The role of lipid peroxidation products and oxidative stress in activation of the canonical wingless-type MMTV integration site (WNT) pathway in a rat model of diabetic retinopathy
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Our recent studies suggest that activation of the wingless-type MMTV integration site (WNT) pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. Here we investigated the causative role of oxidative stress in retinal WNT pathway activation in an experimental model of diabetes.
Cultured retinal pigment epithelial cells and retinal capillary endothelial cells were treated with a lipid peroxidation product, 4-hydroxynonenal (HNE), and an antioxidant, N-acetyl-cysteine (NAC). In vivo, rats with streptozotocin-induced diabetes were treated by NAC for 8 weeks. Activation of the canonical WNT pathway was measured by TOPFLASH assay and by western blot analysis of WNT pathway components and a WNT target gene, Ctgf. Oxidative stress in the retina was evaluated by immunostaining of HNE and 3-nitrotyrosine.
Levels of phosphorylated and total LDL receptor-related protein (LRP)6, and cytosolic β-catenin, as well as transcriptional activity of T cell factor (TCF)/β-catenin were significantly increased by HNE. The production of connective tissue growth factor (CTGF) was also upregulated by HNE. NAC blocked the WNT pathway activation induced by HNE. Furthermore, LRP6 stability was increased by HNE and decreased by NAC. Retinal levels of HNE and 3-nitrotyrosine were significantly increased in diabetic rats, compared with those in non-diabetic rats. In the same diabetic rat retinas, levels of LRP6, cytosolic β-catenin and CTGF were significantly increased. NAC treatment reduced HNE and 3-nitrotyrosine levels and attenuated the upregulation of LRP6, β-catenin and CTGF in diabetic rat retina.
Lipid peroxidation products activate the canonical WNT pathway through oxidative stress, which plays an important role in the development of retinal diseases.
KeywordsDiabetic retinopathy Inflammation LRP Oxidation Peroxidation Retina WNT
Age-related macular degeneration
Bovine retinal capillary endothelial cells
Connective tissue growth factor
Forkhead box class O
Glycogen synthase kinase 3β
Heavily oxidised glycated LDL
LDL receptor-related protein
T cell factor
Wingless-type MMTV integration site
Lipid peroxidation refers to the oxidative degradation of lipids. Reactive oxygen species including superoxide anion radical (O2−), hydrogen peroxide (H2O2), hydroxyl radical (OH⋅), nitric oxide (NO⋅) and peroxynitrite (ONOO−⋅) are most commonly involved in the initiation of lipid peroxidation . The most intensively investigated lipid peroxidation product is 4-hydroxynonenal (HNE), a major oxidation product of membrane lipids containing polyunsaturated n-6 acyl groups. HNE was initially identified as a toxic endproduct of lipid peroxidation, but later emerged as a signalling mediator, which is involved in cell signal transduction and regulation of gene expression . Increasing evidence has shown that tissue and blood levels of HNE are higher in a number of human diseases, including chronic inflammation, atherosclerosis, neurodegenerative diseases, diabetes and different types of cancer. It has been suggested that excessive HNE production could interfere with normal signalling pathways and have a potential pathogenic role in these diseases . Besides the polyunsaturated fatty acids, cholesterol may also undergo free radical-mediated oxidation. One of the most important products of cholesterol peroxidation is 7-ketocholesterol, which is a main component of oxidised LDL and responsible for the cytotoxicity of oxidised LDL .
The retina is highly susceptible to oxidative stress and lipid peroxidation, as it has high levels of polyunsaturated fatty acids, the highest oxygen consumption per gram of tissue in the body and is also exposed to light . Diabetic retinopathy and age-related macular degeneration (AMD) are leading causes of blindness in the USA [6, 7]. Although several lines of evidence have shown that lipid peroxidation products are important contributors to development of these diseases [8, 9, 10], the precise molecular mechanism that mediates the signalling transduction is still unclear.
The WNT signalling pathway is versatile, regulating cell proliferation and differentiation, apoptosis, stem cell maintenance, angiogenesis, inflammation, fibrosis and carcinogenesis . WNT signalling had been shown to be a key regulator of retinal development and has been implicated in certain retinal diseases . WNT ligands are secreted cysteine-rich glycosylated proteins, which bind to a receptor complex comprising the frizzled receptors and the LDL receptor-related protein (LRP) 5 or 6. WNT ligand binding leads to phosphorylation and activation of LRP6, which is essential for WNT signalling. It also leads to inactivation of ‘destructive complex’, which is composed of glycogen synthase kinase-3β (GSK3β), axin and adenomatous polyposis coli. Inactivation of the ‘destructive complex’ prevents the proteosomal degradation of the transcriptional factor β-catenin and promotes its accumulation and nuclear translocation. Once β-catenin translocates into the nucleus, it associates with the T cell factor (TCF) and regulates transcription of the WNT target genes, including VEGF and CTGF [13, 14, 15].
