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Blood, 15 April 2002, Vol. 99, No. 8, pp. 2637-2646
PLENARY PAPER
Interactions of STAT5b-RAR , a novel acute promyelocytic
leukemia fusion protein, with retinoic acid receptor and STAT3
signaling pathways
Shuo Dong and
David J. Tweardy
From the Section of Infectious Disease, Department of
Medicine, Baylor College of Medicine, Houston, TX, and the Shanghai
Institute of Hematology, Shanghai Rui-Jin Hospital, Shanghai, Peoples
Republic of China.
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Abstract |
Signal transducer and activator of transcription (STAT) 5b-retinoic
acid receptor (RAR)  is the fifth fusion protein identified in acute
promyelocytic leukemia (APL). Initially described in a patient with
all-trans retinoic acid (ATRA)-unresponsive disease, STAT5b-RAR resulted from an interstitial deletion on chromosome 17. To determine the molecular mechanisms of myeloid leukemogenesis and
maturation arrest in STAT5b-RAR+ APL and its
unresponsiveness to ATRA, we examined the effect of STAT5b-RAR on
the activity of myeloid transcription factors including RAR/retinoid
X receptor (RXR) , STAT3, and STAT5 as well as its molecular
interactions with the nuclear receptor corepressor, SMRT, and nuclear
receptor coactivator, TRAM-1. STAT5b-RAR bound to retinoic
acid response elements (RAREs) both as a homodimer and as a heterodimer
with RXR and inhibited wild-type RAR/RXR transactivation.
Although STAT5b-RAR had no effect on ligand-induced STAT5b
activation, it enhanced interleukin 6-induced STAT3-dependent reporter
activity, an effect shared by other APL fusion proteins including
promyelocytic leukemia-RAR and promyelocytic leukemia zinc finger
(PLZF)-RAR. SMRT was released from STAT5b-RAR/SMRT complexes by
ATRA at 10 6 M, whereas TRAM-1 became associated with
STAT5b-RAR at 107 M. The coiled-coil domain of STAT5b
was required for formation of STAT5b-RAR homodimers, for the
inhibition of RAR/RXR transcriptional activity, and for stability
of the STAT5b-RAR/SMRT complex. Thus, STAT5b-RAR contributes to
myeloid maturation arrest by binding to RARE as either a homodimer or
as a heterodimer with RXR resulting in the recruitment of SMRT and
inhibition of RAR/RXR transcriptional activity. In addition,
STAT5b-RAR and other APL fusion proteins may contribute to
leukemogenesis by interaction with the STAT3 oncogene pathway.
(Blood. 2002;99:2637-2646)
© 2002 by The American Society of Hematology.
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Introduction |
Nonrandom chromosomal translocations play a
critical role in the pathogenesis of human blood
malignancies.1 Five different chromosomal translocations
have been reported and characterized so far in acute promyelocytic
leukemia (APL), a disease effectively treated by agents that target the
resultant chimeric transcription factor.2-4 In the great
majority of patients, there is a specific chromosomal translocation
t(15;17)(q22;q21), which involves the PML (promyelocytic
leukemia) gene located on chromosome 15 and the RARA
(retinoic acid receptor ) gene located on chromosome 17.5,6 The wild-type PML is a component of a nuclear
structure referred to as PML nuclear body or POD (PML oncogenic
domain). Almost all patients with t(15;17) APL respond well to
differentiation therapy with all-trans retinoic acid
(ATRA).4 Several variant chromosomal translocations occur
in a small subset of APLs. Two variant translocations that result in
ATRA-responsive APL are t(5;17)(q35;q21) and t(11;17)(q13;q21), which
involve the RAR locus and a known gene NPM
(nucleophosmin) on chromosome 57 and NuMA
(nuclear mitotic apparatus protein) on chromosome 11,8 respectively. One additional variant translocation t(11;17)(q23;q21) has been found that fuses the RAR locus with the PLZF
(promyelocytic leukemia zinc finger) gene on chromosome
11q23.9 In contrast to patients with APL with t(15;17),
t(5;17), and t(11;17), those with t(11;17)(q23;q21) have a poor
response to ATRA.3 Very recently, a fifth fusion gene
STAT5b-RARA has been identified in one patient with
ATRA-unresponsive APL.10 This fusion gene occurred as a
result of an interstitial deletion within chromosome 17 and represents
the first stable chromosomal abnormality described in a malignancy that
involves a member of the STAT protein family.
Seven signal transducer and activator of transcription (STAT) proteins
have been identified in mammalian cells, STAT1, 2, 3, 4, 5a, 5b, and 6;
each is tyrosine-phosphorylated by JAKs following the binding of
cytokine to its receptor.11 Thus far, more than 40 different polypeptide ligands have been shown to cause STAT protein
activation.11,12 On tyrosine phosphorylation, STAT proteins form homodimers or heterodimers through reciprocal
intermolecular interactions involving the SH2 domain of one STAT
protein binding to the phosphorylated tyrosine of its partner.
