PTHrP, PTH, and the PTH/PTHrP Receptor in Endochondral ... · PTHRP, PTH AND THE PTH/PTHRP RECEPTOR...

PTHrP, PTH, and the PTH/PTHrP Receptor inEndochondral Bone Development

Ernestina Schipani and Sylvain Provot

INTRODUCTIONSkeletal development proceeds viatwo mechanisms, intramembra-nous and endochondral bone for-mation (Erlebacher et al., 1995).The former, in which mesenchymalcells develop directly into osteo-blasts, is involved in the formationof the flat skull bones. The latter,accounting for the development ofmost other bones, involves a two-stage mechanism, whereby chon-drocytes form a matrix template,the growth plate, in which osteo-blasts differentiate and initiate theossification process (Fig. 1). An un-derstanding of this process at themolecular level is emerging (Kro-nenberg, 2003).

During endochondral bone devel-opment, growth plate chondrocytesundergo well-ordered and con-trolled phases of cell proliferation,

differentiation, and apoptosis (Bal-lock and O’Keefe, 2003; Eames etal., 2003; Kronenberg, 2003;Shum et al., 2003). Round prolifer-ative chondrocytes synthesize typeII collagen and form a columnarlayer, then stop proliferating andbecome prehypertrophic chondro-cytes that mature into post-mitotic,hypertrophic cells. Hypertrophicchondrocytes express predomi-nantly type X collagen and mineral-ize the surrounding matrix. Thisunique maturation/differentiationprocess is followed by the death ofhypertrophic chondrocytes, bloodvessel invasion, and finally replace-ment of the cartilaginous matrixwith trabecular bone. Calcified car-tilage is resorbed by osteoclasts,and then replaced by bone (the pri-mary spongiosa). With continuingresorption of the primary spon-

giosa, the primary center splits intotwo opposite growth plates; in eachof these, the maturation of carti-lage and subsequent remodelinginto bone continues as long as newchondrocytes are generated in thegrowth plates. As chondrocyte pro-liferation fuels longitudinal bonegrowth during postnatal life, thephyses are separated by an in-creasing amount of space that be-comes filled with bone marrow. Hy-pertrophic chondrocytes play apivotal role in coordinating chon-drogenesis and osteogenesis, ashypertrophic chondrocytes providea scaffold for subsequent formationof trabecular bone. In addition, hy-pertrophic chondrocytes modulatethe formation of the bone collar, theprecursor of cortical bone, in theadjacent perichondrium.

This review will summarize thecritical role of parathyroid hor-mone-related peptide (PTHrP),parathyroid hormone (PTH), andthe PTH/PTHrP receptor in endo-chondral bone development.

PTHRP, PTH AND THEPTH/PTHRP RECEPTORPTH, an 84–amino acid polypep-tide, is a major regulator of mam-malian mineral ion homeostasis inpostnatal life (Fig. 2A and C). Thetwo major target organs of PTH ac-tion are bone and kidney (Kronen-berg et al., 1993). In the kidney,PTH acts at two sites: in the proxi-mal tubule, PTH activates 1-alphahydroxylase, the enzyme respon-

Ernestina Schipani and Sylvain Provot are from the Endocrine Unit, MGH-Harvard Medical School, Boston, Massachusetts.

*Correspondence to: Ernestina Schipani, Endocrine Unit, Wellman Building Room 501, Massachusetts General Hospital, 50 BlossomStreet, Boston, MA 02114. E-mail: [email protected]

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.10028

Endochondral bone development is a fascinating story of proliferation,maturation, and death. An understanding of this process at the molecularlevel is emerging. In particular, significant advances have been made inunderstanding the role of parathyroid-hormone-related peptide (PTHrP),parathyroid hormone (PTH), and the PTH/PTHrP receptor in endochondralbone development. Mutations of the PTH/PTHrP receptor have beenidentified in Jansen metaphyseal chondrodysplasia, Blomstrand’s lethalchondrodysplasia, and enchondromatosis. Furthermore, geneticmanipulations of the PTHrP, PTH, and the PTH/PTHrP receptor genes,respectively, have demonstrated the critical role of these proteins inregulating both the switch between proliferation and differentiation ofchondrocytes, and their replacement by bone cells. A future area ofinvestigation will be the identification of downstream effectors of PTH,PTHrP, and PTH/PTHrP receptor activities. Furthermore , it will be ofcritical importance to study how these proteins cooperate and integratewith other molecules that are essential for growth plate development.Birth Defects Research (Part C) 69:352–362, 2003.© 2003 Wiley-Liss, Inc.

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WBirth Defects Research (Part C) 69:352–362 (2003)

© 2003 Wiley-Liss, Inc.

sible for hydroxylating 25-hy-droxyvitamin D, and inhibits phos-phate reabsorption by blockingsodium-dependent phosphate co-transport; in the distal tubule, itstimulates calcium absorptionagainst an electrochemical gradi-ent. In bone, PTH-mediated acti-vation of osteoblasts leads to in-creased osteoclast number andresorptive activity. The primaryphysiological consequences of in-creasing serum PTH are, therefore,an increase in serum calcium, a de-crease in serum phosphate, and anincrease in circulating 1, 25-dihy-droxyvitamin D3.

