The Nedd4-2/Ndfip1 axis is a negative regulator of IgE-mediated mast cell activation

Cross-linkage of the high-affinity immunoglobulin E (IgE) receptor (FceRI) on mast cells by antigen ligation has a critical role in the pathology of IgE-dependent allergic disorders, such as anaphylaxis and asthma. Restraint of intracellular signal transduction pathways that promote release of mast cell-derived pro-inflammatory mediators is necessary to dampen activation and restore homoeostasis. Here we show that the ligase Nedd4-2 and the adaptor Ndfip1 (Nedd4 family interacting protein 1) limit the intensity and duration of IgE-FceRI-induced positive signal transduction by ubiquitinating phosphorylated Syk, a tyrosine kinase that is indispensable for downstream FceRI signalosome activity. Importantly, loss of Nedd4-2 or Ndfip1 in mast cells results in exacerbated and prolonged IgE-mediated cutaneous anaphylaxis in vivo. Our findings reveal an important negative regulatory function for Nedd4-2 and Ndfip1 in IgE-dependent mast cell activity.Kwok Ho Yip, Natasha Kolesnikoff, Nicholas Hauschild, Lisa Biggs, Angel F. Lopez, Stephen J. Galli, Sharad Kumar, Michele A. Grimbaldesto

Interrelationships between the extracellular matrix and the immune microenvironment that govern epithelial tumour progression

Solid tumours are composed of cancer cells characterised by genetic mutations that underpin the disease, but also contain a suite of genetically normal cells and the extracellular matrix (ECM). These two latter components are constituents of the tumour microenvironment (TME), and are key determinants of tumour biology and thereby the outcomes for patients. The tumour ECM has been the subject of intense research over the past two decades, revealing key biochemical and mechanobiological principles that underpin its role in tumour cell proliferation and survival. However, the ECM also strongly influences the genetically normal immune cells within the microenvironment, regulating not only their proliferation and survival, but also their differentiation and access to tumour cells. Here we review recent advances in our knowledge of how the ECM regulates the tumour immune microenvironment and vice versa, comparing normal skin wound healing to the pathological condition of tumour progression.Natasha Kolesnikoff, Chun-Hsien Chen, and Michael Susithiran Samue

The microRNA-200 Family Regulates Epithelial to Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is an essential process in development and is proposed to be an initiating step in cancer metastasis. This review discusses recent findings by Gregory et al (2008) who show that EMT is regulated by members of the miRNA-200 family and miR-205. These miRNAs maintain the epithelial phenotype by targeting the transcriptional repressors, ZEB1 and SIP1, which downregulate E-cadherin. Manipulation of miR-200s and miR-205 levels was sufficient to induce EMT or MET (mesenchymal-epithelial transition) in cell culture models. Analysis of miRNA levels in breast cancer cell lines revealed a loss of the miR-200s in mesenchymal but not epithelial cell lines. Furthermore, examination of ductal (epithelial) and metaplastic (mesenchymal-like) breast cancers showed loss of the miR-200s in the metaplastic tumours, indicating that a loss of the miR-200s may promote EMT in tumours.Paterson Emily L., Kolesnikoff Natasha, Gregory Philip A., Bert Andrew G., Khew-Goodall Yeesim and Goodall Gregory J

Specificity protein 1 (Sp1) maintains basal epithelial expression of the miR-200 family: implications for epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is required for the specification of tissues during embryonic development and is recapitulated during the metastatic progression of tumors. The miR-200 family plays a critical role in enforcing the epithelial state with their expression lost in cells undergoing EMT. EMT can be mediated by activation of the ZEB1 and ZEB2 (ZEB) transcription factors, which repress miR-200 expression via a self-reinforcing double negative feedback loop to promote the mesenchymal state. However, it remains unclear what factors drive and maintain epithelial-specific expression of miR-200 in the absence of EMT-inducing factors. Here, we show that the transcription factor Specificity Protein 1 (Sp1) binds to the miR-200b∼200a∼429 proximal promoter and activates miR-200 expression in epithelial cells. In mesenchymal cells, Sp1 expression is maintained, but its ability to activate the miR-200 promoter is perturbed by ZEB-mediated repression. Reduction of Sp1 expression caused changes in EMT-associated markers in epithelial cells. Furthermore, we observed co-expression of Sp1 and miR-200 during mouse embryonic development wherein miR-200 expression was only lost in regions with high ZEB expression. Together, these findings indicate that miR-200 family members require Sp1 to drive basal expression and to maintain an epithelial state.Natasha Kolesnikoff, Joanne L. Attema, Suraya Roslan, Andrew G. Bert, Quenten P. Schwarz, Philip A. Gregory and Gregory J. Goodal

A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition

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/delta EF1 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 double-negative 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.Cameron P. Bracken, Philip A. Gregory, Natasha Kolesnikoff, Andrew G. Bert, Jun Wang, M. Frances Shannon, and Gregory J. Goodal

Role of the miR-200 family in mediating EMT in response to TGF-beta

Presentation SY-10 at Symposium 3: microRNA regulation of cytokine gene expressionPhilip A. Gregory, Cameron P. Bracken, Andrew G. Bert, Emily L. Paterson, Natasha Kolesnikoff, Gelareh Farshid, Yeesim Khew-Goodall, Gregory J. Goodal

The skin immune atlas: Three-dimensional analysis of cutaneous leukocyte subsets by multiphoton microscopy

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, and 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. Although 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

Mechanisms of vitamin D₃ metabolite repression of IgE-dependent mast cell activation

Abstract not available.Kwok-Ho Yip, Natasha Kolesnikoff, Chunping Yu, Nicholas Hauschild, Houng Taing, Lisa Biggs, David Goltzman, Philip A. Gregory, Paul H. Anderson, Michael S. Samuel, Stephen J. Galli, Angel F. Lopez, and Michele A. Grimbaldesto