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306 nature genetics • volume 30 • march 2002

A mutant PTH/PTHrP type I receptor in enchondromatosis

Sevan Hopyan1–4, Nalan Gokgoz2, Raymond Poon1, Robert C. Gensure5, Chunying Yu1, William G. Cole3,6,7,Robert S. Bell3,4,8, Harald Jüppner5, Irene L. Andrulis2,7,8, Jay S. Wunder2–4,8 & Benjamin A. Alman1,3,4,6

1Program in Developmental Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada. 2Program in Molecular Biology andCancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G1X5, Canada. 3Department of Surgery, Division ofOrthopaedics, University of Toronto, Toronto, Ontario, Canada. 4Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada. 5Endocrineand Pediatric Endocrine Units, Departments of Medicine and Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston,Massachusetts, USA. 6Division of Orthopaedics, The Hospital for Sick Children, Toronto, Ontario, Canada. 7Department of Molecular and Medical Genetics,University of Toronto, Toronto, Ontario, Canada. 8University Musculoskeletal Oncology Unit and Division of Orthopaedics, Mount Sinai Hospital, Toronto,Ontario M5G1X5, Canada. Correspondence should be addressed to J.S.W. (e-mail: wunder@mshri.on.ca).

Enchondromas are common benign cartilage tumors of bone.They can occur as solitary lesions or as multiple lesions in enchon-dromatosis (Ollier and Maffucci diseases). Clinical problems causedby enchondromas include skeletal deformity and the potential formalignant change to chondrosarcoma1–3. The extent of skeletalinvolvement is variable in enchondromatosis and may include dys-plasia that is not directly attributable to enchondromas4. Enchon-dromatosis is rare, obvious inheritance of the condition is unusualand no candidate loci have been identified. Enchondromas areusually in close proximity to, or in continuity with, growth-platecartilage. Consequently, they may result from abnormal regulationof proliferation and terminal differentiation of chondrocytes in the

adjoining growth plate. In normal growth plates, differentiationof proliferative chondrocytes to post-mitotic hypertrophic chon-drocytes is regulated in part by a tightly coupled signaling relayinvolving parathyroid hormone related protein (PTHrP) andIndian hedgehog (IHH)5–9. PTHrP delays the hypertrophic differ-entiation of proliferating chondrocytes, whereas IHH promoteschondrocyte proliferation. We identified a mutant PTH/PTHrPtype I receptor (PTHR1) in human enchondromatosis that signalsabnormally in vitro and causes enchondroma-like lesions in trans-genic mice. The mutant receptor constitutively activates Hedge-hog signaling, and excessive Hedgehog signaling is sufficient tocause formation of enchondroma-like lesions.

Published online: 19 February 2002, DOI: 10.1038/ng844

Fig. 1 PTHR1 mutation and functional analysis. a, Heterozy-gous C→T change and corresponding amino-acid substitu-tion. b, Confocal immunofluorescent subcellular localizationof C-terminal c-Myc–tagged wildtype PTHR1 (WT) and R150CPTHR1 in COS-7 cells using 9E10 α-c-Myc antibody, after cellu-lar permeabilization. c, Western blot of total lysate or biotinimmunoprecipitates from c-Myc–tagged WT and R150CPTHR1 expressing COS-7 cells probed with 9E10 (α-Myc-epi-tope) and α-actin antibodies showing decreased membraneexpression of R150C PTHR1 relative to PTHR1. Intact cellswere biotin-labeled before protein extraction. d, IP3[3H]Thymidine assay in embryonic stem cells lacking PTHR1. e, Cyclic AMP enzymeimmunoassay in COS-7 cells comparing R150C variant with empty vector (pcDNA1),WT, other known inactivating (P132L14,15) and activating mutants (H223R13, T410P12) and a silent polymorphism (E546K) found in one affected individual29, in thepresence of 10–6 M PTHrP (1-34) or carrier alone. f, cAMP accumulation in response to increasing PTHrP dose. g, Competitive radioreceptor binding assay using [125I]-labeled rat [Nle8,21, Tyr34]PTH (1-34) amide and increasing concentrations of human [Tyr34]PTH (1-34) amide as competitor showed markedly reduced apparent bind-ing affinity of R150C PTHR1 for ligand compared with WT (Kd=1.24 ± 0.10 nM for WT, Kd=39.19 ± 3.9 nM for R150C PTHR1). h, Cyclic AMP accumulation normalized tocell-surface expression (as determined by Scatchard transformation). i, Basal cAMP level normalized to surface expression is higher in cells expressing R150C PTHR1than in those expressing WT. Assays were carried out three times in duplicate with DNA from two separate bacterial colonies.

