Elizabeth L. Rugg

The keratins and their disorders

Diseases caused by mutations in gene encoding keratin intermediate filaments (IF) are characterized by a loss of structural integrity in the cells expressing those keratins in vivo. This is manifested as cell fragility, compensatory epidermal hyperkeratosis, and keratin filament aggregation in some affected tissues. Keratin disorders are a novel molecular category including quite different phenotypes such as epidermolysis bullosa simplex (EBS), bullous congenital ichthyosiform erthroderma (BCIE), pachyonychia congenital (PC), steatocystoma multiplex, ichthyosis bullosa of Siemens (IBS), and white sponge nevus (WSN) of the orogenital mucosa.

Ovol2 Suppresses Cell Cycling and Terminal Differentiation of Keratinocytes by Directly Repressing c-Myc and Notch1

Ovol2 belongs to the Ovo family of evolutionarily conserved zinc finger transcription factors that act downstream of key developmental signaling pathways including Wg/Wnt and BMP/TGF-β. We previously reported Ovol2 expression in the basal layer of epidermis, where epidermal stem/progenitor cells reside. In this work, we use HaCaT human keratinocytes to investigate the cellular and molecular functions of Ovol2. We show that depletion of Ovol2 leads to transient cell expansion but a loss of cells with long term proliferation potential. Mathematical modeling and experimental findings suggest that both faster cycling and precocious withdrawal from the cell cycle underlie this phenotype. Ovol2 depletion also accelerates extracellular signal-induced terminal differentiation in two- and three-dimensional culture models. By chromatin immunoprecipitation, luciferase reporter, and functional rescue assays, we demonstrate that Ovol2 directly represses two critical downstream targets, c-Myc and Notch1, thereby suppressing keratinocyte transient proliferation and terminal differentiation, respectively. These findings shed light on how an epidermal cell maintains a proliferation-competent and differentiation-resistant state.

Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re‐epithelialization of the wound area is rapid (typically <11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration.

Connective Tissue Fibroblast Properties Are Position-Dependent during Mouse Digit Tip Regeneration

A key factor that contributes to the regenerative ability of regeneration-competent animals such as the salamander is their use of innate positional cues that guide the regeneration process. The limbs of mammals has severe regenerative limitations, however the distal most portion of the terminal phalange is regeneration competent. This regenerative ability of the adult mouse digit is level dependent: amputation through the distal half of the terminal phalanx (P3) leads to successful regeneration, whereas amputation through a more proximal location, e.g. the subterminal phalangeal element (P2), fails to regenerate. Do the connective tissue cells of the mammalian digit play a role similar to that of the salamander limb in controlling the regenerative response? To begin to address this question, we isolated and cultured cells of the connective tissue surrounding the phalangeal bones of regeneration competent (P3) and incompetent (P2) levels. Despite their close proximity and localization, these cells show very distinctive profiles when characterized in vitro and in vivo. In vitro studies comparing their proliferation and position-specific interactions reveal that cells isolated from the P3 and P2 are both capable of organizing and differentiating epithelial progenitors, but with different outcomes. The difference in interactions are further characterized with three-dimension cultures, in which P3 regenerative cells are shown to lack a contractile response that is seen in other fibroblast cultures, including the P2 cultures. In in vivo engraftment studies, the difference between these two cell lines is made more apparent. While both P2 and P3 cells participated in the regeneration of the terminal phalanx, their survival and proliferative indices were distinct, thus suggesting a key difference in their ability to interact within a regeneration permissive environment. These studies are the first to demonstrate distinct positional characteristics of connective tissue cells that are associated with their regenerative capabilities.

Coherent Movement of Cell Layers during Wound Healing by Image Correlation Spectroscopy

We have determined the complex sequence of events from the point of injury until reepithelialization in axolotl skin explant model and shown that cell layers move coherently driven by cell swelling after injury. We quantified three-dimensional cell migration using correlation spectroscopy and resolved complex dynamics such as the formation of dislocation points and concerted cell motion. We quantified relative behavior such as velocities and swelling of cells as a function of cell layer during healing. We propose that increased cell volume ( approximately 37% at the basal layer) is the driving impetus for the start of cell migration after injury where the enlarged cells produce a point of dislocation that foreshadows and dictates the initial direction of the migrating cells. Globally, the cells follow a concerted vortex motion that is maintained after wound closure. Our results suggest that cell volume changes the migration of the cells after injury.

