From the Program in Molecular Cardiology, University of North Carolina, Chapel Hill, North Carolina 27599-7295
Received for publication, September 26, 2001, and in revised form, February 19, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The growth-stimulating effects of thrombin are
mediated primarily via activation of a G protein-coupled receptor,
PAR-1. Because PAR-1 has no intrinsic tyrosine kinase activity, yet
requires tyrosine phosphorylation events to induce mitogenesis, we
investigated the role of the Janus tyrosine kinases (JAKs) in
thrombin-mediated signaling. JAK2 was activated rapidly in rat vascular
smooth muscle cells (VSMC) treated with thrombin, and signal
transducers and activators of transcription (STAT1 and STAT3) were
phosphorylated and translocated to the nucleus in a
JAK2-dependent manner. AG-490, a JAK2-specific inhibitor,
and a dominant negative JAK2 mutant inhibited thrombin-induced ERK2
activity and VSMC proliferation suggesting that JAK2 is upstream of the
Ras/Raf/MEK/ERK pathway. To elucidate the functional significance of
JAK-STAT activation, we studied the effect of thrombin on heat shock
protein (Hsp) expression, based upon the following: 1) reports that
thrombin stimulates reactive oxygen species production in VSMC; 2) the putative role of Hsps in modulating cellular responses to reactive oxygen species; and 3) the presence of functional STAT1/3-binding sites
in Hsp70 and Hsp90 In addition to regulating hemostasis and thrombosis, the
serine protease thrombin also promotes the inflammatory response and
wound healing (1) where it is mitogenic for lymphocytes, fibroblasts,
vascular endothelial and smooth muscle cells (2-4). Many of the
functions of thrombin are mediated via activation of protease-activated
receptor(s), PAR-1, PAR-3, or PAR-4 (5-7). Thrombin cleaves the N
terminus of its PAR-1 receptor between Arg41 and
Ser42 to create a new N terminus
42SFLLRN47 that acts as a tethered ligand and
activates the receptor (5). Increased Ser/Thr kinase activity in
response to the stimulation of PAR-1 and other G protein-coupled
receptors is well demonstrated (8-10). Thrombin also stimulates the
expression of nuclear proteins that constitute the transcription factor
AP-1, which participates in transactivation of several early growth
response genes implicated in
VSMC1 proliferation (9,
11).
PAR-1, angiotensin II (Ang II) receptor, and other G
protein-coupled receptors, which do not themselves possess intrinsic tyrosine kinase activity, require tyrosine kinase activity to induce
mitogenesis (8, 12-14). These observations suggest that G
protein-coupled receptors may utilize cytoplasmic protein tyrosine kinases such as Janus kinases (JAKs) and Src kinases to initiate mitogenesis. In fact, both JAK and Src kinases play important roles in
VSMC proliferation induced by Ang II (10, 15).
JAKs are 1 of 11 mammalian nonreceptor tyrosine kinase families that
were initially identified as essential mediators of cellular signaling
induced by the interaction of cytokines with their cognate receptors
(16). There are four members of the JAK family, JAK1, JAK2, JAK3, and
TYK2. Targeted gene disruption studies in mice demonstrate that JAKs
are essential for cytokine-induced signaling (17, 18). In interactions
of cytokines with their cognate receptors, receptor dimerization
induced by ligand binding to cell surface receptors leads to the
activation of one or more of the JAK family of kinases associated with
the transmembrane receptor. This, in turn, leads to phosphorylation of
tyrosine residues in the receptor cytoplasmic domains, which provide
docking sites for signal transducers and activators of transcription
(STATs) and other proteins that contain phosphotyrosine-binding motifs (19). STATs, upon phosphorylation by JAKs on tyrosine residues, undergo
homo- or heterodimerization with other STAT family members and migrate
to the nucleus. Within the nucleus, STAT dimers bind to target genes to
enhance transcription (20, 21). In addition to tyrosine
phosphorylation, STAT proteins undergo serine phosphorylation in a
mitogen-activated protein kinase (MAP kinase)-dependent
manner. In fact, both serine and tyrosine phosphorylation of STAT
proteins is necessary for maximal activation of transcription (22, 23). Activation of STAT proteins has been reported in cells treated with
various cytokines, growth factors, insulin, and Ang II (24-26).
Heat shock proteins (Hsps), initially identified by their enhanced
synthesis in cells exposed to elevated temperatures, have been
subsequently shown to accumulate in response to various stresses including cardiac hypertrophy, ischemic preconditioning, oxidative stress, and aging (27). Expressed constitutively, Hsps function as
molecular chaperones under physiologic conditions. During stress, Hsps
prevent protein aggregation, either through refolding of denatured
proteins or by promoting their degradation through a proteolytic
pathway (28). Induction of Hsps on exposure to a stressor confers
protection against exposure to a subsequent stressor in various cell
types (29, 30). Overexpression of individual Hsps also protects against
thermal and ischemic stress and apoptosis (31, 32). Hsps may also
regulate stress-responsive signaling pathways such as activation of
c-Jun N-terminal kinase1 (JNK1) and p38 (33). Induction of several Hsps
has been reported in VSMC (34) and may contribute to VSMC proliferation
leading to the onset of vascular diseases such as atherosclerosis.
Because thrombin-activated PAR-1 requires tyrosine phosphorylation
events to induce mitogenesis, we investigated the effect of thrombin on
the activation of members of the JAK family and their substrates,
STATs. We show that thrombin causes activation of JAK2 and tyrosine
phosphorylation and nuclear translocation of STAT1, -2, and -3. We also
demonstrate that inhibition of JAK2 activity attenuates
thrombin-induced ERK2 activity and VSMC proliferation. Furthermore, our
results show that thrombin induces Hsp70 and Hsp90 expression in VSMC
via activation of the JAK-STAT pathway. Thus, the JAK-STAT pathway may
be an important physiologic mediator of thrombin-induced events in
VSMC.
Materials--
Thrombin and AG-490 were purchased from
Calbiochem. H9C2 cells were obtained from American Type Culture
Collection. Antibodies used are as follows: anti-JAK1, JAK2, TYK2,
STAT1, STAT2, STAT3, FLAG, and anti-phosphotyrosine (4G10) (Upstate
Biotechnology, Inc., Lake Placid, NY, and Santa Cruz Biotechnology, San
Diego, CA); anti-phosphospecific JAK2 (BIOSOURCE
International, Camarillo, CA); anti-phosphospecific and
-nonphosphospecific ERK1/2 (New England Biolabs, Beverly, MA);
anti-Hsp70 (Affinity BioReagents, Golden, CO) and anti-Hsp90
(StressGen, Victoria, Canada). [14C]Chloramphenicol (55 mCi/mmol), [methyl-3H]thymidine (70 Ci/mmol),
and [ Cell Culture--
VSMC were isolated from the thoracic aortas of
200-250-g male Harlan Sprague-Dawley rats (8). Cells were maintained
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin at
37 °C in a humidified 95% air, 5% CO2 atmosphere. All
experiments were conducted using VSMC between passage numbers 7 and 20 that were growth-arrested by incubation in DMEM containing 0.1% calf serum for 72 h.
[3H]Thymidine Incorporation--
VSMC, grown
to ~70% confluence in 60-mm dishes, were quiesced by incubating in
DMEM containing 0.1% calf serum for 72 h. Quiesced VSMC were
exposed to 1.0 unit/ml thrombin for 24 h after pretreatment with a
JAK2-specific inhibitor, AG-490, for 16 h. Cells were labeled with
[methyl-3H]thymidine for 4 h, and its
incorporation into DNA was measured as described previously (9).
