Epithelial to mesenchymal transition occurs during embryologic development to allow tissue remodeling and is proposed to be a key step in the metastasis of epithelial-derived tumors. The miR-200 family of microRNAs plays a major role in specifying the epithelial phenotype by preventing expression of the transcription repressors, ZEB1/DEF1 and SIP1/ZEB2. We show here that miR-200a, miR-200b, and the related miR-429 are all encoded on a 7.5-kb polycistronic primary miRNA (pri-miR) transcript. We show that the promoter for the pri-miR is located within a 300-bp segment located 4 kb upstream of miR-200b. This promoter region is sufficient to confer expression in epithelial cells and is repressed in mesenchymal cells by ZEB1 and SIP1 through their binding to a conserved pair of ZEB-type E-box elements located proximal to the transcription start site. These findings establish a doublenegative feedback loop controlling ZEB1-SIP1 and miR-200 family expression that regulates cellular phenotype and has direct relevance to the role of these factors in tumor progression.

Background Mast cells have gained notoriety based on their detrimental contributions to IgE-mediated allergic disorders. Although mast cells express the vitamin D receptor (VDR), it is not clear to what extent 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), or its predominant inactive precursor metabolite in circulation, 25-hydroxyvitamin D3 (25OHD3), can influence IgE-mediated mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo. Objective We sought to assess whether the vitamin D3 metabolites, 25OHD3 and 1α,25(OH)2D3, can repress IgE-dependent mast cell activation via mast cell-CYP27B1 and -vitamin D receptor activity. Methods We measured the extent of vitamin D3 suppression of IgE-mediated mast cell degranulation and mediator production in vitro, as well as the vitamin D3-induced curtailment of PCA responses in WBB6F1-KitW/W-v or C57BL/6J-KitW-sh/W-sh mice engrafted with mast cells that did or did not express VDR or CYP27B1. Results Here we show that mouse and human mast cells can convert 25OHD3 to 1α,25(OH)2D3 via 25-hydroxyvitamin D-1α–hydroxylase (CYP27B1) activity, and that both of these vitamin D3 metabolites suppressed IgE-induced mast cell-derived pro-inflammatory and vasodilatory mediator production in a VDR-dependent manner in vitro. Furthermore, epicutaneously applied vitamin D3 metabolites significantly reduced the magnitude of skin swelling associated IgE-mediated PCA reactions in vivo; a response that required functional mast cell-VDRs and mast cell-CYP27B1. Conclusion Taken together, our findings provide a mechanistic explanation for the anti-inflammatory effects of vitamin D3 on mast cell function by demonstrating that mast cells can actively metabolize 25OHD3 to dampen IgE-mediated mast cell activation in vitro and in vivo.

Site-specific differences in skin response to pathogens and in the course of cutaneous inflammatory diseases are well appreciated. The composition and localization of cutaneous leukocytes has been studied extensively using histology and flow cytometry. However, the precise three-dimensional (3D) distribution of distinct immune cell subsets within skin at different body sites requires visualization of intact living skin. We used intravital multiphoton microscopy in transgenic reporter mice in combination with quantitative flow cytometry to generate a 3D immune cell atlas of mouse skin. The 3D location of innate and adaptive immune cells and site-specific differences in the densities of macrophages, T cells and mast cells at four defined sites (ear, back, footpad, tail) is presented. The combinatorial approach further demonstrates an as yet unreported age-dependent expansion of dermal gamma-delta T cells. Localization of dermal immune cells relative to anatomical structures was also determined. While dendritic cells were dispersed homogeneously within the dermis, mast cells preferentially localized to the perivascular space. Finally, we show the functional relevance of site-specific mast cell disparities using the passive cutaneous anaphylaxis model. These approaches are applicable to assessing immune cell variations and potential functional consequences in the setting of infection as well as the pathogenesis of inflammatory skin conditions.

The majority of human cancers originate from epithelial cells[1], with their local invasion and metastasis accounting for 90% of cancer-related death[2]. The progression of metastasis is a complex event thought to incorporate the reversible developmental process of epithelial to mesenchymal transition (EMT). This involves cancerous epithelial cells transitioning into motile mesenchymal cells, which invade distant sites in the body where they lodge and undergo mesenchymal to epithelial transition (MET) before proliferating into secondary tumours[3]. Essential to the maintenance of the polarised epithelial monolayer is the adherens junction protein, E-cadherin, which has been described as a tumour suppressor because its down-regulation is associated with invasion and metastasis[4]. During EMT, the increase of transcription factors, such as ZEB1, SIP1, Snail, and Slug, strongly represses the transcription of the Ecadherin gene, facilitating loss of the strong cell-cell interactions characteristic of epithelial cells. First discovered in 1993, microRNAs (miRNAs) are an abundant class of noncoding, 18-25 nt, single-stranded oligoribonucleotides that function post-transcriptionally to negatively regulate the translation of messenger RNA (mRNA). Target recognition is based on complementary binding to the 3' untranslated region (3'UTR) of the target mRNA. Expression of miRNAs can vary from ubiquitous to highly site and/or temporal specific. They are predicted to regulate up to 30% of genes in eukaryotes[5] and have regulatory roles in cellular processes, such as proliferation[6,7,8], differentiation[9], apoptosis[10,11], metabolism[12], embryogenesis and developmental timing[13,14,15,16]. Consistent with their roles in maintaining normal cell function, the aberrant expression of miRNAs has been linked to oncogenesis[17] and, more recently, metastasis[18,19,20,21,22]. Since EMT is an essential developmental process and is implicated in metastasis, we postulated that miRNAs may be involved in its regulation. Evidence that EMT is required for cancer metastasis is increasing with investigations into the signalling mechanisms driving EMT. A potent inducer of EMT is the cytokine transforming growth factor-β (TGF-β), which has been implicated in regulating transcription factors including Snail, Slug, ZEB1, SIP1, and basic-helix-loop-helix (bHLH) factors, such as Twist[23,24]. However, knowledge of the role of miRNAs and their potential target genes in EMT is limited. Previous studies have shown that

No abstract

Background: Epithelial-mesenchymal transition (EMT) is a key process in embryonic development and cancer metastasis. Results: Sp1 activates miR-200 transcription in epithelial cells and prevents EMT. Conclusion: miR-200 family members require Sp1 to drive basal expression and maintain an epithelial state. Significance: Defining the mechanisms controlling the epithelial state has implications for understanding early differentiation and for designing interventions to prevent cancer metastasis.

ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.

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.