Growth factor receptor signalling in human lens cells: role of the calcium store

Abstract

In the human lens, stimulation of tyrosine-kinase coupled growth factor receptors such as epidermal growth factor receptor (EGFR) can induce calcium release from endoplasmic reticulum (ER) stores. The present study investigated the impact of calcium store inactivation on EGFR signalling, cell growth and death in a well-characterised human lens cell line (FHL124). FHL124 cells were routinely cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% foetal calf serum (FCS) and seeded on 24-well plates (DNA and protein synthesis), tissue culture dishes (growth assay, western immunoblot), and glass coverslips (immunocytochemistry). DNA and protein synthesis rates were quantified by measuring the incorporation of 3H-thymidine and 35S-methionine into FHL124 cells in serum-free EMEM or EMEM supplemented with thapsigargin (Tg) (100 n m and 1 μ m). Longer-term growth was assessed by quantifying the increase in area over time of a circular patch of seeded cells. EGFR was identified using anti-EGFR mouse monoclonal antibody and visualised by fluorescence microscopy with ALEXA 488 conjugated secondary antibody. Programmed cell death was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay method. Activation of the mitogen-activated protein kinase (MAPK) signalling protein extracellular signal-regulated kinase (ERK) and the cell cycle proteins CDK2 and P27 kip1 were detected by western immunoblot techniques. Inactivation by ≥100 n m Tg inhibited both protein and DNA synthesis although the effect on the latter was greatest. The cell cycle activator CDK2 was reduced by Tg, while the inhibitor P27 kip1 was increased along with the percentage of apoptotic cells. A single, maximal epidermal growth factor (EGF) (10 ng ml −1) exposure induced receptor internalization and increased ERK phosphorylation. Both internalisation and ERK activation were unaffected by the presence of Tg. However, reduced internalisation and ERK activation followed repeated EGF applications in the presence of Tg. Additionally, ERK activation by submaximal EGF concentrations was reduced by store depletion. An intact endoplasmic reticulum calcium store therefore plays a significant role in human lens cell survival and growth.