Retracted Article
Total Page：16
File Type：pdf, Size：1020Kb
Page 1 of 31 Diabetes Activation of aldose reductase by interaction with tubulin and involvement of this mechanism in diabetic cataract formation Juan F. Rivellia, Verónica S. Santandera, Sofía O. Perettia, Noelia E. Monesteroloa, Ayelen D. Nigraa, Gabriela Previtalia, Marina R. Amaidena, Carlos A. Arceb, Emiliano Primoa, Angela T. Lisaa, Juan Piec and César H. Casalea a- Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Río Cuarto, 5800-Córdoba, Argentina. b- Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), UNC- CONICET, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000-Córdoba, Argentina. c- Departments of Pharmacology Physiology and Pediatrics, Medical School, University of Zaragoza, Zaragoza, Spain. Corresponding author: César H. Casale. Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales,ARTICLE Universidad Nacional de Río Cuarto, Río Cuarto, 5800-Córdoba, Argentina. Tel.: +54 358 4676422; fax: +54 358 4676232; E-mail: [email protected] ABSTRACT Our previous studies have shown that high levels of glucose induce inhibition of RETRACTEDNa+,K+-ATPase (NKA) via stimulation of aldose reductase (AR), polymerisation of microtubules, and formation of an acetylated tubulin/NKA complex. Inhibition of AR eliminated the effect of high glucose on NKA activity. In this study, we investigated the mechanism of regulation of AR activity by tubulin. Purified tubulin and AR were used. The results indicate that: (i) tubulin and AR interact with each other directly; (ii) tubulin/AR interaction results in a 6-fold increase of AR activity under microtubule growing conditions; (iii) AR interacts preferentially with tubulin that contains 3-nitro-L- tyrosine (3-NTyr); (iv) free tyrosine and 3-nitro-tyrsine are able to block tubulin/AR interaction and thereby prevent AR activation; (v) exposure of cultured COS cells to high glucose concentrations promotes microtubule polymerisation and NKA inhibition, and both these promoting effects are inhibited by addition of free Tyr or 3-NTyr; (vi) 1 For Peer Review Only Diabetes Page 2 of 31 treatment of experimental (STZ-induced) diabetic rats with 3-NTyr prevented cataract formation, suggesting that this complication of diabetes involves tubulin/AR interaction and AR activity. Taken together, these findings indicate that AR activity is controlled by association/dissociation of the tubulin/AR complex, that Tyr and 3-NTyr block such effect by preventing tubulin/AR complex formation, and that AR activity can be reduced by 3-NTyr or other compounds that inhibit tubulin/AR interaction. INTRODUCTION The major pathogenic pathway in diabetes whereby hyperglycemia causes damage in tissues with insulin-independent glucose transport is activation of the enzyme aldose reductase (AR) (1). AR activation is associated with various complications of diabetes (e.g., cataract formation, retinopathy) (2). In spite of over five decades of research, no AR inhibitor has been found that combines selectivity, efficacy, and safety for human therapeutic application (3). We demonstrated previously that acetylated tubulin (AcTub) is capable of associating with Na+,K+-ATPase (NKA) to form a complex, and that such association results in inhibition of NKA enzyme activity (4, 5, 6, 7, 8,ARTICLE 9). We demonstrated recently that high glucose concentrations induce polymerisation of microtubules, additional formation of AcTub/NKA complex, and inhibition of enzyme activity. The increase in microtubule content appeared to result from increased levels of sorbitol caused by AR activation. AR activity increased when AR was associated with microtubules, resulting in an "upregulation cycle" between microtubule formation and AR activation. On the RETRACTEDbasis of these findings, we proposed that glucose triggers a synergistic effect between tubulin and sorbitol that leads to AR activation (by association with tubulin), microtubule polymerisation, and consequent NKA inhibition (10). The objective of the present study was to elucidate the mechanism whereby tubulin/AR interaction regulates AR activity. We present evidence, using a diabetic rat model, that AR interacts directly with tubulin (primarily the 3-NTyr-tub isotype) and that consequent AR activation occurs through tubulin polymerisation. Free Tyr or 3- NTyr inhibited tubulin/AR complex formation, AR activation, and various pathological events (including cataract formation) induced by high glucose concentrations. We propose a novel mechanism of NKA and AR regulation by tubulin under high-glucose 2 For Peer Review Only Page 3 of 31 Diabetes conditions, and demonstrate that AR activity can be regulated by drugs that inhibit tubulin/AR association. RESEARCH DESIGN AND METHODS Cell culture and treatment with glucose, Tyr, or 