Cell Proliferation Assay--
VSMC were plated in 60-mm dishes
at an initial concentration of 3,500 cells/cm2 and grown in
DMEM supplemented with 10% fetal bovine serum for 48 h. Cells
were growth-arrested by incubating in DMEM containing 0.1% calf serum
for 48 h and then either left untreated or exposed to AG-490 for
16 h before treatment with 1.0 unit/ml thrombin for 48 h. The
cells were washed with phosphate-buffered saline, trypsinized, and
diluted with isotonic solution, and the increase in cell number was
directly measured with a Coulter counter (model ZM, Coulter Corp.,
Hialeah, FL).
Immunoprecipitation, ERK2 Activity Assay, and Western
Blotting--
Growth-arrested VSMC were treated with 1.0 unit/ml
thrombin in the presence and absence of AG-490 for the specified times at 37 °C. The cells were lysed in a buffer containing 20 mM HEPES, pH 7.4, 2 mM EGTA, 1 mM
dithiothreitol, 50 mM Electrophoretic Mobility Shift Assay--
Nuclear
extracts were prepared from growth-arrested VSMC that were either
treated or untreated with thrombin (35). DNA binding was performed by
incubating 5 µg of nuclear protein in a total volume of 20 µl of
reaction mixture containing 10 mM HEPES, pH 7.9, 50 mM KCl, 4% glycerol, 1 mM dithiothreitol, 1 mM EDTA, 1 µg of poly(dI-dC), and 100,000 cpm of
32P-labeled double-stranded Hsp70
( Transient Transfection of H9C2 Cells--
Dominant negative JAK2
( Transient Transfection and CAT Assay--
The Hsp70 CAT reporter
constructs LSN ( Statistical Analysis--
Differences were analyzed with one-way
analysis of variance, and post-hoc analysis was performed using
Student-Newman-Keuls method. Values of p < 0.05 were
considered statistically significant.
Thrombin-induced Mitogenesis Is Inhibited by AG-490--
To
understand the role of tyrosine phosphorylation in thrombin-induced
VSMC mitogenesis, we have investigated the effect of thrombin on the
JAK-STAT pathway. Initially, growth-arrested rat VSMC were treated with
1.0 unit/ml thrombin in the presence and absence of AG-490, a specific
inhibitor of JAK2. As shown in Fig. 1A, thrombin induced a
4.6-fold increase in DNA synthesis as measured by thymidine uptake
after 24 h, an effect that was significantly inhibited in a
dose-dependent manner by AG-490 (p < 0.05). AG-490 per se had no significant effect on DNA
synthesis even at a concentration of 50 µM.
Measuring its effect on cell counts corroborated the inhibitory effect
of AG-490 on thrombin-induced DNA synthesis. AG-490 significantly
inhibited the thrombin-induced increase in VSMC proliferation in a
dose-dependent manner (Fig. 1B,
p < 0.05). AG-490 alone had no significant effect on
VSMC proliferation, indicating that it is not cytotoxic at the
concentrations used in this experiment. AG-490 also significantly
inhibited PAR-1-derived agonist peptide (SFLLRNP)-induced VSMC DNA
synthesis (not shown) suggesting that the effects of thrombin-induced
JAK2 activation are mediated via PAR-1 activation. The effect of AG-490
on thrombin-induced VSMC proliferation is similar to its effect on VSMC
growth induced by Ang II, another G protein-coupled receptor agonist
(10). These results suggest that JAK2 plays a role in VSMC
proliferation induced by the activation of G protein-coupled receptors
and led us to test the activation of specific members of the JAK-STAT pathway in response to thrombin stimulation.
Thrombin Stimulates JAK2 and TYK2 Kinase Activity in Rat
VSMC--
To assess the contributions of the different JAK kinases to
thrombin-induced tyrosine phosphorylation, we immunoprecipitated tyrosine-phosphorylated proteins after thrombin stimulation and probed
for the presence of phosphorylated JAK proteins (Fig.
2). By using an anti-JAK1 antibody, we
found that JAK1 was transiently phosphorylated at 15 min after
stimulation with thrombin (Fig. 2). In contrast, we observed a biphasic
increase in JAK2 tyrosine phosphorylation following treatment with
thrombin (Fig. 2). This was confirmed by Western blot analysis of
thrombin-stimulated cell lysates with a phospho-specific JAK2 antibody
(Fig. 3A), which demonstrated
maximal stimulation of JAK2 at 1 min (8.7 ± 4.0-fold increase)
(Fig. 3B). Finally, we also measured thrombin-induced TYK2
phosphorylation (Fig. 2). In contrast to the rapid activation of JAK2,
peak activation of TYK2 was observed after 15 min of exposure to
thrombin. These results were corroborated by additional experiments in
which thrombin-stimulated VSMC lysates were immunoprecipitated with an
anti-TYK2 antibody and analyzed by Western blotting with the monoclonal
anti-phosphotyrosine antibody (Fig. 3C). TYK2 tyrosine phosphorylation was maximum (5.0 ± 1.0-fold increase) at 15 min after thrombin stimulation (Fig. 3D). JAK3 protein was not
observed in VSMC lysates, which is consistent with reports that the
expression of this protein is confined to lymphoid and myeloid cells
(39, 40). The differences in the time course of tyrosine
phosphorylation of various JAKs suggest that they may participate in
different stress-mediated events in VSMC.
The increase in tyrosine phosphorylation of JAK2 and TYK2 in response
to thrombin treatment was not due to an increase in the levels of these
protein as determined by Western blotting (Fig. 3, A and
C), indicating that thrombin altered tyrosine
phosphorylation of JAKs without affecting steady-state protein levels.
Similar to the observation of Abe and Berk (41), pretreatment of VSMC with 50 µM AG-490 for 16 h completely inhibited
thrombin-induced tyrosine phosphorylation of JAK2, whereas it had no
effect on c-Src, a non-JAK cytosolic tyrosine kinase (not shown).
Because JAK2 phosphorylation was most pronounced following treatment
with thrombin and inhibition of the JAK2 phosphorylation with AG-490 blocked thrombin-induced proliferation of VSMC, we chose to investigate further the role of JAK2 in thrombin-induced mitogenesis.
Thrombin-induced JAK2 Activation Leads to Tyrosine Phosphorylation
and Nuclear Translocation of STAT1, STAT2, and STAT3 in Rat
VSMC--
To determine whether thrombin-induced JAK2 activation leads
to tyrosine phosphorylation of STAT proteins, the JAK substrates, we
measured tyrosine phosphorylation and nuclear translocation of these
proteins. First, thrombin-treated VSMC lysates were immunoprecipitated with an anti-phosphotyrosine antibody, and the Western blots were probed with polyclonal antibodies against STAT1
Immunoprecipitation/immunoblotting experiments revealed an increase in
tyrosine phosphorylation of STAT3 within 1 min in response to treatment
with thrombin that was sustained throughout the 60-min treatment period
(Fig. 4, D and E). As with STAT1, STAT3 also rapidly translocated to the nucleus following treatment with thrombin, whereas the levels of this protein in cytosolic fractions were similar
to control values (Fig. 4F). Peak nuclear translocation of
STAT3 protein was observed at 10 min after treatment with thrombin (6.63 ± 1.52-fold increase) (Fig. 4G). Increased
tyrosine phosphorylation and nuclear translocation of STAT2 was also
observed in VSMC treated with 1.0 unit/ml thrombin (not shown). These
experiments demonstrate that thrombin causes tyrosine phosphorylation
and nuclear translocation of STAT proteins in VSMC. Inhibition of JAK2
tyrosine phosphorylation by pretreatment of VSMC with 50 µM AG-490 for 16 h blocked tyrosine phosphorylation
and nuclear translocation of STAT1, STAT2, and STAT3 (not shown),
indicating that JAK2 kinase activity is required for phosphorylation of
these proteins.
