Human glutathione S-transferase A (GSTA) family genes are regulated by steroidogenic factor 1 (SF-1) and are involved in steroidogenesis.

Steroidogenic factor 1 (SF-1) is a master regulator for steroidogenesis. In this study, we identified novel SF-1 target genes using a genome-wide promoter tiling array and a DNA microarray. SF-1 was found to regulate human glutathione S-transferase A (GSTA) family genes (hGSTA1-hGSTA4), a superfamily of detoxification enzymes clustered on chromosome 6p12. All hGSTA genes were up-regulated by transduction of SF-1 into human mesenchymal stem cells, while knockdown of endogenous SF-1 in H295R cells down-regulated all hGSTA genes. Chromatin immunoprecipitation assays, however, revealed that SF-1 bound directly to the promoters of hGSTA3 and weakly of hGSTA4. Chromosome conformation capture assays revealed that the coordinated expression of the genes was based on changes in higher-order chromatin structure triggered by SF-1, which enables the formation of long-range interactions, at least between hGSTA1 and hGSTA3 gene promoters. In steroidogenesis, dehydrogenation of the 3-hydroxy group and subsequent Δ(5)-Δ(4) isomerization are thought to be enzymatic properties of 3ß-hydroxysteroid dehydrogenase (3ß-HSD). Here, we demonstrated that, in steroidogenic cells, the hGSTA1 and hGSTA3 gene products catalyze Δ(5)-Δ(4) isomerization in a coordinated fashion with 3ß-HSD II to produce progesterone or Δ(4)-androstenedione from their Δ(5)-precursors. Thus, hGSTA1 and hGSTA3 gene products are new members of steroidogenesis working as Δ(5)-Δ(4) isomerases.

Protein expression profiling of lens epithelial cells from Prdx6-depleted mice and their vulnerability to UV radiation exposure.

Oxidative stress is one of the causative factors in progression and etiology of age-related cataract. Peroxiredoxin 6 (Prdx6), a savior for cells from internal or external environmental stresses, plays a role in cellular signaling by detoxifying reactive oxygen species (ROS) and thereby controlling gene regulation. Using targeted inactivation of the Prdx6 gene, we show that Prdx6-deficient lens epithelial cells (LECs) are more vulnerable to UV-triggered cell death, a major cause of skin disorders including cataractogenesis, and these cells display abnormal protein profiles. PRDX6-depleted LECs showed phenotypic changes and formed lentoid body, a characteristic of terminal cell differentiation and epithelial-mesenchymal transition. Prdx6(-/-) LECs exposed to UV-B showed higher ROS expression and were prone to apoptosis compared with wild-type LECs, underscoring a protective role for Prdx6. Comparative proteomic analysis using fluorescence-based difference gel electrophoresis along with mass spectrometry and database searching revealed a total of 13 proteins that were differentially expressed in Prdx6(-/-) cells. Six proteins were upregulated, whereas expression of seven proteins was decreased compared with Prdx6(+/+) LECs. Among the cytoskeleton-associated proteins that were highly expressed in Prdx6-deficient LECs was tropomyosin (Tm)2beta. Protein blot and real-time PCR validated dramatic increase of Tm2beta and Tm1alpha expression in these cells. Importantly, Prdx6(+/+) LECs showed a similar pattern of Tm2beta protein expression after transforming growth factor (TGF)-beta or H(2)O(2) treatment. An extrinsic supply of PRDX6 could restore Tm2beta expression, demonstrating that PRDX6 may attenuate adverse signaling in cells and thereby maintain cellular homeostasis. Exploring redox-proteomics (Prdx6(-/-)) and characterization and identification of abnormally expressed proteins and their attenuation by PRDX6 delivery should provide a basis for development of novel therapeutic interventions to postpone ROS-mediated abnormal signaling deleterious to cells or tissues.

Aldose reductase inhibitor counteracts the attenuated adhesion of human corneal epithelial cells induced by high glucose through modulation of MMP-10 expression.

