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1.
Antioxidants (Basel) ; 11(10)2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36290696

RESUMO

The ocular lens has a very high content of the antioxidant glutathione (GSH) and the enzymes that can recycle its oxidized form, glutathione disulfide (GSSG), for further use. It can be synthesized in the lens and, in part, transported from the neighboring anterior aqueous humor and posterior vitreous body. GSH is known to protect the thiols of the structural lens crystallin proteins from oxidation by reactive oxygen species (ROS) so the lens can maintain its transparency for proper visual function. Age-related lens opacity or senile cataract is the major visual impairment in the general population, and its cause is closely associated with aging and a constant exposure to environmental oxidative stress, such as ultraviolet light and the metabolic end product, H2O2. The mechanism for senile cataractogenesis has been hypothesized as the results of oxidation-induced protein-thiol mixed disulfide formation, such as protein-S-S-glutathione and protein-S-S-cysteine mixed disulfides, which if not reduced in time, can change the protein conformation to allow cascading modifications of various kinds leading to protein-protein aggregation and insolubilization. The consequence of such changes in lens structural proteins is lens opacity. Besides GSH, the lens has several antioxidation defense enzymes that can repair oxidation damage. One of the specific redox regulating enzymes that has been recently identified is thioltransferase (glutaredoxin 1), which works in concert with GSH, to reduce the oxidative stress as well as to regulate thiol/disulfide redox balance by preventing protein-thiol mixed disulfide accumulation in the lens. This oxidation-resistant and inducible enzyme has multiple physiological functions. In addition to protecting structural proteins and metabolic enzymes, it is able to regulate the redox signaling of the cells during growth factor-stimulated cell proliferation and other cellular functions. This review article focuses on describing the redox regulating functions of GSH and the thioltransferase enzyme in the ocular lens.

2.
Sci Rep ; 9(1): 8459, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186457

RESUMO

The paradigm that cataracts are irreversible and that vision from cataracts can only be restored through surgery has recently been challenged by reports that oxysterols such as lanosterol and 25-hydroxycholesterol can restore vision by binding to αB-crystallin chaperone protein to dissolve or disaggregate lenticular opacities. To confirm this premise, in vitro rat lens studies along with human lens protein solubilization studies were conducted. Cataracts were induced in viable rat lenses cultured for 48 hours in TC-199 bicarbonate media through physical trauma, 10 mM ouabain as Na+/K+ ATPase ion transport inhibitor, or 1 mM of an experimental compound that induces water influx into the lens. Subsequent 48-hour incubation with 15 mM of lanosterol liposomes failed to either reverse these lens opacities or prevent the further progression of cataracts to the nuclear stage. Similarly, 3-day incubation of 47-year old human lenses in media containing 0.20 mM lanosterol or 60-year-old human lenses in 0.25 and 0.50 mM 25-hydroxycholesterol failed to increase the levels of soluble lens proteins or decrease the levels of insoluble lens proteins. These binding studies were followed up with in silico binding studies of lanosterol, 25-hydroxycholesterol, and ATP as a control to two wild type (2WJ7 and 2KLR) and one R120G mutant (2Y1Z) αB-crystallins using standard MOETM (Molecular Operating Environment) and Schrödinger's Maestro software. Results confirmed that compared to ATP, both oxysterols failed to reach the acceptable threshold binding scores for good predictive binding to the αB-crystallins. In summary, all three studies failed to provide evidence that lanosterol or 25-hydroxycholesterol have either anti-cataractogenic activity or bind aggregated lens protein to dissolve cataracts.


