increased expression levels of ADAMTS-1 do not alter the invasive capacity of human trophoblastic cells in vitro. A possible explanation for these contradicting observations involves auto-proteolytic processing that can account for pro- or anti-metastatic effects depending on the cleavage site [46]. It is proposed that proteolytic cleavage of the substrate binding site in ADAMTS-1 impairs substrate binding and unveils anti-angiogenic TSP-1 motifs. Recent studies characterizing the role of ADAMTS-12 in tumor progression demonstrated an anti-tumorigenic role for this subtype [48]. Llamazares et al showed ADAMTS-12 to inhibit HGF- mediated scattering of MDCK cells and also prevented bovine aortic endothelial cells from forming tubules in response to VEGF. Additionally, subcutaneous injection of SCID mice with the highly aggressive lung carcinoma A549 cell line over-expressing ADAMTS-12 repressed tumor growth. More recently, El Hour et al [49] characterized the role of ADAMTS-12 in regulating tumorigenesis using a malignant keratinocyte model system in ADAMTS-12 mice. Although this study did not specifically address cell invasion, it did shed light on the importance of how ADAMTS-12 in the tumor microenvironment inhibits key aspects of tumorigenesis, such as cell proliferation and angiogenesis. In lieu of novel data describing ADAMTS-12 as a putative tumor suppressor gene expressed predominantly in the stroma [49,50], further studies are required to evaluate the biological and clinical significance of (dys)regulated expression levels of ADAMTS-12 in the onset and/or progression of cancer to the later stages of the disease state, and to delineate the function of this ADAMTS subtype in processes such as cell migration, invasion, and growth in other in vivo tumor model systems. Our demonstration that ADAMTS-12 modulates both cell- ECM binding and cell invasion through an RGD-dependent mechanism is an intriguing finding as neither the disintegrin-like domain, the TSP-repeat domains nor any other domain of any characterized ADAMTS metalloproteinase contain RGD-like motifs [8]. It is therefore unlikely that ADAMTS-12 promotes an invasive phenotype directly through an RGD-dependent interaction, but rather regulates the function or expression of proteins capable of controlling RGD-mediated cell-matrix adhe- sion and cell invasion. Our work demonstrated that ADAMTS-12 regulates cell-ECM binding to specific ECM substrates and cell attachment facilitated by specific integrin cell adhesion molecules. Specifically, we showed that ADAMTS-12 expressing trophoblas- tic cells bind more readily to collagen II, collagen IV and vitronectin and up-regulate the expression of av and avb3 integrins on their cell surface. Furthermore, this up-regulation is associated with an increase in integrin signaling activity and cell invasion. Collagen IV is a basement membrane protein whereas collagen II is a fibrillar collagen predominantly found in cartilage connective tissue and interacts predominantly with b1 or b2 integrins [51,52]. While we did not observe any changes in b1 or b2 integrin-mediated binding in ADAMTS-12 transfected JEG-3 cells, this conflicting finding may be explained by the ability of EVTs to synthesize the collagen II degrading protease, MMP-13 [53]. Collagen II remodeling by MMP-13 may result in the uncovering of cryptic integrin-binding sites that may lead to increased cell-matrix adhesion by integrins other than b1 or b2. Collagen interactions mediated by other integrins absent in our microplate integrin adhesion assay may provide additional explanations as to why we observed increases in cell binding to collagens II and IV. For example, a10b1 and a11b1 integrins have been shown to bind to these collagens in vitro [54]. Lastly, the association of ADAMTS-12 with the ECM might lead to proteolytic-independent conformational ECM changes that may also result in uncovering cryptic integrin binding sites. ADAMTS-12 and Trophoblast Invasion Vitronectin is an RGD-containing multifunctional protein found in serum and extracellular matrices, and is secreted by trophoblastic cells during the first trimester of pregnancy [55,56]. It has been demonstrated that integrin receptors containing RGD binding motifs (ie. avb3, avb5, a5b1 and aIIbb3) bind to vitronectin [52]. The avb3 integrin has been shown to play important roles in promoting both trophoblast and cancer cell invasion [34,57–61]. Binding of avb3 integrin to its ligands results in the activation of mitogen-activated protein kinase (MAPK) and focal adhesion kinase (FAK) [56]. Kabir-Salmani et al [35] demonstrated that IGF-I-mediated migration of trophoblasts is avb3 integrin-dependent and is associated with avb3 integrin heterodimer co-localization with phosphorylated FAK, paxillin and viniculin at focal adhesions. These data, along with the findings in our study, provide strong evidence in assigning a key role for the avb3 integrin in promoting an invasive phenotype in human trophoblasts. Activation of FAK is facilitated by many b1 and b3-containing integrins and is therefore not a singular avb3 event [62]. Analysis of FAK activation in our cell culture system should be interpreted only as an indicator for the acquisition of an invasive phenotype facilitated by ADAMTS-12. In light of these observations, avb3 integrin appears to play key roles in facilitating cell invasion processes in both physiological and pathological contexts. Recent findings that have shown ADAMTS proteinases to regulate cell motility through Ras-MAPK signaling events by mechanisms independent of their MMP-like domain support our findings [63]. Exactly how ADAMTS-12 regulates integrin expression remains to be elucidated. Our data would suggest that ADAMTS-12 acts in a cell automomous manner regulating ECM activity independent of its endogenous proteolytic function, however more studies are required to fully interrogate the mechanisms by which ADAMTS-12 regulates av integrin transcription and function. In summary, we have determined that ADAMTS-12, indepen- dent of its proteolytic activity, plays a critical role in human trophoblastic cell invasion in vitro. This study not only provides an insight into the molecular mechanism underlying epithelial cell invasion but also extends our understanding of the cell biology and complexity of the ADAMTS gene family. Furthermore, the trophoblastic cell cultures examined in these studies offer an ideal model to further dissect the contributions of the distinct domains of ADAMTS-12 in regulating epithelial cell invasion. Supporting Information Figure S1 Exogenous ADAMTS-12 does not alter cellular aggregation. Photomicrographs show JEG-3 cells stably trans- fected with pcDNA3-ADAM-TS12 (A12FL), pcDNA3-ADAM- TS12-MUT (A12Mut) or pcDNA3-LacZ (LacZ) that were cultured in 20 ml hanging drops for 2 or 8 h prior to being inverted and mounted with a glass cover slip. The histogram quantitatively describes cellular aggregation assayed at 2 h as determined by grouping cell aggregates into three categories (,5 cells/aggregate, 5–15 cells/aggregate, .15 cells/aggregate). Hanging drop assays were done in triplicate and the experiment was performed on three independent occasions (n = 3). (TIFF) Figure S2 ADAMTS-12 does not regulate the expression of a subset of ECM proteins. Ethidium bromide-stained photographs of RT-PCR products generated using primers specific to the ECM proteins vitronectin (V), fibronectin (F), laminin (L) and tenascin (T) and the housekeeping gene GAPDH from cDNA’s made from (A) JEG-3 cells stably transfected with ADAMTS-12 (A12FL) or control LacZ or from (B) cDNA’s made PLoS ONE | www.plosone.org 13 April 2011 | Volume 6 | Issue 4 | e18473