(Received for publication, June 29, 1994; and in revised form, December 2, 1994)
From the
The mechanisms by which the binding of growth hormone (GH) to
its cell surface receptor elicits changes in gene transcription are
largely unknown. The transcription factor Stat1/p91 has been shown to
be activated by GH. Here we show that acute phase response factor or
Stat3 (or an antigenically related protein), is also activated by GH.
Stat3 has been implicated in the interleukin-6-dependent induction of
acute phase response genes. GH promotes in 3T3-F442A fibroblasts the
tyrosyl phosphorylation of a protein immunoprecipitated by antibodies
to Stat3. This protein co-migrates with a tyrosyl phosphorylated
protein from cells treated with leukemia inhibitory factor, a cytokine
known to activate Stat3. Tyrosyl phosphorylated Stat3 is also observed
in response to interferon-. Stat3 is present in GH-inducible
DNA-binding complexes that bind the sis-inducible element in
the c-fos promoter and the acute phase response element in the
-macroglobulin promoter. The ability of GH to activate
both Stat1 and Stat3 (i.e. increase their tyrosyl
phosphorylation and ability to bind to DNA) suggests that gene
regulation by GH involves multiple Stat proteins. Shared transcription
factors among hormones and cytokines that activate JAK kinases provide
an explanation for shared responses, while the ability of the different
ligands to differentially recruit various Stat family members suggests
mechanisms by which specificity in gene regulation could be achieved.
GH ()is an important mediator of body growth and
regulates both cellular differentiation and proliferation (reviewed in (1) ), functions that necessitate the coordinated regulation of
gene expression. The GH receptor, a member of the cytokine receptor
superfamily(2) , transmits an intracellular signal through
activation of the cytoplasmic tyrosine kinase JAK2(3) .
However, molecular mechanisms linking the activated GH receptor-JAK2
complex to gene transcription are largely unknown.
The Stat (Signal Transducers and Activators of Transcription) family of transcription factors provides a
signaling pathway linking activated receptor-tyrosine kinase complexes
to the nuclear apparatus for transcriptional regulation. Stat proteins
were first identified as signaling molecules for the Tyk2/JAK1-coupled
receptor for IFN-(4, 5) . In response to
IFN-
, p91/Stat1
, p84/Stat1
(an alternatively spliced
form of Stat1
), p113/Stat2, and a 48-kDa non-Stat protein form the
heteromeric IFN-stimulated gene factor-3 complex which binds to an
IFN-stimulated response element in IFN-
induced
genes(6, 7) . Stat1
(henceforth referred to as
Stat1) has been identified as a component of DNA-binding complexes
formed in response to multiple growth and differentiation factors which
signal via JAK kinases (e.g. IFN-
, IL-6, LIF, oncostatin
M) (8, 9, 10, 11, 12, 13, 14) .
Stat3/APRF (15, 16, 17, 18, 19) was
originally identified as a regulator of transcription of acute phase
response genes activated by IL-6 (20) and Stat5/mammary gland
factor is a transcription factor for prolactin (21) . Stat4 is
a newly cloned protein whose function is not yet known (17) .
Based primarily upon studies of Stats1-3, the following model of Stat protein signaling has been proposed (reviewed in (22) ). Unstimulated Stat proteins are monomeric cytoplasmic proteins. When cells are stimulated with ligand, Stat proteins are thought to associate with phosphorylated tyrosyl residues of an activated receptor-tyrosine kinase complex through Stat Src homology (SH)-2 domains and to become tyrosyl phosphorylated. The tyrosyl phosphorylated Stat proteins form homo- and heteromeric complexes that translocate to the nucleus, where they bind specific DNA sequences and presumably regulate the transcription of appropriate target genes.
