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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2862-2870
Expression of Human Inducible Nitric Oxide Synthase Gene in T-Cell
Lines Infected With Human T-Cell Leukemia Virus Type-I and Primary
Adult T-Cell Leukemia Cells
By
Naoki Mori,
Youichi Nunokawa,
Yasuaki Yamada,
Shuichi Ikeda,
Masao Tomonaga, and
Naoki Yamamoto
From the Department of Preventive Medicine and AIDS Research,
Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan;
the Department of Laboratory Medicine, Nagasaki University School of
Medicine, Nagasaki, Japan; the Department of Hematology, Atomic Disease
Institute, Nagasaki University School of Medicine, Nagasaki, Japan;
City of Sasebo General Hospital, Sasebo, Japan; and Suntory Institute
for Biomedical Research, Osaka, Japan.
 |
ABSTRACT |
We examined the expression of messenger RNA (mRNA) of the human
inducible nitric oxide synthase (hiNOS) gene in a panel of human T-cell lines. Reverse transcriptase-polymerase chain reaction showed that human T-cell leukemia virus type-I (HTLV-I)-infected T-cell lines (MT-1, SLB-1, and C5/MJ) expressed mRNA for the hiNOS, but
TL-Om1 or uninfected Jurkat, H9, and CCRF-CEM did not. The MT-1, SLB-1,
and C5/MJ cell lines are infected with HTLV-I and express the viral
transactivator Tax, whereas TL-Om1 cells, although derived from adult
T-cell leukemia (ATL) leukemic cells, do not express Tax. There was,
thus, a correlation between Tax and hiNOS mRNA expression. The
transcriptional regulatory region of the hiNOS gene was
activated by Tax in Jurkat, in which endogenous hiNOS is induced by
Tax. Deletion analysis showed that the region of hiNOS encompassing
nucleotides  159 to 111 contained the minimum Tax-responsive
elements. Mutations in the NF- B element at position 115 and
106 bp in the hiNOS promoter were still activated by Tax, and a Tax
mutant defective for activation of the NF-B pathway retained the
ability to activate the hiNOS promoter. In addition, overexpression of
the dominant-negative mutants of IB and I B failed to
reduce Tax-induced activation of hiNOS gene. Furthermore, hiNOS
mRNA was detected in leukemic cells from ATL patients. Our results show
that the hiNOS promoter contains a minimum Tax-responsive element
located between nucleotides 159 and 111, and imply that the
expression of the hiNOS gene is involved in the pathogenesis of
HTLV-I-associated diseases.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN T-CELL LEUKEMIA virus type-I
(HTLV-I) is the etiologic agent of adult T-cell leukemia
(ATL).1,2 HTLV-I may also be associated with several
chronic inflammatory diseases of presumed autoimmune etiology, such as
HTLV-I-associated myelopathy/tropical spastic
paraparesis,3,4 HTLV-I-associated
arthropathy,5 uveitis,6
polymyositis,7 and Sjögren's syndrome.8
The possible roles of abnormal immune activation and cytokine
overproduction in the pathogenesis of diseases caused by HTLV-I has
been suggested, but the mechanism by which they cause these diseases
remains unknown.
Nitric oxide (NO) is an intercellular and intracellular messenger
engaged in the function of virtually every mammalian organ system. Its
functional repertoire includes inflammation and antimicrobial defense,
in addition to neurotransmission and vascular homeostasis.9 The enzyme, NO synthase (NOS), synthesizes NO from L-arginine. Three
isoforms of NOS have been identified, which conform to two biochemical
profiles. The neuronal and endothelial isoforms are calcium/calmodulin-dependent and are expressed constitutively (cNOS),
whereas the macrophage isoform is calcium-independent and is expressed
after transcriptional induction by several stimuli (iNOS).10,11 Induction of iNOS has been reported in human
cells, including macrophages,12 neutrophils,13
hepatocytes,14 vascular smooth muscles,15
megakaryoblasts,16 chondrocytes,17
keratinocytes,18 and the human colonic adenocarcinoma cell
line.19 In human T cells, however, little is known about
the expression of human iNOS (hiNOS) mRNA.
hiNOS may contribute to the pathogenesis of HTLV-I-associated diseases
if it were expressed in HTLV-I-infected T cells. In the present study,
we showed that hiNOS mRNA is expressed in leukemic cells of ATL
patients as well as in some HTLV-I-infected T-cell lines. We have
previously shown that interleukin (IL)-1 induces hiNOS gene
expression via NF-B activation in human tumor cell lines.20 The Tax protein encoded by the pX region of HTLV-I is essential for viral gene expression by driving its transcription through the cAMP-responsive element (CRE) in the viral long terminal repeat.21 Furthermore, Tax is known to transactivate many
cellular genes through a number of cis-acting DNA elements including
NF-B and serum responsive element (SRE).21 The hiNOS
promoter contains consensus binding sequences for several of these
factors, implying but not confirming that they contribute to
Tax-induced hiNOS activation. The studies described here were designed
to identify the molecular mechanism of hiNOS activation after
HTLV-I infection in T cells. We report for the first time that Tax
induces hiNOS promoter in T cells.
