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Rajakrishnan Veluthakal, Anjan Kowluru, Renu Kowluru; Diabetic retinopathy and regulation of p38 MAP kinase by Tiam1-Rac1 signaling module. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4922.
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Emerging evidence in multiple tissues, including retina, implicates generation of reactive oxygen species and the ensuing oxidative stress as triggers for mitochondrial stress and cell apoptosis. We recently reported novel roles for phagocyte-like NADPH oxidase [Nox2] in retinal mitochondrial dysfunction and cell death leading to the development of diabetic retinopathy. Herein, we investigated contributory roles of p38 MAP kinase [p38 MAPK] as a potential downstream signaling event in Rac1-mediated induction of metabolic stress leading to capillary cell apoptosis.
Activation of p38 MAPK was quantified in retinal endothelial cells incubated with low (5 mM) or high (20 mM) glucose for up to 96 hours (a duration where mitochondrial dysfunction and capillary cell apoptosis can be observed) by Western blotting using anti-phospho-Thr180/Tyr 182 antibody. The role of Tiam1-Rac1 mediated activation of p38 MAPK was confirmed by incubating the cells with NSC23766 (20 µM). Since S-palmitoylation is a requisite post-translational modification of Rac1, which regulates its activation and trafficking, the role of palmitoylation in p38 MAPK activation was confirmed using 2-bromopalmitate (100 µM), an inhibitor of S-palmitoylation. The in vitro results were confirmed in the retina from rats diabetic for 2 months.
Activation of p38 MAPK in retinal endothelial cells was observed as early as 3 hours after high glucose exposure, and the activation continued until 96 hours. Further, NSC23766 markedly attenuated glucose-induced activation of p38 MAPK, suggesting a regulatory role for Tiam1-Rac1 in this signaling axis. In addition, 2-bromopalmitate, also inhibited glucose-induced p38 MAPK activation. Consistent with the in vitro results, p38 MAPK activation was significantly higher in the retina from diabetic rats compared to age-matched normal rats.
Our findings suggest that Tiam1-Rac1 mediated activation of Nox2 and p38 MAPK constitute early signaling events leading to mitochondrial dysfunction and the development of diabetic retinopathy. These results also provide the first evidence to implicate novel roles for protein palmitoylation in this signaling cascade.
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