Inhibition of JAK2 Kinase Activity Partially Blocks ERK1/2
Activation in VSMC--
Because JAK2 has been suggested to be upstream
of the MAP kinase cascade (10, 41, 42) and MAP kinase activation is
required for thrombin-induced DNA synthesis in VSMC (8), we have
investigated whether JAK2 activation is required for thrombin-induced
stimulation of ERK1/2. ERK1/2 activation was measured in Western blots
using a phospho-specific antibody and also by an immunocomplex kinase assay. Consistent with our previous work (8), thrombin activated ERK1/2
kinases in a biphasic manner with peaks at 5 and 60 min (Fig.
5, A and B). AG-490
markedly inhibited thrombin-induced activation of ERK1/2 in Western
blots (Fig. 5A) and ERK2 activity in immunocomplex kinase
assays (Fig. 5B) (4.3 ± 0.7 at 60 min versus 2.1 ± 0.6 and 1.5 ± 0.4 with 10 and 50 µM AG-490, respectively, p < 0.05).
However, no detectable change was observed in the steady-state ERK1/2
protein levels following treatment with thrombin, either in the
presence or absence of AG-490 or with AG-490 alone (Fig. 5C). AG-490 caused a concentration-dependent
inhibition of thrombin-induced JAK2 phosphorylation (Fig.
6A) and ERK2 activation (Fig.
5B and 6B). In contrast, ERK2 activation induced
by 1 µM phorbol 12-myristate 13-acetate (PMA) and 10%
fetal bovine serum was not significantly affected by 50 µM AG-490. ERK activation in VSMC induced by PMA and
serum is consistent with the previous reports (9, 43). Together, these
results suggest that thrombin-induced JAK2 is specifically inhibited by
AG-490, and thrombin-induced ERK1/2 activation is partially regulated
through JAK2 kinase in VSMC.
It is possible that any given inhibitor may have pleiotropic effects on
cell physiology. Recently, AG-490 was shown to inhibit activation of
JAK3 induced by interleukin-2 in antigen-activated human T cells (44).
However, we did not detect JAK3 in VSMC with or without thrombin
treatment. To provide an alternative demonstration of the role of JAK2
inhibition, H9C2 rat myoblast cells were transiently transfected with a
dominant negative JAK2, which lacks the kinase domain (37) and a
FLAG-ERK2. The rationale for using this clonal muscle cell line instead
of VSMC is the relative ease of transfection. As expected, transfection
of dominant negative JAK2 markedly inhibited thrombin-induced JAK2
phosphorylation (Fig. 7A),
whereas a vector control had no such effect (not shown). Dominant
negative JAK2 transfection also had no effect on steady-state ERK2
protein levels (Fig. 7B). Dominant negative JAK2 markedly reduced thrombin-induced FLAG-ERK2 activation (4.40 ± 0.53 versus 2.00 ± 0.27 at 60 min of thrombin treatment,
p < 0.05) confirming the regulatory role of JAK2 in
thrombin-induced ERK2 activation (Fig. 7, C and
D).
Antioxidants Inhibit Thrombin-stimulated JAK2
Phosphorylation--
Reactive oxygen species
(ROS)-dependent activation of JAK2 has been reported in rat
VSMC treated with Ang II (45). Because thrombin also generates ROS in
VSMC (58), we investigated whether antioxidants inhibit
thrombin-induced activation of JAK2. Pretreatment of VSMC with various
antioxidants (diphenyleneiodonium, an inhibitor of flavin-containing
enzymes, N-acetyl-L-cysteine, and pyrrolidine dithiocarbamate) significantly inhibited thrombin-induced JAK2 phosphorylation without affecting steady-state protein levels (Fig.
8), indicating that one mechanism by
which thrombin induces JAK2 activation is via generation of ROS.
Thrombin-induced Heat Shock Protein (Hsp70 and Hsp90) Expression Is
Mediated through the Activation of the JAK-STAT Pathway--
G
protein-coupled receptor agonists such as Ang II (46) and thrombin (9)
and receptor tyrosine kinase agonists such as platelet-derived growth
factor-BB (PDGF-BB) (47) are known to regulate VSMC proliferation
through the induction of ROS. Accumulation of Hsps has been reported in
cardiac tissue during ischemia and reperfusion, conditions known to
produce ROS (48, 49). In addition, Hsp70 and Hsp90
Next, we investigated whether induction of Hsp70 and Hsp90 in
thrombin-treated VSMC is mediated via the transcriptional activity of
the STAT proteins. For this, an electrophoretic mobility shift assay
was performed by incubating nuclear proteins from thrombin-treated VSMC
with a synthetic Hsp70-STAT oligonucleotide corresponding to Activation of the Hsp90 Thrombin-mediated tyrosine phosphorylation of various proteins
such as insulin-like growth factor-1 receptor (8) and epidermal growth
factor receptor (51) has been attributed to the activation of cytosolic
tyrosine kinase, c-src. Stimulation of the Ras/MAP kinase
pathway by the activation of G protein-coupled receptors has also been
linked to Src kinases (14). In contrast, we show that JAK2, a non-Src
family cytosolic tyrosine kinase, is involved in thrombin-induced
activation of ERK1/2 kinases, VSMC proliferation, and expression of
Hsp70 and Hsp90.
The involvement of JAK kinases in signaling pathways induced by the
activation of cytokine receptors and receptor tyrosine kinases is well
documented (16, 53). Recently, it was shown that activation of the G
protein-coupled receptor, Ang II AT1, leads to
phosphorylation of tyrosine 319 in the C-terminal intracellular domain
and subsequent binding of SHP-2 phosphotyrosine phosphatase and the
JAK2 tyrosine kinase complex (54). This suggests that G protein-coupled
receptors possess mechanisms similar to those of cytokine and growth
factor receptors for signal transduction involving cytosolic tyrosine
kinases such as JAK2. Here we demonstrate that thrombin causes JAK2 and
TYK2 activation in rat VSMC, and we have investigated the role of JAK2
in thrombin-induced cellular signaling using a specific pharmacologic
inhibitor of JAK2. AG-490 inhibited both thrombin and PAR-1-derived
peptide-induced DNA synthesis in VSMC. In addition, JAK2 coprecipitates
with PAR-1 in VSMC treated with
thrombin,2 suggesting a
physical association between JAK2 and PAR-1. It remains to be
determined whether the association of JAK2 with PAR-1 is similar to
that described between JAK2 and Ang II AT1.
We found JAK2-dependent rapid tyrosine phosphorylation and
nuclear translocation of STAT1, STAT2, and STAT3 proteins in VSMC. Tyrosine phosphorylation of STATs was also reported in Ang II-treated VSMC (25). In agreement with our findings, Ang II-induced STAT1 tyrosine phosphorylation is mediated by JAK2 (54). Recently, it has
been shown that JAK2, TYK2, and STATs are also activated in response to
oxidants in several cell types, including VSMC ((41, 55, 56).2 We and others (47, 57, 58) have demonstrated that
growth factors such as PDGF-BB and G protein-coupled receptor agonists such as Ang II and thrombin stimulate VSMC growth through the production of ROS. We also found that JAK2 activation induced by
thrombin is sensitive to antioxidants. Similar results with regards
to JAK2 sensitivity to antioxidants were reported
recently in VSMC treated with Ang II (45). Together these
results suggest that in addition to receptor-associated stimulation,
another possible mechanism for the activation of JAK-STAT pathway by
thrombin is via the generation of intracellular ROS.
