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IMMUNOBIOLOGY
From the Centre de Recherche en Infectiologie, Centre
Hospitalier Universitaire de Québec, Pavillon CHUL, and
Département de Biologie médicale, Faculté de
Médecine, Université Laval, Ste-Foy, QC, Canada.
Although protein tyrosine phosphatase (PTP) inhibitors used in
combination with other stimuli can induce interleukin 2 (IL-2) production in T cells, a direct implication of nuclear factor of
activated T cells (NFAT) has not yet been demonstrated. This study
reports that exposure of leukemic T cells and human peripheral blood
mononuclear cells to bis-peroxovanadium (bpV) PTP inhibitors markedly
induce activation and nuclear translocation of NFAT. NFAT activation by
bpV was inhibited by the immunosuppressive drugs FK506 and cyclosporin
A, as well as by a specific peptide inhibitor of NFAT activation.
Mobility shift assays showed specific induction of the NFAT1 member by
bpV molecules. The bpV-mediated NFAT activation was observed to be
important for the up-regulation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) and the IL-2 promoter; NFAT1 was
demonstrated to be particularly important in bpV-dependent positive
action on HIV-1 LTR transcription. The active participation of
p56lck, ZAP-70, p21ras,
and calcium in the bpV-mediated signaling cascade leading to NFAT
activation was confirmed, using deficient cell lines and dominant-negative mutants. Finally, overexpression of wild-type SHP-1
resulted in a greatly diminished activation of NFAT by bpV, suggesting
an involvement of SHP-1 in the regulation of NFAT activation. These
data were confirmed by constitutive NFAT translocation observed in
Jurkat cells stably expressing a dominant-negative version of SHP-1.
The study proposes that PTP activity attenuates constitutive kinase
activities that otherwise would lead to constant NFAT activation and
that this activation is participating in HIV-1 LTR stimulation by PTP inhibition.
(Blood. 2001;97:2390-2400) T-cell activation results in the induction of
several genes that play crucial roles for the subsequent functions that
these cells accomplish in the immune system. The induction of different types of transcription factors is responsible for the transcriptional activation of these immune response genes. Paradoxically, these same
factors positively modulate human immunodeficiency virus type-1 (HIV-1)
replication, the causal agent of acquired immune deficiency syndrome
(AIDS).1 In fact, HIV-1 transcription is very intimately
linked to T-cell activation, due to the overlapping of the signal
transduction requirement between T-cell lymphokine gene expression and
HIV-1 long terminal repeat (LTR) transactivation.2,3 This
is consequential to the HIV-1 LTR architecture sharing many different
motifs found in regulatory regions of genes induced following T-cell
activation.4 One of the factors binding to these motifs is
the well-known nuclear factor-kappa B (NF- The nuclear factor of activated T cells (NFAT) is another family of
Rel-related transcription factors known to be activated early in time
following T-cell activation. Several NFAT family members are present in
human T cells, such as NFAT1, NFAT2, and NFAT4.8 NFAT
factors are generally sequestered in the cytoplasm and translocated to
the nucleus on an increase in intracellular calcium
content.9 Modulation of intracellular calcium triggers conformational changes in calmodulin and increases its binding to the
calcineurin serine/threonine phosphatase, leading in turn to its
activation. The ensuing NFAT dephosphorylation by calcineurin renders
the nuclear-localizing sequence accessible, allowing nuclear translocation.9 This mechanistic description of the
activation steps of NFAT has also been well established by the