AIMS: We investigated the preventive effect of aldose reductase inhibitor (ARI) on the adhesion of SV40-transformed human corneal epithelial cells (HCECs) exposed to high glucose, and the underlying mechanism focusing on the role of matrix metalloproteinase (MMP)-10. METHODS: HCECs were cultured in medium containing a normal (5.5 mM) or high (31.2 mM) concentration of D-glucose in the presence or absence of ARI, fidarestat. Cell attachment ability was evaluated by short-term adhesion assay. The levels of intracellular polyol were measured by liquid-gas chromatography. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were used to determine the expression levels. RESULTS: Decreased attachment activity and increased accumulation of polyol induced by exposure to high glucose were abrogated by ARI. Supply of recombinant MMP-10 decreased integrin alpha3beta1-expression and cell adhesion. The expression level of MMP-10 was enhanced at both protein and mRNA levels by exposure to high glucose, while that of integrin alpha3beta1 was decreased at the protein level, but remained unchanged at the mRNA level. These alterations in the expression levels of MMP-10 and integrin alpha3beta1 were normalized by ARI. CONCLUSIONS: ARI counteracts the decreased adhesion of HCECs induced by high glucose exposure, through the modulation of the expression of MMP-10.

TAT-mediated peroxiredoxin 5 and 6 protein transduction protects against high-glucose-induced cytotoxicity in retinal pericytes.

AIMS: Hyperglycemia-induced oxidative stress is implicated in pericyte apoptosis seen in diabetic retinopathy. The six mammalian Peroxiredoxins (PRDXs) comprise a novel family of antioxidative proteins that negatively regulate oxidative stress-induced apoptosis by controlling reactive oxygen species (ROS) levels. MAIN METHODS: Sprague-Dawley rats were used to detect the retinal expressions of PRDXs1-6. Pig pericytes cultured in high-glucose medium were used to monitor the protective effect of PRDX5 and 6 against high-glucose-associated change. Recombinant PRDX5 and 6 proteins were linked to the Trans-Activating Transduction (TAT) domain from HIV-1 TAT protein for their efficient delivery into cells/tissues. KEY FINDINGS: We found higher expression of PRDX5 and 6 mRNAs and PRDX5 and 6 proteins in retina than the other Prdxs (Prdx1-4). Western blotting affirmed the intracellular presence of TAT-linked proteins and revealed the efficient transduction of TAT-HA-PRDX5 and 6 in these cells. Extrinsic supply of TAT-HA-PRDX5 and 6 proteins inhibited the oxidative stress-induced DNA damage after high-glucose exposure in pig pericytes. The cell survival and apoptosis assay revealed that extrinsic supply of TAT-HA-PRDX5 and 6 proteins was responsible for inhibiting hyperglycemia-induced pericyte apoptosis. SIGNIFICANCE: Results suggest that delivery of PRDX5 and 6 might protect hyperglycemia-induced pericyte loss to inhibit oxidative stress.

Analysis of the effect of intravitreal bevacizumab injection on diabetic macular edema after cataract surgery.

PURPOSE: To determine the feasibility and clinical effectiveness of intravitreal bevacizumab combined with cataract surgery for management of the postoperative increase of retinal thickness in patients with diabetic maculopathy. DESIGN: Prospective, randomized, masked cohort study. PARTICIPANTS: Forty-two eyes with diabetic macular edema (DME) of 42 patients with type 2 diabetes mellitus. METHODS: Patients were randomly assigned to receive either cataract surgery only (control; 21 eyes) or combined with intravitreal injection of 1.25 mg bevacizumab (21 eyes). Efficacy measures included best-corrected visual acuity (BCVA) testing, optical coherence tomography (OCT), and ophthalmoscopic examination. MAIN OUTCOME MEASURES: Retinal thickness (RT) on OCT and BCVA were measured at baseline and 1 and 3 months after surgery. RESULTS: There were no significant differences in RT, BCVA, severity of cataract, or systemic condition between the control and bevacizumab groups at the baseline. One and 3 months after surgery, the control group showed a significant increase in RT, whereas the bevacizumab group showed a significant decrease. Although postoperatively the eyes in both groups showed a significant improvement of BCVA, bevacizumab-treated eyes showed significantly better results (mean logarithm of the minimum angle of resolution, 0.38) than the control group (0.51) at month 3. There was a significant relationship between RT and visual acuity (VA) postoperatively in the control (P = 0.0001) and bevacizumab (P = 0.0141) groups. No systemic or ocular adverse events were observed. CONCLUSIONS: Short-term results suggest that intravitreal bevacizumab has the potential not only to prevent the increase in RT, but also reduce the RT of eyes with DME after cataract surgery. Further improvement of VA in bevacizumab-treated eyes may be dependent on a reduction in central RT. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Attenuation of aldose reductase gene suppresses high-glucose-induced apoptosis and oxidative stress in rat lens epithelial cells.