A biphasic increase in ERK1/2 activity was observed in rat VSMC treated
with thrombin. Inhibition of JAK2 activity by AG-490 pretreatment
partially inhibits thrombin-induced ERK1/2 activity. This is in
contrast to the complete inhibition of ERK1/2 phosphorylation by AG-490
in VSMC treated with either Ang II or PDGF-BB (10). This inhibition of
ERK1/2 activity was shown to be a consequence of the inhibitory effect
of AG-490 on JAK2 stimulation, blocking the association between JAK2
and Raf1 and subsequent Raf1 tyrosine phosphorylation. Abe and Berk
(41) reported that H2O2-mediated ERK1/2
phosphorylation is partially dependent on JAK2 activation. Taken
together, these results place JAK2 upstream of Ras in the Ras/Raf/MEK/ERK pathway and, thus, implicate JAK2 in regulation of
early growth response genes and cell proliferation. This argument is
supported by a recent report (59) that activation of p38 MAP kinase,
another member of the MAP kinase superfamily, is
JAK2-dependent. Because inhibition of JAK2 stimulation only
partially inhibits thrombin-induced ERK1/2 activation, a parallel
pathway may exist to activate ERK1/2 in thrombin-treated cells similar
to that proposed for H2O2-treated fibroblasts
(41). Cross-talk between the JAK-STAT and ERK pathways suggests that
inhibition of JAK2 activity might block not only direct tyrosine
phosphorylation but also serine phosphorylation of STATs by ERK1/2.
However, emerging evidence indicates that regulation of STAT proteins
by ERK1/2 depends on the nature of stimulus. In contrast to the reports
that maximal activation of transcription by STATs requires serine
phosphorylation by MAP kinases in addition to tyrosine phosphorylation
(22, 23), ERK1/2 activation has been shown to inhibit
interleukin-6-induced JAK-STAT signaling (60). Further experiments are
required to define the role of ERK1/2 in thrombin-induced activation of
STATs.
Thrombin is known to stimulate generation of ROS, the cellular effects
of which are modulated by Hsps. We therefore hypothesized that thrombin
might induce Hsp production in VSMC. This hypothesis was supported by
previous reports documenting the presence of functional STAT-binding
sites in Hsp70 and Hsp90 promoters (50). Our results demonstrate
time-dependent induction of Hsp70 and Hsp90 proteins in
thrombin-treated VSMC. Pretreatment with 50 µM AG-490
inhibits the induction of these proteins suggesting that Hsp synthesis
is regulated via the JAK-STAT pathway.
Although Hsps were initially characterized by their induction in
response to stress, emerging evidence indicates that these proteins
play a role in cellular signaling and cell proliferation (61).
Abrogation of Hsp70 expression in tumor cells inhibits cell
proliferation and induces apoptosis (62). Prior induction of Hsp70 has
been shown to play a role in the resumption of proliferation after
acute heat treatment (63). Similarly Hsp90 is known to interact with
many signaling molecules, particularly kinases and ligand-regulated
transcription factors (64, 65). Decreased Hsp90 protein levels have
been linked to a slow rate of cell division (66). More recently it was
demonstrated that Hsp90 plays a crucial role in the maturation and
regulation of eukaryotic translation initiation factor kinase Gcn2 (67)
and proper functioning of the centrosome (52). Induction of VSMC growth
by ROS has been linked in part to autocrine/paracrine effects of
proteins, including Hsp90 Electrophoretic mobility shift assays demonstrate that STAT1 and
STAT1/STAT3 heterodimers recognize the STAT binding domain of
the Hsp70 promoter, whereas STAT1 and STAT3 are the selective proteins
that bind the STAT-binding element of the Hsp90 In summary, we have shown that the JAK-STAT pathway plays an important
role in thrombin-induced VSMC proliferation. In addition, enhanced
expression of Hsp70 and Hsp90 via the JAK-STAT pathway indicates that
this pathway modulates cellular responses to generation of ROS in VSMC
treated with thrombin. Together with the extensive work done on the
JAK-STAT pathway in VSMC mitogenesis (10, 34, 41), our results suggest
that this pathway plays a significant role in the progression of
pathophysiologic vascular diseases such as atherosclerosis.
promoters. Indeed, thrombin up-regulated Hsp70
and Hsp90 protein expression via enhanced binding of STATs to cognate
binding sites in the Hsp70 and Hsp90 promoters. Together, these results
suggest that JAK-STAT pathway activation is necessary for
thrombin-induced VSMC growth and Hsp gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3,000 Ci/mmol) were obtained from
PerkinElmer Life Sciences. Diphenyleneiodonium,
N-acetyl-L-cysteine, and pyrrolidine
dithiocarbamate were obtained from Sigma.
-glycerophosphate, 1% Triton
X-100, 20 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM
sodium orthovanadate, and 400 µM phenylmethylsulfonyl
fluoride (9). For immunoprecipitation, cell lysates containing equal
amounts of proteins were incubated with appropriate antibodies
overnight at 4 °C. The antibody-protein complexes were incubated
with protein A-Sepharose CL-4B/protein A/G plus agarose beads for
2 h at 4 °C, and antibody-protein complexes bound to the beads
were pelleted at 2,000 × g for 2 min. The beads were
washed three times with lysis buffer and once with phosphate-buffered
saline and resuspended in Laemmli sample buffer. The samples were
resolved on 7.5% SDS-polyacrylamide gels. ERK2 activity assay and
Western blotting were performed as described previously (9).
122GATCCGGCGAAACCCCTGGAATATTCCCCGACCT
90)
or Hsp90
(
643GCCTGGAAACTGCTGGAAAT
623)
oligonucleotide for 20 min at room temperature. Canonical
double-stranded oligonucleotides for SP1 (5'-ATTCGATCGGGGCGGGGCGAGC-3')
and a high affinity double-stranded STAT1-binding sequence SIEm67
(5'-GATCTGATTACGGGAAATG-3') (36) were used in competition studies. For
identifying bands containing specific STAT proteins, the samples were
incubated with STAT1 or STAT3 antibody for 30 min before the
DNA-binding reaction was performed. Protein-DNA complexes were resolved
on a 4% polyacrylamide gel, and the dried gel was exposed to X-Omat AR
x-ray film with intensifying screen at
70 °C·.
JAK2) that lacks the C-terminal kinase domain was kindly provided
by Dr. S. Watanabe (University of Tokyo) (37). H9C2 cells were grown to
60-70% confluence in DMEM containing 10% fetal bovine serum and were
transfected with either 10 µg of control vector or
JAK2 and
FLAG-ERK2 (kindly provided by Dr. M. J. Weber, University of
Virginia, Charlottesville, VA) plasmid DNA. Transient transfection was
done using FuGENE (Roche Molecular Biochemicals) according to the
manufacturer's instructions. Cells were quiesced with DMEM containing
0.1% calf serum 24 h after transfection. After quiescing for
20 h, cells were treated with 1.0 unit/ml thrombin for the
indicated times.
188 to +1) and LSNP (
100 to +1) were kindly
provided by Richard Morimoto (Northwestern University, Evanston, IL).
The 5' Hsp90
CAT reporter constructs A (
1044 to +36) and C (
299
to +36) were kindly provided by David Latchman (University College
London, London, UK). For transfection, VSMC were grown to 70-80%
confluence in 100-mm dishes containing DMEM with 10% fetal bovine
serum. Transfection of VSMC with Hsp90
CAT reporter and
JAK2
plasmid DNA was performed by the calcium precipitate method (9). Cells
were cotransfected with
-galactosidase expression vector to
normalize for transfection efficiency. Cells were quiesced with DMEM
containing 0.1% calf serum 16 h after transfection. After being
quiescent for 36 h, cells were either untreated or treated with
thrombin for 6 h. In experiments with AG-490, cells were treated
with the inhibitor for 16 h before thrombin treatment. Cell
lysates were prepared as described previously (9), and CAT activity was
measured (38). In brief, 100 µg of protein was incubated with 4 µl
of 40 mM acetyl-CoA and 4 µl of 50 µCi/ml
[C14]chloramphenicol in a total volume of 150 µl at
37 °C for 2-4 h. Acetylated and nonacetylated chloramphenicol were
extracted with ethyl acetate and separated by thin layer chromatography on Silica Gel 1B plates using chloroform/methanol mixture (19:1) as
solvent. Air-dried silica plates were subjected to autoradiography, and
the acetylated [C14]chloramphenicol was quantified using
an Instant Imager (Packard Instrument Co.).