Our previous studies have shown that the WNT pathway is activated in retina from human patients with diabetic retinopathy and in those from animal models of diabetes . The WNT pathway is also activated in an animal model of AMD . Moreover, blockade of WNT signalling attenuates retinal inflammation and neovascularisation in diabetic retinopathy and in animal models of AMD. These observations suggest that WNT pathway activation in diabetic retinopathy and AMD contributes to retinal inflammation and neovascularisation. The mechanism responsible for WNT pathway activation in these disease conditions is not clear.
The present study investigated the causative roles of oxidative stress and subsequent lipid peroxidation products in WNT pathway activation in AMD and diabetic retinopathy.
Materials and antibodies
HNE was purchased from Calbiochem (Madison, WI, USA). N-Acetyl-cysteine (NAC), hydrogen peroxide (H2O2), 7-ketocholesterol and cycloheximide were purchased from Sigma (St Louis, MO, USA). Normal human LDL and heavily oxidised glycated LDL (HOG-LDL) were isolated and prepared following a documented protocol . Antibodies against β-catenin, total-LRP6 and connective tissue growth factor (CTGF) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies against phosphorylated-LRP6 (pLRP6) were from Cell Signaling (Danvers, MA, USA) and an anti-HNE adducts antibody was from Alpha Diagnostic (San Antonio, TX, USA). Antibodies for 3-nitrotyrosine and β-actin, and horseradish peroxidase-conjugated secondary antibodies were obtained from Abcam (Cambridge, MA, USA). The 2F1 monoclonal antibody was customer-generated through a contract with Protein-Tech (Chicago, IL, USA). Conditioned media containing wingless-type MMTV integration site family, member 3 (WNT3A) were prepared from mouse L cells (ATCC, Manassas, VA, USA) stably producing WNT3A. Control conditioned media were obtained from parental L cells.
ARPE19 cell line was purchased from ATCC and cultured according to the manufacturer’s protocol. Cells in the 15th to 30th passages were used for all experiments. Primary bovine retinal capillary endothelial cells (BRCEC) were isolated and cultured as described previously . Cells in the fifth to sixth passages were used for all experiments.
Cell viability assay
ARPE19 cells in 96-well plates were treated with different stressors. Cell viability was measured using a kit (Cell Counting Kit-8; Dujindu, Rockville, MD, USA) according to the manufacturer’s protocol.
Dual-luciferase reporter assay
The TOPFLASH, FOPFLASH plasmids from the TCF Reporter Plasmid Kit (Millipore, Temecula, CA, USA) and renilla luciferase pRL-TK vectors were co-transfected into ARPE19 cells according to manufacturer’s protocol. TOPFLASH, FOPFLASH activity was measured using dual luciferase reporter system (Promega, Madison, WI, USA) and normalised to renilla luciferase activity.
Cells were fractionated using FractPrep (BioVision, Mountain View, CA, USA) following the manufacturer’s protocol.
Western blot analysis
Western blot analysis was performed as described previously .
Animals and induction of diabetes in rats
Female Brown Norway rats (8 weeks of age) were purchased from Harlan (Indianapolis, IN, USA). Care, use and treatment of all animals in this study were in strict agreement with the guidelines for the care and use of laboratory animals set forth by the University of Oklahoma. Experimental diabetes was induced as described previously .
Immunohistochemistry was performed as described previously . Antibodies for 3-nitrotyrosine and HNE were used at a dilution of 1:200 and the antibody for β-catenin at a dilution of 1:300, with incubation overnight. After thorough washes with PBS, immunosignals were developed using a kit (Vectastain ABC; Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s protocol.
Data are presented as means ± SD. Comparisons were performed by two-tailed paired Student’s t test. A value of p < 0.05 was considered statistically significant.
7-Ketocholesterol, H2O2 and HNE activated the canonical WNT pathway
The canonical WNT pathway was activated by HNE
In the canonical WNT pathway, β-catenin is phosphorylated by GSK3β in the absence of WNT ligands and then degraded in the cytosol. GSK3β activity is regulated by phosphorylation, e.g. phosphorylation at residue serine 9 (Ser9) is inhibitory to the kinase activity of GSK3β . As shown in Fig. 2d, e, HNE induced an increase in phosphorylation of GSK3β at Ser9, indicating a decreased kinase activity of GSK3β. Consistently, cytosolic β-catenin levels were increased significantly in cells treated with HNE, compared with those in untreated cells (Fig. 2f, g), indicating stabilisation of β-catenin.