PTHrP was first discovered as thecause of humoral hypercalcemia ofmalignancy (HMM) syndrome (Broa-dus and Stewart, 1994). This syn-drome is characterized by serum lev-els of calcium and phosphate that are

essentially indistinguishable fromthose observed in patients with hy-perparathyroidism (i.e., hypercal-cemia and hypophosphatemia), butwith low or normal PTH levels.PTHrP and PTH share a limited se-quence homology, with only eightidentical residues in the first 34amino acids (Fig. 2A). Unlike PTH,PTHrP synthesis is not regulated byserum calcium, and circulating lev-els of PTHrP are very low in healthyadults. PTHrP is produced in a largevariety of normal adult and fetal tis-sues, including cartilage, heart,kidney, hair follicles, placenta,breast, lungs, and many epithelialsurfaces. This tissue distributionpattern suggests that PTHrP servesbiological functions other thanthose linked to the regulation ofmineral ion metabolism (Fig. 2C).Indeed, the hypothesis that PTHrP

plays a broad role as an autocrine/paracrine factor has now been con-firmed by studies that have showndramatic developmental abnormal-ities in mice genetically modified toeither overexpress or not expressPTHrP (see below).

Despite their limited sequence ho-mology, PTH and PTHrP bind and ac-tivate the same PTH/PTHrP receptorwith almost indistinguishable highaffinity (Fig. 2B) (Jüppner et al.,1991; Abou-Samra et al., 1992;Schipani et al., 1993). As discussedin detail below, the PTH/PTHrP re-ceptor mediates both the endocrineactions of PTH and the autocrine/paracrine actions of PTHrP (Kronen-berg et al., 1998). This places thePTH/PTHrP receptor as a central reg-ulator of both mineral ion homeosta-sis and bone development. Consis-tent with the ability of the PTH/PTHrPreceptor to recognize PTH andPTHrP, mRNA transcripts that en-code the PTH/PTHrP receptor arefound in a wide variety of fetal andadult tissues, with the highest levelof expression seen in kidney, bone,and cartilage (Urena et al., 1993;Lee et al., 1994, 1995, 1996).

Together with the receptors forcalcitonin (Lin et al., 1991) and se-cretin (Ishihara et al., 1991), thePTH/PTHrP receptor belongs to a dis-tinct group of G protein–coupled re-ceptors termed Family B (Gardellaand Juppner, 2001). All members ofthis secretin/calcitonin/PTH receptorfamily have seven membrane-span-ning domains, and a relatively longamino-terminal extracellular domain(approximately 160 amino acids)that contains six conserved, func-tionally important cysteine residues,and up to four potential N-linked gly-cosylation sites. Approximately 45amino acid residues, which are dis-persed throughout the transmem-brane domains and in the amino-ter-minal extracellular portion, arestrictly conserved in all members ofthis receptor family, and are likely tohave important functions in ligandbinding, signal transduction, or both.

Like all members of the Family Breceptors, the PTH/PTHrP receptoris coupled to signal effector mole-cules by heterotrimeric (��) gua-nine nucleotide binding proteins (G

Figure 1. Endochondral bone formation. A: Representative pictures of tibia, at differentstages of embryonic mouse development, are shown. The lower panels correspond to ahigher magnification view of the upper panels. See text for more details. RC: roundproliferative chondrocytes; CL: columnar layer; HC: hypertrophic chondrocytes; CB:cortical bone; TB: trabecular bone. Arrow heads on the far left panels indicate the tibia.B: Schematic representation of a mouse tibia at late stage of fetal development. Char-acteristic markers for bone, periarticular, flat, prehypertrophic, and hypertrophic chon-drocytes are noted.

ENDOCHONDRAL BONE DEVELOPMENT AND THE PTH FAMILY 353

Birth Defects Research (Part C) 69:352–362, (2003)

proteins; Fig. 2B). Upon binding of itsligand, the PTH/PTHrP receptor canactivate adenylate cyclase (AC)through G�s, and phospholipase C�(PLC�) through G�q/11 (Bringhurstet al., 1993; Iida-Klein et al., 1997).Activated AC then stimulates the for-mation of 3�,5�-adenosine mono-phosphate (cAMP), which in turn ac-tivates protein kinase A (PKA).Activated PLC� stimulates the for-mation of diacylglycerol (DAG) and1,4,5-inositol triphosphate (IP3). Inturn, DAG activates protein kinase C(PKC), and the production of IP3leads to an increase in the intracellu-lar free Ca��. The PTH/PTHrP recep-tor can also stimulate the extracellu-

lar influx of Ca�� through regulationof calcium channels (Swarthout etal., 2002). Furthermore, recentstudies have also indicated that thePTH/PTHrP receptor can activateprotein kinase C through a PLC-inde-pendent pathway (Whitfield et al.,2001). While the best-characterizedsecond messenger of the PTH/PTHrPreceptor is undeniably cAMP, activa-tion of the phospholipase C and pro-tein kinase C pathways by PTH islikely to play a significant role in renalphosphate transport (Iida-Klein etal., 1997), chondrocyte differentia-tion (Guo et al., 2002), and osteo-blast proliferation (Carpio et al.,2001).