Dimerization is followed by rapid translocation to the nucleus, binding
to target DNA, and induction of gene expression. STAT5 was originally
identified in sheep as a prolactin-induced mammary gland transcription
factor.13 STAT3 was originally termed acute-phase response
factor (APRF) because it was first identified as a transcription factor
that bound to interleukin 6 (IL-6)-responsive elements within the
promoters of various acute-phase protein genes.14 STAT5a
and STAT5b are encoded by 2 highly homologous genes located in close
proximity to each other and to STAT3 on mouse chromosome 11 and human
chromosome 17.15-17 STAT protein activation, especially
STAT5 and STAT3, has been implicated in cell transformation and
carcinogenesis. In addition to their constitutive activation in solid
tumors, aberrant activation of STAT5 and STAT3 has been
reported in a variety of hematopoietic cancers including acute and
chronic myelogenous and lymphocytic leukemias and
lymphomas.18
Retinoic acid receptor is a member of a superfamily of nuclear
hormone receptors, which affects many physiologic processes including
differentiation and growth arrest of various cell types including
hematopoietic cells.19 In normal myeloid cells, RAR dimerizes with retinoid X receptor (RXR); the dimers bind to retinoic acid response elements (RAREs) located in promoter/enhancer regions of specific genes. Recent models suggests that in the absence
of ligand (retinoic acid, RA), RAR/RXR heterodimers associate at
a 1:1 ratio with nuclear receptor transcriptional repressor complex
corepressor (CoR) composed of SMRT/NCoR, Sin3, and histone deacetylase
(HDAC) resulting in repression of basal transcription.20,21 Physiologic levels of RA induce
dissociation of the CoR complex followed by recruitment of the
transcriptional activation complex coactivator (CoA), consisting of
CBP/p300, P/CAF, SRC-1, TIF2 (GRIP1/SRC-2), and p/CIP
(TRAM-1/ACTR/AIB1/RAC3/SRC-3). Binding of the CoA complex results in
the activation of gene expression and normal
differentiation.22-24 In ATRA-responsive APL with
t(15;17), the PML-RAR fusion protein binds RARE as a homodimer and
recruits 2 CoR complexes with higher affinity than wild-type
RAR.25,26 Complete dissociation of CoR from PML-RAR
does not occur at physiologic levels of RA, but rather requires higher
levels of ligand achieved during treatment with ATRA.25
Several hypotheses have been proposed to explain ATRA unresponsiveness
in some variant APL including altered stoichiometry and stability of
the CoR-fusion protein interaction26,27 and resistance of
the APL-fusion gene products to ATRA-induced
proteolysis.28
The molecular bases for leukemogenesis, arrested differentiation, and
ATRA unresponsiveness in STAT5b-RAR+ APL are unknown. To
address these issues, we examined the effect of STAT5b-RAR on
myeloid leukemogenic and differentiation pathways involving
RAR/RXR, STAT3 and STAT5 as well as the interaction between
STAT5b-RAR and the nuclear receptor coregulators, CoR and CoA. We
determined that STAT5b-RAR binds RARE as either a homodimer or as a
heterodimer with RXR and modulates the transcriptional activities of
RAR/RXR and STAT3 but not STAT5. STAT5b-RAR was insensitive to
ATRA-induced proteolysis in transient expression COS-7 and HeLa cells.
Finally, the ability of STAT5b-RAR to enhance STAT3 activity is
shared by other APL fusion proteins including PML-RAR and
PLZF-RAR and may therefore represent an important new pathway
contributing to leukemogenesis in APL.
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Materials and methods |
Cell lines and reagents
COS-7, 293T, HepG2, and HeLa cells were cultured in Dulbecco
modified Eagle medium (DMEM; Life Technologies, Grand Island, NY) with 10% fetal bovine serum, penicillin (100 U/mL), and
streptomycin (100 U/mL). Human IL-2 and IL-6 were purchased from Roche
Molecular Biochemicals (Indianapolis, IN) and R & D Systems
(Minneapolis, MN), respectively. ATRA was obtained from Sigma (St
Louis, MO) and the ovine prolactin (NIDDK-oPRL-20) was obtained
from National Institute of Diabetes and Digestive and Kidney Diseases
(NIDDK; Bethesda, MD).