NAC attenuated HNE-induced activation of the canonical WNT pathway
Next, we examined the protein stability of LRP6 in the cells treated with HNE and HNE plus NAC. We found that the half-life of LRP6 is approximately 3 h in untreated control cells. The HNE treatment significantly inhibited degradation of LRP6, prolonging its half-life. This stabilisation of LRP6 by HNE was reversed by NAC, suggesting that oxidative stress induces WNT pathway activation via stabilisation of LRP6 (Fig. 3e, f).
Effect of NAC on WNT ligand-induced WNT signalling
Level of CTGF was upregulated by HNE and inhibited by NAC
β-Catenin was induced by HNE and HOG-LDL, and blocked by NAC treatment in endothelial cells
Causative effects of oxidative stress on the WNT pathway in the retina of diabetic rats
Our previous studies have shown that activation of the WNT pathway plays a pathogenic role in diabetic retinopathy and AMD [16, 17]. Accumulating evidence has demonstrated that oxidative stress and lipid peroxidation are causative factors of retinal diseases including diabetic retinopathy and AMD [8, 9, 10]. Leakage and subsequent oxidation of plasma lipoproteins in the retina may be implicated . These observations prompted us to hypothesise that oxidative stress is a major cause of the WNT pathway activation in these disease conditions. The present study demonstrates that oxidative stress is responsible, at least in part, for the WNT pathway activation in retina of diabetic rats. Our results also show that several oxidants, including lipid peroxidation products, activate the WNT pathway in cultured retinal cells and endothelial cells. Mechanism studies indicate that these oxidants activate the canonical WNT pathway via stabilisation of LRP6 and increasing of LRP6 levels. These results for the first time elucidate the mechanism for WNT pathway activation in diabetic retinopathy and AMD.
The WNT signalling pathway participates in angiogenesis during embryogenesis and in pathological processes involving chronic inflammation and the development of tumours . Recent evidence has linked the WNT pathway to vascular growth in murine and human retina . Previously, we have shown that the WNT pathway is activated in retina from human patients with diabetic retinopathy and in animal models including streptozotocin-induced diabetic rats, Akita mice and oxygen-induced retinopathy mice . The WNT pathway is also activated in VLDL receptor knockout mice, a genetic model of AMD . In addition, activation of the WNT pathway alone in retinal pigment epithelial cells and in retina of normal rats upregulated angiogenic and inflammatory factors, including vascular endothelial growth factor, TNF-α, intercellular adhesion molecule 1 and nuclear factor kappa B . A WNT signalling inhibitor ameliorated inflammation and neovascularisation in animal models of oxygen-induced retinopathy and AMD [16, 30]. These results, together with the incipient investigation, established the causative role of the WNT pathway in retinal neovascularisation and inflammation in diabetic retinopathy and AMD. Although compelling evidence revealed the role of oxidative stress and lipid peroxidation products in retinal diseases [8, 9, 10], no further studies were conducted to investigate the relationship between oxidative stress and WNT pathway activation in the retina. Here, we unmasked the intrinsic cause of WNT activation under these pathological conditions. As shown here, hydrogen peroxide, one of the initiators of lipid peroxidation, and different lipid peroxidation products (7-ketocholesterol, HNE, HOG-LDL) all activated the WNT pathway. Moreover, the anti-oxidant NAC blocked WNT pathway activation. These results established the causative role of oxidative stress in WNT pathway activation in diabetic retinopathy and AMD.
Oxidative stress is known to cause damage to nucleic acid, proteins and lipids . One of the prevalent aldehydes generated is HNE, which is highly reactive and considered an indicator of oxidative stress and messenger in cell signalling. Due to chemical modification, HNE is able to invoke different cell signalling pathways depending on its concentration. At low and non-cytotoxic concentrations, HNE regulates cell proliferation and signal transduction, whereas at high concentrations it induces differentiation and apoptosis . It is well accepted that HNE participates in the cell signalling that regulates inflammation, which represents the main driving force in many chronic human diseases. A large volume of data has clearly proved the involvement of HNE in human pathological conditions . For instance, HNE-derived protein modifications are involved in lipofuscinogenesis and contribute to cell damaging effects of lipofuscin in retinal diseases such as AMD . Moreover, concentrations of HNE in the plasma were significantly elevated in patients with diabetic retinopathy in comparison to diabetic patients without retinopathy . Therefore, we chose HNE as a representative lipid peroxidation product for this study.
