Hlx homeobox transcription factor negatively regulates interferon-production in monokine-activated natural killer cells
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From www.bloodjournal.org by guest on May 20, 2015. For personal use only.
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,
4Divisionof 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
Natural killer (NK) cells contribute to host activated NK cells, but with delayed kinet- of the active Y693-phosphorylated form
immunity, including tumor surveillance, ics compared to IFN-␥. Ectopic Hlx expres- of STAT4. Thus, Hlx expression in acti-
through the production of interferon sion decreases IFN-␥ synthesis in primary vated NK cells temporally controls and
gamma (IFN-␥). Although there is some human NK cells and IFN-␥ promoter activ- limits the monokine-induced produc-
knowledge about molecular mechanisms ity in an NK-like cell line. Hlx protein tion of IFN-␥, in part through the tar-
that induce IFN-␥ in NK cells, consider- levels inversely correlate with those of geted depletion of STAT4. (Blood. 2007;
ably less is known about the mechanisms STAT4, a requisite factor for optimal IFN-␥ 109:2481-2487)
that reduce its expression. Here, we inves- transcription. Mechanistically, we pro-
tigate the role of the Hlx transcription vide evidence indicating that Hlx overex-
factor in IFN-␥ production by NK cells. pression accelerates dephosphorylation
Hlx expression is induced in monokine- and proteasome-dependent degradation © 2007 by The American Society of Hematology
Introduction
Innate immunity is characterized by the ability of immune cells to numerous trans-acting factors have been implicated as positive
rapidly detect an invading pathogen and to restrict its dissemination regulators of IFN-␥ gene expression8; however, the negative
while targeting compromised host cells for elimination. This regulation of this process is far less understood at a molecular level.
complex task is achieved in part through the production of soluble The H2.0-like homeobox 1 (HLX1, HB24, Hlx) gene encodes a
cytokines and chemokines by natural killer (NK) cells.1 NK cells highly conserved putative homeobox transcription factor9 that is
elaborate interferon gamma (IFN-␥) in response to stimulation with preferentially expressed by T-helper 1 (TH1) polarized CD4⫹ T
monokines, particularly IL-12 in combination with IL-15, IL-18, or cells.10,11 Ectopic expression of Hlx during TH2 differentiation
IL-1.2 IFN-␥ signals through a heterodimeric receptor and STAT1 leads to increased IFN-␥ mRNA and protein expression.10-12 In this
to promote the maturation and activation of monocytes, leading to study, we report that Hlx is a negative regulator of IFN-␥
improved antigen presentation and the establishment of macro- production by NK cells and that its inhibitory function is achieved
phage effector functions.3 at least in part through the proteasomal degradation of STAT4, a
The importance of IFN-␥ is illustrated by naturally occurring key transcription factor for IFN-␥ mRNA synthesis.13,14
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 addi- Materials and methods
tion, mice lacking IFN-␥ responsiveness exhibit increased inci-
dence of tumors, consistent with a role for IFN-␥ in tumor Human NK cell isolation and cell lines
surveillance by immune cells.5 Although the beneficial roles of Human NK cells were isolated from peripheral blood (American Red
IFN-␥ are unquestionable, excessive IFN-␥ can be detrimental to Cross) in accordance with The Ohio State University Institutional
the host. In fact, high levels of IFN-␥ are implicated in the etiology Review Board. NK-92, Phoenix-Ampho, and DERL-7 cells were
of inflammatory bowel disease.6 In addition, unabated IFN-␥ cultured as described.15,16
production leads to enhanced apoptosis of hematopoietic stem cells
and impaired NK cell development.7 Therefore, it is not surprising Murine NK cell culture
that IFN-␥ synthesis is subject to stringent control in vivo, with Hlx⫹/⫺ mice were maintained on an FVB/N background. Dams from
multiple checkpoints including transcriptional control.8 Indeed, Hlx⫹/⫺ breedings were killed at E13.5, and fetal livers were isolated. DNA
Submitted October 10, 2006; accepted November 6, 2006. Prepublished online The publication costs of this article were defrayed in part by page charge
as Blood First Edition Paper, November 16, 2006; DOI 10.1182/blood-2006-10- payment. Therefore, and solely to indicate this fact, this article is hereby
050096. 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 2481From www.bloodjournal.org by guest on May 20, 2015. For personal use only.
