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The Journal of Immunology, 1998, 160: 1875-1885.
Copyright © 1998 by The American Association of Immunologists

Involvement of Extracellular Signal-Regulated Kinase Module in HIV-Mediated CD4 Signals Controlling Activation of Nuclear Factor-{kappa}B and AP-1 Transcription Factors1

Laurence Briant2,*, Véronique Robert-Hebmann*, Virginie Sivan*, Anne Brunet{dagger}, Jacques Pouysségur{dagger} and Christian Devaux*

* Centre de Recherches de Biochimie Macromoléculaire, Laboratoire d’Immunologie des Infections Rétrovirales, Institut de Biologie, Montpellier, France; and {dagger} Centre de Biochimie, Nice, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the molecular mechanisms by which the HIV-1 triggers either T cell activation, anergy, or apoptosis remain poorly understood, it is well established that the interaction of HIV-1 envelope glycoproteins with cell surface CD4 delivers signals to the target cell, resulting in activation of transcription factors such as NF-{kappa}B and AP-1. In this study, we report the first evidence indicating that kinases MEK-1 (MAP kinase/Erk kinase) and ERK-1 (extracellular signal-regulated kinase) act as intermediates in the cascade of events that regulate NF-{kappa}B and AP-1 activation upon HIV-1 binding to cell surface CD4. We found that CEM cells transfected with dominant negative forms of MEK-1 or ERK-1 do not display NF-{kappa}B activation after HIV-1 binding to CD4. In contrast, NF-{kappa}B activation was observed in these cells after PMA stimulation. Although the different cell lines studied expressed similar amounts of CD4 and p56lck, HIV-1 replication and HIV-1-induced apoptosis were slightly delayed in cells expressing dominant negative forms of MEK-1 or ERK-1 compared with parental CEM cells and cells expressing a constitutively active mutant form of MEK-1 or wild-type ERK-1. In light of recently published data, we propose that a positive signal initiated following oligomerization of CD4 by the virus is likely to involve a recruitment of active forms of p56lck, Raf-1, MEK-1, and ERK-1, before AP-1 and NF-{kappa}B activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CD4 molecule is a 58-kDa transmembrane glycoprotein expressed predominantly at the plasma membrane of mature peripheral Th lymphocytes (reviewed in 1 . The native molecule contains four extracellular domains (D1–D4)3 showing structural homology with Ig V{kappa} regions and a cytoplasmic tail at the COOH extremity (2, 3). This protein serves as a coreceptor for the TCR/CD3 complex by stabilizing the MHC-TCR/CD3 interactions (4, 5), and acts as a signal-transducing molecule by virtue of the association of its cytoplasmic tail with the p56lck protein tyrosine kinase (6, 7). Additionally, CD4 is the primary receptor for the HIV-1 that binds the Ig V{kappa}-like CDR2 homology region within the domain 1 (D1) of the molecule (8, 9, 10).

The ability of HIV-1 to remain latent or establish productive infection in T lymphocytes is determined, at least in part, by the activation status of the infected cell (11, 12). Several years ago, evidence was reported that HIV-1 itself can trigger T cell activation by interaction of its envelope glycoproteins (gp120/gp160) with CD4 (13, 14). More recently, we and others (15, 16, 17, 18) demonstrated that the binding of heat-inactivated HIV-1 (iHIV-1) or gp120/anti-gp120 immune complexes to the cell surface CD4 molecule enhances the DNA-binding activity of NF-{kappa}B and AP-1 transcription factors in CD4+ T lymphoblastoid cell line and primary T lymphocytes. In contrast, stimulation of NF-{kappa}B was not found in T cell lines expressing a truncated form of CD4 that lacks the cytoplasmic domain (15), indicating that the CD4 molecule transduces a signal leading to NF-{kappa}B activation only when the molecule contains a cytoplasmic tail capable of interacting with a second messenger.

Although the role of CD4 in positive and negative signals transduction in T cells is well documented, the cascade of biochemical events leading to NF-{kappa}B and AP-1 transcription factor activation upon HIV-1 binding to CD4 remains poorly understood. The first event that triggers activation signals following HIV-1 binding to CD4 is most likely the formation of CD4 homodimers or oligomers, which probably involves contact between regions localized in D1 (at the CDR3-like loop), D3, and D4 (19, 20, 21). The second intermediate of this cascade is most likely p56lck. Stimulation of p56lck activity and autophosphorylation at amino acid 394 upon HIV-1 binding to CD4 is well documented (22, 23, 24). Moreover, we have observed recently that HIV-1 binding to CD4 expressed at the surface of HeLa cells transfected with wild-type CD4 and an inactive p56lck did not induce NF-{kappa}B nuclear translocation, whereas NF-{kappa}B activation was evidenced in cells transfected with wild-type forms of CD4 and p56lck (L. Briant, V. Robert-Hebmann, C. Acquaviva, A. Pelchen-Matthews, M. Marsh, and C. Devaux, manuscript in preparation), an observation that corroborates results from Merzouki and coworkers (25). The next cellular intermediates involved in the signaling cascade(s) triggered by CD4 engagement with HIV-1 are not clearly identified. CD4 ligation by HIV-1 envelope was shown to stimulate phosphorylation of the CD4 molecule by protein kinase C (13), to provoke a rise in intracellular calcium levels and induce hydrolysis of phosphatidylinositol to inositol triphosphate (14), and to stimulate the activity of phosphatidylinositol-3-kinase (26, 27) and phosphatidylinositol-4-kinase (28). A recent study by Popik and Pitha indicated that Raf-1 contributes to such signal transduction by direct association with p56lck (29). Involvement of Raf-1 suggested that the dual-specificity kinases MEKs (MAP kinase/Erk kinases, also named MAPKK) and serine/threonine kinases ERKs (extracellular signal-regulated kinases, also named p42/p44 MAPKs) may possibly be involved in this cascade. Activation of MEK-1 is triggered by phosphorylation of two serine residues by Raf (30). MEK-1 in turn phosphorylates and activates ERK-1 and ERK-2, a critical step before their translocation in the nucleus (31, 32, 33). Once in the nucleus, ERKs phosphorylate and thereby regulate several transcription factors such as Elk-1 and participate in c-fos transcriptional regulation (34). Furthermore, the ERK module was shown recently to be involved in NF-{kappa}B-dependent gene expression (35).

