TY - JOUR
AU - Kuo, Hung-Chih
AB - Abstract The trans-spliced noncoding RNA RMST (tsRMST) is an emerging regulatory lncRNA in the human pluripotency circuit. Previously, we found that tsRMST represses lineage-specific transcription factors through the PRC2 complex and NANOG in human pluripotent stem cells (hESCs). Here, we demonstrate that tsRMST also modulates noncanonical Wnt signaling to suppress the epithelial-to-mesenchymal transition (EMT) and in vitro differentiation of embryonic stem cells (ESCs). Our results demonstrate that disruption of tsRMST expression in hESCs results in the upregulation of WNT5A, EMT, and lineage-specific genes/markers. Furthermore, we found that the PKC inhibitors Go6983 and Go6976 inhibited the effects of WNT5A, indicating that WNT5A promotes the EMT and in vitro differentiation although conventional and novel PKC activation in hESCs. Finally, we showed that either antiserum neutralization of WNT5A or Go6983 treatment in tsRMST knockdown cells decreased the expression of mesenchymal and lineage-specific markers. Together, these findings indicate that tsRMST regulates Wnt and EMT signaling pathways in hESCs by repressing WNT5A, which is a potential EMT inducer for promoting in vitro differentiation of hESCs through PKC activation. Our findings provide further insights into the role of trans-spliced RNA and WNT5A in hESC differentiation, in which EMT plays an important role. In hESCs, tsRMST forms a complex with NANOG and SUZ12 to repress noncanonical Wnt ligand WNT5A. When the tsRMST complex is downregulated, differentiation-related transcription factors are activated to promote differentiation, and activated WNT5A further strengthens differentiation via EMT through activating the PKC cascade. Open in new tabDownload slide Open in new tabDownload slide Trans-spliced RNA, Pluripotency, WNT5A, EMT Significance Statement Although trans-splicing events have been described for various species, their functions remain unclear. Here, our study demonstrate that tsRMST contributes to the regulation of early lineage differentiation of hESCs through other mechanism involved WNT5A-mediated EMT. These findings of tsNCRMS are particular interesting as it is the first reported trans-spliced lincRNA that has function associated with noncanonical WNT regulation, EMT and hESC differentiation. These results therefore illustrate the functional importance of trans-splicing, even though it is a small class of post-transcriptional events. Introduction Human embryonic stem cells (hESCs) were derived from the pluripotent inner cell mass of preimplantation human blastocysts generated through in vitro fertilization. They are capable of unlimited self-renewal, and give rise to cell types representing all three embryonic germ layers and germline lineages in vitro [1]. Accordingly, elucidation of the cellular and molecular mechanisms that control pluripotency and self-renewal in hESCs would contribute to the understanding of mechanisms underlying early lineage specification. Functional analysis of the transcription factors (TFs) Oct4 [2], Nanog [3, 4], and Sox2 [5], as well as their downstream effectors, revealed that they are essential for pluripotency maintenance in embryonic stem cells (ESCs). In addition to the intrinsic TFs, it was also demonstrated that various signal transduction pathways, such as FGF, IGF, TGF/Nodal/activin, and WNT, contribute to the maintenance of the undifferentiated status in hESCs through collaborative activities between them and their downstream target genes [6]. These findings reveal that the specialized regulatory network in ESCs may contribute to pluripotency and self-renewal by activating molecular pathways required for self-renewal and repressing genes required for differentiation. Noncoding RNAs, including small noncoding RNAs (<200 nucleotides) and long noncoding RNAs (>200 nucleotides), form a group of RNAs with little or no protein coding potential. Accumulating evidence has revealed that they are important players in the regulation of various biological processes. For example, microRNA (miRNA), a type of small noncoding RNA, can directly regulate the expression of their target genes via RNA-induced silencing complex-mediated mRNA decay [7]. Conversely, long noncoding RNA (lncRNAs), which are spliced and processed in a similar manner to their coding counterparts, can regulate gene expression by diverse routes. For example, they can regulate gene expression either in cis [8] or in trans [9–11] in the nucleus. Furthermore, they can also act as a competitor [12] or reservoir [13] of miRNAs to indirectly regulate gene expression in the cytosol. The importance of lncRNAs in regulating pluripotency and early lineage differentiation has started to become apparent. Wang et al. demonstrated that lncRNA-ROR contributes to pluripotency maintenance by stabilizing the pluripotency master genes OCT4, NANOG, and SOX2 through inhibition of miR-145 in hESCs [12]; Ng et al. showed that lncRNA-N1 and lncRNA-N3 contribute to the suppression of neural lineage differentiation through interaction with components of the PRC2 complex [14]. It has also been reported that lncRNA-ROR, lncRNA-SFMBT2, and lncRNA-VLDLR may be part of the machinery of pluripotency reprogramming [15]. Trans-splicing, which joins exons from separate pre-mRNAs, is one means by which alternative splicing can generate diverse transcripts from a limited number of genes. The most well-known trans-splicing event