Assuntos
Catarata/tratamento farmacológico , Lanosterol/farmacologia , Cristalino/efeitos dos fármacos , Cadeia B de alfa-Cristalina/genética , Animais , Catarata/metabolismo , Catarata/patologia , Cristalinas/genética , Modelos Animais de Doenças , Humanos , Hidroxicolesteróis/metabolismo , Lanosterol/efeitos adversos , Cristalino/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/farmacologia , Oxisteróis/efeitos adversos , Oxisteróis/farmacologia , Ratos
3.
Exp Eye Res ; 161: 36-42, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28579033

RESUMO

Oxidative stress is a known risk factor in senile cataract formation. In recent years, it has been suggested that oxidation may also be associated with cataract induced by hyperglycemia, but this concept has not been well examined or validated. Since thioltransferase (TTase) is one of the key enzymes that regulates redox homeostasis and protects against oxidative stress in the lens, we have used TTase gene knockout (KO) mice as a model to examine this new concept. Lenses from 4 months old TTase KO and wild-type (WT) mice were incubated in TC199 culture medium containing 30 mM glucose for 48 h. Each lens was assessed for opacity, graded by LOCSII system, and the wet weight was recorded after which it was homogenized in lysis buffer and analyzed for water-soluble protein and free glutathione (GSH). In vivo studies were carried out using 4 months old TTase KO and WT mouse groups. Each mouse received two consecutive days of intraperitoneal streptozotozin (STZ) injections to induce diabetes. The lenses were examined weekly for 4 weeks using a slit-lamp biomicroscope, and then extracted and analyzed for levels of GSH, water-soluble protein, ATP and protein-GSH mixed disulfide (PSSG). TTase KO lenses cultured in high glucose developed a mild cortical opacity but slightly more than that of the WT lenses. Both groups had similar contents of soluble proteins and GSH. Exposure to high glucose did not change the soluble protein level but did suppress GSH by 20% in lenses with or without TTase. STZ-induced diabetic KO mice also developed a higher degree of mild cortical lens opacity compared to that of the diabetic WT controls. Similar 15-20% losses in lens GSH and ATP were found after one-month induced diabetes in WT and KO mice. There was a 20% greater amount of PSSG in the lenses of TTase KO than the WT control. Under diabetic condition, both groups displayed more glutathionylated proteins in the beta-actin (42 kDa) and lens crystallin proteins (18-22 kDa) regions, and some additional modified proteins at 15-17 kDa and 60-70 kDa, with a total 20-30% PSSG increment in both groups. In conclusion, we have found that hyperglycemia induced some oxidative stress-associated biochemical changes with mild lens opacity in both WT and KO mice. However, these changes were only marginally higher in the TTase KO mouse than that of the WT control, suggesting that TTase deletion may only play a minor role in the early stage of hyperglycemia-induced cataract formation in the mice.


Assuntos
Catarata/metabolismo , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Glutarredoxinas/genética , Estresse Oxidativo/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Glicemia/metabolismo , Western Blotting , Catarata/etiologia , Catarata/patologia , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/patologia , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Técnicas de Inativação de Genes , Glutationa/análogos & derivados , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Hiperglicemia/complicações , Cristalino/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Penicilamina/metabolismo
4.
Exp Eye Res ; 130: 58-65, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25479045

RESUMO

Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase's distribution in the eye is known, we focused on this study by investigating the Grx2 distribution in the ocular tissues in comparison to the lens. Fresh porcine eyes were dissected into cornea, iris, ciliary body, the lens, vitreous humor, retina, and optic nerve. Each tissue (pooled from three eyes) was homogenized and processed for mitochondrial isolation. The mitochondrial fraction was analyzed for Grx2 protein using Western blotting with anti-Grx2 antibody, and Grx2 activity using the published procedure. The eye tissues were also measured for Grx2 mRNA expression by RT-PCR with GAPDH as the control. Grx2-rich mouse liver and purified recombinant mouse Grx2 were used as positive controls for the above analyses. It was found that Grx2 was present in all the tested ocular tissues, except vitreous humor. In comparison with the mouse liver, the protein levels of Grx2 in porcine ciliary body and the lens were 27-fold and 0.75-fold, respectively. Comparing to the lens, Grx2 protein was highest in the ciliary body (13.5-fold), followed by retina (9.2-fold), iris and optic nerve (2-fold), and cornea (1.2-fold). Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.9 mU/mg protein), followed by ciliary body (3.1 mU/mg), the lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Grx2 gene expression in these ocular tissues was further confirmed by RT-PCR analysis. Grx2 mRNA expression showed the highest in ciliary body, followed by retina, optic nerve, cornea, iris, and the lens. No Grx2 mRNA, protein or enzyme activity could be found in the vitreous humor. The results indicate that Grx2 level was higher in eye tissues rich in vasculature and mitochondria (i.e. ciliary body and retina), corroborating with the levels of mRNA expression and Grx2 activity. The rich presence of Grx2 in these tissues is also consistent with their known sensitivity to oxidative stress.


