The metalloproteinase ADAM-12 regulates bronchial epithelial cell proliferation and apoptosis

Abstract

Objectives: The ADAMs (a disintegrin and metalloproteinase) enzymes compose a family of membrane-bound proteins characterized by their multi-domain structure and ADAM-12 expression is elevated in human non-small cell lung cancers. The aim of this study was to investigate the roles played by ADAM-12 in critical steps of bronchial cell transformation during carcinogenesis.

Materials and methods: To assess the role of ADAM-12 in tumorigenicity, BEAS-2B cells were transfected with a plasmid encoding human full-length ADAM-12 cDNA, and then the effects of ADAM-12 overexpression on cell behaviour were explored. Treatment of clones with heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) neutralizing antibodies as well as an EGFR inhibitor allowed the dissection of mechanisms regulating cell proliferation and apoptosis.

Results: Overexpression of ADAM-12 in BEAS-2B cells promoted cell proliferation. ADAM-12 overexpressing clones produced higher quantities of HB-EGF in their culture medium which may rely on membrane-bound HB-EGF shedding by ADAM-12. Targeting HB-EGF activity with a neutralizing antibody abrogated enhanced cell proliferation in the ADAM-12 overexpressing clones. In sharp contrast, targeting of amphiregulin, EGF or transforming growth factor-alpha failed to influence cell proliferation; moreover, ADAM-12 transfectants were resistant to etoposide-induced apoptosis and the use of a neutralizing antibody against HB-EGF activity restored rates of apoptosis to be similar to controls.

Conclusions: ADAM-12 contributes to enhancing HB-EGF shedding from plasma membranes leading to increased cell proliferation and reduced apoptosis in this bronchial epithelial cell line.

Figure 1

Characterization and selection of transfectants. (a) Semi‐quantitative PCR analysis of transfected clones. ADAM‐12 mRNA expression is higher in cells transfected with ADAM‐12 cDNA‐containing pcDNA3 plasmid (BA) than in cells transfected with the empty vector used as control (BV). L: 200 bp molecular weight marker (Smart Ladder, Eurogentec, Belgium). (b) Real‐time quantitative PCR measuring ADAM‐12 expression in clones. ADAM‐12 expression is higher in ADAM‐12 transfected clones than in control clones. Results are the mean ± SEM of relative gene expression expressed in folds (2^−ΔΔCt) compared with BEAS‐2B gene expression (n = 3). *P < 0.05, Mann–Whitney test. (c) ADAM‐12 protein production was determined by flow cytometry. ADAM‐12 protein production is higher in ADAM‐12 transfected clones than in control clones. Results are expressed as mean ± SEM of ΔMFI (difference between mean fluorescence intensity versus control isotype) (n = 3). **P < 0.01, BEAS‐2B versus BA clones; *P < 0.05, BV27 versus BA17, Mann–Whitney test.

Figure 2

In vitro proliferation of BEAS‐2B clones. (a) Cells cultured in complete culture medium were incubated (in triplicate) in 24‐well plates (10⁴ cells/well) and cell number was determined by measuring DNA concentration at 1‐day interval (n = 3); *P < 0.05, BA17 and BA36 versus BV27 and parental BEAS‐2B (B2B) cells, Mann–Whitney test. Results shown are those of a representative assay out of three different experiments. (b and c) Clones were grown in 24‐well dishes in soft agar and cell growth was determined by counting number of colonies developed after 9 days of incubation. ADAM‐12 overexpression significantly enhanced growth abilities of cells without any anchorage support (***P < 0.001 versus BV27 and BEAS‐2B cells). Results are expressed as mean number of colonies counted per field at 2× magnification, Mann–Whitney test (results are representative of three independent experiments).

Figure 3

Mature HB‐EGF expression in clones overexpressing ADAM‐12 and effects of HB‐EGF inhibition on cell proliferation. (a) Mature (soluble) HB‐EGF protein (sHB‐EGF) production measured by Western blot analysis in conditioned media of transfected cells (top panel). Data expressed as relative sHB‐EGF band intensities, considering BV27 intensity as 100% (lower panel). (b–d) Cells were grown either in standard media or in media supplemented with a specific inhibitor of EGF receptor tyrosine kinase (AG1478) (b) or an anti‐HB‐EGF neutralizing antibody (c), or neutralizing antibodies against amphiregulin (AR), EGF, TGF‐α (d). Results are representative of three independent experiments. In (d), results are shown for BA36 clone, as a representative example. **P < 0.01, ***P < 0.001 versus stimulation with inhibitors. °P < 0.05 versus BV27; °°P < 0.01 versus BV27, Mann–Whitney test.

Figure 4

Representative results of TUNEL assay, measuring role of ADAM‐12, in cell resistance to etoposide‐induced apoptosis, effects of general caspase inhibition and HB‐EGF neutralization. (a) Cells were immunostained with FITC‐12‐dUTP to investigate baseline or etoposide‐induced apoptosis (presented data are representative of two independent experiments). Results are expressed as percentage of mean number of TUNEL‐positive cells compared to total cell number for each group ± SEM. **P < 0.01, compared to parental or control transfected cells; °°P < 0.01, compared to baseline apoptosis in parental BEAS‐2B cells, Mann–Whitney test. (b) Cells cultured in presence of a general caspase inhibitor and subjected to apoptosis inducing conditions showed reduced apoptotic levels when compared to standard apoptosis inducing experiments (*P < 0.05, ***P < 0.001). (c) Inhibition of HB‐EGF by a neutralizing antibody enhanced apoptotic levels of ADAM‐12 overexpressing cells. Apoptotic levels in parental and control cells were not different when cells were cultured with etoposide alone or with etoposide and anti‐HB‐EGF neutralizing antibody. (d) Representative microphotographs of TUNEL staining in ADAM‐12 overexpressing clones and controls. White arrows indicate examples of cells positive for TUNEL staining.