| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Correspondence
Manuel Fresno
mfresno{at}cbm.uam.es
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Stevenson, 2003). Among the viral factors involved, the protein Tat has been studied widely. Tat is a small (72–101 aa) regulatory viral protein required for efficient transcription and virus replication (Jeang et al., 1999). Tat has been shown to interact with several members of the transcriptional machinery during the process of initiation and elongation of viral transcription (Cullen, 1998). Furthermore, Tat not only affects viral transactivation but also mediates alterations of multiple cellular processes. Among these, Tat has been involved in the aberrant expression of several cytokine and chemokine genes such as tumour necrosis factor 
(Buonaguro et al., 1992), transforming growth factor 
(Zauli et al., 1992), interleukin 6 (IL-6) (Ambrosino et al., 1997), IL-2 (Vacca et al., 1994; Westendorp et al., 1994), gamma interferon (Zagury et al., 1998), IL-8 (Ott et al., 1998), monocyte chemoattractant protein 1 (MCP-1) (Abraham et al., 2005) and MCP-2 (Izmailova et al., 2003). Many of these effects are most likely mediated by alterations of gene expression by Tat. In this regard, a direct interaction of the viral protein with several transcription factors including Oct, Sp1 (Jeang et al., 1993), Egr (Yang et al., 2002), E2F (Ambrosino et al., 2002) and nuclear factor of activated T cells (NFAT) (Macián & Rao, 1999) has been demonstrated. In addition, indirect mechanisms have been proposed to explain alterations in the activity of NF-
B (Biswas et al., 1995; Demarchi et al., 1999) and activator protein 1 (AP-1) (Li et al., 1997; Kumar et al., 1998) transcription factors.
Cytokine transcription is controlled by, among others, the transcription factors NF-B, AP-1 and NFAT (Ghosh et al., 1998; Macián et al., 2001). AP-1 dimers are composed of members of the Jun, Fos or ATF families. Their activation is regulated by transcriptional and post-transcriptional mechanisms, including specific interactions with transcriptional co-activators (Karin et al., 1997). In contrast to AP-1, NFAT proteins are functional as monomers and their activation is tightly regulated by intracellular calcium concentrations. Thus, the phosphatase calcineurin, which dephosphorylates NFAT inducing its translocation to the nucleus and increasing its DNA binding and transactivating activities, is activated by calcium increases (Im & Rao, 2004).
The interplay between families of transcription factors represents an additional mechanism of gene regulation. Interactions between NFAT and AP-1 proteins take place on composite elements present in, among others, the IL-2, IL-4, IL-5, CD40L and granulocyte–macrophage colony-stimulating factor (GM-CSF) and cyclo-oxygenase 2 (Cox-2) promoters (Kel et al., 1999; Íñiguez et al., 2000; Macián et al., 2001; Johnson et al., 2004). The NFAT distal site of the human IL-2 promoter allowed the identification of the NFAT/AP-1 binding complex (Shaw et al., 1988). These cooperative interactions have an important functional significance, as they allow the integration of signals by the binding of different family (Rel-ZIP) transcription factors to specific DNA-response elements. They increase the functional diversity of the transcription factors, as these supracomplexes show DNA-binding and transactivating capacities distinct from either transcription factor partner alone (Nolan, 1994).
As the Tat viral protein has been shown to affect cytokine transcription and NFAT, we considered it to be of interest to study how this viral protein can affect its functional cooperation with AP-1. Here, we have shown that Tat interacts with NFAT, favouring its association with c-Jun at composite NFAT/AP-1 elements and increasing its transactivating activity. Furthermore, we showed that Tat strongly increases NFAT/AP-1 cooperative DNA binding and transactivation without having any significant effect on the individual binding of these factors to DNA.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 25 ng ml–1; Sigma Chemicals) and calcium ionophore A23187
[GenBank]
(Io, 1 µM; Sigma Chemicals) as indicated. None of the agents affect the viability of the cells at the concentrations used.
Plasmid constructs.