Recent findings suggest that Stat family members play a role in the
regulation of genes whose transcription is modulated by GH, providing
the first insight into how GH might regulate gene transcription. GH has
been found to promote the tyrosyl phosphorylation of Stat1 in 3T3-F442A
preadipocytes and rat liver. In these systems, Stat1 was also present
in a GH-inducible factor (GHIF) complex that
binds the high affinity sis-inducible element (SIE) of the
c-fos promoter(23, 24) . In IM-9 cells, a
93-kDa tyrosyl phosphorylated protein antigenically related to, but
distinct from, Stat1 was present in GH-induced DNA-binding complexes
capable of binding to a response region from IFN-
induced
genes (25) . Its identity is not yet known.
Additional insight into how GH might regulate gene transcription is provided by the evidence presented here that GH activates Stat3/APRF (or an antigenically related protein) in addition to Stat1. The finding that GH stimulates tyrosyl phosphorylation of Stat3 and Stat3 binding to DNA suggests that Stat3 serves as a signaling molecule for GH that links GH receptor binding and subsequent JAK kinase activation at the membrane to regulation of gene transcription in the nucleus. Shared transcription factors among hormones and cytokines that activate JAK kinases provide an explanation for shared responses, while the ability of the different ligands to differentially recruit various Stat family members suggests mechanisms by which specificity in gene regulation could be achieved.
In our previous investigation of GH-dependent tyrosyl
phosphorylation of Stat1 in 3T3-F442A cells, we observed
co-immunoprecipitation of Stat1 and a second, more rapidly migrating,
tyrosyl phosphorylated protein (p85)(23) . The appearance of
this 85-kDa phosphoprotein was GH-dependent. Because p85 was not
recognized by Stat1 in Western blots (Fig. 1, lanes
G-L, and (23) ), we speculated that p85 was associated
with Stat1. However, experiments using serial dilutions of
Stat1 (Fig. 1) revealed that the amount of p85 immunoprecipitated by
Stat1 from GH-treated cells diminished throughout the dilution
series, while the amount of tyrosyl phosphorylated Stat1 remained
constant. This argues against the presence of p85 in the
Stat1
immunoprecipitates because of its association with Stat1. Rather, it
suggests that p85 is antigenically related to Stat1.
Figure 1:
Serial dilution of Stat1
diminishes precipitation of p85 but not Stat1. Whole cell lysates of
control (lanes A, C, E, G, I, and K) or GH-stimulated
(500 ng/ml, 15 min, 37 °C) (lanes B, D, F, H, J, and L) 3T3-F442A fibroblasts were immunoprecipitated with the
indicated dilutions of
Stat1 and immunoblotted with
PY (lanes A-F). The blot was re-probed with
Stat1 (lanes
G-L).
p85 migrates in
SDS-polyacrylamide gels at a position similar to that reported for the
recently identified Stat3(15, 16, 18) . To
determine if p85 might be Stat3, whole cell lysates were prepared from
3T3-F442A fibroblasts incubated with or without GH for 15 min. Proteins
were immunoprecipitated with Stat3 and analyzed by
PY
immunoblot. GH strongly promotes tyrosyl phosphorylation of proteins
immunoprecipitable by
Stat3 (Fig. 2, lane D). The
response to GH was compared to that of LIF (a cytokine reported to
promote tyrosyl phosphorylation of Stat3, (18) ) and IFN-
(a cytokine reported not to affect the phosphorylation of Stat3, (16) and (18) ). As expected, LIF promotes tyrosyl
phosphorylation of a protein immunoprecipitable by
Stat3 (Fig. 2, lane F) which co-migrates with the GH-induced
phosphoprotein. Surprisingly, IFN-
also promotes tyrosyl
phosphorylation of a protein recognized by
Stat3 that co-migrates
with the GH- and LIF-induced 85-kDa phosphoprotein, although IFN-
is significantly less effective than GH or LIF. As reported previously,
immunoprecipitated Stat3 appears on
PY immunoblots as a doublet
(p89/p87, (15) ; p89/p92, (16) ). The doublet is
clearly evident in the immunoprecipitates from IFN-
stimulated
cells (lane H); it is also seen in the immunoprecipitates of
LIF and GH stimulated cells with shorter exposures of the immunoblot.