THE PTH/PTHRPRECEPTOR IN HUMANCHONDRODYSPLASIAS

The critical role of the PTH/PTHrPreceptor in endochondral bone de-velopment is highlighted by the dis-covery that two devastating chon-drodysplasias, Blomstrand’s lethalchondrodysplasia and Jansen’s me-taphyseal chondrodysplasia, arecaused by mutations of this protein.More recently, mutant PTH/PTHrPreceptors have been also identifiedin some cases of human enchon-dromatosis.

Blomstrand’s LethalChondrodysplasia (BLC)

Blomstrand et al. (1985) reportedthe first case of BLC, and severalother cases then followed (Oostraet al., 2000). The disease is charac-terized by prenatal lethality, pre-mature and abnormal bone miner-alization and ossification, andshortened limbs (Fig. 3B). Endo-chondral bone formation is mark-edly advanced in BLC fetuses, andthe columnar proliferative layer inthe mutant growth plate is virtuallyabsent. In two recent cases of BLC,defects in tooth and mammarygland development were noted(Wysolmerski et al., 2001). The dis-ease appears to have an autosomalrecessive pattern of inheritance, asmost BLC cases are derived fromconsanguineous parents.

Genetic studies of BLC patientsled to the determination that BLC iscaused by inactivating mutations inthe PTH/PTHrP receptor (Fig. 3A)(Jobert et al., 1998; Karaplis et al.,1998; Zhang et al., 1998; Karp-erien et al., 1999). Thus far, threetypes of mutant PTH/PTHrP recep-tors have been identified, and allthree have been studied in vitro us-ing recombinant expression sys-tems. The first mutant, �373-383-PPR, lacks a region of the fifthtransmembrane domain of thePTH/PTHrP receptor due to a singlenucleotide change that affectsmRNA splicing (Jobert et al., 1998).Despite having normal cell-surfaceexpression, �373-383-PPR doesnot bind or respond to PTH orPTHrP. The second identified muta-

Figure 2. PTH, PTHrP, and the PTH/PTHrP-receptor. A: Schematic representation of theparathyroid hormone (PTH) and PTH related peptide (PTHrP). The three domains, definedon the basis of degree of homology between the two ligands, are indicated. The purplerectangles represent the regions of greater homology (dark purple) and lesser homology(light purple). The C-terminal part of these two ligands diverged. B: Downstream effec-tors of the PTH/PTHrP receptor. PTH and PTHrP share the same, unique seven-trans-membrane G-protein–coupled receptor, and the PTH/PTHrP receptor. Upon binding of itsligand, the PTH/PTHrP receptor can activate adenylate cyclase (AC) through G�s, andphospholipase C� (PLC�) through G�q/11. Activated AC then stimulates the formation of3�, 5�-adenosine monophosphate (cAMP), which in turn activates protein kinase A (PKA).Activated PLC� stimulates the formation of diacylglycerol (DAG) and 1,4,5-inositoltriphosphate (IP3). In turn, DAG activates protein kinase C (PKC), and the production ofIP3 leads to an increase of intracellular free Ca��. The PTH/PTHrP receptor can alsostimulate extracellular influx of Ca�� through regulation of calcium channels. C: Targettissues and function for PTH and PTHrP. See text for more details.

354 SCHIPANI AND PROVOT


tion arose from a single nucleotideexchange that led to the P132L mu-tation in the N-terminal extracellu-lar domain (P132L-PPR) (Karapliset al., 1998; Zhang et al., 1998).When studied in COS cells, this re-ceptor shows slightly decreased ex-pression, decreased specific ligandbinding, and decreased cAMP for-mation upon exposure to agonist li-gands. A homozygous deletion inexon EL2 was identified in a thirdBLC patient (Karperien et al.,1999). The resultant protein, �365-593-PPR, lacks transmembrane do-mains 5, 6, and 7, the connectingloops, and the cytoplasmic “tail,”and does not display any measur-able response to PTH. Notably, thedegree of skeletal abnormalitiesobserved in BLC patients can becorrelated with the severity of thePTH/PTHrP receptor mutation thatthey carried: P132L-PPR (the least

deleterious mutation) resulted inless severe defects than did either�365-593-PPR or �373-383-PPR.

