Rho protein-mediated changes in the structure of the actin cytoskeleton regulate human inducible NO synthase gene expression

doi:10.1016/S0014-4827(03)00129-0

Experimental Cell Research

Volume 287, Issue 1, 1 July 2003, Pages 106-115

https://doi.org/10.1016/S0014-4827(03)00129-0 Get rights and content

Abstract

Rho proteins (Rho, Rac, Cdc 42) are known to control the organization of the actin cytoskeleton as well as gene expression. Inhibition of Rho proteins by Clostridium difficile toxin B disrupted the F-actin cytoskeleton and enhanced cytokine-induced inducible nitric oxide synthase (iNOS) expression in human epithelial cells. Also specific inhibition by Y-27632 of p160ROCK, which mediates Rho effects on actin fibers, caused a disruption of the actin cytoskeleton and a superinduction of cytokine-induced iNOS expression. Accordingly, direct disruption of the actin cytoskeleton by cytochalasin D, latrunculin B, or jasplakinolide enhanced cytokine-induced iNOS expression. The transcription factor serum response factor (SRF) has been described as mediating actin cytoskeleton-dependent regulation of gene expression. Direct targets of SRF are activating protein 1 (AP1)-dependent genes. All compounds used inhibited SRF- and AP1-dependent reporter gene expression in DLD-1 cells. However, the enhancing effect of the actin cytoskeleton-disrupting compounds on human iNOS promoter activity was much less pronounced than the effect on iNOS mRNA expression. Therefore, besides transcriptional mechanisms, posttranscriptional effects seem to be involved in the regulation of iNOS expression by the above compounds. In conclusion, our data suggest that Rho protein-mediated changes of the actin cytoskeleton negatively modulate the expression of human iNOS.

Introduction

The inducible NO synthase (iNOS),¹ the high-output NOS, is normally absent from resting cells [1]. After activation of cells by different inducers (bacterial lipopolysaccharides, cytokines) iNOS is expressed. NO synthesized by iNOS can have beneficial effects, such as antimicrobial, antiatherogenic, and antiapoptotic actions. Also iNOS-generated NO has been shown to be involved in skin wound healing [2] and protection of liver cells against different types of stress [3]. In contrast, inappropriate iNOS induction can have detrimental consequences, such as cellular injury in arthritis, colitis, or septic shock [1], [4].

The regulation of iNOS expression is cell- and species-specific, with a wide variety of signal transduction pathways involved [1]. It has generally been believed that the expression of iNOS is regulated mainly at the level of transcription. The human iNOS promoter contains potential binding sites for a number of transcription factors, such as STAT-1 (signal transducer and activator of transcription 1), AP1 (jun/fos transcription factor), and NF-κB (nuclear factor κB). These transcription factors are known to participate in the induction of iNOS expression by cytokines [1], [5]. However, the steady-state level of a particular mRNA depends not only on its rate of synthesis, but also on its rate of degradation. Several authors have presented data showing that regulation of mRNA stability contributes to iNOS expression [6], [7].

Small GTP-binding proteins (G-proteins) are monomeric proteins with molecular masses of 20–40 kDa. Five subfamilies are known [8]. Two of these subfamilies, Ras and Rho proteins, have been described as regulators of gene expression [8]. Accumulating data point to the activation of the Jun N-terminal kinase (JNK) and the p38 mitogen-activated protein kinase (p38 MAPK) by Rho proteins (mostly Cdc42 and Rac), whereas Ras proteins have been shown to control activation of the p42/44 MAPK cascade [8]. Rho proteins have been reported to activate the transcription factors serum response factor (SRF) and NF-κB [8]. In addition, the Rho family of small G-proteins is known to regulate diverse cellular processes involving the cytoskeleton. These include actin polymerization, F-actin bundling, myosin-based contractility, focal adhesion formation, and cytokinesis [8]. A direct Rho A target, which mediates Rho-induced assembly of focal adhesions and stress fibers, is p160ROCK [8], which can be specifically inhibited by compound Y-27632 [9]. Recent data published by Sotiropoulos et al. [10] showed regulation of SRF activity by changes in actin dynamics. Therefore, reorganization of the actin cytoskeleton by Rho proteins may also be important for Rho-dependent gene regulation. In addition, several authors have described the regulation of gene expression by compounds like cytochalasin D and latrunculin B, which directly disrupt the F-actin fibers [11], [12].

