ADAMTS-12 and Trophoblast Invasion Figure 4. Exogenous ADAMTS-12 expression specifically increases the invasive capacity of JEG-3 cells, independent of its intrinsic proteolytic activity. (A) A representative Western blot depicting ADAMTS-12 protein levels in JEG-3 cells stably transfected with LacZ, A12FL or A12Mut. (B) A Western blot depicting ADAMTS-1 protein levels in JEG-3 cells stably transfected with A1 or LacZ. Expression levels of each ADAMTS were standardized to b-actin. Invasion assays were also performed using these cell lines and are represented as histograms (A and B). The results are presented as mean 6 S.E.M; n$10 in the bar graphs (* = P#0.05, where * denotes a significantly different value compared to LacZ). doi:10.1371/journal.pone.0018473.g004 inhibiting peptide, control peptide, or culture media alone. RGD peptide treatment significantly abrogated cell-ECM binding in all ADAMTS-12 and LacZ transfected cell populations, whereas ECM binding affinities of cells cultured in untreated media or with the control peptide were not affected (Figure 5B). Interestingly, the extent of RGD peptide-mediated cell-ECM binding inhibition was significantly more pronounced in ADAMTS-12 expressing cells than in LacZ expressing cells cultured on their own native ECM (p#0.01 versus p#0.05). Similar to our previous findings, exogenous ADAMTS-12 altered cell-binding affinities to native ECM deposited by JEG-3 cells transfected with A12FL or LacZ, though in this experiment it was shown that ADAMTS-12- mediated cell-ECM adhesion is in part regulated through an RGD-dependent mechanism. This finding sheds new light on the putative mechanism by which ADAMTS-12 modulates cell-ECM interaction, namely through an RGD sequence molecular event most likely involving members of the integrin cell adhesion molecule family. Cell invasion is regulated in part by cell-ECM interactions mediated primarily through integrin receptors expressed on the cell surface [33]. Our findings demonstrating that ADAMTS-12 could modulate JEG-3 cell-ECM adhesion in an RGD dependent manner suggested that ADAMTS-12-mediated cell invasion could also be regulated through a similar RGD/integrin-dependent mechanism. To test this hypothesis, we cultured A12FL, A12Mut, and LacZ expressing JEG-3 cells in Matrigel-coated Transwell invasion chambers in the presence of the RGD inhibiting peptide. Cells were also cultured in the presence of a control peptide that served as a technical control, or were cultured in complete DMEM media. There were no significant differences in the invasive indices of LacZ transfected JEG-3 cells when cultured in the presence of either the RGD or control peptides (Figure 5C). However, the invasive indices of JEG-3 cells stably transfected with A12FL or A12Mut cultured in the presence of the RGD peptide were significantly less than the invasive indices of the same cells cultured in presence of the control peptide or media alone (Figure 5C). ADAMTS-12 promotes an invasive EVT-like cell-ECM binding phenotype Different integrin heterodimer combinations allow for cells to acquire distinct ECM substrate preferences, and studies have shown in both cancer and trophoblast models that specific integrins regulate cell invasion [34]. Using fluorimetric 96-well microplate arrays pre-coated with specific ECM proteins (collagen I, collagen II, collagen IV, fibronectin, laminin, tenascin, and vitronectin) and monoclonal antibodies to integrin subtypes (a1, PLoS ONE | www.plosone.org 8 April 2011 | Volume 6 | Issue 4 | e18473