Stat3 immunoprecipitates from LIF-stimulated cells contain a
tyrosyl phosphorylated protein that migrates at a position appropriate
for LIF receptor component gp130 (lane F, diffuse band most
proximate to Stat3). This is consistent with the reported ability of
Stat3/APRF to associate with gp130(13) .
Figure 2:
GH promotes tyrosyl phosphorylation of
Stat3. 3T3-F442A fibroblasts were incubated for 15 min at 37 °C
with vehicle (lanes A, B, and I), GH (500 ng/ml, lanes C, D, and J), LIF (65 ng/ml, lanes E, F, and K), or IFN- (10 ng/ml, lanes G, H, and L). Lysates were prepared and proteins immunoprecipitated with
Stat1 (lanes A, C, E, and G) or
Stat3 (lanes B, D, F, and H-L) and immunoblotted with
PY. Lanes A-H, cells were lysed using a nondenaturing
buffer containing 0.1% Triton X-100 as described(23) . Lanes I-L, cells were lysed by boiling in 50 mM Tris,
137 mM NaCl (pH 7.5) containing 0.75% SDS, 100 mM dithiothreitol, 100 µg/ml leupeptin, 100 µg/ml aprotinin.
The lysates were diluted 10-fold with lysis buffer (23) prior
to immunoprecipitation.
Cell lysates were
also subjected to immunoprecipitation with Stat1 (Fig. 2, lanes A, C, E, and G). 91- and 85-kDa tyrosyl
phosphorylated proteins are present in the
Stat1
immunoprecipitates prepared from GH, IFN-
, and LIF-treated cells.
Consistent with p85 in the
Stat1 precipitate being Stat3, the
amount of tyrosyl phosphorylated 85-kDa protein immunoprecipitated by
Stat1 and by
Stat3 showed the same relationship for GH versus LIF versus IFN-
-treated cells, and p85
co-migrated with the phosphoprotein immunoprecipitated with
Stat3.
Re-probing the blot in Fig. 2with
Stat3 revealed the
co-migration of Stat3 with the 85-kDa tyrosyl phosphorylated proteins
immunoprecipitated by either
Stat1 or
Stat3 (data not shown).
The latter blot also revealed that ligand treatment affects the level
of tyrosyl phosphorylation of Stat3 and not the amount of Stat3.
To
confirm that the tyrosyl phosphorylated protein immunoprecipitated by
Stat3 was precipitated through direct binding to the antibody
rather than through association with another protein recognized by the
antibody,
Stat3 immunoprecipitations were performed using cell
lysates boiled in the presence of SDS and dithiothreitol to dissociate
protein complexes (Fig. 2, lanes I-L). A tyrosyl
phosphorylated protein co-migrating with
Stat3 is evident in the
lysates from both GH- and LIF-stimulated cells. A longer exposure of
the autoradiogram also revealed the presence of two bands co-migrating
with the Stat3 doublet when cells were treated with IFN
(data not
shown). These proteins co-migrated with proteins recognized by antibody
when the Western blot was reprobed with
Stat3. The amount of Stat3
precipitated under these conditions was less than the amount
precipitated under nondenaturing conditions, presumably because of the
reduced ability of
Stat3 to immunoprecipitate SDS-denatured Stat3
(data not shown). The identity of the band migrating just above Stat3
in Fig. 2, lanes I-L, is unknown. It is believed not to
be Stat1 because in contrast to Stat1, it appears in
PY Western
blots in the absence of GH.
Stat3 containing DNA-binding complexes
have been shown to interact with the c-fos SIE(16) .
Our previous data indicate that GH promotes the formation of
SIE-binding complexes containing multiple components(23) . It
was therefore logical to ask if Stat3 might be part of GH-inducible
SIE-binding complexes. When nuclear extracts from control and
GH-stimulated 3T3-F442A fibroblasts are incubated with a high affinity
SIE probe in an electrophoretic mobility shift assay, GH promotes the
formation of SIE-binding complexes containing three bands (Fig. 3A, lane B), a complex designated GHIF.