is spliced leader splicing, which joins different genes with the same 5′ cap and leader sequences, in unicellular organisms, nematodes, and trypanosomes [16–19]. In higher eukaryotes, two well-studied examples of trans-splicing genes, mod (mdg4) and lola, play roles in apoptosis and axon guidance in Drosophila, respectively [20, 21]. In human, the trans-splicing events JAZF1-JJAZ1 and SLC45A3-ELK4 can be translated into proteins associated with antiapoptotic effects and prostate cancer, respectively [22, 23]. Although trans-splicing events have been described for various species, their functions remain unclear. Recently, we demonstrated that tsRMST, a trans-spliced lncRNA, is highly expressed in hESCs and downregulated during in vitro differentiation. Further, gain- and loss-of-function studies revealed that tsRMST contributes to pluripotency maintenance in hESCs. Further mechanistic analysis demonstrated that tsRMST exerts its function through association with the PRC2 complex and NANOG to repress somatic lineage-related transcription factors [24]. However, it remains unknown whether tsRMST participates in other processes that regulate the pluripotency machinery. In this study, we first explored downstream signaling pathways of tsRMST by microarray and Ingenuity pathway analysis (IPA), which highlighted the repressive role of tsRMST on the noncanonical WNT signaling and EMT pathways. We then confirmed that tsRMST forms a repressive complex with the PRC2 complex component SUZ12 to suppress noncanonical WNT5A expression. Furthermore, we demonstrated that WNT5A promotes the epithelial-to-mesenchymal transition (EMT) process in differentiating hESCs, and promotes EMT and in vitro differentiation through PKCs. Together, our results reveal the important role of trans-spliced lncRNA in regulating the machinery associated with the in vitro differentiation of hESCs. Materials and Methods Cell Culture and Treatment Mouse embryonic fibroblasts (MEFs) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), × 1 nonessential amino acids (NEAA, Invitrogen), 2 mM L-glutamine (Invitrogen), and ×1 penicillin/streptomycin (Invitrogen). Human ESC H9 (WiCell Bank, WI, U.S.) was grown on MEF feeders (2 × 104 cells/cm2) in DMEM/F12 media plus 20% Knockout Serum Replacement (Invitrogen) and 4 ng/ml bFGF (Sigma-Aldrich). For WNT5A/B treatment of hESCs, hESCs were incubated in cultured medium containing 100 ng/ml recombinant human WNT5A or WNT5B (R&D) for 3 days, and then harvested for analysis. For PKC inhibition, hESCs were incubated in freshly prepared media containing 10 nM Go6983 or Go6976 (Sigma), which was exchanged daily. Lentivirus-Mediated Gene Expression and Short Hairpin RNA Knockdown The tsRMST and NANOG transcripts were cloned from the hESC H9 cDNA library, and subcloned into lentiviral plasmid FUW for expression. The lentiviral plasmid pLKO_1 (U6p-shRNA) was obtained from the National RNAi Core Facility (Taipei, Taiwan), and construction of a tsRMST-targeted shRNA was performed according to a protocol provided by the same facility, and following the guidelines of the National Institutes of Health. Targeting sequences are listed in Supporting Information Table S4. RNA Extraction and qRT-PCR Total RNA isolated using TRI Reagent (Applied Biosystems) was treated with DNase I (NEB) to remove genomic DNA contamination, and then reverse transcribed using SuperScript III transcriptase (Life technologies) to generate a cDNA library. The qRT-PCR assays were performed using the KAPA SYBR Fast Kit (KAPA Biosystems). All primers used are listed in Supporting Information Table S4. All qRT-PCR reactions were performed in biological triplicate. Microarray and IPA Analysis Total RNA (10 μg) purified by TRI Reagent (Applied Biosystems) was used to generate biotin-labeled cRNA probes, which were then hybridized to an Affymetrix Human Genome Plus 2.0 Array (Affymetrix). Probe signal intensities were detected using an Affymetrix GeneChip Scanner 7G, and analyzed using GeneSpring XI software (Agilent). The raw microarray data were deposited in the GEO with accession number GSE32503. Genes with a fold-change > 2+/−2 and a p value < .05 were applied to ingenuity pathway analysis with default settings (QIAGEN). Immunoblotting Western blotting was performed as previously described [25] and separate blots were used for individual antibodies. The signal intensity was measured by ImageJ and normalized to background on the same blot. The primary antibodies used in study are listed in Supporting Information Table S3. The secondary antibodies were as follows: HRP-donkey anti-mouse (1:6000, Sigma) and HRP-goat anti rabbit (1:6000, Sigma). Signals were detected and recorded with an ECL detection system (Thermo) and a luminescent image analyzer (LAS-4000 mini, Fujifilm). Alkaline Phosphatase and Immunocytochemical Staining Alkaline phosphatase (AP) staining was performed using the vector blue Alkaline Phosphatase substrate kit (Vector laboratories) according to the manufacturer's instructions. For immunocytochemical staining, cells were cultured on cover slides, fixed in 4% paraformaldehyde (PFA), and permeabilized with 0.05% TX-100. After blocking with 2% donkey serum (Invitrogen), cells were incubated with the appropriate primary antibodies (listed in Supporting Information Table S3). Cells were washed with PBST, and then incubated with the corresponding fluorescein-conjugated secondary antibodies. Stained samples were mounted using Vector