(Received for publication, March 7, 1995; and in revised form, May 31, 1995)
From the
The activation of a latent DNA binding factor by interleukin-4
(IL-4), the IL-4 nuclear activated factor (IL-4 NAF), occurs within
minutes of IL-4 binding to its receptor. Molecular characterization of
IL-4 NAF by ultraviolet light cross-linking experiments revealed a
single protein of 120-130 kDa in contact with the DNA target
site. Glycerol gradient sedimentation analysis indicated a molecular
mass of IL-4 NAF consistent with a monomer that is capable of binding
DNA. The IL-4 NAF target site is a palindromic sequence that is also
recognized by the interferon-induced transcription factor,
p91/STAT1. However, IL-4 NAF and p91/STAT1
display
distinguishable DNA binding specificities that may generate one level
of specificity in the expression of target genes. Previous studies
suggested the involvement of the insulin receptor substrate-1 (IRS-1)
in the IL-4 signal transduction pathway. Although IRS-1 is involved in
the stimulation of mitogenesis, our results demonstrate that activation
of IL-4 NAF is independent of IRS-signaling proteins. The results of
this study indicate that IL-4 stimulates bifurcating signal pathways
that can direct mitogenesis via the IRS-signaling proteins and specific
gene expression via the IL-4 NAF.
Cells respond to extracellular stimuli with distinct biological
changes that can dictate proliferation, differentiation, or death.
Within minutes of extracellular stimulation, signals are transmitted
from the cell surface to the nucleus culminating in an alteration of
specific gene expression. Interleukin-4 (IL-4) ()is a
polypeptide cytokine that initially was identified by its potent effect
on B lymphocyte proliferation ((1) , and for review, see (2) ). It is now known that the physiological effects of IL-4
are not restricted to B cells but in fact are elicited in a variety of
cell types. Activated T lymphocytes and mast cells produce and secrete
IL-4, which can function in an autocrine manner or can stimulate other
cells of both hematopoietic and nonhematopoietic lineages.
The
actions of IL-4 appear to be dependent on the stimulation of specific
gene expression. IL-4 induces the transcription of genes involved in
immune recognition, including the immunoglobulin E receptor
(FcRII) (3, 4) and the major histocompatibility
complex class II genes (for review, see (5) and (6) ).
Furthermore, IL-4 stimulates the transcriptional activation of
immunoglobulin constant region genes IgE and IgG
and
thereby promotes Ig class switching (for review, see (7) ).
Some of the transcriptional effects of IL-4 are either analogous or
antagonistic in comparison with the effects of another cytokine,
interferon-
(IFN-
). We have found that IL-4 may exert these
effects by activating a DNA binding factor that recognizes the same DNA
sequence that is recognized by an IFN-
-induced factor (8) .
Recent studies in the IFN- system have identified
a receptor to nucleus signal pathway that involves the activation of a
latent DNA binding factor composed of a 91-kDa protein (for review, see
Refs. 9 and 10). This protein is a member of the newly emerging family
of latent cytoplasmic transcription factors that are activated by
tyrosine phosphorylation and have been termed signal transducers and
activators of transcription (STAT)(10) . The 91-kDa protein
that is tyrosine phosphorylated in response to IFN-
has been
designated STAT1
. Following phosphorylation of STAT1
, it
translocates to the nucleus and binds as a dimer to the
IFN-
-activated site (GAS), a palindromic DNA sequence in the
promoter of genes transcriptionally responsive to
IFN-
(11) .
A GAS-binding factor distinct from
STAT1 is activated by tyrosine phosphorylation following
stimulation with IL-4 that we termed the IL-4 nuclear activated factor
(IL-4 NAF)(8, 12, 13) . Recently, a gene
encoding an IL-4-induced STAT (STAT6) has been cloned that may serve as
the component of IL-4 NAF activity(14) . It remains to be
determined how transcription factors such as STAT1
and STAT6 bind
to the same DNA target site but elicit differential gene transcription.
In this report, we characterize the nature of the DNA binding component
of IL-4 NAF and in addition compare the GAS-binding specificities of
IL-4 NAF with the IFN-
-activated STAT1
.
