The PA System Modulates ESC Differentiation Figure 6. inhibition of uPA by amiloride treatments regulates ESC skeletal myogenesis. EBs from wild type CGR8 ES cells were induced to differentiate to skeletal myotube and treated or not by 100 mM amiloride for different period of time as in figure 4. (A) mRNAs were extracted and analyzed by real time RT-PCR for the expression of the skeletal myogenic markers MyH1 (left panel) and Myogenin (right panel). 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. (B) MyH1 protein expression at day 24 of differentiation of CGR8 cells treated or not by amiloride, as indicated, was analyzed by Western blotting with anti-MyH1 (a-MyH1) antibodies. Membranes were reprobed with a- ERK antibodies as loading control. (C) Myotube formation of CGR8 treated or not by amiloride, as indicated, was analyzed by immunofluorescence experiments with anti-MyH1 antibodies (right panels) and nuclei were counterstained with DAPI (left panels), representative field are shown. (D) Differentiated cells at day 24 were analyzed for Myogenin expression by flow cytometry, two different experiments are shown, controls giving either 28.7% (upper panels) or 16.2% (lower panels) myogenin positive cells. doi:10.1371/journal.pone.0049065.g006 embryonic germ layers. Interestingly, during this period, whereas endogenous PAI-1 is not expressed, maximum uPA enzymatic activity is detected. During this early period, we found that uPA inhibition decreases adipogenesis. To explain these inhibitory effects one could hypothesize that the PA system regulates ESC differentiation by interfering with extracellular matrix deposition. However our preliminary results show that even if the PA system modulates collagen-1 deposition, there is no direct correlation between this effect and the PA-mediated effects on ESC adipogenesis. Interestingly, the PA system has also been implicated in the activation of three major intracellular transduction path- ways: ERK, PI3K/PKB and JAK/STAT pathways [34], them- selves involved in adipogenesis. One can hypothesize that one or several of these pathways are implicated in ESC differentiation in response to the PA system. It has been already shown that PAI-1 prevents the cooperation of avb3 integrin with insulin signaling in NIH3T3 fibroblasts, resulting in a decrease in insulin-induced PKB phosphorylation [35]. Since, insulin is required for the terminal step of differentiation of ESC towards adipocytes [30], this pathway could be involved in the effects that we observed in response to PA interference during ESC adipogenesis. Whether the early (d0–d7) and late (d7–d24) effects are regulated by common or different molecular mechanisms requires further studies. However, it is very likely that they correspond to the specific targeting of successive early precursors of the adipocyte lineage. Indeed, despite the fact that such precursors have not be precisely identified nor characterized, we know that PPARgamma, the master gene of terminal adipocyte differentiation, is not present before day 15 of ESC differentiation [30]. Therefore we believe that the PA modulation of the ESC experimental model could constitute a powerful tool to characterize the early precursors of the adipogenic lineage. For example, our results suggest that during the early period, one can specifically target the adipocyte lineage by an amiloride treatment, potentially allowing PLOS ONE | www.plosone.org 10 November 2012 | Volume 7 | Issue 11 | e49065