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Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USADepartment of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USADepartment of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
The 61st Annual Montagna Symposium on the Biology of Skin, “Keeping It All Together: Adhesion, the Cytoskeleton and Signaling in Morphogenesis and Tissue Function,” was held in Gleneden Beach, Oregon, 11–15 October 2012. Approximately 100 scientists, clinicians, residents, postdoctoral fellows, students, industry representatives, and representatives from the National Institutes of Health attended the 4-day, five-session meeting to explore five themes and sessions, chaired by an international program committee: Adhesion, Junctions, and Cell Polarity in Tissue Morphogenesis (Carien Niessen, Ph.D.); Dynamic Mechanisms for Regulating Adhesion Strength and Cell Behavior (Andrew Kowalczyk, Ph.D.); Cell Matrix and Migration in Wound Healing and Disease (John McGrath, M.D.); Coordinating Adhesion and Signaling in Epithelia (Kathleen Green, Ph.D.); and Diseases of Adhesion: From Bench to Bedside (Masayuki Amagai, M.D.).
Fiona Watt opened the meeting with insights into the functions of the plakin family, a group of proteins that crosslink cytoskeletal elements to each other and to the plasma membrane. Dr Watt focused on envoplakin and periplakin, first identified in cornified envelopes of the epidermis and later found in desmosomes, and showed that mice lacking envoplakin, periplakin, and the cell envelope protein involucrin exhibit barrier defects and signs consistent with atopic dermatitis. Yet, in a DMBA/TPA (7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate) carcinogenesis mouse model, TPA heightened inflammatory response, increased innate immunity, and unexpectedly decreased tumor burden. Dr Watt’s talk highlighted a theme explored throughout the meeting, that is, epithelial adhesion and barrier functions communicate with the immune system to maintain tissue homeostasis.
Carien Niessen demonstrated key roles of epidermal polarity proteins and other junction molecules in cell growth, metabolism, and inflammation. By using targeted mouse models, Niessen found that classical cadherins regulate epidermal integrity and tight junction/barrier function through mechanisms that involve regulation of EGFR receptor activity and the actin cytoskeleton, respectively. This latter cytoskeleton-dependent regulation of barrier may be mediated by a protein kinase C gamma, which also balances the ratio of asymmetric and symmetric divisions required to generate different epidermal cell-fate lineages.
The structure of the resulting skin barrier can now be probed with exciting new technology, as presented by Akiharu Kubo, who used time-of-flight secondary ion mass spectrometry to reveal a novel polarized organization of three distinct layers in the stratum corneum, each contributing its own barrier properties to the epidermis. Kubo and colleagues propose that these layers may correspond to filaggrin degradation-dependent metabolic processes, as the innermost layer, containing mature filaggrin, is impaired in filaggrin-deficient mice.
Functional aspects of desmosomes that, by anchoring intermediate filaments to the plasma membrane, form a supracellular scaffolding that is remarkably resistant to mechanical stresses in skin and the heart were addressed. Terry Lechler showed that desmosomes control the redistribution of microtubules during differentiation into noncentrosomal arrays. These cortical microtubules in turn recruit myosin II to the cell cortex to engage adherens junctions (AJs), resulting in increased mechanical integrity of cell sheets and associated increased tight junction function and barrier properties. Peter Koch presented a novel transcriptional regulatory switch controlling expression of desmocollin 3 (Dsc3), normally associated with increased differentiation, and Dsc2, which is basally expressed and associated with invasive growth and tumors. He demonstrated that the transcription factor Lef1 represses Dsc3, unless plakoglobin (PG) is present, and then PG/Lef together activate its expression. PG alone or the transcription factor c-Rel, in contrast, activates Dsc2. Regulation through these pathways is likely important during wound healing, whereas disregulation is associated with skin tumor invasion.