Jansen’s MetaphysealChondrodysplasia (JMC)

Jansen’s metaphyseal chondrodys-plasia (JMC) is a rare autosomaldominant disorder characterized byshort-limbed dwarfism secondaryto severe abnormalities of thegrowth plate, and hypercalcemia(Fig. 3B) (Jansen, 1934; Jüppnerand Schipani, 1997). Clinical find-ings in JMC patients include severeshort stature, disproportionatelyshort limbs, and micrognathia(Frame and Poznanski, 1980). Thelaboratory findings in JMC patientsare also reminiscent of primary hy-perparathyroidism: severe andasymptomatic hypercalcemia, hy-pophosphatemia, decreased tubu-

lar reabsorption of phosphate, in-creased urinary excretion of cAMP,and elevated circulating levels of1,25-(OH)2VitD (Rao et al., 1979;Kruse and Schütz, 1993; Parfitt etal., 1996).

The pathogenesis of JMC hasbeen obscure for many years. Al-though JMC and hyperparathyroid-ism share many symptoms, para-thyroid gland abnormalities werenot detected in JMC patients, andcirculating levels of PTH and PTHrPwere either normal or undetect-able. Although early investigatorspostulated the existence of an un-known calcium-regulating agent, itwas later considered that the samesymptomatology—hypercalcemiain the absence of elevated PTH—might be observed if the patientshad a mutation in the PTH/PTHrPreceptor that led to ligand-indepen-dent (constitutive) activation of thereceptor (Schipani et al., 1995; Ya-suda et al., 1996). Accordingly,genomic DNA from a JMC patientwas screened for mutations in thecoding exons for the PTH/PTHrP re-ceptor, and a heterozygous muta-tion that changed residue 223 fromhistidine to arginine was found (Fig.3A). Since the original report in1995, the genomic DNA from nineother JMC patients has been exam-ined: seven patients had the H223Rheterozygous nucleotide exchange,and two patients had distinct het-erozygous mutations in the PTH/PTHrP receptor (T410P and I458R;as shown in Fig. 3A) (Schipani etal., 1996, 1999). None of these mu-tations has been detected in thegenomic DNA taken from a largenumber of healthy individuals, andthere is only one familial case inwhich the affected mother of a JMCpatient also had the mutation(Schipani et al., 1996). Thus, whileit appears that at least the H223Rmutation has a dominant mode ofinheritance, the dataset suggeststhat the three JMC mutations nor-mally arise as new germline muta-tions or as spontaneous somaticmutations that appear early in life.

When the corresponding mutantreceptors (H223R-, T410P-, andI458R-PPR) were examined invitro, it was found that cells ex-pressing these receptors demon-

Figure 3. The PTH/PTHrP receptor in human diseases. A: Schematic representation ofthe PTH/PTHrP receptor showing the mutations identified in patients with Jansen’s me-taphyseal chondrodysplasia (in green), Blomstrand’s lethal chondrodysplasia (in red),and enchondromatosis (in yellow). Mutations are missense point mutations unless spec-ified. See text for more details. B: View of a patient with Jansen’s metaphyseal chondro-dysplasia at the age of 22 years (left panel), and a postmortem view of a baby withBlomstrand’s lethal chondrodysplasia (generous gift of Dr. Caroline Silve, right panel).



strated increases in basal cAMP sig-naling that correlated with theamount of DNA transfected, indi-cating that the mutant receptorswere indeed constitutively active(Schipani et al., 1995, 1996,1999). Relative to those that ex-pressed the wild-type PTH/PTHrPreceptor, cells expressing eitherthe H223R-PPR or T410P-PPR mu-tation mounted submaximal re-sponses to either PTH or PTHrP (ap-proximately two-fold above basal).In contrast, cells expressing theI458R-PPR mutation showed thesame maximal cAMP accumulationin response to PTH or PTHrP as didthe wild-type receptor. While noneof the three JMC receptors acti-vated the phospholipase C pathwayin the absence of exogenous PTH,both T410P-PPR and I458R-PPRwere able to stimulate a normalphosphoinositol response to ago-nist ligands. In contrast, PTH didnot elicit increases in phosphoinosi-tol production from cells containingthe H223R-PPR mutation. In com-petition assays using radiolabeledPTH analogs, it is possible to dem-onstrate that all three of the JMCconstitutively active receptors bindPTH with either normal or modestlyenhanced affinity. Despite the sub-tle differences in the interactions ofeach of the three receptors withPTH, the corresponding JMC pa-tients did not show any obvious dif-ferences in their clinical or bio-chemical presentations. It thusappears that the PTH-independentactivation of the cAMP pathway, ev-idenced by these mutant receptors,is the origin of the pathology of JMC(Schipani et al., 1995, 1996,1999).