3-NTyr COS cells were grown in DMEM at 37 °C in a water-saturated atmosphere of air/CO2 (19:1). For treatment with glucose, Tyr, or 3-NTyr, cells cultured to 90% confluence were rinsed with HEPES-FBS buffer (25 mM HEPES, pH 7.4, supplemented with 1 mM sodium pyruvate, 0.22% sodium carbonate, 10 mM glutamine, 100 mM NaCl, 10% FBS, 10 IU/ml penicillin, and 100 µg/ml streptomycin) and incubated for 2 h. Animals and in vivo experiments Induction of diabetes in rats by streptozotocin (STZ). Diabetes was induced in a rat model as described by Lin et al. (11). In brief, 7-wk-old Wistar rats with ad libitum access to food were injected i.p. with a single dose of STZARTICLE (70 mg/kg body wt, in 0.1 mM citrate buffer, pH 4.5). In successful cases, the blood glucose concentration 7-8 days after injection was 300-400 mg/dl. Control animals were injected with an equal volume of citrate buffer. Tyr and 3-NTyr treatment. To investigate the effects of Tyr and 3-NTyr on diabetic cataract formation, rats were assigned randomly to one of the following groups: RETRACTEDcontrol, diabetic, diabetic treated with 3-NTyr (30 mg . kg-1. day-1), and diabetic treated with Tyr (90 mg . kg-1. day-1). Tyr and 3-NTyr were mixed with the water supply. Treatments were started on day 7 after STZ injection and continued until day 90. Rats were sacrificed, and blood was obtained by cardiac puncture for HbA1c assay. Eyes were examined with an ophthalmoscope once per wk. Pupils were dilated by a drop of a 1:1 mixture of 1% tropicamide and 10% phenylephrine hydrochloride. The degree of cataract maturity was classified as grade 0, clear; grade 1, peripheral vesicles and opacities; grade 2, central opacities; grade 3, diffused opacities; grade 4, mature cataract; grade 5, hypermature cataract. Rat lens culture and analysis of lens opacity 3 For Peer Review Only Diabetes Page 4 of 31 For ex vivo examination of lens opacity, lenses were dissected from 6-wk-old male rats. Each isolated lens was incubated in 2 ml of 199 medium with antibiotics and 20 mM glucose in 24-well plates under 95% air/5% CO2 atmosphere for 2 days at 37 °C. The medium was changed every day and supplemented with Tyr or 3-NTyr (500 µM) in addition to 20 mM glucose. All the reagents used in lens culture were filtered (pore diameter 0.2 µm). Lenses were examined for the development of generalized opacity under an optical microscope with a CCD camera. The opaque area intensities of the lens were quantified using the Scion imaging software programme (12). To determine AR activity, 20 lenses from each treatment were homogenized in 4 ml Tris- HCl buffer (20 mM Tris-HCl, pH 7) using a glass-teflon homogenizer. The homogenate was centrifuged at 10,000 x g for 20 min at 4 °C. The resulting supernatant was used as an enzyme source (13). Isolation of membrane fraction from COS cells Confluent cells from four 150-cm2 flasks were washed once with TBS-PMSF (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and 0.1 mM PMSF), harvested in TBS-PMSF, centrifuged at 2,000 x g for 10 min at 4 ºC, resuspended in hypotonic solution (10 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 0.1 mMARTICLE PMSF), and stirred for 30 min on ice. The suspension was homogenized in a glass Dounce homogenizer (40 strokes), the homogenate was centrifuged at 100,000 x g for 30 min at 4 ºC, and the resulting pellet was resuspended in 3 ml TBS-PMSF. Isolation of cytosolic and cytoskeletal tubulin RETRACTED COS cells were washed at room temperature with microtubule-stabilising buffer (90 mM Mes, pH 6.7, 1 mM EGTA, 1 mM MgCl2, 10% (v/v) glycerol) and extracted with 2.5 ml of the same buffer containing 10 µM taxol, 0.5% (v/v) Triton X-100, and protease inhibitors (10 µg/ml aprotinin, 0.5 mM benzamidine, 5 µg/ml o- phenanthroline, 0.2 mM PMSF) for 3 min at 37 ºC with gentle agitation. The detergent extract (cytosolic tubulin fraction including membrane fraction) was separated, and the diluted proteins were concentrated by the chloroform/methanol method of Wessel and Flügge (14). The cytoskeletal tubulin fraction (which remained bound to the dish) was washed with pre-warmed microtubule-stabilising buffer and resuspended in sample buffer (15). The cytosolic fraction was resuspended in an identical volume of sample buffer. Both fractions were immediately subjected to immunoblotting. 4 For Peer Review Only Page 5 of 31 Diabetes Tubulin purification and incorporation of 3-NTyr Brains from 30-60-day-old rats were homogenized at 4 ºC in one volume of MEM buffer (0.1 M Mes/NaOH, pH 6.7, containing 1 mM EGTA and 1 mM MgCl2). The homogenate was centrifuged at 100,000 x g for 45 min, and the pellet was discarded. Tubulin was purified as described by Sloboda et al. (16). The concentration was adjusted to 1 mg/ml, and the tubulin was used immediately.