demonstration that immunosuppressor drugs cyclosporin A (CsA) and FK506
both inhibited calcineurin phosphatase activity.10,11 NFAT
often associates with the newly synthesized AP-1 complex, thereby
acquiring high transactivating potential on binding to its consensus
sequence 5'-(T/A)GGAAA(A/N)(A/T/C)-3'.9 Certain
NFAT-binding sites suggested to be cooperative with AP-1 are known to
resemble NF- Activation of T cells is a very complex process that involves
cell-to-cell interactions of several cell surface molecules. Although
multimerization of the T-cell receptor (TCR) induces a cascade of
intracellular events, optimal T-cell activation requires signaling
through other co-receptors.14 Several second messengers modulate signals induced from the membrane to the nucleus, but one
common theme is an increase in intracellular tyrosine phosphorylation levels.15 Different studies have shown the importance of
this increase in phosphotyrosine levels, which initially depends on 2 specific protein tyrosine kinases (PTKs), p56lck and
p59fyn.16,17 Another important PTK
involved in the TCR-initiated cascade includes ZAP-70.18 A
balance between PTK and protein tyrosine phosphatase (PTP) activities
controls intracellular tyrosine phosphorylation levels.19
PTPs are therefore very important modulators of the T-cell activation
cascade generally being considered as inhibitors of T-cell
activation.20 This has been clearly indicated by studies
of the PTP SHP-1.21 In fact, inhibition of PTP in the T
cell induces cellular activation and IL-2 production.22,23 PTPs also play an important role in the activation of the NF- We were thus interested in defining the cellular factor responsible for
the NF- Cell lines
Preparation of PBMCs
Plasmids and antibodies The plasmids pLTR-LUC (wild-type LTR) and pm BLTR-LUC
(NF-B-mutated LTR) were provided by Dr K. L. Calame (Columbia
University, New York, NY).37 The pB-TATA-LUC plasmid
(Dr W. C. Greene, The J. Gladstone Institutes, San Francisco, CA)
contains the HIV-1 B enhancer region and a TATA box upstream of the
luciferase reporter gene.38 pIL-2-LUC and pNFAT-LUC
contain the complete 320-base pair (bp) IL-2 promoter and the minimal
IL-2 promoter with 3 tandem copies of the NFAT1-binding site,
respectively (Dr G. Crabtree, Stanford University Medical
School).39 The pLTR/N17 plasmid coding for the
dominant-negative mutant p21rasN17 under the
control of murine leukemia virus LTR was obtained from Dr John Telford
(Immunobiological Research Institute, Siena, Italy).40 The
human p56lck-encoding pEFneoLckWT and the empty
pEFneo vectors41 were obtained from Dr Clement Couture
(Lady Davis Institute, Montreal, Canada). pcDNA3-dnNFAT expresses a
dominant-negative NFAT mutant (Dr Roger J. Davis, University of
Massachusetts Medical School, Worcester, MA).42 The
NFAT-inhibiting pVIVIT-GFP expression vector and the empty
vector43 were supplied by Dr Anjana Rao (Harvard Medical School, Boston, MA). The VIVIT-GFP fragment was excised from this vector and cloned in the pRc/ -actin vector with HindIII/NotI restriction sites (pRc/-actin VIVIT-GFP). Vectors encoding for wild-type or dominant-negative (C453S) SHP-1 plus the vector pSFFVneo were provided by Dr Matthew L. Thomas (Washington University School of
Medicine).36 Vectors encoding for wild-type and
kinase-dead ZAP-70, plus the empty vector,44 were obtained
from Dr Arthur Weiss (Howard Hughes Medical Center). The pREP4-NFAT1
and pREP4-NFAT2 expression vectors along with the pREP4 vector were
supplied by Dr Tim Hoey (Tularik, San Francisco, CA). Rabbit antisera
specific for NFAT1, NFAT4, or all NFAT members (panNFAT)8
were obtained from Dr Nancy Rice (National Cancer Institute, Frederick,
MD). Anti-NFAT2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies from the anti-CD3 OKT3 hybridoma were
