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Mol Vis. 2000 Nov 6;6:222-31.

Subcellular localization of the expressed 18 kDa FGF-2 isoform in corneal endothelial cells.

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1
Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

Abstract

PURPOSE:

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.

PMID:
11073556
[Indexed for MEDLINE]
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