Heparan sulfate in skeletal development, growth, and pathology: the case of hereditary multiple exostoses

Abstract

Heparan sulfate (HS) is an essential component of cell surface and matrix-associated proteoglycans. 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 those proteins as well as 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.

Keywords: cell surface proteoglycans; ectopic cartilage; growth plate; heparan sulfate; hereditary multiple exostoses; signaling proteins.

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 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.