purified with mAbTrap protein G affinity columns (Pharmacia LKB
Biotechnology AB, Uppsala, Sweden). Purified anti-CD28 antibodies (clone 9.3) were given by Dr Jeffrey A. Ledbetter (Bristol-Myers Squibb, Seattle, WA).45
Transfections and reporter gene assays Transient transfections of T-cell lines using the DEAE-Dextran method were performed as previously described.26 Electroporation of PBMCs was conducted according to the protocol of Hughes and Pober.46 Stably transfected Jurkat cells were obtained by electroporation. Briefly, 107 cells were transfected in 400 µL of medium with 20 µg pNFAT-LUC or pIL-2-LUC (250 V and 960 µF). After 24 hours, cells were diluted at 5 × 104 cells/mL in the presence of 1 mg/mL G418. Pooled G418-resistant cells were identified as either J-NFAT-LUC or J-IL-2-LUC. Transient transfection of 293T cells were performed by a calcium phosphate transfection protocol. Stably or transiently transfected cells were next seeded at a density of 105 cells/well in 96-well plates and left unstimulated or treated for 8 hours (unless specified) with bpV[HOpic], bpV[bipy], bpV[pic], PHA-P, phorbol 12-myristate 13-acetate (PMA; Sigma), ionomycin (Iono; Calbiochem), anti-CD3 antibody (clone OKT3), or anti-CD28 antibody (clone 9.3) in a final volume of 200 µL. For some experiments, prior to activation, cells were pretreated with FK506 (Fujisawa, Osaka, Japan) or CsA (Sigma) for 15 minutes at 37°C or with sodium salicylate (NaSal; Laboratories Mat, Beauport, QC, Canada) for 1 hour at 37°C. Cell viability was estimated by the (3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.47 Luciferase activity was determined with a Dynex 96-well plate luminometer device (Chantilly, VA).26IL-2 production Following a 24-hour stimulation, supernatants from J-IL-2-LUC cells were removed, and quantification of secreted IL-2 was carried out, using a commercial cytokine-matched antibody pair enzyme-linked immunosorbent assay (ELISA) kit (Endogen, Woburn, MA).Preparation of nuclear extracts and electrophoretic mobility shift assay Jurkat cells or PBMCs were left untreated or incubated for 1 hour at 37°C with bpV[pic] or PMA/Iono. Cells were next washed with ice-cold PBS, and nuclear extracts were prepared according to the microscale preparation protocol.48 Electrophoretic mobility shift assay (EMSA) was performed as previously described,26 using the following double-stranded oligonucleotides: the consensus NFAT-binding site from the murine IL-2 promoter (5'-TCGAGCCCAAAGAGGAAAATTTGTTTCATG-3'), the consensus NF-B-binding site (5'-ATGTGAGGGGACTTTCCCAGGC-3'), the wild-type
HIV-1 enhancer (5'-CAAGGGACTTTCCGCTGGGGACTTTCCAGGG-3'), and the
B-mutated HIV-1 enhancer
(5'-CAACTCACTTTCCGCTGCTCACTTTCCAGGG-3'). Supershift assays were performed by preincubation of nuclear extracts with 1 µL antibody for 30 minutes on ice.
bpV induction of wild-type and NF- B
translocation49 led to a partial loss of the bpV-dependent
induction of HIV-1 LTR-driven luciferase activity (Figure 1A). Because
PHA acts mainly on NFAT,27 NaSal did not affect
PHA-mediated LTR activation; however, FK506 and CsA both inhibited this
induction of the LTR. Surprisingly, FK506 and CsA also blocked
bpV-mediated induction of HIV-1 LTR activity. Furthermore, when either
FK506 or CsA was added in combination with NaSal, greater inhibition of
HIV-1 LTR activity was apparent in bpV-treated cells. No detectable
toxicity was measured on cell incubation with any combination of
these inhibitors (data not shown). Inability to completely inhibit
bpV-mediated HIV-1 LTR activation by the different combinations is
probably due to residual NF-B activity. The bpV molecule bpV[pic]
was used for subsequent experiments, being representative of the
other bpV compounds.
