Hereditary Multiple Exostoses and Heparan Sulfate Polymerization $

Biochimica et Biophysica Acta 1573 (2002) 346–355 www.bba-direct.com Review Hereditary multiple exostoses and heparan sulfate polymerization $

Beverly M. Zak1, Brett E. Crawford1, Jeffrey D. Esko*

Glycobiology Research and Training Center, Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, CMM-East Room 1054, La Jolla, CA 92093-0687, USA

Received 6 December 2001; received in revised form 9 April 2002; accepted 12 April 2002

Abstract

Hereditary multiple exostoses (HME, OMIM 133700, 133701) results from mutations in EXT1 and EXT2, genes encoding the copolymerase responsible for heparan sulfate (HS) biosynthesis. Members of this multigene family share the ability to transfer N- acetylglucosamine to a variety of oligosaccharide acceptors. EXT1 and EXT2 encode the copolymerase, whereas the roles of the other EXT family members (EXTL1, L2, and L3) are less clearly defined. Here, we provide an overview of HME, the EXT family of proteins, and possible models for the relationship of altered HS biosynthesis to the ectopic bone growth characteristic of the disease. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Chondrocyte; Chondrosarcoma; Bone development; Biosynthesis; EXT

1. Hereditary multiple exostoses (HME) and feet are also often involved. This benign condition has an estimated prevalence of 1 in 50,000 among the general HME is an autosomal dominant disease that clinically population [3]. Occasionally, HME leads to serious compli- manifests as cartilaginous capped bony outgrowths (osteo- cations, such as compression of nerves and soft tissues, chondromas) located at the growth plates of long bones [1]. causing extreme pain. The growths may also lead to The growth plate consists of a rich extracellular matrix of occlusion of blood vessels and interfere with joint move- collagen and chondroitin sulfate proteoglycans, with embed- ment. Each of these complications may necessitate surgical ded chondrocytes in various stages of maturation. A sur- removal of the exostosis, but the growths can recur. Patients rounding sheath of perichondral and periosteal cells plays often have multiple surgeries on the same area, which may important roles in controlling bone growth. Bone elongation be due to independent exostoses or continued growth of occurs by the process of endochondral ossification. After tissue that had not been completely removed. Malignant degradation of the cartilaginous scaffold constructed by the transformation of the benign tumor to a chondrosarcoma or chondrocytes in the growth plate, infiltrating osteoblasts lay an osteosarcoma occurs in approximately 1–2% of patients, down new trabecular bone [2]. The normal growth plate, and the risk of malignant transformation increases with age located at the epiphysis of long bones, has a highly [3]. organized structure (Fig. 1A). In contrast, the osteochon- dromas of HME consist of a disorganized collection of chondrocytes leading to random ossification (Fig. 1B). 2. Genetics of HME HME usually presents early in life with 80% of patients diagnosed before the age of 10 [1]. The most commonly HME has been linked to mutations at three genetic loci: affected bones include the femur, tibia and fibula, but the EXT1, which maps to 8q24.1 [4], EXT2, which maps to humerus, radius, ulna, ribs, scapula and bones of the hands 11p13 [5] and EXT3, which is located on the short arm of chromosome 19, although its exact position has not been mapped [6]. The majority of HME patients have mutations $ For additional information on HME, please contact The MHE in EXT1 (60–70%) or EXT2 (30–40%). Most HME cases Coalition http://www.geocities.com/mhecoalition). * Corresponding author. Tel.: +1-619-822-1100; fax: +1-619-534-5611. arise from missense or frameshift mutations in these genes, E-mail address: [email protected] (J.D. Esko). leading to synthesis of abnormally truncated forms of the 1 Both authors contributed equally to this work. EXT proteins (Fig. 2) [7,8]. Interestingly, while mutations in

Fig. 1. Histology of growth plates and an exostosis. (A) Histological section of a newborn mouse metatarsal growth plate stained with hematoxylin and eosin. Chondrocytes of the resting zone give rise to proliferative chondrocytes that ultimately lead to an increase in bone length. These cells enlarge into prehypertrophic and ultimately hypertrophic chondrocytes, which then apoptose and undergo replacement by new trabecular bone at the ossification center. Arrows indicate the perichondrium and periosteum, fibrous tissue containing cells that surround cartilage and bone, respectively. (B) Stained section of a human exostosis. Notice the lack of uniformity of the chondrocytes (Ch) and the disorganized trabecular bone (Tb).

