Purpose: In the mammalian lens, the expression of the beta- and gamma-crystallin families is thought to be limited to fiber cells. However, several studies detected these proteins or their mRNAs in human lens epithelial cells. To resolve this apparent discrepancy, 14 crystallin mRNAs were examined and the expression and subcellular distribution of selected crystallin proteins in lens epithelial cells determined.
Methods: Transcript levels were analyzed by quantitative real-time PCR using mRNA from P3 rat lens epithelia cultured for 0 or 20 hours or 4 or 7 days in basal medium or with added FGF2. Antibodies to betaB1-, gammaS-, alphaA-, and alphaB-crystallins were used for Western blot analysis of proteins extracted from adult mouse, human, bovine, rabbit, and rat lens epithelial and fiber cells. Rat lenses or lens epithelia were rapidly fixed in situ, 30 minutes after death, or after dissection from the lens, and the intracellular distributions of crystallins were examined by immunostaining and confocal microscopy.
Results: Four patterns of crystallin gene expression were detected in cultured lens epithelia. Transcripts encoding most beta- and gamma-crystallins were detectable and, in some cases, abundant at the time of explantation. Changes in crystallin protein levels in P3 epithelia cultured in basal or FGF-supplemented medium generally reflected the changes in their mRNAs. betaB1- and gammaS-crystallins were abundant in adult human, mouse, rat, rabbit, and bovine lens epithelial cells. The alpha-, beta- and gamma-crystallins were found in distinct subcellular locations in adult lens epithelial cells. These proteins dramatically relocalized during fiber cell differentiation and after death and/or dissection of the lens epithelium.
Conclusions: BetaB1- and gammaS-crystallins are normally abundant in adult mammalian lens epithelial cells. Complex programs of transcription and degradation regulate the accumulation of crystallin mRNAs in lens epithelial cells after stress, at different ages, and during cell differentiation. Because crystallins selectively localize in distinct subcellular compartments during differentiation or stress, they may function to protect lens cells from injury. After stress, most alphaA- and alphaB-crystallin subunits are not in the same macromolecular complexes.