From the Department of Medicine, Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California 90048
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ABSTRACT |
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Leukemia inhibitory factor (LIF) is a pleiotropic
neuroimmune cytokine that promotes corticotroph cell differentiation
and induces proopiomelanocortin (POMC) mRNA expression and
adrenocorticotropin hormone (ACTH) secretion. However, molecular
mechanisms for this induction remain elusive. We therefore developed
ACTH-secreting AtT20 transformants for wild-type or mutated STAT3, a
cytokine signaling molecule, to address whether STAT3 is a determinant of LIF-mediated ACTH regulation. We show that these mutants act in a
dominant negative manner by blocking endogenous STAT3 tyrosine phosphorylation or STAT3 DNA binding. Attenuation of STAT3 activity in
the dominant negative AtT20 clones prevented LIF from promoting transcriptional activation of the POMC promoter (2.1-fold), whereas this LIF action was enhanced (7.7-fold; p < 0.05) in
wild-type STAT3-overexpressing clones in comparison to mock-transfected cells (4.5-fold). However, wild-type or dominant negative
STAT3-overexpressing clones showed comparable (4-fold) POMC induction
after treatment with cyclic adenosine monophosphate (cAMP), an
alternate inducer of POMC transcription, indicating the STAT3
specificity for LIF signaling. Moreover, dominant negative inactivation
of STAT3 activity resulted in abrogation of LIF-induced POMC mRNA
levels and ACTH secretion, confirming the in vivo role of
STAT3 in LIF-mediated corticotroph action. Chemical or molecular
blockade of the mitogen-activated protein kinase pathway did not affect
LIF-mediated corticotroph function. These results indicate that STAT3
is a critical intrapituitary component of the LIF-mediated
neuroimmunoendocrine interface in corticotroph cells.
The coordinated neuroendocrine stress response comprises both
classic endocrine as well as neuroimmune regulatory pathways (1).
Pituitary corticotroph cell function is controlled by complex
hypothalamic (2) and peripheral paracrine signals (3), all ultimately
impacting on proopiomelanocortin
(POMC)1 gene expression and
adrenocorticotropin hormone (ACTH) secretion. Several cytokines are
both peripheral and central modulators of neuroendocrine function and
serve as mediators of the immune neuroendocrine system interactions (1,
3-5). The pituitary is an abundant source of growth factors that
regulate the hypothalamo-pituitary-adrenal axis in vivo and
pituicyte ACTH production in vitro (6-9). Leukemia inhibitory factor (LIF) is a pleiotropic neuroimmune cytokine whose
gene expression has been demonstrated in human fetal, adult and murine
pituitary cells (10, 11). LIF is a component of the interleukin-6
(IL-6) cytokine family, which also includes IL-6, IL-11, ciliary
neutrophic factor, oncostatin M, and cardiotrophin-1. Signaling
initiated by these cytokines is transduced by activation of composite
receptors that share a common transmembrane gp130 protein subunit
(12-16). LIF ligand binding is associated with gp130 and LIF receptor
subunit heterodimerization and subsequent activation of downstream
tyrosine kinases (17-18). The cytokine receptors lack intrinsic kinase
activity. Subsequent to LIF receptor dimerization, JAK2 is activated by
tyrosine autophosphorylation (19). These events lead to tyrosine
phosphorylation, auto- or hetero-dimerization, and nuclear
translocation of STAT3 (signal transducer and activator of
transcription) and, to a lesser extent, STAT1 In AtT20 murine corticotroph cells, LIF increases POMC mRNA levels
and ACTH secretion, and synergizes with corticotropin releasing hormone
(CRH) to trigger these effects (20). Recently, we delineated elements
of the POMC promoter important for a component of LIF and CRH synergy
on POMC transcription (23). However, identification of a specific
LIF-induced transcription factor that activates POMC transcription has
been elusive. We have recently shown that overexpression of SOCS-3, a
cytokine-inducible signaling inhibitor related to JAB, SSI, or CIS (24,
25), inhibited LIF activation of POMC gene expression in
vitro (26). As SOCS-3 was also potently induced by LIF, SOCS-3 was
implicated as a negative intracellular regulator of pituitary LIF
signaling. SOCS-3 overexpression in corticotroph cells abrogated
LIF-induced gp130 and STAT3 phosphorylation, suggesting STAT3 as a
critical regulator of pituitary LIF function.
