Heparan sulfate-dependent mechanisms of normal and pathological skeletogenesis
Skeletogenesis of the axial and appendicular elements occurs through endochondral ossification, a complex process partially regulated by heparan sulfate proteoglycans (HSPGs) and their heparan sulfate (HS) chains. Negatively charged HS interacts with a number of growth factors including members of the bone morphogenetic protein, Wnt, hedgehog, and fibroblast growth factor families, all of which influence endochondral ossification. These interactions control factor distribution and bioavailability, providing tissue and cell-specific signaling events. The importance of HSPGs in skeletal development is reiterated by the skeletal disorder Hereditary Multiple Exostoses (HME) caused by heterozygous mutations in HS-polymerizing enzymes EXT1 or EXT2. HME patients develop ectopic cartilaginous outgrowths that form next to growth plates of long bones adjacent to joints. The goal of this investigation was to better understand the connection between loss of HS and exostosis formation and to specifically address a few poorly understood aspects of HME. We hypothesized that loss of HS would allow pro-chondrogenic signaling factors to signal more robustly outside typical distribution regions, potentially initiating ectopic chondrocyte differentiation. In order to address cell origin and track early exostosis formation, an in vivo model was developed with conditional deletion of Ext1 in a population of joint-forming epiphyseal cells. Ex vivo and in vitro models of HS deficiency allowed us to investigate how this change influences chondrogenesis as well as cell signaling events. We uncovered a specific population of perichondrial cells that contribute to exostosis development, at least partially due to an increase in BMP2 signaling in this area. Biochemical analysis of HS-BMP interactions demonstrated that interfering with HS increased the amount of "free" and available ligand, likely promoting receptor interaction and subsequent signaling. Another interesting characteristic of HME is that many exostoses express very low to no HS even within a heterozygous patient (which should express 50% of normal HS levels). We hypothesized that extracellular degradation of existing HS chains may further impact the level of functional HSPGs within exostosis tissue. In fact, antagonizing HS does increase levels of heparanase (HPSE), an endoglucoronidase that cleaves HS chains from the cell surface into short fragments. This suggests a regulatory mechanism between HS and HPSE and signifies that an important inverse regulatory loop exists by which a drop in HS would trigger an increase in HPSE, leading to very steep decreases in overall HS levels. In turn, HPSE caused increased proliferation and migration of chondrocyte progenitors, as well as promoting their chondrogenic differentiation. Additionally, HPSE expression in human exostosis tissue was extensive and was not contingent on chondrocyte maturation stage as seen in control growth plates. Together, it is clear that wild-type levels of HS are necessary for normal signaling processes and chondrocyte differentiation within appropriate areas. Targeting HPSE or taking other pro-HS approaches could prove fruitful in developing an HME therapy.
Molecular biology|Cellular biology|Developmental biology
Huegel, Julianne, "Heparan sulfate-dependent mechanisms of normal and pathological skeletogenesis" (2013). ETD Collection for Thomas Jefferson University. AAI3605998.