Human immunodeficiency virus type 1 viral protein R (Vpr) induces CCL5 expression in astrocytes via PI3K and MAPK signaling pathways

Abstract

Background: Neurocognitive impairments remain prevalent in HIV-1 infected individuals despite current antiretroviral therapies. It is increasingly becoming evident that astrocytes play a critical role in HIV-1 neuropathogenesis through the production of proinflammatory cytokines/chemokines. HIV-1 viral protein R (Vpr) plays an important role in neuronal dysfunction; however, its role in neuroinflammation is not well characterized. The major objective of this study was to determine the effect of Vpr in induction of proinflammatory chemokine CCL5 in astrocytes and to define the underlying mechanism(s).

Methods: SVGA astrocytes were either mock transfected or were transfected with a plasmid encoding HIV-1 Vpr, and the cells were harvested at different time intervals. The mRNA level of CCL5 expression was quantified using real-time RT-PCR, and cell culture supernatants were assayed for CCL5 protein concentration. Immunocytochemistry was performed on HIV-1 Vpr transfected astrocytes to check CCL5 expression. Various signaling mechanisms such as p38 MAPK, PI3K/Akt, NF-κB and AP-1 were explored using specific chemical inhibitors and siRNAs.

Results: HIV-1 Vpr transfected astrocytes exhibited time-dependent induction of CCL5 as compared to mock-transfected astrocytes at both the mRNA and protein level. Immunostained images of astrocytes transfected with HIV-1 Vpr also showed much higher accumulation of CCL5 in comparison to untransfected and mock-transfected astrocytes. Pre-treatment with NF-κB (SC514) and PI3K/Akt (LY294002) inhibitor partially abrogated CCL5 mRNA and protein expression levels as opposed to untreated controls after HIV-1 Vpr transfection. Specific siRNAs against p50 and p65 subunits of NF-κB, p38δ MAPK, Akt-2 and Akt-3, and AP-1 transcription factor substantially inhibited the production of CCL5 in HIV-1 Vpr transfected astrocytes.

Conclusion: These results demonstrate the ability of HIV-1 Vpr to induce CCL5 in astrocytes in a time-dependent manner. Furthermore, this effect was observed to be mediated by transcription factors NF-κB and AP-1 and involved the p38-MAPK and PI3K/Akt pathway.

Figure 1

Time-dependent induction of CCL5 by HIV-1 Vpr in SVGA astrocytes. SVGA cells were mock transfected or transfected with a plasmid encoding HIV-1 Vpr. Cells were harvested at 1, 3, 6, 12, 24, 48 and 72 h post-transfection, and CCL5 expression levels were determined using real-time RT-PCR. The supernatants were also collected at 6, 12, 24, 48 and 72 h post-transfection, and protein concentration of CCL5 was determined using Bioplex assay. (A) mRNA expression levels calculated relative to mock-transfected controls; (B) protein concentration for CCL5. The bars represent the mean ± SE of three independent experiments done in triplicate. Statistical significance was determined using Student’s t-test, **p < 0.01, *p < 0.05.

Figure 2

Immunocytochemistry for HIV-1 Vpr-mediated induction of CCL5 in astrocytes. SVGA astrocytes were cultured on cover slips and were either mock transfected (D-F) or were transfected with a plasmid encoding Vpr (G-I). Non-transfected cells were used as control (A-C). The cells were stained for nucleus (blue); CCL5 (green) and GFAP (red), and the images were captured using a Leica TCS SP5 II on an inverted microscope platform with a 40× zoom oil emersion lens. The Image J software was used to get the merged images, and the Multi Measure tool in Image J was used to quantify the intensities. (J) The image intensities are plotted relative to GFAP. The bars represent the mean ± SE of three independent images. Statistical significance was determined using Student’s t-test, **p < 0.01.

Figure 3

Involvement of NF-κB in HIV-1 Vpr-mediated upregulation of CCL5 in astrocytes. SVGA astrocytes were pre-treated with the chemical inhibitors or transfected with siRNA targeting the NF-κB pathway, followed by mock transfection or transfection with a plasmid encoding Vpr. (A) and (B) Effect of chemical inhibitor of the NF-κB pathway, SC514 on mRNA expression and protein concentration for CCL5, respectively. (C) and (D) Effect of siRNA against p50 and p65 subunits of NF-κB on CCL5 mRNA expression and protein concentration, respectively. The bars represent the mean ± SE of three independent experiments done in triplicate. Statistical significance was determined using Student’s t-test, **p < 0.01, *p < 0.05.

Figure 4

Role of p38-MAPK and AP-1 in HIV-1 Vpr-mediated induction of CCL5 in astrocytes. SVGA astrocytes were pre-treated with the chemical inhibitors for the JNK-MAPK (SP600125) and p38-MAPK (SB203580) pathway 1 h before mock transfection or transfection with a plasmid encoding HIV-1 Vpr. (A) and (B) Effect of chemical inhibitors on mRNA expression and protein concentration for CCL5, respectively. SVGA cells were also transfected with siRNA targeting p38 isoforms (α, β, γ, δ) and AP-1 followed by mock transfection or transfection with a plasmid encoding Vpr. (C) and (F) Effect of siRNAs on CCL5 mRNA expression; (D) and (G) effect on CCL5 protein concentration, respectively. (E) Specificity of p38 isoform siRNAs against respective subunits; (H) inhibition of AP-1 (c-fos) with the siRNA directed toward the p38δ subunit of p38-MAPK. The bars represent the mean ± SE of three independent experiments done in triplicate. Statistical significance was determined using Student’s t-test, **p < 0.01, *p < 0.05.

Figure 5

Activation of NF-κB involves PI3K/Akt signaling in HIV-1 Vpr-mediated induction of CCL5 in astrocytes. SVGA astrocytes were pre-treated with the chemical inhibitor for the PI3K/Akt pathway (LY294002) 1 h before mock transfection or transfection with a plasmid encoding HIV-1 Vpr. (A) and (B) Effect of LY294002 on mRNA expression and protein concentration for CCL5, respectively. SVGA cells were also transfected with siRNA targeting Akt isoforms (Akt-1, Akt-2 and Akt-3) followed by mock transfection or transfection with a plasmid encoding Vpr. (C) and (D) Effect of siRNAs on CCL5 mRNA expression and protein concentration, respectively. (E) Specificity of siRNA for Akt isoforms against individual subunits. (F) Inhibition of NF-κB (p50) nuclear translocation with LY294002. The bars represent the mean ± SE of three independent experiments done in triplicate. Statistical significance was determined using Student’s t-test, **p < 0.01, *p < 0.05

Figure 6

Schematic of the signaling pathways involved in the induction of CCL5 by HIV-1 Vpr in astrocytes. HIV-1 Vpr activates p38-MAPK- and PI3K/Akt-related signaling pathways in SVGA astrocytes leading to the activation of transcription factors AP-1 and NF-κB, respectively. NF-κB and AP-1 bind to the promoter region of CCL5 and increase its production in astrocytes. Blue color represents the use of chemical inhibitor, while green color represents the use of siRNA. Involvement of particular signaling is shown in darker shades, while lighter shades imply no involvement.