Jurkat cells were tested for a bpV[pic] response with the
p NFAT-dependent luciferase expression is markedly stimulated by the bpV compound Because NFAT was the obvious candidate in bpV[pic] activation, transfections were performed in Jurkat cells with pNFAT-LUC containing 3 tandem repeats of the human IL-2-derived NFAT-binding site, the human IL-2 minimal promoter, and the luciferase reporter gene. A strong induction of NFAT-dependent luciferase activity was mediated by the PHA/PMA combination (Figure 2A). Interestingly, a more potent induction was observed on bpV[pic] stimulation of transfected Jurkat cells. Time kinetic analyses showed a transient increase in luciferase activity that peaked from 6 to 8 hours for PHA/PMA (53.5-fold and 61.1-fold increase), whereas maximal activation was reached during the 8- to 12-hour period for bpV[pic] (92.2-fold and 73.2-fold increase). For the subsequent experiments, the 8-hour time frame was chosen for treatment of transfected cell lines. Similar analyses were performed with freshly isolated PBMCs electroporated with pNFAT-LUC and showed bpV[pic]-mediated NFAT activation (albeit at a lower extent than in PMA/Iono-treated PBMCs) (Figure 2B). In addition, time kinetics were different in PBMCs in comparison to Jurkat cells, showing a much slower and sustained induction.
Stably pNFAT-LUC-transfected Jurkat cells (J-NFAT-LUC) were also tested with the various NFAT inducers with or without FK506 pretreatment. J-NFAT-LUC cells were highly responsive to PHA in the absence or presence of PMA (48.9- and 72.1-fold, respectively), whereas PMA alone had no effect (Figure 2C). An important induction of luciferase activity in J-NFAT-LUC cells was observed with bpV[pic] (113.3-fold) and was further enhanced in the presence of PMA (208.1-fold). After pretreatment with subcytotoxic FK506 concentrations, NFAT activation was abrogated for all tested agents, including bpV[pic]. Finally, to confirm NFAT activation by bpV[pic], Jurkat cells were transfected with pNFAT-LUC and a vector that encodes a GFP protein fused to VIVIT, a highly specific NFAT-inhibiting peptide.43 The expression of this NFAT inhibitor caused a strong inhibition of NFAT activation by bpV[pic] and the other agents (Figure 2D). Similar inhibitions of NFAT activation were also obtained when the dominant-negative mutant dnNFAT42 was instead transfected in Jurkat cells (data not shown). These results hence demonstrated for the first time that PTP inhibitors directly activate NFAT in a more pronounced fashion in comparison to frequently used NFAT activators. Nuclear translocation of NFAT by bpV[pic] Nuclear translocation of NFAT induced by bpV[pic] was evaluated by EMSA. A strong NFAT-specific signal was obtained with PMA/Iono-treated nuclear extracts (Figure 3A, lane 2). bpV[pic] also led to the induction of a strong signal, which was competed by increasing concentrations of unlabeled NFAT oligonucleotide (compare lane 3 with lanes 4-6) but not by excess of unlabeled NF-B oligonucleotide (lane
7). For identification of this signal, bpV-treated Jurkat nuclear
extracts were preincubated with NFAT member-specific antibodies. The
signal induced by bpV[pic] was completely abrogated by the addition
of anti-NFAT1 antibodies with a concomitant supershift (Figure 3B,
lanes 2 and 3). No such effect could be observed with anti-NFAT2 or
anti-NFAT4 antibodies (lanes 4 and 5). The presence of a weak
supershift in anti-NFAT2-incubated nuclear extracts might, however, be
reminiscent of a minimal representation of this member in the shifted
complex. The fast migrating band in these EMSA experiments likely
represented degraded NFAT.
