X Gu1, E P Kay. 1. Doheny Eye Institute, Los Angeles, CA 90033, USA.
Abstract
PURPOSE: Corneal endothelial modulation factor (CEMF) released by inflammatory cells induces de novo synthesis of fibroblast growth factor (FGF)-2, which is a morphogen and a potent mitogen of corneal endothelial cells (CECs). Four isoforms of FGF-2 have been found in the nucleus, cytoplasm, or extracellular matrix (ECM) in different cell lines. In the present study, the profiles of the isoforms of FGF-2 that are induced by CEMF were investigated, and whether the differential localization of the isoforms of FGF-2 plays a role in CECs proliferation and subsequent modulation was examined. METHODS: Nuclear, cytoplasmic, and ECM fractions of normal and modulated CECs were separated, and FGF-2 isoforms were further purified by heparin-Sepharose column. The molecular sizes of the isoforms were determined by immunoblot analysis, using a specific antibody directed against FGF-2. Cell proliferation was determined by cell counting. Cellular localization of FGF-2 was determined by immunofluorescence staining during different stages of cell growth. RESULTS: To confirm that CEMF modulated CECs under the conditions used in this study, its effect on cell proliferation and cell shape was determined: CEMF-treated cells showed enhanced cell proliferation profiles and fibroblastlike morphology. In rapidly growing normal CECs, FGF-2 was predominantly present in the nucleus. As the cells reached confluence, the staining potential in the nucleus was markedly reduced. Cytoplasmic staining of FGF-2 was barely detectable, regardless of cell stages. In CEMF-modulated cells, the rapidly growing cells showed strong staining of FGF-2 in the nucleus, whereas cytoplasmic and ECM staining was weak. When modulated cells reached confluence, the staining of FGF-2 in the nuclei remained strong, whereas ECM staining was significantly increased. Immunoblot analysis of the subcellular fraction showed that the 24-kDa FGF-2 was predominantly present in the nucleus, whereas the 18-kDa form was the major molecule in cytoplasmic and ECM fractions in normal and modulated cells. CONCLUSIONS: These findings indicate that 24-kDa nuclear FGF-2 may be involved in cell proliferation in growing CECs. The persistent nuclear localization and simultaneous ECM localization of FGF-2 are induced by CEMF, and these FGF-2 isoforms seem to play a role in cell proliferation and modulation.
PURPOSE: Corneal endothelial modulation factor (CEMF) released by inflammatory cells induces de novo synthesis of fibroblast growth factor (FGF)-2, which is a morphogen and a potent mitogen of corneal endothelial cells (CECs). Four isoforms of FGF-2 have been found in the nucleus, cytoplasm, or extracellular matrix (ECM) in different cell lines. In the present study, the profiles of the isoforms of FGF-2 that are induced by CEMF were investigated, and whether the differential localization of the isoforms of FGF-2 plays a role in CECs proliferation and subsequent modulation was examined. METHODS: Nuclear, cytoplasmic, and ECM fractions of normal and modulated CECs were separated, and FGF-2 isoforms were further purified by heparin-Sepharose column. The molecular sizes of the isoforms were determined by immunoblot analysis, using a specific antibody directed against FGF-2. Cell proliferation was determined by cell counting. Cellular localization of FGF-2 was determined by immunofluorescence staining during different stages of cell growth. RESULTS: To confirm that CEMF modulated CECs under the conditions used in this study, its effect on cell proliferation and cell shape was determined: CEMF-treated cells showed enhanced cell proliferation profiles and fibroblastlike morphology. In rapidly growing normal CECs, FGF-2 was predominantly present in the nucleus. As the cells reached confluence, the staining potential in the nucleus was markedly reduced. Cytoplasmic staining of FGF-2 was barely detectable, regardless of cell stages. In CEMF-modulated cells, the rapidly growing cells showed strong staining of FGF-2 in the nucleus, whereas cytoplasmic and ECM staining was weak. When modulated cells reached confluence, the staining of FGF-2 in the nuclei remained strong, whereas ECM staining was significantly increased. Immunoblot analysis of the subcellular fraction showed that the 24-kDa FGF-2 was predominantly present in the nucleus, whereas the 18-kDa form was the major molecule in cytoplasmic and ECM fractions in normal and modulated cells. CONCLUSIONS: These findings indicate that 24-kDa nuclear FGF-2 may be involved in cell proliferation in growing CECs. The persistent nuclear localization and simultaneous ECM localization of FGF-2 are induced by CEMF, and these FGF-2 isoforms seem to play a role in cell proliferation and modulation.