We previously reported that HIV-1 binding to CD4 induces phosphorylation of ERK-2 (28, 36). More recently, we observed the phosphorylation of ERK-1 following cross-linking of CD4 by HIV-1 envelope glycoprotein 120 (gp120)/anti-gp120 immune complexes (L.B., unpublished observations). In the present study, we investigated the involvement of MEK-1 and ERK-1 as possible intermediates in the cascade of events, resulting in activation of NF-{kappa}B and AP-1 upon iHIV-1 binding to CD4. To this end, we constructed a panel of CD4-positive T lymphoblastoid cell lines, derived from the parental CEM cell line, stably transfected with MEK-1 or ERK-1 expression vectors, allowing constitutive expression of these molecules as transdominant negative or constitutively active kinases that may be discriminated from the endogenous forms by the presence of a hemagglutinin (HA) tag at the NH2 extremity. The present study demonstrates that both NF-{kappa}B and AP-1 activation generated upon HIV-CD4 interaction require functional expression of MEK-1 and ERK-1 molecules. Furthermore, cells expressing dominant negative forms of MEK-1 or ERK-1 intermediates presented delayed HIV particle production, suggesting that the ERK cascade is involved in T cell signaling pathways up-regulated upon HIV-CD4 binding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

The CD4+ lymphoblastoid cell line CEM was obtained from American Type Culture Collection (Bethesda, MD). The cells were cultured to a density of 5 x 105 cells/ml in RPMI 1640 medium supplemented with a penicillin-streptomycin antibiotic mixture, glutamine (Axcell-Novotec, Lentilly, France) and 10% FCS (ATGC-Botechnologie, Noisy-le-Grand, France), in a 5% C02 atmosphere. Transfectant lymphoblastoid cell lines expressing the tagged wild-type or mutated forms of the ERK-1 or MEK-1 proteins were obtained by electroporation of CEM cells with 20 µg of the pCDNA expression (CMV promoter) vectors encoding either HA/p44 mapk or HA/p44T192A mapk fusion proteins (37, 38), or pECE expression (SV40 promoter) vectors containing the mutated constructs of MEK-1 (39, 40). All vectors used for transfections contained a sequence coding the nine residues corresponding to the major epitope from influenza virus HA-1 at the NH2 terminus of each recombinant kinase. Forty-eight hours after electroporation, 1 mg/ml of G418 (Life Technologies, Eragny, France) was added to the culture medium, and resistant cells were isolated by limiting dilution. Stably transfected lines were maintained in culture medium supplemented with 1 mg/ml of G418.

Antibodies

mAb 12CA5, raised against a peptide corresponding to the major epitope from influenza HA-1 protein, was purchased from Boehringer Mannheim Corp. (Indianapolis, IN). Rabbit anti-p56lck polyclonal Ab and anti-MEK-1 affinity-purified rabbit Ig (C-18) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ERK-1 mAb (MK12) was from Transduction Laboratories (Lexington, U.K.). Anti-actin mAb (C4) was purchased from ICN Biomedicals (Costa Mesa, CA). Anti-CD4 mAb (BL4) was kindly provided by M. Hirn (Coulter-Immunotech, Marseille, France).

Virus production

Viral stock was prepared from culture supernatants of chronically infected CEM cells, as previously described (41), and kept frozen at -80°C until use. After thawing, 100 µl of these stock viruses corresponding to 100 TCID50 (50% tissue culture infectious dose) were used for infection assays. iHIV-1 was obtained by incubation of infectious HIV at 56°C for 30 min.

HIV infection assays

Cells (5 x 105) were incubated for 30 min at 4°C in flat-bottom 96-microwell plates (Costar, Badhoevedorp, The Netherlands) with 100 µl of HIV-1 at 1000 TCID50/ml. Thereafter, the cells were washed five times and cultured in 24-microwell plates (Costar). The amount of virus produced by CEM cells was monitored twice per week by measuring RT activity in 1 ml of cell-free culture supernatants using a synthetic template primer that permitted the RT to neosynthesize radioactive DNA, as previously described (41).

Electrophoretic mobility shift assay (EMSA)

Nuclear extracts were prepared according to the described method (42). Briefly, 2 x 106 cells were incubated for 16 h at 37°C in presence of 100 TCID50 of iHIV or PMA at 20 ng/ml. Thereafter, the cells were washed extensively with PBS, pH 7.8, transferred into 1.5-ml Eppendorf tubes, and microfuged at 4°C for 15 s. The pellet was resuspended in 800 µl of A buffer (containing 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 4 µg/ml leupeptin, and 10 mM HEPES, pH 7.8). After 15 min on ice, 50 µl solution of 10% Nonidet P-40 was added to the sample, and cells were homogenized by vortexing and microfuged at 4°C for 30 s. The pellets were resuspended in 50 µl of B buffer (containing 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 4 µg/ml leupeptin, 10% glycerol, and 50 mM HEPES, pH 7.8). The nuclear extracts were microfuged at 4°C for 5 min, and the supernatants were stored at -80°C until used. The NF-{kappa}B, AP-1, and Sp-1 mobility shift assays were performed using 2 µg protein of nuclear extracts, 1 x 105 cpm of radiolabeled double-stranded probe (NF-{kappa}B, AP-1, or Sp-1) in C buffer (containing 100 mM KCl, 1 mM DTT, 1 mM ZnSO4, 20% glycerol, 0.01% Nonidet P-40, and 50 mM HEPES, pH 7.9), supplemented with BSA, tRNA, and poly(dI:dC) in a final volume of 20 µl. After 20 min at room temperature, the mixture was run at 120 V in a 10% polyacrylamide gel.