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MATERIALS AND METHODS |
Cell lines.
Human T-cell lines Jurkat, H9, CCRF-CEM, and HTLV-I-infected T-cell
lines MT-1, SLB-1, C5/MJ, TL-Om1 were grown in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum (FBS),
penicillin G (50 U/mL), and streptomycin (50 µg/mL) in a humidified
incubator containing 5% CO2 in air. JPX-9 and JPX-9/M (kindly provided by Dr M. Nakamura, Tokyo Medical and Dental
University, Tokyo, Japan) are subclones of Jurkat cells, expressing Tax
and nonfunctional Tax, respectively, under the control of
metallothionein promoter.22
Patient samples.
The study protocol was approved by the Human Ethics Review Committees
of the participating institutions. Leukemic cells from eight patients
diagnosed with acute-type (n = 5; patients 3, 5 to 8) and chronic-type
ATL (n = 3; patients 1, 2, and 4) were analyzed. The diagnosis was
based on clinical features, hematological findings, and serum
anti-HTLV-I antibodies. Monoclonal HTLV-I provirus integration into
the DNA of leukemic cells was confirmed by Southern blot hybridization
in all cases (data not shown). Mononuclear cells were isolated by
Ficoll/Hypaque (Pharmacia LKB, Uppsala, Sweden) with density gradient
centrifugation, washed with phosphate-buffered saline (PBS), and
further incubated at 37°C for 2 hours in plastic culture dishes to
remove adherent cells. This solution was then removed from the dishes,
and cells were washed with PBS.
Determination of nitrite and nitrate concentrations.
To assess the amount of NO produced, the culture supernatants were
assayed by measuring the accumulated stable degradation products,
nitrite, and nitrate, using the 2,3-diaminonaphthalene kit (Dojin
Chemicals, Kumamoto, Japan). Fluorescence intensity was measured
with a Fluoroscan II (Labsystems, Helsinki, Finland).
Reverse transcriptase-polymerase chain reaction (RT-PCR).
Total RNA was extracted by using Trizol (GIBCO-BRL, Gaithersburg, MD),
according to the protocol provided by the manufacturer. First-strand
complementary DNA (cDNA) was synthesized in a 20 µL reaction volume
using an RNA-PCR kit (Takara Shuzo, Kyoto, Japan) with random primers.
Thereafter, cDNA was amplified for 30, 28, and 28 cycles for hiNOS,
Tax, and -actin, respectively. The oligonucleotide primers
used were as follows: for hiNOS, sense 5'-CGGTGCTGTATTTCCTTACGAGGCGAAGAAGG-3', antisense
5'-GGTGCTGCTTGTTAGGAGGTCAAGTAAAGGGC-3'; for Tax, sense
5'-CCCACTTCCCAGGGTTTGGACAGA-3', antisense
5'-CTGTAGAGCTGAGCCGATAACGCG-3'; for -actin, sense
5'-GTGGGGCGCCCCAGGCACCA-3', antisense
5'-CTCCTTAATGTCACGCACGATTTC-3'. Product sizes were: 259, 203, and 548 bp, respectively. Cycling conditions were as follow:
denaturing at 94°C (45 seconds for hiNOS and 30 seconds for Tax and
-actin), annealing at 60°C (45 seconds for hiNOS and 30 seconds
for Tax and -actin), and extension at 72°C (120 seconds for
hiNOS and 90 seconds for Tax and -actin). The PCR products were
fractionated on 2% agarose gel and visualized by ethidium bromide staining.
Detection of iNOS protein by fluorescence-activated cell sorting
(FACS) analysis.
The presence of iNOS was analyzed by flow cytometry of permeabilized
cells. Briefly, 106 cells were fixed with cold 4%
paraformaldehyde for 20 minutes, then washed with PBS containing 0.1%
sodium azide and suspended in 50 µL of permeabilization buffer (PBS
with 0.1% sodium azide, 1% heat-inactivated FBS, and 0.1% saponin).
Cells were incubated at 4°C with an fluorescein isothiocyanate
(FITC)-labeled anti-iNOS monoclonal antibody (MoAb) (FITC-macNOS, clone
6; Transduction Laboratories, Lexington, KY) and IgG2a-FITC
as control isotype for 45 minutes, washed with permeabilization buffer,
suspended in PBS, and analyzed by flow cytometry. The anti-iNOS MoAb
(clone 6) is directed against a protein fragment corresponding to the amino acids 961 to 1,144 of murine iNOS and recognize the human enzyme.
