Heparan sulfate in skeletal development, growth, and ...

Heparan Sulfate in Skeletal Development, Growth, and Pathology:

The Case of Hereditary Multiple Exostoses

Julianne Huegel, Federica Sgariglia, Motomi Enomoto-Iwamoto, Eiki Koyama,

John P. Dormans and Maurizio Pacifici

Translational Research Program in Pediatric Orthopaedics, Division of Orthopaedic Surgery, The

Children’s Hospital of Philadelphia, Philadelphia, PA 19104

Running title: Heparan sulfate in development and skeletal dysplasia

Key words: Heparan sulfate; cell surface proteoglycans; growth plate; signaling proteins; ectopic

cartilage; Hereditary Multiple Exostoses

• Heparan sulfate proteoglycans and a number of growth factors they control are expressed

and active in the growth plate and surrounding perichondrium

• Congenital mutations in HS-synthesizing and modifying enzymes and HSPG expression

cause severe skeletal and craniofacial phenotypes

• Recent developments in understanding Hereditary Multiple Exostoses (HME) suggest

that aberrant growth factor signaling plays a major role in exostosis initiation and growth

Funding: NIH RC1AR058382 and R01AR061758.

Correspondence to:

Julianne Huegel

[email protected]

Abramson Research Center Room 904

3615 Civic Center Blvd

Philadelphia, PA 19104

267-425-2077

Review Developmental DynamicsDOI 10.1002/dvdy.24010

Accepted Articles are accepted, unedited articles for future issues, temporarily published onlinein advance of the final edited version.© 2013 Wiley Periodicals, Inc.Received: May 14, 2013; Revised: Jun 21, 2013; Accepted: Jun 22, 2013

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Abstract

Heparan sulfate (HS) is an essential component of cell surface and matrix-associated

proteoglycans (HSPGs). Due to their sulfation patterns, the HS chains interact with numerous

signaling proteins and regulate their distribution and activity on target cells. Many of these

proteins, including bone morphogenetic protein family members, are expressed in the growth

plate of developing skeletal elements, and several skeletal phenotypes are caused by mutations in

HS-synthesizing and modifying enzymes. The disease we discuss here is Hereditary Multiple

Exostoses (HME), a disorder caused by mutations in HS synthesizing enzymes EXT1 and EXT2,

leading to HS deficiency. The exostoses are benign cartilaginous-bony outgrowths, form next to

growth plates, can cause growth retardation and deformities, chronic pain and impaired motion,

and progress to malignancy in 2-5% of patients. We describe recent advancements on HME

pathogenesis and exostosis formation deriving from studies that have determined distribution,

activities and roles of signaling proteins in wild type and HS-deficient cells and tissues. Aberrant

distribution of signaling factors combined with aberrant responsiveness of target cells to those

same factors appear to be a major culprit in exostosis formation. Insights from these studies

suggest plausible and cogent ideas about how HME could be treated in the future.

Introduction

Heparan sulfate (HS) is a versatile and essential component of cell surface and matrix-

associated proteoglycans (HSPGs) (Bernfield et al., 1999). Due to their specific chemistry and

highly negative charge, the HS chains can bind to a number of proteins, including growth factors,

signaling proteins, integral membrane receptors, chemokines, and extracellular matrix proteins

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(Hacker et al., 2005; Bishop et al., 2007). Studies have indicated that in particular, the HS chains

endow the proteoglycans with the key ability to regulate the distribution and availability of the

growth and signaling proteins and their respective interactions, function and bioactivity on target

cells (Bernfield et al., 1999; Lin, 2004; Umulis et al., 2009). Because many of these proteins,

including members of the hedgehog, bone morphogenetic protein (BMP), fibroblast growth

factor (FGF) and Wnt families, are expressed in the growth plate (Kronenberg, 2003), it is

evident that HS influences many important processes in skeletogenesis and skeletal growth and

morphogenesis. Mice deficient in Ext1, an essential HS polymerizing enzyme, show altered

patterns of Indian hedgehog (Ihh) diffusion, increasing the range of signaling and resulting in

significant changes in growth plate morphology (Koziel et al., 2004; Hilton et al., 2005). N-

sulfotransferase 1 (Ndst1) is another critical enzyme in HS assembly, establishing tissue-specific

sulfation patterns by replacing acetyl groups with sulfate modifications. Loss of Ndst1 also

causes severe changes in Hedgehog distribution and growth plate function (Yasuda et al., 2010).

The significance of HS chains and HSPGs in skeletogenesis is reiterated by the fact that there are

a number of skeletal and craniofacial phenotypes related to genetic mutations in HS-synthesizing

and modifying enzymes and in HSPG expression (Bishop et al., 2007). A case in point is

Hereditary Multiple Exostoses (HME), a pediatric autosomal-dominant disorder during which

cartilage outgrowths called exostoses form next to the growth plate of skeletal elements such as

long bones, ribs and pelvis and protrude into the adjacent perichondrium and neighboring tissues

(Porter and Simpson, 1999; Hecht et al., 2005). In turn, the exostoses can cause skeletal

deformities, chronic pain and early onset osteoarthritis, among a variety of other pathological

events (Dormans, 2005; Jones, 2011). HME –also called Multiple Osteochondroma (MO) or

Multiple Hereditary Exostoses (MHE) - is estimated to affect 1 in 50,000 children, and is almost

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100% penetrant. The majority of cases of HME are caused by loss-of-function mutations in

EXT1 and EXT2, which encode for Golgi-associated glycosyltransferases. After a number of

other enzymes create a linkage tetrasaccharide on a serine residue of the core protein, Ext1 and

Ext2 are collectively responsible for polymerization of the HS chain (Esko and Selleck, 2002).

The number and type of EXT mutations are many and lead to varying degrees of HS deficiency

(Jennes et al., 2009). What is not fully understood is whether and how the HS deficiency leads to

exostosis formation, whether it can account for all the various symptoms and complications of

the syndrome, and what could be done therapeutically to treat or reverse it. Several recent studies

reported by this and other groups have aimed to decipher the possible roles of HS deficiency on

exostosis formation and HME pathogenesis in general, and have also provided clues on the

genesis of other complications of the disease. In so doing, these studies have contributed to

further understand also the normal roles of HS and HSPGs in skeletal development and growth.

This review article summarizes the studies and provides a critical assessment of current findings

and underlying hypotheses.

HME Pathology and Population Studies

HME presents with predominantly orthopedic manifestations. The exostoses are the most

evident trait of the syndrome from which it takes its name, and are composed of a growth plate-

like cartilaginous cap overlaying a bony base (Fig. 1) (Jones, 2011). Because of their location,

size, number and interactions, the exostoses can cause: compression of nerves, blood vessels, and

tendons with consequent pain and impairment of motion; skeletal deformities and growth

retardation by interfering with normal growth plate function; and early onset osteoarthritis (Fig 2,

A-B). Patients with HME often show slightly shortened stature, bowing and shortening of

forearm elements, changes in angulations of the knee and fingers, and limb-length inequalities.

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Previous hypotheses suggested that exostoses growing along the bones of the forearm affected

their shape and introduced a “steal phenomenon” that caused bone shortening. However, recent

experiments show no change in overall bone volume and no correlation between forearm

shortening and the presence of an osteochondroma. This work indicates that HS loss may be

important for directing the ratio between peripheral and longitudinal growth (Jones et al., 2013).

Exostoses can also sometimes occur in craniofacial elements such as the mandible (Ruiz and

Lara, 2012). Chronic pain and restrictions in activity are common consequences of the disease.

