hK5 and hK7, two serine proteinases abundant in human skin, are inhibited by LEKTI domain 6

BACKGROUND: Several skin diseases and atopic disorders including Netherton syndrome and atopic dermatitis have been associated with mutations and deviations of expression of SPINK5, the gene encoding the human 15-domain serine proteinase inhibitor LEKTI. The biochemical mechanisms underlying this phenomenon have not yet been fully clarified. OBJECTIVES: To identify target proteinases of LEKTI important for processes of desquamation and inflammation of the skin which will enable the development of specific drugs. METHODS: The inhibitory activities of LEKTI domains 6 and 15 were tested on a number of commercially available serine proteinases and also on the purified kallikreins hK5 and hK7. In addition, recombinant hK5 was used. RESULTS: LEKTI domain 6 is a potent inhibitor of hK5 and hK7, whereas LEKTI domain 15 exhibits inhibitory activity on plasmin. hK5 and hK7 in particular are relevant to skin disorders. CONCLUSIONS: The inhibition of hK5 and hK7 by LEKTI domain 6 indicates an important regulatory role of LEKTI in processes of skin desquamation and inflammation, which may explain the severe pathological symptoms associated with abnormalities of SPINK5 and/or its expression. Thus, LEKTI represents a potential drug for the treatment of these disorders.     

SPINK5 knockdown in organotypic human skin culture as a model system for Netherton syndrome: effect of genetic inhibition of serine proteases kallikrein 5 and kallikrein 7

Netherton syndrome (NS; OMIM 256500) is a genetic skin disease resulting from defects in the serine protease inhibitor Kazal‐type 5 (SPINK5) gene, which encodes the protease inhibitor lympho‐epithelial Kazal type inhibitor (LEKTI). We established a SPINK5 knockdown skin model by transfecting SPINK5 small interfering RNA (siRNA) into normal human epidermal keratinocytes, which were used together with fibroblast‐populated collagen gels to generate organotypic skin cultures. This model recapitulates some of the NS skin morphology: thicker, parakeratotic stratum corneum frequently detached from the underlying epidermis and loss of corneodesmosomes. As enhanced serine protease activity has been implicated in the disease pathogenesis, we investigated the impact of the kallikreins KLK5 [stratum corneum trypsin‐like enzyme (SCTE)] and KLK7 [stratum corneum chymotrypsin‐like enzyme (SCCE)] on the SPINK5 knockdown phenotype by generating double knockdowns in the organotypic model. Knockdown of KLK5 or KLK7 partially ameliorated the epidermal architecture: increased epidermal thickness and expression of desmocollin 1 (DSC1), desmoglein 1 (DSG1) and (pro)filaggrin. Thus, inhibition of serine proteases KLK5 and KLK7 could be therapeutically beneficial in NS.     

LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a putative serine protease inhibitor encoded by serine protease inhibitor Kazal-type 5 (SPINK5). It is strongly expressed in differentiated keratinocytes in normal skin but expression is markedly reduced or absent in Netherton syndrome (NS), a severe ichthyosis caused by SPINK5 mutations. At present, however, both the precise intracellular localization and biological roles of LEKTI are not known. To understand the functional role of LEKTI, we examined the localization of LEKTI together with kallikrein (KLK)7 and KLK5, possible targets of LEKTI, in the human epidermis, by confocal laser scanning microscopy and immunoelectron microscopy. In normal skin, LEKTI, KLK7, and KLK5 were all found in the lamellar granule (LG) system, but were separately localized. LEKTI was expressed earlier than KLK7 and KLK5. In NS skin, LEKTI was absent and an abnormal split in the superficial stratum granulosum was seen in three of four cases. Collectively, these results suggest that in normal skin the LG system transports and secretes LEKTI earlier than KLK7 and KLK5 preventing premature loss of stratum corneum integrity/cohesion. Our data provide new insights into the biological functions of LG and the pathogenesis of NS.     