Western blot analysis.
Cellular lysates were fractionated by 10% sodium dodecyl
sulfate-polyacrylamide gel, electrophoretically transferred to
polyvinylidine difluoride membranes (Immobilon-P; Millipore Corp,
Bedford, MA), and then analyzed for immunoreactivity with a mouse
anti-Tax antibody, Lt-4 (provided by Dr Y. Tanaka, University of the
Ryukyus, Okinawa, Japan),23 and horseradish
peroxidase-conjugated sheep antimouse IgG (Amersham Life Science Inc,
Arlington Heights, IL) with an enhanced chemiluminescence detection
system (ECL; Amersham Life Science Inc).
Plasmids.
To construct the hiNOS reporter gene constructs, the 3.2 kb putative
promoter region cloned into pUC118 was liberated as a Kpn
I/Xba I fragment and ligated into the Kpn I/Nhe
I site of a pGL3-basic plasmid (Promega, Madison, WI), which contained
the luciferase gene as a reporter and the gene for the poly(A)
signal sequence of late SV40 (pGLNOSa).20 A series of
deletion constructs were prepared by cloning various restriction
fragments of the hiNOS promoter into the pGL3-basic plasmid described
previously.20 The four shortest constructs (pGLNOS-0.37,
0.33, 0.16, 0.11) were constructed by PCR using upstream primers
that begin at specific nucleotides within the promoter, and a common
downstream primer containing Nco I site of the pGL3-basic. The
PCR fragments were cloned into Kpn I/Nco I site within
the plasmid. The forward upstream primers containing the Kpn I
site were designed as follows:
5'-GGGAGGTACCAGAGCTCCCTGCTGAGGAAA-3' for pGLNOS-0.37,
5'-GGGAGGTACCAAGTGAGGCCATGTGGCTT-3' for pGLNOS-0.33, 5'-GGGAGGTACCCCTGGCAGTCACAGTCATA-3' for pGLNOS-0.16,
5'-GGGAGGTACCACTCCCTTTGGAAACC-3' for pGLNOS-0.11. The
sequence of the common downstream primer was
5'-TGGCGTCTTCCATGGTGGCTTTACCAAC-3'. The mutated construct in the putative NF-B binding site was generated from the pGLNOSa plasmid using a site-directed mutagenesis kit.20 The
pH2R40M (+Tax) plasmid (kindly provided by Dr M. Hatanaka, Shionogi
Institute for Medical Science, Osaka, Japan) was a Tax expression
plasmid containing the SV40 sequences including the SV40 promoter and the SV40 polyadenylation signal, an R fragment of HTLV-I long-terminal repeat, and a neomycin-resistant gene under control of the SV40 promoter.24 The pH2Rneo (Tax) plasmid was used as a
negative control. Tax wild-type (TaxMT-2) and mutant expression vectors (TaxM22 and Tax703) were generously provided by Dr K. Matsumoto (Osaka
Red Cross Blood Center, Osaka, Japan). These genes were cloned into pH
Pr0.1-neo, which has a -actin promoter for protein expression.
The Tax mutants TaxM22 and Tax703 have been characterized previously.25 NF-B p65 expression vector was reported
previously.26 Deletion mutants of IB
(IB N)27 and IB (IBN)28
lacking the NH2-terminal 36 amino acids and 23 amino acids,
respectively, were cloned into the pCMV4 polylinker (kindly provided by
Dr D.W. Ballard, Vanderbilt University School of Medicine, Nashville, TN). B-LUC, WT-LUC (provided by Dr J. Fujisawa, Kansai Medical University, Osaka, Japan), and pCArG-LUC (provided by Dr K. Shimotohno, Kyoto University, Kyoto, Japan) are luciferase expression plasmids regulated by five copies of the B element from the IL-2 receptor (IL-2R) gene, five copies of the 21-bp viral
enhancer (CRE), a monomer of the CArG box from the c-fos gene, respectively.
Cell transfection and luciferase assays.
Electroporation of cells was performed as previously
described.29 After 24 hours of incubation, cell lysates
were prepared and luciferase activity was measured. Relative luciferase
activities were normalized to the amount of intracellular protein.
Results were confirmed by at least three independent transfections.
DNA fragmentation.
106 cells were pelleted and lysed for 10 minutes at 4°C
in 10 mmol/L Tris-HCl, pH 7.4, 0.5% Triton X-100, 10 mmol/L EDTA.
After centrifugation at 15,000 rpm for 20 minutes, the supernatant was treated for 1 hour at 37°C with RNase and then treated with 0.4 mg/mL of proteinase K for 1 hour at 37°C. DNA was precipitated with
0.5 mol/L NaCl and 1 vol of isopropanol at 20°C overnight. Purified DNA was dissolved in 20µL of TE (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0) buffer, loaded on a 2% agarose gel and separated
by electrophoresis. Separated DNA was then visualized by staining with
0.5 µg/mL ethidium bromide.
| |
RESULTS |
Constitutive expression of hiNOS mRNA in Tax-expressing T-cell lines
infected with HTLV-I.
