Graduation Year

2007

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Molecular Medicine

Major Professor

John R. Hassell, Ph.D.

Keywords

Cartilage, Endochondral, FGF, FGFR, HSPG2

Abstract

Perlecan is the major heparan sulfate proteoglycan (HSPG) in growth plate cartilage and is critical for growth plate chondrocyte proliferation and proper skeletal development. Its core protein and attached chondroitin sulfate (CS) and heparan sulfate (HS) chains mediate interactions with many diverse proteins. Fibroblast growth factor (FGF)-2 and FGF-18 are other regulators of chondrocyte proliferation in the growth plate. Additionally, FGF-18 controls the hypertrophy and cartilage vascularization necessary for endochondral ossification. The research presented in this dissertation aimed to identify known and novel perlecan-binding proteins that are endogenous to the growth plate and to characterize their interactions with perlecan. FGF-2 (known to bind HSPGs) bound to perlecan in both a cationic filtration (CAF) assay and an immunoprecipitation (IP) assay primarily via the HS chains on perlecan.

When digested with chondroitinase ABC to remove its CS chains, perlecan augmented binding of FGF-2 to the FGFR-1 and FGFR-3 receptors and increased FGF-2 -stimulated proliferation in BaF3 cells expressing these FGF receptors. Thus, growth plate perlecan binds to FGF-2 by its HS chains but can only deliver FGF-2 to FGF receptors when its CS chains are removed. FGF-18 (known to bind to heparin and to heparan sulfate from some sources) bound to growth plate perlecan. This binding was unchanged by chondroitinase or heparitinase digestion of perlecan, indicating that perlecan GAGs are not involved in FGF-18 binding. FGF-18 bound equally to recombinant domains I-III of perlecan (Alt1) and to full-length perlecan purified from the growth plate. Additionally, FGF-18 bound equally to recombinant domain III of perlecan, to Alt1 and to Alt2 (a domain I-III variant with no heparan sulfate). Therefore, binding sites for FGF-18 are present in domain III of perlecan.

Affinity chromatography isolated histone H3 as a perlecan-binding protein from the chondrocyte matrix. CAF assays confirmed the interaction as specific, dependent primarily on HS chains of perlecan, although CS chains and the perlecan core were also involved. Immunohistochemistry detected perlecan and histone H3 colocalized in growth plate cartilage. These results can help us better understand the growth factor-independent control that perlecan exerts on endochondral ossification and, therefore, long bone growth.

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