A recent national cohort study in the Netherlands found a significantly lower outcome in

physical functioning and resultant role limitations, social functioning, vitality, pain, and general

health perception in patients with HME compared with three separate reference groups (Goud et

al., 2012). Unfortunately, as these symptoms and difficulties progress, patients must undergo

surgery, with 70% of patients undergoing an operation by the time they reach 18 years of age. In

children, surgery can be dangerous as it can cause irreversible damage to the adjacent growth

plate. Although no new exostoses form after puberty when the growth plates close, existing

exostoses can continue to grow and cause further pain and complications, leading to a surgical

rate of 67% in adults (Goud et al., 2012).

One potentially serious complication of HME is the transformation of benign exostoses to

malignant chondrosarcomas, a life-threatening progression of this syndrome that occurs in about

2 to 5% of patients (Fig. 2, C-D). For patients whose exostoses undergo malignant degeneration,

the mean age at time of diagnoses- 35 years old- is younger than that for general HME patients

(Bjornsson et al., 1998). Another serious complication of HME is the formation of exostoses on

the surface of the vertebrae, which can compress the spinal cord or nerve roots (Fig. 2, E-F).

Spinal cord compression due to exostoses has been seen to manifest as motor or sensory deficits

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including gait disturbance, weakness or numbness, amplified reflex responses and spasticity, and

incontinence (Zaijun et al., 2011; Bari et al., 2012). Vertebral exostoses can also impinge on the

esophagus, impairing normal swallowing (Perrone, 1967). Additionally, exostoses on both

vertebral as well as costal surfaces can interfere with lung function and can cause spontaneous

hemothorax, pneumothorax, and pericardial effusion, typically leading to immediate surgical

intervention (Assefa et al., 2011).

It is interesting that, although HS production in HME patients is likely to decrease in all

tissues, the only truly apparent and diagnostic phenotype are the exostoses themselves.

However, as indicated above, HME patients can suffer from a variety of less obvious problems

that can include wound healing delay, learning disabilities, dental problems and others (Hosalkar

et al., 2007; Wiweger et al., 2012). This clinical and biomedical complexity, though still not fully

understood and clear, certainly relates well with the fact that HSPGs regulate numerous, if not

most, physiologic processes in the growing and adult organism. Thus, a generalized deficiency

in HS could, and should, have broad and widespread consequences the severity of which would

reflect the specific importance and roles that HS and HSPG have in different tissues and organs

and distinct biological contexts and processes (Bishop et al., 2007).

HS Synthesis and HME Genetics

Heparan sulfate (HS) constitutes the glycosaminoglycan moiety of such cell surface and

matrix proteoglycans as syndecans, glypicans and perlecan (Bernfield et al., 1999). The HS

chains are composed of repeating D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine

(GlcNAc) residues that are assembled into linear polysaccharides in a biosynthetic process

including initiation, elongation, and modification steps. Synthesis is initiated in the endoplasmic

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reticulum by the addition of a xylose to a serine residue in the proteoglycans core protein by

xylosyltransferase. The linker tetrasaccharide is completed in the Golgi apparatus by the

addition of two galactose residues and a glucuronic acid, carried out by galactyltransferases I and

II and glucuronosyltransferase I, respectively. HS elongation is then performed in a stepwise

manner by the Golgi-associated heterodimer complexes of EXT1 and EXT2 that are ubiquitously

expressed type-II transmembrane glycoproteins (Esko and Selleck, 2002). Both proteins and

expression of both alleles are required to produce and maintain normal physiologic HS levels and

homeostasis. During assembly, the nascent chains undergo extensive modifications that include

N-deacetylation/N-sulfation, epimerization, and O-sulfation, requiring a number of enzymatic

reactions and providing a range of HS structural variability between and within tissues that

confer structural and protein-binding specificity (Bulow and Hobert, 2006).

EXT1 and EXT2, originally considered to be tumor-suppressor genes, are part of a family

of EXT proteins that also includes three EXTL (exostosin-like) members. The latter proteins

have roles in HS synthesis as well, including addition of the first carbohydrate residue to the

tetrasaccharide linkage (Kitagawa et al., 1999; Kim et al., 2001; Busse et al., 2007; Okada et al.,

2010). Mutations in either EXT1 or EXT2 are responsible for about 90% of HME cases, with the

majority (~65%) seen in EXT1, the protein believed to have enzymatic activity within the

EXT1/EXT2 complex (EXT2 plays a structural role and may also have chaperone functions)

(McCormick et al., 1998). To date, over 650 unique mutations have been found in these two

genes, most of which are nonsense, frame shift, or splice-site mutations (Jennes et al., 2009;

Ciaverella et al., 2012). These inactivating mutations result in premature termination of EXT

proteins, causing premature degradation and nearly complete loss of function (Wuyts and Van

Hul, 2000). Very recently, a family of patients negative for EXT1 or EXT2 mutations was

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shown to have intronic rearrangement within the first intron of EXT1, suggesting a possible

mechanism for HME that would not be detected with conventional diagnostic techniques

(Waaijer et al., 2013).

Several studies have been carried out with the goal of establishing a relationship between

genotype and phenotype, but no firm relationship has been obtained yet. Interestingly, there is a

wide spectrum of variation in disease presentation within families and between individuals with

the same mutation, suggesting additional genetic, hormonal, or environmental influences in

phenotype specification and severity. However, some general correlations have been determined

in a number of HME population studies. Due to its enzymatic function, EXT1 mutations are

correlated to more severe presentations of the disease (Francannet et al., 2001; Porter et al.,

2004). Additionally, male patients typically show a more severe clinical presentation, which is

hypothesized to be caused by a later growth plate closure, allowing more time for exostosis

formation. Accordingly, patients with a greater number of exostoses (>20) usually have more

disabilities and deformities (Alvarez et al., 2006; Pedrini et al., 2011).

Animal Models of HME

The development of skeletal elements in the skull, trunk and limbs initiates with the

formation of ecto-mesenchymal and mesenchymal cell condensations at prescribed times and

locations. Several condensations located in the skull region undergo intramembranous

ossification and produce skeletal elements such as the calvaria and jaw. The remaining and more

numerous condensations undergo endochondral ossification during which the condensed

mesenchymal cells differentiate into chondrocytes and become organized into growth plates

closely surrounded by perichondrial tissues. The growth plate chondrocytes proliferate, undergo

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hypertrophy and are replaced by endochondral bone and marrow, thus sustaining skeletal growth

until the end of puberty and producing definitive skeletal elements throughout the body that

include ribs, vertebrae and long bones (Kronenberg, 2003). As pointed out above, the growth

chondrocytes express both cell surface and matrix-associated HSPGs including syndecan-3,

glypicans-5 and perlecan that are required for function, including regulation of growth factor

distribution and action and relationships of growth plate with surrounding tissues and most

importantly perichondrium (Arikawa-Hirasawa et al., 1999; Viviano et al., 2005; Habuchi et al.,

2007; Yasuda et al., 2010). As pointed out above, the exostoses exhibit an intriguing growth

plate-like organization in which their main axis of elongation is at about 90° angle compared to

that of the adjacent native growth plate. Understanding the role of HS and HSPGs in the growth

plate is thus critical for elucidating the processes by which exostoses can form and grow next to,

but never within, the growth plates and protrude into perichondrium and surrounding tissues.

A number of zebrafish models have been developed to assess changes in HSPGs and their

affect on cartilage development. Dackel (dak/ext2) mutants lack HS chains and showed a severe

cartilage phenotype, with disorganized cells that failed to flatten and intercalate into stacks and

lost expression of collagen10a1. However, these cells are able to express markers of both early

chondrocytes as well as perichondrium, suggesting that they are capable of forming components

of an exostosis. Interestingly, pinscher mutants (pic/slc35b2) exhibit an even more severe

phenotype, with thickened perichondrium and reduced matrix deposition. Pic mutants are unable

to transport sulphate donors into the Golgi and produce sulphate-less GAGs (including keratin

sulfate and chondroitin sulfate). This demonstrates the necessity of GAG chains in maintaining

proper chondrocyte and perichondrial cell phenotype and morphology (Wiweger et al., 2011).

