From the
The cytoplasmic receptor sequences required for the
transcriptional control via the IL-6 response element (IL-6RE) and the
hematopoietin receptor response element (HRRE) in hepatoma cells were
defined by transient expression of wild-type and mutant
granulocyte-colony stimulating factor receptor-gp130 chimeric
receptors. gp130 generated two separate transcriptional signals, one of
which was directed to IL-6RE and required an intact box 3 motif, and
another, which was directed to HRRE and was box 3-independent. The
activation of DNA-binding of STAT3 required the same gp130 domains as
the IL-6RE response. A box 3-independent activation of STAT proteins
was achieved by overexpression of the kinases JAK2 or TYK2. The
increase in the DNA-binding activity of STAT proteins, however, did not
result in a corresponding increase in transcription via either IL-6RE
or HRRE. The data indicate that activation of the DNA-binding potential
of STAT proteins via gp130 is not sufficient to achieve transcriptional
up-regulation of specific target genes.
The receptor for interleukin-6 (IL-6R)
gp130
contains in its cytoplasmic domain several functional elements
including box 1, 2, and 3 motifs (12, 13) and binding
sites for Syp (14) and Shc(15) . These elements are necessary
for the activation of JAKs, stimulation of proliferation, and/or
induction of several immediate response
genes(4, 5, 12) . Although a causal relationship
between box 3 function, STAT3 activation, and IL-6-responsive gene
regulation was expected(8, 14) , we provide evidence
that the activation of STAT proteins was facilitated but not absolutely
dependent on the presence of a box 3 motif, and that elevated
DNA-binding activity of STAT proteins is not sufficient to account for
the transcription of target genes via IL-6RE.
To determine G-CSFR-gp130
protein expression, 20 µl of whole cell lysate from 5
Gene-regulatory function of the receptor
mutants was tested in H-35 cells (Fig. 2D). The M3
mutation in gp130(133) inactivated the regulation of the IL-6RE-CAT
gene but not of the HRRE-CAT gene. The P14/16A mutant box 1 (M1)
abolished all signaling function of gp130(133), i.e. activation of STAT proteins in L-cells and transcriptional
regulation of IL-6RE- and HRRE-CAT genes in H-35 cells, even though the
M1 protein was abundantly expressed in transfected L-cells (data not
shown; see also Fig. 4C below).
Similar signal reconstitution in H-35 cells (Fig. 4B)
confirmed the findings made with L-cells and revealed additional
important features. 1) Transfected STATs and/or JAKs had no significant
effect on action of the endogenous IL-6R or transfected
G-CSFR-gp130(133wt); 2) G-CSFR-gp130(133M1) was inactive under all
conditions; 3) G-CSFR-gp130(133M3) produced a 2-5-fold
transcriptional stimulation through IL-6RE in the presence of
overexpressed STAT3, JAK2, or TYK2. However, even under optimal
reconstitution conditions, the box 3-deficient G-CSFR-gp130 yielded a
maximal IL-6RE response that was only a fraction of that obtained with
the wild-type G-CSFR-gp130(133). The data from Fig. 4(A and B) indicate that transcriptional regulation through
IL-6RE by gp130 was not strictly correlated with relative amounts of
activated STATs. We hypothesize that an additional regulatory factor
(or factors) is activated by gp130(133), probably by the action of
receptor-associated kinase(s). This factor could not be experimentally
identified by binding to SIE or IL-6RE (Fig. 2A; data
not shown) and thus was termed ``SIF-independent gene
regulator'' or ``SIG.''
Evidence for a functional
entity such as SIG in hepatoma cells was provided by the box 3- and
STAT3-independent transcriptional regulation through HRRE ( Fig. 1and Fig. 2, A and D). The HRRE
signal was, however, dependent on box 1, suggesting that box
1-interacting JAKs may act as potential mediators of that hypothetical
signal pathway. The relatively low HRRE regulating activity of
G-CSFR-gp130(40) conceivably was due to reduced association with and/or
activation of JAKs. Overexpression of JAK2 (Fig. 3, A and B) or TYK2 (data not shown) clearly rectified this
inefficient kinase action. Furthermore, cotransfection of the
G-CSFR-gp130(40) with JAK2 yielded a 20-fold increase in transcription
through HRRE that was independent of cotransfected STAT3 (Fig. 4C).
