Background
Airway remodeling, a fundamental pathogenic feature of asthma, is characterized by matrix deposition and enhanced smooth muscle mass in the airways. For patients with recurring episodes of asthma, structural changes are one of the most important reasons for the deterioration of lung function, which is now becoming a life-threatening challenge in the treatment of asthma [
1,
2]. Grainge [
3] had confirmed that collagen deposition and goblet cell hyperplasia contributed to airway remodeling in mild asthma, but detailed mechanism remained to be elucidated.
TGF-β1, a pleiotropic cytokine that had been evidenced to be involved in the synthesis of matrix molecules in the ASM cells, especially on the synthesis of collagen types I, III, IV, VII and X, fibronectin and proteoglycans [
4], has been implicated in the pathogenesis of airway remodeling in asthma [
5‐
7]. For years, the mechanisms underlying the development of fibrosis have been extensively studied and multiple signal pathways are involved in, such as the integrin α5β6 [
8], phosphatidylcholine-specific phospholipase C, protein kinase C-delta [
9], the integrin receptor α5β1 [
10], the canonical Smad-dependent signaling pathway [
11] and p38 MAPK [
12]. In recent years, a series of observations reported that Angiotensin II could be a putative mediator in increasing TGF-β1 and Col-I deposition [
13‐
15]. Although the roles of these signaling pathways have been well established by ample experimental studies, no specific inhibitor applicable in asthma has been described.
HuR, the sole ubiquitous member of the Hu RNA-binding protein family, can bind to a subset of short-lived mRNAs that harbor AU-rich elements (AREs) in their 3′ untranslated regions (UTR), which is called post-transcriptional gene regulation that coordinating the process of mRNA splicing, transport, turnover, and translation in multiple development processes and diseases [
16]. These mRNAs include c-fos, VEGF, TNF, α,β-catenin, c-myc, cyclooxygenase 2, myogenin, MyoD, and granulocyte/macrophage colony-stimulating factor (GM-CSF) [
17‐
19]. Fan [
20] has showed that HuR critically regulates the epithelial response by associating with multiple functionally related ARE-bearing inflammatory transcripts, and Zhang [
21] also reported that HuR participated in ASM proliferation by mediating CyclinD1 expression. In particular, TGF-β1 3′UTR was reported to be a putative target of HuR in human cancer cells [
22]. However, the underlying relationship between HuR and TGF-β1 in regarding to airway remodeling has been reported in few studies. So it is still a challenge to explore the possible pathogenic mechanisms of refractory airway remodeling.
In our study, we found a novel HuR/TGF-β1 feedback circuit that modulating airway remodeling in airway smooth muscle cells and in asthmatic mouse firstly. In vitro, we detected that HuR and TGF-β1 demonstrated high expression in a time-dependent manner under the stimulation of PDGF, a strong stimulus for asthmatic response. Besides, α-SMA and Col-I simultaneously exhibited over-expression. Furthermore, knockdown of HuR led to an increase of ASM cells apoptosis and down-regulation of TGF-β1, α-SMA and Col-I. Moreover, the half-life of TGF-β1 was shorter compared with the control. However, interfering TGF-β1 with siRNA can obviously decrease HuR and Col-I expression. But exogenous TGF-β1 could recover HuR and Col-I expression. In vivo, OVA-induced mice showed widely infiltration of inflammatory cells surrounding the bronchioles in comparison with PBS-induced mice. Sirius red staining distinguished higher deposition of collagen type I and III around the bronchiole in OVA-induced mice then in PBS-induced mice. RT-PCR, western blotting and immunohistochemistry all showed higher levels of HuR, TGF-β1 and α-SMA in OVA -induced mice than PBS-induced mice. Thus we hypothesized that a HuR/TGF-β1 feedback is involved in airway remodeling and targeting them might have considerable potential for the control of asthma.