Transplant experiments show that most dak mutant cells can be rescued by surrounding WT

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cells, taking on a proper flat and intercalated phenotype. However, these cells occasionally grow

out from the edge of developing cartilage, oriented perpendicular to the WT cells; this growth

mimics developing exostoses in humans that consistently form along the side of the growth plate,

extending into the perichondrium. This model suggests that the location of mutant cells within a

cartilage element may dictate their responsiveness to changes in growth factor distribution or

ability to contact neighboring WT cells (Clement et al., 2008).

Over the last decade or so, several mammalian models of HME have also been developed

to uncover and understand exostosis pathogenesis as well as the roles of HS in normal

mammalian skeletal development (Table 1). Ext1-null mice are embryonic lethal at E8.5,

indicating the essential importance of HSPGs for sustaining and regulating critical

developmental stages (Lin et al., 2000). Indeed, as early as day E10.5, Ext1 expression becomes

conspicuous in the developing limb buds (Stickens et al., 2000). Interestingly, mice

heterozygous-null for Ext1- originally created as a model of HME- were found to be largely

normal and to lack an obvious skeletal phenotype (Hilton et al., 2005). However, upon closer

examination their growth plates exhibited subtle changes including an increase in collagen II

expression, a decrease in collagen X expression, and increased BrdU incorporation in the

proliferative zone, collectively suggesting altered chondrocyte proliferation and maturation

patterns. Because the overall length of limb bones in adult mutant mice were similar to those in

wild-type mice, it was proposed that the above effects of Ext1 and HS deficiency would be

compensated over time (Hilton et al., 2005). Mice lacking Ext2 were also found to be embryonic

lethal and the embryos actually failed to undergo gastrulation, likely as a result of a disruption of

several signaling pathways critical to mesoderm development and formation of extra-embryonic

structures (Stickens et al., 2005). Heterozygous null Ext2+/-

mice showed growth plate

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disturbances similar to those in their Ext1+/-

counterparts, with a disorganized proliferative zone

and changes in Ihh expression domains. Of significant interest was the fact that a small

percentage of the Ext2+/-

mice did develop small exostosis-like outgrowths along the ribs near

the costochondral junction, supporting the idea that a partial loss of Ext function and HS

production may be sufficient for exostosis formation (Stickens et al., 2005).

Intriguingly, however, the HS levels observed in surgical retrieval specimens of human

exostosis cartilage were found to be very low and apparently lower than the 50% levels

presumably caused by a single heterozygous EXT mutation (Hecht et al., 2005). Loss-of-

heterozygosity could explain such low HS levels, but LOH has actually been documented in only

a few cases (Bovee et al., 1999) though it may have been underestimated originally because of

the mixed nature of cells within exostoses (Jones, 2011). Thus, it is possible that secondary

mutations in other HS-related genes, action of HS degrading enzymes such as heparanase

(Trebicz-Geffen et al., 2008), or other mechanisms may play roles in reducing HS levels further.

To test these ideas, we recently created and analyzed double heterozygous Ext1+/-

;Ext2+/-

mice

(double hets) (Zak et al., 2011). Because a full complement of Ext proteins is needed for normal

levels of HS synthesis, it was predicted that these compound mice would have a significant

decrease in HS levels (about 25%) compared to single heterozygous mutant (about 50%) and

wild types (100%). Indeed, we found that in addition to developing rib exostosis-like outgrowths

as seen in single Ext2+/-

heterozygous mice, almost half of the compound Ext1+/-

;Ext2+/-

mutants

exhibited stereotypic exostoses next to the growth plates of long bones and deformations in their

pelvis. The exostoses displayed a growth plate-like arrangement of chondrocyte zones with

typical expression patterns of zone markers such as Sox9, Col2, Indian hedgehog (Ihh) and

Col10. The HS levels in growth plates and exostoses of these mice were significantly reduced

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compared to those in wild-type littermates as indicated by immunostaining, and the HS chains

that were produced by cultured compound mutant chondrocytes were shorter than those

produced by WT chondrocytes. We also found evidence of changes in chondrocyte response to

growth factors important for normal skeletal development (Zak et al., 2011). These phenotypes

strongly support the idea that significant, but not necessarily complete, loss of Ext expression and

HS production is sufficient for formation of multiple stereotypic exostoses. Thus, it appears that

exostosis initiation and frequency are inversely related to overall Ext expression and function and

increase almost linearly in single het vs double hets vs WT. This finding may explain the broad

range of phenotype severities seen in HME patients in whom EXT protein function and HS

production may vary depending on the nature of the EXT mutation and other concurrent genetic

modulations.

Other mouse models of HME have provided additional information about exostosis

pathogenesis and the role of HS in normal growth plate development. A hypomorphic, gene-

trapped Ext1Gt/Gt

mouse line expressing a truncated form of Ext1 displayed shortened skeletal

elements and fused vertebrae at E15.5 (Koziel et al., 2004). These changes were accompanied

by increased distribution and signaling of Ihh within the growth plate and delayed hypertrophic

differentiation, reiterating the idea that Ext expression and HS production are needed to regulate

action and distribution of signaling proteins within the growth plate. Postnatal, chimeric and

conditional inactivation of Ext1 in chondrocytes in mouse growth plate cartilage was found to

result in the formation of numerous exostoses throughout the appendicular skeleton, indicating

that a full Ext loss leads to aggressive exostosis formation (Jones et al., 2010). Conditional

deletion of Ext1 from limb mesenchyme utilizing Prx1-Cre transgenic mice caused severe limb

skeletal defects including joint fusion and variable numbers of developing digits. These effects

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were at least partially attributed to broader and non-physiologic distribution of bone

morphogenetic proteins (BMPs) and BMP signaling activity on targets (Matsumoto et al., 2010).

HS and Signaling Proteins

The above studies clearly indicate that HS and HSPGs are critical to determine and

supervise the distribution of signaling proteins, their range of action, and the effects exerted on

their targets. They also make it plain that HS deficiencies can have profound repercussions on

those mechanisms and could directly contribute to pathologic changes. Interactions between

growth factors and HS have been thoroughly studied for many years in a multitude of organ

systems, cell types, and in vivo scenarios. One major overall insight stemming from all this

work is that however, those interactions and relationships are contextually dependent and can

specifically vary depending on the developmental stage of the tissue or organ, the types of cells

involved, the presence of other systemic and local factors, etc. (Rider and Mulloy, 2010). One

well-characterized HS interaction is the one with members of the fibroblast growth factor (FGF)

family. In this relationship, HS is most often required for effective signal transduction as it acts

as a FGF co-receptor (Hacker et al., 2005). Early stage Ext2 null embryos do not in fact respond

to FGF signaling, conceivably an explanation for their early embryonic lethal phenotype

(Shimokawa et al., 2011). Fibroblasts isolated from Ext1Gt/Gt

mice show decreased amounts of

cell surface HS as expected and, in addition, exhibit a reduced signaling response to FGF2 and

consequent decrease in proliferation after growth factor stimulation (Osterholm et al., 2009).

Hedgehog signaling is another important regulator of axial and limb skeletal

development. Since primary cilia are largely responsible for this signaling pathway, there is

much evidence that they are also essential for skeletal development (Huangfu and Anderson,

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2005). Conditional deletion of the primary cilium component Kif3a in chondrocytes resulted in

both limb and cranial skeletal abnormalities, including exostosis-like cartilaginous masses

forming near the growth plate. We showed that unexpectedly, the mutant Kif3a-null growth

plates displayed a drastic reduction in HSPG expression (and in particular syndecan-3 and

perlecan) and a concomitant broader distribution of Ihh within the growth plate and all along the

adjacent perichondrium. This was associated, and probably directly caused, ectopic hedgehog

signaling all along the perichondrium, ectopic chondrogenesis and then local formation of

ectopic exostosis-like cartilaginous masses, suggesting a role for hedgehog proteins and

defective Kif3a-related mechanisms in exostosis formation as well (Koyama et al., 2007).