2482 BECKNELL et al BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6
was extracted from embryonic tissue for genotyping (Extract-N-Amp Cruz Biotechnology, Santa Cruz, CA), anti–-actin (1:1000; Santa Cruz
Tissue PCR Rapid Genotyping Kit; Sigma, St Louis, MO).17 Fetal liver Biotechnology), anti-Ku70 (1:1000; Santa Cruz Biotechnology), and
cells were cultured in 96-well, round-bottom plates (104 cells/well) in anti-Brg1 (1:1000).
DMEM with 10% FBS (Invitrogen, Carlsbad, CA), antibiotic/antimycotic,
glutamine, pegylated rat stem cell factor (100 ng/mL; Amgen, Thousand Reporter assays
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, Five million DERL-7 were transfected by nucleofection (Amaxa, Cologne,
Basel, Switzerland). Successful derivation of NK cells was confirmed Germany) using Solution V and DNA as follows: 5 g empty pGL3-Basic
cytometrically with NK1.1-APC and DX5-PE, CD19-biotin, CD14-biotin, (Promega, Madison, WI) or the IFN-␥ firefly luciferase vector (a kind gift of
CD3-biotin, Ter119-biotin, and streptavidin-PerCP-Cy5.5 (BD PharMin- Howard Young, National Cancer Institute22), 5 g pcDNA3.1-Hlx or empty
gen, San Diego, CA). pcDNA3.1 vector (Invitrogen), 50 ng TK-renilla luciferase vector (pRL-
TK; Promega), and program O-17. After electroporation, cells were
Retroviral infection of primary NK and NK-92 cells immediately placed in 10% RPMI containing IL-12/IL-18 and were
incubated for 6 hours at 37°C. Cells were subsequently washed with
The entire Hlx open-reading frame, containing its native start and stop ice-cold PBS and lysed in 140 L 1 ⫻ passive lysis buffer (Promega), and
codons, was amplified from human leukocyte cDNA and introduced into luminescence was analyzed with the dual-luciferase reporter system
pCR2.1-TOPO (Invitrogen). Forward and reverse primers for PCR were (Promega). All assays were performed in triplicate, and all firefly luciferase
modified at their 5⬘ ends with BamHI and EcoRI sites, respectively. After values were normalized for transfection efficiency by subtracting the renilla
bidirectional sequencing, the Hlx cDNA was mobilized to the PINCO luciferase activity derived from the cotransfected pRL-TK plasmid. The
retroviral transfer vector as a BamHI-EcoRI fragment. Alternatively, for activity of the pGL3 basic reporter vector alone was subtracted from that of
IFN-␥ reporter assays, Hlx was subcloned into pcDNA3.1 (Invitrogen). the vector with the IFN-␥ promoter, and the mean ⫾ SD of the triplicate
Retrovirus was produced, and cells were infected and purified as described.16 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 Data were analyzed using a Student 2-tailed t test. P values below .05 were
containing 10% FBS and IL-12 (10 ng/mL; Genetics Institute, Cambridge, considered statistically significant.
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 Results
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). Hlx expression is induced in monokine-activated NK cells
For proteasomal inhibition, MG-132 (Calbiochem), epoxomicin (Sigma),
and lactacystin were reconstituted according to the manufacturer’s instruc- We first investigated the relationship between Hlx protein and
tions and incubated at 20 M final concentration with rested PINCO-Hlx– IFN-␥ mRNA levels in primary human NK cells over time after
infected NK-92 cells for 1 hour at 37°C before IL-12/IL-18 was added for treatment with IL-12 and IL-18 (IL-12/IL-18). Hlx protein expres-
the indicated times. To inhibit protein translation, cycloheximide (Sigma) sion was readily detectable by immunoblotting in resting NK cells,
was reconstituted in 100% ethanol and incubated at 20 g/mL final increased on IL-12/IL-18 treatment, peaked at 72 hours, and was
concentration with rested PINCO-Hlx–infected NK-92 cells for 30 minutes maintained after 96 hours of stimulation (Figure 1A, upper panel).
at 37°C before IL-12/IL-18 was added for the indicated times.