Plasmids
The PML-RAR, PLZF-RAR, PLZF, RAR, and RXR
expression vectors were described previously.29-31 The
(RARE)3-tk-luciferase reporter5 and the APRE-luciferase
reporter construct,32 which has 4 copies of acute phase
response elements (APREs) were kindly provided by Dr A. Dejean (Paris,
France) and Dr I. Matsumura (Osaka, Japan), respectively. The
 -casein luciferase reporter gene is from -casein gene promoter
region ( 2300 to +490; a gift from Dr J. Rosen, Houston,
TX).33 The human expression vectors for STAT5b,
IL-2-receptor (IL-2R) chain and  common chain were provided by
Dr W. J. Leonard (National Institutes of Health [NIH], Bethesda,
MD).16,34-36 The human JAK3 and the Nb2 PRL-R expression vector are from Dr O'Shea (NIH) and Dr L. Yu-Lee (Houston, TX), respectively. GST-TRAM-1 (residues 577-821) in pGEX-5X-2 and GST-SMRT (residues 983-1172) in pGEX-5X-1 both containing the receptor interaction domain were produced by polymerase chain reaction (PCR)
with pfu DNA polymerase (Stratagene, La Jolla, CA) using TRAM-1
expression vector (kindly provided by Dr W. W. Chin, Boston, MA)24 and SMRT expression vector20 as
template, respectively. The STAT5b-RAR (in pSG5 vector) (Stratagene)
expression vectors was constructed by fusing human
STAT5b16 and RAR using fusion-PCR technology37 based on the published
sequence.10 A similar PCR approach also is applied to
construct a series of mutant STAT5b-RARs, STAT5b-RAR( N),
STAT5b-RAR(CC), and STAT5b-RAR(DBD). All plasmid
constructs were confirmed by DNA sequencing and by in vitro translation
and immunoblotting.
Cell transfections
For transient transfections, COS-7 and 293T cells were grown in
6-well (35-mm diameter) tissue culture plates to 50% to 80% confluence. Twelve hours later, the cells were transiently transfected with the indicated expression vectors and reporter genes by standard calcium phosphate coprecipitation method.38 The amounts of
plasmid DNA used per well were 1 µg reporter vector, 1 to 4 µg
expression vector, and 1 µg -galactosidase expression vector
(Promega, Madison, WI) as transfection control. For HepG2 and
HeLa cell, the GeneJuice transfection reagent (Novagen, Madison,
WI) was used according to the manufacturer's instruction.
Luciferase activity was measured in a luminometer (Luminoskan
Ascent, Labsystems, Franklin, MA), expressed in arbitrary units and
normalized according to the internal control. Each point is the mean of
at least 3 independent experiments.
In vitro translation
The TNT-coupled rabbit reticulocyte lysate (Promega) system was
used for in vitro translated proteins according to the manufacturer's instructions. The relative quantity of in vitro translated proteins was
estimated as described.30,31 Briefly, parallel translation reactions were performed in the presence of [-35S]
methionine (NEN, Boston, MA) and the proteins were visualized by
autoradiography after separation on a 10% sodium dodecyl
sulfate-polyacrylamide gel.
Gel-shift DNA-binding assays
The 293T, COS-7 and HepG2 cell lines were transiently
transfected in 6-well plates using 2 to 4 µg plasmids. Forty-eight
hours later, cells were either not treated or treated for 30 minutes with cytokines, IL-2 (50 ng/mL), prolactin (500 ng/mL), IL-6 (25 ng/mL), and the whole cell extracts (WCEs) were prepared as reported previously.39 About 20 µg WCE was incubated with duplex
oligonucleotides in binding buffer (13 mM Hepes, pH 8.0, 65 mM NaCl, 1 mM DTT, 0.14 mM EDTA, 8% glycerol) and separated on 5% polyacrylamide gels. Duplex oligonucleotide probes included the high-affinity serum-inducible element (hSIE), prolactin response element (PRE) contained within the -casein promoter,13 and the APRE,
an IL-6 response element within the rat 2-macroglobulin
promoter.32 For supershift, 1 µg antibody was included
in this reaction system when needed. Gel shift assays using in vitro
protein were performed as described previously.30 Briefly,
in vitro translated proteins were preincubated for 15 minutes at room
temperature in the following buffer: 20 mM Hepes, pH 7.4, 50 mM KCl, 1 mM 2-mercaptoethanol, 10% glycerol, 1 µg poly (dI-dC)
(Pharmacia), and 100 µg bovine serum albumin (BSA).
[-32P] adenosine triphosphate (ATP; NEN) end-labeled
duplex oligonucleotide probe was added and samples incubated at room
temperature for 30 minutes and at 4°C for 30 minutes. Protein-DNA
complexes were separated on 6% polyacrylamide gels equilibrated in
0.25 times tris-borate-EDTA buffer (TBE). Gels were dried,
exposed to PhosphorImager plates and images developed and quantitated
using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and
ImageQuant software.
