Kathleen J Green - US grants

The proper development of skin during embryogenesis, and its maintenance throughout life are essential for the protective and mechanical functions it serves. A variety of birth defects including blistering and keratinization disorders and dysplasias, are manifested as skin malformations. Undoubtedly many more epidermal abnormalities are lethal to the developing embryo. Defining the elements involved in the morphogenesis of a stratified epithelium will enable us to better understand how normal functions are perturbed and perhaps increase our ability to diagnose and ultimately prevent such defects. The goal of this proposal is to investigate the structure and expression of two human desmosomal proteins involved in the development and differentiation of skin. Desmoplakins (DPs) I and II are closely related molecules localized in the cytoplasmic plaque of the desmosome, a junction thought to play a crucial role in cell-cell adhesion. Desmosomes also appear to act as specific attachment sites for intermediate filaments (IF). Because of their extreme insolubility their structure and sequence have not been determined and it has been difficult to define their functional relationships to other desmosomal components and IF. DPI appears to be present in all desmosomes, whereas DPII appears to be present only in stratified tissues. Understanding the relationship between DPII expression and stratification has been hampered by the lack of a DPII specific probe. Generation of such a probe is a primary goal of this research. I have isolated several cDNA clones from human foreskin and bovine tongue lambda gt11 libraries. My results suggest that in bovine tongue, there are two messages for the DPs, and these messages are transcribed from a single gene. This possibility will be tested for the human system by Northern and Southern hybridization experiments. The structure of the human DP cDNAs (mRNAs) will be determined by restriction mapping, cross- hybridization experiments and DNA sequence analysis. This may allow identification of sequences which could serve as a DPII specific probe. Genomic clones will also be isolated and the initial characterization of gene structure and flanking regions will be carried out to identify possible mechanisms of transcriptional and post-transcriptional regulation. The spatial distribution and expression of the DPs at the message and protein levels will be studied in human embryonic, fetal and adult skin by in situ hybridization, Northern analysis, immunolocalization and immunoblotting to identify differences in DPI and DPII expression. Finally, the expression of DPI and II will be studied during differentiation and stratification in cultured human keratinocytes. This work will surely increase our understanding of the elements involved in the successful morphogenesis and differentiation of human skin.

The autoantibodies in the blistering disease pemphigus foliaceus (PF) have been shown to bind to a transmembrane desmosomal glycoprotein called desmoglein I (DGI). It has been suggested that these antibodies might directly inhibit cell adhesion by binding to the extracellular domain of DGI, thereby causing acantholysis. Although desmosomes are one of the most prominent types of adhesive junctions in epithelia, until recently very little was known regarding the nature of the molecules responsible for desmosome-mediated adhesion. Recently, sequence analysis has demonstrated that DGI is related to the calcium-dependent class of cell adhesion molecules known as cadherins. In spite of this, DGI has not yet been demonstrated experimentally to be an adhesion molecule. In this proposal a combined cell biological and molecular genetic approach will be taken to study the function of the PF antigen, desmoglein I, and its relationship to the disease process. The first aim will be to investigate whether DGI is sufficient for mediating adhesion in cultured fibroblasts that are normally non- adherent. Constructs encoding full length DGI will be transfected into non-adherent fibroblasts, and the ability of these cells to adhere in the presence of calcium will be assessed. If DGI alone is not sufficient, the possibility that additional cytoplasmic proteins are required will be tested by co-expression experiments. As an alternative approach, in aim two antisense mRNA experiments will be carried out to test whether DGI is necessary for adhesion or plaque integrity in human keratinocytes. In aim three, the ability of latex beads coated with extracellular domains of desmosomal cadherins to bind to desmosome-containing cells and induce signals necessary for induction of plaque assembly will be investigated. In the fourth aim, deletion mutants and chimeric molecules combining specific portions of DGI with portions of E-cadherin will be expressed in L cells and/or junction bearing cells, and the fat of expressed protein, and effect on endogenous junctions and filament systems will be assessed. Finally, the relationship of DGI autoantibodies to acantholysis in PF will be determined. the goal of this final aim will be to a) generate fusion proteins encoding specific regions in the extracellular domain of DGI, b) affinity purify individual IgG that bind these epitopes from PF patient sera, and c) assess the ability of these antibodies to cause acantholysis in vivo and in vitro. This work should provide insights into the function of the PF antigen and may provide a groundwork for the development of a plasmapheresis-based therapy for PF.

