(Received for publication, March 29, 1995; and in revised form, June 12, 1995)
From the
We recently reported that angiotensin II (AII), acting through
the STAT (Signal Transducers and Activators of Transcription) pathway,
stimulated a delayed SIF (sis-inducing factor)-like DNA
binding activity (maximal at 2-3 h) (Bhat, G. J., Thekkumkara, T.
J., Thomas, W. G., Conrad, K. M., and Baker, K. M.(1994) J. Biol.
Chem. 269, 31443-31449). Using a cell line transfected with
the AT receptor (T3CHO/AT
), we further
characterized the AII-induced SIF response and explored the possible
reasons for the delay in stimulated SIF activity. In cells transfected
with a chloramphenicol acetyltransferase reporter plasmid, under the
control of a SIE (sis-inducing element), AII markedly
stimulated chloramphenicol acetyltransferase activity. The delayed SIF
activation by AII was not due to a requirement for the release of other
SIF inducing factors into the medium and contrasts with the rapid (5
min) induction elicited by the cytokine, interleukin-6 (IL-6).
Interestingly, both agents stimulated tyrosine phosphorylation of
Stat92 and predominantly the formation of SIF complex A. We tested the
hypothesis that AII initially activated an inhibitory pathway, which
was responsible for delaying the maximal SIF stimulation until 2 h.
Pretreatment of cells for 15 min with AII resulted in significant
inhibition of the IL-6-induced nuclear SIF response (10 min) and Stat92
tyrosine phosphorylation, which was blocked by EXP3174, an AT
receptor antagonist. This inhibition was transient with return of
the IL-6-induced SIF response at 2 h, suggesting that the delayed
maximal activation of SIF by AII occurs following an initial transient
inhibitory phase. Pretreatment of cells with phorbol 12-myristate
13-acetate for 15 min, to activate protein kinase C, resulted in
inhibition of the IL-6-induced SIF response (10 min). However,
down-regulation of protein kinase C activity prevented phorbol
12-myristate 13-acetate, but not AII mediated inhibition of the
IL-6-induced SIF response. Although the mechanism is not clear, the
results presented in this paper raise the interesting possibility that
the activation of SIF/Stat92 by AII is characterized by an initial
inhibitory phase, followed by the induction process. The observation
that AII and IL-6 utilize similar components of the STAT pathway and
that AII can cross-talk with IL-6 signaling through inhibition of
IL-6-induced SIF/Stat92, implies a modulatory role for AII in cellular
responses to cytokines.
Angiotensin II (AII) ()stimulates a variety of
physiological responses related to the regulation of blood pressure,
salt, and fluid homeostasis(1, 2) . In addition, AII
promotes growth responses in many cells, including cardiomyocytes,
cardiac fibroblasts, and vascular smooth muscle
cells(3, 4, 5, 6, 7, 8) .
Angiotensin II exerts effects through specific G-protein coupled
receptors, predominantly the AT
receptor subtype. AT
receptors couple to intracellular calcium mobilization,
activation of tyrosine kinases such as p125
,
p46
, and p54
, and induction of
serine/threonine kinases, including protein kinase C (PKC) and
mitogen-activated protein kinases (2, 9, 10, 11, 12, 13, 14) .
Angiotensin II may act directly through these signaling pathways or
indirectly via the release of growth factors such as PDGF and
TGF-
, as demonstrated for rat vascular smooth muscle
cells(4, 7) . Like other growth factors, AII induces a
rapid increase of the growth associated nuclear proto-oncogenes
c-myc, c-fos, and c-jun and several cellular
genes including tenascin, fibronectin, and
collagen(4, 15, 16, 17, 18, 19) .
These studies indicate that AII can induce rapid changes in gene
expression and function, that may ultimately lead to increased cell
growth(20) .
Growth factors and cytokines transduce
signaling through a common pathway (STAT pathway) from receptor to the
nucleus(21) . The c-fos regulatory element SIE (sis-inducing element) has been used extensively to study the
activation of STAT pathways by many ligands, including PDGF, epidermal
growth factor, IFN-, and
IL-6(22, 23, 24, 25, 26, 27, 28) .
Binding of the ligand to the receptor activates tyrosine kinases, which
phosphorylate monomeric STAT proteins in the cytoplasm, leading to
dimerization and formation of complexes referred to as SIF (sis-inducing factor)(29) . SIF subsequently
translocates to the nucleus and interacts with SIE, or SIE-like
elements, in the promoter of genes to induce expression. Depending upon
the ligand, SIF appears in three different forms: complex A, B, and
C(22, 24) . PDGF and epidermal growth factor induce
all three complexes; IL-6 induces mainly complex A and IFN-
mainly
complex C.
