From the Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263 and the § Oncology Gene Therapy Program, The Toronto Hospital, Toronto, Ontario M5G 2M1, Canada
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ABSTRACT |
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Interleukin (IL)-6 is a major regulator of
hepatic acute-phase plasma protein (APP) genes. The membrane-proximal
133-amino acid cytoplasmic domain of glycoprotein (gp) 130, containing
one copy of the Box3 motif, is sufficient to transmit a productive signal to endogenous APP genes in rat hepatoma H-35 cells. In contrast,
a mutant gp130 domain lacking the Box3 motif activates Janus kinases to
a normal level but fails to activate signal transducer and activator of
transcription 3 and to up-regulate a number of APP genes, including
thiostatin, fibrinogen, hemopexin, and haptoglobin. However, in the
absence of Box3, gp130 still stimulates the expression of
Interleukin (IL)1-6 is a
pleiotropic cytokine that elicits a wide variety of biologic activities
in different cell types (1-3). In liver, IL-6 is a prominent inducer
of the acute-phase plasma proteins (APPs) which function to protect the
host against the harmful consequences of inflammation and to restore
normal homeostasis (3-5). IL-6 acts in concert with other factors such
as IL-1, tumor necrosis factor, and glucocorticoids to modulate the
expression of APP genes. Based on the requirement of IL-1 for maximal
expression, APP genes are classified into type I and type II genes (6). For type I APP genes such as The receptor for IL-6 consists of a ligand-binding subunit, IL-6
receptor- The current model of IL-6 signaling suggests that STAT proteins are
recruited to the receptor via a phosphotyrosine-SH2 domain interaction
and serve as the direct downstream targets of the Janus family of
tyrosine kinases (JAKs) (20, 32, 33). Phosphorylated STATs form dimers
and acquire DNA binding activity. After translocation from the
cytoplasm to the nucleus, STAT dimers bind to specific regulatory
elements to transactivate gene expression. Other signaling pathways
have been described to have modulatory effects on the JAK/STAT pathway.
For example, it has been demonstrated that the mitogen-activated
protein kinase (MAPK) targets a specific serine residue at position 727 on STAT3 (34, 35). This serine phosphorylation has been proposed to
have an enhancing effect on the transactivation potential of STAT3
(35).
Previously, we characterized the signaling capability of gp130 in
hepatoma cells by the transient transfection approach, and we showed
that gp130 cytoplasmic domain engages at least two pathways that differ
in their requirement for the Box3 motif of gp130 (36, 37). In this
experimental system, the Box3-dependent pathway appears to
involve STAT3 in the regulation of some APP gene promoters when
presented as part of episomal constructs (38). However, a direct
assessment of the precise role for Box3 and STAT3 in the control of
endogenous APP genes has not yet been possible. Unfortunately, a STAT3
knock-out mouse is not available to address this question as
STAT3-deficient embryos die during early stage of development (39). On
the other hand, the Box3-independent pathway signals to an artificial
element, the hematopoietin receptor response element (HRRE), which
corresponds to the core IL-6RE of the rat
Receptor Constructs--
The chimeric receptor containing the
extracellular domain of human G-CSF receptor and the transmembrane and
the first 133 amino acids of the intracellular domain of gp130
(designated as G-gp130(133)WT), as well as the corresponding construct
with a tyrosine mutation in the Box3 sequence (designated as
G-gp130(133)M3), have been described previously (36). The chimeric
receptor construct containing the full-length 277 residue, wild-type
gp130 cytoplasmic domain with C-terminal FLAG epitope, has been
described (41). The equivalent FLAG epitope (DYKDDDDK) was also
introduced at the end of G-gp130(133)WT, yielding
G-gp130(133)WT-FLAG.
Cells and Treatments--
Parental rat hepatoma H-35 cells
(clone T-7-18; Ref. 6) and their derivatives were cultured in
Dulbecco's modified Eagle's medium with 10% fetal calf serum. To
generate stable receptor-expressing cells, the chimeric receptors
G-gp130(133)WT, G-gp130(133)WT-FLAG, and G-gp130(133)M3 were cloned
into the retroviral vector, MINV (42). MINV contains an internal
ribosome entry site upstream of the neomycin-resistant gene, permitting
co-expression of the chimeric receptors and the neo gene
product driven by the viral long terminal repeat. Replication-deficient
recombinant viruses with amphotropic host range were produced from
stably transduced PA317 packaging cells generated by infection with
virus harvested from transiently transfected GP+E-86 ecotropic
packaging cells. Virus-containing supernatant was collected from
confluent cultures 24 h after medium change, filtered through a
0.45-µm membrane, and added to H-35 cell cultures. Viral infection
was repeated twice more on the same cell population after 3 and 6 days.
Cells with stably integrated proviruses were selected in medium
containing 2 mg/ml G418 for 4 weeks. G418-resistant cells expressing
the chimeric receptors without FLAG-tag were subjected to limiting dilution to establish clonal cell lines. H-35 cells transduced with
G-gp130(277)-FLAG has been reported (41). Non-cloned pools of cells
expressing the FLAG-tagged receptors were used for a few comparative
analyses (Fig. 4, B and C).
Cells were treated with 1 µM dexamethasone (Sigma), 100 ng/ml IL-6 (Genetics Institute) and G-CSF, and 10 ng/ml IL-1 Receptor Binding Assay--
Human G-CSF was radiolabeled with
125I using nonporous polystyrene beads immobilized with
chloramine T (Pierce) to a specific activity of 81,000 cpm/ng.
