Heparan and chondroitin sulfate on growth plate perlecan mediate binding and delivery of FGF-2 to FGF receptors.

Fibroblast growth factor (FGF)-2 regulates chondrocyte proliferation in the growth plate. Heparan sulfate (HS) proteoglycans bind FGF-2. Perlecan, a heparan sulfate proteoglycan (HSPG) in the developing growth plate, however, contains both HS and chondroitin sulfate (CS) chains. The binding of FGF-2 to perlecan isolated from the growth plate was evaluated using cationic filtration (CAF) and immunoprecipitation (IP) assays. FGF-2 bound to perlecan in both the CAF and IP assays primarily via the HS chains on perlecan. A maximum of 123 molecules of FGF-2 was calculated to bind per molecule of 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 also increased FGF-2 stimulation of [(3)H]-thymidine incorporation in BaF3 cells expressing these FGF receptors. These data show that 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.

[1]  R. Timpl,et al.  Perlecan Maintains the Integrity of Cartilage and Some Basement Membranes , 1999, The Journal of cell biology.

[2]  J. Hassell,et al.  Identification of Sites in Domain I of Perlecan That Regulate Heparan Sulfate Synthesis* , 1997, The Journal of Biological Chemistry.

[3]  S. Pawlowski,et al.  Fibroblast growth factor 2 control of vascular tone , 1998, Nature Medicine.

[4]  D. Noonan,et al.  The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule. , 1991, The Journal of biological chemistry.

[5]  D. Hill,et al.  Cell Cycle-Dependent Localization of Irnmunoreactive Basic Fibroblast Growth Factor to Cytoplasm and Nucleus of Isolated Ovine Fetal Growth Plate Chondrocytes , 1992 .

[6]  S. Rennard,et al.  Isolation of a heparan sulfate-containing proteoglycan from basement membrane. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[7]  M. Nugent,et al.  A Simple Assay for Evaluating Inhibitors of Proteoglycan-Ligand Binding , 2004, Annals of Biomedical Engineering.

[8]  J. Melrose,et al.  Not All Perlecans Are Created Equal , 2002, The Journal of Biological Chemistry.

[9]  J. Uitto,et al.  The Protein Core of the Proteoglycan Perlecan Binds Specifically to Fibroblast Growth Factor-7* , 2000, The Journal of Biological Chemistry.

[10]  A. Yayon,et al.  Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis , 1994, Cell.

[11]  P. Marie,et al.  FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. , 2002, Genes & development.

[12]  K. White,et al.  Mutations that cause osteoglophonic dysplasia define novel roles for FGFR1 in bone elongation. , 2005, American journal of human genetics.

[13]  D. Church,et al.  Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia , 1994, Cell.

[14]  R. Timpl,et al.  Characterization of recombinant perlecan domain I and its substitution by glycosaminoglycans and oligosaccharides. , 1997, European journal of biochemistry.

[15]  M. Ito,et al.  Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation. , 2000, The Journal of clinical investigation.

[16]  R. Timpl,et al.  Chondroitin Sulfate Perlecan Enhances Collagen Fibril Formation , 2006, Journal of Biological Chemistry.

[17]  W. Wilcox,et al.  Dyssegmental dysplasia, Silverman-Handmaker type, is caused by functional null mutations of the perlecan gene , 2001, Nature Genetics.

[18]  R. Iozzo.  Basement membrane proteoglycans: from cellar to ceiling , 2005, Nature Reviews Molecular Cell Biology.

[19]  T. Koob,et al.  Changes in perlecan during chondrocyte differentiation in the fetal bovine rib growth plate , 2006, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[20]  R. Stevenson,et al.  Structural and functional mutations of the perlecan gene cause Schwartz-Jampel syndrome, with myotonic myopathy and chondrodysplasia. , 2002, American journal of human genetics.

[21]  S. Ledbetter,et al.  Domain structure of the basement membrane heparan sulfate proteoglycan. , 1987, Biochemistry.

[22]  D. Schoenfeld,et al.  Regulation of growth-plate chondrocytes by insulin-like growth-factor I and basic fibroblast growth factor. , 1993, The Journal of bone and joint surgery. American volume.

[23]  J. Hecht,et al.  Expression of the Heparan Sulfate Proteoglycan, Perlecan, during Mouse Embryogenesis and Perlecan Chondrogenic Activity In Vitro , 1999, The Journal of cell biology.

[24]  J. Turnbull,et al.  Interactions of heparin/heparan sulfate with proteins: appraisal of structural factors and experimental approaches. , 2004, Glycobiology.

[25]  R. Iozzo,et al.  Developmental expression of perlecan during murine embryogenesis , 1997, Developmental dynamics : an official publication of the American Association of Anatomists.