Materials
Anti-HuR (ab17397, Abcam), TGF-β1 (ab92486,Abcam), α-SMA (ab5694, Abcam), collagen I (ab34710, Abam), and actin (ab8226, Abcam) antibodies were available for western blotting, immunohistochemistry and immunofluoresence. Reverse kit was purchased from Takara (Japan). PDGF-BB was purchased from Peprotech and recombinant human TGF-β1 was bought from Invitrogen. Small interference RNA duplexes against HuR and TGF-β1 plus negative control were synthesized in Genepharm (Shanghai, China). Lip3000 and Trizol was purchased from Invitrogen. TGF-β1 and human Col-I ELISA kit were bought from Bioworld. All other reagents were enrolled from domestic companies with good reputations unless specifically stated.
Real-time PCR
Cells were harvested and total RNA was isolated with Trizol. In mice, the total RNA was extracted from the right lungs using Trizol (Invitrogen, USA). Reverse transcription of 1 μg RNA was carried out according to the instructions of Takara RT kit (Japan). Quantitect SYBR Green kit (USA) was used for amplification and fluorescence was detected by using ABI Prism 7700 Detection System. GAPDH was run as internal control and 2
−ΔΔCt method was performed for analysis (n = 3). To test the TGF-β1 mRNA stability, ASM cells transfected with ConsiRNA or HuRsiRNA were further treated with PDGF or without for 6 h. Then actinomycinD (5 μg/ml) was added in every well immediately for 0, 4, 8 and 12 h and RNA was extracted at indicated time to examine the RNA abundance for stability analysis. Primers sequences were listed at Table
1.
Table 1
Primers and sequences used in this study
GAPDH real time primer | F-5′-GCAAGTTCAACGGCACAG-3′ |
| R-5′-GCCAGTAGACTCCACGACATA-3′ |
HuR real time primer | F-5′-GGCGAGCATACGACA3′ |
| R-5′-TATTCGGGATAAAGTAGC3′ |
TGF-β1 real time primer | F-5′-CCCACTGATACGCCTGAG-3′ |
| R-5′-TGAAGCGAAAGCCCTGTA-3′ |
Col-I real time primer | F-5′-CACTCAGCCCTCTGTGCCT-3′ |
| R-5′-ACCTTCGCTTCCATACTCG-3′ |
ConsiRNA | F-5′-UUCUCCGAACGUGUCACGUTT-3′ |
| R-5′-ACGUGACACGUUCGGAGAATT-3′ |
HuRsiRNA | F-5′-CAACAAGUCCCACAAAUAAUU-3′ |
| R-5′-AAUUAUUUGUGGGACUUGUUG-3′ |
Western blotting
Harvested cells were rinased with PBS and lysed by RIPA containing 50 mM Tris, 150 mM NaCl, 1 % Triton X-100, 1 % Sodium deoxycholate, 0.1 % SDS plus protease inhibitor PMSF. Lung tissues were homogenized with ice-cold RIPA plus PMSF. Protein concentration was tested by the BCA protein assay kit. Protein samples (30 μg per lane) were submitted to electrophoresis on 10 % SDS-polyacrylamide gel and resolved to PVDF membrane. After blocking for 1 h (5 % non-fat milk in Tris buffered saline plus 0.1 % Tween 20), the membranes were incubated in blocking buffer at 4 °C overnight with anti-HuR (1:2000), anti-TGF-β1 (1:1000), anti-α-SMA (1:150) and anti-Col I (1:1000). After three times washing next day, the membranes were probed with HRP (1:5000 in blocking buffer) linked secondary antibodies, and visualized with ECL reagent (Thermo Scientific).
Immunofluorescence
After the ASM cells were stimulated with PDGF for 0, 6, 12 and 24 h respectively, cells were fixed in 4 % paraformaldehyde for 15 min and permeabilized for 20 min in phosphate-buffered saline containing 0.5 % Triton X-100. After incubation in blocking buffer (goat serum) for 1 h at 37 °C, cover slips were incubated in a 1:50 dilution of anti-TGF-β1 and anti-HuR prepared in blocking buffer overnight. Cover slips were washed with blocking buffer next day and incubated for at least 1 h with TRITC-labeled goats anti-rabbit IgG or anti-mouse IgG. Then cover slips were washed for thee times with blocking buffer and cells were dyed nucleus with DAPI for 15 min. After carefully washing, cell images were acquired with a fluorescence inverted microscope (Olympus BX50).
