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Dmitry V Ivanov, Xenia Dvoriantchikova, Andrea Rachelle Santos, Galina Dvoriantchikova; The Role Of Protein Kinase C (PKC) In Heat Shock Protein 70 (HSP70)-Mediated Neurotoxicity. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1348.
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© ARVO (1962-2015); The Authors (2016-present)
Sterile inflammation, characterized by innate immune responses that occur in the absence of live pathogens, is an unavoidable consequence of ischemia-reperfusion (IR) injury in the retina. It is known that toll-like receptor 4 (TLR4) triggers an innate immune response in tissue and contributes to damage and sterile inflammation in the retina mediated by IR. However, the mechanism of TLR4 activation under sterile conditions in ischemic tissue was poorly understood. We performed this study to clarify the mechanism. To this end, we focused on the extracellular heat shock protein 70 (HSP70), the prototypic ligand of TLR4.
Retinal IR injury was induced by unilateral elevation of intraocular pressure. To test the role of PKC in IR injury, ischemic retinas were treated with the PKC inhibitors (polymyxin B and Go6976) and retinal damage was evaluated by direct counting of neurons in the ganglion cell layer of flat-mounted retinas 7 days after IR. Primary retinal ganglion cells (RGCs) isolated from retinas according to the two-step immunopanning protocol and glial cells obtained by the “shaking method” were used for in vitro experiments. Quantitative RT-PCR, ELISA and western blot analysis were used to study production of pro-inflammatory factors in ischemic retinas and in primary RGC and glial cultures.
We noted that PKC was involved in TLR4signaling and found that PKC inhibitors promote neuroprotection by reducing the pro-inflammatory activity in ischemic tissue. To put all of the pieces in the signaling cascade together, we performed an in vitro study. We found that PKC is critical to mediate HSP70-dependent pro-inflammatory response. At the same time, the contamination of HSP70 preparations with low-dose endotoxin is not critical to mediate production of pro-inflammatory factors. We also found that extracellular HSP70 can promote neuronal death at least by mediating production of cytotoxic levels of tumor necrosis factor-alpha (TNF) predominantly due to the TLR4/MyD88/PKC signaling cascade.
Thus, our findings suggest that PKC acts as a switch to amplify the pro-inflammatory activity of HSP70/TLR4signaling, which is sufficient to mediate neuronal death.
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