Kallikrein-Binding Protein Protects Retinal Müller Cells Against H2O2-Induced Cell Death by Inhibiting Calcium Overload

Abstract

Purpose:: Kallikrein-binding protein (KBP) belongs to the SERPIN (serine proteinase inhibitor) family, in which some neurotrophic factors have been identified. We have shown previously that KBP levels in the vitreous are decreased in a diabetic animal model and in diabetic patients, which may contribute to retinal degeneration in diabetes. It has been suggested that oxidative stress in diabetes plays a pathogenic role for retinal degeneration. Our purpose is to investigate whether KBP protects retina against oxidative stress-induced damage and the mechanism of action.

Methods:: Retinal Müller cell is an important cell type supporting neuronal cells in the retina. H₂O₂ is an oxidant and also a product of oxidative stress, which can trigger cell death in various types of cells including glial cells. Exposure of cultured Müller cells to H₂O₂ (400 µM) was used to induce cell death. MTT and flow cytometry were used to quantify the cell viability and necrotic/apoptotic cells. FITC labeled-KBP was used for the binding and competitive biding assay. The fluorescence of the probe CM-H2DCFDA and Fluo-4/AM were measured to determine the intracellular ROS level and calcium concentration. Phosphorylated AKT and total AKT were measured using Western blotting.

Results:: H₂O₂ induced Müller cell death mostly through necrosis. KBP showed a dose-dependent protective effect on retinal Müller cells against H₂O₂-induced necrosis. Similarly, KBP protected Y79 retinoblastoma cells under the H₂O₂ insult but increased cell death in bovine retinal capillary endothelial cells (BRCEC), suggesting that the protective effect of KBP is cell type-specific. The protective effect was not via decreasing intracellular ROS or H₂O₂ levels. The intracellular calcium overload and activated AKT were stimulated by H₂O₂. Blocking PI3K/AKT pathway inhibited H₂O₂-induced calcium overload. KBP blocked the calcium overload induced by H₂O₂ but not the AKT activation. Similarly, the same protective effect was achieved by a calcium chelator BAPTA/AM. KBP displayed saturable and reversible binding to Müller cells, suggesting that the protective effect of KBP may be mediated via a specific receptor on the cell surface.

Conclusions:: H₂O₂-induced retinal Müller cell death is through the PI3K/AKT/Calcium/Necorsis pathway. KBP protects Müller cell by inhibiting calcium overload, downstream of PI3K/AKT.

Keywords: Muller cells • retinal degenerations: cell biology • neuroprotection