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Manuela Bartoli, Menaka Thounaojam, Diana Gutsaeva, Wan Jin Jahng; TLR-4 phosphorylation at tyrosine 674 is necessary for NF-kB and inflammasome activation in the diabetic retina and in retinal endothelial cells exposed to glucidic stress. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4046.
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Up-regulation of Toll-like receptor 4 (TLR4) expression and signaling has been implicated in the pathogenesis of diabetic retinopathy (DR), however the molecular mechanisms of TLR4 activation in the diseased retina are poorly understood and often referring to promiscuous downstream signaling events. We have conducted studies assessing the occurrence and biological significance of TLR4 phosphorylation at tyrosine 674, which is located in the highly biologically active toll-IL-1 resistance (TIR) domain of this molecule.
Western blotting analysis was conducted to measure phospho-Tyr (674)-TLR4 in diabetic and non-diabetic human postmortem retinas and in retinas of streptozotocin-induced diabetic rats (STZ-rats) as compared to their respective normoglycemic controls. The occurrence of TLR4 phosphorylation at Tyr674 was also measured by immunoblotting in human retinal endothelial cells exposed to glucidic stress (HG=25mM D-glucose) or to normal glucose levels (NG=5mM D-glucose) or the osmotic control (LG=25mM L-glucose). HuREC were transfected with TRL4-Tyr-(674)-Ala mutants and exposed to the different glucose conditions for 48 hours. Downstream TLR4-mediated signaling events were measured by assessing NF-kB and p38MAPK phosphorylation /activation and expression of IL-1beta in cells exposed to the different treatments.
TLR4 expression and phosphorylation at Tyr 674 was significantly up-regulated in the diabetic rat retinas as well as in human postmortem diabetic retinas as compared to their respective normoglycemic controls. Importantly, HG stimulated TLR4 expression and phosphorylation at Tyr674 and consequent phosphorylation/activation of NF-kB and p38MAPK as well as production of mature IL-1beta. In addition blockade of TLR4 phosphorylation at Tyr674, achieved by transfection of HuREC with Tyr674-Ala mutants, resulted in signficantly decreased phosphorylation/activation of NF-kB and p38MAPK in these cells as well as decreased levels of mature IL-1beta.
Our data suggest that monitoring and study of TLR4 phosphorylation at Tyr 674 in response to hyperglycemia may offer a more specific diagnostic and therapeutic target for the prevention and treatment of DR and, potentially, of other ischemic retinopathies involving TLR4 activation
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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