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Regulation of thioredoxin by ceramide in retinal pigment epithelial cells.

ABSTRACT: The purpose of this study was to determine the expression, regulation and signaling of a key redoxin family member thioredoxin 1 (Trx1) in normal, oxidant-stimulated and growth factor-pretreated RPE cells. Trx1 is expressed in early passage, human RPE cell cultures. RPE cells exposed to C(2)-ceramide for 24h showed no significant change in expression of Trx1 vs. controls with and without pretreatment for 24h with hepatocyte growth factor (HGF). Neither hypoxia from 1% O(2) or from CoCl(2) exposure resulted in any alteration in Trx1 expression in RPE cells. C(2)-ceramide treatment caused translocation of Trx1 from cytosol to the nucleus, which was abolished by pre-treatment of cells with a p38 MAPK-specific inhibitor. Furthermore, the gene and protein expression of thioredoxin interacting protein (Txnip) increased with ceramide treatment and was significantly (p<0.001) elevated with HGF preincubation vs. untreated controls. Prominent protection from ceramide-induced RPE cell death by exogenous rTrx1 was demonstrated. Although Trx1 directly interacts with its inhibitor, Txnip, p38 inhibition does not appear to have a role in this interaction. We found no direct interaction between apoptosis signal regulating kinase (ASK-1) and Txnip under the same experimental conditions. In summary, our data demonstrate the expression of Trx1 and Txnip in human RPE cells. Ceramide treatment results in translocation of Trx1 to the nucleus, and upregulation of Txnip expression; exogenous rTrx1 protects from ceramide-induced cell death. These results suggest that Trx1 and Txnip play an important role in the response of RPE to ceramide toxicity.

SUBMITTER: Sreekumar PG

PROVIDER: S-EPMC2693936 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Regulation of thioredoxin by ceramide in retinal pigment epithelial cells.

Sreekumar Parameswaran G PG Ding Yi Y Ryan Stephen J SJ Kannan Ram R Hinton David R DR

Experimental eye research 20081101 3

The purpose of this study was to determine the expression, regulation and signaling of a key redoxin family member thioredoxin 1 (Trx1) in normal, oxidant-stimulated and growth factor-pretreated RPE cells. Trx1 is expressed in early passage, human RPE cell cultures. RPE cells exposed to C(2)-ceramide for 24h showed no significant change in expression of Trx1 vs. controls with and without pretreatment for 24h with hepatocyte growth factor (HGF). Neither hypoxia from 1% O(2) or from CoCl(2) exposu ...[more]

PMID: 18996115

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Project description:Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood-retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1?, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 ?M, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

Dataset Information

Regulation of thioredoxin by ceramide in retinal pigment epithelial cells.

Publications

Regulation of thioredoxin by ceramide in retinal pigment epithelial cells.

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