Analysis of SPINK 5, KLK 7 and FLG genotypes in a French atopic dermatitis cohort

The role of a genetically impaired epidermal barrier as a major predisposing factor in the pathogenesis of atopic disorders is currently under closer investigation. Variants on three candidate genes (SPINK5, KLK7 and FLG) have been associated with atopic dermatitis. A functional relevance has already been established for filaggrin variants, but not for SPINK5 and KLK7 polymorphisms. The objectives of this study were to confirm the association between SPINK5, KLK7, FLG variants and atopic dermatitis and to assess how variants influence selected phenotypic traits. This cross-sectional study was carried out over 20 months in 99 children and adults with atopic dermatitis (median age 7 years). The following items were analysed: SCORAD, TEWL, ichthyosis vulgaris, presence of asthma, total IgE serum levels. The SPINK5 E420K SNP, the KLK7 4bp insertion polymorphism and the filaggrin mutants (R510X and 2282del4) were analysed as described previously. The control group for genetic analysis was recruited in an ethnically matched, phenotypically anonymous cohort (n=102). The allelic frequencies were 0.525 for SPINK5, 0.26 for KLK7 polymorphisms, 0.101 and 0.075 for 2282del4 and R501X FLG mutants, respectively. The association of atopic dermatitis with filaggrin variants was confirmed, but not that of SPINK5 or KLK7 polymorphisms. SCORAD and TEWL measurements were not influenced by any of the variants. The SPINK5 polymorphism was associated with high IgE serum levels (p=0.011). Abnormal barrier genes do not influence the severity of atopic dermatitis. The SPINK5 gene polymorphism may modulate systemic immune effects favouring the IgE response to atopens. TEWL does not allow the characterization of subsets of patients with or without abnormal barrier genes.     

Mechanisms for a novel immune evasion strategy in the scabies mite sarcoptes scabiei: a multigene family of inactivated serine proteases

Parasitic infestation of the skin by the mite Sarcoptes scabiei is a significant problem worldwide, particularly in socially disadvantaged communities. A multigene family of at least 24 homologs of a serine protease allergen have been identified in S. scabiei. Surprisingly, the products of all but one of these genes are predicted to be catalytically inactive, due to mutations at a critical triad of amino acids at the active site. We discuss the possibility that these genes for inactivated proteases have been conserved because they mediate a novel host defense evasion strategy that the mite has evolved as an adaptation to parasitism of the epidermis. The identification of this family, and elucidation of its value to the parasite, may present an unanticipated approach to protective vaccination.     

NKp46 regulates the production of serine proteases and IL‐22 in human mast cells in urticaria pigmentosa

NKp46 (natural cytotoxic receptor 1/CD335) is expressed on natural killer cells and Th2‐type innate lymphocytes. However, NKp46 expression in human mast cells has not yet been reported. Here, we explored the expression of, and possible role played by, NKp46 in such cells. NKp46 protein was expressed in human mast cells in urticaria pigmentosa principally of the tryptase‐positive/chymase‐negative type (MCT), but not in human non‐neoplastic skin mast cells of the tryptase‐positive/chymase‐positive (MCTC) type. NKp46 expression was also evident in the human neoplastic mast cell line HMC1.2. NKp46 knockdown changed the phenotype of this cell line from MCT to MCTC and downregulated GrB production, but did not influence IL‐22 production. An agonistic anti‐NKp46 antibody upregulated production of GrB and IL‐22, but did not change the MCT‐like phenotype of HMC1.2 cells. NKp46 was thus involved in the production of serine proteases and IL‐22 in human mast cells.     

