The Pilot and Feasibility Program awards four annual grants to Northwestern University faculty. The program is funded through designated grant support of the Administrative Core, as well as from the Feinberg School of Medicine Dean’s office and the Department of Dermatology. Awards are established on an annual basis by the Pilot and Feasibility committee. If sufficient scientific progress is demonstrated over the grant year, it is possible that an award may be renewed for no more than one additional year. The studies are designed to:
- Foster research about keratinocytes leading to sufficient preliminary data for procurement of federal funding
- Attract established investigators to pursue research related to skin biology
- Encourage collaborative opportunities between the Department of Dermatology and other departments within the medical school and the university
- Provide mentorship in grant writing as well as feedback regarding the conduct of scientific investigation for more junior scientists
All recipients of Pilot and Feasibility awards automatically become Core Members during their funding period and retain membership if they continue research in cutaneous biology. Submissions to the Pilot and Feasibility Committee are requested campus-wide every spring. Starting with the 2014-2015 academic year, it has been determined that one of the four awards will be granted to a project focusing on a problem which addresses minority or sex issues in cutaneous biology research.
Keratinocyte Mitochondria as Systemic Oxygen Sensor
This proposal explores the consequences of mitochondrial loss of function in keratinocytes. The major function of mitochondria in cellular homeostatis has historically been the generation of energy through oxidative phosphorylation. However, mitochondria can also serve as a signaling organelle. The projects described in Dr. Chandel's Lab are driven by the hypothesis that when cells encounter stress the
mitochondria serve as key regulators of biological outcomes that include the induction of adaptive genes, cellular proliferation, senescence and death. One stress studied is how cells respond to decreased oxygen levels (hypoxia). Multi-cellular organisms have evolved multiple mechanisms to respond to hypoxia. Healthy individuals typically encounter hypoxia at high altitudes, where at least three prominent
physiological responses take place: neurotransmitter release by the carotid body to increase breathing; pulmonary vascular constriction to shunt blood to better oxygenated regions of the lung; and production of the hormone erythropoietin (EPO) in the liver to enhance red blood cell mass and hemoglobin concentration in the blood. At the molecular level the physiological responses to hypoxia are mediated by the transcription factor hypoxia inducible factor, HIF-1. A recent study demonstrated that when mice with a keratinocyte-specific deletion of HIF-1 were exposed to hypoxia, the predicted increase in plasma EPO levels was blunted and induction of EOP expression in the kidney was lost. This was suprising because it suggested that the keratinocytes were sensing the hypoxia to activate HIF-1 and regulate EPO production
in the kidney. But how cells sense the decrease in oxygen to activate HIF dependent gene expression is not fully understood. This study will test whether mitochondria function as oxygen sensors in the keratinocytes to induce HIF-1 to regulate EPO production in mice.
- To determine whether loss of TFAM in keratinocytes abolishes the hypoxic increase in HIF-1 in vitro.
- To determine whether loss of TFAM in keratinocytes blunts the hypoxic increase in plasma EPO levels in vivo.
Information generated from the Pilot and Feasibility Study will:
- Further our understanding of the role(s) of HIF-1 in cutaneous wound healing.
- Provide a rationale for the use of mitchondrial targeted antioxidants as therapy to prevent local inflammation in the skin, since HIF-1 impacts on skin inflammation.
- Justify the development of novel topical therapies for treating anemia, if the mitochondrial ROS/HIF pathway in the skin can stimulate systemic erythropoietin levels.
The Role of Keratinocytes as Independent Thermosensors
This proposal begins to investigate the possibility that keratinocytes or other skin cells can function as sensory transducers, independent of nerves, in response to changes in temperature. Thermosensitive channels in skin keratinocytes and/or sensory neurons innervating the skin open in response to heating or cooling and mediate our perception of ambient temperature. Dr. Garcia-Añoveros's Lab has found that some basal cells of the epidermis, as well as some cells of hair follicles, express the TRPML3 channel. It has also been observed that this channel may open in response to changes in temperature. This study aims at elucidating the function of TRPML3 channels in the epidermis and hair follicle, and may lead to further studies of how the skin, including the vasculature and eccrine glands, detect and respond to changes in temperature.