We have recently shown (30) that AII stimulates
the STAT pathway and induces predominantly SIF complex A in both
neonatal rat cardiac fibroblasts and CHO-K1 cells expressing AT receptors (T3CHO/AT
). Like growth factors,
activation of SIF by AII was post-translational and required the
actions of tyrosine kinases. Angiotensin II-induced SIF complexes
contained tyrosine phosphorylated Stat91, with activation occurring in
the cytoplasm followed by translocation to the nucleus. However, with
respect to the time course of SIF activation, the AII-induced response
significantly differed from that of growth factor/cytokine responses.
Unlike the rapid induction of SIF by cytokines and growth factors
(maximal in less than 30 min), AII-induced activity although detectable
at 30 min, was maximal at 2-3 h(30) . In the present
study, using T3CHO/AT
cells, we explored the possible
reasons for the delayed maximal SIF activation by AII. Our data
indicate that AII directly activates SIF activity and that the delayed
maximal activation of SIF is not due to the secondary release of other
SIF inducing factors into the medium. We also provide evidence that AII
evokes a PKC independent, transient inhibitory effect on the rapid SIF
induction by IL-6. We propose that similar transient inhibitory
mechanisms may be responsible for delaying the maximal SIF activation
by AII (2 h).
Figure 1:
Effect of
different agents on the induction of SIF activity in T3CHO/AT cells. Cells were treated with AII (100 nM) for 2 h (lane 2); PDGF (10 ng/ml) for 30 min (lane 3); IL-6
(10 ng/ml) for 30 min (lane 4); and TGF-
(5 ng/ml) for 30
min (lane 5). Nuclear extracts were prepared and analyzed in
an electrophoretic mobility shift assay using
P-labeled
SIE. The positions of SIF complexes A, B, and C are indicated. These
data are representative of three separate experiments. Ctl,
untreated control (lane 1).
Figure 2:
Angiotensin II directly induces SIF
activity through the AT receptor. Cells were treated for 2
h with AII (100 nM, lane 2), conditioned medium was
removed and added to quiescent cells. The incubation was continued for
30 min or 2 h in the presence (lanes 5 and 7) or
absence (lanes 4 and 6) of a 100-fold excess of
EXP3174, an AT
receptor antagonist. Nuclear extracts were
prepared and analyzed in an electrophoretic mobility shift assay using
P-labeled SIE. Lane 3 represents cells pretreated
with EXP3174 (EXP) and stimulated with AII for 2 h, as a positive
control for the specificity of AII action. This experiment was repeated
three times. Ctl, untreated control (lane
1).
Figure 3:
SIF
activation by AII requires continuous exposure where as activation by
IL-6 is rapid. A, time course of activation of SIF by AII: as
a positive control for AII induced SIF, cells were stimulated with AII
(100 nM) for 2 h (lane 2). Alternatively, cells were exposed
to AII for varying time periods, 5 min (lane 3), 15 min (lane 4), 35 min (lane 5), 1 h (lane 6),
followed by the addition of a 100-fold excess of EXP3174 for a total
incubation period of 2 h. Nuclear extracts were prepared and analyzed
in a gel mobility shift assay using P-labeled SIE. This
experiment was repeated three times. Ctl, untreated control (lane 1). B, time course of activation of SIF by
IL-6: cells were treated with IL-6 (10 ng/ml) for the indicated times,
nuclear extracts were prepared and analyzed in a gel mobility shift
assay using
P-labeled SIE (lanes 2-6). This
representative experiment was reproduced four times. Ctl,
untreated control (lane 1).
Figure 4:
Angiotensin II-induced SIF complex A
contains Stat92 or a related protein. Nuclear extracts from AII (100
nM, 2 h) or IL-6 (10 ng/ml, 10 min) treated cells were
incubated with P-labeled SIE. For supershift assays,
nuclear extracts from similarly treated cells were incubated with 2
µg of anti-Stat92 antibody (lanes 3 and 7),
incubated on ice for 1 h, and complexes resolved in a gel mobility
shift assay. The SIF complex A (SIF A) and supershifted
complexes (SS) are indicated. Supershifted complexes were
competed with a 100-fold excess of unlabeled (competitor) SIE (lanes 4 and 8). This blot is representative of three
experiments. Ctl, untreated control (lane 1); SS, supershifted complex; NRA, non-related antibody; com. SIE, competitor SIE.
Figure 5:
Angiotensin II pretreatment inhibits the
rapid induction of SIF by IL-6. Cells were treated with AII alone (100
nM) for 25 min (lane 2) and 2 h (lane 3),
and IL-6 alone (10 ng/ml) for 10 min (lane 4). Alternatively,
cells were pretreated with AII (100 nM) for 15 min followed by
IL-6 (10 ng/ml) for 10 min (lane 5), or pretreated with
EXP3174 (1 10
M) for 30 min and then
sequentially with AII (100 nM) for 15 min and IL-6 (10 ng/ml)
for 10 min (lane 6). Nuclear extracts were prepared and
analyzed in a mobility shift assay using
P-labeled SIE.