EMSAs were also performed with freshly isolated PBMCs or PHA/IL-2-stimulated PBMCs rested for 3 days before bpV treatment. Treatment of starved or fresh PBMCs with bpV[pic] resulted in nuclear translocation of NFAT (Figure 3C, lanes 2 and 9). Specificity of the signal was demonstrated through competition experiments (data not shown). Supershift analyses again indicated specific supershifting with anti-NFAT1 and panNFAT (recognizing all NFAT members) sera in both PBMC nuclear extracts (Figure 3C, lanes 4, 7, 11, and 14). The remaining fast migrating band observed in lane 4 might be reminiscent of bpV[pic] induction of another NFAT member that would be induced in PBMCs, but its identification has yet to be determined. Overall, these experiments suggested that bpV[pic] activates NFAT through its nuclear translocation in both Jurkat and PBMCs and that most of these translocated factors consist of NFAT1. NFAT is important for HIV-1 LTR activation by bpV[pic] Because NFAT might be an important factor in bpV[pic]-mediated HIV-1 LTR activation (Figure 1), this assumption was directly tested with the VIVIT NFAT inhibitor. However, to avoid a potential quenching effect on the activation of the HIV-1 LTR from the NF-B-binding sites present in the cytomegalovirus promoter of the original plasmid,
the VIVIT-GFP fragment was cloned upstream of the -actin promoter
(pRc/-actin VIVIT-GFP). In fact, the addition of pRc/-actin had
little effect on the activation of the HIV-1 LTR by bpV compound when
compared to Jurkat cells transfected with pLTR-LUC alone (26-fold
compared to 24-fold) (data not shown). Jurkat cells were thus
transfected with pLTR-LUC along with the empty pRc/-actin or
pRc/-actin VIVIT-GFP vectors. Data showed that the
NF-B-activating PMA agent was not affected by the addition of the
VIVIT-GFP expression vector, whereas this vector led to a near 60%
decrease in luciferase activity in both bpV[pic]- and PHA-stimulated
cells (Figure 4A).
To corroborate these results, EMSA analyses were performed, using an
HIV-1 enhancer probe. A specific broad signal was observed with
extracts from bpV[pic]-stimulated Jurkat cells (Figure 4B, lane 1).
Because it seemed possible that this signal resulted from
superimposition of NFAT and NF- Because this NFAT member has previously been shown to act negatively on HIV-1 LTR activity,50 NFAT1 was directly tested for its LTR-activating potential by co-transfecting 293T cells with pLTR-LUC and NFAT1 or NFAT2 expression vectors (Figure 4C). LTR activity was increased by both NFAT1 and NFAT2, and, importantly, this induction was more pronounced in NFAT1-transfected cells (10.3- versus 3-fold). These data hence suggested that NFAT1 expression can up-regulate HIV-1 LTR activity and is important in the induction of the HIV-1 LTR by our PTP inhibitor. bpV molecules in combination with other agents activate
transcription of the IL-2 promoter and IL-2 production. The pervanadate PTP inhibitor has previously been shown to up-regulate the expression of IL-2 in the presence of other activators.22-24,51
Because IL-2 transcription is dependent on NFAT-binding sites, we
wanted to determine whether bpV[pic] could equally induce IL-2 gene
expression in an NFAT-dependent manner. As demonstrated in Figure
5A, PHA/PMA treatment of
pIL-2-LUC-transfected Jurkat cells revealed a maximal 3-fold
induction of luciferase activity. Importantly, bpV[pic] in
combination with PMA could equally activate IL-2 promoter-dependent luciferase activity (5.4-fold). Neither PHA, PMA, nor bpV[pic] alone
resulted in IL-2 promoter activation in this transient transfection system (data not shown). The kinetic of luciferase induction observed with bpV[pic]/PMA was fairly similar to PHA/PMA, except that maximal luciferase induction reached its plateau between 8 and 12 hours. In
stably pIL-2-LUC-transfected Jurkat cells (J-IL-2-LUC), PHA/PMA- and bpV[pic]/PMA-mediated activation of the IL-2 promoter were both
FK506 sensitive (Figure 5B).