Previous studies
have indicated the involvement of a common signaling molecule in the
response to either IL-4 or insulin (for review, see (15) ). A
prominent tyrosine phosphorylated protein originally called the IL-4
phosphorylated substrate (4PS) appears following IL-4 stimulation of
hematopoietic cells. Recently the gene encoding this protein has been
cloned and found to be related to the insulin receptor substrate-1
(IRS-1); it has been designated as IRS-2. ()The
IRS-signaling proteins contain multiple sites of tyrosine
phosphorylation that can act as binding sites for signaling molecules
that have Src homology 2 domains such as the growth factor
receptor-bound protein 2 (Grb2) and phosphatidylinositol 3-kinase (for
review, see (16) ). In this manner, the IRS-signaling proteins
act to amplify the signals initiated at the receptor. The mitogenic
response of cells to insulin or IL-4 appears to require IRS
molecules(17) . A cell line that lacks IRS-signaling proteins
and does not respond mitogenically to IL-4 can be reverted to a
responsive phenotype by complementation with the IRS-1 gene. In this
study we test the involvement of IRS-1 in the activation of IL-4 NAF
and the stimulation of DNA synthesis by IL-4.
Figure 1:
Characterization of IL-4 NAF in HeLa
cells. A, gel mobility shift analysis of IL-4-treated HeLa
cells. Nuclear extracts were prepared from HeLa cells either untreated (lane1) or treated for 15 min with IL-4 (20 ng/ml) (lanes2 and 3). Gel shift analysis was
performed as described previously with an end-labeled double-stranded
oligonucleotide representing the GAS site from the FcRI gene.
Migration of IL-4 NAF (lane2) is depicted by an arrow. To demonstrate binding specificity a 50-fold molar
excess of unlabeled oligonucleotide was included in the binding
reaction (lane3). B, photoaffinity
cross-linking of IL-4 NAF. Proteins chromatographed on P-11
phosphocellulose were fractionated as material that bound IL-4 NAF and
eluted at 300 mM KCl (B) or as unbound material that
eluted at 400 mM KCl (U). A preparative gel shift
assay was performed with a bromodeoxyuridine-substituted Fc
RI GAS
probe. The gel was exposed to UV light and autoradiographed. Gel slices
were excised and analyzed on a 9% SDS-polyacrylamide gel. The position
of protein standards is shown (in kDa), and migration of the IL-4 NAF
DNA binding component cross-linked to DNA is depicted by an arrow. C, glycerol gradient sedimentation analysis.
Partially purified IL-4 NAF or molecular weight standards were layered
onto 25-50% glycerol gradients for sedimentation analysis. Every
other collected fraction from the IL-4 NAF gradient was analyzed by a
gel shift assay. A sample of IL-4 NAF activity prior to sedimentation
is shown as a control (S). The peak migration of protein
standards, as determined by Coomassie Blue staining, is shown above the lanes. A competition analysis with a 50-fold molar
excess of unlabeled GAS oligonucleotide is shown with fraction 15 and 25 in the rightmostlanes to
demonstrate binding specificity.
The molecular composition of IL-4
NAF is distinct from the IFN--induced STAT1
factor that binds
to the GAS site. We previously demonstrated that STAT1
is not a
component of the IL-4 NAF complex(8) . To further characterize
this complex, IL-4 NAF was partially purified from HeLa cells by
standard chromatography methods. Photoaffinity cross-linking was
performed with fractions from a P-11 phosphocellulose column to
identify the molecular weight of the IL-4 NAF DNA binding component (Fig. 1B). A bromodeoxyuridine-substituted probe was
used in a preparative gel shift analysis with a partially purified IL-4
NAF fraction that eluted at 300 mM KCl or a control fraction
that eluted at 400 mM KCl that did not contain IL-4 NAF.
Proteins contacting the Fc
RI GAS oligonucleotide were cross-linked
to the DNA by exposing the gel to UV light. After autoradiography, gel
slices representing the IL-4 NAF complex or an adjacent control lane
corresponding to a DNA binding reaction with a chromatography fraction
that did not contain IL-4 NAF were excised and analyzed by a
SDS-polyacrylamide gel. The protein contacting the GAS is visible by
virtue of its covalent attachment to the radiolabeled DNA. The control
gel slice did not reveal any protein cross-linked to the DNA (U). The IL-4 NAF complex contained a protein that
specifically cross-linked to the DNA (B). Relative mobility of
the DNA-protein complex predicts a molecular size of the IL-4 NAF DNA
binding component to be 120-130 kDa, taking into account the
molecular weight of the cross-linked oligonucleotide. These results
suggest that the DNA binding component of IL-4 NAF is a single protein
subunit.