shield H-2000 mounting media, and images were captured using a fluorescence microscope (Leica). ChIP-qPCR H9 hESCs and tsRMST knockdown H9 were treated with 1% (v/v) formaldehyde for 8 min at room temperature, and the cross-linking reaction was then quenched with 125 mM glycine. Chromatin was sonicated to an average size of 100–150 bp. Each reaction contained 5 × 105 cells and 2 μg antibody. The isolated DNA was purified using a QIAquick PCR purification kit (Qiagen). The purified DNA was quantified by qPCR using SYBR® FAST 2X qPCR Master Mix (KAPA) and an ABI PRISM© 7900 Sequence Detection System (Applied Biosystems), with H9 hESC DNA as standard. Enrichment (% Input) was determined as ChIP DNA/ input DNA × 100%. Primers are listed in Supporting Information Table S4. Primers for qPCR were confirmed to be specific by the presence of a single band on a 2% agarose gel following electrophoresis, and of a single peak in the dissociation curve. Each experiment was repeated at least three times, with each replicate producing a similar pattern. The results are expressed as means ± SE of three qPCR triplicates. Promoter Reporter Assay WNT5A and WNT5B promoters ( ± 0.5 Kb from transcription start site) or promoters lacking NANOG binding regions were cloned into the pGL3 vector (Promega) to generate reporter plasmids, as shown in Supporting Information Fig. S2. pGal vector containing the β-galactosidase gene was used as an internal standard to normalize transfection efficiency. Reporter plasmid (0.5 μg) and pGal (0.1 μg) were cotransfected into Hela cells with Lipofetamine 2000 (Invitrogen), and luciferase activity was examined at 2 days after transfection using the Luciferase assay system (Promega). Luminescence was measured using a Luminescence Counter (PerkinElmer). The activity of β-galactosidase was examined using the β-galactosidase Enzyme Assay System (E2000, Promega) and signals were measured by TopCount NXT Microplate Scintillation. Relative luciferase units were calculated as the activity of luciferase/β-galactosidase. Results Disruption of tsRMST Expression Activates WNT Signaling and EMT Pathways in hESCs To determine whether or not tsRMST plays a role in pluripotency maintenance by targeting signaling pathways in hESCs, we compared the gene expression profiles of hESCs with and without tsRMST knockdown at 2 days post-transfection. Microarray analysis and IPA revealed a panel of tsRMST-repressed genes (fold change > 4) in tsRMST knockdown hESCs (H9-shTS2 hESCs) (Supporting Information Table S1). Notably, the majority of the tsRMST-repressed genes were annotated as being part of pluripotency related networks by IPA (Fig. 1A). Of these networks, Wnt signaling and EMT pathways were shown to be the most significantly repressed by tsRMST (Fig. 1A and Supporting Information Table S2). The majority of tsRMST-repressed genes in the Wnt signaling pathway are signaling ligands, including WNT5A, WNT5B (noncanonical Wnt agonists), TGFB2 (TGF agonist), DKK1, and DKK2 (Wnt antagonists) (Fig. 1B); in EMT, these genes include those encoding the core transcription factors of EMT (SANI2, TWIST1, ZEB1) and the signaling ligands TGFB2, WNT5A, and WNT5B (Fig. 1B). Further qRT-PCR analysis confirmed that the expression levels of WNT5A, WNT5B, TWIST1, and SNAI2 were significantly increased in H9-shTS2 hESCs (fold change > 10), whereas levels of TGFB2, ZEB1, DKK1, and DKK2 were only slightly increased as compared to those in control hESCs (Fig. 1C, 1D). To exclude the possibility of off-target effects of shTS2, we rescued H9-shTS2 ESCs by overexpressing tsRMST (Supporting Information Fig. S1A): the expression levels of WNT5A, WNT5B, TWIST1, and SNAI2 were significantly downregulated in tsRMST-rescued hESCs (shTS2-res) (Supporting Information Fig. S1B, S1C). The upregulation of SNAI2, TWIST1, WNT5A, and WNT5B expression in H9-shTS2 hESCs was also confirmed by Western blotting analysis (Fig. 1E). ICC staining demonstrated that SNAI2+, TWIST1+, and WNT5A+ cells were increased in H9-shTS2 hESCs. The enhanced expression of SNAI2 and TWIST1 in H9-shTS2 hESCs suggested activation of EMT; we thus confirmed that CDH2+ cells were increased and CDH1+ cells were decreased in H9-shTS2 hESCs by Western blotting and ICC staining (Fig. 1F). Collectively, these results suggest that tsRMST suppresses the expression of genes involved in the noncanonical WNT and EMT pathways in hESCs. Figure 1 Open in new tabDownload slide Disruption of tsRMST expression activated WNT signaling and epithelial-to-mesenchymal transition (EMT) pathways in human embryonic stem cells (hESCs). (A): Ingenuity canonical pathway analysis of genes repressed by tsRMST (Fold change > 4). p value was calculated by Fisher's exact test. (B): Heatmap analysis was performed to show the fold change of genes activated in H9-shTS2 cells. H9 cells transfected with shLuc were used as a control (H9-shLuc). (C, D) qRT-PCR analysis of the expression of (C): noncanonical Wnt agonists (WNT5A, WNT5B), Wnt antagonists (DKK1, DKK2), TGFB2, (D): epithelial markers (CDH1), and mesenchymal cell markers (CDH2, ZEB1, ZEB2, TWIST1, SNAI1, SNAI2, and VIM) in H9 hESCs at 48 hours after shTS2 transfection. Error bars represent the standard deviation; p-values were obtained by 2-tailed independent t-test (*, p < .05; **, p < .01; ***, p < .001). (E): Immunoblotting detection of WNT5A, WNT5B, CDH2, TWIST1, and SNAI2 proteins in H9-shTS2 cells. (F): Immunocytochemical detection of