p53 Transactivation of the HIV-1 Long Terminal Repeat Is Blocked
by PD 144795, a Calcineurin-Inhibitor with Anti-HIV Properties*
Antonio
Gualberto
§,
Gracia
Marquez¶,
Modesto
Carballo¶,
Geri L.
Youngblood
,
Stephen W.
Hunt III**,
Albert S.
Baldwin
, and
Francisco
Sobrino¶
From the
Department of Physiology and Biophysics and
Ireland Cancer Center, CWRU School of Medicine, Cleveland, Ohio 44106, the ¶ Department of Biochemistry, University of Seville School of
Medicine, 41009 Seville, Spain, the
Lineberger Comprehensive
Cancer Center and Department of Biology, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599, and the
** Departments of Immunopathology and Molecular Biology, Parke-Davis
Pharmaceutical Research, Division of Warner-Lambert Co.,
Ann Arbor, Michigan 48105
 |
ABSTRACT |
Previous reports have indicated that
benzothiophenes exhibit broad anti-inflammatory properties and inhibit
human immunodeficiency virus-type 1 (HIV-1) replication. We show that
the immunosuppressant cyclosporin A (CsA) and
benzothiophene-2-carboxamide, 5-methoxy-3-(1-methyl ethoxy)-1-oxide (PD
144795) block the induction of p53 and NF-
B binding to the HIV-1
long terminal repeat (LTR) by the T cell receptor activator
phytohemagglutinin. CsA and PD 144795 also inhibit the induction by
phytohemagglutinin of the transcription mediated by an HIV-1 LTR
fragment containing the p53 and NF-
B sites. These effects of PD
144795 on HIV-1 transcription correlate with its ability to inhibit the
phosphatase activity of calcineurin and are similar to those previously
described for CsA. Moreover, a constitutive active form of calcineurin
is able to induce expression from the HIV-1 LTR in a p53- and
NF-
B-dependent manner and PD 144795 is able to block
this induction. These results demonstrate that the DNA binding of p53
to the HIV-1 LTR can be modulated by calcineurin and provide a
framework to understand the anti-HIV properties of benzothiophene
derivatives.
 |
INTRODUCTION |
Benzothiophene derivatives have been shown to have both
anti-inflammatory and
anti-HIV1 effects.
Originally, these compounds were shown to block expression of cellular
adhesion molecules and to exhibit anti-inflammatory properties (1).
Recently, benzothiophene derivatives were shown to block HIV-1
transcription in response to tumor necrosis factor
stimulation of
promyelocytes (2). Additionally, these compounds blocked constitutive
HIV-1 transcription in chronically infected cells and induced a latency
state in cytokine-activated cells. The benzothiophene derivatives did
not block the activation of NF-
B in response to tumor necrosis
factor
treatment and did not block Tat function (2). In this
report, we have studied the effects of PD 144795 (Parke-Davis
Pharmaceuticals) (Fig. 1) on HIV-1 LTR-directed transcription in Jurkat
T cells.
The expression of HIV-1 genes is controlled in part by the interaction
of sequence-specific transcription factors with the LTR region of the
provirus. NF-
B, Sp1, and other sequence-specific transcription
factors have been shown to control transcription initiation directed by
HIV LTR (3, 4). We have recently identified an inducible form of p53 in
Jurkat T cells that directly interacts with a specific DNA-binding site
positioned immediately downstream to the most 5' Sp1-binding site in
the HIV-1 LTR (5, 6). We have also shown that this DNA element mediates
the induction of the HIV-1 LTR transcriptional activity by tumor
promoting mutant forms of p53 (5). These results provide a mechanism to
explain the dramatic increase in HIV-1 replication observed after the overexpression of mutant p53 in cells that completely lack expression of this protein (7).
It is well known that the replication of the HIV-1 virus in lymphocytes
correlates with the activation/proliferation status of the infected
cell (8, 9). Treatment of HIV-1-infected Jurkat cells with T cell
receptor (TCR) or protein kinase C activators induces HIV-1 replication
(10, 11). Previous reports have indicated that ligands of the TCR
activate LTR-dependent transcription through a
CsA-sensitive mechanism (12-14). CsA, through its interaction with
cyclophilin (reviewed in Ref. 15), acts as an strong inhibitor of the
serine/threonine phosphatase CN (16, 17). This enzyme is a
heterotrimeric complex consisting of a 59-kDa catalytic subunit, calcineurin A subunit, a calcium-binding regulatory subunit of 19 kDa,
calcineurin B subunit, and a 17-kDa calcium-binding protein, calmodulin.
CN plays a critical role in the regulation of
calcium-dependent signaling pathways that are necessary for
T cell activation (16, 18, 19). This enzyme regulates the nuclear
translocation of NF-ATp, a transcription factor implicated in the
expression of several cytokines (20). Also, CN inhibitors are known to decrease HIV-1 viral replication and to inhibit HIV-1 LTR-mediated transcription (12, 21). Inhibition of NF-
B binding to the LTR region
of the HIV-1 provirus has been suggested as the mechanism by which CN
inhibitors would repress the transcriptional activity mediated by this
promoter (14, 22). Intriguingly, the relatively large number of genes
whose expression is modulated by CN inhibitors in T cells (23, 24)
suggests that, in addition to NF-AT and NF-
B, other transcription
factors could be affected by these drugs.
In this study we have investigated the effect of CsA and PD 144795 on
the transcriptional activity mediated by p53 and NF-
B. Using several
approaches we demonstrate that calcineurin is implicated in the
regulation of p53 transcriptional activity, that PD 144795 is an
inhibitor of calcineurin, and that the DNA binding and transactivation of p53 to the HIV-1 LTR can be modulated by calcineurin inhibitors. These results offer insight into the understanding of the molecular basis for the inflammatory and anti-HIV properties of benzothiophene derivatives.
 |
EXPERIMENTAL PROCEDURES |
PD 144795--
Compound PD 144795 (Fig.
1) was synthesized by Parke-Davis
Pharmaceutical Research as described previously (1).
Electrophoretic Mobility Shift Assays--
The LTR B sequences
were as described (5). DNA oligonucleotides were prepared with an
Applied Biosystems 391EP DNA synthesizer using the phosphoramidite
method, and purified by Sep-Pak C18 cartridges (Waters
Associates). p53 gel shift mobility assays were prepared as follows: a
10-µl reaction volume containing the binding reaction buffer (20 mM Hepes pH 7.5, 400 mM NaCl, 5 mM MgCl2, 1 mM ZnCl2, 1 mM
-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride,
5% glycerol) and 1-2 µg of protein nuclear extract was mixed with 2 µg of poly(dI·dC) and 0.2 ng of radiolabeled probes and incubated
at room temperature for 30 min. Reactions were loaded onto a 3.8%
polyacrylamide gel previously prerun for 15 min. The NF-
B HIV-1 EMSA
sequence was as follows (double strand): CGCTGGGGACTTTCCAGGG. NF-
B
EMSA reactions were prepared as described previously (25). The octamer
site (underlined) was from sequences
60 to
41 of the HLA DR
gene
(double strand): AGTAATTGATTTGCATTTTA (46).
Plasmids, Cell Culture, Cell Extracts, Transfections, and CAT
Assays--
The
121,+232 HIV-1 LTR CAT reporter plasmid construct
was a gift from B. Stein. PG13 and pSR
-
CAM-AI were a gift from B. Vogelstein and E. A. O'Neill, respectively. NF-
B/p53E1BTATA
CAT, HIV1
p53CAT, and HIV1
NF-
BCAT have been previously
described (5, 26). Plasmids were prepared with Qiagen columns according to the directions of the manufacturers.
Jurkat T leukemia cells from ATCC were cultured at 0.1-0.5 × 105 cells/ml in
-minimal essential medium, 2% heat
inactivated fetal calf serum (Irvine), and penicillin/streptomycin.
When incubations were performed, cells were centrifuged, resuspended in
new media at 1 × 106 cells/ml, and incubated as
indicated. Incubations were stopped by centrifugation and cells
were processed for the preparation of nuclear extracts as described
(25) but a final concentration of 1 mM phenylmethylsulfonyl
fluoride was used. Extracts were aliquoted and stored at
80 °C.
For transfection experiments, Jurkat or CEM lymphocytes were
transfected by electroporation at 5 × 106 cells, 0.5 ml of phosphate-buffered saline and resuspended in 10 ml of media.
Incubations were carried out for 24-48 h. Cells were lysed by a 3-s
ultrasonic vibration at 4 °C using a Branson Sonifier at setting 3. Equal amounts of protein, as determined by the Bradford assay
(Bio-Rad), were then analyzed for chloramphenicol acetyltransferase
(CAT) activity using the fluor diffusion assay (27).
Calcineurin Phosphatase Assay--
Cell extracts were prepared
as follows: 106 Jurkat lymphocytes per experimental
condition were washed in phosphate-buffered saline and lysed in 50 µl
of buffer containing 50 mM Tris, pH 7.5, 0.1 mM
EGTA, 1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 5 µg of protease
inhibitors leupeptin, aprotinin, and soybean trypsin inhibitor. Cells
were disrupted by freeze/thaw and extracts cleared by centrifugation at
10,000 × g for 10 min. Peptide substrate (Peninsula
Laboratories) was as in Hubbard and Klee (28). CN activity was
determined in the presence of 100 nM calmodulin and 5 µM 32P-labeled phosphopeptide in a 50-µl
reaction mixture containing 20 mM Tris, pH 8.0, 100 mM NaCl, 6 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mM CaCl2,
and 20 µl of cell lysate. Reactions were incubated at 30 °C for 15 min and stopped by the adition of 5% trichloroacetic acid. Phosphate
was isolated by Dowex AG 1-X8 chromatography and quantitated by
scintillation counting. Alkaline phosphatase activity was measured as
previously reported (29). Lactate dehydrogenase activity was measured
using a spectrophotometric assay (Boehringer Mannheim).
 |
RESULTS AND DISCUSSION |
CsA and PD 144795 Block p53 and NF-
B Binding to the HIV-1
LTR--
The HIV-1 LTR contains a binding site for a
proliferation-associated form of p53 (5). The p53 protein bound to this
site is in a proliferative or mutant-like unfolded state based on
recognition by specific monoclonal antibodies. This DNA region mediates
the induction of the HIV-1 LTR by tumor necrosis factor
and by
transforming mutant forms of p53. Importantly, tumor necrosis factor
induces a physical interaction between p53 and Sp1 and this
interaction is required for the induction of HIV-1 LTR-mediated
transcription by this cytokine (6). We investigated whether or not
p53/Sp1 binding to the HIV-1 LTR is also induced by activation of the T
cell receptor. For that purpose, we performed EMSA using an oligonucleotide probe (LTR B),
CAGGGAGGCGTGGCCTGGGCGGGACTGGGG, that
contains the composite p53 (underlined) and Sp1 (italics) site in the
HIV-1 LTR, and nuclear extracts prepared from Jurkat T cells either
unstimulated or stimulated with PHA (Fig.