Although LIF action occurs mainly through the JAK-STAT pathway, other
subcellular signaling pathways are activated by IL-6-related cytokines
including phospholipase C, phosphoinositol 3-kinase, phosphotyrosine
phosphatase D, pp120, the insulin receptor substrate 1, or several
components of the mitogen-activated protein kinases cascade pathway,
including SHC, GRB2, Raf-1, ERK1, and ERK2 (27-32). Activation of
these widespread signal transducing molecules implies substantial
convergence between cytokine-activated pathways and receptor tyrosine
kinase-activated pathways. Nevertheless, JAK proteins appear to be key
determinants for this convergence as their disruption results in
failure of downstream phosphorylation events (31, 33). Furthermore,
convergence between both pathways may also occur at the level of STAT
proteins. Indeed, activation of Raf-1 by interferon To address molecular mechanisms responsible for LIF induction of the
neuroendocrine stress response, we tested the involvement of STAT3 or
MAPK in this LIF response. Inactivating STAT3 mutants include a
phenylalanine substitution at a critical tyrosine carboxyl-terminal residue, Tyr-705 (STAT3F), and a mutant containing two alanine substitutions at positions important for STAT3 DNA binding (Glu-434 and
Glu-435) (36). We therefore isolated stable mutant STAT3 AtT20
transformants and show that they behave as dominant negative inhibitors
of endogenous corticotroph cell STAT3. Suppression of STAT3 activity is
here shown to abrogate LIF action on POMC transcription and ACTH
secretion. However, chemical or molecular blockade of the MAPK pathway
did not impact LIF-induced POMC promoter activity. These results thus
indicate the key role of STAT3 in the LIF-mediated neuroendocrine
pituitary stress response.
Cell Culture--
AtT20 cells, obtained from the American Type
Culture Collection (Rockville, MD), were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml streptomycin, 100 units/ml penicillin, and 0.25 µg/ml amphotericin B (Life Technology,
Inc.).
Transformation of AtT20 Cells--
To isolate stable
transformants expressing wild-type or inactivated Stat3,
pCAGGS-Neo-HA-Stat3WT, pCAGGS-Neo-HA-Stat3F, and pCGGS-Neo-HA-Stat3D
(36) were transfected in AtT20 cells by calcium precipitation and
transformants selected with G418 (1 mg/ml). STAT3F presents a
substitution of the residue Tyr-705 with a phenylalanine and, STAT3D
presents two substitutions of the glutamic acid residues 434 and 435 with two alanine (36).
Immunoprecipitation and Western Blot Analysis--
AtT20 cells
were grown in 10-ml dishes to 50% confluence and serum-deprived
16 h before treatment with LIF (R&D Systems, Minneapolis, MN).
Cells were then washed once with phosphate-buffered saline (pH 7.0),
once with lysis buffer (50 mM Hepes, 150 mM
NaCl, 10 mM EDTA, 10 mM
Na4P2O7, 100 mM NaF, 2 mM sodium orthovanadate, pH 7.4) and lysed in 500 µl of
lysis buffer containing 1% Triton X-100 and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 2 µM leupeptin). After a 10-min incubation at 4 °C, the
lysate was collected and centrifuged at 13,000 × g for
10 min at 4 °C to remove insoluble material. STAT3 proteins were
immunoprecipitated by incubating the solubilized cells for 2 h at
4 °C with a monoclonal anti-hemagglutinin antibody, 12CA5 (Roche
Molecular Biochemicals) prebound to Sepharose-protein G beads (Sigma)
or a polyclonal anti-STAT3 antibody (Santa Cruz) prebound to Sepharose
A beads (Sigma). Immune complexes were collected by centrifugation,
washed twice with a buffer containing 30 mM Hepes, 30 mM NaCl, and 0.1% Triton X-100, pH 7.4, and boiled for 3 min in 40 µl of SDS-sample buffer. Samples were separated on 7.5%
SDS-polyacrylamide gel and electroblotted on polyvinylidene difluoride
membrane. The membrane was blocked for 16 h at 4 °C in 20 mM Tris, pH 7.4, 500 mM NaCl, and 0.1% Tween
20 supplemented with 3% bovine serum albumin, blotted for 3 h at
room temperature with polyclonal anti-STAT3 or monoclonal
anti-phosphotyrosine antibodies (Santa Cruz). Bound antibodies were
detected with horseradish peroxidase-conjugated anti-mouse or
anti-rabbit IgG followed by ECL detection (Amersham Pharmacia Biotech).
