PIAS1 Negatively Regulates EMT Figure 3. PIAS1 suppresses the loss of E-cadherin levels associated with TGFb-induced EMT. A) Expression of wild type and the SUMO ligase mutant (CS) PIAS1 in stable NMuMG cells. Lysates of NMuMG cells stably transfected with the control vector expressing the resistance marker alone or together with wild type (WT) or SUMO E3 ligase mutant (CS) PIAS1 were subjected to anti-FLAG immunoprecipitation (a-FLAG IP) followed by anti-PIAS1 immunoblotting (upper panel), or were immunoblotted for PIAS1 and actin, the latter to serve as loading control (lower two panels). Wild type and SUMO ligase mutant PIAS1 (CS) are expressed at similar levels. B and C) PIAS1 acts in a SUMO E3 ligase dependent manner to suppress TGFb-reduction in E-cadherin protein level. B) Lysates of NMuMG cells stably transfected with the control plasmid or a plasmid encoding WT or CS PIAS1 as in A and left untreated or incubated with TGFb for 48 h were subjected to E-cadherin and actin immunoblotting. Scans are from a representative experiment that was repeated 4 times. C) Each column is the mean (6 SEM, n = 4) of percent reduction by TGFb of actin-normalized E- cadherin levels relative to the basal levels of the respective transfectant obtained from immunoblots including those shown in B (see MATERIALS and METHODS). D) Representative E-cadherin immunofluorescence images of vector control (2), wild type (WT) or SUMO E3 ligase mutant (CS) PIAS1 expressing NMuMG stable cells that were left untreated (I) or incubated with 20 pM (II) or 100 pM (III) TGFb for 48 h. E) Quantification of E-cadherin immunofluorescence from images as shown in D. Intensity of E-cadherin immunofluorescence per cell was obtained and analyzed (see MATERIALS and METHODS). Each column is the mean (6 SEM, n = 4) of percent reduction in E-cadherin levels by 20 pM (clear column) or 100 pM (grey column) TGFb. * indicates statistical significant difference (p,0.05) as compared to the vector control. doi:10.1371/journal.pone.0013971.g003 The finding that sumoylation contributes to SnoN’s ability to inhibit TGFb-induced EMT supports the conclusion that SnoN mediates the ability of the SnoN SUMO E3 ligase PIAS1 to suppress EMT. To further investigate the role of PIAS1-SnoN sumoylation pathway in the regulation of EMT, we examined the codependence of PIAS1 and SnoN in inhibiting TGFb-induced downregulation of E-cadherin promoter. First, we asked whether expression of the SUMO loss of function SnoN (EdA) mutant might act in a dominantly interfering manner to block the function of the SUMO E3 ligase PIAS1 in NMuMG cells. Co- immunoprecipitation studies revealed that the SnoN (EdA) mutant was as effective as the wild type SnoN in associating with PIAS1 (Figure 7A). Importantly, in reporter analysis assays, we found that expression of the SnoN (EdA) mutant, but not the wild type SnoN, blocked the ability of PIAS1 to mitigate TGFb repression of E- cadherin-promoter activity (Figure 7B). Quantitative immunoflu- orescence analysis showed that PIAS1 was expressed at similar levels in the control NMuMG cells as well as cells expressing wild type SnoN or the SnoN (EdA) mutant (Figures S4Ai and S4Aii). In other experiments, we examined the effect of expression of the SUMO E3 ligase inactive PIAS1 (CS) mutant on the ability of exogenous SnoN to block TGFb-repression of E-cadherin promoter transcription. Co-immunoprecipitation experiments demonstrated that SnoN interacts with PIAS1 (CS) as effectively as with wild type PIAS1 (Figure 7C). In reporter assays, we found that expression of the PIAS1 (CS) mutant, but not the wild type PIAS1, reversed the ability of SnoN to counter TGFb-repression of E-cadherin transcription (Figure 7D). Immunofluorescence analysis showed that SnoN was expressed at similar levels in control NMuMG cells as well as those expressing wild type PIAS1 or the PIAS1 (CS) mutant (Figures S4Bi and S4Bii). Together, these findings support the conclusion that SnoN plays a critical role in the ability of PIAS1 to block TGFb-repression of E- cadherin promoter activity during EMT. Collectively, our findings point to an important functional link involving TGFb, PIAS1 and SnoN in the regulation of EMT. Discussion In this report, we identify a novel mechanism that regulates EMT. We have found that TGFb reduces the levels of the SUMO E3 ligase PIAS1 in cells undergoing EMT. Expression of PIAS1 antagonizes the ability of TGFb to induce EMT. Importantly, the SUMO E3 ligase activity of PIAS1 is critical for its suppression of EMT. TGFb stimulation leads to the inhibition of sumoylation of the PIAS1 substrate SnoN. Loss of function mutations of SnoN sumoylation abrogated the ability of SnoN to inhibit TGFb- induced EMT. Collectively, these data suggest that PIAS1-induced sumoylation of SnoN antagonizes EMT. Conversely, TGFb leads to a reduction in the level of PIAS1 and sumoylated SnoN to facilitate the induction of EMT. The identification of PIAS1 downregulation as a critical mechanism for TGFb-induced EMT bears significant implications for our understanding of TGFb responses. Since EMT comprises the loss of apical-basal polarity and cell-cell adhesion of epithelial cells, PIAS1 inhibition of EMT suggests that PIAS1 may play a critical role in maintaining cell polarity and cell-cell contact in epithelial tissues. In other words, the PIAS1 sumoylation pathway may operate as a checkpoint that must be overcome by TGFb signaling in order for EMT to proceed. In future studies, it will be important to determine the role of PIAS1 activity in organizing the morphogenesis of epithelial tissues during development and in the control of metastasis of epithelial tumors. Because TGFb regulates a diverse set of biological responses including cell proliferation, differentiation and apoptosis, our findings suggest that PIAS1 may also influence other TGFb-regulated responses in addition to EMT. PIAS1 interacts with a number of proteins including transcrip- tional regulators and signal transducers, many of which have been identified as PIAS1 SUMO substrates [46]. The finding that TGFb triggers the downregulation of PIAS1 in cells undergoing EMT uncovers a novel mechanism that modulates PIAS1 actions. The increase in polyubiquitinated PIAS1 conjugates in cells undergoing EMT suggests that TGFb signaling may lead to alteration in the expression or activity of components of the ubiquitin machinery that ultimately may regulate PIAS1 in these cells. It will be important in future studies to identify the ubiquitin ligases or deubiquitinases that may couple TGFb signaling to the control of PIAS1 ubiquitination and consequent degradation. An important question raised by our study is how PIAS1- induced sumoylation promotes the ability of SnoN to antagonize TGFb-induced EMT. Sumoylation impacts the subcellular localization, stability, or transcriptional activity of protein substrates. However, sumoylation does not appear to affect the stability or subcellular localization of SnoN [38]. Therefore, it will be important in future studies to determine the molecular basis underlying the ability of sumoylation of SnoN to inhibit EMT. In summary, our study defines an important and novel role for the SUMO E3 ligase PIAS1 as a suppressor of EMT. In particular, our data indicate that SnoN, a PIAS1 SUMO ligase substrate, might contribute to the ability of PIAS1 to inhibit EMT. On the other hand, TGFb inhibits the PIAS1-SnoN sumoylation pathway in cells undergoing EMT. As EMT is critical for proper development and in the progression of cancer, it will be important in future studies to identify the role of PIAS1 and sumoylated SnoN in these biological processes. PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e13971