 1998 by The Endocrine Society

ARTICLES

The Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Mediates Actions of Both Ligands in Murine Bone1

B. Lanske2, P. Divieti, C. S. Kovacs3, A. Pirro, W. J. Landis, S. M. Krane, F. R. Bringhurst and H. M. Kronenberg

Endocrine Unit (B.L., P.D., C.S.K., A.P., F.R.B., H.M.K.), Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114; Department of Orthopedic Surgery (W.J.L.), Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Arthritis Unit (S.M.K.), Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Henry M. Kronenberg, M.D., Endocrine Unit, Wellman 5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: kronenberg.henry{at}mgh.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH and PTH-related peptide (PTHrP) have been shown to bind to and activate the same PTH/PTHrP receptor. Recent studies have demonstrated, however, the presence of additional receptors specific for each ligand. We used the PTHrP and PTH/PTHrP receptor gene knock-out models to investigate whether this receptor mediates the actions of both ligands in bone.

The similar phenotype of the PTHrP (-/-) and PTH/PTHrP receptor (-/-) animals in the growth plate of the tibia suggests that this receptor mediates the actions of PTHrP. Electron microscopic studies have confirmed the accelerated differentiation and disordered organization of chondrocytes, with the accumulation of large amounts of dispersed glycogen granules in the cytoplasm of proliferative and maturing cells of both genotypes.

The contrasting growth plate mineralization patterns of the PTHrP (-/-) and PTH/PTHrP receptor (-/-) mice, however, suggest that the actions of PTHrP and the PTH/PTHrP receptor are not identical.

Studies using calvariae from PTH/PTHrP receptor (-/-) embryos demonstrate that this receptor solely mediates the ability of PTH and PTHrP to stimulate adenylate cyclase in bone and to stimulate bone resorption.

Furthermore, we show that osteoblasts of PTH/PTHrP receptor (-/-) animals, but not PTHrP (-/-) animals, have decreased levels of collagenase 3, osteopontin, and osteocalcin messenger RNAs.

The PTH/PTHrP receptor, therefore, mediates distinct physiologic actions of both PTH and PTHrP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH and PTH-related peptide (PTHrP) can both activate the PTH/PTHrP receptor. PTH, the major peptide regulator of mineral ion homeostasis in mammals (1), acts on two major target organs, kidney and bone, and signals by activating both adenylate cyclase (AC) (2, 3) and phospholipase C (PLC) (4). PTH stimulates bone resorption by triggering a complex cascade in which stimulation of cells of the osteoblast lineage leads indirectly to increased development of osteoclasts and activation of mature osteoclasts (5, 6). PTH also has several other effects on osteoblasts that include stimulation of collagenase 3 (interstitial collagenase, matrix metalloproteinase 13) synthesis (7) and inhibition of type I collagen synthesis (8).

PTHrP is secreted by many cancers and causes the hypercalcemia of malignancy by mimicking the actions of PTH (9). The physiological roles of PTHrP differ from those of PTH, however. Unlike PTH, which is expressed almost exclusively in the parathyroid gland and, to a much lesser extent, in the hypothalamus, PTHrP is expressed in many fetal and adult tissues (10, 11, 12), where it is likely to subserve a variety of local functions as varied as cellular differentiation and smooth muscle relaxation (13). Despite the apparently contrasting roles of PTH and PTHrP, the ability of aminoterminal fragments of both PTH and PTHrP to activate a common PTH/PTHrP receptor (14, 15) suggested that the diverse actions of both peptides might be mediated by the same PTH/PTHrP receptor.

Expression of complementary DNAs (cDNAs) encoding the PTH/PTHrP receptors from different mammalian species showed conclusively that a single receptor binds both ligands, PTH and PTHrP, with indistinguishable affinity (14, 16, 17). Furthermore, the PTH/PTHrP receptor activates both the AC and PLC second-messenger pathways. The cloned PTH/PTHrP receptor is not only found in the major target organs of PTH action, i.e. kidney and bone, but also in nearly all other tissues examined (18, 19, 20). These observations suggest that the common PTH/PTHrP receptor might mediate not only the endocrine functions of PTH but also the autocrine/paracrine actions of PTHrP.

The idea that one receptor might mediate the actions of two distinct ligands with apparently distinct physiologic roles raises a series of questions. Does the receptor respond to changes in levels of both ligands, and, if so, do both ligands have significant functional overlap? Does the PTH/PTHrP receptor mediate all of the actions of each ligand? Mounting evidence suggests that both PTH and PTHrP activate additional receptors (21, 22, 23, 24). For example, PTHrP has been shown to play an important role in regulating calcium homeostasis during fetal life. Placental perfusion studies have demonstrated that a specific midregion portion of PTHrP that does not bind the PTH/PTHrP receptor stimulates transplacental calcium transport (25, 26). Furthermore, the recently cloned PTH-2 receptor activates AC, in response to aminoterminal fragments of PTH, in ways that closely resemble the response of the PTH/PTHrP receptor (24).