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We speculated that abnormal IHH/PTHrP signaling contributes tothe genesis of enchondromas by delaying terminal chondrocytedifferentiation. Key components of the IHH-PTHrP pathway werefound to be expressed in human enchondromas and chondrosar-comas, and primary cultures from these lesions provided evidenceof increased signaling (S. Hopyan and colleagues, manuscript inpreparation). We hypothesized that mutant PTHR1 mightaccount for these observations. Tο test this possibility, we under-took single-strand conformation polymorphism (SSCP) analysisand manual sequencing to screen cartilage-tumor specimens forsequence alterations in PTHR1. We found a heterozygous C→Tchange (Fig. 1a) resulting in an R150C substitution in the extracel-lular domain of PTHR1 in enchondroma specimens from two ofsix individuals with enchondromatosis (Ollier disease). These twoaffected individuals were unrelated males with mild to moderatedisease severity. In one of these men, the variant PTHR1 allele(hereafter referred to as ‘R150C PTHR1’) was carried in the germline and was inherited from the father, who has mild skeletal dys-plasia but no evidence of enchondromas, similar to a scenarioreported previously10. In the other affected male, the R150CPTHR1 allele probably represented a somatic change, as normalbone adjacent to the enchondroma specimen did not contain thevariant allele. It is possible that this second affected individual is amosaic carrier of the R150C PTHR1 allele, but we were not able totest this possibility because enchondroma tissue from only one sitewas available for study. The R150C change was not found in DNAsamples from 100 unaffected individuals, nor in 50 sporadic chon-drosarcoma specimens.

Activated PTHR1 mediates its actions through at least two sec-ond messengers, cyclic adenosine monosphosphate (cAMP) andinositol triphosphate (IP3)11–14. To assess these signaling pathways,

we transfected COS-7 cells with cDNA sequences encoding eitherwildtype PTHR1 or the mutant R150C PTHR1, or c-Myc–taggedversions of these receptors. PTHR1 mutations previously identifiedin individuals with Jansen metaphyseal chondrodysplasia12,13 andBlomstrand chondrodysplasia14,15 served as controls. We firstassessed expression levels and the subcellular localization of thewildtype and R150C PTHR1 proteins. We tested both receptors bynonquantitative immunofluorescence and found that both pro-teins can localize to the cell surface (Fig. 1b). However, Westernanalysis of anti-biotin immunoprecipitates of lysates from intactbiotinylated cells expressing either receptor protein revealed thatR150C PTHR1 was located at the membrane to a lesser extent thanPTHR1, even though similar concentrations of both receptor pro-teins were detected in total cell lysates (Fig. 1c).

To assess agonist-induced second-messenger accumulation,we carried out cAMP and IP3 assays using COS-7 cells andembryonic stem cells, respectively, lacking both copies of thegene PTHR1 (ref. 9). We did not observe agonist stimulation ofIP3 accumulation when R150C PThR1 was transfected (Fig. 1d);this finding is consistent with prolonged differentiation delay ofgrowth-plate chondrocytes16. Cells transfected with the R150CPTHR1 mutant cDNA showed significantly lower basal and ago-nist-stimulated cAMP accumulation than cells transfected withPTHR1 (Fig. 1e,f). These findings could be related to the lowercell-surface expression of the mutant receptor (Fig. 1c). In agree-ment with this hypothesis, radioreceptor studies using [125I]-labeled rat [Nle8,21, Tyr34]PTH (1-34) amide and increasingconcentrations of human [Tyr34]PTH (1-34) amide as competi-tor showed markedly reduced apparent binding affinity of

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Fig. 2 Newborn mouse growth plates. a,b, α-c-Myc immunohistochemistryrevealed expression of the c-Myc–tagged R150C (and WT; not shown) PTHR1transgene (brown color) under the control of Col2A1 regulatory elements ingrowth-plate chondrocytes (a), but not in the surrounding perichondriumwhere Col2A1 is not expressed, nor in a negative control growth plate (b).c,d, Trichrome-stained WT (c) and R150C PTHR1 (d) transgenic proximalhumeral growth plates at identical magnification showed a difference in theheight of the hypertrophic zone (between dark lines). e,f, α-COL10A1immunostaining of WT (e) and R150C PTHR1 (f) transgenic distal femoralgrowth plates highlighted the relatively short hypertrophic zone (darkbrown color and arrows) in R150C PTHR1 mice. g, Western blot from trans-genic fetal-limb lysates showing similar level of c-Myc–tagged receptor pro-tein expression in WT and R150C PTHR1 founder lines.