Oligonucleotides

ERK-specific oligonucleotides are: ERK1.1, 5'-CCACCGGGACCTGAAGC-3'; ERK1.2, 5'-GTCCAGATAGTGCTTGCC-3'; and ERK1.3, 5'-CTTGATGGCCACTCTAG-3'. MEK-specific primers are MEK1.1, 5'-ACCTTGAATACCACTCC-3' and MEK1.2, 5'-CACCTTGAATACCACTCC-3'. HA-specific primer is 5'-GTTCCTGATTATGCTAGCC-3'. Cellular oligonucleotide primers are TKI, 5'-GAGTACTCGGGTTCGTGAAC-3', and TKII, 5'-GGTCATGTGTGCAGAAGCTG-3'. Double-stranded oligonucleotides used for EMSA are: LTR, 5'-1 (NF-{kappa}B sequence from HIV-1; sense strand only, 5'-GCTGG GGACT TTCCA GGGAG GCGT-3'); AP-1 (AP-1 sequence from HIV-1; sense strand only, 5'-CAGGG GTCAG ATATC CACTG ACCTT-3'); and Sp-1 (Sp-1 sequence from HIV-1; sense strand only, 5'-GGAGG CGTGG CCTGG GCGGG ACTGG GGAGT GGCGA-3'). Oligonucleotides were purchased from Eurogentec (Seraing, Belgium).

RT-PCR amplifications

PCR detection of reverse-transcribed RNAs was performed according to a previously published procedure, with slight modifications (41). Briefly, total RNA was extracted in guanidium thiocyanate from 4 x 106 cells and resuspended in 40 µl H2O/0.1% diethylpyrocarbonate. To reduce the amount of DNA originating from lysis, supernatants were treated with RNase-free DNase (Boehringer Mannheim Corp.; 10 U/ml) for 30 min at 20°C, and then for 5 min at 65°C. To 2 µg of RNA sample (10 µl) was added 200 ng of oligo(dT) primer (1 µl) for 10 min at 65°C. Each sample was made up with reaction buffer (50 mM Tris-HCl, pH 8.3, 30 mM KCl, 8 mM MgCl2, 9 mM DTT, and 320 nM dNTPs) to a final volume of 25 µl, supplemented with 20 U of RNase inhibitor (Boehringer Mannheim Corp.) and 25 U of avian myeloblastosis virus RT (Boehringer Mannheim Corp.), and incubated for 90 min at 42°C. PCR were conducted on 4 µl of sample supplemented with an amplification mixture containing 20 pmol of each of the oligonucleotide primers and 2 U of Taq DNA polymerase. The amplification reaction was run in a PHC2 thermal cycler (Techne, Cambridge, U.K.). The amplified products were electrophoresed in a 2% agarose gel, blotted for 2 h onto Hybond N+ membrane (Amersham, Les Ullis, France), and hybridized with {alpha}-32P-labeled specific probe.

Western blotting analysis

Transfectant cells were washed twice in PBS and lysed in 50 mM Tris-HCl, pH 8, 1% Triton X-100, 100 mM NaCl, 1 mM MgCl2, 2 mM benzamidine, 2 µg/ml leupeptin, and 150 µM PMSF. Cell lysates were electrophoresed in 12.5% SDS-PAGE and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, St Quentin en Yvelines, France). The blots were saturated for 1 h in PBS, 10% milk, and 0.05% Tween-20. After 1-h incubation at 20°C with the appropriate mAb, the blots were washed three times with PBS and 0.05% Tween-20 and incubated for 30 min with 1/5000 dilution of goat anti-mouse (GAM) or goat anti-rabbit Ig peroxidase conjugate (Immunotech). After three washes, bound mAb were detected by incubating the membrane for 1 min with enhanced chemoluminescence reagent (Amersham). The membrane was then exposed for 0.5 to 5 min to Hyperfilms (Amersham).

ERK activity assays

ERK activity was measured using the MAP kinase assay kit from New England Biolabs (Beverly, MA), which allows the selective immunoprecipitation of active ERK from cell lysates using a phosphospecific mAb to ERK, followed by phosphorylation of the ERK-specific substrate Elk-1 (provided under the form of an Elk-1 fusion protein). Phosphorylation of Elk-1 was measured by Western blotting using a phosphospecific Elk-1 Ab.

Flow cytometry

Cells (1 x 105) were incubated for 1 h at 4°C with PBS containing 0.2% BSA (PBS-BSA) or PBS-BSA supplemented with anti-CD4 mAb at concentrations necessary for saturation of cell surface CD4. After washing three times with PBS-BSA, bound mAb was revealed by addition of 50 µl of a 1/50 dilution of fluoresceinated GAM Ig (Immunotech). After 30-min staining, cells were washed with PBS-BSA, and fluorescence intensity was measured on an EPICS XL4-C cytofluorometer (Coulter, Coultronics, Margency, France). The percentage of apoptotic cells was also assessed by flow-cytometry analysis using the impermeant DNA intercalant dye YOPRO-1 (10 mM) (EX max/EM max (nm) = 491/509; Molecular Probes, Eugene, OR), as described (43).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Obtention and characterization of CEM T cell lines stably expressing epitope-tagged ERK-1 and MEK-1

To analyze the involvement of ERK-1 and MEK-1 protein kinases in T cell activation induced by HIV-1 binding to the CD4 receptor, we produced a series of stably transfected CEM T cell lines expressing HA epitope-tagged ERK-1 and MEK-1 kinases: 1) CEM cells transfected by an HA-tagged wild-type ERK-1 (CEM + ERK-1 WT), which was described previously to respond to mitogen in a similar fashion as the endogenous kinase when transfected in Chinese hamster lung fibroblasts (38). 2) CEM cells transfected by a vector encoding the ERK-1 T192A mutant form (CEM + ERK-1 T192A). The ERK-1 T192A mutant protein displays a point mutation that abolishes phosphorylation of one activating site of the kinase (44) and was shown previously in fibroblasts to exert a dominant negative effect when overexpressed (37). 3) CEM cells transfected by a vector encoding the S222A mutant form of MEK-1 (CEM + MEK-1 S222A). The Ser-222 residue represents one key Raf1/MEK kinase-1-dependent phosphorylation site, critical for the activation of MEK-1. Mutant form S222A of MEK-1 was shown to exert a dominant negative effect on MEK-1 (39). 4) CEM cells transfected by the MEK-1 SSDD vector that encodes another mutant isoform of MEK-1 displaying a double substitution (S218D and S222D, respectively), demonstrated to exceed by up to fivefold the full activation of the wild-type kinase (CEM + MEK-1 SSDD). The MEK-1 SSDD mutant was shown to induce activation of ERK in resting cells (40), as expected from a constitutively active protein kinase.

