The PA System Modulates ESC Differentiation Figure 4. inhibition of uPA by amiloride treatments interfere with ESC adipogenesis. EBs from wild type CGR8 ES cells were induced to differentiate and treated or not by 100 mM amiloride for different period of time: from days 0 to 24 [A], from days 0 to 3 [A(d0–d3)], from days 3 to 7 [A(d3–d7)], from days 7 to 14 [A(d7–d14)] or 24 [A(d7–d24)]. (A) Scheme of the different amiloride treatments. (B and C) ELISA assay quantification of mouse activated uPA into the 24hrs conditioned medium of 106 cells RA-treated (C, adipogenic conditions) or not (B, skeletal myogenic conditions) and treated or not by amiloride. (D to G) Effects of amiloride treatments on ESC adipogenesis. mRNAs from retinoic acid- treated (adipogenic conditions) EBs were extracted and analyzed by real time RT-PCR for the expression of the adipogenic markers adiponectin (D), aP2 (E) and PPARgamma (F). Results are expressed in arbitrary units, with the values of untreated CGR8 at day 24 taken as 1, and are the means 6 S.E.M. of at least 3 independent experiments. Significance to Ct values is given as: *P,0.05, **P,0.01 and ***P,0.001. (G) Adipocyte formation of CGR8 treated or not by amiloride, as indicated, was visualized by Oil RedO staining, representative fields are shown. doi:10.1371/journal.pone.0049065.g004 as day 12 in the NoRA condition. This effect is reproduced (although to a lesser extend in the NoRA condition) by the A(d7– d24) treatment. These results suggest that uPA inhibition by amiloride leads to ECM accumulation in differentiating ESCs. We can hypothesize that this effect is mediated by interference with the uPA-plasmin-matrix metalloproteases cascade which induces ECM degradation [33]. The early amiloride treatments A(d0–d3) and A(d3–d7) have no significant effect on ECM deposition, examined at day 24; whereas they augment collagen 1 deposition at day 12. It is important to note that all the amiloride treatments are equally inhibitor regarding adipocyte differentiation; therefore it is possible that the collagen 1 regulation by amiloride contributes to its effect on differentiation but is neither sufficient nor necessary. Construction of an ESC Line Expressing Ectopically PAI-1 under an Inducible Control In order to by-pass a potential effect of amiloride unrelated to uPA inhibition, we undertook a genetic approach. We used a sophisticated Tet-On inducible system adapted to mouse ESCs described in Iacovino et al. [22]. Upon cre-lox recombination, the mouse ESC line A2lox.cre, allows the reproducible targeting of a locus on the X-chromosome, in the region upstream of the Hprt gene, giving robust doxycycline-inducible expression of genes of interest in both mESCs and differentiated cells. We first analyzed the time-course expression of the endogenous PA components in A2lox.cre ESCs before and after differentiation. Similar results to what observed in CGR8 cells were obtained (Data S5). The human PAI-1 cDNA was cloned into the targeting vector and we isolated A2lox.cre ESC clones expressing human PAI-1 after doxycycline induction. We further characterized the clone 3 that expresses high levels of hPAI-1. As expected, clone 3 do not express human PAI-1 protein without doxycycline in- duction, either intracellularly (not shown) or within its conditioned medium, and before or after differentiation with or without RA treatment (Fig. 5 A and B, black curves). By contrast, significant human PAI-1 protein levels are constantly produced after induction during the 24 days of the differentiation protocol PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49065