Sustained serine proteases activity by prolonged increase in pH leads to degradation of lipid processing enzymes and profound alterations of barrier function and stratum corneum integrity

We showed recently that short-term increases in stratum corneum (SC) pH are accompanied by minor alterations in permeability barrier homeostasis and SC integrity/cohesion. Since prolonged SC neutralization more closely mirrors clinical situations (i.e., neonatal skin, occupational dermatitis conditions), we assessed here whether sustained elevations of SC pH by long-term application of 1,1,3,3-tetramethylguanidine superbase provoke profound alterations in SC function. Sustained SC neutralization altered not only barrier recovery kinetics but also basal permeability barrier function. These abnormalities were attributable to a decrease in beta-glucocerebrosidase (beta-GlcCer'ase) and acidic sphingomyelinase (aSMase) catalytic activity and enzyme degradation consequent to a pH-induced sustained serine protease (SP) activity. The role of SP in this process was shown by the normalization of enzyme activities/content by co-applied SP inhibitors (SPI). To address whether lipid-processing enzymes are potential substrates for the stratum corneum chymotryptic enzyme (SCCE), protein extracts from human SC were treated for 2 h at 37 degrees C with recombinant active SCCE at pH 7.2. Recombinant SCCE induced a significant decrease in the immunoblotting of both beta-GlcCer'ase or aSMase compared with control experiments performed in the absence of the active SCCE. Finally, with sustained SC neutralization, SC integrity/cohesion deteriorated, attributable to SP-mediated degradation of corneodesmosomes (CD) as well as CD constituent proteins, desmoglein 1. These abnormalities were again reversed by co-applied SPI. In conclusion, prolonged SC neutralization provokes profound abnormalities in SC function, due to pH-induced high SP activity that, in turn, degrades lipid processing enzymes and CD proteins.     

Expression profile of the amino acid transporters SLC7A5, SLC7A7, SLC7A8 and the enzyme TDO2 in basal cell carcinoma

Basal cell carcinoma (BCC) is the most common non-malignant skin cancer and the number of patients with BCC is increasing worldwide. Despite variations between carcinomas, some tumour-specific changes can be found across all cases. These changes may include expression of tumour specific factors that could help doctors diagnose a BCC or predict its development, which consequently could lead to the development of new target medicines. In our study, we compared gene expressions in basal cell carcinomas with healthy skin. Gene expression is the process by which specific genes are activated to produce a required protein. Essentially, a cell will express certain genes depending on what the role of the cell will be. The authors of this study used microarray, a method allowing analysis of thousands of genes in one single sample. Among the results they found higher expression of genes involved in transport of amino acids (e.g. SLC7A5, SLC7A7 and SLC7A8) and amino acid metabolism (TDO2) to be of further interest. Tumour cells often depend on altered energy usage and need access to building blocks for growth. When comparing the basal cell carcinomas with healthy skin, the genes SLC7A5, SLC7A8 and TDO2 were significantly upregulated (increased) in the carcinomas. The authors could not prove that there was higher gene expression of SLC7A7. Looking at the cellular origin of the four genes, they found that proteins SLC7A5 and SLC7A8 were expressed by the tumour cells while TDO2 was mainly found in the surrounding tissue and in immune cells. The SLC7A7 protein was located in stratum granulosum, which is a thin layer of cells in the outermost layer of skin called the epidermis. SLC7A7 is not likely involved in carcinogenesis (cancer development). In conclusion, the authors identified several proteins with a potential role in BCC that may be potential targets for special drugs, but further research is needed.     

Morinda officinalis extract exhibits protective effects against atopic dermatitis by regulating the MALAT1/miR-590-5p/CCR7 axis

Background

Atopic dermatitis (AD) is a chronic inflammatory skin disease with a genetic predisposition, and the traditional Chinese medicine Morinda officinalis and its roots are characterized with anti-inflammatory effects and have been used for the treatment of various disease. However, it is still largely unknown whether Morinda officinalis extract (MOE) can be used for the treatment of AD.

Objectives

In our study we aimed to determine whether MOE could ameliorate 2,4-dinitrochlorobenzene (DNCB)-induced AD and elucidate molecular mechanisms.

Methods

We established an AD mouse model by using DNCB. Skin pathological analysis and ELISA assay were used to detect the effect of MOE on the inflammation of AD model mouse skin and the expression changes of inflammatory factors, and further functional verification was performed in TNF-α/IFN-γ-induced HaCaT cells.

