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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2622.)
© 2006 American Heart Association, Inc.

Vascular Biology

The Role of Human Antigen R, an RNA-binding Protein, in Mediating the Stabilization of Toll-Like Receptor 4 mRNA Induced by Endotoxin

A Novel Mechanism Involved in Vascular Inflammation

Feng-Yen Lin; Yung-Hsiang Chen; Yi-Wen Lin; Jen-Sung Tsai; Jaw-Wen Chen; Hsiao-Jung Wang; Yuh-Lien Chen; Chi-Yuan Li; Shing-Jong Lin

From the Graduate Institute of Medical Sciences (F.Y.L.), Graduate Institute of Life Sciences (Y.W.L.), Departments of Anesthesiology (C.Y.L.), Departments of Cardiovascular Surgery (J.S.T.), Tri-service General Hospital, National Defense Medical Center; Institute of Clinical Medicine (Y.H.C., H.J.W., S.J.L.), and Cardiovascular Research Center (J.W.C., S.J.L.), National Yang-Ming University; Division of Cardiology (J.W.C., S.J.L.), Taipei Veterans General Hospital; Department of Medical Education and Research (C.Y.L.), Buddhist Tzu-Chi General Hospital; Department of Anatomy and Cell Biology (Y.L.C.), College of Medicine, National Taiwan University, Taipei, Taiwan.

Correspondence to Dr Shing-Jong Lin, Division of Cardiology, Taipei Veterans General Hospital, 201, Sec. 2, Shi-Pai Rd., Taipei 112, Tawian. E-mail sjlin{at}vghtpe.gov.tw; Dr Yuh-Lien Chen, ylchen@ha.mc.ntu.edu.tw; Dr Chi-Yuan Li, cyli@ndmctsgh.edu.tw

	Abstract

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Objective— Lipopolysaccharide (LPS) interacts with toll-like receptor 4 (TLR4) and induces proliferation of vascular smooth muscle cells (VSMCs) which plays a causal role in atherogenesis. The role of TLR4 expression and regulation in LPS-stimulated VSMCs remains unclear. TLR4 mRNAs often contain AU-rich elements (AREs) in their 3' untranslated regions (3'UTR) which have a high affinity for RNA-binding proteins. It is not know whether the RNA-binding protein, human antigen R (HuR), regulates TLR4 expression in human aortic smooth muscle cells (HASMCs).

Methods and Results— Stimulation of HASMCs with LPS significantly increased the cytosolic HuR level in vitro. Immunoprecipitation and RT-PCR demonstrated that LPS markedly increased the interaction of HuR and 3'UTR of TLR4 mRNA. The reporter plasmid, which contains the 3'UTR of TLR4 mRNA, significantly increased luciferase reporter gene expression in LPS-induced HASMCs. These data suggest that the 3'UTR of TLR4 mRNA confers LPS responsiveness and that HuR modulates 3'UTR-mediated gene expression. Knock-down of HuR inhibited LPS-induced TLR4 mRNA stability in HASMCs and luciferase reporter gene expression in CMV-Luciferase-TLR4 3'UTR-transfected HASMCs. In addition, inhibition of NADPH oxidase activity by diphenylene iodonium, knock-down of Rac1 gene expression by siRNA, and decrease of p38 MAPK activity by SB203580 significantly decreased the cytosolic HuR level, which mediates TLR4 mRNA stability.

Conclusion— Activation of NADPH oxidase and the MAPK-signaling pathway contribute to HuR-mediated stabilization of TLR4 mRNA induced by LPS in HASMCs. In the balloon injured rabbit aorta model, systemic inflammation induced by LPS caused intimal hyperplasia and increased TLR4 and HuR expression.

We investigated the posttranscriptional regulation in LPS-induced TLR4 expression in HASMCs and balloon-injured rabbit aorta. These results demonstrate that activation of NADPH oxidase and the MAPK-signaling pathway contribute to HuR-mediated stabilization of TLR4 mRNA induced by LPS in smooth muscle cells.

Key Words: LPS • toll-like receptor • human antigen R • inflammation • vascular smooth muscle cell (VSMC)

	Introduction

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Epidemiological research suggests that failure of coronary artery bridge grafts or restenosis is brought about by chronic inflammation¹ induced by endotoxin.² LPS-induced systemic inflammatory responses could increase neointimal formation after balloon injury and stent implantation,³ and the resulting proliferation of vascular smooth muscle cells (VSMCs) may play a key role in atherogenesis. Toll-like receptor 4 (TLR4) mediates the cellular activation by LPS. When cells are stimulated by endotoxin, TLR4 leads to activation of p44/p42 mitogen-activated protein kinase (MAPK)⁴ and proliferation⁵ in VSMCs.

