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. 2014;13(23):3640-4.
doi: 10.4161/15384101.2014.952959.

HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels

Anna Paola Bruno  1 , Francesca Isabella De Simone, Vittoria Iorio, Margot De Marco, Kamel Khalili, Ilker Kudret Sariyer, Mario Capunzo, Stefania Lucia Nori, Alessandra Rosati
Affiliations

HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels

Anna Paola Bruno et al. Cell Cycle. 2014.

Abstract

BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.

Keywords: BAG3; BAG3, Bcl-2-Associated Athanogene 3; HIV-1; LC3, microtubule-associated protein 1 light chain 3; Tat; Tat, Trans-Activator of Transcription;; autophagy; glioblastoma.

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Figures

Figure 1.
Figure 1.
BAG3 protein levels increase in Tat- overexpressing cells. U87MG and A-172 glioblastoma cell lines were transfected with increasing concentrations of an HIV-1 Tat expressing plasmid or an empty vector. BAG3 protein expression levels were monitored using Western blot analysis (A), and BAG3 mRNA levels were analyzed using RT-PCR (B); the graphs depict mean levels (± SD), and data are representative of 3 independent experiments. *P = 0.05 to 0.01; **P = 0.01 to 0.001; ***P < 0.001.
Figure 2.
Figure 2.
Autophagy activation in Tat-transfected cells. (A) U87MG, A-172 and NHA cells were transfected with a HIV-1 Tat- expressing plasmid or an empty vector. LC3-II levels were analyzed using Western blotting, and GAPDH levels were detected to monitor equal loading conditions. (B) A-172 and U87MG cells were transfected with a GFP-LC3 expressing plasmid in presence of a Tat- expressing plasmid or an empty vector as control. Lysotracker Deep Red was used for staining lysosomal compartments. Cells were then analyzed with a confocal microscope. Images were obtained from DAPI staining, GFP-LC3 and Lysotracker deep red fluorescence. Yellow region indicate co-localization. (C) U87MG and A-172 cells were plated at 30% of confluence, transfected with a BAG3siRNA or a non-targeted (NT) siRNA. After 72 h, cells were transfected with a Tat- expressing plasmid or with an empty vector as described above. Apoptotic cell death was analyzed by flow cytometry. Graph depicts mean ± SD percentage of sub-G0/G1 cells. Data are representative of 3 independent experiments. (D) A-172 were transfected with BAG3siRNA or NT siRNA as described above. Rapamycin- treated cells were used as a control of autophagy induction. LC3-II levels were analyzed by protein gel blotting. *P = 0.05 to 0.01; **P = 0.01 to 0.001; ***P < 0.001.
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