Assuntos
Corpo Ciliar/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glutarredoxinas/genética , Mitocôndrias/enzimologia , Retina/enzimologia , Animais , Western Blotting , Cristalino/enzimologia , Camundongos , Mitocôndrias Hepáticas/enzimologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência , Suínos , Corpo Vítreo/enzimologia
5.
Invest Ophthalmol Vis Sci ; 56(1): 598-605, 2014 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-25537203

RESUMO

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.


Assuntos
Envelhecimento/metabolismo , Catarata/metabolismo , Glutarredoxinas/metabolismo , Cristalino/metabolismo , Estresse Oxidativo , Tiorredoxinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Catarata/patologia , Feminino , Humanos , Immunoblotting , Cristalino/patologia , Masculino , Pessoa de Meia-Idade
6.
J Biol Chem ; 289(52): 36125-39, 2014 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-25362663

RESUMO

Glutaredoxin 2 (Grx2) is an isozyme of glutaredoxin1 (thioltransferase) present in the mitochondria and nucleus with disulfide reductase and peroxidase activities, and it controls thiol/disulfide balance in cells. In this study, we investigated whether Grx2 gene deletion could induce faster age-related cataract formation and elucidated the biochemical changes effected by Grx2 gene deletion that may contribute to lens opacity. Slit lamp was used to examine the lenses in Grx2 knock-out (KO) mice and age-matched wild-type (WT) mice ages 1 to 16 months. In the Grx2 null mice, the lens nuclear opacity began at 5 months, 3 months sooner than that of the control mice, and the progression of cataracts was also much faster than the age-matched controls. Lenses of KO mice contained lower levels of protein thiols and GSH with a significant accumulation of S-glutathionylated proteins. Actin, αA-crystallin, and ßB2-crystallin were identified by Western blot and mass spectroscopy as the major S-glutathionylated proteins in the lenses of 16-month-old Grx2 KO mice. Compared with the WT control, the lens of Grx2 KO mice had only 50% of the activity in complex I and complex IV and less than 10% of the ATP pool. It was concluded that Grx2 gene deletion altered the function of lens structural proteins through S-glutathionylation and also caused severe disturbance in mitochondrial function. These combined alterations affected lens transparency.


Assuntos
Catarata/genética , Glutarredoxinas/genética , Trifosfato de Adenosina/metabolismo , Animais , Cistina/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas do Olho/metabolismo , Deleção de Genes , Glutationa/metabolismo , Cápsula do Cristalino/metabolismo , Cápsula do Cristalino/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout
7.
J Biol Chem ; 289(21): 14812-28, 2014 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-24727547

RESUMO

Glutaredoxin-2 (Grx2) modulates the activity of several mitochondrial proteins in cardiac tissue by catalyzing deglutathionylation reactions. However, it remains uncertain whether Grx2 is required to control mitochondrial ATP output in heart. Here, we report that Grx2 plays a vital role modulating mitochondrial energetics and heart physiology by mediating the deglutathionylation of mitochondrial proteins. Deletion of Grx2 (Grx2(-/-)) decreased ATP production by complex I-linked substrates to half that in wild type (WT) mitochondria. Decreased respiration was associated with increased complex I glutathionylation diminishing its activity. Tissue glucose uptake was concomitantly increased. Mitochondrial ATP output and complex I activity could be recovered by restoring the redox environment to that favoring the deglutathionylated states of proteins. Grx2(-/-) hearts also developed left ventricular hypertrophy and fibrosis, and mice became hypertensive. Mitochondrial energetics from Grx2 heterozygotes (Grx2(+/-)) were also dysfunctional, and hearts were hypertrophic. Intriguingly, Grx2(+/-) mice were far less hypertensive than Grx2(-/-) mice. Thus, Grx2 plays a vital role in modulating mitochondrial metabolism in cardiac muscle, and Grx2 deficiency leads to pathology. As mitochondrial ATP production was restored by the addition of reductants, these findings may be relevant to novel redox-related therapies in cardiac disease.


