Department of Medicine, Molecular Biology, and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Interleukin-1 (IL-1
) induces the inducible
nitric oxide synthase (iNOS), resulting in the release of nitric oxide
(NO) from glomerular mesangial cells. In this study, we demonstrated
that disruption of F-actin formation by sequestration of G-actin with the toxin latrunculin B (LatB) dramatically potentiated IL-1
-induced iNOS protein expression in a dose-dependent manner. LatB by itself had
little or no effect on iNOS expression. Staining of F-actin with
nitrobenzoxadiazole (NBD)-phallacidin demonstrated that LatB significantly impaired F-actin stress fiber formation. Jasplakinolide (Jasp), which binds to and stabilizes F-actin, suppressed iNOS expression enhanced by LatB. These data strongly suggest that actin
cytoskeletal dynamics regulates IL-1
-induced iNOS expression. We
demonstrated that LatB decreases serum response factor (SRF) activity
as determined by reporter gene assays, whereas Jasp increases SRF
activity. The negative correlation between SRF activity and iNOS
expression suggests a negative regulatory role for SRF in iNOS
expression. Overexpression of a dominant negative mutant of SRF
increases the IL-1
-induced iNOS expression, providing direct
evidence that SRF inhibits iNOS expression.
inducible nitric oxide synthase; serum response factor; latrunculin B; jasplakinolide; mesangial cell
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INTRODUCTION |
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NITRIC OXIDE (NO) is
involved in many physiological and pathological functions (2,
36). It mediates blood vessel relaxation, functions as a
neurotransmitter, and mediates macrophage cytotoxicity during host
defense and leads to tissue injury in some inflammatory and autoimmune
diseases. NO is produced by NO synthase through oxidation of one of the
amide nitrogens of L-arginine. Three isoforms exist in
mammalian cells. Type I [neuronal NO synthase (nNOS)] and type III
[endothelial NO synthase (eNOS)] were first identified in neurons and
endothelial cells, respectively. Both nNOS and eNOS are constitutively
expressed in cells and are calcium and calmodulin dependent. Type II
[inducible NO synthase (iNOS)] is generally expressed only following
stimulation of most mammalian cell types with proinflammatory agents
(2), including interleukin-1 (IL-1
), tumor necrosis
factor-
, and interferon-
, or pathophysiological processes
(28), including ischemia and stroke. iNOS is
subject to transcriptional regulation. Murine, rat, and human iNOS
promoters have been cloned (8, 10, 65). Functional
characterization of the promoters with several cell types has
established that nuclear factor-
B (NF-
B) is required for the
induction of iNOS by cytokines and bacterial products such as
lipopolysaccharide (25, 64). Our laboratory has shown that
mitogen-activated protein (MAP) kinase pathways, p38MAPK
and JNK, are also required for IL-1
-induced iNOS expression in rat
mesangial cells (16). Other regulators found to be
critical in iNOS expression include C/EBP
(11, 18),
IRF-1 (24, 33, 54), and Oct-1 (63). On the
other hand, iNOS is subject to immunological suppression, for example,
by transforming growth factor-
(41, 58), IL-4, and
IL-13. Activation of phosphatidylinositol 3-kinase has been shown to
mediate the inhibitory effect of IL-13 (9, 40, 49, 62).
Serum response factor (SRF) is an important regulator of many genes associated with cell growth and differentiation (55). SRF was first identified based on its ability to mediate serum and growth factor activation of the c-fos protooncogene (57). Subsequently, it was found that SRF binds to the c-fos promoter at the serum response element (SRE). The c-fos SRE is comprised of a core sequence CC(A/T)6GG, which binds SRF, and a nearby Ets motif (C/A)(C/A)GGA(A/T), which binds the ternary complex factors (TCFs). The full regulatory properties of the c-fos SRE require binding of both SRF and TCF proteins. Human SRF contains a central DNA-binding and dimerization domain (37), termed the MADS box domain, which is highly conserved in a group of DNA binding proteins (43), the amino-terminal domain, containing Ser103 phosphorylation sites that facilitate SRF DNA binding activity (20, 31), and the carboxy-terminal activation domain that binds a diversity of protein factors such as the RAP74 subunit of TFIIF (23), and ATF6 (66). Three TCF proteins, Elk-1, SAP-1, and SAP2/ERP/NET, have been identified (44, 55). Each contains an Ets domain, which binds to DNA in the Ets motif of the c-fos SRE, a conserved 20-residue region, which interacts with SRF, and a conserved regulatory region, which contains copies of S/T-P consensus sequence for MAP kinase phosphorylation. TCFs respond to MAP kinase signaling pathways and cooperate with the SRF carboxy-terminal activation domain to regulate gene transcription.