Electromobility Shift Assay--
Whole cell extracts (20 µg)
were preincubated in a final volume of 20 µl (10 mM
Hepes, pH 7.9, 80 mM NaCl, 10 mM glycerol, 1 mM dithiothreitol, 1 mM EDTA) with 2 µg of
poly(dI-dC)-poly(dI-dC) at room temperature for 15 min. A
32P-labeled double-stranded oligonucleotide SIE67 used as
probe (40,000 cpm, 5 fmol/reaction) was then added for 20 min at room temperature. In competition experiments, 100-fold molar excess unlabeled competitor oligonucleotides were added to the preincubation reaction. For supershift experiments, extracts were preincubated with 2 µg of polyclonal STAT3 antibody (Santa Cruz) for 1 h at 4 °C.
Protein-DNA complexes were resolved on a 6% nondenaturing polyacrylamide gel containing 2.5% glycerol in 0.5× Tris borate/EDTA at 4 °C, dried, and autoradiographed. Nucleotide sequences for SIE
and AP-1 are: SIE, 5'-cgcgTcATTTcccgTAAATcA-3'; AP-1, 5'-cgcTT- gATgAgTcAgccggAA-3'.
Transfection and Luciferase Assay--
AtT20 cells were plated
in 2-ml dishes (100,000 cells/well) and allowed to adhere for 16 h. Cells were transfected as described (20) with 0.5 µg per dish of
rat POMC promoter ( Northern Blot Analysis--
Total RNA from AtT20 cells
transfected with either STAT3wt, F, or D and treated with or without
0.5 nM LIF for 24 h, was prepared using Trizol reagent
(Life Technologies, Inc.). 10 µg of RNA/lane was size-fractionated
under denaturing conditions using formaldehyde-agarose (1.2%) gel,
transferred to nitrocellulose, and hybridized with a
32P-labeled probe POMC probe (2.5 × 106
cpm/2 ml hybridization solution) (Stratagene) according to the manufacturer.
ACTH Assay--
ACTH was measured by a radioimmunoassay kit
purchased from Diagnostic Products Corp. (Los Angeles, CA).
Isolation of Pituicyte Transformants Stably Expressing Dominant
Negative Forms of STAT3--
The IL-6 family of cytokines activate the
JAK-STAT pathway in several systems, and LIF is a powerful activator of
the JAK-STAT pathway, in particular, in AtT20 cells. We therefore
isolated pituicyte transformants stably expressing wild-type or
dominant negative forms of STAT3 to test whether STAT3 mediates LIF
induction of corticotroph function in these cells. Plasmids encoding
hemagglutinin peptide (HA)-tagged STAT3F (HA-STAT3F) and HA-tagged
STAT3D (HA-STAT3D) as well as HA-tagged wild-type STAT3 (HA-STAT3wt)
were introduced into AtT20 murine corticotroph pituitary cells. These
cells have been shown to be an excellent model for studying the role of
cytokine action in the ACTH stress response (20, 23). Several different clones selected by G-418 resistance were established; each expressed HA-STAT3F, HA-STAT3D, or HA-STAT3wt. Expression levels of transfected STAT3F or D mutants or STAT3wt, respectively, were determined by
immunoprecipitation of HA-STAT3 with anti-HA monoclonal antibody (12CA5) followed by immunoblot analysis with polyclonal anti-STAT3 antibody (Fig. 1). Three independent
clones for STAT3wt transformants (clones 1, 2, and 3) and for each
dominant negative form of STAT3F (clones 2, 4, and 5) or STAT3D (clones
2, 4, and 6), respectively, were selected. Each clone was shown to
express similar levels of HA-STAT3 protein and was used in the ensuing
experiments.
To confirm the dominant negative action of HA-STAT3F on endogenous
STAT3 function, cell lysates were immunoprecipitated with STAT3
antibody, followed by immunoblotting with anti-phosphotyrosine antibody
(Fig. 2A). This Western blot
showed the inability of both endogenous STAT3 and exogenous HA-STAT3F
to be phosphorylated after 1 nM LIF treatment for 5 or 10 min in the HA-STAT3F transfected clones. However, in the HA-STAT3wt or
HA-STAT3D transfected clones, similar LIF treatments enhanced tyrosine
phosphorylation of STAT3, as expected. These results indicated that
HA-STAT3F protein behaves in a dominant negative manner by blocking
LIF-induced endogenous STAT3 phosphorylation.