In vitro protein-binding assays
The SMRT protein was expressed in Escherichia coli
DH5 as a GST fusion product and purified by standard
methodology. Twelve microliters of -35S]
methionine-labeled in vitro translated proteins (STAT5b-RAR or
STAT5b-RAR [CC]) was incubated with 1 µg GST-SMRT fusion protein conjugated to glutathione Sepharose (Amersham-Pharmacia Biotech, Piscataway, NJ) in binding buffer (20 mM Tris, pH 8.0, 150 mM KCl, 1 mM EDTA, 4 mM MgCl2, 0.2% NP-40, 10%
glycerol) at 4°C for 2 hours without or with ATRA (Sigma). Bound
proteins were washed 3 times with binding buffer, eluted by boiling in
sample buffer, and resolved by 10% SDS-polyacrylamide gel
electrophoresis (PAGE). Gels were dried, exposed, and analyzed by
PhosphorImager. For in vitro coimmunoprecipitation, in vitro translated
SMRT protein was incubated with 35S-labeled proteins, PLZF,
RAR, and STAT5b, respectively, in binding buffer for 1 hour at
4°C. Immune complexes were isolated by further incubation with SMRT
antibody presorbed on protein A/G (Santa Cruz Biotechnology, Santa
Cruz, CA), washed 3 times in binding buffer and analyzed
by SDS-PAGE.
Immunoblotting
Whole cell lysates were prepared in lysis buffer (20 mM Hepes,
pH 7.9, 420 mM NaCl, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol [DTT], 20% glycerol, 0.5 mM phenylmethylsulfonyl
fluoride [PMSF]). Equivalent amounts of total cellular protein were
electrophoresed on 7.5% SDS-polyacrylamide gel and transferred to
polyvinylidene difluoride (PVDF) membrane (Millipore).
Probing of PVDF membranes with primary antibodies and detection of
horseradish peroxidase-conjugated secondary antibodies by enhanced
chemiluminescence as directed (Amersham-Pharmacia Biotech). Antibodies
used in this study are as follows: antihuman-RAR (C-20)
antihuman-STAT5b (N-20), antihuman-STAT5b (C-17), and antihuman-SMRT (N-20), all from Santa Cruz Biotechnology, and antihuman--actin (monoclonal; Sigma).
| |
Results |
STAT5b-RAR binds RARE as a homodimer and preferentially as a
heterodimer with RXR
The APL fusion proteins previously identified and characterized
contribute to leukemogenesis by binding to RAREs either as homodimers
or heterodimers with RXR, thereby repressing gene transcription
essential for myeloid differentiation.3 To determine if
STAT5b-RAR is capable of binding to RAREs, in vitro translated STAT5b-RAR protein was examined by gel-shift assay using a series of
RARE duplex oligonucleotides. As shown in Figure
1A, STAT5b-RAR alone bound to all
RAREs tested and this binding could be competitively inhibited by
100-fold excess unlabeled RARE (Figure 1E). The shifted band
corresponded to a homodimer of STAT5b-RAR with migration characteristics similar to PML-RAR and PLZF-RAR using
RAR/RXR as a size reference (Figure
2A). The STAT5b-RAR homodimer binding preferences overall are similar to PML-RAR and PLZF-RAR (Figure 1A-C).30,31,40 However, a few slight differences in
binding preferences were observed: STAT5b-RAR and PLZF-RAR bound
to RARE-p21-WAF less efficiently than PML-RAR, and STAT5b-RAR
bound to CRBPII and HOXA1 less efficiently than either PLZF-RAR
or PML-RAR.
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| Figure 1.
RARE binding by homodimers of APL fusion proteins.
Binding of homodimers of STAT5b-RAR (A), PML-RAR (B), and
PLZF-RAR (C) and heterodimers of RAR/RXR (D) to a series of
RAREs. Gel-shift assays were performed with in vitro translated
proteins using the following radiolabeled probes: synthetic RARE, DR1,
2, 3, 4 and 5G,40 2 RARE (DR5T) from the
human RARB gene,30 2 RARE from the human
RARA gene,59 the natural enhancer elements from
the rat cellular retinal-binding protein type I gene
(CRBPI),60 the rat cellular retinal-binding
protein type II gene (CRBPII),61 the murine
cellular RA-binding protein gene (CRABPII),62
HOXA1 gene,63 HOXB1
gene64 and p21-WAF1
gene.65 Equivalent amounts of in vitro translated
protein and labeled oligonucleotide were added to each binding
reaction. The results of quantitative analysis using ImageQuant
software are shown below each autoradiogram. In panel E, radiolabeled
DR5G was incubated with in vitro translated STAT5b-RAR and the
indicated fold excess unlabeled DR5G followed by gel-shift assay.
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| Figure 2.
RARE binding by heterodimers of APL fusion proteins.
(A) Binding of heterodimers containing RXR and STAT5b-RAR,
PLZF-RAR, PML-RAR, or wild-type RAR to 2 RAREs, DR5G and DR5T.
(B) STAT5b-RAR/RXR heterodimer formation and RARE binding with
increasing concentration of RXR. In vitro translated STAT5b-RAR
protein (2.0 µL) was incubated without or with in vitro translated
RXR protein (0, 0.05, 0.1, 0.2, 0.5, 1.0, and 2.5 µL). Gel-shift
assays were performed using 2 RAREs, DR5G and DR5T. The location of the
STAT5b-RAR homodimer band is indicated by the filled triangle; the
location of the STAT5b-RAR/RXR heterodimer composed of one
molecule of STAT5b-RAR plus one molecule of RXR is indicated by
the unfilled triangle. The arrow indicates the position of the RXR
homodimer binding to DR5G. (C) STAT5b-RAR/RXR heterodimer can
bind a series of RAREs. STAT5b-RAR/RXR heterodimer is indicated
by the filled triangle; RXR homodimer is indicated by the unfilled
triangle.
