Stress Responses in Myotonic Dystrophy Lens Cells Involve FGFR Activation and an Increased Ca2+ Influx

Abstract

Purpose:: Although cataract is a characteristic feature of myotonic dystrophy type 1 (DM), little is known of the underlying mechanisms. Recent work has shown that DM1 lens cells have reduced growth and an impaired ability to survive stress (Human Molecular Genetics, Vol. 15, 2006). We have therefore investigated the mechanisms of proliferation and cell death in lens cell lines derived from cataract patients with and without DM.

Methods:: A total of 4 cell lines were derived by SV40 transformation of lens capsulorhexis specimens obtained immediately following cataract surgery. Rhexis samples were collected in culture medium and cells were infected with adenovirus carrying SV40 large T. Two age matched lines from routine cataracts (CCat1 and 2) and two from DM cataracts (DMCat1 and 2) were used in this study. Cells were counted at each passage using a haemocytometer. Cell growth and proliferation were estimated using a total protein assay (BCA). Cell death was assessed by measuring the release of LDH. Conditioned media (CM) were harvested from both cell types at regular intervals and their effect on cell growth was determined using the untransformed lens cell line FHL-124. Levels of pFGFR and pERK were measured by Western blotting. The calcium responses elicited on exposure of FHL-124 cells to the CM were monitored by confocal microscopy on Fluo-4 loaded cells.

Results:: Surprisingly, CM from the DM cell lines induced a greater growth stimulation in FHL-124 cells than the corresponding CM from the two control cell lines. This was inhibited by SU5402 and Wortmanin. Western blotting showed that CM also induced phosphorylation of FGFR and increased the level of phosphor-ERK. CM from the DM cell lines induced an increase in intracellular calcium that was greater than that produced by the control cells. The Ca²⁺ increase persisted in the presence of thapsigargin but was abolished in the absence of external Ca²⁺, indicating the involvement of channel activation. The cell lines that showed the greatest growth stimulation also induced the largest Ca²⁺ influx.

Conclusions:: Under stress conditions, induced by serum deprivation, both DM and control cells produce survival factors that induce FGFR phophorylation and Ca²⁺ channel activation. Despite producing a greater amount of survival factors this is not sufficient to overcome the stress from the increased CTG repeat load in the DM cells.

Keywords: cataract • growth factors/growth factor receptors • calcium