(Received for publication, October 15, 1995; and in revised form, October 28, 1995)
From the
Adult rat ventricular myocytes and cardiac microvascular
endothelial cells (CMEC) both express an inducible nitric oxide
synthase (iNOS or NOS2) following exposure to soluble inflammatory
mediators. However, NOS2 gene expression is regulated differently in
response to specific cytokines in each cell type. Interleukin-1
(IL-1
) induces NOS2 in both, whereas interferon
(IFN
)
induces NOS2 expression in myocytes but not in CMEC. Therefore, we
examined the specific signal transduction pathways that could regulate
NOS2 mRNA levels, including activation of 44- and 42-kDa
mitogen-activated protein kinases (MAPKs; ERK1/ERK2) and STAT1
, a
transcriptional regulatory protein linked to cell membrane receptors.
Although IL-1
treatment increased ERK1/ERK2 activities in both
cell types, IFN
activated these MAPKs only in myocytes. STAT1
phosphorylation, consistent with IFN
-induced signaling, was
readily apparent in both cell types, and binding of activated
STAT1
from cytoplasmic or nuclear fractions from IFN
-treated
adult myocytes to a sis-inducible element could be
demonstrated by gel-shift assay. The farnesyl transferase inhibitor
BZA-5B blocked activation of ERK1/ERK2 and induction of NOS2 by
IFN
and IL-1
in myocytes. IL-1
and IFN
-induced NOS2
gene expression in myocytes was also down-regulated by both protein
kinase C (PKC) desensitization and by the PKC inhibitor
bisindolylmaleimide, implicating PKC-linked activation of Ras or Raf in
the induction of NOS2 by IL-1
and IFN
in cardiac muscle
cells. In CMEC, the MAPK kinase inhibitor PD 98059 blocked activation
of ERK1/ERK2 and down-regulated IL-1
-mediated NOS2 induction,
whereas activation of ERK2 in the absence of cytokines by okadaic acid,
an inhibitor of phosphoserine protein phosphatases, also induced NOS2
mRNA. These data demonstrate that ERK1/ERK2 activation appears to be
necessary for the induction of NOS2 by IL-1
and IFN
in
cardiac myocytes and CMEC. In the absence of ERK1/ERK2 activation by
IFN
in CMEC, phosphorylation of STAT1
is not sufficient for
NOS2 gene expression. These overlapping yet distinct cellular responses
to specific cytokines may serve to target NOS2 gene expression to
specific cells or regions within the heart and also provide for rapid
escalation of NO production if required for host defense.
Both cardiac myocytes and microvascular endothelial cells
isolated from adult rat ventricular muscle express the
cytokine-inducible form of nitric oxide synthase (iNOS or NOS2) ()both in vivo and in primary culture, although the
regulation of NOS2 gene expression in response to specific cytokines is
regulated differently in these two cell types. Interleukin-1
(IL-1
) treatment induces NOS2 in both cell types, whereas
interferon
(IFN
) induces NOS2 in ventricular myocytes but
not in CMEC. However, IFN
does augment NOS2 induction by IL-1
in CMEC(1, 2, 3, 4, 5) . To
gain insight into the mechanisms regulating NOS2 induction by cytokines
in both cell types, we studied two distinct signal transduction
pathways: activation of p44/p42 mitogen-activated protein kinases
(MAPKs; or extracellular signal-regulated kinases,
ERK1/ERK2)(6, 7, 8, 9, 10) ,
and the tyrosine phosphorylation of STAT1
(signal transducer and
activator of transcription-1
;
-activating factor; GAF; a
91-kDa protein, p91) (11) . The murine macrophage NOS2 gene
promoter region has been shown to contain two AP-1 sites for which
trans-acting transcriptional factors are regulated by MAPKs and three
IFN
-activated sites for STAT1
binding(12) .
The 44- and 42-kDa MAPK (ERK1/ERK2) isoforms are ubiquitously expressed serine/threonine protein kinases, activated by dual specificity MAPK kinases (MEK1/MEK2) in response to diverse stimuli. A number of receptor tyrosine kinases, cytokine receptors, and heterotrimeric G proteins have been shown to activate MEK1/MEK2 and MAPKs(10, 13, 14) . In neonatal rat cardiac myocytes, several endogenous hypertrophic stimuli have also been shown to activate MAPKs(15, 16, 17, 18, 19, 20) . Among other actions, activated MAPKs translocate to the nucleus(21, 22) , where they can phosphorylate downstream kinases that directly activate transcription factors.
