A comparative analysis of alphaA- and alphaB-crystallin expression during the cell cycle in primary mouse lens epithelial cultures.
AlphaA- and alphaB-crystallins are small heat shock proteins and molecular chaperones that prevent non-specific aggregation of denaturing proteins. Previous work in our laboratory has shown that lens epithelial cells derived from alphaA-/- mice exhibit slower growth, whereas alphaB-/- lens epithelial cells hyperproliferate at a higher rate in culture [Andley et al., J. Biol. Chem. 273 (1998) 31252; FASEB J. 15 (2001) 221]. Although both have been implicated in apoptosis and cell proliferation, direct analysis of their expression during the cell cycle has not been investigated. This study was undertaken to define the expression levels of alphaA and alphaB-crystallins during the cell cycle. Primary lens epithelial cell cultures derived from wild type mice were synchronized by serum starvation, and pulsed with bromodeoxyuridine (BrdU) at different times after re-stimulation with serum. Dual parameter flow cytometric studies with BrdU and propidium iodide (PI)-labeled cells were performed. Cells entered S phase 14 hr after serum re-stimulation. The duration of the S phase was 6 hr, and the total cell cycle transit time was between 24-27 hr. Enhanced expression of cyclin A, a protein essential for DNA synthesis was used as an additional marker to define the initiation of the S phase. Immunoblotting analysis demonstrated that the expression of alphaA and alphaB-crystallin was up to 10-fold higher in cells synchronized in G0 phase than in G1 phase. The levels of the proteins increased three-fold again as the cells entered the S phase and progressed to mitosis, but did not rise to the levels observed in G0 phase. This increase in expression of alphaA-crystallin resulted in part from enhanced synthesis during the S phase, as shown by an increase in [35S]methionine-labeling and immunoprecipitation of the radiolabeled alphaA-crystallin. The results were further confirmed by flow cytometric analysis using DNA content and alphaA-crystallin expression. The increase in alphaB-crystallin in S phase was paralleled by an increase in gene expression as shown by real-time RT-PCR analysis. These results demonstrate for the first time that in lens epithelial cells, alphaA and alphaB-crystallin levels are modulated during the cell cycle. Since the absence of alphaA and alphaB- crystallin in lens epithelial cells has been associated with disturbance of the tubulin cytoskeleton during mitosis, and with increased cell death or genomic instability, our results indicating that the alphaA- and alphaB-crystallin expression increases prior to mitosis are significant. The differential expression of these crystallins in the cell cycle may be important for optimal lens epithelial growth and lens transparency.
Bai
Fang
F
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8096, St Louis, MO 63110, USA.
Xi
Jinghua
J
Higashikubo
Ryuji
R
Andley
Usha P
UP
D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D002478:Cells, Cultured; D019925:Cyclin A / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D007903:Lens Capsule, Crystalline / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D016192:Resting Phase, Cell Cycle / Q000502:physiology; D016196:S Phase / Q000502:physiology; D038202:alpha-Crystallin A Chain / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000378:metabolism*
D019925:Cyclin A; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain