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IMMUNOBIOLOGY
Hlx homeobox transcription factor negatively regulates interferon-␥ production in
monokine-activated natural killer cells
Brian Becknell,1,2 Tiffany L. Hughes,2 Aharon G. Freud,1,2 Bradley W. Blaser,1,2 Jianhua Yu,3 Rossana Trotta,3
Hsiaoyin C. Mao,3 Marie L. Caligiuri de Jesús,4 Mohamad Alghothani,4 Don M. Benson Jr,4 Amy Lehman,5 David Jarjoura,5
Danilo Perrotti,6,8 Michael D. Bates,7 and Michael A. Caligiuri2-4,8
1Medical
Scientist Program, 2Integrated Biomedical Science Graduate Program, 3Department of Molecular Virology, Immunology, and Medical Genetics,
of Hematology/Oncology, 5Department of Epidemiology and Biometrics, 6Human Cancer Genetics Program, Department of Internal Medicine, College
of Medicine and Public Health, The Ohio State University, Columbus; 7Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital
Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, OH; 8Comprehensive Cancer Center, The Ohio State University,
Columbus
4Division
Natural killer (NK) cells contribute to host
immunity, including tumor surveillance,
through the production of interferon
gamma (IFN-␥). Although there is some
knowledge about molecular mechanisms
that induce IFN-␥ in NK cells, considerably less is known about the mechanisms
that reduce its expression. Here, we investigate the role of the Hlx transcription
factor in IFN-␥ production by NK cells.
Hlx expression is induced in monokine-
activated NK cells, but with delayed kinetics compared to IFN-␥. Ectopic Hlx expression decreases IFN-␥ synthesis in primary
human NK cells and IFN-␥ promoter activity in an NK-like cell line. Hlx protein
levels inversely correlate with those of
STAT4, a requisite factor for optimal IFN-␥
transcription. Mechanistically, we provide evidence indicating that Hlx overexpression accelerates dephosphorylation
and proteasome-dependent degradation
of the active Y693-phosphorylated form
of STAT4. Thus, Hlx expression in activated NK cells temporally controls and
limits the monokine-induced production of IFN-␥, in part through the targeted depletion of STAT4. (Blood. 2007;
109:2481-2487)
© 2007 by The American Society of Hematology
Introduction
Innate immunity is characterized by the ability of immune cells to
rapidly detect an invading pathogen and to restrict its dissemination
while targeting compromised host cells for elimination. This
complex task is achieved in part through the production of soluble
cytokines and chemokines by natural killer (NK) cells.1 NK cells
elaborate interferon gamma (IFN-␥) in response to stimulation with
monokines, particularly IL-12 in combination with IL-15, IL-18, or
IL-1␤.2 IFN-␥ signals through a heterodimeric receptor and STAT1
to promote the maturation and activation of monocytes, leading to
improved antigen presentation and the establishment of macrophage effector functions.3
The importance of IFN-␥ is illustrated by naturally occurring
mutations in IFN-␥ receptor subunits and STAT1 in humans, who
are highly susceptible to systemic disease after mycobacterial
infection or vaccination with bacillus Calmette-Guerin.4 In addition, mice lacking IFN-␥ responsiveness exhibit increased incidence of tumors, consistent with a role for IFN-␥ in tumor
surveillance by immune cells.5 Although the beneficial roles of
IFN-␥ are unquestionable, excessive IFN-␥ can be detrimental to
the host. In fact, high levels of IFN-␥ are implicated in the etiology
of inflammatory bowel disease.6 In addition, unabated IFN-␥
production leads to enhanced apoptosis of hematopoietic stem cells
and impaired NK cell development.7 Therefore, it is not surprising
that IFN-␥ synthesis is subject to stringent control in vivo, with
multiple checkpoints including transcriptional control.8 Indeed,
numerous trans-acting factors have been implicated as positive
regulators of IFN-␥ gene expression8; however, the negative
regulation of this process is far less understood at a molecular level.
The H2.0-like homeobox 1 (HLX1, HB24, Hlx) gene encodes a
highly conserved putative homeobox transcription factor9 that is
preferentially expressed by T-helper 1 (TH1) polarized CD4⫹ T
cells.10,11 Ectopic expression of Hlx during TH2 differentiation
leads to increased IFN-␥ mRNA and protein expression.10-12 In this
study, we report that Hlx is a negative regulator of IFN-␥
production by NK cells and that its inhibitory function is achieved
at least in part through the proteasomal degradation of STAT4, a
key transcription factor for IFN-␥ mRNA synthesis.13,14
Submitted October 10, 2006; accepted November 6, 2006. Prepublished online
as Blood First Edition Paper, November 16, 2006; DOI 10.1182/blood-2006-10050096.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
© 2007 by The American Society of Hematology
BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6
Materials and methods
Human NK cell isolation and cell lines
Human NK cells were isolated from peripheral blood (American Red
Cross) in accordance with The Ohio State University Institutional
Review Board. NK-92, Phoenix-Ampho, and DERL-7 cells were
cultured as described.15,16
Murine NK cell culture
Hlx⫹/⫺ mice were maintained on an FVB/N background. Dams from
Hlx⫹/⫺ breedings were killed at E13.5, and fetal livers were isolated. DNA
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BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6
BECKNELL et al
was extracted from embryonic tissue for genotyping (Extract-N-Amp
Tissue PCR Rapid Genotyping Kit; Sigma, St Louis, MO).17 Fetal liver
cells were cultured in 96-well, round-bottom plates (104 cells/well) in
DMEM with 10% FBS (Invitrogen, Carlsbad, CA), antibiotic/antimycotic,
glutamine, pegylated rat stem cell factor (100 ng/mL; Amgen, Thousand
Oaks, CA), murine IL-7 (10 ng/mL; Peprotech, Rocky Hill, NJ), human
Flt-3 ligand (100 ng/mL; Amgen), and human IL-2 (1000 IU/mL; Roche,
Basel, Switzerland). Successful derivation of NK cells was confirmed
cytometrically with NK1.1-APC and DX5-PE, CD19-biotin, CD14-biotin,
CD3-biotin, Ter119-biotin, and streptavidin-PerCP-Cy5.5 (BD PharMingen, San Diego, CA).
Cruz Biotechnology, Santa Cruz, CA), anti–␤-actin (1:1000; Santa Cruz
Biotechnology), anti-Ku70 (1:1000; Santa Cruz Biotechnology), and
anti-Brg1 (1:1000).
Reporter assays
The entire Hlx open-reading frame, containing its native start and stop
codons, was amplified from human leukocyte cDNA and introduced into
pCR2.1-TOPO (Invitrogen). Forward and reverse primers for PCR were
modified at their 5⬘ ends with BamHI and EcoRI sites, respectively. After
bidirectional sequencing, the Hlx cDNA was mobilized to the PINCO
retroviral transfer vector as a BamHI-EcoRI fragment. Alternatively, for
IFN-␥ reporter assays, Hlx was subcloned into pcDNA3.1 (Invitrogen).
Retrovirus was produced, and cells were infected and purified as described.16
Five million DERL-7 were transfected by nucleofection (Amaxa, Cologne,
Germany) using Solution V and DNA as follows: 5 ␮g empty pGL3-Basic
(Promega, Madison, WI) or the IFN-␥ firefly luciferase vector (a kind gift of
Howard Young, National Cancer Institute22), 5 ␮g pcDNA3.1-Hlx or empty
pcDNA3.1 vector (Invitrogen), 50 ng TK-renilla luciferase vector (pRLTK; Promega), and program O-17. After electroporation, cells were
immediately placed in 10% RPMI containing IL-12/IL-18 and were
incubated for 6 hours at 37°C. Cells were subsequently washed with
ice-cold PBS and lysed in 140 ␮L 1 ⫻ passive lysis buffer (Promega), and
luminescence was analyzed with the dual-luciferase reporter system
(Promega). All assays were performed in triplicate, and all firefly luciferase
values were normalized for transfection efficiency by subtracting the renilla
luciferase activity derived from the cotransfected pRL-TK plasmid. The
activity of the pGL3 basic reporter vector alone was subtracted from that of
the vector with the IFN-␥ promoter, and the mean ⫾ SD of the triplicate
values of the difference is shown.
Monokine stimulation of NK cells
Statistical analysis
Human NK cells were stimulated at 2 million/mL in RPMI medium
containing 10% FBS and IL-12 (10 ng/mL; Genetics Institute, Cambridge,
MA), IL-15 (100 ng/mL; Amgen), or IL-18 (50 ng/mL; R&D Systems,
Minneapolis, MN). Before stimulation at 1 million/mL, NK-92 cells were
washed extensively with PBS and rested for 24 hours in RPMI containing
10% FBS in the absence of IL-2. Murine NK cells were stimulated at 2
million/mL in DMEM containing 10% FBS with the cytokines murine
IL-12 (10 ng/mL; Genetics Institute) and human IL-15 (3 ␮g/mL; Amgen).
For proteasomal inhibition, MG-132 (Calbiochem), epoxomicin (Sigma),
and lactacystin were reconstituted according to the manufacturer’s instructions and incubated at 20 ␮M final concentration with rested PINCO-Hlx–
infected NK-92 cells for 1 hour at 37°C before IL-12/IL-18 was added for
the indicated times. To inhibit protein translation, cycloheximide (Sigma)
was reconstituted in 100% ethanol and incubated at 20 ␮g/mL final
concentration with rested PINCO-Hlx–infected NK-92 cells for 30 minutes
at 37°C before IL-12/IL-18 was added for the indicated times.
Data were analyzed using a Student 2-tailed t test. P values below .05 were
considered statistically significant.
Retroviral infection of primary NK and NK-92 cells
Detection of IFN-␥ and Y693 pSTAT4
Intracellular staining for IFN-␥ was performed as described.18 Y693
pSTAT4 was detected with Y693 pSTAT4 or IgG2b isotype control mAb
(BD PharMingen).19 Human IFN-␥ protein was detected by ELISA.18
QRT-PCR
Total RNA was extracted and reverse transcribed.18 Sequences of all
QRT-PCR primers and probes are available on request. QRT-PCR reactions
were performed and analyzed by the ⌬Ct method after normalizing to
internal 18S control reactions, as described.20 Results—expressed as the
mean ⫾ SEM of triplicate wells—were the n-fold difference of transcript
levels in an 18S-normalized sample compared with calibrator cDNA.
Calibrator cDNA was unstimulated NK (Figure 1A), unstimulated CD56dim
NK (Figure 1C), or unstimulated PINCO NK-92 (Figure 3C).
Detection of Hlx protein and immunoblotting
Hlx protein was detected using affinity-purified rabbit antisera raised
against a GST-Hlx fusion protein (Abgent, San Diego, CA). To construct
the GST-Hlx fusion protein, a human Hlx cDNA fragment corresponding to
amino acids 333 to 488 was PCR amplified and cloned into the BamHI and
EcoRI sites of pGEX-6P-1 (Amersham Biosciences, Freiburg, Germany).
Affinity-purified Hlx antiserum was used at 1:1000 dilution. Immunoblotting was performed with whole cell lysates18 or nuclear and
cytosolic protein.21 Antibodies were anti–pSTAT4 Y693 (1:5000 mouse
IgG2b monoclonal; BD PharMingen), anti–total STAT4 (1:1000; Santa
Results
Hlx expression is induced in monokine-activated NK cells
We first investigated the relationship between Hlx protein and
IFN-␥ mRNA levels in primary human NK cells over time after
treatment with IL-12 and IL-18 (IL-12/IL-18). Hlx protein expression was readily detectable by immunoblotting in resting NK cells,
increased on IL-12/IL-18 treatment, peaked at 72 hours, and was
maintained after 96 hours of stimulation (Figure 1A, upper panel).
In contrast, IFN-␥ mRNA expression—measured by quantitative
reverse transcription–polymerase chain reaction (QRT-PCR)—
peaked at 24 hours and subsequently declined (Figure 1A, lower
panel). Maximal induction of Hlx expression required costimulation with different cytokines (eg, IL-12/IL-18) (Figure 1B)
that, as reported,2 synergistically promoted high IFN-␥ production. Treatment with individual monokines led to modest or
undetectable changes in Hlx protein levels (Figure 1B), suggesting that increased Hlx expression may have a role in the
regulation of IFN-␥ production.
Because combination monokine treatment (eg, IL-12/IL-18 or
IL-12/IL-15) strongly stimulates IFN-␥ production by CD56bright
but not CD56dim human NK cells,2,20 we investigated whether the
expression of Hlx was restricted to a specific CD56 NK subset.
Indeed, though Hlx mRNA and protein levels were comparable in
resting CD56 NK subsets, induction of Hlx mRNA and protein
expression occurred preferentially in IL-12/IL-18– and IL-12/IL15–stimulated CD56bright NK cells (Figure 1C). Thus, Hlx expression is induced by monokine costimulation and enhanced within
the CD56bright NK subset; however, higher levels of Hlx correlated
with decreased IFN-␥ production by NK cells.
Hlx negatively regulates IFN-␥ production in
monokine-activated NK cells
Based on the kinetics of Hlx expression relative to IFN-␥ transcription, it is conceivable that Hlx acts as a negative regulator of IFN-␥
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BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6
0
Time (h)
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72
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96
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HLX
42 kDa
β-actin
15000
IFN-γ mRNA
2483
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UN
12+15
12+18
UN
12+15
12+18
A
Hlx INHIBITS IFN-␥ PRODUCTION BY NK CELLS
10000
50 kDa
Hlx
42 kDa
β−actin
P < .0001
5000
P < .0002
Hlx mRNA
1000
0
0
48
24
72
96
Time (h)
B
100
10
1
UN
12
15
18
12+15 12+18 15+18
50 kDa
Hlx
UN
12+15
CD56bright
UN
12+15
CD56dim
β−actin
42 kDa
CD56bright
Figure 1. Monokine-dependent Hlx expression by human
NK cells is delayed with respect to IFN-␥ transcription. (A) Delayed induction of Hlx protein with
respect to IFN-␥ production in primary NK cells. (upper) Total NK cells were stimulated with IL-12/IL-18 for indicated time points, and Hlx protein levels were analyzed by
immunoblotting. ␤-Actin levels were analyzed to ensure equal loading (n ⫽ 4 experiments). (lower) In parallel, IFN-␥ mRNA levels were analyzed by QRT-PCR. Mean ⫾ SEM
from 4 separate experiments is shown. (B) Maximal Hlx protein induction requires monokine costimulation. Total NK cells were stimulated for 72 hours, as indicated, and Hlx
and ␤-actin protein levels were analyzed by immunoblotting (n ⫽ 3 experiments). (C) Preferential induction of Hlx protein in the CD56bright NK subset. (upper) FACS-purified
unstimulated NK subsets were lysed directly (UN) or after stimulation with the indicated monokine combinations (72 hours). Lysates were analyzed for Hlx and ␤-actin protein
by immunoblotting (n ⫽ 3 experiments). (lower) FACS-purified NK subsets were lysed immediately for RNA (UN) or stimulated with IL-12/IL-15 for 12 hours before lysis. Hlx
mRNA levels were analyzed by QRT-PCR. Mean ⫾ SEM from 5 separate donors is shown.
production in CD56bright NK cells. Thus, IFN-␥ production was
assessed by intracellular staining in IL-12/IL-18–stimulated primary human NK cells previously transduced with the EGFP
(enhanced green fluorescence protein)–expressing PINCO-Hlx or
empty PINCO retrovirus (Figure 2). Interestingly, a significant
decrease in the proportion of IFN-␥⫹ cells was observed in the
Hlx-expressing EGFP⫹CD56bright NK cells (CD56bright PINCO-Hlx
compared with CD56bright PINCO; P ⬍ .02; n ⫽ 3 experiments)
and in the CD56dim fraction (CD56dim PINCO-Hlx compared with
CD56dim PINCO; P ⬍ .006; n ⫽ 3 experiments). In similar experiments, human NK-92 cells, which as reported have a phenotype
that resembles the CD56bright NK cell subset,23 were infected with
the Hlx or the empty retrovirus and were EGFP-sorted. As
expected, anti-Hlx Western blots performed on subcellular fractions from EGFP⫹ PINCO– and EGFP⫹ PINCO-Hlx–transduced
NK-92 cell lysates showed that Hlx was indeed overexpressed and
primarily localized in the nuclear compartment (Figure 3A). Note
that Brg1 and Ku70 were detected as controls for purity of the
nuclear fraction and equal loading, respectively (Figure 3A).
Consistent with the data obtained with primary CD56bright NK cells,
Hlx overexpression led to a marked decrease in IFN-␥ protein
levels in IL-12/IL-18–stimulated NK-92 cells after 24 hours of
stimulation (Figure 3B). Moreover, levels of IFN-␥ mRNA, albeit
similar in unstimulated vector- and Hlx-transduced NK-92 cells,
dramatically increased over time after IL-12/IL-18 stimulation of
vector-transduced cells but not in NK-92 cells overexpressing Hlx
(Figure 3C). Thus, our data not only indicated that sustained Hlx
expression impairs the ability of NK cells to produce IFN-␥ in
response to monokine costimulation, they also suggested that a
monokine-induced increase of Hlx level is most likely required to
temporally control the expression and, therefore, the production of
IFN-␥ by activated NK cells. Indeed, Hlx-deficient murine NK
cells show enhanced IFN-␥ mRNA and protein production after in
vitro monokine costimulation with IL-12 and IL-15 compared with
wild-type and heterozygous control NK cells (Figure 3D and data
not shown).
Hlx represses IFNG promoter activity in NK cells
Figure 2. Hlx inhibits IFN-␥ production by primary CD56bright NK. PINCO- or
PINCO-Hlx–infected primary human NK cells were stimulated for 24 hours with
IL-12/IL-18 and were subjected to staining for CD56 and IFN-␥. (left) Infected cells
were gated on CD56brightEGFP⫹ or CD56dimEGFP⫹. (right) IFN-␥ staining in each
population compared with isotype controls. Results shown are representative of 3
experiments (CD56bright PINCO-Hlx compared with CD56bright PINCO; P ⬍ .02).
Because Hlx overexpression in NK-92 cells resulted in decreased
IFN-␥ mRNA levels and because Hlx is a homeobox transcription
factor, we reasoned that Hlx may regulate the transcriptional
activity of the IFNG promoter in NK cells. To begin to test this
hypothesis, we sought to determine whether Hlx could inhibit
IFNG promoter activity in vivo. We made use of luciferase reporter
assays in the DERL-7 NK-like cell line,24 which is easily transduced by electroporation.15,25 DERL-7 was electroporated with a
previously characterized full-length human IFNG promoter-firefly
luciferase reporter construct containing an approximately 3.6-kb
IFNG 5⬘ flanking sequence and an 0.8-kB enhancer element from
the first intron of IFNG following the luciferase open-reading
frame (Figure 4A).22,26 After electroporation, cells were treated
with IL-12/IL-18 for 6 hours. After normalizing for transfection
efficiency, we determined that Hlx overexpression repressed IFNG
promoter activity (Figure 4B). However, on subsequent deletion
analysis of the 3.6-kb IFNG promoter and the 0.8-kb intron 1
enhancer element within the construct, we were unable to map Hlx
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BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6
BECKNELL et al
Figure 3. Hlx inhibits IFN-␥ mRNA and protein expression in NK-92 cells. (A)
Overexpression of Hlx protein in NK-92 cells. FACS-purified NK-92 cells transduced
with PINCO or PINCO-Hlx were subjected to nucleocytoplasmic fractionation and
immunoblotting for Hlx. Enrichment of nuclear protein was confirmed by Brg1
staining. Equal loading was confirmed by Ku70 staining. (B) Inhibition of IFN-␥
production by Hlx. PINCO- or PINCO-Hlx–infected NK-92 cells were stimulated with
IL-12/IL-18, and supernatants were harvested after 24 hours for IFN-␥ ELISA. Shown
are the mean ⫾ SEM from 5 separate experiments. (C) Time course of IFN-␥ mRNA
in PINCO- compared with PINCO-Hlx–transduced NK-92 in response to IL-12/IL-18.