Results

Our in vivo experiments confirmed that MOE remarkably reduced DNCB-induced AD lesions and symptoms, such as epidermal and dermal thickness and mast cell infiltration and inflammatory cytokines secretion in the mice models. In addition, the underlying mechanisms by which MOE ameliorated AD had been uncovered, and we verified that MOE inhibited MALAT1 expression in AD, resulting in attenuated expression of C-C chemokine receptor type 7 (CCR7) regulated by MALAT1-sponge miR-590-5p in a competing endogenous RNA (ceRNA) mechanisms-dependent manner, thereby inhibiting TNF-α/IFN-γ-induced cellular proliferation and inflammation.     

Comparative analysis of armadillo family proteins in the regulation of a431 epithelial cell junction assembly, adhesion and migration

p0071 is an armadillo family protein related to both the adherens junction protein p120ctn and to the desmosomal proteins plakophilins 1-3. p0071 assembles into both adherens junctions and desmosomes, suggesting that this protein may regulate the balance between adherens junction and desmosome formation. Furthermore, this subfamily of proteins may also regulate cell functions directly influenced by intercellular junctions, including the strength of cell adhesion and the ability of cells to migrate. These possibilities were tested by expressing exogenous p0071 in A431 epithelial cells and monitoring the effects on adhesive junction assembly in comparison to other closely related armadillo family proteins. In this model system, p0071 specifically enhanced adherens junction assembly but dramatically compromised desmosome assembly, resulting in keratin filament retraction from regions of cell-cell contact. Protein interaction studies revealed that p0071 bound to the first 160 amino-terminal residues of desmoplakin and also interacted directly with plakoglobin, suggesting that p0071 may regulate desmosome assembly by controlling plakoglobin availability. Using an in vitro assay to measure the strength of cell-cell contacts, both plakophilin-1 and p120ctn were found to increase the strength of adhesion. Interestingly, p0071 expression caused no overall changes in adhesive strength, but dramatically inhibited the ability of A431 cells to close an in vitro wound. These results suggest that p120ctn/plakophilin family proteins interact with intercellular junction binding partners to differentially modulate the adhesive and migratory behavior of epithelial cells.     

Differential expression of two new members of the p53 family, p63 and p73, in extramammary Paget's disease

Background.  The proteins p53, p63 and p73 are known to be overexpressed and to play important roles in the pathogenesis of many tumours, but the expression of p63 and p73 has not previously been investigated in extramammary Paget's disease (EMPD).
Aim.  To investigate the potential contribution of p53, p63 and p73 in the pathogenesis of EMPD.
Methods.  In total, 35 paraffin wax-embedded tissue samples from patients with EMPD were examined using immunohistochemical staining for p53, p63 and p73.
Results.  All of the 35 EMPD specimens, including all 6 invasive EMPD and 2 metastatic lymph-node specimens, showed nuclear overexpression of both p53 and p73. The expression levels (percentage of positive cells) of p53 and p73 (90.66 ± 12.53% and 80.20 ± 13.07%) in EMPD were significantly higher than those of normal skin. There was a significant correlation between the expression levels of p53 and p73 in EMPD. In 29 of 35 EMPD specimens, there was no nuclear expression of p63, and weak or moderate staining was found in only 6 specimens. The expression level of p63 in EMPD was significantly less than that in normal skin.
Conclusions.  Our study shows that the concordant overexpression of p53 and p73 and the decreased expression of p63 may play a pivotal role in the pathogenesis of EMPD. The decreased expression of p63 may play a more important role in the pathogenesis of EMPD than the overexpression of p53 and p73.     