To determine whether hiNOS expression in T cells correlates with HTLV-I
infection, RNA was extracted from HTLV-I-infected or uninfected human
T-cell lines, and expression of hiNOS gene was analyzed by
RT-PCR using hiNOS-specific primers. Three HTLV-I-infected cell lines
expressed the viral transactivator Tax (Fig
1, lane 4, C5/MJ; lane 5, SLB-1; lane 6, MT-1). The hiNOS transcript was also detected in these three cell
lines, whereas it was undetectable in the uninfected lines, Jurkat, H9,
and CCRF-CEM (lanes 1 to 3). The other infected cell line, TL-Om1,
without Tax transcript, also did not express hiNOS (lane 7). These
results show a plausible correlation between hiNOS expression and the
viral transactivator Tax expression, suggesting that Tax is responsible
for the stimulated transcription of hiNOS gene similar to other
Tax-responsive cellular genes.
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| Fig 1.
Detection of hiNOS mRNA in Tax-expressing T-cell lines
infected with HTLV-I by RT-PCR analysis. RT of cellular RNA (1 µg
total RNA) and PCR were performed. One tenth of the PCR mixture was
analyzed by ethidium bromide staining of 2% agarose gel. Agarose gel
electrophoresis of DNA amplified by hiNOS, Tax, and -actin primers.
M: size markers. The band of specific mRNA for hiNOS is 259 bp, Tax of
HTLV-I is 203 bp, and -actin as internal control is 548 bp. The
three T-cell lines, Jurkat, H9, and CCRF-CEM, are negative for HTLV-I.
Other T-cell lines are positive for HTLV-I. Arrows indicate the
position of the specifically amplified DNA.
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Detection of iNOS protein and NO oxidation products nitrite and
nitrate in Tax-expressing T-cell lines infected with HTLV-I. Cell lines detected hiNOS transcript were analyzed for the
intracytoplasmic expression of iNOS proteins by direct
immunofluorescence FACS analysis on permeabilized cells. Tax-expressing
T-cell lines infected with HTLV-I were significantly labeled with the
anti-iNOS MoAb (clone 6) directly conjugated to FITC. The percentage of
positive cells was 68%, 43%, and 13% in C5/MJ, SLB-1, and MT-1
cells, respectively. Furthermore, the supernatants from C5/MJ, SLB-1,
and MT-1 cells cultured for 48 hours contained readily detectable
levels of the NO oxidation products nitrite and nitrate release (9.6, 6.4, and 3.1 µmol/L, respectively).
Expression of hiNOS mRNA in ATL.
RT-PCR assay was performed to evaluate the expression of hiNOS
gene on leukemic T cells derived from patients with ATL. In eight of
eight cases, we observed a significant expression of hiNOS mRNA (Fig
2), indicating that this gene is abnormally
expressed in ATL. Furthermore, similar results were obtained with fresh leukemic cells in the absence of any culture (data not shown), indicating that hiNOS mRNA expression was not a result of stimulation by components in the culture medium. In contrast, hiNOS was not expressed in peripheral blood lymphocytes (PBL) from three normal donors. The intensity of the RT-PCR product of hiNOS did not correlate with the level of expression of Tax in ATL.
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| Fig 2.
Detection of hiNOS and Tax mRNA in fresh ATL samples by
RT-PCR analysis. Lanes 1-3, normal PBL RNA; Lanes 4-11, PBL RNA samples
from patients with ATL. Bottom, ethidium bromide staining of the RT-PCR
performed with -actin primers. Arrows indicate the position of the
specifically amplified DNA.
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Tax induces expression of hiNOS gene.
Expression of hiNOS in HTLV-I-infected T cells strongly
suggested that its expression might be directly transactivated by the
tax gene product of HTLV-I, because Tax can activate the
transcription of cellular genes as well as the HTLV-I genome. To
further evaluate the effect of Tax on the expression of hiNOS,
we next used two inducible Jurkat Tax transfectants, JPX-9, which
generates wild-type Tax, and JPX-9/M, which produces a nonfunctional
Tax mutant after the addition of CdCl2.22
Western blot analysis showed that the addition of CdCl2 to
the culture medium induced Tax protein in JPX-9 cells (Fig
3A). Expression of hiNOS gene was
assayed by RT-PCR performed on total RNA extracted after a 48-hour
culture of cells either in medium alone or in medium containing 20 µmol/L CdCl2. As shown in Fig 3B, hiNOS mRNA expression
was detected in JPX-9 treated with CdCl2 but not in the
mutant JPX-9/M. These data suggest that the Tax protein is involved in
the high levels of hiNOS transcript observed in HTLV-I-infected T
cells.
