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Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway.

Wang H, Zhu X, Zhu Y, Liu J, Hu X, Wang Y, Peng S, Chen Y, Chen R, Ding F, Liu R - Biomed Res Int (2014)

Bottom Line: Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation.The molecular mechanism of this activation, however, is far from clear.In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China ; Department of Neurobiology, Xuzhou Medical College, Xuzhou, Jiangsu 221009, China.

ABSTRACT
Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation. The molecular mechanism of this activation, however, is far from clear. Here, we show that the protein kinase D3 (PKD3) is essential for prostratin-induced transcription activation of latent HIV-1 provirus. First, silencing PKD3, but not the other members of PKD family, blocked prostratin-induced transcription of HIV-1. Second, overexpressing the constitutively active form of PKD3, but not the wild-type or kinase-dead form of PKD3, augmented the expression of HIV-1. Consistent with this observation, we found that prostratin could trigger PKD3 activation by inducing the phosphorylation of its activation loop. In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation. Finally, the activation effect of PKD3 on HIV-1 transcription was shown to depend on the presence of κB element and the prostratin-induced activation of NF-κB, as indicated by the fact that silencing PKD3 blocked prostratin-induced NF-κB activation and NF-κB-dependent HIV-1 transcription. Therefore, for the first time, PKD3 is implicated in the transcription activation of latent HIV-1 provirus, and our results revealed a molecular mechanism of prostratin-induced HIV-1 transcription via PKCε/PKD3/NF-κB signaling pathway.

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Prostratin enhances the transcription initiation of latent HIV-1 provirus. (a) Time course of prostratin-induced HIV-1 expression. HeLa cells with an integrated HIV-LTR-luciferase reporter gene (HIV-LTR-Luc) were treated with 2 μM of prostratin for 0–6 hrs, and the cell lysates were prepared for the detection of luciferase activity. The expression levels of luciferase gene were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (b) Titration assay of prostratin-induced transcription activation of latent HIV-1 provirus. 2D10 cells, a Jurkat-based cell line containing the latent HIV-1 provirus with eGFP in place of Nef, were treated with indicated concentration of prostratin for 16 hrs. The GFP-positive cells were measured by flow cytometry and plotted as percentage of total cells based on 3 independent experiments. (c) Effect of prostratin treatment on transcription initiation and elongation of HIV-1. The schematics of the HIV-LTR-luciferase reporter gene (HIV-LTR-Luc) and the locations of qRT-PCR primers for detecting transcripts corresponding to the region of initiation (+1~+59, TAR) or elongation (+496~593, luciferase) are illustrated in top panel. HeLa cells with integrated HIV-LTR-luciferase gene were treated with 2 μM of prostratin for 4 hrs. The transcription levels were detected by qRT-PCR and plotted based on 3 independent experiments, with the level of untreated cells set to 1.0.
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fig1: Prostratin enhances the transcription initiation of latent HIV-1 provirus. (a) Time course of prostratin-induced HIV-1 expression. HeLa cells with an integrated HIV-LTR-luciferase reporter gene (HIV-LTR-Luc) were treated with 2 μM of prostratin for 0–6 hrs, and the cell lysates were prepared for the detection of luciferase activity. The expression levels of luciferase gene were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (b) Titration assay of prostratin-induced transcription activation of latent HIV-1 provirus. 2D10 cells, a Jurkat-based cell line containing the latent HIV-1 provirus with eGFP in place of Nef, were treated with indicated concentration of prostratin for 16 hrs. The GFP-positive cells were measured by flow cytometry and plotted as percentage of total cells based on 3 independent experiments. (c) Effect of prostratin treatment on transcription initiation and elongation of HIV-1. The schematics of the HIV-LTR-luciferase reporter gene (HIV-LTR-Luc) and the locations of qRT-PCR primers for detecting transcripts corresponding to the region of initiation (+1~+59, TAR) or elongation (+496~593, luciferase) are illustrated in top panel. HeLa cells with integrated HIV-LTR-luciferase gene were treated with 2 μM of prostratin for 4 hrs. The transcription levels were detected by qRT-PCR and plotted based on 3 independent experiments, with the level of untreated cells set to 1.0.

Mentions: To study the molecular mechanism of HIV-1 transcription activation by prostratin, we employed a HeLa-based cell line with an integrated HIV-LTR-luciferase reporter gene (HIV-LTR-Luc) and a Jurkat-based cell line containing a transcriptionally latent HIV-1 provirus with eGFP in place of Nef (J-Lat clone 2D10) [24] as model systems. In HIV-LTR-Luc cells, 2 μM prostratin treatment could progressively induce the luciferase activity to about 20- fold by 6 hrs as indicated by luciferase assay (Figure 1(a)). In J-Lat 2D10 cells, prostratin treatment for 16 hrs induced a concentration-dependent increase of the GFP-positive cells to 54% in 0.5 μM prostratin treated cells, as indicated by flow cytometry analysis (Figure 1(b)). Hence, we used 2 μM prostratin treating for 6 hrs for HeLa cells with HIV-LTR-Luc reporter gene and 0.5 μM prostratin for 16 hrs for 2D10 cells.


Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway.