The Hippo/Warts pathway is a transcriptional effector pathway regulated by junction proteins, which controls organ size and is also important in cancer. In an unbiased small interfering RNA screen, Valera Vasioukhin and colleagues found that the AJ protein α-catenin, whose deletion from the hair follicle stem cell niche results in prominent inflammation and development of squamous cell carcinoma tumors, interacts with Yap1, an effector in the Hippo signaling pathway. Their work showed that α-catenin controls the nuclear localization and transcriptional activity of Yap1 by preventing a Src–Yap complex from entering the nucleus where it can drive tumorigenesis. This work is a remarkable example of extrajunctional functions of AJ proteins, which have recently been shown to transduce mechanical signals from the environment to cells, to change their behavior. In this vein, Aaron Mertz presented his work on mapping traction stress exerted by cells on their substrate to examine how mechanically sensitive E-cadherin-based cell–cell adhesions communicate to their cell-matrix counterparts to control mechanics in keratinocytes. Mertz demonstrated that the formation of E-cadherin adhesions in colonies of live differentiating keratinocytes corresponds to the transition of cell–extracellular matrix force from a disorganized arrangement to myosin-II-dependent concentration of force at the colony’s periphery.
Christina Van Itallie described the application of a proteomic approach for characterizing the tight junction-ome, in which administration of biotin to cells expressing a promiscuous biotin ligase fused to a protein of interest is used to define proteins in close proximity (Roux et al. (2012), J Cell Biol, 196:801-10). By using this method, Dr Van Itallie identified hundreds of proteins in the vicinity of the tight junction proteins ZO-1 and occludin, enriched in different categories.
Andrew Kowalczyk described how the cadherin-binding protein p120 catenin covers up a newly discovered sequence motif important in the cadherin tail for preventing its endocytosis and subsequent degradation. This motif is not present in any other cell-surface receptors, so it may be specific to cadherin regulation. Dr Kowalczyk showed that endothelial cell migration, but not proliferation or barrier function, was affected by mutations that block normal endocytic cycling, implicating cadherin endocytosis as a critical factor in intercellular junction plasticity essential for endothelial and epithelial migration.
Two speakers discussed the formation and stabilization of intercellular junctions from different perspectives. Sergey Troyanovsky focused on microfilament-associated AJs, demonstrating that ectodomain interactions are sufficient for cadherin clustering at sites of cell–cell contact, but these clusters require reinforcement by additional interactions with the actin cytoskeleton. David Garrod addressed adhesive strength regulation, introducing the concept of “Hyperadhesion,” an adhesive state unique to desmosomes and characterized by strong adhesion and resistance to chelation of extracellular calcium (calcium independence). Hyperadhesion can be reversed by activation of protein kinase C alpha, such as the one that occurs at the leading edge of wounds.
Mechanical force is critical for linking form and function during morphogenesis, and it serves as a mechanism for cells to sense and respond to their environment during tissue homeostasis and in disease. Caitlin Collins used magnetic tweezers to examine mechanochemical signaling in response to tensional forces on endothelial cell PECAM-1. Her data revealed a global mechano-transduction response to forces produced by blood flow through an integrin-RhoA pathway depending on phosphoinositide 3-kinase.
Sanjeevi Sivansankar and Christopher Chen described novel approaches for measuring adhesion and cell mechanics. Dr Sivansankar presented his work on the use of atomic force microscopy to examine how cadherins convert between different functional conformations in response to forces that mimic physiological stress. These “catch,” “slip,” and “ideal” bonds allow E-cadherin molecules to withstand tensile force and tune the mechanical properties of AJs (Rakshit et al. (2012), Proc Natl Acad Sci USA 109:18815–20). Dr Chen described strategies for measuring adhesion and cell mechanics, using subcellular dimension pillars of different compliance and height to study cell behavior, the differences between two-dimensional and three-dimensional environments, and how cells can be driven toward different cell lineages by cooperative action of matrix and growth factor signals. Dr Chen closed by demonstrating an angiogenesis model whereby endothelial cells are seeded into microfabricated channels and exposed to nearby gradients to promote vessel sprouting.
Anand Reddi presented work that demonstrated microRNA-9 expression correlating with membrane α-catenin loss and β-catenin nuclear translocation, and with expression of ATP-binding cassette transporters, which have been associated with expansion of a side population of cancer stem cells in a model of squamous cell carcinoma.