Preincubation of the nuclear extracts with Stat3 (Fig. 3A, lane D) results in a ``supershift''
of the complex such that the most slowly migrating band (A),
and to a lesser extent the middle band (B), is diminished. The
most rapidly migrating band (C) is not detectably altered by
the antibody. These data suggest that bands A and B contain Stat3.
Figure 3:
The GH-inducible SIE binding complex
contains Stat3. A, 3T3-F442A fibroblasts were incubated for 30
min with vehicle (lane A), GH (500 ng/ml, lanes
B-D), LIF (65 ng/ml, lanes E-G), or IFN-
(10 ng/ml, lanes H-J). Nuclear extracts from cells were
preincubated for 20 min at room temperature with
Stat1 (1:1000
dilution) (lanes C, F, and I) or
Stat3 (1:100
dilution) (lanes D, G, and J). Electrophoretic
mobility shift assays were performed using a high affinity SIE probe
(m67). B, 3T3-F442A fibroblasts were incubated with vehicle (lane A) or GH (500 ng/ml, lanes B-E). Nuclear
extracts were incubated with nonimmune rabbit serum (1:40 dilution, lane C), a 100-fold excess of unlabeled high affinity SIE
probe (lane D) or a 100-fold excess of unlabeled wild-type SIE
probe (lane E).
Preincubation of the nuclear extracts with Stat1 (used at a
dilution that preferentially interacts with Stat1, see Fig. 1)
diminishes all three bands. In contrast to results with
Stat3,
Stat1 preferentially reduce bands B and C (Fig. 3A,
lane C). The partial reduction of band A is thought to reflect
cross-reactivity of
Stat1 with Stat3 since a similar complex was
not observed using a different Stat1 antibody (data not shown). LIF and
IFN-
also induce SIE-binding complexes which migrate as three
bands similar to those of the GH-induced complexes (Fig. 3A,
lanes E and H, respectively), (
)although the
relative amounts of the three bands differ for each ligand. Whereas GH
favors the formation of band B, LIF favors bands A and B, and IFN-
favors band C, suggesting that IFN-
, GH, and LIF do not induce
identical SIE-binding complexes. As with the extracts from GH-treated
cells, preincubation of the extracts from either LIF- or
IFN-
-treated cells with
Stat3 results in a supershift such
that bands A and B are reduced, while preincubation with
Stat1
leads to a shift of the complex with reduction primarily of bands B and
C.
The DNA-binding complexes induced by GH are unaffected by
preincubation with non-immune serum (Fig. 3B, lane C),
indicating that the abilities of Stat1 and
Stat3 to affect
the mobility of the SIE complex are specific. GHIF binding is blocked
by the addition of unlabeled oligonucleotide (m67) based on the high
affinity SIE probe (Fig. 3B, lane D), indicating
specificity of binding. The GHIF complex also recognizes the wild-type
SIE sequence of the human c-fos gene since unlabeled
oligonucleotide based on the wild-type SIE reduces GHIF binding (Fig. 3B, lane E). Further evidence that GH induces the
binding of Stat1 and Stat3 to the wild-type SIE is provided by the
finding that GH promotes the formation of complexes bound to wild-type
SIE which contains three bands (Fig. 4, A and B,
lane B) that co-migrate in electrophoretic mobility shift assays
with the three GH-dependent bands formed using the high affinity SIE
probe. The DNA-binding complexes formed using the wild-type SIE probe
are present at a substantially reduced level compared to those formed
using the high affinity m67 SIE probe (data not shown), as expected
from its lower affinity for the sis-inducible factor. GHIF
binding to the wild-type SIE probe is blocked by the addition of either
unlabeled oligonucleotide based on the wild-type SIE sequence (Fig. 4A, lane D) or the high affinity SIE sequence
(m67) (Fig. 4A, lane C), indicating specificity of
binding. Consistent with the presence of Stat3 in the GHIF complex that
binds the wild-type SIE probe, incubation of the nuclear extracts from
GH-treated cells with
Stat3 (Fig. 4B, lane D)
prior to addition of the wild-type SIE probe results in a supershift of
