a4, a5, av, avb3), and b (b1, b2, b3, b4, b6, avb5, a5b1) integrins/subunits, that are immobilized onto goat-anti-mouse antibody coated microtiter plates. Goat anti-mouse antibody coated wells served as negative assay controls. Following the manufacturers protocol, 16105 cells suspended in DMEM (100 ml) were plated into the 96-wells and incubated for 2 h. Unbound cells were washed away, and adherent cells were lysed and integrin binding function/expression was detected using a fluorescent DNA-binding dye provided with the integrin binding kit using a fluorescence plate reader (485/530 nm). Integrin binding assays were performed on three independent occasions (n = 3). Statistical Analysis Statistical differences between the absorbance values were assessed by the analysis of variance (ANOVA) using GraphPad Prism 4 computer software (San Diego, CA, USA), and significant differences between the means were determined using Dunnett’s test [16]. Cellular invasion indices were analyzed by one-way ANOVA followed by the Tukey multiple comparison test [22]. The differences were accepted as significant at P,0.05. Results Characterization of the ADAMTS subtypes present in human placental tissue ADAMTS-1, -2, -4, -5 (also known as ADAMTS-11), -6, -7, -9, and -12 mRNA levels were detected in first trimester human placenta (Figure 1A). In contrast, ADAMTS-3, -8 and -10 mRNA were not detected in these placental tissues but were readily detectable in our positive controls (data not shown). This repertoire of ADAMTS subtypes was maintained in both highly invasive EVTs and poorly invasive JEG-3 cells, with the exception of ADAMTS-5, which was not detected in either trophoblastic cell type examined in these studies (Figure 1A). However, ADAMTS- 12 mRNA levels in EVTs were significantly higher than those detected in JEG-3 cells. In contrast, ADAMTS-1, -2, -7, and -9 mRNA levels were significantly lower in EVTs, whereas there was no significant difference between the levels of the ADAMTS-4 and -6 mRNA transcripts present in these two trophoblastic cell types. High ADAMTS-12 expression is observed in invasive human trophoblastic cells As previous work has failed to detect ADAMTS-12 in normal human tissues and cells [21], we performed Western blot analysis to confirm that this ADAMTS subtype was expressed in first trimester human placenta in vivo and in trophoblastic cells in vitro. Major ADAMTS-12 protein species (83, 50 and 35 kDa), corresponding to a C-terminal fragments generated by post- translational cleavage of the ADAMTS-12 zymogen [21] were detected in the total protein lysates prepared from these placental tissues or cultures of EVTs or JEG-3 cells (Figure 1B). Further- more, and in agreement with our PCR data, ADAMTS-12 protein expression levels were found to be significantly higher in EVTs as compared to JEG-3 cells. Cytokines regulate ADAMTS-12 mRNA levels in EVTs TGF-b1 and IL-1b are spatiotemporally expressed at the maternal-fetal interface and have been shown to be potent regulators of human trophoblastic cell invasion in vitro [3,15]. In view of these observations, we examined the ability of these two cytokines to regulate ADAMTS-12 mRNA levels in primary cultures of EVTs in a time- and dose-dependent manner. ADAMTS-12 mRNA was detected in all of the EVT cultures ADAMTS-12 and Trophoblast Invasion (Figures 2A and 2B). The addition of vehicle (ethanol) to the culture medium of EVTs, which served as a negative control, had no significant effect on ADAMTS-12 mRNA levels in these cells at any of the time points examined (data not shown). A significant decrease in ADAMTS-12 mRNA levels was detected in EVTs cultured in the presence of TGF-b1 (1 ng/ml) for 24 h with the levels of this mRNA transcript continuing to decrease until the termination of these studies at 48 h (Figure 2A). The addition of increasing concentrations of TGF-b1 to the culture medium of these cells demonstrated that ADAMTS-12 mRNA levels in EVTs were regulated in a dose-dependent manner (Figure 2A). A function-perturbing monoclonal antibody directed against TGF-b1 abolished the TGF-b1-mediated de- crease in ADAMTS-12 mRNA levels in these primary cell cultures (Figure 2A). In contrast, IL-1b (100 IU) caused a continuous and significant increase in ADAMTS-12 mRNA levels in EVTs over time in culture (Figure 2B). Maximum levels of this mRNA transcript were subsequently detected in EVTs cultured in the presence of the highest concentration (1000 IU) of this cytokine examined in these studies (Figure 2B). A function-perturbing monoclonal antibody directed against IL-1b was also found to attenuate the increase in ADAMTS-12 mRNA levels observed in EVTs cultured in the presence of this cytokine (Figure 2B). Our initial findings demonstrated that similar to many other normal and malignant epithelial cells examined to date [21], ADAMTS-12 expression levels are higher in human trophoblastic cells with an invasive phenotype. The ability of cytokines to differentially regulate ADAMTS-12 mRNA levels in primary cultures of EVTs provided further evidence that ADAMTS-12 contributes to the highly regulated invasion of human trophoblas- tic cells. In view of these observations, we hypothesized that altered expression levels of ADAMTS-12 would, in turn, modulate the invasive capacity of these cells. Silencing ADAMTS-12 expression reduces the invasive capacity of EVTs In order to decrease ADAMTS-12 expression in primary cultures of EVTs, we obtained 4 distinct siRNA sequences complementary to the human ADAMTS-12 mRNA transcript. Transient transfection with these siRNAs demonstrated that only one siRNA (herein referred to as A12i) significantly decreased ADAMTS-12 mRNA and protein expression levels in these cell cultures after 24 h (Figure 3A). In contrast, there was no significant difference between ADAMTS-12 mRNA and protein levels in the two control cultures; EVTs transfected with a non-silencing (NS), scrambled siRNA or cultured in the presence of transfection reagent alone (EVT; Figure 3A). To verify the knockdown specificity of A12i and to determine whether there were compensatory changes in the levels of other ADAMTS subtypes, PCR was performed using template cDNA generated from EVTs transfected with either the A12i or control siRNA and the primers specific for each ADAMTS subtype previously identified in these cells. Additionally, A12i did not affect the levels of ADAMTS-1, - 2, -4, -6, and -7 mRNA transcripts (Figure 3B). These data confirm specificity of the siRNA used to knock-down endogenous ADAMTS-12 mRNA levels in EVT cultures, and also demon- strate that a reduction in ADAMTS-12 expression is not accompanied by compensatory changes in the expression of other ADAMTS subtypes. We next tested whether a reduction in ADAMTS-12 expression levels in EVTs would result in a concomitant decrease in their invasive capacity. Matrigel-coated Transwell invasion chamber assays revealed that the number of cells that penetrated the Matrigel barrier and adhered to the underside of the Millipore PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e18473