It has been shown that SRF also plays a major role in the control of
muscle gene expression and muscle differentiation (38, 51). Functional SREs are found in the promoters of several
muscle-specific genes such as smooth muscle -actin, SM22
, and
cardiac
-actin. In contrast to the c-fos promoter, the
SREs in these muscle-specific genes do not have nearby Ets motifs and
therefore are not likely to bind TCF. SRF, in muscle-specific
promoters, functions independently of TCF. The regulation of
muscle-specific gene expression appears to be mediated by the
interaction of the SRF MADS box with striated muscle tissue-restricted
factors such as the basic helix-loop-helix protein MyoD (15,
48) and homeodomain protein Nkx-2.5 (5). Target
deletion of the SRF gene in mice has revealed that SRF null mutant is
embryonic lethal due to a failure of gastrulation and mesoderm
differentiation (1).
The regulation of SRF activity has been the effort of several studies. At the c-fos SRE, serum activates SRF activity through two pathways (22). In the TCF-dependent pathway, the activity of the TCF is regulated by the MAP kinase signaling pathway. In the TCF-independent pathway, the Rho family GTPases are involved. Expression of constitutively active forms of both RhoA and other Rho family GTPase such as Cdc42 and Rac1 can activate SRF (19). Cdc42 and Rac1 function independently of RhoA. At the SRE of muscle-specific promoters, activation of SRF is also dependent on functional RhoA in the absence of TCF (4, 59).
Rho-like GTPases have been shown to be key regulators in signaling pathways that link extracellular growth signals or intracellular stimuli to the assembly and organization of the actin cytoskeleton (50). In an effort to elaborate the mechanism of SRF activation and its relationship to other Rho GTPase-controlled processes, one laboratory has reported that signal-regulated activation of SRF is mediated by changes in actin dynamics (53), a process in which polymers (F-actin) assemble spontaneously via noncovalent interactions between the monomeric subunits (G-actin) and are highly dynamic structures with subunit turnover at both ends. Depletion of G-actin is both necessary and sufficient for activation of both SRF reporters and a subset of SRF target genes.
Mesangial cells serve multiple functions within the glomerulus,
including regulation of glomerular filtration, elaboration of
extracellular matrix, and phagocytosis of immune complexes. Our
laboratory has previously reported that IL-1 induces iNOS protein
expression with concomitant synthesis of NO in renal mesangial cells
(16). In this report we demonstrated that actin dynamics controls iNOS expression and that its signaling pathway is mediated by SRF.
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MATERIALS AND METHODS |
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Plasmids.
pCGN is a cytomegalovirus promoter-driven expression vector. pCGN-SRF
or pCGN-SRFpm1 was obtained by subcloning, respectively, the gene
for human wild-type SRF or its dominant negative mutant (SRFpm1) into
the pCGN vector. SRFpml is a triple-point mutant in human SRF that
converts Arg143, Lys145, and Leu146
to Ile143, Ala145, and Gly146
(7). Expressed SRF or SRFpm1 are fused to the influenza
virus hemagglutinin antigen at the amino terminus. The reporter plasmid c-fos-luc contains a single copy of the human
c-fos SRE region (53 to +45) subcloned upstream of firefly
luciferase in the pGL3 vector (Promega). The above constructs
(pCGN-SRF, pCGN-SRFpm1, and c-fos-luc) were
kindly provided by Dr. Robert J. Schwartz at Baylor College of
Medicine, TX. The reporter plasmid pRL-TK (Promega) contains the herpes
simplex virus thymidine kinase (TK) promoter region upstream of
Renilla luciferase.
Cell culture. Primary mesangial cell cultures were prepared from male Sprague-Dawley rats as described previously (17). Cells were grown in mesangial cell medium [RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 0.3 IU/ml insulin, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 µg/ml amphotericin B, and 15 mM HEPES, pH 7.4]. Cells were used at passages 3 through 8.
