Muc5ac Is The Target Of MCLCA3in Mucus Overproduction In Asthma

BackgroundThe bronchus asthma is a kind of complex airway chronic inflammation disease,which is characterized by episodic occurrence of dyspnea, coughing, wheezing andchest tightness. Asthma attacks with one or more of these symptoms which areassociated with reversible airway obstruction and airway hyperresponsiveness [1].These reversible airway obstruction and AHR are reported to result from chronicinflammation of the bronchial mucosa [2,3], which is distinguished by differentdegree of epithelial mucus overproduction, goblet cell metaplasia and fibrosis [4]. Inconclusion, the airway epithelium plays an important role in the mechanism of airwaydefense. Evidence has shown that airway epithelium is the fundamentally disorderedin patients with chronic asthma [5].CLCA is a family of calcium activated chloride channel proteins and mCLCA3isone member of the family [6,7]. At present, there are15CLCA which have beenfound in6mammalian apecies [8]. These include4human homologues (hCLCA1,hCLCA2, hCLCA3and hCLCA4alias hCaCC2),6murine homologues (mCLCA1,mCLCA2, mCLCA3alias gob-5, mCLCA4, mCLCA5and mCLCA6),2bovinemembers (bCLCA1alias CaCC and bCLCA2alias Lu-ECAM-1),1porcine memberpCLCA1,1equine member eCLCA1, and1rat homologue [8]. The link betweenCLCA gene expression and airway diseases is very interesting because each of these diseases is associated with airway mucus excess production, and above one of CLCAmember is selectively expressed in the mucous cells [10-12]. Initial studies haveindicated that CLCA family of proteins evoke a calcium-activated chlorideconductance when transfected into HEK293cells. However, other studies havesuggested that the CLCA family of proteins were secreted soluble molecules [8,13].Whether the CLCA proteins function as chloride channels is not yet clear.mCLCA3and its human ortholog hCLCA1have been identified as clinicallyrelevant molecules in diseases with secretory function disorders, including asthma andcystic fibrosis [8]. It was reported that mCLCA3was abundantly expressed in thesmall intestine, colon, stomach, and uterus, with slight expression in tracheal tissue[15]. In the research of Atsushi Nakanishi, mCLCA3was detected with similarexpression levels in gastrointestinal and uterus tissues from AHR-model and normalmice. However, it was strongly expressed in lung tissues from AHR-model mice,whereas signal was scarcely detected inthe normal lung. It indicated that mCLCA3was induced specifically in lung tissue with OVA challenge. On the basis of thesestudies, intratracheal administration of adenovirus-expressing antisense mCLCA3RNA into AHR-model mice efficiently suppressed mucus overproduction. In contrast,over expression of mCLCA3in airway epithelia by using an adenoviral vectorexacerbated the mucus secretion [18]. Thus, mCLCA3plays an important role inmucus production and goblet cell metaplasia. Gene transfer of mCLCA3or hCLCA1into NCI-292cell line in vitro induced mucus production which further confirmed it[18,19].To better understand the relevance of mCLCA3and mucus production withoutthe influence of OVA-challenge, we perform a transfer of pcDNA3.1-mCLCA3vectorrespectively into lung of normal mice and AHR-model mice under the help of PEI.We will observe the change of airway inflammation and Muc5ac expression. Purpose: To observe the role of mCLCA3in mucus production and airwayinflammation in asthma.Method: Mice were divided into three groups:①normal mice+negative controlplasmid group,②normal mice+pcDNA3.1-mCLCA3group,③asthmatic mice.We instilled the plasmids and transfection reagents into lung of each mouse viaintranasal instillation. Real-time PCR was used to detect the expression of mCLCA3,Muc5ac, chemokines CCL2, CCL5, CCL11and Th2cytokines IL13, IL4, IL5, andTh1cytokine IFN-γmRNA. Immunohistochemistry and Western-blotting wasapplied to detect the expression of mCLCA3in lung. We observe the airwayinflammation of each mouse with inflammation score and cell counts in BALF.Result: mCLCA3only present in the airway epithelial. Result of real time PCR andwestern-blotting suggested that mCLCA3expression in asthmaticgroup and normal+pcDNA3.1-mCLCA3group was obviously higher than the normal group, P<0.05.Compare of asthmatic group and normal+pcDNA3.1-mCLCA3group, expression ofmCLCA3mRNA had no remarkable difference, P﹥0.05. Whereas the western blotindicated that expression of mCLCA3protein in asthmatic group was remarkablyhigher than normal+pcDNA3.1-mCLCA3group, P＞0.05. Real time PCR ofMuc5ac and IL13indicated that expression of mCLCA3mRNAin asthmatic groupand normal+pcDNA3.1-mCLCA3group was remarkablyhigher than normal group.No difference was detected in asthmatic group and normal+pcDNA3.1-mCLCA3group, P﹥0.05. Correlation analysis indicated that mCLCA3mRNA level waspositively correlated with Muc5ac and IL13mRNA. And the level of IL13mRNAwas also positively correlated with Muc5ac mRNA. Compared with normal group,there was no significant increase of CCL2, CCL5, CCL11, IL4, IL5and IFN-γ innormal+pcDNA3.1-mCLCA3group.Conclusion: mCLCA3promote the expression of Muc5ac, inducing mucus overproduction in asthma.