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| Fig 3.
Effect of Tax expression on hiNOS mRNA induction. (A)
JPX-9 cells were incubated with various concentrations of
CdCl2 for 48 hours and then collected for preparation of
whole-cell extracts. Whole-cell extracts isolated from JPX-9 cells were
subjected to immunoblotting with anti-Tax antibody. (B) PCR analysis of
reverse-transcribed hiNOS mRNA in JPX-9 and JPX-9/M cells cultured in
medium alone () or medium containing 20 µmol/L CdCl2
(+) was performed with the hiNOS oligonucleotide primer pair.
Amplified products were electrophoresed.
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Analysis of hiNOS promoter activity in HTLV-I-infected T-cell lines.
To determine the promoter activity of hiNOS, we transiently
transfected the pGLNOSa plasmid, which contains the 3.2 kb putative promoter of hiNOS, into T-cell lines. The promoter activity was expressed as the fold induction compared with that of HTLV-I negative control Jurkat. The reporter activity of the pGLNOSa construct was
17.9-fold higher in C5/MJ, 16.1-fold higher in SLB-1, and 9-fold higher
in MT-1, compared with Jurkat (Fig 4). In
contrast, when the same construct was transfected into another HTLV-I
negative T-cell lines (H9 and CCRF-CEM) and an HTLV-I-infected T-cell
line, TL-Om1, which does not express Tax, the level of reporter
activity was similar to that seen in Jurkat (Fig 4). These results are in agreement with the data of hiNOS mRNA expression shown in Fig 1.
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| Fig 4.
Reporter assays of the hiNOS promoter construct pGLNOSa
in HTLV-I-infected and uninfected T-cell lines. Five micrograms of the
pGLNOSa construct were transfected. Luciferase activity was normalized
to protein content. The promoter activity is represented as fold
induction relative to that of the HTLV-I negative control Jurkat. Data
represent the mean ± SEM of three experiments.
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Transactivation of hiNOS promoter by HTLV-I Tax.
To examine whether the hiNOS promoter responds to Tax, we assayed
luciferase activity in the cells. After transfecting the pGLNOSa
plasmid with or without a Tax expression plasmid, we measured the
luciferase activity expressed in cells. Luciferase expression was
enhanced 6.1-fold by the Tax plasmid (Fig
5A). Therefore, the promoter of hiNOS was
transactivated by the viral transactivator Tax. This could be one
mechanism by which HTLV-I-infected cells express the hiNOS
gene.
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| Fig 5.
(A) Activation of the hiNOS promoter by HTLV-I
tax gene. The described hiNOS promoter constructs (1 µg) were
transiently cotransfected with 5 µg of pH2R40M (+Tax) or with 5 µg of pH2Rneo (Tax) into Jurkat cells. Luciferase activity
normalized by protein content was expressed as the mean ± SEM of at
least three experiments. The relative luciferase activity was
calculated from the luciferase activity of each reporter plasmid
relative to the value of pGLNOS-0.11, cotransfected with pH2Rneo and
assigned a value of 1.0. The induction ratio represented the average
stimulated value divided by average unstimulated value. (B) Effect of
point mutation on the inducibility of luciferase activity. pGLNOSa or
pGLNOSa bearing a 3-bp mutation in the NF-B site (pGLNOSBm) were
cotransfected into Jurkat cells with 5 µg of pH2R40M (+Tax) or 5 µg of pH2Rneo (Tax). The NF-B site (115 to 106;
GGGACACTCC) in the hiNOS promoter was mutated to CTCACACTCC.
Tax-dependent increase in reporter gene activity was expressed as a
percentage of the value of pGLNOSa cotransfected with pH2R40M. Data
represent the mean ± SEM of three experiments.
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To identify the regions of hiNOS promoter that are important for the
Tax-induced activity in Jurkat, a series of deletion constructs were
established with progressively smaller fragments of the 5'
flanking sequence, and then transiently transfected into Jurkat cells
(Fig 5A). The luciferase activity fluctuated between 1,088 and
419: the expression level reached the maximum with construct
pGLNOS-1.1 and decreased about twofold with construct pGLNOS-0.4. These
results suggested that there are several positive regulatory elements
present between 1,088 and 419. The pGLNOS-1.8, 1.1, 0.4, 0.37, 0.33, and 0.16 deletion constructs manifested Tax-induced
luciferase activity. With pGLNOS-0.11, in which the region between
nucleotides 159 to 111 of pGLNOS-0.16 construct is
deleted, no induction of luciferase activity by Tax was observed, indicating that this region of the hiNOS promoter is essential for
Tax-induced transcriptional activity. Furthermore, these serial deletions at the 5' end of the hiNOS construct were transfected into the C5/MJ cells as a representative of HTLV-I-positive cell lines
(Fig 6A). Removal of the region between
nucleotides 159 and 111 in the pGLNOS-0.16 construct
reduced the reporter activity (Fig 6A), indicating that this region of
the hiNOS promoter is required for its basal transcriptional activity
in HTLV-I-positive cells.
