Human immunodeficiency virus type 1 (HIV-1) tat induces nitric-oxide synthase in human astroglia - PubMed (original) (raw)

Human immunodeficiency virus type 1 (HIV-1) tat induces nitric-oxide synthase in human astroglia

Xiaojuan Liu et al. J Biol Chem. 2002.

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection is known to cause neuronal injury and dementia in a significant proportion of patients. However, the mechanism by which HIV-1 mediates its deleterious effects in the brain is poorly defined. The present study was undertaken to investigate the effect of the HIV-1 tat gene on the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astroglia. Expression of the tat gene as RSV-tat but not that of the CAT gene as RSV-CAT in U373MG astroglial cells led to the induction of NO production and the expression of iNOS protein and mRNA. Induction of NO production by recombinant HIV-1 Tat protein and inhibition of RSV-tat-induced NO production by anti-Tat antibodies suggest that RSV-tat-induced production of NO is dependent on Tat and that Tat is secreted from RSV-tat-transfected astroglia. Similar to U373MG astroglial cells, RSV-tat also induced the production of NO in human primary astroglia. The induction of human iNOS promoter-derived luciferase activity by the expression of RSV-tat suggests that RSV-tat induces the transcription of iNOS. To understand the mechanism of induction of iNOS, we investigated the role of NF-kappaB and C/EBPbeta, transcription factors responsible for the induction of iNOS. Activation of NF-kappaB as well as C/EBPbeta by RSV-tat, stimulation of RSV-tat-induced production of NO by the wild type of p65 and C/EBPbeta, and inhibition of RSV-tat-induced production of NO by deltap65, a dominant-negative mutant of p65, and deltaC/EBPbeta, a dominant-negative mutant of C/EBPbeta, suggest that RSV-tat induces iNOS through the activation of NF-kappaB and C/EBPbeta. In addition, we show that extracellular signal-regulated kinase (ERK) but not that p38 mitogen-activated protein kinase (MAPK) is involved in RSV-tat induced production of NO. Interestingly, PD98059, an inhibitor of the ERK pathway, and deltaERK2, a dominant-negative mutant of ERK2, inhibited RSV-tat-induced production of NO through the inhibition of C/EBPbeta but not that of NF-kappaB. This study illustrates a novel role for HIV-1 tat in inducing the expression of iNOS in human astrocytes that may participate in the pathogenesis of HIV-associated dementia.

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Figures

Fig. 1

Fig. 1. Expression of RSV-tat induces the production of NO and the expression of iNOS protein in human U373MG astroglial cells

Cells plated at 50–60% confluence in 6-well plates were transfected with different amounts of either RSV-CAT or RSV-tat using LipofectAMINE Plus (Invitrogen) as described under “Materials and Methods.” After 24 h of transfection, cells were incubated under serum-free conditions. A, after 24 h of incubation, supernatants were used for nitrite assay. Data are the mean ± S.D. of three different experiments. B, cell homogenates were electrophoresed, transferred onto nitrocellulose membrane, and immunoblotted with antibodies against mouse macrophage iNOS as mentioned under “Materials and Methods.” C, cells plated at 50–60% confluence in 100-mm dishes were transfected with different amounts of either RSV-CAT or RSV-tat. After 24 h of transfection, cells were incubated under serum-free conditions. After 24 h of incubation, cells were taken out directly by adding Ultraspec-II RNA reagent (Biotecx Laboratories Inc.) to the plates for isolation of total RNA, and Northern blot analysis for iNOS mRNA was carried out as described under “Materials and Methods.”

Fig. 2

Fig. 2. Expression of RSV-tat induces the production of NO through the secretion of tat in human U373MG astroglial cells

A, cells plated in 6-well plates were transfected with 0.2 _μ_g of RSV-tat. After 24 h of transfection, cells were incubated under serum-free conditions in the presence of different concentrations of anti-Tat antibodies and control IgG. After 24 h of incubation, supernatants were used for nitrite assay. Data are the mean ± S.D. of three different experiments. B, cells were treated with different concentrations of recombinant HIV-1 Tat protein under serum-free conditions. After 24 h, supernatants were used for nitrite assay. Data are the mean ± S.D. of three different experiments.

Fig. 3

Fig. 3. Expression of RSV-tat induces the production of NO in human primary astroglia

Cells plated at 50–60% confluence in 6-well plates were transfected with different amounts of either RSV-CAT or RSV-tat. After 24 h of transfection, cells were incubated under serum-free conditions. After 24 h of incubation, supernatants were used for nitrite assay. Data are the mean ± S.D. of three different experiments.

