Donna Brennan

Noncanonical Hedgehog signaling.

The notion of noncanonical hedgehog (Hh) signaling in mammals has started to receive support from numerous observations. By noncanonical, we refer to all those cellular and tissue responses to any of the Hh isoforms that are independent of transcriptional changes mediated by the Gli family of transcription factors. In this chapter, we discuss the most recent findings that suggest that Patched1 can regulate cell proliferation and apoptosis independently of Smoothened (Smo) and Gli and the reports that Smo modulates actin cytoskeleton-dependent processes such as fibroblast migration, endothelial cell tubulogenesis, axonal extension, and neurite formation by diverse mechanisms that exclude any involvement of Gli-dependent transcription. We also acknowledge the existence of less stronger evidence of noncanonical signaling in Drosophila.

Suprabasal Dsg2 expression in transgenic mouse skin confers a hyperproliferative and apoptosis-resistant phenotype to keratinocytes.

Desmoglein 2 (Dsg2), a component of the desmosomal cell-cell adhesion structure, has been linked to invasion and metastasis in squamous cell carcinomas. However, it is unknown whether – and if so how – Dsg2 contributes to the malignant phenotype of keratinocytes. In this study, we addressed the consequences of Dsg2 overexpression under control of the involucrin promoter (Inv-Dsg2) in the epidermis of transgenic mice. These mice exhibited epidermal hyperkeratosis with slightly disrupted early and late differentiation markers, but intact epidermal barrier function. However, Inv-Dsg2 transgene expression was associated with extensive epidermal hyperplasia and increased keratinocyte proliferation in basal and suprabasal epidermal strata. Cultured Inv-Dsg2 keratinocytes showed enhanced cell survival in the anchorage-independent state that was critically dependent on EGF receptor activation and NF-κB activity. Consistent with the hyperproliferative and apoptosis-resistant phenotype of Inv-Dsg2 transgenic keratinocytes, we observed enhanced activation of multiple growth and survival pathways, including PI 3-kinase/AKT, MEK-MAPK, STAT3 and NF-κB, in the transgenic skin in situ. Finally, Inv-Dsg2 transgenic mice developed intraepidermal skin lesions resembling precancerous papillomas and were more susceptible to chemically induced carcinogenesis. In summary, overexpression of Dsg2 in epidermal keratinocytes deregulates multiple signaling pathways associated with increased growth rate, anchorage-independent cell survival, and the development of skin tumors in vivo.

Deregulation of keratinocyte differentiation and activation: a hallmark of venous ulcers.

Epidermal morphology of chronic wounds differs from that of normal epidermis. Biopsies of non‐healing edges obtained from patients with venous ulcers show thick and hyperproliferative epidermis with mitosis present in suprabasal layers. This epidermis is also hyper‐keratotic and parakeratotic. This suggests incomplete activation and differentiation of keratinocytes. To identify molecular changes that lead to pathogenic alterations in keratinocyte activation and differentiation pathways we isolated mRNA from non‐healing edges deriving from venous ulcers patients and determined transcriptional profiles using Affymetrix chips. Obtained transcriptional profiles were compared to those from healthy, unwounded skin. As previously indicated by histology, we found deregulation of differentiation and activation markers. We also found differential regulation of signalling molecules that regulate these two processes. Early differentiation markers, keratins K1/K10 and a subset of small proline‐rich proteins, along with the late differentiation marker filaggrin were suppressed, whereas late differentiation markers involucrin, transgultaminase 1 and another subset of small proline‐rich proteins were induced in ulcers when compared to healthy skin. Surprisingly, desomosomal and tight junction components were also deregulated. Keratinocyte activation markers keratins K6/K16/K17 were induced. We conclude that keratinocytes at the non‐healing edges of venous ulcers do not execute either activation or differentiation pathway, resulting in thick callus‐like formation at the edge of a venous ulcers.

Delineation of diversified desmoglein distribution in stratified squamous epithelia: implications in diseases

Abstract:  Desmogleins play critical roles in cell adhesion and skin blistering diseases, as they are the target antigens of autoimmune antibodies and bacterial toxins. We recently cloned several novel members of the desmoglein gene family, bringing the number of desmogleins to four in the rat and human genomes and six in the mouse. Here, we have produced a monoclonal antibody to a cytoplasmic epitope of Dsg4, assessed its specificity and compared it to several existing Dsg1–3 antibodies. We also demonstrated cross‐reactivity of commercially available and often used Dsg1 antibodies. Using these tools, we delineated the unique expression patterns of each desmoglein isoform in various human and mouse stratified squamous epithelia, including skin, hair, palm, and oral mucosa. Interestingly, in the epidermis, the expression of each desmoglein correlates with their gene arrangement in the cadherin locus. In human, Dsg4 was detected primarily in the granular and cornified cell layers of the epidermis, while present throughout all differentiated layers of the oral mucosa and palm, and in the matrix cells of anagen hair bulb. Similar pattern of expression for Dsg4 was observed in mouse, with the exception that it was expressed at significantly lower levels in the mouse epidermis. These results demonstrate the complexity of desmoglein gene expression and provide additional insights into the correlation between tissue expression patterns and disease phenotypes.