To confirm the dominant negative inactivating action of HA-STAT3D, DNA
binding was tested by probing with the STAT inducible element (SIEm67)
contained within the c-fos promoter. This element is readily recognized
by either STAT1 or STAT3 (35). After electrophoretic mobility shift
assay with extracts derived from mock-transfected AtT20 cells treated
for 15 min with 1 nM LIF, three specific complexes were
visualized and were effectively competed by 100-fold molar excess of
unlabeled SIE probe, but not by an exogenous double-stranded oligonucleotide (Fig. 2B). These three complexes correspond
to the previously described SIF (Sis-inducible factor) A (STAT3
homodimer), SIF B (STAT1/STAT3 heterodimer), and SIF C (STAT1
homodimer) (19, 21). In the HA-STAT3wt transfected clone, 15 min of 1 nM LIF treatment induced the formation of a single specific
complex. STAT3 was shown to be a component of this complex as addition of STAT3 antibody to cell extracts, prior to addition of the SIE probe,
inhibited complex formation and induced a faint supershift. However, in
the HA-STAT3D transfected clones, this complex was clearly not induced
by LIF treatment. This abrogation of DNA binding demonstrated the
dominant negative action of HA-STAT3D on endogenous STAT3 action in the
HA-STAT3D transfected clones.
Dominant Negative HA-STAT3F and HA-STAT3D Block LIF-induced POMC
Promoter Activity--
As LIF activates rat POMC expression through
the
To confirm that the HA-STAT3F and D clones were functionally viable,
and yet failed to respond specifically to LIF because of lack of
inducible STAT3, cells were also treated with dibutyryl cAMP, which
stimulates POMC expression through an AP-1 like element in the first
exon of the POMC gene (37), and whose recognition motif is also present
within the POMC promoter fragment ( Dominant Negative HA-STAT3F and HA-STAT3D Block LIF Induction of
Endogenous POMC mRNA Expression--
To test the in
vivo significance of the preceding results, POMC Northern blots
were also performed on mRNA extracted from HA-STAT3wt, F, or D
clones stimulated with 1 nM LIF for 24 h (Fig. 4). As expected, LIF treatment for
24 h in HA-STAT3wt clones enhanced POMC mRNA expression
(2.24 ± 0.21-fold induction) (Fig. 4C). This induction
was observed within 3 h of treatment (Fig. 4A) and was maximal after 24 h. This time point was also utilized to compare LIF effects in wild-type and dominant negative STAT3 clones (Fig. 4,
B and C). In HA-STAT3F and D clones, induction of
POMC mRNA expression was decreased, respectively, to 1.42-fold ± 0.07 (p < 0.01) and to 1.59-fold ± 0.16 (p < 0.02) versus HA-STAT3wt. These results
thus confirmed the in vivo importance of STAT3 in transducing neuroendocrine LIF signaling to endogenous POMC.
Dominant Negative HA-STAT3F and HA-STAT3D Block LIF Induction of
ACTH Secretion--
The physiologic relevance of STAT3 disruption in
the HA-STAT3 clones was also assessed by measuring ACTH levels in
conditioned medium after 24 h treatment with 1 nM LIF
(Fig. 5). In mock-transfected AtT20 cells
as well as in HA-STAT3 wt clones, LIF stimulated ACTH basal levels
2.4 ± 0.2-fold and 2.7 ± 0.1-fold, respectively. However,
in HA-STAT3F and D dominant negative clones, this induction was
attenuated (respectively, 2 ± 0.08 and 1.9 ± 0.09)
(p < 0.01) in comparison to both mock-transfected
AtT20 cells and HA-STAT3wt clones.