EXT1 occur throughout the entire length of the gene, have not yet been done to determine if EXT mutations occur mutations in EXT2 concentrate toward its N-terminus, in tumors of other origin. implying specific functions for this part of the protein. Multiple cartilaginous exostoses also occur in Langer– Giedion syndrome (LGS, trichorhinophalangeal syndrome 3. EXTs as key enzymes in heparan sulfate (HS) type II, OMIM 150230), which also includes craniofacial synthesis dysmorphisms. The genetic defects giving rise to LGS include translocations, deletions and insertions at 8q24.1 Although HME had been linked to the EXT loci, it was and can include loss of EXT1 [9]. only recently that the proteins encoded by these genes were EXT1 and EXT2 have been identified as tumor suppres- correlated with HS biosynthesis. The assembly of HS was sor genes since loss of heterozygosity at these loci occurs in recently reviewed in depth elsewhere [12]. Briefly, HS syn- HME patients whose benign tumors transform into chon- thesis initiates by the formation of a tetrasaccharide (glucur- drosarcomas [10]. Loss of heterozygosity at the EXT1 locus onic acid–galactose–galactose–xylose, GlcAh3Galh3Galh is also implicated in hepatocellular carcinoma [11]. Studies 4Xyl-) at specific serine residues of proteoglycan core pro-

Fig. 2. EXT mutations. Etiologic mutations identified in human HME patients are listed above the line [7,8], and mutations identified in CHO HS deficient mutants (pgsD) are listed below the line [21]. Nonsense and frameshift mutations resulting in a truncated protein are in red, missense mutations and in-frame deletions are in yellow. Potential DXD motifs are boxed in blue. Numbers refer to the amino acid sequence based on human EXT1 and EXT2. 348 B.M. Zak et al. / Biochimica et Biophysica Acta 1573 (2002) 346–355 teins (Fig. 3). This intermediate serves as a primer for the activities were encoded by EXT1. The essential role of addition of N-acetylglucosamine (GlcNAca1,4), the first EXT1 in HS biosynthesis has also been demonstrated in committed step in HS synthesis. Chain polymerization then vivo through the generation of EXT1-deficient mice [23] takes place by the alternating addition of GlcAh1,4 and and in mutants of Drosophila melanogaster in the homol- GlcNAca1,4 from the corresponding nucleotide sugars ogous gene tout-velu (ttv) [24–26]. (UDP-GlcA and UDP-GlcNAc, respectively). The addition EXT2 was linked to HS synthesis by a more direct of the initiating GlcNAc residue is catalyzed by a different biochemical approach. Lind et al. [27] purified the copoly- enzyme (or set of enzymes) than that involved in chain merase through a series of chromatographic steps and polymerization [13]. As polymerization proceeds, a series of showed that the ability to catalyze the transfer of GlcA modifications takes place that include N-deacetylation of and GlcNAc was associated with a single f 70 kDa clustered sets of GlcNAc residues, followed by sulfation of protein. This protein was later sequenced and identified as the resulting