Primary cilia are also at least partially responsible for organizing chondrocytes into their growth-

plate specific columnar structures. Substantiating the role of the primary cilia in HME, this

polarity has been found to be lost in human osteochondroma cells, suggesting significant changes

in cell adhesion and motility and related cell-matrix and cell-cell communication mechanisms

(de Andrea et al., 2010).

Bone morphogenetic proteins (BMPs), a subfamily of the TGFβ superfamily of secreted

proteins, also regulate a number of stages in skeletal development. Depletion of HS from the

surface of C2C12 cells enhanced BMP2 bioactivity while inhibiting its internalization (Jiao et

al., 2007). Disruption of HS chains with exogenous heparinase also increased pSmad1/5/8

signaling in human mesenchymal stem cells (Manton et al., 2007). Several members of both the

BMP and FGF families are expressed in growth plate and/or perichondrium and have been

shown to be part of interactive loops regulating Ihh and PTHrP expression and overall growth

plate activities (Zou et al., 1997; Pathi et al., 1999). Misexpression of Ihh causes changes in the

expression of pro-chondrogenic BMPs as well as their anti-chondrogenic antagonists Noggin and

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Chordin, altering the differentiation of cells in the growth plate as well as the bordering

perichondrium (Pathi et al., 1999).

During mesenchymal condensation in early skeletal development, Wnt ligands induce an

accumulation of β-catenin in the cytoplasm. In turn, β-catenin translocates to the nucleus and

binds to transcription factors, controlling downstream gene transcription to determine an

osteogenic lineage. Ablation of β-catenin initiates cartilage formation while transgenic

overexpression of Wnt signaling promotes osteoblast differentiation (Day et al., 2005). Wnt

ligands also bind tightly to HSPGs at the cell surface, which act to maintain the solubility of

hydrophobic Wnt and stabilize its activity (Fuerer et al., 2010; Kikuchi et al., 2011). The Wnt/β-

catenin signaling pathway also plays important roles in cartilage maintenance and growth plate

function (Yuasa et al., 2009). Interestingly, postnatal β-catenin ablation in cartilage causes

exostosis-like cartilage masses to form off of the growth plate as well as in the periosteum.

Additionally, cartilage tissue collected from osteochondromas of HME patients showed little to

no β-catenin positive cells, potentially extending the lifespan of exostosis chondrocytes (Cantley

et al., 2012). In sum, the above and related studies have provided clear evidence for essential

roles that HS-dependent mechanisms have to orchestrate and coordinate the different functions

and processes within the growth plate as they relate to the function and action of signaling

proteins and factors (Minina et al., 2002).

Signaling Proteins and Exostosis Initiation

The above studies hint to the possibility that a decrease in Ext expression/HS levels and a

concomitant increase in signaling protein distribution/action may initiate ectopic chondrogenesis

and exostosis formation. To test these possibilities more directly, we recently resorted to in vitro

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studies in which we isolated mouse embryo limb mesenchymal cells and grew them in

chondrogenic conditions in micromass culture (Huegel et al., 2013). Cultures were maintained in

absence or presence of Surfen, a small molecule heparan sulfate antagonist (Schuksz et al.,

2008). Indeed, Surfen treatment caused a dose-dependent increase in the number of alcian blue-

positive cartilaginous nodules (Fig 3, A-C). Interestingly, the nodules in Surfen-treated cultures

had lost their typical round, individual morphology and fused with one another, indicating that

the nodules were unable to maintain their border and circumscribed perimeter normally occupied

by flat perichondrium-like cells (Ahrens et al., 1979). Isolated RNA from the micromass cultures

was subjected to qPCR. Surfen treatment clearly caused a significant increase in expression of

characteristic chondrogenic genes including aggrecan, collagen type II, Runx2 and Sox9,

confirming that loss of HS function stimulates chondrogenic differentiation of progenitor cells

(Fig 3D). The same effects were observed when Ext1fl/fl

limb bud cells in micromass cultures

were treated with a Cre-expressing adenovirus, thus reducing Ext expression and HS production;

the effects and responses were abrogated and reduced by addition of the BMP antagonist Noggin

(not shown) (see (Paine-Saunders et al., 2002)).

As discussed previously, HS mediates the interaction of chondrogenic factors with target

cells, limiting their availability, distribution and action (Bernfield et al., 1992; Lin, 2004). Thus,

we reasoned that Surfen could have stimulated chondrogenesis by increasing availability and

action by signaling proteins. For proof-of-principle, we chose BMP signaling since it has strong

pro-chondrogenic activity (Weston et al., 2000). Indeed, BMP signaling -as indicated by

pSmad1/5/8 phosphorylation levels- was greatly enhanced by Surfen treatment of limb

mesenchymal micromass cultures and its increase preceded the increase in cartilage nodule

formation. These responses were abrogated by addition of the BMP antagonist Noggin. Reporter

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plasmid assays confirmed that cells treated with Surfen were more sensitive to both endogenous

and exogenous BMP2 (Huegel et al., 2013). To verify these observation in vivo, we

conditionally deleted Ext1 in perichondrium flanking the upper portions of the growth plate in

Ext1fl/fl

;Gdf5Cre mouse embryos. We found that many mutant perichondrial cells became

positive for pSmad1/5/8 signaling in the nucleus, while most cells in controls were negative.

Limb explants treated with Surfen showed similar changes in signaling (Fig 4, C-D). This

ectopic BMP signaling was followed by formation of ectopic cartilage within perichondrium (Fig

4, A-B). Collectively, these studies suggest that Ext1 and HS are critical regulators of the

perichondrium phenotype -allowing it to act as an anti-chondrogenic border around the growth

plate- and are also essential to restrain and contain cartilage growth. In an Ext1- and HS-deficient

environment, BMP signaling would be enhanced and mis-regulated, leading to abnormal

behavior and growth of chondrocytes and enhanced chondrogenic response of perichondrium.

Other Modifiers of HS

Another interesting aspect of the biology of HS chains and HSPGs is that the chains can

be modified extracellularly by the action of enzymes such as heparanase (HPSE) and sulfatases

(Ai et al., 2007; Fux et al., 2009). Indeed, increased expression of HPSE has been described in

exostosis tissue (Trebicz-Geffen et al., 2008; Yang et al., 2010). Responsible for cleaving HS

chains into small fragments, the endoglucuronidase HPSE has pro-proliferative activity and is

implicated in a range of cancers by assisting in the structural remodeling of the extracellular

matrix during cellular invasion and release of growth factors (Ilan et al., 2006). This invasive

characteristic is also seen in tooth development when dental follicular cells penetrate through the

epithelial root sheath to differentiate into cementoblasts, which coordinates with up-regulated

HPSE expression (Hirata and Nakamura, 2006). Additionally, increased levels of HPSE are

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present at the chondro-osseous junction in developing bones, suggesting that HPSE plays a role

in late chondrocyte differentiation during endochondral ossification (Brown et al., 2008). It also

suggests that regulated degradation of HS chains could promote factor release and signaling

during other key points of the bone formation process.

Sulfatases modify extracellular HS chains by selectively cleaving 6-O-sulfate groups,

altering the structural pattern and heterogeneity and impacting interaction with signaling

molecules (Ai et al., 2007). The idea that the HS sulfation pattern can be fluid after synthesis

provides another level of cell or tissue-specific regulation of HSPG function. During Xenopus

embryogenesis, Sulf1 enzyme expression is highly regulated, and acts negatively on both BMP

and FGF signaling, allowing for the formation of morphogen gradients vital to axis patterning,

somitogenesis, and other key developmental processes (Freeman et al., 2008). More recently,

articular cartilage in Sulf1-/-

mice was shown to display a spontaneous osteoarthritis phenotype

with decreased BMP and upregulated FGF signaling (Otsuki et al., 2010). Evidently, Sulf

expression is also necessary for maintaining articular cartilage homeostasis by regulating

signaling between chondrocytes.