- To determine the spatial and temporal expression of TRPML3 during epidermal and hair follicle development.
- To determine whether the cold-to-warm current that we have detected in keratinocytes is mediated by TRPML3.
- To determine the effects of deleting the Trpml3 gene is skin.
The Effect of IL-4 Receptor Signaling on Inflammation and Skin Barrier Function in Atopic Dermatitis
PI: Douglas A. Kuperman, PhD, Assistant Professor in the Department of Medicine, Division of Allergy-Immunology
This study proposes to test the in vivo relevance of IL-4 and IL-13 acting on keratinocytes in the context of atopic dermatitis. Atopic dermatitis occurs as a result of inappropriate immune responses to common elements in the environment. The T-helper type II (Th2) lymphocyte is a critical contributor to the pathologic inflammatory responses observed in these patients. For example, atopic dermatitis is characterized by increased levels of the Th2 cytokines, IL-4 and IL-13, in the skin. When keratincytes are cultured with either IL-4 or IL-13 they respond by producing a variety of pro-inflammatory mediators and they lose expression of filaggrin as well as loricrin, which are important proteins that maintain skin barrier function. However, there have been no studies performed to test the in vivo relevance of IL-4 and IL-13 signal by ligation of a common receptor, IL-4Rα. Dr. Kuperman's hypothesis is that IL-4Rα signaling specifically in keratinocytes contributes to inflammation and failure of skin barrier function in a murine model of atopic dermatitis.
- To generate a mouse strain that is deficient in IL-4Rα exclusively in keratinocytes.
- To determine the in vivo requirement of keratinocyte-specific expression of IL-4Rα in the development of skin pathology associated with experimental atopic dermatitis.
Nanoparticle Delivery of Oligonucleotides Targeting Missense Mutations in Keratinocytes
This proposal will test the function of oligomers in keratinocytes, initially to suppress survivin expression in keratinocytes and then to test the functional effect of targeting a common missense mutation of keratin 14 to reverse the keratin filament defect. Controlling gene expression in primary cultured cells, such as human keratinocytes, is difficult because the epidermal barrier precludes the entry of suppressive nucleic acids. Methods to introduce gene regulation agents such as oligonucleotides have met with limited success. The proposed work will develop oligonucleotide-nanoparticle antisense agents as methods for controlling gene expression in keratinocytes. This work will take advantage of newly discovered conjugate properties of the nanomaterials, including enhanced stability, high penetration ability, and lack of toxicity. The ability of these agents to work on genes of relevance in skin biology models will be tested.
- Optimize the keratinocyte and epidermal uptake of oligonucleotide-gold nanoparticle conjugates (DNA-Au NPs) and determine their utility in suppressing gene expression in vitro and in mouse skin
- Investigate the ability of DNA-Au NPs to selectively suppress mutant keratin 14 expression in keratinocytes from a patient with epidermolysis bullosa simplex (Dowling-Meara).
Mechanisms Underlying Adverse Cutaneous Effects Of The Anti-Cancer Drug, Bortezomib
This proposal will incorporate a series of studies involving the proteosome inhibitor Bortezomib (BZ), an anti-cancer drug used in the treatment of multiple myeloma. Unfortunately, it has side effects, particularly in the skin. Dr. Brown hypothesizes that this relates to mast cell recruitment to the skin. She therefore will test this hypothesis in aim 1 and will also assess whether mast cell stabilizers might have utility in blocking the toxic effect of BZ. In aim 2, Dr. Brown proposes to assay mast cell recruitment to the skin in patients being treated with BZ.
- To evaluate the role of mast cells in BZ-dependent cutaneous effects (KO mice).
