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

Abstract

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

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Identifiers

ORCID for Donna Davies: orcid.org/0000-0002-5117-2991

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