This blot is representative of three experiments. Ctl,
untreated control (lane 1); EXP,
EXP3174.
Figure 6: Angiotensin II pretreatment inhibits IL-6- induced Stat92 tyrosine phosphorylation. A, phosphotyrosine blots of immunoprecipitated Stat92. Samples corresponding to those described in the legend to Fig.6were immunoprecipitated with anti-Stat92 antibody (1 µg) for 12 h at 4 °C and the resulting immunoprecipitates resolved on an 8% polyacrylamide-SDS gel. Proteins were transferred to nitrocellulose and probed with antiphosphotyrosine antibody and the presence of tyrosine-phosphorylated proteins detected by chemiluminescence. Migration of differentially phosphorylated Stat92 protein is indicated by an arrow. B, the same blot in A was stripped and reprobed with Stat92 antibody. The major band seen below the 55-kDa marker corresponds to immunoglobulin heavy chain (IgG). This experiment was repeated three times.
Figure 7:
Inhibition of IL-6 induced SIF by AII is
transient. Cells were treated with AII for 2 h (100 nM, lanes 2 and 3), IL-6 (10 ng/ml, lanes 4 and 5), or sequentially with AII and IL-6 (lanes 6 and 7), or sequentially with AII, EXP3174, and IL-6 (lane
8) for the indicated times. Nuclear extracts were prepared and
analyzed in a gel mobility shift assay using P-labeled
SIE. This experiment was performed four times. Ctl, untreated
control (lane 1).
Figure 8:
Inhibition of IL-6-induced SIF by AII is
mimicked by PMA. Cells were treated with IL-6 (10 ng/ml) for 10 min (lane 2), or sequentially with PMA (100 nM) for 15
min and then with IL-6 (10 ng/ml) for 10 min (lane 3), or
sequentially with AII (100 nM) for 15 min and then with IL-6
(10 ng/ml) for 10 min (lane 4). Nuclear extracts were prepared
and analyzed in a gel mobility shift assay using P-labeled
SIE. Lanes 5-8 represent experiments performed using PKC
down-regulated cells, with identical treatment conditions as described
for lanes 1-4. For PKC down-regulation, the cells were
exposed for 24 h to PMA (250 nM) before treating with
agonists. This experiment was repeated three times. Ctl,
untreated control (lanes 1 and 5).
Figure 9:
Gene transcription is activated by
AII-induced SIF. T3CHO/AT cells were transfected with CAT
reporter plasmid pBLCAT2, or this plasmid carrying three copies of wild
type SIE (SIE/pBLCAT2) or three copies of mutant SIE (m.SIE/pBLCAT2).
Twenty-four hours after transfections, cells were serum starved for 3 h
and treated with AII (100 nM) or EXP3174 (100 µM)
as indicated, for 12 h. Cell extracts were prepared and CAT activity
measured using thin layer chromatography. This experiment was repeated
three times. EXP, EXP3174.
SIF complexes (A, B, and C) contain tyrosine-phosphorylated
STAT proteins. Complex A contains dimerized Stat92, complex B is a
heterodimer of Stat92 and Stat91, and complex C is a homodimer of
Stat91. We have previously demonstrated that stimulation of the AII
G-protein coupled receptor (AT) induces SIF activity
(mainly complex A) and activation of Stat91(30) . We now
identify a more significant contribution of Stat92 to AII stimulated
SIF formation consistent with the predominance of complex A. Activation
of Stat92 by AII was delayed compared to its rapid induction by IL-6
which parallels our initial observation (30) that nuclear SIF
activity stimulated by AII was delayed (maximal at 2-3 h).
Angiotensin II directly activated SIF activity through the AT
receptor and the delayed activation was not due to a requirement
for the release of other SIF inducing factors. We hypothesized that AII
signaling was bifunctional, capable of activating an inhibitory pathway
that prevented/suppressed SIF/Stat92 activation during the initial
phase (0-30 min) after AII exposure, followed by a stimulatory
pathway that induced significant levels of SIF/Stat92 activity at 2 h.
In agreement with this speculation, detectable levels of SIF activity
were only observed after a 30-min exposure to AII (Fig.3A). To test this hypothesis, we determined the
capacity of AII to modulate the rapid induction of SIF/Stat92 by IL-6.