Assuntos
Glutarredoxinas/metabolismo , Glutationa/metabolismo , Miocárdio/metabolismo , Fosforilação Oxidativa , Trifosfato de Adenosina/metabolismo , Animais , Complexo I de Transporte de Elétrons/metabolismo , Fibrose/genética , Glutarredoxinas/genética , Hipertensão/genética , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/ultraestrutura , Miocárdio/patologia , Tamanho do Órgão/genética , Oxirredução
8.
Free Radic Biol Med ; 57: 92-104, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23291592

RESUMO

Thioredoxin (Trx) is an important redox regulator with cytosolic Trx1 and mitochondrial Trx2 isozymes. Trx has multiple physiological functions in cells and its bioavailability is negatively controlled through active-site binding to a specific thioredoxin-binding protein (TBP-2). This paper describes the delicate balance between TBP-2 and Trx and the effect of overexpression of TBP-2 in human lens epithelial cells. Cells overexpressing TBP-2 (TBP-2 OE) showed a sevenfold increase in TBP-2 and a nearly 40% suppression of Trx activity but no change in Trx expression. The TBP-2 OE cells grew slower and their population decreased to 30% by day 7. Cell cycle analysis showed that TBP-2 OE cells arrested at the G2/M stage and that they displayed low expression of the cell cycle elements P-cdc2(Y15), cdc2, cdc25A, and cdc25C. Furthermore, TBP-2 OE cells were more sensitive to oxidation. Under H2O2 (200µM, 24h) treatment, these cells lost 80% viability and became highly apoptotic. Brief oxidative stress (200µM, 30min) to TBP-2 OE cells disrupted the Trx antiapoptotic function by dissociating the cytosolic and mitochondrial Trx-ASK binding complexes. The same H2O2-treated cells also showed activated ASK (P-ASK), increased Bax, lowered Bcl-2, cytochrome c release, and elevated caspase 3/7 activity. We conclude from these studies that high cellular levels of TBP-2 can potentially suppress Trx bioavailability and increase oxidation sensitivity. Overexpression of TBP-2 also causes slow growth by mitotic arrest and apoptosis by activating the ASK death pathway.


Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cristalino/metabolismo , Tiorredoxinas/metabolismo , Proteínas de Transporte/genética , Caspases/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Citocromos c/metabolismo , Ativação Enzimática , Células Epiteliais/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Pontos de Checagem da Fase M do Ciclo Celular , Mitocôndrias/metabolismo , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Proteína X Associada a bcl-2/metabolismo
9.
J Biol Chem ; 288(12): 8365-8379, 2013 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-23335511

RESUMO

Glutathionylation has emerged as a key modification required for controlling protein function in response to changes in cell redox status. Recently, we showed that the glutathionylation state of uncoupling protein-3 (UCP3) modulates the leak of protons back into the mitochondrial matrix, thus controlling reactive oxygen species production. However, whether or not UCP3 glutathionylation is mediated enzymatically has remained unknown because previous work relied on the use of pharmacological agents, such as diamide, to alter the UCP3 glutathionylation state. Here, we demonstrate that glutaredoxin-2 (Grx2), a matrix oxidoreductase, is required to glutathionylate and inhibit UCP3. Analysis of bioenergetics in skeletal muscle mitochondria revealed that knock-out of Grx2 (Grx2(-/-)) increased proton leak in a UCP3-dependent manner. These effects were reversed using diamide, a glutathionylation catalyst. Importantly, the increased leak did not compromise coupled respiration. Knockdown of Grx2 augmented proton leak-dependent respiration in primary myotubes from wild type mice, an effect that was absent in UCP3(-/-) cells. These results confirm that Grx2 deactivates UCP3 by glutathionylation. To our knowledge, this is the first enzyme identified to regulate UCP3 by glutathionylation and is the first study on the role of Grx2 in the regulation of energy metabolism.


