a2, a3, a4, a5, av, avb3, b1, b2, b3, b4, b6, avb5, a5b1) we sought to determine differences in cell-binding affinities to these defined substrates elicited by ADAMTS-12. Wildtype EVTs isolated from first trimester chorionic villous explants served as a biological control for ECM binding affinities associated with an invasive phenotype. EVTs were shown to bind significantly to all the ECM proteins analyzed (Figure 6A), however, the binding affinities to collagen II, fibronectin and vitronectin were strongest. LacZ expressing JEG-3 cells bound to collagen I, collagen II, fibronectin, laminin and tensacin, but bound with low affinities to collagen IV and vitronectin (Figure 6A). Similar to EVTs, A12FL and A12Mut expressing JEG-3 cells bound significantly to all the ECM proteins. Notably, their binding affinity to collagen II, collagen IV and vitronectin were significantly greater than LacZ expressing cells (Figure 6A). Integrin cell adhesion was also shown to be different between ADAMTS-12 (A12FL and A12Mut) and LacZ expressing JEG-3 cells. Whereas EVTs bound with great affinity to all a integrin subunits (Figure 6B), LacZ-expressing JEG-3 cells failed to bind to a1, a4, and av antibodies (Figure 6B). Upon exogenous expression of A12FL or A12Mut, a modest increase in binding to a3 was observed while binding to the av antibody was greatly increased as was binding to avb3 (Figure 6B). Additionally, decreases in binding to the b2 antibody and to avb5 and a5b1 antibodies were observed. Taken together, these data demonstrate that ADAMTS-12, regardless of a functional catalytic ADAMTS-12 and Trophoblast Invasion domain, regulates specific cell-ECM binding differences resem- bling those of an invasive phenotype. av integrin expression is controlled by ADAMTS-12 Since studies have shown that the av integrin subunit, and in particular, the avb3 integrin heterodimer plays an important role in EVT invasion [34,35] and in light of our findings showing a significant increase in avb3 mediated cell adhesion (Figure 6), we investigated whether ADAMTS-12 regulates the expression of these integrin subunits. In EVTs where endogenous ADAMTS-12 was knocked down using siRNA, av integrin mRNA and protein levels showed a correlative decrease in expression compared to controls (Figure 7A). b1 and b3 integrin subunit levels were unaffected by a reduction in ADAMTS-12 expression (Figure 7A). Gain of ADAMTS-12 function in JEG-3 cells (A12FL and A12Mut) lead to an increase in mRNA and protein expression of the av subunit, while b1 and b3 integrin subunit protein levels remained unchanged, although b3 integrin subunit mRNA levels in A12FL and A12Mut cells showed a modest decrease in expression (Figure 7B). Since phosphorylation of FAK (phospho-397) is an indicator of activated integrin signaling [33], we performed Western blot analysis on protein lysates harvested from EVTs transfected with ADAMTS-12 siRNA or JEG-3 cells expressing A12FL or A12Mut and assayed for phospho-397 FAK and total FAK expression (Figure 7C). Loss-of ADAMTS-12 in EVTs showed a Figure 6. ADAMTS-12 modulates ECM binding mimicking that of an invasive phenotype. (A) Histogram depicting binding affinities of EVTs and JEG-3 cells expressing LacZ, A12FL or A12Mut to specific human ECM proteins. A fluorimetric ECM protein binding assay utilizing a 96-well microtiter plate coated with collagen I, collagen II, collagen IV, fibronectin, laminin, tenascin and vitronectin was used to analyze cell-ECM binding. Pre-coated BSA served as a negative control. (B) Histograms showing a/b integrin binding affinities of EVTs and LacZ-, A12FL- and A12Mut-expressing JEG-3 cells. Cell-integrin adhesion was determined in 96-well plates coated with monoclonal antibodies specific to a (a1, a2, a3, a4, a5, av, avb3) or b (b1, b2, b3, b4, b6, avb5, a5b1) integrins. ECM and integrin adhesion was quantified by lysing bound cells, incubating with the nucleic acid binding CyQuant GR dye, and reading fluorescence at 485/530 nm. Dotted line provides reference to baseline measurements (n = 3; * = P#0.05, where * denotes a significantly different value compared to LacZ control values). doi:10.1371/journal.pone.0018473.g006 PLoS ONE | www.plosone.org 10 April 2011 | Volume 6 | Issue 4 | e18473