Purpose: EGF has been identified as a major signalling factor in the human lens. The present study investigated the role of downstream ERK proteins in EGF signalling responses in human lens epithelial cells. Methods: The human lens cell line FHL 124 was routinely cultured and seeded onto glass coverslips or 35 mm culture dishes. Anterior and equatorial regions of the native human lens epithelium were prepared separately from human donor lenses by carefully dissecting and removing the central region. Western blot analysis of total and phosphorylated ERK (pERK) was carried out on cell lysates. Immunocytochemistry was also used to assess pERK intracellular distribution patterns. Results: Western blot analysis showed that addition of 10ng/ml EGF to FHL 124 cells induced a significant increase in pERK levels. The maximum levels were found between 10 and 60 minutes. Immunocytochemistry revealed that at 10 minutes the distribution was largely nuclear, while at 30 minutes pERK could be seen throughout the cell. Pre-incubation of FHL 124 cells with the specific EGF receptor inhibitor AG1478 was found to ablate the EGF-induced increase in pERK. In the anterior and equatorial epithelia the background level of pERK showed regional differences, such that greatest levels were seen in the equatorial region. A similar distribution pattern was also observed with the total levels of ERK. When stimulated with 10ng/ml EGF an increase of pERK was observed in both regions. Conclusion: Both FHL 124 cells and native lens epithelial cells respond to EGF by inducing phosphorylation of ERK proteins. Native epithelial cells demonstrate intrinsic ERK phosphorylation. Levels are lowest in the anterior epithelium and highest in the equatorial region and these differences correlate with availability of signalling molecules. The levels in both regions can be further elevated by exposure to EGF.
|Journal||Investigative Ophthalmology and Visual Science|
|Publication status||Published - 2002|