Departments of 1 Pediatrics, 3 Surgery, and 4 Environmental and Occupational Health, University of Pittsburgh School of Medicine and Graduate School of Public Health, Pittsburgh, Pennsylvania 15261; 5 Drug Discovery Program, H. Lee Moffitt Cancer Center, Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida 33612; and 2 Department of Chemistry, Yale University, New Haven, Connecticut 06520
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interleukin (IL)-1
is an important early mediator of inflammation in pulmonary artery
smooth muscle cells. We previously reported that a
geranylgeranyltransferase inhibitor elevated basal levels of inducible
nitric oxide synthase (iNOS) and enhanced IL-1
-mediated induction,
suggesting that Rac or Rho small G proteins are candidates for
antagonism of such induction. In this study, overexpression of
constitutively active Rac1 or its dominant negative mutant did not
affect IL-1
induction of iNOS. Alternatively, treatment with
Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho, was associated with superinduction of iNOS, suggesting an inhibitory role for Rho. IL-1
activated the three mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2, c-Jun
NH2-terminal kinase/stress-activated protein kinase, and p38) and the
Janus kinase (JAK)-signal transducer and activator of transcription
pathways. The former two pathways were not associated with
IL-1
-mediated iNOS induction, whereas the latter two appeared to
have inhibitory roles in iNOS expression. These data suggest that a
broad intracellular signaling response to IL-1
in rat pulmonary
artery smooth muscle cells results in elevated levels of iNOS that is
opposed by the geranylgeranylated small G protein Rho as well as the
p38 and JAK2 pathways.
inducible nitric oxide synthase; interleukin-1; Rho; mitogen-activated protein kinase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
INTERLEUKIN (IL)-1
is an important early mediator of inflammation in a variety of cell
types including pulmonary artery smooth muscle. IL-1
mediates
inflammation, at least in part, by inducing expression of inducible
nitric oxide (NO) synthase (iNOS), with concordant enhanced synthesis
of NO, a critical effector molecule. In particular, we have shown that
IL-1
alone, in sufficient quantities, in cultured rat pulmonary
artery smooth muscle cells (RPASMCs) is capable of 1)
inducing iNOS (33, 47); 2) increasing
production of superoxide anion as well as of NO, resulting in the
production of peroxynitrite (7); and 3)
producing sufficient NO to be toxic to cocultures of rat pulmonary
artery endothelial cells (22, 43).
IL-1 results in activation of a complex array of intracellular
signaling molecules (35) including several GTPases and
serine/threonine kinases in a tissue- and cell-specific fashion
(38, 39). Relatively little is known, however, regarding
the mechanism by which IL-1
affects iNOS expression in pulmonary
vascular smooth muscle. In this regard, we used novel synthetic
isoprenoid inhibitors to show that small GTP-binding proteins of the
Ras/Rho superfamily are involved in the regulation of iNOS
(16) and the synthesis of superoxide anion
(6) by IL-1
in RPASMCs. Inhibition of geranylgeranyltransferase resulted in superinduction of iNOS
(16) and inhibition of superoxide anion (6),
perhaps accounting for the inhibition in growth and promotion of
apoptosis in these cells by IL-1
(40). In the
present study, we focused on the role of Rho, a geranylgeranylated
protein, that was coimmunoprecipitated with the IL-1 receptor after
IL-1 stimulation in HeLa cells (39) and appeared to act as
a negative regulator of iNOS expression in human tumor cell lines
(19) and systemic vascular smooth muscle cells
(32).
IL-1 stimulation has been associated with several of the
mitogen-activated protein (MAP) kinase (MAPK) cascades involved in the
transmission of signal from the cell surface to the nucleus. The main
signaling cascades are referred to by their ultimate protein kinase and
include extracellular signal-regulated kinase (ERK) 1/ERK2
(p44MAPK/p42MAPK) kinase, p38 kinase, and c-Jun
NH2-terminal kinase (JNK)-stress-activated protein kinase (SAPK).
Although these pathways have not been demonstrated to be involved in
IL-1
-mediated effects on pulmonary vascular smooth muscle, it is
noteworthy that p38 and ERK contribute to such effects in human
(24) or canine (20) airway or myometrial (3) smooth muscle. Signal transducer and activator of
transcription (STAT) proteins are critical signaling molecules in most
cytokine-mediated responses. Activation of STAT proteins usually
involves a cytokine receptor-associated kinase [Janus kinase (JAK)],
with subsequent serine phosphorylation by less well-defined kinases
(8). In general, it has not been possible to place STATs
clearly in the MAPK cascade (12), although the
coactivation and contributions of these pathways are evident. In
particular, the JAKs, which act through the associated STAT proteins
(9), have, until recently, not been associated with
IL-1
signaling (10). Although the mechanism of STAT
involvement in IL-1 signaling remains obscure, two recent reports
(31, 44) have suggested an involvement of STAT in the IL-1
receptor signaling pathway. Both the MAPK and STAT pathways are
components of the signaling pathways of IL-1
induction of iNOS,
including p38 in cardiac myocytes (24), mesangial cells
(18), chondrocytes (2) and islet cells
(26); JNK/SAPK in mesangial cells (18);
ERK1/ERK2 in rat islet cells (26) and cardiac myocytes
(24); and JAK-STAT in interferon (IFN)-
-primed rat
islet cells (21). Accordingly, we examined the
contributions of these complex pathways to iNOS regulation in RPASMCs.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All experiments were performed in triplicate unless noted. Representative experimental results are shown.