The availability of gene knock-out mice missing either the PTHrP gene (27) or the PTH/PTHrP receptor gene (28) provides a means for assessing the functional interactions of PTH and PTHrP with the PTH/PTHrP receptor in intact animals. Both PTHrP (-/-) and PTH/PTHrP receptor (-/-) mice have dramatically accelerated differentiation of chondrocytes during endochondral ossification in developing growth plates (29, 30). These results suggest that the PTH/PTHrP receptor, expressed in growth plate chondrocytes (29), mediates the actions of PTHrP to slow chondrocyte differentiation (28).

Here, we use the gene knock-out mice to explore further the interactions of PTH, PTHrP, and the PTH/PTHrP receptor in bone. We demonstrate that: 1) the PTH/PTHrP receptor mediates the bone-resorbing actions of PTH; 2) the chondrocytes of PTHrP (-/-) and PTH/PTHrP receptor (-/-) mice closely resemble each other at the ultrastructural level but differ substantially in their patterns of matrix mineralization; and 3) the effect of the ablation of the PTH/PTHrP receptor gene has greater effects on expression of selected genes in osteoblasts than does the ablation of the PTHrP gene. These results suggest that osteoblasts respond physiologically to circulating PTH, as well as to locally produced PTHrP. Thus, the PTH/PTHrP receptor mediates physiologic actions of both PTH and PTHrP and may integrate responses to both ligands.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Riboprobe. Complementary 35S-labeled riboprobes (complementary RNAs) were transcribed from plasmids encoding type I collagen (31), cartilage matrix protein (CMP; kindly provided by P. F. Goetinck, Massachusetts General Hospital, Boston, MA) (32), matrix gla protein (MGP; kindly provided by G. Karsenty, Baylor University, Houston, TX) (33), osteocalcin (OC; kindly provided by G. Karsenty) (34), osteopontin (35), and collagenase 3 (kindly provided by Y. Eeckhout, Brussels, Belgium) (36). Sense and antisense probes were synthesized from the linearized plasmids using 35S-uridine 5'-triphosphate (1289 Ci/mmol) (NEN, Boston, MA) and Sp6, T3, and T7 RNA polymerases (Promega Corp., Madison, WI). The following reagents were purchased: Scintillation fluid (Scint-A, Packard Instrument Co., Inc., Downers Grove, IL), paraformaldehyde (PFA), VectaBond-coated glass slides, silver nitrate, sodium thiosulfate, Permount (Fisher Scientific International, Inc., Fairlawn, NJ), cacodylic acid, Alizarin Red S, isobutylmethylxantine, naphthol AS-MX phosphate, Nuclear Fast Red, Fast Blue RR salt (Sigma Chemical Co., St. Louis, MO), glutaraldehyde, osmium tetroxide (Polysciences, Warrington, PA), LR White resin (E. F. Fullam, Inc., Latham, NY), uranyl acetate (Electron Microscopy Sciences, Ft. Washington, PA), lead citrate (Ted Pella, Inc., Redding, CA), mouse tumor necrosis factor-{alpha} (TNF-{alpha}) (TNF-M, Genzyme, Cambridge, MA), cAMP RIA, 45CaCl2 (NEN), Antibiotic/Antimycotic solution (Gibco, Grand Island, NY), NTB-2 photoemulsion (Kodak, New Haven, CT), and x-ray film (Hyperfilm; ß-max, Amersham, Arlington Heights, IL). Rat PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and human (h) PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) were synthesized in the Peptide and Oligonucleotide Core Laboratory of the Massachusetts General Hospital Endocrine Unit. Recombinant hPTH(1–84) was a gift of Chugai Pharmaceutical Co. (Tokyo, Japan).

Animals
Transgenic mice were derived from matings of mice heterozygous for the ablation of the PTHrP and PTH/PTHrP receptor gene. Each strain had similar genetic backgrounds: the original SvJ129/C57Bl6 knock-out heterozygotes were crossed into C57 for several generations, and then these were crossed into Black Swiss for two generations. Genotypes of mice were confirmed by Southern blot or PCR of genomic DNA, as described previously (27, 28). Animals were maintained in facilities operated by the Office of Laboratory Animal Research of the Massachusetts General Hospital, in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and were employed using protocols approved by the institution’s subcommittee on animal care.

Preparation of genomic DNA
Genomic DNA was isolated from tail clips by standard methods, as described (26).