RNA-interference
According to the manufacturer’s instructions, the trypsinized ASM cells were resuspended at a density of (0.25-1) × 106/ml in 6-well plate. 5 μl Lip3000 was diluted in 125 μl OPTI-MEM and 2.5 μg DNA plus 5 μl P3000 were diluted in 125 μl OPTI-MEM. Then two compounds were well mixed and incubated 10 min at room temperature to form transfected complexes. Then total 250 μl complexes were dispensed into a culture plate containing 1750 μl complete DMEM medium and mixed with the cell suspension gently. Additional experiments were performed after transfection for 48 h.
Flow cytometry and CCK8 assay
Apoptosis assay: ASM cells were divided into Consi, Consi + PDGF, HuRsi and HuRsi + PDGF groups. ASM cells were then harvested, washed and resuspended with PBS. Apoptotic cells were determined with an Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit (Invitrogen) according to the manufacturer’s protocol. Briefly, the cells were washed and subsequently incubated in 100 μl of 1X Annexin binding buffer containing 5 μl of Annexin V-FITC and 2 μl of propidium iodide (PI) for 15 min in the dark. Then, apoptosis was analyzed using a FACScan laser flow cytometer. Data were analyzed by using FlowJo software.
Cell proliferation assay: ASM cells were divided into Consi, Consi + PDGF, TGFsi, TGFsi + PDGF groups. 10 μl CCK8 was added to each well. Cells were further cultured for 1 h, and then when each well turned to orange, the optical density(OD) was measured at 450 nm using a multiscan reader. The average OD of four wells for each group was calculated.
ELISA
The concentration of TGF-β1 and Col-I in the cultured serum was measured by ELISA-kits. The protocols were followed according to the manufacturer’s instructions. Briefly, the cultured serum samples collecting from the Consi and HuRsi group after cultured for indicated time were added in triplicate to 96-well plates with 100 μl per well. The appropriate biotinconjugated antibodies were added to each well. The samples were incubated at room temperature for 2 h. The wells were then aspirated, and each well was washed five times. The substrate solutions were added to each well, and were incubated for 30 min at room temperature in the dark. The optical density (OD) of each well was determined using a microplate reader that was set to 450 nm. A standard curve was created of the average of the OD duplicate readings. Data was analyzed by CurveExpert 1.3 and SPSS 19.0.
Bronchoalveolar lavage fluid (BALF) and cell collection
All mice were sacrificed within 24 h after the last treatment. The left major bronchus was tied with a string, which was inserted with a 24-gauge needle, and the BALF was obtained by the infusion and collection of 0.5 ml of saline. The infusion and collection steps were repeated for 3 times. The BALF was centrifuged at 2000 rpm for 10 min at 4 °C.The different cell counts in the BALF were carried out as described [
24]. In brief, the pellet was resuspended with 0.5 ml of saline and the different cell counts were performed with Giemsa staining. The different cell counts were determined by light microscopy from a count of at least 400 cells. The percentages of macrophages, eosinophils, lymphocytes and neutrophils were determined by counting their numbers in randomly selected high-power fields and by dividing this number by the total number of cells per high-power field. All of the counts were performed by the same observer in a blinded manner and in a randomized order at the end of the study.
Histology
The lungs were dissected from the chest cavity after the lavage. The left lungs were immediately fixed in 4 % paraformaldehyde and paraffin-embedded, and tissue sections (5 mm) were prepared. To assess airway remodeling, the sections were stained with periodic acid Schiff stain (PAS) for goblet cells and with Sirius red staining for collagen deposition, as described previously [
25]. Briefly, goblet cell upregulation within the airway epithelia was assessed by measuring the length of the airway basement membrane that was covered by goblet cells. Peribronchial collagen thickness was measured using Image-Pro Plus software (version 6, Media Cybernetics, USA). Three bronchioles were selected at randomly from each section and the mean depth of collagen in the basement membrane was determined from five measurements around the bronchiole. For immunohistochemical analysis, the sections were initially incubated with anti-α-SMA rabbit monoclonal antibody (1:150; Abcam), anti-HuR rabbit monoclonal antibody (1:500; Abcam) and anti-TGF-β1 rabbit polyclonal antibody (1:100; Abcam) at 4 °C overnight, then were incubated with HRP-conjugated goat anti-rabbit (1:50; Abcam) for 30 min at 37 °C. Positive staining was detected with HRP-conjugated streptavidin, visualized with 3,3′-diaminobenzidine and counterstained with hematoxylin [
26]. Finally, the sections were mounted, cover-slipped, and examined under a light microscope (Olympus BX50). The extent of positive area were analyzed by using Image-Pro Plus 4.5.