free amino groups, epimerization of the flank- EXT2 [28]. EXT2 expression in yeast (which lack endog- ing GlcA to L-iduronic acid (IdoA) and sulfation at C2 of the enous GlcNAc and GlcA transferase activities) confirmed uronic acids and C6 and C3 of the glucosamine residues that the protein has both enzyme activities [29]. Although [12,14]. The arrangement of the variably sulfated residues mutants in EXT2 have not yet been characterized, gene and uronic acid epimers along the chain gives rise to binding silencing techniques have recently demonstrated that dimin- sites for various ligands, such as growth factors, extracellular ished EXT2 expression in cultured mammalian cells blocks enzymes and viral adhesins [15]. HS synthesis (B. Crawford and J. Esko, unpublished Surprisingly, the correlation between the EXT loci and results), indicating that both EXT1 and EXT2 are necessary HS came from studies of Herpes simplex virus (HSV) [16]. components of the system. As discussed below, an ortholog In 1994, Shieh and Spear [17] demonstrated that HSV-1 of EXT2 exists in Drosophila, but mutants have not yet utilized cell surface HS for viral attachment, mediated by been described. specific viral glycoproteins present on the viral envelope. Chinese hamster ovary cell mutants defective in HS synthesis were resistant to viral attachment and invasion and 4. Structure–function studies of EXT1 and EXT2 exogenous heparin blocked attachment of virions to a variety of cell types [18]. Tufaro et al. [19] took advantage Analysis of the primary sequence of EXT1 and EXT2 of these findings and used the cytotoxic effect of HSV reveals many shared characteristics that suggest related infection to identify mouse fibroblast cell lines resistant to functions (Fig. 4) and evolutionary origin (Fig. 5). Both infection, which also turned out to be deficient in HS EXTs appear to be type II transmembrane proteins with a synthesis. By screening a HeLa cDNA library for restoration single 17 residue transmembrane domain (sequence element of viral susceptibility, McCormick et al. [20] discovered a 2) and a short amino-terminal cytoplasmic tail. Interesting- gene with complementing activity that also restored HS ly, the cytoplasmic tail of EXT1 and ttv are identical synthesis. This gene turned out to be EXT1, which had been (MQAKKRY, element 1), suggesting a conserved function. previously identified as the gene mutated in HME [4]. Several cysteine residues with perfectly conserved spacing Subsequently, the hamster EXT1 homolog was cloned by suggest that EXT1 and EXT2 proteins share a common correction of a CHO cell mutant defective in HS polymer- structural fold (elements 4, 5, 9, 14). rib-1, the most closely ization [21]. Prior studies of this mutant had demonstrated related Caenorhabditis elegans ortholog of EXT1, lacks that a single protein catalyzed the transfer of both GlcNAc elements 1 and 2 and is truncated after element 9. Due to the and GlcA units to nascent chains [22], suggesting that both lack of homologous proteins with known three-dimensional