The cytokines used to induce iNOS expression in different cell models are known to activate small G-proteins of the Ras/Rho family [13]. Indirect inhibition of the activity of these proteins with statins enhanced cytokine-induced iNOS expression in human epithelial cells [14]. This statin-related enhancement of iNOS expression was blocked by addition of geranylgeranylpyrophosphate (GGPP), but not farnesylpyrophosphate [14], pointing to an involvement of the Rho family of small G-proteins, as their activity depends on posttranslational geranylation.

In the current study, we sought to investigate the molecular mechanism of the negative regulation of iNOS expression by small G-proteins of the Rho family in human epithelial cells. Our data demonstrate that the negative regulation of iNOS induction in human cells seems to be related to a Rho A-mediated reorganization of the actin cytoskeleton. This effect may involve an activation of transcription factors SRF and AP1 and transcriptional and posttranscriptional mechanisms.

Section snippets

Reagents

Trypsin, glutamine, and pyruvate solutions, agarose, tRNA, cytochalasin D, TRITC-labeled phalloidin, formaldehyde, TPA (12-O-tetradecanoyl-phorbol-13-acetate), Triton X-100 and BSA were purchased from Sigma, Deisenhofen, Germany. Isotopes were obtained from NEN/Dupont, Köln, Germany. Restriction enzymes, Taq polymerase, Klenow DNA polymerase, T7-Sequencing Kit, dNTPs, and NTPs were purchased from Amersham-Biosciences, Freiburg, Germany. T3 and T7 RNA polymerase, RNase A, RNase T1, DNase I, and

Calculations

All data are presented as means ± SEM. Differences between groups were tested for statistical significance using factorial ANOVA (StatView, SAS Institute Inc.) followed by Fisher’s PLSD test.

TcdB enhances cytokine-induced iNOS expression in human cells

Previous research in our laboratory had demonstrated that cytokine-induce iNOS expression was enhanced by statins in human and murine cells. This effect of statins had been attributed to an indirect inhibition of the activity of Rho proteins caused by reduced cellular GGPP levels [14]. Here we show in human epithelial DLD-1 cells that direct inhibition of the Rho proteins with TcdB [20] also enhanced cytokine-induced iNOS mRNA and NO production (Fig. 1A, 1B and 1D, [14]). TcdB did not induce

Conclusion

Our data demonstrate that TcdB and compound Y-27632 potentiate cytokine-induced iNOS expression via inhibition of Rho A-dependent reorganization of the actin cytoskeleton. Compounds directly disrupting the actin cytoskeleton such as cytochalasin D, latrunculin B, and jasplakinolide mimicked this effect on iNOS expression. All of these compounds seem to exert a dual effect on the iNOS gene: increased promoter activity and stabilization of the mRNA.

Acknowledgements

The expert technical assistance of I. Ihrig-Biedert and K. Masch is gratefully acknowledged. The plasmid pNOS(16)Luc was a generous gift of Dr. D.A. Geller, Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA. This work was supported by Grant 8312-38 62 61 from the Innovation Foundation of the State of Rhineland-Palatinate (to H.K. and U.F.), Grant K1 1020/4-1 from the Deutsche Forschungsgemeinschaft (to H.K.), and by the Collaborative Research Center SFB 553 (Project A7 to

References (40)

H Kleinert et al.
Regulation of the expression of nitric oxide synthase isoforms
B Stallmeyer et al.
The function of nitric oxide in wound repairinhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization
J. Invest. Dermatol.
(1999)
Y.M Kim et al.
Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-α-induced apoptosis by inducing heat shock protein 70 expression
J. Biol. Chem.
(1997)
F Rodriguez-Pascual et al.
Complex contribution of the 3′-untranslated region to the expressional regulation of the human inducible nitric-oxide synthase geneinvolvement of the RNA-binding protein HuR
J. Biol. Chem.
(2000)
A Sotiropoulos et al.
Signal-regulated activation of serum response factor is mediated by changes in actin dynamics
Cell
(1999)
C.P Mack et al.
Smooth muscle differentiation marker gene expression is regulated by RhoA-mediated actin polymerization
J. Biol. Chem.
(2001)
P Chomczynski et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987)
H Kleinert et al.
Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor nuclear factor-κB
Mol. Pharmacol.
(1996)
C von Eichel-Streiber et al.
Large clostridial cytotoxinsa family of glycosyltransferases modifying small GTP-binding proteins
Trends Microbiol.
(1996)
M Coue et al.
Inhibition of actin polymerization by latrunculin A
FEBS Lett.
(1987)

C.J Sympson et al.

Cytochalasin D-induced actin gene expression in murine erythroleukemia cells

Exp. Cell Res.