Monolayer cells (~1.5 × 106) cultured on 6-well
plates were incubated with 50 pM 125I-G-CSF in
binding medium (0.5% bovine serum albumin in phosphate-buffered saline) for 4 h at 4 °C. Specific binding was determined by
competition with 100 nM unlabeled G-CSF during incubation.
Cells were then washed twice with binding medium and lysed in 1 M NaOH, 0.1% SDS. The radioactivity in cell lysates was
measured by a gamma counter (Beckman Instruments, Inc.). Bound
radioactivity inhibited by excess unlabeled G-CSF was calculated in
counts/min and represents the value for specific G-CSF binding.
Experiments were done in triplicate.
Electrophoretic Mobility Shift Assay (EMSA)--
Total protein
extracts were prepared from treated cells and used for EMSA as
published previously (43). Protein samples were preincubated in binding
buffer for 15 min on ice. The double-stranded and end-labeled
oligonucleotide representing the sequence of SIEm67 (44) was added to
the reaction. After incubation for 15 min at room temperature, the
DNA-protein complexes were resolved on a 4% nondenaturing
polyacrylamide gel. Gel was dried and exposed to x-ray film.
Northern Blot Analysis--
Total RNA was prepared by Trizol
according to manufacturer (Life Technologies, Inc.). Equal amounts of
RNA samples (5 or 10 µg) were resolved in 1.5% agarose gels
containing formaldehyde and then transferred to Nytran-plus membranes
(Schleicher & Schuell). The cDNAs encoding APP genes (Tst, Immunoprecipitation and Western Blotting--
Cells were washed
twice with ice-cold phosphate-buffered saline and lysed in RIPA buffer.
Lysates were incubated with specific antibodies to JAK1, JAK2 (Upstate
Biotechnology, Inc.), TYK2, SHP-2 (Santa Cruz Biotechnology), or FLAG
(M2 antibody, Eastman Kodak Co.) for 16 h at 4 °C.
Immunocomplexes were recovered by incubation with protein G-conjugated
Sepharose (Amersham Pharmacia Biotech) for 2 h at 4 °C with
agitation. Beads were washed three times with lysis buffer and boiled
in SDS sample buffer. The immunoprecipitates were separated on a 6%
SDS-polyacrylamide gel and transferred to a Protran membrane
(Schleicher & Schuell). Depending upon the experimental setup, the
membrane was first incubated with a mixture of the
anti-phosphotyrosine-specific antibodies, 4G10 (Upstate Biotechnology,
Inc.) and PY20 (Transduction Laboratories), then stripped and reprobed
for individual JAK members, SHP-2 or FLAG, to verify equal loading.
Results were visualized by enhanced chemiluminescence reaction (ECL)
according to manufacturer (Amersham Pharmacia Biotech). For detection
of active MAPK, equal amounts of total lysates were applied on 10% SDS
gels, and transferred proteins reacted with an antibody that recognizes
phosphothreonine 202 and phosphotyrosine 204 on ERK1/2 (New England
Biolab). The level of ERK proteins was subsequently visualized by
reaction with anti-ERK1/2 antibodies (New England Biolab).
Analysis of Cell Proliferation and Cell Culture
Morphology--
Subconfluent H-35 cells were plated into a 96-well
plate. Twenty-four hours after plating, cells were treated with
cytokines for 24 h in serum-free medium followed by addition of
0.4 µCi of [3H]thymidine for 16 h. Cells were
harvested, and the incorporated radioactivity was quantitated by a
liquid scintillation counter (Wallac). In parallel, cells were cultured
in Dulbecco's modified Eagle's medium containing 0.5% serum for 2 days and then changed to medium with 5% serum with or without G-CSF
for 4 days. The cell numbers were determined by counting viable cells
with a hemocytometer after trypan blue staining. Cell culture
morphology was determined by growing cells to confluent monolayers that
were then treated with cytokines with or without dexamethasone for
24 h. The morphologic changes were observed under a phase-contrast
microscope (Nikon).
Generation of H-35 Cells Stably Expressing Chimeric
G-CSFR-gp130 Receptors--
Previously, by constructing
G-CSFR-gp130 chimeric receptors, we have reconstituted signaling
events in transiently transfected hepatoma cells, which were similar to
those initiated by endogenous gp130 (26). Deletion analysis of gp130
indicated that the membrane-proximal 133 amino acids of gp130
were sufficient to induce transcription of co-transfected
chloramphenicol acetyltransferase reporter-gene constructs through the
IL-6RE and some APP gene promoters (26, 36). Within this region, a
single tyrosine residue (Tyr-126) in a Box3 sequence (YXXQ)
was found to be critical for the regulation. Since the transient
transfection approach has its technical limitation in reproducing
physiologically normal conditions, we assessed the signaling
specificity conferred by the Box3 motif on the regulation of endogenous
APP genes in hepatic cells, by generating lines of rat hepatoma H-35
cells stably expressing the chimeric receptor, G-gp130(133)WT, and its
Box3 mutant counterpart, G-gp130(133)M3, using the retroviral vector
MINV. From several clonal lines of transduced H-35 cells obtained, one
representative line of each receptor type was selected. Northern blot
analysis indicated equal levels of expression of the messenger RNAs
encoding the expected bicistronic receptor-neo gene
transcripts (Fig. 1A). The two
G-gp130 lines also expressed comparable G-CSF binding activity on the cell surface as shown by the 125I-G-CSF binding assay (Fig.