The purpose of this proposal is to generate funds to support the travel, registration and subsistence for participants in the Gordon Research Conference on Intermediate Filaments, which will be held July 24-29, 1994 at the Tilton School, Tilton, New Hampshire. Intermediate filaments (IFs) are prominent constituents of the cytoskeleton and karyoskeleton of most eukaryotic cells, and virtually fill the cytoplasm of terminally differentiating epidermal keratinocytes and the axons of vertebrate and invertebrate neurons. The intermediate filament (IF) gene family comprises five different categories which are expressed in a tissue specific manner. In spite of this heterogeneity, IF diameter (8-10nm) and structural appearance upon assembly in vitro is quite similar regardless of the tissue source. The heterogeneity of IFs suggests a cell type specific function for this class of polypeptides. Elucidating these functions has been a major challenge for researchers over the years. However, the recent discovery that mutations in IF genes can lead to human disease highlights the importance of the IF network in maintaining tissue integrity, and has ushered this field into an exciting new era. This conference will bring together an international field of participants from both academia and industry in an atmosphere conducive to the free exchange of ideas. Themes to be developed include: 1) the evolution of IF genes and their role in development, 2) the structure, assembly and dynamics of IFs and nuclear lamins, 3) IF-cell surface interactions, 4) homologues and analogues of IFs in plants and lower organisms, 5) the involvement of IFs in pathology and disease as related to cancer, 6) a workshop on homologous recombination technology, 7) epidermal diseases of keratin, and 8) the involvement of IFs in neurological diseases. As in previous years, there will be informal poster presentations and a session dedicated to the discussion of selected posters, including the winning entries in a young scientist competition to be instituted this year.

DESCRIPTION: Desmosomes are the most prominent intercellular junctions in the epidermis where they provide an attachment site for the keratin intermediate filament (IF) cytoskeleton. During the current funding period we demonstrated that the obligate desmosomal component desmoplakin (DP) is required for linking IF to the desmosome and thus integrates the IF cytoskeleton into a supracellular network that joins all cells within the epidermis. The importance of DP in the epidermis was underscored by the discovery of an inherited form of palmoplantar keratoderma caused by haploinsufficiency of DP. Nevertheless, a number of questions remain which are key to understanding the etiology of human diseases targeting adhesive junctions. In the next funding period we propose to: 1) Determine the basis for tissue- and differentiation-specific interactions between IF and the DP C-terminus by a) carrying out yeast two hybrid analysis of associations between DP and simple epithelial versus epidermal keratins, by b) performing high resolution structural analysis of the DP C-terminus, and by c) analyzing the role of DP phosphorylation in desmosome assembly in living cells and tissues; 2) Determine how the DP N-terminus collaborates with cell type- and differentiation-specific armadillo proteins in desmosome assembly by defining the repertoire of DP binding partners in the arm family in cells and in vitro; 3) Address the importance of the IF-desmosome connection in keratinocyte behavior and mechanical integrity by using laser tracking and mechanical stretch assays coupled with gene microarray analysis to study cells inducibly expressing a dominant negative DP mutant that severs the DP-IF connection; 4) Address whether DP drives desmosome assembly in a classic cadherin dependent fashion by a) using fluorescently-tagged junction markers to establish the temporal and spatial events linking desmosome and adherens junction assembly in living cells, b) determining whether adherens junction proteins form a complex with DP, and c) examining whether desmosomal cadherins bind directly to downstream regions of the DP N-terminus, contributing to later stages of desmosome assembly. These experiments will provide new insight into how desmosomes are assembled and regulated, and will establish novel approaches towards defining the role of the DP-IF connection in keratinocyte integrity, intracellular signaling and behavior.