Fig. 3. Heparan sulfate biosynthesis. HS is synthesized by the formation of a linkage region on a proteoglycan protein core, followed by polymerization and sulfation of the chain. The first committed reaction for HS assembly is the attachment of the first GlcNAc unit (filled squares) catalyzed by the initiating GlcNAc transferase (aGlcNAcTI), possibly represented by the genes EXTL2 and EXTL3. The copolymerase complex elongates the chain by alternating additions of h1,4GlcA and a1,4GlcNAc residues catalyzed by EXT1 and EXT2. Further modifications include N-deacetylation/N-sulfation of GlcNAc residues, epimerization, and various O-sulfation reactions [12,14]. High affinity FGF-2 binding requires 2-O-sulfation of iduronic acid, whereas binding to FGF receptors also requires of GlcA 6-O-sulfation. The tentative identification and site of action of each EXT isoform is described in the text. B.M. Zak et al. / Biochimica et Biophysica Acta 1573 (2002) 346–355 349

Fig. 4. EXT family members have common domains. The carboxyl terminal portion of the EXTs is most highly conserved. Conserved sequence elements: 1, perfectly conserved cytoplasmic tail of EXT1 orthologs (MQAKKRY); 2, a 17 amino acid type II transmembrane domain; 3, a coiled-coil motif; 4, CX4CX5C; 5, ACL; 6, DXD(1); 7, GX2H; 8, DXD(2); 9, CX10LPF; 10, approximately 100 amino acid region unique to EXTL3 orthologs; 11, DXD(3); 12, seven amino acid insert unique to EXTL2; 13, DXD(4); 14, CX11PL; 15, C; 16, KK. structures, modeling of EXT1 and 2 has not been possible. yeast has both activities [29]. The GlcA transferase domain Therefore, the functional attributes of these conserved ele- of EXT1 appears to reside in the amino terminal part of the ments are unknown. protein containing the first and second DXDs. Data support- Many glycosyltransferases have been shown to contain an ing this idea emerged from two kinds of experiments. First, aspartic acid–any amino acid–aspartic acid (DXD) motif, a subset of pgsD mutants containing point mutations near critical for catalytic function [30–33]. The DXD motif may the second DXD motif (element 8) specifically lack GlcA be essential for binding the UDP-sugar donor through the transferase activity [21]. Secondly, etiologic mutations in coordination of a divalent cation [34]. Analysis of glycosyl- this region lack GlcA transferase activity, suggesting that transferases by hydrophobic cluster analysis (HCA) have the active site is nearby [37]. Point mutations in EXT1 that shown that active site DXD motifs are generally preceded by specifically eliminate the GlcNAc transferase activity have a hydrophobic region, predicted to form a h strand, followed not yet been found, making it difficult to assign the location by another cluster of hydrophobic residues [35]. EXT1 and of its active site. ttv contain four conserved DXD motifs (Figs. 2 and 4, EXT1 and EXT2 can form complexes. The two proteins elements 6, 8, 11, 13). The first and third of these motifs co-immunoprecipitate and colocalize in the Golgi [37,38]. have the clusters of hydrophobic residues characteristic of When either EXT1 or EXT2 are overexpressed by trans- functional DXD motifs. However, mutations in EXT1 that fection, the bulk of the protein remains in the endoplasmic abrogate HS biosynthesis cluster around the second DXD reticulum. When expressed together, they move to the Golgi [21], which does not have the expected hydrophobic cluster. [29,37], where polymerization and sulfation are thought to Recent studies of etiological mutations of EXT1 (i.e., those occur [39]. Importantly, coexpression greatly enhances associated with HME) have shown that a mutation in the first GlcA transferase activity in mammalian cells [37], and both DXD motif (D164H) abrogates activity, as measured by the GlcNAc and GlcA transferase activities when coexpressed inability of the transgene to restore HS biosynthesis in EXT1 in yeast [29]. These observations suggest that the active site deficient cell lines [36]. Thus, both the first and second DXD for GlcNAc transferase may reside in EXT2 or that it motifs may be necessary for activity. depends on the association of EXT subunits into an oligo- EXT1 has both GlcNAc and GlcA transferase activities. meric complex. One class of HS-deficient CHO mutants (pgsD) has dra- EXT2 also can catalyze the transfer of GlcNAc and GlcA matically reduced or undetectable EXT1 mRNA levels and to oligosaccharide acceptors in vitro, but the location of the lack both GlcNAc and GlcA transferase activities [21], functional domains in EXT2 is unknown. Of the various suggesting that EXT1 has the capacity to catalyze both conserved DXD motifs, only one is present in EXT2 (Fig. 4, reactions. Additionally, recombinant EXT1 expressed in element 11). In spite of its dual catalytic properties, EXT2 350 B.M. Zak et al. / Biochimica et Biophysica Acta 1573 (2002) 346–355

EXT1 contains more robust activity than EXT2 when expressed independently, but both subunits apparently can catalyze the individual reactions. Mapping studies indicate the location of the GlcA transferase domain in EXT1, but comparable studies have not yet been done in EXT2, and the location of the GlcNAc transferase site remains unknown. Optimal activity appears to require both subunits suggesting some form of allosteric control over the activity.