A
number of cytokines (e.g. IFNs, IL-6, leukemia inhibitory
factor, and colony stimulating factor 1) and growth factors (e.g. epidermal growth factor and platelet-derived growth factor) have
been shown to tyrosine phosphorylate STAT1 through the activation
of Janus family kinases, JAK1 and
JAK2(11, 23, 24, 25) . The STAT
family of signal transduction proteins are substrates for the JAK
kinases, with specific STAT isoforms acting to provide specificity for
cytokine receptor-mediated signaling. Depending on the identity of the
activated cytokine receptor and JAK recruited to the membrane, specific
STAT isoforms form either heterodimers or homodimers and bind to
promoter elements of specific genes. With IFN
signaling, activated
STAT1
forms homodimers, translocates to the nucleus, and binds to
IFN
-activated site elements of IFN
-responsive
genes(26, 27) .
In this report, we present evidence
that activation of ERK1/ERK2 (MAPKs) is essential for the induction of
NOS2 gene expression in response to IL-1 and IFN
in adult rat
ventricular myocytes and cardiac microvascular endothelial cells.
Activation of STAT1
itself is not sufficient for NOS2 gene
expression, although it can act synergistically to increase NOS2 mRNA
in the presence of activated ERK1/ERK2 in both cell types.
CMEC from adult rat hearts were isolated as described by
Nishida et al.(29) . Briefly, after removing the
atria, valvular tissue, and right ventricle, the left ventricle was
immersed in 70% ethanol for 10 s to devitalize epicardial mesothelial
and endocardial endothelial cells. After peeling off the outer
ventricular wall, the remaining tissue was finely minced and treated
with collagenase and trypsin in Ca-free Hanks'
balanced salt solution (Life Technologies, Inc.). Dissociated cells
were washed and resuspended in DMEM containing 20% fetal calf serum and
antibiotics and plated on laminin (1 µg/cm
)-coated
dishes. After reaching confluency, CMEC were serum-starved for 24 h
before treatment with reagents for MAPK and STAT1
assays. For
northern analyses, confluent cells were serum-starved for 4 h before
treatment with cytokines or okadaic acid for 16 h.
Figure 1: Activation of ERK1/ERK2 by phorbol esters in adult ventricular myocytes. Adult rat ventricular myocytes were cultured for 24 h in defined medium before treatment with 200 ng/ml TPA or equal amounts of solvent dimethyl sulfoxide (DMSO) for the indicated times. Total cell lysates were analyzed by in-gel MBP kinase assay as described under ``Experimental Procedures.'' Lane 0 represents baseline ERK1/ERK2 activity in untreated myocytes at time 0. Cell lysates prepared from NIH 3T3 cells that were confluent and serum-starved for 24 h and then exposed to 10% fetal calf serum for 10 min were used as positive controls for ERK1/ERK2 activation (3T3). The positions of ERK1/ERK2 (p44/p42 MAPKs) on the gel are shown.
Earlier reports from this laboratory have
documented that cytokines (e.g. IFN, IL-1
, and
IFN
+ IL-1
) can induce the expression of NOS2 in adult
rat ventricular myocytes(1) . We examined the ability of these
cytokines to activate MAPK in these cells, as shown in Fig. 2A. Untreated cardiac myocytes cultured in
serum-free defined medium have some detectable ERK1/ERK2 activity at
baseline using the in-gel MAPK assay technique, and this activity could
be enhanced by a 15-min exposure of these cells to either cytokine and
to the
-adrenergic agonist phenylephrine as a positive control.
Both rmIFN
(500 units/ml) and rhIL-1
(4 ng/ml) activated
MAPKs over control cells. To verify these results obtained by the
in-gel MAPK assay, the activation of ERK1/ERK2 by IFN
was further
studied by an immune complex MAPK assay. Cell lysates prepared after a
15-min exposure to IFN
were immunoprecipitated by anti-ERK2
antibodies. Immune complexes were then incubated with a reaction
mixture containing MBP and [
-
P]ATP. The
results shown in Fig. 2B confirm that ERK1/ERK2
activity is increased in IFN
-treated cells when compared with
untreated cells. As expected, an increase in c-fos mRNA levels
could be detected within 30 min of addition of IFN
to myocyte
primary isolates (Fig. 2C).