Histology and tissue preparation
Fresh tissues were obtained by cesarean sections from fetuses derived from heterozygous interbreeding at different days of gestation. For regular histology and in situ hybridization, fetuses were fixed in 10% buffered formalin, pH 7.2, and embedded in paraffin. Sections, 5- to 10-µm thick were obtained with a Reichert Jung Model 2045 microtome, collected on VectaBond-coated glass slides, and stained with hematoxylin-eosin or further processed for in situ hybridization. For enzyme staining, hindlimbs were fixed in 10% buffered formalin, pH 7.2, overnight at 4 C, rinsed several times in PBS, and then transferred into 30% sucrose in PBS. Limbs were then mounted on a cryostat support in a drop of mounting medium and slowly frozen in a mixture of 100% ethanol and dry ice. Sections of fresh frozen bones, of 10 µm, were obtained with a Reichert Frigocut cryotome and stained for alkaline phosphatase.

Differential staining of bone
Alizarin red S. After bone sections were deparaffinized and hydrated, they were stained with 1% Alizarin red S (pH 6.4) for 3 min, washed with water, and immediately photographed.

von Kossa. Tissue sections were deparaffinized, rehydrated in decreasing concentrations of ethanol (100, 96, 90, 80, 70%), and equilibrated in distilled water. Subsequently, sections were placed in a 1% aqueous silver nitrate solution and exposed to strong light for 15–60 min. After several washes in distilled water, the sections were treated with sodium thiosulfate for 5 min and then washed again with distilled water. Sections were counterstained with 1% methyl green for 3 min, rinsed three times in n-butanol, dehydrated, and mounted with Permount.

In situ hybridization
In situ hybridization was performed as described previously (29, 37). In brief, tissue sections were deparaffinized, rehydrated in decreasing concentrations of ethanol (100, 96, 90, 80, 70%), and then postfixed with 4% PFA for 15 min. After several washes in PBS, the sections were digested with 10 µg/ml proteinase K (15 min at room temperature). Subsequently, the sections were incubated in 4% PFA/PBS (10 min). The wash step in PFA/PBS was followed by an incubation in 0.2% HCl for 10 min. Again, sections were washed in PBS before being acetylated with 0.25% acetic acid in the presence of 0.1 M triethanolamine (10 min). Sections were then dehydrated in increasing concentrations of ethanol and air-dried. Hybridization was performed in a humidified chamber in a solution containing 50% formamide, 10% dextran sulfate, 1 x Denhardt’s solution, 600 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 50 mM dithiothreitol, 0.25% SDS, and 200 µg/ml transfer RNA. After hybridization at 55 C for 16 h, sections were washed briefly with 5 x SSC at 50 C and then incubated in 50% formamide/2 x SSC for 30 min, followed by an incubation in 1 x TNE buffer (10 mM Tris-HCl, pH 7.6, 500 mM NaCl, 1 mM EDTA). Sections were then treated with 10 µg/ml ribonuclease A in TNE (37 C, 30 min) and then washed in 1 x TNE. The final wash steps were done once in 2 x SSC and twice in 0.2 x SSC (each: 20 min at 50 C). Hybridization of the messenger RNA (mRNA) with the specific 35S-labeled riboprobes was detected as follows: sections were dehydrated using increasing concentrations of ethanol and then air-dried. To estimate the intensity of expression for each mRNA, sections were exposed to x-ray film overnight at room temperature. Slides were then dipped into NTB-2 and stored at 4 C for the time needed (estimated by autoradiography). After developing the photoemulsion, sections were counterstained with hematoxylin-eosin and mounted. For semiquantitative analysis, sections from several animals of the same genotype (3, 4, 5, 6, 7) were processed at least twice with the same probe, to avoid artifacts. Additionally, comparisons across genotypes were always performed by processing samples at the same time with the same reagents.

cAMP production in calvarial bones
Calvarial bones were isolated from E18.5 mouse fetuses, as described (38). Briefly, frontal and parietal bones of calvariae were aseptically dissected, cleaned of adherent tissues, and cut into four pieces. Each piece was processed separately. Calvariae were washed once with 0.5 ml of ice-cold assay buffer [130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 1 mM NaPi (pH 7.4)] and then incubated in 0.5 ml of the same buffer containing 0.1% heat-inactivated BSA, 1 mM isobutylmethylxantine, 5 mM glucose, with the appropriate treatment at 37 C for 15 min. The four pieces of each calvariae were incubated with vehicle alone (assay buffer), 10-7 M rat PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), 10-7 M hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), or 10-5 M forskolin. At the end of the incubation, the reaction was terminated by addition of 0.5 ml of cold 90% 2-propanol in 0.5 M HCl. Bones were incubated for 16–18 h at 4 C. Propanol extraction was repeated, and the combined extracts were evaporated by vacuum centrifugation. The dried extracts were redissolved in acetate buffer (50 mM Na acetate/0.05% Na azide, pH 6.2) for measurement of cAMP by a specific RIA, as already described (38). Bones were washed twice with 1 ml acetone and once with 0.5 ml ether and were air-dried and weighed. The results were normalized for the bone weight, and the data was expressed as picomoles of cAMP produced per µg of dry bone.