The expression of the wild-type and mutant forms of ERK-1 and MEK-1 in the different transfected cell lines was first assessed by RT-PCR analysis. mRNAs containing an HA sequence were detected in all transfected cell lines, but not in the parental CEM cell line (Fig. 1Go, A and B). In addition, mRNAs encoding ERK-1 or MEK-1 proteins were detected in all cell lines, including the parental CEM line (Fig. 1Go, C and D). Next, expression of the recombinant kinases was analyzed further by immunoblotting using a mAb (12CA5) designed against the HA tag. This mAb discriminates the recombinant molecules from the endogenous ones. The anti-HA mAb 12CA5 specifically detected proteins of apparent m.w. of 46 to 47 kDa in cell lines transfected with ERK-1 (Fig. 2GoA) or MEK-1 constructs (Fig. 2GoB). In untransfected CEM cells, no band was found running at 46 to 47 kDa. As a control, the total amount of ERK-1 or MEK-1 (recombinant + endogenous kinase) found in the CEM extracts was evaluated using ERK-1 (Fig. 2GoC)- or MEK-1-specific reagents (Fig. 2GoD); ERK-1 or MEK-1 proteins were detected in each type of cell, including the parental CEM.



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  		FIGURE 1. Expression of MEK-1 and ERK-1 mRNAs in transfected CEM cells. CEM cells were electroporated with expression vectors coding ERK-1 or MEK-1 HA epitope-tagged kinases, selected for G418 resistance, and isolated by limiting dilution. ERK-1 (A) and MEK-1 (B) epitope-tagged expression was detected (lanes 3 and 4) by PCR amplification of reverse-transcribed total mRNA with TAG-HA/ERK1.3 and TAG-HA/MEK1.2 primer pairs, respectively. As control, cells were analyzed for expression of the endogenous ERK-1 (C) and MEK-1 (D) using the ERK1.1/ERK1.2 and MEK1.1/MEK1.2 oligonucleotide primer pair, respectively. PCR amplification products were electrophoresed, blotted, hybridized with specific {alpha}-32P-labeled specific probes, and visualized by autoradiography. PCR amplification of reverse-transcribed thymidine kinase (TK) RNA is also shown as control (E and F). A control PCR amplification was performed with RNA from untransfected CEM (lane 2). Lane 1 corresponds to an RNA-free sample submitted to RT-PCR amplification.

 


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  		FIGURE 2. Expression of MEK-1 and ERK-1 proteins in transfected CEM cells. Expression of HA-tagged kinases and endogenous kinases in transfected CEM cells was evaluated by Western blot analysis. One hundred micrograms of Triton X-100 soluble proteins were resolved by SDS-PAGE (10% acrylamide gel) and immunoblotted with appropriated Ab. Expression of ERK-1 (A) and MEK-1 (B) HA-tagged proteins in transfected CEM cells was detected using the 12CA5 mAb specific for the HA epitope. Expression of total ERK-1 (expressed + endogenous) was detected using the ERK-1-specific MK12 mAb (C). Expression of total MEK-1 was detected using MEK-1-specific C-18 rabbit Ig (D). Immunoblots using an anti-actin Ab are shown as control (E, F).

 
Having previously determined that NF-{kappa}B and AP-1 transcription factor activation induced upon HIV-CD4 interaction requires the expression of the cytoplasmic tail of CD4 (15) and of the p56lck tyrosine kinase (L. Briant, V. Robert-Hebmann, C. Acquaviva, A. Pelchen-Matthews, M. Marsh, and C. Devaux, manuscript in preparation), it remained to be controlled whether the transfected cell lines expressed both CD4 and p56lck proteins. As shown in Figure 3Go, a strong expression of the CD4 Ag was detected by flow cytometry on the parental CEM cells and on each type of CEM transfected by a kinase expression vector. It is worth noting that expression of CD4 was about two- to fourfold lower in CEM + MEK-1 SSDD cells than in the parental cell line. Expression of the p56lck protein was detected in the parental and transfected CEM cell lines by immunoblotting (Fig. 4Go), suggesting that CD4-dependent signals should be transduced correctly through CD4/p56lck complexes in these cells.



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  		FIGURE 3. Cell surface expression of CD4 in CEM parental cell lines and in cells expressing the ERK-1 or MEK-1 HA-tagged proteins. Cells were incubated with medium alone (to determine the background fluorescence) or medium containing the BL4 anti-CD4 mAb. mAb binding to CD4 was detected by a FITC-labeled GAM Ig. The fluorescence intensity was recorded in the log mode on an EPICS XL4 cytofluorometer. The CD4-negative A2.01 T cell line (15), which derives from CD4-positive A3.01 subline of CEM, was used as control.

 


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  		FIGURE 4. Analysis of lck protein expression. Lysates from CEM cells (lane 1), p56lck-negative MT2 cells (50) (lane 2), and transfected CEM cell lines (lanes 3-6) containing 50 µg of total cellular protein extract were electrophoresed onto SDS-PAGE and blotted onto polyvinylidene difluoride membrane. The membrane was incubated successively with an anti-p56lck mAb and GAM Ig peroxidase conjugate. Bound mAb was detected by chemiluminescence after incubation with an enhanced chemoluminescence reagent.

 
These results indicate that the four transfected cell lines expressed both the endogenous and recombinant kinase. Since these cells also exhibit comparable amounts of CD4 and p56lck, they represent appropriate tools to investigate the role of MEK-1 and ERK-1 in CD4 signal transduction.