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Previous studies have demonstrated that TLR4 is expressed abundantly in failing myocardium⁶ and in macrophages infiltrated into human and murine lipid-rich atherosclerotic lesions.⁷ The functional expression of TLR4 and subsequent augmentation of intimal hyperplasia have recently been described.⁸ Hypoxia diminishes TLR4 expression through reactive oxygen species (ROS) generated by mitochondria.⁹ Antecedent resuscitated hemorrhagic shock influences the TLR4 mRNA steady-state level.¹⁰ Although TLR4 expression affects cell responsiveness under endotoxin stimulation, and TLR4 expression under LPS stimulation is controlled by transcriptional and posttranscriptional mechanisms,¹⁰ TLR4 expression and related mechanisms in VSMCs are still unclear.

The basal expression of proteins associated with inflammatory responses, immunoregulation, oncogenesis, and cell growth is normally very low,¹¹ possibly because of repression of transcription through the potentially unstable expression of mRNAs.¹² Unstable mRNAs often contain AU-rich elements (AREs) in their 3' untranslated region (UTR). The characteristic motif is AUUUA, but the ARE size and AU content may vary. The ARE-mRNA database (http://rc.kfshrc.edu.sa/ared) has clustered ARE into three groups depending on the number of motifs in the ARE stretch; previous reports have demonstrated that ARE-encoded biological diversity results in the occurrence of some diseases.¹³

ARE-regulated mRNA stability is mediated by RNA-binding proteins, such as human antigen R (HuR), AU-binding factor 1 (AUF 1), and tristetraprolin (TTP).¹⁴ HuR is a ubiquitous protein belonging to the embryonic lethal abnormal vision family of RNA-binding proteins, predominantly nuclear proteins, which shuttle between the nucleus and cytoplasm. HuR regulates cyclin A, cyclin B1,¹⁵ and p21¹⁶ mRNA stability during cell proliferation. AUF1, a member of the heteronuclear ribonucleoprotein (hnRNP) family (also known as hnRNP D), is another RNA-binding protein, which exists in four isoforms (37, 40, 42, and 45 kDa). Although AUF1 is thought to destabilize mRNA,¹⁷ the AUF1 isoforms have different roles in the regulation of mRNA turnover.¹⁸ TTP is critically implicated in inflammation and is a member of zinc finger proteins that bind to AREs and destabilize mRNAs of tumor necrosis factor (TNF-).¹⁹ However, TTP knockout causes a severe inflammatory syndrome in vivo,²⁰ and it is possible that the effects of TTP are countered by HuR.²¹

Our previous data showed that LPS-enhanced TLR4 expression in human aortic smooth muscle cells (HASMCs) is mediated by mRNA stabilization, although we have not yet clarified whether RNA-binding proteins are involved in this process. The aim of this study was to explore the cellular events involved in, and the mechanisms underlying, the LPS-enhanced TLR4 mRNA stability in HASMCs. We used an animal model to confirm that LPS affects the expression of RNA-binding proteins associated with TLR4 expression in the neointima of the balloon-injured rabbit aorta. Our results indicate that HuR is involved in mediating the stabilization of TLR4 mRNA induced by LPS.

	Materials and Methods

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Cell Culture
The HASMCs and THP-1 cells were used for this study (please see the data supplement, available online at http://atvb.ahajournals.org).

Quantitative Real Time and Traditional Polymerase Chain Reaction
Total RNA was isolated using a TRIzol reagent kit (Invitrogen), according to the manufacturer’s instructions. The detailed method was published on http://atvb.ahajournals.org.

Actinomycin D Chase Experiments
Actinomycin D chase experiments were performed for mRNA stability (please see the online data supplement).

Confocal Microscopy
The expression of HuR was observed with confocal microscopy (please see the online data supplement).

Western Blot Analysis
Western blot analysis was conducted to determine the levels of HuR and AUF1 in HASMCs (please see the online data supplement).

Knockdown Gene Expression With Interference RNA
Knockdown gene expression by transfection was measured with small interfering RNA (siRNA) (please see the online data supplement).

Cross-Linking Immunoprecipitation Assay of RNA-Protein Interaction
To determine whether HuR interacts directly with the 3'UTR of TLR4 mRNA, immunoprecipitation and RT-PCR were carried out (please see the online data supplement).