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| Fig 6.
(A) Reporter assay of hiNOS promoter deletion constructs
in C5/MJ, an HTLV-I-infected T-cell line. The relative luciferase
activity was calculated from the luciferase activity of each reporter
plasmid relative to the value of pGLNOS-0.11 and assigned a value of
1.0. (B) Effect of point mutation on hiNOS basal transcriptional
activity in C5/MJ cells. Values represent the mean ± SEM of three
experiments.
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NF-B binding site is not essential for activation by Tax.
Between 115 and 106 bp of the hiNOS promoter fragment,
there is a consensus sequence for the NF-B binding site,
5'-GGGACACTCC-3', which is reportedly essential for the
cytokine-induced expression of hiNOS gene.20 Since
the Tax-responsive cellular enhancer also contains an
NF-B-responsive element, this site may be a Tax-responsive element.
Furthermore, because the NF-B binding site at 115 and
106 bp is destroyed in the reporter construct pGLNOS-0.11, the
results of the deletion analysis suggested that the NF-B site was
involved in Tax transactivation of the hiNOS promoter. Substitution
mutations were introduced into this site of the pGLNOSa plasmid to
examine its role on Tax transactivation. However, the substitution in
the NF-B site (Fig 5B, pGLNOSBm) did not affect Tax-induced
activation. To evaluate that the NF-B site within the hiNOS promoter
is functional, the DLD-1 cells, a human colorectal adenocarcinoma cell
line, were transfected with the wild-type pGLNOSa construct. The
transfected cells showed the inducibility of the reporter activity
after IL-1 stimulation (2.1-fold induction). These cells transfected
with the mutant pGLNOSBm construct completely failed to respond to
IL-1. These results show that the NF-B binding site is
dispensable for Tax-induced expression. Furthermore, we transfected
pGLNOSBm into C5/MJ cells. However, mutation in NF-B site did not
abrogate hiNOS promoter activity (Fig 6B).
Tax mutants that are defective for activation of the
NF-B, CRE, and SRE pathways activate the hiNOS
promoter.
Tax is known to activate promoters containing CRE, SRE, or NF-B
elements. To confirm that NF-B binding site within the hiNOS
promoter was not required for Tax activation, Tax mutants that failed
to activate NF-B (TaxM22)-CRE, (Tax703)-, or SRE (Tax703)-dependent
promoters25 were analyzed to examine their ability to
activate the pGLNOSa promoter construct (Fig
7B). Both TaxM22 and Tax703 activated the
pGLNOSa promoter to levels similar to those observed with the wild-type
Tax (Fig 6B). Controls for these experiments showed that Tax703, but
not TaxM22, transactivated B-LUC (Fig 7A). Conversely, Tax mutant
M22, but not 703, induced luciferase expression from WT-LUC and
pCArG-LUC (Fig 7A).
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| Fig 7.
Tax transactivation of the hiNOS promoter is independent
of NF-B element. (A) Reporter luciferase plasmids with B
(B-LUC), CRE element (WT-LUC), or CArG box (pCArG-LUC) were used as
control promoters for Tax mutants. Luciferase activity was determined
after incubation for 24 hours and normalized for protein content. The
activity of Tax mutants, M22 and Tax703, relative to that of the
wild-type Tax, was calculated by comparing luciferase activity in the
presence of M22 or Tax703 with that in the presence of wild-type Tax.
(B) Jurkat cells were transfected with pGLNOSa together with expression
plasmids encoding Tax, its indicated mutants, or the parental
expression vector with no insert (Control). Data represent the mean ± SEM of three experiments.
|
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Overexpression of dominant-negative mutants of
IB and
IB fail to reduce Tax-induced
activation of hiNOS gene.