the upper two bands. Consistent with the presence of Stat1 in the GHIF
complex that binds the wild-type SIE probe, incubation of the nuclear
extracts from GH-treated cells with
Stat1 (Fig. 4B,
lane C) prior to addition of the wild-type SIE probe
preferentially supershifted the lower two bands. The three bands
present in wild-type SIE-binding complexes induced by GH were also
present when the cells were incubated with LIF (Fig. 4B,
lane E). In contrast, only the two lower bands were visible when
the cells were incubated with IFN
(Fig. 4B, lane
H). The relative ratios of the different bands for each ligand
were the same as those observed using the m67 high affinity SIE probe (Fig. 3A). As expected, incubation of the nuclear
extracts from LIF and IFN
-treated cells with
Stat1 prior to
addition of the wild-type SIE probe preferentially decreased the
intensity of the lower two bands (Fig. 4B, lanes F and I) while incubation of the nuclear extract from LIF-treated
cells with
Stat3 preferentially decreased the intensity of the
upper two bands (Fig. 4B, lane G). Incubation of the
nuclear extract from IFN
-treated cells with
Stat3 had no
detectable effect on the band pattern, consistent with the absence of
band A.
Figure 4:
GH induces Stat3 binding to the wild-type
SIE. A, 3T3-F442A fibroblasts were incubated for 30 min with
vehicle (lane A) or GH (500 ng/ml, lanes B-D).
Nuclear extracts were incubated with a 100-fold excess of unlabeled
high affinity SIE probe (lane C) or wild-type (wt)
SIE probe (lane D). B, 3T3-F442A fibroblasts were
incubated for 30 min with vehicle (lane A), GH (500 ng/ml, lanes B-D), LIF (65 ng/ml, lanes E-G), or
IFN- (10 ng/ml, lanes H-J). Nuclear extracts from
cells were preincubated for 20 min at room temperature with
Stat1
(1:1000 dilution) (lanes C, F, and I) or
Stat3
(1:20 dilution) (lanes D, G, and J). Electrophoretic
mobility shift assays were performed using the wild-type SIE
probe.
Stat3 was originally identified as an IL-6 dependent
transcription factor that binds to an APRE in a variety of promoters of
genes encoding acute phase response proteins. If the protein activated
by GH is authentic Stat3, one would predict that, like IL-6, GH would
promote binding of Stat3-containing nuclear protein complexes to the
APRE. Fig. 5illustrates that GH does promote the binding of
nuclear proteins to an oligonucleotide probe corresponding to the APRE
in the rat -macroglobulin gene. As in the experiment
using the SIE probes, multiple DNA-binding complexes were formed, with
two (designated bands A and B) being readily visible
in Fig. 5(lane B) and three visible in other
experiments not shown. The same bands were present in extracts from
IL-6-treated cells (Fig. 5, lane E). Consistent with
the presence of Stat3 in bands A and B, band A was substantially
reduced and band B was partially reduced in intensity when nuclear
extracts from GH and IL-6-treated cells were pretreated with
Stat3
prior to addition of the oligonucleotide probe. Preincubation of
nuclear extracts with
Stat3 caused the appearance of a faint band
migrating above band A for both GH and IL-6 treated cells, consistent
with
Stat3 altering the mobility of bands A and/or B. This band,
denoted by the arrow, is visible in Fig. 5for IL-6 treated
cells and in the original autoradiograph for the GH-treated cells (Fig. 5, lane C). The fact that band B is not
diminished by
Stat3 to the same extent as band A suggests that
this band may contain factors not present in band A. A smaller band A
signal for the IL-6-treated cells compared to GH-treated cells is
consistent with the decreased ability of IL-6 compared to GH to
stimulate tyrosyl phosphorylation of Stat3 in these cells (data not
shown). Specificity of binding is indicated by the observation that
GHIF binding is blocked by the addition of unlabeled oligonucleotide
based on the
-macroglobulin APRE sequence (Fig. 5, lanes D and G).
