Subcellular localization of the expressed 18 kDa FGF-2 isoform in corneal endothelial cells.
OBJECTIVE
To determine the subcellular localization of 18 kDa FGF-2 after synthesis and before secretion into the extracellular matrix.


METHODS
Corneal endothelial cells (CEC) were transfected with an expression vector coding for green fluorescent protein (GFP) and 18 kDa FGF-2. Expression of the fusion protein was determined by immunoblot analysis and the subcellular localization of the fusion protein was examined by immunocytochemical analysis.


RESULTS
When the expression of the fusion protein was determined by immunoblot analysis, the expressed fusion protein had a molecular weight of 45 kDa, resulting from the 27 kDa GFP and 18 kDa FGF-2. Following a 90 min exposure of cells to the vector, the expressed 18 kDa FGF-2 was completely translocated to the nucleus within a 24 h incubation. When cells were further incubated for another 24 h, one-half of the fusion protein was retro-transported from the nucleus to the cytoplasm, largely to the membrane and focal adhesion site, while the other half remained in the nucleus. During a 72 h incubation, the fusion protein was completely translocated to the cytoplasm, where it was diffusely distributed and its staining potential was greatly lost. Transfected cells showed both a slight increase in cell proliferation and a down-regulation in the expression of the high affinity receptors of FGF.


CONCLUSIONS
These results indicate that the 18 kDa FGF-2 is directly translocated from its synthetic site to the nucleus. The nuclear 18 kDa FGF-2 is then retro-transported to membrane/focal adhesion sites, after which the molecule may be secreted. When 18 kDa FGF-2 remains in the nucleus, there is a slight stimulatory activity of cell proliferation and a down-regulation of its receptor. These data suggest an intracellular action of 18 kDa FGF-2 through mechanisms independent of the receptor-mediated signaling pathways.
Choi
J
J
Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
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M K
MK
Kay
E P
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D000818:Animals; D001693:Biological Transport, Active; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008970:Molecular Weight; D008979:Moloney murine leukemia virus / Q000235:genetics; D020033:Protein Isoforms / Q000378:metabolism; D011817:Rabbits; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions; D014162:Transfection
D008164:Luminescent Proteins; D020033:Protein Isoforms; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins