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*
AIDS Immunophatogenesis Unit, DIBIT, and
Division of Infectious Diseases, Centro San Luigi, San Raffaele Scientific Institute, Milan, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, Sp1, NF-ATc, and
cAMP response element binding protein, and repressors, such as leader
binding protein-1 (LBP-1) (5, 6) (also known as late SV40
factor (LSF)) and ying yang-1 (YY1), have been described to modulate
HIV-LTR-driven transcription, respectively (2, 3). In
contrast to an abundant literature concerning induction and modulation
of positive transcription factors, mostly focusing on activation of
NF-B (7, 8), little is known about the expression and
modulation of negative transcription factors. In this regard, LBP-1 is
a nuclear factor that was initially identified as a 63-kDa polypeptide
called LSF (9, 10), because of its ability to
stimulate transcription from the SV40 major late promoter
(10), which is rapidly phosphorylated upon mitogenic
stimulation of human T cells (11). Sequence analysis
revealed that LSF is identical with LBP-1c, which is one of the four
proteins (LBP-1a, -b, -c, and -d) encoded by the cDNAs generated by
alternative splicing of two related genes (one gene encoding the LBP-1a
and -b and the other encoding the LBP-1d and -c products, respectively)
(12). LBP-1 binds to the -16 to +27 HIV-LTR sequence,
including the initiation site and the TATA box (13).
Mutations of distinct sites in the -2 to +21 region of the HIV-LTR,
encompassing the LBP-1 (LSF)-DNA binding element, did not affect either
the basal or Tat-induced HIV-LTR-driven transcription in transient
transfection experiments (14). However, it was later
demonstrated that LBP-1 (LSF) indeed repressed HIV expression by in
vitro transcriptional analysis (12). In the same study,
however, immunodepletion of LBP-1 (LSF) failed to show any effect,
suggesting that another factor(s) could bind to the same region of the
viral LTR (12).
YY1 is a multifunctional, nuclear zinc finger, 68-kDa protein
(15, 16, 17, 18) that can activate, repress, or initiate the
transcription of several cellular genes, including IFN-
, ß-casein,
c-fos, and c-myc, as well as the promoters of
several viruses, including CMV, Moloney murine leukemia virus, human
papilloma virus, and HIV-1 (19, 20). Of interest, YY1 was
shown to bind the same region (-17 to +27) in the HIV-LTR recognized
by LBP-1 (21). Recently, a heterodimeric complex composed
of the two transcription factors, YY1 and LBP-1 (LSF), was demonstrated
to bind to the -17 to +27 HIV-1-LTR element (22). In
addition, YY1 and LBP-1 were shown to cooperate in repressing HIV-LTR
expression in in vitro cotransfection experiments (22).
In contrast to the solid in vitro evidence of the involvement of YY1 and LBP-1 (LSF) in the modulation of HIV-LTR, no information on the state of expression and DNA binding of these transcriptional regulators in cells of HIV-infected individuals has been reported to date. Because LBP-1 (LSF)-DNA binding activity in T lymphocytes can be enhanced by mitogenic stimulation in vitro (11), we investigated whether in vivo IL-2 administration could somehow affect both LBP-1 (LSF) and YY1-DNA expression and binding activities in PBMC. In this regard, IL-2 is a cytokine responsible for the activation, proliferation, and differentiation of T lymphocytes and other immune cells (23) that has been previously shown to increase the absolute number of circulating CD4+ T cells in HIV+ individuals in a stable manner to normal or near normal levels (24, 25, 26, 27). However, transient peaks of HIV-1 replication after infusion of IL-2 in the absence of antivirals have been clearly documented (24), and since then, administration of the cytokine has been always associated with potent antiretroviral therapy (ART) (28). In the present study we investigated the state of activation of YY1 and LBP-1 (LSF) before, during, and after administration of IL-2/ART to HIV-infected individuals in vivo. We have observed that YY1- and LBP-1 (LSF)-DNA bindings are profoundly down-modulated by IL-2 administration; we also found that a proteolytic activity is induced in PBMC by administration of this cytokine in vivo, and it is probably responsible for the diminished expression of the two cellular transcription factors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six HIV-infected individuals (three women and three men; age,
21–60 years; average, 40 years) were chosen among patients enrolled in
a randomized phase II study of administration of rIL-2 (Proleukin,
Chiron, Emeryville, CA) after obtaining signed informed consent. All
HIV-seropositive individuals had CD4+ T cell
count between 200–500 cells/mm3 and were
antiviral-experienced at study entry. After enrollment, they received a
mixture of antivirals, consisting of a combination of a protease
inhibitor (PI) (Saquinavir) plus two reverse transcriptase inhibitors
(RTI) together with IL-2. All tested individuals but one received 3
million international units (MIU) of IL-2 twice daily s.c. for 5 days
of a 5-wk treatment cycle for a total of 12 cycles; one individual
received ART plus 15 MIU by continuous infusion for 5 days for two
cycles with an 8-wk interval, followed by four cycles of 7.5 MIU twice
daily for 5 days. The cumulative amount of IL-2 administered over 12 mo
was equal for all patients. These individuals belonged to an open-label
trial designed as a four-arm study enrolling 15 individuals/arm, in
which the control group received antiretrovirals (two RTI and one PI)
only. The results of the trial, fully supporting the superiority of
IL-2-containing regimens vs ART alone in terms of reconstitution of
physiologic levels of circulating CD4+ T
lymphocytes (Table I
), are being reported
separately (our manuscript in preparation). PBMC were isolated from
either peripheral venous blood of HIV-infected individuals or buffy
coats of healthy normal donors by Ficoll-Hypaque gradient sedimentation
(Pharmacia Biotech, Uppsala, Sweden), as previously described
(29). Aliquots of 1 x 106
cells were washed twice with RPMI 1640 (Life Technologies-BRL, Grand
Island, NY) and spun at 13,000 x g for 2–3 min, and
the pellets were stored at -80°C for further analyses.
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Affinity-purified rabbit polyclonal Ab raised against a C-terminal epitope of human YY1 (sc-281) was purchased from Santa Cruz Biotechnology (S. Cruz, CA); rabbit polyclonal Ab against human actin (A2066) was purchased from Sigma (St. Louis, MO).
Whole-cell extracts (WCE) and EMSA
WCE were prepared by repeated freeze-thaw cycles, as previously described (30). Briefly, the cell pellets were resuspended in high salt buffer C containing 0.1% Nonidet P-40, a mixture of protease inhibitors that included leupeptin (10 µg/ml), pepstatin A (10 µg/ml), aprotinin (33 µg/ml), E-64 (10 µg/ml), Pefabloc 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF; 1 mM), di-isopropylfluorophosphate (3 mM), and the phosphatase inhibitors sodium vanadate (1 mM), and sodium fluoride (50 mM). Following three cycles of freezing and thawing, cellular debris were pelleted by centrifugation at 1200 x g for 15 min at 4°C. The resulting supernatants (WCE) were stored at -80°C. The protein concentration was measured using a kit based on the Bradford method (Bio-Rad, Hercules, CA). EMSA was performed as previously described (30), with minor modifications, in that binding buffers appropriate for each specific oligonucleotide were used. In particular, for the LSF-280 probe, corresponding to the LSF binding site (+260 to +301) within the SV40 major late promoter (5'-ACA CAC ATT CCA CAG CTG GTT CTT TCC GCC TCA GAA GGT ACC TAA C-3') (10), a binding buffer containing 20% glycerol, 1.2% Nonidet P-40, 127 mM KCl, 8 mM Tris-HCl (pH 7.9), 20 mM HEPES (pH 7.9), 0.18 mM EDTA, and 0.86 mM DTT was used (11). For the UCR probe, corresponding to the YY1 binding site within the Moloney murine leukemia virus promoter (5'-CTG CAG TAA CGC CAT TTT GCA AGG CAT GAA-3') (18), a binding buffer containing 10% glycerol, 10 mM Tris-HCl (pH 7.9), 100 mM KCl, 5 mM MgCl2, and 1 mM DTT was adopted. For the prolactin-responsive element probe, located within the ß-casein promoter (TAG ATT TCT AGG AAT TCG) (31), a binding buffer containing 10 mM Tris (pH 7.5), 100 mM KCl, 5 mM MgCl2, 1 mM DTT, and 10% glycerol was used. The DNA-protein complexes were resolved on PAGE as previously described (30).
Immunoblot analyses
Immunoblot analyses were performed as previously described (30). Anti-YY1 and anti-actin Abs were diluted 1/2000 and 1/500, respectively, following the manufacturer’s instructions. HRP-conjugated anti-mouse or anti-rabbit secondary Abs were diluted 1/5,000 and 1/15,000, respectively. The signal was revealed by the enhanced chemiluminescence system (ECL, Amersham, Aylesbury, U.K.) following the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Binding of YY1/LBP-1 (LSF) complex to the HIV-1-LTR element has
been previously demonstrated using WCE obtained from uninfected cells
(22). Therefore, we have incubated WCE obtained from PBMC
of HIV+ individuals isolated both before and
during in vivo IL-2 administration with either the canonical YY1 DNA
binding element, UCR, present in the Moloney murine leukemia virus
promoter (18), or the canonical LBP-1 (LSF)-DNA binding
element, LSF-280, of the SV40 major late promoter. Strong binding of
YY1 to the UCR element was demonstrated before the initiation of IL-2
therapy (time zero; Fig. 1A),
whereas IL-2 administration was associated with a profound
down-modulation of YY1/UCR binding activity, which, however, returned
to a detectable level at suspension of cytokine administration
(intercycle; Fig. 1A). Of interest, the transient
disappearance of YY1 binding to the UCR during IL-2 administration was
much more evident in an individual (patient 1) who showed a greater
increase in circulating CD4+ T cells after five
cycles (1728 vs 477 cells/µl at baseline; with an absolute increase
of 3.6-fold, respectively) in response to IL-2 than in a second
individual (patient 2), who only moderately responded after 10 cycles
(with 731 vs 320 cells/µl at baseline; with an absolute increase of
2.2-fold, respectively). Of note, the lack of YY1 binding was already
present on day 1 of the sixth cycle of IL-2 administration in patient
1, whereas no substantial modulation of YY1 binding was simultaneously
observed in patient 2 (Fig. 1A). To exclude nonspecific degradation due
to improper preparation of WCE, selected time points have been tested
for DNA binding activity of the transcription factor STAT5, which we
have recently demonstrated to be constitutively activated in the
majority of HIV+ individuals (32).
No significant difference was observed before or after IL-2
administration in both patients (Fig. 1B). The analysis of
four other individuals revealed that three of them responded to IL-2
therapy similarly to patient 1, whereas weak binding activity and no
substantial modulation by IL-2 were observed in one individual (patient
6), as shown in Fig. 1C. These results demonstrate that IL-2
administration either directly or indirectly causes a reversible
disappearance of YY1-DNA binding. We next examined whether the LBP-1
(LSF)-DNA binding activity was also affected by IL-2 administration by
using the radiolabeled LSF280 oligonucleotide and WCE of patient 1. As
shown in Fig. 2, down-regulation of LBP-1
(LSF)-DNA binding activity mirrored that of YY1-UCR binding both during
cycle 1 and after cycle 6.
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IL-2 induces a proteolytic activity responsible for YY1-DNA binding down-modulation
We investigated whether the disappearance of YY1 DNA binding
activity in WCE of IL-2-treated individuals was caused by a proteolytic
activity. To test this hypothesis, WCE from a normal healthy donor
seronegative for HIV was mixed with UCR probe alone or in the presence
of increasing amounts of WCE from patient 1 obtained on day 5/cycle 1
either at room temperature or at 37°C for 30 min. Normal WCE showed a
band in EMSA corresponding to YY1 binding at both room temperature and
37°C (Fig. 3A, lanes
1 and 6, respectively), whereas WCE from the
IL-2-treated individual showed a band that migrated much faster than
that observed with the WCE of a normal donor (Fig. 3A,
lane 1 vs lane 11, respectively), probably
corresponding to a truncated form of YY1 (YY1
). At room temperature,
mixing different amounts of the WCE of the HIV+
and HIV- individuals resulted in a decrease in
the intensity of the YY1 band at all concentrations of IL-2-treated WCE
added, and, in parallel, in the appearance of a band migrating at the
same level of the IL-2-treated patient’s WCE (Fig. 3A,
lanes 2–5). At 37°C, the disappearance of the upper band
corresponding to full-length YY1 was almost complete at the highest
concentrations of IL-2-treated WCE (Fig. 3A), as
demonstrated by quantitative analysis performed with a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA; Fig. 3B).