In contrast, IFN-␥ mRNA expression—measured by quantitative
reverse transcription–polymerase chain reaction (QRT-PCR)—
Detection of IFN-␥ and Y693 pSTAT4
peaked at 24 hours and subsequently declined (Figure 1A, lower
Intracellular staining for IFN-␥ was performed as described.18 Y693 panel). Maximal induction of Hlx expression required costimu-
pSTAT4 was detected with Y693 pSTAT4 or IgG2b isotype control mAb lation with different cytokines (eg, IL-12/IL-18) (Figure 1B)
(BD PharMingen).19 Human IFN-␥ protein was detected by ELISA.18 that, as reported,2 synergistically promoted high IFN-␥ produc-
tion. Treatment with individual monokines led to modest or
QRT-PCR undetectable changes in Hlx protein levels (Figure 1B), suggest-
Total RNA was extracted and reverse transcribed.18 Sequences of all ing that increased Hlx expression may have a role in the
QRT-PCR primers and probes are available on request. QRT-PCR reactions regulation of IFN-␥ production.
were performed and analyzed by the ⌬Ct method after normalizing to Because combination monokine treatment (eg, IL-12/IL-18 or
internal 18S control reactions, as described.20 Results—expressed as the IL-12/IL-15) strongly stimulates IFN-␥ production by CD56bright
mean ⫾ SEM of triplicate wells—were the n-fold difference of transcript but not CD56dim human NK cells,2,20 we investigated whether the
levels in an 18S-normalized sample compared with calibrator cDNA. expression of Hlx was restricted to a specific CD56 NK subset.
Calibrator cDNA was unstimulated NK (Figure 1A), unstimulated CD56dim Indeed, though Hlx mRNA and protein levels were comparable in
NK (Figure 1C), or unstimulated PINCO NK-92 (Figure 3C).
resting CD56 NK subsets, induction of Hlx mRNA and protein
expression occurred preferentially in IL-12/IL-18– and IL-12/IL-
Detection of Hlx protein and immunoblotting
15–stimulated CD56bright NK cells (Figure 1C). Thus, Hlx expres-
Hlx protein was detected using affinity-purified rabbit antisera raised sion is induced by monokine costimulation and enhanced within
against a GST-Hlx fusion protein (Abgent, San Diego, CA). To construct the CD56bright NK subset; however, higher levels of Hlx correlated
the GST-Hlx fusion protein, a human Hlx cDNA fragment corresponding to with decreased IFN-␥ production by NK cells.
amino acids 333 to 488 was PCR amplified and cloned into the BamHI and
EcoRI sites of pGEX-6P-1 (Amersham Biosciences, Freiburg, Germany). Hlx negatively regulates IFN-␥ production in
Affinity-purified Hlx antiserum was used at 1:1000 dilution. Immuno- monokine-activated NK cells
blotting was performed with whole cell lysates18 or nuclear and
cytosolic protein.21 Antibodies were anti–pSTAT4 Y693 (1:5000 mouse Based on the kinetics of Hlx expression relative to IFN-␥ transcrip-
IgG2b monoclonal; BD PharMingen), anti–total STAT4 (1:1000; Santa tion, it is conceivable that Hlx acts as a negative regulator of IFN-␥From www.bloodjournal.org by guest on May 20, 2015. For personal use only.
BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6 Hlx INHIBITS IFN-␥ PRODUCTION BY NK CELLS 2483
A Time (h) 0 24 48 72 96
C CD56bright CD56dim
50 kDa
12+18
HLX
12+15
12+18
12+15
β-actin
UN
UN
42 kDa
15000
50 kDa Hlx
β−actin
IFN-γ mRNA
42 kDa
10000
5000 P < .0001 P < .0002
1000
Hlx mRNA
0
0 24 48 72 96 100
Time (h)
10
B UN 12 15 18 12+15 12+18 15+18
1
UN 12+15 UN 12+15
50 kDa CD56bright CD56dim
Hlx
42 kDa β−actin
Figure 1. Monokine-dependent Hlx expression by human CD56bright
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 Consistent with the data obtained with primary CD56bright NK cells,
assessed by intracellular staining in IL-12/IL-18–stimulated pri- Hlx overexpression led to a marked decrease in IFN-␥ protein
mary human NK cells previously transduced with the EGFP levels in IL-12/IL-18–stimulated NK-92 cells after 24 hours of
(enhanced green fluorescence protein)–expressing PINCO-Hlx or stimulation (Figure 3B). Moreover, levels of IFN-␥ mRNA, albeit
empty PINCO retrovirus (Figure 2). Interestingly, a significant similar in unstimulated vector- and Hlx-transduced NK-92 cells,
decrease in the proportion of IFN-␥⫹ cells was observed in the dramatically increased over time after IL-12/IL-18 stimulation of
Hlx-expressing EGFP⫹CD56bright NK cells (CD56bright PINCO-Hlx vector-transduced cells but not in NK-92 cells overexpressing Hlx