-Galactosidase assay was
performed following manufacturer's protocol (Promega).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 1.
Thrombin-induced VSMC DNA synthesis and
proliferation is inhibited by AG-490. A,
growth-arrested VSMC were pretreated with the JAK2-specific inhibitor,
AG-490, for 16 h prior to exposure to 1.0 unit/ml thrombin for
24 h. DNA synthesis is expressed as [3H]thymidine
incorporation in cpm/dish. Data represent the means ± S.D. of two
experiments, each with four replicates. B, growth-arrested
VSMC were either left untreated or exposed to AG-490 for 16 h
before treatment with 1.0 unit/ml thrombin for 48 h. Increase in
cell number was directly measured with a Coulter counter. Data
represent the means ± S.D. of two experiments, each with four
replicates. The asterisk represents significant differences
compared with control, and the double asterisks represent
significant differences compared with thrombin treatment
(p < 0.05). DMSO, dimethyl sulfoxide.
View larger version (25K):
[in a new window]
Fig. 2.
Thrombin activates JAK family kinases in rat
VSMC. VSMC were growth-arrested for 72 h and treated with 1.0 unit/ml thrombin for the indicated times. Cells were harvested in
kinase lysis buffer, and lysates containing an equal amount of protein
were immunoprecipitated (IP) with anti-phosphotyrosine
antibody (4G10). Western blot (WB) analysis was performed
with anti-JAK1, anti-JAK2, or anti-TYK2 antibody. The results presented
are representative of an experiment that was repeated at least three
times.
View larger version (26K):
[in a new window]
Fig. 3.
Rapid tyrosine phosphorylation of JAK2 and
delayed activation of TYK2 in VSMC treated with thrombin.
Growth-arrested VSMC were treated with 1.0 unit/ml thrombin for the
indicated times and harvested in lysis buffer. A, cell
lysates containing equal amounts of protein were analyzed by Western
blotting (WB) with anti-phosphotyrosine-specific JAK2
antibody (top). Thrombin had no effect on JAK2 protein
levels as observed in Western blot analysis with anti-JAK2 antibody
(bottom). B, densitometric analysis of JAK2
tyrosine phosphorylation (mean ± S.D., n = 3).
C, cell lysates containing equal amounts of protein were
immunoprecipitated (IP) with anti-TYK2 antibody, and Western
analysis was performed with anti-phosphotyrosine antibody (4G10)
(top) or anti-TYK2 antibody (bottom). No
difference in TYK2 protein levels was observed in the Western blot
probed with anti-TYK2 antibody. D, densitometric analysis of
TYK2 tyrosine phosphorylation (mean ± S.D., n = 3).
/
, STAT2, or STAT3. All three STAT proteins were tyrosine-phosphorylated in response
to thrombin treatment. STAT1
was rapidly tyrosine-phosphorylated within 1 min, an effect that was sustained for 60 min (Fig.
4A). The increase in tyrosine
phosphorylation of STAT1
in response to treatment with thrombin was
much less pronounced than that of STAT1
. To determine whether
tyrosine phosphorylation of STAT proteins in response to treatment with
thrombin was accompanied by translocation into the nucleus, Western
blot analyses of nuclear and cytosolic fractions of thrombin-treated
VSMC were performed (Fig. 4B). Thrombin induced nuclear
translocation of STAT1 in 5 min (3.03 ± 0.25-fold increase), an
increase that was sustained for 60 min (Fig. 4C), whereas no
discernible change was observed in the protein levels in cytosolic
fractions (Fig. 4D).
View larger version (32K):
[in a new window]
Fig. 4.
Thrombin induces tyrosine phosphorylation and
nuclear translocation of STAT1 and STAT3. Growth-arrested VSMC
were treated with 1.0 unit/ml thrombin for the indicated times, and
cell lysates were prepared. A, cell lysates with equal
amounts of protein were immunoprecipitated (IP) with
anti-phosphotyrosine antibody, and Western blot (WB)
analysis was performed with anti-STAT1 antibody. B, nuclear
(top) or cytosolic (bottom) fractions prepared
from thrombin-treated VSMC, containing equal amounts of protein, were
analyzed by Western blotting with anti-STAT1 antibody. C,
densitometric analysis of nuclear translocated STAT1 p84/p91 protein
levels (mean ± S.D., n = 3). D,
lysates containing equal amounts of protein were immunoprecipitated
with anti-phosphotyrosine antibody and analyzed by Western blotting
with anti-STAT3 antibody. E, lysates containing equal
amounts of protein were analyzed by Western blotting with
anti-phosphotyrosine-specific STAT3 antibody. F, nuclear
(top) or cytosolic (bottom) fractions from
thrombin-treated VSMC containing equal amounts of protein were analyzed
by Western blotting with anti-STAT3 antibody. G,
densitometric analysis of nuclear translocated STAT3 protein levels
(mean ± S.D., n = 3).
View larger version (33K):
[in a new window]
Fig. 5.
AG-490 causes partial inhibition of
thrombin-induced ERK1/2 activation. Growth-arrested VSMC were
pretreated with AG-490 for 16 h and then treated with 1.0 unit/ml
thrombin for the indicated times. A, cell lysates containing
equal amounts of protein were analyzed by Western blotting
(WB) with phosphospecific anti-ERK1/2 antibody.
B, lysates containing equal amounts of protein were
immunoprecipitated with anti-ERK2 antibody. ERK2 activity was measured
through immunocomplex kinase assay using myelin basic protein
(MBP) as a substrate. C, Western blot analysis of
cell lysates with anti-ERK1/2 antibody did not show any difference in
ERK1/2 protein levels. Results shown represent an experiment that was
repeated at least twice with similar results. DMSO, dimethyl
sulfoxide.
View larger version (27K):
[in a new window]
Fig. 6.
AG-490 inhibits thrombin-induced JAK2
phosphorylation and ERK2 activation. A, growth-arrested
VSMC were pretreated with various concentrations of AG-490 for 16 h and then treated with 1.0 unit/ml thrombin for 1 min. Cell lysates
containing equal amounts of protein were analyzed by Western blotting
(WB) with anti-phosphotyrosine-specific JAK2 antibody.
B, ERK2 activity was measured from lysates of cells treated
with thrombin for 60 min in the presence and absence of AG-490 as
described for Fig. 5. C, growth-arrested VSMC were
pretreated with 50 µM AG-490 for 16 h and then
treated with 1.0 unit/ml thrombin, 10% fetal bovine serum, or 1 µM PMA for 5 min. ERK2 activity was measured as described
above. MBP, myelin basic protein; DMSO, dimethyl
sulfoxide.
View larger version (22K):
[in a new window]
Fig. 7.
Dominant negative JAK2
( JAK2) inhibits thrombin-induced ERK2
activation. H9C2 cells were transfected with either vector control
or
JAK2 and FLAG-ERK2 plasmids. Cells were grown in DMEM
supplemented with 10% fetal bovine serum for 36 h and
growth-arrested for 20 h. Cells were treated with 1.0 unit/ml
thrombin for the indicated times, and lysates containing equal amounts
of protein were analyzed by Western blotting (WB) with
anti-JAK2 antibody (A). B, lysates containing
equal amounts of protein were immunoprecipitated (IP) with
anti-FLAG antibody, and Western blotting was performed with anti-ERK2
antibody (C). ERK2 activity was measured in cell lysates
immunoprecipitated with anti-FLAG antibody as described for Fig. 5.
MBP, myelin basic protein. D, densitometric
analysis of ERK2 activity (mean ± S.D., n = 3).
The asterisk represents significant difference compared with
control, and the double asterisks represent significant
differences compared with thrombin treatment (p < 0.05).
View larger version (18K):
[in a new window]
Fig. 8.