Fig. 3 Enchondromas in PTHR1 transgenic mice. a–f, Mouse enchondromaswere evident in R150C PTHR1 (b (arrows),d) but not WT (a), proximal humeriiat 8.5 wk as multiple metaphyseal cartilage rests. These rests persisted to atleast 38 wk (c,e), occurred in several bones including the distal femur (20 wk; f)and consisted of islands of chondrocytes in a hyaline cartilage matrix, similar tohuman enchondromas.

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R150C PTHR1 for ligand, compared with wildtype PTHR1(Kd=1.24 ± 0.10 nM for wildtype, Kd=39.19 ± 3.9 nM for R150CPTHR1; Fig. 1g). When DEAE-dextran was used for transfec-tions instead of Fugene 6, specific radioligand binding was com-parable for COS-7 cells expressing either receptor, but theapparent agonist-binding affinity of the mutant PTHR1remained significantly lower than that of the wildtype receptor.When corrected for reduced cell-surface expression (as deter-mined by Scatchard transformation), cells expressing the mutantPTHR1 showed similar to slightly elevated maximal agonist-induced cAMP accumulation compared with that of cellsexpressing the wildtype receptor (Fig. 1h). In addition, after cor-rection for cell-surface expression, the R150C mutant revealedevidence of increased basal cAMP accumulation (Fig. 1i), sug-gesting some degree of constitutive activity. This finding is remi-niscent of the constitutive activity of mutant PTHR1 that causesJansen metaphyseal chondrodysplasia12,13. Of note, it has beensuggested that Jansen disease and Ollier disease share some radi-ographic and histologic features17. Increased cAMP signalingactivity by PTHR1 is consistent with slowed chondrocyte differ-entiation12,13 and is therefore possibly consistent with the forma-tion of enchondromas.

To evaluate the effect of the R150C PTHR1 mutation in vivo, weused the mouse type II collagen (Col2A1) promoter andenhancer18,19 to drive expression of PTHR1 or R150C PTHR1 inthe growth plates of transgenic mice (Fig. 2a,b). Transgene expres-sion levels in wildtype and R150C founder lines were comparable

(Fig. 2g). Mice expressing wildtype PTHR1 in separate founderlines did not have any morphological abnormalities compared withwildtype littermates. At birth, growth plates of mice from three sep-arate founder lines expressing R150C PTHR1 were of similar over-all size, but had shorter hypertrophic zones, than those of miceexpressing PTHR1 (Fig. 2c,d). This difference was greatest ingrowth plates that contribute the most to longitudinal growth, suchas the proximal humerus versus the distal humerus, and was con-firmed by immunostaining against Type X collagen (COL10A1) tohighlight the hypertrophic zone (Fig. 2e,f)20.

Beyond eight weeks of age, features similar to those of humanenchondromatosis were evident in two of these R150C PTHR1founder lines. Paraffin-embedded sections stained with safranin-Ο to highlight cartilage revealed cellular cartilage islands, orrests, in the metaphyses of several long bones (Fig. 3), includingthe proximal humerus (Fig. 3a–e), the distal femur (Fig. 3f) andthe tibia. Columns of cartilage commonly connected the rests tothe adjoining growth plate, as is often found in human enchon-dromatosis (Fig. 3b,c). We saw regions of varying cellularity, withproliferating cells and occasional binucleate lacunae arranged inlobular patterns in a hyaline cartilage matrix that was partially orcompletely surrounded by bony matrix (Fig. 3d,e), similar tohuman enchondromas2. Another similarity to human enchon-dromatosis was the persistance of these rests well into adulthood(Fig. 3c,e).

The paucity of hypertrophic cells in the growth plates ofR150C PTHR1 mice persisted beyond adolescence (Fig. 4a,b)and may have resulted in the inclusion of cells which were stillcompetent to proliferate within the metaphyseal trabecular coresof cartilage (thin, red longitudinal streaks in Fig. 3a,b), wherethey could give rise to enchondromas. Moreover, the rests proba-bly originated from abnormal differentiation rather than a defectof resorption. Chondroclasts are cells which contribute toresorption of calcified cartilage at the cartilage–bone interfaceand are distinguished by their ability to retain an acid phos-phatase stain following tartrate challenge (TRAP stain)21. Wefound chondroclasts in equal numbers in PTHR1 and R150CPTHR1 mice (9 ± 2 TRAP-positive cells observed on two adja-cent, central-longitudinal, proximal humeral growth-plate sec-tions of three transgenic offspring of each of two wildtype andtwo R150C founder lines; Fig. 4c,d).