Cell transfection and immunoblotting.
Primary rat mesangial cells were transiently transfected using
Superfect reagent (Qiagen). Cells were plated on 60-mm-diameter dishes
2 days before transfection. At 60-90% confluence, cells were
transfected with either pCGN-SRF (2.5, 5, or 7.5 µg) or pCGN-SRFpm1 (2.5, 5, or 7.5 µg). Cells were then cultured in the mesangial cell
medium containing 100 U/ml IL-1 (Roche Molecular Biochemicals) for
15 h. Cell lysates were prepared as described previously
(16). Thirty micrograms of protein from each sample
were analyzed by standard immunoblotting procedures. The expressed iNOS
was blotted with anti-iNOS antibody (BD Transduction Laboratories) at
1:2,000 dilution and horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham Pharmacia Biotech) at 1:5,000 dilution. The overexpressed SRF and SRFpm1 were blotted with anti-hemagglutinin (HA)
antibody (Babco) at 1:1,000 dilution and horseradish
peroxidase-conjugated goat anti-mouse IgG (Amersham Pharmacia
Biotech) at 1:5,000 dilution. For detection, SuperSignal WestDura
extended duration substrate (Pierce) was used.
Dual-luciferase reporter gene assay. c-Fos-luc (2.5 µg) and 0.25 µg of pRL-TK for each 35-mm-diameter dish were used to cotransfect rat mesangial cell transiently by the Superfect reagent (Qiagen) method. pRL-TK was used to normalize for transfection efficiency. Cells were then cultured for 18 h in the mesangial cell medium modified to contain 0.5% FBS. After serum starvation, cells were either left untreated or treated with actin-binding drugs [0.5 µM latrunculin B (Calbiochem), 0.5 µM jasplakinolide (Molecular Probes), 0.2 µM cytochalasin D (Calbiochem), or 30 nM swinholide A (Kamiya Biomedical) in the mesangial cell medium containing 10% FBS for 22 h]. Cells lysates were prepared and assayed for luciferase activity using the dual-luciferase reporter assay system (Promega).
Fluorescence microscopy.
Rat mesangial cells grown on a glass coverslip were either left
untreated or were treated with 0.5 µM latrunculin B and/or IL-1
(100 U/ml) for 1 h. Actin in the cell was stained with
nitrobenzoxadiazole (NBD)-phallacidin (Molecular Probes). Cells in the
coverslip were washed twice with prewarmed PBS, pH 7.4, and then fixed
in 3.7% formaldehyde solution in PBS for 10 min at room temperature.
After each coverslip was washed three times in PBS, it was placed in a
glass petri dish and was permeabilized with acetone at
20°C for 5 min. Cells were then washed twice with PBS and were stained with 165 nM
NBD-phallacidin in PBS for 20 min. To reduce nonspecific background
staining, the fixed cells were preincubated with PBS containing 1% BSA
for 20 min before the NBD-phallacidin solution was added. Cells were
washed three times with PBS before being mounted on a slide with the
cell side down in a 1:1 solution of PBS and glycerol. Stained F-actin
was imaged using a Nikon epifluorescent microscope.
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RESULTS |
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Effect of increased G-actin on IL-1-induced iNOS expression.
The toxin latrunculin B disrupts the actin cytoskeleton by sequestering
G-actin monomers (increasing cellular G-actin) and therefore
inhibiting actin polymerization. We made a surprising observation that
stimulation of latrunculin B-treated rat mesangial cells with IL-1
produced a dramatic increase in iNOS expression over and above
that in cells not treated with latrunculin B (Fig. 1A). Furthermore, the
peak response for protein expression was shifted from 24 h in
cells not treated with latrunculin B to 9-12 h in cells treated
with latrunculin B (Fig. 1, A and B). Latrunculin B by itself had little or no effect on iNOS expression (Fig.
1C). These results imply that a link exists between the
actin cytoskeleton and iNOS expression.
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|
Jasplakinolide suppresses iNOS expression enhanced by latrunculin
B.
To further confirm that latrunculin B enhances IL-1-induced iNOS
expression through the mechanism of actin cytoskeleton disruption, we
tested the effect of jasplakinolide on iNOS expression. In contrast to
latrunculin B, jasplakinolide is a potent inducer of actin
polymerization by binding to and stabilizing F-actin. The effects of
the toxin lead to a reduction of monomeric G-actin. Cells were treated
with latrunculin B alone or both latrunculin B and jasplakinolide. In
the presence of IL-1
, the enhanced iNOS expression by latrunculin B
was dramatically suppressed by adding jasplakinolide (Fig.