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To assure that the proteolytic cleavage of YY1 indeed occurred in
individuals receiving IL-2, Western blot analyses of WCE from patient 1
was performed using an anti-YY1 rabbit polyclonal Ab. The single
sharp band corresponding to YY1 and visible on day 0 was no longer
detectable on days 3 and 5 of the first cycle of IL-2 administration
and on days 1, 3, and 5 of cycle 6, respectively (Fig. 4, upper panel). Several faint
bands smaller than the expected 68 kDa were recognized by the
anti-YY1 Ab, further supporting proteolytic cleavage of the
transcription factor. After stripping the anti-YY1 Ab, the membrane
was reprobed with an anti-actin polyclonal rabbit Ab. At all time
points, a single band was clearly detectable (Fig. 4, lower
panel), demonstrating that the IL-2-induced degradation was
relatively specific for YY1. In addition, Western blot analyses were
performed using WCE obtained from PBMC of HIV-seronegative healthy
donors that were either left unstimulated or were stimulated in vitro
for 1, 3, and 5 days with IL-2 (20 U/ml). The expression of YY1 protein
during this in vitro culture period remained unaltered (data not
shown).
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We finally evaluated whether LBP-1 (LSF) was proteolytically
cleaved by IL-2 administration by performing WCE-mixing EMSA. A
concentration-dependent disappearance of the upper canonical band was
indeed observed as a function of increasing the concentration of WCE
from the HIV-infected individual. However, in contrast to what was
observed for YY1, the disappearance of LBP-1 (LSF)/DNA binding was
clearly concentration dependent at both room temperature and 37°C,
although the effect was much stronger at 37°C than at room
temperature (Fig. 5A).
Quantitative analysis indeed demonstrated an almost complete lack of
LBP-1 (LSF)-DNA binding at the two highest concentrations of
IL-2-treated patients’ WCE at 37°C (Fig. 5B).
Unfortunately, we could not assess LBP-1 (LSF) expression, because the
amount of WCE obtained from the HIV-infected patients required for the
detection of the LBP-1 isoforms was not sufficient to produce a
detectable signal in Western blot experiments (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Thus, the down-modulation of YY1 and LBP-1 (LSF) seems more
likely caused by a soluble factor(s) induced by IL-2 administration or
by a different activation state of the PBMC rather than by a different
redistribution of cell subsets.
Evidence that WCE of IL-2-treated individuals contain a proteolytic
activity that is responsible for YY1 degradation are here shown based
on the results of the EMSA mixing experiments, in which the DNA binding
of normal WCE was lost when IL-2-treated WCE was added to the reaction
before the addition of the probe. By Western blot and EMSA
analyses, we demonstrated that YY1 cleavage was relatively
specific, because actin was intact and STAT5 binding activity
unaltered, respectively, at all time points of IL-2 administration
tested. In support of this hypothesis, a higher reduction of
full-length YY1 binding to the DNA was observed when the extracts were
incubated at 37°C. Moreover, quantitative analysis of the EMSA bands
revealed that in contrast to the full-length YY1-UCR, the appearance of
the truncated YY1-UCR band was dependent on the amounts of IL-2
extracts added at both room temperature and 37°C, very likely as a
consequence of the contribution of the truncated YY1 present in the
IL-2-treated WCE. Conversely, quantitative analysis of the LBP-1
(LSF)-DNA binding activity demonstrated that the disappearance of the
LBP-1 (LSF)-DNA binding was concentration dependent at both
temperatures, although the effect was more evident at 37°C. One
possible explanation is that the proteolytic activity required for YY1
degradation is not identical with that responsible for LBP-1 (LSF)
cleavage and is still fully active at room temperature. Alternatively,
the concentration-dependent decrease in LBP-1 (LSF)-DNA binding
activity at room temperature might also depend upon a specific
inhibitor of LBP-1 (LSF)-DNA binding induced in WCE of HIV-infected
individuals receiving IL-2. In this regard, LBP-1d, lacking the DNA
binding domain, was, however, shown to be capable of squelching the
binding of the other LBP-1 isoforms by protein-protein interaction
(12). Thus, we cannot rule out the possibility that in
vivo IL-2 administration may result in a superinduction of either the
LBP-1d isoform or analogous inhibitors.
The nature of the proteolytic activity present in IL-2-treated WCE is unknown at present, but it is very unlikely attributable to the HIV-associated protease for several reasons. First, the IL-2-treated patients were all receiving two RTI and one PI. Although beyond the scope of the present study, this antiviral regimen resulted in a sustained decrease in viremia (from 947 to <400 copies/ml at baseline and after 2 mo of therapy in patient 1, respectively; our manuscript in preparation). Furthermore, no increases in plasma viremia were observed during IL-2 administration. Second, the incubation of WCE from a normal healthy donor with WCE obtained from an HIV+ individual receiving IL-2 but in whom YY1 was not degraded produced no reduction in either YY1- or LBP-1 (LSF)-DNA binding activities, suggesting that the effect was strictly dependent on IL-2 administration. Finally, only a minority of cells (1:100–10,000) are known to be infected in individuals at any particular time point (33, 34), and it is extremely unlikely that the viral protease present in these cells may override the pattern of protein expression of uninfected cells.
