The protein tyrosine phosphatase Pez regulates TGFβ, epithelial–mesenchymal transition, and organ development

Epithelial–mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFβ signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFβ3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFβ and EMT

Identification of an enhancer that increases miR-200b~200a~429 gene expression in breast cancer cells

The miR-200b~200a~429 gene cluster is a key regulator of EMT and cancer metastasis, however the transcription-based mechanisms controlling its expression during this process are not well understood. We have analyzed the miR-200b~200a~429 locus for epigenetic modifications in breast epithelial and mesenchymal cell lines using chromatin immunoprecipitation assays and DNA methylation analysis. We discovered a novel enhancer located approximately 5.1kb upstream of the miR-200b~200a~429 transcriptional start site. This region was associated with the active enhancer chromatin signature comprising H3K4me1, H3K27ac, RNA polymerase II and CpG dinucleotide hypomethylation. Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated miR-200b eRNA. Over-expression of miR-200b eRNA had little effect on miR-200b~200a~429 promoter activity and its production did not correlate with miR-200b~200a~429 gene expression. While additional investigations of miR-200b eRNA function will be necessary, it is possible that miR-200b eRNA may be involved in the regulation of miR-200b~200a~429 gene expression and silencing. Taken together, these findings reveal the presence of a novel enhancer, which contributes to miR-200b~200a~429 transcriptional regulation in epithelial cells.Joanne L. Attema, Andrew G. Bert, Yat-Yuen Lim, Natasha Kolesnikoff, David M. Lawrence, Katherine A. Pillman, Eric Smith, Paul A. Drew, Yeesim Khew-Goodall, Frances Shannon, Gregory J. Goodal

Pyruvate carboxylase: its interactions with acetyl CoA / by Yee Sim Khew-Goodall

Bibliography: leaves 148-157viii, 157, [126] leaves : ill ; 28 cm.Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 198

The Protein Tyrosine Phosphatase Pez Is a Major Phosphatase of Adherens Junctions and Dephosphorylates β-Catenin

Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified β-catenin, a component of adherens junctions, as a substrate of Pez by a “substrate trapping” approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of β-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including β-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro “wound” assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion

A perspective on regulation of cell-cell adhesion and epithelial-mesenchymal transition: Known and novel

A number of recent reviews in the field have described many of the known growth factors and signalling pathways that may be involved in regulating epithelial-mesenchymal transitions. This perspective will focus on some aspects of posttranslational regulation of cell-cell adhesion that are less well understood and their potential role in initiating epithelial-mesenchymal transitions. In addition, a potential novel intermediate in the signalling pathway of epithelial-mesenchymal transition will also be described

Down-Regulation of the miRNA-200 Family at the Invasive Front of Colorectal Cancers with Degraded Basement Membrane Indicates EMT Is Involved in Cancer Progression

Cancer progression is a complex series of events thought to incorporate the reversible developmental process of epithelial-to-mesenchymal transition (EMT). In vitro, the microRNA-200 family maintains the epithelial phenotype by posttranscriptionally inhibiting the E-cadherin repressors, ZEB1 and ZEB2. Here, we used in situ hybridization and immunohistochemistry to assess expression of miR-200 and EMT biomarkers in formalin-fixed paraffin-embedded human colorectal adenocarcinomas. In addition, laser capture microdissection and quantitative real-time polymerase chain reaction were employed to quantify levels of miR-200 in the normal epithelium, tumor core, invasive front, and stroma. We find that miR-200 is downregulated at the invasive front of colorectal adenocarcinomas that have destroyed and invaded beyond the basement membrane. However, regional lymph node metastases and vascular carcinoma deposits show strong expression of miR-200, suggesting this family of miRNAs is involved in the recapitulation of the primary tumor phenotype at metastatic sites. In contrast, adenomas and adenocarcinomas with intact basement membranes showed uniform miR-200 expression from the tumor core to the tumor-host interface. Taken together, these data support the involvement of EMT and mesenchymal-to-epithelial transition (MET) in the metastasis cascade and show that miR-200 is downregulated in the initial stages of stromal invasion but is restored at metastatic sites