Constitutively active MEK-1 and wild-type ERK-1 induce AP-1 activation and NF-{kappa}B translocation when overexpressed in CEM cells

In several cell types, the ERK pathway was demonstrated to be involved in AP-1 transcriptional regulation, and recently, the ERK module was suggested to play a pivotal role in triggering NF-{kappa}B/Rel DNA-binding protein activity (35). To determine the involvement of MEK (the upstream activator of ERK) and ERK in NF-{kappa}B activation, we first examined by gel shift experiments (EMSA) whether the constitutively active mutant of MEK-1 (SSDD; S218D/S222D) could be sufficient to induce NF-{kappa}B and AP-1 DNA-binding activity. As expected, AP-1 activation level was increased in nuclear extracts from unstimulated CEM + MEK-1 SSDD cells (Fig. 5GoA, lane 7) compared with extracts from untransfected CEM (Fig. 5GoA, lane 1). Next, the biologic consequences of expression of constitutively active MEK-1 SSDD in CEM cells were assessed by evaluating NF-{kappa}B activity. A major increase in the shift of labeled NF-{kappa}B oligonucleotide was observed when the probe was incubated with nuclear extracts from transfected cells compared with that from untransfected cells (Fig. 5GoB, lanes 7 and 1, respectively). These results suggested that constitutive activation of MEK-1 triggers permanent AP-1 and NF-{kappa}B activation.



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  		FIGURE 5. Effect of iHIV/CD4 interaction on AP-1 and NF-{kappa}B nuclear translocation in CEM-transfected cell lines expressing MEK-1 mutant kinases. Effect of MEK-1 mutant isoform expression on iHIV/CD4-induced DNA-binding protein translocation was analyzed using an EMSA. Nuclear extracts were prepared from CEM (lanes 1–3) or CEM-transfected cells expressing the HA-tagged MEK-1 S222A (lanes 4–6) or HA-tagged MEK-1 SSDD (lanes 7–9), cultured for 16 h in medium alone or medium containing 20 µg/ml PMA or a concentration of iHIV-1 corresponding to 100 x TCID50 of HIV (iHIV). Nuclear extracts were reacted with AP-1 (A)- or NF-{kappa}B (B)-labeled dsDNA probes. EMSA performed in the presence of an Sp-1 probe (C), which allows detection of a constitutively expressed Sp-1 nuclear factor, is shown as control.

 
To further investigate the role of MEK-1/ERK-1 intermediates in the signal-transduction pathway resulting in AP-1 and NF-{kappa}B activation, we analyzed the effect of ERK-1 overexpression on the activation of these DNA-binding factors. When nuclear extracts were prepared from cells transfected with a vector encoding the wild-type ERK-1 protein kinase (CEM + ERK-1 WT), a significant increase in the shift of the labeled probe was observed compared with the basal NF-{kappa}B translocation detected in untransfected CEM (Fig. 6GoA, lanes 7 and 1, respectively). Similar observations were made using an AP-1 probe (data not shown). It is worth noting that the NF-{kappa}B translocation level was apparently lower in CEM + ERK-1 WT (Fig. 6GoA, lanes 7–9) than in cells expressing MEK-1 SSDD mutant (Fig. 5GoB, lanes 7–9), an observation compatible with the fact that MEK-1 SSDD mutant is constitutively active, whereas ERK-1 WT is not.



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  		FIGURE 6. Effect of iHIV/CD4 interaction on NF-{kappa}B translocation in CEM and CEM-transfected cell lines expressing the HA-tagged ERK-1 kinases and inhibition of the endogenous ERK-1 by the dominant negative mutant ERK-1 T192A. A, Nuclear extracts were prepared from CEM (lanes 1–3) or CEM-transfected cells expressing the HA-tagged ERK-1 T192A (lanes 4–6) or HA-tagged ERK-1 WT (lanes 7–9), cultured for 16 h in medium alone or medium containing PMA or iHIV. NF-{kappa}B DNA-binding protein activation was assessed by EMSA, as in Figure 5Go. B, Relative abundance of expressed ERK-1 T192A, ERK-1 WT, and endogenous ERK-1 in transfected CEM cells was evaluated by Western blot analysis. Soluble proteins (30, 10, and 5 µg, respectively) extracted from CEM (lanes 1–3), CEM + ERK-1 T192A (lanes 4–6), and CEM + ERK-1 WT (lanes 7–9) were resolved by SDS-PAGE (15% acrylamide gel) and immunoblotted using the ERK-1-specific MK12 mAb (C). Cells (CEM, CEM + ERK-1 T192A, and CEM + ERK-1 WT) were cultured in medium alone (-) or medium containing PMA (+). A phosphospecific Ab to ERK was used to immunoprecipitate active ERK-1 from CEM, CEM + ERK-1 T192A, and CEM + ERK-1 WT cell lysates. ERK-1 activity (upper panel) was estimated by measuring the phosphorylation of the ERK-specific substrate Elk-1, as described in Materials and Methods. Detection of ERK-1 in each sample is shown as control (lower panel).

 
These results suggest that MEK-1 and ERK-1 may function as cellular intermediates involved in signaling pathways leading to AP-1 and NF-{kappa}B activation in T lymphoblastoid cell lines.

Implication of MEK-1 and ERK-1 in AP-1 activation triggered by HIV-1 binding to CD4

We previously demonstrated that HIV-1 envelope binding to CD4 induced AP-1 activation in primary lymphocytes (18). Recently, we observed that HIV-CD4 interaction enhances ERK-2 activity in T lymphoblastoid cell lines and in primary lymphocytes (28, 36). It remained to be defined whether kinases from the MEK and ERK families could be involved in AP-1 activation induced upon CD4 ligation.

There is much evidence in the literature indicating that proteins of the MEK and ERK families are involved in AP-1 DNA-binding protein activation induced by a variety of stimuli. Furthermore, as indicated above, the AP-1 transcription factor was found to be activated upon HIV-CD4 interaction. Thus, the functional biochemical machinery of the various transfectant cell lines included in the present study was assessed by testing the capacity of MEK-1 and ERK-1 transdominant negative mutants to interfere with AP-1 activation. When nuclear extracts from CEM + MEK-1 S222A cells (a cell line expressing the S222A MEK-1 transdominant negative mutant) exposed to iHIV-1 (iHIV-1 was used at a concentration of virus equivalent to 100 TCID50 of HIV-1) were incubated with a labeled double-stranded oligonucleotide corresponding to the AP-1 binding site from the HIV-LTR, no shift was observed by EMSA (Fig. 5GoA, lane 5). In contrast, a significant AP-1 shift was observed when this cell line was stimulated with PMA (Fig. 5GoA, lane 6). Similar results were obtained using the cell line expressing the ERK-1 T192A isoform (data not shown).