1B). The number of binding sites in each case was estimated
to be about 1,500 sites per cell. With the latter development of H-35
cell lines transduced with FLAG-tagged receptors, we could also
visualize the level of receptor proteins by Western blotting (Fig.
4B, bottom).
Role of Box3 in the JAK-STAT Pathway--
Signaling by
hematopoietin receptors is initiated by members of the JAK family (19,
46). Immunoprecipitation and Western blot analyses showed that JAK1 was
induced to a comparable level by G-CSF in cells expressing either the
wild-type or the M3 mutant G-gp130 receptor (Fig.
2, upper panel). TYK2 (Fig. 2,
lower panel) and JAK2 (data not shown) were likewise
stimulated by G-CSF to a similar extent despite a relatively higher
basal level in untreated cells. Importantly, the activation of JAKs by
G-CSF was similar to that induced by IL-6.
Next, we examined the effects of the Box3 mutation on the activation of
STAT proteins which are recognized downstream targets of JAKs.
Expression of the chimeric receptors did not noticeably alter the
magnitude or the kinetics of STAT activation induced by IL-6 (Fig.
3, upper panel). A strong
activation of SIE-binding activities that resolved into three bands was
observed 15 min after IL-6 treatment. As shown previously, these bands
represented STAT3 homodimer (top band), STAT3/STAT1
heterodimer (middle band), and STAT1 homodimer (bottom
band), respectively (47). Only the STAT homodimer was maintained
during longer treatment. Upon exposure to G-CSF, G-gp130(133)WT
mediated a characteristic pattern of STAT activation similar to IL-6,
albeit lower in magnitude, probably reflecting the effect of deleting
three of the four Box3 motifs (Fig. 3, lower panel). In
contrast, the Box3 mutant receptor failed to stimulate any significant
amount of detectable SIE binding activity. These results indicate that
although Box3 is not required for JAK activation, it is indispensable
for the activation of STATs.
Cooperation of Box3-dependent and Box3-independent
Pathways in the Regulation of APP Genes--
STAT3 was originally
purified as a protein binding to the IL-6RE of the
The lower APP stimulation by G-gp130(133)WT than by endogenous IL-6R
was attributed to the reduced activation of STAT3 by the chimeric
receptor. To confirm in the G-CSFR-gp130 context the relevance of four
versus one Box3 elements in determining quantitative level
APP induction, we compared APP response in pool cultures of H-35 cells
transduced with G-g130(277) or G-gp130(133), both tagged at the C
terminus with FLAG epitope. In separate experiments (not presented), we
have established that the FLAG epitope has no appreciable influence on
signaling gp130 cytoplasmic domains. The FLAG epitope allowed
immunodetection of both receptor proteins and their ligand-induced
phosphorylation (Fig. 4B). FLAG antibody reaction with
G-gp130(133) yielded an approximately 2-fold higher signal than with
G-gp130(277). However, the signal with antiphosphotyrosine antibody was
severalfold lower with G-gp130(133), which is in agreement with the
reduced number of phosphorylation sites. Treatment of these cells for
24 h with G-CSF resulted in a stimulated production of thiostatin
in G-gp130(277) cells that was similar to IL-6 treatment (Fig.
4C, samples 1 and 2). In contrast,
G-gp130(133)WT cells responded to G-CSF by a production of Tst that was
only 15% that to IL-6 (samples 4 and 5). An
equivalently low Tst induction by G-CSF was noted in cells expressing
G-gp130(133) without FLAG epitope (Fig. 4A and Fig.
5B). Cells treated with the
combination of G-CSF and IL-6 indicated an enhanced response (Fig.
5B, samples 3 and 6) that, in the case
of G-gp130(133)WT, amounted to an additive action.
A stimulatory action of G-gp130(133)M3 on the expression of the four
APPs in Fig. 4A was not detectable, and an inhibitory effect, if any, was not identifiable due to the low to non-detectable basal expression of these APPs. In testing the response of
G-gp130(133)M3 cells (Fig. 4C) (or recently established H-35
cells expressing G-gp130(133)M3-FLAG (data not shown)) with the
combination of IL-6 and G-CSF, the stimulatory action of IL-6 was
reduced rather than enhanced (Fig. 4C, sample 9). This
suggests that the signaling retained by G-gp130(133)M3 was opposing, in
part, the signaling of the Box3-dependent pathway that is
effective on the Tst gene.
Previously, by transient transfection of reporter gene constructs, we
identified the distal response element of the rat AGP gene as a genetic
target of the Box3-independent gp130 signaling pathway. We have also
observed that the rat MAPK Activity Is Prolonged in the Box3 Mutant--
We then sought
to identify the downstream signaling molecules in the Box3-independent
pathway which seem to exert both a stimulatory (
In hepatic cells, IL-6 regulates the expression of a set of early
response genes including egr-1 and junB, which
are the possible targets of MAPK-responsive transcription factors.
Therefore, we investigated the regulation of these genes by the
chimeric receptors. Northern blot analysis showed that the induction of
egr-1 and junB mRNAs after 30 min of G-CSF
treatment was not detectably affected by the Box3 mutation (Fig. 6,
lower panel). The magnitudes of the responses were
comparable between cells expressing the two different chimeric
receptors. Unexpectedly, however, time course analysis revealed that
G-CSF induced an extended period of MAPK activity in cells expressing
the Box3 mutant gp130 (Fig. 7A). While MAPK activity (like
phosphorylated STAT3) declined after 10-20 min treatment and reached
essentially basal level after 45 min in cells with G-gp130(133)WT, an
elevated level of MAPK activity was still maintained in M3 cells after
1 h. Concomitantly, in parallel to the MAPK activity, RNA level of
egr-1 was increased for an extended period in M3 cell (Fig.