Luciferase Reporter Assay and Transfection
Functional analysis of the 3'UTR of TLR4 mRNA was performed using plasmids containing the 3'UTR and luciferase reporter gene (from pGL-Basic vector (Promega) (please see the online data supplement).

Animal Balloon Injury Experiment
The animals were randomly divided into five groups. Group 1 served as the control; group 2 (LPS) and 4 (ED+LPS) received intravenous injections of LPS (220 ng/kg) through the ear vein; groups 3 (ED) and 4 received the balloon injury treatment. In addition, group 4 received intravenous LPS immediately and again 1 week after balloon injury (please see the online data supplement).

Immunohistochemical Staining
The expression of TLR4 and HuR were observed with immunohistochemical staining (please see the online data supplement).

Statistical Analyses
Values are expressed as the mean±SEM. Data were analyzed using Student t test, and one- or two-way ANOVA followed by the Dunnett test. A probability value <0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
LPS Prolongs TLR4 mRNA Stability and Promotes TLR4 mRNA Expression
HASMCs were treated with 25 ng/mL LPS for 2 hours and then actinomycin D for 1 hour. The t_1/2 of mRNA deduced for the various conditions indicated that LPS stimulation rapidly increased the stability of TLR4 mRNA in HASMCs (LPS group: 309.2±22.2 minutes versus control group: 58.9±6.9 minutes). Under 25 ng/mL of LPS stimulation, the expression of TLR4 mRNA was elevated at 1 hour (149.5±15.9% of control) and reached maximal level (210.3±13.2% of control) at 2 hours, suggesting that LPS significantly induces TLR4 mRNA expression in HASMCs (please see the online data supplement).

LPS Triggers a Distinct Increase in Cytoplasmic HuR
HuR was found predominantly in the nucleus in nontreated HASMCs. Treatment with 25 ng/mL LPS caused a marked accumulation of cytoplasmic HuR over time (Figure 1A); in contrast, AUF1 expression was found predominantly in the nucleus, and its distribution remained unchanged following LPS treatment (Figure 1B). In Western blot analysis, LPS markedly increased the cytoplasmic level of HuR but not AUF1 (Figure 1C); and the level of nuclear HuR did not decrease concomitantly with the increase in cytoplasmic HuR. The heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 and ß-actin were used to supervise the process of protein extraction, and the extracted protein was loaded on gels. To identify the TTP expression in HASMCs, traditional RT-PCR and quantitative real-time PCR were used to measure TTP mRNA expression in HASMCs after 100 ng/mL LPS treatment. TTP mRNA was not expressed in quiescent or LPS-stimulated HASMCs; in contrast, as reported previously,²² the addition of 100 ng/mL LPS rapidly increased the expression of TTP mRNA by THP-1 cells (Figure 1D). These observations suggest that LPS treatment significantly increases cytoplasmic HuR accumulation because of the export of nuclear HuR but does not influences the distribution of AUF1 or expression of TTP.

View larger version (41K): [in this window] [in a new window]   Figure 1. Subcellular distribution of HuR and AUF1 in HASMCs detected by immunofluorescence and Western blot analysis. A and B, HuR and AUF1 was identified with immunocytofluorescence and observed by confocal microscope. C, HuR and AUF1 protein expression in the HASMC cytoplasm and nucleu. ß-actin and hnRNP C1/C2 were used as internal controls. D, Traditional RT-PCR and quantitative real time PCR were used to identify TTP mRNA expression in HASMCs and THP-1 cells. The data are presented as means±SEM and represent the results from three independent experiments. *P<0.01 compared with the unstimulated group.

Knockdown of HuR Gene Inhibits LPS-Induced TLR4 mRNA Expression
Western blot analysis revealed the effective reduction of HuR in the HuR siRNA-transfection group compared with the negative control siRNA-transfection group and the naive control group (Figure 2A). LPS-prolonged TLR4 mRNA stability and LPS-induced TLR4 mRNA expression were blocked completely by HuR siRNA (Figure 2B and 2C); this effect was not observed in the siRNA negative control, suggesting the critical role of HuR in the regulation of TLR4 mRNA.

View larger version (18K): [in this window] [in a new window]   Figure 2. Silencing of HuR expression by siRNA inhibited LPS-induced TLR4 mRNA expression in HASMCs. A, After silence of HuR expression by siRNA, the intracellular total HuR level was measured by Western blot analysis. ß-actin was used to monitor the equality of sample loading. B and C, The half-life and expression level of TLR4 mRNA were analyzed by an actinomycin D chase experiment and quantitative real time PCR, respectively. The half-life of TLR4 mRNA was calculated according to the mRNA decay rate. The data are presented as means±SEM and represent the results from four independent experiments. *P<0.01 compared with the unstimulated group, P<0.01 compared with the LPS-treated group.