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Very intriguingly, gel-shift assays containing both STAT5b-RAR and
RXR resulted in the appearance of an additional prominent band
(STAT5b-RAR/RXR heterodimer) with mobility characteristics intermediate between RAR/RXR heterodimer and STAT5b-RAR
homodimer (Figure 2A). Increasing the amount of RXR while keeping
the amount of STAT5b-RAR constant (Figure 2B) resulted in the
disappearance of STAT5b-RAR homodimer band and increased the
prominence of the STAT5b-RAR/RXR heterodimer band suggesting that
STAT5b-RAR prefers to bind RARE as a heterodimer with RXR (one
molecule of STAT5b-RAR plus one molecule of RXR) rather than as a
homodimer. We and others have demonstrated
previously30,31,40 and confirmed in this study (Figure 2A
and data not shown) that when PML-RAR or PLZF-RAR binds RARE in
combination with RXR, each does so as a single heterodimer as well
as a higher order multimeric complex. In contrast, STAT5b-RAR and
RXR bind RAREs virtually exclusively as a single heterodimer over a
wide range of ratios and irrespective of the RARE (Figure 2C).
The STAT5b coiled-coil domain is responsible for STAT5b-RAR
homodimer formation and inhibition of RAR/RXR-mediated
transcriptional activity
STAT5b-RAR retains 3 complete domains of STAT5b, the N-terminal
oligomerization domain, the coiled-coil domain, and the DNA-binding domain, in addition to a truncated SH2 domain (Figure
3A). The N-terminal and coiled-coil
domains each have been demonstrated to mediate protein-protein
interactions.11,41 To investigate whether or not either of
these 2 domains or the DNA binding domain is important for the
oncogenic functions of STAT5b-RAR, we compared the activities and
functions of wild-type STAT5b-RAR with mutants of STAT5b-RAR in
which the N-terminal, coiled-coil, or DNA-binding domain was deleted
(Figure 3A). In gel-shift assays, STAT5b-RAR(N), STAT5b-RAR(DBD), and STAT5b-RAR(linker and part of SH2)
each bound RARE alone as a homodimer or as a heterodimer with RXR (Figure 3B and data not shown). In contrast, STAT5b-RAR(CC) could
not bind RARE as a homodimer, but rather it bound RARE only as a
heterodimer with RXR. These findings indicate that the coiled-coil domain of STAT5b, and not the N-terminal or DNA-binding domains, is
important for STAT5b-RAR homodimer formation. These results are
reminiscent of those for PML-RAR in which the coiled-coil domain of
PML was found to be responsible for the formation of PML-RAR homodimers.40,42,43
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| Figure 3.
Requirement of the coiled-coil domain of STAT5b-RAR for RARE binding
and transcription activation.
(A) Schematic illustration of wild-type and mutated STAT5b-RAR
constructs. (B) Gel-shift assays with wild-type and mutant
STAT5b-RAR using 2 RAREs, DR5G and DR5T. (C) Transactivational
activities of wild-type and mutant STAT5b-RAR in COS-7 cells.
Following transfection with the indicated constructs, cells were
cultured in medium with 106 M ATRA for 24 hours.
Luciferase activity was normalized for transfection efficiency using a
-galactosidase reporter plasmid. The results presented are the
mean ± SD of triplicate wells and are representative of 3 separate experiments.
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Previously, PML-RAR and PLZF-RAR were demonstrated to have a
dominant-negative effect on wild-type RAR/RXR transcriptional activity.3,5,29 To determine whether or not STAT5b-RAR behaves similarly, we examined the effect of STAT5b-RAR on
RA-dependent, RAR/RXR-mediated transactivation of the
RARE3-tk-luc reporter gene construct (Figure 3C). Transfection of the
reporter construct alone or with RAR resulted in RA-dependent
transactivation. Transfection with STAT5b-RAR alone or with RAR
inhibited RA-dependent transactivation. Transfection of
STAT5b-RAR(CC) did not inhibit but rather augmented RA-dependent
RAR/RXR-mediated transcriptional activity similar to RAR,
indicating that inhibition of RAR/RXR activity requires the
coiled-coil domain of STAT5b-RAR and that in the absence of its
coiled-coil domain, STAT5b-RAR functions like wild-type RAR.