H2O2 is a reactive oxygen species as well as an initiator of lipid peroxidation , and can alone cause a significant increase of HNE adducts . It is accepted that H2O2 modulates signalling pathways partly through its effects on lipid peroxidation . Our results show that H2O2 induced over-activation of the canonical WNT pathway, indicating: (1) that free radicals in oxidative stress and subsequent lipid peroxidation are initial causative factors for activation of the canonical WNT pathway; and (2) the possible involvement of HNE in oxidative stress-mediated signalling. It is well accepted that oxidative stress activates forkhead box class O (FOXO) signalling; indeed, some publications have concluded that FOXO and TCF proteins compete for the limited pool of β-catenin under oxidative stress [34, 35, 36, 37]. However, the interactions between the WNT signalling pathway and oxidative stress remain controversial. Previously, Shin et al. reported that WNT signalling was inhibited by a high concentration of H2O2 . However, Funato et al. showed that a low concentration of H2O2 induced rapid stabilisation of β-catenin and activation of TCF in NIH3T3 or HEK293 cells, providing new insights into the cross-talk between redox and the WNT/β-catenin pathway . With regard to HNE, it is well established that HNE interferes with many signalling pathways, but previous reports appear contradictory in some aspects. For example, HNE (5 μmol/l) was found to induce early neuronal cell death. Against this, Dozza et al. observed that HNE (10 μmol/l) provoked significant inhibition of GSK3β in human neuroblastoma IRM-32 cells . The disparity may be ascribed to the concentration of oxidants and incubation time, as well as different cell types in these studies.
Conjugation with glutathione is the main pathway for HNE metabolism . A deficiency of glutathione has been found in several diseases including inflammation, diabetes, cystic fibrosis, HIV, etc. NAC is the most effective and versatile antioxidant among other commonly used antioxidants, such as vitamin C, vitamin E and lipoic acid, since NAC supplies the cysteine required for glutathione synthesis and replenishment, reduces disulphide bonds in proteins and scavenges free radicals . Our in vitro experiments showed that HNE-induced WNT pathway activation was attenuated by NAC treatment, mainly through blocking LRP6 phosphorylation and stabilisation induced by HNE, indicating that NAC renders more glutathione to conjugate HNE and prevents its interactions with target proteins. Additionally, NAC also blocked WNT ligand-induced WNT activation. Similarly, a previous study reported that WNTs and related signalling proteins are the major targets of glutathione action , strongly supporting the notion that WNT signalling pathway is redox-sensitive and that oxidative stress is a cause of WNT pathway activation. Moreover, placebo-controlled trials have shown that oral administration of NAC is beneficial in several diseases . Our in vivo study presented here showed that NAC treatment inhibits WNT pathway activation in retina of diabetic rats and downregulates expression of a target gene of WNT signalling. These findings suggest that blockade of the WNT pathway by NAC may be a possible molecular mechanism underlying its beneficial effects.
Regarding the mechanism underlying HNE-induced activation of the WNT pathway, we showed that HNE increases phosphorylation of LRP6, an initial step in WNT pathway activation. However, the mechanism by which HNE increases phosphorylated LRP6 levels remains unclear. It is known that WNT ligands lead to WNT–frizzled–LRP6 complex formation and LRP6 phosphorylation [41, 42, 43]. The hydroxyl group in HNE makes it highly reactive with thiol of cysteine in proteins . Moreover, some studies have suggested that proteins cross-link via bridging by HNE and subsequently become more resistant to proteolysis . Proteasome itself is a known target of HNE, and HNE contributes to protein accumulation via impairment of ubiquitin and/or proteasome-dependent intracellular proteolysis . Thus, one possible scenario is that HNE modifies LRP6 or proteasome and inhibits LRP6 degradation. This assumption is supported by our LRP6 stability assay, which showed that HNE inhibits LRP6 degradation rather than upregulating its production. The increased stability of LRP6 by HNE could contribute to the elevation of LRP6 protein levels by HNE. Moreover, HNE can react with nucleic acid and acts as a strong mutagen through a clastogenic action. HNE covalently binds to DNA and DNA-associated histones, which can affect the conformation of histones . A documented study has reported that the ability of HNE to alter DNA×histone interactions could contribute to the vulnerability of DNA to oxidation, which has been observed in the brain of Alzheimer patients . The covalent and non-covalent modifications of DNA and histone proteins, which is a well-known form of epigenetic regulation, is one of the mechanisms that directly influence chromatin structure . Therefore, it is possible that epigenetic alteration may contribute to the increase of LRP6 protein content/phosphorylation by HNE. Since LRP6 phosphorylation itself involves multiple mechanisms and many proteins [48, 49], the molecular mechanism underlying the HNE-mediated LRP phosphorylation warrants further investigation.
In conclusion, our study established oxidative stress and lipid peroxidation products as activators of the canonical WNT pathway, a pathway that is pivotal in the pathogenesis of diabetic retinopathy and AMD. Furthermore, this study showed that the effect of HNE on the WNT signalling pathway could be through stabilisation of the WNT co-receptor, thus elucidating a pathogenic mechanism of HNE in chronic diseases.
This study was supported by National Institutes of Health grants EY018659, EY012231, EY019309, P20RR024215, EY17313, a grant from the Oklahoma Center for the Advancement of Science and Technology (OCAST) and a research award from the ADA.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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