Enchondromatosis

Enchondromas are common benigncartilage tumors of bone that canoccur as solitary lesions or, in en-chondromatosis, as multiple le-sions. Recently, the heterozygousmissense mutation R150C has beenidentified in the PTH/PTHrP recep-tor (R150C-PPR) of two patientswith enchondromatosis (Fig. 3A)(Hopyan et al., 2002). One patienthad inherited the mutation from thefather, who had only a very mild

chondrodysplasia; in the secondcase the mutation was just limitedto the tumor tissue. When tested invitro, the mutant R150C-PPR ap-peared to be very poorly ex-pressed, and displayed a severeimpairment of both binding to PTHand cAMP production upon chal-lenge with the agonist, very likely aresult of impaired cell surface ex-pression. However, cells trans-fected with the mutant R150C-PPRappeared to have higher basalcAMP levels than controls, whenbasal cAMP values were correctedfor the level of receptors on the cellsurface. The mutant R150C-PPR isthus constitutively active. Interest-ingly, the patient heterozygous forthe R150C mutation, unlike the pa-tients with Jansen’s disease, did notdisplay any obvious sign of chon-drodysplasia besides the enchon-dromas, and was not hypercalce-mic. Of course, a variety ofconsiderations could explain theobvious differences. The R150Cmutation could affect receptor cellsurface expression in a more se-vere manner than the Jansen mu-tations, or the Jansen mutations sofar identified could be intrinsicallymore potent in terms of constitutiveactivity. Alternatively, it is possiblethat constitutive activity is not theonly critical feature of the R150C-PPR, and that other signaling prop-erties, as yet not explored, couldalso be affected by the R150C mu-tation, in addition to the increase ofbasal cAMP levels. The mechanismthat leads to formation of enchon-droma in the absence of any obvi-ous sign of chondrodysplasia in pa-tients carrying the R150C mutationis still largely unknown. In order toaddress this question, the effect ofthe R150C mutation on IndianHedgehog (Ihh) activity has beeninvestigated in vitro (Hopyan et al.,2002). Ihh is a morphogen that hasa critical role in endochondral boneformation (see below). Transcrip-tional assays in HeLa cells co-trans-fected with a Hedgehog-responsivereporter gene and either R150C-PPR or the wild-type PTH/PTHrP re-ceptor showed that only the mutantreceptor, and not the wild-type re-ceptor, resulted in a constitutiveactivation of the reporter. The find-

ing suggests that the R150C muta-tion overactivates the Ihh signalingpathway. This result is somehowsurprising, especially in light of invitro and in vivo data showing anegative regulation of Ihh by PTHrP(see below). More studies will benecessary in order to better under-stand how the R150C-PPR mutantleads to activation of the Ihh path-way and to formation of enchondro-mas.

The discovery of mutant PTH/PTHrP receptors as causes of thesehuman diseases clearly under-scores the critical developmentalrole of the PTH/PTHrP receptor inendochondral bone formation. Nu-merous genetic models have beengenerated, which have been instru-mental in understanding the role ofPTHrP, PTH, and their receptor dur-ing development. Some of thesemodels are described in the nextparagraph.

GENETIC MANIPULATIONSOF PTHRP, PTH, AND THEPTH/PTHRP RECEPTOR INENDOCHONDRAL BONE

Genetically modified animals havedramatically demonstrated the crit-ical developmental role of PTHrP. Inthe growth plate, PTHrP mRNA isexpressed by perichondrial cellsand proliferating chondrocytes inthe periarticular region, whereasthe PTH/PTHrP receptor mRNA isexpressed at low levels by prolifer-ating chondrocytes in columns andat higher levels by prehypertrophicchondrocytes (Lanske et al., 1996;Vortkamp et al., 1996). Homolo-gous ablation of the PTHrP gene inmice (PTHrP–/–) results in animalsthat die during the perinatal period(Karaplis et al., 1994). These miceshow severe abnormalities in thebones that form through the endo-chondral process; in particular,they display a dramatic shorteningof the snout, mandible, and ex-tremities, and a reduced diameterof the ribcage. The contracted andhypoplastic character of the ribcagein these mice is the one feature thatis mostly responsible for their in-ability to survive after birth.PTHrP–/– mice also show severe ab-normalities in mammary gland epi-



thelial development, suggestingthat PTHrP plays a crucial role inepithelial–mesenchyme interac-tions (Wysolmerski et al., 1995,1998).

The premature mineralizationand ossification seen with PTHrP–/–

mice appears to be related to thepremature transition of prolifera-tive chondrocytes to hypertrophicchondrocytes in the fetal growthplate (Fig. 4). Consistent with thesefindings, the study in which PTHrPoverexpression was targeted to tis-sues expressing the type II colla-gen promoter (TgPTHrP) showsthat the transgenic animals areborn with shortened limbs (Fig. 4).This is apparently due to delayedmineralization and deceleratedchondrocyte maturation in the fetalgrowth plate (Weir et al., 1996).

As previously discussed, the well-ordered and controlled prolifera-tion, differentiation, and apoptoticdeath of the growth plate chondro-

cytes is crucial for proper control ofbone elongation, since it sets thestage for the timing of the replace-ment of the cartilage matrix with atrabecular bone matrix. In particu-lar, the switch from a proliferativeto a postproliferative state deter-mines the number of chondrocytesin the proliferative versus the hy-pertrophic pool. The animal modelsdescribed above demonstrate thatPTHrP is critically involved in thisswitch.