The reversible modulation of both cell-cell and cell-substrate adhesive contacts if thought to play an important role during epithelial tissue remodeling. During the migratory phase of remodeling, a dramatic reduction in the number of cell-cell junctions known as desmosomes has been reported. However, the mechanisms governing desmosome disappearance or reassembly during this process are unknown. One example of remodeling that contributes to the progression of periodontal disease, which is a major health problem in the U.S., is the inward migration of junctional epithelium along the tooth surface that occurs following dissolution of gingival connective tissue. In order to understand how modulation of desmosomes may impact on oral epithelial cell migration, the molecular mechanisms that regulate desmosome assembly and dissolution must be elucidated using well-defined in vitro and cell culture models. In this project we will continue our efforts to define protein-protein interactions in the desmosome and t investigate how adhesive junctions are modulated in oral epithelial cell cultures. The specific aims are: 1) To determine the protein-protein interactions involved in establishing the structure of the desmosomal plaque and ensuring segregation of desmosomal and adherens junction components into distinct membrane domains, 2) To investigate intercellular junction dynamics and the role of the associated cytoskeleton and underlying extracellular matrix during migration of oral epithelial cells, using a combination of live cell observations and molecular genetic manipulation of oral epithelial ells, 3) To examine the contribution of proteinases present in the gingival microenvironment to junction dissolution and to define whether specific desmosomal cadherins are substrates for these proteinases. These studies will interface extensively with other project leaders who will provide reagents (i.e., IFAP300 from Dr. Goldman and laminin-5 peptides from Drs. Jones and Stack) and expertise (generation of fluorescently labeled probes for living cell observations, Dr. Goldman; zymographic analysis, Dr. Stack). This work will provide important insights into mechanisms by which cell-cell adhesive junctions are assembled and modulated in migrating oral epithelial cells, and will provide a basis for the design of therapeutic approached to curb the progression of periodontal disease.

Patients with the blistering disease pemphigus foliaceus (PF) have circulating autoantibodies whose antigenic target is a transmembrane desmosomal cadherin called desmoglein 1 (Dsg1). It has been hypothesized that PF IgG might directly inhibit cell adhesion by binding to the extracellular domain of Dsg1 antibodies directly cause PF blisters, nor had Dsg1's role in adhesion been investigated. Over the last funding period substantial progress has been made to define specific epitopes against which PF antibodies are directed, to determine that binding of PF antibodies to these epitopes is dependent on their correct conformation and to demonstrate that anti-Dsg1 antibodies are pathogenic in the disease. However, the precise mechanism by which anti-Dsg1 results in acantholysis is not known. Elucidating this process will depend on first understanding how the desmosomal cadherin adhesive complex is assembled, maintained and regulated. Our aims for the next five years are: 1. To define the contribution of Dsg1 and other desmosomal cadherins to cell-cell adhesion in a fibroblast reconstitution system and to test the hypothesis that the cytoskeletal linker, desmoplakin, is required for desmosomal cadherin-mediated adhesion. 2. To establish the organization of the desmosomal cadherin extracellular domains at the adhesive interface of desmosomes by cross-linking keratinocyte cell surface molecules and analyzing cross-links by peptide sequencing. 3. To investigate the basis and biological significance of the unusual 6:1 stoichiometry exhibited by the plakoglobin:Dsg1 complex and whether plakoglobin:Dsg1 interactions are regulated by phosphorylation of the Dsg1 cytoplasmic tail, and 4. To examine the consequences of ectopically expressing Dsg1 or dominant negative plakoglobin mutants on adhesion, stratification and keratinocyte differention in organotypic raft cultures.

The objective of this proposal is to continue an interdisciplinary program for training predoctoral students at Northwestern University in the area of carcinogenesis which will provide research and training opportunities in this area of distinction at Northwestern University. This program has served as a focus for students, postdoctoral fellows, and faculty interested in studying various aspects of carcinogenesis and providing a stimulating forum for their interaction. In this grant, we have expanded the number of faculty by the addition of individuals who have consistently contributed to the program. The faculty identified in this proposal have active research programs in the areas of: 1) mechanisms of chemical carcinogenesis; 2) DNA damage and repair; 3) tumor viruses; 4) gene expression; 5) differentiation; 6) membrane alterations in transformation; 7) peroxisome proliferators and cancer induction; 8) genetic analysis of the malignant phenotype; and 9) modulation of carcinogenesis utilizing transgenic models. The program during the past 10 years was based on the strong existing graduate programs in Molecular and Cell Biology, Medicine, Microbiology-Immunology, Pathology, Tumor Cell Biology, Biochemistry, Cell Biology and Molecular Biology, and Molecular Pharmacology and Biochemistry. In this application, we add faculty from the departments of Chemistry (Evanston), Urology, and Pediatrics. Thus, graduate students admitted to any of these departments or programs and working with preceptors who are on this training grant, will be considered for support by the training grant. Despite the large number of programs from which trainees were drawn, the training will be coordinated to produce PhDs who are well trained in the study of carcinogenesis. The Integrated Graduate Program in the Life Sciences has markedly increased as the quality and number of excellent students recruited into the carcinogenesis program. Carcinogenesis training consists of one and a half years of course work in the basic life sciences and carcinogenesis followed by three years of thesis research. Integrative aspects of this program include weekly carcinogenesis group meetings and a carcinogenesis seminar program and an annual symposium given by invited speakers. These features collectively have resulted in increased interactions between preceptors and trainees, and to a higher level of collaboration among investigators involved in carcinogenesis research at Northwestern University.