Bone resorption bioassay
Bone resorption was quantitated by the release of previously incorporated 45Ca from fetal mouse calvarial bones in vitro. Calvaria from E18.5 homozygous and heterozygous PTH/PTHrP receptor knock-out embryos and normal littermates were obtained, as previously described (38), after injecting the pregnant mothers with 50 µCi 45CaCl2 sc on E16.5. Frontal and parietal bones of calvariae were aseptically dissected, cleaned of adherent tissues, and cut into 4 pieces. Subsequently, these bones were precultured in 2 ml DMEM containing 1 mM calcium, 2 mM phosphate, 5% heat-inactivated horse serum, and 1% antibiotic/antimycotic solution, on a rocking platform at 50 oscillations/min in a 37 C incubator under 5% CO2 in air. After 24 h, the medium was replaced with 3 ml of medium that contained 10-7 M rat PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), 10-7 M hPTH(1–84), or 10-8 M TNF-{alpha}. After an additional 72 h, the bones were removed, rinsed in saline, and placed in scintillation fluid for determination of released radioactivity. Bones and medium were counted, and the percentage of total bone 45Ca released into the medium during the 72-h treatment period was calculated. Results were expressed as the mean ± SE of the mean of the percentage of 45Ca released for groups of 3 homozygous, 7 wild-type, or 16 heterozygous bones.

Electron microscopy
Femoral and tibial epiphyseal growth plate cartilage and adjacent perichondrium from normal E18.5 day-old fetal PTH/PTHrP receptor homozygous mutant embryos and normal littermates were examined after dissection of whole mouse legs. The specimens were fixed in a mixture having final concentrations of 2.5% glutaraldehyde-4% PFA in 0.1 M cacodylate buffer, pH 7.4, for 3 h at 4 C. Tissues were dehydrated and embedded in LR White resin. Some specimens were postfixed in 1% osmium tetroxide for an additional 2 h at 4 C before dehydration. Samples were initially sectioned at 1-µm thickness and stained with toluidine blue for light microscopy using a Leitz Ortholux II. Tissue regions to be analyzed by electron microscopy were thin-sectioned (~80 nm), stained with uranyl acetate and lead citrate, and examined at 60 kV in a Philips EM300 electron microscope.

Alkaline phosphatase staining
Frozen sections were stained in a 50-ml solution containing 2 ml Naphthol AS-MX phosphate concentrate (pH 8.6), 48 ml distilled water, and 25 mg Fast Blue RR salt at room temperature for 15 min to 1 h. After the slides were washed in distilled water for 1 min, they were washed in tap water for 5 min. Counterstaining was performed for 8 min in Nuclear Fast Red, and sections were washed for 1 min in distilled water. Subsequently, sections were mounted in buffered glycerol.

Statistical analysis
Data from the bone resorption assays were analyzed using SYSTAT 5.2.1 for Macintosh (SYSTAT Inc., Evanston, IL). ANOVA was used for the initial analysis; Tukey’s test was used in the posthoc analysis to determine which pairs of means differed significantly from each other. Two-tailed probabilities are reported, and all data are presented as mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of PTHrP on chondrocyte differentiation via the PTH/PTHrP receptor
Electron microscopy was performed to characterize the abnormal chondrocytes at the ultrastructural level in PTH/PTHrP receptor-ablated animals. Clusters of persisting nonhypertrophic chondrocytes were identified among the hypertrophic chondrocytes of PTH/PTHrP receptor-ablated animals in the absence of a defined junction between proliferative and hypertrophic cells, features similar to those reported previously in PTHrP (-/-) mice (30). In addition, large numbers of dispersed glycogen granules were found in the cytoplasm of proliferating and maturing chondrocytes in both knock-out models (30) (Fig. 1Go). The accumulation of glycogen is consistent with an alteration in the energy metabolism of the cells. The similarity of the phenotypes of the two knock-outs confirms further the hypothesis that the actions of PTHrP on chondrocytes are mediated by the PTH/PTHrP receptor. Further studies, however, have identified differences in the pattern of mineralization in the growth plate in the two knock-out models.



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  		Figure 1. Electron micrograph of the tibial epiphyseal proliferative and maturation zone of a wild-type (panels A and C) and PTH/PTHrP receptor (-/-) mutant (panels B and D) at E18.5. Accumulation of electron dense glycogen granules is seen in the cytoplasm of the mutant chondrocytes (panels B and D, white arrowheads). ER, Endoplasmic reticulum; C, cisternae of ER; N, nucleus. (A and B, 1:750; BC and D, 1:8000).