HuR Interacts With the 3'UTR of TLR4 mRNA
Based on the cytoplasmic localization of HuR in LPS-treated HASMCs and the specific region of ARE recognized by HuR,²³ we postulated that the HuR might interact with the 3'UTR of TLR4 mRNA and assessed this possibility using immunoprecipitation and RT-PCR. Protein fractions were subjected to immunoprecipitation with anti-HuR antibody or control pre-immune mouse serum and subjected to polyacrylamide gel electrophoresis. Anti-HuR antibody was efficient in the immunoprecipitation process (Figure 3A). Treatment with LPS markedly increased the HuR interaction with 3'UTR of TLR4 mRNA (Figure 3B). These findings indicate that LPS increases the HuR interaction with 3'UTR of TLR4 mRNA in HASMCs.

View larger version (22K): [in this window] [in a new window]   Figure 3. HuR interacts with 3'UTR of TLR4 mRNA. A, The cytoplasmic fractions were analyzed with immunoprecipitation and quantitative real time PCR. B, 5 µL of immunoprecipitated materials was used in quantitative real time PCR reactions to detect the presence of 3'UTR of TLR4 mRNA. The data are presented as mean±SEM and represent the results from four independent experiments. *P<0.01 compared with the unstimulated group.

The 3'UTR of TLR4 mRNA and HuR siRNA Confer LPS Responsiveness
To investigate whether the 3'UTR promotes TLR4 mRNA expression, a reporter plasmid containing the 3'UTR and luciferase reporter gene were transfected into HASMCs. A schematic representation of the various plasmids containing luciferase and the 3'UTR of TLR4 mRNA are shown in Figure 4A. The CMV-Luciferase plasmid-transfected group had a higher basal luciferase activity than the control groups (naive cells and pcDNA3.1 vector-transfected cells). Treatment with 25 ng/mL LPS caused a slight increase in luciferase activity compared with unstimulated cells in the CMV-luciferase plasmid-transfected group and a significant increase in luciferase activity in the CMV-Luciferase-TLR4 3'UTR sense plasmid-transfected group (Figure 4B). In contrast, LPS treatment did not change the basal luciferase activity in the CMV-Luciferase-TLR4 3'UTR antisense plasmid-transfected group.

View larger version (24K): [in this window] [in a new window]   Figure 4. The 3'UTR flanking sequence of TLR4 mRNA and HuR siRNA conferred LPS-responsiveness in HASMCs. A, Schematic representation of the various plasmids containing luciferase and 3'UTR of TLR4 mRNA. B, HASMCs were transfected with CMV-Luciferase-TLR4 3'UTR sense or CMV-Luciferase-TLR4 3'UTR antisense plasmids. Equal amounts of the empty vector (pcDNA3.1) or luciferase reporter gene containing plasmid (CMV-Luciferase) were used as controls. Uniform transfection efficiencies were confirmed using a ß-galactosidase reporter plasmid. The luciferase activity was quantified by luminometry. Data are expressed as relative luciferase units. The data are presented as mean±SEM and represent the results of three separate experiments. *P<0.05 compared with the CMV-Luciferase group, P<0.05 compared with the CMV-Luciferase-3'UTR+LPS group, #P<0.05 compared with the CMV-Luciferase-3'UTR group. C, HASMCs were cotransfected with CMV-Luciferase-TLR4 3'UTR sense or CMV-Luciferase-TLR4 3'UTR antisense plasmid, ß-galactosidase reporter plasmid, and HuR siRNA. The luciferase activity was quantified. Data are presented as mean±SEM and represent the results of three independent experiments. *P<0.05 compared with the CMV-Luciferase-3'UTR group, P<0.05 compared with the CMV-Luciferase-3'UTR+LPS group.

HASMCs were cotransfected with the HuR RNAi and CMV-Luciferase-TLR4 3'UTR sense plasmid followed by LPS treatment. HuR-specific but not the negative control siRNA effectively blocked the luciferase activity in CMV-Luciferase-TLR4 3'UTR sense plasmid-transfected cells stimulated with LPS (Figure 4C). These findings suggest that the 3'UTR of TLR4 mRNA confers LPS responsiveness and that HuR modulates the 3'UTR-mediated gene expression in HASMCs.

