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. 2011 Mar;6(1):121-9.
doi: 10.1007/s11481-010-9217-8. Epub 2010 Apr 23.

Activation of Egr-1 expression in astrocytes by HIV-1 Tat: new insights into astrocyte-mediated Tat neurotoxicity

Yan Fan  1 , Wei Zou, Linden A Green, Byung Oh Kim, Johnny J He
Affiliations

Activation of Egr-1 expression in astrocytes by HIV-1 Tat: new insights into astrocyte-mediated Tat neurotoxicity

Yan Fan et al. J Neuroimmune Pharmacol. 2011 Mar.

Abstract

Human immunodeficiency virus type 1 (HIV-1) Tat plays an important role in HIV-associated neuropathogenesis; the underlying mechanisms are still evolving. We have recently shown that HIV-1 Tat induces expression of glial fibrillary acidic protein (GFAP), a characteristic of HIV-1 infection of the central nervous system. We have also shown that the Tat-induced GFAP expression in astrocytes is regulated by p300 and that deletion of the early growth response 1 (Egr-1) cis-transacting element within the p300 promoter abolishes Tat-induced GFAP expression. In this study, we further examined the relationship between Tat and Egr-1 in astrocytes. We found increased Egr-1 protein expression in Tat-expressing human astrocytoma cells and mouse primary astrocytes. Using the Egr-1 promoter-driven firefly luciferase reporter gene assay and the site-directed mutagenesis, we demonstrated that Tat increased Egr-1 expression by transactivating the Egr-1 promoter and involving specific serum response elements within the promoter. Consistent with these data, we showed that Tat transactivation of the Egr-1 promoter was abrogated when astrocytes were cultured in serum-reduced media. Taken together, these results reveal that Tat directly transactivates Egr-1 expression and suggest that Tat interaction with Egr-1 is probably one of the very upstream molecular events that initiate Tat-induced astrocyte dysfunction and subsequent Tat neurotoxicity.

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Figures

Figure 1
Figure 1. Egr-1 up-regulation in Tat-expressing U373 cells, U87 cells and primary astrocytes
Cells were transiently transfected with pCDNA3 or pcTat.Myc and harvested after 72 h; Cell lysates of (A) U373 and (B) U87 cells were analyzed for Tat and Egr-1 expression, with β-actin included as a loading control. Primary astrocytes were prepared from brain-targeted inducible Tat transgenic mice and cultured in the absence or presence of 5ug/ml doxcycline (Dox) for up to 3 days (C) then analyzed for Tat and Egr-1 expression. Total RNA was isolated and subjected to RT-PCR using Tat-specific primers with GAPDH used as a loading control. As a control, primary astrocytes were also prepared from wild-type C57/BL6 mice, treated with Dox and analyzed for Egr-1 expression (D). Meanwhile, U373 cells were infected with increasing amounts of VSV-G pseudotyped HIV-GFP virus and harvested after 72 h. Cell lysates were analyzed for Egr-1, GFAP, and Tat expression (E), along with p24 and GFP to ensure infection efficiency. β-actin was included as a loading control. Data is representative of at least three independent experiments.
Figure 2
Figure 2. Egr-1 promoter activity and expression of Egr-1 protein increases in the presence of a specific amount of Tat
U373 cells were transiently transfected with Egr-1 promoter-driven luciferase reporter plasmid pEgr-1p-Luc or control plasmid pGL3 along with increasing amounts of pcTat.Myc and harvested 72h post-transfection. Half of the cells were to detect Egr-1 promoter activity (A) with pCMV-βgal used to normalize for variations in transfection efficiency. The remaining transfected cells were separated and used for Triton-soluble (TS) and Triton-insoluble (TIS) fractions and analyzed for Egr-1, GFAP and Tat expression, with β-actin included as a loading control (B).
Figure 3
Figure 3. Analysis of the functional elements of the human Egr-1 promoter in response to Tat expression
The full length human Egr-1 promoter was cloned from the genomic DNA of U87 cells and inserted into pGL3, and then used to create a panel of Egr-1 promoter mutations (A). Cells were transiently transfected each pEgr-1p-luc mutant plasmid and either pcTat.Myc or pcDNA3, then harvested 72h post-transfection. Cell lysates were used to determine luciferase activity (B), with pCMV-βgal included to normalize the transfection efficiency. Data is representative of at least three independent experiments.
Figure 4
Figure 4. Tat-induced Egr-1 up-regulation is modulated by the serum response element during starvation
U373 cells were transfected with either pEgr-1p-700-luc or pEgr-1p-200-luc along with pcTat.Myc or pcDNA3. At 16h post-transfection the media was replaced with DMEM containing reduced concentrations of serum and incubated for additional 48 hr. Cell lysates were then used to determine luciferase activity, with pCMV-βgal included to normalize the transfection efficiency. Data is representative of at least three independent experiments.
Figure 5
Figure 5. The Egr-1 transcriptional network altered by HIV-1 Tat in astrocytes
HIV-1 Tat protein in astrocytes can be derived from direct HIV infection of these cells themselves. It can also be from HIV-infected microglia/macrophages, followed by its uptake by astrocytes. Tat then transactivates Egr-1 expression, which in turn transactivates p300 expression, which in turn transactivates GFAP expression. Activation of GFAP expression or expression of other target genes of Egr-1 and p300 leads to changes in astrocyte proliferation, survival and function and as a result, impairs neuronal integrity and survival.
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