It is possible that IL-4 NAF is a multimeric protein complex and the UV cross-linked protein is the subunit associated with DNA. To determine the native molecular weight of IL-4 NAF, glycerol gradient sedimentation analysis was performed (Fig. 1C). Partially purified IL-4 NAF from a SP Sepharose column or molecular weight standards were centrifuged through 25-50% glycerol gradients. The sedimentation coefficients of protein standards with known molecular mass were estimated by their position in the gradient and detected by SDS-polyacrylamide electrophoresis followed by staining with Coomassie Blue. IL-4 NAF activity was tested by a gel shift assay and found predominantly between fractions 15 and 17. This peak fraction represents a protein with a relative size of 140-150 kDa. A slower migrating protein-DNA complex in fraction 25 was not specific since it did not compete for binding with unlabeled specific oligonucleotide (rightmostlane). The mass of the IL-4 NAF complex predicted by this sedimentation analysis is similar to the size of the DNA binding component identified by UV cross-linking. It appears that IL-4 NAF predominantly exists as a monomer under these experimental conditions. However, it is possible that IL-4 NAF exists as a dimer and dissociates during glycerol gradient sedimentation.
Figure 2:
Analysis of IL-4 NAF DNA binding
specificity. A, single-stranded sequences representing the
FcRI GAS site (-33 to -14) with two, three, four, or
five nucleotides (underlined) between the GAA palindrome (arrows) are shown. B, nuclear extracts from HeLa
cells untreated (lane1) or IL-4-treated (20 ng/ml)
for 15 min (lanes2-10) were used in a
competitive gel shift analysis. A 100- or 200-fold molar excess of
unlabeled double-stranded oligonucleotide was included in the binding
reaction before incubation with the native Fc
RI radiolabeled GAS
probe. The spacer designations refer to nucleotide number between the
inverted repeat (shown in Fig. 2A). Migration of the
IL-4 NAF complex is shown by an arrow. C, nuclear
extracts from untreated (lane1) or 15 min IFN-
treated (1000 units/ml) HeLa cells (lanes2-10)
were analyzed in a competitive gel shift assay with a 100- or 200-fold
molar excess of the double-stranded oligonucleotides described in A.
To
determine if the DNA binding specificity of STAT1 was distinct
from IL-4 NAF, nuclear extracts from IFN-
-treated cells were
subjected to a similar analysis (Fig. 2C). We found
that STAT1
bound most efficiently to the DNA with three
nucleotides between the inverted repeat (lanes5 and 6) and with less efficiency to the DNA with two nucleotides
between the repeats (lanes3 and 4). The DNA
sequence with four or five nucleotides between the repeats did not
compete efficiently (lanes7-10). Therefore,
the DNA binding specificities of STAT1
and IL-4 NAF are distinct.
This finding would predict that IFN-
-induced STAT1
and
IL-4-induced IL-4 NAF can recognize both overlapping and distinct
subsets of genes.
Figure 3:
Activation of IL-4 NAF is independent of
IRS-signaling proteins. Murine myeloid cell lines were untreated
(-), treated with IL-3, or treated with IL-4 (200 ng/ml) for 15
min. Nuclear extracts were prepared from 32D cells (lanes1-3), 32D+IRS-1 cells (lanes4-6), 32D+INS-R (lanes7-9), or 32D+INS-R/IRS-1 (lanes10-12) and analyzed by a gel shift assay with a
FcRI GAS probe. The migration of protein-DNA complexes is depicted
by arrows.
Since the 32D myeloid cells that lack IRS-signaling
proteins responded to IL-4 with the activation of IL-4 NAF, the ability
of IL-4 to signal mitogenesis in these cells was analyzed.
IRS-deficient cells (32D), IRS-1-expressing cells (32D+IRS-1), and
a positive control cell line containing 4PS/IRS-2 (FDC-P1)
were untreated or treated with IL-4, and DNA synthesis was measured by
[
H]thymidine incorporation (Fig. 4A). In FDC-P1 cells, IL-4 stimulated DNA
synthesis by increasing [
H]thymidine
incorporation 20-fold over unstimulated cells. 32D cells did not
undergo a substantial increase in DNA synthesis, even though IL-4 NAF
was activated. IRS-1-expressing 32D cells stimulated DNA synthesis as
efficiently as FDC-P1 cells. Therefore in the absence of IRS-signaling
proteins, IL-4 NAF activation alone is not sufficient to signal
mitogenesis.
Figure 4:
A, IL-4-induced DNA synthesis.
FDC-P1, 32D, and 32D+IRS-1 cells were starved for 6 h before
treatment with IL-4 (100 ng/ml) or media for 16 h. DNA synthesis was
measured by incubation of the cells with
[H]thymidine for 5 h.