(1993)

M.R Bubb et al.

Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin

J. Biol. Chem.

(1994)

M.R Bubb et al.

Effects of jasplakinolide on the kinetics of actin polymerizationan explanation for certain in vivo observations

J. Biol. Chem.

(2000)

T Henics

Microfilament-dependent modulation of cytoplasmic protein binding to TNFalpha mRNA AU-rich instability element in human lymphoid cells

Cell Biol. Int.

(1999)

G Zambetti et al.

Disruption of the cytoskeleton with cytochalasin D induces c-fos gene expression

Exp. Cell Res.

(1991)

D Rajotte et al.

Contribution of both STAT and SRF/TCF to c-fos promoter activation by granulocyte–macrophage colony-stimulating factor

Blood

(1996)

T Kizaki et al.

Negative regulation of LPS-stimulated expression of inducible nitric oxide synthase by AP-1 in macrophage cell line J774A.1

Biochem. Biophys. Res. Commun.

(2001)

K.D Kröncke et al.

Inducible nitric oxide synthase in human diseases

Clin. Exp. Immunol.

(1998)

H Kleinert et al.

Cytokine induction of NO synthase II in human DLD-1 cellsroles of the JAK-STAT, AP-1 and NF-κB-signaling pathways

Br. J. Pharmacol.

(1998)

Y Vodovotz et al.

Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor-β1

J. Exp. Med.

(1993)

Cited by (45)