Wang H, Zhu X, Zhu Y, Liu J, Hu X, Wang Y, Peng S, Chen Y, Chen R, Ding F, Liu R - Biomed Res Int (2014)

Prostratin enhances the transcription initiation of latent HIV-1 provirus. (a) Time course of prostratin-induced HIV-1 expression. HeLa cells with an integrated HIV-LTR-luciferase reporter gene (HIV-LTR-Luc) were treated with 2 μM of prostratin for 0–6 hrs, and the cell lysates were prepared for the detection of luciferase activity. The expression levels of luciferase gene were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (b) Titration assay of prostratin-induced transcription activation of latent HIV-1 provirus. 2D10 cells, a Jurkat-based cell line containing the latent HIV-1 provirus with eGFP in place of Nef, were treated with indicated concentration of prostratin for 16 hrs. The GFP-positive cells were measured by flow cytometry and plotted as percentage of total cells based on 3 independent experiments. (c) Effect of prostratin treatment on transcription initiation and elongation of HIV-1. The schematics of the HIV-LTR-luciferase reporter gene (HIV-LTR-Luc) and the locations of qRT-PCR primers for detecting transcripts corresponding to the region of initiation (+1~+59, TAR) or elongation (+496~593, luciferase) are illustrated in top panel. HeLa cells with integrated HIV-LTR-luciferase gene were treated with 2 μM of prostratin for 4 hrs. The transcription levels were detected by qRT-PCR and plotted based on 3 independent experiments, with the level of untreated cells set to 1.0.
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Related In: Results  -  Collection

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fig1: Prostratin enhances the transcription initiation of latent HIV-1 provirus. (a) Time course of prostratin-induced HIV-1 expression. HeLa cells with an integrated HIV-LTR-luciferase reporter gene (HIV-LTR-Luc) were treated with 2 μM of prostratin for 0–6 hrs, and the cell lysates were prepared for the detection of luciferase activity. The expression levels of luciferase gene were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (b) Titration assay of prostratin-induced transcription activation of latent HIV-1 provirus. 2D10 cells, a Jurkat-based cell line containing the latent HIV-1 provirus with eGFP in place of Nef, were treated with indicated concentration of prostratin for 16 hrs. The GFP-positive cells were measured by flow cytometry and plotted as percentage of total cells based on 3 independent experiments. (c) Effect of prostratin treatment on transcription initiation and elongation of HIV-1. The schematics of the HIV-LTR-luciferase reporter gene (HIV-LTR-Luc) and the locations of qRT-PCR primers for detecting transcripts corresponding to the region of initiation (+1~+59, TAR) or elongation (+496~593, luciferase) are illustrated in top panel. HeLa cells with integrated HIV-LTR-luciferase gene were treated with 2 μM of prostratin for 4 hrs. The transcription levels were detected by qRT-PCR and plotted based on 3 independent experiments, with the level of untreated cells set to 1.0.
Mentions: To study the molecular mechanism of HIV-1 transcription activation by prostratin, we employed a HeLa-based cell line with an integrated HIV-LTR-luciferase reporter gene (HIV-LTR-Luc) and a Jurkat-based cell line containing a transcriptionally latent HIV-1 provirus with eGFP in place of Nef (J-Lat clone 2D10) [24] as model systems. In HIV-LTR-Luc cells, 2 μM prostratin treatment could progressively induce the luciferase activity to about 20- fold by 6 hrs as indicated by luciferase assay (Figure 1(a)). In J-Lat 2D10 cells, prostratin treatment for 16 hrs induced a concentration-dependent increase of the GFP-positive cells to 54% in 0.5 μM prostratin treated cells, as indicated by flow cytometry analysis (Figure 1(b)). Hence, we used 2 μM prostratin treating for 6 hrs for HeLa cells with HIV-LTR-Luc reporter gene and 0.5 μM prostratin for 16 hrs for 2D10 cells.

Bottom Line: Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation.The molecular mechanism of this activation, however, is far from clear.In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China ; Department of Neurobiology, Xuzhou Medical College, Xuzhou, Jiangsu 221009, China.

ABSTRACT
Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation. The molecular mechanism of this activation, however, is far from clear. Here, we show that the protein kinase D3 (PKD3) is essential for prostratin-induced transcription activation of latent HIV-1 provirus. First, silencing PKD3, but not the other members of PKD family, blocked prostratin-induced transcription of HIV-1. Second, overexpressing the constitutively active form of PKD3, but not the wild-type or kinase-dead form of PKD3, augmented the expression of HIV-1. Consistent with this observation, we found that prostratin could trigger PKD3 activation by inducing the phosphorylation of its activation loop. In addition, we identified PKCε of the novel PKC subfamily as the upstream kinase for this phosphorylation. Finally, the activation effect of PKD3 on HIV-1 transcription was shown to depend on the presence of κB element and the prostratin-induced activation of NF-κB, as indicated by the fact that silencing PKD3 blocked prostratin-induced NF-κB activation and NF-κB-dependent HIV-1 transcription. Therefore, for the first time, PKD3 is implicated in the transcription activation of latent HIV-1 provirus, and our results revealed a molecular mechanism of prostratin-induced HIV-1 transcription via PKCε/PKD3/NF-κB signaling pathway.

Show MeSH
Related in: MedlinePlus