title: Distribution and putative roles of fibroblast growth factor-2 isoforms in corneal endothelial modulation.
abstract: OBJECTIVE
  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.
authors.affiliation: Doheny Eye Institute, Los Angeles, CA 90033, USA.
authors.forename: X
authors.initials: X
authors.lastname: Gu
authors.forename: E P
authors.initials: EP
authors.lastname: Kay
mesh_terms: D000818:Animals; D002455:Cell Division / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000145:classification / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006133:Growth Substances / Q000494:pharmacology; D015151:Immunoblotting; D009504:Neutrophils / Q000502:physiology; D011817:Rabbits; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D006133:Growth Substances; D016222:Fibroblast Growth Factor 2