[
H]Thymidine incorporation was calculated as a
-fold induction of cpm in IL-4-stimulated cells over unstimulated cells
and is depicted as a bar graph for each cell line. B, activation of MAPK. 32D+INS-R/IRS-1 cells were stimulated
with either IL-3 (lanes1 and 6), IL-4 (200
ng/ml) (lanes2 and 7), insulin (250 ng/ml) (lanes3 and 8), or unconditioned media (lanes4 and 5) for either 10 min (lanes1-4) or 2 h (lanes5-8). 25
µg of protein lysate were electrophoresed and immunoblotted with
antibody that recognizes MAPK. The mobilities of p44 MAPK and p42 MAPK
are indicated by arrows. C, in vitro MAPK
assay. 32D+INS-R/IRS-1 cells were stimulated as in B for
15 min, lysed, and immunoprecipitated with antibody to MAPK. MAPK
immunoprecipitates were used in an in vitro kinase assay with
MBP as substrate. Phosphorylated MBP is shown by an arrow.
Many signaling pathways that control cellular proliferation or differentiation initiate a cascade of events that activate the MAPKs (also known as extracellular signal regulated kinases) (for review, see (26) and (27) ). For this reason, we analyzed the activation of two well characterized MAPKs of 42 (p42) and 44 kDa (p44) following IL-4 stimulation (Fig. 4B). Latent MAPK is unphosphorylated, and activation requires tyrosine and threonine phosphorylation by a dual specificity kinase. The phosphorylated MAPK migrates more slowly than unphosphorylated MAPK during electrophoresis in SDS-polyacrylamide gels and can be visualized by immunoblot assays(28) . This assay was employed to detect activated MAPK following treatment of 32D+INS-R/IRS-1 cells with IL-3, IL-4, or insulin for either 10 min or 2 h. IL-3 stimulation resulted in a sustained activation of MAPK as phosphorylated levels of MAPK were still high at 2 h (lanes1 and 6). Insulin activated MAPK but produced only a short-lived response (lane3versus8). This assay did not detect IL-4 activation of MAPK. An alternative method to evaluate MAPK activation was performed with an in vitro kinase assay using MBP as a substrate molecule (Fig. 4C). Radiolabeled MBP was quantified, and results demonstrated that IL-3 and insulin reproducibly stimulated activation of MAPK, whereas IL-4 did not.
Binding of IL-4 to specific cell surface receptors stimulates the activation of a latent DNA binding factor, IL-4 NAF(8, 12, 13) . Activation of IL-4 NAF occurs in both hematopoietic and nonhematopoietic cell lineages and likely mediates specific effects on gene expression. To characterize this transcription factor, we have used UV cross-linking experiments to determine the molecular size of its DNA binding component. A single protein of molecular size 120-130 kDa was identified by this technique (Fig. 1B). Recently a gene encoding an IL-4-induced GAS-binding factor was cloned from a hematopoietic line(14) . The cDNA of this gene predicts a 94-kDa protein with significant homology to a family of transcription factors known as STATs and has been termed IL-4 STAT or STAT6(10, 14) . It remains to be determined whether IL-4 NAF and STAT6 are identical; however, the activities indicate that they represent the same factor. The reason for the greater molecular mass detected by our biochemical studies could reflect protein modification and awaits further study.
Recent studies indicate that the STAT family members STAT1 and
STAT6 bind to their target DNA sites as
dimers(11, 14) . Since the IL-4 NAF target site has an
inverted repeat sequence (palindrome), the IL-4 NAF may bind the site
as a dimer. To determine if the molecular mass of IL-4 NAF corresponded
to a dimer, a glycerol gradient sedimentation analysis was performed.
The results of this study indicated that the native molecular weight of
IL-4 NAF was similar to that of the DNA binding component, and
suggested that it exists as a monomer in solution (Fig. 1C). It is possible that IL-4 NAF exists as a
dimer in solution, but it dissociates to monomers during glycerol
gradient sedimentation. If this is the case, our results would suggest
that an IL-4 NAF monomer can bind to DNA or that the monomers can
interact to form dimers by association with the DNA target.
Distinct
members of the STAT family of transcription factors are activated by
different cytokines and growth factors, and although the STATs appear
to recognize a similar GAA palindromic sequence, the cytokines elicit
different biological responses and gene expression. To understand the
specificity of gene activation induced by distinct extracellular
ligands, we analyzed the DNA binding specificites of STAT1 and
IL-4 NAF. The GAA inverted repeats found in various promoters differ in
the number of nucleotides that separate the repeat (8) . For
this reason, we tested inverted repeat sequences separated by two,
three, four, or five nucleotides for their ability to be recognized by
STAT1
or IL-4 NAF (Fig. 2). IL-4 NAF was found to bind
preferentially to inverted repeats separated by three or four
nucleotides, whereas STAT1
predominantly binds to an inverted
repeat separated by three nucleotides. Inverted repeats with a spacing
of three or four nucleotides are common in the promoters of the
IL-4-responsive genes Fc
RIIb and C
1(8) . Our finding
that the DNA binding properties of IL-4 NAF and STAT1
are distinct
suggests that nucleotide spacing can generate one level of specificity
in the expression of target genes by each factor. The ability of
transcription factors to discriminate between similar repeat sequences
separated by different distance is best exemplified with retinoid
receptors(29) . In this system, DNA binding specificity is
determined by the number of nucleotides between direct repeat
sequences. Our studies indicate that a similar mechanism is utilized by
the DNA binding factors that recognize the GAA inverted repeat of the
GAS.