Figure 5:
GH
induces Stat3 binding to the rat
-macroglobulin APRE. 3T3-F442A fibroblasts
were incubated for 30 min with vehicle (lane A), GH (500
ng/ml, lanes B-D), or IL-6 (25 ng/ml, lanes E-G).
Nuclear extracts from cells were preincubated for 20 min at room
temperature with
Stat3 (1:100 dilution) (lanes C and F) or a 100-fold excess of unlabeled APRE probe (lanes D and G). Electrophoretic mobility shift assays were
performed using the rat
-macroglobulin
probe.
As currently understood, activation of Stat proteins by
ligands requires tyrosyl phosphorylation and results in increased
binding to specific DNA sequences. Recognition that both Stat1 (23, 24) and Stat3 (this paper) (or antigenically
regulated proteins) are thus activated by GH suggests two interacting
pathways linking the GH receptor and the nucleus, since Stat proteins
have been shown to bind both membrane receptors and specific DNA
sequences and to function as transcription factors (reviewed in (22) ). Stats 1 and 3 are the first identifiable transcription
factors shown to be regulated by GH. Like GH, other hormones and
cytokines (e.g. IFN, IFN
, prolactin, LIF, oncostatin
M, IL-6, ciliary neurotrophic factor, and epidermal growth factor) have
recently been shown to utilize JAK tyrosine kinases and Stat proteins
in their signaling
pathways(8, 11, 12, 13, 14, 27, 29) ,
a finding that provides an explanation for why many of these
cytokines/hormones elicit similar responses. However, it also raises
the question of how different ligands elicit specific responses. One
source of specificity is the cell-specific distribution of the
receptors. Specificity could also arise from cell-specific expression
of various Stat proteins which would offer a potential explanation for
why GH stimulates Stat1 and 3 in 3T3-F442A fibroblasts ((23) ,
this paper) but apparently not in IM-9 lymphocytes(25) . The
finding that IFN-
appears to activate Stat3 in 3T3-F442A
fibroblasts (this paper) and NIH3T3 cells (19) but not in HepG2 (16) or IM-9 cells (25) is also consistent with cell
specific expression of either Stat3 or an accessory protein.
Alternatively, assay sensitivity may have been greater in the
fibroblast studies, or the protein recognized by
Stat3 in the
3T3-F442A and/or NIH3T3 cells may be antigenically related to Stat3
rather than authentic Stat3.
Cell specificity of receptor and/or
Stat expression cannot account entirely for the specificity of cytokine
responses. Although GH, IFN-, and LIF all activate JAK2 tyrosine
kinase in 3T3-F442A fibroblasts, they clearly differ in their ability
to activate Stat1 and 3, as judged by both extent of tyrosyl
phosphorylation and relative amounts of the three bands in the high
affinity SIE-binding complexes. Among the three ligands, GH is by far
the most potent in activating JAK2 in 3T3-F442A cells, while LIF and
IFN-
are significantly less potent. (
)In contrast, LIF
is the most potent activator of Stat3, while IFN-
is the least
potent. The lack of correlation between the activation of JAK2 and
Stat3 by GH, LIF, and IFN-
indicates that JAK2 activation is
unlikely to be sufficient for ligand-dependent stimulation of Stat3 in
3T3-F442A fibroblasts, even though co-expression of JAK2 and Stat3 in
COS cells appears to be sufficient for binding of Stat3 to the
c-fos SIE(19) . JAK2 could be the activating Stat3
kinase for GH, LIF and IFN-
if Stat3 activation was facilitated by
binding of Stat3 to phosphorylated tyrosines in the different
receptors. Phosphorylated tyrosines in the receptors for GH, LIF and
IFN-
are likely to have different affinities for the SH2 domain of
Stat3. In support of this idea, a tyrosyl phosphorylated protein
corresponding in size to LIF receptor component gp130 is precipitated
by Stat3 antibodies (this paper and (13) ) and Stat1 has been
shown to bind to the epidermal growth factor receptor by a mechanism
involving the SH2 domain of Stat1(30) . However, we do not
detect tyrosyl phosphorylated bands corresponding in size to the GH
receptor or to JAK2 in the
Stat3 immunoprecipitates from
GH-treated cells, suggesting a lower affinity of Stat3 for these
proteins compared to LIF receptor components, fewer receptors for GH
compared to LIF, or fewer Stat3 binding sites per receptor.
