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.