Various combinations of activators were next tested on J-IL-2-LUC cells. As described elsewhere52 and in agreement with our above results, PHA, Iono, or PMA alone did not result in any activation of the IL-2 promoter, whereas bpV[pic] had a modest effect (data not shown). However, when combining bpV[pic] with these agents, activation of the IL-2 promoter was observed. With the bpV[pic]/PHA and bpV[pic]/Iono combination, activation of the IL-2 promoter was demonstrated (21- and 49-fold increases, respectively) (Figure 5C). Stronger promoter activation (143-fold) was achieved when combining bpV[pic] and PMA. However, the strongest increase was obtained when combining bpV[pic] to PMA/Iono (220-fold increase). As previously observed in Jurkat cells,53 simultaneous stimulation through TCR (anti-CD3 antibody) and CD28 resulted in a modest increase in IL-2 promoter activity, whereas stimulation through each of the receptors alone showed no promoter modulation (data not shown). However, addition of bpV[pic] to either anti-CD3, anti-CD28 antibodies, or both strongly stimulated IL-2 promoter-driven luciferase activity (36-, 119-, and 156-fold activation, respectively). IL-2 production in the supernatant of these stimulated J-IL-2-LUC cells was also measured and fairly reflected IL-2 promoter activity (Figure 5D). Indeed, when IL-2 production was plotted against luciferase activity, a strong correlation was obtained (data not shown). Our results therefore demonstrated the induction of IL-2 expression and secretion by bpV molecules when used in combination with a variety of agents, including antigen-mimicking agents (anti-CD3/anti-CD28). NFAT activation by bpV[pic] is dependent on TCR-proximal signaling events We next wanted to characterize the signaling pathway activated by the bpV compounds leading to NFAT activation. The implication of the PTKs p56lck and ZAP-70 in bpV-induced NFAT activation was examined. When transfected with the pNFAT-LUC plasmid, p56lck-deficient JCaM-TAg cells were unresponsive to most tested activators, including bpV[pic] (Figure 6A). To demonstrate that this lack of bpV-mediated NFAT induction was p56lck dependent, we co-transfected JCaM-TAg cells with pNFAT-LUC and the p56lck-encoding vector pEFneoLckWT. PHA/PMA-induced NFAT activation was then restored in JCaM-TAg cells, consistent with the p56lck-dependent nature of TCR signaling. This same p56lck dependency was observed for bpV[pic]-induced luciferase activity in JCaM-TAg. The ZAP-70-deficient P116 cell line was then similarly investigated. On transfection with the pNFAT-LUC reporter vector plus a control vector, no NFAT activation was observed for most of the tested stimuli (Figure 6B). However, when a wild-type ZAP-70 expression vector was added, response to these stimuli, including bpV[pic], was restored. ZAP-70 kinase activity was required for bpV-dependent signaling to be conveyed, as no NFAT activation was restored in P116 cells transfected with an expression vector encoding kinase-inactive ZAP-70 (Figure 6B). PMA/Iono was always a potent activator of NFAT in all these tested cell lines, and no important differences following transfections of the different vectors were apparent (Figure 6A,B).
More downstream effectors of NFAT activation were also evaluated for their importance in bpV[pic]-induced cascade. Intracellular calcium, which is known to be crucial for NFAT activation was investigated through the use of Jurkat-derived cell lines defective for capacitative calcium entry. After transfection with pNFAT-LUC, compared with Jurkat cells, these defective cells were not as responsive to the NFAT activators PMA/Iono, PHA, and bpV[pic] (data not shown). Furthermore, similar to the TCR-mediated cascade, the bpV[pic]-activated pathway also involved a functional p21ras protein as assessed by the use of the p21rasN17 dominant-negative mutant (data not shown). These results demonstrated that several effectors involved in TCR-dependent signaling were similarly required in the cascade initiated by bpV compounds culminating in the activation of NFAT. Possible involvement of SHP-1 in NFAT activation by bpV[pic] Because of its previously reported negative regulatory role in proximal TCR-mediated biochemical events,36,54 we reasoned that SHP-1 might be the bpV-targeted PTP. We further assumed that overexpression of wild-type SHP-1 should down-modulate bpV-dependent activation of NFAT. We tested this hypothesis by co-transfecting Jurkat cells with the pNFAT-LUC plasmid plus increasing doses of wild-type or dominant-negative SHP-1-encoding vectors. As shown in Figure 7A, NFAT activation by several different agents, including bpV[pic], was strongly reduced following overexpression of wild-type SHP-1. However, overexpression of the SHP-1 mutant resulted, at the lowest quantity of plasmid, in small increases of NFAT activation (Figure 7B). To corroborate these results, nuclear extracts were prepared from the Jurkat-derived J.SHP-1C/S cell line stably transfected with a vector expressing a dominant-negative form of the SHP-1 protein (termed SHP-1C/S).36 EMSA analysis showed a NFAT-specific signal with nuclear extracts from untreated J.SHP-1C/S cells, which was competed with cold NFAT oligonucleotide (Figure 7C, compare lanes 5 and 6). No such complex was apparent in untreated Jurkat cells (lane 1). Furthermore, when PMA/Iono stimulation was performed, the NFAT-specific signal was stronger in J.SHP-1C/S than in Jurkat cells (compare lanes 3 and 7). These results suggested that the bpV compound might target SHP-1 and that this PTP would act as a regulator of NFAT activity.