NANOG, TWIST1, SNAI2, CDH2, WNT5A, and CDH1 expression in H9-shLuc and H9-shTS2 cells (bar: 20 μm) Figure 1 Open in new tabDownload slide Disruption of tsRMST expression activated WNT signaling and epithelial-to-mesenchymal transition (EMT) pathways in human embryonic stem cells (hESCs). (A): Ingenuity canonical pathway analysis of genes repressed by tsRMST (Fold change > 4). p value was calculated by Fisher's exact test. (B): Heatmap analysis was performed to show the fold change of genes activated in H9-shTS2 cells. H9 cells transfected with shLuc were used as a control (H9-shLuc). (C, D) qRT-PCR analysis of the expression of (C): noncanonical Wnt agonists (WNT5A, WNT5B), Wnt antagonists (DKK1, DKK2), TGFB2, (D): epithelial markers (CDH1), and mesenchymal cell markers (CDH2, ZEB1, ZEB2, TWIST1, SNAI1, SNAI2, and VIM) in H9 hESCs at 48 hours after shTS2 transfection. Error bars represent the standard deviation; p-values were obtained by 2-tailed independent t-test (*, p < .05; **, p < .01; ***, p < .001). (E): Immunoblotting detection of WNT5A, WNT5B, CDH2, TWIST1, and SNAI2 proteins in H9-shTS2 cells. (F): Immunocytochemical detection of NANOG, TWIST1, SNAI2, CDH2, WNT5A, and CDH1 expression in H9-shLuc and H9-shTS2 cells (bar: 20 μm) TsRMST Suppresses WNT5A and WNT5B Expression through Association with the PRC2 Component SUZ12 Previously, we showed that tsRMST repressed gene expression through association with pluripotency-associated transcription factors and a PRC2 component, and subsequent tri-methylation of H3K27 on the promoter regions of target genes [24]. To determine whether WNT5A, WNT5B, SNAI2, and TWIST1 are directly repressed by tsRMST, we first examined whether the promoter regions of these genes are occupied by NANOG and SUZ12 and the H3K27me3 modification. Based on the Encode ChIP-seq data [26], we found that the promoters of WNT5A and WNT5B were modified by H3K27me3 and occupied by NANOG and SUZ12 (Fig. 2A, 2B), thereby meeting the characteristics expected for tsRMST-regulated genes. This observation is consistent with our finding that WNT5A and WNT5B are not expressed in hESCs (Fig. 1E, 1F). Furthermore, ChIP-qPCR experiments confirmed that knockdown of tsRMST decreased NANOG and SUZ12 occupancy and H3K27me3 modification on the promoters of WNT5A and WNT5B (Fig. 2A, 2B). To determine if NANOG directly regulates WNT5A and WNT5B, we performed promoter reporter assays using luciferase reporters containing WNT5A or WNT5B promoters. We found that coexpression of tsRMST and NANOG greatly reduced the luciferase activity of reporters containing WNT5A or WNT5B promoters in Hela cells, but not mutant reporter lacking NANOG binding sites (Supporting Information Fig. S2). Unlike WNT5A and WNT5B, the SNAI2 and TWIST1 promoters exhibited low H3K27me3, suggesting that SNAI2 and TWIST1 may not be repressed by the PRC2 complex in hESCs (Fig. 2C). Consistent with the low levels of H3K27me3 modification at these promoters, low expression of TWIST1 was observed in hESCs, whereas SNAI2 expression was absent (Fig. 1E, 1F). SNAI2 may be post-transcriptionally repressed in hESCs (for example, by microRNAs). Thus, the above results suggest that tsRMST is directly targeted to WNT5A and WNT5B; tsRMST most likely modulates the expression of SNAI2 and TWIST1 through an indirect regulatory mechanism. Figure 2 Open in new tabDownload slide tsRMST suppresses WNT5A and WNT5B expression through association with PRC2 component SUZ12. ChIP-qPCR analysis of the H3K27me3 modification and the occupancy of NANOG and SUZ12 on the promoters of genes repressed by tsRMST: (A): WNT5A (Chr3:55499743-55515426), (B): WNT5B (Chr12:1739978-1756378), (C): SNAI2 (Chr8:49830239-49833999) and TWIST1 (Chr7:19155091-19157295). ENCODE ChIP-Seq data of NANOG and SUZ12 occupancy and the H3K27me3 modification were aligned to the promoter regions, as indicated by the yellow bars. The promoter regions were defined as the region from −0.5 to + 0.5 kb of the transcription start sites. For each figure, the y-axis represents the intensity of ChIP-Seq reads. The highest NANOG binding peaks on the promoter regions of WNT5A and WNT5B are shown as red horizontal bars (WNT5A: Chr3:55514895-55515679; WNT5B: Chr12:1740006-1740815). ChIP fragments containing the highest NANOG binding peak (labeled as 0) or its four flanking regions (labeled as −1, −2, 1, and 2, which are located within −0.5 to + 0.5 kb of position 0 [shown as yellow bars]) in shLuc- and shTS2-transduced hESCs were quantified by qPCR and normalized with input genome used in ChIP. Error bars represent the standard deviation, and p-values were obtained by 2-tailed independent t-tests. (*, p < .05; **, p < .01; ***, p < .001). Figure 2 Open in new tabDownload slide tsRMST suppresses WNT5A and WNT5B expression through association with PRC2 component SUZ12. ChIP-qPCR analysis of the H3K27me3 modification and the occupancy of NANOG and SUZ12 on the promoters of genes repressed by tsRMST: (A): WNT5A (Chr3:55499743-55515426), (B): WNT5B (Chr12:1739978-1756378), (C): SNAI2 (Chr8:49830239-49833999) and TWIST1 (Chr7:19155091-19157295). ENCODE ChIP-Seq data of NANOG and SUZ12 occupancy and the H3K27me3 modification were aligned to the promoter regions, as indicated by the yellow bars. The promoter regions were defined as the region from −0.5 to + 0.5 kb of the transcription start sites. For each figure, the y-axis represents the intensity of ChIP-Seq reads. The highest NANOG binding peaks on the promoter regions of