Figure 2:
Activation of ERK1/ERK2 and c-fos induction by cytokines in ventricular myocytes. A,
analysis of ERK1/ERK2 activity. Adult ventricular myocytes were treated
for 15 min with defined medium alone (lane C), rmIFN (500
units/ml, IFN), rhIL-1
(4 ng/ml, IL-1), or the
-adrenergic agonist phenylephrine (10 µM) with 1
µM propranolol (Phe). Total cell lysates (100
µg) were analyzed by in-gel MBP kinase assay, and the positions of
the p42/p44 MAPKs are identified. The experiment was performed three
times with similar results. B, immune complex assay of
ERK1/ERK2 activity. Myocyte cell lysates prepared from rmIFN
(500
units/ml)-treated (IFN) or untreated (lane C) cells
were immunoprecipitated with anti-ERK2 antibodies. Immune complexes
thus obtained were incubated in a kinase buffer containing
[
-
P]ATP and MBP. Phosphorylated MBP was
analyzed by SDS-PAGE and autoradiography. C, c-fos mRNA levels in IFN
-treated myocytes. In addition to
activation of ERK1/ERK2, changes in levels of c-fos mRNA were
examined by Northern blot. Total RNA was extracted from myocytes
exposed to IFN
for 0, 15, and 30 min, and 15-µg samples were
analyzed using
P-labeled c-fos and
glyceraldehyde-phosphate dehydrogenase
probes.
Figure 3:
Activation of ERK1/ERK2 and c-fos induction by cytokines in CMEC. A, ERK1/ERK2 activation.
Confluent 24 h serum-starved CMEC were treated with DMEM (lane
C), rhIL-1 (4 ng/ml, IL-1), rmIFN
(500
units/ml, IFN), or a combination of cytokines (IFN,
IL-1) for 15 min. Total cell lysates (60 µg) were analyzed by
in-gel MBP kinase assay, and the positions of p42/p44 MAPKs are
identified. The experiment was performed three times with similar
results. B, c-fos mRNA levels in IFN
-treated
CMEC. c-fos mRNA levels were examined by Northern blot in
confluent serum-starved CMEC exposed to 500 units/ml of rmIFN
for
0, 15, or 30 min. Total RNA was extracted, and 15 µg were analyzed
using
P-labeled c-fos and
glyceraldehyde-phosphate dehydrogenase
probes.
Figure 4:
STAT1 (p91) phosphorylation in
ventricular myocytes and CMEC. Cell lysates prepared from
cytokine-treated (15 min) or untreated cells were immunoprecipitated
with anti-STAT1 (p91/84) antibodies. Immunoprecipitates were analyzed
by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies as
described under ``Experimental Procedures.'' A and B, phosphorylation of STAT1
(p91) in ventricular
myocytes. A, cells were treated with defined medium alone (lane C), with phenylephrine (Phe), or with 500
units/ml rmIFN
(IFN). B, myocytes were treated
with 500 units/ml rmIFN
(IFN), 4 ng/ml rhIL-1
(IL-1), or a combination of these two cytokines (IL-1,
IFN). C, phosphorylation of STAT1
(p91) in CMEC.
Confluent serum-starved cells were exposed to defined medium (lane
C), 500 units/ml of rmIFN
(IFN), 4 ng/ml of
rhIL-1
(IL-1), or a combination of 500 units/ml
rmIFN
with 4 ng/ml rhIL-1
(IL-1,
IFN).
To determine whether this phosphorylated STAT isoform can bind DNA,
electrophoretic mobility gel-shift assays were carried out (Fig. 5). A protein from the nuclear as well as cytoplasmic
fractions prepared from IFN-pretreated adult ventricular myocytes
bound the radiolabeled oligonucleotide designed to bind STAT1
,
whereas no protein from control cytoplasmic or nuclear fractions bound
this oligonucleotide. The identity of factor(s) responsible for
altering the mobility of the oligonucleotide was confirmed by
supershift of this protein by anti-STAT1
(p91) antibodies. Thus,
in cardiac myocytes IFN
induces activation of ERK1/ERK2 and
activation and binding of STAT1
to DNA elements.