compared with CD56bright PINCO; P ⬍ .02; n ⫽ 3 experiments) (Figure 3C). Thus, our data not only indicated that sustained Hlx
and in the CD56dim fraction (CD56dim PINCO-Hlx compared with expression impairs the ability of NK cells to produce IFN-␥ in
CD56dim PINCO; P ⬍ .006; n ⫽ 3 experiments). In similar experi- response to monokine costimulation, they also suggested that a
ments, human NK-92 cells, which as reported have a phenotype monokine-induced increase of Hlx level is most likely required to
that resembles the CD56bright NK cell subset,23 were infected with temporally control the expression and, therefore, the production of
the Hlx or the empty retrovirus and were EGFP-sorted. As IFN-␥ by activated NK cells. Indeed, Hlx-deficient murine NK
expected, anti-Hlx Western blots performed on subcellular frac- cells show enhanced IFN-␥ mRNA and protein production after in
tions from EGFP⫹ PINCO– and EGFP⫹ PINCO-Hlx–transduced vitro monokine costimulation with IL-12 and IL-15 compared with
NK-92 cell lysates showed that Hlx was indeed overexpressed and wild-type and heterozygous control NK cells (Figure 3D and data
primarily localized in the nuclear compartment (Figure 3A). Note not shown).
that Brg1 and Ku70 were detected as controls for purity of the
nuclear fraction and equal loading, respectively (Figure 3A). Hlx represses IFNG promoter activity in NK cells
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 trans-
duced 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
Figure 2. Hlx inhibits IFN-␥ production by primary CD56bright NK. PINCO- or with IL-12/IL-18 for 6 hours. After normalizing for transfection
PINCO-Hlx–infected primary human NK cells were stimulated for 24 hours with efficiency, we determined that Hlx overexpression repressed IFNG
IL-12/IL-18 and were subjected to staining for CD56 and IFN-␥. (left) Infected cells promoter activity (Figure 4B). However, on subsequent deletion
were gated on CD56brightEGFP⫹ or CD56dimEGFP⫹. (right) IFN-␥ staining in each
population compared with isotype controls. Results shown are representative of 3 analysis of the 3.6-kb IFNG promoter and the 0.8-kb intron 1
experiments (CD56bright PINCO-Hlx compared with CD56bright PINCO; P ⬍ .02). enhancer element within the construct, we were unable to map HlxFrom www.bloodjournal.org by guest on May 20, 2015. For personal use only.
2484 BECKNELL et al BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6
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, EGFP-
sorted and stimulated with IL-12/IL-18. Before stimulation, Hlx-
and 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
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 to reverse the loss of Y693 pSTAT4 protein through the application
with PINCO or PINCO-Hlx were subjected to nucleocytoplasmic fractionation and of MG-132, an inhibitor of the 26S proteasome subunit. Indeed,
immunoblotting for Hlx. Enrichment of nuclear protein was confirmed by Brg1
MG-132 rescued the loss of Y693 pSTAT4 after short-term (3 or 6
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 hours) treatment of Hlx-expressing NK-92 cells with IL-12/IL-18
IL-12/IL-18, and supernatants were harvested after 24 hours for IFN-␥ ELISA. Shown (Figure 5C). Similar results were obtained with the additional
are the mean ⫾ SEM from 5 separate experiments. (C) Time course of IFN-␥ mRNA proteasome inhibitors epoxomicin and lactacystin (data not shown).
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 Interestingly, proteasome inhibition also rescued expression of the
triplicate PCR reactions. (D) Increased IFN-␥ production by Hlx⫺/⫺ NK in response to unphosphorylated STAT4 protein, particularly after 6-hour stimula-
monokine costimulation. Fetal liver–derived NK of indicated genotypes were sub- tion with IL-12/IL-18 (Figure 5C). The E3 ubiquitin ligase SLIM
jected 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
has been shown to catalyze polyubiquitylation of Y693 pSTAT4 in
NK cells derived from 9 or more livers of each genotype are shown. *P ⬍ .001 Hlx⫹/⫹ vitro and to attenuate STAT4 protein expression in vivo.30 There-
compared with Hlx⫺/⫺. **P ⬍ .005 Hlx⫹/⫺ compared with Hlx⫺/⫺). fore, we sought to determine whether SLIM protein levels were
repressive activity to any particular region (data not shown). Thus,
the mechanism of IFNG promoter regulation by Hlx is likely A 3.6 kB 0.8 kB
complex and dependent on multiple cis-regulatory elements.