Cell culture. RPASMCs were isolated from explants of the intrapulmonary arteries of adult male Sprague-Dawley rats. The explants were placed endothelium side down in flasks containing low-glucose DMEM-Ham's F-12 medium (1:1; GIBCO BRL, Life Technologies, Grand Island, NY), 10% fetal bovine serum (HyClone, Logan, UT), 4 mM L-glutamine, 5 U/ml of penicillin, and 5 µg/ml of streptomycin (Sigma, St. Louis, MO), grown to confluence, and subpassaged. The cells were confirmed as smooth muscle by positive immunostaining for the smooth muscle isoforms of actin and myosin.
Chemical inhibitors. The MAPK kinase 1 inhibitor PD-98059 was purchased from New England BioLabs (Beverly, MA) and was used at a concentration of 10 µM in all experiments. The JAK2 inhibitor AG-490 (tyrphostin) and the p38 inhibitor SB-203580 were purchased from Calbiochem (La Jolla, CA). AG-490 was used at a concentration of 10 µM for all experiments. SB-20350 was used at a concentration of 10 µM for all experiments. The geranylgeranylation inhibitors GGTI-298 and GGTI-2166 and the farnsyltransferase inhibitor FTI-277 were produced by A. Hamilton. They were used at concentrations of 10, 15, and 10 µM, respectively.
Western blotting.
The cell monolayers were lysed in ice-cold buffer [50 mM Tris, pH 8.0;
110 mM NaCl; 5 mM EDTA; 1% Triton X-100; and the protease inhibitors
antipain, pepstatin, leupeptin, chymostatin, and phenylmethylsulfonyl fluoride (PMSF)], scraped into 1.5-ml sample tubes, and centrifuged to
remove cellular debris. Protein levels of the supernatants were then
determined. After being boiled in Laemmli buffer, 8-25 µg of the
whole cell extract were separated on 7.5% polyacrylamide gels,
transferred to nitrocellulose, and immunoblotted with an affinity-purified rabbit polyclonal antibody to murine macrophage iNOS
(Transduction Laboratories, Lexington, KY). The immunoblot was
developed with Renaissance brand chemiluminescence reagent (NEN Life Science Products, Boston, MA) with secondary antibody, peroxidase-labeled goat anti-rabbit IgG (Sigma). Western blotting for
p38 and phospho-p38 was performed with rabbit polyclonal anti-p38 and
anti-phospho-p38 antibodies (New England Biolabs), the secondary antibody, and enhanced chemiluminescence as described above. For iNOS
Western blotting, the cells were exposed to IL-1 at a concentration of 10 nM (R&D Systems, Minneapolis, MN) for 24 h; for kinase
assays, exposure to IL-1
was 15 min. Antibodies to c-Myc 9E10
epitope and Rho were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). A nonradioactive JNK assay kit was purchased from New
England Biolabs.
Preparation of nuclear extracts. Nuclear extracts were prepared as described by Dignam et al. (15) with some modifications. Cultured cells were rinsed twice with ice-cold PBS, scraped into 1 ml of PBS, and microcentrifuged at 4,500 rpm for 5 min. The pelleted cells were resuspended in 1 ml of buffer A (10 mM Tris, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM PMSF, and 0.1 mM Na3VO4). The cells were microcentrifuged at 4,500 rpm for 5 min, resuspended in 0.5 ml of buffer A, and recentrifuged at 4,500 rpm for 5 min. The cells were resuspended and gently mixed for 10 min at 0°C in 100 ml of buffer A with 0.1% Triton X-100 to lyse the cell membranes. The nuclei were recovered by centrifugation at 7,000 rpm for 5 min. The nuclear proteins were extracted at 0°C by gently mixing the nuclei in 75 ml of buffer C (20 mM Tris, pH 7.5, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.1% Triton X-100, 25% glycerol, 0.5 mM dithiothreitol, 0.5 mM PMSF, and 0.1 mM Na3VO4). The supernatants were collected after 25 min of microcentrifugation at 14,500 rpm for 15 min. The supernatants were diluted 1:2 in a buffer containing 1 part buffer C to 2 parts buffer D (20 mM Tris, pH 7.5, 1.5 mM MgCl2, 25 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.5 mM dithiothreitol, 0.5 mM PMSF, and 0.1 mM Na3VO4). Protein concentration was measured with the Bradford assay.
Electrophoretic mobility shift assay.
Nuclear extracts (2.5 µg) were incubated with 1 µl (~50,000
counts/min) of 32P-labeled high-affinity serum-inducible
element (hSIE) oligonucleotide (~2 pM) for 30 min at room temperature
in a buffer containing 2 µl (2 mg) of poly(dI-dC) (Boehringer
Mannheim), 4 µl of buffer X (10 mM Tris, pH 7.5, 10%
glycerol, 1 mM EDTA, 0.5 mM dithiothreitol, and 1% Nonidet P-40), 0.5 ml of buffer D, and the remainder water (final volume 20 µl). hSIE preferentially binds STAT3 and STAT1 (46).