Later studies with the PTHrP–/–

mice have shown that PTHrP is oneof the mediators of Ihh activity inthe growth plate. As previouslymentioned, Ihh is a member of afamily of proteins important for em-bryonic patterning that are highlyexpressed in the transition zone be-tween proliferating and hypertrophiccells and in hypertrophic chondro-cytes. It appears that Ihh inhibitschondrocyte differentiation by in-creasing PTHrP synthesis by the peri-

articular chondrocytes (Lanske et al.,1996; Vortkamp et al., 1996), andthereby delays the mineralization ofthe cartilage matrix. Overexpressionof Ihh protein by the injection of arecombinant retrovirus into embry-onic chick limbs delays hypertrophyof growth plate chondrocytes, asdoes addition of an active NH2-termi-nal fragment of Sonic Hedgehog(Shh, a relative of Ihh known tomimic Ihh actions in chondrocytes)to embryonic mouse limbs in vitro.These gain-of-function phenotypesare associated with an increase inPTHrP mRNA expression in the peri-chondrial region at the end of longbones. Consistent with these find-ings, the Shh fragment has no effecton PTHrP–/– mouse limbs. Further-more, mice homozygous for a nullmutation in the Ihh gene (Ihh–/–)have no detectable PTHrP mRNA intheir growth plate, and hypertrophicchondrocytes predominate in theIhh–/– growth plate late in fetal devel-opment, in association with a dra-matic inhibition of chondrocyte pro-liferation (St-Jacques et al., 1999).Taken together, these data suggestthat Ihh delays the switch from pro-liferation to hypertrophy of chondro-cytes by stimulating PTHrP produc-tion in the periarticular region of thegrowth plate. In addition, Ihh is alsoa potent stimulator of chondrocyteproliferation (St-Jacques et al.,1999; Long et al., 2001b). BecauseIhh stimulates PTHrP expression,which in turn keeps the chondrocytesin the proliferative pool, and therebydelays Ihh production, a negativeIhh/PTHrP feedback loop is then es-tablished in the growth plate (Fig. 6).Consistent with this model, expres-sion of a constitutively active PTH/PTHrP receptor in chondrocytes ofIhh–/– mice prevented prematurechondrocyte hypertrophy (Karp etal., 2000). Interestingly, it did notrescue the decreased chondrocyteproliferation. These experimentsdemonstrate that the molecularmechanism preventing chondrocytehypertrophy is distinct from thatwhich drives proliferation. Ihh posi-tively regulates PTHrP, which is suf-ficient to prevent chondrocyte hy-pertrophy and maintain a normaldomain of cells competent to un-dergo proliferation. In contrast, Ihh

Figure 4. Role of PTHrP and the PTH/PTHrP receptor in endochondral bone development.A: Histology of tibia obtained from wild-type (WT), PTHrP–/–, PPR–/– mice, or transgenicmice overexpressing in cartilage PTHrP (TgPTHrP) and a constitutively active form of thePTH/PTHrP receptor (TgPPR). Representative sections of E18.5 (PPR–/–) or newborn tibia(WT, PTH–/–;PTHrP–/–, TgPTHrP, and TgPPR) stained with hematoxylin and eosin (H&E)are shown. B: Higher magnification view of the panels presented in (A).



is necessary for normal chondrocyteproliferation in a pathway that can-not be rescued by PTHrP signaling.

Consistent with the notion that thePTH/PTHrP receptor mediates theaction of PTHrP in growth plate de-velopment, ablation of the PTH/PTHrP receptor mimics the effect ofthe PTHrP ablation on chondrocytedifferentiation (Lanske et al., 1996).Like PTHrP–/– mice, PPR–/– mice diearound birth, and show dramaticskeletal abnormalities secondary toacceleration of chondrocyte hyper-trophy (Fig. 4). These findings arestrikingly reminiscent of those re-ported in Blomstrand’s chondrodys-plasia. Conversely, consistent withthe observations in patients withJMC, transgenic mice, in which theH223R-PPR was targeted to thegrowth plate by placing its expres-sion under the control of the �1 (II)collagen promoter (TgPPR), showdelayed mineralization and deceler-ated chondrocyte maturation in skel-etal segments that are formed by en-dochondral bone development (Fig.4) (Schipani et al., 1997). This phe-

notype is very similar to the pheno-type of TgPTHrP mice with targetedoverexpression of PTHrP in thegrowth plate (Fig. 4) (Weir et al.,1996). The striking similarity be-tween the two animal models indi-cates that the PTH/PTHrP receptor isthe main mediator of PTHrP action inthe developing endochondral bone(Chung et al., 2001).

The remarkable parallelism be-tween human and mouse models isfurther underlined by the pheno-type of transgenic mice expressingthe R150C-PPR in the chondrocyticgrowth plate under the control ofthe collagen type II promoter(Hopyan et al., 2002). Consistentwith the findings in humans, longbones in these transgenic mutantmice were not shorter than con-trols, even though the hypertrophiclayer appeared to be reduced insize. More importantly, in adult-hood they showed persistence ofcartilage islands in the bony diaph-yses that were phenotypically sim-ilar to the human enchondromas.