John McGrath reported newly discovered mutations involved in inherited blistering skin diseases, including the first announcement of a new type of epidermolysis bullosa (EB). One of these mutations is in a protein called Slac2-b or exophilin-5, a downstream effector of a small Rab GTPase, Rab27B, involved in intracellular trafficking. Preliminary data indicate an unexpected role for this protein in keratinocyte cell migration and adhesion; Slac2-b appears to colocalize with β4 integrin at the margins of spreading keratinocytes where there is cell adhesion to a laminin-332 matrix. Ultrastructurally visualized disruption of the keratin filament network in the lower epidermis accounts for the clinical features of trauma-induced skin fragility similar to some other intraepidermal forms of EB simplex.
Although adhesion complexes are important for stable anchorage of keratinocytes to the underlying dermis, Jonathan Jones presented data indicating that α6β4 integrin steers migrating keratinocytes through a Rac/cofilin pathway, and also regulates the translation of the α3 integrin subunit in keratinocytes through an Akt/phosphoinositide 3-kinase/4EBP1 pathway. This regulates α2 integrin mRNA stability or transcription and velocity of migratory cells. His work provided another illustration of important functions of adhesion receptors that transcend their well-known roles in adhesion.
Mike DiPersio then discussed two outcomes resulting from the increased α3β1 expression occurring in response to epidermal wounding. By regulating secretion of angiogenic factors, α3β1 stimulates endothelial cells to promote angiogenesis. It also regulates basement membrane reassembly by controlling BMP-1-dependent processing of LM-332 γ2 chain and by governing expression of fibulin, needed for incorporation of LM-332 into the basement membrane. These data may help explain the basis of defects in newly described patients with mutations in the α3 gene, leading to epidermal blistering and congenital kidney defects.
Reinhard Fässler presented recent findings about kindlin, a target for the human skin fragility disease, Kindler syndrome, showing its importance not only for activation of β1 integrin but also regulation of its cell-surface presentation. Interference with the integrin–kindlin complex by mutating the kindlin-binding site on β1 interrupts normal cycling of β1 integrin, which normally is internalized and recycled to the surface many times before lysosomal degradation. Fässler’s lab determined that this occurs through interference with molecular machinery involving the sorting nexin, SNX17, which normally prevents targeting of ubiquitinated integrin into the lysosomal degradation pathway.
The next two presentations provided a new appreciation of mechanisms that integrate adhesion receptors and the cytoskeleton to drive epithelial migration. Greg Weber described the insights his studies of collective migration of Xenopus gastrula mesendoderm provide into why directional migration requires the maintenance of cell–cell adhesions. Single cells are unable to migrate directionally, but this attribute can be restored by tugging on a cadherin-coated bead stuck to a cell. Such mechano-responsive behavior requires PG-dependent recruitment of keratin intermediate filaments to the rear of the cell at sites of increased intercellular stress, and this subcellular asymmetry seems to be driven by force transmitted through cadherin, leading to degradation and inhibition of the small GTPase Rac. Martine Dunnwald’s talk on regulation of keratinocyte migration by IFN regulatory factor-6 revealed decreased directionality and velocity of Irf6-deficient keratinocytes compared with wild-type cells. Dr Dunnwald’s data suggested that this may be achieved through another small GTPase, Rho, and a GTPase-activating protein Arhgap29, which was decreased in IRf6-deficient mouse skin.
Sabine Werner identified an inverse gradient of the transcription factors Nrf2 and Nrf3 in the epidermis, with Nrf2 predominating suprabasally and Nrf3 basally. Proper balance of these factors is critical for normal skin homeostasis, controlling inflammation in wounded skin and protection from UVB-induced apoptosis through loss from the basement membrane (anoikis). Increasing Nrf2, for instance through the application of sulfurophane or reservatol, can be protective against insults such as UVB and skin cancer. Conversely, too much chronically expressed Nrf2 has adverse effects, including hyperkeratosis and scaling resembling Lamellar ichthyosis.