ROS mediate thrombin-induced JAK2 tyrosine
phosphorylation. Growth-arrested VSMC were pretreated with 10 µM diphenyleneiodonium (DPI), 100 µM pyrrolidine dithiocarbamate (PDTC), or 10 mM N-acetyl-L-cysteine
(NAC) for 30 min and then treated with 1.0 unit/ml thrombin
for 1 min. Lysates containing equal amounts of protein were analyzed by
Western blotting (WB) with either
anti-phosphotyrosine-specific JAK2 (A) or JAK2
(B) antibody. C, densitometric analysis of JAK2
tyrosine phosphorylation (mean ± S.D., n = 3).
promoters contain
functional STAT-binding sites (50). Therefore, we investigated whether
treatment of VSMC with thrombin leads to the accumulation of these
proteins. Thrombin-stimulated expression of Hsp70 protein was evident
at 2 h, and a 3.36 ± 0.78-fold increase was observed at
24 h (Fig. 9A).
Pretreatment of VSMC with 50 µM AG-490 prior to exposure to thrombin abolished the agonist-induced increase in Hsp70
steady-state protein levels (3.50 ± 0.50 versus
1.30 ± 0.17, p < 0.05) (Fig. 9B). A
biphasic increase in Hsp90 protein levels was also observed in VSMC in
response to thrombin, with an initial peak at 4 h (2.46 ± 0.74-fold increase), and a second peak at 24 h (3.87 ± 0.71-fold increase) (Fig. 9C). As with Hsp70, increased
levels of Hsp90 were ablated in response to the inhibition of JAK2
tyrosine phosphorylation with 50 µM AG-490 (3.67 ± 0.83 versus 1.33 ± 0.06, p < 0.05)
(Fig. 9D). AG-490, by itself, had no marked
effect on Hsp70 and Hsp90 protein levels.
View larger version (30K):
[in a new window]
Fig. 9.
AG-490 inhibits thrombin-induced Hsp70 and
Hsp90 protein levels. A, growth-arrested VSMC were
treated with 1.0 unit/ml thrombin for the indicated times, and Hsp70
protein levels were analyzed by Western blotting with anti-Hsp70
antibody (top). Densitometric analysis of Hsp70
protein levels was performed (mean ± S.D., n = 3)
(bottom). B, cells were pretreated with AG-490
for 16 h and treated with thrombin for 24 h (top).
Cell lysates containing equal amounts of protein were analyzed by
Western blotting (WB) with anti-Hsp70 antibody.
Densitometric analysis of Hsp70 protein levels was done
(mean ± S.D., n = 3) (bottom).
C, cell lysates from growth-arrested VSMC treated with 1.0 unit/ml thrombin for the indicated times were analyzed by Western
blotting with anti-Hsp90 antibody (top).
Densitometric analysis of Hsp90 protein levels was performed
(mean ± S.D., n = 3) (bottom).
D, cells were pretreated with AG-490 for 16 h and
treated with thrombin for 24 h. Cell lysates containing equal
amounts of protein were analyzed by Western blotting with anti-Hsp90
antibody (top). Densitometric analysis of Hsp90
protein levels was done (mean ± S.D., n = 3)
(bottom). The asterisk represents significant
difference compared with control, and the double asterisks
represent significant differences compared with thrombin treatment
(p < 0.05). DMSO, dimetyl sulfoxide.
122 to
90 base pairs of the Hsp70 promoter. Three shifted bands were
observed with nuclear extracts from thrombin-treated VSMC, each of
which was competed with an excess of unlabeled specific oligonucleotide, but not with a nonspecific one (Fig.
10). The faster migrating two bands
were partially abolished by preincubation of complexes with either
anti-STAT1 or anti-STAT3 antibodies, indicating that these complexes
likely contain STAT1/STAT3 heterodimers. The slower migrating band was
abolished by anti-STAT1 antibody, but not by anti-STAT3 antibody,
demonstrating the presence of STAT1 protein in this complex.
Electrophoretic mobility shift assay of VSMC nuclear extracts with the
STAT-binding region of Hsp90
synthetic oligonucleotide (
643 to
623 of Hsp90
promoter) demonstrated two shifted bands, the
intensity of which was enhanced in response to thrombin (Fig.
11). The bands were competed with an
excess of unlabeled specific oligonucleotide but not with a nonspecific
one. The faster migrating band was competed with an unlabeled high
affinity STAT1-binding sequence (SIEm67 oligonucleotide) from the
c-fos promoter and was abolished by addition of an
anti-STAT1 antibody. In contrast, the slower migrating band was
abolished by anti-STAT3 antibody but not by anti-STAT1 antibody. These
results indicate that the faster migrating band has STAT1 protein and the slower migrating band has STAT3 protein in the complex. None of the
antibodies tested produced supershifts in electrophoretic mobility
shift assays.
View larger version (56K):
[in a new window]
Fig. 10.
Thrombin induces DNA binding of Hsp70 STAT
sequence. Nuclear extracts from VSMC, either untreated
(1st lane 1) or treated with 1.0 unit/ml thrombin
for 10 min (2nd to 6th lanes), were
subjected to an electrophoretic mobility shift assay using a labeled
Hsp70 STAT probe. To determine specificity of Hsp70 STAT binding
complex, nuclear extracts were preincubated with unlabeled specific or
nonspecific competitors. The specific competitors used were 100-fold
molar excess of Hsp70 STAT (3rd lane),
and the nonspecific competitor was 100-fold molar excess of SP1
consensus oligonucleotide (4th lane). For the
characterization of protein components of thrombin-induced binding
complex, nuclear extracts were preincubated with anti-STAT1
(5th lane) or anti-STAT3 (6th
lane) antibody.
View larger version (48K):
[in a new window]
Fig. 11.
Thrombin induces DNA binding of Hsp90 STAT
sequence. Nuclear extracts from VSMC, either untreated (1st
lane) or treated with 1.0 unit/ml thrombin for 10 min
(2nd to 7th lanes), were subjected to
an electrophoretic mobility shift assay using a labeled Hsp90 STAT
probe. To determine specificity of Hsp90 STAT binding complex, nuclear
extracts were preincubated with unlabeled specific or nonspecific
competitors. The specific competitors used were 100-fold molar excess
of Hsp90 STAT and STAT1-inducible element (SIE)
(3rd and 4th lanes, respectively), and the
nonspecific competitor was 100-fold molar excess of SP1 consensus
oligonucleotide (5th lane). For the characterization of
protein components of thrombin-induced binding complex, nuclear
extracts were preincubated with anti-STAT1 (6th lane) or
anti-STAT3 (7th lane) antibody (Ab).
Promoter by Thrombin Is
JAK2-dependent--
To investigate whether
thrombin-induced Hsp90 expression was mediated via a direct effect of
activated STAT proteins on its promoter, VSMC were transfected with an
Hsp90
promoter-reporter construct either containing Hsp90 A (
1044
to +36) or lacking Hsp90 C (
299 to +36), a functional STAT-binding
site. The Hsp90 A construct, besides possessing a STAT3-like binding
site, also binds activated STAT1 protein (50). The reporter construct
Hsp90 A was activated 2-4-fold by thrombin, whereas deletion of
sequences containing the functional STAT-binding site abolished the
activation of this promoter by thrombin (Fig.
12A). Thrombin-induced
Hsp90
promoter activity was also abolished in VSMC pretreated with
50 µM AG-490, indicating that phosphorylation of STAT
proteins by JAK2 kinase is necessary for maximal promoter activity
(Fig. 12B). To confirm the role of JAK-STAT pathway in
thrombin-induced Hsp90 expression, VSMC were cotransfected with Hsp90 A
promoter-reporter construct and a dominant negative JAK2. Again,
thrombin-induced Hsp90
promoter activity was completely abolished in
the presence of dominant negative JAK2 (Fig. 12C). Together,
these results indicate that the G protein-coupled receptor agonist
thrombin causes activation of the JAK-STAT pathway in rat VSMC, and
this pathway plays an important role in thrombin-induced VSMC
proliferation and expression of proliferation-associated Hsps.
