Loss of thiol repair systems in human cataractous lenses.

PURPOSE: The purpose of this study was to investigate the thiol repair systems of thioltransferase (TTase) and thioredoxin (Trx) and oxidation-damaged proteins in human cataractous lenses. METHODS: Cataractous lenses in humans (57-85 years of age) were classified into cortical, nuclear, mixed, mature, and hypermature cataract types by using a lens opacity classification system, and were obtained by extracapsular cataract extraction (ECCE) procedure. Cortical and nuclear cataracts were grouped by decreasing order of visual acuity into optical chart reading (R), counting fingers (CF), hand motion (HM), and light perception (LP). ECCE lens homogenate was analyzed for glutathione (GSH) level and enzyme activities of TTase, glutathione reductase (GR), Trx, and thioredoxin reductase (TR). Cortical and nuclear cataractous lenses (8 of each) with visual acuity better than HM were each dissected into cortical and nuclear portions for measurement of glyceraldehyde 3-phosphate dehydrogenase (G3PD) activity. Clear lenses (in humans 49-71 years of age) were used as control. RESULTS: Compared with control, all cataractous lenses lost more than 80% GSH and 70% GR; TR and Trx activity; and 40% to 70% TTase activity, corroborated with the loss in visual acuity. Among cataracts with R and CF visual acuity, cortical cataract lost more cortical G3PD activity (18% of control) than that of nuclear cataract (50% of control), whereas GSH depletion and TTase inactivation were similar in both cataracts. CONCLUSIONS: Thiol repair systems were damaged in all types of cataracts. Cortical and nuclear cataracts showed differential G3PD inactivation in the cortex, implying those 2 type of cataracts might be formed through different mechanisms.

Regulation of cytosolic phospholipase A2 (cPLA2alpha) and its association with cell proliferation in human lens epithelial cells.

PURPOSE: To investigate the molecular mechanism for cytosolic phospholipase A2 (cPLA(2)α) regulation and its association to platelet-derived growth factor (PDGF)-induced cell proliferation. METHODS: cPLA(2)α was examined using human lens epithelial (HLE) B3 cells. Reactive oxygen species (ROS) generation induced by PDGF was analyzed by luminescence assay. Cell proliferation was measured by cell counting and by BrdU assay. Human cPLA(2)α gene was cloned via RT-PCR followed by site-directed mutagenesis to construct HLE B3 cells expressing either inactive cPLA(2)α enzyme with S228A mutation (S228A), or cPLA(2)α truncated at the calcium-binding C2 domain (C2D). Activity of cPLA(2)α was measured by arachidonic acid (AA) release from cell membranes using [(3)H]-arachidonic acid prelabeled cells. The effect of intracellular calcium level on cPLA(2)α function was examined by treating cells with ionomycin (calcium influx), thapsgargin (endoplasmic reticulum [ER] calcium store release) or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA; calcium chelator). Activation of extracellular signal-regulated kinases (ERK), JNK, p38, or Akt was detected by Western blot analysis using specific antibodies. RESULTS: S228A mutant showed suppressed PDGF-induced reactive oxygen species generation, ERK and JNK activation (no effect on p38 or Akt), and cell proliferation in comparison with the vector alone (Vec) control. Calcium-binding C2 domain cells lost the ability of membrane translocation and activation of cPLA(2)α. PDGF cell signaling was calcium-dependent, and the calcium was supplied either from the external flux or endoplasmic reticulum store. However, enrichment of cellular calcium not only augmented PDGF function, but also demonstrated a cPLA(2)α-dependent calcium-signaling cascade that led to cell proliferation. CONCLUSIONS: cPLA(2)α is regulated by calcium mobilization and mitogen-activated protein kinases (MAPK) activation. Both PDGF mitogenic action and calcium signaling are cPLA(2)α-dependent.

Effect of age on the thioltransferase (glutaredoxin) and thioredoxin systems in the human lens.

PURPOSE: To investigate the effect of age on the key oxidation repair enzymes of the thioltransferase (TTase) and thioredoxin (TRx) systems in the human lens. METHODS: Twenty-three normal human lenses (donor ages, 19-77 years) were grouped into second, third, fifth, sixth, and seventh decades and analyzed for TTase, TRx, glutathione reductase (GR), thioredoxin reductase (TR), and glyceraldehyde-3-phosphate dehydrogenase (G3PD) activities, as well as the glutathione (GSH) pool. Additionally, 19 contralateral lenses of the donor eyes were each divided into cortex and nucleus for enzyme distribution studies. RESULTS: All the enzymes showed similar activity in the cortex and nucleus, regardless of age, but were inactivated to various extents in the older lenses. In the TTase system, both TTase and GR showed activity loss over the five decades, with 70% remaining in the seventh decade, whereas the GSH pool was depleted extensively, with only 35% left in the older lenses. In the TRx system, TRx activity was not affected as much as TR for which only 70% of the activity was found in the seventh decade compared with the second to third decades. Overall, G3PD was more sensitive to age because only 50% activity remained after the sixth decade. CONCLUSIONS: With increasing age there is a gradual activity loss in both the TTase and the TRx systems and a lowered GSH pool. These alterations, compounded with the age-related loss in G3PD activity, may lead to redox and energy imbalance, likely contributing to a higher risk to cataract formation in the aging population.

Effect of thioltransferase (glutaredoxin) deletion on cellular sensitivity to oxidative stress and cell proliferation in lens epithelial cells of thioltransferase knockout mouse.

PURPOSE: To examine the physiological function of the thioltransferase (TTase)/glutathione (GSH) system in the lens using TTase knockout mouse (TTase(-/-)) lens epithelial cells (LECs) as a model. METHODS: Primary LEC cultures were obtained from wild-type (TTase(+/+)) and TTase(-/-) mice. Characterization and validation of the cells were determined by immunoblotting for TTase and alpha-crystallin proteins and by immunohistochemistry for glutathionylated proteins. Cell proliferation was examined by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and BrdU analysis, and cell apoptosis after H(2)O(2) stress was assessed by fluorescence-activated cell sorter analysis. Reloading of TTase protein into the TTase(-/-) cells was achieved with reagent. RESULTS: Primary LEC cultures obtained from wild-type (TTase(+/+)) and TTase(-/-) mice were characterized and found to contain lens-specific alpha-crystallin protein. Western blot analysis confirmed the absence of TTase protein in the TTase(-/-) cells and its presence in the wild-type cells. TTase(-/-) LECs had significantly lower levels of glutathione (GSH) and protein thiols with extensive elevation of glutathionylated proteins, and they exhibited less resistance to oxidative stress than did TTase(+/+) cells. These cells were less viable and more apoptotic, and they had a reduced ability to remove H(2)O(2) after challenge with low levels of H(2)O(2). Reloading of purified TTase into the TTase(-/-) cells restored the antioxidant function in TTase(-/-) cells to a near normal state. CONCLUSIONS: These findings confirm the importance of TTase in regulating redox homeostasis and suggest a new physiological function in controlling cell proliferation in the lens epithelial cells.