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E.F. Wawrousek, V. Morozov; Secondary Lens Fiber Cell Disintegration in A–/B–Crystallin Double Knockout Mice – The Role of Caspases . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2017.
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© ARVO (1962-2015); The Authors (2016-present)
The lenses of α–crystallin null mice (DKO) are opaque, and secondary fiber cells undergo a disintegration process. We hypothesized that in the normal lens, α–crystallin plays a role in suppressing caspase activity, thus preventing the final steps of the apoptosis–like differentiation process, and maintaining cellular integrity after subcellular organelle destruction. Previously we showed that both Caspase 3 and 6 activities are elevated in DKO lenses compared to wild type (WT).
αA/αB–Crystallin double knockout (DKO) mice were previously generated in this lab, and shown to exhibit disintegrative morphological changes in secondary lens fiber cells and elevated caspase activities. To assess nuclear DNA fragmentation, TUNEL assay was performed on frozen sections of WT and DKO lenses. To assess crystallin binding to executioner caspases, pull–down assays were used. Polyhistidine–tagged caspase 6 and caspase 3 were incubated with lens extract from WT mice, and then bound to cobalt columns. Proteins eluted from the columns were analyzed by western blot using antibodies to αA– and αB–crystallin.
Pull–down assays, demonstrated interaction of caspase 6 with αA–crystallin, but not with αB–crystallin. In this type of assay, caspase 3 shows no interaction with either αA– or αB–crystallin, which is surprising since αB has been shown to bind and inhibit autocatalytic maturation of partially processed procaspase 3. TUNEL labeling revealed a higher level of DNA fragmentation in the secondary fiber lens cells of DKO mice, compared to WT mice.
In normal lenses, α–crystallin interferes with the apoptosis–like maturation program, stopping it after organelle removal, but before cellular disintegration. Our data, along with data of others, suggest that α–crystallin interferes with the caspase cascade. The increased activities of apoptotic executioner caspases in DKO lenses lacking α–crystallin contribute to the observed cell disintegration, which is inhibited by α–crystallin in the normal lens. Moreover, interaction between caspase 6 and αA–crystallin suggests a role for αA–crystallin in regulation of apoptosis.
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