Fig 1: ADAMTS-12 blocks digestion of Fibulin-2 by ADAMTS-5(A) Fibulin-2 was incubated with ADAMTS-5 in the presence (+) or absence (−) of ADAMTS-12 at 0, 8 and 16 h, and results were examined by western blot. (B) Presence of ADAMTS-12 decreases the ability of SK-BR-3 overproducing ADAMTS-5 to invade. Left, representative pictures showing the invasive SK-BR-3 cells using Matrigel-coated invasion chambers. SK-BR-3c indicates cells transfected with an empty vector; ADAMTS-5, SK-BR-3 cells expressing exogenous ADAMTS-5; ADAMTS-12 indicates cells expressing exogenous ADAMTS-12, and ADAMTS-5 + ADAMTS-12 indicates SK-BR-3 cells expressing both metalloproteases as is indicated in the center panel (β-actin was employed as loading control). Right, quantification of fold-change in invasion capacity of SK-BR-3 cells under the different conditions assayed. (C) Proposed models for the influence of the examined ADAMTS metalloproteases in the progression of breast cancer. Left, secretion of ADAMTS-4 and particularly of ADAMTS-5 by tumor cells can contribute to cleave Fibulin-2 thus leading to cancer cell spreading and an increase of the migratory potential of the fibroblasts. Right, ADAMTS-12 may act as a tumor-protective metalloprotease through the interaction with Fibulin-2. This interaction may impede digestion of Fibulin-2 by aggrecanases thus hindering progression of tumor cells.
Fig 2: Fibulin-2 cleavage by ADAMTS-4 and ADAMTS-5(A) Left, in vitro digestion of Fibulin-2 by aggrecanases. Fibulin-2 was incubated with ADAMTS-1 (Fib.2 + ADAMTS-1), ADAMTS-4 (Fib.2 + ADAMTS4) or ADAMTS-5 (Fib.2 + ADAMTS-5) for 16 h. Fib.2 indicates Fibulin-2 incubated alone (without metalloproteases, control). Right, an independent experiment was perfomed to evaluate final degradation products. To this end, Fibulin-2 was incubated for 24 h with ADAMTS-1 (Fib.2 + ADAMTS-1), ADAMTS-4 (Fib.2 + ADAMTS-4) or ADAMTS-5 (Fib.2 + ADAMTS-5). Main proteolytic products were detected with an anti-Fibulin-2 antibody and are indicated with arrows. Molecular weight markers are indicated on the left. (B) Left, MCF-7 cells were transfected with a plasmid containing the full-length cDNA for Fibulin-2 (Fib.2) or co-transfected with this plasmid and plasmids containing the full-length cDNA for ADAMTS-4 (Fib.2 + ADAMTS-4), or ADAMTS-5 (Fib.2 + ADAMTS-5). SK-BR-3 produces fibulin-2 endogenously and they were transfected with an empty vector (C, Control), or with vector containing the full-length cDNAs for ADAMTS-4 or ADAMTS-5. Right, expression of exogenous ADAMTS-4 and ADAMTS-5 was analyzed by using an anti-FLAG antibody (bottom panels). Intervening irrelevant lanes are not shown. Molecular weight markers are indicated on the left and β-actins were used as loading control.
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