The cytoplasmic inhibitor IB is responsible for
sequestering NF-B subunits in the cytoplasm by masking the nuclear
localization signals. Previous studies showed that both IB and
IB mutants in their NH2-terminal region escape from
proteolytic breakdown and prevent NF-B activation in Tax-expressing
cells.27,28 To further confirm the role of NF-B in the
regulation of hiNOS, transient cotransfections were performed
with a mutant of IB (IBN) or IB (IBN)
lacking the NH2-terminal amino acids. As shown in Fig
8, both IBN and IBN
abolished p65-dependent transcription of B-LUC, containing a
luciferase gene under the control of the NF-B binding site
of IL-2R gene. Furthermore, both IBN
and IBN also inhibited Tax-induced transcription from B-LUC
(Fig 8). To evaluate the specificity of this interaction, either
IBN or IBN effector plasmid was cotransfected with Tax and a distinct Tax-responsive reporter construct. In sharp contrast
to -LUC, both IBN and IBN failed to significantly downregulate transcription directed from WT-LUC, which responds to Tax
by five repeats of a 21-bp viral enhancer (CRE). Tax-induced transcription from the hiNOS promoter was not reduced in cells expressing both IBN and IBN (Fig 8). Considered
together, these results show that Tax activation of the hiNOS promoter
does not depend on the NF-B pathway. Thus, Tax activation of the
hiNOS promoter is likely to involve a previously uncharacterized
Tax-responsive element.
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| Fig 8.
Failure of repression of Tax-dependent transcription from
the hiNOS promoter by the dominant-negative mutants of IB and
IB. Jurkat cells were transfected with 5 µg of a p65 or Tax
expression vector, 5 µg of IB N or IBN effector
plasmid, and 5 µg of a luciferase reporter construct under
transcriptional control of either the B-LUC, WT-LUC, or pGLNOSa.
Input DNA for all transfections was normalized by addition of a blank
pCMV4 expression vector. The resulting activities were plotted as fold
induction relative to that with the corresponding reporter in the
absence of p65 or Tax. Data represent the mean ± SEM of three
experiments.
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Effect of NO on apoptosis in an HTLV-I-infected T-cell line.
Human Epstein-Barr virus-infected B lymphocytes and Burkitt lymphoma
cell lines constitutively express hiNOS.30 NO appears to
play a role in the inhibition of apoptosis of lymphocytes and prevention of viral reactivation in these cells. In addition, spontaneous expression of a functional hiNOS that has an antiapoptotic role has been reported in B-cell chronic lymphocytic leukemia cells.31 In agreement with previous reports, we have shown
that the addition of NG-monomethyl-L-arginine (L-NMMA), a
NOS inhibitor, enhanced DNA fragmentation in C5/MJ, HTLV-I-infected
T-cell line (Fig 9). These results suggest
that NO released by HTLV-I-infected T cells may contribute to their
survival in vivo. Failure of cells to undergo apoptosis is a factor
contributing to the development of some forms of cancer. Thus, elevated
NO production mediated by Tax may inhibit apoptotic death of leukemic
cells in ATL patients, thereby contributing to leukemogenesis.
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| Fig 9.
Effect of NO on apoptosis in an HTLV-I-infected T-cell
line. C5/MJ cells were incubated for 72 hours in the presence (+) or
absence () of L-NMMA (1 mmol/L). Cellular DNA was prepared and
resolved by electrophoresis on a 2% agarose gel.
|
|
| |
DISCUSSION |
The major finding of the present study was the demonstration of
overexpression of hiNOS gene in HTLV-I-infected T-cell lines and primary ATL cells compared with uninfected T-cell lines and normal
PBL. The expression in HTLV-I-infected cells suggests that the gene
might play a role in the pathogenesis of HTLV-I-associated diseases.
Recently, Tax has been shown to induce hiNOS mRNA expression and NO
production in a human monoblast cell line.32 In the present study, we investigated the ability of Tax to transactivate the hiNOS
promoter in T cells. Our results showed that Tax can induce hiNOS mRNA
and activate the hiNOS promoter, and the minimum Tax-responsive region
of this promoter was narrowed to a 48-bp sequence located between
nucleotides 159 and 111. Because Tax has previously been
shown to function through NF-B element, we suggest that the NF-B
element located between nucleotide 115 and 106 is involved in Tax activation. However, Tax can transactivate promoters containing a mutation in the NF-B site (pGLNOSBm). To confirm this, we showed that a Tax mutant defective for activation of NF-B-dependent promoter was capable of activating the hiNOS
promoter. We have also shown that overexpression of the
dominant-negative mutants of IB and IB failed to reduce
Tax-induced activation of hiNOS gene. These results show that
Tax activation of the hiNOS promoter does not depend on the NF-B pathway.