Concluding Remarks and Future Directions

The above studies have provided major new insights into the interplays amongst HS

biology, growth plate function and exostosis formation as well as the complexities and subtleties

of these mechanisms. It appears plausible and likely therefore that exostosis formation is the

ultimate outcome of changes in HS-dependent signaling pathways, including BMP and Ihh

pathways, converging to create pro-chondrogenic responses and proliferative environments along

the border of growth plates. Exostosis formation would thus result from increased distribution of

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these factors, increased responsiveness of the cells to these factors, and reduced capacity of

growth plate cartilage and/or perichondrium to remain distinct and phenotypically stable.

Other aspects of exostosis biology remain to be clarified. For instance, exostoses have

been shown to often consist of a mix of HS-expressing and HS-null cells (Jones, 2011),

suggesting that the cell population within each exostosis may be varied and phenotypically

diverse and may have varying developmental origins. With regard to the latter, different

hypotheses have been put forward over the years regarding the origin of exostosis forming cells.

One hypothesis is that the cells represent borderline growth plate chondrocytes that would

misbehave as a result of Ext/HS deficiency and lack of Ext tumor suppressor function, thus

behaving as benign tumor cells and solely responsible for exostosis formation (Fig 5A). A

second hypothesis is that the cells would originate in perichondrium and would be progenitors in

nature (Fig 5B). The cells would lose their fibrogenic/progenitor phenotype and become

reassigned to the chondrogenic lineage, and would undergo de novo chondrogenesis and give

rise to the exostoses. The third possibility is that the cells would originate in the groove of

Ranvier, a specialized region of perichondrium near the epiphysis which is rich in progenitor

cells and may contain a specialized stem cell niche (Fig 5C). Data in favor of, and against, these

various hypotheses exist in the literature, thus requiring further work and more refined tools to be

clarified and defined in a conclusive manner. As pointed out above, it may be that these

hypotheses are not mutually exclusive and that exostosis formation could co-involve growth

plate and perichondrial/groove cells. Their common denominators would be: enhanced

responsiveness to growth factors; greater availability of those factors to act; increased enzymatic

degradation of existing HS chains by heparanase; and inclusion of a mixed cell population (Fig

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5D). Based on our studies, we believe the contribution of perichondrial/groove cells may be

preponderant.

An equally critical issue to be addressed in the future is whether and how exostosis

formation could be prevented or even reversed therapeutically. As discussed above, symptomatic

exostoses are removed surgically at present, but surgery is dangerous, can have complications

and cannot be used to remove each exostosis since the number of exostoses is usually very high

in each patient. Hence, biological solutions are needed to aid surgery. Given that the exostoses

likely represent a de novo chondrogenic process involving growth plate chondrocytes,

perichondrial cells or both, it is plausible to assume that anti-chondrogenic tools could be

effective to block exostosis formation. Powerful anti-chondrogenic mechanisms include the

retinoid pathway, Wnt signaling, and BMP antagonists such as Noggin and Gremlin. We have

recently used acute activation of retinoid signaling via nuclear retinoic acid receptor-selective

synthetic agonists to prevent and block heterotopic ossification, an ectopic endochondral

processes triggered by trauma or gain-of-function activin receptor 1a mutations (Shimono et al.,

2011). It is conceivable that such therapy may work in HME as well, and we will be testing this

thesis in one of our HME mouse models in the near future. Another possibility is that

microRNAs could be used to treat HME as it is being currently tested in other cancer fields. A

recent study has shown that the miR transcriptome in human exostosis tissue differs from that of

normal human cartilage pointing to multiple putative therapeutic targets (Zuntini et al., 2010).

Last but not least, the identification of Surfen as an HS antagonist shows that HS can be affected

and modulated by pharmacological means. Thus, it is possible that HS agonists could be

identified and would increase HS bioactivity, reduce turnover or increase EXT expression.

Whatever their mechanisms of action, the drugs would then be tested for ability to increase or

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restore HS function/levels in HME mouse models and eventually patients, reduce exostosis

formation and ameliorate other symptoms. Our genetic studies show that exostosis formation

does not require a complete loss of Ext function to occur. It may be that pharmacological

prevention of exostosis formation may require a significant, but not a major and complete,

increase in HS function and levels as well.

Acknowledgements

The original studies described in this review were supported by NIH grants

RC1AR058382 and R01AR061758. We are grateful to Dr. David Kingsley (Stanford University)

for providing the Gdf5-Cre mice and Dr. Yu Yamaguchi (Sanford-Burnham Medical Research

Institute) for providing the Ext1fl/fl

mice. We also thank Ms. Jennifer Talarico for helping with the

clinical images.

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Table 1. Summary of current models of EXT deficiency in mice.

*Note that original nomenclature has been replicated in this table. The term exostosis is considered

synonymous with a number of other terms including osteochondroma and chondroma.

Genotype Skeletal phenotype Detection of exostoses* Reference

Ext1+/-

Minor alterations in growth plate

chondrocytes None

Hilton, 2005

Ext2+/-

Minor alterations in growth plate

chondrocytes

Small exostoses in ribs of <20% of

mice Stickens, 2005

Ext1+/-;Ext2+/- Bowed forearms, shortened HS

chains

Stereotypic exostoses in long bones

in 50% of mice; exostoses display

growth plate-like characteristics Zak, 2011

Ext1Gt/Gt

Shortened skeletal elements,

delayed chondrocyte

differentiation, prenatal lethal at

E15 None Koziel, 2004

Col2-rtTA-

Cre;Ext1e2neofl/e2neofl Shortened limbs

Aggressive epiphyseal

osteochondromas in all mice

Jones, 2009

Jones, 2012

Col2-CreERT;Ext1fl/fl

(stochastic model)

Bowed forearms, scoliosis,

short stature

Overgrowth of growth plate cartilage

to form cartilage-capped bony

protrusions

Matsumoto,

2010a

Prx1Cre;Ext1fl/fl

Severe skeletal abnormalities,

joint fusion, shortened limbs,

missing carpals/metacarpals,

postnatal lethal None

Matsumoto,

2010b

GDF5Cre;Ext1fl/fl

Shortened skeletal elements,

joint fusion in digits, postnatal

lethal Small exostoses in all mice

Mundy, 2010

Huegel, 2013

Col2CreER;ββββ-catenin fl/fl

Changes in chondrocyte

proliferation and differentiation

Lateral outgrowth of the growth

plate to form chondroma-like

masses Cantley, 2013

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Figure legends

FIGURE 1: Histological comparison of a typical human growth plate and an exostosis.

(A) Longitudinal section through a normal growth plate shows the stratified organization of the

chondrocytes and their distinct morphologies in each zone. Note that the chondro-osseous border

is located at the bottom of the picture. (B) The exostosis contains similar diverse populations of

chondrocytes but absence of clear organization. Note that the chondro-osseous border is located

on the left reflecting the 90° orientation of the exostosis relative to the longitudinal axis of the

adjacent growth plate. The cartilaginous portions stains strongly with Safranin-O, while the

adjacent bone is stained with fast green. (B, Inset) A macroscopic view of exostosis tissue

removed from a patient. Stereotypic exostoses are continuous with normal medullary and cortical

bone and are covered by perichondrial tissue. Bar for A and B, 75 µm; bar for inset, 2 mm.

