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
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.
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-α).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
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).
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).
The expression of TLR4 and HuR were observed with immunohistochemical staining (please see the online data supplement).
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.
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 t1/2 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 β-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.
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.
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.
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.
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 t1/2 (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.
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 α-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 α-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.
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-α,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-α 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, p22phox 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-κB 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.
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).
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.
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