Figure 8. ADAMTS-12 promotes cell invasion through an avb3 integrin mediated mechanism. (A) A histogram representing the number of JEG-3 cells stably transfected with LacZ, A12FL or A12Mut that invaded through Matrigel-coated Transwell invasion chambers. Cells were cultured in complete DMEM media alone (-) or containing a function-perturbing antibody against the av integrin subunit (av Abi; 1:50 dilution) for 48 h. (B) A histogram showing the invasiveness of the aforementioned JEG-3 cells in Matrigel-coated Transwell invasion chambers. Cells were cultured in complete DMEM media containing 5 nM of an avb3 integrin-specific bi-cyclic RGD peptide (cyclic RGD), control peptide (peptide con) or media alone (-) for 48 h. All assays were performed in triplicate and performed on three independent occasions (n = 3; * = P#0.05, compared to controls). doi:10.1371/journal.pone.0018473.g008 invasion compared to LacZ, with A12FL demonstrating a slightly more invasive phenotype over A12Mut. The effect on cell invasion of a bi-cyclic RGD peptide that demonstrates preference towards the avb3 integrin heterodimer had a similar affect to that of the av perturbing antibody (Figure 8B). Specifically, peptide treatment did not affect LacZ cell invasion, but significantly inhibited A12FL and A12Mut invasion. Interestingly, the number of avb3 peptide- treated cells that invaded through the Matrigel-coated insert were higher in the A12FL population than in the A12Mut population, suggesting that a component of ADAMTS-12-mediated invasion is dependent on its catalytic domain. Any advantage that A12Mut provided in JEG-3 cell invasion was completely abolished with bi- cyclic RGD peptide treatment. Discussion Multiple ADAMTS (ADAMTS-1, -2, -4, -5, -6, -7, -9, and -12) were detected in tissue samples of first trimester human placenta. With the exception of ADAMTS-5, this repertoire of ADAMTS ADAMTS-12 and Trophoblast Invasion subtypes was maintained, albeit at differing levels, in cultures of highly invasive EVTs propagated from these tissues and in poorly invasive JEG-3 cells. Although the overall biological significance of this expression pattern of ADAMTS proteins in the human placenta remains to be elucidated, our studies demonstrate that ADAMTS-12 plays a non-redundant role in human trophoblastic cell invasion in vitro. In addition, ADAMTS-12 mRNA levels in EVTs are differentially regulated by TGF-b1 and IL-1b in a concentration- and time-dependent manner. Thus, ADAMTS-12 is tightly regulated in human trophoblastic cells and signaling pathways that control invasion act, at least in part, by downregulating ADAMTS-12 gene expression. In agreement with our findings, ADAMTS-2 mRNA has been detected in first trimester placenta [36], with mRNA transcripts encoding ADAMTS-1, -4, -5, -6, -7, -9, and -10 mRNA also being found in human term placenta [37–41]. In addition to these ADAMTS subtypes, we found that ADAMTS-12 is present in placenta obtained during the first trimester of pregnancy and in primary cultures of EVTs propagated from these tissues. Cal et al [21] failed to detect ADAMTS-12 mRNA transcripts in normal human tissues that included term placenta, but found it exclusively in fetal lung. Additionally, ADAMTS-12 expression has been detected in normal cartilage where it is proposed to regulate chondrocyte differentiation [42]. Differences in placental ADAMTS-12 mRNA levels are likely attributable to the changes in the subpopulations of cytotrophoblasts that constitute this dynamic tissue throughout gestation. In particular, EVTs predominate during the first trimester with the number of these cells declining sharply thereafter and being absent in term placental tissues [1]. Failure to detect ADAMTS-5 in human trophoblastic cells in vitro suggests that the expression of this ADAMTS subtype in the placenta is restricted to one or more of the other cellular compartments that comprise this dynamic tissue, particularly the mesenchymal core and/or vasculature. Molecular weight discrepancies between endogenous ADAMTS-12 protein species in EVTs and first trimester placenta (35 kDa major product) compared to exogenous ADAMTS-12 in JEG-3 cells (83 kDa major product) were observed. This could in part be explained by the different cell types analyzed and variability in expression of proteolytic processing enzymes. Artificially high levels of over-expressed ADAMTS-12 may saturate proteolytic processes resulting in a decrease in these cleavage products. In contrast to its restricted expression in normal human tissues, ADAMTS-12 mRNA is readily detectable in the cartilage and synovium of patients with arthritis and in cancer cell lines of diverse origin with levels of this mRNA transcript being present at higher levels in gastric carcinomas compared to matched normal tissue controls [21]. These later findings suggest a putative role(s) for ADAMTS-12 in the development of an invasive cellular phenotype, and indeed our studies revealed that manipulating the function of this ADAMTS subtype ascribed a pro-invasive phenotype in human trophoblastic cells. Altered expression levels of other ADAMTS subtypes have also been detected in human carcinomas but their individual contribution(s) to the onset and progression of cancer remains unclear [7,9,10,43–45]. For example, ADAMTS-1 mRNA levels have been shown to be either increased [41] or decreased [9] in breast carcinomas. Higher levels of this ADAMTS subtype have also been associated with pancreatic and hepatocellular cancer [43] whereas ADAMTS-1 mRNA levels are unchanged in the onset and progression of kidney cancer [46] and decreased in lung carcinomas [41]. Exogenous expression of ADAMTS-1 has been shown to decrease the experimental metastasis of Chinese hamster ovary cells [46] but increase the metastatic potential of mammary and lung cancer cell lines in vivo [47] whereas our study demonstrates that PLoS ONE | www.plosone.org 12 April 2011 | Volume 6 | Issue 4 | e18473