Assuntos
Glutarredoxinas/fisiologia , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Prótons , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Ciclo do Ácido Cítrico , Diamida/farmacologia , Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Homeostase , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Musculares/metabolismo , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Consumo de Oxigênio , Cultura Primária de Células , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 3
10.
Invest Ophthalmol Vis Sci ; 53(11): 7276-85, 2012 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-23010639

RESUMO

PURPOSE: To study the effect of age on the morphologic and biochemical alterations induced by in vivo exposure of ultraviolet radiation (UV). METHODS: Young and old C57BL/6 mice were exposed to broadband UVB+UVA and euthanized after 2 days. Another batch of UV-exposed young mice was monitored for changes after 1, 2, 4, and 8 days. Age-matched nonexposed mice served as controls. Lens changes were documented in vivo by slit-lamp biomicroscopy and dark field microscopy photographs ex vivo. Lens homogenates were analyzed for glutathione (GSH) level, and the activities of thioredoxin (Trx), thioltransferase (TTase), and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Glutathionylated lens proteins (PSSGs) were detected by immunoblotting using GSH antibody. Western blot analysis was also done for the expression levels of TTase and Trx. RESULTS: Both age groups developed epithelial and superficial anterior subcapsular cataract at 2 days postexposure. The lens GSH level and G3PD activity were decreased, and PSSGs were elevated in both age groups, but more prominent in the older mice. TTase and Trx activity and protein expression were elevated only in the young mice. Interestingly, lens TTase and Trx in the young mice showed a transient increase, peaking at 2 days after UV exposure and returning to baseline at day 8, corroborated by lens transparency. CONCLUSIONS: The lenses of old mice were more susceptible to UV radiation-induced cataract. The upregulated TTase and Trx likely provided oxidation damage repair in the young mice.


Assuntos
Envelhecimento/metabolismo , Catarata/metabolismo , Cristalinas/biossíntese , Estresse Oxidativo , Regulação para Cima , Animais , Western Blotting , Catarata/etiologia , Catarata/patologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Seguimentos , Glutarredoxinas/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Cristalino/metabolismo , Cristalino/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tiorredoxinas/biossíntese , Raios Ultravioleta/efeitos adversos
11.
Exp Eye Res ; 101: 36-43, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22710095

RESUMO

In sugar cataract formation in rats, aldose reductase (AR) activity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 h in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galactose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose/galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-ß, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osmotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-ß and the altered cytotoxic signaling that is observed during sugar cataract formation.


Assuntos
Aldeído Redutase/metabolismo , Catarata/metabolismo , Diabetes Mellitus Experimental/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Estresse Fisiológico , Fator de Crescimento Transformador beta/metabolismo , Aldeído Redutase/antagonistas & inibidores , Animais , Western Blotting , Catarata/patologia , Diabetes Mellitus Experimental/patologia , Eletroforese em Gel de Poliacrilamida , Galactose/farmacologia , Glucose/farmacologia , Glutationa/metabolismo , Hiperglicemia/metabolismo , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Masculino , Técnicas de Cultura de Órgãos , Pressão Osmótica , Ratos , Ratos Sprague-Dawley , Sorbitol/metabolismo
12.
Invest Ophthalmol Vis Sci ; 53(1): 248-52, 2012 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-22167097