5. EXT L1–3

EXT1 and 2 are members of the exostosin family of genes (EXT), which contains three other members known as the EXT-like genes (EXTL1-3) due to sequence similarities to EXT1 and EXT2 (Figs. 4 and 5). While the link between EXT1 and 2 to HME is clear, the other known members of the EXT family have not yet been implicated. A single EXT- like ortholog exists in Drosophila and C. elegans (discussed below). Based on the level of amino acid conservation, it was hypothesized that all members of this family possess glycosyltransferase activities similar to those detected in EXT1 and EXT2 [42]. EXTL1 is more closely related to EXT1 than to other Fig. 5. Phylogenetic analysis of EXT family members. Nucleotide EXT family members (Figs. 4 and 5). Orthologs of the sequences of the various EXTs were aligned with ClustalX. Based on this alignment, a phylogenetic tree was generated with PAUP * 4.0b8. Relative mammalian EXTL1 are absent from the C. elegans and D. transferase activities are listed to the right of each EXT family member, (+) melanogaster genomes, suggesting that EXTL1 may not be activity detected, ( À ) no activity detected, (NT) not tested. aGlcNAcTI an essential component of the HS biosynthetic machinery. activity is measured by the transfer of GlcNAc from UDP-GlcNAc to Biochemical characterization of a soluble protein A fusion h linkage region oligosaccharides terminating in GlcA 1,3 [13]. aGlcNAcTII of EXTL1 demonstrated GlcNAc transferase activity with and hGlcATII are measured by the transfer of GlcNAc or GlcA, respectively, to N-acetylheparosan oligosaccharides [22]. m = mouse, acceptors that resemble intermediates in chain polymeriza- h = human, d = Drosophila. tion [43]. Assuming that protein expression levels were equivalent, the activity detected from EXTL1 was approximately one-tenth of the activity of a protein A-EXT1 does not appear to play a redundant role to EXT1 in HS chimera. Genetic experiments are needed to conclusively polymerization. Transfection of mammalian cells with demonstrate a role for EXTL1 in HS polymerization. EXT2 has very little effect on measured enzyme activities The role of EXTL2 is equally enigmatic. It was first and paradoxically decreases GlcA transferase activity in described as an a-GalNAc transferase [44]. The protein COS [28] and CHO cells (B. Crawford and J. Esko, was purified from fetal bovine serum [44], mouse mas- unpublished results). Additionally, transfection of EXT1- tocytoma [45], and human sarcoma conditioned medium deficient cell lines with EXT2 does not restore HS synthesis [46] based on its ability to transfer aGalNAc to a linkage [21,37]. region tetrasaccharide forming the product, GalNAca1, Simmons et al. [40] have used a yeast two-hybrid system 3GlcAh1,3Galh1,3Galh1,4Xyl-. Glycans with this structure to screen for proteins that interact with EXT1 and EXT2. have not been described in native proteoglycans, but have Tumor necrosis factor type I associated protein (TRAP-1) been detected in the oligosaccharides synthesized on h-D- was found to interact with the carboxyl terminus of EXT1 xylosides, which act as artificial primers of GAG synthesis and EXT2, and polypeptide GalNAc transferase T5 was [47–50]. When the enzyme was purified sufficiently to found to interact with EXT2. The biological relevance of yield peptide sequence, it was identified as EXTL2 [42]. these potential protein interactions is presently unclear as Based on homology to other EXTs, recombinant EXTL2 the GalNAc transferase has no direct connection to glyco- was tested for aGlcNAc transfer and found to have V 10% saminoglycan biosynthesis. Instead, it may be involved in activity compared to the transfer of aGalNAc [42,51]. posttranslational modification of the enzyme with O-linked Thus, the primary function of this enzyme in vivo remains oligosaccharides [41]. unclear. In summary, the evidence suggests that the HS copoly- EXTL3 appears to be a bifunctional aGlcNAc transferase, merase may be a complex containing EXT1 and 2, in which with the capacity to transfer GlcNAc to oligosaccharides that both subunits are essential for activity. Experimentally, represent the linkage region and to intermediates in chain B.M. Zak et al. / Biochimica et Biophysica Acta 1573 (2002) 346–355 351 polymerization [43]. These activities are significantly higher 6. EXT orthologs in C. elegans and D. melanogaster than transferase activities associated with EXTL1 and EXTL2 [42].TheC. elegans gene rib2 is most closely As mentioned previously, EXT orthologs have been homologous to vertebrate EXTL3 (Figs. 4 and 5) [51],in identified in C. elegans and D. melanogaster. These ortho- contrast to the idea that it represents an EXT2 ortholog. When logs can be organized by homology to their mammalian expressed in COS-1 cells and assayed for enzyme activity, counterparts based on sequence (Figs. 4 and 5). Drosophila rib2 like EXTL3 transferred aGlcNAc to acceptors that contains orthologs of the mammalian EXT1 (ttv), EXT2 mimic both initiation and polymerization reactions [51].A (dEXT2), and EXTL3 (dEXTL3). ttv was discovered through Drosophila ortholog also exists (dEXTL3). Together, these a screen