The reporter plasmid pLTR-luc, containing the long terminal repeat (LTR) from HIV-1 subtype B, was a generous gift from Dr J. L. Virelizier (Schwartz et al., 1990). The plasmid pB-CONA-luc contained three tandem copies of the B site of the immunoglobulin 
chain promoter cloned upstream of the conalbumin (CONA) transcription start site (Arenzana-Seisdedos et al., 1993). The pNFAT-luc reporter plasmid was a gift from Dr Crabtree (Durand et al., 1988) and contained three tandem copies of the NFAT/AP-1 distal site of the human IL-2 promoter fused to the minimal human IL-2 promoter. The p–73col-luc plasmid included the AP-1-responsive regions (–73/+63 bp) of the human collagenase promoter fused to the luciferase gene (Deng & Karin, 1993). The p275-Cox-2-luc plasmid containing the –170 to +104 region of the human Cox-2 promoter fused to the luciferase gene and their AP-1 and NFAT mutated versions have been described previously (Íñiguez et al., 2000). The plasmid pEF-BOS-NFATc2 contained the gene encoding the influenza virus haemagglutinin (HA)-tagged NFATc2 (NFAT1) and was a generous gift from Dr J. M. Redondo (Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain), whilst pSH107-NFATc1 (NFAT2) was a gift from Dr G. Crabtree. RSV-c-Jun was provided by Dr A. Muñoz (Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Madrid, Spain) and HA-tagged pCMV-c-Jun-HA by Dr C. Weiss (Weiss et al., 2003). pCMV-Tat was a gift from Dr J. Alcamí (Instituto de Salud Carlos III, Madrid, Spain) and contained full-length HIV-1 Tat (86 aa) under the control of the cytomegalovirus (CMV) immediate-early promoter (Schwartz et al., 1990). pcDNA3-Tat plasmids carrying the Y26A and Y47N mutations were a generous gift of Dr B. Berkhout (Verhoef & Berkhout, 1999). The pcDNA3 plasmid (Invitrogen) was used as a control in the transfection of expression plasmids or to adjust the quantities of DNA transfected. The promoter region of IL-4 containing a pure NFAT site (pIL-4-luc) was a gift from Dr R. Davis (Howard Hughes Medical Institute, University of Massachusets Medical School, Worcester, USA) (Aune & Flavell, 1997). Tat-HA was generated from pSV2-tat72 (obtained through the AIDS Research and Reference Reagent Program, NIAID, NIH) from Dr A. Frankel (Frankel & Pabo, 1988). The plasmid pRL-tk-luc (Promega) was used to evaluate transfection efficiency.
Plasmid pGEX2TK-Tat was obtained from E. Muñoz (Universidad de Córdoba, Spain). Purification of the GST–Tat fusion protein was performed as described previously (Blazquez et al., 1999). The purity and integrity of the proteins were routinely checked by Western blotting, using the anti-Tat polyclonal antiserum.
Western blot analysis.
Nuclear extracts of Jurkat cells and total extracts of COS-7 cells transfected with the various plasmids obtained as described previously (González et al., 2001) were separated by SDS-PAGE. For the detection of NFATc2 and c-Jun, 6 % polyacrylamide gels were electrophoretically transferred to nitrocellulose filters (Bio-Rad); for the detection of Tat, 17 % polyacrylamide gels were transferred to ProBlott PTMP membranes (Applied Biosystems) and processed as described previously (González et al., 2001). The anti-NFATc2 672 serum (a generous gift from Dr J. M. Redondo) was used at a 1 : 3000 dilution and the anti-c-Jun (Santa Cruz Biotechnology), anti-HA antibodies, anti-Tat serum (obtained through Dr B. Cullen, AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH; Hauber et al., 1987) and the monoclonal antibody against HIV-1 Tat (aa 1–9) (Advanced Biotechnologies) were diluted 1 : 1000.
Electrophoretic mobility-shift assays (EMSAs).
Nuclear extracts were obtained from Jurkat cells or from COS-7 cells transfected with pSH107-NFATc1, RSV-c-Jun and/or Tat-HA and gel retardation assays were performed as described previously (Navarro et al., 1998). The sequences of the oligonucleotides used as probes in EMSAs were: 5'-gatcGGAGGAAAAACTGTTTCATACAGAAGGCGT-3' (distal NFAT site of the human IL-2 promoter); 5'-gatcGCCCAAAGAGGAAAATTTGTTTCATACAG-3' (distal NFAT site of the murine IL-2 promoter); 5'-gatcATAAAATTTTCCAATGTAAA-3' (mouse P sequence of the IL-4 promoter); and 5'-CGCTTGATGAGTCAGCCGGAA-3' (AP-1 consensus oligonucleotide). The lower-case letters indicate the restriction enzyme site Bsp143I, which was added to the promoter sequence.
The identity of the retarded complexes was determined by supershift assays with specific anti-NFATc1 serum 676 (a gift from Dr J. M. Redondo), anti-c-Fos family sera (González et al., 2001) or anti-c-Jun (Santa Cruz Biotechnology).
Luciferase assays.
Transcriptional activity in Jurkat cells (2x106) was measured in reporter gene assays after transient transfection of cells with 0.5 µg luciferase reporter plasmid together with 10 ng pRL-tk-luc in a final volume of 1 ml OptiMem (Íñiguez et al., 2000). Cells were treated for 6 h and then harvested and lysed, and the luciferase activity was measured in a luminometer following the instructions in the Dual-luciferase Assay System kit (Promega). Data are represented in relative firefly luciferase units normalized by the relative Renilla luciferase units obtained in the control samples of each transfection (RLUff/ren). Every experiment was carried out in duplicate.
Immunoprecipitation assays.