Shown are results of 1 of 4 representative experiments; error bars represent SD of
triplicate PCR reactions. (D) Increased IFN-␥ production by Hlx⫺/⫺ NK in response to
monokine costimulation. Fetal liver–derived NK of indicated genotypes were subjected to 24-hour stimulation with IL-12 and IL-15, followed by intracellular staining for
IFN-␥. Mean ⫾ SEM percentages of IFN-␥⫹ NK1.1⫹ cells from 9 experiments with
NK cells derived from 9 or more livers of each genotype are shown. *P ⬍ .001 Hlx⫹/⫹
compared with Hlx⫺/⫺. **P ⬍ .005 Hlx⫹/⫺ compared with Hlx⫺/⫺).
repressive activity to any particular region (data not shown). Thus,
the mechanism of IFNG promoter regulation by Hlx is likely
complex and dependent on multiple cis-regulatory elements.
Hlx promotes proteasome-dependent STAT4 degradation
after monokine stimulation
We further hypothesized that Hlx might negatively regulate IFN-␥
mRNA levels by inhibiting the function or activity of a positive
regulator of IFN-␥ production. STAT4 is a known trans-activator
of the IFN-␥ promoter in NK cells.13,14 STAT4 is activated by
tyrosine phosphorylation at tyrosine residue 693 (Y693) by Jak2/
Tyk2 kinases within seconds of IL-12 stimulation, resulting in its
rapid nuclear translocation and direct association with multiple
cis-regulatory elements within the IFN-␥ promoter.26-28 In IL-12/IL18–stimulated primary human NK or NK-92 cells, we observed
that the increase of Hlx expression was accompanied by a decrease
in total STAT4 protein levels (Figure 5A), suggesting that Hlx may
negatively regulate IFN-␥ gene transcription by suppressing STAT4
expression. Thus, levels of total STAT4 protein were also evaluated
in NK-92 cells transduced with Hlx or empty retrovirus, EGFPsorted and stimulated with IL-12/IL-18. Before stimulation, Hlxand vector-transduced NK-92 cells expressed similar levels of
unphosphorylated STAT4 (lower, faster-migrating band at 0 hour;
Figure 5B). Similarly, both populations demonstrated comparable
induction of Y693 pSTAT4 levels after IL-12/IL-18 stimulation
(upper, slower-migrating band at 0.5 hours; Figure 5B). However,
Hlx overexpression inhibited long-term monokine-induced STAT4
activation as levels of Y693 pSTAT4 progressively decreased over
time in Hlx-expressing NK-92 cells but not in vector-transduced
NK-92 cells (Figure 5B). In parallel experiments, we did not
observe an effect of Hlx on total and tyrosine-phosphorylated
STAT1 and STAT5, indicating that Hlx exerts its effect specifically
on STAT4. We also found that retroviral expression of Hlx in
primary NK reduced the proportion of Y693 pSTAT4⫹ cells after
IL-12/IL-18 treatment (from 78.0% ⫾ 5.4% in vector-transduced
cells to 67.1% ⫾ 5.4% in Hlx-transduced cells; n ⫽ 5 independent
experiments; P ⬍ .005).
Interestingly, at later time points, we observed that ectopic Hlx
expression not only interfered with STAT4 activation, it also
significantly reduced the level of unphosphorylated STAT4 protein
(Figure 5B). This reduction in total STAT4 protein levels led us to
test whether Hlx overexpression is associated with a decrease in
STAT4 mRNA; however, no difference in STAT4 transcript levels
was detectable by QRT-PCR in Hlx compared with vector-infected
NK-92 cells during time-course experiments in IL-12/IL-18 (n ⫽ 4
experiments; data not shown). Alternatively, it has been reported
that Y693 pSTAT4 undergoes proteasome-dependent degradation
in T lymphocytes after IL-12 treatment.29,30 We therefore attempted
to reverse the loss of Y693 pSTAT4 protein through the application
of MG-132, an inhibitor of the 26S proteasome subunit. Indeed,
MG-132 rescued the loss of Y693 pSTAT4 after short-term (3 or 6
hours) treatment of Hlx-expressing NK-92 cells with IL-12/IL-18
(Figure 5C). Similar results were obtained with the additional
proteasome inhibitors epoxomicin and lactacystin (data not shown).