The activation of NF- The immunosuppressors FK506 and CsA were initially observed to diminish
bpV-mediated activation of the HIV-1 LTR or the isolated enhancer
region. Because these calcineurin inhibitors have been shown to also
affect NF- We have also determined that bpV compounds led to NFAT
translocation. This was observed with nuclear extracts from
bpV-stimulated Jurkat and PBMCs. A more detailed analysis revealed that
NFAT1 was highly prevalent in these extracts. This is consistent with earlier demonstration of the dominance of NFAT1 in the DNA-binding activity of nuclear extracts from activated peripheral blood T lymphocytes.8 Through specific competition experiments and supershift assays, we have further shown that there was an important induction of an HIV-1 enhancer-bound NFAT complex by bpV compounds and
that again NFAT1 was abundant in this complex (data not shown). This
latter result is unexpected, as this same NFAT member has been proposed
to negatively regulate the HIV-1 enhancer.50 However, the
results obtained in pLTR-LUC-transfected Jurkat cells with the
VIVIT-GFP inhibitor strongly suggest a positive contribution of NFAT1
in the activation of HIV-1 LTR by bpV compounds. We have also shown
that NFAT1 could positively modulate HIV-1 LTR activity in 293T cells.
Differences in experimental settings could account for this existing
discrepancy between our result and the results of Macian and
Rao.50 In fact, most of the experiments performed in their
study were focused on the use of a reporter vector containing one
NF- Our results have also demonstrated that bpV led to the activation of
the IL-2 promoter and an increase in secreted IL-2 levels when added
with PMA, Iono, anti-CD3, anti-CD28 antibodies, and PHA. Similar
observations have already been described with
pervanadate.22,24,51 The activation of the IL-2 promoter
by bpV[pic] was FK506 sensitive, which further suggests the
implication of NFAT in IL-2 expression in Jurkat cells as it has
recently been demonstrated by Chow et al.42 In fact, using
their dominant-negative mutant of NFAT, our results indicated that
bpV-mediated IL-2 promoter induction was similarly sensitive to its
expression (data not shown). Hence, our results suggest that the
multifactorial requirement for IL-2 expression is in part greatly
helped by the bpV compound through concomitant activation of the
NF- Implication of both ZAP-70 and p56lck PTKs
in this bpV-activated signaling cascade has been determined. This
suggests that the point of entry of the PTP inhibitors is membrane
proximal and likely results from the usual signaling pathway induced by
TCR multimerization. This assumption is further supported by previous studies that show pervanadate-induced tyrosine phosphorylation of
several membrane-associated signal transducers, including
p56lck and ZAP-70.24,61 However,
the importance of the TCR in the bpV-mediated induction of NFAT is not
clear, as TCR-negative J.RT3 cells demonstrated reproducible levels of
bpV-induced NFAT activation (data not shown). Intracytoplasmic
association of p56lck with different cell surface molecules
makes their possible implication an interesting
alternative.62,63 Preliminary results also suggest that CD45, the required PTP for TCR-dependent signaling events, is not
essential for NFAT activation by bpV compounds (data not shown). CD45
is thus an unlikely target for bpV molecules in NFAT activation, which
comes in contrast with its previously reported implication in the