This study illustrates that gp130, like
other hematopoietin receptors (4, 5, 12), can activate various STAT
proteins. The prominence of this response and the specificity of STAT-3
for DNA sequences related regulation of genes by IL-6 have led to a
model proposing STAT-3 to function as both signal transducers and activators of transcription(4, 5) . However, the
data of this study indicate that STAT3 does not act as sole activator
of transcription and that other activities controlled by gp130, such as
the hypothetical SIG, are likely involved. Contributing activities may
include the action of serine kinase noted to enhance DNA binding of
STAT proteins(23, 24, 25) . Moreover, the
current model of the JAK-STAT pathway from receptor to the regulated
gene will need modifications based on the two observations that 1) STAT
activation does not strictly depend on phosphotyrosine residues on the
receptor molecule(26) , and 2) regulation of the prototypical
IL-6-responsive
We thank Dr. D. M. Wojchowski for providing
pEF-BOS-JAK2, Dr. J. J. Krolewski for the TYK2 cDNA, Dr. D. J. Tweardy
for sheep anti G-CSFR, and Marcia Held for secretarial assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)is composed of the 80 kDa ligand-binding subunit
(IL-6R
) (1) and the signal-transducing subunit,
gp130(2) . After ligand binding, the IL-6R subunits are believed
to aggregate into a hexameric complex consisting of two molecules each
of IL-6, IL-6R
, and gp130(3) . Signal transduction has been
shown, or predicted, to involve activation of receptor-associated
protein tyrosine kinases belonging either to the JAK (4, 5) or to the Bruton's tyrosine kinase (BTK)
families(6, 7) . These kinases phosphorylated both
themselves and the cytoplasmic portion of the receptor. The
phosphotyrosine residues serve in part as recognition sites for
SH2-domain-containing signaling proteins, in particular STAT3 (or ARPF) (8) and to a lesser extent, STAT1(9) . STAT proteins are
also phosphorylated that then dimerize, translocate to the nucleus, and
bind to specific DNA sequences(10) . Binding to the high
affinity form of SIE of the c-fos gene and related
interferon-
activating site sequences provide the identifying
feature of the IL-6-regulated STATs(9, 11) .
Cells
Rat hepatoma H-35, L-, and COS-1 cells
were cultured as described(13) . A clonal line of L-cells stably
transfected with an expression vector for human
G-CSFR-gp130(277)(13) , and pSV2neo was selected and maintained
in medium containing 0.4 mg/ml G418. Hormonal treatments were carried
out in serum-free medium containing 1 µM dexamethasone
alone or in combination with 100 ng/ml human IL-6 (Genetics Institute)
or human G-CSF (Immunex Corp.).
Expression Vectors and CAT Reporter Gene
Constructs
Expression vectors encoding the chimeric human
receptor G-CSFR-gp130(277) and the truncated forms with cytoplasmic
domains of 230, 165, 133, 109, 90, 65, 40, and 1 (cyto) residues,
respectively, have been described(13) . Tyrosine 125 was mutated
to alanine (Y125A) in box 3a (termed M3) of G-CSFR-gp130(277)
and(133) , and the prolines 14 and 16 were mutated to leucines
(P14/16L) in box 1 (termed M1) of G-CSFR-gp130(133) by using the in
vitro mutagenesis kit from Clontech. Rat STAT1 cDNA (a 2,300-bp EcoRI-XhoI fragment) and rat STAT3 cDNA (a 3,000-bp EcoRI-EcoRV fragment).(
)
were
inserted into the NotI site of pSV-Sport 1 (Life Technologies,
Inc.). The 4,176-bp human TYK2 cDNA (provided by Dr. J. J. Krolewski) (16) was inserted into the KpnI-NotI sites of
pDC302(17) . The expression vector for mouse JAK2 in pEF-BOS has
been provided by Dr. D. M. Wojchowski(18) . The CAT reporter
gene constructs pHRRE-CAT and pIL-6RE-CAT have been
described(19) .
Cell Transfection and Analysis
Plasmid DNA was
transfected by the DEAE-dextran method (20) optimized for each
application (see figure legends). Cell cultures were subdivided,
treated, and analyzed for CAT activity as
described(13, 21) . STAT protein activation was
determined in whole cell extracts prepared from cells after treatment
with cytokines for 15 min at 37 °C by EMSA(9) . The high
affinity SIEm67 oligonucleotide served as binding
substrates(9) . STAT3-containing complexes were identified by
``supershift'' assay using 1 µg of rabbit antibodies
against STAT3 (Santa Cruz Biotechnology).
10
transfected cells was mixed with 25 µl of SDS buffer
and electrophoresed in a 6% SDS-polyacrylamide gel. The proteins were
transferred to Immobilon membrane (Millipore), incubated with sheep
anti-human G-CSFR (provided by Dr. D. J. Tweardy), and processed for
chemiluminescent reaction according to the supplier (DuPont NEN).