For the immunoprecipitation assays, COS-7 cells were transfected with 5 µg pEF-BOS-NFATc2 and/or pCMV-Tat DNA as described previously (González et al., 2001). Cell extracts were separated by SDS-PAGE for Western blot detection of overexpressed proteins and the same extracts were used for the immunoprecipitation assays. Cellular extracts were supplemented with BSA (1 mg ml–1, essentially -globulin-free; Sigma Chemicals) and incubated overnight at 4 °C with anti-NFATc2 672 serum (1 : 200 dilution). Protein A–Sepharose (Sigma Chemicals) was added to the extracts and after 2 h of incubation at 4 °C, immune complexes were pelleted and washed five times with 15 ml lysis buffer each. Immunoprecipitates were analysed by Western blotting.
GST pull-down assays.
For GST pull-down assays, transfected COS-7 cells were lysed in ice-cold buffer [20 mM HEPES/KOH (pH 7.9), 120 mM KCl, 1 mM MgCl2, 17 % (v/v) glycerol, 1 mM EDTA, 0.25 % NP-40, 2 mM DTT, 0.5 mM PMSF, 2 µg pepstatin ml–1, 2 µg leupeptin ml–1, 2 µg aprotinin ml–1, 10 mM Na2MoO4] for 30 min. The extracts were pre-cleared with GST adsorbed to glutathione–Sepharose (Amersham Biosciences) before affinity chromatography with GST–Tat adsorbed to glutathione–Sepharose for 2 h at 4 °C. Complexes were washed four times in lysis buffer (without NP-40) and analysed by Western blotting.
All of the experiments shown are representative of at least three experiments performed in order to guarantee the reproducibility of the results.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
B transcription factors, which are involved in the regulation of cytokine transcription in T cells. Jurkat cells [either wild-type or stably expressing Tat (Jurkat-Tat+)] (González et al., 2001) (Fig. 1b) were transiently transfected with plasmids expressing the luciferase gene under the control of target sequences of these transcription factors. Transfected cells were then stimulated with PMA and Io alone or in combination. As expected, PMA increased NF-B- and AP-1-dependent promoters (Fig. 1a). Io alone did not induce the NF-B, AP-1 and NFAT/AP-1 sites, whereas it weakly activated the IL-4 promoter, which is dependent on a pure NFAT site (Aune & Flavell, 1997). Combined treatment with PMA/Io was required to activate the NFAT/AP-1 reporter. Next, we compared transcription induced by these stimuli in Jurkat cells stably expressing Tat with wild-type cells. As expected, in Tat-expressing cells, LTR transcription was strongly increased. Transcription dependent on NF-B or AP-1 sites was not significantly altered in Jurkat-Tat+ cells. In contrast, transcription dependent on the NFAT/AP-1 site was enhanced 
twofold in Jurkat-Tat+ cells after stimulating the cells with a potent stimulus such as PMA/Io (Fig. 1a). These differences were not due to altered expression of NFAT and c-Jun in Tat-expressing cells (Fig. 1b).
|
).
|
).
Next, we tested whether this effect of Tat was related to its ability to transactivate the LTR. For this, we transfected Jurkat cells with equal amounts of various plasmids encoding Tat mutants. Point mutations that drastically affected this Tat activity did not affect its ability to enhance NFAT/AP-1-dependent transcription in the presence of PMA/Io (Fig. 3). Thus, the Y26A mutation, which was completely inactive in activating HIV-1 LTR transcription (Verhoef & Berkhout, 1999), did not affect the ability of Tat to transactivate NFAT/AP-1 sites.
|
). Contamination of the nuclear extract with cytosol was excluded by testing these nuclear extracts by Western blot analysis with antibodies against the cytoplasmic protein marker lactate dehydrogenase (data not shown). In this regard, although the presence of a certain amount of NFATc2 in the nucleus of unstimulated Jurkat-Tat– and Jurkat-Tat+ cells is intriguing, similar observations have already been described in Raji and Jurkat cells in which partially dephosphorylated forms of NFATc2 were constitutively found in unstimulated cells (Park et al., 1995). A suggested explanation is that transformation-associated activation in cell lines may result in nuclear localization of NFAT. Taken together, the above results suggested that the effects of Tat were not mediated by alterations in the calcium-signalling pathway or in NFAT translocation to the nucleus.
|
) but not by PMA or Io alone (not shown) led to the formation of a specific complex. CsA inhibited the translocation of NFAT to the nucleus. Interestingly, the amount of complex bound was strongly increased (by fivefold, as determined by densitometric scanning) in Jurkat-Tat+ cells (Fig. 5a). Since this site is a composite element, we decided to investigate whether individual binding was being affected by Tat. Thus, we used oligonucleotides able to bind NFAT (corresponding to the NFAT site of the IL-4 promoter) or AP-1 exclusively in EMSAs. Interestingly, neither of these two sites showed an augmented binding in Jurkat-Tat+ cells when compared with Jurkat-Tat– cells, either in basal or in stimulated conditions (Fig. 5b and c).
|
). Thus, when Jurkat cells were treated with Io, which only activates NFAT, the induced binding of NFAT to the probe was similar in nuclear extracts of stimulated Jurkat-Tat– and Jurkat-Tat+ cells (Fig. 5d). However, when Io was used in combination with PMA (which activates AP-1), NFAT could bind to this probe alone or combined with AP-1, forming two different complexes that migrated differently and could be identified with specific antibodies. Interestingly, in Jurkat-Tat+ cells, only the formation of the slower-migrating complex (NFAT/AP-1) was strongly increased (by 20-fold by densitometric scanning) (Fig. 5d). The most likely explanation is that Tat was interacting with either NFAT or AP-1 (or both), favouring their cooperative binding to these NFAT/AP-1 composite DNA elements.