7B).
Box3 Mediates a Growth Inhibitory Signal in H-35 Cells--
IL-6
is known to be an important growth factor for cells such as melanoma
and plasmacytoma (48, 49). On the other hand, in a few cell lines, for
example myeloid leukemic M1 cells, IL-6 exhibits a growth inhibitory
effect. These observations suggest that the effects of IL-6 on cell
proliferation are determined by cellular context. We examined the
influence of IL-6 on the growth of hepatic cells, and we tested whether
the growth response would be affected by the Box3 mutation. Thymidine
incorporation assays showed that IL-6 inhibited proliferation of H-35
cells (Fig. 8A). The results
of G-CSF treatment demonstrated that the membrane-proximal 133 amino
acids were sufficient to confer, at least in part, the inhibitory
response. The Box3 mutation completely abolished the negative effect of
gp130 on cell growth. Cell counts from long term cultures indicated
reduced numbers of G-gp130(133)WT cells following G-CSF treatment but
no observable growth inhibition by G-CSF on G-gp130(133)M3 cells (Fig.
8B).
Box3-lacking gp130 Induces Distinct Morphologic Changes--
MAPK
has been described to associate with microtubules and to participate in
the control of the cytoskeletal structure (50, 51). As shown in Fig.
9A, treatment of M3 cells
(panel 11), but not WT cells (panel 5), for
24 h with G-CSF resulted in distinct changes in cell morphology.
The compact single cell monolayers (panels 4 and
10) tended to desegregate, and the cells developed a rounded
shape. Notable, the G-CSF-induced changes became apparent only when the
cells were maintained in the presence of dexamethasone (compare
panels 8 and 11). No significant changes were
observed with IL-6 treatment (panels 6 and
12).
To determine whether enhanced ERK activity or loss of STAT3 activation
was responsible for the M3 culture phenotype, the cells were treated
with G-CSF and dexamethasone either in the presence of the MEK-1
inhibitor PD98059 (to reduce or inhibit ERK activation) or in the
presence of IL-6 (to co-activate STAT3) (Fig. 9B). Although we used a concentration of PD98059 that effectively suppressed ERK
activation by G-CSF and IL-6 (data not shown), the
G-CSF-dependent morphologic change of the M3 cell culture
was maintained. In contrast, IL-6 treatment significantly suppressed
the G-CSF effect, suggesting that loss of STAT3 rather than the
enhanced ERK activation was critical in gaining the M3 phenotype.
Analyses of APPs released by M3 cells treated with G-CSF and IL-6 in
the presence of PD98059 revealed that the inhibitory signal of
G-gp130(133)M3 on IL-6-stimulated Tst expression was relieved (Fig.
4C, samples 11 and 13). The PD98059-sensitive
effect on Tst was similarly observed in the presence of
dexamethasone (data not shown), indicating that the ERK-dependent inhibitory process was not influenced by
steroid treatment. However, G-CSF induced expression of
Our earlier investigations focusing on the transcriptional control
through isolated APP gene promoter elements have demonstrated that
IL-6-type cytokine receptors engage separate signal-transducing pathways and that the Box3-dependent pathway is responsible
for regulation of the IL-6RE in the APP gene promoters (36). Together with the observations that the Box3 motif of gp130 is a binding site
for STAT3 and that STAT3 exists in protein complexes associated with
the IL-6RE, it seems reasonable to assume that STAT3 is the principal
regulatory factor controlling induction of those APP genes that contain
IL-6REs (17, 18, 33, 52). Data from this report are consistent with
this notion, but they also suggest that the Box3-independent signal
appreciably contributes to the regulatory process. Our major findings
on the relationship between Box3 and APP gene regulation are as
follows: 1) the Box3 motif in gp130 is necessary for maximal expression
of most, if not all, APP genes, and this is tightly linked to the
activation of STAT3; and 2) APP gene exhibits varying degrees of
dependence on the Box3 signal. Some genes including Tst, Fb, and
hemopexin have an obligate requirement for STAT3. In contrast,
The Box3-derived signal, as manifested by and often equated to STAT3
activation, appears to be critical for the immediate induction of APP
genes. Although loss of the Box3 signal eliminated the capability of
gp130 to mediate immediate induction of APP genes, during long term
treatment (hours to days), the Box3-independent signal seems to be
capable of inducing relevant levels of expression of genes like
The structure/function relationship of gp130 has been assessed in other
experimental cell systems including the murine pro-B cell line BAF-B03
(60), the myeloid leukemic cell line M1 (61), and the rat
pheochromocytoma cell line PC12 (62). IL-6 displays distinct biological
activities in these cells. In BAF/B03 cells, gp130 mediates a
proliferative signal stimulating cell growth. Conversely, IL-6 inhibits
the growth of M1 and PC12 cells and promotes their differentiation. In
response to IL-6, M1 cells are arrested at
G0/G1 phase and express macrophage-like
morphologic changes and inducible nitric oxide synthase, whereas PDC12
cells develop neurite outgrowth when pre-treated with nerve growth
factor. Here we observed that besides the induction of APP genes, IL-6 causes growth inhibition of H-35 cells. This biological response of
H-35 cells has not been previously reported. For all of the IL-6
effects described above, the minimal receptor domain required was
mapped to the membrane-proximal 133 amino acids in the cytoplasmic region of gp130, in which a single Box3 motif was found to be necessary
for generating an effective signal. Inactivation of all functional Box3
sequences, as in the case of G-gp130(133)M3, abolished all of the above
biologic activities, except for BAF/B03 cells in which proliferation
can be maintained if the cells do not undergo IL-3 starvation. Since in
each of these cell systems the mutation of Box3 parallels the loss of
STAT3 activity, a pivotal role of STAT3 in regulating diverse cellular
functions in different cellular contexts is strongly suggested.