2A). Incubation with PHA
induced the appearance of two complexes, which we called PIC 1 and 2 (Fig. 2A). Subsequent experiments indicated that maximal
complex formation by PHA was obtained at a 5-h incubation time (not
shown). To confirm that these complexes contain p53 and Sp1, extracts
were incubated with antibodies against these proteins prior to the
mobility shift assay. Monoclonal anti-p53 PAb 421 and polyclonal
anti-Sp1 demonstrated that p53 was present in PIC 1 and 2 while Sp1 was
detected only in PIC 1 (Fig. 2B). A supershift was obtained
with the anti-Sp1 antibody, whereas disruption of Sp1/p53 binding to
the LTR was observed with the anti-p53 antibody PAb 421. We have
previously shown that PAb 421 specifically blocks p53 interaction with
LTR B p53/Sp1 composite site (6).

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Fig. 2.
Characterization of the PHA-induced
nucleoprotein complexes and inhibition of complex formation by
cyclosporin A and PD 144795. A, PHA induces the formation of
specific nucleoprotein complexes on the HIV-1 LTR p53·Sp1 composite
site. Jurkat cells cultured in -minimal essential medium with 2%
fetal calf serum and penicillin/streptomycin, were resuspended in new
media and incubated for 4 h in the presence or absence of 5 µg/ml PHA. Cells were then harvested and processed for the
preparation of nuclear extracts. 2 µg of nuclear extracts were
assayed by EMSA using 0.2 ng of the LTR B probe and 2 µg of
poly(dI·dC) as indicated under "Experimental Procedures."
B, identification of p53 and Sp1 in the PICs. Jurkat cells
were incubated with PHA as in A and nuclear extracts
prepared. 2 µg of nuclear extracts were incubated for 45 min at
4 °C with 0.2 µg of serum (NA), monoclonal anti-p53 antibodies PAb 240 (240, conformational), 421 (421, C-terminal), 1620 (1620, conformational),
1801 (1801, N-terminal); or polyclonal anti-Sp1
(Sp1), and then assayed by EMSA as in A.
C, CsA and PD 144795 inhibit the formation of PICs. Jurkat
cells cultured as in part A were preincubated for 30 min
with 0.03% Me2SO (NA), 0.5 µg/ml CsA, 100 ng/ml rapamycin (Rap), 3 µM PD 144795 (144795), or 100 µg/ml pentoxifilline, and then incubated
for 4 h in the presence of 5 µg/ml PHA. Equal volumes of
Me2SO were added in each condition. Nuclear extracts were
prepared and assayed for binding to the LTR B probe. D,
binding of the nuclear extracts from the Me2SO-
(NA), CsA-, and PD 144795-treated cells to an Oct-1
probe. Conditions as in C. E, effect of PD 144795 (144795) on the induction of p53/Sp1 binding to the HIV-1
LTR by PHA and PMA. Jurkat cells cultivated as in A were
preincubated for 30 min in the presence or absence of 3 µM PD 144795, and then incubated with 5 µg/ml PHA or 50 ng/ml PMA. Nuclear extracts were prepared and assayed for nucleoprotein
complex formation as in A.
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Subsequent experiments were directed to determine if the PHA-inducible
binding of p53/Sp1 to the HIV-1 LTR is sensitive to pharmacological
modification. We performed EMSA with nuclear extracts of Jurkat T cells
incubated with PHA and exposed to a series of pharmacological agents.
Incubation of Jurkat cells for 30 min with CsA or PD 144795 blocked the
induction of PICs by PHA, however, incubation with pentoxifylline (data
not shown), an NF-
B inhibitor (30), or the immunosuppressant
rapamycin (15) had no effect (Fig. 2C). Other
5-methoxybenzothiophene derivatives had minor or no effect on PIC
formation (not shown). Also, titration experiments indicated that
inhibition of the induction of the p53·Sp1 complexes was reached at 3 µM PD 144795 and 0.5 µg/ml CsA (not shown). As a
control, the nuclear extracts used in Fig. 2C were tested
for their ability to bind the HLA DR
octamer site using an
oligonucleotide sequence from this gene (46). No effect of CsA or PD
144795 was detected on Oct-1 binding (Fig. 2D). The identity
of the Oct-1 complex was determined by supershift with an anti-Oct-1
antibody (not shown). CsA and PD 144795 also inhibited the induction of p53·Sp1 complexes in another T cell line, CEM, when these cells were
incubated in the presence of the calcium ionophore A23187 (47) (not
shown). Finally, the effect of CsA and PD 144795 was specific for the
TCR-dependent signal and no effect of these compounds was
detected when Sp1·p53 complexes were induced by the incubation of the
Jurkat cells with phorbol myristate acetate (PMA) (Fig. 2E).