Cytoplasmic Domain of gp130 Mediates Two
Signals
The chimeric G-CSFR-gp130 receptor form was used to
define the signaling reactions initiated by the cytoplasmic domain of
gp130 independently of the resident receptor subunits for IL-6-type
cytokines(13, 21) . Transfection of G-CSFR-gp130 with
full-length or progressively truncated cytoplasmic domains into H-35
cells allowed the mapping of the regions required for gene activation
via two characteristic elements, IL-6RE and HRRE (Fig. 1).
G-CSFR-gp130(133) was the minimal construct generating a significant
transcriptional response via IL-6RE. Deletion of the carboxyl-terminal
box 3 repeated motifs b, c, and d (Fig. 1) diminished the
magnitude of regulation 2-fold. Removal of residues 133-109,
containing the box 3a sequence, eliminated all detectable IL-6RE
response. However, regulation via HRRE by the same receptors was
maintained. The HRRE response decreased to 5-10% only after
removal of box 2 and was lost with deletion of box 1.
Figure 1:
Differential regulation of IL-6
responsive gene elements by gp130 motifs. The chimeric receptor forms
of G-CSFR-gp130 indicated at the top (1 µg/ml) were cotransfected
with pIL-6RE-CAT (6 µg/ml) and HRRE-CAT (6 µg/ml) into H-35
cells. Subcultures were treated with G-CSF for 24 h. The CAT activities
were normalized to the internal transfection marker pIE-MUP and
expressed relative to the control culture in each experimental
series.
Box 3 Is Necessary for the Activation of STAT3 and
Transcription through IL-6RE
To identify whether gene regulation
by the G-CSFR-gp130 is correlated with the ability to activate STAT
proteins, the receptors were transfected into L-cells. This cell type
has a higher transfection efficiency than H-35 cells, thus yielding a
receptor protein expression detectable by Western blotting activation
of endogenous STAT proteins(19) , low level regulation of
cotransfected IL-6RE-containing CAT gene constructs (see Fig. 4A and Ref. 21). Wild-type G-CSFR-gp130(277)
mediated a G-CSF-specific activation of STAT proteins producing a SIF
complex that comigrated on EMSA with SIF-A induced by the endogenous
IL-6R (Fig. 2A). STAT3 contributed essentially to all
DNA-binding activity detected by EMSA as shown by an antibody-mediated
supershift assay (Fig. 2B).
Figure 4:
Effect of ectopically expressed STAT
proteins on gene regulation. A, L-cells were transfected with
G-CSFR-gp130(133wt) (1.5 µg/ml), Hp(IL-6RE)CAT (2 µg/ml), and,
where indicated, with expression vectors for STAT1 (1.5 µg/ml),
STAT3 (1.5 µg/ml), or JAK2 (0.1 µg/ml). Subcultures of the
transfected cells were used to determine the activation of STAT
proteins by EMSA after 15 min of G-CSF treatment and increased
expression of CAT activity after 24 h of treatment. All analyses were
normalized to the expression of the internal transfection marker
pIE-MUP (13, 21). B, gene regulation in H-35 cells. Various
receptor constructs (1 µg/ml), pIL-6RE-CAT (6 µg/ml), and
expression vectors for STAT1 (3 µg/ml), STAT3 (3 µg/ml), JAK2
(0.1 µg/ml), or TYK2 (0.1 µg/ml) were cotransfected as
indicated. The subcultures were treated with the indicated agonists,
and the magnitude of stimulation of CAT enzyme activity was calculated
relative to the control in each series. Mean and S.D. of three separate
experiments are shown. C, activation of HRRE signal by JAK2.
H-35 cells were transfected with G-CSFR-gp130(40) (1 µg/ml),
HRRE-CAT (5 µg/ml), and expression vectors for STAT3 (3 µg/ml)
and JAK2 (0.1 µg/ml) as indicated. The relative changes in CAT
activities were quantitated.
Figure 2:
Correlation of STAT protein activation and
transcriptional regulation through the IL-6RE. A, activation
of STAT protein in L-cells. The G-CSFR-gp130 receptor forms indicated
at the top (3 µg/ml) were transfected into L-cells. Subcultures
were treated for 15 min with or without G-CSF, extracted, and analyzed
by EMSA. Extracts from IL-6-treated L-cells and a G-CSF-treated
G-CSFR-gp130(277) stable L-cell line served as standards. B,
identification of STAT3-containing SIF complex. The extract from
G-CSFR-gp130(133wt)-transfected and G-CSF-treated culture from A was analyzed by EMSA with or without inclusion of anti-STAT3
antibodies in the binding reaction. C, equal amounts of whole
cell extracts from L-cells transfected with the indicated receptors
from A were analyzed by Western blot for the expression of
chimeric G-CSFR proteins. The patterns show the characteristic two
forms of the mature chimeric G-CSFR proteins. D, gene
regulating activities of the G-CSFR-gp130 receptor forms. The receptors
indicated at the top were cotransfected into H-35 cells together with
either Hp(IL-6RE)-CAT or HRRE-CAT. Subcultures were treated with G-CSF,
and the effects on the CAT activities relative to control cells were
determined. The relative changes are given above the
autoradiograms.
