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A-Expressing Human Lens Epithelial Cell Lines and in A Knockout Mouse Lenses
1 From the Division of Gastrointestinal and Liver Diseases, University of Southern California Keck School of Medicine, Los Angeles; 2 National Eye Institute, Bethesda, Maryland; and 3 Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri.
PURPOSE. To study the mechanism of regulation of GSH in HLE-B3 cells expressing
A-crystallin (A) and in A knockout mouse lenses.
METHODS. GSH levels and maximal rates of GSH synthesis were measured in
immortalized, A-transfected HLE-B3 cells containing varying amounts
of A. The mRNA and protein for the rate-limiting enzyme for GSH
synthesis, 
-glutamylcysteine synthetase (GCS), were also determined
in A- and mock-transfected cells by Northern blot analysis and
Western blot analysis of heavy (GCS-HS) and light (GCS-LS) subunits.
The effect of absence of A and B on lens GSH concentrations was
evaluated in whole lenses of A knockout and B knockout mice as a
function of age. GCS-HS mRNA and protein were determined in young,
precataractous and cataractous A knockout lenses.
RESULTS. GSH levels were significantly higher in HLE-B3 cells expressing A-
compared with mock-transfected cells and were correlated positively
with A content. Mean rate of GSH synthesis was also higher in
A-expressing cells than in mock controls (0.84 vs. 0.61 nmol ·
min-1 per mg protein, respectively). GCS-HS mRNA and
GCS-LS mRNA were approximately twofold higher in A-expressing cells,
whereas the heavy and light GCS subunit proteins increased by 80% to
100%. In A(-/-) mouse lenses, GSH level was not different
from that of wild type up to 2 months from birth, after which it
dropped to 
50% of controls. On the other hand, GCS-HS and GCS-LS
proteins showed a significant decrease before cataract formation as
early as 15 days after birth. GSH level in cataract-free B(-/-)
lenses was similar to that of wild type for up to 14 months.
CONCLUSIONS. Expression of A caused an increase in cellular GSH, in part, because
of an increase in mRNA and protein of both GCS subunits. GSH levels
decreased with increasing age in cataractous A(-/-) lenses but not
in the noncataractous B(-/-) lenses. It is suggested that neonatal
precataractous lenses (with normal GSH and decreased GCS) may maintain
their GSH level by other compensatory mechanisms such as increased GSH
transport.
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