A HuR/TGF-β1 Feedback Circuit Regulates Airway Remodeling In Airway Smooth Muscle Cells

BackgroundBronchial asthma is one of the most common diseases in respiratory system.Interestingly,the prevalence and morbidity of asthma go higher year by year.In the worldwide,approximately 300 million patients are suffered from asthma and in China the amount is more than 15 million.Thus,it is time to seek for effective measures and more attention should be paid to the exploration of fundamental mechanism.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.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.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,has been implicated in the pathogenesis of airway remodeling in asthma.In particular,TGF-(31 3’ UTR was reported to be a putative target of HuR in human cancer cells.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,α strong stimulus for asthmatic response.Besides,a-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,a-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 a-SMA in OVA-induced mice than PBS-induced mice.Thus we hypothesized that a HuR/TGF-(31 feedback is involved in airway remodeling and targeting them might have considerable potential for the control of asthma.Objective1.This study explored the association between HuR and TGF-β1 in asthmatic airway remodeling to determine whether a HuR/TGF-β1 feedback regulated the development of airway remodeling.2.This study also aims to determine the effect of HuR/TGF-β1 feedback at OVA-driven mice on airway remodeling.Methods1.Cultured human ASM cells were stimulated by PDGF for 0,6,12 and 24 h.Western blotting,RT-PCR and immunofluoresence were used to detect the expression of HuR,TGF-β1,α-smooth muscle actins(α-SMA)and collagen type I(Col-I).Knockdown of HuR,flow cytomerty was used to detect the morphological change and western blotting for functionally change of ASM cells.Furthermore,the interference of TGF-β1 and exogenous TGF-β1 were implemented to testify the influence on HuR.2.A murine OVA-driven allergic model based on sensitization and challenge was developed to verify the role of HuR/TGF-β1 on airway remodeling.This study evaluated the expression of HuR,TGF-β1,α-SMA and Col-I using Western blot,RT-PCR,Immunofluoresence in cultured airway smooth muscle cells.Results1.PDGF treatment elevated the expression of TGF-β1 plus Col-I and α-SMA,cultured ASM cells were treated with 20ng/ml PDGF for 0,6,12 and 24 h.Compared with control,PDGF treatment for 6,12 and 24 h elevated TGF-β1mRNA levels by 27%,60%and 87%separately(P<0.05).Similar alterations could also be shown at TGF-β1 protein levels,evidenced by 31%,69%and 106%elevation(P<0.05).Likewise,Col-I and a-SMA,were also increased in a time-dependent manner(both P<0.05).Collectively,these results above provided direct evidence that PDGF treatment promoted TGF-β1 along with Col-I and α-SMA expression.2.Annexin V shown the proportion of apoptosis in the Consi group after cultured for 12 h were 1.236%,while in the HuRsi group was 3.315%(P<0.05).Western blotting dedicated that the relative expression of HuR protein in the HuRsi group was reduced 44.1%compared with the Consi group.mRNA levels showed that a 28.8%difference between the Consi and HuRsi groupwas found.The results above showed that HuR could protect ASM cells from apoptosis to a certain extent.3.Western blotting analysis demonstrated that HuR silencing decreased HuR expression to very low level,simultaneously the TGF-β1protein content was also decreased.The concentration of TGF-β1 in the cultured serum also showed the same alteration.We further investigated decreased mRNA stability occurred in the transfected ASM cells.Treatment with actinomycinD revealed that there was a tiny but consistent decrease in TGF-β1 mRNA half-life in the HuRsi group,indicating that HuR might enhance the TGF-β1 mRNA stability.Furthermore,the western blotting and the cultured medium showed lower expression of Col-I and a-SMA in the HuRsi group than in the Consi group.4.ASM cells in TGFsi group owned lower survival rate than the Consi group on the PDGF stimulation for 12 h.The relative expression of TGF-β1 protein in TGFsi group was reduced 23.8%compared with the Consi group.The TGF-β1-blockaded ASM cells were treated with 4 ng/ml TGF-β1 for 12 h,HuR elevated 89%under the stimulation of extra TGF-β1,while PDGF treatment alone elevated 40%expression of HuR.These data further corroborated our hypothesis that a HuR/TGF-β1 feedback existed among ASM cells to regulate airway remodeling,especially in the expression of Col-I and a-SMA.5.We showed that chronic exposure to OVA induced a robust airway inflammation,Elevations in the total cell numbers,and the percentages of macrophages,eosinophils,lymphocytes and neutrophils(7.56 ± 1.53 X 106/ml,28.20 ± 1.95%,35.40 ±2.02%,26.70 ±2.38%and 7.08 ±0.74%,respectively)were observed in the BALF of OVA-exposed mice compared with the values that were observed in PBS controls(3.69±0.98 × 106/ml,68.88±2.73%,6.25±0.95%,18.12± 1.58%and 3.02 ±0.63%,respectively).In our study,we interestingly found large amount of type I and III collagen deposition surrounding the peribronchioles,Furthermore,PAS staining revealed that OVA induced goblet cell hyperplasia in the airway epithelium while PBS showed little change.These results clearly indicate that OVA effectively promotes airway remodeling in mice.6.To further understand the HuR/TGF-β1 feedback in vivo,we found that chronic OVA-challenged mice also highly expressed HuR,TGF-(31 and a-SMA in mRNA levels and protein levels of lung tissues.These results of immunohistochemistry showed a significant increase of three proteins in OVA-induced mice,which was well consistent with the results of western blotting.Overall,these data illustrated that OVA-induced airway remodeling is associated with the high expression of HuR,TGF-β1and α-SMA.Conclusions1.A HuR/TGF-β1 feedback circuit regulates airway remodeling by modulating Col-I and a-SMA in cultured cells.2.Increased expression of HuR,TGF-β1 and α-SMA in lung tissue of OVA-induced mice accompanied with large amounts of collagen deposition.