This probe was end labeled by a fill-in reaction with the Klenow
fragment of DNA polymerase I. For supershift assays, nuclear extracts
were incubated on ice for 1 h with 2 µl of anti-STAT3 antibody
(200 µg/0.1 ml), 2 µl of anti-STAT1 p84/p91 antibody (100 µg/ml),
1 µl of anti-STAT3 antibody, or 1 µl of anti-STAT3
antibody.
DNA-protein complexes were resolved on a 4% nondenaturing polyacrylamide gel in 0.4× Tris-borate-EDTA running buffer. After electrophoresis, the gels were dried, subjected to autoradiography, and
analyzed with a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
Kinase assays.
Cells in 75-cm2 culture flasks were rinsed once with
ice-cold PBS and then solubilized on ice for 5 min in 350 µl of lysis buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM -glycerophosphate, 1%
Triton X-100, and 10% glycerol) plus inhibitors (1 mM dithiothreitol, 2 mM Na3VO4, 1 mM PMSF, 1 mg/ml of leupeptin, 5 mg/ml of antipain, and 1 mg/ml of aprotinin). The lysates were
sonicated briefly and spun for 5 min at 10,000 rpm to remove debris.
The cleared lysates were assayed for protein concentration with the
Bradford assay. From each condition, 250 µg of protein were incubated
for 4 h on a rocking table at 4°C with agarose-conjugated
antibodies. For the MAPK assay, 10 µl of goat anti-rat ERK1 and ERK2
antibodies were used for each reaction. For the p38 kinase assay, 20 µl of goat anti-mouse p38 antibody were used. For the p46/p54 kinase assay, 20 µl of rabbit anti-human JNK1 antibody were used for each
reaction. All antibodies were obtained as agarose conjugates (Santa
Cruz Biotechnology). After incubation, the beads were washed three
times with lysis buffer, three times with LiCl wash buffer (500 mM
LiCl, 100 mM Tris-Cl, pH 7.6, and 0.1% Triton X-100 plus the
inhibitors as above), and three times with assay buffer (20 mM MOPS, pH
7.2, 2 mM EGTA, 10 mM MgCl2, and 0.1% Triton X-100 plus
the inhibitors as above). After the final wash, the beads were left
suspended in an equal volume of assay buffer to which 100 µg of
substrate and a radioactive ATP mix [50 mM MgCl2 and 0.5 mM (6,000 counts · min
1 · pmol
1)
[
-32P]ATP] were added. For p42/p44 MAPK and p38 MAPK,
myelin basic protein was used (Sigma). For the p46/p54 kinase
assay, glutathione S-transferase-c-Jun fusion protein was
used. Reaction mixtures were incubated for 20 min at 30°C, with
mixing every 2 min. The reaction was stopped with the addition of 30 µl of 5× Laemmli buffer. The samples were boiled for 2 min, spun
briefly at 3,000 rpm, and separated electrophoretically on a
polyacrylamide gel. The gels were dried, exposed to a phosphor screen
overnight, imaged on a Storm 860 phosphorimager (Molecular
Dynamics), and then exposed to Kodak BioMax MR film (Eastman
Kodak, Rochester, NY).
C3 exoenzyme treatment.
Cells were treated with C3 exoenzyme with the scrape-loading technique
of Malcolm et al. (28). RPASMCs were grown to confluence in 100-mm culture dishes (Corning, Corning, NY), and the monolayer was
rinsed once in 5 ml of sterile PBS and once in 2 ml of scrape-loading buffer (114 mM KCl, 15 mM NaCl, and 5.5 mM MgCl2). Each
dish was then given 0.5 ml of scrape-loading buffer or 0.5 ml of
scrape-loading buffer containing 5 µg/ml of Clostridium
botulinum ADP-ribosyltransferase C3 (Sigma). The cells were gently
scraped up into the buffer, pipetted up and down to ensure even
dispersal throughout the mix, and then replated into six-well plates
(Costar, Corning), plating the cells from one 100-mm dish into one
well. Each well was given 3 ml of growth medium without serum; after 15 min, serum was added at the concentration found in regular growth
medium. The cells were allowed to recover in growth medium overnight.
Unscraped cells were serum starved overnight while the scraped cells
recovered in growth medium. The next day, all cells were given IL-1
in basal medium.
ADP-ribosylation assay. Control and C3-treated cells were washed and lysed separately. The level of ADP-ribosylation was determined by incubation of the cell homogenates with additional C3 transferase (5 µg/ml) in the presence of 5 µCi of [32P]NAD for 90 min at 30°C. The reaction was terminated by the addition of Laemmli sample buffer. After normalization for protein content, 100 µg of protein were loaded per lane, separated by SDS-PAGE, transferred to nitrocellulose membrane, and subjected to autoradiography.
