Regulation Of Cell Proliferation And Apoptosis By ERK Signaling Pathway In Airway Smooth Muscle Cells Of Asthmatic Rats

Asthma is characterized by airway hyperresponsiveness, chronic inflammation and airway remodeling. Severe asthmatics develop irreversible airway obstruction and persistent airway hyperresponsiveness[1-3] . The airways include a variety of structural changes such as subepithelial fibrosis, mucous cell hyperplasia/metaplasia, and increased smooth muscle mass, which are collectively referred to as airway wall remodeling. It has been previously reported that proliferation of airway smooth muscle cells (ASMC) was an important contributor to the airway remodeling by abnormal proliferation[4-6]. So, investigation on abnormal proliferation of ASMC is very important to understand the pathogenesis of airway remodeling in asthmatics, inhibition of abnormal proliferation of ASMC may be one of the effective methods to reduce airway wall remodeling. We had previously demonstrated that the functional influence and biological effect of asthmatic ASMC by various mitogens such as growth factor, cytokine and so on. But little is known about various of bioinformation involved in the regulation of asthmatic ASMC intracellular signaling transduction.A critical role for extracellular signal-regulated kinase(ERK) in cell proliferation and differentiation has been suggested in many cell types, which is an important intracellular signaling pathway and plays a key role in regulating signaling transduction between extracellular signal and nuclear reaction[7]. ERK, being a key enzyme in this pathway, has a variety of subtypes. ERK kinase 1 and 2 (ERK1/2) have been demonstrated to be primarily distributed in smooth muscle cells. ERK signaling pathway has also been reported as a vital signaling pathway mediating vascular smooth muscle cel(lVSMC)abnormal proliferation[8]. ERK are the regulators in VSMC phenotype variation, migration, proliferation, apoptosis and externalization[9,10]. Based on those reports, it is speculated that ERK signaling pathway may be involved in regulating the abnormal proliferation of chronic asthmatic ASMC. So far, little is known about the regulation of ERK1/2 in asthmatic ASMC abnormal proliferation. In this study, we focused on the endogenous proliferation activity and the regulation of cell proliferation by ERK signaling pathway in ASMC from chronic asthmatic rats with the chronic asthmatic model and cultured ASMC in vitro. Thus the results further provided experimental evidence for the significance of ERK signaling pathway in chronic asthmatic ASMC proliferation. This study was therefore undertaken to explore the role of ERK signaling pathway in pathogenesis of asthmatic airway remodeling from the view point of the intracellular signal transduction, and the results should be helpful for further finding the new therapic targets to aim directly at airway remodeling in asthma.Methods1. Airway remodeling was detected in chronic asthmatic rats by using image analysis system. The expressions of ERK and PCNA in lung tissue from chronic asthmatic rats were observed by immuocytochemistry staining. The expressions of ERK1/2, phosphorylated of ERK1/2(p-ERK1/2)and PCNA were detected in airway smooth muscle (ASM) by immunofluorescence double staining with confocal microscopy, and the expressions of protein or mRNA of ERK and PCNA in ASM were also detected by immunoblotting and hybridization in situ,respectively. Thus this study was designed to investigate the expressions of ERK in ASM in rat model of chronic asthma.2. The rat model of chronic asthma was established. Extracellular signal-regulated kinase (ERK) agonist epidermal growth factor (EGF) and inhibitor PD98059 were used to investigate the regulative effect of ERK signaling pathway on airway smooth muscle cells (ASMC) proliferation in chronic asthmatic rats. The ASMC proliferations were examined with flow cytometry analysis, methyl thiazolyl tetrazolium (MTT) colorimetric assay, [3H]thymidine incorporation and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of cyclin D1 and CDK2 were detected by immunocytochemical staining. The expressions of ERK mRNA, ERK protein, p-ERK1/2 protein were observed by RT-PCR and Western blotting. Thus this study was designed to investigate the endogenous proliferation activity and the regulation of cell proliferation by ERK signaling pathway in ASMC from chronic asthmatic rats.3. Primary cultures of ASMC were established and cells between passages 4 and 7 were used for experiments. ASMC were treated with ERK activator epidermal growth factor (EGF) and inhibitor PD 98059. Apoptosis of ASMC were detected by Situ end labeling and Annexin-V FITC PI double staining. The expressions of bcl-2 and bax were detected by immunocytochemical staining. The levels of caspase-3 protein were detected by Western blotting. The expressions of ERK mRNA, ERK protein, p-ERK1/2 protein were observed by RT-PCR and Western blotting. Thus this study was designed to investigate the regulation of cell apoptosis by ERK signaling pathway in ASMC from chronic asthmatic rats.4. ASMC were transfected with ERK sense, antisense and mismatched oligodeoxynucleotides (ODN). Proliferations of ASMC were detected by flow cytometry analysis, MTT colorimetric assay, and [3H] thymidine incorporation. Apoptosises of ASMC were detected by in situ end labeling and Annexin-V FITC PI double staining. The expressions of ERK mRNA, ERK