Quantitative export of FGF‐2 occurs through an alternative, energy‐dependent, non‐ER/Golgi pathway

Abstract

Although basic fibroblast growth factor (bFGF/FGF-2) is found outside cells, it lacks a conventional signal peptide sequence; the mechanism underlying its export from cells is therefore unknown. Using a transient COS-1 cell expression system, we have identified a novel membrane-associated transport pathway that mediates export of FGF-2. This export pathway is specific for the 18-kD isoform of FGF-2, is resistant to the anti-Golgi effects of Brefeldin A, and is energy-dependent. In FGF-2-transfected COS-1 cells, this ER/Golgi-independent pathway appears to be constitutively active and functions to quantitatively export metabolically-labeled 18-kD FGF-2. Co-transfection and co-immunoprecipitation experiments, using a vector encoding the cytoplasmic protein neomycin phosphotransferase, further demonstrated the selectivity of this export pathway for FGF-2. When neomycin phosphotransferase was appended to the COOH-terminus of 18-kD FGF-2, the chimera was exported. However, the transmembrane anchor sequence of the integral membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) blocked export. The chimeric protein localized to the plasma membrane with its FGF-2 domain extracellular and remained cell-associated following alkaline carbonate extraction. Taken together, the data suggest that FGF-2 is "exported" from cells via a unique cellular pathway, which is clearly distinct from classical signal peptide-mediated secretion. This model system provides a basis for the development and testing of therapeutic agents which may block FGF-2 export. Such an intervention may be of considerable use for the treatment of angiogenesis-dependent diseases involving FGF-2.

Angiogenesis induced in muscle by a recombinant adenovirus expressing functional isoforms of basic fibroblast growth factor

“…The underlying export mechanism of aFGF, bFGF and FGF-9 is not well known. Florkiewicz et al 27 have…”

Delivery of FGF-2 but not VEGF by encapsulated genetically engineered myoblasts improves survival and vascularization in a model of acute skin flap ischemia

“…Natural release of FGF-2 from cells in the absence of a signal peptide has been shown to occur using a unique export pathway which is independent of the ER/Golgi system. 41 In vitro bioactivity of VEGF and FGF-2 produced by C 2 C 12 cells was assessed in cocultures with BME cells in a three-dimensional collagen gel system. This system assays directly for cytokine-induced angiogenic activity, since the target cells are quiescent endothelial cells, which, in the absence of an angiogenic stimulus, do not invade the collagen gel or form tube-like structures.…”

Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.

“…Standards for the multiple human and rodent FGF-2 isoforms were prepared from extracts of COS-1 cells transfected with expression vectors encoding human or rat FGF-2 as previously described (Florkiewicz and Sommer, 1989 (Florkiewicz et al, 1995). Protein concentrations were determined using the BCA protein assay reagent (Pierce Chemical, Rockford, IL).…”

“…The high molecular weight FGF-2 isoforms localize to the nucleus while the 18-kDa isoform is predominantly cytoplasmic or extracellular (Brigstock et al, 1990;Florkiewicz et al, 1991a). The 18-kDa isoform is believed to be exported independently of the ER golgi pathway (Florkiewicz et al, 1995) and predominates in FGF-2 bound to the extracellular matrix (Brigstock et al, 1991). The nuclear forms of FGF-2 can also be phosphorylated, leading to consideration of FGF-2 as a transacting nuclear factor, regulated by phosphorylation/dephosphorylation (Vilgrain and Baird, 1991).…”