- To assess BZ effects on mast cells recruitment and function in the skin of multiple myeloma patients undergoing BZ treatmen
The regulation of cell migration and intercalation through keratinocytes
PI:Brian Mitchell, PhD, Assistant Professor of Cell and Molecular Biology
This proposal aims to develop the experimentally pliable skin of Xenopus embryos as a model system for dissecting the molecular mechanisms via which cells break through and re-establish junctional barriers. Xenopus skin is an ideal system for this analysis since it consists of two distinct layers, the outer keratinocytes, and an inner basal layer composed of precursor cells with the ability to differentiate into either multi-ciliated cells or ionocytes. During development, these inner layer cells undergo a stereotyped migration event in which they intercalate into the keratinocyte layer. Dr. Mitchell proposes to define the complex cell biological processes which underlie how skin cells migrate in a directed manner, break down cell-cell junctions of the outer keratinocytes and stop their migration and form new cell-cell junctions.
- To determine how small GTPases regulate cytoskeletal dynamics during migration and the termination of this migration.
- To determine the role of the microtubule binding protein CLAMP on the initiation of multi-ciliated cell intercalation.
- To determine what which genes are upregulated in both multi-ciliated cells and ionocytes during the intercalation of these diverse cell types.
Keratinocyte innate immune responses after herpes simplex virus infection
PI:William J. Muller, MD, PhD, Assistant Professor of Pediatrics
Evidence from studies in mice and in tissue culture models suggests that the interaction of herpes simplex virus with one of its principal receptors, the herpes virus entry mediator (HVEM), influences early events in innate immune signaling. Our overall hypothesis is that virus interaction with HVEM alters this signaling to promote viral replication and establishment of infection. Using virus which is altered to abolish interaction with HVEM, we will use molecular techniques to measure the induction and translation of innate genes in human keratinocytes after infection and how this is influenced by HVEM signaling. We will then confirm and extend these results in assays using wild-type virus and cells lacking HVEM signaling; either murine cells with disruptions in the HVEM gene, or human cells with HVEM expression knocked down.
- Measure the induction and translation of innate genes in human keratinocytes after infection with wildtype HSV-1 and HSV-2, compared to mutants of these viruses unable to engage HVEM.
- Measure the induction and translation of innate genes in murine keratinocyte cultures derived from wildtype mice and mice lacking HVEM (Tnfrsf14-/-mice), and in human keratinocyte cultures for which HVEM is knocked down, after infection with wild-type HSV.
Innate immune host defense function of Keratinocytes
PI:Christian Stehlik, PhD, Assistant Professor of Medicine, Rheumatology Division
The goal of this study is to characterize a novel innate immune function of Keratinocytes by testing our hypothesis that select NLRs are able to promote inflammasome activation in Keratinocytes upon sensing pathogen infection of the skin and to subsequently recruit phagocytes. We further hypothesize that dysregulation of this system contributes to the excessive production of IL-1β in inflammatory skin disease.
- To determine inflammasome activation in Keratinocytes upon bacterial infection.
- To determine if select NLRP mutations cause chronic inflammasome activation in Keratinocytes.
Study of keratinocyte biology from kidney transplant patients with different skin types.
CO-I: June K. Robinson, MD, Research Assistant Professor of Dermatology
Dr. Johnson theorizes that keratinocytes from non-Hispanic White, Hispanic, and African American kidney transplant patients with medium to dark skin color will display differences when compared with keratinocytes from patients with light skin color. Dr. Johnson hypothesizes that the differences in keratinocyte biology from kidney transplant patients with different skin types will significantly correspond with each group’s propensity to develop post-transplant squamous cell carcinoma (SCC) and metastatic SCC. The proposed aim of this study is to understand biological differences between keratinocytes from patients with different skin types to elucidate underlying causes of the lower incidence and rate of SCC formation but slightly higher mortality rate in transplant patients. To complete the aim of this project Dr. Robinson will coordinate the clinical collection of biopsies and patient information and Dr. Johnson will coordinate the laboratory studies. This project will formally begin a new collaboration between Drs. Johnson and Robinson and will advance the SDRC initiative to focus on differences between keratinocytes of Caucasians and individuals with skin of color.