Effects of ATRA on the interaction of STAT5b-RAR with CoR SMRT
and CoA TRAM-1 and on STAT5b-RAR protein degradation
RAR/RXR and the APL fusion proteins PML-RAR and
PLZF-RAR suppress transcription by associating with CoR and CoA
depending on the concentration of ATRA.3 To determine the
ATRA concentration dependence of the interactions of STAT5b-RAR with
CoR and CoA, we examined the composition of complexes containing
STAT5b-RAR, GST-SMRT, and GST-TRAM-1 in varying concentrations of
ATRA (Figure 4A). SMRT dissociated from
STAT5b-RAR at pharmacologic concentrations of ATRA
(106 M) similar to that required for its dissociation
from PML-RAR. This ATRA concentration is one log greater than that
required to cause SMRT dissociation from RAR/RXR
(107 M) and one log lower than that required for SMRT
dissociation from PLZF-RAR (105 M).
Immunoprecipitation assays (Figure 4B) demonstrated that although
SMRT can form a complex with PLZF and RAR, as shown previously,25 it does not bind STAT5b. Complete
recruitment of the CoA TRAM-1 to STAT5b-RAR occurred at
107 M ATRA similar to that for PLZF-RAR (Figure 4A),
but one log greater that that for PML-RAR and RAR/RXR
(108 M).
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| Figure 4.
Interactions of STAT5b-RAR, PML-RAR, and PLZF-RAR and
wild-type RAR/RXR with the CoR SMRT and CoA TRAM-1.
(A) The in vitro translated proteins indicated on the left were
incubated with or without SMRT or TRAM-1 as indicated and the
radiolabeled RARE, DR5G. The location of the complex containing the
chimeric or wild-type receptor plus SMRT/TRAM-1 is indicated by the
solid triangle; the location of the chimeric or wild-type receptor
alone is indicated by the open triangle. (B) SDS-PAGE and
autoradiography were performed on in vitro translated and
35S-methionine-labeled PLZF, RAR, or STAT5b alone or
following incubation with SMRT and immunoprecipitation with SMRT
antibody as indicated.
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The coiled-coil domain of PML-RAR APL fusion proteins contributes to
the stability of the APL fusion protein/SMRT complex.26 To
determine if this is the case for STAT5b-RAR, we examined the
stability of the binding of in vitro translation proteins STAT5b-RAR
and STAT5b-RAR(CC) with GST-SMRT in a GST pull-down assay under
varying concentrations of ATRA (Figure
5A,B). STAT5b-RAR dissociated from
GST-SMRT beginning at 106 M ATRA, whereas
STAT5b-RAR(CC) dissociated from GST-SMRT beginning at
107 M ATRA, one log lower. These findings indicate that
the coiled-coil domain plays an important role in the stability of
STAT5b/SMRT-RAR complexes.
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| Figure 5.
Effect of ATRA on interactions
STAT5b-RAR with GST-SMRT and on STAT5b-RAR stability.
Radiolabeled wild-type or mutant STAT5b-RAR was incubated with
GST-SMRT in the presence of the indicated concentrations of ATRA. After
absorption with glutathione Sepharose, the proteins were separated and
analyzed by autoradiography (A) and PhosphorImager analysis (B). COS-7
cells (C) and HeLa cells (D) were transiently transfected with
STAT5b-RAR or PLZF-RAR and incubated without (lanes 1 and 3) or
with (lanes 2 and 4) 106 M ATRA for 24 hours. Cells were
lysed; proteins were separated by SDS-PAGE and immunoblotted with
RAR antibody (upper panel) as well as -actin antibody (bottom
panel), which was used as loading control. Densitometry analysis is
presented below each immunoblot. The results shown are representative
of up to 3 separate experiments. The empty triangle to the right of
each upper panel indicates presumed proteolytic fragments of
PLZF-RAR.
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Exposure to ATRA induces intracellular degradation of the
PML-RAR and PLZF-RAR fusion proteins.44,45 We
examined whether or not ATRA exposure similarly resulted in degradation
of STAT5b-RAR. COS-7 and HeLa cells were transiently transfected
with expression constructs containing STAT5b-RAR or PLZF-RAR and
the cells incubated in 106 M ATRA for 24 hours. As
previously described,44 PLZF-RAR protein levels sharply
decreased in cells incubated with ATRA (Figure 5C,D). In contrast, the
level of STAT5b-RAR protein in cells incubated with ATRA was
virtually unchanged.
STAT5b-RAR enhances STAT3 transcriptional activity but has no
effect on STAT5 transcriptional activity
Aberrant STAT3 activation has been demonstrated to occur in human
leukemias and lymphomas,18 to be critical for v-Src
transformation,46,47 and alone to be able to transform
mouse and rat fibroblasts.41 To examine whether or not
STAT5b-RAR modulated STAT3 activity, we used the HepG2 cell line in
which the IL-6 receptor signaling pathway is intact together with an
APRE-luciferase reporter gene construct.32 IL-6 exposure
of HepG2 cells increased APRE-luciferase reporter gene activity
120-fold through the activation of endogenous STAT3 (Figure
6A and data not shown). This activation
was inhibited 75% by cotransfection of HepG2 cells with STAT5b (Figure
6A, lane 2). Cotransfection of HepG2 cells with STAT5b-RAR augmented
IL-6-induced reporter-construct activity 8-fold (Figure 6A, lane 3).
