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Redis R.S.,University of Houston | Vela L.E.,University of Houston | Lu W.,University of Houston | Ivan C.,University of Houston | And 41 more authors.
Molecular Cell | Year: 2016

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA. Redis et al. report that the two alleles of the lncRNA, CCAT2, induce distinct metabolic phenotypes. By interacting with the CFIm complex with allele-specific affinities, CCAT2 regulates the alternative splicing of GLS, resulting in the preferential expression of the more aggressive splice isoform. © 2016 Elsevier Inc.

De Koning H.D.,Radboud University Nijmegen | De Koning H.D.,Radboud Institute for Molecular Life science RIMLS | De Koning H.D.,Nijmegen Center for Immunodeficiency and Autoinflammation
Clinical and Translational Allergy | Year: 2014

Schnitzler's syndrome is an autoinflammatory disorder characterized by the association of a monoclonal IgM (or IgG) gammopathy, a chronic urticarial rash, and signs and symptoms of systemic inflammation, including fever, arthralgias and bone pain. It was first described in 1972. This review summarizes the clinical features, efficacy of therapies, and follow-up data of the 281 cases that have been reported to date. Also, the results of skin histology, bone imaging, laboratory investigations, and studies of the pathogenesis will be discussed, including the pivotal role of interleukin-1 beta in this disorder. © 2014 de Koning; licensee BioMed Central Ltd.

Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2008

The objective of this project is to create a validated in vitro model system that can replace transgenic or knockout mouse approaches, thereby reducing the number of experimental animals used in the field of skin biology and dermatology. We aim to construct lentiviral vectors with three different promoters to direct gene expression in specific epidermal cell types. Both controlled overexpression and knockdown by siRNA will be pursued. Advanced technology (pDeCap vector) will be used to minimize interferon responses. These new vectors will be used in combination with reconstructed skin models to obtain physiologically relevant phenotypes in vitro. To obtain proof of concept we will apply this system to the recently discovered cystatin M/E pathway, for which we will follow a conventional transgenic and knockout strategy in parallel, to allow comparison and validation. BACKGROUND: The use of knockout or transgenic mouse models is a cornerstone of modern biomedical research and has provided mechanistic insights in biology and pathology. The scientific, medical and commercial importance of these technologies has to be weighed against ethical considerations and societal concern on the use of experimental animals. We are currently using knockout and transgenic approaches to investigate the role of various genes in skin biology and skin diseases, involving hundreds of genetically modified mice per year with minor to severe phenotypes. We reasoned that if a generally applicable strategy could be developed for gene knock-down or overexpression in a model that closely resembles normal human skin, this would lead to a significant reduction of animal use and discomfort. STRATEGY AND VALIDATION: Several state-of-the art technologies will be combined to create a novel in vitro system that is functionally equivalent to transgenic or knockout mouse approaches. This will result in a 3-D tissue culture model of reconstructed human skin in which lentiviral vectors are used for gene knockdown (by delivery of siRNA) or overexpression of certain genes. We will create vectors that use stratum-specific promoters of the involucrin, cytokeratin 10 and cytokeratin 14 genes to direct expression to the correct cell layer (granular, spinous and basal). The pDeCap vector technology will be used to avoid interferon responses. To obtain proof of concept, we will apply this new technology to elucidate a new biochemical pathway that we have recently discovered in human epidermis, involving the cystatin M/E-cathepsin V-cathepsin L-transglutaminase-3 pathway. We have already studied part of this pathway in knockout mouse models, which enables us to compare and validate the proposed technology with previous data obtained from experimental animals.DELIVERABLES: We aim to develop a technology that has the potential to significantly reduce the use of genetically modified animals. The results and research tools (vectors) will be made available to the scientific community without restriction. Dissemination of knowledge will be achieved by scientific publications and by organizing an international meeting on skin models within the European Epidermal Barrier Research Network (E2BRN) and the European Society of Dermatological Research (ESDR). When proven successful for skin research, this approach might initiate applications in other tissues that can be studied in vitro. Areas of application range from basic research to target validation in pharmaceutical industry.