 
Contrasting growth plate mineralization phenotypes of PTHrP and PTH/PTHrP receptor(-/-) mice
In contrast to the PTHrP (-/-) mice, in which the growth plates show an accelerated mineralization in the zone of hypertrophic chondrocytes (29), PTH/PTHrP receptor-ablated animals exhibited growth plates with a quite different mineralization pattern. Alizarin red S staining (Fig. 2Go, A and B), as well as von Kossa staining (data not shown) of nondemineralized PTH/PTHrP receptor (-/-) hindlimb sections, showed (surprisingly) a marked reduction of mineralization in the growth plate of the tibia and failure of mineralization in the bone collar flanking the epiphyseal growth plate. Abnormalities of mineralization could result directly from the absence of PTH/PTHrP receptors in chondrocytes or indirectly from interference with the function of the PTH/PTHrP receptor on other cells. To evaluate possible local effects of one postulated regulator of mineralization, the activity of alkaline phosphatase was examined and found to be unaffected in the PTH/PTHrP receptor (-/-) growth plate (Fig. 2Go, C and D). Furthermore, other markers of chondrocyte differentiation (collagen type II, collagen type X, and CMP) were distributed similarly in the PTHrP (-/-) and PTH/PTHrP receptor (-/-) growth plate (data not shown).



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  		Figure 2. Mineralization and alkaline phosphatase activity. Alizarin red staining of the proximal tibia of a wild-type (A) and PTH/PTHrP receptor homozygous mutant (B) at E18.5. The mutant growth plate shows a marked decrease in mineralization and lacks a mineralized bone collar flanking the epiphyseal region (A, arrowhead). Alkaline phosphatase enzyme actvity (blue) is indistinguishable in wild-type (C) and mutant (D) animals. F, Von Kossa staining (black) of the tibia of a double homozygous [PTH/PTHrP receptor (-/-)/PTHrP (-/-)] embryo is identical to that of a PTH/PTHrP receptor (-/-) mutant (E) alone. HY, hypertrophic chondrocytes; PS, primary spongiosa (A and B, 1:100; C, D, E, and F, 1:40).

 
The mineralization defects could be the consequence of altered mineral ion homeostasis. The ionized calcium levels in PTH/PTHrP receptor (-/-) mice are even lower than those of PTHrP (-/-) mice (26), and they might be low enough to interfere with mineralization of the growth plate. Alternatively, the differing patterns of mineralization of the PTHrP (-/-) mice (increased) and the PTH/PTHrP receptor (-/-) mice (decreased) could involve actions of PTHrP not mediated by the PTH/PTHrP receptor. To evaluate this possibility, the mineralization of growth plates in mice doubly homozygous for deletion of both the PTHrP and the PTH/PTHrP receptor genes was examined. These double homozygotes had a pattern of decreased mineralization of the growth plates that closely resembled that of the PTH/PTHrP receptor (-/-) mouse (Fig. 2Go, E and F). This observation, thus, is consistent with the hypothesis that the PTH/PTHrP receptor mediates the actions of PTHrP on the growth plate but that other actions of the PTH/PTHrP receptor, probably resulting from PTH stimulation, affect the mineralization phenotype, as well.

PTH/PTHrP receptor regulates bone resorption
The low levels of blood calcium in PTH/PTHrP receptor (-/-) mice may, in part, reflect the absence of effects of PTH and PTHrP, acting on the PTH/PTHrP receptor, to increase bone resorption. To assess this possibility, we first examined the accumulation of cAMP, in response to PTH and PTHrP in total calvariae during in vitro incubation. Calvariae from either heterozygous or wild-type animals showed an 8- to 9-fold increase in cAMP accumulation after challenge with rat PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) (P < 0.001 vs. respective basal values) (Fig. 3Go). In contrast, calvariae from homozygous mice showed no response to either peptide [P = not significant (NS)]. Exposure to forskolin led to an 8- to 12-fold increase of cAMP accumulation, a response which was indistinguishable in calvariae from homozygous, heterozygous, and wild-type fetuses (P = NS). The loss of responsiveness to PTH and PTHrP of PTH/PTHrP receptor (-/-) calvariae shows that the ability of PTH and PTHrP to stimulate AC in bone is mediated solely by the PTH/PTHrP receptor.



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  		Figure 3. cAMP accumulation in calvariae of wild-type (WT, n = 5), heterozygous (HET, n = 19), and homozygous (HOM, n = 6) PTH/PTHrP receptor knock-out animals at E18.5. Mutant bones did not respond to treatment with either 10-7 M rPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) or 10-7 M hPTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ). Forskolin (Fsk; 10-5 M) was used as positive control and showed an 8-fold increase of cAMP in the homozygous mutants.

 
To test whether PTH-induced bone resorption is dependent on the expression of the cloned PTH/PTHrP receptor, 45Ca release from calvaria of PTH/PTHrP receptor (-/-), (+/-), and (+/+) littermates was measured after treatment with vehicle, rat PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), hPTH(1–84), or TNF-{alpha} (Fig. 4Go). Though there was a roughly 2-fold increase in 45Ca release from calvariae of PTH/PTHrP receptor (+/-) or (+/+) mice after treatment with either PTH fragment (P < 0.02 vs. respective basal values), calvariae from PTH/PTHrP receptor (-/-) mice did not show any response to PTH. Calvaria from the latter animals, however, responded to treatment with TNF-{alpha} with a 2.5-fold increase in 45Ca release, a response indistinguishable from the TNF response in PTH/PTHrP receptor (+/+) and (+/-) mice (P = NS). Thus, the calvariae from PTH/PTHrP receptor (-/-) mice can release 45Ca, but they fail to do so in response to PTH. The PTH/PTHrP receptor, thus, mediates PTH-stimulated bone resorption.