Although IL-6 through wild-type gp130 induces growth inhibition of both
M1 and H-35 cells, comparison of the expression profiles of the
growth-related genes reveals some major differences. For example,
egr-1 and junB are stimulated rapidly in H-35
cells, with a peak at 30 min and returning to basal level by 1 h.
In contrast, elevated levels of both mRNAs are maintained for at least 24 h in M1 cells. These observations suggest that the
regulation of growth inhibition may be different in M1 cells and H-35
cells. Therefore, H-35 cells can serve as an alternative model for the study of IL-6-mediated growth control.
Inasmuch as cytokine receptors with a single Box3 motif are known, such
as those for G-CSF, leptin, and thrombopoietin, receptors containing
multiple copies of Box3 seem to be more common. In addition to gp130,
the receptors for OSM and IL-10 contain two copies, whereas that for
LIF contains three copies of the Box3 motif (26-29, 63). The reason
for the presence of multiple copies of the Box3 is not well understood.
In the H-35-derived cell line, G-gp130(133)WT, the level of APP gene
expression induced by G-CSF is consistently lower than that elicited by
G-gp130(277)WT or IL-6 (Fig. 4C). This correlated with the
level of STAT3 activation, which is also lower following G-CSF
treatment (Fig. 3). It is not likely due to a lower level of expression
of the chimeric receptor (Fig. 4B), since the activation
levels of JAKs and MAPK stimulated by G-CSF is equal to, if not better
than, those induced by IL-6. Thus, we argue that the reduced G-CSF
response may be related to the fact that there is only a single Box3 in
the chimeric receptor, compared with four copies in the full-length
gp130. Conceivably, the presence of multiple Box3 sequences might
confer a "dose effect" on the signaling capacity of the receptor
affecting some biologic activities. Of course, we cannot exclude the
possibility that signaling molecules other than STAT3 might also bind
to Box3, and different Box3 motifs may have discrete preferred
substrates. However, to date, no other Box3-interacting proteins have
been reported.
One of the intriguing findings in our study is that inactivation of the
Box3 motif leads to prolonged MAPK activation. This observation has not
been reported in similar studies with other cell systems. It has been
described that SHP-2 acts upstream of the Ras-Raf-MAPK signaling
cascade and that Tyr-118 in the cytoplasmic domain of gp130 is required
for the association of SHP-2 to the receptor as well as for the
activation of SHP-2 (41, 60). Mutation of the SHP-2 site abrogates the
activation of MAPK (60). Notably, the SHP-2-binding site is in close
proximity to Tyr-126 of the Box3 motif. We hypothesize that binding of
STAT3 to the first Box3 motif would impede SHP-2 association to gp130
by physical exclusion. Thus, tyrosine mutation of Box3 provides an
unobstructed access of SHP-2 to gp130 leading to prolonged MAPK
activity. If so, it is reasonable to assume that this interference
between STAT3 and SHP-2 would not occur with Box3 motifs located more distantly on gp130. This also implies that the multiple copies of Box3
may not be functionally identical. The first Box3 could exert a
specific inhibitory effect on SHP-2, thereby regulating the duration of
MAPK activation. It is known that the duration of MAPK activation has a
determining effect on phenotypic outcome. For example, in PC12 cells,
epidermal growth factor stimulates a transient activation of MAPK
promoting cell growth. However, sustained MAPK activation by nerve
growth factor causes neuronal differentiation (62). Similarly, in F-36P
erythroleukemia cells, transient MAPK activity by IL-3 favors cell
proliferation, whereas more robust MAPK activation by thrombopoietin
elicits megakaryocytic differentiation (64). We observed that the Box3
mutation resulted in prolonged ERK activity, sustained Egr-1
expression, and morphological changes in H-35 cells. Evidently, two
processes govern gp130 signal specificity, one determines the
activation of specific signaling molecules (qualitative aspect) and the
other monitors the length of their activation time (quantitative aspect).
Finally, although STAT3 has been established to be a critical
transcription factor for APP gene induction in gp130 signaling, other
factors appear to be engaged which contribute, both positively and
negatively, to APP gene regulation. The modulatory actions of these
factors may occur at several levels, perhaps affecting concentration or
activity of the IL-6 receptor subunits and its signal-transducing
mechanisms, or modifying transcriptional and post-transcriptional
events underlying APP gene expression (65). By eliminating Box3, we
remove the STAT3 pathway from gp130 signal transduction and enhance
transcriptional responses transmitted by the Box3-independent pathway.
Based on the recent observations that prevention of SHP-2 recruitment
to gp130 essentially removes the MAPK pathway and simultaneously
enhances the effect of the STAT3-dependent pathway on APP
gene expression, a significant cross-modulation between the two
pathways seems to exist (41). Currently, work is underway to identify
effects in addition to the MAPKs and the downstream targets involved in
the Box3-independent signaling pathway of gp130 and to understand how
these factors cooperate with STAT3 to execute proper control of APP
gene expression.