FIGURE 2: Images from patients with Hereditary Multiple Exostoses. (A) Frontal and

(B) lateral X-ray images of the forearm of a 14 year-old HME patient reveal the presence of a

large exostosis at the distal end of the radius (arrowheads). (C) X-ray image and (D) CT scan

reveal the presence of a chondrosarcoma lesion near the scapula in a 17 year-old patient

(arrows). The humerus also contains exostoses (arrowheads). (E-F) These intraoperative CT

scans demonstrate the presence of an exostosis lesion within the spinal canal at the T12 level

(arrowhead).

FIGURE 3: Pharmacological interference with HS stimulates chondrogenesis. (A-C)

Mouse embryo limb bud mesenchymal cells in micromass cultures were treated with vehicle

(control) or 5 or 10 µM Surfen for 5 to 7 days. Note that the treated cultures display a much

larger number of alcian blue-positive cartilaginous nodules and that several of them are fused

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into each other. (D) qPCR analysis shows that expression of indicated cartilage marker genes is

up-regulated by Surfen in a dose-dependent manner in the micromass cultures. Vertical bars in

each histogram indicate standard deviations (Huegel et al., 2013).

FIGURE 4: Heparan sulfate inhibition leads to ectopic BMP signaling and cartilage

formation. (A-B) Histological images of Safranin-O/fast green-stained epiphyses showing that

ectopic cartilage was forming along the flanking perichondrium in Surfen-treated explants

(outlined in D), while the chondro-perichondrial border was continuous and intact in controls.

(C-D) Immunostaining images showing that phosphorylated Smad1/5/8 staining is apparent in

the perichondrium flanking the epiphysis of Surfen treated explants (arrowheads in B), while

there is background staining in control long bones (Huegel et al., 2013).

FIGURE 5: Schematic summarizes current hypotheses regarding the origin of exostosis-

forming cells. Those cells are currently thought to be: (A) growth plate chondrocytes themselves

(blue); (B) perichondrial cells (red); or (C) cells originating in the groove of Ranvier (purple).

Data in favor and against each of these theses have been reported in recent years. Our own work

indicates that perichondrium, including the groove of Ranvier, could represent a source of these

cells. It is also possible that the exostosis-founding cells could reside in growth plate or

perichondrium at the onset of exostosis formation, and would subsequently recruit cells from

surrounding sites to sustain and boost the outgrowth process. Thus, exostosis formation could

involve more than one source of cells (D). Other factors may also play a role in initiation and

growth of ectopic cartilage, including increased range and responsiveness of growth factor

signaling as well as upregulated heparanase.

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Ahrens PB, Solursh M, Reiter RS, Singley CT. 1979. Position-related capacity for differentiation of limb

mesenchyme in cell culture. Dev. Biol. 69:436-450.

Ai X, Kitazawa T, Anh-Tri D, Kusche-Gullberg M, Labosky PA, Emerson CP. 2007. SULF1 and SULF2

regulate heparan sulfate-mediated GDNF signaling for esophageal innervation. Development

134:3327-3338.

Alvarez C, Tredwell S, De Vera M, Hayden M. 2006. The genotype-phenotype correlation of hereditary

multiple exostoses. Clin. Genet. 70:122-130.

Arikawa-Hirasawa E, Watanabe H, Takami H, Hassell JR, Yamada Y. 1999. Perlecan is essential for

cartilage and cephalic development. Nature Genet. 23:354-358.

Assefa D, Murphy RC, Bergman K, Atlas AB. 2011. Three faces of costal exostoses: case series and review

of literature. Pediatr. Emerg. Care 27.

Bari MS, Jahangir Alam MM, Chowdhury FR, Dhar PB, Begum A. 2012. Hereditary multiple exostoses

causing cord compression. J. Coll. Physicians Surg. Pak. 22:797-799.

Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J, Zako M. 1999. Functions of cell

surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68:729-777.

Bernfield M, Kokenyesi R, Kato M, Hinkes MT, Spring J, Gallo RL, Lose EJ. 1992. Biology of the syndecans:

a family of transmembrane heparan sulfate proteoglycans. Annu. Rev. Cell Biol. 8:365-393.

Bishop JR, Schuksz M, Esko JD. 2007. Heparan sulphate proteoglycans fine-tune mammalian physiology.

Nature 446:1030-1037.

Bjornsson J, McLeod RA, Unni KK, Ilstrup DM, Pritchard DJ. 1998. Primary Chondrosarcoma of Long

Bones and Limb Girdles. Cancer 83.

Bovee JV, Cleton-Jansen A-M, Wuyts W, Caethoven G, Taminiau AH, Bakker E, Hul WV, Cornelisse CJ,

Hogendoorn PC. 1999. EXT-mutation analysis and loss of heterozygosity in sporadic and

hereditary osteochondromas and secondary chondrosarcoma. Am. J. Hum. Genet. 65:689-698.

Brown A, Alicknavitch M, D'Souza SS, Daikoku T, Kirn-Safran CB, Marchetti D, Carson DD, Farach-Carson

MC. 2008. Heparanase expression and activity influences chondrogenic and osteogenic

processes during endochondral bone formation. Bone 43:689-699.

Bulow HE, Hobert O. 2006. The molecular diversity of glycosaminoglycans shapes animal development.

Annu. Rev. Cell Dev. Biol. 22:375-407.

Busse M, Feta A, Presto J, Wilen M, Gronning M, Kjellen L, Kusche-Gullberg M. 2007. Contribution of

EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation. J. Biol. Chem. 282:32802-32810.

Cantley L, Saunders C, Guttenberg M, Candela ME, Ohta Y, Yasuhara R, Kondo N, Sgariglia F, Asai S,

Zhang X, Qin L, Hecht JT, Chen D, Yamamoto M, Toyosawa S, Dormans JP, Esko JD, Yamaguchi Y,

Iwamoto M, Pacifici M, Enomoto-Iwamoto M. 2012. Loss of B-catenin induced multifocal

perisoteal chondroma-like masses in mice. Am. J. Path. 182:917-927.

Ciaverella M, Coco M, Baorda F, Stanziale P, Chetta M, Bisceglia L, Palumbo P, Bengala M, Raiteri P,

Silengo M, Caldarini C, Facchini R, Lala R, Caveliere ML, De Brasi D, Pasini B, Zelante L, Guarnieri

V, D'Agruma L. 2012. 20 novel point mutations and one large deletion in EXT1 and EXT2 genes:

report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple

exostosis. Gene 515:339-348.

Clement A, Wiweger M, von der Hardt S, Rusch MA, Selleck S, Chien C-B, Roehl HH. 2008. Regulation of

zebrafish skeletogenesis by ext2/dackel and papst1/pinscher. PLoS Genetics 4:e1000136.

Day TF, Guo X, Garrett-Beal L, Yang Y. 2005. Wnt/•-catenin signaling in mesenchymal progenitors

controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell

8:739-750.

of 34



evel

opm

enta

l Dyn

amic

s

26

de Andrea CE, Wiweger M, Prins F, Bovee JVMG, Romeo S, Hogendoorn PC. 2010. Primary cilia

organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in

osteochondroma. Lab. Invest. 90:1091-1101.

Dormans JP. 2005. Pediatric Orthopaedics: Core Knowledge in Orthopaedics. Philadelphia: Elsevier

Mosby.

Esko JD, Selleck SB. 2002. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu.

Rev. Biochem. 71:435-471.

Francannet C, Cohen-Tagugi A, Le Merrer M, Munnick A, Bonaventure J, Legeai-Mallet L. 2001.

Genetype-phenotype correlation in hereditary multiple exostoses. J. Med. Genet. 38:430-434.

Freeman SD, Moore WM, Guiral EC, Holme AD, Turnbull JE, Pownall ME. 2008. Extracellular regulation of

developmental cell signaling by XtSulf1. Dev. Biol. 320:436-445.

Fuerer C, Habib SJ, Nusse R. 2010. A study on the interactions between heparan sulfate proteoglycans

and Wnt proteins. Dev. Dyn. 239:184-190.