Fig. 4

Fig. 4. Expression of RSV-tat induces iNOS promoter-derived luciferase activity in human U373MG astroglial cells

Cells plated in 6-well plates were cotransfected with 0.5 _μ_g of phiNOS(7.2)Luc (a construct containing the human iNOS promoter fused to the luciferase gene) and different amounts of either RSV-CAT or RSV-tat. All transfections also included 50 ng/_μ_g pRL-TK (as transfection efficiency control). After 24 h of transfection, cells were incubated under serum-free conditions for 18 h. Firefly (ff-Luc) and Renilla (r-Luc) luciferase activities were obtained by analyzing total cell extract as described under “Materials and Methods.” Data are the mean ± S.D. of three different experiments.

Fig. 5

Fig. 5. Expression of RSV-tat induces activation of NF-κ_B and C/EBP_β in human U373MG astroglial cells

Cells plated in 6-well plates were cotransfected with 0.5 _μ_g of either pBIIX-Luc (A) or pC/ EBP_β_-Luc (B) and different amounts of either RSV-CAT or RSV-tat. All transfections also included 50 ng/_μ_g pRL-TK. After 24 h of transfection, cells were incubated under serum-free conditions for 18 h. Firefly (ff-Luc) and Renilla (r-Luc) luciferase activities were obtained by analyzing total cell extract. Data are the mean ± S.D. of three different experiments.

Fig. 6

Fig. 6. Effect of wild-type p65 and C/EBP_β_ and dominant-negative mutants of p65 (Δp65) and C/EBP_β_ (ΔC/EBP_β_) on RSV-_tat_-induced production of NO in human U373MG astroglial cells

Cells plated in 6-well plates were cotransfected with 0.2 μ_g of RSV-tat and 0.5 μ_g of an empty vector, p65, C/EBP_β, Δp65, or ΔC/EBP_β. After 24 h of transfection, cells were incubated under serum-free conditions for another 24 h. Supernatants were used for the nitrite assay. Data are the mean ± S.D. of three different experiments.

Fig. 7

Fig. 7. Expression of RSV-tat induces activation of ERK in human U373MG astroglial cells

Cells were transfected with different amounts of RSV-tat. After 24 h of transfection, cells were incubated under serum-free conditions. After 18 h of incubation, activities of ERK and p38 MAPK were assayed as described under “Materials and Methods.”

Fig. 8

Fig. 8. Role of ERK and p38 MAPK in RSV-_tat_-induced NO production in human U373MG astroglial cells

A, cells were transfected with 0.2 _μ_g of RSV-tat. After 24 h of transfection, cells were incubated under serum-free conditions in the presence or absence of different concentrations of PD98059 and SB203580. After 24 h of incubation, supernatants were used for the nitrite assay. Data are the mean ± S.D. of three different experiments. B, cells were cotransfected with 0.2 _μ_g of RSV-tat and 0.5 _μ_g of either an empty vector or ΔERK1, ΔERK2, or Δp38. After 24 h of transfection, cells were incubated under serum-free conditions for another 24 h. Supernatants were used for the nitrite assay. Data are the mean ± S.D. of three different experiments.

Fig. 9

Fig. 9. Effect of PD98059 on RSV-_tat_-induced activation of NF-κ_B and C/EBP_β in human U373MG astroglial cells

Cells plated in 6-well plates were cotransfected with 0.2 _μ_g of RSV-tat and 0.5 _μ_g of either pBIIX-Luc (A) or pC/EBP_β_-Luc (B). All transfections also included 50 ng/_μ_g pRL-TK. After 24 h of transfection, cells were incubated in the presence or absence of different concentrations of PD98059 under serum-free conditions for 18 h. Firefly (ff-Luc) and Renilla (r-Luc) luciferase activities were obtained by analyzing total cell extract. Data are the mean ± S.D. of three different experiments.

Fig. 10

Fig. 10. Effect of ΔERK2, the dominant-negative mutant of ERK2, on RSV-_tat_-induced activation of NF-κ_B and C/EBP_β in human U373MG astroglial cells

Cells were cotransfected with 0.2 _μ_g of RSV-tat, 0.5 _μ_g of either an empty vector or ΔERK2, and 0.5 _μ_g of either pBIIX-Luc (A) or pC/EBP_β_-Luc (B). After 24 h of transfection, cells were incubated under serum-free conditions for 18 h. Firefly (ff-Luc) and Renilla (r-Luc) luciferase activities were obtained by analyzing total cell extract. Data are the mean ± S.D. of three different experiments.

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