The chemical chaperone trimethylamine N‐oxide ameliorates the effects of mutant keratins in cultured cells

were negative for all corticosteroids. Due to their anti-inflammatory and immune-modifying effects corticosteroids are first-line therapy in any medical specialty treating inflammatory, allergic or autoimmune diseases including disseminated encephalomyelitis. In recent years corticosteroids have been reported to elicit a variety of adverse drug reactions, contact hypersensitivity representing the most common form. In contrast, generalized delayed-type hypersensitivity reactions are quite rare. The histopathological features and the comorbidity seen in AGEP (e.g. multiple sclerosis, psoriasis, inflammatory bowel disease, etc.) suggest an underlying tendency for a Th1-dominated immune response pattern. Here, previous reports provide evidence for a key role of drug-specific T cells in AGEP. AGEP induced by corticosteroids was first reported in a woman after subcutaneous injection of dexamethasone by Demitsu et al. In 2001, Mussot-Chia et al. – similar to our case – published a case of AGEP following high-dose methylprednisolone administration in a young woman with multiple sclerosis. Furthermore, in 2006 Buettiker et al. reported two cases of AGEP induced by oral prednisolone. Our patient cross-reacted to methylprednisolone (class A) and prednicarbate (class D2). The corresponding sensitization was apparently acquired upon the former use of corticosteroid eye drops. However, dexamethasone treatment was tolerated well and may be used in further treatment of disseminated encephalomyelitis. To our knowledge, this is the second documented case of a patient developing AGEP following group A corticosteroid treatment in disseminated encephalomyelitis. This case is instructive for any physician treating inflammatory, allergic or autoimmune-mediated diseases to keep in mind that besides their anti-inflammatory potential corticosteroids may trigger severe adverse cutaneous reactions. M. B Ä R L . JOHN S . WONSCH I K J . S CHM I TT W. KEMPT ER A . BAUER M. MEUR ER Department of Dermatology, Carl Gustav Carus Medical School, University of Technology Dresden, Fetscherstraße 74, 01307 Dresden, Germany E-mail: Michael.Baer@uniklinikum-dresden.de

Diagnosis and confirmation of epidermolytic palmoplantar keratoderma by the identification of mutations in keratin 9 using denaturing high‐performance liquid chromatography

Summary Background Epidermolytic palmoplantar keratoderma (EPPK) is one of a number of disorders characterized by diffuse thickening of palm and sole skin. Although EPPK is not a life‐threatening condition, palmoplantar keratoderma can be associated with cancer and heart disease and therefore differential diagnosis is important so that adequate surveillance can be provided for the more serious conditions. Most cases of EPPK are caused by mutations in the gene encoding the palm‐ and sole‐specific keratin 9 (K9), and this provides an option for molecular diagnosis of this condition.

Immunohistochemistry of angiogenesis mediators before and after pulsed dye laser treatment of angiomas.

Tissue effects of vascular lesion laser treatment are incompletely understood. Injury caused by pulsed dye laser (PDL) treatment may result in altered expression of mediators associated with angiogenesis.

20-P003 Cell volume and tissue shape direct cell migration in a skin explant model

The posterior lateral line primordium (pLLp) migrates caudally and periodically deposits neuromasts under the skin in the zebrafish trunk and tail. Each neuromast, formed within the migrating pLLp, has a central atoh1-positive hair cell determined by Notch mediated-lateral inhibition. The generation of new neuromasts and their deposition as the pLLp migrates caudally is coordinated by mutually antagonistic signaling centers; a Wnt signaling center at the leading edge and a FGF signaling center in the adjacent trailing domain, which determines both the morphogenesis of epithelial rosettes and expression of atoh1 in the forming neuromasts. We have now shown that the central atoh1 expressing cell in the neuromast also plays a critical role in regulating FGF signaling. When Notch signaling fails, too many cells express atoh1 and this eventually leads to failure of FGF signaling and unregulated Wnt signaling. This eventually leads to collapse and disorganization of the migrating pLLp. Computational modeling reveals how interaction between these three signaling systems and differential regulation of chemokine receptors regulates morphogenesis and migration of the pLLp. The modeling also predicts a key role played by negative feedback in the self-organization of this remarkable system.