Interestingly, proteasome inhibition also rescued expression of the
unphosphorylated STAT4 protein, particularly after 6-hour stimulation with IL-12/IL-18 (Figure 5C). The E3 ubiquitin ligase SLIM
has been shown to catalyze polyubiquitylation of Y693 pSTAT4 in
vitro and to attenuate STAT4 protein expression in vivo.30 Therefore, we sought to determine whether SLIM protein levels were
A
0.8 kB
3.6 kB
IFNG promoter
luc
IFNG intron 1
B
Relative IFNG
promoter activity
2484
40
30
20
10
0
Vector
Hlx
Figure 4. Hlx inhibits IFNG promoter activity in DERL-7 cells. (A) The human
IFNG luciferase construct used in this study consisted of 3.6 kB of a 5⬘ flanking
sequence upstream of the transcriptional start site, the firefly luciferase open-reading
frame, and 0.8 kB of the intron 1 enhancer element. (B) DERL-7 cells with Hlx or
empty vector were transfected with the IFNG luciferase construct, and luciferase
activity was measured after 6-hour stimulation with IL-12/IL-18. Mean ⫾SD of 1 of 3
representative experiments is shown.
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BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6
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0
0.5 1
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_
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Hlx INHIBITS IFN-␥ PRODUCTION BY NK CELLS
EtOH
Hlx
80 kDa
STAT4
STAT4
42 kDa
Actin
β-Actin
12 h
24 h
Figure 5. Hlx overexpression leads to proteasome-dependent degradation of STAT4. (A) Endogenous Hlx levels increased as total STAT4 levels declined. Primary
human NK or NK-92 cells were harvested at the indicated time points after IL-12/IL-18 stimulation, and Hlx, STAT4, and ␤-actin protein levels were determined by
immunoblotting. (n ⫽ 4 experiments in primary NK, n ⫽ 2 experiments in NK-92). (B) Hlx overexpression decreased total and Y693 pSTAT4 levels in NK-92 after IL-12/IL-18
treatment. FACS-purified PINCO- or Hlx-expressing NK-92 cells were harvested at indicated time points after IL-12/IL-18 stimulation. Total STAT4 and ␤-actin levels were
assessed by immunoblotting (n ⫽ 4 experiments). (C) Proteasome inhibition rescued loss of STAT4 in NK-92 cells overexpressing Hlx. Cells were preincubated for 1 hour with
20 mM MG-132 (MG), an equal amount of DMSO vehicle, or medium alone (—), followed by IL-12/IL-18 treatment for the indicated times. Total protein was harvested, and total
STAT4 and ␤-actin levels were assessed by immunoblotting (n ⫽ 3 experiments). (D) Diminished Y693 pSTAT4 levels in Hlx-overexpressing NK-92 cells despite inhibition of
new protein synthesis. PINCO- or Hlx-expressing NK-92 cells were preincubated for 30 minutes with 10 ␮M CHX, followed by IL-12/IL-18 treatment for the indicated times.
Total cellular protein was harvested, and total STAT4 and ␤-actin levels were assessed by immunoblotting (the arrowhead indicates the position of the 80 kDa marker). (E)
Comparison of CHX compared with ethanol (EtOH) carrier effects on STAT4 expression in PINCO- (P) and Hlx-expressing NK-92 after IL-12/IL-18 stimulation for the indicated
times (n ⫽ 3 experiments).
altered by Hlx overexpression by immunoblotting, but no change in
SLIM expression was observed (data not shown).
To confirm that the loss of STAT4 protein was caused by
decreased protein stability, new protein synthesis was inhibited
using cycloheximide (CHX), and the kinetics of STAT4 degradation were monitored in NK-92 expressing Hlx compared with
empty vector. As expected, in the presence of CHX, Hlx overexpression resulted in decreased Y693 pSTAT4 levels beginning after 6
hours of IL-12/IL-18 stimulation compared with empty vector
(Figure 5D). Surprisingly, however, CHX treatment resulted in an
accumulation of unphosphorylated STAT4 in cells expressing Hlx
and empty vector but with more rapid kinetics with Hlx overexpression (Figure 5D). Thus, it appears that Hlx overexpression accelerates the dephosphorylation of Y693 STAT4 and that its subsequent
degradation is reliant on new protein synthesis. To confirm these
findings were specific to CHX treatment, we treated Hlx and vector
expressing NK-92 with CHX or ethanol carrier for 12 hours or 24
hours and repeated the STAT4 Western blot (Figure 5E). Again, we
found that the inhibitory effect of Hlx overexpression on Y693
pSTAT4 levels was insensitive to CHX treatment. Furthermore, we
confirmed the stabilizing influence of CHX treatment on the levels
of unphosphorylated STAT4, regardless of Hlx or vector expression.
