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G.F. Weber, A.S. Menko; Apoptosis–Related Bcl–2 and Caspase–Dependent Pathways Signal Initiation of Lens Differentiation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2408.
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Purpose: Previously, we have shown expression and activation of pro– and anti–apoptotic pathways in the chick embryo lens temporally precedes nuclear and organelle loss by two days. In this study, we have investigated the function of these apoptotic–related pathways as an initiating signal for lens cell differentiation. Methods: Studies used microdissected chick lenses or primary quail lens cultures. Caspase activity was analyzed using a commercial kit. Apoptotic–related pathways were induced with either staurosporine or anisomyosin, and Z–VAD–FMK was used to inhibit caspases. Fluorescence labeling of F–actin and DNA were used to examine cell morphology. Lens differentiation markers were examined by immunoblot analysis. Results: Although caspases signal organelle and nuclear loss in the lens, these proteases are also necessary for the differentiation of cells that do not lose subcellular structures. We have found that caspase 3–like activity in the E10 chick embryo lens peaks in the equatorial epithelium far before the development of an organelle free zone at E12. The level of caspase 3–like activity involved in differentiation was lower than occurs in apoptotic cells. This led us to investigate whether the mitochondrial death pathway signals the differentiation of lens epithelial cells into fiber cells. Inhibition of endogenous caspases in lens cell cultures attenuated the formation of differentiated lentoid structures. Most significantly, lens epithelial cells were induced to differentiate by exposure to the apoptogen staurosporine. Short term (4 hr) incubation with 250nM staurosporine initially caused cell contraction, but the cells quickly recovered, Δ–crystallin expression was induced, and 100% of the cells formed lentoids. Lower concentrations of staurosporine (50nM) not only induced expression of Δ–crystallin but also caused elongation of lens epithelial cells, their nuclei and actin filaments, similar to the phenotype of cortical fiber cells in the embryonic lens. Conclusions: We have shown that low level activation of apoptosis–related pathways is necessary and sufficient to induce differentiation of lens epithelial cells. Our findings demonstrate a distinct non–apoptotic role for the canonical mitochondrial death pathway in signaling the differentiation of lens cells.
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