Fig. 4 Growth plates in PTHR1 transgenic mice. a,b, A paucity of hypertrophicchondrocytes in R150C PTHR1 growth plates (b) persisted relative to WTgrowth plates (a) and may have allowed proliferating chondrocytes to havebeen left behind in the metaphysis to form enchondromas. c,d, Tartrate-resis-tant acid phosphatase staining specifically highlighted equal numbers of chon-droclasts (red-brown color) at the cartilage–bone interface in WT (c) and R150CPTHR1 (d) bones, suggesting the metaphyseal cartilage rests were not causedby a resorption defect.

Fig. 5 a, The R150C PTHR1 mutant constitutively activates a Hedghehog-responsive Gli2-luciferase reporter construct. b–f, Enchondromas arise inCol2A1-Gli2 transgenic mice (c–f) but not in wild type littermates (b). Picturedhere are enchondromas in the distal femur at 13 wk (b–d) and 16 wk (e) and inthe proximal humerus at 43 wk (f).

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To investigate whether mutant PTHR1 enhances Hedgehogsignaling, as suggested by our studies using primary cultures ofhuman enchondromas (S. Hopyan and colleagues, manuscriptin preparation), we transfected HeLa cells with a Hedgehog-responsive Gli2-luciferase reporter gene. Co-transfection of cellswith R150C PTHR1, but not wildtype PTHR1, resulted in consti-tutive activation of the reporter (Fig. 5a). There was no differ-ence in activation of a control TCF-responsive reporter(pTOPFLASH) between the mutant and wildtype receptor. Todetermine whether excessive Hedgehog signaling is sufficient tocause enchondroma formation, we generated additional trans-genic mice expressing the Hedgehog transcriptional regulatorGli2 (refs 22,23; as has been done previously (ref. 24) to induceexcessive Hedgehog signaling in vivo) under the control of theCol2A1 promoter. Offspring of three founder lines expressing theCol2A1-Gli2 transgene, but not their wildtype littermates, devel-oped ectopic cartilage rests similar to those of R150C PTHR1mice (Fig. 5b–f). These rests were multifocal and persisted to atleast 43 weeks of age.

Together, these findings indicate that the R150C PTHR1 muta-tion probably contributes to the pathogenesis of at least some casesof enchondromatosis by slowing chondrocyte differentiation, lead-ing to incomplete endochondral ossification. Moreover, thePTHR1 mutant described here seems to augment Hedgehog sig-naling. In agreement with this observation, overexpression of thetranscription factor Gli2, which is down-stream of the Ihh receptorcomplex Ptch/Smoh, is sufficient to cause enchondroma-likelesions in mice. Cases of enchondromatosis without a demonstra-ble PTHR1 mutation, as well as solitary enchondromas, may ariseowing to similar molecular mechanisms. The persistence ofgrowth-plate tissue in the form of enchondromas beyond adoles-cence, when growth-plate tissue has normally disappeared inhumans, may allow for accumulation of secondary genetic eventsthat contribute to the development of chondrosarcoma.

MethodsTissue specimens. We obtained tissues from the National Cancer Institute ofCanada Sarcoma Tissue Bank housed at Mount Sinai Hospital, the MountSinai Hospital Bone Bank and The Hospital for Sick Children. We obtainedconsent for each specimen according to each institution’s policies.

Mutational analysis. We carried out single-strand conformation poly-morphism (SSCP) analysis and manual sequencing as previouslydescribed25. Primer sequences are available upon request.

PTHR1 functional analysis. We used non-PCR based site-directedmutagenesis (Site-Directed Mutagenesis Kit, Clontech) of the cDNAencoding PTHR1 in the pcDNA1 vector to generate R150C and the otherpublished mutations of PTHR1 (refs 12–15). The mutant contructs wereverified by automated sequencing of both strands. We carried out confo-cal immunofluorescent subcellular localization using carboxy-terminalc-Myc–tagged versions of the wildtype and variant PTHR1 and α-c-Myc9E10 antibody (Santa Cruz) after cell-membrane permeabilization with0.2% Triton X100 for 3 min at room temperature. (Tagging the C termi-nus of PTHR1 has been shown to not interfere with protein expression,ligand binding or signaling capability26.) We carried out western blots aspreviously described27. COS7 cells were plated at a density of 5 × 104 cellsml–1 and transfected the next day with the aid of a transfection reagent(Fugene 6, Roche or DEAE-Dextran). Forty hours after transfection, cellswere serum-starved for 2 h and then treated with either 10–6 M PTHrP(1-34) or PTH (1-34) in DMEM containing 0.1% FBS carrier or carrieralone for 15 or 45 min for cAMP assays and for 40 min for IP3 assays. Wecarried out cAMP assays by either enzymeimmunoassay (RPN 225,Amersham) or radioimmunoassay (as previously described28) withCOS7 cells, and IP3 assays using a tritiated IP3 assay system (TRK 1000,Amersham) with Pthr1–/– cells. Intact COS-7 cells expressing c-Myc–tagged wildtype or R150C PTHR1 were biotin-labeled before lysis.

