• Influence of HIV-1-TAT on Gene Expression in Retinal Pigment Epithelium

      Hu, Huankai; Department of Biochemistry and Molecular Biology (2005-09)
      Specific Aims 1. Characterization of Tat-transgenic mouse We generated a transgenic Tat-mouse line in which the expression of the transgene was restricted to macrophages, B cells, and dendritic cells. To characterize these transgenic mice, we performed the genotyping of mouse by genomic PCR of Tat to confirm the integration HIV-1 Tat transgene into the mouse genome. Then we confirmed the expression of HIV-1 Tat in peritoneal macrophages and in the ocular tissue. There is evidence in the literature showing that HIV-1 Tat decreases glutathione levels in the liver and in the erythrocytes by down-regulation of the GCS light subunit30. Therefore, we monitored the levels of GSH and expression of GCS-LS in RPE in control and Tat-transgenic mice. 2. Investigation of influence of HIVl-Tat on the x'c system in retinal pigment epithelium The profile of gene expression affected by HIV-1 Tat in ARPE-19 cells(Table 1), shows that xCT is up-regulated about two-fold. xCT is the light chain of cystine/glutamate exchanger x"Cj which plays an essential role in supplying cysteine for de novo synthesis of glutathione. HIV-1 Tat is associated with decreased levels of glutathione seen in AIDS patients. Since we hypothesized that oxidative stress caused by HIV-Tat is involved in the pathogenesis of non-infectious AIDS retinopathy, it would be of interest to investigate the influence of HIV-1 Tat on the expression and activity of this transporter. Therefore, we first used control and Tat-expressing ARPE-19 cells to assess the influence of HIV-1 Tat on the levels o f glutathione, expression of enzymes related to glutathione synthesis and degradation, and the expression and activity o f x'c. Subsequently, the results were confirmed with ocular tissues collected from control mice and Tat-transgenic mice. 3. Study of the effects of HIYl-Tat on uptake of deltorphin II in RPE According to the profile of gene expression affected by HIV-1 Tat in ARPE-19 cells (Table 1), OATP-A is down-regulated about three-fold. Since OATP-A transports opioid peptides such as DPDPE, deltorphin II, and Leu-enkephalin 87, we investigated the influence of HIV-1 Tat on deltorphin II uptake in ARPE-19 cells. Deltorphin II is a natural 5 opioid QO peptide agonist, which binds 8 opioid receptor and plays an important role in analgesia . 4. Investigation of effects of HIVl-Tat on the expression and activity of creatine transporter in RPE The genechip analysis showed that CRT1 is upregulated by HIV-1 Tat in ARPE-19 cells. Retina contains high concentrations of creatine and phosphocreatine and CRT1 in RPE is responsible for accumulating creatine from the circulation into the subretinal space. Since creatine plays an essential role in energy homeostasis in the retina, it was interesting to study the regulation o f creatine transporter by the HIV-1 Tat in RPE using Tat expressing ARPE-19 cells and Tat-transgenic mouse model. 5. Investigation of the influence of Tat-(l-72) versus Tat-(l-lOl) on the regulation of expression of transporters in RPE During the course of the present project, we demonstrated that the expression of Tat-(1- 72) in ARPE-19 cells led to marked changes in the expression o f various genes related to oxidative stress, excitotoxicity, and apoptosis. We confirmed these findings in transgenic mice expressing the same one-exon Tat. We employed the one-exon Tat in these studies based on the evidence that most of the biological effects of Tat reside in the first 72 amino acids. But, HIV-1 infected individuals are exposed to full-length Tat coded by both exons rather than to truncated Tat coded only by the first exon. Therefore, to understand the in vivo relevance of our findings to AIDS patients, it is essential to know whether the biochemical changes observed in ARPE-19 cells in response to the expression of Tat-(l-72) are also seen in response to the expression of the full-length Tat coded by the two exons. For this purpose, we will generate a stable ARPE-19 cell line that expresses the two-exon Tat, i.e., Tat-(l-lO l), and compare the Tat-induced changes in various transport systems in this cell line with those observed in the cell line expressing the one-exon Tat.