Edel A. O'Toole

Inflammatory Skin and Bowel Disease Linked to ADAM17 Deletion

We performed genetic and immunohistochemical studies in a sister and brother with autosomal recessive neonatal inflammatory skin and bowel lesions. The girl died suddenly at 12 years of age from parvovirus B19-associated myocarditis; her brother had mild cardiomyopathy. We identified a loss-of-function mutation in ADAM17, which encodes a disintegrin and metalloproteinase 17 (also called tumor necrosis factor α [TNF-α]-converting enzyme, or TACE), as the probable cause of this syndrome. Peripheral-blood mononuclear cells (PBMCs) obtained from the brother at 17 years of age showed high levels of lipopolysaccharide-induced production of interleukin-1β and interleukin-6 but impaired release of TNF-α. Despite repeated skin infections, this young man has led a relatively normal life. (Funded by Barts and the London Charity and the European Commission Seventh Framework Programme.).

The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, is mutated in inherited anonychia

Anonychia and hyponychia congenita (OMIM 206800) are rare autosomal recessive conditions in which the only presenting phenotype is the absence or severe hypoplasia of all fingernails and toenails. After determining linkage to chromosome 20p13, we identified homozygous or compound heterozygous mutations in the gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, in eight affected families. Rspo4 expression was specifically localized to developing mouse nail mesenchyme at embryonic day 15.5, suggesting a crucial role in nail morphogenesis.NOTE: In the version of this article initially published, the national origin of the three consanguineous families (P2–P4) was incorrectly described as Indian. The national origin of all three families (P2–P4) is Pakistani. This error has been corrected in the HTML and PDF versions of the article.

Extracellular matrix and keratinocyte migration.

We are just beginning to understand some of the cellular mechanisms involved in human keratinocyte migration on extracellular matrix. Extracellular matrix components have differing effects on keratinocyte motility. Signalling through integrin receptors and secretion of collagenase are both components of this process. An understanding of the effect of extracellular matrix on keratinocyte migration has direct relevance to the problem of wound re‐epithelialization and will assist in the development of therapeutic efforts to enhance wound healing artificially.

Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of basement-membrane type VII collagen

Type VII collagen (ColVII) is the main component of anchoring fibrils, attachment structures within the lamina densa of the basement membrane that are responsible for attachment of the epidermis to the dermis in skin. Mutations in the human ColVII gene, COL7A1, cause the severe inherited blistering disorder recessive dystrophic epidermolysis bullosa (RDEB) affecting skin and mucosae, associated with a greatly increased risk of skin cancer. In this study, we examined the effect of loss of ColVII on squamous cell carcinoma (SCC) tumourigenesis using RNAi in a 3D organotypic skin model. Our findings suggest that loss of ColVII promotes SCC migration and invasion as well as regulating cell differentiation with evidence for concomitant promotion of epithelial-mesenchymal transition (EMT). Immunostaining of RDEB skin and a tissue array of sporadic cutaneous SCCs confirmed that loss of ColVII correlates with decreased involucrin expression in vivo. Gene-expression-array data and immunostaining demonstrated that loss of ColVII increases expression of the chemokine ligand-receptor CXCL10-CXCR3 and downstream-associated PLC signalling, which might contribute to the increased metastatic potential of SCCs with reduced or absent ColVII expression. Together, these findings may explain the aggressive behaviour of SCCs in RDEB patients and may also be relevant to non-RDEB skin cancer, as well as other tumours from organs where ColVII is expressed.

Functional studies of human skin disease- and deafness-associated connexin 30 mutations

Connexin 30 (Cx30) is a component of the gap junction complex. Dominant and recessive mutations in the GJB6 gene encoding Cx30 are associated with a variety of human inherited diseases primarily affecting the epidermis, hair, nail, and/or the inner ear. The underlying mechanism of disease associated with different GJB6 mutations such as the disruption of gap junction mediated intercellular communication is unknown. Towards understanding these disease mechanisms, transfection studies were performed in a keratinocyte cell line and in HeLa cells using EGFP tagged wildtype Cx30 and mutant Cx30 constructs harbouring dominant disease-associated GJB6 mutations. For all three of the skin disease-associated Cx30 mutations investigated, impaired trafficking of the protein to the plasma membrane was observed thus preventing the formation of functional Cx30 gap junctions. In contrast, the deafness-associated mutation T5M-Cx30/EGFP trafficked to the membrane but defective channel activity was observed following dye transfer studies.