To check whether similar effects of Tat were observed with NFATc1, another member of the NFAT family present in T lymphocytes, we transfected COS-7 cells with NFATc1 and c-Jun with or without Tat and performed EMSAs with the NFAT/AP-1 composite element of the human IL-2 promoter. COS-7 cells did not express NFAT (Fig. 6, lane 1); a complex with the NFAT/AP-1 probe was formed only when the RSV-c-Jun and pSH107-NFATc1 expression vectors were simultaneously transfected (Fig. 6, lane 2). Simultaneous Tat-HA transfection seemed to increase the amount of complex detected, which ran slightly slower (Fig. 6, lane 4). More interestingly, anti-HA (Tat) antibodies as well as anti-NFAT 676 serum (Fig. 6, lanes 5 and 6, respectively) supershifted the specific complex, whereas anti-c-Jun inhibited it (Fig. 6, lane 7). Addition of a control rabbit serum had no effect on the complexes (not shown). This suggests that a complex between NFAT, c-Jun and Tat may be formed on the DNA probe.
|
100-fold increase in the transactivation of the human IL-2 NFAT/AP-1 reporter gene (Fig. 7a). The co-expression of Tat together with NFATc2 increased the observed transcriptional induction further up to 440-fold over the basal level. Transfection of c-Jun, the main component of AP-1, was able to augment the activity of the reporter twofold and also that of NFAT by the same extent (210-fold over the basal level). Co-transfection of Tat modestly increased c-Jun activity (twofold). More interestingly, Tat synergistically increased with c-Jun and NFATc2 transactivation up to 2600-fold over the basal level (or 12-fold over optimal doses of NFAT and c-Jun together) (Fig. 7a). There were no significant differences in the amounts of various proteins in transfected cells that could explain these differences (Fig. 7b), thus supporting a strong cooperative effect of Tat with NFAT and c-Jun.
|
, lanes 2 and 3) and NFAT (Fig. 8a, lanes 4 and 5) were detected in COS-7 cells only when transfected with the corresponding expression plasmids. Antibodies against NFATc2 co-precipitated Tat only when both expression plasmids were co-transfected (Fig. 8b, lane 5) and non-specific binding of Tat to anti-NFATc2 was excluded, as no Tat protein was immunoprecipitated in the absence of NFATc2 (Fig. 8b, lanes 2 and 3). Control of Tat and NFATc2 migration in the gels and of the specificity of the antibodies were determined by running total extracts of COS-7 cells transfected with Tat or NFAT (Fig. 8b, lane C). To corroborate this further, we performed pull-down assays of COS-7 cells transfected with either c-Jun or NFATc2. GST–Tat pulled down NFAT but not c-Jun (Fig. 8c).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Stevenson, 2003). However, the underlying mechanisms are unclear. This may stem from the fact that Tat may exert some effects on HIV-1-infected cells as well as on neighbouring bystander cells. Tat may alter cytokine secretion at two levels: (i) by acting in neighbouring cells as a paracrine molecule, and (ii) by affecting transcription in cells infected by HIV-1. In order to investigate how intracellular Tat affects cytokine expression, we analysed its effects on the activity of several of the most important factors controlling cytokine transcription, NFAT, AP-1 and NF-B (Ghosh et al., 1998; Macián et al., 2001). A previous report showed enhanced transactivation of the NFAT/AP-1 composite element of the human IL-2 gene (Macián & Rao, 1999). Our results are in agreement with these results, but provide additional evidence of the underlying mechanism. Thus, we showed that the expression of HIV-1 Tat in T cells was able to increase strongly NFAT and c-Jun cooperative interactions, resulting in enhanced binding to DNA and transcriptional activity. In the Cox-2 promoter, containing a composite NFAT/AP-1 element (Íñiguez et al., 2000), the enhancing effect of Tat was severely diminished if either the NFAT or AP-1 site was mutated. Signal transduction events that led to either NFAT or AP-1 activation, including translocation of NFAT to the nucleus, were not affected by intracellular Tat expression, nor did Tat affect the amount of c-Jun and NFAT in these cells.