Upon HIV-1 binding to CD4, a signal is delivered to T cells that lead to AP-1 activation. Our results suggest that activation of MEK-1 and ERK-1 is necessary for triggering this signal.

Implication of MEK-1 and ERK-1 in NF-{kappa}B activation after HIV-1 binding to CD4

There is but little evidence in the literature suggesting that NF-{kappa}B nuclear translocation could be triggered by activated ERK. In light of our previous data indicating that HIV-1 envelope binding to CD4 enhances ERK-2 activity and induces NF-{kappa}B nuclear translocation in CEM cells and primary lymphocytes (28, 36, 15, 18), it became interesting to study whether the ERK pathway may act as a link between CD4 signal and NF-{kappa}B nuclear translocation.

The ability of MEK-1 and ERK-1 transdominant negative mutants to interfere with NF-{kappa}B nuclear translocation induced by iHIV-CD4 engagement or PMA was investigated. As shown in Figures 5GoB and 6A, a shift of labeled NF-{kappa}B oligonucleotide was observed when mixed with nuclear extracts from untransfected CEM cells exposed to iHIV-1 (lane 2). In contrast, after similar treatments with iHIV-1, NF-{kappa}B translocation was detected neither in CEM + MEK-1 S222A cells expressing a transdominant negative form of MEK-1 kinase (Fig. 5GoB, lane 5) nor CEM + ERK-1 T192A expressing a transdominant negative form of ERK-1 (Fig. 6GoA, lane 5), although CEM + ERK-1 T192A expressed slightly more surface CD4 than the parental CEM. To confirm that the lack of NF-{kappa}B nuclear translocation in these cell lines can be ascribed directly to expression of the mutated kinase, we exposed these cell lines to 20 µg/ml of PMA. Lane 6 in Figures 5GoB and 6A shows that a shift of labeled NF-{kappa}B oligonucleotide was generated when ERK-1 and MEK-1 transdominant negative kinase-expressing cell lines were exposed to phorbol esters, indicating that the absence of shift of labeled NF-{kappa}B probe following HIV-CD4 binding was not due to a defect in the ability of the cell to activate this DNA-binding protein, but rather to a specific blockade of the CD4 signal transduced through the ERK pathway. Such observation was reproducible using two different clones of each type (Fig. 7Go, A and C).



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  		FIGURE 7. Effect of iHIV/CD4 interaction on NF-{kappa}B translocation in several clones of each type. Nuclear extracts were prepared from two clones of each type: CEM + ERK-1 T192A (A), CEM + ERK-1 WT (B), CEM + MEK-1 S222A (C), and CEM + MEK-1 SSDD (D), cultured for 16 h in medium alone (lanes 1 and 4) or medium containing iHIV (lanes 2 and 5) or PMA (lanes 3 and 6). NF-{kappa}B DNA-binding protein activation was assessed by EMSA, as in Figure 5Go.

 
These results indicate that MEK-1 and ERK-1 activation is necessary for triggering the signal pathways that lead to NF-{kappa}B activation in T lymphoblastoid cell lines following HIV-1 binding to cell surface-expressed CD4.

Direct evidence that inhibition of endogenous ERK-1 by the ERK-1 T192A dominant negative mutant accounts for the lack of NF-{kappa}B activation upon CD4 signaling in CEM + ERK-1 T192A cells

To further investigate the possibility that CD4 signal cannot be transduced through the ERK pathway in CEM + ERK-1 T192A cells because the endogenous ERK-1 activation does not occur when ERK-1 T192A mutant is expressed, the relative abundance of the expressed and endogenous ERK-1 in CEM + ERK-1 T192A cells, as well as the ability of r ERK-1 T192A to inhibit the endogenous ERK-1 activity, were analyzed. A mAb raised against the C-terminal region of ERK-1 was used to compare the levels of expression of the ectopically expressed ERK-1 with that of the endogenous one. Using highly resolutive conditions for protein migration during electrophoresis, we found (Fig. 6GoB) that this anti-ERK-1 mAb recognized a single protein of 44 kDa in control CEM cells and a doublet of 44 and 46 kDa in transfected CEM + ERK-1 T192A and CEM + ERK-1 WT cells, corresponding respectively to the detection of the endogenous kinase and of the tagged ERK-1 molecule. The levels of expression of the ectopically expressed and endogenous ERK-1 in CEM + ERK-1 T192A and CEM + ERK-1 WT cells were apparently similar. Next, the ability of rERK-1 T192A to inhibit the endogenous ERK-1 was measured by in vitro phosphorylation of the ERK-specific substrate, Elk-1. As shown in Figure 6GoC, the phosphospecific Elk-1 Ab allowed identification of phosphorylated Elk-1 in samples reacted with ERK immunoprecipitated from extracts of CEM or CEM + ERK-1 WT cells treated by PMA. In contrast, Elk-1 was not phosphorylated when samples derived from CEM + ERK-1 T192A cells treated by PMA.

These results demonstrate that ERK-1 activity is necessary for transducing the signal that led to NF-{kappa}B activation in T lymphoblastoid cell lines following HIV-1 binding to cell surface-expressed CD4. In contrast to iHIV-1, PMA can trigger NF-{kappa}B activation in CEM + ERK-1 T192A cells, although the endogenous ERK-1 is inactive.

HIV replication and HIV-1-induced apoptosis are delayed in cells expressing a trandominant negative mutant of MEK-1 or ERK-1 protein kinases

