Three cellular sources of
perlecan were examined in this study, namely human umbilical arterial endothelial cells (HUAEC), a transformed human umbilical venous endothelial cell line (C 1 1 STH) and a human colon
carcinoma cell line (WiDr). Perlecans were immunopurified from
conditioned media of the above cells and the purity of the
perlecan preparations was examined by composite
agarose polyacrylamide gel electrophoresis (CAPAGE) and semi-dry immunoblotting with
monoclonal antibodies directed to either the
perlecan core
protein (mAb A76) or heparan sulphate (HS) side-chain (mAb10E4). The ability of each
perlecan species to bind
fibroblast growth factor-l (FGF-1) was examined using a biosensor (BIAcore). The bioactivity of
perlecan FGF-1 interactions was also analysed using BaF3 cells transfected with
fibroblast growth factor receptors FGFR1b and 1c. CAPAGE demonstrated subtle differences between the perlecans, indicating they had differing charge to mass ratios with C 11 STH
perlecan being slightly more mobile in CAPAGE than the HUAEC and WiDr sample. BIAcore biosensor analysis demonstrated distinct differences in the ability of
perlecan preparations to bind FGF-1; HUAEC and C 11 STH
perlecan showed similar high binding responses as compared to WiDr
perlecan, which bound
FGF-1 very poorly. Binding of
FGF-1 to endothelial perlecans was shown to be HS-dependent. Interestingly, HUAEC
perlecan stimulated the growth of FGFR1b and FGFR1c expressing cells in the presence of
FGF-1 comparable to
heparin, whereas C 11 STH
perlecan showed only very limited stimulation of FGFR 1b cells and was incapable of stimulating FGFR1c cells. WiDr
perlecan exhibited no stimulation of growth in either cell line. Collectively the data presented herein indicate that. different cell types express perlecans which vary in the
growth factor binding capabilities, which may suggest differences in their HS chain substructure. This may represent a subtle mechanism whereby cells can modulate the responsiveness of
perlecan to a range of biologically important
ligands and thus in a broader context may have important implications for cell signalling.