2-macroglobulin and synergizes with IL-1 to
up-regulate
1-acid glycoprotein. The Box3 motif is not
required for activation of the SH2-containing protein tyrosine
phosphatase 2 or the mitogen-activated protein kinase (MAPK), nor is
the immediate induction of egr-1 and junB
significantly altered. Surprisingly, gp130 without any functional Box3
stimulates prolonged activation of MAPK, leading to an extended period
of up-regulation of egr-1 and to an extracellularly regulated kinase-mediated reduction in the IL-6-stimulated production of thiostatin. IL-6 reduces proliferation of H-35 cells through signaling by the Box3. In addition, cells expressing Box3-deficient gp130 showed distinct morphologic changes upon receptor activation. Taken together, these results indicate that Box3-derived and
Box3-independent signals cooperate in the control of hepatic APP genes
and that Box3 may be involved in the modulation of MAPK activity in
gp130 signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
1-acid glycoprotein (AGP),
IL-1 synergizes with IL-6 and glucocorticoids in stimulating gene
expression. In contrast, maximal expression of type II APP genes
depends on IL-6 and glucocorticoids, and IL-1 inhibits expression of
some type II APP genes such as thiostatin (Tst) and fibrinogen (Fb). The IL-6 responsive elements (IL-6RE) of a number of APP gene promoters
have been mapped (6-16). These elements are both necessary and
sufficient to confer IL-6 responsiveness. With the IL-6RE as binding
substrate, IL-6-induced transcription factors have been purified, and
the corresponding cDNAs were subsequently cloned (17, 18). These
factors were identified as members of the Signal Transducer and
Activator of Transcription family, STAT1, STAT3, and STAT5 which are
also involved in the signaling of many other hematopoietic cytokines
(19, 20).
, and a signal-transducing subunit, gp130 (21, 22). Upon
ligand binding, two of each receptor subunit form a hexameric complex
with two IL-6 molecules and initiate downstream signaling events (23,
24). Signal transduction depends on the homodimerization of the
cytoplasmic domains of gp130 (25, 26). Comparison of the cytoplasmic
regions between gp130 and other IL-6-type cytokine receptors including
the receptors for leukemia inhibitory factor (LIF), oncostatin M (OSM),
and granulocyte colony-stimulating factor (G-CSF) reveals three
conserved sequence motifs, namely Box1, Box2, and Box3 (26-29). The
Box3 motif, with a consensus amino acid sequence of YXXQ,
occurs in multiple copies in gp130, LIF receptor, and OSM receptor and
represents a binding site for STAT1 and STAT3 (30, 31).
2-macroglobulin gene (
2-MG) and to the
"distal regulatory element" in the
1-acid
glycoprotein (AGP) gene promoter (40). The chromosomal genes regulated
by this proposed pathway have not yet been identified. Here we assessed
the importance of the Box3 motif in controlling endogenous APP genes by
generating stable hepatoma cell lines expressing chimeric G-CSFR-gp130
receptor. We also attempted to elucidate the components of the
Box3-independent signaling pathway in these cells. Our results indicate
that both Box3-dependent and Box3-independent pathways
contribute to the regulation of APP genes. The data also suggest a
functional role of Box3 in modulating the duration of MAPK activity.
EXPERIMENTAL PROCEDURES
(Immunex Corp.), alone or in combinations in serum-free medium. Optimal inhibition of ERK activation was achieved by treatment with 25 µM PD98059 (Calbiochem).
Fb,
hemopexin, haptoglobin,
2-MG, and AGP) or the early
response genes (junB and egr-1) were labeled by
random-prime method with a commercial kit (Amersham Pharmacia Biotech).
Hybridization was carried out in buffer containing 4× SSC, 0.1 M sodium phosphate, 1× Denhardt's solution, 0.2% SDS, 200 µg/ml sheared/denatured salmon sperm DNA at 65 °C for
overnight. Membranes were then washed in 2× SSC, 0.1% SDS and exposed
to x-ray film at
70 °C. Ethidium bromide-stained pattern of 28 S rRNA band served as marker for RNA loading.
2-MG and Tst secreted into the medium were quantitated
by immunoelectrophoresis (45) or determined by Western blotting using
reaction with combined goat antibodies against rat Tst and
2-MG (Roswell Park Cancer Institute).
RESULTS
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Fig. 1.
Expression of the chimeric receptor
G-CSFR-gp130 in rat hepatoma H-35 cells. A, equal
aliquots of RNA from the indicated cell lines were analyzed by Northern
blotting using a cDNA encoding the extracellular domain of G-CSFR
as a probe (top panel). The ethidium bromide
(EtBr) staining of 28 S rRNA indicates equal loading
(bottom panel). B, specific binding of
125I-G-CSF on the stable cell lines was determined. The
means and standard deviation of experiments in triplicate are
shown.
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Fig. 2.
Box3 motif is not required for JAK
activation. Cells were either untreated or treated with the
indicated cytokines (G, G-CSF; 6, IL-6) for 10 min. Cell lysates were used for immunoprecipitation (IP)
with anti-JAK1 or anti-JAK2 antibodies. Immunoprecipitates were
recovered and subjected to Western blot analysis with
anti-phosphotyrosine (anti-PY) antibody. Membranes were then
stripped and re-probed with anti-JAK antibodies.
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Fig. 3.