In contrast, cotransfection with RAR had minimal effect. To assess
whether or not the ability of STAT5b-RAR to augment STAT3
transcriptional activity is limited to STAT5b-RAR, we examined other
APL fusion proteins in a similar fashion in HepG2 cells (Figure 6A,
lanes 5 and 6). In addition to STAT5b-RAR, both PML-RAR and
PLZF-RAR enhanced IL-6-mediated STAT3 transcriptional activity 8- to 26-fold.
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| Figure 6.
Effect of STAT5b-RAR and other APL fusion proteins on
the STAT3 transcriptional and DNA binding activity.
(A) Transactivation activities of STAT5b, STAT5b-RAR, RAR,
PML-RAR, and PLZF-RAR in HepG2 cells. HepG2 cells were
transiently transfected with 500 ng of APRE-luciferase reporter gene,
500 ng of -galactosidase expression vector, and 2.0 µg STAT5b,
STAT5b-RAR, RAR, PML-RAR, and PLZF-RAR. Transfected cells
were stimulated with IL-6 (25 ng/mL) for 24 hours. Luciferase activity
was measured and normalized for transfection efficiency using a
-galactosidase reporter construct. Data represent the mean ± SD of 5 separate experiments. The luciferase activity shown in lane 1 is increased 120-fold over the activity of identical cells incubated
without IL-6 (not shown). (B) Gel-shift assays with WCEs from HepG2
cells transiently transfected with STAT5b or STAT5b-RAR. Transfected
cells were incubated without or with IL-6 (25 ng/mL) for 30 minutes.
The APRE (upper panel) and the hSIE (middle panel) were used as duplex
oligonucleotide probes in this study. The location of the specific
STAT3/DNA complex (upper panel and middle panel) and STAT5b/DNA (upper
panel, lane 5) are indicated by the solid triangle and arrow,
respectively; the empty triangle indicates the location of a
nonspecific band. Levels of protein expression in the transiently
transfected cells were determined by immunoblotting with the antibody
against the N-terminal region of STAT5b (bottom panel).
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To begin to explore the mechanism of the enhanced STAT3 transcriptional
activity, we assessed STAT3 DNA binding activity and Ser727
phosphorylation status in extracts of transfected versus nontransfected
HepG2 cells. Cotransfection of STAT5b or STAT5b-RAR did not affect
IL-6-stimulated binding to APRE or hSIE nor did it affect
levels of Ser727 phosphorylation (Figure 6B and data not shown).
Gene deletion mouse models48 and studies of dominant
negative mutant constructs in cell lines49 support an
important role for STAT5b in myeloid cell development. Consequently,
the leukemogenic effect of STAT5b-RAR could also be mediated,
in part, through its interference with normal STAT5b function. To
investigate this possibility, we examined the effect of STAT5b-RAR
on STAT5 activity downstream of the prolactin and IL-2Rs following
reconstitution of these signaling pathways in COS-7 and 293T cells
(Figure 7A and 7C). Transactivation of a
reporter construct containing a -casein promoter was increased
following transient transfection of prolactin
receptor-reconstituted COS-7 and IL-2R-reconstituted 293T cells with
STAT5b. Transient transfection of these cells with
STAT5b-RAR, however, did not increase reporter construct activation
nor did it inhibit the augmentation seen with transient overexpression
of STAT5b. Similar results were obtained in IL-2R-reconstituted COS-7
cells (data not shown). In addition, transient coexpression of the
fusion protein did not affect STAT5 DNA binding activity induced by
prolactin in prolactin receptor-reconstituted COS-7 cells (Figure 7B)
or by IL-2 in IL-2R-reconstituted COS-7 cells (Figure 7D).
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| Figure 7.
Effect of STAT5b-RAR on STAT5b transcriptional and DNA binding
activity.
(A) COS-7 cells were transiently transfected with the human prolactin
receptor, a -casein-luciferase reporter gene construct, and STAT5b
with or without STAT5b-RAR. Transfected cells were stimulated
without or with ovine prolactin (500 ng/mL) for 24 hours. Luciferase
activity was measured and normalized for transfection efficiency using
a -galactosidase reporter construct. Data presented represent the
mean ± SD of 3 separate experiments. (B) Gel-shift assays were
performed using the PRE and WCEs of transiently transfected COS-7 cells
that were incubated without or with prolactin (500 ng/mL) for 30 minutes. The location of the specific STAT5b/DNA complex is indicated
with the solid triangle; a nonspecific band is indicated with the empty
triangle. The level of protein expression, determined by immunoblotting
with antibody against STAT5b, is shown in the bottom panel. (C) 293T
cells were transiently transfected with the human
-casein-luciferase reporter gene construct and expression
constructs for IL-2R, C, Jak3, and STAT5b with or
without STAT5b-RAR. Transfected cells were incubated without or with
IL-2 (50 ng/mL) for 24 hours. Luciferase activity was measured and
normalized for transfection efficiency using a -galactosidase
reporter construct. Data presented represent the mean ± SD of 4 separate experiments. (D) Gel-shift assays were performed using the PRE
and WCEs from IL-2R reconstituted and transiently transfected COS-7
cells that were incubated without or with IL-2 (50 ng/mL) for 30 minutes. The location of the specific STAT5b/DNA complex is indicated
with the solid triangle; a nonspecific band is indicated with the empty
triangle. The arrow indicates the position of the supershifted band.