DESCRIPTION (provided by applicant): Desmogleins are members of the cadherin superfamily of cell adhesion molecules. Along with desmocollins (Dscs), desmogleins (Dsgs) make up the adhesive core of the desmosome through their extracellular domains, and anchor intermediate filaments to the plasma membrane through interactions between their cytoplasmic domains and armadillo/plakin proteins. Autoantibodies directed against desmogleins are causative in the blistering diseases known as pemphigus; however, the molecular mechanism underlying pathogenesis in pemphigus is poorly understood. Furthermore, there are three Dsg genes and three Dsc genes, expressed in a differentiation-dependent manner in the epidermis, and it is unclear why multiple genes are required. To understand the underlying basis of human autoimmune and inherited disorders targeting the desmogleins, it will be crucial to determine the functions of desmosomal cadherins as well as how the adhesive complex is assembled, maintained and regulated. Our aims for the next funding period are: 1) To determine the molecular basis of desmosomal cadherin-mediated adhesion and pairing preferences for Dscs and Dsgs by using a tetracycline-regulatable system in which adhesive function can be reconstituted in L929 cells, and to develop this system into a test for the pathogenic activity of pemphigus antibodies, 2) To determine how Dsgs/Dscs collaborate with armadillo proteins to form distinct, differentiation-specific protein complexes using a combination of in vitro and high resolution structural studies to establish the identity and affinity of desmosomal cadherin-armadillo binding partners, and cellular reconstitution techniques to determine how interactions translate into structurally distinct desmosomes, 3) To determine how desmosomal cadherins and their associated proteins are coordinated spatially and temporally during their assembly into keratinocytes by time lapse analysis of living cells using fluorescently-labeled probes, and 4) To determine the function of Dsg1 and biological significance of Dsg1 as a caspase substrate for keratinocyte adhesion, differentiation and apoptosis by defining UV-dependent Dsg1 processing events that occur in vitro and in cultured keratinocytes and by defining the roles of wild type Dsg1 its cleavage products and cleavage resistant forms during differentiation in cultured human keratinocytes and transgenic mice.

[unreadable] DESCRIPTION (PROVIDED BY APPLICANT): [unreadable] This proposal requests support for the 13th EPITHELIAL DIFFERENTIATION & KERATINIZATION Gordon Research Conference to be held July 13-18th, 2003 at Tilton School, N.H. This conference has been held every other year since 1979 and continues to engage scientists in academia and industry to focus on basic science and applied research in epithelial biology. The specific aims are to 1) to build on a solid tradition of defining the most important problems and opportunities at the frontiers of epithelial biology and facilitating interactions and discussion that explore new research areas, 2) to nurture the development of young investigators who will in turn continue this tradition, 3) to ensure that the field remains vibrant and receptive to concepts and advances in related fields by bringing in and encouraging cross talk among investigators in these fields. The proposed schedule for 2003 reflects the continued, remarkable progress in cutaneous biology and other areas of epithelial morphogenesis and homeostasis. 4) outstanding speakers and chairpersons will participate in the following sessions: 1) Plenary Session on Development, Differentiation and Disease in Stratified Epithelia, 2) Cell Fate and Stem Cell Biology, 3) Morphogenesis of Epidermis and Appendages, 4) Epithelial Cytoskeleton and Polarity: Biological Structure and Imaging in the Epithelium, 4) Adhesion Signaling in Epithelial Differentiation, Migration and Disease, 5) Epithelial Defense, 6) Signaling in Epithelial Development, Differentiation, and Disease. In addition to these major sessions, two Issues Rising Sessions highlighting poster abstracts and two afternoon workshops on stem cells and epidermal appendages will cap off the program. Funds are requested to support conference fees, and travel expenses of the participants. (ie. speakers, chairpersons, graduate students, and post docs. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): By tethering the intermediate filament (IF) cytoskeleton to the plasma membrane, the desmosome plaque component desmoplakin (DP) strengthens adhesion mediated by the transmembrane desmosomal cadherins. Mutations in DP or its associated armadillo proteins result in potentially lethal disorders of the skin and heart. The loss of mechanical tissue integrity caused by desmosome dysfunction is commonly thought to underlie disease pathogenesis. However, in addition to their mechanical functions, desmosomal molecules provide signaling cues to regulate IF attachment, drive junction assembly, and guide epidermal morphogenesis. The mechanisms by which desmosomes govern signaling pathways to control tissue homeostasis and disease pathogenesis are poorly understood. We propose that the DP N-terminus and associated armadillo proteins in the plakophilin (PKP) family act as scaffolds to harness the activities of signaling mediators when and where they are needed for junction assembly and epidermal differentiation. Our goal is to determine how DP/PKP deficiency and mutations affecting DP-PKP interactions contribute to disease pathogenesis by interfering with structural and signaling functions by: 1) Determining the independent and cooperative roles of DP and PKPs in desmosome assembly and cell- cell adhesion and the effect of human disease mutations that interfere with DP-PKP interactions on these processes, 2) Testing whether DP and PKPs form scaffolds for PKC and small GTPase (RhoA) signaling mediators to integrate effector pathways that control desmosome function and cytoskeletal remodeling, and 3) Elucidating how DP works in conjunction with PKPs to promote epidermal morphogenesis and homeostasis. We will use an shRNA-dependent knock down approach combined with analysis of tissues and cells from mouse models and human patients with DP and PKP deficiencies to establish the respective roles of DP, PKPs and related signaling pathways in differentiation using 2D submerged, 3D in vitro and in vivo transplanted cultures. Desmosome-associated signaling mediators hold promise as targets for the design of small molecule therapies to ameliorate diseases caused by mutations or autoimmune antibodies that co-opt downstream pathways associated with these structural proteins.