Discussion
In this study, we found that Hlx is a novel negative regulator of
IFN-␥ production in NK cells after monokine costimulation. The
delayed kinetics of Hlx protein induction with respect to IFN-␥
itself are consistent with a model in which Hlx serves as a feedback
inhibitor of IFN-␥ synthesis in activated CD56bright NK. This
Hlx-mediated negative feedback pathway is likely important in
vivo, given the potentially deleterious effects of unchecked IFN-␥
production on the host.6,7 The process of Hlx protein induction after
monokine costimulation is likely complex. Indeed, Hlx regulation
must occur at transcriptional and posttranscriptional levels because
parallel mRNA and protein expression studies have established
long lags between monokine costimulation and the onset of Hlx
mRNA expression (approximately 12 hours; data not shown) and
between the onset of detectable Hlx mRNA and appreciable Hlx
protein expression (Figure 1A). In future studies, it will be
insightful to analyze the HLX promoter itself and to determine the
direct influence of monokines on its activity and the potential
effects of NK-derived cytokines that have been shown to inhibit
IFN-␥ production, such as transforming growth factor-␤ and
TWEAK,15,31 on Hlx mRNA and protein expression.
In addition, we identified the STAT4 transcription factor as a
key molecular target of Hlx action in NK cells. Multiple lines of
evidence have established the role of STAT4 as a requisite, direct
transactivator of the IFNG promoter in NK cells after monokine
stimulation.13,14,27 Constitutive STAT4 activation is strongly implicated in autoimmune disease, including rheumatoid arthritis and
inflammatory bowel disease.6,32 The inhibitory effect of Hlx on
STAT4 levels identifies Hlx as a potentially relevant molecular
target to exploit in autoimmunity. Ultimately, the physiological and
pathophysiological roles of Hlx in the attenuation of STAT4
transcriptional activity and IFN-␥ production must be determined
in vivo.
Mechanistically, we found that Hlx promotes the degradation of
Y693 pSTAT4. Previous work has identified SLIM as an E3
ubiquitin ligase with substrate specificity toward Y693 pSTAT4.30
However, this study did not explore the possibility that Y693
pSTAT4 undergoes dephosphorylation before its degradation by
the proteasome. In our study, through the use of CHX, we
implicated Hlx specifically in the dephosphorylation of pSTAT4
Y693 (Figure 4E), and we demonstrated that subsequent degradation of unphosphorylated STAT4 requires new protein synthesis.
These findings suggest that the process of Y693 pSTAT4 degradation is complex, consisting of a CHX-insensitive dephosphorylation step and a CHX-sensitive degradation step. The specific role of
Hlx in Y693 pSTAT4 dephosphorylation remains unclear. Although
the Hlx protein structure lacks sequence homology to known
tyrosine phosphatase domains, it is conceivable that Hlx may
recruit a protein tyrosine phosphatase to DNA-bound Y693 pSTAT4
dimers or tetramers. In addition, it is possible that Hlx may
associate directly with DNA through its homeodomain and may
displace DNA-bound Y693 pSTAT4 multimers, rendering them
more susceptible to dephosphorylation and degradation. Moreover,
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BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6
BECKNELL et al
we have not fully rejected the hypothesis that Hlx functions as a
transcription factor and that it is in fact the transcriptional target(s)
of Hlx, translated before CHX and monokine treatment, that trigger
Y693 pSTAT4 dephosphorylation after IL-12/IL-18 stimulation.
Indeed, it is conceivable that SLIM is one such target of Hlx.
Besides identifying a role for STAT4 in Hlx-dependent regulation of IFN-␥, our work identifies the IFNG promoter as a potential
direct target of Hlx transcriptional repression. However, in preliminary experiments, we were unable to identify the specific region
required for Hlx repression (data not shown). This difficulty may be
attributable to a complex requirement for multiple cis-regulatory
motifs throughout the IFNG promoter for Hlx repression of IFN-␥
to occur. Therefore, in subsequent work, it will be essential to
distinguish the effects of Hlx on STAT4 from its direct effect, if
any, at the IFNG promoter. Stimulation of Hlx overexpressing
NK-92 cells with IL-2 or IL-15, alone or in combination with
IL-18, did not influence IFN-␥ mRNA or protein levels compared
with cells infected with empty retroviral vector (data not shown).
Because STAT4 is activated minimally under these conditions (data
not shown), this finding is consistent with a model in which the
effects of Hlx are mediated primarily by STAT4. STAT4 is required
for IFN-␥ production in response to IL-12,13,14 making it prohibitive to explore the consequence of Hlx overexpression in STAT4deficient NK cells on IFN-␥ production after IL-12/IL-18 treatment
or to determine the effect of Hlx on the activity of IFNG promoter
constructs lacking STAT4 enhancer elements. Given these challenges, it may be insightful instead to perform a detailed structure–
function analysis of Hlx itself so as to identify Hlx mutants that
maintain IFN-␥ repression without affecting STAT4 levels and
vice versa.