In Jurkat cells, a strong activation (210-fold) of the NFAT/AP-1 reporter was observed after overexpressing both NFATc2 and c-Jun. Despite this huge activation, Tat increased this transactivation further (up to 2600-fold). Taken together, these results suggest a mechanism involving direct interactions between Tat and the NFAT/AP-1 complex, which take place only at these composite elements. This hypothesis was supported further by: (i) the enhanced binding of the NFAT/AP-1 complex to the composite element in the IL-2 promoter observed in Jurkat-Tat+ cells compared with wild-type cells or after the addition of recombinant Tat to the nuclear extracts of Jurkat wild-type cells and in COS-7 cells overexpressing NFAT, c-Jun and Tat; (ii) the ability of anti-Tat antibodies to supershift or to prevent complexes bound to the NFAT/AP-1 DNA probe in COS-7 cells overexpressing NFAT, c-Jun and Tat. Moreover, the existence of a direct interaction between NFAT and Tat was demonstrated by co-immunoprecipitation assays in cellular extracts of COS-7 cells when both proteins were co-expressed. These data confirm those obtained previously by Macián & Rao (1999) using cellular extracts of HEK293 cells co-transfected with both expression plasmids. In addition, we found that GST–Tat pulled down NFAT but not c-Jun from transfected COS-7 cells. Although the direct NFAT–Tat interaction leads to increased transactivation in Gal4–NFAT assays (Macián & Rao, 1999), this could not explain satisfactorily the increased transactivation with the NFAT/AP-1 reporter plasmid, as these effects were less pronounced than those observed with the combined NFAT/AP-1 site. Moreover, this single interaction with NFAT was not sufficient to explain the increased binding of the NFAT/AP-1 complex to this composite element, as Tat does not affect the binding of NFAT to sequences where this transcription factor binds independently from AP-1. Therefore, we concluded that the NFAT–Tat interaction strongly potentiates its cooperation with AP-1.
Binding of AP-1 to the NFAT/AP-1 distal site of the human IL-2 gene promoter is strongly dependent on its interaction with NFAT. For NFATc1, this interaction takes place through amino acids near the N-terminal region (Peterson et al., 1996) where the interaction between NFATc2 and Tat has been localized (Macián & Rao, 1999). Therefore, we believe that Tat interaction with NFAT may affect NFAT–AP-1 contacts and, subsequently, binding of the complex to DNA. In addition, the NFAT–AP-1 interaction at this composite element differs from other protein–protein interactions by its marked electrostatic character (Sun et al., 1997) and Tat has strongly charged regions (Truant & Cullen, 1999). Consequently, alteration of electrostatic interactions in the NFAT/AP-1 complex as a result of the binding of Tat to NFAT could be a possible mechanism to explain the observed effects.
We did not detect alterations by transient or stable expression of Tat either in the binding or in the transactivation activities of AP-1. HIV-1 Tat was not able to interact physically with c-Jun, nor to potentiate its transactivating activity (not shown) or affect AP-1 binding. In spite of this, Tat may interact with AP-1 through interaction with other transcription factors. In this regard, Lim & Garzino-Demo (2000) found that Tat interaction with Sp1 at the human MCP-1 gene promoter may serve as a platform to recruit and stabilize the interaction of AP-1 and NF-B proteins to this promoter. In addition, HIV-1 Tat can activate the Jun kinase enzyme (JNK), which is required for AP-1 activation (Biswas et al., 1995; Li et al., 1997; Kumar et al., 1998). However, these effects were observed with extracellular Tat and Tat-neutralizing antibodies inhibited JNK activation. In our experiments, Tat secretion by Jurkat-Tat+ cells could be insufficient to achieve the extracellular concentration required to act on neighbouring cells and affect JNK activation.
The selective effect of Tat on DNA target sequences where NFAT and AP-1 bind cooperatively may have important functional implications, as many genes (e.g. IL-2, Cox-2, IL-4, IL-5, CD40L, GM-CSF) are regulated by NFAT/AP-1 composite elements with defined structural requirements (Kel et al., 1999; Macián et al., 2001). The distal NFAT/AP-1 site of the human IL-2 promoter is a representative example of nuclear factor cooperation; NFAT increases the affinity of AP-1 10-fold (Peterson et al., 1996) and the NFAT/AP-1/DNA complex is 10 times more stable than the NFAT/DNA complex (Jain et al., 1993). In this way, the cooperative binding of both transcription factors allows the complete function of this response element. The effect of Tat on the cooperative interactions between such transcription factors and DNA may represent a tuned HIV-1 strategy to regulate the expression of certain genes depending on those composite elements that are critically involved in the immune response, such as IL-2 and the above-mentioned proteins. In addition, our results show that these effects take place at very low levels of expression of the viral protein in the cell, close to the physiological situation. Interestingly, these effects of Tat do not seem to be related to its ability to transactivate the LTR. Thus, theoretically HIV-1 strains partly defective in active virus replication could still modulate NFAT/AP-1 transcription and produce cytokine dysregulation.