Box3 is indispensable for the activation of
STAT proteins. Cells were treated with cytokines for the indicated
times. Whole cell lysates were prepared and subjected to EMSA using SIE
as a probe.
2-MG
gene promoter (also called the acute-phase response factor (APRF)). By
transient transfection, we demonstrated that STAT3 is able to
transactivate certain APP gene promoters. It has also been shown that
Box3 acts as a receptor-binding site for STAT3, thus implicating an
essential role for Box3 in the control of APP genes. To test this
functional link, we examined the effects of the Box3 mutation on the
expression of the endogenous APP genes in the stable G-gp130 cell
lines. Northern blot analysis of four representative APP genes,
including Tst,
Fb, hemopexin, and haptoglobin, showed an
up-regulation of RNA levels in response to G-CSF in cells expressing
G-gp130(133)WT (Fig.
4A). The M3 mutant cells
were unresponsive to similar treatment, and no induction of these APPs
was detected. The IL-6 response of the two different cell lines was,
however, comparable.
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Fig. 4.
Box3-dependent regulation of APP
expression. A, cells were treated with cytokines in the
presence of dexamethasone for 16 h. Total RNA was prepared, and
equal aliquots were analyzed using specific cDNA probes for APP
genes ( -FB,
-fibrinogen; TST, thiostatin;
HPX, hemopexin; and HP, haptoglobin). Equal
loading is demonstrated by ethidium bromide (EtBr) staining
of 28 S rRNA band. B, H-35 cells transduced with
G-gp130(277)WT-FLAG or G-gp130(133)WT-FLAG were treated for 15 min and
receptors immunoprecipitated (i.p.) with anti-FLAG
antibodies. The proteins following separation were analyzed on Western
blot (w.b.) first with anti-phosphotyrosine (PY)
followed by anti-FLAG antibodies. Equal protein loading is demonstrated
by the comparable staining of nonspecific protein bands. C,
cells were incubated for 24 h in serum-free medium containing IL-6
or G-CSF but in the absence of dexamethasone. Some cultures received in
addition 25 µM PD98059. Tst produced by each culture
during the subsequent 24-h treatment period was determined by
immunoelectrophoresis, normalized to equal cell number in the culture,
and expressed relative to the value obtained for IL-6-treated cells in
each culture series (= 100%) (mean ± S.D.; n = 3-5).
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Fig. 5.
Box3-independent regulation of
1-acid glycoprotein and
2-macroglobulin. A,
cells were treated with indicated combinations of cytokines in the
presence of dexamethasone for 48 h. Equal aliquots of total RNA
were analyzed with cDNA probes for AGP or
2-MG.
B, cells were treated in two time periods of 24 h each
with serum-free medium containing 1 µM dexamethasone and
the cytokines as indicated. The amount of
2-MG produced
during the second 24-h period was determined by immunoelectrophoresis
(left panel). The relative change in concentration of
2-MG and Tst was visualized by Western blotting using
equal aliquots of culture medium and reaction with
anti-
2-MG and Tst antibodies combined (right
panel).
2-MG promoter is unique among
other APP gene promoters in its responsiveness to some Box3-deficient
hematopoietin receptors such as
IL-2R.2 These observations
prompted us to examine the influence of the Box3 mutation on the
regulation of the endogenous AGP and
2-MG genes. In
G-gp130(133)WT cells, G-CSF treatment led to increased RNA expression
of both AGP and
2-MG (Fig. 5A). IL-1
synergized with G-CSF in the stimulation of AGP but exerted an
inhibitory effect on
2-MG expression. In G-gp130(133)M3
cells, G-CSF alone had no detectable stimulatory effect, but synergized
prominently with IL-1 to enhance AGP expression. In contrast, the
expression of
2-MG was stimulated by G-CSF alone.
Interestingly, the G-CSF effect was not suppressed by IL-1 suggesting
IL-1 action was directed toward suppressing the
Box3-dependent stimulatory signal of gp130. Treatment with
IL-6 yielded similar results in the two stable G-gp130 lines. IL-6
alone stimulated the expression of both AGP and
2-MG. As
described previously, IL-1 synergized with IL-6 to up-regulate AGP
expression but reduced the IL-6-induced expression of
2-MG. The differential regulation of
2-MG
and thiostatin provided evidence that the gp130-derived signals did not
act uniformly on type II APPs. The signals triggered by the combination
of G-CSF and IL-6 proved to act additively on
2-MG gene
in both G-gp130(133)WT and G-gp130(133)M3 cells (Fig. 5B,
left panel), whereas the expression of Tst in the latter cell line
was substantially reduced below that of IL-6 alone (Fig. 5B,
right panel). Collectively, these results indicated that, in
the absence of STAT3 activation, the Box3-independent pathway did
contribute to the control of a subset of APP genes as represented by
AGP and
2-MG.
2-MG)
and an inhibitory (Tst) effect. G-CSF treatment resulted in the
phosphorylation of SHP-2 to similar levels in the two stable G-gp130
cell lines, regardless of the Box3 mutation (Fig.
6, upper panel). Unlike gp130,
we did not observe association of SHP-2 with the chimeric receptors,
possibly due to reduced affinity of SHP-2 for the chimeric receptor as
a result of the cytoplasmic truncation. MAPK has been shown to be the
downstream effector molecule of SHP-2 and thus was expected to be
activated in a similar fashion. As predicted, G-CSF induced ERK1/2
phosphorylation to a similar level as IL-6 did (Fig. 6, upper
panel). No significant difference in the magnitude of ERK1/2
activation through the chimeric receptors between the two G-gp130 cell
lines was observed.