Genome-wide analysis for nanofiber induced global gene expression profile: A study in MC3T3-E1 cells by RNA-Seq
2023, Colloids and Surfaces B: Biointerfaces
Nanofibers are one of the attractive biomaterials that can provide unique environments to direct cell behaviors. However, how nanofiber structure affects the global gene expression of laden cells remains unclear. Herein, high-throughput mRNA sequencing (RNA-seq) is applied to analyze the transcriptome of the MC3T3-E1 cells (a model osteoblast cell line) cultured on electrospun nanofibers. The cell-adhesive poly(L-lactide) nanofibers and membranes are developed by the mussel-inspired coating of gelatin-dopamine conjugate under H₂O₂-mediated oxidation. The MC3T3-E1 cells cultured on nanofibers exhibit elongated morphology and increased proliferation compared with those on membranes. The differences in global gene expression profiles are determined by RNA-seq, in which 905 differentially expressed genes (DEGs) are identified. Significantly, the DEGs related to cytoskeleton, promotion of cell cycle progression, cell adhesion, and cell proliferation, are higher expressed in the cells on nanofibers, while the DEGs involved in cell-cycle arrest and osteoblast mineralization are up-regulated in the cells on membranes. This study elucidates the roles of nanofiber structure in affecting gene expression of laden cells at the whole transcriptome level, and it will lay the foundation for understanding nanofiber-guided cell behaviors.
ROCK induced inflammation of the microcirculation during endotoxemia mediated by nitric oxide synthase
2011, Microvascular Research
Citation Excerpt :
The response to Rho and ROCK inhibition is conflicting, with evidence for both increased and decreased iNOS expression. One study using hearts from diabetic rats, indicated that activation of the Rho/ROCK pathway is involved in iNOS up-regulation (Soliman et al. 2008) and this has been supported in a number of models (Ikeda et al., 2001; Machida et al., 2008; Tiftik et al., 2008; Witteck et al., 2003) whereas decreased iNOS expression has been observed in preadipocytes (Dobashi et al., 2008). This conflicting evidence may be due to the different in vitro and in vivo models and may explain the lack of response with the higher dose of fasudil (10 mg kg− 1) in our model.
Sepsis may be modeled using lipopolysaccharide (LPS), which alters levels of nitric oxide (NO), synthesized via endothelial and inducible nitric oxide synthase (eNOS and iNOS). This study aimed to determine whether the Rho kinase (ROCK) inhibitor fasudil protected against LPS-induced (endotoxemia) macromolecular leak and leukocyte adhesion via NOS pathways. Male Wistar rats (283 ± 8 g, n = 36) were anaesthetized with thiopental and the mesentery prepared for fluorescent intravital microscopy (IVM). Animals received either (i) LPS alone (150 μg kg^− 1 h⁻¹ i.v., n = 6); (ii) fasudil (FAS, 3 mg kg^− 1 i.v., n = 6) or (iii) fasudil (10 mg kg^− 1 i.v., n = 6), immediately prior to LPS administration, (iv) fasudil (FAS, 3 mg kg^− 1 i.v., n = 6) alone or (v) fasudil (FAS, 10 mg kg^− 1 i.v., n = 6) alone, or (vi) saline alone (1 ml kg^− 1 h^− 1 i.v, n = 6) for 4 h (240 min). LPS increased macromolecular leak (cumulative normalized grey levels, arbitrary units) from post capillary venules (< 40 μm) and this was reduced by 3 mg kg^− 1 fasudil, however, 10 mg kg^− 1 was less effective (t = 240 min, control: 3.3 ± 1.7; LPS: 15.1 ± 2.0; LPS + 3 mg kg^− 1 fasudil: 3.3 ± 1.1 (p < 0.05), LPS + 10 mg kg^− 1 fasudil: 8.4 ± 3.2 NS). The numbers of leukocytes adhering for > 1 min/100 μm venule were reduced by fasudil (t = 240 min, control: 1.8 ± 0.7; LPS: 7.0 ± 1.0; LPS + 3 mg kg^− 1 fasudil: 1.75 ± 0.25, p < 0.05; LPS + 10 mg kg^− 1 fasudil: 1.8 ± 0.8, p < 0.05). Immunohistochemistry demonstrated that fasudil increased endothelial cell expression of eNOS during sepsis, and decreased LPS-induced up-regulation of iNOS. Inhibition of ROCK in rats increases eNOS and decreases iNOS during endotoxemia, concomitantly reducing microvascular inflammation. Thus, targeting the ROCK pathway during sepsis could have therapeutic potential for reducing inflammation via a NO dependent mechanism.
Regulation of the expression of inducible nitric oxide synthase
2010, Nitric Oxide - Biology and Chemistry
Nitric oxide (NO) generated by the inducible isoform of nitric oxide synthase (iNOS) is involved in complex immunomodulatory and antitumoral mechanisms and has been described to have multiple beneficial microbicidal, antiviral and antiparasital effects. However, dysfunctional induction of iNOS expression seems to be involved in the pathophysiology of several human diseases. Therefore iNOS has to be regulated very tightly.
Modulation of expression, on both the transcriptional and post-transcriptional level, is the major regulation mechanism for iNOS. Pathways resulting in the induction of iNOS expression vary in different cells or species. Activation of the transcription factors NF-κB and STAT-1α and thereby activation of the iNOS promoter seems to be an essential step for the iNOS induction in most human cells. However, at least in the human system, also post-transcriptional mechanisms involving a complex network of RNA-binding proteins build up by AUF1, HuR, KSRP, PTB and TTP is critically involved in the regulation of iNOS expression. Recent data also implicate regulation of iNOS expression by non-coding RNAs (ncRNAs).
Transcriptional and post-transcriptional regulation of iNOS expression in human chondrocytes
2010, Biochemical Pharmacology
Citation Excerpt :
This implies that iNOS induction in chondrocytes needs some “superconfluency-derived” stress signals to enable the cytokines to activate iNOS expression. As iNOS expression is regulated by Rho-mediated changes in the structure of the actin cytoskeleton [34], it is tempting to speculate that the above-mentioned stress signals may relate to superconfluency-induced changes in the structure of the actin cytoskeleton. In addition, there is evidence that the Rho-mediated modulation of the actin cytoskeleton is involved in the expressional changes essential for chondrogenesis [35].
