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. 2008 Jul;216(1):128-34.
doi: 10.1002/jcp.21382.

HIV-1 Tat inhibits NGF-induced Egr-1 transcriptional activity and consequent p35 expression in neural cells

Nune Darbinian  1 , Armine Darbinyan, Marta Czernik, Francesca Peruzzi, Kamel Khalili, Krzysztof Reiss, Jennifer Gordon, Shohreh Amini
Affiliations

HIV-1 Tat inhibits NGF-induced Egr-1 transcriptional activity and consequent p35 expression in neural cells

Nune Darbinian et al. J Cell Physiol. 2008 Jul.

Abstract

Infection with HIV-1 causes degeneration of neurons leading to motor and cognitive dysfunction in AIDS patients. One of the key viral regulatory proteins, Tat, which is released by infected cells, can be taken up by various uninfected cells including neurons and by dysregulating several biological events induces cell injury and death. In earlier studies, we demonstrated that treatment of neuronal cells with Tat affects the nerve growth factor (NGF) signaling pathway involving MAPK/ERK. Here we demonstrate that a decrease in the level of Egr-1, one of the targets for MAPK, by Tat has a negative impact on the level of p35 expression in NGF-treated neural cells. Further, we demonstrate a reduced level of Egr-1 association with the p35 promoter sequence in NGF-treated cells expressing Tat. As p35, by associating with Cdk5, phosphorylates several neuronal proteins including neurofilaments and plays a role in neuronal differentiation and survival, we examined kinase activity of p35 complexes obtained from cells expressing Tat. Results from H1 kinase assays showed reduced activity of the p35 complex from Tat-expressing cells in comparison to that from control cells. Accordingly, the level of phosphorylated neurofilaments was diminished in Tat-expressing cells. Similarly, treatment of PC12 cells with Tat protein or supernatant from HIV-1 infected cells decreased kinase activity of p35 in these cells. These observations ascribe a role for Tat in altering p35 expression and its activity that affects phosphorylation of proteins involved in neuronal cell survival.