De Koning H.D.,Radboud University Nijmegen | De Koning H.D.,Radboud Institute for Molecular Life science RIMLS | De Koning H.D.,Nijmegen Center for Immunodeficiency and Autoinflammation | Van Vlijmen-Willems I.M.J.J.,Radboud University Nijmegen | And 11 more authors.
British Journal of Dermatology | Year: 2015

Summary Background Schnitzler's syndrome (SchS) is an autoinflammatory disease characterized by a chronic urticarial rash, a monoclonal component and signs of systemic inflammation. Interleukin (IL)-1β is pivotal in the pathophysiology. Objectives Here we investigated the cellular source of proinflammatory mediators in the skin of patients with SchS. Methods Skin biopsies of lesional and nonlesional skin from eight patients with SchS and healthy controls, and patients with cryopyrin-associated periodic syndrome (CAPS), delayed-pressure urticaria (DPU) and cold-contact urticaria (CCU) were studied. We studied in vivoIL-1β, IL-17 and antimicrobial protein (AMP) expression in resident skin cells and infiltrating cells. In addition we investigated the in vitro effect of IL-1β, IL-17 and polyinosinic-polycytidylic acid (poly:IC) stimulation on cultured epidermal keratinocytes. Results Remarkably, we found IL-1β-positive dermal mast cells in both lesional and nonlesional skin of patients with SchS, but not in healthy control skin and CCU, and fewer in CAPS. IL-17-positive neutrophils were observed only in lesional SchS and DPU skin. In lesional SchS epidermis, mRNA and protein expression levels of AMPs were strongly increased compared with nonlesional skin and that of healthy controls. When exposed to IL-1β, poly:IC or IL-17, patient and control primary human keratinocytes produced AMPs in similar amounts. Conclusions Dermal mast cells of patients with SchS produce IL-1β. This presumably leads to activation of keratinocytes and neutrophil influx, and further amplification of inflammation by IL-17 (from neutrophils and mast cells) and epidermal AMP production leading to chronic histamine-independent neutrophilic urticarial dermatosis. What's already known about this topic? Interleukin (IL)-1β is pivotal in the pathophysiology of Schnitzler's syndrome (SchS). Dermal mast cells produce IL-1β in cryopyrin-associated periodic syndrome, which has clinical similarities to SchS. What does this study add? Dermal mast cells produce IL-1β in SchS. Dermal neutrophils produce IL-17 in SchS. Antimicrobial protein expression is increased in lesional epidermis in SchS. © 2015 British Association of Dermatologists.

Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2001

The kidney plays a vital role in the Ca2+ homeostasis by determining the excretion of Ca2+ from the body. The fine-tuning of the Ca2+ excretion takes place in the distal convoluted and connecting tubule, where Ca2+ is reabsorbed via an active transcellular pathway. This transcellular transport is realized in three steps consisting of Ca2+ entry across the apical plasma membrane; cytosolic diffusion of Ca2+ bound to calbindin-D28K; and extrusion across the basolateral membrane by the Na+-Ca2+ exchanger and the Ca2+- ATPase. The molecular nature of the apical Ca2+ entry pathway has recently been identified by our group and named epithelial Ca2+ channel (ECaC). This channel belongs to a new family of ion channels and exhibits the defining properties for being the gatekeeper in transcellular Ca2+ reabsorption. The present proposal is a logic and timely continuation of this research program and investigates for the first time the in vivo regulation of this new Ca2+ channel. With respect to the expression of ECaC in rat kidney the below mentioned key questions will be addressed. They are related to alterations in Ca2+ reabsorption observed in daily clinical routine but their detailed molecular mechanism remains largely unknown. |i) What is the effect of the main calciotropic hormone, 1,25-dihydroxyvitamin D3? |Vitamin D3 is a key regulator of Ca2+ reabsorption and is frequently subscribed to patients with chronic renal failure.|ii) What is the effect of treatment with furosemide, thiazides and cyclosporin A? |These frequentiy used drugs have numerous effects on Ca2+ metabolism and can promote or inhibit renal Ca2+ excretion. The diuretics provide, therefore, therapeutic opportunities to correct imbalances of plasma Ca2+ levels.|iii) What is the effect of chronic metabolic acidosis? |Chronic metabolic acidosis is frequently observed in chronic renal failure in patients on and off dialysis and is accompanied by hypercalciuria. Recent studies indicate that the increased Ca2+ excretion is, in addition to an increased bone resorption, also due to a direct reduction of distal Ca2+ reabsorption. |In summary, these studies will further advance our understanding of renal handling in general and will, in particular, provide a molecular basis for achieving a better understanding of Ca2+ (mal)reabsorption. Ultimately, this could advance the therapeutic options to control renal Ca2+ excretion.

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