AIMS: A major contributory factor to diabetic cataract formation is increased aldose reductase (AR) activity in the polyol pathway. We investigated the effects of aldose reductase inhibition by RNA interference (RNAi) of the aldose reductase gene and administration of an aldose reductase inhibitor (ARI) on the changes induced by high glucose levels in rat lens epithelial cells (RLECs). METHODS: Small interfering RNAs (siRNAs) were designed to target the coding sequence of rat AR-siRNA. RLECs were cultured in either normal or high d-glucose. Western analysis was performed to monitor AR expression. MTS (3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt) and TUNEL assays were used to detect apoptotic cell death. Intracellular reactive oxygen species (ROS) were assessed by using DCFH-DA. Activation of nuclear factor-kappaB (NF-kappaB) was measured by an ELISA-based detection method. RESULTS: Both siRNA and ARI suppressed increased levels of ROS, activation of NF-kappaB, and apoptotic cell death induced by high glucose levels. Inhibition of rAR expression by siRNA and inhibition of AR activity by ARI also suppressed sorbitol accumulation. CONCLUSIONS: Both inhibition of rAR expression by rAR siRNA and inhibition of rAR activity by an ARI appeared effective in diminishing the changes of RLECs associated with high glucose levels.

Role of the polyol pathway in high glucose-induced apoptosis of retinal pericytes and proliferation of endothelial cells.

PURPOSE: The selective degeneration of pericytes and the proliferation of endothelial cells (ECs) appear to be associated with microaneurysm formation, an initial deficit in the early stage of diabetic retinopathy. The preventive effect of aldose reductase (AR) inhibitor (ARI) for high glucose-induced pericyte loss and EC growth was investigated. METHODS: The effect of high glucose (30 mM) exposure in the presence or absence of ARI for the cell growth of porcine pericytes and ECs was examined with the use of an in vitro coculture system to mimic the interaction between pericytes and ECs. To determine the role of transforming growth factor (TGF)-beta, its amount in culture media was measured, and the effects of the treatment of TGF-beta or neutralizing antibody on EC growth were examined. RESULTS: Abundant expression of AR and increased levels of polyol and apoptosis induced by high glucose were observed in pericytes, but not in ECs. ECs overexpressing AR cultured in high-glucose medium showed decreased cell viability. The growth-inhibitory effect of ECs on coculture with pericytes was attenuated by exposure to a high glucose concentration. Biochemical assays disclosed that the levels of active TGF-beta in media were linked to EC growth. Supply of active TGF-beta to coculture medium containing 30 mM D-glucose restored the inhibitory activity on EC growth. CONCLUSIONS: ARI rescued pericytes from high glucose-induced apoptosis and maintained the levels of TGF-beta, resulting in the prevention of cocultured EC growth.

TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity.

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-beta 1 and of alpha-smooth muscle actin and beta ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-beta1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.

Epithelial cell density in cataractous lenses of patients with diabetes: association with erythrocyte aldose reductase.

In the present study we evaluated the cell density of lens epithelium and its relation to the degree of erythrocyte aldose reductase (AR) in patients with type 2 diabetes. This prospective clinical study included 46 eyes of patients with type 2 diabetes and 48 eyes of patients without diabetes mellitus (DM). Flat preparations of lens epithelial cells (LECs) attached to the anterior capsule were studied. Multiple regression analysis was performed to evaluate the association between lens cell density and age, gender, type of cataract, duration of diabetes, diabetic retinopathy (DR), the levels of glycosylated hemoglobin (HbA1c) and erythrocyte AR. The mean density of LECs of patients with type 2 diabetes was 4,141+/-508cells/mm(2), which was significantly lower than that of patients without DM (4,560+/-458cells/mm(2); p<0.0001). Multiple regression analysis revealed that the level of erythrocyte AR was correlated with the reduction of LECs in the eyes of patients with type 2 diabetes. The correlation between the density of LECs and the amount of erythrocyte AR was significant in the diabetic group with a high value of HbA1c (>6.5%) or with DR. These results suggest that the polyol pathway via AR may be associated with the reduction of epithelial cell density in the eyes of patients with DM.

Age-dependent retinal capillary pericyte degeneration in galactose-fed dogs.

