INTERACTION OF HIGH-MOLECULAR-WEIGHT BASIC FIBROBLAST GROWTH-FACTOR WITH ENDOTHELIUM - BIOLOGICAL-ACTIVITY AND INTRACELLULAR FATE OF HUMAN RECOMBINANT M(R) 24,000 BFGF

The single-copy gene of human basic fibroblast growth factor (bFGF) en codes four co-expressed isoforms, with an apparent molecular weight (M (r)) of 24 kD, 22.5 kD, 22 kD, and 18 kD, co-translated from a single mRNA. As a tool for the study of the role exerted by the different bFG F isoforms in the biology of endothelial cells, human recombinant 24-k D bFGF was produced and purified from transformed Escherichia coli cel ls. To this purpose, the novel CUG start codon present in human bFGF c DNA and responsible for the synthesis of 24-kD bFGF was mutagenized to the classic AUG start codon. Transient expression of the mutagenized cDNA in simian COS-1 cells, followed by immunolocalization and subcell ular fractionation, resulted in the synthesis of high levels of 24-kD bFGF, which localizes in the cell nucleus as an intact protein. When t he same 24-kD bFGF cDNA was expressed in E. coli, the recombinant prot ein was purified to homoge neity by heparin-Sepharose and ion-exchange chromatography. Recombinant 24-kD bFGF was similar to recombinant 18- kD bFGF in receptor-binding activity and in inducing cell proliferatio n, plasminogen activator production, and chemotactic movement in cultu red endothelial cells. In agreement with the in vitro observations, 24 -kD bFGF and 18-kD bFGF exerted a similar angiogenic response when ass ayed in vivo in the rabbit cornea. Experiments performed with the radi olabeled molecule demonstrated that 24-kD bFGF has an intrinsic abilit y to bind to high-affinity receptors when added to endothelial GM 7373 cell cultures. Receptor-bound 24-kD bFGF is internalized within the c ell and associates with the nucleus with kinetics similar to 18-kD bFG F. Internalized 24-kD bFGF is first processed to the 18-kD form via a chloroquine-insensitive pathway and then to smaller fragments into the lysosomal compartment. At variance with the data obtained in transfec ted COS-1 cells, only limited amounts of exogenous internalized 24-kD bFGF associates with the nucleus in the intact form, mostly of the nuc lear-bound molecule being represented by the processed 18-kD protein a nd by smaller degradation products. In conclusion, human recombinant 2 4-kD bFGF exerts a biological response in endothelial cells similar to 18-kD bFGF both in vitro and in vivo. Our data point to a different i ntracellular behavior of the high-molecular-weight bFGF isoform when a dded exogenously to cultured cells or when produced endogenously in tr ansfected cells. (C) 1994 Wiley-Liss, Inc.