Adenoviral-mediated transfer of small G proteins. To determine the proper level of adenovirus to use for our cell type, we used an adenovirus expressing LacZ (a gift from Dr. Bruce Johnson, University of Pittsburgh, Pittsburgh, PA). We found that a multiplicity of infection (MOI) of 1:1,000 was sufficient to demonstrate viral expression in 100% of the cells as determined by X-Gal staining at 48 h. Cells were grown to 80% confluence in 10-cm petri dishes. They were infected in DMEM with a MOI of 1:1,000. A volume sufficient to cover the cells (typically 250 µl for a 25-cm2 flask) containing the virus was added to the cells, and the flasks were rocked every 15 min to ensure dispersion of the virus over the surface of the dish. After 1 h, basal medium was added to increase the volume of the medium to 5 ml/T25 flask. Experiments were performed after 48 h of exposure to the virus in basal medium.
Data analysis. Pixel density determination and analysis of autoradiographs were performed with EagleSight software (Stratagene, La Jolla, CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rho proteins but not Rac1 affect IL-1-mediated iNOS induction in
RPASMCs.
From an original report by Finder et al. (16) and
observations by others (19, 32), it appears that a
candidate small GTPase that is geranylgeranylated at its isoprenyl
moiety (e.g., Rho superfamily) is involved in the negative regulation
of iNOS. Accordingly, we investigated the contribution of two members
of the Rho family (Rac and Rho) to IL-1
-mediated regulation of iNOS in RPASMCs. Adenoviral-mediated infection of RPASMCs with either a
dominant negative (N17) mutant of Rac1 or a constitutively active form
(V12) of Rac1 did not affect either the basal levels of iNOS or the
increase in immunoreactive iNOS after IL-1
(Fig.
1, top). Transgene expression
was demonstrated by presence of the epitope tag c-Myc via Western
blotting (Fig. 1, bottom). Infection of RPASMCs with
adenoviral vectors containing cDNA for a reporter gene,
-galactosidase, of a similar MOI resulted in quantitative transgene
expression (data not shown).
|
|
IL-1 stimulates p44/p42 MAPK activation, but this is not
required for iNOS induction.
Exposure of RPASMCs to IL-1
resulted in activation of ERK1/ERK2 MAPK
as noted by an increase in immunoprecipitated p44/p42MAPK
that was sensitive to the MAPK kinase 1 inhibitor PD-98059 (Fig. 3, bottom). Although IL-1
increased immunoreactive iNOS in these same cells, this increase was
not sensitive to PD-98059 (Fig. 3, top), suggesting that
ERK1/ERK2 kinase was not critical for IL-1
-mediated regulation of
iNOS expression.
|
|
IL-1, p38 kinase cascade, and iNOS expression in RPASMCs.
Exposure of RPASMCs to IL-1
resulted in an increase in p38 kinase as
ascertained by the increase in immunoreactive phosphorylated p38 (Figs.
5 and
6). Neither GGTI-298 (Fig. 5) nor
SB-203580 (Fig. 6) affected the phosphorylation status of p38 under
basal or IL-1
-stimulated conditions. It is possible that GGTI-298,
like SB-203580 (10), decreases the activity of p38 kinase
without affecting phosphorylation status. Regardless, GGTI-298 (Fig. 5)
and SB-203580 (Fig. 6) were each associated with an increase in iNOS
expression after IL-1
, suggesting a possible negative role for p38
in the regulation of iNOS in RPASMCs.
|
|
IL-1 and inhibition of geranylgeranyltransferase
activate STAT3 in RPASMCs.
As shown in Fig. 7, a 15-min
incubation of RPASMCs with either IL-1
or GGTI-298 resulted in the
nuclear translocation of STAT1 and/or STAT3 as detected by the reduced
mobility of a probe containing consensus sequences for STAT1/3 binding,
i.e., hSIE (46). A combination of IL-1
and GGTI-298,
however, did not lead to greater nuclear translocation than either
agent alone. Excess cold hSIE oligonucleotide inhibited the formation
of the nuclear complex, suggesting a degree of specificity of such
translocation (data not shown). Inclusion of antiserum to STAT3 but not
to STAT1 in the binding reactions resulted in a further reduction in
the mobility of the complex after either IL-1
or GGTI-298 (Fig.
8). The combination of IL-1
and
GGTI-298, however, did not result in enhanced intensity of the
supershift complex compared with either agent alone (Fig. 8).
Therefore, it appears that GGTI-298, like IL-1
, can induce STAT3
homodimers, and this induction is associated with enhanced iNOS
expression. The specificity of these observations was supported by the
lack of effect of FTI-277 on STAT activation (data not shown). The
nuclear translocation of STAT3 was insensitive to the JAK inhibitor
AG-490, suggesting that tyrosine phosphorylation of STAT3 by JAK2 after
IL-1
exposure in RPASMCs was not critical. The JAK2 inhibitor AG-490
did not affect expression of iNOS under control and IL-1
conditions
and with treatment with GGTI-298, suggesting that the STAT3 activation by either IL-1 or GGGTI-298 was not JAK2 mediated.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IL-1 is a critical sentinel inflammatory cytokine in the
pulmonary circulation. By affecting various cell types in the pulmonary vascular wall, IL-1
is capable of altering pulmonary vasoregulation, remodeling of the vascular wall, and/or the response to other inflammatory mediators. It is apparent that pulmonary vascular smooth
muscle cells are an important target for IL-1
. Although the
intracellular signaling pathways responsible for IL-1
-mediated changes in pulmonary vascular smooth muscle metabolism are unclear, induction of iNOS appears to be an important consequence. The present
study is an extension of the original observations by Finder et al.