Levels of protein expression, determined by immunoblotting with
antibody against STAT5b, are shown in the bottom panel for lanes 1 through 6.
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Discussion |
To understand the contribution to the pathogenesis of APL of a
chromosomal abnormality resulting in a new RAR-containing fusion
protein, it is necessary to evaluate several potential consequences of
the abnormality including the effect of the resultant fusion protein on
RAR function, the effect of the fusion protein on the function of
the normal allele of the fusion partner, and the effect of the
reciprocal fusion protein.3
In the studies outlined in this report, we demonstrated that
STAT5b-RAR can bind RARE as a homodimer and can recruit SMRT. SMRT
remained bound to STAT5b-RAR at physiologic concentrations of ATRA.
Because STAT5b by itself did not bind SMRT, these findings suggest that
the STAT5b portion of the fusion protein confers an allosteric change
in the RAR portion of the fusion protein, similar to the non-RAR
portions of other APL fusion proteins, thereby increasing its affinity
for SMRT.3 This alone or together with the potential for
each homodimer of STAT5b-RAR to bind 2 molecules of SMRT may result
in superrepression of gene transcription by
RAR/RXR.26,50 In addition to binding RARE as a homodimer, STAT5b-RAR preferentially bound RARE as a heterodimer with RXR. This feature is unique for STAT5b-RAR among APL fusion proteins and
may contribute to its oncogenic potential by sequestering RXR and
other essential transcription cofactors. The effect of the interstitial
deletion and creation of the STAT5b-RAR on one allele of STAT5b has
the effect of reducing by one half the amount of STAT5b expression.
This by itself, however, would not be expected to affect myeloid
development because deletion of both alleles of STAT5b in mice did not
affect the myeloid lineage51 presumably because of its
functional redundancy in myelopoiesis shared with STAT5a. The STAT5b
portion of the fusion protein contains a truncated SH2 that includes
the phosphotyrosine binding pocket.39 Consequently, STAT5b-RAR might retain the ability to bind activated STAT5a/b and
act as a dominant negative. Our findings, however, that STAT5b-RAR did not affect ligand-induced STAT5 transcriptional activity or DNA
binding activity do not support this possibility. Finally, because
STAT5b-RAR was the result of an interstitial deletion, no reciprocal
fusion protein was created.
Recent evidence suggests that dimerization of PML-RAR is
critically important for its oncogenic activity including inhibition of
RA-mediated myeloid differentiation.26,42,43,50 Deletion of the coiled-coil domain within the PML portion of PML-RAR
abrogated dimerization and relieved the inhibitory effects of the
fusion protein on RA-induced differentiation.26,40,43
Similarly, we have demonstrated that the coiled-coil domain within the
STAT5b portion of STAT5b-RAR is required for homodimerization in the presence of RARE, for its inhibitory effect on RAR/RXR
transcriptional activity in COS-7 cells, and for its ability to stably
interact with SMRT.
Our results demonstrated that STAT5b-RAR augmented STAT3
transcriptional activity, whereas STAT5b inhibited it. Inhibition of
STAT3 by STAT5b may be due to competition for binding to the APRE
reporter construct coupled with the failure of STAT5b, relative to
STAT3, to recruit the basal transcription machinery to the reporter
construct. Alternatively, although STAT5b does not interact with the
CoR SMRT, its ability to bind the APRE site and yet reduce transcriptional activity suggests it may bind and recruit another unidentified CoR.
In addition to STAT5b-RAR, we demonstrated that other APL fusion
proteins including PML-RAR and PLZF-RAR also had this enhancing
effect on STAT3 transcriptional activity. STAT protein activation,
especially STAT3, has been implicated in cell models of transformation
and carcinogenesis. STAT3 was shown to be constitutively activated in
cells transformed by oncoproteins such as v-Src.52,53 In
addition, use of dominant-negative STAT3 constructs has shown that
STAT3 is essential for fibroblast transformation by
v-Src.46,47 Overexpression of a constitutively active form
of STAT3 in immortalized rat or mouse fibroblasts induced their
transformation and conferred the ability to form tumors in nude mice
indicating that STAT3 alone can function as an oncogene.54
Aberrant activation of STAT3 also has been demonstrated in various
human blood mal |
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Abstract
Introduction