Role of MAPK in LIF Induction of POMC Promoter Activity--
The
IL-6 cytokine family, which transduces through the gp130 subunit, may
also activate Ras-MAPK pathways (27, 29-32). We therefore tested
whether LIF induces MAPK activation in AtT20 cells. Using LIF-activated
AtT20 cell extracts in a Western blotting experiment using an
anti-tyrosine-phosphorylated form of MAPK antibody, treatment for 10 min with 1 nM LIF was shown to induce MAPK phosphorylation
2-fold (data not shown). To test whether the Ras-MAPK pathway might be
a functional component of LIF function in AtT20 cells, we blocked MAPK
by either using a chemical inhibitor of mitogen-activated protein
kinase kinase (MAPKK), PD 98059, which inhibits MAPK activation and
subsequent phosphorylation of MAPK substrates, or, a cDNA encoding
a dominant negative form of MAPK (TAYF). Pretreatment of AtT20 cells
for 1 h with PD 98059 (5-100 µM) did not affect
either LIF-induced POMC promoter activity (Table
I) or LIF-induced STAT3 binding to the
SIE probe (data not shown). Cotransfection of various concentrations of
a dominant negative form of MAPK (T183A/Y185F) with the POMC promoter
luciferase construct also did not alter LIF-induced POMC promoter
activity (Table II). Thus, MAPK does not
appear to be implicated in LIF corticotroph function despite the weak
induction of ERK2 tyrosine phosphorylation by LIF in these cells.
We have previously shown that LIF stimulated POMC promoter
activity, POMC mRNA expression, and ACTH secretion (20). These results now demonstrate that disruption of STAT3 expression in AtT20
corticotroph cells significantly abrogates these LIF responses. We used
two forms of mutated STAT3, which were both expected to behave as
signaling inhibitors via different mechanisms (36). Both mutants of
STAT3, STAT3F with a phenylalanine substitution at tyrosine 705 and
STAT3D mutated at positions important for DNA binding, acted in a
dominant negative manner in these cells. Endogenous STAT3 was not
tyrosine-phosphorylated in HA-STATF clones, nor did STAT3 bind the SIE
probe from the c-Fos promoter in the HA-STAT3D clones. This dominant
negative action implies that the mutants compete with endogenous STAT3
protein for recruitment to activated gp130-JAK complex in the HA-STAT3F
clones. In the HA-STAT3D clones, endogenous homodimeric STAT3 DNA
binding is abrogated. This result is also consistent with previous
studies using similar STAT3 mutants and showing STAT3 involvement in
IL-6-induced growth arrest and terminal differentiation in M1 cells or
in LIF-induced ES cell differentiation (36, 38). Furthermore, these
HA-STAT3F and D AtT20 transformants were shown, in a separate study, to inhibit LIF-induced SOCS-3 mRNA
expression.2
In the DNA binding assay showing disrupted endogenous STAT3 DNA binding
activity in the HA-STAT3D clones, a single complex was consistently
observed in the assay using HA-STAT3wt extracts, and was identified as
a STAT3 homodimer because addition of STAT3 antibody blocked its
formation and also caused a band supershift. This complex was
identified as SIF A (Sis-inducible factor) and is composed of a STAT3
homodimer, as distinct from SIF B and C composed, respectively, of
STAT1 and 3 heterodimer or of STAT1 homodimer (40). SIF B and C were
not observed in the electrophoretic mobility shift assay assay using
HA-STAT3wt extracts, whereas both these complexes were evident using
mock-transfected AtT20 cell extracts. This may be explained by the
strong overexpression of HA-STAT3wt competing with endogenous STAT1 for
SIE binding. Furthermore, SIF C (STAT1 homodimer), which was evident in
AtT20 extracts, was not regulated by LIF treatment. This observation also points to the major role of STAT3 for LIF signaling in these cells
as compared with other STAT family members.
Using the rat POMC promoter ( The abrogation of LIF effects by STAT3 disruption differed
quantitatively on POMC promoter activity, and on POMC mRNA
expression or ACTH secretion; LIF effects on endogenous POMC expression
and ACTH secretion were not as marked as those observed in the POMC promoter studies. This could be explained by the sensitivity of the
luciferase assay for studying POMC promoter activity, especially in the
mock-transfected AtT20 and HA-STAT3wt cells, than the in vivo assays. We utilized a 770-base pair fragment of the POMC promoter to study LIF action on luciferase reporter expression. However, the POMC promoter containing potential LIF-regulated elements
may be longer in vivo, and thus may require transcription factors other than STAT3 (20).