3). Conceivably, this is because
jasplakinolide antagonizes the effect of latrunculin B on the
steady-state levels of G-actin. Together, the effects of latrunculin B
and jasplakinolide on IL-1
-induced iNOS expression strongly suggest
that the actin cytoskeleton is involved in this process. Figure 3,
together with Fig. 1C, also shows that, in the absence of
IL-1
, latrunculin B and jasplakinolide do not affect iNOS
expression, suggesting that disruption of the actin cytoskeleton by
itself is not sufficient for iNOS expression and that other signaling
pathways stimulated by IL-1
are required.
|
SRF activity negatively correlates with IL-1-induced iNOS
expression.
We next asked the question as to how disruption of the actin
cytoskeleton upregulates IL-1
-induced iNOS expression. Recently, it
was reported that actin cytoskeleton dynamics regulates the activity of
SRF (53). In addition, we and others previously demonstrated that serum-free medium, in which a basal level of SRF
activity in the cell is low, significantly potentiated IL-1
-induced iNOS expression compared with the medium containing 10% serum, in
which the SRF activity in the cell is dramatically activated. These
results made us hypothesize that SRF may play an inhibitory role in
IL-1
-induced iNOS expression. To test this hypothesis, we designed
the experiments to examine the correlation between iNOS expression and
SRF activity. We transiently transfected rat mesangial cells with
SRF-controlled reporter plasmid, c-fos-luc, which
contains the human c-fos SRE cis-acting element
followed by firefly luciferase. Cells were then either left untreated
or treated with actin-binding drugs, 0.5 µM latrunculin B, and/or 0.5 µM jasplakinolide. Cell lysates were prepared, and firefly luciferase
activity was measured and normalized to internal control Renilla luciferase activity (Fig.
4A). We also used the same
lysates for the reporter assay to determine the levels of iNOS
expression by Western blot analysis as shown in Fig. 3A.
Latrunculin B strongly decreased SRF activity but increased
IL-1
-induced iNOS. Treatment with both latrunculin B and
jasplakinolide caused a significant increase in SRF activity but a
decrease in IL-1
-induced iNOS compared with the treatment with
latrunculin B alone. These data clearly show that SRF activity
negatively correlates with IL-1
-induced iNOS expression. To ensure
that these results are not merely due to the side effects of
latrunculin B and jasplakinolide, we examined the effects of two other
drugs, swinholide A and cytochalasin D. Swinholide A sequesters G-actin
as dimers and severs F-actin (3, 29), while cytochalasin D
caps actin filaments, promotes ATP hydrolysis on G-actin, and induces
dimerization of the ATP-actin monomer (6). Both swinholide
A (30 nM) and cytochalasin D (0.2 µM) decreased SRF activity but
increased IL-1
-induced iNOS (Fig. 4, B and C).
These data suggest that SRF plays an inhibitory role in IL-1
-induced
iNOS expression. Our data also showed that jasplakinolide by itself
strongly activated SRF as expected, but it had little effect on
IL-1
-induced iNOS expression. The explanation for the latter is as
follows. In the cells not treated with drugs, basal SRF activity is
activated by 10% FBS. Serum-activated SRF has enough activity to fully
inhibit IL-1
-induced iNOS expression; therefore, further activation
of SRF by jasplakinolide does not contribute more to this inhibitory
effect.
|
Overexpression of dominant negative mutant of SRF increased
IL-1-induced iNOS expression.
To further test the idea that SRF inhibits IL-1
-induced iNOS
expression, we transiently transfected rat mesangial cells with plasmids encoding HA-tagged versions of wild-type SRF or dominant negative mutant SRFpm1. This mutant SRFpm1 contains triple mutations within the MADS box and is defective in DNA binding but capable of
heterodimerizing with the wild-type SRF monomer. Increasing amounts of
DNA (2.5, 5, or 7.5 µg for each 60-mm-diameter plate) were used.
After stimulation with IL-1
for 15 h, iNOS expression in the
cell lysates was examined by Western blot analysis. As shown in Fig.
