6929

TCF3

epithelial-mesenchymal transition (EMT), a process whereby renal tubular epithelial cells lose phenotype and gain fibroblast-like characteristics, has been demonstrated to contribute significantly to the development of renal fibrosis. The immunosuppressant cyclosporine A (CsA) has been shown to induce renal fibrosis, a major complication of CsA therapy. The mechanisms that drive CsA-induced fibrosis remain undefined, however, CsA has been demonstrated to induce EMT in human renal proximal tubular epithelial cells (RPTEC). E2A transcription factors were identified as being upregulated by CsA treatment. To further examine the role of E2A proteins in EMT, E12 and E47 were overexpressed, alone and in combination, in human RPTEC. Both E12 and E47 elicited EMT effects on tubular epithelial cells with E47 more potent in inducing the fibroblast-like phenotype. These results indicate the important role of the E2A gene products in the progression of CsA-induced EMT and provide novel insights into CsA-induced renal fibrosis.

E12/E47 proteins (encoded by E2A gene) are members of the class I basic helix-loop-helix (bHLH) transcription factors (also known as E proteins). E47 has been described as repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). We reported previously that EMT mediated by E47 in MDCK cells occurs with a concomitant overexpression of Id1 and Id3 proteins. Id proteins belong to class V of HLH factors that lack the basic domain; they dimerise with E proteins and prevent their DNA interaction, thus, acting as dominant negative of E proteins. Here, we show that E47 interacts with Id1 in E47 overexpressing MDCK cells that underwent a full EMT as well as in mesenchymal breast carcinoma and melanoma cell lines. By conducting chromatin immunoprecipitation assays we demonstrate that E47 binds directly to the endogenous E-cadherin promoter of mesenchymal MDCK-E47 cells in a complex devoid of Id1. Importantly, our data suggest that both E47 and Id1 are required to maintain the mesenchymal phenotype of MDCK-E47 cells. These data support the collaboration between E47 and Id1 in the maintenance of EMT by mechanisms independent of the dominant negative action of Id1 on E47 binding to E-cadherin promoter. Finally, the analysis of several N0 breast tumour series indicates that the expression of E47 and ID1 is significantly associated with the basal-like phenotype supporting the biological significance of the present findings.

The p21-activated kinase 5 (PAK5) is overexpressed in advanced cancer and the transcription factor E47 is a direct repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). However, the relationship between PAK5 and E47 has not been explored. In this study, we found that PAK5-mediated E47 phosphorylation promoted EMT in advanced colon cancer. PAK5 interacted with E47 and phosphorylated E47 on Ser39 under hepatocyte growth factor (HGF) stimulation, which decreased cell-cell cohesion, increased cell migration and invasion in vitro and promoted metastasis in a xenograft model. Furthermore, phosphorylation of E47 facilitated its accumulating in nucleus in an importin alpha-dependent manner, and enhanced E47 binding to E-cadherin promoter directly, leading to inhibition of E-cadherin transcription. In contrast, PAK5-knockdown resulted in blockage of HGF-induced E47 phosphorylation, attenuated association of E47 with importin alpha and decreased E47 binding to E-cadherin promoter. In addition, we demonstrated a close correlation between PAK5 and phospho-Ser39 E47 expression in colon cancer specimens. More importantly, high expression of phospho-E47 was associated with an aggressive phenotype of colon cancer and nuclear phospho-E47 staining was found in certain cases of colon cancer with metastasis. Collectively, E47 is a novel substrate of PAK5, and PAK5-mediated phosphorylation of E47 promotes EMT and metastasis of colon cancer, suggesting that phosphorylated E47 on Ser39 may be a potential therapeutic target in progressive colon cancer.

Several E-box-binding transcription factors regulate individual and collective cell migration and enhance the motility of epithelial cells by promoting epithelial-mesenchymal transition (EMT). Here, we characterized the role of a subset of these transcription factors and the EMT proteome in branching morphogenesis of mammary epithelial tissues using a three-dimensional organotypic culture model of the mammary duct. We found that the transcription factors Snail1, Snail2, and E47 were transiently upregulated at branch sites; decreasing the expression of these transcription factors inhibited branching. Conversely, ectopic expression of Snail1, Snail2, and E47 induced branching in the absence of exogenous stimuli. These changes correlated with the expression of mesenchymal markers and repression of E-cadherin, which was essential for branching. Snail1 and Snail2 also promoted cell survival at branch sites, but this was not sufficient to induce branching. These findings indicate that Snail1, Snail2, and E47 can promote collective migration during branching morphogenesis of mammary epithelial tissues through key regulators of EMT.

Previous reports have demonstrated that hepatitis B virus (HBV) X protein (HBx) represses E-cadherin expression to induce epithelial-mesenchymal transition (EMT), an essential component of cancer progression to more aggressive phenotypes characterized by tumour invasion, migration and metastasis; however, the underlying mechanism for this phenomenon is still unclear. In this study, we found that ectopic expression of HBx in human hepatocytes using overexpression and 1.2-mer WT HBV replicon systems upregulated levels of the transcriptional repressors E12 and E47, resulting in inactivation of the E-cadherin promoter, containing three E-box motifs, and subsequent repression of its expression. E12/E47 knockdown using a specific small interfering RNA almost completely abolished the potential of HBx to repress E-cadherin expression. HBx inhibited the ubiquitin-dependent proteasomal degradation of E12/E47 without affecting their expression at the transcriptional level. Upregulation of E12/E47 by HBx ultimately led to EMT in human hepatocytes, as demonstrated by morphological changes, altered protein levels of EMT markers, including E-cadherin, plakoglobin, fibronectin, vimentin and N-cadherin, and increased capacity for cell detachment and migration.