Investigative ophthalmology & visual science · 2000
Hepatocyte growth factor function and c-Met expression in human lens epithelial cells.
СтатьяПоддержка исследования: вне правительства СШАПоддержка службы здравоохранения СШАНазвание и аннотация: Перевод в очереди
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Wormstone I M, Tamiya S, Marcantonio J M, Reddan J R
Аннотация
PURPOSE: Hepatocyte growth factor (HGF) and its receptor c-met perform a multitude of functions. However, despite the significant degree of study of HGF and c-met in numerous tissues and cell types, relatively few investigations have been performed on the lens. In the current study, therefore, the role of HGF and the receptor c-met in human lens epithelial cells was investigated.
METHODS: Anterior epithelium and capsular bags were prepared from human donor eyes and maintained in Eagle's minimum essential medium (EMEM) in a 5% CO(2) atmosphere at 35 degrees C. In addition, the human lens cell line FHL124, was routinely cultured and seeded onto glass coverslips (c-met immunodetection), 12-well plates (DNA and protein synthesis), and tissue culture dishes (migration). c-Met was detected by immunocytochemistry and fluorescence-activated cell scanning (FACS). HGF was measured using enzyme-linked immunosorbent assay (ELISA) techniques. Proliferation and protein synthesis were determined by [(3)H]thymidine and (35)S-methionine incorporation into DNA and proteins, respectively. Migration was assessed using a scratch-wound assay and time-lapse video microscopy.
RESULTS: HGF was detected at all stages of culture of capsular bags in protein-free medium. Moreover, c-met was present on the native epithelium and after mechanical trauma was seen to be upregulated. Immunolocalization and FACS analysis demonstrated c-met expression on FHL124 cells throughout the whole population. Furthermore, FACS analysis showed that serum-maintained cells sustained a higher level of receptor expression relative to serum-deprived cells. Additionally, HGF was found to stimulate proliferation, protein synthesis, and migratory responses.
CONCLUSIONS: c-Met receptors are expressed in native epithelium, capsular bag cultures, and FHL124 cells. Receptor is distributed across the entire cell population; however, this expression is environmentally and mechanically sensitive. HGF is also present in capsular bags at all stages of culture. In addition, HGF can stimulate migration, proliferation, and protein synthesis. It therefore appears that a multifunctional autocrine loop involving HGF and c-met is in place and could be important in the development of posterior capsule opacification.
METHODS: Anterior epithelium and capsular bags were prepared from human donor eyes and maintained in Eagle's minimum essential medium (EMEM) in a 5% CO(2) atmosphere at 35 degrees C. In addition, the human lens cell line FHL124, was routinely cultured and seeded onto glass coverslips (c-met immunodetection), 12-well plates (DNA and protein synthesis), and tissue culture dishes (migration). c-Met was detected by immunocytochemistry and fluorescence-activated cell scanning (FACS). HGF was measured using enzyme-linked immunosorbent assay (ELISA) techniques. Proliferation and protein synthesis were determined by [(3)H]thymidine and (35)S-methionine incorporation into DNA and proteins, respectively. Migration was assessed using a scratch-wound assay and time-lapse video microscopy.
RESULTS: HGF was detected at all stages of culture of capsular bags in protein-free medium. Moreover, c-met was present on the native epithelium and after mechanical trauma was seen to be upregulated. Immunolocalization and FACS analysis demonstrated c-met expression on FHL124 cells throughout the whole population. Furthermore, FACS analysis showed that serum-maintained cells sustained a higher level of receptor expression relative to serum-deprived cells. Additionally, HGF was found to stimulate proliferation, protein synthesis, and migratory responses.
CONCLUSIONS: c-Met receptors are expressed in native epithelium, capsular bag cultures, and FHL124 cells. Receptor is distributed across the entire cell population; however, this expression is environmentally and mechanically sensitive. HGF is also present in capsular bags at all stages of culture. In addition, HGF can stimulate migration, proliferation, and protein synthesis. It therefore appears that a multifunctional autocrine loop involving HGF and c-met is in place and could be important in the development of posterior capsule opacification.