G-CSFR-gp130(133) was 3
times less active than the full-length form (Fig. 2A).
Deletion to residue 109 abrogated detectable STAT protein activation.
The functionally relevant sequence within the 133-residue form was
determined by mutation. The Y125A mutant box 3 (M3) abolished STAT
activation (Fig. 2A) but did not affect receptor protein
expression as demonstrated by Western blotting (Fig. 2C). By contrast, the M3 mutation in full-length
gp130(277) did not influence STAT activation (Fig. 2A).
Thus, the repeated box 3 motifs (b, c, and d) were functionally
redundant(14) .
Box 3-independent STAT Activation by JAK2
If
active STAT proteins cause transcriptional regulation, a change in
their cellular concentration should entail a corresponding change in
the transcriptional activity of IL-6RE-containing genes. To assess
whether G-CSFR-gp130 could activate excess amounts of ectopically
expressed STAT proteins and thus prominently alter the intracellular
concentrations of active STAT proteins, expression vectors for
G-CSFR-gp130s forms and for rat STAT1 and STAT3 were cotransfected into
COS-1 cells (Fig. 3A). COS-1 cells were used because of
high expression of transfected vectors and the low level contribution
of endogenous STATs, hence permitting clear demonstration of the
specificity of STAT protein activation. Transfected G-CSFR-gp130 alone
produced a minor increase of the endogenous SIF-C protein-DNA complex (Fig. 3A). Transfected rat STAT1 and 3 were strongly
activated, producing rat SIF-C and SIF-A complexes, respectively, which
showed different mobilities from the corresponding endogenous SIF
complexes (Fig. 3A). Inclusion of JAK2 caused two
notable effects: an increase in basal level of activated STAT3 (Fig. 3A) or STAT1 (not shown), and a further induction
following G-CSF treatment. Excess levels of JAK2 were capable of
increasing basal levels of activated STATs even in the absence of
ligand-receptor interaction. This JAK2-mediated, receptor-dependent
activation of STAT3 did not require box 3. In fact, box 1 was
sufficient, as demonstrated by the data obtained with G-CSFR-gp130(40) (Fig. 3A). The resulting intensities of SIF complexes
exceeded by a factor of 1,000-10,000 those achieved in COS cells
not supplemented with STAT proteins.
Figure 3:
Modulation of SIF by overexpression of
STATs and JAKs. A, the indicated receptors (2 µg/ml) were
transfected into COS-1 cells alone or with STAT1 (2 µg/ml), STAT3
(2 µg/ml), or JAK2 (0.15 µg/ml). Subcultures were treated as
indicated, extracted, and analyzed by EMSA. B, as in A, COS-1 cells were cotransfected with the indicated receptor
forms (2 µg/ml) STAT3 (2 µg/ml) TYK2 (0.15 µg/ml) and
analyzed as in A.
TYK2 has been noted to interact
with gp130(8) . Therefore, we tested whether TYK2 was also
effective in activating STAT proteins (Fig. 3B). In the
absence of any transfected kinase, the box 3-specific activation of
coexpressed rat STAT3 by G-CSFR-gp130(133) was detectable, probably
mediated by the resident COS cell kinases. Overexpressed TYK2, like
JAK2, promoted a several hundred-fold increase of STAT3 activation
independently of agonist treatment and presence of box 3. Neither TYK2
nor JAK2 was effective with transfected G-CSFR-gp130(133M1), suggesting
that the kinase action was linked to box 1 element in agreement with
the published observations for other hematopoietin
receptors(4, 5, 12, 22) .
STAT3 and JAK2 Contribute to but Are Not Sufficient for
Gene Regulation
The prominent activation of ectopically
expressed STAT proteins provided the experimental condition to assess
the link between activation of STAT proteins and transcriptional
induction of genes. G-CSFR-gp130(133) and STAT proteins were introduced
into L- and H-35 cells and their effects on cotransfected IL-6RE-CAT
gene determined (Fig. 4, A and B). Although a
prominent STAT1 and -3 activation was achieved in L-cells (Fig. 4A), the IL-6RE response after 24 h G-CSF
treatment was not appreciably elevated above the control culture. JAK2
alone raised basal activity 2-fold and enhanced the G-CSF effect
severalfold. Surprisingly, the combination of JAK2 and the STATs did
not increase transcription proportionally to the STAT protein activity.
-fibrinogen gene does not appear to involve
STAT3(27) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.