We carried out western-blot analysis using an α-Myc-epitope antibody(9E10) on α-biotin immunoprecipitates and total lysates, to determine cellsurface and total cellular levels of the receptors. We carried out radiorecep-tor-binding assays using rat [Nle8,21, Tyr34]PTH (1-34) amide as radioli-gand as previously described28. Transfection efficiency with Fugene 6 wastypically over 40%, as assessed by co-transfection with green fluorescentprotein cDNA, and was controlled for by co-transfecting cells with β-galac-tosidase cDNA and normalizing assay results to β-galactosidase activitymeasured at 415 λ (β Galactosidase Assay Kit, Stratagene). To assess Hedge-hog signalling in vitro, we co-transfected HeLa cells expressing PTCH1 andthe Drosophila smoothened homolog (SMOH) with a Hedgehog-responsivemouse Gli2 promoter fused to the firefly luciferase gene and normalizedluciferase activity to β-galactosidase activity. Cells were treated with either 5µg ml–1 Shh-N in 0.1% FBS or carrier alone.

Transgenic mice. We cloned human wildtype PTHR1 and R150C PTHR1and mouse full-length Gli2 cDNAs by blunt-end ligation into the NotI sitesof the mouse Col2A1 promoter- and enhancer-containing constructpKN185 (refs 18,19) and excised the linear transgene construct by diges-tion with NdeI and HindIII prior to microinjection into pronuclei of fertil-ized ICR eggs. Genomic DNA was extracted from tails and screened bySouthern blot for integration of the transgene.

Immunohistochemistry. We dewaxed, hydrated and epitope-exposed 5-µm paraffin-embedded sections fixed in 4% paraformaldehyde using heat-induced epitope retrieval. Sections were blocked for endogenous peroxi-dase and biotin, incubated for 1 h at room temperature with primary anti-body (9E10 α-c-Myc), detected with a mouse-on-mouse immunostainingkit (Vector Labs) and visualized using diaminobenzidene. We obtained α-human recombinant COL10A1 antibody from Quartett Immunodiagnos-tika Biotechnologie GMBH.

TRAP staining. We used a TRAP staining kit (Sigma) on paraffin-embed-ded sections to identify chondroclasts.

AcknowledgmentsWe thank affected individuals and their families for their cooperation, C.-C.Hui for Gli2 and Gli2-luciferase cDNA, U.-I. Chung for PTHR1–/–

embryonic stem cells, Y. Yamada for Col2A1 promoter/enhancer cDNA, T.Ingolia (Ontogeny) for Shh-N protein, A. Karaplis for c-Myc–tagged PTHR1cDNA (Hkrk), F. de Sauvage (Genentech) for Smo cDNA, S. Tondat and L.Schwartz for pronuclear microinjections, L. Morikawa for histology, M. Hofor anti-c-Myc immunohistochemistry, M. To for assistance with cAMPassays, S. Cheon for assistance with SSCP analysis, A. Davis for help withstatistical analysis, J. Ahier for patient contact, members of the Andrulis andAlman labs for support, A. Gross, C. Hutchison and J. Wright for tissuespecimens; and J. Aubin, U. Chung, J. Dennis, S. Egan, J. Haigh, C.-C. Hui,H. Kronenberg and J. Rossant for critical advice. This work was supported bygrants from the National Cancer Institute of Canada with funds from theTerry Fox Run (to B.A.A., I.L.A. and R.S.B.), the Canadian Institutes ofHealth Research (to J.S.W., B.A.A. and W.G.C.) and The Hospital for SickChildren Research Institute Trainees’ Start-Up Fund (to S.H.). S.H. issupported by a Clinician-Scientist Fellowship from the Research TrainingCentre of The Hospital for Sick Children Research Institute, and B.A.A. issupported by the Canadian Research Chair Program.

Received 27 September 2001; accepted 15 January 2002.

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