Fux L, Ilan N, Sanderson RD, Vlodavsky I. 2009. Heparanase: Busy at the cell surface. Trends Biochem.

Sciences 34:511-519.

Goud AL, de Lange J, Scholtes VA, Bulstra SK, Ham SJ. 2012. Pain, physical and social functioning, and

quality of life in individuals with multiple hereditary exostoses in The Netherlands: a national

cohort study. J. Bone Joint Surg. Am. 94:1013-1020.

Habuchi H, Nagai N, Sugaya N, Atsumi F, Stevens RL, Kimata K. 2007. Mice deficient in heparan sulfate 6-

O-sulfotransferase-1 exhibit defective heparan sulfate biosynthesis, abnormal placentation, and

late embryonic lethality. J. Biol. Chem. 282:15578-15588.

Hacker U, Nybakken K, Perrimon N. 2005. Heparan sulphate proteoglycans: the sweet side of

development. Nature Rev. Mol. Cell Biol. 6:530-541.

Hecht JT, Hayes E, Haynes R, Cole GC, Long RJ, Farach-Carson MC, Carson DD. 2005. Differentiation-

induced loss of heparan sulfate in human exostosis derived chondrocytes. Differentiation

73:212-221.

Hilton MJ, Gutierrez L, Martinez DA, Wells DE. 2005. EXT1 regulates chondrocyte proliferation and

differentiation during endochondral bone development. Bone 36:379-386.

Hirata A, Nakamura H. 2006. Localization of Perlecan and Heparanase in Hertwig's Epithelial Root Sheath

During Root Formation in Mouse Molars. Histochem. Cytochem. 54:1105-1113.

Hosalkar H, Greenberg J, Gaugler RL, Garg S, Dormans JP. 2007. Abnormal scarring with keloid formation

after osteochondroma excision in children with multiple hereditary exostoses. J. Pediatr.

Orthop. 27:333-337.

Huangfu D, Anderson KV. 2005. Cilia and Hedgehog responsiveness in the mouse. Proc. Natl. Acad. Sci.

USA 102:11325-11330.

Huegel J, Mundy C, Sgariglia F, Nygren P, Billings PC, Yamaguchi Y, Koyama E, Pacifici M. 2013.

Perichondrium phenotype and border function are regulated by Ext1 and heparan sulfate in

developing long bones: A mechanism likely deranged in Hereditary Multiple Exostoses. Dev. Biol.

377:100-112.

Ilan N, Elkin M, Vlodavsky I. 2006. Regulation, function and clinical significance of heparanase in cancer

metastasis and angiongenesis. Int. J. Biochem. Cell Biol. 38:2018-2039.

Jennes I, Pedrini E, Zuntini M, Mordenti M, Balkassmi S, Asteggiano CG, Casey B, Bakker S, Sangiorgi L,

Wuyts W. 2009. Multiple osteochondromas: mutation update and description of the multiple

osteochondromas mutation database (MOdb). Hum. Mutat. 30:1620-1627.

Jiao X, Billings PC, O'Connell MP, Kaplan FS, Shore E, Glaser DL. 2007. Heparan sulfate proteoglycans

(HSPGs) modulate BMP2 osteogenic bioactivity in C2C12 cells. J. Biol. Chem. 282:1080-1086.

Jones KB. 2011. Glycobiology and the Growth Plate: Current Concepts in Multiple Hereditary Exostoses.

J. Pediatr. Orthop. 31:577-586.

of 34



evel

opm

enta

l Dyn

amic

s

27

Jones KB, Datar M, Ravichandran S, Jin H, Jurrus E, Whitaker R, Capecchi MR. 2013. Toward an

understanding of the short bone phenotype associated with multiple osteochondromas. J.

Ortho. Res. 31:651-657.

Jones KB, Piombo V, Searby C, Kurriger G, Yang B, Grabellus F, Roughley PJ, Morcuende JA, Buckwalter

JA, Capechhi MR, A. V, Sheffield VC. 2010. A mouse model of osteochondromagenesis from

clonal inactivation of Ext1 in chondrocytes. Proc. Natl. Acad. Sci. USA 107:2054-2059.

Kikuchi A, Yamamoto H, Sato A, Matsumoto S. 2011. New insights into the mechanism of Wnt signaling

pathway activation. Int. Rev. Cell Mol. Biol. 291:21-71.

Kim BT, Kitagawa H, Tamura J, Saito T, Kusche-Gullberg M, Lindahl U, Sugahara K. 2001. Human tumor

suppresor EXT gene family members EXTL1 and EXTL3 encode alpha 1,4-N-

acetylglucosaminaltransferases that likely are involved in heparan sulfate/heparin biosynthesis.

Proc. Natl. Acad. Sci. USA 98:7176-7181.

Kitagawa H, Shimakawa H, Sugahara K. 1999. The tumor suppressor EXT-like gene EXTL2 encodes an

alpha1,4-Nacetylhexosaminyltransferase that transfers N-acetylgalactosamine and N-

acetylglucosamine to the common glycosaminoglycan-protein linkage region. The key enzyme

for the chain initiation of heparan sulfate. J. Biol. Chem. 274:13933-13937.

Koyama E, Young B, Nagayama M, Shibukawa Y, Enomoto-Iwamoto M, Iwamoto M, Maeda Y, Lanske B,

Song B, Serra R, Pacifici M. 2007. Conditional Kif3a ablation causes abnormal hedgehog signaling

topography, growth plate dysfunction, and excessive bone and cartilage formation during

mouse skeletogenesis. Development 134:2159-2169.

Koziel L, Kunath M, Kelly OG, Vortkamp A. 2004. Ext1-dependent heparan sulfate regulates the range of

Ihh signaling during endochondral ossification. Dev. Cell 6:801-813.

Kronenberg HM. 2003. Developmental regulation of the growth plate. Nature 423:332-336.

Lin X. 2004. Functions of heparan sulfate proteoglycans in cell signaling during development.

Development 131:6009-6021.

Lin X, Wei G, Shi Z, Dryer L, Esko JD, Wells DE, Matzuk MM. 2000. Disruption of gastrulation and heparan

sulfate biosynthesis in EXT1-deficient mice. Dev. Biol. 224:299-311.

Manton KJ, D.F.M. L, Cool SM, Nurcombe v. 2007. Disruption of heparan and chondroitin sulfate

signaling enhances mesenchymal stem cell-derived osteogenic differentiation via bone

morphogenetic protein signaling pathways. Stem Cells 25:2845-2854.

Matsumoto Y, Matsumoto K, Irie F, Fukushi J-I, Stallcup WB, Yamaguchi Y. 2010. Conditional ablation of

the heparan sulfate-synthesizing enzyme Ext1 leads to dysregulation of bone morphogenetic

protein signaling and severe skeletal defects. J. Biol. Chem. 285:19227-19234.

McCormick C, Leduc Y, Martindale D, Mattison K, Esford L, Dyer A, Tufaro F. 1998. The putative tumor

suppressor EXT1 alters the expression of cell-surface heparan sulfate. Nat. Genet. 19:158-161.

Minina E, Kreschel C, Naski MC, Ornitz DM, Vortkamp A. 2002. Interaction of FGF, Ihh/Pthlh, and BMP

signaling integrates chondrocyte proliferation and hypertrophic differentiation. Dev. Cell 3:439-

449.

Okada M, Nadanaka S, Shoji N, Tamura J, Kitagawa H. 2010. Biosynthesis of heparan sulfate in EXT1-

deficient cells. Biochem. J. 428:463-471.

Osterholm C, Barczyk MM, Busse M, Gronning M, Reed RK, Kusche-Gullberg M. 2009. Mutation in the

heparan sulfate biosynthesis enzyme EXT1 influences growth factor signaling and fibroblast

interactions with the extracellular matrix. J. Biol. Chem. 284:34935-34943.