WNT5A and WNT5B are shown as red horizontal bars (WNT5A: Chr3:55514895-55515679; WNT5B: Chr12:1740006-1740815). ChIP fragments containing the highest NANOG binding peak (labeled as 0) or its four flanking regions (labeled as −1, −2, 1, and 2, which are located within −0.5 to + 0.5 kb of position 0 [shown as yellow bars]) in shLuc- and shTS2-transduced hESCs were quantified by qPCR and normalized with input genome used in ChIP. Error bars represent the standard deviation, and p-values were obtained by 2-tailed independent t-tests. (*, p < .05; **, p < .01; ***, p < .001). WNT5A Promotes EMT and In Vitro Differentiation of hESCs Since the above results indicate that tsRMST can directly suppress WNT5A and WNT5B expression, it is possible that tsRMST exerts its function through the WNTs. To test this hypothesis, we first examined the functional role of WNT5A/5B in hESCs. To this end, we treated hESCs with recombinant WNT5A or WNT5B proteins and subjected the WNT5A/5B-treated hESCs to various assays. AP staining showed that treatment with WNT5A, but not WNT5B, resulted in loss of AP expression in hESCs (Fig. 3A). Next, qRT-PCR and ICC analysis were performed to reveal that WNT5A treatment decreased expression of pluripotent genes (OCT4 and NANOG) but increased the expression of differentiation markers (Brachyury, MIXL1, GATA4, and SOX17) (Fig. 3B, 3E). In contrast, WNT5B treatment has only minor effects on the expression of pluripotent markers and did not induce differentiation marker expression in hESCs (Fig. 3B, 3E). Furthermore, the expression of mesenchyme-related genes, including SNAI1, SNAI2, TWIST1, ZEB1, ZEB2, CDH2, and VIM were all upregulated, whereas the epithelium-associated gene, CDH1, was down-regulated in hESCs treated with WNT5A, but not with WNT5B (Fig. 3C). Finally, data derived from Western blot and ICC analysis showed that WNT5A treatment also promoted the expression of CDH2 and SNAI2 and repressed that of CDH1 in hESCs (Fig. 3D, 3E). Together, these results show that treatment with WNT5A, but not WNT5B, upregulated EMT-related genes and promoted in vitro differentiation of hESCs, which resembles the effects of tsRMST knockdown in hESCs. Figure 3 Open in new tabDownload slide WNT5A promotes epithelial-to-mesenchymal transition (EMT) and in vitro differentiation of human embryonic stem cells (hESCs). Human ESCs H9 were treated with 100 ng/ml WNT5A or WNT5B proteins for 3 days and subjected to (A): Alkaline phosphatase staining (Scale bar: 200 μm) and (B): qRT-PCR analysis of pluripotent genes (NANOG and OCT4), differentiation-related genes (Brachyury, MIXL1, GATA4, SOX17, PAX6, and SOX1), (C): mesenchymal markers (SNAI1, SNAI2, TWIST1, ZEB1, ZEB2, CDH2, and VIM), and epithelial markers (CDH1). Error bars represent the standard deviation, and p-values were obtained by 2-tailed independent t-tests. (*, p < .05; **, p < .01; ***, p < .001). (D): Immunoblotting detection of CDH2 and SNAI2 expression in hESCs treated with WNT5A or WNT5B ligands. (E): Immunocytochemical detection of the protein products of pluripotency genes (NANOG and OCT4), differentiation-related genes (Brachyury, GATA4, and PAX6), and EMT-related genes (CDH1, CDH2, and SNAI2) in hESCs treated with WNT5A or WNT5B (scale bar: 20 μm). Figure 3 Open in new tabDownload slide WNT5A promotes epithelial-to-mesenchymal transition (EMT) and in vitro differentiation of human embryonic stem cells (hESCs). Human ESCs H9 were treated with 100 ng/ml WNT5A or WNT5B proteins for 3 days and subjected to (A): Alkaline phosphatase staining (Scale bar: 200 μm) and (B): qRT-PCR analysis of pluripotent genes (NANOG and OCT4), differentiation-related genes (Brachyury, MIXL1, GATA4, SOX17, PAX6, and SOX1), (C): mesenchymal markers (SNAI1, SNAI2, TWIST1, ZEB1, ZEB2, CDH2, and VIM), and epithelial markers (CDH1). Error bars represent the standard deviation, and p-values were obtained by 2-tailed independent t-tests. (*, p < .05; **, p < .01; ***, p < .001). (D): Immunoblotting detection of CDH2 and SNAI2 expression in hESCs treated with WNT5A or WNT5B ligands. (E): Immunocytochemical detection of the protein products of pluripotency genes (NANOG and OCT4), differentiation-related genes (Brachyury, GATA4, and PAX6), and EMT-related genes (CDH1, CDH2, and SNAI2) in hESCs treated with WNT5A or WNT5B (scale bar: 20 μm). WNT5A Promotes EMT and Differentiation via Novel and Conventional PKC Activation in hESCs As a noncanonical Wnt ligand, WNT5A transduces signals in a context-dependent manner. For instance, WNT5A can regulate planar cell polarity (PCP) through RAC-JNK activation, or activate phospholipase C (PLC) to activate both novel and conventional PKCs, or inhibit β-catenin/TCF by activation of CaMKII-NLK or Calcineurin-NFAT cascades (Fig. 4A). To identify which of the above signaling cascades was activated by WNT5A in hESCs, we treated hESCs with WNT5A and used Western blot to determine which of the above cascades was activated. We found that phosphorylated JNK, CaMKII, and β-catenin were not increased by WNT5A treatment of hESCs (Fig. 4B), whereas phosphorylated novel PKC (PKC-σ) and conventional PKC (PKCα) were increased (Fig. 4C). This suggests that WNT5A may exert its functional effects through the PLC-PKC axis. To further confirm that WNT5A induces differentiation and EMT in hESCs through PKC activation, we treated WNT5A-treated hESCs with pan-PKC inhibitor Go6983 or conventional PKC inhibitor Go6976 (see the Materials and Methods