Among many genes transcriptionally regulated by YY1, the 
-actin
genes have been shown to be repressed by this transcription factor in
analogy with the effect on the HIV-LTR (35). Of interest,
proteolytic degradation of YY1 has been correlated with myogenic
differentiation (35). In these cells, cleavage of YY1
during the myoblast-myotube transition allowed the expression of the
sarcomeric -actin genes, thus facilitating muscle development
(35). In the same system the
Ca2+-dependent cysteine protease m-calpain has
been clearly demonstrated as the enzyme responsible for the
post-translational mechanism of YY1 down-regulation (35).
Of note, the levels of this endopeptidase were also found increased
after activation of T lymphocytes (36). Furthermore, in
vitro activation-induced programmed cell death in PBMC from
HIV+ individuals has been shown to be
specifically inhibited by calpain inhibitor II, suggesting that a
calpain-dependent apoptosis might contribute to HIV-associated
immunodeficiency (37). Although we did not observe YY1
degradation after IL-2 stimulation of patients’ PBMC in vitro, we
speculate that activation of circulating T cells of
HIV+-infected individuals by in vivo
administration of IL-2 may increase the concentration of m-calpain,
resulting in cleavage of YY1 and/or LBP-1 (LSF).
In conclusion, our findings are of potential interest for understanding the biochemical and immunological correlates of IL-2 administration to HIV-infected individuals. In this regard, it has been previously emphasized that one potential drawback of IL-2 administration observed in HIV-infected individuals was the transient increase in plasma HIV RNA levels frequently observed at the end of each cycle of i.v. infusion of IL-2 in the absence of ART (24). Our findings may provide a biochemical correlate of this phenomenon, i.e., the decreased expression or activity of two HIV transcriptional repressors such as YY1 and LBP-1 (LSF). Of note is the fact that increases in plasma viremia were not observed in any of our patients who received IL-2 by either s.c. or continuous infusion routes in the presence of ART (our manuscript in preparation). In addition, the copy numbers of both spliced and unspliced RNA, measured by competitive RT-PCR (38), remained unchanged in patients 2 (with 450 and 500 copies of unspliced RNA and 526 and 500 copies of fully spliced RNA/µg of total RNA after IL-2 vs baseline, respectively; data not shown), but were below the threshold of detection in patient 1 (40 copies of RNA/µg of total RNA) both before and after IL-2 treatment (data not shown), leaving open the question of whether IL-2 and IL-2-induced modulation of YY1 and LBP-1 (LSF) can significantly regulate HIV transcription in vivo in the presence of potent antivirals. Despite the fact the IL-2 is being considered as one of the most promising therapeutic agents to be associated with antiviral mixtures, little is known of its biological effects on HIV-infected individuals. Here, by showing a potential correlation between in vivo effects of IL-2 and down-modulation of YY1 and LBP-1 (LSF), we provide evidence that molecular markers can be found and should be further investigated to define novel sensitive correlates of the response to immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chiara Bovolenta, P2/P3 Laboratories, San Raffaele Scientific Institute, via Olgettina n. 58, 20132 Milan, Italy. E-mail address:
3 Abbreviations used in this paper: LTR, long terminal repeat; ART, antiretroviral therapy; YY1, ying yang-1; LBP-1, leader binding protein-1; LSF, late SV40 factor; PI, protease inhibitor; RTI, reverse transcriptase inhibitor; MIU, millions of international units; WCE, whole cell extract region; UCR, upstream conserved
Received for publication May 18, 1999. Accepted for publication September 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel transcription factors. Microbiol. Rev. 59:481.
), that binds to the immunoglobulin 3' enhancer and the immunoglobulin heavy-chain µ E1 site. Proc. Natl. Acad. Sci. USA 88:9804.- but not of IFN--induced JAK/STAT pathway in a subset of U937 clones prevents the antiretroviral effect of IFN- against HIV-1. J. Immunol. 162:323.This article has been cited by other articles:
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