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First published online 25 September 2007
doi: 10.1242/jcs.005751

Journal of Cell Science 120, 3544-3552 (2007)
Published by The Company of Biologists 2007
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The ADAMTS12 metalloproteinase exhibits anti-tumorigenic properties through modulation of the Ras-dependent ERK signalling pathway

María Llamazares1,*, Alvaro J. Obaya2, Angela Moncada-Pazos1, Ritva Heljasvaara1,{ddagger}, Jesús Espada1, Carlos López-Otín1 and Santiago Cal1,§

1 Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncologia, Universidad de Oviedo, 33006-Oviedo, Asturias, Spain
2 Departamento de Biología Funcional (Fisiología), Instituto Universitario de Oncologia, Universidad de Oviedo, 33006-Oviedo, Asturias, Spain


Figure 1
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  		Fig. 1. Characterization of MDCK ADAMTS12 stable transfectants. (A) Domain organization of ADAMTS12 showing the position of the FLAG epitope. (B) RT-PCR and western blot analysis of the ADAMTS12-expressing clones TS12-4 and TS12-22, using the H-142 antibody against ADAMTS12. The cMDCK clone was used as a negative control in these assays. In RT-PCR experiments, amplification of mRNA encoding beta-actin was used to ascertain RNA integrity and ensure equal loading. (C) Immunostaining of non-permeabilized cMDCK, TS12-4 and TS12-22 cells using an anti-FLAG antibody. Bar, 10 µm.

 

Figure 2
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  		Fig. 2. MDCK ADAMTS12-expressing clones are refractory to the scattering effect of HGF stimulation. The control cMDCK clone shows reduced cell-cell adhesion and has a spindle-shape morphology upon treatment with HGF. By contrast, TS12-4 and TS12-22 still form epithelial-like colonies after 16 hours of incubation with HGF. A MDCK clone that expresses a catalytically inactive mutant version of ADAMTS12 (TS12-MUT) is also refractory to the scattering effect. Bar, 50 µm.

 

Figure 3
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  		Fig. 3. ADAMTS12 negatively regulates the HGF signalling pathway. (A) Levels of different components of the HGF pathway (E-cadherin, vimentin, ERK, P-ERK, AKT, P-AKT and P-Met) were evaluated by western blot in the selected cMDCK, TS12-4 and TS12-22 clones, in both non-stimulated (– HGF) and stimulated (+ HGF) conditions. Ectodomain shedding of Met was also analyzed in the conditioned medium from these clones (Met). (B) Immunostaining of E-cadherin and vimentin in the selected clones without (– HGF) and with (+ HGF) stimulation by hepatocyte growth factor. Bar, 10 µm.

 

Figure 4
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  		Fig. 4. MDCK cells expressing ADAMTS12 fail to form tubular structures in a 3D collagen matrix. Selected clones expressing ADAMTS12, TS12-4 and TS12-22 were grown within a collagen gel for seven days. Morphological changes such as long extensions (arrows) from the cysts are clearly observed in cMDCK and MT1-MDCK clones. Bar, 50 µm.

 

Figure 5
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  		Fig. 5. Cells expressing a truncated ADAMTS12 lacking thrombospondin domains (ADAMTS-{Delta}TS12) undergo an EMT following HGF stimulation. (A) Domain organization of ADAMTS-{Delta}TS12. The position of the HisTag epitope is indicated. (B) Expression analysis of ADAMTS-{Delta}TS12 by RT-PCR and western blot using an antibody against the HisTag. In both cases, the cMDCK clone was used as a control. Truncated ADAMTS12 is located in the conditioned medium of cells that stably express this form of the enzyme. In RT-PCR, amplification of the mRNA encoding beta-actin was used to ascertain RNA integrity and ensure equal loading. (C) Scattering assay (2D), tubulogenesis assay (3D) and immunostaining of E-cadherin and vimentin of the ADAMTS-{Delta}TS12-expressing cells in the absence (– HGF) or presence (+ HGF) of hepatocyte growth factor. Bars, 30 µm (left, centre); 10 µm (right).

 

Figure 6
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  		Fig. 6. Purification process and enzymatic activity of ADAMTS12. (A) Western blot analysis of ADAMTS12 during the purification process. c-EBNA and EBNA-TS12 indicate cell-layer-associated fractions from 293-EBNA cells transfected with an empty vector, and the equivalent fraction from 293-EBNA cells expressing ADAMTS12, respectively. Recombinant purified ADAMTS12 is also indicated. (B) Following incubation of aggrecan with recombinant ADAMTS4 (lane 2), purified ADAMTS12 (lane 3) or an equivalent fraction purified from c-EBNA cells (lane 1), samples were deglycosylated and the aggrecan degradation products were detected by using the BC-3 antibody. In lane 4, purified ADAMTS12 was previously incubated with the metalloproteinase inhibitor ilomastat (+Ilo). Molecular mass markers (kDa) in both panels are indicated on the left.

 

Figure 7
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  		Fig. 7. Effect of ADAMTS12 on tubulogenesis of BAE-1 cells. BAE-1 cells were cultured within a collagen gel for 2 days in the absence (a) or presence (b–f) of 100 ng/ml VEGF. 10 µl of the cell-layer-associated fraction from 293-EBNA cells that express ADAMTS12 (EBNA-TS12) (c), or the same amount of the equivalent fraction from c-EBNA cells (d), were added to the culture. BAE-1 cells were grown in the presence of 10 µl of purified ADAMTS12 (e), or the same amount of the equivalent fraction purified from c-EBNA cells (f). Addition of 50 µl of conditioned medium from cells that express truncated ADAMTS12 (g) or from control cells transfected with an empty vector (h) does not affect the ability of BAE-1 cells to form a capillary network in the presence of VEGF. Bar, 30 µm.

 

Figure 8
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  		Fig. 8. Inhibition of subcutaneous tumour growth by ADAMTS12. (A) SCID mice were injected with parental A549 cells or with cells from an A549 clone (A549-TS12) expressing ADAMTS12. Tumour growth was followed until day 37, and tumour volumes were calculated after measurements were taken with a calliper. (B) Representative subcutaneous tumours at day 37 following injection are shown for mice that received A549 or A549-TS12 cells

 

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© The Company of Biologists Ltd 2007