Alternatively, a protein other than or in addition to JAK2 may be
required for activation of Stat3 by GH.
Signalling specificity in
response to various cytokines/hormones that activate JAK kinases and
Stat proteins is most likely related to, at least in part, the ability
of Stat proteins to form homo and
heterodimers(4, 31) . Different combinations of Stat
proteins could provide specificity for these different ligands, as has
been shown for other transcription factors(32) . Supporting
this is the finding that, relative to each other, the intensities of
the three ligand-induced bands observed in the SIE electrophoretic
mobility shift assay were different for LIF, IFN-, and GH-treated
cells. These three ligand-induced bands are hypothesized by us and
others (33, 16) to contain Stat3 homodimers (band A),
Stat1/Stat3 heterodimers (band B), and Stat1 homodimers (band C).
However, it should be noted that in contrast to what one might expect
if Stat1 and Stat3 formed heterodimers, little or no Stat1 was
precipitated by
Stat3 (Fig. 2) and at a concentration of
Stat3 that preferentially precipitates Stat1, only a small amount
of Stat3 was detected in the
Stat1 precipitate (Fig. 1, lane B). Preliminary evidence indicates that this inability to
detect Stat1/Stat3 heterodimers by immunoprecipitation may be due at
least in part to dissociation of Stat complexes during the
solubilization and subsequent immunoprecipitation steps. An inability
to detect Stat1/Stat3 heterodimers, assuming they exist, could also be
explained if the ratio of heterodimers to homodimers and monomers is
low or if the Stat antibodies immunoprecipitate Stat monomers and/or
homodimers more effectively than heterodimers.
Stat3 was originally
identified as a protein that binds to an IL-6 response element in the
promoter of several IL-6 inducible acute phase response genes in
liver(15, 20) . This element can confer IL-6
responsiveness to a heterologous promoter(34) . Thus, Stat3 is
thought to play a role in the regulation of these genes by IL-6. The
finding here that GH can also induce binding of Stat3 containing
complexes to APRE raises the possibility that GH might also regulate
the expression of acute phase response genes in liver. Similarly, it
seems likely that Stat3 plays a role in the GH-dependent regulation of
c-fos, since Stat3 binds to the SIE of the c-fos gene
(this paper and (16) and (19) ) and since GH activates
c-fos transcription(26) . The SIE has been shown to
mediate induction of c-fos transcription in response to
platelet-derived growth factor (NIH3T3 fibroblasts, (28) ) and
epidermal growth factor (COS cells overexpressing Stat1, (30) ), two growth factors shown recently to activate Stats 1
and 3(16, 19, 33, 35) . Consistent
with the SIE and the proteins that bind it playing a critical role in
the regulation of c-fos gene expression, Curran and colleagues ()have observed that mutation of the SIE obliterates normal
expression of Fos in transgenic mice and Fos inducibility in
fibroblasts derived from them. Stimulation of Stat3 DNA binding to the
SIE alone would not necessarily be expected to be sufficient for full
induction of c-fos, since the serum response element of the
c-fos promoter can mediate induction of c-fos by
GH(36) . Whether proteins that bind to the SIE regulate
c-fos expression independently or must act in concert with
factors that bind to other response elements (e.g. serum
response element) remains to be determined. It seems likely, however,
that Stat1 and -3 participate in the regulation of genes other than
c-fos. Thus, the finding that multiple Stat proteins are
activated by GH not only provides insight into the molecular mechanism
by which GH and other members of the cytokine receptor family are
likely to regulate gene expression, but also suggests strategies to
identify new target genes for GH and additional GH-dependent
transcription factors.