View larger version (30K):
[in a new window]
Fig. 12.
JAK2 inactivation inhibits thrombin-induced
Hsp90 promoter activity. AG-490 inhibits thrombin-induced Hsp90
promoter activity. A, VSMC were transiently transfected with
an Hsp90 CAT reporter construct containing 1044 to +36 (A)
or lacking
299 to +36 (C) STAT binding region, or vector
lacking any insert, growth-arrested, and were either untreated or
treated with thrombin for 6 h. Cell lysates were prepared, and
lysates containing equal amounts of protein were assayed for CAT
activity. To normalize for transfection efficiency, cells were also
cotransfected with a
-galactosidase construct. B, VSMC
transfected with Hsp90 CAT reporter constructs were growth-arrested,
pretreated with AG-490 for 16 h, and treated with thrombin in the
presence and absence of AG-490. C, VSMC transfected with
Hsp90 CAT reporter and either vector or
JAK2 DNA were
growth-arrested and treated with thrombin for 6 h. Autoradiograms
shown represent an experiment that was repeated at least twice with
similar results. Fold activation shown is based on the quantitation of
radioactivity measured by an Instant Imager. DMSO, dimethyl
sulfoxide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and cyclophilins (34). AlThough we have
not addressed the role of Hsps in VSMC growth, these reports suggest
that Hsps are likely to play a role in thrombin-induced VSMC proliferation.
promoter. Hsp70 and
Hsp90
promoter constructs containing functional STAT-binding sites
are activated by thrombin, and pretreatment with AG-490 or
cotransfection with dominant negative JAK2 blocks the activity of these
promoters. This strongly suggests that JAK2-mediated tyrosine
phosphorylation is required for activation of the Hsp70 and Hsp90
promoters in VSMC. In close proximity to the STAT binding regions, the
Hsp70 and Hsp90
promoters also contain binding sites for the
stress-activated transcription factor, HSF1 (50). Activation of HSF-1
has been reported in heart tissue perfused with
H2O2 (48). Therefore, a concomitant role for
HSF1 in thrombin-induced expression of Hsp70 and Hsp90 is possible in
view of the stimulation of ROS in thrombin-treated VSMC. Consistent
with this hypothesis, overexpression of STAT1 and HSF1 has an additive
effect on Hsp70 promoter activity in HepG2 cells, suggesting that
protein-protein interactions between these nuclear proteins may play a
role in the regulation of Hsp70 transcriptional activity (50).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Chris Horaist and Joann Aaron for editorial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL57352.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.
To whom correspondence should be addressed: Dept. of Medicine,
3033 Old Clinic Bldg., CB 7005, University of North Carolina, Chapel
Hill, NC 27599-7005. Tel.: 919-966-4468; Fax: 409-966-5775; E-mail:
mrunge@med.unc.edu.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M008802200
2 N. R. Madamanchi, S. Li, C. Patterson, and M. S. Runge, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: VSMC, vascular smooth muscle cells; JAK, Janus kinases; Hsp, heat shock protein; ROS, reactive oxygen species; STATs, signal transducers and activators of transcription; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; Ang II, angiotensin II; PMA, phorbol 12-myristate 13-acetate; PDGF-BB, platelet-derived growth factor-BB.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Coughlin, S. R. (1993) Thromb. Haemostasis 66, 184-187 |
2. |
Seuwen, K.,
Kahan, C.,
Hartmann, T.,
and Pouyssegur, J.
(1990)
J. Biol. Chem.
265,
22292-22299 |
3. | Graham, D. J., and Alexander, J. J. (1990) J. Vasc. Surg. 11, 307-313[CrossRef][Medline] [Order article via Infotrieve] |
4. | McNamara, C. A., Sarembock, I. J., Gimple, L. W., Fenton, J. W. I., Coughlin, S. R., and Owens, G. K. (1993) J. Clin. Invest. 91, 94-98[Medline] [Order article via Infotrieve] |
5. | Vu, T. K. H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068[Medline] [Order article via Infotrieve] |
6. | Ishihara, H., Connolly, A. J., Zeng, D., Kahn, M. L., Zheng, Y. W., Timmons, C., Tram, T., and Coughlin, S. R. (1997) Nature 386, 502-506[CrossRef][Medline] [Order article via Infotrieve] |
7. | Kahn, M. L., Zheng, Y.-W., Huang, W., Bigornia, V., Zeng, D., Moff, S., Farese Jr, R. V., Tam, C., and Coughlin, S. R. (1998) Nature 394, 690-694[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Rao, G. N.,
Delafontaine, P.,
and Runge, M. S.
(1995)
J. Biol. Chem.
270,
27871-27875 |
9. |
Rao, G. N.,
Katki, K. A.,
Madamanchi, N. R.,
Wu, Y.,
and Birrer, M. J.
(1999)
J. Biol. Chem.
274,
6003-6010 |
10. |
Marrero, M. B.,
Schieffer, B.,
Li, B.,
Sun, J.,
Harp, J. B.,
and Ling, B. N.
(1997)
J. Biol. Chem.
272,
24684-24690 |
11. | Grand, J. A. R., Turnell, A. S., and Grabham, P. W. (1996) Biochem. J. 313, 353-368[Medline] [Order article via Infotrieve] |
12. |
Weiss, R. H.,
and Nuccitelli, R.
(1992)
J. Biol. Chem.
267,
5608-5613 |
13. |
Molly, C. J.,
Taylor, D. S.,
and Weber, H.
(1993)
J. Biol. Chem.
268,
7338-7345 |
14. |
Luttrell, L. M.,
Hawes, B. E.,
van Biesen, T.,
Luttrell, D. K.,
Lansing, T. J.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
19443-19450 |
15. |
Schieffer, B.,
Paxton, W. G.,
Chai, Q.,
Marrero, M. B.,
and Bernstein, K. E.
(1996)
J. Biol. Chem.
271,
10329-1033 |
16. | Aringer, M., Cheng, A., Nelson, J. W., Chen, M., Sudarshan, C., Zhou, Y.-J., and O'Shea, J. J. (1999) Life Sci. 64, 2173-2186[CrossRef][Medline] [Order article via Infotrieve] |
17. | Parganas, E., Wang, D., Stravopodis, D., Topham, D. J., Marine, J.-C., Teglund, S., Vanin, E. F., Bodner, S., Colamonici, O. R., van Deursen, J. M., Grosveld, G., and Ihle, J. N. (1998) Cell 93, 385-395[Medline] [Order article via Infotrieve] |
18. | Rodig, S. J., Meraz, M. A., White, J. M., Lampe, P. A., Riley, J. K., Arthur, C. D., King, K. L., Sheehan, K. C. F., Yin, L., Pennica, D., Johnson, E. M., Jr., and Schreiber, R. D. (1998) Cell 93, 373-383[Medline] [Order article via Infotrieve] |
19. | Hilton, D. J. (1999) Cell. Mol. Life Sci. 55, 1568-1577[CrossRef][Medline] [Order article via Infotrieve] |
20. | Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415-1421[Medline] [Order article via Infotrieve] |
21. |
Huang, Y.-Q.,
Li, J.-J.,
and Karpatkin, S.
(2000)
J. Biol. Chem.
275,
6462-6468 |
22. | Wen, Z., Zhong, Z., and Darnell, J. E., Jr. (1995) Cell 82, 241-250[Medline] [Order article via Infotrieve] |
23. | Zhang, X., Blenis, J., Li, H.-C., Schindler, C., and Chen-Kiang, S. (1995) Science 267, 1990-1994[Medline] [Order article via Infotrieve] |
24. | Levy, D. E. (1999) Cell. Mol. Life Sci. 55, 1559-1567[CrossRef][Medline] [Order article via Infotrieve] |
25. | Marrero, M. B., Schieffer, B., Paxton, W. G., Heerdt, L., Berk, B. C., Delafontaine, P., and Bernstein, K. E. (1995) Nature 375, 247-250[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Venema, R. C.,
Venema, V. J.,
Eaton, D. C.,
and Marrero, M. B.