RESUMO

PURPOSE: To determine the protection factor (PF) for glutaredoxin-1 (Grx1) with regard to UVR-induced cataract by comparison of in vivo ultraviolet radiation (UVR) lens toxicity between double knockout Grx1⁻/⁻ and Grx1⁺/⁺ mice. METHODS: Twenty Grx1⁺/⁺ mice and 20 Grx1⁻/⁻ mice were unilaterally exposed in vivo to UVR for 15 minutes. Groups of four animals each received 0.0, 2.1, 2.9, 3.6, and 4.1 kJ/m(2) UVR-300 nm. At 48 hours after UVR exposure, light-scattering in the exposed and contralateral nonexposed lenses was measured quantitatively. Macroscopic lens changes were documented with dark-field illumination photography. RESULTS: UVR-300 nm induced subcapsular and cortical cataract in Grx1⁻/⁻ and Grx1⁺/⁺ mice. In both Grx1⁻/⁻ and Grx1⁺/⁺, the light-scattering intensified with increased in vivo exposure doses of UVR-300 nm. The intensity of forward light-scattering was higher in the lenses of Grx1⁻/⁻ mice than in the lenses of Grx1⁺/⁺ mice. The threshold dose for in vivo UVR-300 nm-induced cataract, expressed as MTD(2.3:16), was 3.8 in the Grx1⁺/⁺ group and 3.0 in the Grx1⁻/⁻ group, resulting in a PF of 1.3. CONCLUSIONS: The PF is an objective relative measure of protective properties. The Grx1 gene is associated with an in vivo PF of 1.3. This result signifies that the presence of the gene allows a 1.3 times longer in vivo exposure to UVR, at equivalent irradiance, than the absence of the gene before early-onset, UVR-induced cataract occurs. This finding indicates the important role of the Grx1 gene in the oxidation defense system of the lens.


Assuntos
Catarata/prevenção & controle , Regulação da Expressão Gênica/fisiologia , Glutarredoxinas/genética , Cristalino/efeitos da radiação , Lesões Experimentais por Radiação/prevenção & controle , Raios Ultravioleta/efeitos adversos , Animais , Catarata/etiologia , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Lesões Experimentais por Radiação/etiologia , Espalhamento de Radiação
13.
Free Radic Biol Med ; 51(11): 2108-17, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21983434

RESUMO

Glutaredoxin belongs to the oxidoreductase family, with cytosolic glutaredoxin 1 (Grx1) and mitochondrial glutaredoxin 2 (Grx2) isoforms. Of the two isozymes, the function of Grx2 is not well understood. This paper describes the effects of Grx2 deletion on cellular function using primary lens epithelial cell cultures isolated from Grx2 gene knockout (KO) and wild-type (WT) mice. We found that both cell types showed similar growth patterns and morphology and comparable mitochondrial glutathione pool and complex I activity. Cells with deleted Grx2 did not show affected Grx1 or thioredoxin expression but exhibited high sensitivity to oxidative stress. Under treatment with H(2)O(2), the KO cells showed less viability, higher membrane leakage, enhanced ATP loss and complex I inactivation, and weakened ability to detoxify H(2)O(2) in comparison with the WT cells. The KO cells had higher glutathionylation in the mitochondrial proteins, particularly the 75-kDa subunit of complex I. Recombinant Grx2 deglutathionylated complex I and restored most of its activity. We conclude that Grx2 has a function that protects cells against H(2)O(2)-induced injury via its peroxidase and dethiolase activities; particularly, Grx2 prevents complex I inactivation and preserves mitochondrial function.


Assuntos
Células Epiteliais/metabolismo , Glutarredoxinas/metabolismo , Cristalino/citologia , Estresse Oxidativo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Glutarredoxinas/deficiência , Peróxido de Hidrogênio/farmacologia , Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos
14.
Invest Ophthalmol Vis Sci ; 52(11): 8231-40, 2011 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-21896865

RESUMO

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.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Epiteliais/enzimologia , Fosfolipases A2 do Grupo IV/metabolismo , Cristalino/enzimologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Western Blotting , Cálcio/metabolismo , Contagem de Células , Linhagem Celular , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosfolipases A2 do Grupo IV/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cristalino/citologia , Mutagênese Sítio-Dirigida , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
J Biol Chem ; 286(38): 33203-12, 2011 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-21768092

RESUMO

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteine-rich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15 KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation. We suggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency.


