HIV-1 Tat-mediated induction of CCL5 in astrocytes involves NF-κB, AP-1, C/EBPα and C/EBPγ transcription factors and JAK, PI3K/Akt and p38 MAPK signaling pathways

Abstract

The incidence of HIV-associated neurological disorders (HAND) has increased during recent years even though the highly active antiretroviral therapy (HAART) has significantly curtailed the virus replication and increased the life expectancy among HIV-1 infected individuals. These neurological deficits have been attributed to HIV proteins including HIV-1 Tat. HIV-1 Tat is known to up-regulate CCL5 expression in mouse astrocytes, but the mechanism of up-regulation is not known. The present study was undertaken with the objective of determining the mechanism(s) underlying HIV-1 Tat-mediated expression of CCL5 in astrocytes. SVGA astrocytes were transiently transfected with a plasmid encoding Tat, and expression of CCL5 was studied at the mRNA and protein levels using real time RT-PCR and multiplex cytokine bead array, respectively. HIV-1 Tat showed a time-dependent increase in the CCL5 expression with peak mRNA and protein levels, observed at 1 h and 48 h post-transfection, respectively. In order to explore the mechanism(s), pharmacological inhibitors and siRNA against different pathway(s) were used. Pre-treatment with SC514 (NF-κB inhibitor), LY294002 (PI3K inhibitor), AG490 (JAK2 inhibitor) and Janex-1 (JAK3 inhibitor) showed partial reduction of the Tat-mediated induction of CCL5 suggesting involvement of JAK, PI3K/Akt and NF-κB in CCL5 expression. These results were further confirmed by knockdown of the respective genes using siRNA. Furthermore, p38 MAPK was found to be involved since the knockdown of p38δ but not other isoforms showed partial reduction in CCL5 induction. This was further confirmed at transcriptional level that AP-1, C/EBPα and C/EBPγ were involved in CCL5 up-regulation.

Figure 1. HIV-1 Tat induces CCL5 in SVGA astrocytes in a time dependent manner.

7×10⁵ SVGA astrocytes were transiently transfected with plasmid encoding HIV-1 Tat for 5 h using Lipofectamine 2000™. (A) The cells were harvested at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h and expression levels of the CCL5 mRNA were determined by RT-PCR. Data presented in the figure is relative to the mock-transfected controls. (B) CCL5 protein concentrations in the supernatants were measured at 6 h, 12 h, 24 h, 48 h, 72 h and 96 h by multiplex cytokine assay. Each experiment was performed in triplicate and each bar in the figure represents the mean ± SE of three individual experiments. Student’s t-test was employed to calculate the significance and p-value was found to be ≤0.0001 in all the cases.

Figure 2. Immunocytochemistry of CCL5 induced by HIV-1 Tat in astrocytes.

(A–I) SVGA astrocytes were grown on a cover slip before transfecting with HIV-1 Tat plasmid. Untransfected control (A–C) and mock-transfected cells (D–F) were used to compare the up-regulation of CCL5 in cells transfected with HIV-1 Tat (G–I). The cells were stained with the primary antibodies against CCL5 and GFAP and secondary antibody labeled with Alexafluor 555 (GFAP) and Alexafluor 488 (CCL5). Finally the cover slips were mounted on medium containing DAPI to stain the nucleus. The merge panels represent the co-localization of CCL5 with GFAP. The images were captured using inverted confocal microscope, Leica TCS SP5 II. (J) The intensity of CCL5 over GFAP was calculated using imageJ software. Student’s t-test was employed to calculate the significance and ** denotes the p-value ≤0.01.

Figure 3. Involvement of NF-κB in HIV-1 Tat mediated production of CCL5 from astrocytes.

(A, B) SVGA astrocytes were treated with 10 µM of NF-κB inhibitor (SC514) prior to transfection with plasmid encoding HIV-1 Tat. (C, D) For knockdown using siRNA, the cells were transfected with the siRNA followed by Tat transfection as mentioned in the Materials and Methods. The expression of CCL5 at the mRNA and protein levels were measured at 6 h and 48 h post-transfection by using RT-PCR (A,C) and multiplex cytokine assay (B, D), respectively. The values represented for mRNA are expressed relative to the mock-transfected controls. Each experiment was performed in triplicate and each bar in the figure represents the mean ± SE of at least three individual experiments. One-way ANOVA was used to perform the statistical analysis and ** denotes p-value ≤0.01 and * denotes p-value of ≤0.05.

