The expression and regulation of ADAM33, a novel asthma susceptibility gene, during myofibrolast differentiation

Abstract

To test the hypothesis that TGFβ may promote the ability of fibroblasts to differentiate into smooth muscle cells in asthma, the expression of five smooth muscle associated genes was examined.  Heavy Chain Myosin, Calponin and Desmin were all shown to be upregulated to a greater extent (p<0.05) in asthmatic (n=7) primary airway fibroblast cultures than in healthy control cells (n=6).  α Smooth Muscle Actin and γ Actin were upregulated maximally at a lower dose of TGFβ in asthmatic cells compared with healthy cells (p<0.05).  Alongside the upregulation of mRNA, an increase in protein for four of these smooth muscle marker genes was detected in TGFβ treated cells by immunohistochemistry.  Desmin protein however, was not detected at the protein level and hence a bona fide phenotype switch from fibroblast to smooth muscle cell was not achieved.

To test the hypothesis that ADAM33 is involved in fibroblast differentiation, the effect of TGFβ on ADAM33 expression was investigated.  In association with the switch towards a myofibroblast phenotype, global ADAM33 mRNA expression as well as that of individual ADAM33 splice variants was down-regulated in a dose dependent manner by TGFβ.  To measure ADAM33 protein expression, antibodies were generated by the immunisation of four rabbits and two chickens with ADAM33 specific peptide sequences.  These antibodies were shown to be active against their respective immunising peptides but failed to detect full length ADAM33.  However, a commercially available anti-ADAM33 antibody preparation was found to specifically detect ADAM33.  ADAM33 protein expression was shown to be down-regulated upon TGFβ treatment as detected by immunofluorescent staining.  In addition products of ADAM33 degradation were shown to accumulate upon TGFβ treatment in primary cells as well as a recombinant model, as detected by western blot.

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