Protein S-glutathionylation is a reversible post-translational modification that is important in redox signal transduction and cellular defense against oxidative stress. S-glutathionylation occurs when a cysteine residue forms a mixed disulfide with glutathione. Glutaredoxin 1 is an enzyme that catalyzes the specific and efficient reversal of glutathionylation (de-glutathionylation). Roles for Grx1 in diabetic retinopathy, a disease characterized by oxidative stress and inflammation, had not been explored prior to the current study. Grx activity was found to be increased in retinae from diabetic rats. Incubation of rat retinal Müller glial cells (rMC-1) in normal glucose medium (5 mM) or diabetic-like glucose medium (25 mM, high glucose) led to corresponding increases in Grx content and activity. High glucose also led to increased nuclear translocation of NFκB and production of ICAM-1 (intercellular adhesion molecule-1), a transcriptional product of NFκB and known pro-inflammatory mediator in diabetic retinopathy. To evaluate the role of Grx1 in mediating these changes, Grx1 in rMC-1 cells was upregulated in normal glucose medium via infection with an adenoviral Grx1 construct (Ad-Grx1). Ad-Grx1 treatment led to increased NFκB activity and ICAM-1 production.

Treatment of rMC-1 cells in high glucose medium with siRNA targeted to Grx1 prevented the increase in Grx1 and coincidentally blocked the increase in ICAM-1. The site of regulation was localized to the cytoplasm, and IκB kinase (IKK) is a master cytosolic regulator of NFκB activation. Inhibition of IKK activity abrogated the increase in ICAM-1 induced by high glucose or by Ad-Grx1. Conditioned medium from the rMC-1 cells over-expressing Grx1 was added to fresh cultures of rMC-1 cells and induced Grx1 and ICAM-1 (autocrine regulation). Similarly, Grx1 and ICAM-1 were elevated in retinal endothelial cells cultured in conditioned medium from the rMC-1 cells over-expressing Grx1 (paracrine regulation). These effects correlate with a novel finding that secretion of IL-6 was elevated in the conditioned medium. Furthermore, IKKβ isolated from Müller cells in normal glucose medium was found to be glutathionylated on Cys179. Hence Grx1-mediated activation of IKK via deglutathionylation may play a critical role in pro-inflammatory responses in diabetic complications in vivo where Grx1 is increased.