NADPH Oxidase and MAPK-Signaling Pathways Mediate LPS-Induced HuR Expression
HASMCs were pretreated with 30 µmol/L diphenylene iodonium (DPI) or transfected with 100 µmol/L Rac1 siRNA, which significantly decreased the LPS-induced lengthening of the TLR4 mRNA t_1/2 (LPS group, 349.3±43.2 minutes; DPI-treated group, 122.9±24.3 minutes; Rac1 siRNA-transfected group, 110.2±21.2 minutes) (Figure 5A). Compared with control, LPS significantly induced cytoplasmic HuR expression. Addition of 30 µmol/L DPI or transfection with 100 µmol/L Rac1 siRNA significantly decreased cytoplasmic HuR expression in HASMCs treated with LPS (Figure 5B). Many RNA-binding proteins that modulate inflammation-related mRNA stability may be regulated by the MAPK pathways.¹⁴ We examined whether HuR expression in LPS-induced HASMCs is regulated by the MAPK-signaling pathways and found that LPS-induced lengthening of TLR4 mRNA stability was significantly reduced by SB203580, a p38 inhibitor (120.9±12.0 minutes), and PD98059, an extracellular signal regulated kinase (ERK) inhibitor (182.9±15.4 minutes), but not by SP600125, a JNK inhibitor (Figure 5C). In addition, SB23580 significantly reduced cytoplasmic HuR expression in HASMCs treated with LPS (Figure 5D). These results suggest that LPS-induced cytoplasmic HuR expression is mediated by an oxidative stress–related mechanism and the p38 MAPK-signaling pathway in HASMCs.

View larger version (37K): [in this window] [in a new window]   Figure 5. NADPH oxidase and MAPK signaling pathways-mediated LPS-induced HuR expression regulates TLR4 mRNA stability. A and C, Actinomycin D chase experiments were used to assess TLR4 mRNA stability. B and D, The expression of cytoplasmic HuR was assessed by Western blot analysis. Negative control siRNA (NC siRNA) was used to validate the knockdown. The bar graphs show the relative intensity of each band relative to ß-actin as measured by densitometry. Data represent the results from three independent experiments. The bar graphs of B and D show the relative intensity of each band relative to ß-actin, which as measured by densitometry. *P<0.05 compared with the unstimulated group and P<0.05 compared with the LPS-treated only group.

HuR Expression in Endothelia-Denuded Abdominal Aorta of Rabbits With Systemic Inflammation
To study whether LPS administration affects HuR expression, which is associated with TLR4 expression, immunohistochemical staining was performed using antibodies against TLR4, HuR, and -actin (to identify smooth muscle cells) on serial sections of abdominal aortas (Figure 6). Compared with sections from the control group, sections showed a slightly thickened intima in the LPS group, markedly thickened intima in the ED group, and severe intimal hyperplesia in the ED+LPS group. Compared with the control group, it also enhances a bit the expression of TLR4 in the ED group; strong TLR4 staining was seen on the luminal surface in the LPS group and in the neointima in the ED+LPS group. Slightly positive HuR staining was seen in the LPS-treated group, and strongly positive HuR staining was seen in the ED+LPS group in the markedly thickened intima. The antibody to -actin to identify smooth muscle cells showed that TLR4 and HuR were expressed predominantly in smooth muscle cells in the ED+LPS group. These results demonstrate that LPS administration increased HuR expression (LPS group versus control group), and significantly severed HuR expression (ED+LPS group versus ED group) in the smooth muscles cells of neointima.

View larger version (70K): [in this window] [in a new window]   Figure 6. Immunohistochemistry to assess TLR4 and HuR expression in rabbit abdominal aorta. The intima was markedly thickened in the ED and ED+LPS groups compared with the control and LPS-treated groups. The black arrowheads indicate smooth muscle cells overlapping with TLR4 (brown signal) and HuR (brown signal) expression. Compared with the control group, it also enhances a bit the expression of TLR4 on the neointima in ED group, stronger TLR4 expression was seen on the luminal surface in the LPS-treated group and on the neointima in the ED+LPS-treated group. HuR expression increased especially in the ED+LPS group compared with the control group. TLR4 and HuR were expressed predominantly in smooth muscle cells, as identified by antibody against -actin (white arrowheads). Corresponding hematoxylin staining was used for nucleus identification. The lumen is uppermost in all sections, and the internal elastic laminae are indicated by arrows. The scale bar indicates 50 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our data revealed that HuR interacts directly with the 3'UTR of TLR4 mRNA to prolong the stability of TLR4 mRNA in LPS-stimulated HASMCs. The Rac1-dependent NADPH oxidase activation and p38 MAPK-signaling pathway play critical roles in LPS-increased HuR activation, which mediates TLR4 mRNA stabilization in HASMCs. In a balloon injured rabbit aorta model, we also demonstrated that LPS increases the expression of TLR4 and HuR. The data provide evidence for a direct involvement of VSMCs in LPS-mediated inflammatory activation, which that may contribute to the progression of cardiovascular disorders.