Disrupting STAT3 signal transduction by either blocking tyrosine
phosphorylation (HA-STAT3F clones) or preventing DNA binding (HA-STAT3D) resulted in decreasing LIF effects. These results also
imply that the DNA-binding event of STAT3 is indispensable for LIF
action, and thus render it unlikely that STAT3 tyrosine phosphorylation
serves as the sole link between the JAK-STAT and known or unknown
pathways (e.g. CREB) involved in POMC transcriptional regulation.
Furthermore, we examined whether other LIF-induced pathways might be
implicated in LIF corticotroph function. LIF weakly induced MAPK
phosphorylation (2-fold) (data not shown). The magnitude of LIF
activation of MAPK differs in various cell lines but is generally low,
and less efficient than oncostatin M (29). The MAPK pathway might be a
component of LIF signaling for several reasons. Our results show that
LIF weakly induces MAPK phosphorylation in AtT20 cells; JAK2 has been
shown to activate both STAT and Ras-dependent or
-independent MAPK pathways (33), and STAT3 contains a serine
phosphorylation site corresponding to a consensus site for MAPK serine
phosphorylation activity (35). Nevertheless, neither coexpression of a
dominant negative form of MAPK nor pretreatment with a specific
inhibitor of MAPKK abrogated POMC promoter activity. We therefore
conclude that, even if LIF weakly stimulates MAPK phosphorylation, it
is not a primary mechanism by which LIF activates corticotroph function.
STAT3 involvement in LIF promotion of the stress response in AtT20
cells was not previously considered likely, mainly because no apparent
STAT3 element has been identified in the POMC promoter. The The dominant negative forms STAT3 transfectants are useful for studying
molecular mechanisms by which LIF specifically determines corticotroph
cell activation in the hypothalamo-pituitary-adrenal axis. The use of a
pituitary cell line lacking endogenous STAT3 was elusive mainly because
the POMC gene is specifically and abundantly expressed in the pituitary
corticotroph cell, which contains tissue-specific POMC promoter
elements (39). Thus, STAT3 appears to be a critical mediator for the
LIF-mediated neuroimmunoendocrine interface in corticotroph cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES
, which regulates
immediate-response gene transcription (20-22).
and oncostatin
M requires expression of the STAT1 transcription factor (34) and,
reciprocally, ERK2 activity is required for stimulation of interferon
- and
-induced gene expression through STAT proteins (35).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
706/+64) fused to a luciferase reporter, and 0.5 µg of pSV-lacZ (Promega) expressing the
-galactosidase protein as
an internal transfection control. In the cotransfection experiments
using the dominant negative form of MAPK cDNA, various
concentrations of the cDNA were also coexpressed. 24 h after
transfection, cells were treated in triplicate for 7 h, washed
once in phosphate-buffered saline, pH 7, lysed in reporter lysis
buffer, and subjected to assay for luciferase (20) or
-galactosidase
activity (Promega).
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Fig. 1.
Screening AtT20 transformants overexpressing
HA-STAT3wt (A), HA-STAT3F (B), or
HA-STAT3D (C). Cells were lysed and HA-STAT3 was
immunoprecipitated with anti-HA antibody. Immunoprecipitates were
resolved on 7.5% acrylamide SDS electrophoresis gels and immunoblotted
with an anti-STAT3 antibody.
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Fig. 2.
HA-STAT3F and HA-STAT3D act in a dominant
negative manner. A, AtT20 clones transfected with
HA-STAT3wt, HA-STAT3F, or HA-STAT3D were serum-starved for 16 h
and treated with 1 nM LIF or vehicle (C) for 5 and 10 min. Cells were lysed and STAT3 proteins immunoprecipitated with
polyclonal anti-STAT3 antibody, separated on an SDS-7.5% acrylamide
electrophoresis gel, and the tyrosine-phosphorylated form of STAT3 was
identified with a monoclonal anti-phosphotyrosine antibody
(upper panel). To confirm equal protein loading
in each lane, the membrane was stripped and reblotted with a polyclonal
anti-STAT3 antibody (lower panel). B
and C, gel-shift analysis with whole cell extracts (20 µg)
derived from mock-transfected AtT20 (B) or HA-STAT3wt
or HA-STAT3D clones (C) treated with 1 nM
LIF or vehicle for 15 and 30 min. 32P-Labeled SIE
oligonucleotide was used as the probe with AtT20 nuclear extracts
(B, lanes 1-5), HA-STAT3wt nuclear
extract (C, lanes 1-6), or HA-STAT3D
nuclear extract (C, lanes 7-12).
