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. 2000 Feb;74(4):1632-40.
doi: 10.1128/jvi.74.4.1632-1640.2000.

The human immunodeficiency virus type 1 Tat protein up-regulates the promoter activity of the beta-chemokine monocyte chemoattractant protein 1 in the human astrocytoma cell line U-87 MG: role of SP-1, AP-1, and NF-kappaB consensus sites

S P Lim  1 , A Garzino-Demo
Affiliations

The human immunodeficiency virus type 1 Tat protein up-regulates the promoter activity of the beta-chemokine monocyte chemoattractant protein 1 in the human astrocytoma cell line U-87 MG: role of SP-1, AP-1, and NF-kappaB consensus sites

S P Lim et al. J Virol. 2000 Feb.

Abstract

It has been shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can specifically enhance expression and release of monocyte chemoattractant protein 1 (MCP-1) from human astrocytes. In this study, we show evidence that Tat-induced MCP-1 expression is mediated at the transcriptional level. Transient transfection of an expression construct encoding the full-length Tat into the human glioblastoma-astrocytoma cell line U-87 MG enhances reporter gene activity from cotransfected deletion constructs of the MCP-1 promoter. HIV-1 Tat exerts its effect through a minimal construct containing 213 nucleotides upstream of the translational start site. Site-directed mutagenesis studies indicate that an SP1 site (located between nucleotides -123 and -115) is critical for both constitutive and Tat-enhanced expression of the human MCP-1 promoter, as mutation of this SP1 site significantly diminished reporter gene expression in both instances. Gel retardation experiments further demonstrate that Tat strongly enhances the binding of SP1 protein to its DNA element on the MCP-1 promoter. Moreover, we also observe an increase in the binding activities of transcriptional factors AP1 and NF-kappaB to the MCP-1 promoter following Tat treatment. Mutagenesis studies show that an upstream AP1 site and an adjacent NF-kappaB site (located at -128 to -122 and -150 to -137, respectively) play a role in Tat-mediated transactivation. In contrast, a further upstream AP1 site (-156 to -150) does not appear to be crucial for promoter activity. We postulate that a Tat-mediated increase in SP1 binding activities augments the binding of AP1 and NF-kappaB, leading to synergistic activation of the MCP-1 promoter.

