Mari Kishibe

Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity

Mutations in SPINK5, encoding the serine protease inhibitor LEKTI, cause Netherton syndrome, a severe autosomal recessive genodermatosis. Spink5−/− mice faithfully replicate key features of Netherton syndrome, including altered desquamation, impaired keratinization, hair malformation and a skin barrier defect. LEKTI deficiency causes abnormal desmosome cleavage in the upper granular layer through degradation of desmoglein 1 due to stratum corneum tryptic enzyme and stratum corneum chymotryptic enzyme–like hyperactivity. This leads to defective stratum corneum adhesion and resultant loss of skin barrier function. Profilaggrin processing is increased and implicates LEKTI in the cornification process. This work identifies LEKTI as a key regulator of epidermal protease activity and degradation of desmoglein 1 as the primary pathogenic event in Netherton syndrome.

Oculocutaneous Albinism Type 4 Is One of the Most Common Types of Albinism in Japan

Oculocutaneous albinism (OCA) is a complex genetic disease with great clinical heterogeneity. Four different types of OCA have been reported to date (OCA1, OCA2, OCA3, and OCA4). MATP was recently reported in a single Turkish OCA patient as the fourth pathological gene, but no other patients with OCA4 have been reported. Here, we report the mutational profile of OCA4, determined by genetic analysis of the MATP gene in a large Japanese population with OCA. Of 75 unrelated patients that were screened, 18 individuals (24%) were identified as having OCA4; they harbored seven novel mutations, including four missense mutations (P58S, D157N, G188V, and V507L) and three frameshift mutations (S90CGGCCA-->GC, V144insAAGT, and V469delG), showing that MATP is the most frequent locus for tyrosinase-positive OCA in Japanese patients. We discuss the functional melanogenic activity of each mutant allele, judging from the relationship between the phenotypes and genotypes of the patients. This is the first report on a large group of patients with OCA4.

Kallikrein 8 Is Involved in Skin Desquamation in Cooperation with Other Kallikreins

Kallikrein type serine proteases, KLK8/neuropsin, KLK6, and KLK7, have been implicated in the proliferation and differentiation of epidermal keratinocytes and in the pathogenesis of psoriasis. However, their mechanistic roles in these processes remain largely unknown. We applied 12-O-tetradecanoylphorbol-13-acetate on the wild type (WT) and the Klk8 gene-disrupted (Klk8-/-) mouse skin, inducing keratinocyte proliferation similar to the human psoriatic lesion. Klk8 mRNA as well as Klk6 and Klk7 mRNA were up-regulated after 12-O-tetradecanoylphorbol-13-acetate application in the WT mice. In contrast, Klk8-/- mice showed minimum increases of Klk6 and Klk7 transcripts, the proteins, and enzymatic activities. Relative to the WT, the Klk8-/- skin showed less proliferation and an increase in the number of cell layers in the stratum corneum. However, overexpression of Klk8 by adenovirus vector in knock-out keratinocytes did not result in an increase in Klk6 or Klk7 mRNA. The inefficient cleavage of adhesion molecules DSG1 and CDSN in Klk8-/- skin contributes to a delay in corneocyte shedding, resulting in the hyperkeratosis phenotype. We propose that in psoriatic lesion, KLK8 modulates hyperproliferation and prevents excessive hyperkeratosis by shedding the corneocytes.

Order and disorder in corneocyte adhesion

Epidermal cornified cells are attached to each other with modified desmosomes, namely corneodesmosomes. Changes in the corneodesmosome degradation process influence the total thickness of the stratum corneum and surface appearance of the skin. The major extracellular constituents of corneodesmosomes are desmoglein 1, desmocollin 1 and corneodesmosin. The intracellular part of corneodesmosomes is cross‐linked into cornified cell envelopes. Corneodesmosomes are degraded from the central surface area of each cell. Peripheral corneodesmosomes retain structural integrity up to the skin surface. A hypothesis where tight junctions in the stratum corneum play a role in this spatial difference in corneodesmosome degradation has recently been proposed. Genetic defects in corneodesmosin and inhibitors for proteases involved in corneodesmosome degradation result in accelerated desquamation and severe barrier impairment, presenting as the inflammatory type of peeling skin syndrome and Netherton syndrome, respectively. Abnormal corneodesmosome degradation is also found in more common skin diseases including ichthyosis vulgaris, atopic dermatitis, psoriasis vulgaris, lichen planus and soap‐induced xerosis.

Tight junctions in the stratum corneum explain spatial differences in corneodesmosome degradation.

Abstract:  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.

Implications of protease M/neurosin in myelination during experimental demyelination and remyelination.

Protease M/neurosin is a serine protease expressed by oligodendrocytes (OLGs) in the central nervous system (CNS). To investigate the role of protease M/neurosin during experimental demyelination and remyelination, mice were fed cuprizone (bis-cyclohexanon oxaldihydrazone). Semi-quantitative RT-PCR analysis and immunohistochemistry revealed that the expressions of protease M/neurosin mRNA and protein were rapidly reduced in demyelination, whereas the expression of protease M/neurosin was increased in pi form of glutathione-S-transferases (GST-pi)-positive OLGs during remyelination. Cultured primary OLGs displayed a strong correlation between protease M/neurosin and myelin basic protein (MBP). After tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma stimulation, these proteins showed colocalization in the oligodendroglial process. The suppression of protease M/neurosin using RNAi reduced the level of MBP mRNA in cultured OLGs. In contrast, the reduced level of protease M/neurosin was not associated with oligodendroglial cell death or differentiation in cultured OLGs. This study identifies that protease M/neurosin in OLGs is closely associated with the expression of the MBP and the PLP gene. Our data emphasize that the maintenance of myelination is an important function of protease M/neurosin in OLGs, suggesting its relation to the oligodendroglial response to myelin disorders.