Controlling the stability of mRNA may modulate gene expression and adjust inflammatory responses efficiently. The stability of mRNA is often modulated by AREs through 3'UTR.¹¹ The characteristic motif of ARE is AUUUA, although the copy numbers and organization of AREs are vary, and there are various classes of ARE.²⁴ We have shown the sequence of 3'UTR of TLR4 mRNA and predicted that the most obvious feature of the nucleotide fragment is the presence of three AU motifs (please see the online data supplement). Our data provide evidence that HuR binds to ARE of TLR4 mRNA and that HuR is an essential regulator of TLR4 expression. In the future, we plan to study the serial deleted constructs to identify the minimal region required for 3'UTR of TLR4 mRNA interaction with HuR; using the site-directed mutagenesis technique to provide evidence of the requirement for the nucleotides in this interaction.

The increased stability of ARE-containing mRNAs has also been linked to an increase in the cytoplasmic level of the endogenous HuR.²⁵ In our LPS-induced HASMCs model, the p38 MAPK signaling pathway is involved in the positive regulation and accumulation of cytoplasmic HuR. Activation of HuR results from the activation of p38 MAPK. MAPKAPK-2, which is phosphorylated by p38 MAPK, regulates the stability of mRNAs for TNF-,²⁶ through their AREs. Importantly, the activation of MAPKAPK-2, which increases the cytoplasmic accumulation of HuR²⁷; dominant negative mutants of MAPKAPK-2 changes the cytoplasmic HuR level²⁷ and blocks cytokine-induced COX-2²⁸ mRNA degradation. HuR-mediated stabilization of TNF- mRNA in LPS-induced macrophages results from an increase in HuR methylation.²⁹ Although our data do not clarify the precise mechanisms responsible for p38 MAPK regulation of the increase in cytoplasmic HuR, we speculate that p38 MAPK regulates protein-arginine methyltransferase and increases the methylation of HuR, which would then modulate the interaction of HuR with its ligands, thus affecting its nucleocytoplasmic shuttling. ERK also regulates transportation of cytokine mRNAs from the nucleus to the cytosol in an ARE-dependent manner.³⁰ Our study showed that ERK does not regulate the cytoplasmic HuR level but decreases the stability of TLR4 mRNA, suggesting the involvement of a mechanism independent of cytoplasmic HuR expression.

Intracellular MAPK signaling pathways are associated with vascular inflammation and that this is modulated by ROS.³¹ In vascular cells, H₂O₂ activates p38 MAPK, JNK/SAPK, and ERK. DPI, NAC, p22^phox siRNA, or catalase may inhibit the activation of p38 MAPK and JNK/SAPK-mediated Rac1-dependent H₂O₂ production.^32,33 The production of ROS and the activation of the p38 MAPK signaling pathways induce the expression of several redox-sensitive genes associated with atherogenesis. The direct interaction of TLR4 with NADPH oxidase is involved in LPS-mediated ROS generation and NF-B activation.³⁴ Our previous study found that apocynin and DPI inhibit LPS-induced MAPK phosphorylation in VSMCs (please see the online data supplement). Treatment with DPI and transfection of Rac1 RNAi may decrease the stability of TLR4 mRNA and downregulate the cytoplasmic expression of HuR suggesting that NADPH oxidase-mediated ROS production contributes to the activation of MAPKs, which is associated with the expression of HuR and stability of TLR4 mRNA in redox-sensitivity vascular inflammation.

In summary, LPS-enhanced TLR4 expression and mRNA stabilization in HASMCs is mediated by HuR expression and that this expression is dependent on the NADPH oxidase activation and p38 MAPK signaling pathways in vitro. Using an inflammatory animal model, involving balloon injured vessels, we also found that LPS increase the expression of TLR4 and HuR in neointima hyperplastic lesion in vivo. Our findings suggest that suppressing HuR activation or therapy with anti-inflammatory agents is a promising means of preventing vascular inflammation.

	Acknowledgments

 
Sources of Funding

This work was supported in part by the C.Y. Foundation for Advancement of Education, Sciences and Medicine, and National Science Council of Taiwan, (NSC 93-2314-B-010-004 and NSC 94-2314-B-010-052), and Taipei Veterans General Hospital (VGH 94-207, V95CI-099, V95ER2-001).

