lysine residues of target substrates. Sumoylation is performed by the sequential action of three sets of enzymes [37]. In the first step, an E1 enzyme covalently binds a SUMO molecule in an ATP- dependent fashion. The SUMO molecule is transferred next to the SUMO E2 conjugating enzyme Ubc9. A SUMO E3 ligase binds to Ubc9 and specific substrates, and thereby facilitates the transfer of a SUMO molecule from Ubc9 to specific lysine residues within substrates. As a SUMO E3 ligase, PIAS1 enhances the sumoyla- tion of transcriptional regulators, including the transcriptional modulator SnoN, which plays a critical role in TGFb responses [38,39]. However, the functional significance of PIAS1 in TGFb- induced EMT induction has remained unknown. In this study, we uncover a novel mechanism that regulates EMT. We show that TGFb reduces the level of the SUMO E3 ligase PIAS1 in cells undergoing EMT. Loss and gain of function experiments suggest that PIAS1 antagonizes EMT. We also find that TGFb leads to inhibition of sumoylation of the PIAS1 substrate SnoN during EMT. Loss of function experiments demonstrate that sumoylation contributes to the ability of SnoN to inhibit EMT. Thus, our study identifies the SUMO E3 ligase PIAS1 as a novel regulator of TGFb-induced EMT. Results TGFb downregulates the SUMO ligase PIAS1 during EMT To identify novel regulators of EMT, we employed mouse mammary epithelial NMuMG cells, widely used for studies of EMT [40,41,42]. We first confirmed that TGFb induced EMT in these cells. NMuMG cells that were untreated or incubated for 2 days with TGFb in the absence or presence of TGFb type I receptor kinase (TbRI) inhibitor SB431542 were assessed for EMT [43,44]. A key molecular hallmark of EMT is loss or mislocaliza- tion of the epithelial cell-cell junction protein E-cadherin. We used an indirect immunofluorescence approach to visualize the localization and quantify the levels of E-cadherin using the Cellomics Kinetic Scan Reader (KSR) and associated Spot- Detector bioapplication [45]. TGFb led to marked reduction of E- cadherin in NMuMG cells, and the TbRI inhibitor completely blocked this effect of TGFb (Figures 1A and 1B). We also monitored actin reorganization from cortical to stress fiber type and cell shape change from cuboidal to fibroblastic like, as additional alterations associated with EMT. We used fluorescently labeled phalloidin and the whole cell dye stain CMFDA to visualize changes in actin reorganization and cell shape, respectively. TGFb stimulation triggered actin stress fiber formation and fibroblastic phenotype that were blocked by TbRI inhibitor (Figures S1A and S1B). Collectively, these data confirm that TGFb induces EMT in these cells. Next, we used a candidate approach to identify potential novel regulators of TGFb-induced EMT. The SUMO E3 ligase PIAS1 can regulate TGFb signaling but the potential role of PIAS1 in EMT remained unidentified. To determine the role of PIAS1 in TGFb-induced EMT, we first measured the levels of PIAS1 in NMuMG cells that were left untreated or that were incubated with TGFb for 2 days. Surprisingly, we found that TGFb dramatically reduced the levels of PIAS1 in NMuMG cells (Figure 1C). The TbRI inhibitor blocked the ability of TGFb to decrease the level of PIAS1 as well as that of the epithelial marker E-cadherin (Figure 1D). We confirmed that TbRI inhibitor blocked TGFb- induced phosphorylation of Smad2 (Figure 1D). Next, we determined if the decrease in the levels of PIAS1 in cells undergoing EMT was at the level of transcription. However, TGFb had little or no effect on the levels of PIAS1 mRNA (Figure 1E). We then considered the possibility that TGFb- PIAS1 Negatively Regulates EMT dependent reduction in the levels of PIAS1 might be due to an increase in PIAS1 ubiquitination and consequent degradation. To test this idea, we incubated control and TGFb-treated NMuMG cells with the proteasome inhibitor MG132 to protect polyubi- quitinated proteins from proteasomal degradation. We found that TGFb induced the polyubiquitination of PIAS1 in NMuMG cells (Figure 1F). These data suggest that TGFb reduces the level of PIAS1 via the ubiquitin-proteasome pathway in cells undergoing EMT. PIAS1 is a member of the PIAS family of SUMO E3 ligases that also includes PIAS2, PIAS3, and PIAS4 [46]. We therefore also determined effect of TGFb on the levels of PIAS 2, 3 and 4 mRNA and protein in NMuMG cells (see Materials and Methods S1). We found that PIAS2, PIAS3 and PIAS4 mRNAs were expressed in control NMuMG cells as determined by RT-PCR analyses, and their levels were not altered upon TGFb treatment (Figure S1C). Immunoblotting analyses revealed that PIAS3 and PIAS4 were expressed in NMuMG cells but there was little or no expression of PIAS2 in these cells (Figure S1D). In contrast to TGFb-induced downregulation of PIAS1 protein in NMuMG cells (Figure 1C), TGFb did not appreciably alter the levels of the PIAS3 and PIAS4 proteins (Figure S1D). Together, these data suggest that TGFb may selectively induce the downregulation of PIAS1 among the PIAS proteins. PIAS1 acts in a SUMO ligase dependent manner to inhibit EMT The finding that TGFb reduces the level of PIAS1 in cells undergoing EMT led us to characterize the function of PIAS1 in EMT. We first tested the effect of expression of exogenous PIAS1 on an E-cadherin promoter driven luciferase reporter gene, whose repression represents a reliable readout of EMT [47,48]. We confirmed that TGFb suppressed expression of the E-cadherin luciferase reporter gene in NMuMG cells (Figures 2Ai and 2Aii). Expression of exogenous PIAS1 inhibited the ability of TGFb to repress the E-cadherin reporter without affecting the basal level of the E-cadherin reporter in NMuMG cells (Figure 2Ai). The cysteine-rich RING type zinc finger domain in PIAS1 is required for its interaction with the E2 SUMO conjugating Ubc9 and hence for the E3 SUMO ligase activity of PIAS1 [35]. We tested the effect of expression of a mutant PIAS1 protein, in which Cysteine 350 within the zinc finger domain was converted to serine, on the E-cadherin-luciferase reporter gene [38]. We confirmed that the wild type and the SUMO ligase mutant PIAS1 were expressed at equivalent levels (see Materials and Methods S1; Figures S2Ai and S2Aii). Importantly, we found that the SUMO ligase mutant PIAS1 failed to antagonize TGFb-inhibition of the E-cadherin reporter gene (Figures 2Ai and 2Aii). We next carried out a dose-response study to further confirm the role and specificity of the SUMO ligase activity in the ability of PIAS1 to inhibit TGFb repression of the E-cadherin promoter. These data showed that PIAS1 antagonized TGFb-induced suppression of the E-cadherin reporter gene in a dose-dependent manner (Figure 2B). In contrast, the SUMO E3 ligase mutant PIAS1 (CS) did not block the ability of TGFb to inhibit the E-cadherin reporter at any concentration (Figure 2B). Collectively, these data suggest that the SUMO ligase activity is critical for PIAS1 to block TGFb- repression of E-cadherin transcription. To characterize the role of endogenous PIAS1 on the ability of TGFb to downregulate E-cadherin transcription, we employed an RNAi approach to induce knockdown of PIAS1 ([38] and Figures S2Bi and S2Bii). Expression of PIAS1 short hairpin RNAs enhanced the ability of TGFb to suppress the E-cadherin promoter suggesting that endogenous PIAS1 counteracts TGFb- PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e13971