Processing, Secretion, and Biological Properties of a Novel Growth Factor of the Fibroblast Growth Factor Family with Oncogenic Potential

Abstract

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.

Retention of Fibroblast Growth Factor 3 in the Golgi Complex May Regulate Its Export from Cells

“…FGF3-related products were readily detected in COS-1 cell lysates, but direct immunoblot analysis of the culture medium failed to detect any exported protein (15). In contrast, when COS-1 cells were transfected with Fgf-4 cDNA, substantial amounts of functional protein could be recovered from the culture fluid (12,21,53). Figure la and b illustrates these findings; in this experiment a combination of immunoprecipitation with specific antisera raised against the C-terminal peptides of mouse FGF3 and FGF4 and subsequent immunoblotting with the same sera were used.…”

“…With FGF4, the major intracellular form is a 22-kDa protein, which, after processing and secondary glycosylation, appears in the culture medium as a series of products of around 21.5 to 23.5 kDa (Fig. lb) (12,21,53). Although these multiple extracellular forms of FGF4 have not been fully characterized, they may reflect 0-linked glycosylation, and it has been reported that the protein can undergo further processing at the amino terminus (36).…”

“…The ability to mediate cell transformation is a property shared by most if not all the FGF family, but it is clear that the transforming potential is markedly dependent on secretion (5,12,21,23,28,33,35,45,47,51,59,61). Although most FGFs have hydrophobic domains at their amino termini and undergo cleavage and glycosylation (22), the two prototypes, FGF1 and FGF2, lack conventional signal peptides and are presumably released into the extracellular compartment by some alternative pathway (27,48).…”

“…An obvious explanation would be that FGF3 has a low affinity for the FGF receptor tyrosine kinases expressed by NIH 3T3 cells, hence its negligible mitogenic activity in standard thymidine incorporation assays. Transformation might therefore require a higher local concentration of FGF3 than, for example, of FGF4, which is a potent mitogen for NIH 3T3 and other cell types (12,15,21,44,53). Alterna-tively, FGF3 might be less efficiently secreted, making it less likely to reach the threshold necessary to trigger the transmembrane receptors.…”

The Int-2/Fgf-3 Oncogene Product Is Secreted and Associates with Extracellular Matrix: Implications for Cell Transformation

“…Such negative results were obtained by using different experimental protocols and different polyclonal antisera, so that the absence of immunoreactive material was unlikely to be attributable to a technical artifact or masking of epitopes. While it is conceivable that the secreted protein is very rapidly degraded or quantitatively sequestered in the extracellular matrix (ECM), other members of the FGF family behave more conventionally and can be detected in conditioned medium by immunological or functional assays (7,19,20,30,37). Since there is increasing evidence that FGFs transform cells via an extracellular autocrine loop (13,30), we felt that it was essential to reevaluate the secretion and subsequent fate of To facilitate these analyses, we focused on two clonal lines of NIH3T3 cells, designated DMI-1 and DMI-3, that produce high levels of Int-2 (14).…”