Statistical analysis
All results were performed at least three independent experiments. All data were processed by SPSS version 19.0 and quantitative data were shown as mean ± standard deviation (SD). A student’s t test (two-tailed) was used to compare two groups and One-way ANOVA for multiple comparisons. Quantity one V4.62 and Graphpad Prism 5.0 were used to quantify relative expression of proteins. Values of P < 0.05 were considered statistically significant.
Discussion
In vitro and in vivo, the present study found that the effects of HuR and TGF-β1 are inter-dependent, and the balance among these interactions plays an important role in mediating airway remodeling. Here, we uncovered a HuR/TGF-β1 feedback that modulates α-SMA and Col-I expression in ASM cells or asthmatic animal mice.
Asthma is a common respiratory disease and the occurrence of which might be associated with the over-expression of asthma-associated genes. Human antigen R (HuR), an ubiquitously expressed RNA-binding protein, is known to regulate the turnover of mRNA for inflammatory genes or cell cycle proteins by binding to adenylate-uridylate-rich elements and related motifs presented in the 3′untranslated region (UTR) of mRNAs. Our previous studies demonstrated that HuR could enhance cyclinD1 mRNA stability by recognizing the 3′UTR, which controlled the proliferation of ASM cells. Airway remodeling, a key feature of asthma, is characterized by matrix deposition and enhanced smooth muscle mass in the airways. For years, multiple intracellular signal pathways have been shown to be involved. p38 mitogen-activated protein kinase (MAPK) signaling has been shown to be a center to the TGF-β1-induced collagen and fibronectin expression in systemic sclerosis (SSc) fibroblasts [
29]. As TGF-β1 induces fibroblasts to synthesize and ECM contract, this cytokine has long been believed to be a central mediator of the fibrotic response [
30]. In light of the analysis above, whether HuR is involved in matrix deposition and how it mediates this event in cultured ASM cells is the focus of our study, and we set to explore the interaction between HuR and TGF-β1 with the goal of defining a new role for HuR in asthma.
Firstly, we found that PDGF stimulation significantly elevated the expression of TGF-β1, α-SMA and Col-I both in the mRNA and protein levels at a time-dependent manner. Furthermore, the result of immunofluorescence of HuR and TGF-β1 were well consistent with western blotting and PCR analysis. These data proved that PDGF stimulation significantly elevated the expression of TGF-β1, α-SMA and Col-I. Secondly, RNA-interference decreased the expression of HuR to a very low level. Suppression of HuR resulted in higher proportion of apoptosis in the HuRsi group than in the Consi group and the contents of TGF-β1, Col-I and α-SMA either in intracelluar or in the cultured medium were reduced in HuRsi group. Furthermore, we detected that the half-life of TGF-β1 mRNA was also much shorter compared with the control, which was similar to some reported studies demonstrating that HuR could regulate target mRNA expression by prolonging or shortening the mRNA half-life [
31‐
33]. Thirdly, to further explore the association between HuR and TGF-β1, TGF-β1 was inhibited by specific small interference RNA. We interestingly found that the knockdown of TGF-β1 also alleviated HuR and Col-I expression in western blotting and extra addition of TGF-β1 could partially recover HuR and Col-I expression in ASM cells, which gives us confidence that a HuR/TGF-β1 may exist to modulate airway remodeling. At last, OVA-driven mice showed widely infiltration of inflammatory cells around the bronchiole compared with PBS-induced mice. Sirius red staining distinguished collagen type I and III deposition surrounding the bronchiole especially in the OVA-induced mice. Immunohistochemistry plus western blotting and RT-PCR showed higher levels of HuR, TGF-β1 and α-SMA in the OVA -induced mice. Thus we hypothesized that a HuR/TGF-β1 feedback is involved in airway remodeling and targeting them may have considerable potential for the control of asthma.