induction of NF- The observed role of p21ras in the bpV-mediated activation of NFAT is not unexpected since several examples in the literature have shown the importance of the Ras signaling pathway in NFAT transcriptional activation.64,65 This is thought to occur by the activation of the AP-1 factor, which would cooperatively act with NFAT.9 AP-1 activation by pervanadate, another PTP inhibitor, has already been described,24 and, based on preliminary results, AP-1 can be equally activated by bpV molecules in T cells (data not shown). The implication of calcium in the process leading to NFAT activation by bpV compounds was also expected, considering its crucial importance in the activation process of NFAT and also given the previously reported induction of intracellular calcium mobilization by PTP inhibitors.22,24,51 Studies have reported that treatment with pervanadate activates second
messengers involved in T-cell activation (eg,
p56lck, p59fyn, PLC On the basis of our results, we propose a model (Figure
8) in which bpV would initiate a cascade
through the targeting of SHP-1. The ensuing activation of the PTKs
p56lck and ZAP-70 would then lead to activation
of AP-1 and permit intracellular calcium release and entry via the
CRAC (Ca2+ release-activated Ca2+)
pumps. This pathway might also be the cascade leading to the activation
of NF-
The demonstration that bpV molecules can activate IL-2 gene
expression and NF-
Submitted June 21, 2000; accepted December 28, 2000.
Supported by grant HOP-15575 from the Canadian Institutes of Health Research HIV/AIDS Research Program (M.J.T.), by a Doctoral Fellowship from the Medical Research Council (MRC) of Canada (J.-F.F.), and by a Scholarship Award (Junior 1) from the Fonds de la Recherche en Santé du Québec (FRSQ) (B.B.). G.A.R. is the recipient of a PhD Fellowship from the FRSQ/Fonds pour la Formation de Chercheurs et l'Aide à la Recherche, and M.J.T. is the recipient of a Canada Research Chair in Human Immuno-Retrovirology.
J.-F.F. and B.B. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michel J. Tremblay, Laboratoire d'Immuno-Rétrovirologie Humaine, Centre de Recherche en Infectiologie, RC709, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 boul. Laurier, Ste-Foy, QC, Canada G1V 4G2; e-mail: michel.j.tremblay{at}crchul.ulaval.ca.
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Top
Abstract
Introduction
) or pretreated with NaSal
(2.5 mM) (
), FK506 (10 ng/mL) (
), CsA (100 ng/mL) (
), or a
combination of NaSal with either FK506 (
) or CsA
(
) for 1 hour at
37°C. Following treatment, cells were either left untreated or
treated for 8 hours with the following agents: PHA (3 µg/mL),
bpV[bipy], bpV[HO pic], or bpV[pic] (10 µM). Jurkat cells were
transiently transfected by the DEAE-Dextran protocol with 15 µg of
either p
105/
) and lysed
after 4, 6, 8, 12, and 24 hours of stimulation. (B) Ficoll
Hypaque-isolated PBMCs were first treated with PHA-L for 20 hours and
then electroporated in the presence of 15 µg of pNFAT-LUC. Following
a 2-hour incubation period, cells were stimulated with bpV[pic] (10 µM) (
) or PMA (20 ng/mL)/Iono (1 µM) (
32P-labeled murine IL-2-derived
NFAT-binding site for 20 minutes in the absence (lane 3) or presence of
10-, 100-, or 200-fold excess of unlabeled NFAT oligonucleotide (lanes
4, 5, and 6, respectively) or 100-fold excess of unlabeled NF-
). After a 24-hour incubation, cells were treated or not with PHA
(3 µg/mL)/PMA (20 ng/mL), bpV[pic] (10 µM), or PMA (20 ng/mL)/Iono (1 µM). Cells were lysed 8 hours after stimulation and
measured for luciferase activity. (B) P116 cells (ZAP-70-deficient)
were transiently transfected by the DEAE-Dextran protocol with 15 µg
of the pNFAT-LUC plasmid plus 15 µg of either the empty vector (
) or ZAP-70 kinase-dead expression vector (
, PLC
activates NF-