The HIV-1 promoter (LTR) contains two NF-{kappa}B binding sites, and viral replication was shown to require NF-{kappa}B nuclear translocation that synergizes with viral Tat transactivator in the stimulation of HIV-1 promoter activity. We have found previously that HIV-1 production is delayed in A2.01/CD4.401 cells (15) and A2.01/CD4.403 cells (C.D., unpublished observations), which express a truncated form of CD4 lacking the cytoplasmic tail. Indeed, these cells lack the ability to activate NF-{kappa}B translocation after HIV-1 binding to their surface CD4 molecule. In this work, we analyzed whether the defect of NF-{kappa}B activation observed in cell lines expressing the transdominant ERK-1 or MEK-1 proteins may affect HIV-1 particles production. Cell lines expressing MEK-1 S222A or ERK-1 T192A dominant negative mutants, and control cell lines were exposed to 100 x TCID50 HIV-1, and virus production in cell-free culture supernatants was measured twice per week. As shown in Figure 8Go, a 3-day delayed virus production (which corresponds to a 10-fold decrease in virus production) was observed in cell lines expressing the transdominant negative mutants of MEK-1 and ERK-1 as compared with untransfected CEM cells expressing the wild-type kinases. In contrast, when cell lines expressed either the ERK-1 WT recombinant molecule or the constitutively active MEK-1 SSDD mutant, no significant difference in RT activity was noticed as compared with the CEM parental cell line. The different capacities of cells to replicate HIV-1 were investigated further by measuring HIV-1-induced apoptosis at days 3, 7, and 10 postinfection in cells expressing the wild-type and mutant forms of MEK-1 and ERK-1. On day 3 postinfection, no significant increase of apoptosis was found in any of the cell line (data not shown). As shown in Figure 9Go, HIV-1-induced apoptosis was found 7 days postinfection in untransfected CEM cells and cell lines expressing either the ERK-1 WT recombinant molecule or the hyperactivated MEK-1 SSDD. In contrast, the increased apoptosis related to HIV-1 infection of cells expressing the transdominant negative mutants of MEK-1 and ERK-1 was absent or minor at day 7 postinfection. However, by day 10 postinfection, all infected cell lines undergo apoptosis.



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  		FIGURE 8. Effect of ERK-1 or MEK-1 HA epitope-tagged kinase expression on HIV-1-productive infection. Cells transfected with ERK-1 or MEK-1 HA epitope-tagged proteins were exposed to 100 µl of HIV-1Lai (103 x TCID50/ml), and virus production was followed by measuring RT activity in cell-free culture supernatant. RT activity less than 1.5 x 103 cpm/ml was considered as negative. Each point is the result of duplicate experiments. Tranfected cell lines expressing the transdominant negative form of ERK ({square}, upper panel), or MEK ({square}, lower panel), or cells expressing the wild-type ERK ({diamond}, upper panel) or active constitutive MEK ({diamond}, lower panel) exposed to HIV-1-infected cells are shown as open symbols. The uninfected corresponding cell lines are represented by black symbols ({blacksquare}, {diamondsuit}). For each experiment, HIV-1-infected CEM cells ({circ}) or uninfected CEM cells (•) are shown as a control.

 


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  		FIGURE 9. Effect of ERK-1 or MEK-1 HA epitope-tagged kinase expression on HIV-1-induced apoptosis. Cells transfected with ERK-1 or MEK-1 HA epitope-tagged proteins were exposed to 100 µl of HIV-1Lai (see legend of Fig. 8Go). The percentage of apoptotic cells in cell cultures was assessed at day 3, day 7, and day 10 of culture by flow-cytometry analysis using the impermeant DNA intercalant YOPRO-1. A, Representative experiment of apoptosis at day 7 postinfection. B, Time course experiment of HIV-1-induced apoptosis in CEM cells expressing the different forms of ERK-1. The results represent the mean of duplicates. CEM ({square}, {blacksquare}), CEM + ERK-1 T192A ({circ}, •), and CEM + ERK-1 WT ({diamond}, {diamondsuit}). Open symbols indicate virus-free cells; closed symbols, HIV-1-infected cells. C, Time course experiment of HIV-1-induced apotosis in CEM cells expressing the different forms of MEK-1. The results represent the mean of duplicates. CEM ({square}, {blacksquare}), CEM + MEK-1 S222A ({triangledown}, {blacktriangledown}), and CEM + MEK-1 SSDD ({triangleup}, {blacktriangleup}). Open symbols indicate virus-free cells; closed symbols, HIV-1-infected cells.

 
These results indicate that the capacity of HIV-1 to efficiently replicate in CEM cells is impaired by expression of MEK-1 or ERK-1 dominant negative mutants. Moreover, they suggest that T cell activation generated upon HIV-CD4 interaction requires a functional ERK module to convey the activation signal downstream to ERK. Thus, ERK can play a role in stimulation of HIV-1 early transcription events.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, we demonstrated that interaction of multimeric HIV-1 envelope gp120 with the CD4 receptor induces T cell activation, resulting both in NF-{kappa}B nuclear translocation and AP-1 activation. Furthermore, analysis of biochemical events generated upon HIV-CD4 interaction was found to activate several cellular kinases, including p56lck, Raf-1, and ERK-2, suggesting a possible involvement of a Raf-1-dependent ERK signaling pathway in this process. The purpose of this study was to investigate the role of MEK and ERK in the signal-transduction pathway(s) leading to AP-1 and NF-{kappa}B activation following engagement of CD4 with HIV-1. We demonstrate in this study that both MEK-1 and ERK-1 are dowstream cellular intermediates in this CD4 receptor-dependent activation cascade that are required for efficient activation of the two DNA-binding proteins.

Although the signaling pathway that leads to AP-1 activation following engagement of CD4 with HIV-1 was not known, it is well established that AP-1 activation resulting from the engagement of several other surface receptors with their extracellular ligands usually involves the Ras, Raf, MEK, and ERK intermediates (reviewed in Refs. 45–47). Accordingly, one could expect MEK and ERK to act as cellular intermediates linking CD4 signals to AP-1 activation. In contrast, observations suggesting that the signaling pathway leading to NF-{kappa}B activation may be somehow linked to the well-defined Raf/MEK/ERK pathway remain limited. During the past few years, PKC-{zeta}, a downstream substrate of Ras, was demonstrated to become activated in response to agents stimulating NF-{kappa}B transcription factor, and Raf-1 was found to induce the dissociation of cytoplasmic NF-{kappa}B/I-{kappa}B complexes (48), suggesting that a Raf-1-dependent pathway may be involved in NF-{kappa}B activation. Recently Berra and coworkers (35) demonstrated that PKC-{zeta} triggers the activation of a number of kinases, and suggested that MEK and ERK may also participate in NF-{kappa}B activation by enhancing AP-1/NF-{kappa}B cross-coupling mechanism. Herein we analyzed NF-{kappa}B nuclear translocation in stably transfected cell lines expressing either constitutively activated or transdominant negative forms of MEK-1 or ERK-1; gel shift experiments demonstrated that a chronic activation of one of these kinases results in the constitutive nuclear translocation of NF-{kappa}B. These observations suggest that MEK-1 and ERK-1 may directly participate in T cell signaling pathways leading to NF-{kappa}B activation. Since MEK and ERK are located downstream of Raf-1 in the classical ERK signaling pathway, our data suggest that a Ras/Raf-1 signal may possibly regulate the inducible NF-{kappa}B DNA-binding activity through MEK and ERK.

