The Role Of Extracellular Signal-regulated Kinase1/2 Signalling Pathway In The Regulation Of Asthmatic Bronchial Smooth Muscle Cells On Their Phenotypic Modulation, Migration And Secretion

Background:Asthma is a chronic inflammatory disorder of airway that is highly prevalent in world and its pathogenesis remains completely uncertain. Despite an increased use of medications that suppress airway inflammation and repress contraction of smooth muscle, some patients with chronic and severe asthma still hardly get effective treatment due to the development of airway remodeling and subsequent poorly reversible airway obstruction. Of the structural changes in the airway wall remodeling process, bronchial smooth muscle cells (BSMCs) are perhaps the most important component. Recent reports show that BSMCs not only contribute to airway remodeling by its increased content, but also, bronchial smooth muscle cells in chronic asthma are capable to undergo phenotypical transition, enabling them to subserve contractile, proliferative, migratory and secretory functional responses that account for airway remodeling and persistent hyperresponsiveness.However, despite consensus toward the importance of BSMCs regarding to the phenotypic plastic, migration and secretion, there is relatively little understanding of the cellular and molecular mechanisms underlying these changes seen in the asthmatic BSMCs. Extracellular signal-regulated kinase1/2 (ERK1/2) signaling pathway is one of the most important signalling pathways that modulate asthma in the chronic airway inflammation and hyperresponsiveness. Nevertheless, there are few reports about the role of ERK1/2 signaling pathway in the regulation of asthmatic BSMCs on their phenotypic modulation and migration. So the aim of this study is to profile the impact of ERK1/2 signaling pathway on the phenotypic transition, migration and secretion of BSMCs in chronic asthmatic model of rats, as well as in passively sensitized human BSMCs by asthmatic serum.Method:Wistar rats underwent OVA intraperitoneal injection and eight weeks inhalation for chronic asthma model. Human BSMCs were sensitized by asthmatic serum. Airway reactivity and relative count of eosinophil in alveolar lavage fluid were measured, and histological sections were investigated for morphometric analysis. The expressions of ERK1/2 and P-ERK1/2 at both protein and mRNA levels were detected by western blot and RT-PCR. Phenotype of BSMCs was established under electron microscrope, also with analysis of phenotypic markers (sm-α-actin for contractile and osteopontin for synthetic) in both protein and mRNA levels by western blot or RT-PCR, respectively. Migration of BSMCs was measured by plate test and transmembrane test. Secretion of BSMCs was measured by ELISA and western blot.Results:1. ERK1/2 was markedly increased in its expression and activity in the rat model of chronic asthma, and the level of P-ERK1/2 closely related to airway thickness.2. The phenotype of BSMCs in chronic asthmatic rats switched from a contractile type to a synthetic type with plentiful synthetic organelles gathering around the nucleus and altered expressions of phenotypic markers. EGF urged the over-synthetic function of BSMCs, while MEK inhibitor PD98059 was capable to reverse the phenotypic change of BSMCs.3. BSMCs of chronic asthmatic rats possessed increased migratory ability. EGF promoted the migration of BSMCs, MEK inhibitor PD98059 was capable to inhibit the change of BSMCs, and ERK1/2 antisense ODN restrained the migration ability of BSMCs.4. BSMCs of chronic asthmatic rats secreted much more growth factors (TGF-β1 , VEGF and CTGF), cytokines (RANTES and EOTAXIN) and extracellular matrix (Fibronectin and CollangenⅠ) than that of normal controls. EGF stimulated the secretion of both two groups, but the response of chronic asthmatic group was more intense. Both PD98059 and antisense oligonucleotide were able to suppress the secretion of BSMCs in chronic ashmatic rats, but antisense oligonucleotide reduced the level of RANTES nearly to that of normal controls, while PD98059 couldn't.5. Local intranasal administration of ERK1/2 antisense oligonucleotides to chronic asthmatic rats led to DNA uptake in lung cells associated with a reduction of intracellular ERK1/2 expression. Such intrapulmonary blockade of ERK1/2 expression caused an abrogation of the increase in the thickness of airway as well as in that of smooth muscle layer. Furthermore, treatment with antisense but not nonsense oligonucleotides inhibited the phenotypic transition of BSMCs in the rat model of chronic asthma.6. Passively sensitized human BSMCs changed further toward the synthetic phenotype as compared to the controls. EGF pushed the phenotypic transition of human BSMCs, and PD98059 inhibited partly the change in human BSMCs. Meanwhile, ERK1/2 antisense ODN was capable to reverse the phenotypic change of passively sensitized human BSMCs.7. The migratory ability was increased in passively sensitized BSMCs as compared to the controls. EGF promoted the migration of human BSMCs, MEK inhibitor PD98059 was capable to inhibit the change of BSMCs, and ERK1/2 antisense ODN suppressed the migration ability of BSMCs.8. Passively sensitized human BSMCs secreted much more growth factors, cytokines and extracellular matrix than that of controls. EGF stimulated the secretion of human BSMCs, and PD98059 was able to suppress the secretion of human BSMCs. While ERK1/2 antisense oligonucleotide induced a dramatic reduction of the secreted factors to levels comparable to that of normal controls.Conclusion:ERK1/2 signalling pathway play an important role on the phenotypic modulation, migration and secretion of bronchial smooth muscle cells in the rat model of chronic asthma, as well as in the model of human BSMCs passively sensitized by asthmatic serum.