Previous reports have shown that CsA inhibits the induction by
calcium-dependent signals of the DNA binding activity of
the transcription factors NF-AT and NF-
B (14, 22, 31-33). An
inhibition of NF-
B by PD 144795 could be critical for HIV-1
replication, as NF-
B is a major regulator of HIV-1 LTR-mediated
transcription (34, 35). We tested whether or not PD 144795 was able to
block the induction by PHA of NF-
B binding to its recognition
sequence in the HIV-1 LTR. Nuclear extracts from Jurkat cells,
incubated as described above, were assayed by EMSA using an
oligonucleotide probe containing the HIV-1 LTR NF-
B II site. A
typical result of these experiments is shown in Fig.
3A. Two specific complexes were identified using this probe. The faster mobility complex contained
NF-
B-p50/p50 homodimers whereas the slower mobility complex was
comprised of NF-
B p50/p65 heterodimers as indicated by supershift
with specific anti-NF-
B antibodies (Fig. 3B). Incubation of Jurkat cells with PHA increased p50/p65 binding while a minor effect
was observed on p50/p50 homodimers. PD 144795, as well as CsA,
inhibited the PHA-induced increase in the levels of p50/p65 heterodimers. No effect of these drugs was observed on the binding of
p50/p50 homodimers. The decrease in p50/p65 binding to a basal or lower
than basal level was observed in several experiments (not shown). A
certain variability in the level of basal p50/p65 binding was observed,
probably resulting from activation of NF-
B by growth factors present
in the serum. CsA and PD 144795 also inhibited NF-
B p50/p65 binding
in CEM lymphocytes when these cells were incubated in the presence of
the calcium ionophore A23187 (47) but not in the presence of PMA (not
shown). In summary, these results indicate that CsA and PD 144795 specifically inhibit the activation by PHA of p53 and NF-
B binding
to the HIV-1 LTR. Intriguingly, these results suggested that both
compounds interfere with a common signal transduction pathway that
regulates transcription factors that are critical for the expression of
the HIV-1 promoter.

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Fig. 3.
Effect of CsA and PD 144795 (Bz) on the
induction of NF- B binding to the HIV-1 LTR by PHA. A,
Jurkat cells were cultured as in Fig. 1, preincubated for 30 min with
CsA (0.5 µg/ml) or PD 144795 (144795, 3 µM)
as indicated, and then incubated for 5 h with 5 µg/ml PHA. Equal
volumes of Me2SO were added in each condition. Nuclear
extracts and mobility shift assays were prepared as in A
using an oligonucleotide probe containing the 3' NF- B-binding site.
B, identification of NF- B p50·p50 and p50·p65
DNA-binding complexes. EMSA was performed using PHA-induced nuclear
extracts and polyclonal NF- B anti-p50 and anti-p65 antibodies.
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CsA and PD 144795 Inhibit the PHA-induced Transcriptional
Activation of the HIV-1 LTR--
To test the sensitivity of the p53
and NF-
B-mediated transcriptional activities to CsA and PD 144795 in vivo, transfection experiments were performed in Jurkat T
cells using the reporter plasmid
121,+232 HIV-1 LTR CAT that contains
the HIV-1 LTR
B II and p53 sites (5). After a 24-h incubation
period, transfected cells were aliquoted and incubated with PHA alone
or in the presence of CsA or PD 144795 for an additional 48 h. The
results of these experiments are shown in Fig.
4A. Incubation of the cells
with PHA and CsA or PD 144795 reduced CAT activity to approximately 50% of PHA alone. However, the PMA induction of the transcriptional activity mediated by this promoter fragment was not altered by the
incubation with CsA or PD 144795. Consistent with the idea of a common
role for PD 144795 and CsA, we observed that the activation of an
interleukin-2 reporter plasmid by calcium-dependent signals was inhibited by PD 144795 and CsA (not shown). These results parallel
the signal-specific inhibition by CsA and PD 144795 of p53 and NF-
B
binding in vitro.

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Fig. 4.
Inhibition by cyclosporin A and PD 144795 of
the transcriptional activity mediated by the HIV-1 LTR p53 and NF- B
sites. A, CsA and PD 144795 (Bz) inhibit the induction by
PHA of the transcriptional activity mediated by the HIV-1 LTR. 3.5 × 107 Jurkat cells were transfected by electroporation
with 35 µg of the reporter plasmid 121,+232 HIV-1 LTR CAT. 8 h
after transfection, cells were aliquoted and incubated for 36 h
with the additions indicated. 5 µg/ml PHA, 50 ng/ml PMA, 1 µg/ml
CsA, and 3 mM PD 144795 were used. Equal amounts of
Me2SO were used in all conditions. PD 144795 and CsA were
added 1 h before the addition of PHA or PMA. After the incubation,
the cells were collected by centrifugation and processed for CAT
activity as indicated under "Experimental Procedures." CAT activity
was normalized to the basal activity of 121,+232 HIV-1 LTR CAT
(approximately 5% acetylation). The figure shows the mean value and
S.D. of three independent experiments. B, CsA and PD 144795 inhibit the induction by PHA of the transcriptional activity mediated
by a minimal promoter construct containing the NF- B and p53 sites.
Jurkat cells were transfected and incubated as in A but
using the reporter plasmid NF- B/p53 E1BTATA CAT. CAT activity was
normalized to the basal activity of this vector (approximately 5%
acetylation). The figure represents three independent experiments.
