| Περίληψη: | The epidermis forms the protective barrier that is essential for survival. Stratum corneum (SC) is the outermost layer of the epidermis, which provides resistance to mechanical stress, while it prevents exposure to chemicals and allergens, entry of microbes and water loss. The SC is composed of multiple layers of terminally differentiated keratinocytes that are embedded in a lipid matrix. Cohesion of corneocytes is achieved via the corneodesmosomes that are specialized structures composed of the corneodesmosomal proteins desmoglein 1 (DSG1), desmocollin 1 (DSC1) and corneodesmosin (CDSN).
Skin desquamation is a physiological process during which the corneocytes of the outer layer of the SC are detached (shed) and replaced by new corneocytes. This process depends on the tightly regulated activity of serine proteases expressed in the epidermis. Serine proteases like the KLK5, KLK7 and KLK14 of the kallikrein-related peptidases (KLKs) family cleave desmosomal proteins, thus, providing the proteolytic activities that regulate the process of skin desquamation (Sotiropoulou et al., 2009; Borgoño et al., 2007; Brattsand et al., 2005; Caubet et al., 2004). In particular, KLK5 is the upstream activator of a regulatory proteolytic cascade in the epidermis, since it is able to autoactivate (Michael et al., 2005). Subsequently, mature (active) KLK5 activates other downstream KLK pro-enzymes, like the KLK7 and KLK14 (Sotiropoulou et al., 2009; Michael et al., 2005; Brattsand et al., 2005).
Peeling Skin Disease (PSD) is a rare autosomal recessive form of congenital ichthyosis, caused by inactivating mutations in the gene encoding CDSN. PSD patients present extensive skin peeling, inflammation, and allergies (Oji et al., 2010; Hacham-Zadeh and Holubar, 1985). Histological examination of skin biopsies reveals acanthosis, hyperkeratosis, parakeratosis, and extensive detachment of the SC from the subjacent stratum granulosum (SG).
Examination of the skin of PSD patients revealed that KLK5 is highly expressed and potentially activated (Oji et al., 2010; Komatsu et al., 2006). The abnormally high KLK5 activity could lead to unregulated cleavage of corneodesmosomes and create the overdesquamating phenotype. Further, it has been suggested that CDSN protects corneodesmosomes from premature proteolysis (Lundström et al., 1994). Therefore, CDSN deficiency, in addition to elevated proteolysis, could account for the pathological overdesquamation in PSD.
Many of the PSD symptoms are reproduced by the mouse model that lacks the Cdsn gene (Cdsn-/-). As in many cases of invalidation of genes expressed in late differentiation of epidermis, Cdsn-/- mice die soon after birth due to extensive desquamation and severe skin barrier defect leading to severe dehydration (Leclerc et al., 2009; Matsumoto et al., 2008). However, Cdsn-/- mice do not recapitulate all major pathological features of the disease, like the epidermal inflammation observed in PSD patients (Zaafouri et al., 2017).
According to all the above, elimination of Klk5 in Cdsn-/- mice could provide evidence regarding the functional role(s) of KLK5 in the pathophysiology of PSD. If KLK5 drives the symptoms of PSD, Cdsn-/-Klk5-/- double knockout (DKO) mice would be expected to show signs of alleviation of the severe PSD symptoms, when compared to Cdsn-/- mice. Nevertheless, it was found that ablation of Klk5 is not sufficient to rescue the lethal desquamating phenotype of the Cdsn-/- mice and Cdsn-/-Klk5-/- mice still exhibited neonatal lethality (Sotiropoulou et al., unpublished results) The macroscopic appearance of Cdsn-/-Klk5-/- mice showed extensive SC detachment with red, shiny, and sticky peeled skin. Histological examination of skin tissue sections from Cdsn-/-Klk5-/- mice revealed persistent SC detachment with no other microscopic alterations. In addition, the function of the epidermal barrier was examined by analyzing the epidermal differentiation profile. It was shown that differentiation markers, such as the involucrin, is normalized in Cdsn-/-Klk5-/- mice, nonetheless, the expression of Dsg1 was not altered in Cdsn-/-Klk5-/- mice.
As ablation of Klk5 reduces the overall proteolytic activity in the epidermis relative to wt mice (Pampalakis et al., 2019), while KLK5 activity is elevated in PSD, we analyzed the proteolytic activities of Cdsn-/-Klk5-/- epidermis by in situ zymography, which revealed that elastinolytic and gelatinolytic activities were increased in the epidermis of Cdsn-/-Klk5-/- mice, while RT-qPCR showed overexpression of Elane. Therefore, deletion of KLK5 does not suppress the abnormally increased overall epidermal proteolysis in Cdsn-/- epidermis. It is hypothesized that other proteolytic enzymes may be activated and cause overdesquamation and enhanced proteolysis in Cdsn-/-Klk5-/- epidermis.
On the other hand, lipids constitute the other major component of the SC that is necessary for maintenance of a normal skin barrier. Abnormal lipid composition and/or organization has been linked to diseased skin (Elias et al., 2014). Here, 1H-NMR spectroscopy and TLC were used to determine qualitative and quantitative lipid alterations in Cdsn-/- and Cdsn-/-Klk5-/- mouse models. Lipid profile analyses revealed abnormal contents of total cholesterol (CH) and free fatty acids (FFAs) in Cdsn-/- that were normalized in Cdsn-/-Klk5-/- neonatal epidermis comparably to wt epidermis.
Finally, analysis of the lipid content was performed in clinical specimens. Specifically, SC flakes from healthy individuals and lesional and non lesional skin from one PSD patient were obtained and their lipid contents were compared. It was found that the lesional skin contained increased FFAs and CH, while the non lesional skin showed expression of FFAs and CH between healthy and lesional SC. These results suggest that CDSN deficiency is linked with alterations in epidermal lipids that become more pronounced in epidermal lesions.
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