Figure 5:
DNA binding of IFN-activated
STAT1
(p91) in ventricular myocytes. DNA binding activity was
detected in both cytoplasmic and nuclear fractions from adult
ventricular myocytes, which were prepared 15 min after exposure to 500
units/ml rmIFN
(IFN) or control medium alone (lane
C). Total proteins (10 µg) were incubated for 30 min at room
temperature with a double-stranded
P-labeled
oligonucleotide probe. In the reaction mix of the last lane,
anti-STAT1
antibodies were added after 20 min, and the reaction
proceeded for an additional 10 min.
Figure 6:
Effect of the farnesyl transferase
inhibitor BZA-5B on ERK1/ERK2 activation and NOS2 mRNA levels in
cytokine-treated ventricular myocytes. A, BZA-5B inhibits
activation of ERK1/ERK2 by IFN. Ventricular myocytes were exposed
to defined medium containing rmIFN
(500 units/ml, IFN) or
defined medium containing 25 µM BZA-5B (BZA) for
15 min. Cells were pretreated for 15 min with 25 µM of
BZA-5B and then with rmIFN
(500 units/ml) for another 15 min (IFN, BZA). Total cell lysates were then analyzed by the
in-gel MBP kinase assay. The positions of p42/p44 MAPKs were identified
by using CMEC exposed to DMEM containing 10% fetal calf serum for 10
min (Serum). The experiment was performed twice with similar
results. B, BZA-5B inhibits NOS2 induction by IFN
.
Ventricular myocytes were exposed to defined medium alone (lane
C) or defined medium containing 500 units/ml of IFN
(IFN) for 16 h. Cells were pretreated with 25 µM BZA-5B for 1 h and then exposed to rmIFN
(500 units/ml) for
16 h (IFN, BZA). Total cellular RNA was then used for Northern
blot analysis using NOS2 cDNA and 18 S rRNA probes. This experiment was
performed twice with similar results. C, BZA-5B inhibits
activation of ERK1/ERK2 by IL-1
. Ventricular myocytes were exposed
to defined medium alone (lane C) or defined medium containing
IL-1
(4 ng/ml) for 5 (IL-1 5`) and 15 min (IL-1
15`). Cells were pretreated for 15 min with 25 µM of
BZA-5B and then with IL-1
(4 ng/ml) for another 15 min (BZA,
IL-1, 15`). Total cell lysates were then analyzed by the in-gel
MBP kinase assay. D, BZA-5B inhibits NOS2 induction by
IL-1
. Ventricular myocytes were exposed to defined medium alone (lane C) or defined medium containing 4 ng/ml of IL-1
(IL-1) for 16 h. Cells were pretreated with 25 µM BZA-5B for 1 h and then exposed to IL-1
(4 ng/ml) for 16 h (IL-1, BZA). Total cellular RNA was then used for Northern
blot analysis using NOS2 cDNA and 18 S rRNA
probes.
Figure 7:
Effect of okadaic acid (OA) and
PD 98059 (MEK inhibitor) on ERK1/ERK2 activation and NOS2 induction in
CMEC. A, ERK1/ERK2 activation by okadaic acid. Confluent CMEC
that had been serum-starved for 24 h were treated with DMEM alone for
15 or 0 min (C15 and CO, respectively) or DMEM
containing 100 nM okadaic acid alone for 5, 15, and 30 min (OA5, OA15, and OA30, respectively). Cells
were pretreated with okadaic acid for 15 min and then IFN for 15
min (OA+IFN). Cell lysates were prepared and analyzed by
in-gel MBP kinase assay. The positions of p42/p44 MAPKs were identified
by using CMEC exposed to DMEM containing 10% fetal calf serum for 10
min (Serum). This experiment was performed three times with
similar results. B, OA induces NOS2 mRNA. Confluent 24 h
serum-starved CMEC were incubated with okadaic acid alone (OA)
or in combination with rmIFN
(500 units/ml) (OA, IFN) for
16 h. Total RNA (15 µg) was analyzed by Northern blot using
radiolabeled NOS2 cDNA and 18 S rRNA probes. Lanes C and IFN represent RNA from untreated or IFN
-treated cells.
This experiment was performed three times with similar results. C, PD 98059 inhibits activation of ERK1/ERK2 by IL-1
.
