Jason Metcalf

THE ROLE OF FIBROBLAST GROWTH FACTOR SIGNALING IN MUCOPOLYSACCHARIDOSIS BONE DEFECTS. Jason Metcalf¹, Matthew Hilton², Fanxin Long², Xiucui Ma³, Mindy Tittiger³, Katherine Ponder³. Department of Biology¹,Department of Molecular Biology and Pharmacology², Department of Hematology³, Washington University Medical School, St. Louis, MO.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases caused by deficiencies in enzymes that degrade glycosaminoglycans (GAGs). The undegraded GAGs accumulate in lysosomes and extracellular space. In some MPS diseases, such as MPS VII, this leads to extremely short bones, but in other types, such as MPS I, bones are normal in length. While both forms of MPS accumulate several subtypes of GAGs, chondroitin sulfate (CS) accumulates in MPS VII but not in MPS I, suggesting that CS may somehow be involved in stunted bone growth. The fibroblast growth factors (FGFs) are a group of signaling molecules that require specific GAGs to stabilize the FGF + receptor complex. Signaling through FGF receptor 3 negatively influences proliferation and differentiation in growth plate, thereby restricting long bone length. We hypothesized that accumulation of CS in the growth plates of MPS VII mice causes short bones by overactivating one or more FGF signaling pathways.

The growth plate is the region of long bones in which the majority of chondrocyte proliferation and bone elongation takes place. To analyze abnormalities in growth plate function, we performed RNA in-situ hybridization on sections of MPS and normal mice tibias, looking for changes in chondrocyte and osteoblast markers, some of which are directly related to FGF signaling. Hypertrophic chondrocyte markers such as collagen X and indian hedgehog were reduced in MPS VII tibias, while osteoblast function appeared to be unchanged for both MPS diseases. Analysis of proliferation in the growth plate showed a marked reduction in MPS VII mice. This suggests that both proliferation and differentiation are being negatively regulated, consistent with overactive FGF receptor 3.

To ascertain which types of GAGs accumulate in the bones of MPS individuals we performed immunohistochemistry for various GAGs on tibia sections at 3 weeks. Normal and MPS I mice showed low levels of CS in the growth plate, while MPS VII mice showed highly elevated levels of CS throughout the entire growth plate and also in the early trabecular bone. This shows that CS is indeed present at abnormally high levels in MPS VII mice in locations relevant to long bone growth. Finally, we performed proliferation assays on cultured Baf3 cells dependent on FGF signaling. We found that both pure CS and GAGs isolated from MPS VII dog growth plates increase proliferation in these cells, suggesting that CS stabilizes the FGF + receptor complex and may be capable of overactivating FGF signaling in MPS VII bones.

These experiments show that CS is building up in MPS VII growth plates, that MPS VII growth plates are abnormal in a fashion consistent with overactivated FGF signaling, and that CS can stimulate FGF. These results suggest that the interaction between CS and FGF may be playing a major role in MPS VII bone disease.