5A, the dominant negative
mutant of SRF significantly increased IL-1
-induced iNOS expression
compared with wild-type SRF. Both the wild-type and dominant negative
mutants of SRF were overexpressed (Fig. 5A). We
cotransfected mesangial cells with SRF and luciferase reporter
constructs. The mutant SRFpm1 caused 90% inhibition of SRF activity
determined by reporter gene assay. We also transiently transfected
cells with SRF wild-type and SRFpm1 mutants and then treated cells with
latrunculin B and jasplakinolide. Figure 5B shows that
either latrunculin B or mutant SRFpm1 increases IL-1
-induced iNOS
expression. Even though the efficiency of transient transfection for
primary mesangial cells is generally low (10-15%), we made the
consistent observation that mutant SRFpm1 increases IL-1
-induced
iNOS expression, providing strong evidence that SRF plays an inhibitory
role in IL-1
-induced iNOS expression.
|
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DISCUSSION |
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We report here two important findings. First, actin cytoskeletal
dynamics regulates IL-1-induced iNOS expression. Second, SRF
mediates the effects of G-actin and is an inhibitory regulator in this
process. We have provided the following evidence. First, disruption of
F-actin polymerization by latrunculin B dramatically upregulates iNOS
expression (Fig. 1). Second, Jasplakinolide, which binds and stabilizes
F-actin filaments, suppresses latrunculin B-enhanced iNOS expression
(Fig. 3). The above observations suggest that actin cytoskeleton
dynamics regulate IL-1
-induced iNOS expression. Third, latrunculin B
inhibits SRF activity as determined by reporter gene assay, whereas
jasplakinolide activates SRF activity (Fig. 4). The negative
correlation between SRF activity and iNOS expression suggests that SRF
plays an inhibitory role in IL-1
-induced iNOS expression. Fourth,
overexpression of the dominant negative SRF mutant by transient
transfections of rat mesangial cell increases iNOS expression,
providing direct evidence that SRF is a negative regulator of iNOS
expression (Fig. 5).
In our rat mesangial cell model system, incubation of cells with
IL-1 for 1 h causes little change in the actin cytoskeleton (Fig. 2). This is consistent with the previous findings by others that
IL-1 induces transient changes in the actin cytoskeleton after
10-30 min of stimulation and that the cytoskeleton returns to
normal after 1 h of treatment (52, 67). We also
observed that IL-1 moderately decreases SRF activity (data not shown). In contrast, others have shown that IL-1 induces SRE-dependent reporter
gene expression in Chinese hamster ovary cells and NIH/3T3 cells in
serum-free medium (60, 61). The difference may be due to a
cell-type-specific response to proinflammatory signals.
Previous studies by others have suggested that cell-matrix interactions
and IL-1-dependent signaling pathways collaborate to regulate cellular
function during inflammation. Several lines of evidence are that
1) cell adhesion to fibronectin modulates a IL-1-dependent
inflammatory response, such as NF-B activity, IL-6 expression, and
induction of collagen (39, 47, 67); 2)
attachment of cells to the extracellular matrix is in part mediated
through integrins, a family of cell surface glycoproteins connecting
the extracellular matrix and the cytoskeleton (30, 34);
and 3) IL-1 has been shown to exert profound effects on fibroblast cytoskeletal organization and cell-matrix interactions at
focal adhesions (46). Our present findings and the
previous observation by others that the cytoskeleton mediates
IL-1-induced iNOS expression (32) have added another line
of evidence to support this interaction. Our finding has clinical
implications. In chronic inflammatory diseases, extracellular matrix
abundance and composition are altered. The matrix alteration may cause
actin cytoskeleton changes within cells, which in turn may mediate a cellular proinflammatory response to cytokines.
