Disruption of the actin cytoskeleton regulates cytokine-induced iNOS expression

Chenbo Zeng and Aubrey R. Morrison

Department of Medicine, Molecular Biology, and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Interleukin-1beta (IL-1beta ) 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-1beta -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-1beta -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-1beta -induced iNOS expression, providing direct evidence that SRF inhibits iNOS expression.

inducible nitric oxide synthase; serum response factor; latrunculin B; jasplakinolide; mesangial cell


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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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-1beta (IL-1beta ), tumor necrosis factor-alpha , and interferon-gamma , 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-kappa B (NF-kappa 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-1beta -induced iNOS expression in rat mesangial cells (16). Other regulators found to be critical in iNOS expression include C/EBPbeta (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-beta (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 alpha -actin, SM22alpha , and cardiac alpha -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-1beta 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.


    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-1beta (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-1beta (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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Effect of increased G-actin on IL-1beta -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-1beta 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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Fig. 1.   Latrunculin B (LatB) dramatically potentiates interleukin-1beta (IL-1beta )-induced inducible nitric oxide synthase (iNOS) protein expression. A: rat mesangial cells were grown in medium containing 10% fetal bovine serum (FBS). Cells were treated with 0.1, 0.5, or 1 µM LatB for 1 h. Cells were then induced with IL-1beta (100 U/ml) for 0, 3, 9, 19, and 24 h. Cell lysates were prepared and subjected to Western blot analysis using anti-iNOS antibody. B: quantitative analysis of Western blot in A. C: LatB by itself had little effect on iNOS expression. Rat mesangial cells were treated with 0.5 µM LatB for 1 h and then were either left untreated or were induced by adding IL-1beta (100 U/ml) into the medium containing 0.5 µM LatB for 0, 6, 12, 18, and 24 h. Cell lysates were prepared and subjected to Western blot analysis.

To test whether the concentrations of latrunculin B that enhance IL-1beta -induced iNOS inhibit actin polymerization in rat mesangial cell, we stained F-actin with NBD-phallacidin, a fluorescent toxin that binds to F-actin. Figure 2 shows that latrunculin B by itself or together with IL-1beta significantly disrupts F-actin filaments with a loss of stress fiber formation, whereas 1 h of IL-1beta treatment has little effect on F-actin structure.


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Fig. 2.   LatB prevents F-actin polymerization. Rat mesangial cells were either untreated (A and C) or treated (B and D) with 0.5 µM LatB for 1 h and then either unstimulated (A and B) or stimulated (C and D) with IL-1beta (100 U/ml) for 1 h. Cells were then fixed with 3.7% formaldehyde solution, washed with acetone, and stained with 165 nM NBD-phallacidin. Stained F-actin was imaged using a Nikon epifluorescent microscope.

Jasplakinolide suppresses iNOS expression enhanced by latrunculin B. To further confirm that latrunculin B enhances IL-1beta -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-1beta , 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-1beta -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-1beta , 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-1beta are required.


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Fig. 3.   Jasplakinolide (Jasp) suppresses IL-1beta -induced iNOS expression enhanced by LatB. Rat mesangial cells were grown in 0.5% FBS for 18 h and then were treated with the medium containing 10% FBS plus IL-1beta (100 U/ml), IL-1beta (100 U/ml) + LatB (0.5 µM), IL-1beta (100 U/ml) + Jasp (0.5 µM), or IL-1beta (100 U/ml) + LatB (0.5 µM) + Jasp (0.5 µM) for 22 h. Cell lysates were prepared and subjected to Western blot analysis using anti-iNOS antibody (B). The quantitative analysis of the Western blot is shown in A.

SRF activity negatively correlates with IL-1beta -induced iNOS expression. We next asked the question as to how disruption of the actin cytoskeleton upregulates IL-1beta -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-1beta -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-1beta -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-1beta -induced iNOS. Treatment with both latrunculin B and jasplakinolide caused a significant increase in SRF activity but a decrease in IL-1beta -induced iNOS compared with the treatment with latrunculin B alone. These data clearly show that SRF activity negatively correlates with IL-1beta -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-1beta -induced iNOS (Fig. 4, B and C). These data suggest that SRF plays an inhibitory role in IL-1beta -induced iNOS expression. Our data also showed that jasplakinolide by itself strongly activated SRF as expected, but it had little effect on IL-1beta -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-1beta -induced iNOS expression; therefore, further activation of SRF by jasplakinolide does not contribute more to this inhibitory effect.


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Fig. 4.   Serum response factor (SRF) activity and IL-1beta -induced iNOS expression are negatively correlated. Rat mesangial cells were cotransfected with c-fos-luc and pRL-TK constructs. After cotransfection, cells were treated with medium containing 10% FBS and IL-1beta (100 U/ml) with or without indicated actin-binding drugs for 22 h. Cell lysates were then prepared and analyzed for luciferase activity by reporter gene assay and iNOS expression by Western blot. The drug concentrations were 0.5 µM latrunculin B (LatB), 0.5 µM jasplakinolide (Jasp), 30 nM swinholide A (Swin), and 0.2 µM cytochalasin D (CytD). Control (CON) was 10% FBS + IL-1beta (100 U/ml).

Overexpression of dominant negative mutant of SRF increased IL-1beta -induced iNOS expression. To further test the idea that SRF inhibits IL-1beta -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-1beta 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-1beta -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-1beta -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-1beta -induced iNOS expression, providing strong evidence that SRF plays an inhibitory role in IL-1beta -induced iNOS expression.


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Fig. 5.   Overexpression of dominant negative mutant of SRF increases iNOS expression. A: rat mesangial cells in each 60-mm dish were transiently transfected with either pCGN-SRF (2.5, 5.0, and 7.5 µg) or pCGN-SRFpm1 (2.5, 5.0, and 7.5 µg). Cells were then cultured in 10% FBS and IL-1beta (100 U/ml) for 15 h. Cell lysates were prepared and subjected to Western blot analysis using anti-iNOS antibody (top). The overexpressed SRF and SRFpml were blotted with anti-HA antibody (bottom). B: cells were transiently transfected with either pCGN-SRF or pCGN-SRFpm1 and maintained in 10% FBS for 24 h. Cells were then treated with 10% FBS + 100 U/ml IL-1beta (CON), 10% FBS + 100 U/ml IL-1beta  + 0.3 µM LatB, or 10% FBS + 100 U/ml IL-1beta  + 0.3 µM Jasp for 24 h. Cell lysates were subjected to Western blot analysis using anti-iNOS antibody.


    DISCUSSION
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We report here two important findings. First, actin cytoskeletal dynamics regulates IL-1beta -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-1beta -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-1beta -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-1beta 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-kappa 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-1beta -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 SM22alpha (27), as well as the IL-2 receptor alpha  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-1beta 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 alpha  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-1beta -induced iNOS expression. It is intriguing to understand how the actin cytoskeleton system relates to IL-1beta -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-1beta -induced iNOS expression. During inflammation, IL-1beta 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-1beta -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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