As noted above, at first glance

the features of the PPR–/– mice aresimilar to those of the PTHrP–/–

mice, but closer examination re-veals that there are indeed differ-ences between these two geneti-cally ablated animals (Lanske et al.,1998). In particular, PPR–/– miceshow decreased trabecular boneformation in the primary spon-giosa, an alteration not seen afterPTHrP ablation. These results sug-gest that another ligand for thePTH/PTHrP receptor plays a role indevelopment. This has been con-firmed by the initial report on micethat lack PTH as a result of homol-ogous recombination (PTH–/–)(Miao et al., 2002): PTH–/– mice areviable but dysmorphic, and theyuniquely demonstrate a modest ex-pansion in the layer of hypertrophicchondrocytes, and reduced me-taphyseal osteoblast and trabecu-lar bone in fetal life and at birth(Fig. 5). The modest expansion inthe layer of hypertrophic chondro-cytes may be secondary to the ab-normal replacement of cartilage bybone, and is therefore consistentwith an important role of PTH in pri-mary spongiosa formation during fe-tal development. Compound PTH–/–

and PTHrP–/– mice mutants displaythe combined cartilaginous and os-seous defects of both single mutants(Fig. 5). These results indicate thatcoordinated action of both PTH andPTHrP are required to achieve nor-mal fetal skeletal morphogenesis.Furthermore, they clearly demon-strate an essential function of PTH atthe cartilage-bone interface, and aunique role for PTH in skeletal devel-opment in utero that complementsthe critical action that has been doc-umented for PTHrP (Miao et al.,2002).

To further explore the role of thePTH/PTHrP receptor in endochondralbone development, an elegant chi-meric model has been developed re-cently that has unveiled another crit-ical role of the Ihh/PTHrP system inendochondral bone development(Chung et al., 1998). Normally,bone collars are formed in the peri-chondrium abutting prehypertrophicand hypertrophic chondrocytes. Inthe growth plate of wt/PPR–/– chi-meric mice, PPR–/– chondrocytes hy-pertrophy ectopically closer to the

Figure 5. Role of PTH and PTHrP in transition from cartilage to bone and in primaryspongiosa formation. A: Sections of newborn tibias from wild-type(WT), PTH–/–, PTHrP–/–,PTH–/–/PTHrP–/– mice stained with H&E. B: (a–d) Undecalcified sections of femur stainedwith von Kossa stain, obtained from newborns PTH–/– or PTHrP–/– animals or PTH;PTHrPdouble-null mouse (PTH–/–;PTHrP–/–); (e–h) enlargement of the primary spongiosastained with H&E of the null animals (with permission from JCI)



articular surface, and bone collarsare formed in the perichondriumadjacent to the ectopically hyper-trophied chondrocytes. A similarchimeric approach has revealedthat ectopic hypertrophic chon-drocytes lacking both the PTH/PTHrP receptor and Ihh no longerinduce ectopic bone collar in theadjacent perichondrium (Chung etal., 2001). These data stronglysuggest that Ihh is indeed in-volved in the formation of thebone collar, and specifies the siteat which perichondrium formsbone. Consistent with this notion,mice lacking Ihh do not show os-teoblasts in either the primaryspongiosa or the bone collar ofbones formed by endochondraldevelopment (St-Jacques et al.,1999).

“HOT AREAS” OF FUTUREINVESTIGATIONS

Signaling PathwaysDownstream of the PTH/PTHrP Receptor

As mentioned above, the PTH/PTHrP receptor can activate morethan one G protein. The in vivoand in vitro characterization ofmutant receptors carrying muta-

tions identified in JMC supportsthe hypothesis that cAMP is a crit-ical mediator of PTH/PTHrP recep-tor activity in the growth plate. Amajor role for cAMP/PKA signalingis also highlighted by the recentobservation, in a chimeric mousemodel, that growth plate chondro-cytes lacking Gs� exhibit acceler-ated differentiation (Drs. Chungand Kronenberg, personal com-munication). Consistent with thismodel, a knock-in mutant mouse,in which the wild-type PTH/PTHrPreceptor had been substitutedwith a mutant receptor (DSEL) im-paired in its ability to increase in-tracellular levels of IP3, displays asignificant delay in chondrocytedifferentiation (Guo et al., 2002).These two downstream effectorsof PTH/PTHrP receptor activity,cAMP and IP3, thus seem to haveopposite or distinct effects ongrowth plate development. IP3signaling via the PTH/PTHrP re-ceptor appears to slow down theproliferation and to hasten the differ-entiation of chondrocytes, actionsthat oppose the dominant effects ofPTH/PTHrP receptors and involvecAMP-dependent signaling path-ways. Interestingly, lack of IP3 sig-naling also increases PTH/PTHrP re-

ceptor levels in chondrocytes (Guo etal., 2002).

