Oxidative Stress–Induced Expression and Modulation of a Tyrosine Phosphatase PRL–1 in Retina

Abstract

Purpose: : PRL– 1, phosphatase of regenerating liver–1, was previously localized to cone photoreceptor outer segments. The tyrosine phosphatase activity of PRL–1 can be reversibly inactivated by hydrogen peroxide (H₂O₂). The purpose of this study was to examine the effects of oxidative stress induced by H₂O₂ or constant light exposure on modulation of PRL–1 in mouse cone photoreceptor–derived 661W cells and rat retinas, respectively.

Methods: : Cultured 661W cells were exposed to 0.1, 0.2, 0.5 or 1 mM H₂O₂ for 30 min; or pretreated with the glutathione (GSH) inhibitor buthionine sulfoximine (BSO, 1 mM) for 17 h followed by exposure to 0.2 mM H₂O₂ for 5, 15, 30 or 60 min. GSH levels were measured using a GSH detection Kit. Albino rats were exposed to constant cool white light (720 Lux) for a period of 0, 2, 5 or 7 days and retinas were collected. PRL–1 mRNA expression was analyzed by quantitative RT–PCR (qRT–PCR). Protein expression and oxidative modulation of PRL–1 were analyzed on Western blots. Expression of another known oxidative response protein, thioredoxin (Trx), and a loading control, γ tubulin, were also analyzed. Protein expression levels were determined by densitometric analysis.

Results: : Densitometric analysis revealed a statistically significant increasing expression of PRL–1 in 661W cells exposed to 0.1 to 1 mM H₂O₂ for 30 min and 0.2 mM of H₂O₂ for 15 to 60 min. In addition, in cells exposed to 0.5 or 1 mM H₂O₂ for 30 min, oxidation of PRL–1 was detected. Oxidation caused formation of an intramolecular disulfide bond involving the cysteine in the catalytic site. Exposure to 0.2 mM H₂O₂ also induced oxidation of PRL–1 in cells pretreated with BSO (which depleted GSH concentrations by 75%), but not in cells without pretreatment. In rats exposed to constant light for 5 or 7 days, increased expression levels of PRL–1 transcripts and protein were detected. Trx levels also showed an increase in response to constant light.

Conclusions: : Our studies have demonstrated that PRL–1 is upregulated in the photoreceptors by oxidative stress. In addition, PRL–1 can be oxidized and thereby inactivated by higher concentrations of H₂O₂. GSH levels modulate the oxidative status of PRL–1. Although the substrate(s) and physiological function of PRL–1 remain to be discovered, oxidative stress increases PRL–1 expression level and inhibits its activity, which in turn, regulates the phosphotyrosine levels in photoreceptor outer segments under some pathological conditions.

Keywords: photoreceptors • oxidation/oxidative or free radical damage • phosphorylation