Additionally, using a series of dominant negative molecules that interfere with the functions of MEK-1 and ERK-1, we investigated whether activation of NF-{kappa}B and AP-1 that follows iHIV-1 binding to CD4 may involve the ERK signaling pathway. We found that activation of transcription factors subsequent to iHIV-1 interaction with CD4 is abolished by transdominant expression of mutated forms of MEK-1 or ERK-1. We also demonstrated that expression of the transdominant mutated forms of ERK-1 (ERK-1 T192A) inhibited the activation of the endogenous ERK-1 by exogenous stimuli. Moreover, a 3-day delay in HIV replication and HIV-1-induced apoptosis was observed in cell lines expressing MEK-1 or ERK-1 transdominant negative kinase. In contrast, no delay was observed in cell lines either overexpressing the wild-type ERK-1 or expressing a constitutively activated MEK-1 protein. Expression of ERK-1 WT was shown previously to lead to an increased recruitment of AP-1 and promotes a significant stimulation of NF-{kappa}B-dependent promoter activity (35). Proteins belonging to the NF-{kappa}B/Rel family of transcription factors appear to be important stimulating factors acting on the two NF-{kappa}B binding sites encountered in the HIV-1 promoter. Altogether, these results indicate that full NF-{kappa}B activation generated upon HIV-CD4 interaction requires functional MEK-1 and ERK-1 intermediates. Thus, ERK is likely to represent a node linking the AP-1 and NF-{kappa}B activation pathways. Our results are in agreement with previous studies showing that p56lck and Raf-1 are involved in T cell activation generated after CD4 ligation by HIV-1 (23, 24, 29). Involvement of Ras and Raf-1-dependent signal-transduction pathways in HIV-1-induced activation of NF-{kappa}B was also demonstrated by Folgueira and coworkers (49) in monocytic cell lineage, by using constitutively active Ras mutants. It is also worth noting that mAb specific for the CDR3-like loop in D1 of CD4 that blocks HIV-1 transcription inhibit ERK activation and NF-{kappa}B nuclear translocation triggered by HIV-1 binding to CD4 (36, 50), suggesting that inhibition of ERK activation that follows mAb treatment accounts for the lack of NF-{kappa}B activation and delayed HIV-1 transcription. A similar antiviral mechanism is probably involved in the inhibition of HIV-1 replication mediated by exocyclic peptides that mimic the CDR3-like loop (51).

Recently, CD4 coreceptors for HIV-1 were identified in CD4+ T lymphocytes and macrophages (52, 53, 54, 55). These coreceptors belong to the superfamily of G protein-coupled seven-transmembrane domain receptors. Heterotrimeric guanine triphosphate-binding protein G is known to stimulate both NH2 Jun terminal kinases (JNK) and ERK, but the subsequent intervening molecules are still poorly defined. Recently, G protein-coupled receptors were shown to be linked to the ERK signaling pathway through phosphatidylinositol 3'-kinase {gamma} (56). We have shown previously by competition studies that NF-{kappa}B and AP-1 activation is abolished by preincubation of iHIV or gp120/anti-gp120 immune complexes with soluble CD4, suggesting that fusin-related signaling pathways are not predominant after HIV-CD4 interaction. However, the interaction of gp120 with the coreceptor is strongly increased after previous contact with CD4 (57, 58). Accordingly, we cannot exclude that T cell signaling generated by HIV-1 glycoproteins may also involve, at least in part, the coreceptor either directly or through cocapping with CD4. This possibility is currently under evaluation in our laboratory. If iHIV-1 turned to modulate ERK activation pathway following ligation with the coreceptor, it would make of ERK a central molecule in T cell activation generated upon HIV-1 binding to its cell surface receptors.

We already suggested that signaling events generated following HIV-1 binding to CD4 directly stimulate HIV life cycle by preparing the host cell to postfusion event, including early gene transcription (15, 18). Besides stimulating virus replication, signaling events generated upon HIV-CD4 interaction that lead to NF-{kappa}B and AP-1 activation may likely contribute to the cell dysfunction. This could occur by activating transcription of cellular genes resulting in the aberrant cytokine expression such as IL-6, IL-10, IFN-{gamma}, and TNF-{alpha} (reviewed in 59 . Additionally, the external glycoprotein gp120 of HIV-1 was suggested to prime, via the activation of cellular kinases, the human CD4+ T lymphocytes for apoptosis (60, 61, 62, 63). Therefore, the activation or alteration of the signaling pathways by binding of HIV-1s to their receptor and/or coreceptor obviously has important consequences for the HIV-induced pathogenicity that remain to be further investigated.


   Acknowledgments
 
We thank C. Acquaviva (student at University of Montpellier) for technical assistance in cell culture, and P. Roux (IGMM-CNRS UMR5535, Montpellier) for helpful discussions.


   Footnotes
 
1 This work was supported in part by institutional funds from the Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), and grants (to C.D.) from Agence Nationale de Recherches sur le SIDA (ANRS) and the FRM-SIDACTION program. L.B. is a fellow of FRM-SIDACTION program. Back

2 Address correspondence and reprint requests to Dr. Laurence Briant, CRBM-CNRS ERS 155, Laboratoire d’Immunologie des Infections Rétrovirales, 4 Boulevard Henri IV, 34060 Montpellier Cedex, France. E-mail address: Back

3 Abbreviations used in this paper: D, domain; CDR, complementarity-determining region; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; GAM, goat anti-mouse; HA, hemagglutinin; iHIV, heat-inactivated HIV; LTR, long terminal repeat; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; NF-{kappa}B, nuclear factor-{kappa}B; RT, reverse transcriptase; TCID50, 50% tissue culture infectious dose; WT, wild-type. Back

Received for publication July 1, 1997. Accepted for publication November 3, 1997.


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