IFNG promoter IFNG intron 1
luc
Hlx promotes proteasome-dependent STAT4 degradation
after monokine stimulation
B
promoter activity
We further hypothesized that Hlx might negatively regulate IFN-␥ 40
Relative IFNG
mRNA levels by inhibiting the function or activity of a positive 30
regulator of IFN-␥ production. STAT4 is a known trans-activator 20
10
of the IFN-␥ promoter in NK cells.13,14 STAT4 is activated by 0
tyrosine phosphorylation at tyrosine residue 693 (Y693) by Jak2/ Vector Hlx
Tyk2 kinases within seconds of IL-12 stimulation, resulting in its Figure 4. Hlx inhibits IFNG promoter activity in DERL-7 cells. (A) The human
rapid nuclear translocation and direct association with multiple IFNG luciferase construct used in this study consisted of 3.6 kB of a 5⬘ flanking
cis-regulatory elements within the IFN-␥ promoter.26-28 In IL-12/IL- 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
18–stimulated primary human NK or NK-92 cells, we observed
empty vector were transfected with the IFNG luciferase construct, and luciferase
that the increase of Hlx expression was accompanied by a decrease activity was measured after 6-hour stimulation with IL-12/IL-18. Mean ⫾SD of 1 of 3
in total STAT4 protein levels (Figure 5A), suggesting that Hlx may representative experiments is shown.From www.bloodjournal.org by guest on May 20, 2015. For personal use only.
BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6 Hlx INHIBITS IFN-␥ PRODUCTION BY NK CELLS 2485
A B D
Primary Human NK PINCO Hlx PINCO Hlx
Time (h) 0 24 48 72 96 0 0.5 1 3 6 12 18 24 0 0.5 1 3 6 12 18 24 Time (h)
0 1 3 6 9 12 24 0 1 3 6 9 12 24
50 kDa Hlx 80 kDa
80 kDa STAT4 STAT4
80 kDa STAT4 42 kDa β−actin β-Actin
42 kDa
42 kDa β-actin
C E _ _ + + _ _ + + CHX
NK-92
IL-12 + IL-18: 3h 6h + + _ _ + + _ _ EtOH
Time (h) 0 24 48 72
- V MG - V MG P Hlx P Hlx P Hlx P Hlx
50 kDa Hlx
80 kDa STAT4
80 kDa STAT4 STAT4
42 kDa β-actin 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 between the onset of detectable Hlx mRNA and appreciable Hlx
SLIM expression was observed (data not shown). protein expression (Figure 1A). In future studies, it will be
To confirm that the loss of STAT4 protein was caused by insightful to analyze the HLX promoter itself and to determine the
decreased protein stability, new protein synthesis was inhibited direct influence of monokines on its activity and the potential
using cycloheximide (CHX), and the kinetics of STAT4 degrada- effects of NK-derived cytokines that have been shown to inhibit
tion were monitored in NK-92 expressing Hlx compared with IFN-␥ production, such as transforming growth factor- and
empty vector. As expected, in the presence of CHX, Hlx overexpres- TWEAK,15,31 on Hlx mRNA and protein expression.
sion resulted in decreased Y693 pSTAT4 levels beginning after 6 In addition, we identified the STAT4 transcription factor as a
hours of IL-12/IL-18 stimulation compared with empty vector key molecular target of Hlx action in NK cells. Multiple lines of
(Figure 5D). Surprisingly, however, CHX treatment resulted in an evidence have established the role of STAT4 as a requisite, direct
accumulation of unphosphorylated STAT4 in cells expressing Hlx transactivator of the IFNG promoter in NK cells after monokine
and empty vector but with more rapid kinetics with Hlx overexpres- stimulation.13,14,27 Constitutive STAT4 activation is strongly impli-
sion (Figure 5D). Thus, it appears that Hlx overexpression acceler- cated in autoimmune disease, including rheumatoid arthritis and
ates the dephosphorylation of Y693 STAT4 and that its subsequent inflammatory bowel disease.6,32 The inhibitory effect of Hlx on
degradation is reliant on new protein synthesis. To confirm these STAT4 levels identifies Hlx as a potentially relevant molecular
findings were specific to CHX treatment, we treated Hlx and vector target to exploit in autoimmunity. Ultimately, the physiological and
expressing NK-92 with CHX or ethanol carrier for 12 hours or 24 pathophysiological roles of Hlx in the attenuation of STAT4
hours and repeated the STAT4 Western blot (Figure 5E). Again, we transcriptional activity and IFN-␥ production must be determined
found that the inhibitory effect of Hlx overexpression on Y693 in vivo.
