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.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
Lipid peroxidation products activate the canonical WNT pathway through oxidative stress, which plays an important role in the development of retinal diseases.
Zhou
T
T
Department of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, Guangzhou, China.
Zhou
K K
KK
Lee
K
K
Gao
G
G
Lyons
T J
TJ
Kowluru
R
R
Ma
J-x
JX
D000111:Acetylcysteine / Q000494:pharmacology; D000447:Aldehydes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D055513:Connective Tissue Growth Factor / Q000378:metabolism; D003930:Diabetic Retinopathy / Q000378:metabolism*; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D007653:Ketocholesterols / Q000494:pharmacology; D026502:LDL-Receptor Related Proteins / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects*; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats; D051820:TCF Transcription Factors / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
D000447:Aldehydes; D007653:Ketocholesterols; D026502:LDL-Receptor Related Proteins; C555475:LRP6 protein, human; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C555477:Lrp6 protein, rat; D051820:TCF Transcription Factors; D051176:beta Catenin; D055513:Connective Tissue Growth Factor; D006861:Hydrogen Peroxide; C027576:4-hydroxy-2-nonenal; C003001:7-ketocholesterol; D000111:Acetylcysteine