Moreover, aside from the impact shown at the cellular level, the described mechanism may play a feedback role in the level of viral transcription in a subtype-specific manner. Indeed, in the HIV-1 LTR, enhancer NFAT (NF-B) and AP-1 binding sites are represented and spaced differently, depending on the viral subtype being considered (Jeeninga et al., 2000; van Opijnen et al., 2004; Centlivre et al., 2005). Thus, the enhancement induced by Tat on the cooperation between NFAT and AP-1 might be relevant for some major subtypes (e.g. A and C) and irrelevant for others (e.g. B).
Moreover, NFAT is rarely able to bind alone to DNA. Rather, it usually forms cooperative interactions with other factors besides AP-1 (Hogan et al., 2003; Macián et al., 2001). Thus, interactions with C/EBP in the peroxisome proliferator activated receptor and insulin-like growth factor promoters (Yang & Chow, 2003) or with IRF-8 in the IL-12 promoter (Zhu et al., 2003) have been described. Tat may modulate (potentiate or inhibit) these interactions by interacting with NFAT. Experiments are in progress to elucidate this. Thus, studying the effects of Tat on synergistic interactions among transcription factors is becoming increasingly relevant to understand fully its physiological role.
In summary, we have shown that Tat favours AP-1 interaction with NFAT. These data contribute to a more complete and complex vision of transcriptional regulation by Tat and are encouraging, bearing in mind that the functional activity of this viral protein may depend on the interactions established around diverse DNA-response elements.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
and Tat modulates MCP-1 gene transcription in astrocytes. J Neuroimmunol 160, 219–227.[CrossRef][Medline]Ambrosino, C., Ruocco, M. R., Chen, X., Mallardo, M., Baudi, F., Trematerra, S., Quinto, I., Venuta, S. & Scala, G. (1997). HIV-1 Tat induces the expression of the interleukin-6 (IL6) gene by binding to the IL6 leader RNA and by interacting with CAAT enhancer-binding protein (NF-IL6) transcription factors. J Biol Chem 272, 14883–14892.
Ambrosino, C., Palmieri, C., Puca, A. & 9 other authors (2002). Physical and functional interaction of HIV-1 Tat with E2F-4, a transcriptional regulator of mammalian cell cycle. J Biol Chem 277, 31448–31458.
Arenzana-Seisdedos, F., Fernandez, B., Dominguez, I., Jacque, J. M., Thomas, D., Diaz-Meco, M. T., Moscat, J. & Virelizier, J. L. (1993). Phosphatidylcholine hydrolysis activates NF-B and increases human immunodeficiency virus replication in human monocytes and T lymphocytes. J Virol 67, 6596–6604.
Aune, T. M. & Flavell, R. A. (1997). Differential expression of transcription directed by a discrete NF-AT binding element from the IL-4 promoter in naive and effector CD4 T cells. J Immunol 159, 36–43.[Abstract]
Biswas, D. K., Salas, T. R., Wang, F., Ahlers, C. M., Dezube, B. J. & Pardee, A. B. (1995). A Tat-induced auto-up-regulatory loop for superactivation of the human immunodeficiency virus type 1 promoter. J Virol 69, 7437–7444.[Abstract]
Blazquez, M. V., Macho, A., Ortiz, C., Lucena, C., Lopez-Cabrera, M., Sanchez-Madrid, F. & Munoz, E. (1999). Extracellular HIV type 1 Tat protein induces CD69 expression through NF-kappaB activation: possible correlation with cell surface Tat-binding proteins. AIDS Res Hum Retroviruses 15, 1209–1218.[CrossRef][Medline]
Buonaguro, L., Barillari, G., Chang, H. K., Bohan, C. A., Kao, V., Morgan, R., Gallo, R. C. & Ensoli, B. (1992). Effects of the human immunodeficiency virus type 1 Tat protein on the expression of inflammatory cytokines. J Virol 66, 7159–7167.
Centlivre, M., Sommer, P., Michel, M. & 8 other authors (2005). HIV-1 clade promoters strongly influence spatial and temporal dynamics of viral replication in vivo. J Clin Invest 115, 348–358.[CrossRef][Medline]
Cullen, B. R. (1998). HIV-1 auxiliary proteins: making connections in a dying cell. Cell 93, 685–692.[CrossRef][Medline]
Demarchi, F., Gutierrez, M. I. & Giacca, M. (1999). Human immunodeficiency virus type 1 Tat protein activates transcription factor NF-B through the cellular interferon-inducible, double-stranded RNA-dependent protein kinase, PKR. J Virol 73, 7080–7086.
Deng, T. & Karin, M. (1993). JunB differs from c-Jun in its DNA-binding and dimerization domains and represses c-Jun by formation of inactive heterodimers. Genes Dev 7, 479–490.
Durand, D. B., Shaw, J. P., Bush, M. R., Replogle, R. E., Belagaje, R. & Crabtree, G. R. (1988). Characterization of antigen receptor response elements within the interleukin-2 enhancer. Mol Cell Biol 8, 1715–1724.
