Binding of human immunodeficiency virus type 1 to CD4 and CXCR4 receptors differentially regulates expression of inflammatory genes and activates the MEK/ERK signaling pathway

Abstract

We have previously shown that binding of human immunodeficiency virus type 1 (HIV-1) virions to CD4 receptors stimulates association of Lck with Raf-1 and results in the activation of Raf-1 kinase in a Ras-independent manner. In the present study, we demonstrate that HIV-1 envelope glycoproteins of both T-cell-tropic and macrophagetropic strains rapidly activate the ERK/mitogen-activated protein (MAP) kinase pathway and the binding of nuclear transcription factors (AP-1, NF-kappaB, and C/EBP) and stimulate expression of cytokine and chemokine genes. The activation of this signaling pathway requires functional CD4 receptors and is independent of binding to CXCR4. Binding of the natural ligand stromal cell-derived factor 1 (SDF-1) to CXCR4, which inhibits entry of T-cell-tropic HIV-1, activates also the ERK/MAP kinase pathway. However, SDF-1 did not affect the CD4-mediated expression of cytokine and chemokine genes. These results provide firm molecular evidence that binding of HIV-1 envelope glycoproteins to CD4 receptor initiates a signaling pathway(s) independent of the binding to the chemokine receptor that leads to the aberrant expression of inflammatory genes and may contribute significantly to HIV-1 replication as well as to deregulation of the immune system.

FIG. 1

Binding of T-cell-tropic NL4-3 or IIIB glycoprotein gp120 to CD4⁺ Jurkat T cells induces nuclear accumulation of transcription factors. Nuclear extracts were prepared from unstimulated (control) cells or cells exposed to NL4-3 (HIV-1) or gp120 IIIB (1 μg/ml) for the indicated times (45). Radiolabeled double-stranded oligonucleotides representing consensus binding sites for AP-1 (A), NF-κB (B), or C/EBP (C) were incubated with nuclear extracts (6 μg), and formed DNA-protein complexes were resolved on a 4% polyacrylamide gel as described in Materials and Methods. Specificity of induction of AP-1 and NF-κB was determined by competition with 10- and 50-fold molar excesses of unlabeled oligonucleotides representing appropriate consensus binding sites. Protein composition of the NF-κB complexes was determined in a supershift assay using specific antibodies against p50 or p65 (data not shown). Specificity of the formation of C/EBP was determined by using end-labeled oligonucleotides with mutated binding sites (mut. C/EBP oligo). NS represents nonspecific binding.

FIG. 2

RT-PCR analysis of cytokine, chemokine, and FasL/Fas mRNA expression stimulated by anti-CD4 antibody. Total RNA was extracted from Jurkat T cells unstimulated (unstim.), treated for 16 h with PHA (5 μg/ml) and TPA (50 ng/ml), or stimulated with anti-CD4 antibody Q4120 (10 μg/ml) for the indicated times. RT-PCR-assisted amplification of cytokine (A) and chemokine and FasL/Fas (B) mRNAs was performed as described in Materials and Methods. PCR products were resolved by electrophoresis in 2% agarose gels and stained with ethidium bromide. Raf-1 and GAPDH represent internal controls. M, 100-bp molecular size markers (600-bp band marked on the left).

FIG. 3

Activation of MEK and ERK/MAP kinases in response to T-cell-tropic gp120 or anti-CD4 antibodies. Jurkat cells were preincubated on ice for 60 min with recombinant gp120 IIIB (1 μg/ml) or anti-CD4 (α-CD4) antibody (5 μg/ml) or left untreated (control) and then incubated at 37°C for the indicated times before lysis. Proteins (50 μg per lane) were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies specific for phosphorylated forms of MEK1/2 (MEK-P) and ERK1/2 (ERK1/2-P) as well as with antibodies specific for total MEK and ERK. The positions of molecular weight markers (in kilodaltons) and kinases are indicated.

FIG. 4

Binding of macrophagetropic viruses to CCR5-negative Jurkat T cells upregulates expression of cytokine, chemokine, and FasL genes. The cells were incubated for 3 h at 37°C with sucrose gradient-purified Ba-L (A) or AD8 (B) virions. Total RNA was isolated, and mRNA expression was analyzed by RT-PCR using primers described in Materials and Methods. GAPDH served as an internal control. PCR products were resolved on 2% agarose gels and stained with ethidium bromide.

FIG. 5

Activation of MEK and ERK/MAP kinases in response to the binding of T-cell-tropic or macrophagetropic HIV-1 or SDF-1. Jurkat cells were preincubated on ice for 60 min with purified virions or SDF-1 (5 μg/ml) or left untreated (control) and then incubated at 37°C for the indicated times before lysis. Proteins (50 μg) were resolved by SDS-PAGE and analyzed as described in the legend to Fig. 3. The positions of phosphorylated forms of MEK1/2 (MEK-P) and ERK1/2 (ERK1/2-P) as well as total MEK and ERK are indicated. Sizes are indicated in kilodaltons.

FIG. 6

Activation of the MEK/ERK kinase pathway by T-cell-tropic or macrophagetropic HIV-1 or anti-CD4 antibody depends on the presence of cytoplasmic domain of CD4 receptor. A2.01/CD4.401 cells expressing truncated CD4 receptors were left untreated (control) or preincubated on ice for 60 min with SDF-1 (5 μg/ml), anti-CD4 antibody (5 μg/ml), gp120 IIIB (1 μg/ml), or purified NL4-3 or Ba-L (5 RT cpm/cell) and then incubated at 37°C for 5 min before lysis. Proteins (50 μg) were resolved by SDS-PAGE and analyzed as described in the legend to Fig. 3. The positions of phosphorylated forms of MEK1/2 (MEK-P) and ERK1/2 (ERK1/2-P) as well as total MEK and ERK are indicated. Sizes are indicated in kilodaltons.

FIG. 7

SDF-1 does not affect the HIV-1-stimulated expression of cytokine and chemokine genes in Jurkat T cells. The cells were left untreated (control) or were stimulated with sucrose gradient-purified NL4-3 (A) or Ba-L virions (B), SDF-1 (5 μg/ml), or a combination of HIV-1 and SDF-1. After 3 h at 37°C, total RNA was isolated and expression of cytokine and chemokine genes was analyzed by RT-PCR as described in the legend to Fig. 2. GAPDH served as an internal control.

FIG. 8

Induction of cytokine and chemokine gene expression is dependent on the affinity of HIV-1 binding to CD4 receptor. (A) The levels of gp120 envelope in pelleted virion preparations (10⁸ RT cpm) of NL4-3 and NL4-3-derived clones NL-D368E and NL-D368P were determined by Western blot analysis using mouse anti-gp120 monoclonal antibodies. (B) Binding of NL4-3 but not NL-D368E and NL-D368P (45) induced expression of cytokine and chemokine genes in Jurkat cells. Purified virions (10⁸ RT cpm) were incubated for 3 h at 37°C, total RNA was isolated, and expression of cytokine and chemokine genes was analyzed by RT-PCR as described in the legend to Fig. 2. GAPDH served as an internal control.