DESCRIPTION (provided by applicant): Members of the cadherin family of cell-cell adhesion molecules are well-established players in tumor progression, but the importance of the desmosomal cadherin sub-class is poorly understood. Of particular interest is the desmoglein (Dsg) subfamily, three of which are distributed in distinct patterns within the self-renewing, complex epithelia of the oral cavity. Our overarching hypothesis is that Dsgs regulate homeostasis by sustaining proliferation in the basal layer while promoting differentiation as cells stratify, and that alterations occurring during tumor progression upset this balance. This proposal focuses on desmoglein 1 (Dsg1), which, among cadherins tested, was uniquely associated with poor patient outcome in head and neck squamous cell carcinoma (HNSCC). Dsg1 is expressed as cells emerge from the basal layer to differentiate and form a protective barrier. We showed that Dsg1 actively participates in epithelial morphogenesis by promoting a differentiation program in epidermal keratinocytes, via suppression of ErbB/MAPK signaling. Importantly, over 90% of HNSCC express elevated ErbB1. We hypothesize that Dsg1 regulates homeostasis in HNSCC by promoting differentiation while limiting tumor progression through an interaction with the ErbB binding protein ERBIN, and that bi-directional interactions between Dsgs and membrane bound sheddases in the ADAM family further regulate tumor cell fate. Our aims are to: 1) test the hypothesis that Dsg1 promotes a program of HNSCC differentiation, to test whether this occurs by attenuating MAPK signaling, and to determine whether Dsg1 loss is associated with suppressed differentiation and increased proliferation/invasion in an animal model and human HNSCCs, 2) determine whether the scaffolding protein, ERBIN, mediates Dsg1-dependent signaling in vitro and in vivo by interfering with a Ras/Raf/Shoc2 complex, and 3) test the hypothesis that Dsgs are both substrates and inhibitors of ADAM sheddase activity and determine the impact of this bi-directional relationship on Dsg-dependent adhesion and signaling in vitro, and tumor progression in vivo. Knowledge gained from this study holds promise for the design of innovative therapies including those that circumvent resistance to drugs targeting upstream players in the ErbB/MAPK pathway.

Progress in our understanding of skin biology and disease has been greatly advanced by the use of epidermal cell cultures. The Keratinocyte Core is designed to support and attract new investigators in skin biology by providing a reliable supply of highly purified preparations of human and mouse epidermal keratinocytes at a reduced cost. In addition, the Core will train researchers in the development and use of these various cell culture systems. In cases where more permanent cultures are required, immortalization and long-term culture techniques will be applied to human and mouse keratinocytes. The Core will serve as a resource for: 1) storing these cells and bulk tissue culture supplies; 2) transmitting expertise and techniques for studying keratinocytes; and 3) providing access to larger equipment suitable for keratinocyte biology (e.g., a UV irradiation lamp box, a humidified variable aerobic workstation for hypoxia studies, and a Nikon inverted microscope integrated for live cell imaging of linear scratch wounds). The Core will also provide training and supplies for a three-dimensional organotypic model for human epidermis that permits analysis of factors controlling the formation and organization of this stratified epithelium during development and in wound healing processes. Collectively, the Core will offer the necessary materials and expertise for NU SDRC investigators to better develop novel cell and molecular based strategies towards curing skin diseases and will help foster new collaborations among groups with the common goal of understanding the biology of epithelial cells.