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  		Figure 4. Bone resorption. 45Ca release from calvariae of PTH/PTHrP receptor wild-type (+/+) (n = 7), heterozygous (±) (n = 16), and homozygous (-/-) mutants (n = 3) after treatment with vehicle, 10-7 M rPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ), 10-7 M hPTH(1–84), and 10-8 M TNF-{alpha}.

 
Osteoblast differentiation in PTHrP and PTH/PTHrP receptor mutants
In situ hybridization was performed on serial sections of bone to determine whether PTHrP and/or the PTH/PTHrP receptor regulate in vivo the expression of the collagen {alpha}1(I), osteopontin, OC, and collagenase III (matrix metalloproteinase 13) genes. The expression of collagen {alpha}1(I) mRNA was indistinguishable in both knock-outs, when compared with normal (data not shown). In contrast to bones from PTHrP (-/-) mice that have been shown to express higher levels of osteopontin mRNA (29) than normal, bones from PTH/PTHrP receptor (-/-) animals exhibited a marked decrease in levels of this mRNA (Fig. 5Go). Similarly, the expression of OC mRNA was elevated in PTHrP (-/-) but was strikingly reduced in PTH/PTHrP receptor (-/-) bones (Fig. 6Go). Further, collagenase-3 mRNA was hardly detectable in receptor mutant animals, but it was expressed normally in PTHrP (-/-) bones (Fig. 7Go). The dramatic effect of ablation of the PTH/PTHrP receptor gene on the levels of mRNA of several genes expressed in osteoblasts (which continue to synthesize type I collagen) suggests that PTHrP must not be the only ligand of this receptor controlling gene expression in these cells; presumably, PTH can stimulate OC, osteopontin, and collagenase 3 mRNA synthesis in the PTHrP (-/-) mice but not in the PTH/PTHrP receptor (-/-) mice.



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  		Figure 5. Osteopontin in situ hybridization of the tibia of a wild-type (A and D), PTHrP (-/-) (B and E), and PTH/PTHrP receptor (-/-) (C and F) animal at E18.5. Osteoblasts of homozygous PTH/PTHrP receptor mutant bones show a decrease in osteopontin mRNA expression. Tibia of PTHrP (-/-) shows an increase in expression, as previously reported (29 ) (A, B, and C, Brightfield view, 1:40; D, E, and F, darkfield view, 1:40).

 


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  		Figure 6. OC in situ hybridization of the tibia of a wild-type (A and D), PTHrP (-/-) (B and E), and PTH/PTHrP receptor (-/-) (C and F) animal at E18.5. Osteoblasts of homozygous PTH/PTHrP receptor mutant bones show a marked decrease in OC mRNA expression (A, B, and C, Brightfield view, 1:40; D, E, and F, darkfield view, 1:40).

 


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  		Figure 7. Collagenase-3 in situ hybridization of the tibia of a wild-type (A and D), PTHrP (-/-) (B and E), and PTH/PTHrP receptor (-/-) (C and F) animal at E18.5. Lack of collagenase 3 mRNA expression in homozygous PTH/PTHrP receptor mutants (A, B, and C, Brightfield view, 1:40; D, E, and F, darkfield view, 1:40).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vitro data have shown that the PTH/PTHrP receptor binds two ligands, PTH and PTHrP, with equal affinity and that it can activate both AC and PLC in response to either ligand (14, 16). The present study demonstrates directly that the PTH/PTHrP receptor is responsible for mediating the actions of these two ligands in vivo. PTH/PTHrP receptor (-/-) fetuses have profound abnormalities in the differentiation pattern of chondrocytes and osteoblasts, and in mineralization of cartilage and bone, that are different from those observed in the PTHrP (-/-) animals. Furthermore, studies using calvariae from PTH/PTHrP receptor (-/-) mice provide evidence that this receptor is responsible for mediating the action of PTH on bone resorption.

Growth plate abnormalities
The pattern of abnormal chondrocyte differentiation is similar in PTHrP (-/-) and PTH/PTHrP receptor (-/-) mice; this similarity suggests that the PTH/PTHrP receptor mediates the actions of PTHrP on chondrocyte differentiation (27, 28). Further evidence for this hypothesis is provided by the finding of large numbers of glycogen granules in the cytoplasm of proliferating and maturing chondrocytes of the PTH/PTHrP receptor (-/-) mice, a phenomenon similar to that previously reported in PTHrP (-/-) animals (30). This glycogen accumulation may reflect abnormal energy metabolism in the chondrocytes. It is possible that glycogen accumulation may reflect altered cAMP-regulated phosphorylation of glycogen synthase and phosphorylase.

