The Release of Fibroblast Growth Factor-1 from NIH 3T3 Cells in Response to Temperature Involves the Function of Cysteine Residues (*)

Fibroblast growth factor (FGF)-1 is released from NIH 3T3 cells in response to heat shock as a biologically inactive protein that is unable to bind heparin and requires activation by (NH4)2SO4 to generate a biologically active extracellular heparin-binding growth factor (Jackson, A., Friedman, S., Zhan, X., Engleka, K. A., Forough, R., and Maciag, T.(1992) Proc. Natl. Acad. Sci. USA 89, 10691-10695). To further study the mechanism of FGF-1 release in response to heat shock (42°C), we examined the kinetics of FGF-1 release from FGF-1-transfected NIH 3T3 cells and observed that the cells require at least 1 h of exposure to heat shock conditions for the release of FGF-1. Interestingly, agents that interfere with the function of the endoplasmic reticulum-Golgi apparatus, exocytosis, and the multidrug resistance pathway (brefelden A, methylamine, and verapamil, respectively) do not inhibit the release of FGF-1 in response to temperature; rather, they exaggerate the release of FGF-1. Because immunoblot analysis of FGF-1 in the conditioned medium of heat-shocked NIH 3T3 cells revealed the presence of a minor band with an apparent molecular weight of a FGF-1 homodimer and because we have previously shown that FGF-1, but not FGF-2, is able to form a homodimer in response to chemical oxidation by CuCl2 (Engleka, K. A., and Maciag, T.(1992) J. Biol. Chem. 267, 11307-11315), we examined whether reducing agents would substitute for (NH4)2SO4 and activate extracellular FGF-1. Indeed, dithiothreitol and reduced glutathione are able to individually generate a FGF-1 monomer as a heparin-binding protein from the conditioned medium of heat-shocked NIH 3T3 cell transfectants. To confirm that cysteine residues are involved in the release of FGF-1 in response to temperature, we used mutagenesis to prepare a human FGF-1 Cys-free mutant in which Cys, Cys, and Cys were converted to serine. Analysis of the release of the FGF-1 Cys-free mutant in NIH 3T3 cells transfected with the FGF-1 Cys-free mutant demonstrated that the FGF-1 Cys-free mutant is not released into the conditioned medium in response to temperature. Interestingly, exposure of the NIH 3T3 cell FGF-1 Cys-free transfectants to brefelden A followed by heat shock also demonstrated the absence of the extracellular FGF-1 Cys-free mutant. Finally, ion-exchange and reverse-phase chromatographies of heat-shocked conditioned medium analyzed by FGF-1 immunoblot analysis were able to resolve FGF-1 as a homodimer under nonreducing conditions and as a monomer under reducing conditions. These data demonstrate that FGF-1 utilizes cysteine residues as an important component of its release from NIH 3T3 cells in vitro in response to temperature and exits the cell as a biologically inactive homodimer with reduced heparin affinity that requires activation by reducing agents to generate heparin binding and biological activities.