100-fold molar excess of unlabeled SIE or unlabeled AP-1
oligonucleotides were used as competitors (B,
lanes 4 and 5, respectively; and
C, lanes 4 and 10, and
lanes 5 and 11, respectively).
Anti-STAT3 antibody was added to the nuclear extracts before addition
of SIE probe (C, lanes 6 and
12).
706/+64 region of the POMC promoter (20), activity of this
promoter was tested in the STAT3 dominant negative clones. Transient
transfections of a POMC promoter-driven luciferase reporter, as well as
of a vector encoding
-galactosidase (internal control), were
performed in mock-transfected AtT20, and HA-STAT3wt, F, and D clones,
respectively (Fig. 3A).
Treatment with 1 nM LIF for 6 h of mock-transfected AtT20 caused a 4.5 ± 0.4-fold (p < 0.01)
luciferase induction. Furthermore, similar LIF treatment of the
HA-STAT3wt clones enhanced (p < 0.05, versus mock-transfected) this induction to 7.7 ± 0.8-fold. However, in the HA-STAT3F and D clones, LIF induction was
significantly abrogated (p < 0.01) as compared with
mock-transfected AtT20 or HA-STAT3wt. Indeed, the HA-STAT3F clones only
showed 1.81 ± 0.1-fold induction (p < 0.01), and
2.55 ± 0.2-fold induction (p < 0.01) was
observed in the HA-STAT3D clones. These results indicated the
requirement for STAT3 in mediating LIF induction of POMC expression at
the transcriptional level.
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Fig. 3.
Dominant negative HA-STA3F and HA-STAT3D
specifically block LIF induction of POMC promoter-driven luciferase
reporter activity. Rat POMC promoter-luciferase transiently
transfected AtT20 cells, HA-STAT3wt, HA-STAT3F, or HA-STAT3D clones
were treated for 6 h with 1 nM LIF (A) or 5 mM dibutyryl cAMP (B). Luciferase activity was
measured as described under "Materials and Methods." The results
are representative of three separate experiments using three
independent clones, and are expressed as -fold induction above
control.
706/+64) (Fig. 3B). 5 mM dibutyryl cAMP treatment for 6 h similarly
stimulated POMC transcription in both mock-transfected AtT20 cells
(4.1 ± 0.2), and in the HA-STAT3wt clones (4.3 ± 0.1) as
well as in the mutant HA-STAT3F and D clones (3.9 ± 0.2 and
4 ± 0.2, respectively). This result indicated that, although the
STAT3 pathway was specifically altered in the HA-STAT3F and D clones,
cAMP known to induce POMC expression through alternate
STAT3-independent transduction signals was still functionally operative
in these cells.
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Fig. 4.
Dominant negative HA-STAT3F and HA-STAT3D
block LIF induction of endogenous POMC mRNA transcription.
A, 5 µg of total mRNA derived from mock-transfected
AtT20 cells treated for 1, 3, 6, or 24 h with 1 nM LIF
or vehicle were loaded per lane and size-fractionated on a denaturing
formaldehyde-agarose (1.2%) gel. After transfer to nitrocellulose
membrane, hybridization was performed with radiolabeled POMC cDNA.
Equal loading and transfer were verified by reprobing the washed
membrane with radiolabeled -actin cDNA. B, 5 µg of
total mRNA derived from HA-STAT3wt, HA-STAT3F, or HA-STAT3D clones
treated for 24 h with 1 nM LIF or vehicle
(C) were loaded per lane, size-fractionated on a denaturing
formaldehyde-agarose (1.2%) gel, transferred, and probed with
radiolabeled POMC cDNA. Equal loading and transfer were verified by
reprobing the washed membrane with radiolabeled
-actin cDNA.
C, scanning quantification of bands corresponding to control
(no treatment) and 24-h LIF treatment of HA-STAT3wt, HA-STAT3F, and
HA-STAT3D clones. These results are representative of five separate
experiments using three independent clones, and are expressed as -fold
induction above control.
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Fig. 5.
Dominant negative HA-STAT3F and HA-STAT3D
block LIF induction of ACTH secretion. AtT20 cells, HA-STAT3wt,
HA-STAT3F, or HA-STAT3D clones were serum-starved for 48 h before
being treated with 1 nM LIF for a subsequent 24 h.