Skin culture models serve as a key tool for understanding normal and diseased skin in well-controlled culture systems that empower investigators to probe skin biology with a rigorous model for mechanistic analysis. 3D skin culture models using primary cells are especially powerful as they closely mimic skin morphogenesis, differentiation, and mechanical properties, leading to a physiologically-relevant tissue architecture. This model is particularly applicable to study mechanisms that govern tissue development, homeostasis and disease. The Skin Tissue Engineering and Morphology (STEM) Core enables SBDRC researchers to apply primary skin culture models in their research program. Towards this aim, the STEM Core provides training, services, specialized equipment, and materials for the initiation, maintenance, processing, and analysis of primary human skin cell cultures, i.e., keratinocytes, melanocytes, fibroblasts, neurons, and immune cells (with the TEST IT Core). The STEM Core also generates short- and long-term mouse keratinocyte cultures for studies aimed at defining the cellular and molecular basis of skin defects evident in engineered mouse models. Users have access to a large supply of primary human keratinocytes isolated from neonatal foreskin, female and male adult skin, and a library of patient keratinocytes. We endeavor to maintain a diverse cell bank with donors of various demographics (age, gender, race, body site, and disease). In collaboration with GET iN Core, the STEM Core optimized methods for viral, pharmacological, and genetic reprogramming of primary skin cells, including induced pluripotent stem cells (iPSCs). Our efforts will also focus on investigator-driven research and development to customize 3D cultures and extend their applicability to address multiple aspects of skin biology from a variety of disciplines including allergy, drug discovery, materials engineering, and chemistry. Training and provision of 3D organotypic models of human epidermis established from control and patient skin cells allows investigators to address skin biology in an architecturally appropriate manner where multiple cell types can interact with one another.

Project Summary/Abstract Melanoma arises from transformation of melanocytes (MCs) in the basal layer of the epidermis where they are surrounded by keratinocytes (KCs). While research has focused predominantly on identifying driver mutations involved in conversion of MCs to melanoma, histologically normal fields of abnormal KCs are also present in sun-exposed epidermis in which melanomas arise. The extent to which melanoma is promoted by altered KCs that surround early lesions is unknown. Our long-term objectives are to define how altered KC:MC communication drives melanoma development and to identify alterations in KCs surrounding pigmented nevi that serve as predictors of malignant transformation and as therapeutic targets. KC:MC interactions occur directly through cadherins and indirectly through secreted factors. Alterations in classic cadherin expression are known to affect melanoma initiation and progression, but little is known about desmosomal cadherins' roles in melanoma. Our data support the idea that the desmosomal cadherin, desmoglein 1 (Dsg1), is critical for MC homeostasis even though it is expressed only in neighboring KCs. Loss of Dsg1 stimulates the production of KC cytokines and other secreted factors that increase MC number, dendricity, pigment production, and pro- tumorigenic cytokine expression. These features resemble the MC response to ultraviolet (UV) light, and we showed that UV results in selective loss of KC Dsg1. Our data also show that Dsg1 is suppressed by secreted factors from melanoma cells, and its expression is decreased in peri-lesional nests surrounding human melanomas and dysplastic nevi. This suggests the existence of a feedback loop that stabilizes a Dsg1-deficient pro-melanomagenic KC:MC unit within the tumor microenvironment. We propose that Dsg1's loss following UV exposure contributes to MC responses to UV, but that chronic damage and prolonged suppression of Dsg1 promotes melanoma development through coordination of pro-tumorigenic paracrine signaling and altered KC:MC contact. We will use candidate and unbiased analysis of Dsg1-deficient 2/3D human models, human tissues and a novel CRISPR/Cas Dsg1-deficient mouse model in conjunction with an HGF animal model of melanoma that closely resembles human pathology to: 1) Determine how Dsg1 loss alters the KC secretome and stimulates MC behaviors resembling the UV response through cooperative paracrine and cell contact- dependent signaling and 2) Determine how a sustained Dsg1-deficient KC:MC unit is established to promote malignant transformation and melanoma development. Understanding KC:MC communication will provide the basis for development of new biomarkers to identify individuals at high risk of developing malignant melanoma, and who may be candidates for prophylactic treatment with agents that restore normal KC:MC communication.