pSTAT4 levels was insensitive to CHX treatment. Furthermore, we Mechanistically, we found that Hlx promotes the degradation of
confirmed the stabilizing influence of CHX treatment on the levels Y693 pSTAT4. Previous work has identified SLIM as an E3
of unphosphorylated STAT4, regardless of Hlx or vector expression. 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
Discussion the proteasome. In our study, through the use of CHX, we
implicated Hlx specifically in the dephosphorylation of pSTAT4
In this study, we found that Hlx is a novel negative regulator of Y693 (Figure 4E), and we demonstrated that subsequent degrada-
IFN-␥ production in NK cells after monokine costimulation. The tion of unphosphorylated STAT4 requires new protein synthesis.
delayed kinetics of Hlx protein induction with respect to IFN-␥ These findings suggest that the process of Y693 pSTAT4 degrada-
itself are consistent with a model in which Hlx serves as a feedback tion is complex, consisting of a CHX-insensitive dephosphoryla-
inhibitor of IFN-␥ synthesis in activated CD56bright NK. This tion step and a CHX-sensitive degradation step. The specific role of
Hlx-mediated negative feedback pathway is likely important in Hlx in Y693 pSTAT4 dephosphorylation remains unclear. Although
vivo, given the potentially deleterious effects of unchecked IFN-␥ the Hlx protein structure lacks sequence homology to known
production on the host.6,7 The process of Hlx protein induction after tyrosine phosphatase domains, it is conceivable that Hlx may
monokine costimulation is likely complex. Indeed, Hlx regulation recruit a protein tyrosine phosphatase to DNA-bound Y693 pSTAT4
must occur at transcriptional and posttranscriptional levels because dimers or tetramers. In addition, it is possible that Hlx may
parallel mRNA and protein expression studies have established associate directly with DNA through its homeodomain and may
long lags between monokine costimulation and the onset of Hlx displace DNA-bound Y693 pSTAT4 multimers, rendering them
mRNA expression (approximately 12 hours; data not shown) and more susceptible to dephosphorylation and degradation. Moreover,From www.bloodjournal.org by guest on May 20, 2015. For personal use only.
2486 BECKNELL et al BLOOD, 15 MARCH 2007 䡠 VOLUME 109, NUMBER 6
we have not fully rejected the hypothesis that Hlx functions as a IL-4 is a potent inhibitor of IFN-␥ production by T cells.35
transcription factor and that it is in fact the transcriptional target(s) Therefore, by decreasing the responsiveness of a CD4⫹ T cell to
of Hlx, translated before CHX and monokine treatment, that trigger IL-4 during early TH2 polarization, Hlx may indirectly promote the
Y693 pSTAT4 dephosphorylation after IL-12/IL-18 stimulation. production of IFN-␥ by this lymphocyte population. If the effect of
Indeed, it is conceivable that SLIM is one such target of Hlx. Hlx on IFN-␥ is attributed entirely to IL-4R␣ regulation, it will be
Besides identifying a role for STAT4 in Hlx-dependent regula- important in the future to determine whether the loss of IL-4R␣
tion of IFN-␥, our work identifies the IFNG promoter as a potential expression eliminates the increase in IFN-␥ seen on ectopic Hlx
direct target of Hlx transcriptional repression. However, in prelimi- expression in TH2 cells. In addition, it will be interesting to
nary experiments, we were unable to identify the specific region determine whether Hlx influences IL-4R␣ expression by NK cells
required for Hlx repression (data not shown). This difficulty may be given that IL-4 plays a very different role in NK cells than in T
attributable to a complex requirement for multiple cis-regulatory cells, namely to costimulate IFN-␥ production.36,37
motifs throughout the IFNG promoter for Hlx repression of IFN-␥ Thus, it is likely that Hlx serves different roles in the T and NK
to occur. Therefore, in subsequent work, it will be essential to lineages, and the extent of its function may be limited to discrete
distinguish the effects of Hlx on STAT4 from its direct effect, if developmental windows. It is not without precedent that transcrip-
any, at the IFNG promoter. Stimulation of Hlx overexpressing tion factors perform divergent functions in multiple lineages.