C, CsA and PD 144795 (Bz) did not affect the transcriptional activity mediated by p53 consensus sites. Normal human lung fibroblasts (WI-38 cells) were transfected by lipofection with 5 µg of PG13. 8 h after transfection, the cells were incubated in new media plus
the additions indicated in the figure. 5 µM
phosphono-L-acetyl aspartate, 1 µg/ml CsA, and 3 µM PD 144795 were used. Cells were incubated for 3 days,
then harvested and CAT activity measured as indicated under
"Experimental Procedures." CAT activity was normalized to the basal
activity of PG13 (approximately 7% acetylation). The figure represents
three independent experiments.
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To define the HIV-1 LTR DNA elements that are targeted by PD 144795 and
CsA, we tested the effect of these compounds on the activity of
NF-
B/p53-E1BTATA CAT, a minimal promoter-reporter plasmid that
contains a 32-base pair HIV-1 LTR fragment comprising the NF-
B II
and the proliferative p53 sites subcloned upsteam of an heterologous
TATA element. PD 144795 and CsA inhibited the PHA, but not the PMA
induction of NF-
B/p53-E1BTATA CAT activity in Jurkat T cells (Fig.
4B). PD 144795 and CsA did not affect the basal activity of
the minimal promoter element at the concentrations used in these
experiments (not shown). As a control we investigated the effect of CsA
and PD 144795 on the transcriptional activity mediated by PG13, a CAT
reporter plasmid containing 13 copies of an RGC consensus
(antiproliferative) p53-binding site. We transfected primary human
fibroblasts with PG13 and incubated these cells with the DNA synthesis
inhibitor phosphono-L-acetyl aspartate because this
compound induces the transcriptional activity mediated by consensus p53
sites.2 Neither CsA nor PD
144795 altered the induction of PG13 activity by
phosphono-L-acetyl aspartate (Fig. 4C). No
effect of PD 144795 or CsA was observed on the basal expression of PG13
(not shown). These experiments further demonstrated that PD 144795 and
CsA target a common signal transduction pathway that mediates the activation of the transcriptional activity of NF-
B and proliferative p53 by ligands of the TCR.
PD 144795 Inhibits Calcineurin Activity--
Since engagement of
the TCR activates HIV-1 LTR-mediated transcription through a
calcineurin-dependent pathway (12-14), and since
benzothiophene showed similar properties to CsA, we tested the effect
of PD 144795 on the activity of calcineurin, the enzymatic activity
that is targeted by CsA (16, 17). Incubation of Jurkat cells with PD
144795 resulted in a dose-dependent inhibition of the
phosphatase activity of calcineurin (Fig.
5A). PD 144795 did not
decrease the activity of the enzyme lactate dehydrogenase, indicating
that this compound was not toxic to the cells (Fig. 5A). PD
144795 and CsA also inhibited the activity of calcineurin in Jurkat
cell extracts (Fig. 5B). However, they did not affect the
activity of the enzyme alkaline phosphatase (Fig. 5B,
inset), indicating that the effect of PD 144795 on
calcineurin was specific.

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Fig. 5.
PD 144795 inhibits the phosphatase activity
of calcineurin and activation by this enzyme of the transcriptional
activity mediated by the HIV-1 LTR. A, incubation of Jurkat
cells with PD 144795 results in a dose-dependent inhibition
of calcineurin. 107 Jurkat cells were incubated for 2 h at 37 °C in media containing the concentrations of PD 144795 indicated in the figure. Cells were harvested by centrifugation and
extracts prepared and calcineurin (CN) and lactate
dehydrogenase (LDH) activity reactions carried out as
indicated under "Experimental Procedures." B, PD 144795 inhibits the phosphatase activity of calcineurin in lymphocyte extracts. Extracts from murine peripheral blood lymphocytes were prepared and assayed for calcineurin activity as indicated under "Experimental Procedures." The extracts were incubated 15 min in
the indicated concentration of PD 144795 or CsA before the addition of
the substrate. Equal amounts of Me2SO were used per reaction. Released phosphate was isolated and quantified by
scintillation counting. The figure represents three independent
experiments. B, inset, CsA and PD 144795 do not inhibit the
activity of alkaline phosphatase. Cell extracts were treated as in
B and assayed for alkaline phosphatase activity.
C, PD 144795 antagonizes the activation by calcineurin of
the transcriptional activity mediated by the HIV-1 LTR. Jurkat and
WI-38 cells (fibroblasts) were co-transfected with 5 µg of 121,+232
HIV-1 LTR CAT and 1 µg of the expression vectors Sr (control,
open bars) and Sr - CAM ( CAM, solid bars). 8 h after transfection cells were placed in new media with
increased concentrations of PD 144795 and incubated for an aditional
48 h. Equal amounts of Me2SO were used in all
conditions. Transfections and CAT assays were carried out as described
in the legend to Fig. 4. The figures represent three independent
experiments.