Lamellar Granule Secretion Starts before the Establishment of Tight Junction Barrier for Paracellular Tracers in Mammalian Epidermis

Defects in epidermal barrier function and/or vesicular transport underlie severe skin diseases including ichthyosis and atopic dermatitis. Tight junctions (TJs) form a single layered network in simple epithelia. TJs are important for both barrier functions and vesicular transport. Epidermis is stratified epithelia and lamellar granules (LGs) are secreted from the stratum granulosum (SG) in a sequential manner. Previously, continuous TJs and paracellular permeability barriers were found in the second layer (SG2) of SG in mice, but their fate and correlation with LG secretion have been poorly understood. We studied epidermal TJ-related structures in humans and in mice and found occludin/ZO-1 immunoreactive multilayered networks spanning the first layer of SG (SG1) and SG2. Paracellular penetration tracer passed through some TJs in SG2, but not in SG1. LG secretion into the paracellular tracer positive spaces started below the level of TJs of SG1. Our study suggests that LG-secretion starts before the establishment of TJ barrier in the mammalian epidermis.

Neuropsin promotes oligodendrocyte death, demyelination and axonal degeneration after spinal cord injury.

Previous studies indicated that the expression of neuropsin, a serine protease, is induced in mature oligodendrocytes after injury to the CNS. The pathophysiology of spinal cord injury (SCI) involves primary and secondary mechanisms, the latter contributing further to permanent losses of function. To explore the role of neuropsin after SCI, histochemical and behavioral analyses were performed in wild-type (WT) and neuropsin-deficient (neuropsin(-/-)) mice using a crush injury model, a well-characterized and consistently reproducible model of SCI. In situ hybridization revealed that neuropsin mRNA expression was induced in the spinal cord white matter from WT mice after crush SCI, peaking at day 4. Neuropsin(-/-) mice showed attenuated demyelination, oligodendrocyte death, and axonal damage after SCI. Although axonal degeneration in the corticospinal tract was obvious caudal to the lesion site in both strains of mice after SCI, the number of surviving nerve fibers caudal to the lesion was significantly larger in neuropsin(-/-) mice than WT mice. Behavioral analysis revealed that the recovery at days 10-42 was significantly improved in neuropsin(-/-) mice compared with WT mice in spite of the severe initial hindlimb impairments due to SCI in both strains. These observations suggest that neuropsin is involved in the secondary phase of the pathogenesis of SCI mediated by demyelination, oligodendrocyte death, and axonal degeneration.

Involvement of corneodesmosome degradation and lamellar granule transportation in the desquamation process

Desquamation in the mammalian skin is a well-balanced process of producing corneocytes and shedding them from the surface of the skin. The corneodesmosome, which is a modified desmosome, is the main adhesive structure in the cornified cell layer. The major extracellular constituents of corneodesmosomes are desmoglein 1, desmocollin 1, and corneodesmosin. Proteases involved in the degradation of corneodesmosomes and their inhibitors are secreted from lamellar granules in the granular cell layer. Genetic defects in corneodesmosin and protease inhibitors result in accelerated desquamation and severe barrier impairment. Abnormalities in transportation and secretion of lamellar granules underlie ichthyosis seen in certain human diseases.

Kallikrein-Related Peptidase 8–Dependent Skin Wound Healing Is Associated with Upregulation of Kallikrein-Related Peptidase 6 and PAR2

Kallikrein-related peptidase 8 (KLK8) is believed to be involved in the maintenance of skin homeostasis and pathogenesis of inflammatory skin diseases. Although previous studies have shown that KLK8 is expressed around incisional wounds, the exact role of KLK8 in wound healing remains obscure. In the present study, we compared wound healing in wild-type (WT) and Klk8 gene-disrupted (kallikrein-related peptidase 8 knockout, Klk8(-/-)) mouse skin. Wound healing in Klk8(-/-) mice was hampered with defective keratinocyte proliferation, differentiation, and migration in the early stages of wound healing. Compared with the prominent induction of Klk6 and protease-activated receptor 2 (PAR2) messenger RNA, and protein in WT mice after wounding, a much lower increase was observed in Klk8(-/-) skin. After skin wounding in WT mice, increased Klk6 was detected from the upper stratum spinosum to the stratum corneum. Moreover, in WT mice, Klk6 protein was processed. PAR2 was diffusely expressed in the cytoplasm of the stratum spinosum at day 7 post wounding in WT mice. These results suggest that Klk8 is involved in the proliferation and migration of keratinocytes through the upregulation and activation of Klk6 in the early stages of wound healing, and possibly in keratinocyte differentiation associated with the upregulation and activation of PAR2 in the late stages of wound healing.