Gold nanoparticles loaded with cobalt schiff base-DNA conjugates for specific and activatable inhibition of Gli transcription factors.
PI: Thomas Meade, PhD, Eileen M. Foell Professor in Cancer Research, Professor of Chemistry, Molecular Biosciences, Neurobiology and Radiology
Gli transcription factors (TFs) represent the terminal step in the Hedgehog (Hh) pathway, which regulates many aspects of embryonic morphogenesis. Aberrant signaling of the Hh pathway is known to promote the establishment and progression of a variety of tumors including basal cell carcinoma (BCC) and medulloblastomas. Due to its involvement in cancer, inhibition of the Hh pathway is of great interest in cancer chemotherapy. For P&F #10, Dr. Meade intends to attach Co (III)-Gli to Au-NPs in order to achieve effective penetration and delivery of the Gli-inhibitor in the skin. This agent would have possible applications in treatment of skin-associated diseases mediated by the Hh pathway. To assess the feasibility of this concept, he proposes to investigate the following: Aim 1: Synthesis and characterization of Co(III)-Gli conjugated Au-NPs (Au-Gli-Co); Aim 2: In vitro validation of the efficacy of Au-Gli-Co at inhibiting the Hh pathway by (1) assessing the efficacy of Au-Gli-Co in vitro, (2) examining the direct target of Au-Gli-Co, and (3) determining tissue penetration. This project involves the development of a unique TF inhibitor that targets the terminal step of the Hh pathway to treat BCC. It employs a new mechanism for inhibiting TFs that confers high specificity and efficacy toward the target. By incorporating a novel and effective delivery mechanism using Au-NPs, Dr. Meade expects Au-Gli-Co to effectively inhibit the Hh pathway. Further, the Au-NP platform will allow improved delivery of the agent through cells and skin, and permit targeted localized delivery of the agent using plasmon activation.
Determining the role of CRAC channels for calcium dynamics and effector function in keratinocytes.
PI: Murali Prakriya, PhD, Associate Professor of Molecular Pharmacology and Biological Chemistry
The hypothesis for this study is that ion channel pores called calcium release activated calcium (CRAC) channels serve as a key route of calcium entry in keratinocytes to regulate gene expression and the release of inflammatory mediators. Dr. Prakriya aims to test this hypothesis using an integrated approach that merges electrophysiology, Ca2+ imaging, immune assays, and mouse genetics. Confirming this hypothesis would resolve important uncertainties about the identity of Ca2+ influx pathways that operate in keratinocytes. Furthermore positive findings will allow the development of novel therapeutic solutions for inflammatory diseases of the skin, and lay a sound foundation on which to build, and finance, the long-term plan for this research. Specific aims of this P&F project include: Aim #1: determine the functional properties and molecular mechanisms of CRAC channels in kertainocytes, and Aim #2: determine the functional significance of CRAC channels for gene expression and generation of inflammatory mediators in keratinocytes. Collectively, this study will illuminate potential physiological roles of CRAC channels in keratinocytes and provide fresh insights in to the mechanism of cytokine and chemokine production in the skin.
Functional Analysis of T Cell Transcription Factors in the Immune Regulation of Skin Inflammation
PI:Liang Zhou, MD, PhD, Assistant Professor of Pathology and Microbiology-Immunology
This study seeks to understand how T helper (Th) 17 and T regulatory (Treg) immune cells are regulated in the skin, both under steady state and psoriatic inflammatory conditions. Dr. Zhou hypothesizes that Ikaros functions as a molecular switch to determine the Treg-Th17 balance via specific DNA-binding zinc fingers, which is important for skin inflammation and the pathogenesis of psoriasis. Specifically, Dr. Zhou proposes to study the crosstalk between keratinocytes and T cells mediated by Ikaros using both in vitro cell culture systems and an in vivo model of psoriasis. Specific aims of this P&F include: Aim #1: determine the molecular mechanisms of action of Ikaros in the Treg /Th17 cell differentiation program, Aim #2: determine the impact of Ikzf1ΔF4 T cells on keratinocytes and Aim #3: Determine the in vivo role of Ikaros using a Th17-mediated murine psoriasis model. Dr. Zhou proposes that this study will represent a unique interface between immunology and keratinocyte biology, and we are positioned with the necessary tools to understand the role of Ikaros in skin inflammation. The findings from the proposed studies will provide novel mechanistic insights into the function of multiple Th17 and Treg cell transcription factors in immune regulation and may identify new therapeutic targets for psoriasis and other inflammatory skin disorders.