(16) that showed that an inhibitor of
geranylgeranyltransferase (GGTI-298) superinduced iNOS after IL-1
in
RPASMCs. We now show that in pulmonary vascular smooth muscle, a
C3-sensitive (e.g., Rho) member of the Rho superfamily is a negative
regulator of IL-1
-mediated induction of iNOS (Fig. 2). The
pleiotropic IL-1
activates MAPK (ERK1/ERK2; Fig. 3); JNK/SAPK (Fig.
4); p38 kinase (Figs. 5 and 6), and JAK-STAT pathways (Figs. 7 and 8)
in RPASMCs. The former two pathways, however, appear unrelated to
IL-1
-mediated iNOS regulation. The p38 pathway may have inhibitory
input because there is an SB-203580 (p38; Fig. 5)-dependent
augmentation of IL-1
-mediated iNOS induction. As for the JAK-STAT
pathway, we conclude that the association of GGTI-298-induced STAT3
homodimer nuclear translocation provides evidence that there is
upstream inhibition, likely Rho mediated, of JAK-STAT activation and
that it is not JAK2 mediated. These findings suggest that the previous report by Finder et al. on the amplification of IL-1
-mediated iNOS
most likely reflects downstream events, in part mediated by STAT3.
Rho as a negative regulator of IL-1-mediated iNOS induction in
RPASMCs.
GGTI-298 by itself can induce iNOS expression in RPASMCs and enhance
IL-1
-mediated iNOS expression (Fig. 5), suggesting that a
geranylgeranylated protein is a negative regulator of such gene regulation. This peptidomimetic inhibitor of protein isoprenylation has
been shown to be extremely selective for geranylgeranyltransferase over
farnesyltransferase (37, 45). We also used GGTI-2166 where
indicated. This agent has been shown to be as potent as GGTI-298 but
even more selective (41). Similarly, the
farnesyltransferase inhibitor FTI-277 is also very selective for
farnesyltransferase over geranylgeranyltransferase (36).
The involvement of a geranylgeranylated protein in this pathway was
supported by the previous observations by Finder et al.
(16) that lovastatin can superinduce iNOS expression in
RPASMCs and that this effect is reversed on the addition of geranylgeraniol. Likely candidate signaling molecules that are geranylgeranylated include members of the Rho family (RhoA, RhoB, and
RhoC; Rac1 and Rac2; Cdc42). We used genetic approaches to discount a
possible role for Rac1 by overexpressing either dominant negative or
constitutively active Rac1 and not affecting iNOS regulation (Fig. 1).
We did observe, however, that C3 exoenzyme, which inactivates Rho
proteins by ADP-ribosylating asparagine at amino acid residue 41 (Fig.
2B), increased basal iNOS expression and greatly enhanced
the effect of IL-1
(Fig. 2A). The similarity in the
morphological changes in RPASMCs exposed to C3 or GGTI-298 (40) is additional evidence that Rho proteins play a
critical role in pulmonary vascular smooth muscle cell biology. It is
unclear which of the Rho proteins or other C3-sensitive members of the Rho superfamily are involved. Nonetheless, these results are quite similar to a very recent report in rat aortic smooth muscle
(32) and suggest similarities rather than differences in
this limited comparison of pulmonary and systemic smooth muscle cells.
Rho proteins have also recently been reported (27, 42) to
be important in the regulation of endothelial NOS in endothelial cells.
IL-1, Rho proteins, MAPK, SAPK, and p38 kinase in iNOS
regulation in RPASMCs.
IL-1
is known to activate MAPKs in various cells including a number
of smooth muscle cell types (3, 20, 25). Rho proteins are
known to be involved in SAPK and p38 kinase pathways (17). Furthermore, ERK1/ERK2 (24, 26), JNK/SAPK
(18), and p38 kinase (2, 18, 24, 26) have
been noted to contribute to IL-1
-mediated increase in iNOS
expression in a variety of cells including mesangial cells, cardiac
myocytes, pancreatic islet cells, and chondrocytes. Although IL-1
activated the kinases ERK1/ERK2 (Fig. 3), JNK/SAPK (Fig. 4), and p38
(Fig. 5), the IL-1
-mediated increase in iNOS was insensitive to
PD-98059 (Fig. 3) and was actually enhanced in the presence of
SB-203580 (Fig. 6), suggesting that ERK1/ERK2 was not important for
this effect and that p38 kinase was an inhibitor of the
IL-1
-mediated increase in iNOS in RPASMCs. Conversely,
IL-1
-mediated activation of JNK/SAPK was unaffected by the presence
of either a farnesyltransferase or geranylgeranyltransferase inhibitor
(Fig. 4) that itself affected iNOS expression, thereby dissociating
these events. Thus although Rho proteins are known to activate p38
kinase in other cell types (17), the lack of effect of
GGTI-298 on the phosphorylation status of p38 (Fig. 5) suggests that
other members of the Rho GTPase family are involved. Indeed, the
superinduction of iNOS by SB-203580 (Fig. 6) in IL-1
-exposed RPASMCs
is relatively unique among all cytokine-induced iNOS regulation where
most investigators have noted a p38-dependent increase (1, 2, 4,
5, 13, 14, 18, 24, 26, 30). This type of cell and cytokine specificity regarding regulation of iNOS is common. For example, within murine macrophages themselves, Chen and Wang
(13) have noted that p38 kinase did not affect
tumor necrosis factor-
or IFN-
induction of iNOS, whereas p38
kinase was inhibitory to IFN-
/lipopolysaccharide induction of iNOS
in these same cells (11). In this latter study, there
appeared to be cross talk between p38 kinases and the JNK/SAPK pathway
that is not likely to account for our results (Fig. 4).