We demonstrated that AII transiently inhibits SIF activation by IL-6 in
T3CHO/AT
cells, suggesting that similar
transient/reversible inhibitory mechanisms may be responsible for the
delayed maximal SIF activation by AII. The delayed SIF activation by
AII is potentially an important observation suggesting that: 1) the
mechanism of the AII-induced Stat92 activation and SIF complex
formation is unique and probably involves additional steps not utilized
by growth factors and cytokines; and that 2) AII activates the
transcription of a subset of genes containing SIE or SIE-like elements
at 2 h, which may differ from those stimulated (30 min) by growth
factors and cytokines. Our observation that AII can cross-talk with a
cytokine signaling pathway raises the possibility that this AII induced
inhibitory pathway is utilized by cells to modulate/inhibit
IL-6-related cytokine responses.
Other mechanisms for the delayed
SIF/Stat92 activation by AII can be envisaged. A recent study suggested
that the interactions of a specific Src homology 2 (SH2) domain on
STATs, with particular phosphotyrosine-containing motifs within the
cytoplasmic domains of activated receptors, was the crucial determinant
by which STAT or STATs were activated by JAK kinases in response to
ligands(39, 40) . While it is unclear how the
AT receptor couples to these activation events, AII
stimulation has been shown to phosphorylate the AT
receptor
on both serine and tyrosine residues(41) . Although the
identity of the phosphorylated tyrosine residues are not yet
determined, tyrosine residues (Tyr-312, Tyr-319, and Tyr-339) within
the cytoplasmic tail of the AT
receptor are potential
candidates and may be accessible to JAKs. A recent study reported that
AII activates JAK2/tyk2 kinases(42) , suggesting that members
of the JAK kinase family may be involved in the activation of STAT
proteins by this peptide. Interestingly, the early activation of JAK2
and tyk2 (within 5 min) coincides with the rapid tyrosine
phosphorylation of Stat91 (within 5 min). However, despite this early
activation of JAK2/tyk2, tyrosine phosphorylation of Stat92 was not
detected until 60 min following exposure to AII(42) . We have
shown that Stat92 is the principal component of AII-stimulated SIF, the
time course of activation of which is confirmed in the preceding report (42) . Thus, the mechanism for AII-induced Stat92 activation
may require other kinases or may involve processes distinct from those
described for growth factors and cytokines. Alternatively, in context
with what is known about STAT signaling, the delayed activation of
Stat92 by AII may be explained if the SH2 domain of Stat92 has low
affinity for the phosphorylated AII receptor.
Our observation that AII can inhibit IL-6 stimulated SIF/Stat92 activation is novel and may occur at multiple levels. It is possible that the interference occurs proximally at the level of the IL-6 receptor or the signal transducer protein gp130. Since the activation of the STAT pathway by IL-6 involves tyrosine phosphorylation and dimerization of gp130(43) , the possibility that AII activates a tyrosine phosphatase which prevents these events, requires further investigation. In support of this hypothesis, there is evidence that a G-protein coupled receptor (the somatostatin receptor) can activate and recruit a tyrosine phosphatase(44) . Similarly, other downstream events such as the tyrosine phosphorylation and activation of JAK kinases and Stat92 may be transiently suppressed by a tyrosine phosphatase.
It has been demonstrated, in many cell types, that AII
rapidly increases intracellular calcium and cellular PKC
activity(9, 11, 14) . Additionally, AII can
modulate levels of cAMP(9, 45, 46) . To
determine the possible role of cAMP, calcium, and PKC in the AII
mediated inhibition of SIF induction by IL-6, we tested the ability of
forskolin, ionomycin, and PMA (which activate cAMP, calcium, and PKC,
respectively) to modulate the SIF induction by IL-6. Pretreatment of
cells with forskolin and ionomycin for 15 min had no effect on the
IL-6-induced SIF response. ()Only PMA inhibited the rapid
induction of SIF by IL-6. Although, PMA mimicked the actions of AII,
the mechanism by which AII inhibited the IL-6-induced SIF/Stat92
activation appeared to be independent of PMA-sensitive isoforms of PKC.
It is possible, however, that a PMA-insensitive isoform of PKC is
involved or that the inhibition may involve other kinases.
Thus, we
have shown that the AT receptor mediates the activation of
SIF complex A and Stat92 or a related protein. Stat92 appears to play a
general role, integrating diverse signals from receptor tyrosine
kinases (PDGF and EGF), from JAK kinase-dependent receptors (IL-6,
IFN-
, IFN-
, and growth hormone) (36, 37) and
from G-protein coupled receptors (AT
), as we have
demonstrated. Although Stat92 is a common major component for both AII
and IL-6-induced SIF, the timing of activation, along with its
interactions with other members of the STAT family may result in
differential gene transcription. We have demonstrated that AII and IL-6
display distinct kinetics of Stat92 activation, providing a basis for
cross-talk between AII and cytokine-mediated cellular processes. Such
cross-talk may occur in cardiac tissues given the presence of receptors
for AII (3, 4, 5) and a variety of
IL-6-related cytokines(47) . The interplay between signaling
pathways for AII and cytokines opens a new area of investigation.