Increased expression of Dsg2 in malignant skin carcinomas: A tissue-microarray based study

Desmoglein 2 (Dsg2), a transmembrane cadherin of the desmosomal cell-cell adhesion structure, is downregulated with epithelial differentiation. We recently demonstrated that overexpression of Dsg2 in epidermal keratinocytes deregulates multiple signaling pathways associated with increased growth rate, anchorage-independent cell survival, and the development of skin tumors. While changes in Dsg2 expression have been observed in neoplastic lesions, the correlation of expression levels and localization of Dsg2 and the state of tumor development has not been fully established. Here we generated a highly sensitive Dsg2 antibody (Ab10) and characterized that antibody along with a previously developed Dsg2 specific antibody 10D2. Using these antibodies in immunostaining of tissue microarrays, we show a dramatic upregulation of Dsg2 expression in certain human epithelial malignancies including basal cell carcinomas (BCC; n = 12), squamous cell carcinomas (SCC; n = 57), carcinomas of sebaceous and sweat glands (n = 12), and adenocarcinomas (n = 3). Dsg2 expression was completely absent in malignant fibrosarcomas (n = 16) and melanomas (n = 15). While Dsg2 expression was consistently strong in BCC, it varied in SCC with a minor correlation between a decrease of Dsg2 expression and tumor differentiation. In summary, we have identified Dsg2 as a potential novel marker for epithelial-derived malignancies.

A role for caveolin-1 in desmoglein binding and desmosome dynamics

Desmoglein-2 (Dsg2) is a desmosomal cadherin that is aberrantly expressed in human skin carcinomas. In addition to its well-known role in mediating intercellular desmosomal adhesion, Dsg2 regulates mitogenic signaling that may promote cancer development and progression. However, the mechanisms by which Dsg2 activates these signaling pathways and the relative contribution of its signaling and adhesion functions in tumor progression are poorly understood. In this study we show that Dsg2 associates with caveolin-1 (Cav-1), the major protein of specialized membrane microdomains called caveolae, which functions in both membrane protein turnover and intracellular signaling. Sequence analysis revealed that Dsg2 contains a putative Cav-1-binding motif. A permeable competing peptide resembling the Cav-1 scaffolding domain bound to Dsg2, disrupted normal Dsg2 staining and interfered with the integrity of epithelial sheets in vitro. Additionally, we observed that Dsg2 is proteolytically processed; resulting in a 95-kDa ectodomain shed product and a 65-kDa membrane-spanning fragment, the latter of which localizes to lipid rafts along with full-length Dsg2. Disruption of lipid rafts shifted Dsg2 to the non-raft fractions, leading to the accumulation of these proteins. Interestingly, Dsg2 proteolytic products are elevated in vivo in skin tumors from transgenic mice overexpressing Dsg2. Collectively, these data are consistent with the possibility that accumulation of truncated Dsg2 protein interferes with desmosome assembly and/or maintenance to disrupt cell–cell adhesion. Furthermore, the association of Dsg2 with Cav-1 may provide a mechanism for regulating mitogenic signaling and modulating the cell-surface presentation of an important adhesion molecule, both of which could contribute to malignant transformation and tumor progression.

Compound Heterozygous Desmoplakin Mutations Result in a Phenotype with a Combination of Myocardial, Skin, Hair, and Enamel Abnormalities

Desmoplakin (DP) anchors the intermediate filament cytoskeleton to the desmosomal cadherins and thereby confers structural stability to tissues. In this study, we present a patient with extensive mucocutaneous blisters, epidermolytic palmoplantar keratoderma, nail dystrophy, enamel dysplasia, and sparse woolly hair. The patient died at the age of 14 years from undiagnosed cardiomyopathy. The skin showed hyperplasia and acantholysis in the mid- and lower epidermal layers, whereas the heart showed extensive fibrosis and fibrofatty replacement in both ventricles. Immunofluorescence microscopy showed a reduction in the C-terminal domain of DP in the skin and oral mucosa. Sequencing of the DP gene showed undescribed mutations in the maternal and paternal alleles. Both mutations affected exon 24 encoding the C-terminal domain. The paternal mutation, c.6310delA, leads to a premature stop codon. The maternal mutation, c.7964 C to A, results in a substitution of an aspartic acid for a conserved alanine residue at amino acid 2655 (A2655D). Structural modeling indicated that this mutation changes the electrostatic potential of the mutated region of DP, possibly altering functions that depend on intermolecular interactions. To conclude, we describe a combination of DP mutation phenotypes affecting the skin, heart, hair, and teeth. This patient case emphasizes the importance of heart examination of patients with desmosomal genodermatoses.