The defect in mineralization in the hypertrophic zone of the cartilage in the growth plate of PTH/PTHrP receptor (-/-) mice and the absence of a mineralized bone collar along the metaphysis in these animals suggest that the phenotype is not simply the result of the lack of response to PTHrP, because PTHrP-ablated animals show accelerated mineralization reaching widely into the hypertrophic zone (29). The mineralization defect in the PTH/PTHrP receptor (-/-) mice seems not to reflect widespread altered expression of chondrocyte-specific genes, because levels of mRNA encoding collagen type II, collagen type X, and CMP were similar in both knock-out mice; and furthermore, alkaline phosphatase enzyme activity was normal. The expression pattern of MGP, a protein that has been shown to be highly abundant in lung and calcifying cartilage (33, 39) reflected the disorganized pattern of chondrocyte differentiation in the growth plate of PTH/PTHrP receptor (-/-) animals (data not shown). Mice homozygous for the deletion of the MGP gene, however, have no detectable abnormalities in growth plate mineralization until one week postnatally (40). Therefore, it is not clear whether MGP contributes to this mineralization defect. The presence of hypocalcemia in the PTH/PTHrP receptor (-/-) mice could contribute to the abnormal mineralization. It has been found, however, that chondrocyte lines established from mice that are missing both copies of the PTH/PTHrP receptor gene show a delay in mineralization, when compared with the heterozygous parental cells, even when the ambient calcium concentration is controlled (J. Guo, MA General Hospital, unpublished; personal communication). This suggests that the defect in mineralization in the PTH/PTHrP receptor (-/-) mice may not be explained solely by the hypocalcemia.

We have no certain explanation for the increased mineralization of the growth plates of PTHrP (-/-) mice. This abnormality may simply reflect the accelerated differentiation of chondrocytes in the mutant mice. With sufficient ambient levels of calcium, chondrocytes in these mice may mineralize prematurely; the low blood calcium in the PTH/PTHrP receptor (-/-) mice may not permit such mineralization. The contrasting mineralization patterns of the PTHrP (-/-) and PTH/PTHrP receptor (-/-) growth plates might also reflect an action of PTHrP not mediated by the PTH/PTHrP receptor. The phenotype of double homozygous knock-out mice, which closely resembles that of the PTH/PTHrP receptor (-/-) mice, does not support this hypothesis, however.

Abnormal osteoblast function
The intense expression of the PTH/PTHrP receptor gene in osteoblasts and a subset of bone stromal cells (18, 20) has suggested a role for the PTH/PTHrP receptor in osteoblast function. Osteoblasts synthesize PTHrP, and PTH is secreted into the circulation from fetal parathyroid glands (41); thus, both ligands might well influence osteoblast function by activating their common receptor. Although PTHrP is produced locally in bone, the PTHrP (-/-) mice had no detectable change in production of mRNA-encoding collagen {alpha}1(I) and collagenase-3. Because the in situ hybridization technique is only semiquantitative, modest changes in expression might have been missed. Nevertheless, the contrasting, dramatic decrease of expression of collagenase-3, osteopontin, and OC mRNA in the osteoblasts of PTH/PTHrP receptor (-/-) mice suggests that a ligand other than PTHrP, presumably PTH, can compensate for the loss of PTHrP in the PTHrP (-/-) mice; in the PTH/PTHrP receptor (-/-) mice, neither ligand can stimulate expression of these genes. These results demonstrate that ligands of the PTH/PTHrP receptor are physiologically important modulators of collagenase-3, osteopontin, and OC expression in vivo. The functional consequences of this modulation are uncertain. Collagenase-3 (interstitial collagenase) cleaves native type I collagen; this cleavage may be involved in osteoclast recruitment or differentiation (7, 42). The phenotype of OC (-/-) mice suggests that OC is a negative regulator of the activity of osteoblasts (43). Osteopontin binds integrins and may be involved in osteoclast function (44). Thus, the PTH/PTHrP receptor activates three genes that are expected to favor activation of osteoclasts and inhibition of the anabolic function of osteoblasts.

The observed dependence of collagenase-3 gene expression on activation of the PTH/PTHrP receptor is generally consistent with prior studies in tissue and organ culture. Treatment of calvarial cultures with PTH for 48 h enhances the degradation of newly synthesized collagen, probably through a cAMP-dependent increase in collagenase production in osteoblasts (45). Furthermore, extensive studies have been performed in UMR 106–01 cells, demonstrating a dramatic increase in collagenase mRNA expression after treatment with PTH (7).