IL-1, JAK-STAT, and iNOS regulation in RPASMCs.
Although IL-1
signaling is now closely associated with various MAPKs
(see above) in contrast to other cytokines, there is only very modest
evidence that it activates STATs (31). In RPASMCs, IL-1
is clearly associated with translocation and activation of STAT3 (Figs.
7 and 8). Because IL-1
-mediated signaling pathways involve complex
cross-reactions between MAPKs and STATs, it becomes difficult to assign
a direct role to STAT activation in altered gene expression.
Nonetheless, it is noteworthy that AG-490, a JAK2 inhibitor,
superinduced iNOS expression, suggesting that the JAK-STAT pathway may
be inhibitory to IL-1
-mediated iNOS gene regulation in RPASMCs. A
similar observation was made in vascular smooth muscle cells exposed to
IFN-
and lipopolysaccharide (29) in which JAK2 was
inhibited pharmacologically (AG-490) and genetically (antisense). These
investigators also noted an increase in iNOS expression after
introducing antibodies to either STAT1 or STAT3. Indeed, the uniqueness
and specificity of this inhibitory response was demonstrated by these
authors in a study in which the same stimulus and signaling pathway
appeared to contribute positively to iNOS induction in RAW 264.7 cells.
A positive regulatory role of STATs for iNOS was also noted in
IFN-
-stimulated C6 glioma cells (34) and DLD-1 cells
(23) and contributed to the IFN-
priming of
IL-1
-mediated iNOS induction in rat islet cells (21). Accordingly, as of now, vascular smooth muscle appears to have a unique
JAK-STAT inhibitory role on cytokine-mediated iNOS induction. The
control of iNOS is thus both species and organ specific. We feel that
the findings in rodent aortic smooth muscle reported elsewhere
(32) are consistent with our own findings in the
pulmonary smooth muscle cell here.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: J. D. Finder, Children's Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213 (E-mail: finder{at}pitt.edu).
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.
Received 10 January 2001; accepted in final form 21 May 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ajizian, SJ,
English BK,
and
Meals EA.
Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-gamma.
J Infect Dis
179:
939-944,
1999[ISI][Medline].
2.
Badger, AM,
Cook MN,
Lark MW,
Newman-Tarr TM,
Swift BA,
Nelson AH,
Barone FC,
and
Kumar S.
SB 203580 inhibits p38 mitogen-activated protein kinase, nitric oxide production, and inducible nitric oxide synthase in bovine cartilage-derived chondrocytes.
J Immunol
161:
467-473,
1998
3.
Bartlett, SR,
Sawdy R,
and
Mann GE.
Induction of cyclooxygenase-2 expression in human myometrial smooth muscle cells by interleukin-1beta: involvement of p38 mitogen-activated protein kinase.
J Physiol (Lond)
520:
399-406,
1999
4.
Bhat, NR,
Zhang P,
and
Bhat AN.
Cytokine induction of inducible nitric oxide synthase in an oligodendrocyte cell line: role of p38 mitogen-activated protein kinase activation.
J Neurochem
72:
472-478,
1999[ISI][Medline].
5.
Bhat, NR,
Zhang P,
Lee JC,
and
Hogan EL.
Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures.
J Neurosci
18:
1633-1641,
1998
6.
Boota, A,
Johnson B,
Lee KL,
Blaskovich MA,
Liu SX,
Kagan VE,
Hamilton A,
Pitt B,
Sebti SM,
and
Davies P.
Prenyltransferase inhibitors block superoxide production by pulmonary vascular smooth muscle.
Am J Physiol Lung Cell Mol Physiol
278:
L329-L334,
2000
7.
Boota, A,
Zar H,
Kim YM,
Johnson B,
Pitt B,
and
Davies P.
IL-1 stimulates superoxide and delayed peroxynitrite production by pulmonary vascular smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
271:
L932-L938,
1996
8.
Bromberg, J,
and
Darnell JE, Jr.
The role of STATs in transcriptional control and their impact on cellular function.
Oncogene
19:
2468-2473,
2000[ISI][Medline].
9.
Cao, Z,
Henzel WJ,
and
Gao X.
IRAK: a kinase associated with the interleukin-1 receptor.
Science
271:
1128-1131,
1996[Abstract].
10.
Caron, E,
and
Hall A.
Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases.
Science
282:
1717-1721,
1998
11.
Chan, E,
Morris K,
Remigio L,
and
Riches D.
Differential regulation by the mitogen-activated protein kinases in the expression of iNOS-NO by lipoarabinomannan (Abstract).
Am J Respir Crit Care Med
161:
A896,
2000.
12.