B Wightman [
34] ever demonstrated that stretch augmented TGF-β1 expression through the enhanced activation of the promoter,which indicating that targeting the posttranscriptional regulatory modules in the TGF-β1 signaling pathway is a promising approach to control airway remodeling by ASM cells. Danna Bai [
27] used RNA-IP to verify that HuR could recognize the 3′UTR of TGF-β1 in cardiac fibroblasts. Their study established that a TGF-β1/HuR feedback circuit regulated the fibrogenic response in fibroblasts. This mechanism should also exist in other organisms on the basis that both HuR and the ARE in the TGF-β1 3′UTR are conserved among various species. Increased TGF-β1 would induce a cascade of the fibrogenic response in both the canonical Smad-dependent signaling pathway and non-Smad pathways by inducing the expression of collagen, fibronectin and other ECM molecules [
4]. However, we have only tested a limited indicator of remodeling, Col-I and α-SMA. Numerous publications have confirmed that proliferative cytokines [
35] and proinflammatory factors [
36] are also involved in airway remodeling. It is therefore an important challenge to test other potential targets that are involved in controlling the process of airway remodeling.
Previous studies have shown that the actions of TGF-β1 on fibroblast proliferation are complex. The proliferation of mink lung fibroblasts is stimulated by low concentrations of TGF-β1 (5–10 ng) but inhibited by higher concentrations [
37]. Treatment of ASM cells with TGF-β1 (4 ng/ml) resulted in a dramatic increase in HuR and Col-I expression compared with the untreated controls or PDGF alone. Our present study established a HuR/TGF-β1 feedback that might regulate the Col-I expression in ASM cells, and suggested that targeting this pathway has considerable potential for controlling the deposition of Col-I in ASM cells. However, the mechanism by which TGF-β1 regulates Col-I and α-SMA expression is still unclear. In mesangial cells, TGF-β1 induction of the type I collagen promoter required the RAS/MEK/ERK MAPK cascade, and in dermal fibroblasts, this response required p38 [
38]. Induction of fibronectin by TGF-β1 in fibroblasts was Smad-independent but required the JNK MAPK cascade and c-jun [
39]. Our next study will further explore the novel pathway between TGF-β1, α-SMA and Col-I in ASM cells.
More than 300 million individuals worldwide are suffered from asthma, and by 2025 the prevalence is predicted to increase by 100 million [
40]. At present, inhaled corticosteroids are the standard therapy for persistent asthma; however, the antioxidant effects of corticosteroids are unsatisfied. Furthermore, this treatment is hindered when steroid dependence or steroid resistance occurs [
41]. Therefore, the development of novel and efficient therapeutic strategies is of great significance in the control of asthma. We have described a HuR/TGF-β1 feedback that mediates airway remodeling in vivo and in vitro. Although experimental studies have adequately demonstrated that blocking these signaling pathways, especially TGF-β1, could effectively decrease airway remodeling, no such chemical inhibitors are clinically available at the present time. Interventions that target TGF-β1 are limited to acute situations, such as immediately after surgery, that would require application of the anti-fibrotic for a limited period. Such strategies might not be appropriate for the treatment of chronic fibrotic diseases that develop over many years, on the basis of the long period of dosing necessary for such diseases.
The post-transcriptional regulation of gene expression includes mRNA transportation, procession, turnover, and mRNA translation.Targeting this pathway has considerable potential to control the deterioration of lung function due to asthma. HuR, as an upstream modulator of TGF-β1, serves to provide a novel insight for searching the precise target of asthmatic airway remodeling. Thus, the future research will focus on silencing HuR in mice to explore a new road being clinically appropriate for controlling asthma.
Acknowledgements
We acknowledge Professor Shuai Wang for providing professional writing services and the facilities supported by the Central Laboratory of Shandong Provincial Hospital.