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First published online 29 August 2006
doi: 10.1242/jcs.03157

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Epilysin (MMP-28) induces TGF-ß mediated epithelial to mesenchymal transition in lung carcinoma cells

Departments of Pathology and Virology, Haartman Institute and Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland

View larger version (70K): [in a new window]   Fig. 1. Epilysin induces EMT in A549 cells. (A) Cell pools stably expressing epilysin were prepared by puromycin selection of A549 cells transfected with expression constructs for either wild-type (Epi) or a catalytically inactive mutant (Epi-E/A) of epilysin. Ctrl denotes empty vector-transfected controls that have undergone the same selection procedure. Conditioned serum-free media from the cell pools were harvested and polypeptides were detected with epilysin specific antibodies (active epilysin: 48 kDa). (B) Immunofluorescence studies were performed with antibodies specific for the extracellular part of E-cadherin. To avoid internalization of the antibodies during the procedure, living cells were incubated on ice with primary antibodies, fixed with PFA and incubated with secondary Alexa-conjugated antibodies. Coverslips were mounted using a mounting medium containing DAPI. E-cadherin: red; DAPI: blue. (C) Immunoblotting of E-cadherin was performed with antibodies recognizing its ectodomain (shed degradation product 80 kDa).

View larger version (25K): [in a new window]   Fig. 2. TGF-ß is upregulated and latent TGF-ß complexes are degraded in the morpholgically altered epilysin-expressing A549 cells. (A) Conditioned serum-free media were harvested from A549 cell pools, and the concentrations of TGF-ß in the untreated (active TGF-ß) or heat-treated (total TGF-ß) media were determined using indicator cells. The results are shown as relative TGF-ß activity where the activity in the medium from the control pool has been set to 1. The asterisk denotes statistically significant difference in the total TGF-ß levels over the control (determined using the Student's t-test, P<0.05). (B,C) Polypeptides in the conditioned media from the cell pools were immunodetected with antibodies specific for latent complexes containing TGF-ß1 (anti-TGF-ß1-LAP) and LTBP-1 as indicated. In panel B the intact large latent TGF-ß1 complexes in the Ctrl and Epi-E/A samples (filled arrow) and proteolytically processed complexes in the Epi sample (unfilled arrow, band encircled) have been indicated. In panel C the intact TGF-ß/LTBP-1 complexes have been indicated with a filled arrow, and the proteolytically processed, truncated forms of LTBP-1 with a bracket.

View larger version (51K): [in a new window]   Fig. 3. Epilysin-induced EMT is prevented by the MMP-inhibitor GM6001 and by anti-TGF-ß antibodies. (A) Epilysin-expressing A549 pools were selected in the presence of GM6001 (1 µM), which prevented the epilysin-induced EMT. The cells were seeded on glass coverslips in 10% serum containing medium with the indicated supplements. After 48 hours the cells were changed to serum free medium containing the same supplements. After 24 hours the amounts of total and active secreted TGF-ß in the media were determined. The TGF-ß levels in the control and E/A pools are shown on the right. (B) Living cells were incubated with anti-E-cadherin antibodies, fixed with PFA, permeabilized and then stained with antibodies against the phosphorylated form of SMAD2. (C) The numbers of E-cadherin and P-SMAD2 positive cells in each culture were counted. The proportions of positive cells in each culture are given.

View larger version (20K): [in a new window]   Fig. 4. MT1-MMP and MMP-9 are upregulated in the epilysin-expressing pools. (A,B) Polypeptides in the conditioned media from the A549 cell pools were immunodetected with antibodies against matrilysin (A) and MT1-MMP (B). (C) The content of gelatinases in the conditioned media from the A549 cell pools was analyzed by gelatin zymography. Bands corresponding to gelatinase B (MMP-9) and gelatinase A (MMP-2) are indicated. Two additional unidentified gelatinolytic bands in the epilysin-expressing pool have been indicated with arrows.

View larger version (52K): [in a new window]   Fig. 5. Recombinant epilysin associates with the surface of epithelial cells through the hemopexin domain. (A) Schematic presentation of cDNA-constructs coding for wild type epilysin (Epi*), a catalytically inactive mutant (Epi-E/A*), the pro and catalytic domains (Cat*) or the hemopexin domain (Pex*) in the pEF1/V5-His vector generating recombinant proteins with C-terminal V5-tags (denoted with *). (B) Transiently transfected A549 cells were changed to serum free medium 24 hours post transfection. The conditioned media (upper panel) were harvested and total cell lysates (lower panel) prepared after 24 hours. Comparable amounts of polypeptides from the media and cell lysates were immunodetected with antibodies specific for the V5-tag. Upper panel: 58 kDa: pro-form of Epi* and Epi-E/A* (unfilled arrow); 48 kDa: mature form of Epi* and Epi-E/A* (filled arrow); 28 kDa: Pex* (filled arrowhead) and 23 kDa: mature form of Cat* (unfilled arrowhead, encircled). Brackets indicate degradation products of Epi*, Epi-E/A* and Pex*. Lower panel: 62 kDa: prepro-form of Epi* and Epi-E/A* (filled arrow); 32 kDa: pro-form of Cat* (unfilled arrow); n.s.: nonspecific band. Ctrl denotes empty vector-transfected control. (C,D) A549 cells were transiently transfected as indicated and living, non-permeabilized cells were stained with antibodies specific for the V5-tag(*) and MT1-MMP. MT1 denotes wild type MT1-MMP and MT1-E/A an inactive mutant of MT1-MMP.

View larger version (44K): [in a new window]   Fig. 6. Cell surface association of epilysin is abolished after EMT. Non-permeabilized, stably transfected A549 cell pools were stained with antibodies specific for epilysin. Phase contrast images show the corresponding cell morphologies. The MMP-inhibitor GM6001 (GM, 1 µM) was included in the incubation medium as indicated.

View larger version (20K): [in a new window]   Fig. 7. Epilysin increases the migration of A549 cells. Cell culture plate inserts with 8 µm pore membranes were coated with type I collagen (0.1 mg/ml). Stable A549 cell pools were added to the upper chamber. The MMP-inhibitor GM6001 (10 µM) was then added to both chambers as indicated. After 4 hours cells that had migrated through the membrane were stained and counted. The asterisk denotes statistically significant variation between indicated samples (Student's t-test, P<0.05).

View larger version (87K): [in a new window]   Fig. 8. Morphologically altered epilysin-expressing A549 cells and MT1-MMP transfected A549 cells have an increased ability to invade type I collagen. Type I collagen gels were made into cell culture plate inserts. (A) Stable A549 cell pools and (B) A549 cells transiently transfected with epilysin and MT1-MMP were seeded to the upper chamber as indicated and medium containing HGF (10 ng/ml) as a chemoattractant was added to the lower chamber. The epilysin-induced morphological changes were not affected by the neutralizing anti-HGF antibodies in the EMT assay (Fig. 3B). The MMP-inhibitor GM6001 (10 µM) was added to both chambers as indicated. The cells were incubated on the collagen gels for 8 days changing the medium every third day. The gels were finally fixed with 3% PFA, dehydrated and embedded in paraffin. Sections were then cut and stained with hematoxylin and eosin. Cells that had invaded into the collagen gel were counted under a light microscope. The asterisks denote statistically significant variation as compared with the control (Student's t-test, P<0.05).

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