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Elevated cell proliferation and VEGF production by high-glucose conditions in Muller cells involve XIAP.

ABSTRACT: PURPOSE: Müller cells have important roles in the pathogenesis of diabetic retinopathy by promoting cell proliferation and inducing the production of vascular endothelial growth factor (VEGF) under hyperglycemic conditions. The objective of this study was to determine the potential mechanism of Müller cell proliferation and VEGF production due to high-glucose conditions. METHODS: Primary cultured rat Müller cells were incubated with medium containing variable concentrations of glucose and/or embelin, a specific inhibitor of X-linked inhibitor of apoptosis protein (XIAP), for 72 h. The proliferation of Müller cells was assessed by the MTT assay. The expression and/or phosphorylation of 146 proteins were assessed using protein pathway array. RESULTS: High concentrations of glucose-induced Müller cell proliferation and altered expression and/or phosphorylation of 47 proteins that have been identified to have key roles in several important signaling pathways (XIAP, VEGF, HIF1α, NFκB, etc) and are involved in the regulation of cell survival, proliferation, or apoptosis. However, Müller cell alterations induced by high-glucose conditions were counteracted by the XIAP inhibitor embelin, and 26 proteins/phosphorylations (out of 47) were restored to their normal levels. Nine proteins, including NFκB p65, p-p38, tumor necrosis factor-α, urokinase-type plasminogen activator, CREB, IL-1β, HCAM, estrogen receptor-α, and p-Stat3, were involved in regulatory networks between XIAP and VEGF. CONCLUSIONS: The current study suggests that XIAP may be a potential regulator that can mediate a series of pathological changes induced by high-glucose conditions in Müller cells. Therefore, embelin could be a potential agent for the prevention and treatment of diabetic retinopathy.

SUBMITTER: Sun Y

PROVIDER: S-EPMC3831121 | biostudies-other | 2013 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundIn various tumour types, elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been observed and XIAP targeting in diverse tumour entities enhanced the susceptibility to chemotherapeutic agents. Therefore, XIAP has been described and reviewed repeatedly as a chemoresistance factor in different tumour entities. However, rather than being an adverse prognostic marker, recent data suggest that elevated XIAP expression may be associated with a favourable clinical outcome. These somewhat conflicting findings, and the fact that in early studies XIAP suppressed apoptosis only when expressed transiently at levels far in excess of its physiological concentration, argue that the function of XIAP as an anti-apoptotic factor in tumour cells is both more complex and diverse than previously appreciated.MethodsTo better understand the impact of long-term elevated XIAP expression on resistance to chemotherapy, we generated cell lines stably overexpressing XIAP. The role of mitochondria was examined by stable expression of Bcl2 or stable knockdown of second mitochondria-derived activator of caspase (SMAC) in combination with up- or downregulation of XIAP expression.ResultsOur data show that long-term expression of XIAP at concentrations comparable to that in tumour cells (two- to five-fold increase) resulted in little or no resistance towards chemotherapeutic drugs. The XIAP overexpression only in conjunction with stable knockdown of a single XIAP-antagonising factor such as SMAC resulted in severe resistance to cytostatic agents demonstrating XIAP as a potent chemoresistance factor only in cells lacking functional XIAP regulatory circuits.ConclusionOur results demonstrated that elevated XIAP expression alone cannot serve as a predictive marker of chemoresistance. Our data suggest that in order to predict the impact of XIAP on chemosusceptibility for a given tumour entity, the expression levels and functional states of all XIAP modulators need to be taken into account.

Project description:Chronic hyperglycemia-induced thioredoxin-interacting protein (TXNIP) expression, associated oxidative/nitrosative stress (ROS/RNS), and mitochondrial dysfunction play critical roles in the etiology of diabetic retinopathy (DR). However, there is no effective drug treatment to prevent or slow down the progression of DR. The purpose of this study is to examine if a combination drug treatment targeting TXNIP and the mitochondria-lysosome pathway prevents high glucose-induced mitochondrial stress and mitophagic flux in retinal Müller glial cells in culture, relevant to DR. We show that diabetes induces TXNIP expression, redox stress, and Müller glia activation (gliosis) in rat retinas when compared to non-diabetic rat retinas. Furthermore, high glucose (HG, 25 mM versus low glucose, LG 5.5 mM) also induces TXNIP expression and mitochondrial stress in a rat retinal Müller cell line, rMC1, in in vitro cultures. Additionally, we develop a mitochondria-targeted mCherry and EGFP probe tagged with two tandem COX8a mitochondrial target sequences (adenovirus-CMV-2×mt8a-CG) to examine mitophagic flux in rMC1. A triple drug combination treatment was applied using TXNIP-IN1 (which inhibits TXNIP interaction with thioredoxin), Mito-Tempo (mitochondrial anti-oxidant), and ML-SA1 (lysosome targeted activator of transient calcium channel MCOLN1/TRPML1 and of transcription factor TFEB) to study the mitochondrial-lysosomal axis dysregulation. We found that HG induces TXNIP expression, redox stress, and mitophagic flux in rMC1 versus LG. Treatment with the triple drug combination prevents mitophagic flux and restores transcription factor TFEB and PGC1α nuclear localization under HG, which is critical for lysosome biosynthesis and mitogenesis, respectively. Our results demonstrate that 2×mt8a-CG is a suitable probe for monitoring mitophagic flux, both in live and fixed cells in in vitro experiments, which may also be applicable to in vivo animal studies, and that the triple drug combination treatment has the potential for preventing retinal injury and disease progression in diabetes.

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Elevated cell proliferation and VEGF production by high-glucose conditions in Muller cells involve XIAP.

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