Confluent CMECs that had been serum-starved for 24 h were exposed to
100 µM of PD 98059, an inhibitor of MEK, for 15 min (PD). The cells were pretreated for 30 min with 100 µM of PD 98059 or vehicle (dimethyl sulfoxide) and then treated with
IL-1
(4 ng/ml) for another 15 min (PD, IL-1 and DMSO,
IL-1). Total cell lysates were analyzed by the in-gel MBP kinase
assay. D, PD 98059 inhibits NOS2 induction by IL-1
.
Confluent 3 h serum-starved CMEC were pretreated for 1 h with 100
µM PD 98059 or vehicle (dimethyl sulfoxide) before
exposing the cells to 4 ng/ml of IL-1
for 16 h. Lane C represents untreated cells. Total cellular RNA was then used for
Northern blot analysis using NOS2 cDNA and 18 S rRNA
probes.
To further
address the role of MAPKs in cytokine-mediated NOS2 induction in CMEC,
we used the MAPK kinase (MEK) inhibitor PD 98059 (39, 40) . This compound is a specific inhibitor of
the activation of MAPK kinases in vitro and in
vivo(41) . At concentrations above 50 µM, PD
98059 has been shown to inhibit both MEK1 and MEK2 by binding to a
regulatory site on the enzyme and preventing activation by c-Raf and
MEK kinase(41) . Pretreatment of CMEC for 30 min with PD 98059
(100 µM) almost completely inhibited IL-1 activation
of ERK1/ERK2 in these cells (Fig. 7C). Pretreatment
with PD 98059 also suppressed IL-1
-mediated induction of NOS2 by
approximately 70% as shown in Fig. 7D.
Figure 8:
Involvement of PKC in cytokine-induced
NOS2 gene expression in ventricular myocytes. A, effect of
down-regulation of PKC by TPA on IL-1-induced NOS2 expression.
Ventricular myocytes were exposed to defined medium alone (lane
C) or left untreated for 24 h before adding 4 ng/ml of IL-1 (IL-1) for a further 16 h. Cells were pretreated with 50 ng/ml
TPA for 24 h and then exposed to 4 ng/ml of IL-1
(TPA,
IL-1) in the same medium for 16 h. The cells in TPA lane
were exposed to 50 ng/ml TPA for 40 h. B, effect of
down-regulation of PKC by TPA on IFN
-induced NOS2 expression.
Ventricular myocytes were exposed to defined medium alone (lane
C) or left untreated for 24 h before adding 500 units/ml of
IFN
(IFN) for a further 16 h. Cells were pretreated with
50 ng/ml of TPA for 24 h and then exposed to 500 units/ml of IFN
(TPA, IFN) in the same medium for 16 h. The cells in lane TPA were exposed to 50 ng/ml TPA for 40 h. C, effect
of BIM on cytokine-induced NOS2. Ventricular myocytes were pretreated
with 500 nM of BIM for 1 h before treating cells with 4 ng/ml
of IL-1
(BIM, IL-1) or with 500 units/ml of IFN
(BIM, IFN) for 16 h. The cells were also treated with 4 ng/ml
of IL-1
alone (IL-1) or 500 units/ml of IFN
alone (IFN) for 16 h. Lane C represents untreated cells. In
all cases, total cellular RNA was used for Northern blot analysis using
NOS2 cDNA and 18 S rRNA probes.
To examine further the role of PKCs in NOS2 induction,
both the nonselective protein kinase inhibitor H7 and
bisindolylmaleimide (BIM), a relatively selective inhibitor of
Ca-dependent PKC
isoenzymes(42, 43) , were used. H7 prevented induction
of NOS2 in cardiac myocytes by IFN
(data not shown). Treatment of
myocytes with 500 nM BIM in combination with IFN
inhibited NOS2 induction by approximately 50% (Fig. 8C). However, BIM had no effect on NOS2 induction
by IL-1
in cardiac myocytes (Fig. 8C) or in CMEC
(data not shown).
The data reported here suggest that in response to IL-1
or IFN
, induction of NOS2 expression in cardiac myocytes and
microvascular endothelial cells, two of the most prevalent cell types
in heart muscle, requires activation of 44- and 42-kDa MAP kinases
(ERK1/ERK2). This conclusion is based on the following observations: 1)
IL-1
and IFN
independently activate ERK1/ERK2 and increase
NOS2 mRNA abundance in cardiac myocytes; 2) IL-1
but not IFN
activates ERK1/ERK2 and increases NOS2 mRNA levels in CMEC; 3)
inhibition of IFN
- and IL-1
-linked signaling proteins leading
to activation of ERK1/ERK2 in cardiac myocytes (i.e. PKCs and Ras) also
inhibited IFN
- and IL-1
-induced NOS2 expression in these
cells; 4) nonreceptor-mediated activation of ERK2, induced by the
phosphoserine protein phosphatase inhibitor okadaic acid, induced NOS2
expression in CMEC; and 5) inhibition of IL-1
-induced activation
of MEK and ERK1/ERK2 in CMEC by PD 98059 also suppressed NOS2 induction
in these cells.