The galactose-fed beagle develops diabetes-like microvascular changes that are histologically and clinically similar in appearance to all stages of human diabetic retinopathy. This animal model is extremely useful for evaluating drugs for the treatment of diabetic retinopathy; however, the time required to develop the various retinal lesions (24-72 months for background to the proliferative stage) may be considered prohibitive. Retinal vascular changes begin with an initial degeneration of capillary pericytes, which has been linked to the aldose reductase catalyzed formation of galactitol. Because aldose reductase-linked sugar cataract formation is known to be age dependent, with the onset and severity of cataract higher in younger diabetic and galactose-fed animals, retinal capillary changes in the eyes of initially 2- versus 9-month-old beagles fed a diet containing 30% galactose were compared. Eyes were enucleated after 36 months of galactose feeding, the intact retinal capillaries were isolated by trypsin digestion, and defined retinal regions were evaluated by computer image analysis. Nicotinamide adenine dinucleotide phosphate-dependent reductase activity, using DL-glyceraldehyde and D-xylose as substrates, was also compared in the lenses and whole retinas of eyes from the 2- and 9-month-old beagles. Significantly (P

Aqueous flow in galactose-fed dogs.

Dogs fed galactose develop diabetes-like ocular complications that include keratopathy, cataracts, and retinopathy. The purpose of this study was to investigate whether galactosemic dogs display reduced aqueous flow similar to that observed in patients with insulin-dependent diabetes mellitus. Twelve male beagles at 9 months of age were divided into three groups of four. The Galactose group was fed diet containing 30% galactose for 97 months and the Reversal group was fed the galactose diet for an initial 38 months then standard dog diet for the remaining period. The Control group was fed standard dog diet for 97 months. Aqueous flow was determined by fluorophotometry in one eye per dog at 96 and 97 months after the initiation of galactose feeding. Intraocular pressure (IOP) was measured once in the morning by pneumatonometry. Anterior chamber depth was measured by A-scan. At the end of the experiment, eyes were enucleated and processed for histological examination. Dogs fed galactose diet for 97 months had significantly (p<0.05) increased body weights but similar IOP and anterior chamber depth compared to the other groups, and significantly (p=0.05) reduced aqueous flow compared to the control group (4.4+/-2.2 vs. 6.8+/-2.4 microl/min, mean+/-standard deviation, respectively). Additionally, aqueous flow decreased in the Reversal group to 3.1+/-1.3 microl/min (p=0.002). This decrease correlated with morphological changes of the ciliary processes. Like patients with insulin-dependent diabetes mellitus, galactose-fed dogs demonstrate reduced aqueous flow. This reduction was irreversible and independent of the retinopathy present. This animal model may be useful for the study of aqueous humor dynamics in diabetes.

Correlation between adult diabetic cataracts and red blood cell aldose reductase levels.

PURPOSE: To investigate the correlation between adult diabetic cataracts and levels of aldose reductase (AR) in red blood cells (RBCs). METHODS: The study involved 337 eyes of 337 patients with diabetes. The extent and severity of lens opacity was assessed according to the Lens Opacities Classification System III (LOCS III). The AR levels within RBCs were determined with an ELISA. The relationship between the AR level in RBCs and the prevalence of nuclear cataract, cortical cataract, and posterior subcapsular cataract in patients with diabetes was examined. RESULTS: There were no significant alterations in AR level in RBCs in patients with a diabetes duration of < or = 10 years and patients < 60 years of age. In each subgroup, a higher amount of AR levels in RBCs significantly correlated with the prevalence of posterior subcapsular cataracts. A significant association between cortical cataract and AR level in RBCs was also seen in a subgroup of patients younger than 60 years. CONCLUSIONS: AR emerges as an important factor affecting the onset of posterior subcapsular cataracts at the early stages of diabetes mellitus. This raises the possibility that AR inhibitors could play a useful role in treatment of adult diabetic cataract through its inhibition of AR activities.

Identification of the nuclear export signal in the helix-loop-helix inhibitor Id1.

Id proteins play important roles in cellular differentiation and proliferation by negatively regulating basic helix-loop-helix transcription factors. Although their intracellular localization may change depending on the biological situation, little is known about the molecular determinants underlying such changes. Here we report the identification of a nuclear export signal (NES) in Id1. The identified NES was different from that of Id2, but had the ability to confine heterologous green fluorescent protein to the cytoplasm. Thus, our results indicate that the intracellular localization of Id1 is regulated differently from that of Id2.

Development- and age-associated expression pattern of peroxiredoxin 6, and its regulation in murine ocular lens.