Assuntos
Catarata/metabolismo , Catarata/patologia , Homeostase , Selenoproteínas/deficiência , Selenoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Cristalino/embriologia , Cristalino/metabolismo , Cristalino/patologia , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Peso Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Células NIH 3T3 , Oxirredução , Estresse Oxidativo , Próstata/metabolismo , Próstata/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Selenoproteína P/metabolismo , Selenoproteínas/química , Selenoproteínas/genética , Resposta a Proteínas não Dobradas
16.
Mol Cell Biochem ; 352(1-2): 181-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21350856

RESUMO

Reactive oxygen species (ROS) produced in macrophages is critical for microbial killing, but they also take part in inflammation and antigen presentation functions. MicroRNAs (miRNAs) are endogenous regulators of gene expression, and they can control immune responses. To dissect the complex nature of ROS-mediated effects in macrophages, we sought to characterize miRNAs that are responsive to oxidative stress-induced with hydrogen peroxide (H(2)O(2)) in the mouse macrophage cell line, RAW 264.7. We have identified a set of unique miRNAs that are differentially expressed in response to H(2)O(2). These include miR-27a*, miR-27b*, miR-29b*, miR-24-2*, and miR-21*, all of which were downregulated except for miR-21*. By using luciferase reporter vector containing nuclear factor-kB (NF-kB) response elements, we demonstrate that overexpression of miR-27b* suppresses lipopolysaccharide-induced activation of NF-kB in RAW 264.7 cells. Our data suggest that macrophage functions can be regulated by oxidative stress-responsive miRNAs by modulating the NF-kB pathway.


Assuntos
MicroRNAs/fisiologia , NF-kappa B/metabolismo , Estresse Oxidativo , Animais , Linhagem Celular , Camundongos , Reação em Cadeia da Polimerase
17.
Invest Ophthalmol Vis Sci ; 52(3): 1723-34, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21087961

RESUMO

PURPOSE: Intracellular reactive oxygen species have been reported to associate with growth factor and integrin signalings in promoting cell adhesion in many cell types. This study is to explore if exogenous H(2)O(2) at low levels can be beneficial to cell adhesion, migration, and wound healing. METHODS: Primary rabbit corneal epithelial cells treated with 0-70 µM H(2)O(2) were tested for viability by MTT assay, adhesion by centrifugation assay, focal contacts of vinculin and F-actin by immunofluorescence, activated Src(pY416), EGF receptor (pY845), vinculin(pY1065), FAK(pY397), and FAK(pY576) by immunoblotting. Cell migration was examined with 0-50 µM H(2)O(2) using the scratch wound technique. Corneal wound healing of ex vivo pig model and in vivo mouse model was examined using H(2)O(2) with and without antioxidant N-acetylcysteine (NAC). RESULTS: Compared with the untreated control, H(2)O(2) at 10-50 µM stimulated cell viability and facilitated adhesion and migration with clear induction of vinculin-rich focal adhesions and F-actin-containing stress fibers by increasing activated Src, FAK(pY576), and vinculin(pY1065). H(2)O(2) also increased phosphorylation of EGFR(Y845) parallel to that of activated Src, but both were eliminated by NAC and PP1 (Src inhibitor). Finally, H(2)O(2) induced faster wound healing in cornea both in vitro and in vivo, but the healing was diminished by NAC. CONCLUSIONS: These findings suggest that H(2)O(2) at low levels promotes cell adhesion, migration, and wound healing in cornea cells or tissue, and the interaction of H(2)O(2) with Src plays a major role.


