ADAM33: a cellular model of over-expression. From full length to soluble

Polymorphic variation in the A Disintegrin And Metalloprotease 33 (ADAM33) genehas been associated with asthma and bronchial hyperresponsiveness. The ADAMfamily of proteins has a multi-domain structure and has diverse biological functionswhich can include growth factor shedding, cell migration and cell adhesion.Evidence for a soluble form of ADAM33 has been found in BAL fluid of asthmaticpatients and there is evidence that ADAM33 has a function in angiogenesis.To determine the possible function(s) of the full length ADAM33 protein, it wasover expressed in HEK293 that do not normally express ADAM33 and thephenotypic consequences analyzed. Cells stably expressing full length ADAM33under the control of a CMV promoter were obtained by selection with the antibiotic,G418. The expression of ADAM33 was confirmed by western blot analysis. Controlcells were transfected with an empty vector control encoding G418 resistance only.Cells were examined for changes in cell phenotype and ability to produce solubleADAM33.Cells stably expressing ADAM33 grew more quickly than mock transfected cellsbut this effect was lost over time. However, processed (ie. active) ADAM33 proteincould not be detected by western blot. In order to determine whether asubpopulation of cells was able to produce active ADAM33, the cells were clonedand re-cloned by limiting dilution. This allowed isolation of a number of ADAM33clones that produced processed protein. Furthermore, these experiments revealedthat ADAM33 expression increased the clonogenic potential of the cells.Supernatant from cloned cells was pulled down with ConA and samples analysed bywestern blot which showed a band at 50-55kDa in the ADAM33 clones using anantibody to the metalloproteinase domain. This soluble ADAM33 was active andpromoted angiogenesis using a HUVEC capillary tube forming assay. SolubleADAM33 was upregulated in the presence of TGF-?2 and its shedding is unliky tobe autocatalytic.Over expression of ADAM33 in HEK293 cells alters the clonogenic potential andsurvival of HEK293 cells in low density cultures. This property may contribute tothe behaviour of mesenchymal cells in asthmatic airways. Soluble ADAM33 isreleased into culture media and has functional activity. Its release is up regulated inthe presence of TGF-?2