James Nelson presented on the structural and functional evolution of epithelia and cell–cell adhesion complexes. Contrary to common belief, epithelial organization first appeared with animals about 600 million years ago. Epithelial tubes with similarities to those found in human kidney are formed by a communal form of the cellular slime mold, Dictyostelium. Just as tube organization requires cadherin–catenin complexes in higher organisms, similar mechanisms lead to formation of tubes in this and other primitive organisms.
Kathleen Green addressed how desmosomes couple cytoskeletal remodeling and Erk inhibition to promote epidermal differentiation and morphogenesis. She reported that the desmosomal cadherin Dsg1, which is first expressed as cells that begin to stratify, promotes a transcriptional program of epidermal differentiation while also controlling the size and shape of keratinocytes during stratification. Two newly discovered binding partners of the Dsg1 cytoplasmic domain drive this program: Erbin, ErbB2-binding protein, and TcTex, a light chain associated with the dynein microtubule motor. Although Erbin is required for Dsg1 to attenuate mitogen-activated protein kinase signaling to promote the expression of differentiation proteins, TcTex binding appears to be required to regulate cortical actin organization in differentiating keratinocytes. The data support supra-adhesive functions of desmosomal cadherins during morphogenesis of stratified epithelia, beyond their roles in maintaining tissue integrity.
Andrea McClatchey’s talk on the ERM protein Ezrin continued the discussion of specialized cortical domains during the establishment of polarity in epithelia. Dr McClatchey demonstrated how restriction of Ezrin within the cell cortex, which occurs with the help of α-catenin and merlin, is important for positioning the centrosome and mitotic spindle in a three-dimensional simple epithelial cyst culture. This cortical system might be important in the transient formation of multipolar spindles leading to aneuploidy, and in mitotic catastrophe or chromosome missegregation promoting tumorigenesis.
Lisa Godsel presented insights into an armadillo family member, plakophilin-2. In addition to its function as a component of intercellular desmosomal junctions, plakophilin-2 also regulates RhoA GTPase signaling and actin remodeling, is important for assembly of cell–cell junctions, and impacts cell–substrate adhesion, focal adhesion structure, and dynamics. Although plakophilin-deficient cells showed reduced spreading, they also exhibited increased invasion associated with upregulation of MMP activity.
Greg Longmore discussed the mechanisms controlling AJ dynamics, continuing the topic of signaling through Rho GTPases. By using a genetic approach coupled with analysis of endocytic recycling in living Drosophila eyes, he demonstrated that Rho1 regulates AJ remodeling in mature epithelia through regulation of both E-cadherin endocytosis and the formation of E-cadherin-containing recycling endosomes. These occur through distinct pathways and are independent of RhoA’s effects upon actomyosin contractility.
Normal epidermal differentiation and homeostasis require basal and suprabasal transcriptional programs to coordinate. To address this challenge, complex epithelia use “Yin–Yang” strategies, such as the transcriptional switch governing expression of different desmocollin isoforms (Koch) or the inverse gradient of Nrf2 and Nrf3 in the epidermis (Werner). Spiro Getsios presented another illustration of this process through his work on Eph receptor tyrosine kinases, activated by ephrin ligands on adjacent cells and regulators of tissue patterning during development and in tumorigenesis. His group showed that downregulation of EphA2 following ephrin-A1 ligand engagement in keratinocytes permits the lower-affinity EphA1 receptor to engage ephrin-A1 ligand and promote keratinocyte differentiation. EphA2 is increased in psoriatic lesions, and pharmacological delivery of an ephrin-A1 ligand mimetic may prove useful for restoring epidermal architecture and homeostasis in inflamed skin.
Eliane Müller presented a genome-wide promoter screen using chromatin immunoprecipitation followed by oligonucleotide “chip” array, to compare targets of the related armadillo proteins PG and β-catenin, both of which have dual roles in intercellular adhesion and transcriptional regulation. The Müller group identified many specific but also overlapping targets, with a preponderance associated with the Wnt pathway, supporting a model in which intercellular adhesion controls Wnt signaling through nuclear accumulation of PG, which, with Lef1, inhibits pro-proliferative genes and enhances genes necessary for cell cycle exit at onset of keratinocyte differentiation.
