Abstract
Abstract: :
Purpose:TAT is a transactivator protein coded by the human immunodeficiency virus (HIV) type 1 genome that is necessary for viral replication. This protein is present in the circulation in HIV-infected persons and is responsible for many of the clinical consequences of HIV infection. The blood and tissue levels of glutathione are reduced in HIV-infected persons. In mammalian cells, the cystine/glutamate transporter xc- is the major supplier of cysteine, the limiting amino acid in the synthesis of glutathione and thus is an important determinant of glutathione status. The retinal pigment epithelium (RPE) normally contains high concentrations of glutathione. The purpose of this study was to investigate the influence of TAT on the activity of xc- in ARPE-19 cells, a human RPE cell line. Methods:TAT-expressing ARPE-19 cells were established by stable transfection. RT-PCR and immunofluorescence were performed to confirm the expression of TAT. The activity of xc- was monitored by the uptake of glutamate in the absence of Na+. Northern blot was used to analyze the changes in steady-state levels of mRNA for xCT, the light chain of xc-, and for 4F2hc, the heavy chain of xc-. Results:RT-PCR and immunofluorescence confirmed the expression of TAT in the stably transfected cells. The transport activity of xc- was 3- to 4-fold higher in TAT-ARPE-19 cells than in control ARPE-19 cells. This stimulatory effect of TAT on xc- was specific because the activities of several other transport systems were not affected by TAT. The TAT-induced xc- activity was associated with a 2.7-fold increase in the maximal velocity of the transport system with no change in substrate affinity. The involvement of xc- in the TAT-induced glutamate uptake was established by substrate specificity studies. The steady-state levels of xCT mRNA increased 2.4-fold and those of 4F2hc mRNA increased 4.0-fold in these cells due to TAT expression. The glutathione status of ARPE-19 cells also influenced the activity of xc-, depletion of glutathione enhancing the activity and supplementation with glutathione suppressing the activity. The stimulatory effect of TAT on xc- activity was, however, not affected by glutathione status. Conclusion:The expression of the HIV-1 protein TAT in ARPE-19 cells upregulates the cystine/glutamate transporter xc-. This influence of TAT on xc- is independent of the glutathione status of the cells.
Keywords: 311 AIDS/HIV • 567 retinal pigment epithelium • 417 gene/expression