RESUMEN
Heparan sulfate moieties of cell-surface proteoglycans modulate the biological responses to fibroblast growth factors (FGFs). We have reported previously that cell-associated heparan sulfates inhibit the binding of the keratinocyte growth factor (KGF), but enhance the binding of acidic FGF to the KGF receptor, both in keratinocytes, which naturally express this receptor, and in rat myoblasts, which ectopically express it (Reich-Slotky, R., Bonneh-Barkay, D., Shaoul, E., Berman, B., Svahn, C. M., and Ron, D. (1994) J. Biol. Chem. 269, 32279-32285). The proteoglycan bearing these modulatory heparan sulfates was purified to homogeneity from salt extracts of rat myoblasts by anion-exchange and FGF affinity chromatography and was identified as rat glypican. Affinity-purified glypican augmented the binding of acidic FGF and basic FGF to human FGF receptor-1 in a cell-free system. This effect was abolished following digestion of glypican by heparinase. Addition of purified soluble glypican effectively replaced heparin in supporting basic FGF-induced cellular proliferation of heparan sulfate-negative cells expressing recombinant FGF receptor-1. In keratinocytes, glypican strongly inhibited the mitogenic response to KGF while enhancing the response to acidic FGF. Taken together, these findings demonstrate that glypican plays an important role in regulating the biological activity of fibroblast growth factors and that, for different growth factors, glypican can either enhance or suppress cellular responsiveness.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Proteoglicanos/metabolismo , Receptores Virales/metabolismo , Animales , Línea Celular , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Sustancias de Crecimiento/metabolismo , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/química , Heparitina Sulfato/aislamiento & purificación , Humanos , Proteoglicanos/química , Proteoglicanos/aislamiento & purificación , RatasRESUMEN
The fibroblast growth factors (FGFs) act through high affinity tyrosine kinase receptors and, in addition, interact with lower affinity receptors that represent cell- or matrix-associated heparan sulfate proteoglycans. These lower affinity receptors modulate the biological activities of FGFs, but the mechanism by which they exert these effects is rather controversial. We have previously shown (Ron, D., Bottaro, D. P., Finch, P. W., Morris, D., Rubin, J. S., and Aaronson, S. A. (1993) J. Biol. Chem. 268, 2984-2988) that heparin potentiates the mitogenic activity of acidic FGF (aFGF) but inhibits that of the keratinocyte growth factor (KGF) in cells that express the KGF receptor (KGFR). Both growth factors bind the KGFR with high affinity. To gain an insight into the mechanism by which heparin modulates the biological activity of aFGF and KGF, we studied the effect of heparin and cell-associated heparan sulfates on the binding of these two growth factors to the KGFR. To work in a well defined system, we expressed functional KGFR in L6E9 myoblasts that lack detectable high affinity binding sites for FGFs. Low concentrations of heparin inhibited the binding of KGF to the KGFR. By contrast, similar concentrations of heparin enhanced the binding of aFGF to this receptor. The effect of heparin was not unique to L6E9 cells expressing the KGFR; it was also observed in Balb/MK cells that naturally express KGFR. Treatment of cells with sodium chlorate, which blocks sulfation of proteoglycans, reduced the binding of aFGF to its low and high affinity binding sites by 95 and 80%, respectively. In contrast, the binding of KGF to its high affinity binding sites was enhanced about 2-fold. Similar results were obtained after degradation of cell-associated heparan sulfates by heparinase and heparitinase. Heparin restored the high affinity binding of aFGF to chlorate-treated cells and completely abolished the high affinity binding of KGF. Binding competition experiments suggest that aFGF and KGF bind to the same population of cell-associated heparan sulfates. In addition, KGF is apparently interacting with an as yet unidentified type of low affinity binding site that is not affected by chlorate or heparan sulfate-degrading enzymes. An important property of the FGF high affinity receptors is their ability to bind more than one ligand with high affinity. Based on the differential effect of cell-associated heparan sulfates on the binding of KGF and aFGF to the KGFR, we propose a regulatory role for cell-associated heparan sulfates as coordinators of the interaction of aFGF and KGF with the KGFR.(ABSTRACT TRUNCATED AT 400 WORDS)