(1998)
J. Biol. Chem.
273,
30795-30800 |
27. |
Benjamin, I. J.,
and McMillan, D. R.
(1998)
Circ. Res.
83,
117-132 |
28. | Gething, M. J., and Sambrook, J. (1992) Nature 355, 33-45[CrossRef][Medline] [Order article via Infotrieve] |
29. | Lowenstein, D. H., Chan, P. H., and Miles, M. F. (1991) Neuron 7, 1053-1060[Medline] [Order article via Infotrieve] |
30. | Amin, V., Cumming, D. V. E., Coffin, R. S., and Latchman, D. S. (1995) Neurosci. Lett. 200, 85-88[CrossRef][Medline] [Order article via Infotrieve] |
31. | Cumming, D. V. E., Heads, R. J., Watson, A., Latchman, D. S., and Yellon, D. M. (1996) J. Mol. Cell. Cardiol. 28, 2343-2349[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Wagstaff, M. J. D.,
Collaco-Moraes, Y.,
Smith, J.,
de Belleroche, J. S.,
Coffin, R. S.,
and Latchman, D. S.
(1999)
J. Biol. Chem.
274,
5061-5069 |
33. |
Gabai, V. L.,
Meriin, A. B.,
Mosser, D. D.,
Caron, A. W.,
Rits, S.,
Shifrin, V. I.,
and Sherman, M. Y.
(1997)
J. Biol. Chem.
272,
18033-18037 |
34. |
Liao, D.-F.,
Jin, J.-Z.,
Baas, A. S.,
Daum, G.,
Gygi, S. P.,
Aebersold, R.,
and Berk, B. C.
(2000)
J. Biol. Chem.
275,
189-196 |
35. | Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract] |
36. | Wagner, B. J., Hayes, T. E., Hoban, C. J., and Cochran, B. H. (1990) EMBO J. 9, 4477-4484[Abstract] |
37. |
Watanabe, S.,
Itoh, T.,
and Arai, K.
(1996)
J. Biol. Chem.
271,
12681-12686 |
38. | Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051[Medline] [Order article via Infotrieve] |
39. | Johnston, J. A., Kawamura, M., Kirken, R. A., Chen, Y, -Q., Blake, T. B., Shibuya, K., Ortaldo, J. R., McVicar, D. W., and O'Shea, J. J. (1994) Nature 370, 151-153[CrossRef][Medline] [Order article via Infotrieve] |
40. | Witthuhn, B. A., Silvennoinen, O., Miura, O., Lai, K. S., Cwik, C., Liu, E. T., and Ihle, J. N. (1994) Nature 370, 153-157[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Abe, J.,
and Berk, B. C.
(1999)
J. Biol. Chem.
274,
21003-21010 |
42. |
Ishida, M.,
Ishida, T.,
Thomas, S.,
and Berk, B. C.
(1998)
Circ. Res.
82,
7-12 |
43. | Pukac, L., Huangpu, J., and Karnovsky, M. J. (1998) Exp. Cell Res. 242, 548-560[CrossRef][Medline] [Order article via Infotrieve] |
44. | Kirken, R. A., Erwin, R. A., Taub, D., Murphy, W. J., Behbod, F., Wang, L., Pericle, F., and Farrar, W. L. (1999) J. Leukocyte Biol. 65, 891-899[Abstract] |
45. | Schieffer, B., Luchtefeld, M., Braun, S., Hilfiker, A., Hilfiker-Kleiner, D., and Drexler, H. (2000) Circ. Res. 87, 1196-1201 |
46. |
Ushio-Fukai, M.,
Alexander, R. W.,
Akers, M.,
Yin, Q.,
Fujio, Y.,
Walsh, K.,
and Griedling, K. K.
(1999)
J. Biol. Chem.
274,
22699-22704 |
47. | Sundaresan, M., Yu, Z.-X., Ferrans, V. J., Irani, K., and Finkel, T. (1995) Science 270, 296-299[Abstract] |
48. |
Nishizawa, J.,
Nakai, A.,
Matsuda, K.,
Komeda, M.,
Ban, T.,
and Nagata, K.
(1999)
Circulation
99,
934-941 |
49. | Plumier, J.-C. L., Robertson, H. A., and Currie, R. W. (1996) J. Mol. Cell. Cardiol. 28, 1251-1260[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Stephanou, A.,
Isenberg, D. A.,
Nakajima, K.,
and Latchman, D. S.
(1999)
J. Biol. Chem.
274,
1723-1728 |
51. |
Vaingankar, S. M.,
and Martins-Green, M.
(1998)
J. Biol. Chem.
273,
5226-5234 |
52. |
Lange, B. M. H.,
Bachi, A.,
Wilm, M.,
and González, C.
(2000)
EMBO J.
19,
1252-1262 |
53. |
Choudhury, G. G.,
Choudhury, N. G.,
and Abboud, H. E.
(1998)
J. Clin. Invest.
101,
2751-2760 |
54. |
Venema, R. C.,
Ju, H.,
Venema, V. J.,
Schieffer, B.,
Harp, J. B.,
Ling, B. N.,
Eaton, D. C.,
and Marrero, M. B.
(1998)
J. Biol. Chem.
273,
7703-7708 |
55. | Simon, A. R., Rai, U., Fanburg, B. L., and Cochran, B. H. (1998) Am. J. Physiol. 275, C1640-C1652[Medline] [Order article via Infotrieve] |
56. |
Carballo, M.,
Conde, M.,
Bekay, R. E.,
Matin-Nieto, J.,
Camacho, M. J.,
Monteseirin, J.,
Conde, J.,
Bedoya, F. J.,
and Sobrino, F.
(1999)
J. Biol. Chem.
274,
17580-17586 |
57. |
Zafari, A. M.,
Ushio-Fukai, M.,
Akers, M.,
Yin, Q.,
Shah, A.,
Harrison, D. G.,
Taylor, W. R.,
and Griendling, K. K.
(1998)
Hypertension
32,
488-495 |
58. |
Patterson, C.,
Ruef, J.,
Madamanchi, N. R.,
Barry-Lane, P.,
Hu, Z.,
Horaist, C.,
Ballinger, A.,
Brasier, A. R.,
Bode, C.,
and Runge, M. S.
(1999)
J. Biol. Chem.
274,
19814-19822 |
59. |
Zhu, T.,
and Lobie, P. E.
(2000)
J. Biol. Chem.
275,
2103-2114 |
60. | Sengupta, T. K., Talbot, E. S., Scherle, P. A., and Ivashkiv, L. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 965, 11107-11112[CrossRef] |
61. | Morano, K. A., and Thiele, D. J. (1999) Gene Expr. 7, 271-282[Medline] [Order article via Infotrieve] |
62. | Wei, Y. Q., Zhao, X., Kariya, Y., Teshigawara, K., and Uchida, A. (1995) Cancer Immunol. Immunother. 40, 73-78[CrossRef][Medline] [Order article via Infotrieve] |
63. | van Dongen, G., and van Wijk, R. (1988) Radiat. Res. 113, 252-267[Medline] [Order article via Infotrieve] |
64. | Aligue, R., Akhavan-Niak, H., and Russell, P. (1994) EMBO J. 13, 6099-6106[Abstract] |
65. | Pratt, W. B. (1998) Proc. Soc. Exp. Biol. Med. 217, 420-434[Abstract] |
66. | Galea-Lauri, J., Latchman, D. S., and Katz, D. R. (1996) Exp. Cell Res. 226, 243-254[CrossRef][Medline] [Order article via Infotrieve] |
67. |
Donzé, O.,
and Picard, D.
(1999)
Mol. Cell. Biol.
19,
8422-8432 |