Our interest in Hlx was prompted by published accounts of its
role as a positive regulator of IFN-␥ production in CD4⫹ T
lymphocytes.10-12 Although the data presented here are not consistent with this role in NK cells, considerable caution should be
exercised in comparing this work with the T-cell studies given the
distinctly different mechanisms of IFN-␥ production in NK and
CD4⫹ T-cell populations. Although NK cells produce IFN-␥ within
minutes of monokine stimulation,20 CD4⫹ T cells must undergo a
process of polarization, entailing cell division and active chromatin
remodeling at the IFNG locus, before IFN-␥ transcription can
occur.33 Ectopic expression of Hlx during TH2 polarization of
CD4⫹ T cells has been shown to promote IFN-␥ production, but
Hlx expression during TH1 polarization and in fully TH1- and
TH2-polarized CD4⫹ T cells reportedly does not influence IFN-␥
synthesis.10-12 These findings have led to the hypothesis that Hlx
functions in a discrete window during early TH-cell differentiation
to promote IFN-␥ production.10-12
Interestingly, recent work from the Zheng34 laboratory clearly
implicates Hlx as a negative regulator of the IL-4 receptor alpha
chain (IL-4R␣). Because IL-4 is required for normal TH2 differentiation, Hlx deficiency results in impaired production of TH2
cytokines, and this phenotype can be reversed by ectopic retroviral
expression of IL-4R␣.34 Apart from promoting TH2 differentiation,
IL-4 is a potent inhibitor of IFN-␥ production by T cells.35
Therefore, by decreasing the responsiveness of a CD4⫹ T cell to
IL-4 during early TH2 polarization, Hlx may indirectly promote the
production of IFN-␥ by this lymphocyte population. If the effect of
Hlx on IFN-␥ is attributed entirely to IL-4R␣ regulation, it will be
important in the future to determine whether the loss of IL-4R␣
expression eliminates the increase in IFN-␥ seen on ectopic Hlx
expression in TH2 cells. In addition, it will be interesting to
determine whether Hlx influences IL-4R␣ expression by NK cells
given that IL-4 plays a very different role in NK cells than in T
cells, namely to costimulate IFN-␥ production.36,37
Thus, it is likely that Hlx serves different roles in the T and NK
lineages, and the extent of its function may be limited to discrete
developmental windows. It is not without precedent that transcription factors perform divergent functions in multiple lineages.
GATA-3 is one transcriptional regulator shown to have apparently
opposing functions in IFN-␥ regulation by NK and CD4⫹ T cells.38
Alternatively, one can envision multiple scenarios that could
account for opposing Hlx activities in CD4⫹ T and NK cells, such
as posttranslational modification of Hlx or unique expression of
Hlx interacting proteins in lineage-specific fashion. It is anticipated
that more detailed studies of the Hlx protein and its interactions
will elucidate its mechanism of action in T- and NK-cell lineages in
the future.
Acknowledgments
This work was supported by National Cancer Institute grants P30
CA16059, CA68458, and CA95426 (M.A.C.) and by National
Institutes of Health grants DK02791 and DK61219 (M.D.B.).
We thank Dr Howard Young (National Cancer Institute, Frederick, MD) for providing the IFNG promoter luciferase construct; Dr
Michael Grusby (Harvard University) for the generous donation of
SLIM antisera, and Dr Denis Guttridge (The Ohio State University)
and Dr Wei-ping Zheng (University of Rochester) for helpful advice.
Authorship
Contribution: B.B. designed and performed the research and wrote
the manuscript. T.L.H. performed the research. A.G.F. designed the
research. B.W.B. performed the research. J.Y designed the research. R.T. designed the research. H.C.M. collected the data.
M.L.C. performed the research. M.A. performed the research.
D.M.B. performed the research. A.L. analyzed the data. D.J.
analyzed the data. D.P. designed the research. M.D.B. designed the
research. M.A.C. designed the research and edited the original and
revised versions of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Michael A. Caligiuri, A458 StarlingLoving Hall, 320 W 10th Ave, Columbus, OH 43210; e-mail:
[email protected].
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2007 109: 2481-2487
doi:10.1182/blood-2006-10-050096 originally published
online November 16, 2006
Hlx homeobox transcription factor negatively regulates interferon-γ
production in monokine-activated natural killer cells
Brian Becknell, Tiffany L. Hughes, Aharon G. Freud, Bradley W. Blaser, Jianhua Yu, Rossana Trotta,
Hsiaoyin C. Mao, Marie L. Caligiuri de Jesús, Mohamad Alghothani, Don M. Benson, Jr, Amy
Lehman, David Jarjoura, Danilo Perrotti, Michael D. Bates and Michael A. Caligiuri
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