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Figures

FIG. 1
FIG. 1
Schematic representations of the MCP-1 promoter. (A) Map of the 5′ flanking region of the hMCP-1 gene showing up to 3 kb upstream of the translational start site. (B) Proximal region of the promoter from nucleotides −213 to +6 from the translational start site (boxed). Putative binding sites for cis-acting factors are underlined.
FIG. 2
FIG. 2
Deletion analysis of the hMCP-1 5′ flanking region. Luciferase assays were performed with extracts of U-87 MG cells transfected with different 5′ deletion constructs. Results are expressed as the induction of luciferase activity of the hMCP promoter constructs over pGL2-Basic and were normalized to units of β-galactosidase activity from cotransfection with a pSV-β-galactosidase plasmid. (A) Luciferase activities from transfected U-87 MG cells were measured 48 h posttransfection; data shown are the means (± standard deviations) of four independent transfection experiments. The average raw luciferase value for pGL2-Basic was 0.05 ± 0.03. (B) Representative experiment showing transfection of U-87 MG cells following treatment with PMA for the indicated times, harvested 48 h posttransfection. The raw luciferase values for pGL2-Basic ranged from 0.1 to 0.24.
FIG. 3
FIG. 3
Deletion analysis of the hMCP-1 5′ flanking region. Luciferase assays were performed with extracts of HeLa cells transfected with different 5′ deletion constructs. Results are expressed as the induction of luciferase activity of the hMCP promoter constructs over pGL2-Basic and were normalized to units of β-galactosidase activity from cotransfection with a pSV-β-galactosidase plasmid. (A) Luciferase activities from transfected HeLa cells were measured 48 h posttransfection; data shown are the means (± standard deviations) of four independent transfection experiments. The average raw luciferase value for pGL2-Basic was 0.01 ± 0.01. (B) A representative experiment showing transfection of HeLa cells following treatment with PMA for the indicated times, harvested 48 h posttransfection. The raw luciferase values for pGL2-Basic ranged from 0 to 0.04 ± 0.04.
FIG. 4
FIG. 4
Tat-mediated induction of hMCP-1 promoter activity. A representative experiment (from three performed) shows luciferase activities of the hMCP-1 deletion constructs following cotransfection in U-87 MG (A) or HeLa (B) cells with the indicated amounts of a Tat expression plasmid. Results are expressed as the induction of luciferase activity of the promoter constructs over pGL2-Basic and were measured 72 h posttransfection. Luciferase activity was normalized to units of β-galactosidase activity from cotransfection with a pSV-β-galactosidase plasmid. The raw luciferase values for pGL2-Basic ranged from 0 to 0.05 ± 0.05.
FIG. 5
FIG. 5
Constitutive hMCP-1 promoter activity in HeLa-tat-III cells. A representative experiment (from three performed) shows luciferase activities of the hMCP-1 deletion constructs following transfection in HeLa-tat-III cells. Results are expressed as induction of luciferase activity of the hMCP promoter constructs over pGL2-Basic and were measured 48 h posttransfection. Luciferase activities were normalized to units of β-galactosidase activity from cotransfection with a pSV-β-galactosidase plasmid. The raw luciferase value for pGL2-Basic was 0.09 ± 0.01.
FIG. 6
FIG. 6
Analysis of transcriptional control elements in the hMCP-1 promoter in U-87 MG cells. Luciferase assays were performed with extracts of U-87 MG cells transfected with hMCP-1 promoter constructs bearing various mutated cis-acting elements. (A) Results are expressed as the percentage of luciferase activity compared to the wild-type construct phMCP213 and in each case were normalized to units of β-galactosidase activity from cotransfection with a pSV-β-galactosidase plasmid. Luciferase activities from transfected U-87 MG cells were measured 48 h posttransfection, and data shown are the means (± standard deviations) of four independent transfection experiments. The average raw luciferase value for pGL2-Basic was 0.31 ± 0.1. (B) Representative cotransfection experiment (from three performed) of the various mutated constructs performed with 1 μg of Tat expression construct. Results are expressed as the percentage of luciferase activity compared to the wild-type construct phMCP213 and in each case were normalized to units of β-galactosidase activity from cotransfection with a pSV-β-galactosidase plasmid. The average raw luciferase values for pGL2-Basic ranged from 0.18 to 0.4 ± 0.03.
FIG. 7
FIG. 7
Analysis of transcriptional control elements in the hMCP-1 promoter in HeLa-tat-III cells. Luciferase assays were performed with extracts of HeLa-tat-III cells transfected with hMCP-1 promoter constructs bearing various mutated cis-acting elements and were measured 48 h posttransfection. Results (average of four experiments) are expressed as the percentage of luciferase activity compared to the wild-type construct phMCP213 and in each case were normalized to units of β-galactosidase activity from cotransfection with a pSV-β-galactosidase plasmid. The average raw luciferase value for pGL2-Basic was 4.5 ± 2.3.
FIG. 8
FIG. 8
Tat-enhanced binding of transcriptional factors to the hMCP-1 promoter region. A radiolabeled DNA fragment containing the region from −213 to +6 bp of the hMCP-1 5′ flanking region was incubated with nuclear extracts from unstimulated U-87 MG cells (lanes 2, 3, and 7), cells treated with 100 nM Tat (lanes 4, 5 and 8), or cells treated with heat-inactivated Tat (lane 9-11). Competition assays were performed with a 100-fold molar excess of unlabeled probe (lanes 3, 5, and 10) or with hMCP213 containing a mutated SP1 site (lanes 7, 8, and 11). Lanes 1 and 6 consist of the probe alone. Arrows indicate specific DNA-protein complexes.
FIG. 9
FIG. 9
SP1 and AP1 bind to the hMCP-1 promoter region. A radiolabeled DNA fragment containing the region from −213 to +6 bp of the hMCP-1 5′ flanking region was incubated with nuclear extracts from U-87 MG cells. Competition assays were performed with a 100-fold molar excess of specific oligonucleotides. Extracts were prepared from unstimulated U-87 MG cells (lanes 2 to 10) or cells treated with 100 nM Tat (lanes 11 to 19). Lane 1 consists of the probe alone. Arrows indicate specific DNA-protein complexes. mut, mutant.
FIG. 10
FIG. 10
Supershifts of protein complexes binding to hMCP-1 promoter region. (A) Nuclear extracts from unstimulated U-87 MG cells (lanes 2 and 3) or cells treated with 100 nM Tat (lanes 4 and 5) were preincubated with polyclonal antibodies to SP1 (lanes 3 and 5) and then gel shifted with a radiolabeled DNA fragment containing the −213 to +6 bp hMCP-1 5′ flanking region. (B) Nuclear extracts from unstimulated U-87 MG cells (lanes 6, 8, 10, 12, and 14) or 40 nM Tat (lanes 7, 9, 11, 13, and 15) were preincubated with polyclonal antibodies to AP1 (lanes 8 and 9), NF-κB p50 (lanes 10 and 11) and p65 (lanes 12 and 13), and C/EBP (lanes 14 and 15) and then gel shifted with a radiolabeled DNA fragment containing the −213 to +6 bp hMCP-1 5′ flanking region. Lane 1 consists of the probe alone. Arrows a and b show positions of supershifted bands with the SP1 and AP1 antibodies, respectively; arrows c, d, and e denote positions of supershifted bands from unstimulated U-87 MG cell extracts with the p50 and p65 antibodies; arrows f, b, and g, represent supershifts with the same antibodies from Tat-treated cell extracts.
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