Frankel, A. D. & Pabo, C. O. (1988). Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189–1193.[CrossRef][Medline]
Ghosh, S., May, M. J. & Kopp, E. B. (1998). NF-B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16, 225–260.[CrossRef][Medline]
González, E., Punzón, C., González, M. & Fresno, M. (2001). HIV-1 Tat inhibits IL-2 gene transcription through qualitative and quantitative alterations of the cooperative Rel/AP1 complex bound to the CD28RE/AP1 composite element of the IL-2 promoter. J Immunol 166, 4560–4569.
Hauber, J., Perkins, A., Heimer, E. P. & Cullen, B. R. (1987). Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc Natl Acad Sci U S A 84, 6364–6368.
Hogan, P. G., Chen, L., Nardone, J. & Rao, A. (2003). Transcriptional regulation by calcium, calcineurin and NFAT. Genes Dev 17, 2205–2232.
Im, S.-H. & Rao, A. (2004). Activation and deactivation of gene expression by Ca2+/calcineurin-NFAT-mediated signaling. Mol Cells 18, 1–9.[Medline]
Íñiguez, M. A., Martinez-Martínez, S., Punzón, C., Redondo, J. M. & Fresno, M. (2000). An essential role of the nuclear factor of activated T cells in the regulation of the expression of the cyclooxygenase-2 gene in human T lymphocytes. J Biol Chem 275, 23627–23635.
Izmailova, E., Bertley, F. M. N., Huang, Q., Makori, N., Miller, C. J., Young, R. A. & Aldovini, A. (2003). HIV-1 Tat reprograms immature dendritic cells to express chemoattractants for activated T cells and macrophages. Nat Med 9, 191–197.[CrossRef][Medline]
Jain, J., Miner, Z. & Rao, A. (1993). Analysis of the preexisting and nuclear forms of nuclear factor of activated T cells. J Immunol 151, 837–848.[Abstract]
Jeang, K.-T., Chun, R., Lin, N. H., Gatignol, A., Glabe, C. G. & Fan, H. (1993). In vitro and in vivo binding of human immunodeficiency virus type 1 Tat protein and Sp1 transcription factor. J Virol 67, 6224–6233.
Jeang, K.-T., Xiao, H. & Rich, E. A. (1999). Multifaceted activities of the HIV-1 transactivator of transcription, Tat. J Biol Chem 274, 28837–28840.
Jeeninga, R. E., Hoogenkamp, M., Armand-Ugon, M., de Baar, M., Verhoef, K. & Berkhout, B. (2000). Functional differences between the long terminal repeat transcriptional promoters of human immunodeficiency virus type 1 subtypes A through G. J Virol 74, 3740–3751.
Johnson, B. V., Bert, A. G., Ryan, G. R., Condina, A. & Cockerill, P. N. (2004). Granulocyte-macrophage colony-stimulating factor enhancer activation requires cooperation between NFAT and AP-1 elements and is associated with extensive nucleosome reorganization. Mol Cell Biol 24, 7914–7930.
Karin, M., Liu, Z. & Zandi, E. (1997). AP-1 function and regulation. Curr Opin Cell Biol 9, 240–246.[CrossRef][Medline]
Kel, A., Kel-Margoulis, O., Babenko, V. & Wingender, E. (1999). Recognition of NFATp/AP-1 composite elements within genes induced upon the activation of immune cells. J Mol Biol 288, 353–376.[CrossRef][Medline]
Kumar, A., Manna, S. K., Dhawan, S. & Aggarwal, B. B. (1998). HIV-Tat protein activates c-Jun N-terminal kinase and activator protein-1. J Immunol 161, 776–781.
Li, C. J., Ueda, Y., Shi, B., Borodyansky, L., Huang, L., Li, Y.-Z. & Pardee, A. B. (1997). Tat protein induces self-perpetuating permissivity for productive HIV-1 infection. Proc Natl Acad Sci U S A 94, 8116–8120.
Lim, S. P. & Garzino-Demo, A. (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, AP1 and NF-B consensus sites. J Virol 74, 1632–1640.
Macián, F. & Rao, A. (1999). Reciprocal modulatory interaction between human immunodeficiency virus type 1 Tat and transcription factor NFAT1. Mol Cell Biol 19, 3645–3653.
Macián, F., López-Rodríguez, C. & Rao, A. (2001). Partners in transcription: NFAT and AP-1. Oncogene 20, 2476–2489.[CrossRef][Medline]
Navarro, J., Punzón, C., Jiménez, J. L., Fernández-Cruz, E., Pizarro, A., Fresno, M. & Muñoz-Fernández, M. A. (1998). Inhibition of phosphodiesterase type IV suppresses human immunodeficiency virus type 1 replication and cytokine production in primary T cells: involvement of NF-B and NFAT. J Virol 72, 4712–4720.
Nolan, G. P. (1994). NF-AT-AP-1 and Rel-bZIP: hybrid vigor and binding under the influence. Cell 77, 795–798.[CrossRef][Medline]
Ott, M., Lovett, J. L., Mueller, L. & Verdin, E. (1998). Superinduction of IL-8 in T cells by HIV-1 Tat protein is mediated through NF-B factors. J Immunol 160, 2872–2880.
