Figure 3. Reduced ADAMTS-12 expression levels decrease EVT invasion. (A) EVTs were transfected with siRNA specific for ADAMTS-12 (A12i), a scrambled control siRNA (NS) or cultured in the presence of transfection reagent alone (EVT) for 48 h. Cultures were harvested for total RNA or protein. The results are presented (mean 6 SEM; n$4) in the bar graphs (* = P#0.05). Representative Western blots prepared using total protein extracts and probed with antibodies directed against either ADAMTS-12 or b-actin are shown. The results are presented (mean 6 SEM; n$4) in the bar graphs (* = P#0.05). (B) Photomicro- graphs of ethidium bromide stained agarose gels containing PCR products generated using primers specific for ADAMTS-1, -2, -4, -5, -6, -7 -12 or GAPDH. (C) EVTs were subjected to Matrigel invasion anlaysis as described in Methods. EVTs cultured in the presence of transfection reagent alone (EVT) were given an arbitrary invasion index = 1.0. Each cell line was plated in triplicate wells with the experiment repeated on at least 3 independent occasions (n = 3). The results are presented as mean 6 S.E.M. in the bar graph (* = P#0.05, compared to EVT control). doi:10.1371/journal.pone.0018473.g003 increase the invasive capacity of human trophoblastic cells in vitro, JEG-3 cells were stably transfected with the mammalian expression vector, pcDNA3-ADAM-TS-1 (A1). Western blot analysis revealed the presence of two distinct ADAMTS-1 protein species in these JEG-3 cell cultures and, albeit at lower levels, in ADAMTS-12 and Trophoblast Invasion those transfected with the control LacZ vector. These 2 isoforms of ADAMTS-1 correspond to the zymogen and mature active form [28]. However, higher ADAMTS-1 expression levels in JEG-3 cells did not result in a concomitant increase in their invasive capacity (p#0.31; Figure 4B). Exogenous ADAMTS-12 expression does not affect cellular aggregation Cell motility is regulated in part by the differential expression and function of cell adhesion molecules expressed on the cell surface [29]. To investigate whether exogenous ADAMTS-12 in JEG-3 cells could affect cell-cell binding affinity, A12FL, A12Mut, and LacZ stably transfected JEG-3 cells were subjected to a cell hanging-drop assay. Figure S1 quantitatively shows that neither full-length nor proteinase-dead ADAMTS-12 alters the aggrega- tive capacity of JEG-3 trophoblastic cells assayed at 2 and 8 h timepoints, suggesting that cell-cell interactions are not modulated by exogenous ADAMTS-12 expression. ADAMTS-12 affects cell-ECM affinity in JEG-3 cells To further elucidate the potential molecular mechanism by which ADAMTS-12 elicits an invasive phenotype in poorly- invasive trophoblastic cells, we analyzed whether cell-ECM binding affinities differed between JEG-3 cells exogenously expressing full-length ADAMTS-12, proteinase-dead ADAMTS- 12 or LacZ. To do this, we cultured A12FL, A12Mut, and LacZ transfected JEG-3 cells in a combinatorial fashion on extracellular matrices deposited by these same JEG-3 transfectants. JEG-3 cells exogenously expressing ADAMTS-12 significantly adhered more readily to their own native ECM (Figure 5A). Likewise, JEG-3 cells exogenously expressing proteinase-dead ADAMTS-12 or LacZ bound with high affinity to their own ECMs. JEG-3 cells expressing ADAMTS-12 adhered to ECM deposited by protein- ase-dead ADAMTS-12 expressing cells and vice versa with high affinity, whereas control LacZ-expressing cells bound with less affinity to ECM deposited by A12FL or A12Mut expressing cells. The ability of the full-length and proteinase-dead ADAMTS-12 expressing JEG-3 cells to bind to ECM deposited by the control LacZ expressing cells were also significantly ablated. A possible explanation for the observed differences in cell-ECM binding could lie in the ability of these cells to differentially synthesize and deposit specific ECM proteins. To investigate this possibility, we analyzed by semi-quantitative PCR analysis the mRNA levels of vitronectin, laminin, tenascin and fibronectin, ECM proteins known to be expressed in the human endometrium [30–32] and therefore would be encountered by invading EVTs (Figure S2). While we did not observe changes in expression levels of these mRNA transcripts in A12FL and LacZ JEG-3 cells, we did note strong differences in vitronectin, laminin and tenascin mRNA levels between primary cultures of EVTs and JEG-3 cells. Specifically, laminin was expressed in greater levels in JEG-3 cells than in EVTs, while vitronectin and tenascin were detected exclusively in EVTs. Taken together, these data suggest that ADAMTS-12 facilitates cell-matrix interactions by regulating cell- ECM adhesion through a mechanism(s) independent of its endogenous proteolytic activity. RGD peptide disrupts ADAMTS-12 mediated cell-ECM binding and invasion In an attempt to elucidate the mechanism by which ADAMTS- 12 regulates cell-ECM binding, we performed native ECM- binding assays as described above for JEG-3 cells stably transfected with ADAMTS-12 or LacZ in the presence of a synthetic RGD PLoS ONE | www.plosone.org 7 April 2011 | Volume 6 | Issue 4 | e18473