Mechanism Of Mucin Production In Airway Epithelial Cell By Aspergillus Fumigatus Extract

Aspergillus is an environmental mold(fungus).Its wide distribution in the environment can cause various human diseases and it poses large threat to the general public.Aspergillus fumigatus(A.fumigatus),due to its unique thermo-tolerance to normal human body temperatures,is one of the most common human pathogens that causes a variety of fungal lung diseases.Mucus overproduction is associated with almost all of A.fumigatus induced airway diseases including allergic bronchopulmonary aspergillosis(ABPA)and fungal asthma.In those diseases,mucus overproduction is a pathogenic hallmark leading to decreased lung function.The major macromolecular components of mucus are high-molecular-weight polymeric gel-forming mucin glycoproteins.In the airway,the major gel-forming mucins are MUC5AC and MUC5B.The mechanism by which Aspergillus fumigatus induces mucin production is not fully understood,and treatment options are limited.Therefore,to understand the underlying molecular mechanism,we have utilized a well-established A.fumigatus extract(AFE)model to elucidate downstream signal pathways that mediate A.fumigatus induced mucin production in airway epithelial cellsNCI-H292 cell is utilized as cell model for mucin research.We use different doses to treat cells at different times in order to optimize the best dose and time by measuring mucin production To measure if the activity of EGFR mediates mucin expression by AFE,we measured the expression of MUC5AC and MUC5B mRNA levels after pretreatment of cells with EGFR inhibitors and neutralizing antibodies,as well as EGFR phosphorylation to investigate the effect of EGFR activity on mucin production.At the same time,in order to study the substances that may induce mucin production in AFE,we used trypsin and related inhibitors to detect the effects of proteases on the expression levels of MUC5AC and MUC5B from both sides.Next,in order to further study the possible pathways through which AFE induces mucin production,related protein inhibitors were used to observe their effects on the expression of MUC5AC and MUC5B mRNA levels.Ca2+ signaling has regulatory significance in all cells,and as a result Ca2+ effects on mucin production was examined.It was found that AFE can induce mucin production in a short time span,and AFE was found to stimulate time-and dose-dependent increases of major airway mucin gene expression(MUC5AC and MUC5B)partly via the elevation of their promoter activities.When AFE was heated,mucin expression is inhibited significantly,this effect was likely mediated by serine proteases in AFE.EGFR inhibitors can inhibit MUC5 AC and MUC5B mRNA expression but does not further increase EGFR-phosphorylation levels.Meanwhile,we found AFE treatment dramatically increased active Ras and further enhanced the downstream Raf1-ERK cascade.However,we found that inhibition of Ca2+ flux significantly repressed ERK activation and mucin induction by AFE.In summary,we have demonstrated that AFE potently induced expression of MUC5AC and MUC5B in airway epithelial cells.Fungal serine proteases play a key role in this process.EGFR activity was required but not sufficient for inducing its activation by AFE.In addition,studies have shown that the fungal protease in AFE induces mucin by activating the Ras/Rafl/ERK signaling pathway,indicating that Ras instead of EGFR may become a new target for the treatment of fungal lung diseases.Ca2+ signaling is an essential determinant of ERK activation and mucin induction.The discovery of this new approach may help us understand the pathogenesis of fungal sensitization in allergic diseases such as asthma.Further extension of this study to in vivo models will advance our understanding of mucin/mucus regulation by A.fumigatus and further establish a Protease/Ras/Rafl/ERK axis as a novel therapeutic target for treating lung diseases caused by environmental mold exposure.