Abstract
Reversible S-glutathionylation of proteins is a focal point of redox signaling and cellular defense against oxidative stress. This post-translational modification alters protein function, and its reversal (deglutathionylation) is catalyzed specifically and efficiently by glutaredoxin (GRx, thioltransferase), a thioldisulfide oxidoreductase. We hypothesized that changes in glutaredoxin might be important in the development of diabetic retinopathy, a condition characterized by oxidative stress. Indeed, GRx protein and activity were increased in retinal homogenates from streptozotocin-diabetic rats. Also, incubation of rat retinal Müller cells (rMC-1) in normal glucose (5 mm) or diabetic-like glucose (25 mm) medium led to selective upregulation of GRx in contrast to thioredoxin, the other thioldisulfide oxidoreductase system. Under analogous conditions, NF-kappaB (p50-p65) translocated to the nucleus, and expression of ICAM-1 (intercellular adhesion molecule-1), a transcriptional product of NF-kappaB, increased. Proinflammatory ICAM-1 is increased in diabetic retinae, and it is implicated in pathogenesis of retinopathy. To evaluate the role of GRx in mediating these changes, intracellular GRx content and activity in rMC-1 cells were increased independently under normal glucose via infection with an adenoviral GRx1 construct (Ad-GRx). rMC-1 cells exhibited adenovirus concentration-dependent increases in GRx and corresponding increases in NF-kappaB nuclear translocation, NF-kappaB luciferase reporter activity, and ICAM-1 expression. Blocking the increase in GRx1 via small interfering RNA in rMC-1 cells in high glucose prevented the increased ICAM-1 expression. These data suggest that redox regulation by glutaredoxin in retinal glial cells is perturbed by hyperglycemia, leading to NF-kappaB activation and a pro-inflammatory response. Thus, GRx may represent a novel therapeutic target to inhibit diabetic retinopathy.
Highlights
Reactive oxygen species are redox signals essential to physiological processes, but they can disrupt normal redox signaling, damage cell components, and irreversibly oxidize cellular proteins when produced in excess [1, 2]
High Glucose Selectively Induces GRx in Muller Cells—To elucidate changes in sulfhydryl homeostasis in the retinal glial (Muller) cells in response to high glucose, the rat Muller cell line was used, and the cells were cultured under conditions mimicking diabetes (i.e. 25 mM glucose in the medium)
Glutaredoxin is elevated in animal models of Parkinson disease, where inactivation of mitochondrial complex I is characteristic of the disease [41]
Summary
Blocking the increase in GRx1 via small interfering RNA in rMC-1 cells in high glucose prevented the increased ICAM-1 expression These data suggest that redox regulation by glutaredoxin in retinal glial cells is perturbed by hyperglycemia, leading to NF-B activation and a pro-inflammatory response. 20 and 28, respectively), suggesting that oxidative signals within cells affect transcriptional activity of NF-B In this regard, many proteins that are implicated in the pathway of regulation of NF-B activity have been reported to have their functions altered by S-glutathionylation in different contexts. The current study was designed to test the hypothesis that the oxidative stress associated with high glucose alters glutaredoxin-regulated redox signaling in retinal glial cells. Knock-down of GRx1 in cells in high glucose prevents the induction of ICAM-1
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