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Thus, our results demonstrate that PD 144795 targets the same enzymatic
activity as CsA, suggesting that inhibition of calcineurin is a common
mechanism of action of both drugs that explains their similar effect on
the transcriptional activity of p53 and NF-
B. Current experiments in
our laboratory indicate that PD 144795 has additional effects on early
events associated with the activation of the TCR. For example, low
concentrations of PD 144795 induced a slow and steady increase in the
intracellular calcium levels in T lymphocytes and inhibited the
PHA-induced rise of intracellular calcium in these
cells.3
The effect of PD 144795 on the activity of calcineurin was further
investigated by experiments of overexpression of this enzyme in
vivo. It has been shown that overexpression of calcineurin in
Jurkat cells renders them more resistant to the effects of CsA and FK
506 (48), another calcineurin-inhibitor agent, and augments both NF-AT-
and NF-interleukin-2A-dependent transcription (18, 19). We
co-transfected
121,+232 HIV-1 LTR CAT with pSR
-
CAM, a pSR
expression vector containing a constitutively active form of
calcineurin, in Jurkat cells and normal human fibroblasts. Calcineurin
induced the transcriptional activity of this HIV-1 LTR fragment in both
cell types (Fig. 5C). A higher induction was obtained in
fibroblasts (15-fold) than in Jurkat cells (6-fold). Incubation of the
cells with PD 144795 inhibited the effect of calcineurin in a
dose-response manner. Fibroblasts were more sensitive than Jurkat cells
to the inhibition by PD 144795. Half-maximal inhibition of CAT activity
by PD 144795 was reached at approximately at 0.2 µM in
normal human fibroblasts and 1 µM in Jurkat cells. A
possible explanation for the cell-type difference in the response to PD
144795 could be the existence of a mutant p53 allele in the Jurkat
cells (36). This mutant p53 protein may already display in basal
conditions the conformation necessary for the interaction with HIV-1
LTR. Alternatively, Jurkat T cells may carry other genetic alterations
that result in a certain degree of constitutive activation of NF-
B
or p53. Interestingly, low concentrations of PD 144795 (0.3 µM) induced a moderate activation of
121,+232 HIV-1 LTR
CAT in both cell types. This paradoxical effect may result from an
indirect activation of endogenous calcineurin by PD 144795 secondary to
an increase in the intracellular calcium levels induced by low
concentrations of the drug. This effect disappeared at higher
concentrations of PD 144795 as it was expected from its inhibitory
effect on calcineurin activity in vivo (Fig. 5A).
The effect of the low concentration of PD 144795 was not observed in
cells transfected with pSR
-
CAM. This calcineurin mutant is not
affected by alterations in the intracellular calcium levels (18).
Finally, since PD 144795 inhibits calcineurin and the transcriptional
activity mediated by NF-
B and p53, we investigated whether or not
calcineurin is involved in the regulation of the transcriptional
activity of NF-
B and p53. For that purpose, we co-transfected CEM
cells with a wild type HIV-1 LTR CAT reporter or HIV-1 LTR CAT plasmids
with mutated NF-
B or p53 sites and the pSR
or pSR
-
CAM
expression vectors. The results of these experiments are shown in the
Fig. 6. As expected, the activated form
of calcineurin induced HIV-1 LTR CAT in this cell type. Importantly, mutagenesis of the NF-
B or p53 sites completely abolished the induction of HIV-1 LTR activity by calcineurin, indicating that the
transcriptional activity of NF-
B and p53 can be modulated in a
calcineurin-dependent manner. Previous reports have
indicated that calcineurin inhibitors modulate NF-
B transcriptional
activity (14, 37-40). The regulation of p53 transcriptional activity
by calcineurin may have important implications for the growth
regulatory functions of this protein. It has been shown that
proliferative forms of p53 may work as positive regulators of cell
growth (Refs. 41-43 and references therein). In addition, recent data
indicates that certain members of the NF-AT(Rel) family of
transcription factors can synergize with NF-
B and Tat in the
transcriptional activation of HIV-1 (44). Since NF-AT activity is
regulated by calcineurin (31, 40, 45), it is possible that an
NF-
B/NF-AT synergistic effect on HIV-1 LTR transcription could be
affected by PD 144795. In our experimental conditions, NF-
B was
solely accounted for by HIV-1
B site nucleoprotein complex formation (Fig. 3 and data not shown). However, we cannot discard that a physical
or functional interaction between NF-
B and certain NF-AT family
members may take place in vivo and could be targetted by PD
144795.

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|
Fig. 6.
The NF- B and p53 sites are required for
the activation of the HIV-1 LTR by calcineurin. CEM cells were
co-transfected by electroporation with 1 µg of the expression vectors
Sr (empty vector, open bars) or Sr - CAM ( CAM,
solid bars) and 5 µg of the CAT reported vectors HIV-1 LTR
(WT), HIV-1 LTR B ( B ), or p53 (p53 ) and incubated for
48 h. CAT assays were carried out as indicated under
"Experimental Procedures." The figure represents three independent
experiments.
|
|
 |
ACKNOWLEDGEMENTS |
We thank B. Stein, G. Nabel, B. Vogelstein,
E. O'Neill, and T. Tlsty for reagents and suggestions.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant AI35098 (to A. S. B.) and Fondo de
Investigaciones Sanitarias, Spain 94/1484 (to F. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Physiology
and Biophysics, CWRU School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-368-3487; Fax: 216-368-3952; E-mail: axg29{at}po.cwru.edu.
1
The abbreviations used are: HIV, human
immunodeficiency virus; NF-
B, nuclear factor
B; PD 144795, benzothiophene-2-carboxamide, 5-methoxy-3-(1-methyl ethoxy)-1-oxide;
LTR, long terminal repeat; CsA, cyclosporin A; TCR, T cell antigen
receptor; CN, calcineurin; NF-AT, nuclear factor of activated T cells;
PHA, phytohemagglutinin; EMSA, electrophoretic mobility shift assay;
CAT, chloramphenicol acetyltransferase; PIC, phytohemagglutinin-induced
complex; PMA, phorbol 12-myristate 13-acetate.
