Search

Novel insights into intra-cellular signalling involved in pemphigus vulgaris (PV), an autoimmune blistering disease of skin and mucous membranes, are now revealing new therapeutic approaches such as the chemical inhibition of PV-associated signals in conjunction with standard immunosuppressive therapy. However, extensive inhibition of signalling molecules that are required for normal tissue function and integrity may hamper this approach. Using a neonatal PV mouse model, we demonstrate that epidermal blistering can be prevented in a dose-dependent manner by clinically approved EGFR inhibitors erlotinib and lapatinib, but only up to approximately 50% of normal EGFR activity. At lower EGFR activity, blisters again aggravated and were highly exacerbated in mice with a conditional deletion of EGFR. Statistical analysis of the relation between EGFR activity and the extent of skin blistering revealed the best fit with a non-linear, V-shaped curve with a median break point at 52% EGFR activity (P = 0.0005). Moreover, lapatinib (a dual EGFR/ErbB2 inhibitor) but not erlotinib significantly reduced blistering in the oral cavity, suggesting that signalling mechanisms differ between PV predilection sites. Our results demonstrate that future clinical trials evaluating EGFR/ErbB2 inhibitors in PV patients must select treatment doses that retain a specific level of signal molecule activity. These findings may also be of relevance for cancer patients treated with EGFR inhibitors, for whom skin lesions due to extensive EGFR inhibition represent a major threat., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

There is mounting evidence that Th2 cytokines adversely affect skin barrier functions and contribute to the pathogenesis of atopic dermatitis (AD). AD is also characterized by abnormal cohesion in the stratum corneum (SC). However, the contribution of Th2 cytokines to this abnormality remains unknown. This study examined the effects of IL-4, a prototypic Th2 cytokine, on the cohesion of the SC. Structural and physiological assessments revealed that repeated intradermal injections of IL-4 compromised the cohesion of the SC of normal hairless mice. Two potential mechanisms were explored to account for the altered cohesion. First, IL-4 decreased the amount of corneodesmosomes and down-regulated the expression of desmoglein 1, but not of corneodesmosin (CDSN) or loricrin expression, in murine skin and in cultured human keratinocytes (KC). IL-4 did not affect the skin surface pH, and in situ zymography revealed no net change in total serine protease activity in the IL-4-treated SC. Yet, IL-4 enhanced expression of kallikrein (KLK)7, while simultaneously down-regulating KLK5 and KLK14. Finally, IL-4 did not alter the expression of the lympho-epithelial Kazal-type inhibitor (LEKTI) in KC. This study suggests that IL-4 abrogates the cohesion of SC primarily by reducing epidermal differentiation., (© 2012 John Wiley & Sons A/S.)

To maintain stratum corneum integrity while simultaneously desquamating at a steady rate, degradation of corneodesmosomes must proceed in a controlled manner. It is unknown why corneodesmosomes are present only at the cell periphery in the upper stratum corneum. To explore this, we studied distributions of three major corneodesmosomal components, corneodesmosin, desmoglein 1 and desmocollin 1 in normal adult human epidermis. Immunofluorescent microscopy studies of skin surface corneocytes detected all three components only at the cell edges. Immunoelectron microscopy revealed selective loss of these components at the central areas starting from the deep cornified layers. We hypothesized that tight junctions (TJs) formed in the superficial granular layer may prevent protease access by functioning as a barrier between the peripheral and the central intercellular spaces in the stratum corneum. Ultrastructural examination demonstrated TJs up to the junctions between the seventh and the eighth deepest cornified layers. Immunoelectron microscopy also detected clusters of occludin and claudin-1 immunolabels at the cell periphery, and kallikrein 7 immunolabels outside of TJs in the lower cornified layers. With colloidal lanthanum nitrate perfusion assay of stripped stratum corneum, the tracer was excluded from TJ domains. Taken together, we propose that TJs inhibit access of proteases to the peripheral corneodesmosomes forming the structural basis for the basket-weave-like appearance of the stratum corneum., (© 2010 John Wiley & Sons A/S.)

Epidermal expression of adhesion molecules such as desmogleins (Dsg) and cadherins is strongly affected by the differentiation status of keratinocytes. We have previously shown that certain protein kinase C (PKC) isoforms differentially alter the growth and differentiation of human epidermal HaCaT keratinocytes. In this paper, using recombinant overexpression and RNA interference, we define the specific roles of the different PKC isoenzymes in modulation of expression of adhesion molecules in HaCaT keratinocytes. The level of Dsg1, a marker of differentiating keratinocytes, was antagonistically regulated by two Ca-independent 'novel' nPKC isoforms; i.e. it increased by the differentiation-promoting nPKCdelta and decreased by the growth-promoting nPKCepsilon. The expression of Dsg3 (highly expressed in proliferating epidermal layers) was conversely regulated by these isoenzymes, and was also inhibited by the differentiation inducer Ca-dependent 'conventional' cPKCalpha. Finally, the expression of P-cadherin (a marker of proliferating keratinocytes) was regulated by all of the examined PKCs, also in an antagonistic manner (inhibited by cPKCalpha/nPKCdelta and stimulated by cPKCbeta/nPKCepsilon). Collectively, the presented results strongly argue for the marked, differential, and in some instances antagonistic roles of individual Ca-dependent and Ca-independent PKC isoforms in the regulation of expression of adhesion molecules of desmosomes and adherent junctions in human epidermal keratinocytes.

Heparanase is a heparan sulphate endo-glycosidase which was previously detected in the outer root sheath of murine hair follicles. Heparanase overexpression was reported to improve mouse hair (re)growth. In this study, we investigated its involvement in human hair biology. Immunofluorescence detection was used to explore heparanase distribution in both anagen and catagen hair follicles. Heparanase functionality was assessed in in vitro cultured hair follicles, in the presence of a heparanase activity inhibitor. Our results showed that heparanase expression was (i) primarily located in the inner root sheath (IRS) of human hair follicle, and there (ii) restricted to anagen phase. Furthermore, inhibition of heparanase in in vitro cultured hair follicles induced a catagen-like process. Hair shaft retreat upward was accompanied by a decrease in Ki67-positive cells, the formation of an epithelial strand as evidenced by K14 keratin expression, and the loss of IRS as assessed by transglutaminase 1 and desmoglein labelling. IRS distribution of heparanase and the induction of catagen-like involution of hair follicles when a potent heparanase inhibitor is added suggest that heparanase is a key actor of IRS differentiation and hair homeostasis.

We have previously demonstrated that serum of patients with pemphigus vulgaris induces reduction of desmoglein 3 (Dsg3) half-life in keratinocytes (FEBS Lett 2006: 580: 3276). This phenomenon seems to occur as a consequence of the progressive depletion of Dsg3 from desmosomes. Here we reported that reduction of full-length Dsg3 may be due to its progressive cleavage, leading to the formation of two fragmentation products with apparent molecular masses of about 60 kDa (fragment 1) and 70 kDa (fragment 2), as revealed by Western blotting. Unexpectedly, analysis of fragmentation pattern suggested cleavage to occur intracellularly. Consistently, fragment 1 was shed and localized within the cytosol, as shown by living cell immunofluorescence microscopy. Total amounts of full-length plakoglobin and Dsg1 were apparently unchanged. Taken together, our findings provide evidence that proteolytic processing of Dsg3 can lead to depletion of Dsg3 from the cell.

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.