[26]  J. Hassell,et al.  Modulation of FGF-2 binding to chondrocytes from the developing growth plate by perlecan. , 2006, Matrix biology : journal of the International Society for Matrix Biology.

[27]  H. Nagai,et al.  Effects of basic fibroblast growth factor (bFGF) on bone formation in growing rats. , 1995, Bone.

[28]  J. Couchman,et al.  Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan. , 1999, Biochemical and biophysical research communications.

[29]  S. Tsang,et al.  Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[30]  R. Miller,et al.  Basic fibroblast growth factor binds to heparan sulfate in the extracellular matrix of rat growth plate chondrocytes. , 1994, Archives of biochemistry and biophysics.

[31]  J G Flanagan,et al.  Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells , 1992, Molecular and cellular biology.

[32]  R. Miller,et al.  Role of heparan sulfate in the terminal differentiation of growth plate chondrocytes. , 1995, Archives of biochemistry and biophysics.

[33]  R. Iozzo.  Perlecan: a gem of a proteoglycan. , 1994, Matrix biology : journal of the International Society for Matrix Biology.

[34]  Gary W. Harding,et al.  Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3 , 1996, Nature Genetics.

[35]  W. Yuan,et al.  Mapping Ligand Binding Domains in Chimeric Fibroblast Growth Factor Receptor Molecules , 1999, The Journal of Biological Chemistry.

[36]  J. Weissenbach,et al.  Perlecan, the major proteoglycan of basement membranes, is altered in patients with Schwartz-Jampel syndrome (chondrodystrophic myotonia) , 2000, Nature Genetics.

[37]  S. Ledbetter,et al.  Perlecan is a component of cartilage matrix and promotes chondrocyte attachment. , 1995, Journal of cell science.

[38]  D. Buttle,et al.  Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. , 1986, Biochimica et biophysica acta.

[39]  D. Gospodarowicz,et al.  Structural characterization and biological functions of fibroblast growth factor. , 1987, Endocrine reviews.

[40]  R. Zeller,et al.  Impaired cerebral cortex development and blood pressure regulation in FGF‐2‐deficient mice , 1998, The EMBO journal.

[41]  Nobuyuki Itoh,et al.  Fibroblast growth factors , 2001, Genome Biology.

[42]  P. Leder,et al.  Ligand specificity and heparin dependence of fibroblast growth factor receptors 1 and 3. , 1992, The Journal of biological chemistry.

[43]  C. Praul,et al.  Basic fibroblast growth factor: An autocrine growth factor for epiphyseal growth plate chondrocytes , 1996, Journal of cellular biochemistry.

[44]  E. Edelman,et al.  Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[45]  G. Dorn,et al.  Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice. , 1995, Molecular biology of the cell.

[46]  R. Timpl,et al.  Structural basis of glycosaminoglycan modification and of heterotypic interactions of perlecan domain V. , 1999, Journal of molecular biology.

[47]  T. Koob,et al.  Isolation and Identification of the Major Heparan Sulfate Proteoglycans in the Developing Bovine Rib Growth Plate* , 2002, The Journal of Biological Chemistry.

[48]  G. R. Dodge,et al.  Primary structure of the human heparan sulfate proteoglycan from basement membrane (HSPG2/perlecan). A chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor. , 1992, The Journal of biological chemistry.

[49]  S. Yamada,et al.  Internalization of basic fibroblast growth factor at the mouse blood–brain barrier involves perlecan, a heparan sulfate proteoglycan , 2002, Journal of neurochemistry.

[50]  Q. Wang,et al.  Differential regulation of endochondral bone growth and joint development by FGFR1 and FGFR3 tyrosine kinase domains. , 2001, Development.

[51]  D. Gospodarowicz,et al.  Fibroblast growth factor: Structural and biological properties , 1987, Journal of cellular physiology. Supplement.

[52]  John M. Whitelock,et al.  The Degradation of Human Endothelial Cell-derived Perlecan and Release of Bound Basic Fibroblast Growth Factor by Stromelysin, Collagenase, Plasmin, and Heparanases (*) , 1996, The Journal of Biological Chemistry.

[53]  B. Olsen.  Life without Perlecan Has Its Problems , 1999, The Journal of cell biology.

[54]  R. Timpl,et al.  Inhibition of glycosaminoglycan modification of perlecan domain I by site‐directed mutagenesis changes protease sensitivity and laminin‐1 binding activity , 1998, FEBS letters.

[55]  Yoshihiko Yamada,et al.  Perlecan is essential for cartilage and cephalic development , 1999, Nature Genetics.

[56]  R. Iozzo,et al.  Isolation and characterization of proteoglycans synthesized by human colon and colon carcinoma. , 1982, The Journal of biological chemistry.

[57]  M. Muenke,et al.  Fibroblast-growth-factor receptor mutations in human skeletal disorders. , 1995, Trends in genetics : TIG.