Extracellular matrix in cutaneous ageing: the effects of 0.1% copper-zinc malonate-containing cream on elastin biosynthesis.

Abstract:  Cutaneous ageing, as visualized at the exposed areas of skin, reflects dramatic alterations in the structure and function of the extracellular matrix of connective tissues. Among them, the elastic fibre network, which is responsible for the physiological elasticity and resilience of normal skin, undergoes degradative changes leading to loss of functional elastic fibres. A potential strategy to counteract these degenerative changes entails topical application of a compound that may lead to regeneration of the elastic fibre network. In this study, we have evaluated the effects of a bi‐metal, 0.1% copper–zinc malonate‐containing cream that has been shown to efface wrinkles in clinical trials. An effect on elastin biosynthesis and elastic tissue accumulation in skin biopsies was observed in 21 female patients with photoaged facial skin, as measured at baseline and at 6 weeks of treatment. Histopathological evaluation revealed evidence of elastic fibre regeneration, including those extending perpendicularly towards the dermo‐epidermal junction within the papillary dermis. Elastin biosynthesis, measured by semi‐quantitative immunofluorescence with an antibody recognizing only the newly synthesized, uncrosslinked tropoelastin molecules, suggested statistically significant enhancement of elastin biosynthesis by the bi‐metal compound when applied twice daily. Accumulation of elastic fibres was confirmed by assay of desmosine, an elastin‐specific crosslink compound. These results suggest that the bi‐metal, 0.1% copper–zinc malonate‐containing cream has the propensity to increase elastin synthesis in human skin in vivo, and that regeneration of elastic fibres may contribute to wrinkle effacement in female patients with photoaged facial skin.

N-cadherin haploinsufficiency increases survival in a mouse model of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) is often detected at a late stage, hence the identification of new therapies that have potential to block tumor progression is critical for this lethal disease. N-cadherin upregulation has been observed in many cancers including PDA, however, a causal role for this cell adhesion receptor in disease progression has yet to be defined. The concomitant expression of oncogenic KrasG12D and mutant p53 (Trp53R172H) in the murine pancreas results in metastatic PDA that recapitulates the cognate features of human pancreatic cancer providing an excellent animal model to identify genes required for tumor progression. Here we determine the consequences of genetically manipulating N-cadherin expression in a mouse model of PDA. Remarkably, mice with reduced N-cadherin expression (that is, Ncad−/+) survived 25% longer (177 vs 142 days, P<0.05) than animals expressing two wild-type N-cadherin (Cdh2) alleles. The survival benefit is likely due to a cumulative effect of N-cadherins role in different aspects of tumorigenesis including tumor-cell survival, growth, migration and invasion. Interestingly, reduced hedgehog signaling may contribute to the better prognosis for the Ncad−/+ mice. Moreover, the matrix metalloproteinase MMP-7, associated with poor prognosis in PDA, was reduced in Ncad−/+ tumors. Finally, Ncad−/+ tumor cells exhibited decreased FGF-stimulated ERK1/2 activation consistent with N-cadherins ability to promote FGFR signaling. These data support a critical role for N-cadherin in PDA and its potential prognostic value. Additionally, this study provides in vivo genetic evidence that the cell-surface protein N-cadherin represents a promising therapeutic target for the treatment of pancreatic cancer.

Superficial Dsg2 Expression Limits Epidermal Blister Formation Mediated by Pemphigus Foliaceus Antibodies and Exfoliative Toxins

Cell-cell adhesion mediated by desmosomes is crucial for maintaining proper epidermal structure and function, as evidenced by several severe and potentially fatal skin disorders involving impairment of desmosomal proteins. Pemphigus foliaceus (PF) and staphylococcal scalded skin syndrome (SSSS) are subcorneal blistering diseases resulting from loss of function of the desmosomal cadherin, desmoglein 1 (Dsg1). To further study the pathomechanism of these diseases and to assess the adhesive properties of Dsg2, we employed a recently established transgenic (Tg) mouse model expressing Dsg2 in the superficial epidermis. Neonatal Tg and wild type (WT) mice were injected with purified ETA or PF Ig. We showed that ectopic expression of Dsg2 reduced the extent of blister formation in response to both ETA and PF Ig. In response to PF Ig, we observed either a dramatic loss or a reorganization of Dsg1-α, Dsg1-β, and, to a lesser extent, Dsg1-γ, in WT mice. The Inv-Dsg2 Tg mice showed enhanced retention of Dsg1 at the cell-cell border. Collectively, our data support the role for Dsg2 in cell adhesion and suggest that ectopic superficial expression of Dsg2 can increase membrane preservation of Dsg1 and limit epidermal blister formation mediated by PF antibodies and exfoliative toxins.