Disclosures

None.

	Footnotes

 
Y.L.C., C.Y.L, and S.J.L. contributed equally to this study.

Original received July 5, 2006; final version accepted September 4, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation. 2002; 105: 1135–1143.[Abstract/Free Full Text]
2. Rice JB, Stoll LL, Li WG, Denning GM, Weydert J, Charipar E, Richenbacher WE, Miller FJ, Jr., Weintraub NL. Low-level endotoxin induces potent inflammatory activation of human blood vessels: inhibition by statins. Arterioscler Thromb Vasc Biol. 2003; 23: 1576–1582.[Abstract/Free Full Text]
3. Danenberg HD, Welt FG, Walker M, 3rd, Seifert P, Toegel GS, Edelman ER. Systemic inflammation induced by lipopolysaccharide increases neointimal formation after balloon and stent injury in rabbits. Circulation. 2002; 105: 2917–2922.[Abstract/Free Full Text]
4. Sasu S, LaVerda D, Qureshi N, Golenbock DT, Beasley D. Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via Toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation. Circ Res. 2001; 89: 244–250.[Abstract/Free Full Text]
5. Stoll LL, Denning GM, Li WG, Rice JB, Harrelson AL, Romig SA, Gunnlaugsson ST, Miller FJ Jr, Weintraub NL. Regulation of endotoxin-induced proinflammatory activation in human coronary artery cells: expression of functional membrane-bound CD14 by human coronary artery smooth muscle cells. J Immunol. 2004; 173: 1336–1343.[Abstract/Free Full Text]
6. Frantz S, Kobzik L, Kim YD, Fukazawa R, Medzhitov R, Lee RT, Kelly RA. Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium. J Clin Invest. 1999; 104: 271–280.[Medline] [Order article via Infotrieve]
7. Xu XH, Shah PK, Faure E, Equils O, Thomas L, Fishbein MC, Luthringer D, Xu XP, Rajavashisth TB, Yano J, Kaul S, Arditi M. Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL. Circulation. 2001; 104: 3103–3108.[Abstract/Free Full Text]
8. Vink A, Schoneveld AH, van der Meer JJ, van Middelaar BJ, Sluijter JP, Smeets MB, Quax PH, Lim SK, Borst C, Pasterkamp G, de Kleijn DP. In vivo evidence for a role of Toll-like receptor 4 in the development of intimal lesions. Circulation. 2002; 106: 1985–1990.[Abstract/Free Full Text]
9. Ishida I, Kubo H, Suzuki S, Suzuki T, Akashi S, Inoue K, Maeda S, Kikuchi H, Sasaki H, Kondo T. Hypoxia diminishes Toll-like receptor 4 expression through reactive oxygen species generated by mitochondria in endothelial cells. J Immunol. 2002; 169: 2069–2075.[Abstract/Free Full Text]
10. Fan J, Kapus A, Marsden PA, Li YH, Oreopoulos G, Marshall JC, Frantz S, Kelly RA, Medzhitov R, Rotstein OD. Regulation of Toll-like receptor 4 expression in the lung following hemorrhagic shock and lipopolysaccharide. J Immunol. 2002; 168: 5252–5259.[Abstract/Free Full Text]
11. Bakheet T, Frevel M, Williams BR, Greer W, Khabar KS. ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins. Nucleic Acids Res. 2001; 29: 246–254.[Abstract/Free Full Text]
12. Wen X, Wu GD. Evidence for epigenetic mechanisms that silence both basal and immune-stimulated transcription of the IL-8 gene. J Immunol. 2001; 166: 7290–7299.[Abstract/Free Full Text]
13. Liu RY, Fan C, Liu G, Olashaw NE, Zuckerman KS. Activation of p38 mitogen-activated protein kinase is required for tumor necrosis factor-alpha-supported proliferation of leukemia and lymphoma cell lines. J Biol Chem. 2000; 275: 21086–21093.[Abstract/Free Full Text]
14. Kracht M, Saklatvala J. Transcriptional and post-transcriptional control of gene expression in inflammation. Cytokine. 2002; 20: 91–106.[CrossRef][Medline] [Order article via Infotrieve]
15. Wang W, Caldwell MC, Lin S, Furneaux H, Gorospe M. HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation. Embo J. 2000; 19: 2340–2350.[CrossRef][Medline] [Order article via Infotrieve]
16. Wang W, Furneaux H, Cheng H, Caldwell MC, Hutter D, Liu Y, Holbrook N, Gorospe M. HuR regulates p21 mRNA stabilization by UV light. Mol Cell Biol. 2000; 20: 760–769.[Abstract/Free Full Text]