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Figures

Fig. 1
Fig. 1
The effect of Tat and NGF on Egr-1 and p35 protein expression. A: Western blot analysis for Egr-1 expression in Tat-producing and control SK-N-MC cells, with and without NGF treatment for 3 h. Anti-Tat antibody was used to confirm the presence of the Tat protein in Tat-producing cells. Grb2 was used as a control for protein loading. B: (Top) Western blot analysis for the detection of p35 protein in SK-N-MC cells after exposure of the cells to NGF (lanes 1–2). (Bottom) Western blot analysis for the detection of Grb2 as a loading control. C: Western blot analysis for the detection of Egr-1 and p35 proteins in PC12 cells upon NGF treatment, in the presence or absence of exogenously added Tat. The level of Grb2 expression serves as a control for protein loading.
Fig. 2
Fig. 2
Effect of Tat, NGF, and Egr-1 siRNA on transcription of the p35 promoter. A: Effect of NGF on transcription of the p35 promoter in SK-N-MC (CFP) and SK-N-MC (CFP-Tat) cells. Luciferase assay of p35 promoter activity upon NGF treatment of the SK-N-MC (CFP) and SK-N-MC (CFP-Tat) cells was performed as described in Materials and Methods Section. B: Effect of Egr-1 silencing on the activity of the p35 promoter in SK-N-MC cells. Cells were co-transfected with the p35-luciferase reporter plasmid and the Egr-1 expression plasmid, and were then treated with NGF for 3 h in the presence of Egr-1-specific siRNA or a control non-targeting siRNA for 72 h. Cells were harvested and protein extracts were analyzed by luciferase assay to determine p35 promoter activity. The histograms show mean relative light units representing luciferase activity and the error bars show the standard deviation for the experiments, which were repeated in duplicate.
Fig. 3
Fig. 3
Effect of Taton Egr-1 binding to the p35 promoter. A:Band-shift assay analysis of protein extracts from Tat-CFP and CFP-producing cells in the presence of NGF. The association of Egr-1 with the DNA results in the formation of DNA-protein complexes (shown by a bracket). The addition of unlabeled competitor (lane2) oranti-Egr-1 antibody (lane3) in CFP-producing cells or the effect of Tat (lane5) was examined. Addition of normal serum as a negative control has no effect on the formation of the Egr-1:DNA complex (lane 4). B: ChIP assay to detect the association of Egr-1 with the p35 promoter in Tat-CFP and CFP-producing cells. SK-N-MC stable cell lines producing CFP or CFP-Tat that express Egr-1 were utilized in ChIP assay as described in Materials and Methods Section. Top part: Immunoprecipitation with antibody to Egr-1, indicating binding of Egr-1 to the p35 promoter region spanning −720 to −477 nt. Middle part: Immunoprecipitation with normal rabbit serum (negative control). Bottom part: Input DNA (positive control). The density of bands (top part) was quantified and is presented as a histogram.
Fig. 4
Fig. 4
Effect of Tat on p35/Cdk5 kinase activity reduces phosphorylation of neurofilaments. A: Suppression of p35/Cdk5-associated phosphorylation of H1 by Tat in NGF treated Tat-producing cells. SK-N-MC cells were treated for 3 h in the presence of NGF, and protein extracts were prepared and immunoprecipitated with p35 antibody and analyzed by kinase assay using purified histone H1 as a substrate. A decrease in the intensity of the band corresponding to the phosphorylated H1 was observed in NGF stimulated cells in the presence of Tat (lane 2). Lane 3 represents the negative control, non-immune mouse serum (NS) used in the precipitation step. Densitometric analysis data for p35 kinase assay are presented. The kinas eassay was performed twice, and the results after normalizing to input H1 are presented in arbitrary units of 1–100.B:Western blot analysis for evaluating levels of phosphorylated NF (top part) in SK-N-MC cells that express either CFP or CFP-Tat grown after serum starvation for 24 h and after NGF treatment of cells for 3 h. Anti-CFP antibody was used to confirm the presence of CFP-Tat protein upon NGF treatment in Tat-producing cells. Expression of Grb2 served as a loading control.
Fig. 5
Fig. 5
Treatment of PC12 cells with exogenous Tat and HIV-1 infected culture media inhibits p35/Cdk5 kinase activity. Approximately 2 × 106 U-937 cells grown in RPMI with 2% fetal bovine serum were infected with HIV-1 JF-RL and after 5 days post-infection, supernatants were collected and used for incubation with PC12 cells. Approximately 2 × 106 PC12 cells maintained in media containing NGF were treated with exogenous Tat protein or conditioned media obtained from infected U-937 cells. Protein extracts were harvested, immunoprecipitated with p35 antibody, and analyzed by kinase assay using purified histone H1 as a substrate. A decrease in the intensity of the band corresponding to the phosphorylated H1 was observed in Tat-treated PC12 cells (lane 2) and after treatment of cells with conditioned media from HIV-1 infected macrophages (lane 3). Lane 4 represents PC12 cells treated with conditioned media from uninfected monocytes. Lane 5 represents the negative control, non-immune mouse serum (NS) used in the precipitation step. Densitometric analysis data for the p35 kinase assay are presented. The kinase assay was performed twice and the results were normalized to total H1 input and presented in arbitrary units.
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References

    1. Adle-Biassette H, Levy Y, Colombel M, Poron F, Natchev S, Keohane C, Gray F. Neuronal apoptosis in HIV infection in adults. Neuropathol Appl Neurobiol. 1995;21:218–227. - PubMed
    1. Al-Sarraj A, Day RM, Thiel G. Specificity of transcriptional regulation by the zinc finger transcription factors Sp1, Sp3, and Egr-1. J Cell Biochem. 2005;94:153–167. - PubMed
    1. Aprea S, Del Valle L, Mameli G, Sawaya BE, Khalili K, Peruzzi F. Tubulin-mediated binding of human immunodeficiency virus-1 Tat to the cytoskeleton causes proteasomal-dependent degradation of microtubule-associated protein 2 and neuronal damage. J Neurosci. 2006;26:4054–4062. - PMC - PubMed
    1. Chapman DL, Wolgemuth DJ. Regulation of M-phase promoting factor activity during development of mouse male germ cells. Dev Biol. 1994;165:500–506. - PubMed
    1. Chen D, Wang M, Zhou S, Zhou Q. HIV-1 Tat targets microtubules to induce apoptosis, a process promoted by the pro-apoptotic Bcl-2 relative Bim. EMBO J. 2002;21:6801–6810. - PMC - PubMed

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