Chondrocytes are important for the development and maintenance of articular cartilage. However, both in osteoarthritis (OA) and rheumatoid arthritis (RA) chondrocytes are involved in the process of cartilage degradation and synthesize important immunomodulatory mediators, including nitric oxide (NO) generated by the inducible NO synthase (iNOS). To uncover the role of iNOS in the pathomechanisms of OA and RA, we analyzed the regulation of iNOS expression using immortalized human chondrocytes as a reproducible model.
In C-28/I2 chondrocytes, iNOS expression was associated with the expression of the chondrocyte phenotype. Peak induction by a cytokine cocktail occurred between 6 and 8 h and declined by 24 h. Inhibition of p38MAPK, NF-κB and the JAK2-STAT-1α pathways resulted in a reduction of iNOS expression. In contrast to other cell types, the cytokine-mediated induction of the human iNOS promoter paralleled the induction rate of the iNOS mRNA expression in C-28/I2 chondrocytes. However, in addition post-transcriptional regulation of iNOS expression by the RNA binding protein KSRP seems to operate in these cells. As seen in other chondrocyte models, glucocorticoids were not able to inhibit cytokine-induced iNOS expression in C-28/I2 cells, due to the lack of the glucocorticoid receptor mRNA expression. In this model of glucocorticoid-resistance, the new fungal anti-inflammatory compound S-curvularin was able to inhibit cytokine-induced iNOS expression and iNOS-dependent NO-production.
In summary, we demonstrate for the first time that differentiated human immortalized C-28/I2 chondrocytes are a representative cell culture model to investigate iNOS gene expression in human joint diseases.
Molecular Regulation of Inducible Nitric Oxide Synthase
2010, Nitric Oxide
This chapter focuses on the molecular mechanisms regulating iNOS expression and the pathways governing iNOS transcription. It reveals how the epigenetic control mechanisms regulate human iNOS expression. Inducible nitric oxide synthase (iNOS) gene expression and NO production can be stimulated in many cell types with suitable agents such as bacterial lipopolysaccharides, cytokines, growth factors, and other compounds. Many signaling pathways are involved in the regulation of iNOS expression, such as IKK-I_kB-NF-_kB, JAK-STATs, PI3K-Akt, MAPKs, as well as the ubiquitin-proteasome degradation pathway. Specific nuclear transcription factors include NF-_kB, STAT-1, IRF-1, AP-1, Oct1, C/EBPβ, and others that interact with their cis-acting DNA binding elements in the iNOS promoter and induce iNOS transcription. The molecular regulation of the rodent and human iNOS genes is complex and involves transcriptional, post-transcriptional, translational, post-translational, and epigenetic control mechanisms. Although transcriptional regulation is the predominant control mechanism, post-transcriptional, translational, and post-translational regulation also play critical roles in iNOS expression. A plethora of cytokines, inflammatory mediators, and transcription factors coordinate the expression of a tightly controlled iNOS gene. Induced NO synthesis has both beneficial and detrimental consequences depending upon the physiologic environment and magnitude of expression. Therefore, understanding the signaling pathways and molecular mechanisms regulating iNOS expression is crucial for the development of therapies to modulate iNOS expression under pathophysiologic conditions.
Regulation of the Expression of Inducible Nitric Oxide Synthase
2010, Nitric Oxide
This chapter reveals how nitric oxide (NO) generated by the inducible isoform of nitric oxide synthase (iNOS) exerts multiple beneficial microbicidal, antiviral, antiparasital, complex immunomodulatory, and antitumoral effects. Aberrant iNOS induction in the wrong place or at the wrong time has detrimental consequences and it is involved in the pathophysiology of several human diseases. Therefore, iNOS has to be regulated very tightly. The inducible isoform of NOS is mainly regulated at the level of expression. The mechanisms regulating iNOS expression involve modulation of promoter activity, mRNA stability and translatability, and protein stability. Modulation of iNOS expression is the major regulation mechanism for iNOS. Both transcriptional and post-transcriptional mechanisms have been shown to regulate iNOS expression. In contrast to the murine system, induction of human iNOS expression normally needs a complex mixture of cytokines. Pathways resulting in the induction of iNOS expression vary in different cells or species. Activation of the transcription factors NF-_kB and STAT-1α and thereby activation of the iNOS promoter seems to be an essential step for iNOS induction in most human cells. However, at least in the human system, post-transcriptional mechanisms, involving a complex network of RNA binding proteins built up by AUF1, HuR, KSRP, PTB, and TTP, are also critically involved in the regulation of iNOS expression.

View all citing articles on Scopus

^☆: This article contains data from the theses of A.W. and Y.Y.

View full text

Regular articleRho protein-mediated changes in the structure of the actin cytoskeleton regulate human inducible NO synthase gene expression ☆

Abstract

Introduction

Section snippets

Reagents

Calculations

TcdB enhances cytokine-induced iNOS expression in human cells

Conclusion

Acknowledgements

J. Invest. Dermatol.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

Anal. Biochem.

Mol. Pharmacol.

Trends Microbiol.

FEBS Lett.

Exp. Cell Res.

J. Biol. Chem.

J. Biol. Chem.

Cell Biol. Int.

Exp. Cell Res.

Blood

Biochem. Biophys. Res. Commun.

Inducible nitric oxide synthase in human diseases

Clin. Exp. Immunol.

Cytokine induction of NO synthase II in human DLD-1 cellsroles of the JAK-STAT, AP-1 and NF-κB-signaling pathways

Br. J. Pharmacol.

Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor-β1

J. Exp. Med.

Regular article
Rho protein-mediated changes in the structure of the actin cytoskeleton regulate human inducible NO synthase gene expression ^☆