17. Brewer G. An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro. Mol Cell Biol. 1991; 11: 2460–2466.[Abstract/Free Full Text]
18. Xu N, Chen CY, Shyu AB. Versatile role for hnRNP D isoforms in the differential regulation of cytoplasmic mRNA turnover. Mol Cell Biol. 2001; 21: 6960–6971.[Abstract/Free Full Text]
19. Lai WS, Carballo E, Strum JR, Kennington EA, Phillips RS, Blackshear PJ. Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. Mol Cell Biol. 1999; 19: 4311–4323.[Abstract/Free Full Text]
20. Carballo E, Lai WS, Blackshear PJ. Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science. 1998; 281: 1001–1005.[Abstract/Free Full Text]
21. Ming XF, Stoecklin G, Lu M, Looser R, Moroni C. Parallel and independent regulation of interleukin-3 mRNA turnover by phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase. Mol Cell Biol. 2001; 21: 5778–5789.[Abstract/Free Full Text]
22. Fairhurst AM, Connolly JE, Hintz KA, Goulding NJ, Rassias AJ, Yeager MP, Rigby W, Wallace PK. Regulation and localization of endogenous human tristetraprolin. Arthritis Res Ther. 2003; 5: R214–R225.[CrossRef][Medline] [Order article via Infotrieve]
23. Fan XC, Steitz JA. Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs. Embo J. 1998; 17: 3448–3460.[CrossRef][Medline] [Order article via Infotrieve]
24. Xu N, Chen CY, Shyu AB. Modulation of the fate of cytoplasmic mRNA by AU-rich elements: key sequence features controlling mRNA deadenylation and decay. Mol Cell Biol. 1997; 17: 4611–4621.[Abstract]
25. Winzen R, Kracht M, Ritter B, Wilhelm A, Chen CY, Shyu AB, Muller M, Gaestel M, Resch K, Holtmann H. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. 1999; 18: 4969–4980.[CrossRef][Medline] [Order article via Infotrieve]
26. Dean JL, Wait R, Mahtani KR, Sully G, Clark AR, Saklatvala J. The 3' untranslated region of tumor necrosis factor alpha mRNA is a target of the mRNA-stabilizing factor HuR. Mol Cell Biol. 2001; 21: 721–730.[Abstract/Free Full Text]
27. Dean JL, Sully G, Clark AR, Saklatvala J. The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilisation. Cell Signal. 2004; 16: 1113–1121.[CrossRef][Medline] [Order article via Infotrieve]
28. Lasa M, Mahtani KR, Finch A, Brewer G, Saklatvala J, Clark AR. Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade. Mol Cell Biol. 2000; 20: 4265–4274.[Abstract/Free Full Text]
29. Li H, Park S, Kilburn B, Jelinek MA, Henschen-Edman A, Aswad DW, Stallcup MR, Laird-Offringa IA. Lipopolysaccharide-induced methylation of HuR, an mRNA-stabilizing protein, by CARM1. Coactivator-associated arginine methyltransferase. J Biol Chem. 2002; 277: 44623–44630.[Abstract/Free Full Text]
30. Dumitru CD, Ceci JD, Tsatsanis C, Kontoyiannis D, Stamatakis K, Lin JH, Patriotis C, Jenkins NA, Copeland NG, Kollias G, Tsichlis PN. TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway. Cell. 2000; 103: 1071–1083.[CrossRef][Medline] [Order article via Infotrieve]
31. Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol. 2000; 20: 2175–2183.[Abstract/Free Full Text]
32. Shin EA, Kim KH, Han SI, Ha KS, Kim JH, Kang KI, Kim HD, Kang HS. Arachidonic acid induces the activation of the stress-activated protein kinase, membrane ruffling and H2O2 production via a small GTPase Rac1. FEBS Lett. 1999; 452: 355–359.[CrossRef][Medline] [Order article via Infotrieve]
33. Viedt C, Soto U, Krieger-Brauer HI, Fei J, Elsing C, Kubler W, Kreuzer J. Differential activation of mitogen-activated protein kinases in smooth muscle cells by angiotensin II: involvement of p22phox and reactive oxygen species. Arterioscler Thromb Vasc Biol. 2000; 20: 940–948.[Abstract/Free Full Text]
34. Park HS, Jung HY, Park EY, Kim J, Lee WJ, Bae YS. Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B. J Immunol. 2004; 173: 3589–3593.[Abstract/Free Full Text]

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