Perichondrium phenotype and border function are regulated by Ext1 and heparan sulfate in developing long bones: A mechanism likely deranged in Hereditary Multiple Exostoses

Abstract

During limb skeletogenesis the cartilaginous long bone anlagen and their growth plates become delimited by perichondrium with which they interact functionally. Yet, little is known about how, despite being so intimately associated with cartilage, perichondrium acquires and maintains its distinct phenotype and exerts its border function. Because perichondrium becomes deranged and interrupted by cartilaginous outgrowths in Hereditary Multiple Exostoses (HME), a pediatric disorder caused by EXT mutations and consequent heparan sulfate (HS) deficiency, we asked whether EXT genes and HS normally have roles in establishing its phenotype and function. Indeed, conditional Ext1 ablation in perichondrium and lateral chondrocytes flanking the epiphyseal region of mouse embryo long bone anlagen – a region encompassing the groove of Ranvier – caused ectopic cartilage formation. A similar response was observed when HS function was disrupted in long bone anlagen explants by genetic, pharmacological or enzymatic means, a response preceded by ectopic BMP signaling within perichondrium. These treatments also triggered excess chondrogenesis and cartilage nodule formation and overexpression of chondrogenic and matrix genes in limb bud mesenchymal cells in micromass culture. Interestingly, the treatments disrupted the peripheral definition and border of the cartilage nodules in such a way that many nodules overgrew and fused with each other into large amorphous cartilaginous masses. Interference with HS function reduced the physical association and interactions of BMP2 with HS and increased the cell responsiveness to endogenous and exogenous BMP proteins. In sum, Ext genes and HS are needed to establish and maintain perichondrium’s phenotype and border function, restrain pro-chondrogenic signaling proteins including BMPs, and restrict chondrogenesis. Alterations in these mechanisms may contribute to exostosis formation in HME, particularly at the expense of regions rich in progenitor cells including the groove of Ranvier.

Aberrant perichondrial BMP signaling mediates multiple osteochondromagenesis in mice

“…One reason for this debate is the difficulty of interrogating the initial phases of osteochondromagenesis using human osteochondroma specimens. Previous data using explant cultures of mouse long bone anlagen support the perichondrial origin of osteochondromas (12). Our observation of the presence of abnormal cell clusters in the perichondrial groove of Ranvier (Figure 1) also suggests that osteochondromas arise from progenitor cells residing in the perichondrium.…”

“…Similar abnormal cell clusters were also observed in the perichondrium of rib bones of Col2a1-Ext1 CKO mice ( Figure 1B). These observations suggest that osteochondromas are derived from aberrant differentiation of cells in the perichondrium, as postulated previously (12)(13)(14).…”

“…There have been several reports suggesting that aberrant BMP signaling plays a role in osteochondromagenesis (12,(27)(28)(29)(30)36). This present study provides multiple lines of functional evidence for this hypothesis.…”

“…For example, conditional inactivation of Ext1 in the limb bud mesenchyme primarily affects the BMP pathway by inducing the spatial expansion of BMP signaling (10). Both enzymatic removal of HS and chemical interference with HS enhance BMP bioactivity (12,27,28). Transgenic mice expressing a constitutively active form of the BMP receptor 1A develop abnormal chondrocytic protrusions in the perichondrium (29).…”

Domains with highest heparan sulfate–binding affinity reside at opposite ends in BMP2/4 versus BMP5/6/7: Implications for function

“…HME is characterized by cartilaginous tumors (called exostoses or osteochondromas) that develop within perichondrium flanking the growth plates of long bones, ribs and other elements, but the underlying mechanisms had long remained unclear (62,63). Using mouse models, we found that conditional Ext1 ablation in perichondrial cells (and concurrent drop in HS levels) caused a sharp decrease in local ERK/FGF signaling and a reciprocal increase in canonical BMP signaling (64,65). These opposing signaling changes were followed by the development of cartilaginous tumors in very good correlation with the fact that normally, BMP signaling is prochondrogenic while ERK/FGF signaling is anti-chondrogenic (66,67).…”