Park, J., Yaseen, N. R., Hogan, P. G., Rao, A. & Sharma, S. (1995). Phosphorylation of the transcription factor NFATp inhibits its DNA binding activity in cyclosporin A-treated human B and T cells. J Biol Chem 270, 20653–20659.
Peterson, B. R., Sun, L. J. & Verdine, G. L. (1996). A critical arginine residue mediates cooperativity in the contact interface between transcription factors NFAT and AP-1. Proc Natl Acad Sci U S A 93, 13671–13676.
Rowland-Jones, S. L. (2003). Timeline: AIDS pathogenesis: what have two decades of HIV research taught us? Nat Rev Immunol 3, 343–348.[CrossRef][Medline]
Schwartz, O., Virelizier, J.-L., Montagnier, L. & Hazan, U. (1990). A microtransfection method using the luciferase-encoding reporter gene for the assay of human immunodeficiency virus LTR promoter activity. Gene 88, 197–205.[CrossRef][Medline]
Shaw, J. P., Utz, P. J., Durand, D. B., Toole, J. J., Emmel, E. A. & Crabtree, G. R. (1988). Identification of a putative regulator of early T cell activation genes. Science 241, 202–205.
Stevenson, M. (2003). HIV-1 pathogenesis. Nat Med 9, 853–860.[CrossRef][Medline]
Sun, L. J., Peterson, B. R. & Verdine, G. L. (1997). Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1. Proc Natl Acad Sci U S A 94, 4919–4924.
Truant, R. & Cullen, B. R. (1999). The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin -dependent nuclear localization signals. Mol Cell Biol 19, 1210–1217.
Vacca, A., Farina, M., Maroder, M., Alesse, E., Screpanti, I., Frati, L. & Gulino, A. (1994). Human immunodeficiency virus type-1 TAT enhances interleukin-2 promoter activity through synergism with phorbol ester and calcium-mediated activation of the NF-AT cis-regulatory motif. Biochem Biophys Res Commun 205, 467–474.[CrossRef][Medline]
van Opijnen, T., Jeeninga, R. E., Boerlijst, M. C., Pollakis, G. P., Zetterberg, V., Salminen, M. & Berkhout, B. (2004). Human immunodeficiency virus type 1 subtypes have a distinct long terminal repeat that determines the replication rate in a host-cell-specific manner. J Virol 78, 3675–3683.
Verhoef, K. & Berkhout, B. (1999). A second-site mutation that restores replication of a Tat-defective human immunodeficiency virus. J Virol 73, 2781–2789.
Weiss, C., Schneider, S., Wagner, E. F., Zhang, X., Seto, E. & Bohmann, D. (2003). JNK phosphorylation relieves HDAC3-dependent suppression of the transcriptional activity of c-Jun. EMBO J 22, 3686–3695.[CrossRef][Medline]
Westendorp, M. O., Li-Weber, M., Frank, R. W. & Krammer, P. H. (1994). Human immunodeficiency virus type 1 Tat upregulates interleukin-2 secretion in activated T cells. J Virol 68, 4177–4185.
Yang, T. T. C. & Chow, C.-W. (2003). Transcription cooperation by NFAT.C/EBP composite enhancer complex. J Biol Chem 278, 15874–15885.
Yang, Y., Dong, B., Mittelstadt, P. R., Xiao, H. & Ashwell, J. D. (2002). HIV Tat binds Egr proteins and enhances Egr-dependent transactivation of the Fas ligand promoter. J Biol Chem 277, 19482–19487.
Zagury, D., Lachgar, A., Chams, V. & 10 other authors (1998). Interferon and Tat involvement in the immunosuppression of uninfected T cells and C-C chemokine decline in AIDS. Proc Natl Acad Sci U S A 95, 3851–3856.
Zauli, G., Davis, B. R., Re, M. C., Visani, G., Furlini, G. & La Place, M. (1992). tat protein stimulates production of transforming growth factor-1 by marrow macrophages: a potential mechanism for human immunodeficiency virus-1-induced hematopoietic suppression. Blood 80, 3036–3043.
Zhu, C., Rao, K., Xiong, H., Gagnidze, K., Li, F., Horvath, C. & Plevy, S. (2003). Activation of the murine interleukin-12 p40 promoter by functional interactions between NFAT and ICSBP. J Biol Chem 278, 39372–39382.
Received 25 October 2005;
accepted 27 January 2006.
This article has been cited by other articles:
|
|
 |
|
  A. Blanco, S. Alvarez, M. Fresno, and M. A. Munoz-Fernandez Extracellular HIV-Tat Induces Cyclooxygenase-2 in Glial Cells through Activation of Nuclear Factor of Activated T Cells J. Immunol., January 1, 2008; 180(1): 530 - 540. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||