Figure 4. Inhibition of p38 MAPK reduces the induction of CCL5 by HIV-1 Tat.

(A–D) SVGA astrocytes were pretreated with 10 µM of p-38 MAPK chemical inhibitor (SB203580) or JNK MAPK chemical inhibitor (SP600125) 1 h prior to the transfection with Tat plasmid. (E, F) For gene knockdown with siRNA, the cells were transfected with siRNA against p38α, p38β, p38γ or p38δ before transfecting with HIV-1 Tat plasmid as mentioned in the Materials and Methods. The expression of CCL5 at the mRNA and protein levels were measured at 6 h and 48 h post-transfection by using RT-PCR (A,C,E) and multiplex cytokine assay (B,D,F), respectively. The values represented for mRNA are expressed relative to the mock-transfected controls. Each experiment was performed in triplicate and each bar in the figure represents the mean ± SE of three individual experiments. One-way ANOVA was used to perform the statistical analysis and ** denotes p-value ≤0.01.

Figure 5. Involvement of C/EBPα, C/EBPγ and AP-1 in HIV-1 Tat mediated induction of CCL5.

SVGA astrocytes were transfected with siRNA against C/EBPα, C/EBPγ and AP-1 (A, B) for 48 h before transfecting with HIV-1 Tat plasmid. The expression of CCL5 was measured at mRNA (A) and protein (B) levels. The values represented for mRNA are expressed relative to the mock-transfected controls. Each experiment was performed in triplicate and each bar in the figure represents the mean ± SE of at least three individual experiments. One-way ANOVA was used to perform the statistical analysis and ** denotes p-value ≤0.01.

Figure 6. Role of PI3K/Akt in HIV-1 Tat mediated up-regulation of CCL5 in astrocytes.

SVGA astrocytes were pretreated with 10 µM of specific PI3K inhibitor (LY294002) (A, B) or transfected with siRNA against different isoforms of Akt (Akt1/2/3) (C, D) prior to transfection with plasmid encoding HIV-1 Tat. Induction of CCL5 was measured at mRNA (A, C) and protein levels (B, D). The values represented for mRNA are expressed relative to the mock-transfected controls. Each experiment was performed in triplicate and each bar in the figure represents the mean ± SE of three individual experiments. One-way ANOVA was used to perform the statistical analysis and ** denotes p-value ≤0.01.

Figure 7. Role of JAK in the induction of CCL5 by HIV-1 Tat in astrocytes.

SVGA astrocytes were pretreated with specific 10 µM JAK2 (AG490) (A,B) or 20 µM JAK3 (Janex-1) inhibitors (C, D) or 25 µM JAK1 (E, F) or siRNA to knock down JAK2 and JAK3 (G,H) prior to the transfection with HIV-1 Tat plasmid. The level of CCL5 induction was measured at mRNA (A, C, E, G) and protein levels (B, D, F, H). The values represented for mRNA are expressed relative to the mock-transfected controls. Each experiment was performed in triplicate and each bar in the figure represents the mean ± SE of at least three individual experiments. One-way ANOVA was used to perform the statistical analysis and ** denotes p-value ≤0.01.

Figure 8. Schematic representation of the signaling pathways involved in HIV-1 Tat mediated up-regulation of CCL5 in astrocytes.

The induction of CCL5 by HIV-1 Tat involved JAK/PI3K/Akt and p38 MAP kinase pathways. These signaling pathways differentially regulated the induction of CCL5 by activating various transcription factors, including NF-κB, C/EBPα, C/EBPγ and AP-1. The target molecules of siRNA are indicated in green color and the involvement of a specific isoform is shown in brighter color and the absence is shown in pale color. The specific inhibitors for their respective targets are shown in red.