Chatterjee-Kishore, M,
van den Akker F,
and
Stark GR.
Association of STATs with relatives and friends.
Trends Cell Biol
10:
106-111,
2000[ISI][Medline].
13.
Chen, CC,
and
Wang JK.
p38 but not p44/42 mitogen-activated protein kinase is required for nitric oxide synthase induction mediated by lipopolysaccharide in RAW 264.7 macrophages.
Mol Pharmacol
55:
481-488,
1999
14.
Da Silva, J,
Pierrat B,
Mary JL,
and
Lesslauer W.
Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes.
J Biol Chem
272:
28373-28380,
1997
15.
Dignam, JD,
Lebovitz RM,
and
Roeder RG.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res
11:
1475-1489,
1983[Abstract].
16.
Finder, JD,
Litz JL,
Blaskovich MA,
McGuire TF,
Qian Y,
Hamilton AD,
Davies P,
and
Sebti SM.
Inhibition of protein geranylgeranylation causes a superinduction of nitric-oxide synthase-2 by interleukin-1beta in vascular smooth muscle cells.
J Biol Chem
272:
13484-13488,
1997
17.
Guan, Z,
Baier LD,
and
Morrison AR.
p38 mitogen-activated protein kinase down-regulates nitric oxide and up-regulates prostaglandin E2 biosynthesis stimulated by interleukin-1beta.
J Biol Chem
272:
8083-8089,
1997
18.
Guan, Z,
Buckman SY,
Springer LD,
and
Morrison AR.
Both p38alpha(MAPK) and JNK/SAPK pathways are important for induction of nitric-oxide synthase by interleukin-1beta in rat glomerular mesangial cells.
J Biol Chem
274:
36200-36206,
1999
19.
Hausding, M,
Witteck A,
Rodriguez-Pascual F, C,
von Eichel-Streiber U.,
Forstermann H,
and
Kleinert
Inhibition of small G proteins of the rho family by statins or Clostridium difficile toxin B enhances cytokine-mediated induction of NO synthase II.
Br J Pharmacol
131:
553-561,
2000
20.
Hedges, JC,
Dechert MA,
Yamboliev IA,
Martin JL,
Hickey E,
Weber LA,
and
Gerthoffer WT.
A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration.
J Biol Chem
274:
24211-24219,
1999
21.
Heitmeier, MR,
Scarim AL,
and
Corbett JA.
Prolonged STAT1 activation is associated with interferon-gamma priming for interleukin-1-induced inducible nitric-oxide synthase expression by islets of Langerhans.
J Biol Chem
274:
29266-29273,
1999
22.
Johnson, BA,
Pitt BR,
and
Davies P.
Pulmonary arterial smooth muscle cells modulate cytokine- and LPS-induced cytotoxicity in endothelial cells.
Am J Physiol Lung Cell Mol Physiol
278:
L460-L468,
2000
23.
Kleinert, H,
Wallerath T,
Fritz G,
Ihrig-Biedert I,
Rodriguez-Pascual F,
Geller DA,
and
Forstermann U.
Cytokine induction of NO synthase II in human DLD-1 cells: roles of the JAK-STAT, AP-1 and NF-kappaB-signaling pathways.
Br J Pharmacol
125:
193-201,
1998[Abstract].
24.
LaPointe, MC,
and
Isenovic E.
Interleukin-1beta regulation of inducible nitric oxide synthase and cyclooxygenase-2 involves the p42/44 and p38 MAPK signaling pathways in cardiac myocytes.
Hypertension
33:
276-282,
1999
25.
Laporte, JD,
Moore PE,
Abraham JH,
Maksym GN,
Fabry B,
Panettieri RA, Jr,
and
Shore SA.
Role of ERK MAP kinases in responses of cultured human airway smooth muscle cells to IL-1.
Am J Physiol Lung Cell Mol Physiol
277:
L943-L951,
1999
26.
Larsen, CM,
Wadt KA,
Juhl LF,
Andersen HU,
Karlsen AE,
Su MS,
Seedorf K,
Shapiro L,
Dinarello CA,
and
Mandrup-Poulsen T.
Interleukin-1beta-induced rat pancreatic islet nitric oxide synthesis requires both the p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases.
J Biol Chem
273:
15294-15300,
1998
27.
Laufs, U,
and
Liao JK.
Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase.
J Biol Chem
273:
24266-24271,
1998
28.
Malcolm, KC,
Elliott CM,
and
Exton JH.
Evidence for Rho-mediated agonist stimulation of phospholipase D in rat1 fibroblasts. Effects of Clostridium botulinum C3 exoenzyme.
J Biol Chem
271:
13135-13139,
1996
29.
Marrero, MB,
Venema VJ,
He H,
Caldwell RB,
and
Venema RC.
Inhibition by the JAK/STAT pathway of IFNgamma- and LPS-stimulated nitric oxide synthase induction in vascular smooth muscle cells.
Biochem Biophys Res Commun
252:
508-512,
1998[ISI][Medline].
30.
Miyazawa, K,
Mori A,
Miyata H,
Akahane M,
Ajisawa Y,
and
Okudaira H.
Regulation of interleukin-1beta-induced interleukin-6 gene expression in human fibroblast-like synoviocytes by p38 mitogen-activated protein kinase.