section). Western blotting analysis revealed that Go6983 can efficiently block the activation of both novel and conventional PKCs, whereas Go6976 can only block conventional PKCs (Fig. 4D). Furthermore, our qRT-PCR analysis demonstrated that the expression levels of mesenchymal markers (SNAI2, ZEB1, ZEB2, TWIST1, CDH2, and VIM) and differentiation markers (Brachyury, MIXL1, GATA4, and SOX17) were decreased by treatment of WNT5A-treated hESCs with either Go6976 or Go6983. Conversely, the expression levels of the pluripotent markers NANOG and OCT4 and epithelial marker CDH1 were increased (Fig. 4E). Together, these results suggest that WNT5A may promote the EMT process and in vitro differentiation of hESCs through conventional and novel PKCs. Figure 4 Open in new tabDownload slide WNT5A promotes epithelial-to-mesenchymal transition (EMT) and differentiation via novel and conventional PKC activation in human embryonic stem cells (hESCs). (A): Diagram showing major signaling pathways activated by noncanonical Wnt ligands. (1) Planar cell polarity activated by RAC-JNK cascade; PLC-β downstream signaling of (2) DAG-dependent novel PKCs, and (3) DAG and Ca2+-dependent classical PKCs; (4) Ca2+-dependent CaMKII-NLK cascade; (5) Calcineurin-NFATdependent β-catenin. (B): Immunoblotting detection of WNT5A-induced phosphorylation of JNK (1), CaMKII (4), β-catenin (5), (C): novel PKC (PKC-δ) (2), and conventional PKC (PKC-α) (3). Results were quantified from three independent treatments. (D): The inhibitory effects of 10 nM conventional PKC inhibitor Go6976 and 10 nM pan-PKC inhibitor Go6983 on PKC-δ and PKC-α. (E): qRT-PCR analysis of differentiation-related genes (Brachyury, MIXL1, GATA4, SOX17, PAX6, and SOX1) and mesenchymal cell markers (SNAI1, SNAI2, TWIST1, ZEB1, ZEB2, CDH2, and VIM) in WNT5A-treated hESCs treated with Go6976 or Go6983. Error bars represent the standard deviation, and p-values were obtained by two-tailed independent t-test (*, p < .05; **, p < .01; ***, p < .001). Figure 4 Open in new tabDownload slide WNT5A promotes epithelial-to-mesenchymal transition (EMT) and differentiation via novel and conventional PKC activation in human embryonic stem cells (hESCs). (A): Diagram showing major signaling pathways activated by noncanonical Wnt ligands. (1) Planar cell polarity activated by RAC-JNK cascade; PLC-β downstream signaling of (2) DAG-dependent novel PKCs, and (3) DAG and Ca2+-dependent classical PKCs; (4) Ca2+-dependent CaMKII-NLK cascade; (5) Calcineurin-NFATdependent β-catenin. (B): Immunoblotting detection of WNT5A-induced phosphorylation of JNK (1), CaMKII (4), β-catenin (5), (C): novel PKC (PKC-δ) (2), and conventional PKC (PKC-α) (3). Results were quantified from three independent treatments. (D): The inhibitory effects of 10 nM conventional PKC inhibitor Go6976 and 10 nM pan-PKC inhibitor Go6983 on PKC-δ and PKC-α. (E): qRT-PCR analysis of differentiation-related genes (Brachyury, MIXL1, GATA4, SOX17, PAX6, and SOX1) and mesenchymal cell markers (SNAI1, SNAI2, TWIST1, ZEB1, ZEB2, CDH2, and VIM) in WNT5A-treated hESCs treated with Go6976 or Go6983. Error bars represent the standard deviation, and p-values were obtained by two-tailed independent t-test (*, p < .05; **, p < .01; ***, p < .001). tsRMST Represses the WNT5A-PKC Signaling Cascade to Prevent EMT and hESC Differentiation To examine whether tsRMST prevents hESC differentiation through inhibiting WNT5A, we treated tsRMST knockdown hESCs (H9-shTS2) with WNT5A antiserum [27] to neutralize secreted WNT5A ligand in H9-shTS2 culture medium. As revealed by Western blotting analysis, neutralization of WNT5A efficiently blocked PKC-α and PKC-δ activation in H9-shTS2 hESCs (Fig. 5A). Further, the qRT-PCR data indicated that treatment with WNT5A antiserum decreased the expression levels of lineage-specific genes, including Brachyury, MIXL1, GATA4, and SOX17, and EMT-related genes, including SNAI1, SNAI2, TWIST1, ZEB1, CDH2, and VIM, in H9-shTS2 hESCs (Fig. 5B, 5C). Of note, the expression levels of the pluripotent genes NANOG and OCT4 were increased in the WNT5A-neutralized H9-shTS2 cells (Fig. 5B). To further confirm that the WNT5A-PKC signaling cascade is downstream of tsRMST, we treated H9-shTS2 hESC with pan-PKC inhibitor Go6983. Our qRT-PCR analysis showed that inhibition of PKC down-regulated the expression of lineage-specific and EMT-related genes (Fig. 5D, 5E). However, the pan-PKC inhibitor could not rescue expression of the pluripotent markers OCT4 and NANOG (Fig. 5D). Thus, these results suggest that tsRMST may impede in vitro differentiation and EMT in hESCs by suppressing the WNT5A-PKC cascade. Figure 5 Open in new tabDownload slide TsRMST represses the WNT5A-PKC signaling cascade to prevent EMT and human embryonic stem cells (hESC) differentiation. (A): Immunoblotting was performed to detect PKC-α and PK·-δ activation in tsRMST knockdown hESCs with or without WNT5A neutralization. (B, C) qRT-PCR was performed to show the effects of WNT5A neutralization on the expression of (B): pluripotency-related genes (NANOG and OCT4), differentiationrelated genes (Brachyury, MIXL1, GATA4, SOX17, PAX6, and SOX1), and (C): EMT-related genes (SNAI1, SNAI2, ZEB, ZEB2, TWIST1, CDH2, and VIM). (D, E) qRT-PCR was performed to show the effect of pan-PKC inhibition on the expression of (D): pluripotency-related genes, differentiation-related genes, and (E) EMT-related genes. Error bars represent the standard deviation, and p-values were obtained by 