Assuntos
Adesão Celular/fisiologia , Movimento Celular/fisiologia , Epitélio Corneano/citologia , Epitélio Corneano/efeitos dos fármacos , Peróxido de Hidrogênio/administração & dosagem , Cicatrização/fisiologia , Actinas/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Epitélio Corneano/metabolismo , Receptores ErbB/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Fosforilação , Coelhos , Suínos , Vinculina/metabolismo
18.
Invest Ophthalmol Vis Sci ; 51(12): 6598-604, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20610843

RESUMO

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.


Assuntos
Envelhecimento/fisiologia , Glutarredoxinas/metabolismo , Cristalino/metabolismo , Tiorredoxinas/metabolismo , Adulto , Idoso , Catarata/enzimologia , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Humanos , Córtex do Cristalino/metabolismo , Núcleo do Cristalino/metabolismo , Pessoa de Meia-Idade , Fatores de Risco , Tiorredoxina Dissulfeto Redutase/metabolismo , Adulto Jovem
19.
Biochim Biophys Acta ; 1797(10): 1705-15, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20547138

RESUMO

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200muM hydrogen peroxide (H(2)O(2)) for 24h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H(2)O(2)-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H(2)O(2) treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. Furthermore, the inactivated complex I in the H(2)O(2)-treated cells could be protected mostly by importing the purified nascent Grx2 protein, but not the Grx2 protein mutated at the active site with C70S, or C73S, or with C70S plus C73S. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I, but not complex II, in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H(2)O(2)-induced apoptosis is likely associated with its ability to preserve complex I.


Assuntos
Apoptose/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Células Epiteliais/efeitos dos fármacos , Glutarredoxinas/metabolismo , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , Western Blotting , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Glutarredoxinas/genética , Humanos , Imunoprecipitação , Cristalino/citologia , Mutação , Oxidantes/farmacologia , Ligação Proteica , Interferência de RNA
20.
Exp Eye Res ; 89(6): 833-9, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19664619

RESUMO

We investigated if the absence of glutaredoxin1, a critical protein thiol repair enzyme, increases lens susceptibility to oxidative stress caused by in vivo exposure to ultraviolet radiation type B (UVR-B). Glrx(-/-) mice and Glrx(+/+) mice were unilaterally exposed in vivo to UVR-B for 15 min. Groups of 12 animals each received 4.3, 8.7, and 14.5 kJ/m(2) respectively. 48 h post UVR-B exposure, the induced cataract was quantified as forward lens light scattering. Cataract morphology was documented with darkfield illumination photography. Glutathione (GSH/GSSG) content was analyzed in Glrx(-/-) and Glrx(+/+) lenses. UVR-B exposure induced anterior sub-capsular cataract (ASC) in Glrx(-/-) and Glrx(+/+) mice. In Glrx(-/-) lenses the opacities extended further towards the lens equator than in wild type animals (Glrx(+/+)). Lens light scattering in Glrx(-/-) mice was increased in all dose groups compared to lenses with normal glutaredoxin1 function. The difference was more pronounced with increasing exposure dose. Lens sensitivity for UVR-B induced damage was significantly higher in Glrx(-/-) lenses compared to Glrx(+/+) lenses. The Glrx gene provides a 44% increase of protection against close to threshold UVR-B induced oxidative stress compared to the absence of the Glrx gene. In conclusion, the absence of glutaredoxin1 increases lens susceptibility to UVR-B induced oxidative stress in the mouse.


Assuntos
Catarata/etiologia , Glutarredoxinas/deficiência , Cristalino/efeitos da radiação , Lesões Experimentais por Radiação/etiologia , Raios Ultravioleta/efeitos adversos , Animais , Catarata/enzimologia , Catarata/patologia , Relação Dose-Resposta à Radiação , Feminino , Glutarredoxinas/fisiologia , Glutationa/metabolismo , Cristalino/metabolismo , Camundongos , Camundongos Knockout , Estresse Oxidativo/efeitos da radiação , Lesões Experimentais por Radiação/enzimologia , Lesões Experimentais por Radiação/patologia , Espalhamento de Radiação
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