The transcription factor nuclear factor erythroid-2–related factor 2 (Nrf2) plays a critical role in reducing oxidative stress by promoting the expression of antioxidant genes. Both individuals with diabetes and preclinical diabetes models exhibit evidence of a defect in retinal Nrf2 activation. We recently demonstrated that increased expression of the stress response protein regulated in development and DNA damage 1 (REDD1) is necessary for the development of oxidative stress in the retina of streptozotocin-induced diabetic mice. In the present study, we tested the hypothesis that REDD1 suppresses the retinal antioxidant response to diabetes by repressing Nrf2 function. We found that REDD1 ablation enhances Nrf2 DNA-binding activity in the retina and that the suppressive effect of diabetes on Nrf2 activity is absent in the retina of REDD1-deficient mice compared with WT. In human MIO-M1 Müller cell cultures, REDD1 deletion prevented oxidative stress in response to hyperglycemic conditions, and this protective effect required Nrf2. REDD1 suppressed Nrf2 stability by promoting its proteasomal degradation independently of Nrf2's interaction with Kelch-like ECH-associated protein 1 (Keap1), but REDD1-mediated Nrf2 degradation required glycogen synthase kinase 3 (GSK3) activity and Ser-351/Ser-356 of Nrf2. Diabetes diminished inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β) at Ser-9 in the retina of WT mice but not in REDD1-deficient mice. Pharmacological inhibition of GSK3 enhanced Nrf2 activity and prevented oxidative stress in the retina of diabetic mice. The findings support a model wherein hyperglycemia-induced REDD1 blunts the Nrf2 antioxidant response to diabetes by activating GSK3, which, in turn, phosphorylates Nrf2 to promote its degradation.
Upregulation of the stress response protein REDD1 (regulated in development and DNA damage response 1) has been implicated in diabetes-induced retinal defects in both preclinical models and diabetic patients. In fact, intravitreal administration of a siRNA targeting the REDD1 mRNA has demonstrated promise for improving visual function in patients with diabetic macular edema. However, the mechanism responsible for increased retinal REDD1 protein content in the context of diabetes has never been established. Herein, we investigated the hypothesis that diabetes promotes REDD1 expression by reducing the rate of REDD1 degradation. In the retina of streptozotocin-induced diabetic mice, REDD1 protein content was increased in the absence of a change in REDD1 mRNA. When diabetic mice were administered antioxidants, neither oxidative stress nor REDD1 expression were increased in the retina. In human MIO-M1 retinal Müller cells, the rate of REDD1 degradation was reduced upon exposure to hyperglycemic conditions. Antioxidant addition to culture medium prevented both the increase in oxidative stress and REDD1 stability in cells exposed to hyperglycemic conditions. Surprisingly, the suppressive effect of oxidative stress on REDD1 degradation was independent of the ubiquitin-proteasome pathway. Rather, REDD1 was rapidly degraded upon activation of chaperone-mediated autophagy (CMA) and exposure to hyperglycemic conditions prevented the suppressive effect of CMA activation on REDD1 expression. Additional mechanistic details of this new regulatory pathway will be presented. Overall, the findings provide evidence that diabetes promotes retinal REDD1 protein content via a post-transcriptional effect and potentially identifies superior therapeutic targets for preventing the pathological increase in REDD1. Disclosure W. P. Miller: None. A. Toro: None. S. Sunilkumar: None. J. Giordano: None. M. D. Dennis: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-14-INI-04 to M.D.D.); National Institutes of Health (R01EY029702 to M.D.D.), (F31EY031199-01 to W.P.M.)
Oxidative stress is a major contributor to the pathophysiology of diabetes. To combat oxidative stress, the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2) promotes the expression of an array of antioxidant gene products. However, in both the retina of diabetic patients and in preclinical diabetes models, there is a failure to properly upregulate Nrf2 activity. We previously demonstrated that retinal REDD1 protein expression is enhanced by diabetes-induced hyperglycemia and necessary for both the rise in retinal reactive oxygen species and the development of visual dysfunction in diabetic mice. In the present study, we investigated the hypothesis that signaling events downstream of REDD1 act to limit the retinal Nrf2 antioxidant response to the diabetic metabolic environment. We found that REDD1 ablation not only enhanced retinal Nrf2 activity, but also prevented the suppressive effect of streptozotocin-induced diabetes as compared to wild type mice. Nuclear Nrf2 protein expression and activity were enhanced in REDD1 knockout human MIO-M1 retinal Müller cells in culture, independent of a change in Nrf2 mRNA abundance. REDD1 deletion prevented oxidative stress in response to hyperglycemic conditions, and this protective effect was absent upon Nrf2 knockdown. REDD1 suppressed Nrf2 stability independent of Keap1 by promoting GSK3-mediated nuclear exclusion and proteasomal degradation via the ubiquitin ligase adapter β transducin repeat containing protein (β-TrCP). In the retina of diabetic REDD1-deficient mice, enhanced GSK3 phosphorylation was associated with a decrease in oxidative stress as compared to diabetic wild type mice. Pharmacological inhibition was used to suppress GSK3 activity in both cells in culture and in the retina of diabetic mice. Remarkably, GSK3 inhibition prevented the suppressive effect on Nrf2. Overall these findings support therapeutic approaches targeting REDD1 to prevent diabetes-induced visual dysfunction. Disclosure W.P. Miller: None. A. Toro: None. J. Giordano: None. M.D. Dennis: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-14-INI-04 to M.D.D.); National Eye Institute (1F31EY031199-01)
This study is a case analysis of Trails East business strategy, including the internal and external environmental factors affecting their operations, and our recommendations for enhancing the performance of the firm. A variety of mitigating factors are addressed in our inquiry including the closure of multiple bus companies following the global pandemic and the current and potential future environmentally based restrictions on emissions and equipment.
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