2-tailed independent t-tests. (*, p < .05; **, p < .01; ***, p < .001). Figure 5 Open in new tabDownload slide TsRMST represses the WNT5A-PKC signaling cascade to prevent EMT and human embryonic stem cells (hESC) differentiation. (A): Immunoblotting was performed to detect PKC-α and PK·-δ activation in tsRMST knockdown hESCs with or without WNT5A neutralization. (B, C) qRT-PCR was performed to show the effects of WNT5A neutralization on the expression of (B): pluripotency-related genes (NANOG and OCT4), differentiationrelated genes (Brachyury, MIXL1, GATA4, SOX17, PAX6, and SOX1), and (C): EMT-related genes (SNAI1, SNAI2, ZEB, ZEB2, TWIST1, CDH2, and VIM). (D, E) qRT-PCR was performed to show the effect of pan-PKC inhibition on the expression of (D): pluripotency-related genes, differentiation-related genes, and (E) EMT-related genes. Error bars represent the standard deviation, and p-values were obtained by 2-tailed independent t-tests. (*, p < .05; **, p < .01; ***, p < .001). Discussion We previously demonstrated that lncRNA-tsRMST forms a repressive complex with NANOG and the PRC2 complex to promote pluripotency maintenance through repressing the transcriptional activation of lineage-specific genes [24]. By performing global gene expression profiling and IPA signaling pathway analysis of hESCs with altered tsRMST expression, we show here that disruption of tsRMST expression in hESCs lead to upregulation of components of the noncanonical WNT and EMT pathways, which promotes in vitro differentiation of hESCs. Furthermore, we demonstrated that treatment with noncanonical WNT5A promotes EMT and in vitro differentiation of hESCs. We provide evidence that WNT5A activates noncanonical Wnt signaling through PKC-α and PKC-δ phosphorylation and subsequently promotes EMT and lineage differentiation during in vitro differentiation of hESCs. Finally, we observed that WNT5A neutralization blocked PKC activation and decreased EMT and lineage specific gene expression in tsRMST knockdown hESCs, indicating that tsRMST prevents hESC differentiation through inhibiting WNT5A. Collectively, these results suggest that tsRMST contributes to the regulation of pluripotency and early lineage differentiation of hESCs through other mechanisms in addition to direct transcriptional regulation of the lineage-specific genes. Therefore, we have identified tsRMST as a key factor for regulating the interplay between intrinsic and extrinsic mechanisms required for the in vitro differentiation of hESCs. The functional roles of Wnt5a in various biological systems have been well documented, and this protein is known to play an important role in regulating cellular functions, including migration, adhesion, and differentiation during developmental processes [28, 29]. Nevertheless, our understanding of the function of Wnt5a signaling in hESCs remains elusive. In this study, our findings that WNT5A treatment does not promote the self-renewal of hESCs suggest that WNT5A does not play a supporting role in pluripotency maintenance and self-renewal of hESCs. This result is consistent with previous reports that WNT5A does not support the proliferation or pluripotency of hESC [30]. Conversely, our data showed that WNT5A promotes EMT and lineage-specific differentiation of hESCs, and supports the hypothesis that lncRNA-tsRMST suppresses hESC differentiation through the WNT5A-mediated signaling cascade. While we demonstrated that WNT5A contributes to in vitro differentiation of hESCs, the precise route by which WNT5A regulates hESC differentiation remains to be defined. Our results raise two possibilities: (i) WNT5A may directly target and down-regulate the pluripotency regulatory circuit of hESC or (ii) it may directly activate differentiation pathways, such as the EMT pathway [31, 32] to augment the intensity of in vitro differentiation (Fig. 3). Conversely, our results showed that PKC inhibition and WNT5A neutralization suppresses in vitro differentiation of hESCs, and WNT5A neutralization further promotes pluripotent marker expression (Fig. 5B, 5D), suggesting that repression of pluripotency is likely independent of the WNT5A/PKC cascade. Given that WNT5A did not activate JNK, CaMKII, NFAT, or β-catenin in hESCs, it is tempting to suggest that an unknown WNT5A downstream signaling cascade may be responsible for direct pluripotency maintenance. Nevertheless, further experiments are required to elucidate the downstream pathway of WNT5A in pluripotency maintenance. The EMT process has been described under various hESC differentiation conditions, and its association with the differentiation of hESCs has also been confirmed [32]. EMT of hESCs involves the E-to-M morphological conversion and marker expression change. Consistent with these findings, we found that the morphological traits of ESCs changed from E to M status, and expression of epithelium genes/markers, such as CDH1, were down-regulated, whereas the mesenchymal genes/markers were upregulated after disruption of tsRMST expression or WNT5A treatment, suggesting that WNT5A signaling is likely a downstream effector of tsRMST for EMT in human pluripotent stem cells. It is well known that induction of EMT during embryonic development involves not only intrinsic transcription factors, such as Snail and Twist, but also extrinsic signaling pathways, such as TGF-β, FGF, EGF, and WNTs. Of these factors, noncanonical WNT5A's role in promoting the EMT of various cancer types [33, 34] and developmental