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Induction of xCT Gene Expression and L-Cystine Transport Activity by Diethyl Maleate at the Inner Blood–Retinal Barrier

¹ From the Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Japan; and the ² Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation (JST); the ³ Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan; and the ⁴ New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

PURPOSE. In this study, the expression and regulation of the L-cystine transporter, system x_c^-, at the inner blood–retinal barrier (inner BRB) was investigated using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as an in vitro model.

METHODS. For the uptake study, TR-iBRB2 cells were cultured at 33°C in the presence or absence of diethyl maleate (DEM), and the uptake rate of [¹⁴C]L-cystine was measured at 37°C. The mRNA levels of system x_c^-, which consists of xCT and 4F2hc, were determined by quantitative real-time RT-PCR analysis with specific primers.

RESULTS. The xCT and 4F2hc mRNAs were expressed in TR-iBRB2 cells. The [¹⁴C]L-cystine uptake by TR-iBRB2 cells appeared to be mediated through a saturable Na⁺-independent process. The corresponding Michaelis-Menten constant was 9.18 µM. At 100 µM DEM, the xCT mRNA level and L-cystine uptake activity in TR-iBRB2 cells were enhanced in a time-dependent manner. Concomitantly, the glutathione concentration in TR-iBRB2 cells was increased. In contrast, the 4F2hc mRNA level was unchanged up to 24 hours and was induced for more than 24 hours by DEM treatment. Under both normal and DEM treatment conditions, the uptake of [¹⁴C]L-cystine was strongly inhibited by L-glutamic acid, L--aminoadipic acid, L-homocysteic acid, and L-quisqualic acid, whereas L-aspartic acid and L-arginine had no effect, which is evidence of the induction of system x_c^-.

CONCLUSIONS. System x_c^--mediated L-cystine uptake appears to be present at the inner BRB. DEM induces L-cystine transport through system x_c^- at the inner BRB by enhanced transcription of the xCT gene.

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