The focus transitioned from bench to bedside, with Masa Amagai’s presentation on Dsg3-specific T-cell transgenic mice with T cells capable of inducing both antibody production (humoral immunity) and interface dermatitis (cellular autoimmunity). Interface dermatitis is a unique T-cell response at the dermal–epidermal interface that can be observed histologically in a number of skin disorders, including paraneoplastic pemphigus, a rare form of pemphigus that exhibits both humoral and cellular responses.
John Stanley reported that antibody phage display has permitted the cloning and characterization of pathogenic and nonpathogenic anti-Dsg monoclonal antibodies from both pemphigus vulgaris and pemphigus foliaceus patients. Data indicate that it is unlikely that pemphigus vulgaris patients have an ongoing loss of B-cell tolerance to Dsg3 but most likely have had some event that has caused loss of tolerance of a few B cells whose clones persist over time. This suggests that if oligoclonal B cells can be eliminated, pemphigus vulgaris disease can be successfully treated.
Satoshi Hirakawa presented how vascular endothelial growth factor-A can induce ectodomain shedding of LYVE-1 lymphatic vessel endothelial hyaluronan receptor, a specific lymphatic vessel marker in the skin. This shedding may contribute to chronic skin alterations such as psoriasis and lymphangiogenesis.
Jouni Uitto and Arnoud Sonnenberg delved into the role of the basement membrane zone and integrin matrix interactions in human disease. Dr Uitto described the classes of EB resulting from targeted mutations in basement membrane components. Identification of specific mutations has formed the basis for prenatal testing and development of novel molecular therapies for these diseases. Dr Sonnenberg provided molecular insights into these disorders. Keratinocytes lacking integrin α3β1 are mobilized to migrate out of the hair follicle in response to TPA treatment, resulting in a more rapid turnover before further mutations occur and a decrease in DMBA/TPA-induced papillomatous tumors.
The meeting closed with hope for patients with disorders caused by defects in structural components of the basement membrane. After describing the history of bone marrow transplantation, Jakub Tolar presented an example of how this approach can be used to treat and cure patients with dystrophic and junctional forms of EB. Healthy donor cells from the hematopoietic graft migrated to injured skin, with simultaneous increase in the production of skin-specific structural proteins deficient in EB. Anchoring fibrils were identified 3 years after bone marrow transplant, and patients experienced significant improvement. Although there is much to learn about what cell types home to the skin and why, this approach holds much promise as a durable, systemic therapy for patients with EB and related disorders.
Society for Investigative Dermatology Eugene M. Farber Travel Awards for Young Investigators
Abhilasha Gupta, Ph.D., Thomas Jefferson University; Aaron F. Mertz, M.Phil., Yale University; Fanny Loschke, M.Sc., University of Leipzig; Andrew Muroyama, Duke University; Nicole Najor, Ph.D., Northwestern University; Anand Reddi, University of Colorado School of Medicine; Emanuel Rognoni, M.S., Max Planck Institute of Biochemistry; Sara N. Stahley, Emory University; Tong San Tan, National University of Singapore.
The Montagna Symposium on the Biology of Skin, directed by Molly F. Kulesz-Martin, Ph.D., is an annual nonprofit scientific meeting, initiated in 1950 by Dr William Montagna, to gather leading cutaneous biologists and dermatologists to discuss new findings, techniques, and goals in skin biology. The Montagna Symposium on the Biology of Skin is supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the National Institute of Aging under award number R13AR009431. Other supporters included the following: Epidermolysis Bullosa Medical Research Foundation; The Procter & Gamble Company; The Company of Biologists; Ashland Specialty Ingredients; Integrated DNA Technologies; CELLnTEC; OHSU Knight Cancer Institute; and The Orentreich Family Foundation. Dr Fiona Watt’s “EMBO Keynote Lecture” was supported by the European Molecular Biology Organization.
*The 61st Annual Montagna Symposium on the Biology of Skin, “Keeping It All Together: Adhesion, the Cytoskeleton, and Signaling in Morphogenesis and Tissue Function,” was held in Gleneden Beach, Oregon, 11–15 October 2012.