IN-VITRO CHANGES IN PLASMA-MEMBRANE HEPARAN-SULFATE PROTEOGLYCANS ANDIN PERLECAN EXPRESSION PARTICIPATE IN THE REGULATION OF FIBROBLAST-GROWTH-FACTOR-2 MITOGENIC ACTIVITY

Fibroblast growth factor 1 (FGF1) and 2 (FGF2) bind to two classes of receptors: the high affinity receptors, a family of four known transme mbrane tyrosine kinases (FGF R1-R4), and the low affinity receptors, c ell surface and basement membrane heparan sulfate proteoglycan (HSPG). During early (first and second) passages of retinal pigmented epithel ial (RPE) cells, both FGF1 and FGF2 exhibited low mitogenic activity, while in later (fifth to ninth) passages the activity of FGF1 remained constant but FGF2 activity increased two- to threefold. We have inves tigated aspects of FGF receptor interactions and the role of heparin/h eparan sulfate which modulates FGF activity on RPE cells during in vit ro senescence. Northern blot analysis demonstrated that FGF receptor t ype 1 (FGF R1) is the major high affinity receptor expressed in RPE ce lls and that its level of expression did not change during serially pa ssage. Both the FGF R1 and the FGF low affinity receptors' binding cha racteristics (i.e., Kd and number of sites per cell) for FGF1 were una ffected by passage number, whereas the capacity of FGF2 binding to FGF R1 and to the low affinity receptors increased by two- and fivefold, respectively, in late passages, although the affinities were unchanged . This change in the capacity of FGF2 to bind to FGF R1 and to HSPG wa s not due to a switch of the IIIc splice form of FGF R1 to the IIIb sp lice form since the exon IIIc was the most predominant splice form of FGF R1 during RPE cell cultures. Furthermore the ratio of the IIIb to the file splice form was not modified during cell subcultures. In para llel in the older RPE cell passages, expression of perlecan, the major FGF low affinity binding site localized on the extracellular matrix o f RPE cells, was much elevated compared to early RPE cell passages. Mo reover, the cell surface of late passage RPE cells had 79% more HSPG t han early passage cells. Therefore, it is suggested that the increase in the number of FGF low affinity receptors present on the cell surfac e or basement membrane could account for a part of the greater prolife rative response of aged RPE cells to FGF2. (C) 1996 Wiley-Liss, Inc.