Additional mRNAs, such as those encoding osteopontin and OC, which appear late during osteoblastic differentiation, are abnormally expressed in mice missing either the PTHrP (29) or the PTH/PTHrP receptor gene. Osteopontin has been shown to be regulated by a number of hormones and growth factors associated with bone formation and bone remodeling, such as transforming growth factor-ß, retinoic acid, and 1,25 dihydroxyvitamin D3 (46, 47). Noda and Rodan (48) showed that PTH suppresses the amount of osteopontin mRNA synthesis in ROS 17/2.8 osteosarcoma cells, possibly via cAMP. In our studies, we confirmed previous observations, by Lee et al. (29), of increased levels of osteopontin mRNA in the growth plates of PTHrP (-/-) mice. These observations are consistent with those of Noda and Rodan (48). In contrast, the expression of osteopontin in osteoblasts from the PTH/PTHrP receptor (-/-) animals was markedly suppressed, inconsistent with the effects of PTH in vitro noted by Noda and Rodan (48). The in vivo phenotype could reflect interactions among different cells, present in the bone, with several different paracrine and hormonal factors.

PTH has also been shown to increase the steady-state level of OC mRNA in ROS 17/2.8 cells (49) by increasing the activity of the rat OC promoter (50). The levels of OC mRNA, which encodes a major noncollagenous protein of bone, are strikingly reduced in the long bones of PTH/PTHrP receptor (-/-) embryos, but they are unchanged, or even increased, in bones of PTHrP (-/-) mice. The decreased levels of OC mRNA in PTH/PTHrP receptor (-/-) mice are thus consistent with the effects of PTH in cultured osteoblast-like cells. The relatively unaffected level of OC mRNA in the PTHrP (-/-) mice may well reflect the actions of PTH, the levels of which are elevated in both the PTHrP (-/-) and the PTH/PTHrP receptor (-/-) mice (C. S. Kovacs, Massachusetts General Hospital, unpublished).

Mounting evidence suggests that both PTH and PTHrP mediate some of their actions through additional receptors (21, 22, 23, 24, 51, 52). The recent cloning of the PTH-2 receptor (24), which is exclusively activated by PTH and not by PTHrP, raises the possibility that this receptor is responsible for mediating actions of PTH in bone. To test this hypothesis, we treated calvariae of wild-type, heterozygous, and homozygous PTH/PTHrP receptor-ablated embryos with rat PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), and forskolin and measured cAMP accumulation. The calvariae from PTH/PTHrP receptor (-/-) mice showed loss of response to both peptides, a result suggesting that the actions of PTH on bone, mediated through the AC pathway, result from binding exclusively to the PTH/PTHrP receptor. This study provides, furthermore, evidence that the PTH/PTHrP receptor also mediates PTH-induced bone resorption. Calvariae from PTH/PTHrP receptor (-/-) mice, treated with either rat PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or hPTH(1–84), in contrast to calvariae from (+/-) or (+/+) littermates, did not release 45Ca. In contrast, TNF-{alpha} treatment of calvariae from PTH/PTHrP receptor (-/-) mice led to 45Ca release indistinguishable from that of littermates, demonstrating that the bone resorption machinery was intact.

These in vivo studies thus demonstrate that the classical PTH/PTHrP receptor has two physiological ligands, PTH and PTHrP. Because the ablation of either the PTHrP or the PTH/PTHrP receptor gene results in a similar abnormality in chondrocyte differentiation and glycogen accumulation, we can conclude that this phenotype is caused by actions of PTHrP on the PTH/PTHrP receptor. In contrast, mice that are missing the PTH/PTHrP receptor gene have abnormalities of collagenase-3, osteopontin, and OC synthesis in osteoblasts that are not found in PTHrP (-/-) mice. PTHrP either has no role in regulating these genes or, perhaps more likely, PTH can compensate for the loss of PTHrP in the PTHrP (-/-) mice. Finally, the studies of calvarial organ cultures demonstrate that the PTH/PTHrP receptor, found in bone only on cells of the osteoblasts lineage (53, 54), is required for stimulation of bone resorption by PTH.


   Acknowledgments
 
The authors wish to thank the following for their valuable suggestions, discussions, and technical support: Drs. Ernestina Schipani, Kae Lee, Michael Mannstadt, Jennifer Guiducci, Peter Carolan, and Karen Hodgens (Children’s Hospital, Department Of Orthopedic Surgery) and Janet Saxton.


   Footnotes
 
1 This work was supported by National Institute of Health Grants DK-47038 and AM-03564. Back

2 Supported, in part, by a fellowship of the Max-Kade Foundation. Current address: Beate Lanske, Ph.D., Molecular Endocrinology, Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried bei München, Germany. Back

3 Supported, in part, by a fellowship from the Medical Research Council of Canada. Current address: Christopher S. Kovacs, M.D., FRCPCF, Faculty of Medicine, Endocrinology, Health Sciences Centre, Memorial University of Newfoundland, 300 Prince Philip Drive, St. John’s, New Foundland A1B 3V6, Canada. Back

Received May 14, 1998.


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 Discussion
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