for mutations that cause segment polarity defects data strongly suggest that EXTL3 is involved in HS polymer- [24] and subsequent studies showed that ttv was essential for ization, but genetic evidence is lacking. EXTL3 also has other hedgehog (Hh) diffusion [59]. Homozygous ttv third instar properties (a receptor for a h-cell regeneration factor, Reg larvae lack detectable HS [26], suggesting that Hh gradient [52], and a factor that can modulate NF-nB activity in formation during disc development depends on cell surface response to TNF-a [53]). The relationship of these other or matrix HS. The Drosophila genome also contains activities to HS synthesis or GlcNAc transferase activity is dEXT2, which shares many of the conserved domains of unclear. vertebrate EXT2, suggesting a related function. Direct One caveat of the biochemical studies described above enzymatic assay of activity for Drosophila dEXTL3 dem- concerns the nature of the assay and the substrates that are onstrates both initiation and polymerization GlcNAc activ- typically employed as acceptors. In practice, chain polymer- ities [60] similar to the behavior of vertebrate EXTL3 and C. ization is not measured directly, but rather inferred from elegans rib2. single sugar transfer reactions to oligosaccharides that con- The C. elegans rib1 and rib2 are most closely related to tain terminal GlcA (for GlcNAc transfer) or GlcNAc (for the mammalian EXT1 and EXTL3, respectively (Figs. 4 and GlcA transfer). These oligosaccharides are typically derived 5). As described above, soluble recombinant forms of the by chemical cleavage of Escherichia coli K5 capsular enzymes were expressed in COS-1 cells and assayed for polysaccharide, which has the identical structure of eukary- enzyme activity [51], revealing that rib2, the EXTL3 otic HS, except that it has none of the modifications ortholog, will transfer aGlcNAc to acceptors involved in (sulfation and IdoA). When assayed with this substrate, chain initiation and polymerization, but GlcA transferase the GlcA transfer reaction has less activity (much lower activity was not detected. rib1, which shows greatest Vmax/Km) compared to the GlcNAc transferase reaction homology to vertebrate EXT1, has no detectable activities [21,54]. This difference in activity is somewhat surprising when assayed under comparable conditions as rib2. Inspec- since copolymerization presumably requires both reactions tion of its structure indicates that rib1 lacks all of the to proceed with comparable efficiency. Prior studies have conserved DXD motifs except for element 8 (Fig. 4).It shown that GlcA transferase prefers substrates that contain also lacks a transmembrane domain and signal sequence, an N-sulfated penultimate GlcN unit, whereas the GlcNAc raising the question as to whether it plays any role in HS transferase does not exhibit any preference [54]. A con- biosynthesis. Future genetic characterization of rib1 and nection between sulfation and polymerization has also been rib2 mutants is needed to yield insight into the role of these observed in microsomal systems that catalyze the polymer- proteins in HS formation. ization of chains [55,56] and in cell culture systems where the overexpression of GlcNAc N-deacetylase/N-sulfotransferases increased the average size of HS chains that were 7. How do mutations in EXT1 or EXT2 relate to HME? produced [57]. Unfortunately, it has not yet been possible to polymerize a chain in a soluble enzyme system. The Although a genetic correlation exists between mutations polymerization reaction as measured in a microsomal sys- in EXT1 and EXT2 and HME, the mechanism by which tem is very sensitive to detergent [58], suggesting that the alterations in HS synthesis leads to ectopic bone growth is coupled reactions might require protein–protein interactions unknown. Several point mutations in EXT1 and EXT2 in in the plane of the membrane. The synergy observed HME patients affect transferase activities [36], and one can between EXT1 and EXT2, their colocalization in the Golgi, predict that many of the frameshift and nonsense mutations and the sensitivity of the system to detergent would seem to would have similar effects (Fig. 2).SinceHMEisa suggest that the physical interaction of these subunits is dominant disorder, apparently loss of only one allele is essential for polymerization to occur. Whether complexes sufficient to cause disease. Interestingly, some EXT1 muta- form with other relevant EXT family members is unclear. tions do not appear to completely abolish enzyme activity The coiled-coil motif in EXTL3 orthologs (element 3, Fig. [36]. Thus, one is left with the notion that partial loss of 4), in the so-called stem region that presumably separates only one allele may be sufficient to cause disease. Embryonic the catalytic domain from the membrane proximal seg- stem cells derived from EXT1 heterozygous mice exhibit ments, may mediate protein–protein interactions with itself one-half the enzyme activity and HS content as wild-type or other EXTs. stem cells, suggesting that the locus is haploinsufficient [23]. 352 B.M. Zak et al. / Biochimica et Biophysica Acta 1573 (2002) 346–355