NK-92 cells with IL-2 or IL-15, alone or in combination with GATA-3 is one transcriptional regulator shown to have apparently
IL-18, did not influence IFN-␥ mRNA or protein levels compared opposing functions in IFN-␥ regulation by NK and CD4⫹ T cells.38
with cells infected with empty retroviral vector (data not shown). Alternatively, one can envision multiple scenarios that could
Because STAT4 is activated minimally under these conditions (data account for opposing Hlx activities in CD4⫹ T and NK cells, such
not shown), this finding is consistent with a model in which the as posttranslational modification of Hlx or unique expression of
effects of Hlx are mediated primarily by STAT4. STAT4 is required Hlx interacting proteins in lineage-specific fashion. It is anticipated
for IFN-␥ production in response to IL-12,13,14 making it prohibi- that more detailed studies of the Hlx protein and its interactions
tive to explore the consequence of Hlx overexpression in STAT4- will elucidate its mechanism of action in T- and NK-cell lineages in
deficient NK cells on IFN-␥ production after IL-12/IL-18 treatment the future.
or to determine the effect of Hlx on the activity of IFNG promoter
constructs lacking STAT4 enhancer elements. Given these chal-
lenges, it may be insightful instead to perform a detailed structure– Acknowledgments
function analysis of Hlx itself so as to identify Hlx mutants that
This work was supported by National Cancer Institute grants P30
maintain IFN-␥ repression without affecting STAT4 levels and
CA16059, CA68458, and CA95426 (M.A.C.) and by National
vice versa.
Institutes of Health grants DK02791 and DK61219 (M.D.B.).
Our interest in Hlx was prompted by published accounts of its
We thank Dr Howard Young (National Cancer Institute, Freder-
role as a positive regulator of IFN-␥ production in CD4⫹ T
ick, MD) for providing the IFNG promoter luciferase construct; Dr
lymphocytes.10-12 Although the data presented here are not consis-
Michael Grusby (Harvard University) for the generous donation of
tent with this role in NK cells, considerable caution should be
SLIM antisera, and Dr Denis Guttridge (The Ohio State University)
exercised in comparing this work with the T-cell studies given the
and Dr Wei-ping Zheng (University of Rochester) for helpful advice.
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 Authorship
process of polarization, entailing cell division and active chromatin
remodeling at the IFNG locus, before IFN-␥ transcription can Contribution: B.B. designed and performed the research and wrote
occur.33 Ectopic expression of Hlx during TH2 polarization of the manuscript. T.L.H. performed the research. A.G.F. designed the
CD4⫹ T cells has been shown to promote IFN-␥ production, but research. B.W.B. performed the research. J.Y designed the re-
Hlx expression during TH1 polarization and in fully TH1- and search. R.T. designed the research. H.C.M. collected the data.
TH2-polarized CD4⫹ T cells reportedly does not influence IFN-␥ M.L.C. performed the research. M.A. performed the research.
synthesis.10-12 These findings have led to the hypothesis that Hlx D.M.B. performed the research. A.L. analyzed the data. D.J.
functions in a discrete window during early TH-cell differentiation analyzed the data. D.P. designed the research. M.D.B. designed the
to promote IFN-␥ production.10-12 research. M.A.C. designed the research and edited the original and
Interestingly, recent work from the Zheng34 laboratory clearly revised versions of the manuscript.
implicates Hlx as a negative regulator of the IL-4 receptor alpha Conflict-of-interest disclosure: The authors declare no compet-
chain (IL-4R␣). Because IL-4 is required for normal TH2 differen- ing financial interests.
tiation, Hlx deficiency results in impaired production of TH2 Correspondence: Michael A. Caligiuri, A458 Starling-
cytokines, and this phenotype can be reversed by ectopic retroviral Loving Hall, 320 W 10th Ave, Columbus, OH 43210; e-mail:
expression of IL-4R␣.34 Apart from promoting TH2 differentiation, michael.caligiuri@osumc.edu.
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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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