Activation of Toll-like Receptors 1, 2, 4, 5, and 7 on Human Melanocytes Modulate Pigmentation

Human melanocytes are not simply pigment-producing cells. It may be part of the inflammatory response, during which the pigmentary system may produce more melanin or suppress melanization. Toll-like receptors (TLRs) have been implicated in both innate host defense against pathogens and inflammatory response. Therefore, it may be possible that activation of TLRs in melanocytes may play a role in the modulation of melanogenesis. In this study, we investigated whether normal human melanocytes expressed TLRs and analyzed pigmentation changes upon TLR stimulation. The expression of TLR1~10 mRNA in cultured human melanocyte was analyzed using RT-PCR, Western blotting and immunocytochemistry. Human melanocytes constitutively express mRNA and protein for TLR2, 3, 4, 5, 7, 9 and 10. Stimulation of TLR1/2 and 4 with Pam3CSK4 and lipopolysaccharide induced pigmentation of melanocytes. Activation of TLR5 and 7 with flagellin and imiquimod treatments reduced pigmentation of melanocytes and zebrafish. In summary, the results provided evidence for TLRs expression in normal human melanocytes. It is speculated that a response of melanocyte to TLR ligands may play a role in the pigmentary change in the skin.     

The diagnostic utility of p63, CK5/6, CK 7, and CK 20 in distinguishing primary cutaneous adnexal neoplasms from metastatic carcinomas

BACKGROUND: Distinguishing primary cutaneous adnexal neoplasms (PCANs) from metastatic carcinomas (MCs) can be difficult. We study the utility of p63, CK 5/6, CK 7, and 20 expression in PCAN vs. MC. METHODS: Twenty-one PCAN with sweat gland differentiation (six benign, 15 malignant), one sebaceous carcinoma, and 15 MC (14 adenocarcinomas, one urothelial carcinoma) to skin were retrieved from the pathology files. Immunostains for p63, CK 5/6, CK 7, and CK 20 were performed and graded as follows: 1, <10; 2, 11-50; and 3 >50% of tumor cells stained. RESULTS: Twenty of 22 PCAN expressed p63 and CK 5/6. Four of 15 and two of 15 MC were positive for CK 5/6 and p63, respectively. Thirteen of 22 PCAN and 13 of 15 MC were positive for CK 7, respectively. All PCAN were negative for CK 20, two of 15 MC were positive. The sensitivity and specificity for the diagnosis of PCAN were 91 and 73% for CK 5/6, 91 and 100% for p63, and 60 and 13% for CK 7, respectively. CONCLUSIONS: For distinguishing PCAN from MC: (1) positivity for p63 and CK 5/6 are relatively specific and sensitive for PCAN, (2) CK 7 and 20 are neither sensitive nor specific, and (3) CK 7 positivity in PCAN was focal with a specific pattern in contrast to the diffuse positivity for MC.     

Spatial analysis of p63, K5 and K7 defines two groups of progenitor cells that differentially contribute to the maintenance of normal sebaceous glands,extraocular sebaceous carcinoma and benign sebaceous tumors

The histogenesis of extraocular sebaceous carcinomas is – in contrast to ocular sebaceous carcinomas – unclear, and information about the exact cellular architecture of these lesions and even of the normal sebaceous gland is still scarce. This study attempts to elucidate the histogenesis of sebaceous tumors, using multicolor immunofluorescence stainings to analyze 21 cases of sebaceous tumors (six each of extraocular sebaceous carcinoma, sebaceous adenoma and sebaceoma, and three cases of steatocystomas) and eight cases of normal sebaceous glands for p63, several keratins, androgen receptor, adipophilin, epithelial membrane antigen (EMA) and Ki‐67. The data of this observational study provide evidence for the existence of two subpopulations of progenitors in normal sebaceous glands: (i) p63⁺ K5⁺ progenitors which generate the K10⁺ luminal cells of sebaceous ducts; and (ii) p63⁺ K5⁺ K7⁺ progenitors which finally generate K7⁺ adipophilin⁺ EMA⁺ sebocytes. Without exception, all types of sebaceous tumors contained p63⁺ K5⁺ cells. Furthermore, these tumors showed a cellular hierarchy and differentiation to adipophilin⁺ and/or EMA⁺ mature sebocytes and to K10⁺ ductal cells through intermediary cells. Notably, a considerable number of sebaceous tumors lack the K7 pathway of cell maintenance in the normal sebaceous lobule. Based on our data, we propose a cellular algorithmic model of the hierarchy of normal sebaceous glands and of sebocytic tumors in which p63⁺ K5⁺ cells play a major role.