The role of ERK1/ERK2 in NOS2 induction was somewhat unexpected, due to recent reports that growth promoting factors known to activate MAPKs in a number of different cell types, such as angiotensin II, basic fibroblast growth factor, and phorbol esters, decrease NOS2 mRNA levels(44, 45, 46, 47, 48) . Although the decline in cytokine-induced NOS activity with growth factors could be correlated with entry into the cell cycle and increased cellular proliferation in some reports, this did not appear to be the explanation in one report of confluent serum-starved rat aortic smooth muscle cells exposed to inflammatory cytokines(47) . However, in PC12 cells nerve growth factor, which is known to induce ERK1/ERK2 in these cells, has been reported recently to increase transcription of several NOS isoforms, including NOS2, suggesting that one or more NOS isoforms could be acting as a growth arrest gene, initiating the switch to cytostasis during differentiation(49, 50, 51) . Also, in inflammatory cells (murine peritoneal macrophages), induction of NOS2 by lipopolysaccharide correlated with ERK2 (p42 MAPK) phosphorylation. Both effects of lipopolysaccharide could be inhibited by the tyrphostin class of tyrosine kinase inhibitors(52) .
Activation of
ERK1/ERK2 MAPKs alone cannot be sufficient for NOS2 induction by
cytokines. Phorbol esters, which activate diacylglycerol-responsive PKC
isoforms and which subsequently can induce Ras/Raf-mediated activation
of MEK1/MEK2 and ERK1/ERK2, do not induce NOS2 expression in myocytes
or in CMEC (data not shown). Presumably, these agents are not able to
simultaneously activate other regulatory factors required for NOS2
promoter activation. IL-1-induced NOS2 gene expression has been
shown to involve PKC-dependent and PKC-independent mechanisms in
different cell types(53, 54) . In cardiac myocytes,
IL-1
-induced NOS2 expression is in part dependent on PKC
activation, whereas in CMEC, IL-1
induction of NOS2 appears to be
mediated by PKC-independent mechanisms. IL-1
, which does not
activate STAT1
but does increase ERK1/ERK2 activities in both cell
types, is also known to activate NF-
B signaling in many different
cell types(52, 55) . This pathway is likely to play a
role in NOS2 induction by IL-1
in both cardiac myocytes and in
CMEC.
In adult cardiac myocytes (i.e. cells that are not
competent to re-enter the cell cycle), IFN activates both
ERK1/ERK2 and STAT1
signaling pathways. The nuclear factor from
IFN
-treated myocytes that bound to a double-stranded
oligonucleotide was positively identified by a STAT1
antibody-induced supershift on gel-shift assay (Fig. 5). This is
presumably mediated by recruitment to type II cytokine receptors of
JAK1 and/or JAK2 phosphotyrosine kinases that subsequently tyrosine
phosphorylate and activate STAT1
(27) . Angiotensin II also
has been shown recently to activate STAT1
/
(p91/p84)
following JAK2 phosphorylation in rat aortic smooth muscle cells and in
neonatal rat cardiac fibroblasts, although the time course of JAK2
tyrosine phosphorylation in smooth muscle cells was significantly
shorter than with activation of this pathway by IFN
(56, 57) . Both epidermal growth factor and
platelet-derived growth factor, cytokines that initiate intracellular
signal transduction at phosphotyrosine kinase receptors, have been
reported to activate STAT1
in Swiss 3T3 cells, and epidermal
growth factor activates STAT3 in rat aortic smooth muscle
cells(56) . However, epidermal growth factor did not activate
STAT1
signaling in adult cardiac myocytes and microvascular
endothelial cells using the experimental conditions we describe here. (
)
The pathway(s) by which IFN activates ERK1/ERK2 in
cardiac myocytes is not known. The ability of the farnesyl transferase
inhibitor BZA-5B to inhibit ERK1/ERK2 activation by IFN
suggests
Ras-mediated membrane recruitment and activation of Raf-1 (MAPK kinase
kinase or MEK kinase). All four Ras proteins and Raf-1 are farnesylated
at CAAX motifs (ras is also myristoylated), although
there appear to be important differences among the Ros proteins in
their sensitivity to this drug(58, 59) . Also,
inactivation of Raf has not been demonstrated to date with BZA-5B.
