The role of Src family kinases in cortical cataract formation.
OBJECTIVE
The goal of this study was to determine the role of Src family kinases (SFKs) in the development of lens cataract. This question was particularly significant, because these tyrosine kinases mediate the stress pathways known to lead to cataract formation. The experiments were focused on whether the inhibition of SFK activity suppresses the formation of lens opacities.


METHODS
A whole-lens culture system was developed, in which cortical opacities formed within 5 days, in embryonic day (E)10 lenses grown in medium containing 10% fetal bovine serum. SFK activity was blocked in the cultured lenses by growth in the presence of the SFK-specific inhibitor PP1. Control cultures were grown in medium without inhibitor or in the presence of PP3, the inactive analogue of PP1. Lenses were cultured for 10 days, observed, and photographed daily. Opacification was quantified with image-analysis software. Tissue architecture was determined after hematoxylin and eosin staining and cellular organization by fluorescent localization of filamentous actin with fluorescein-conjugated phalloidin.


RESULTS
Almost all lenses in the control cultures developed cortical opacities covering approximately 50% of the lens area by day 10. Similar to control cultures, PP1-treated lenses showed mild posterior opacities during the first 5 days in culture, but then became strikingly transparent. Only 7% of the PP1-treated lenses showed development of cortical cataract, and the average area of opacity was just 0.5% by culture day 10. In all cultured lenses, even in the presence of the PP1 inhibitor, the bow region of the lens extended to the posterior pole, and distribution of nuclei from the posterior pole toward the anterior aspects of the lens suggested that newly added fiber cells were misdirected. However, neither this feature, nor the presence of vacuoles appeared to correlate with the development of opacity in the cultured lenses. Instead, the lens opacities appeared to result from gross abnormalities in the shape and organization of cells in the equatorial and cortical fiber zones, as observed by F-actin staining. Culturing the lenses in the presence of the SFK inhibitor prevented these lens cell aberrations as well as the development of lens opacity.


CONCLUSIONS
The formation of cataract can involve activation of SFK-mediated pathway(s) leading to disorganization of developing lens fiber cells, and inhibiting these tyrosine kinases blocks cataract progression.
Zhou
Jian
J
Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA.
Menko
A Sue
AS
D000818:Animals; D002386:Cataract / Q000196:embryology / Q000201:enzymology* / Q000517:prevention & control; D002642:Chick Embryo; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007904:Lens Cortex, Crystalline / Q000187:drug effects / Q000196:embryology / Q000201:enzymology*; D023421:Models, Animal; D009924:Organ Culture Techniques; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D013997:Time Factors; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D004791:Enzyme Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D019061:src-Family Kinases