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Fig. 6.
Box3-independent signaling in H-35
cells. Upper panel, lysates were prepared from cells
treated with cytokines for 10 min and subjected to SHP-2
immunoprecipitation (IP) followed by Western blotting
(WB) for anti-phosphotyrosine (pY). In parallel,
activation of MAPK was analyzed by directly probing lysates with an
antibody specific for phospho-ERK1/2. Bottom panel, total
RNA was extracted from cells 30 min after cytokine treatments and
analyzed for the levels of junB and egr-1 with
respective cDNA probes. G, G-CSF; 6,
IL-6.
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Fig. 7.
Box3 mutation sustains prolonged MAPK
activation. A, equal amounts of protein lysates from
cells treated with G-CSF for different times were examined in duplicate
for phosphotyrosine-STAT3 (PY-STAT3) and STAT3, and for
ERK1/2 and total ERK1/2 (note predominance of ERK2). B,
cells were untreated or treated with G-CSF or IL-6 for different times.
RNA expression of egr-1 was analyzed by Northern
blotting.
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Fig. 8.
Box3 is required to inhibit proliferation in
H-35 cells. A, cells were treated with G-CSF or IL-6
for 24 h and subjected to thymidine incorporation assay. A
representative experiment done in triplicate is shown. , no
cytokines;
, G-CSF;
, IL-6. B, in parallel, cells were
treated with cytokines for 4 days, and cell number was counted after
trypan blue staining. The experiment was done in triplicate.
, no
cytokines; shaded box, G-CSF.
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Fig. 9.
Distinct morphological
changes induced by Box3-deficient gp130. A,
cells were treated with G-CSF or IL-6 with or without dexamethasone
(Dex) for 24 h. B, G-gp130(133)M3 cells were
treated for 24 h with serum-free medium containing dexamethasone,
alone or in combinations with G-CSF, IL-6, and PD98059 as indicated.
Cell culture morphologies were observed under a phase-contrast
microscope.
2-MG in M3 cells was still detectable in the presence of
PD98059 (data not shown), suggesting that this stimulatory signal was
either independent of ERK pathway or less susceptible to suppression by
PD98059 than the inhibitory signal.
DISCUSSION
2-MG expression can be regulated, at least partially, in
the absence of detectable active STAT3. 3) As highlighted by our
analysis of AGP gene expression, IL-1 exerts a considerable synergistic
effect with the gp130 signal independent of Box3; and 4) with removal
of a Box3 motif, gp130 signaling toward the ERK pathway is enhanced
that, in part, attenuates the effect of the Box3-dependent
pathway as observed for Tst expression.
2-MG. The factors responding to the Box3-independent signal are still poorly understood. Since MAPK activation, in particular that of ERKs, is largely unaffected by the Box3 mutation, the downstream targets of ERKs are appealing candidates for effectors of APP gene regulation in the absence of the Box3 motif. MAPK is known
to phosphorylate and modulate the activities of transcription factors
such as STAT3, C/EBP
, CREB, SRF, AP-1, ATF-2, and Elk-1 (35, 53,
54). It is conceivable that the factors other than STATs also act
directly on the APP promoters and may explain the modulatory effect
attributed to growth factors that engage ERKs (55, 56). However, at
present, there is no evidence that any of these non-STAT factors
account significantly for IL-6-enhanced expression of type II APPs (57,
58). On the other hand, the transcription factors, whose synthesis is
regulated by the Box3-independent signal (Fig. 6), may also contribute
indirectly to the APP gene regulation in the later period, in part,
through stimulating proliferative activity of the cells. Increased
proliferation of the hepatic cells has been noted to lower cytokine
regulation of APP genes (56, 59).
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ACKNOWLEDGEMENTS |
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We thank Immunex Corp. and Genetics Institute for providing cytokines; Dr. David Gearing for chimeric receptor constructs; Dr. Olivier Robledo for 125I-labeled G-CSF; Erin Kinzie for technical assistance; and Lucy Scere and Marcia Held for secretarial assistance.
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FOOTNOTES |
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* This work was supported by NCI Grant CA26122 from the National Institutes of Health (to H. B.), Boehringer Ingelheim Fonds (to J. R.), and in part by shared resources of RPCI Cancer Center Support Grant CA16056.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.
Current address: Dept. de Biologie Moléculaire, Sciences II,
Université de Genève, 30, Quai Ernest Ansermet, CH-1211,
Genève-4, Switzerland.
¶ To whom correspondence should be addressed. Tel.: 716-845-4587; Fax: 716-845-8389; E-mail: baumann{at}sc3101.med.buffalo.edu.
2 J. Ripperger and G. H. Fey, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
IL, interleukin;
AGP, acid glycoprotein;
APP, acute-phase protein;
EMSA, electrophoretic
mobility-shift assay;
ERK, extracellularly regulated kinase;
Fb, fibrinogen;
G-CSF, granulocyte colony-stimulating factor;
JAK, Janus
kinase;
LIF, leukemia inhibitory factor;
2-MG,
2-macroglobulin;
MAPK, mitogen-activated protein kinase;
OSM, oncostatin M;
RE, response element;
SHP-2, SH2-containing
phosphatase 2;
STAT, signal transducer and activator of transcription;
Tst, thiostatin;
gp, glycoprotein;
SIE, sis-inducible element.
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REFERENCES |
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