However, the ability of phorbol ester pretreatment (i.e. PKC
desensitization) and of bisindolylmaleimide to block IFN
-mediated
induction of NOS2 suggests that activation of a
diacylglycerol-regulated PKC isoform is required(60) . Type II
cytokine receptors (i.e. type II interferon receptors
/
) are not trimeric G protein-coupled receptors that could
initiate phospholipid signaling by activating phospholipase C
isoforms. Nor are they phosphotyrosine kinases that could recruit
proteins with src-homology (SH2) domains, such as phospholipase C
isoforms or phosphoinositide 3-kinase(61, 62) .
However, after receptor activation and oligomerization, phosphotyrosine
kinases such as JAK could phosphorylate tyrosine residues on the
cytosolic domain of these receptors, which could then initiate
phospholipid signaling and PKC activation after binding and activation
of phospholipase C
and other proteins(27) . Although not
proven by the data reported here, it is likely that activation of
phospholipid signaling and STAT1
recruitment and phosphorylation
together act to induce NOS2 expression with IFN
in ventricular
myocytes.
In microvascular endothelial cells, IL-1-mediated
activation of ERK1/ERK2 could be blocked by the MEK inhibitor PD 98059
and the extent of NOS2 induction reduced by 70%, suggesting that CMEC
IL-1
-induced NOS2 gene expression is at least partially dependent
on Ras/Raf-mediated signaling. Although IFN
alone does not induce
ERK1/ERK2 phosphorylation or increase NOS2 mRNA abundance in CMEC, we
have shown previously that this cytokine accelerates the time course
and extent of NOS2 mRNA accumulation and protein activity in
combination with IL-1
(3, 4) . The ability of
IFN
to also potentiate the increase in NOS2 mRNA accumulation
induced by the phosphoserine protein phosphatase inhibitor okadaic
acid, which acts directly to inhibit dephosphorylation of MEKs and
ERK1/ERK2 downstream from PKC, supports the notion that IFN
signaling is mediated by a non-ERK1/ERK2-dependent signaling pathway in
these cells, one of which is presumably mediated by STAT1
. The
suppression of IFN
-induced NOS2 expression in cardiac myocytes
also exposed to okadaic acid emphasizes the differences among cell
types in the balance of activities of protein kinases and
phosphatases(63) .
The apparent redundancy of IL-1 and
IFN
signaling in ventricular myocytes, at least with respect to
NOS2 expression coupled with important differences in signaling
initiated by these cytokines in other cell types such as CMEC, termed
pleiotropy by Taniguchi(27) , is likely necessary to provide
specificity and to regulate the intensity of host defense mechanisms.
If the phenotype of these endothelial cells is low passage, confluent
primary cultures that are representative of the capillary endothelium in vivo, which does express NOS2 abundantly in several
experimental animal models(3, 64) , then cell
type-specific cytokine signaling for NOS2 induction probably occurs in situ within cardiac muscle as well. This would make sense
biologically because selectively increased microvascular endothelial
production of NO or related congeners would elicit both local
vasodilation and increased vascular permeability, among other actions
that are necessary for the early stages of an inflammatory response.
Unrestricted production of NO by infiltrating inflammatory cells and/or
by microvascular endothelial cells can directly impair the contractile
function of adjacent cardiac myocytes, as has been shown in short term
primary heterotypic culture models(4) . In addition, high
levels of NOS2 induction in the heart, which appear to occur with high
blood and tissue levels of cytokines, as occurs in the systemic
inflammatory response syndrome, and which may occur in cardiac
allograft rejection as well, will result in global dysfunction of the
heart and is often clearly detrimental to the organism(65) .
Therefore, the selective activation of ERK1/ERK2 we observed in
response to IFN
in cardiac myocytes in vitro but not in
microvascular endothelial cells illustrates one mechanism by which the
expression could be limited to a specific cell type within the heart
and other tissues.