J Biol Chem
273:
24832-24838,
1998
31.
Morton, NM,
de Groot RP,
Cawthorne MA,
and
Emilsson V.
Interleukin-1beta activates a short STAT-3 isoform in clonal insulin-secreting cells.
FEBS Lett
442:
57-60,
1999[ISI][Medline].
32.
Muniyappa, R,
Xu R,
Ram JL,
and
Sowers JR.
Inhibition of Rho protein stimulates iNOS expression in rat vascular smooth muscle cells.
Am J Physiol Heart Circ Physiol
278:
H1762-H1768,
2000
33.
Nakayama, DK,
Geller DA,
Lowenstein CJ,
Chern HD,
Davies P,
Pitt BR,
Simmons RL,
and
Billiar TR.
Cytokines and lipopolysaccharide induce nitric oxide synthase in cultured rat pulmonary artery smooth muscle.
Am J Respir Cell Mol Biol
7:
471-476,
1992[ISI][Medline].
34.
Nishiya, T,
Uehara T,
Edamatsu H,
Kaziro Y,
Itoh H,
and
Nomura Y.
Activation of Stat1 and subsequent transcription of inducible nitric oxide synthase gene in C6 glioma cells is independent of interferon-gamma-induced MAPK activation that is mediated by p21ras.
FEBS Lett
408:
33-38,
1997[ISI][Medline].
35.
O'Neill, LA,
and
Greene C.
Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants.
J Leukoc Biol
63:
650-657,
1998[Abstract].
36.
Qian, Y,
Vogt A,
Sebti SM,
and
Hamilton AD.
Design and synthesis of non-peptide Ras CAAX mimetics as potent farnesyltransferase inhibitors.
J Med Chem
39:
217-223,
1996[ISI][Medline].
37.
Sebti, S,
and
Hamilton AD.
Inhibitors of prenyl transferases.
Curr Opin Oncol
9:
557-561,
1997[Medline].
38.
Singh, R,
Huang S,
Guth T,
Konieczkowski M,
and
Sedor JR.
Cytosolic domain of the type I interleukin-1 receptor spontaneously recruits signaling molecules to activate a proinflammatory gene.
J Clin Invest
100:
419-428,
1997
39.
Singh, R,
Wang B,
Shirvaikar S,
Khan S,
Kamat S,
Schelling J,
Konieczkowski M,
and
Sedor J.
The IL-1 receptor and Rho directly associate to drive cell activation and inflammation.
J Clin Invest
103:
1561-1570,
1999
40.
Stark, WW, Jr,
Blaskovich MA,
Johnson BA,
Qian Y,
Vasudevan A,
Pitt B,
Hamilton AD,
Sebti SM,
and
Davies P.
Inhibiting geranylgeranylation blocks growth and promotes apoptosis in pulmonary vascular smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
275:
L55-L63,
1998
41.
Sun, J,
Blaskovich MA,
Knowles D,
Qian Y,
Ohkanda J,
Bailey RD,
Hamilton AD,
and
Sebti SM.
Antitumor efficacy of a novel class of non-thiol-containing peptidomimetic inhibitors of farnesyltransferase and geranylgeranyltransferase I: combination therapy with the cytotoxic agents cisplatin, Taxol, and gemcitabine.
Cancer Res
59:
4919-4926,
1999
42.
Takeuchi, S,
Kawashima S,
Rikitake Y,
Ueyama T,
Inoue N,
Hirata K,
and
Yokoyama M.
Cerivastatin suppresses lipopolysaccharide-induced ICAM-1 expression through inhibition of Rho GTPase in BAEC.
Biochem Biophys Res Commun
269:
97-102,
2000[ISI][Medline].
43.
Thomae, KR,
Joshi PC,
Davies P,
Pitt BR,
Billiar TR,
Simmons RL,
and
Nakayama DK.
Nitric oxide produced by cytokine-activated pulmonary artery smooth muscle cells is cytotoxic to cocultured endothelium.
Surgery
119:
61-66,
1996[ISI][Medline].
44.
Tsukada, J,
Waterman WR,
Koyama Y,
Webb AC,
and
Auron PE.
A novel STAT-like factor mediates lipopolysaccharide, interleukin 1 (IL-1), and IL-6 signaling and recognizes a gamma interferon activation site-like element in the IL1B gene.
Mol Cell Biol
16:
2183-2194,
1996[Abstract].
45.
Vasudevan, A,
Qian Y,
Vogt A,
Blaskovich MA,
Ohkanda J,
Sebti SM,
and
Hamilton AD.
Potent, highly selective, and non-thiol inhibitors of protein geranylgeranyltransferase-I.
J Med Chem
42:
1333-1340,
1999[ISI][Medline].
46.
Wagner, BJ,
Hayes TE,
Hoban CJ,
and
Cochran BH.
The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter.
EMBO J
9:
4477-4484,
1990[Abstract].
47.
Wong, HR,
Finder JD,
Wasserloos K,
Lowenstein CJ,
Geller DA,
Billiar TR,
Pitt BR,
and
Davies P.
Transcriptional regulation of iNOS by IL-1 in cultured rat pulmonary artery smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
271:
L166-L171,
1996