protein, p-ERK1/2 protein and PCNA protein were observed by RT-PCR and Western blotting, respectively. Thus this study was designed to investigate the effect of ERK antisense oligodeoxynucleotides on proliferation and apoptosis of airway smooth muscle cells (ASMCs) from chronic asthmatic rats.Results1. The thickening of smooth muscle and structural remodeling in airway were observed in chronic asthmatic rats by image analysis. The enhanced expressions of ERK and PCNA appeared obviously increased in same lung tissue and the expressions of protein or mRNA of ERK and PCNA were significantly increased in ASM.2. Compared with control group, the percentage of G0/G1 phase were significantly decreased and the percentage of S + G2/M phase were significantly increased, absorbance ( A490 ) value, DNA synthesis value,the expression of PCNA protein,the expression of cyclin D1 protein and the expression of CDK2 protein in ASMC from chronic asthmatic group were significantly increased. The expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from chronic asthmatic group were significantly increased compared to those in control group. After treatment with PD98059,the percentage of S + G2/M phase, A490 value, DNA synthesis value,the expression of PCNA protein,the expression of cyclin D1 protein and the expression of CDK2 protein in ASMC from chronic asthmatic group were significantly decreased,the expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from chronic asthmatic group were significantly decreased compared to those before treatment. After treatment with EGF, the percentage of S + G2/M phase, A490 value, DNA synthesis value,the expression of PCNA protein,the expression of cyclin D1 protein and the expression of CDK2 protein in ASMC from chronic asthmatic group were significantly increased compared to those before treatment. PD98059 inhibited markedly the effect of EGF.3. Compared with control group, the apoptotic index and the percentage of the early apoptotic cells in ASMC from chronic asthmatic group were significantly decreased. The expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from chronic asthmatic group were significantly increased compared to those in control group. After treatment with PD98059,the apoptotic index, the percentage of the early apoptotic cells, the expression of bax and the expression of caspase-3 protein in ASMC from chronic asthmatic group were significantly increased compared to those before treatment, the expressions of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK were also significantly down-regulated. Compared with those before treatment, apoptotic index and the percentage of the early apoptotic cells were significantly increased in EGF-treated cells. PD98059 inhibited markedly the effect of EGF.4. The percentage of G0/G1 phase were significantly increased, the percentage of S + G2/M phase, A490 value, DNA synthesis value, the expression of PCNA protein, the expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from antisense oligodeoxynucleotides group were significantly decreased compared to those of chronic asthmatic group. The apoptotic index and the percentage of the early apoptotic cells in ASMC from antisense oligodeoxynucleotides group were significantly increased compared to those of chronic asthmatic group. However, the sense and the mismatched did not have these effects. Conclusions1. The thickening of smooth muscle and structural remodeling in airway were observed in asthmatic rat model. It indicated the rat model of chronic asthma was established.2. The enhanced expressions of ERK and PCNA appeared obviously in same lung tissue and the expressions of protein or mRNA of ERK and PCNA were significantly increased in ASM from asthmatic rat model. It indicated ERK signaling pathway might be an important pathway in regulating cell proliferation of ASM resulting in asthmatic airway remodeling.3. The endogenous proliferation activity of ASMC from chronic asthmatic rats was significantly increased and the expressions of protein or mRNA of ERK and the activation ratio of ERK in chronic asthmatic rats were significantly increased compared with that from normal ones. ERK1/2 participate in this process by inducing enhanced expression of cyclinD1 and CDK2,promote asthmatic ASMC from G0/G1 into S phase, leading to cell proliferation.ERK signaling pathway might play an important role in regulating ASMC proliferation, leading to asthmatic airway remodeling.4. Apoptotic activation is significantly decreased in ASMC from chronic asthmatic group while the endogenous proliferation activation is significantly increased. The regulation of cell apoptosis was possibly related to ERK signaling pathway. The bcl-2 family and caspase-3 may participate in the regulation mechanism of cell apoptosis by ERK signaling pathway in ASMC from chronic asthmatic group.5. ERK antisense ODN could inhibit the proliferation and increase the apoptosis in cultured ASMC from chronic asthmatic rats.So, our results suggest that ERK signaling pathway is crucial for the regulation of ASMC abnormal proliferation. It plays a pivotal role on regulation of the proliferation and apoptosis. ERK1/2 are involved in the regulation of asthmatic ASMC proliferation and apoptosis via cell cycle, bcl-2 family and caspace-3. The results should be helpful for further providing the novel clue for further elucidating the pathogenesis in asthma and finding the treatment in severe asthmatics.