Conditioned medium ACTH was measured by radioimmunoassay. These results
are representative of three separate experiments using three
independent clones, and are expressed as -fold induction above
control.
Blockade of MAPK pathway does not alter LIF-induced POMC promoter
activity
Blockade of MAPK pathway does not alter LIF-induced POMC promoter
activity
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
706/+64), we previously showed that LIF
stimulated basal POMC expression and potently enhanced CRH action on
rat POMC transcription (20). A common POMC promoter element, which does
not contain a STAT-binding site, also mediated a component of the
synergy between both ACTH-inducing peptides. Moreover, disruption of
this element (
173/
160), within the full-length POMC promoter
(
706/+64), only partially blocked LIF and CRH-mediated effects (23).
Thus, additional mechanisms may also be involved in this synergistic
induction of POMC expression. The effects of STAT3 disruption in AtT20
cells also support this hypothesis, as LIF stimulation of POMC-promoter
activity was reduced from 4.5-fold in mock-transfected or 7.7-fold in
HA-STAT3wt clones to 1.8- or 2.5-fold in HA-STATF or D clones,
respectively. Furthermore, overexpression of STAT3wt in HA-STAT3wt
clones induced LIF action in comparison with mock-transfected AtT20
cells (7.7-fold versus 4.5-fold, respectively;
p < 0.05). This observation also further confirmed the
STAT3-specificity of LIF action, as was the demonstration that, in the
STAT3 dominant negative clones, POMC transcription was briskly induced
via an alternate pathway. CRH-like receptor binding ligands induce cAMP
and activate CREB protein, which also induces POMC transcription (37,
41-42). We therefore stimulated the STAT3 dominant negative clones, as
well as HA-STAT3wt and mock-transfected AtT20 cells with dibutyryl cAMP
to increase steady state levels of cAMP, and showed that POMC was
effectively induced in all clones.
173/
160
element of POMC promoter shown to mediate LIF and CRH transcriptional
synergy does not contain an evident STAT3 binding motif. Nevertheless,
we now show that STAT3 disruption significantly blocks LIF
transcriptional activation of POMC and ACTH secretion. Direct
interactions between STAT3 and POMC promoter remain, however, to be
elucidated. STAT3 might interact directly with the POMC promoter by
binding to a non-classical STAT binding site. In HepG2 cells, STAT3 and
a CRE-like transcription factor interaction were stimulated by
interleukin-6 and shown to bind to a JRE-IL6 response element, which
differed from a classical high affinity STAT binding site but which
contained a low affinity STAT-binding site overlapping with an Ets-like
and a CRE-like site (43). Interactions between STAT3 and CRE-like
binding proteins on the POMC promoter will be important to explore.
Indeed, CRH stimulates POMC transcriptional activation by activating a
CRE binding protein (37). Furthermore, interaction between STAT and
other transcription factors have been reported elsewhere (44, 45).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Toshio Hirano (Osaka University Medical School, Japan) for providing the plasmids encoding wild-type HA-STAT3, and the HA-STAT3F and HA-STAT3D mutants, and to Dr. Larner (Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, MD) for providing the plasmid encoding for mutated MAPK (TAYF).
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK33802 and the Doris Factor Molecular Endocrinology Laboratory.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a doctoral fellowship from the Ministère de
l'Education Nationale, de l'Enseignement Supérieur et de la
Recherche, France.
§ To whom correspondence should be addressed: Academic Affairs, Cedars-Sinai Medical Center, 8700 Beverly Blvd., 2015, Los Angeles, CA 90048. Tel.: 310-855-4691; Fax: 310-967-0119; E-mail: Melmed{at}CSMC.edu.
2 C. J. Auernhammer, C. Bousquet, and S. Melmed, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: POMC, proopiomelanocortin; ACTH, adrenocorticotropin hormone; CRE, cAMP response element; CREB protein, cAMP response element-binding protein; CRH, corticotropin releasing hormone; ERK, extracellular regulated kinase; HA, hemagglutinin; IL-6, interleukin 6; IL-11, interleukin 11; JAK, Janus kinase; LIF, leukemia inhibitory factor; MAPK, mitogen-activated protein kinase; SIE, Sis-inducible element; SIF, Sis-inducible factor; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription.
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