Further studies will now be re-quired in order to understand howthe different signaling pathwaysdownstream of the PTH/PTHrP re-ceptor are connected and inte-grated, and to identify the crucialdownstream effectors of PTH/PTHrP receptor activity in chon-drocytes. In this regard, ourknowledge of potential down-stream targets of cAMP/PKA sig-nals in chondrocytes is expanding.It has been shown that the masterchondrogenic factor Sox9 (Bi etal., 1999, 2001; Akiyama et al.,2002;) is phosphorylated by PKAupon PTHrP treatment (Huang etal., 2001). This phosphorylation re-sults in an increased DNA affinity ofSox9, and thus increases Sox9 tran-scriptional activity (Huang et al.,2000). Sox9 is required during se-quential steps of the chondrocyte dif-ferentiation pathway; it is critical forcommitment of mesenchymal cellstowards chondrocytes, positivelyregulates chondrocyte proliferation,and delays their terminal differentia-tion. Sox9 is highly expressed byproliferative chondrocytes in vivo;however, it is currently not clearwhether this factor directly or indi-rectly regulates their proliferation.Another known target of cAMP/PKAsignaling is the cAMP response ele-ment binding protein (CREB). In pri-mary cultures of chondrocytes, it hasbeen shown that PTHrP inducesphosphorylation of CREB, whichbinds and activates the transcriptionof the Cyclin D1 promoter, and acti-vates cell division (Beier et al., 2001;Jonescu et al., 2001). Interestingly,however, in vivo, chondrocytes lack-ing PTHrP or its receptor do not dis-play any decrease in CREB phos-phorylation in comparison to normalcells, and transgenic mice express-ing a dominant-negative CREB havea phenotype that is very differentfrom that of the PTHrP–/– mouse(Long et al., 2001a). This indicatesthat the PTHrP/cAMP/PKA signal maypromote chondrocyte proliferationindependently of PKA/CREB signal-ing, through a yet uncharacterizedsignaling pathway.

Figure 6. Schematic representation of feedback loops and biological activities of PTHrP,Ihh, BMPs, and FGFs in the fetal growth plate. Ihh induces PTHrP expression at thearticular region, and PTHrP in turn represses Ihh expression, generating a negativefeedback loop. Ihh also induces the expression of BMPs, which themselves induce Ihhexpression in a positive feedback loop. Conversely, FGFs repress Ihh expression. PTHrP,Ihh, and BMPs are positive modulators of proliferation, and negatively affect maturation;the effect of Ihh on maturation is exclusively PTHrP dependent, while Ihh induces pro-liferation independently of PTHrP; and the effects of BMPs on proliferation and maturationcan occur independently of the PTHrP/Ihh axis. FGFs are negative modulators of prolif-eration, and positively affect terminal maturation; these effects can occur independentlyof PTHrP/Ihh action.



Integration andCooperation of the PTH/PTHrP Receptor SignalingSystem with OtherSignaling Systems duringEndochondral BoneDevelopment

In recent years, it has become pro-gressively clear that endochondralbone development is the result of acomplex and integrated network ofnumerous endocrine and paracrineactivities. PTHrP, PTH, and the PTH/PTHrP receptor are part of thesecritical signaling systems, but alarge number of other factors havebeen extensively investigated bothin vitro and in vivo. For example,numerous studies have establishedthat fibroblast growth factor (FGF)signaling, and bone morphogeneticprotein (BMP) signaling, respec-tively, interact with the Ihh/PTHrPpathway (Minina et al., 2001,2002; Ornitz and Marie, 2002). It iswell known that gain-of-functionmutations of the FGF receptors arethe cause of the most commonforms of human chondrodysplasias(Vajo et al., 2000). Consistent withthese findings, FGF signaling re-presses chondrocyte proliferation,and accelerates terminal differenti-ation of hypertrophic chondrocytes(Ornitz and Marie, 2002). Studiesinvolving organ culture of bone ex-plants in vitro suggest that the ef-fect of FGFs on chondrocyte prolif-eration is independent of thesuppression of Ihh activity (Mininaet al., 2002). However, it is still anopen question whether the effectsof the FGF signaling pathway inchondrocytes in vivo are, at least inpart, dependent on the Ihh/PTHrPsystem. Differently from FGFs andsimilarly to Ihh/PTHrP, BMPs arepositive modulators of chondrocyteproliferation, and they negativelyregulate chondrocyte terminal dif-ferentiation (Minina et al., 2001,2002). Consistent with these find-ings, a positive autoregulatoryfeedback loop between BMPs andIhh/PTHrP exists (Minina et al.,2001). Interestingly, a role forBMPs in chondrocyte proliferationand maturation that is independent

of Ihh/PTHrP has also been sug-gested (Minina et al., 2001).

It will be exciting and challengingto dissect how all these differentpathways interact and cooperate inorder to generate the complex net-work of actions that determine andtightly regulate endochondral bonedevelopment.

ACKNOWLEDGMENTSWe thank Dr. Henry M. Kronenbergfor critical review of the manu-script. We also thank Dr. TatsuyaKobayashi for providing histologicalslides and for helpful discussion.

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