Studies elucidating the molecular mechanisms involved in the
transcriptional regulation of iNOS have been reported for murine macrophages. Two regions in the murine macrophage iNOS promoter are
essential for conferring inducibility of iNOS to lipopolysaccharide and
interferon (IFN)- .33,34 An NF-B element, which exists within 0.1-kb from the transcriptional start site of murine
iNOS gene, was found to bind members of the NF-B/Rel family
of proteins in response to lipopolysaccharide.35 Further
upstream (positions 923 to 913), an IFN--responsive
element, was shown to bind IRF-1 upon stimulation of RAW 264.7 cells
with IFN-.36 On the other hand, the putative promoter
region of hiNOS has been cloned.37,38 There are,
however, major differences in the sequence and potential cis-acting
elements between human and rodent iNOS 5' flanking regions.37,38 We have previously shown that the promoter
regions respond to IL-1 via NF-B activation in a human colorectal
adenocarcinoma cell line.20 In contrast, de Vera et
al39 reported that the functional promoter of hiNOS does
not exist within the 3.8 kb DNA sequence in human liver epithelial cell
line. More recently, the same group reported multiple NF-B elements
responsible for cytokine-induced transcriptional activation of the gene
that were all localized in the 5' flanking region upstream of
4.7 kb.40 In the present study, we showed that
Jurkat cells transfected with a series of hiNOS-luciferase gene
constructs exhibited significant activity in the first 3.2 kb of the
5' flanking region upon stimulation with Tax similar to the human
tumor cell line stimulated with IL-1. Transient expression of the
luciferase reporter gene linked to serially deleted sequences of the
5' flanking region of the hiNOS gene has shown several positive
regulatory elements involved in the Tax-inducible expression of the
hiNOS gene in Jurkat cells. A distal region between 1,088 and
419 was shown to be involved in the Tax-inducible expression of
the hiNOS gene. Through computer search, several DNA sequences within
1,088 to 419 were found to be similar to the binding
sites of certain known transcription factors. These sites included six
IFN--responsive elements at 1,074 to 1,067,
977 to 970, 881 to 874, 864 to
857, 840 to 833, and 474 to 467. A
proximal region between 159 and 111 was identified as the
minimum sequence for the Tax-inducible expression of the hiNOS gene.
Interestingly, a unique NF-B element in the hiNOS promoter is not
involved in Tax activation. Homology search showed the presence of some
putative elements between nucleotides 159 and 111,
including a tumor necrosis factor-responsive element and
IFN--responsive elements.37,38 Whereas our initial
studies could not identify the specific elements required for
transcriptional activation by Tax, the present results showed that the
hiNOS promoter contains important Tax-responsive regions located
between nucleotides 159 and 111.
Apparently, there is no doubt that the viral transactivator Tax can
transactivate the hiNOS promoter. However, the question is whether Tax
is the only mechanism for constitutive expression of hiNOS gene in ATL.
The intensity of the RT-PCR product of hiNOS did not correlate with the
level of expression of Tax in ATL in vivo. Although the accumulation of
hiNOS mRNA was obviously induced by Tax in JPX-9, the level appeared to
be much lower than those in the cell lines infected with HTLV-I, such
as C5/MJ and SLB-1. These findings indicate that factors of either
viral or cellular origin other than Tax are also involved in the
constitutive expression of hiNOS in HTLV-I-infected cells.
Our experiments point to an antiapoptotic role for NO in
HTLV-I-infected T cells. The inhibition of apoptosis is potentially deleterious in supporting tumor cell growth and survival. It is also
interesting to note that transgenic animals for the tax gene show chronic inflammatory arthropathy.41 Furthermore,
increased iNOS expression and NO production have been noted in induced
and spontaneous animal models of arthritis, whereas inhibitors of NOS
can reduce arthritis in these conditions.42 Other studies have shown high levels of NO catabolites in serum and synovial fluid of
patients with inflammatory arthritis.43 In addition, synovial tissue and peripheral blood mononuclear cells from humans with
inflammatory arthritis express iNOS mRNA and protein, and generate
NO.44 Further investigation of hiNOS gene
expression using Tax transgenic animals could provide the basis for our
understanding the precise role of hiNOS in the pathogenesis of
HTLV-I-associated diseases. NO-related therapies may be useful in the
treatment of ATL and HTLV-I-associated diseases.
| |
ACKNOWLEDGMENT |
We are grateful to Drs W.C. Greene, D.W. Ballard, M. Hatanaka, J. Fujisawa, K. Shimotohno, and K. Matsumoto for providing plasmids, Dr Y. Tanaka for providing anti-Tax MoAb, Dr M. Nakamura for providing JPX-9
and JPX-9/M, and Fujisaki Cell Center, Hayashibara Biochemical
Laboratories, Inc (Okayama, Japan) for providing Jurkat, MT-1, and
C5/MJ cell lines. We also thank Dr Y. Yamasaki for providing the blood
samples of patients and M. Yamamoto and M. Sasaki for the excellent
technical assistance.
| |
FOOTNOTES |
Submitted November 16, 1998; accepted June 15, 1999.
Supported in part by a Grant-in-Aid for Encouragement of Young
Scientists from the Ministry of Education, Science, Sports and Culture
of Japan.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Naoki Mori, MD, Department of Preventive
Medicine and AIDS Research, Institute of Tropical Medicine, Nagasaki
University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; e-mail:
n-mori{at}net.nagasaki-u.ac.jp.
| |
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ABSTRACT
INTRODUCTION