Regulation of Expression of Creatine Transporter in ARPE-19 Cells by HIV-1 TAT Protein

Abstract

Abstract: : Purpose: Previous studies using microarray analysis indicated that heterologous expression of HIV-1 TAT protein in the human retinal pigment epithelial cell line ARPE-19 induces the expression of the gene coding for the creatine transporter. Creatine plays a crucial role in energy storage in cells. The purpose of this study was to corroborate these results with RT-PCR, northern blot, and functional assays. Methods: Control and HIV-1 TAT-expressing ARPE-19 cells were used. RNA was prepared from these cells and the steady state levels of creatine transporter mRNA were quantified by northern blot and by semi-quantitative RT-PCR. Functional activity of the creatine transporter was monitored by the Na⁺- and Cl^?-dependent uptake of creatine. Results: The steady state levels of creatine transporter mRNA increased 3-fold in HIV-1 TAT-expressing cells compared to control cells as assessed by RT-PCR and northern blot. The increase in mRNA was accompanied by an increase in creatine uptake. Creatine uptake increased 2.5-fold in HIV-1 TAT-expressing cells compared to control cells. However, there was no difference between control cells and HIV-1 TAT-expressing cells in the basic functional characteristics of creatine uptake. In both cell lines, the uptake of creatine was Na⁺- and Cl^?-dependent with a K_m value of 30 ± 2 ?M for creatine. The dependence of creatine uptake on Na⁺ showed a sigmoidal relationship in both cell lines with a Hill coefficient of 2.3 ± 0.1. The dependence of creatine uptake on Cl^? was hyperbolic with a Hill coefficient of 0.9 ± 0.1. The K_0.5 values for the activation of creatine uptake by Na⁺ and Cl^? were 56 ± 5 and 61 ± 10 mM, respectively. Even though the basic characteristics of creatine uptake remained the same in both cell lines, the maximal velocity for the creatine uptake process increased 3.3-fold in HIV-1 TAT-expressing cells compared to control cells (3.3 ± 0.2 versus 1.0 ± 0.1 nmol/mg protein/45 min). Conclusions: The expression of HIV-1 TAT in ARPE-19 cells leads to an induction of the gene coding for the creatine transporter as detected by an increase in steady state mRNA levels and in transport function. Microarray analysis has indicated that several proapoptotic genes are upregulated in TAT-expressing ARPE-19 cells compared to control cells. The upregulation of the creatine transporter may be a compensatory mechanism to counteract the influence of proapoptotic factors on cell survival.

Keywords: AIDS/HIV • gene/expression • retinal pigment epithelium