The activation of IL-4 NAF requires tyrosine phosphorylation
and occurs within minutes of IL-4 binding to specific cell surface
receptors(8, 12, 13) . The IL-4 receptor
possesses a single transmembrane domain, but it does not possess
intrinsic kinase activity(2, 15) . Recent studies have
demonstrated activation of the Janus kinase (JAK) family of cytoplasmic
protein tyrosine kinases, JAK1 and JAK3, following IL-4
treatment(30, 31) . In addition, dimerization of the
IL-4 receptor with the IL-2 receptor chain is believed to
initiate signal activation (32, 33) . Several
observations have suggested the involvement of the IRS signaling system
in the IL-4 signal pathway. Following IL-4 stimulation, 4PS/IRS-2 is
tyrosine-phosphorylated and associates with the p85 regulatory subunit
of phosphatidylinositol 3-kinase believed to be involved in mediating a
mitogenic response(23, 24) . In addition, murine
myeloid 32D cells that lack IRS signaling proteins are not responsive
mitogenically to IL-4(17) . For these reasons, we sought to
determine if IRS-signaling proteins are required for activation of IL-4
NAF. Our results demonstrate that activation of IL-4 NAF is independent
of IRS molecules (Fig. 3). While the IRS molecules clearly play
a role in the ability of IL-4 to fully induce mitogenesis (Fig. 4A; (17) ), it was not required for
signal activation of the transcription factor IL-4 NAF. Although a
small increase in mitogenesis was induced by IL-4 in the absence of IRS
molecules (
10% of control), activation of IL-4 NAF does not appear
to be sufficient to stimulate proliferation.
Many extracellular stimuli that trigger cellular proliferation activate the MAPK pathway (for review, see (26) and (27) ). In fact, constitutive activation of MAPK kinase, the regulator of MAPK, is sufficient for cellular transformation and tumorigenesis(34, 35) . Conversely, negative interfering mutants of MAPK kinase inhibit proliferation and revert transformed cells(34) . We examined the effects of IL-3, IL-4, and insulin on MAPK in the murine myeloid cells that express IRS-1 and found that IL-4 does not activate MAPK, in accordance with other observations (Fig. 4, B and C)(36, 37) . Insulin and IL-3 were used as positive controls for the activation of MAPK(19, 36, 38) . Insulin activated MAPK for a short (10 min) duration, but IL-3 treatment produced a sustained activation of MAPK (>2 h) (Fig. 4B). IL-3-induced mitogenesis may depend on prolonged activation of MAPK.
The results of this study suggest that bifurcating pathways emanate from a ligand bound IL-4 receptor to produce specific biological responses such as proliferation and differentiation (Fig. 5). IL-4 signals a direct receptor to nucleus pathway that entails the activation of a latent DNA binding factor, IL-4 NAF, which functions to regulate specific gene expression necessary for differentiation. A second signal pathway involves the activation of IRS-signaling proteins and their association with specific targets such as phosphatidylinositol 3-kinase that function to signal mitogenesis. It is possible that signals from both pathways intersect at a common point to produce a mitogenic response. In addition, unidentified pathways may be stimulated following activation of this complex signaling network that contribute to the pluripotent effects of IL-4 on various cell types.
Figure 5: Illustrative model of the IL-4 signal transduction pathway. Bifurcating IL-4 signals emanate from a ligand-bound IL-4 receptor. IL-4 binding activates JAK1 and JAK3 and the subsequent tyrosine phosphorylation of IL-4 NAF/STAT. Activated IL-4 NAF/STAT translocates to the nucleus and binds to a specific target site (GAS) in the promoters of regulated genes. IL-4 also stimulates the tyrosine phosphorylation of IRS proteins and their association with signaling molecules that include phosphatidylinositol 3-kinase and Grb2 (39) that may be involved in mitogenesis.
Note Added in Proof-Subsequent to this study a specific antibody that recognizes STAT6 became available (Santa Cruz Biotechnology). This antibody recognizes the IL-4 NAF complex in a gel mobility assay indicating that STAT6 is a component of IL-4 NAF.