Peroxiredoxin (PRDX) 6 is a unique member of the PRDX family. Its antioxidant and signaling properties are related to its expression level in cells. We studied development- and age-associated changes in PRDX6 expression in the murine lens. We also investigated the effects of dexamethasone (Dex), transforming growth factor-beta1 (TGF-beta1) and tumor necrosis factor-alfa (TNF-alpha) on PRDX6 expression. Expression levels of PRDX6 mRNA in whole lenses isolated from postnatal day (PD) 1- to 18-month-old mice, and the effects of Dex, TGF-beta1 and TNF-alpha on the expression of PRDX6 in lens epithelial cells (LECs), were monitored using real-time reverse transcriptase-polymerase chain reaction (PCR) or Western blot. Localization of PRDX6 was studied using in situ hybridization and immunohistochemistry. PRDX6 expression gradually increased in the lenses of 4-week- to 6-month-old mice and declined thereafter. In situ hybridization and immunohistochemistry revealed that PRDX6 was localized in the cytoplasm of LECs and in lens fibers. Intense PRDX6 staining was present in the whole lens on gestational days 14 and 18. The lenses of PD1 mice showed diminished nuclear fiber staining, while those of 4-week-old mice revealed lack of nuclear fiber staining but intense staining of the germinative zone. LECs treated with TNF-alpha or Dex showed higher PRDX6 expression, while TGF-beta1 down regulated expression. Thus, our results provide a topographic basis for understanding the role of PRDX6 in the lens.

Characteristics of acid extrusion from Chinese hamster ovary cells expressing different prostaglandin EP receptors.

Acid extrusion responses to prostaglandin E2 were investigated in Chinese hamster ovary (CHO) cells heterologously expressing human EP1, EP2, and EP3I receptors (hEP1, hEP2 and hEP3I) by using a microphysiometer that detected small pH changes in the extracellular microenvironment. In the cells expressing hEP1, which is known to increase intracellular Ca2+, prostaglandin E2 (1 and 10 nM) slowly accelerated acid extrusion, but at higher concentrations an initial transient phase (approximately 5 times greater than the basal acidification) overlapped the slowly developing phase. In contrast, the cells expressing hEP2, which evokes cAMP production, showed dual responses to prostaglandin E2: an initial reduction followed by an acceleration of acid extrusion. In the cells expressing hEP3I, which is known to produce both a decrease in cAMP and a modest increase in intracellular Ca2+, acid extrusion was gradually accelerated by prostaglandin E2 and reached a plateau at around 2 min. Elimination of extracellular Ca2+ diminished the responses to prostaglandin E2 in hEP1 cells, but had little effect on the responses in hEP2 and hEP3I cells. Forskolin mimicked the dual effects of prostaglandin E2 observed in the hEP2 cells. Pretreatment with pertussis toxin inhibited the response to prostaglandin E2 in hEP3I cells, but the responses in hEP1 and hEP2 cells were not affected. Na+/H+ exchanger (NHE) inhibitors (EIPA and HOE642) suppressed all the responses induced by prostaglandin E2 in hEP1, hEP2, and hEP3I cells. These results suggest that EP receptor subtypes regulate acid extrusion mainly via NHE-1 through distinct signal transduction pathways in CHO cells.

Vlgr1 knockout mice show audiogenic seizure susceptibility.

Susceptibility to audiogenic seizures, which are reflex seizures provoked by loud noise, can be induced in rodents by acoustic priming (exposing animals to strong auditory stimuli at an early developmental stage). Some strains of mice and rats are susceptible to audiogenic seizures without priming and these have been used as good experimental models with which to study epilepsies. Here we identified Vlgr1d and Vlgr1e, novel alternatively-spliced variants of Vlgr1b/MGR1, which, upon sequence analysis, were shown to be transcripts from a locus previously characterized as mass1. Vlgr1 (Vlgr1b, Vlgr1d and Vlgr1e) mRNA is expressed predominantly in the neuroepithelium of the developing mouse brain. Our protein-tagged experiment suggested that Vlgr1d and Vlgr1e are secretory molecules, while Vlgr1b is a receptor. Knockout mice lacking exons 2-4 of Vlgr1 were susceptible to audiogenic seizures without priming, although there were no apparent histological abnormalities in their brains. Ninety-five percent of these knockout mice exhibited wild running, a feature typical of the preconvulsive phase of audiogenic seizures triggered by loud noise (11 kHz, 105 dB), and 68% exhibited tonic convulsions at 3 weeks after birth. Our monogenic mice, which have a unique genetic background, serve as a useful tool for further studies on seizures.