Therefore, mild mutations in the gene may indeed lower the 8. Is there a role for FGF in HME? amount of HS expressed. Since exostoses appear to represent a benign outgrowth FGFs and FGF receptors (FGFRs) regulate endochondral from the growth plate, it would appear that subtle changes in bone development [68].Inmice,FGFR1,2and3are HS expression in this location could have profound effects expressed in prebone cartilage rudiments, but the expression on chondrocyte growth and/or differentiation. HS proteo- pattern changes when endochondral ossification begins. glycans (HSPGs) act as coreceptors for several growth FGFR3 is only expressed in resting cartilage [69] and signal- factors including fibroblast growth factors (FGFs) and bone ing through this receptor prevents chondrocyte proliferation morphogenic proteins (BMPs). Also, HSPGs affect signal- [69]. Constitutively active mutants of FGFR3 cause achon- ing by hedgehog proteins (including Ihh) and members of droplasia (dwarfism) in mice and humans [70–72], whereas the Wnt family of growth factors in an as yet undefined FGFR3 null mutations in mice cause skeletal overgrowth manner [61]. These observations have suggested that a lack [73]. Together, these findings suggest that a primary role for of HSPGs in HME may alter proper growth factor signaling FGFR3 may be to inhibit chondrocyte proliferation. leading to the aberrant bony growths [62]. The dependence of FGF signaling pathways on HS has A model often proposed suggests the involvement of the been the object of many studies [65]. Specific modifications Ihh/parathyroid hormone-related peptide (PTHrP) signaling of the HS chain are needed for appropriate interactions with pathway. This complex paracrine feedback loop has been the ligand and receptor (Fig. 3), including 2-O-sulfation of shown to control the rate of growth plate chondrocyte iduronic acid and 6-O-sulfation of N-sulfated glucosamine proliferation and differentiation, and it appears to involve residues [74–76]. Thus, mutations in EXT1 and EXT2 one or more BMPs [2,63]. The growth plate at the end of could decrease HS synthesis, leading to diminished FGF long bones is surrounded by perichondrium and is com- signaling and abnormal chondrocyte proliferation at sites of prised of proliferating chondrocytes, pre-hypertrophic exostosis formation. Previous studies of human chondro- chondrocytes, and hypertrophic chondrocytes (Fig. 1). cytes from HME patients have found abnormalities in Hypertrophic chondrocytes undergo apoptosis and are collagen production, cytoskeletal elements and chondrocyte resorbed, giving way to trabecular bone formation by differentiation [77], possibly indicative of inappropriate osteoblasts that infiltrate from the vascular beds. Chondro- progression through the cell cycle. If correct, the defect cytes in the growth plate committed to hypertrophy secrete must be rather focal since endochondral bone development Ihh, which is thought to diffuse and bind to Ihh receptors continues to occur in HME patients. With the exception of located on cells in the perichondrium. Signaling results in exostoses, no other symptoms have been described in HME the release of PTHrP, which acts on proliferating and patients, suggesting that specific cell types in the growth prehypertrophic chondrocytes to maintain the proliferative plate may be exquisitely sensitive to HS levels. state and decrease the terminal commitment to hypertrophy Animal models are being used to gain more insight into [2]. Since HS can bind hedgehog in the cellular micro- this problem. Mice with inactivated alleles of EXT1 fail to environment [64], a decrease in HS could affect this develop past E7 due to defective gastrulation [23]. However, signaling pathway. HS may act as a sink in the extrac- heterozygous mice, which presumably would exhibit the ellular matrix, regulate the rate of Ihh diffusion [24,59],or same state of deficiency as HME patients, only rarely may be directly involved as a coreceptor on the cell develop obvious exostoses. This difference may highlight surface, analogous to the role of HS in FGF signaling the variation in bone development in humans and animal [65]. In either case, perturbing HS levels might prevent models: the mouse growth plate, unlike the human, does not Ihh diffusion or proper signaling, thus interrupting the close once maturity is achieved, and environmental factors pathway. Notably, EXT1 and EXT2 mRNA expression (e.g., loading) differ significantly between mice and coincides with Ihh expression in the prehypertrophic humans. The degree of penetrance (clinically evident exo- chondrocytes [66]. Also, deletion of EXT1 in mice results stosis formation) also varies dramatically across a kindred in failure to immobilize Ihh in the cell matrix, thus carrying a mutated EXT gene [3], suggesting that genetic affecting mesoderm differentiation in early development modifiers may exist as well. Recent studies of EXT1- [23]. deficient mice shows that exostoses arise at a higher If hedgehog signaling were interrupted as the model frequency in a C57B1/6 background, consistent with this suggests, the downstream effect would be a lack of PTHrP idea (D. Stickens, B. Zak, G. Evans, D. Wells, J. Esko, and persistent terminal differentiation of prehypertrophic unpublished results). chondrocytes. Thus, the absence of HS would seem to favor Although information about HS synthesis is rapidly differentiation rather than proliferation. The primary pheno- emerging, the data is raising many questions. Why does type in PTHrP null mice is premature terminal differentia- HME only associate with mutations in EXT1 and EXT2, tion of chondrocytes into hypertrophic chondrocytes, which and not other members of the EXT gene family? What is the results in dwarfism [67]. Thus, it is unclear how this model structure and function of the EXT3 locus? 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