ADAMTS-12 and Trophoblast Invasion Figure 7. ADAMTS-12 regulates av integrin expression and FAK activity. (A) Representative ethidium bromide stained agarose gels containing RT-PCR products (i) and Western blot autoradiographs (ii) respectively depicting mRNA and protein levels of ADAMTS-12, av-, b1-, and b3- integrin subunits in EVTs untreated (-) or transfected with an siRNA oligonucleotide directed against ADAMTS-12 (A12i) or control non-silencing siRNA (NS). GAPDH and b-actin serve as endogenous mRNA and protein controls respectively. (B) Ethidium bromide stained gels and Western blot autoradiographs depicting mRNA (i) and protein levels (ii) of the genes described above in JEG-3 cells stably transfected with pcDNA3.1 constructs expressing LacZ, A12FL or A12Mut. EVTs served as an ADAMTS-12 positive control. (C) Representative autoradiographs of Western blots depicting phospho-397 FAK (p397) and total FAK (FAK) protein levels in the EVTs (i) and JEG-3 cells (ii) described above. The molecular weight markers (kDa) are shown to the left. doi:10.1371/journal.pone.0018473.g007 marked decrease in FAK phosphorylation whereas overexpression of ADAMTS-12 lead to an increase in FAK phosphorylation. Although total FAK levels in EVTs remained unchanged, we did observe a proportional increase in total FAK compared to phosphorylated FAK in JEG-3 cells overexpressing A12FL and A12Mut. To investigate whether a physical interaction between ADAMTS-12 and avb3 integrin occurs, we imunoprecipitated av integrin from protein lysates obtained from A12FL-expressing JEG- 3 cells and probed for ADAMTS-12. IP analysis suggested that no physical interaction between ADAMTS-12 and avb3 occurs (Figure S3). Verification of av pull-down was achieved by re-probing the immunoblot with a monoclonal av-integrin antibody. ADAMTS-12 controls cell invasion through an avb3 integrin mediated mechanism Seeing that ADAMTS-12 regulates the expression and function of avb3 integrin, and that avb3 has been previously shown to promote an invasive phenotype in human EVTs, we next tested whether ADAMTS-12-mediated cell invasion is facilitated by avb3. Treatment of LacZ, A12FL or A12Mut expressing JEG-3 cells with a function-perturbing av integrin antibody cultured in Matrigel-coated Transwells significantly inhibited A12FL- and A12Mut-mediated invasion, but had no affect on invasion of LacZ expressing cells (Figure 8A). In concordance with our previous data, A12FL and A12Mut showed a significant increase in cell PLoS ONE | www.plosone.org 11 April 2011 | Volume 6 | Issue 4 | e18473