Hydrogen peroxide inhibits human keratinocyte migration

BACKGROUND Reepithelialization is an important component of wound healing. In the first 48 hours keratinocyte migration and proliferation are important events in this process. Although the literature agrees that the risk/benefit of antiseptics has not been established, hydrogen peroxide is still commonly used in the management of acute and chronic wounds. OBJECTIVE The purpose of this study was to evaluate the effect of hydrogen peroxide on human keratinocyte migration and proliferative potential. METHODS The viability and proliferative potential of human keratinocytes in the presence of hydrogen peroxide was assessed by trypan blue exclusion, cell morphology, substratum attachment, and thymidine incorporation. Using concentrations of hydrogen peroxide that do not affect keratinocyte viability, keratinocyte migration was evaluated by a standard motility assay. RESULTS Hydrogen peroxide in concentrations ≤700 fan was found to have no effect on keratinocyte viability. At these low concentrations, however, hydrogen peroxide had a profound inhibitory effect upon keratinocyte migration on extracellular matrix and decreased the proliferative potential of the cells in a concentration‐dependent fashion. CONCLUSION Hydrogen peroxide, in very low concentrations (1000‐fold less than the “everyday use” dilution) inhibits keratinocyte migration and proliferation.

LEKTI demonstrable by immunohistochemistry of the skin: a potential diagnostic skin test for Netherton syndrome

Background   Netherton syndrome (NS) is a rare autosomal recessive condition characterized by ichthyosiform erythroderma, trichorrhexis invaginata and atopic manifestations. Confirming the diagnosis may be difficult in the early stages. Mutations in the SPINK5 gene which encodes for the serine protease inhibitor LEKTI are associated with NS. These mutations create premature termination codons which result in absent or abnormal expression of LEKTI in patients with NS.

Development and characterization of a recombinant truncated type VII collagen "minigene": implication for gene therapy of dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder of skin caused by mutations in the type VII collagen gene. The lack of therapy for DEB provides an impetus to develop gene therapy strategies. However, the full-length 9-kilobase type VII collagen cDNA exceeds the cloning capacity of current viral delivery vectors. In this study, we produced a recombinant type VII minicollagen containing the intact noncollagenous domains, NC1 and NC2, and part of the central collagenous domain using stably transfected human 293 cell clones and purified large quantities of the recombinant minicollagen VII from culture media. Minicollagen VII was secreted as correctly-folded, disulfide-bonded, helical trimers resistant to protease degradation. Purified minicollagen VII bound to fibronectin, laminin-5, type I collagen, and type IV collagen. Furthermore, retroviral-mediated transduction of the minigene construct into DEB keratinocytes (in which type VII collagen was absent) resulted in persistent synthesis and secretion of a 230-kDa recombinant minicollagen VII. In comparison with parent DEB keratinocytes, the gene-corrected DEB keratinocytes demonstrated enhanced cell-substratum adhesion, increased proliferative potential, and reduced cell motility, features that reversed the DEB phenotype toward normal. We conclude that the use of the minicollagen VII may provide a strategy to correct the cellular manifestations of gene defects in DEB.

Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours

The molecular mechanisms that underlie the development of squamous cell skin cancers (SSC) are poorly understood. We have used oligonucleotide microarrays to compare the differences in cellular gene expression between a series of keratinocyte cell that mimic disease progression with the aim of identifying genes that may potentially contribute towards squamous cell carcinoma (SCC) progression in vivo, and in particular to identify markers that may serve as potential therapeutic targets for SCC treatment. Gene expression differences were corroborated by polymerase chain reaction and Western blotting. We identified Axl, a receptor tyrosine kinase with transforming potential that has also been shown to have a role in cell survival, adhesion and chemotaxis, was upregulated in vitro in SCC-derived cells compared to premalignant cells. Extending the investigation to tumour biopsies showed that the Axl protein was overexpressed in vivo in a series of SCCs.

Connexin interaction patterns in keratinocytes revealed morphologically and by FRET analysis

Multiple connexins, the major proteins of gap junctions, have overlapping expression in the human epidermis and are postulated to have a key role in keratinocyte differentiation and homeostasis. The functional importance of connexins in the epidermis is emphasised by the association of mutations in four human connexins with various hyperproliferative skin disorders. As immunohistochemistry demonstrated overlapping expression of specific connexins in keratinocytes, we performed colocalisation analyses and applied a modified FRET methodology to assess possible heteromeric interactions between different combinations of four wild-type (wt) and mutant connexins. The data generated indicate that there is evidence for multiple connexin interactions at the plasma membrane between (wt)Cx26, (wt)Cx30 and (wt)Cx31 in keratinocytes and thus, the potential for the formation of a large number of different channel types each with different channel properties. In addition, we demonstrate that the inherent in vitro trafficking defect of the skin disease mutations (D50N)Cx26 and (G11R)Cx30 can be overcome partially by the coexpression of different wild-type connexins but this rescue does not result in large gap junction aggregates at the plasma membrane. These data indicate that skin disease associated Cx26 or Cx30 mutations are likely to disrupt a number of different channel types important in distinct aspects of keratinocyte biology.