Elevated Stratum Corneum Hydrolytic Activity in Netherton Syndrome Suggests an Inhibitory Regulation of Desquamation by SPINK5-Derived Peptides

Department of Dermatology, School of Medicine, Kanazawa University, Kanazawa, Japan.
Journal of Investigative Dermatology (Impact Factor: 7.22). 04/2002; 118(3):436-43. DOI: 10.1046/j.0022-202x.2001.01663.x
Source: PubMed


Netherton syndrome is a congenital ichthyosis associated with erythroderma, hair shaft defects, and atopic features. The mutations of the secretory serine protease inhibitor Kazal-type 5 gene have been identified in Netherton syndrome patients; however, the actual physiologic substrates of the serine protease inhibitor Kazal-type 5 proprotein are unknown, and how the genetic defects cause characteristic skin phenotype remains uncertain. Here, we describe the serine protease inhibitor Kazal-type 5 gene mutations, including two novel non-sense mutations, and genotype-phenotype correlation in three Netherton syndrome patients in two unrelated Japanese families. Furthermore, based on the reappraisal of the structure of the serine protease inhibitor Kazal-type 5 proprotein, demonstration of the presence of carboxypeptidase in normal keratinocytes, and the observation of mRNA localization of the serine protease inhibitor Kazal-type 5 transcripts in the uppermost epidermis as well as pilosebaceous units, we propose a hypothetical model of proteolytic processing of the serine protease inhibitor Kazal-type 5 proprotein in the epidermis and inhibitory regulation of corneocyte desquamation by a set of serine protease inhibitor Kazal-type 5-derived peptides. This hypothesis is supported by the marked increase of trypsin-like hydrolytic activity demonstrated in stratum corneum samples from our Netherton syndrome patients. The findings in this study suggest that the defective inhibitory regulation of desquamation due to the serine protease inhibitor Kazal-type 5 gene mutations may cause over-desquamation of corneocytes in Netherton syndrome, leading to severe skin permeability barrier dysfunction.

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    • "Thus, KLK and SPINK5 proteins co-localize and interact with each other at the SC [14]. Nonetheless, expression of KLKs is usually found throughout the stratum spinosum (SS) and SG [5] and, on the other hand, expression of SPINK5 is limited at the very surface of SG [12]. GATA3 plays a very important role for the ubiquitous expression of KLKs [15], whereas the major regulators of local expression of SPINK5 are yet known. "
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    ABSTRACT: ObjectivesSerine protease inhibitor Kazal type-5 (SPINK5) plays a crucial role in deciding the timing of desquamation of the skin. Its gene expression is limited at the very surface of the stratum granulosum (SG), whereas expression of kallikreins (KLKs) encoding proteases is usually found throughout the stratum spinosum and SG.MethodsTo explore the difference in expression regulation of these proteases/inhibitors, the function of SPINK5 promoter was examined using luciferase assay.ResultsLuciferase assay targeting the SPINK5 promoters (nucleotide −676/−532 and −318/−146 from the major transcription start site) showed high intensity in NHEK human keratinocyte. These two sites had neither common cis-elements nor GATA3 element but electrophoretic mobility shift assay showed similar retardation bands. Moreover, DNA footprinting did not display specific protected bands. Thus, we could not identify cis-element(s) that controlled these elements. Differentiation induced by high Ca2+ medium failed to alter their luciferase activities. Transfection of GATA3 expressing vector significantly but slightly increased them and that of vector expressing its dominant negative form decreased.ConclusionsAlthough GATA3 is reportedly important for inhibition of proliferation and induction of differentiation of keratinocytes, its effect on SPINK5 expression was indirect and GATA3 alone was insufficient for final differentiation of keratinocytes where full SPINK5 expression was observed.
    Environmental Health and Preventive Medicine 06/2014; 19(4). DOI:10.1007/s12199-014-0393-7
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    • "Several skin disorders are characterized by elevated protease activity (Komatsu et al. , 2002, 2008 ; Descargues et al. , 2006 ; Yamasaki et al. , 2007 ; Cork et al. , 2009 ). Thus far, treatment has mainly been non-specifi c and aimed at the symptoms rather than the cause, with unwanted side effects such as atrophy after prolonged use of glucocorticoids. "
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    ABSTRACT: SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH.
    Biological Chemistry 04/2012; 393(5):369-77. DOI:10.1515/hsz-2011-0238 · 3.27 Impact Factor
    • "It is expressed in epidermal and mucosal surfaces, tonsils, and thymus. In the epidermis, LEKTI is strongly expressed in the uppermost spinous and granular layers, and is considered to play an essential role in skin barrier formation through inhibiting activities of several proteases[45] and may play a role in local anti-inflammatory and/or antimicrobial effects. Chavanas et al., reported pathogenic mutations of SPINK5 in NS.[3] "
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    ABSTRACT: Netherton syndrome (NS) is a severe autosomal recessive ichthyosis. It is characterized by congenital ichthyosiform erythroderma, trichorrhexis invaginata, ichthyosis linearis circumflexa, atopic diathesis, and frequent bacterial infections. The disease is caused by mutations in the SPINK5 (serine protease inhibitor Kazal-type 5) gene, a new type of serine protease inhibitor involved in the regulation of skin barrier formation and immunity. We report one Chinese adult with NS. The patient had typical manifestation of NS except for trichorrhexis invaginata with an atopic diathesis and recurrent staphylococcal infections since birth. To evaluate the gene mutation and of its product activity of SPINK5 gene in confirmation of the diagnosis of one Chinese adult with NS. To screen mutations in the SPINK5 gene, 33 exons and flanking intron boundaries of SPINK5 were amplified with polymerase chain reaction (PCR) and used for direct sequencing. In addition, immunohistochemical staining of LEKTI (lymphoepithelial Kazal-type-related inhibitor) with specific antibody was used to confirm the diagnosis of NS. The results were compared with that of healthy individuals (twenty-five blood samples). A G318A mutation was found at exon 5 of patient's SPINK5 gene which is a novel missense mutation. The PCR amplification products with mutation-specific primer were obtained only from the DNA of the patients and their mother, but not from their father and 25 healthy individuals. Immunohistochemical studies indicated there was no LEKTI expression in NS patient's skin and there was a strong LEKTI expression in the normal human skin. In this report, we describe heterozygous mutation in the SPINK5 gene and expression of LEKTI in one Chinese with NS. The results indicate that defective expression of LEKTI in the epidermis and mutations of SPINK5 gene are reliable for diagnostic feature of NS with atypical clinical symptoms.
    Indian Journal of Dermatology 03/2012; 57(4):265-8. DOI:10.4103/0019-5154.97660
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