Abstract
Abstract: :
Purpose: In the lens, the ß– and γ–crystallins are thought to be expressed exclusively in fiber cells, although recent studies have detected these proteins and their mRNAs in the lens epithelium. To resolve their expression and possible function, we quantified all crystallin mRNAs in lens epithelial cells and examined the expression of ßB1–, γS–, αA–, and αB–crystallins in epithelial explants and during postnatal development. Methods:Transcript levels were analyzed by quantitative PCR using mRNA from P3 rat lens epithelia cultured for 0, 20 hr, 4 or 7 days in defined medium or with FGF2 (100 ng/ml). ßB1–, γS–, αA– and αB–crystallins from rat, mouse, human, bovine and rabbit lens epithelial and fiber cells were quantified by Western blotting. Rat lens epithelia were fixed immediately or 15 minutes after death and the intracellular distribution of crystallins was examined by immunostaining and confocal microscopy. Results: Four distinct patterns of crystallin gene expression were detected in cultured lens epithelia. Transcripts encoding γA–E and ßA1/3, ßA4 and ßB1–3 were detectable (sometimes abundant) at explantation (T0), decreased during culture in defined medium and increased greatly after treatment for 4 or 7 days with FGF2. Both ßA2– and γS–crystallin mRNAs were low at T0, increased substantially in defined medium and even more after treatment with FGF2. Transcripts encoding αA– and αB–crystallins were both high at T0, but αA mRNA declined in defined medium and increased in FGF2, while αB mRNA increased in defined medium and declined after treatment with FGF2. γS–crystallin levels increased significantly after culture for 4 or 7 days and increased linearly in both epithelial and fiber cells during postnatal development. In contrast, ßB1–crystallin levels were unchanged in cultured epithelia, but increased postnatally. ßB1– and γS–crystallins were abundant in adult human, mouse, rat, rabbit and bovine lens epithelia. In adult rat lens epithelial cells, αA–, αB–, and ßB1–crystallins localized mostly to the cytoplasm, while γS–crystallin was mostly nuclear. Within 15 min after death, αB– and ßB1–crystallins were mostly nuclear and γS–crystallin nearly all nuclear, while αA–crystallin remained in the cytoplasm. Conclusions: ßB1– and γS–crystallin are normally abundant in mammalian lens epithelial cells; other ß– and γ–crystallins are probably present there as well. The expression patterns of crystallin mRNAs in lens epithelial cells are complex and change differentially with stress (culture), developmental age, and differentiation. Crystallins selectively localize to the nucleus after stress.
Keywords: crystallins • stress response • gene/expression