Sexually dimorphic effects of topical glucocorticoids
PI: Irina Budunova, MD, PhD, Associate Professor in Dermatology and Urology
Nuclear Polarity during Epidermal Differentiation
PI: Steven T. Kosak, PhD, Assistant Professor in Cell and Molecular Biology
Nuclear organization is involved in the regulation of gene expression, and dynamic changes to this organization are thought to be necessary to achieve the precise and coordinated modifications in gene expression that occur during normal cell growth. The nucleus is generally studied as an independent structure: however, it is important to remember that within a cell the nucleus must communicate with other organelles and extracellular signals. Furthermore, most cells are not symmetric structures, and cell polarity is involved in many cellular functions including proliferation, differentiation, and morphogenesis1. Therefore, in order to fully examine nuclear organization, Dr. Kosak and his team will study the nucleus in the context of an asymmetric cell and incorporate the idea of nuclear polarity, which requires defining nuclear organization with respect to structures and processes on the nuclear exterior. With their SDRC Pilot and Feasibility Award they propose to examine a previously unstudied aspect of nuclear organization in human cells, nuclear polarity, and to determine how regulation of this form of nuclear organization relates to changes in gene expression during epidermal differentiation.
Targeting a bacterial Ras-Specific protease to treat skin diseases
Rat sarcoma (Ras) oncoprotein is a ubiquitous small GTPase in eukaryotic cells that is a critical node that senses incoming signals and subsequently activates downstream target proteins. These targets include mitogen activated protein kinase (MAPK) signaling cascades that ultimately turn on additional genes directing cell proliferation, differentiation and survival. Ras is also pivotal to the host response to infection coordinating signals from surface receptors that bind bacterial antigens to active cytokine and chemokine expression and macrophage maturation. Ras enzymatic activity is regulated by cycling between an inactive (GDP-bound) state and an active (GTP-bound) state. Constitutively activating mutations that mimic or stabilize the GTP-bound state are found in ~30% of human malignancies, inc. skin melanomas. Germline mutations in Ras or genes connected to RAS are also implicated in developmental RASopathies. Despite the potential for this important protein to be a therapeutic target for these diseases, no inhibitors that directly target RAS have been successfully developed for clinical use and RAS has been labeled “undruggable”.
Lipid Antigen-Specific T Cells in Chronic Skin Inflammatory Diseases
The goal of the proposed research is to investigate the potential role of group 1 CD1-restricted autoreactive T cells in the development of chronic skin inflammation. Dr. Wang has significant expertise in the field of CD1 biology, which has been the primary focus of her research for past twenty years. Her lab has generated multiple novel mouse models that have contributed significantly to the study of group 1 and group 2 CD1-restricted T cells in the context of autoimmunity and infectious disease. Recently, her lab has generated double transgenic mice that expressed the human group 1 CD1 molecules and TCR specific to group 1 CD1 and species-conserved self lipid antigens. Using this mouse model, they demonstrated that autoreactive group 1 CD1-restricted T cells have similar developmental requirements, surface phenotypes and functional properties to those of CD1d-restricted NKT cells. Interestingly, these mice spontaneously developed dermatitis when they were on the Apo-E deficient background. They have just obtained a R21 to investigate how group 1 CD1 autoreactive T cells contribute to chronic skin inflammation in the animal model. They are very interested to extend their study into patients with chronic skin inflammation, such as atopic dermatitis and psoriasis. They anticipate that these studies may lead to the identification of new biomarkers or new therapeutic targets for treating these diseases.
For additional information on the P&F Program, contact Robert M. Lavker, PhD.