2
A. Gualberto, unpublished data.
3
R. Montaño and F. Sobrino, unpublished
data.
 |
REFERENCES |
-
Boschelli, D. H.,
Kramer, J. B.,
Khatana, S. S.,
Sorenson, R. J.,
Connor, D. T.,
Ferin, M. A.,
Wright, C. D.,
Lesch, M. E.,
Imre, K.,
Okonkwo, G. C.,
Schrier, D. J.,
Conroy, M. C.,
Ferguson, E.,
Woelle, J.,
Saxena, U.
(1995)
J. Med. Chem.
38,
45597-45614
-
Butera, S. T.,
Roberts, B. D.,
Critchfield, J. W.,
Fang, G.,
McQuade, T.,
Gracheck, S. J.,
Folks, T. M.
(1995)
Mol. Med.
1,
758-767[Medline]
[Order article via Infotrieve]
-
Jones, K. A.,
and Peterlin, B. M.
(1994)
Annu. Rev. Biochem.
63,
717-743[CrossRef][Medline]
[Order article via Infotrieve]
-
Nabel, G.,
and Baltimore, D.
(1987)
Nature
326,
711-713[CrossRef][Medline]
[Order article via Infotrieve]
-
Gualberto, A.,
Hixon, M. L.,
Finco, T. S.,
Perkins, N. D.,
Nabel, G. L.,
Baldwin, A. S., Jr.
(1995)
Mol. Cell. Biol.
15,
3450-3459[Abstract]
-
Gualberto, A.,
and Baldwin, A. S., Jr.
(1995)
J. Biol. Chem.
270,
19680-19683[Abstract/Free Full Text]
-
Duan, L.,
Ozaki, I.,
Oakes, J. W.,
Taylor, J. P.,
Khalili, K.,
Pomerantz, R. J.
(1994)
J. Virol.
68,
4302-4313[Abstract]
-
McDougal, J. S.,
Mawle, A.,
Cort, S. P.,
Nicholson, J. K.,
Cross, G. D.,
Scheppler-Campbell, J. A.,
Hicks, D.,
Sligh, J.
(1985)
J. Immunol.
135,
3151-3162[Abstract/Free Full Text]
-
Folks, T.,
Kelly, J.,
Benn, S.,
Kinter, A.,
Justement, J.,
Gold, J.,
Redfield, R.,
Sell, K. W.,
Fauci, A. S.
(1986)
J. Immunol.
136,
4049-4053[Abstract/Free Full Text]
-
Zagury, D.,
Bernard, J.,
Leonard, R.,
Cheynier, R.,
Feldman, M.,
Sarin, P. S.,
Gallo, R. C.
(1986)
Science
231,
850-853[Medline]
[Order article via Infotrieve]
-
Harada, S.,
Koyanagi, Y.,
Nakashima, H.,
Kobayashi, N.,
and Yamamoto, N.
(1986)
Virology
154,
249-258[Medline]
[Order article via Infotrieve]
-
Tong-Starkesen, S. E.,
Luciw, P. A.,
Peterlin, B. M.
(1989)
J. Immunol.
142,
702-707[Abstract/Free Full Text]
-
Siekevitz, M.,
Josephs, S. F.,
Dukovich, M.,
Peffer, N.,
Wong-Staal, F.,
and Greene, W. C.
(1987)
Science
238,
1575-1578[Medline]
[Order article via Infotrieve]
-
Schmidt, A.,
Hennighausen, L.,
and Siebenlist, U.
(1990)
J. Virol.
64,
4037-4041[Medline]
[Order article via Infotrieve]
-
Fruman, D. A.,
Burakoff, S. J.,
and Bierer, B. E.
(1994)
FASEB J.
8,
391-400[Abstract/Free Full Text]
-
Liu, J.,
Farmer, J. D., Jr.,
Lane, W. S.,
Friedman, J.,
Weissman, I.,
Schreiber, S. L.
(1991)
Cell
66,
807-815[Medline]
[Order article via Infotrieve]
-
Friedman, J.,
and Weissman, I.
(1991)
Cell
66,
799-806[Medline]
[Order article via Infotrieve]
-
O'Keefe, S. J.,
Tamura, J.,
Kincaid, R. L.,
Tocci, M. J.,
O'Neil, E. A.
(1992)
Nature
357,
692-694[CrossRef][Medline]
[Order article via Infotrieve]
-
Clipstone, N. A.,
and Crabtree, G. R.
(1992)
Nature
357,
695-697[CrossRef][Medline]
[Order article via Infotrieve]
-
Rao, A.
(1995)
J. Leukocyte Biol.
57,
536-542[Abstract]
-
Karpas, A.,
Lowdell, M.,
Jacobson, S. K.,
Hill, F.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
8351-8355[Abstract]
-
Emmel, E. A.,
Verweij, C. L.,
Durand, D. B.,
Higgins, K. M.,
Lacy, E.,
Crabtree, G. R.
(1989)
Science
246,
1617-1620[Medline]
[Order article via Infotrieve]
-
Gunter, K. C.,
Irving, S. G.,
Zipfel, P. F.,
Siebenlist, U.,
Kelly, K.
