Abstract
Purpose. The lens epithelium can be separated into two regions, the nondividing central zone and the equator, the site of all division in the normal lens. In the present study, the distribution of epithelial growth factor (EGF)/epithelial growth factor receptor (EGFR) signaling components was investigated and related to mitotic distribution in the lens.
Methods. Anterior and equatorial regions of the native epithelium were prepared separately from donor lenses. In vitro capsular bags were prepared from donor eyes and cultured. Receptor distribution was determined by immunocytochemistry and RT-PCR. Western blot analysis of phospholipase C (PLC)-γ and extracellular signal-regulated kinase (ERK; total and active) was performed on cell lysates. Function was determined by calcium imaging of Fura-2-AM–loaded cells and also, in the case of capsular bags, by cell growth.
Results. Immunocytochemistry and RT-PCR showed an even distribution of EGFR across the native epithelium. Whole lenses, however, exhibited only a calcium response to EGF (10 ng/mL) at the equatorial region. Western blot analysis demonstrated significantly greater expression of PLCγ and ERK (total and active) in the equator than in the central region. Addition of EGF increased growth rates of cells in capsular bags and an EGFR inhibitor decreased rates. EGF also induced a calcium response in posterior capsule cells in the capsular bags.
Conclusions. EGFR is evenly distributed across the entire epithelium, whereas related calcium signaling and expression of PLCγ and ERK have a marked bias to the equator. Therefore, levels of downstream enzyme components rather than changes in receptor expression dictate EGFR signaling output in the normal lens. In the wounded lens (capsular bag) EGFR signaling persists in cells growing on the posterior capsule.
Methods. Anterior and equatorial regions of the native epithelium were prepared separately from donor lenses. In vitro capsular bags were prepared from donor eyes and cultured. Receptor distribution was determined by immunocytochemistry and RT-PCR. Western blot analysis of phospholipase C (PLC)-γ and extracellular signal-regulated kinase (ERK; total and active) was performed on cell lysates. Function was determined by calcium imaging of Fura-2-AM–loaded cells and also, in the case of capsular bags, by cell growth.
Results. Immunocytochemistry and RT-PCR showed an even distribution of EGFR across the native epithelium. Whole lenses, however, exhibited only a calcium response to EGF (10 ng/mL) at the equatorial region. Western blot analysis demonstrated significantly greater expression of PLCγ and ERK (total and active) in the equator than in the central region. Addition of EGF increased growth rates of cells in capsular bags and an EGFR inhibitor decreased rates. EGF also induced a calcium response in posterior capsule cells in the capsular bags.
Conclusions. EGFR is evenly distributed across the entire epithelium, whereas related calcium signaling and expression of PLCγ and ERK have a marked bias to the equator. Therefore, levels of downstream enzyme components rather than changes in receptor expression dictate EGFR signaling output in the normal lens. In the wounded lens (capsular bag) EGFR signaling persists in cells growing on the posterior capsule.
| Original language | English |
|---|---|
| Pages (from-to) | 1427-1435 |
| Number of pages | 9 |
| Journal | Investigative Ophthalmology and Visual Science |
| Volume | 45 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - 2004 |