Otsuki S, Hanson SR, Miyaki S, Grogan SP, Kinoshita M, Asahara H, Wong C-H, Lotz M. 2010. Extracellular

sulfatases support cartilage homeostasis by regulating BMP and FGF signaling pathways. Proc.

Natl. Acad. Sci. USA 107:10202-10207.

of 34



evel

opm

enta

l Dyn

amic

s

28

Paine-Saunders S, Viviano B, Economides AN, Saunders S. 2002. Heparan sulfate proteoglycans retain

Noggin at the cell surface. A potential mechanism for shaping bone morphogenetic protein

gradients. J. Biol. Chem. 277:2089-2096.

Pathi S, Rutenberg JB, Johnson RL, Vortkamp A. 1999. Interaction of Ihh and BMP/Noggin signaling

during cartilage differentiation. . Dev. Biol. 209:239-253.

Pedrini E, Jennes I, Tremosini M, Milanesi A, Mordenti M, Parra A, Sgariglia F, Zuntini M, Campanacci L,

Fabbri N, Pignotti E, Wuyts W, Sangiorgi L. 2011. Genotype-phenotype correlation study in 529

patients with Hereditary Multiple Exostoses: identification of "protective" and "risk" factors. J.

Bone Joint Surg. (in press).

Perrone JA. 1967. Dysphagia, due to massive cervical exostoses. Arch. Otolaryngol. Head Neck Surg.

86:346-347.

Porter DE, Lonie L, Fraser M, Dobson-Stone C, Porter JR, Monaco AP, Simpson AH. 2004. Severity of

disease and risk in malignant change in hereditary multiple exostoses. J. Bone Joint Surg. Br.

86:1041-1046.

Porter DE, Simpson AHRW. 1999. The neoplastic pathogenesis of solitary and multiple

osteochondromas. J. Pathol. 188:119-125.

Rider CC, Mulloy B. 2010. Bone morphogenetic protein and growth differentiation factor cytokine

families and their protein antagonists. Biochem. J. 429:1-12.

Ruiz LP, Lara JC. 2012. Craniomaxillofacial features in hereditary multiple exostosis. J. Craniofac. Surg.

23:e336-338.

Schuksz M, Fuster MM, Brown JR, Crawford BE, Ditto DP, Lawrence R, Glass CA, Wang LC, Tor Y, Esko JD.

2008. Surfen, a small molecule antagonist of heparan sulfate. Proc. Natl. Acad. Sci. USA

105:13075-13080.

Shimokawa K, Kimura-Yoshida C, Nagai N, Mukai K, Matsubara K, Watanabe H, Matsuda Y, Mochida K,

Matsuo I. 2011. Cell surface heparan sulfate chains regulate local reception of FGF signaling in

the mouse embryo. Dev. Cell 21:257-272.

Shimono K, Tung W-E, Macolino C, Chi A, Didizian JH, Mundy C, Chandraratna RAS, Mishina Y, Enomoto-

Iwamoto M, Pacifici M, Iwamoto M. 2011. Potent inhibition of heterotopic ossification by

nuclear retinoic acid receptor-• agonists. Nature Med. 17:454-460.

Stickens D, Brown D, Evans GA. 2000. EXT genes are differentially expressed in bone and cartilage during

mouse embryogenesis. Dev. Dynam. 218:452-464.

Stickens D, Zak BM, Rougier N, Esko JD, Werb Z. 2005. Mice deficient in Ext2 lack heparan sulfate and

develop exostoses. Development 132:5055-5068.

Trebicz-Geffen M, Robinson D, Evron Z, Glaser T, Fridkin M, Kollander Y, Vlodavsky I, Ilan N, Law KF,

Cheah KSE, Chan D, Werner H, Nevo Z. 2008. The molecular and cellular basis of exostosis

formation in hereditary multiple exostoses. Int. J. Exp. Path. 89:321-331.

Umulis D, O'Connor MB, Blair SS. 2009. The extracellular regulation of bone morphogenetic protein

signaling. Development 136:3715-3728.

Viviano BL, L. S, Pflederer C, Paine-Saunders S, Mills K, Saunders S. 2005. Altered hematopoiesis in

glypican-3-deficient mice results in decreased osteoclast differentiation and a delay in

endochondral ossification. Dev. Biol. 282:152-162.

Waaijer CJ, Winter MGT, Reijnders CMA, de Jong D, Ham SJ, Bovee JV, Szuhai K. 2013. Intronic deletion

and duplication proximal of the EXT1 gene: a novel causative mechanism for multiple

osteochondromas. Genes Chromosomes Cancer 52:431-436.

Weston AD, Rosen V, Chandraratna RAS, Underhill TM. 2000. Regulation of skeletal progenitor

differentiation by the BMP and retinoid signaling pathways. J. Cell Biol. 148:679-690.

of 34



evel

opm

enta

l Dyn

amic

s

29

Wiweger M, Avramut CM, de Andrea CE, Prins F, Koster AJ, Raveli RBG, Hogendoorn PC. 2011. Cartilage

ultrastructure in proteoglycan-deficient zebrafish mutants brings to light new candidate genes

for human skeletal disorders. J. Pathol. 223:531-542.

Wiweger MI, Zhao Z, van Merkesteyn RJ, Roehl HH, Hogendoorn PC. 2012. HSPG-deficient zebrafish

uncovers dental aspect of multiple osteochondromas. PLoS ONE 7:e29734.

Wuyts W, Van Hul W. 2000. Molecular basis of multiple exostoses: mutations in the EXT1 and EXT2

genes. Hum. Mutat. 15:220-227.

Yang L, Hui WS, Chan WCW, Ng VCW, Yam THY, Leung HCM, Huang J, Shum DKY, Lie Q, Cheung KMC,

Cheah KSE, Luo Z, Chan D. 2010. A splice-site mutation leads to haploinsufficiency of Ext2 mRNA

for a dominant trait in a large family with multiple osteochondromas. J. Orthop. Res. 28:1522-

1530.

Yasuda T, Mundy C, Kinumatsu T, Shibukawa Y, Shibutani T, Grobe K, Minugh-Purvis N, Pacifici M,

Koyama E. 2010. Sulfotransferase Ndst1 is needed for mandibular and TMJ development. J.

Dent. Res. 89:1111-1116.

Yuasa T, Kondo N, Yasuhara R, Shimono K, Mackem S, Pacifici M, Iwamoto M, Enomoto-Iwamoto M.

2009. Transient activation of Wnt/•-catenin signaling induces growth plate closure and articular

cartilage thickening in postnatal mice. Am. J. Path. 175:1993-2003.

Zaijun L, Xinhai Y, Zhipeng W, Wending H, Quan H, Zhenhua Z, Dapeng F, Jisheng Z, Wei Z, Jianru X. 2011.

Outcome and prognosis of myelopathy and radiculopathy from osteochondroma in the mobile

spine: a report on 14 patients. J. Spinal Disord. Tech. In Press.

Zak BM, Schuksz M, Koyama E, Mundy C, Wells DE, Yamaguchi Y, Pacifici M, Esko JD. 2011. Compound

heterozygous loss of Ext1 and Ext2 is sufficient for formation of multiple exostoses in mouse ribs

and long bones. Bone 48:979-987.

Zou H, Wieser R, Massague J, Niswander L. 1997. Distinct roles of type I bone morphogenetic protein

receptors in the formation and differentiation of cartilage. Genes Dev 11:2191-2203.

Zuntini M, Salvatore M, Pedrini E, Parra A, Sgariglia F, Magrelli A, Taruscio D, Sangiorgi L. 2010.

MicroRNA profiling of multiple osteochondromas: identification of disease-specific and normal

cartilage signatures. Clin. Genet. 78:507-516.

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Heparan sulfate in skeletal development, growth, and ...

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