(1989)
J. Immunol.
142,
3286-3291[Abstract/Free Full Text]
-
Zipfel, P. F.,
Irving, S. G.,
Kelly, K.,
and Siebenlist, U.
(1989)
Mol. Cell. Biol.
9,
1041-1048[Medline]
[Order article via Infotrieve]
-
Beg, A. A.,
Finco, T. S.,
Nantermet, P. V.,
Baldwin, A. S., Jr.
(1993)
Mol. Cell. Biol.
13,
3301-3310[Abstract]
-
Perkins, N. D.,
Edwards, N. L.,
Duckett, C. S.,
Agranoff, A. B.,
Schmid, R. M.,
Nabel, G. J.
(1993)
EMBO J.
12,
3551-3558[Abstract]
-
Neumann, J. R.,
Morency, C. A.,
and Russian, K. O.
(1992)
Biotechniques
5,
444-448
-
Hubbard, M. J.,
and Klee, C. B.
(1989)
in
Molecular Neurobiology (Wheal, H., and Chad, J., eds), pp. 135-157, Oxford University Press, Oxford, United Kingdom
-
Feldbush, T. L.,
and Lafrenz, D.
(1991)
J. Immunol.
147,
3690-3695[Abstract/Free Full Text]
-
Biswas, D. K.,
Dezube, B. J.,
Ahlers, C. M.,
Pardee, A. B.
(1993)
J. AIDS
6,
778-786[Medline]
[Order article via Infotrieve]
-
Park, J.,
Yaseen, N. R.,
Hogan, P. G.,
Rao, A.,
Sharma, S.
(1995)
J. Biol. Chem.
270,
20653-20659[Abstract/Free Full Text]
-
Luo, C.,
Burgeon, E.,
Carew, J. A.,
McCaffrey, P. G.,
Badalian, T. M.,
Lane, W. S.,
Hogan, P. G.,
Rao, A.
(1996)
Mol. Cell. Biol.
16,
3955-3966[Abstract]
-
Ho, S.,
Clipstone, N.,
Timmermann, L.,
Northrop, J.,
Graef, I.,
Fiorentino, D.,
Nourse, J.,
and Crabtree, G. R.
(1996)
Clin. Immunol. Immunopathol.
80,
40-45[CrossRef]
-
Garcia, J.,
and Gaynor, R.
(1994)
Prog. Nucleic Acids Res. Mol. Biol.
49,
157-196[Medline]
[Order article via Infotrieve]
-
Alcami, J.,
Lain de Lera, T.,
Folgueira, L.,
Pedraza, M. A.,
Jacque, J. M.,
Bachelerie, F.,
Noriega, A. R.,
Hay, R. T.,
Harrich, D.,
Gaynor, R. B.,
Virelizier, J.-L.,
Arenzana-Seisdedos, F.
(1995)
EMBO J.
14,
1552-1560[Abstract]
-
Cheng, J.,
and Haas, M.
(1990)
Mol. Cell. Biol.
10,
5502-5509[Medline]
[Order article via Infotrieve]
-
Brini, A. T.,
Harel-Bellan, A.,
and Farrar, W. L.
(1990)
Eur. Cytokine Net.
1,
131-139[Medline]
[Order article via Infotrieve]
-
Granelli-Piperno, A.,
Nolan, P.,
Inaba, K.,
and Steinman, R. M.
(1990)
J. Exp. Med.
172,
1869-1872[Abstract]
-
Frantz, B.,
Nordby, E. C.,
Bren, G.,
Steffan, N.,
Paya, C. V.,
Kincaid, R. L.,
Tocci, M. J.,
O'Keefe, S. J.,
O'Neill, E. A.
(1994)
EMBO J.
13,
861-870[Abstract]
-
McCaffrey, P. G.,
Kim, P. K.,
Valge-Archer, V. E.,
Sen, R.,
Rao, A.
(1994)
Nucleic Acids Res.
22,
2134-2142[Abstract]
-
Zhang, W.,
Hu, G.,
Estey, E.,
Hester, J.,
and Deisseroth, A.
(1992)
Oncogen
7,
1645-1647[Medline]
[Order article via Infotrieve]
-
Ullrich, S. J.,
Anderson, C. W.,
Mercer, W. E.,
Appella, E.
(1992)
J. Biol. Chem.
267,
15259-15262[Free Full Text]
-
Milner, J.
(1994)
Semin. Cancer Biol.
5,
211-219[Medline]
[Order article via Infotrieve]
-
Kinoshita, S.,
Su, L.,
Amano, M.,
Timmerman, L. A.,
Kaneshima, H.,
Nolan, G. P.
(1997)
Immunity
6,
235-244[Medline]
[Order article via Infotrieve]
-
Ruff, V. A.,
and Leach, K. L.
(1995)
J. Biol. Chem.
270,
22602-22607[Abstract/Free Full Text]
-
Peterlin, B. M.,
Hardy, K. J.,
and Larsen, A. S.
(1987)
Mol. Cell. Biol.
7,
1967-1972[Medline]
[Order article via Infotrieve]
-
Weiss, A.,
Imboden, J.,
Shoback, D.,
and Stobo, J.
(1984)
Proc. Natl. Acad. Sci. U. S. A.
81,
4169-4173[Abstract]
-
Fruman, D. A.,
Klee, C. B.,
Bierer, B. E.,
Burakoff, S. J.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
3689-3690
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