processes [28, 35] is well established. As described, WNT5A can promote the EMT process through various downstream pathways, including the PCP pathway [36], PLC-PKC cascade [33], canonical Wnt signaling [37], and the newly discovered Fyn-Stat3 cascade [38]. In line with previous studies, our findings demonstrate that WNT5A-mediated induction of hESC differentiation depends on the WNT-PKC signaling cascade. However, further examination of phosphorylated JNK indicates that JNK, which is a well-known EMT inducer [36], is not the main effector of EMT in WNT5A-treated hESCs. This conflict may be resolved by referring to a recent report showing that PKC-α induces EMT-mediator SNAI1 expression in hESCs [32]. Our demonstration that SNAI1 is upregulated in lncRNA-tsRMST knockdown cells further supports this possibility. Since SNAI1 is a critical downstream effector of Wnt-induced mesoderm differentiation [39], it is thus tempting to suggest that the WNT5A/PKC-α/SNAI1/EMT axis may play an important role in regulating in vitro lineage differentiation of hESCs. The regulatory roles of lncRNA in signaling pathways, especially canonical Wnt signaling, have been revealed in previous studies. For instance, it has been demonstrated that lncRNA-P21 and lncRNA-mrhl can repress Wnt signaling by inhibiting β-catenin [40, 41]. In contrast, lncRNA-HOTAIR and lncRNA-LALR1 can activate Wnt signaling by repressing Wnt repressors WIF-1 and AXIN1 [42, 43]. Unlike canonical Wnt signaling, the regulatory roles of lncRNAs in noncanonical Wnt signaling remain unclear. The only lncRNA previously reported to participate in noncanonical Wnt signaling is lncRNA-MALAT1, which mediates noncanonical WNT5A-induced migration in glioblastoma [44]. Therefore, our discovery of the regulatory role of lncRNA-tsRMST in noncanonical Wnt signaling provides further insights into the functional role of lncRNA in regulating signaling pathways. Our results also show that noncanonical WNT5A, like canonical Wnts, induces mesendoderm differentiation; this suggests that WNT5A may directly activate canonical Wnts (as suggested by a study of osteogenesis in mouse [45]) or form a regulatory loop with canonical Wnts to indirectly promote mesendoderm differentiation [46]. In our study, we showed that β-catenin was not activated in hESCs treated with WNT5A, and this suggested that WNT5A likely promotes the differentiation of hESCs through the noncanonical WNT5A-PKC pathway, but not through the canonical WNT pathway. Nevertheless, our results do not exclude the possibility that tsRMST may regulate or be regulated by other external signaling factors, such as FGF and BMP, that promote mesendodermal differentiation, and future efforts are needed to explore these possibilities. In summary, our previous [24] and current findings suggest that the function of lncRNA-tsRMST may be twofold: first, degradation of lncRNA-tsRMST may initially upregulate the expression of lineage-specific transcription factors, such as GATA4, GATA6, and WNT5A, in hESCs, and second, WNT5A subsequently further enhances in vitro differentiation by promoting EMT in the already differentiated hESCs (Fig. 6). These findings underline the contribution of a trans-spliced lncRNA to human ESC pluripotency and early lineage differentiation, and suggest that trans-spliced lncRNAs may serve as an important factor for regulation of genes and signaling pathways, and as a potential target for the development of pluripotent stem cell technologies. Figure 6 Open in new tabDownload slide TsRMST represses differentiation-related transcription factors and signaling pathways to inhibit in vitro differentiation of human embryonic stem cells (hESCs). In hESCs, tsRMST forms a complex with NANOG and SUZ12 to repress the expression of differentiation-related transcription factors and noncanonical Wnt ligand WNT5A. When the tsRMST complex is downregulated (by tsRMST knockdown or differentiation), differentiation-related transcription factors are activated to promote differentiation, and activated WNT5A further strengthens differentiation via EMT through activating the PKC cascade. Figure 6 Open in new tabDownload slide TsRMST represses differentiation-related transcription factors and signaling pathways to inhibit in vitro differentiation of human embryonic stem cells (hESCs). In hESCs, tsRMST forms a complex with NANOG and SUZ12 to repress the expression of differentiation-related transcription factors and noncanonical Wnt ligand WNT5A. When the tsRMST complex is downregulated (by tsRMST knockdown or differentiation), differentiation-related transcription factors are activated to promote differentiation, and activated WNT5A further strengthens differentiation via EMT through activating the PKC cascade. Acknowledgments This work was supported by the Ministry of Science and Technology [103-2321-B-001-065] and National Health Research Institutes [NHRI-EX104-10320SI]. 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Google Scholar Crossref Search ADS WorldCat © 2016 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - The Trans-Spliced Long Noncoding RNA tsRMST Impedes Human Embryonic Stem Cell Differentiation Through WNT5A-Mediated Inhibition of the Epithelial-to-Mesenchymal Transition
JF - Stem Cells
DO - 10.1002/stem.2386
DA - 2016-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-trans-spliced-long-noncoding-rna-tsrmst-impedes-human-embryonic-fCyMimU20h
SP - 2052
EP - 2062
VL - 34
IS - 8
DP - DeepDyve
ER -