It was surprising that SRF inhibited IL-1-induced iNOS expression,
because it has been reported that SRF acts as a transcriptional activator (37). Injection of anti-SRF antibodies into rat
fibroblasts strongly inhibits serum-induced c-fos expression
(13). The carboxy-terminal region of SRF is responsible
for this transcriptional activation, since it functions as a
transactivation domain when fused to the DNA-binding domain of the
yeast protein GAL4 (21). In addition to c-fos,
SRF also constitutively activates the muscle-specific genes such as
SM22
(27), as well as the IL-2 receptor
chain gene
(35). Although SRF is widely accepted as a transcriptional activator, several reports suggest an inhibitory role for SRF in
transcriptional regulation. Although low amounts of SRF activate in
vitro transcription from a c-fos promoter construct, high
amounts of SRF inhibit it (45). Overexpression of SRF in
vivo by transfection also inhibits transcription of the
c-fos gene (12). The suggested explanation for
the above observations is that saturating amounts of SRF titrate
coactivators involved in transcription machinery and thereby inhibit
transcription. Another observation is that, in muscle cells, SRF is
constitutively active in muscle-specific genes but remains inactive in
the c-fos gene, raising a possibility that there is negative
regulation of SRF before activation (15, 56). It has been
generally assumed that muscle-specific factors may interact with SRF,
rendering SRF constitutive activity in muscle genes but inhibiting SRF
in the c-fos gene (56). Moreover, the amino
terminus of SRF is found to mediate the repression of transcription in
GAL4-SRF fusions and may be the structural basis for the inhibitory
function of SRF (21).
In the current study, we report that SRF inhibits iNOS expression
stimulated by IL-1 in the rat mesangial cell. This could occur
either directly or indirectly. In the indirect model, the target genes
of SRF and their downstream components may play inhibitory roles on the
iNOS promoter. These target genes include the immediate early genes
(e.g., c-fos, egr-1), cytoskeletal actin,
vinculin, SRF itself, and IL-2 receptor
chain (26,
42). Further studies of these target genes and their downstream
components will be helpful to elucidate the SRF inhibitory mechanism.
In the direct inhibition model, SRF may physically interact with the
transcriptional machinery in the iNOS promoter, leading to inhibition.
We searched for SREs for human, mouse, and rat iNOS promoter sequences
available in the GenBank database and did not find this
cis-acting element. We cannot exclude the possibility that
an SRE exists in the region beyond the published sequences. Moreover,
we speculate that SRF may function even without binding to DNA, because
free SRF has been reported to inhibit SRE-independent transcription
(12, 45).
We have demonstrated that disruption of the actin cytoskeleton enhances
IL-1-induced iNOS expression. It is intriguing to understand how the
actin cytoskeleton system relates to IL-1
-induced iNOS expression.
We have reported here that SRF is one such link between the actin
cytoskeleton and iNOS expression. The mechanism by which the actin
cytoskeleton regulates SRF activity has been reported in a recent paper
(53). Actin polymerization inhibitors such as latrunculin
B or Clostridium botulinum C2 toxin prevent SRF activation.
Actin polymerizing agents such as jasplakinolide induce SRF activity.
Overexpression of vasodilator-stimulated phosphoprotein (VASP),
Wiskott-Aldrich syndrome protein (WASP), and diaphanous family
proteins, which promote actin polymerization, increases SRF activity.
Moreover, overexpression of G-actin causes decreased SRF activity.
These data strongly suggest that depletion of G-actin levels induces
SRF activation.
Based on our data and that of others, we propose the following model.
Disruption of the actin cytoskeleton increases the G-actin level, which
in turn decreases SRF activity. Decreased SRF activity relieves its
inhibition and therefore upregulates IL-1-induced iNOS expression.
During inflammation, IL-1
induces iNOS expression and NO production.
It was reported (14) that NO itself may increase G-actin
levels via cGMP-dependent protein kinase in human cervical epithelial
cells. In mesangial cells, we speculate that NO may shift the
steady-state actin toward G-actin, which further upregulates IL-1
-induced iNOS and NO production, a positive feedback loop. High
output of NO produced in mesangial cells plays an important role in the
kidney during inflammation. NO can affect contraction of mesangial
cells, which may regulate the glomerular filtration rate during
inflammation. Further dissection of the signaling pathway from the
actin cytoskeleton to iNOS expression may lead to a new finding of the
mechanism by which iNOS expression is regulated and a better
understanding of iNOS-involved physiological and pathological processes.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney DiseasesGrant DK-50606. C. Zeng was the recipient of a research Award from the National Kidney Foundation of Missouri and Metro East.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. R. Morrison, Barnes/Jewish Hospital, 216 South Kingshighway, 822 Yalem Research Bldg., St. Louis, MO 63110 (E-mail: morrison{at}pcg.wustl.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 9 January 2001; accepted in final form 3 May 2001.
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