SASPase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing.
ABSTRACT: The stratum corneum (SC), the outermost layer of the epidermis, acts as a barrier against the external environment. It is hydrated by endogenous humectants to avoid desiccation. However, the molecular mechanisms of SC hydration remain unclear. We report that skin-specific retroviral-like aspartic protease (SASPase) deficiency in hairless mice resulted in dry skin and a thicker and less hydrated SC with an accumulation of aberrantly processed profilaggrin, a marked decrease of filaggrin, but no alteration in free amino acid composition, compared with control hairless mice. We demonstrated that recombinant SASPase directly cleaved a linker peptide of recombinant profilaggrin. Furthermore, missense mutations were detected in 5 of 196 atopic dermatitis (AD) patients and 2 of 28 normal individuals. Among these, the V243A mutation induced complete absence of protease activity in vitro, while the V187I mutation induced a marked decrease in its activity. These findings indicate that SASPase activity is indispensable for processing profilaggrin and maintaining the texture and hydration of the SC. This provides a novel approach for elucidating the complex pathophysiology of atopic dry skin.
Project description:Ichthyoses are a heterogeneous group of inherited cornification disorders characterized by generalized dry skin, scaling and/or hyperkeratosis. Ichthyosis vulgaris is the most common form of ichthyosis in humans and caused by genetic variants in the FLG gene encoding filaggrin. Filaggrin is a key player in the formation of the stratum corneum, the uppermost layer of the epidermis and therefore crucial for barrier function. During terminal differentiation of keratinocytes, the precursor profilaggrin is cleaved by several proteases into filaggrin monomers and eventually processed into free amino acids contributing to the hydration of the cornified layer. We studied a German Shepherd dog with a novel form of ichthyosis. Comparing the genome sequence of the affected dog with 288 genomes from genetically diverse non-affected dogs we identified a private heterozygous variant in the ASPRV1 gene encoding "aspartic peptidase, retroviral-like 1", which is also known as skin aspartic protease (SASPase). The variant was absent in both parents and therefore due to a de novo mutation event. It was a missense variant, c.1052T>C, affecting a conserved residue close to an autoprocessing cleavage site, p.(Leu351Pro). ASPRV1 encodes a retroviral-like protease involved in profilaggrin-to-filaggrin processing. By immunofluorescence staining we showed that the filaggrin expression pattern was altered in the affected dog. Thus, our findings provide strong evidence that the identified de novo variant is causative for the ichthyosis in the affected dog and that ASPRV1 plays an essential role in skin barrier formation. ASPRV1 is thus a novel candidate gene for unexplained human forms of ichthyoses.
Project description:Photoaging occurs by chronic skin exposure to the sun and ultraviolet irradiation and leads to skin aging accompanied by a lack of skin hydration. We previously demonstrated the photoprotective effect of fermented Cyclopia intermedia (honeybush) extract on the skin. In this study, we evaluated the skin hydration effects of scaled-up fermented honeybush extract (HU-018) against ultraviolet B (UVB) radiation in HaCaT immortalized human keratinocytes and hairless mice. Pretreating HaCaT cells with HU-018 attenuated the decreased hyaluronic acid (HA) levels and mRNA expression of genes encoding involucrin, filaggrin, and loricrin by UVB irradiation. HU-018 treatment also ameliorated the decreased stratum corneum (SC) hydration and the increased levels of transepidermal water loss (TEWL) and erythema index (EI) in hairless mice after UVB exposure. Microarray analysis revealed changes in gene expression patterns of hyaluronan synthase 2 (Has2), transforming growth factor-beta 3 (TGF-?3), and elastin induced by HU-018 in UVB-irradiated mice. Consistently, the mRNA expression of Has2, TGF-?3, and elastin was increased by HU-018 treatment. Moreover, HU-018 restored the increased epidermal thickness and collagen disorganization in skin tissue of UVB-irradiated mice. HU-018 treatment also decreased matrix metalloproteinase-1 (MMP-1) expression and increased procollagen type-1, elastin, and TGF-?1 expression. In conclusion, we found that HU-018 promoted skin hydration processes in UVB-irradiated keratinocytes and hairless mice by modulating involucrin, filaggrin, loricrin, and HA expression and ameliorating visible signs of photoaging. Thus, HU-018 may be a good skin hydration agent for skin care.
Project description:Recently, loss-of-function mutations in FLG, the human gene encoding profilaggrin and filaggrin, have been identified as the cause of the common skin condition ichthyosis vulgaris (which is characterised by dry, scaly skin). These mutations, which are carried by up to 10% of people, also represent a strong genetic predisposing factor for atopic eczema, asthma and allergies. Profilaggrin is the major component of the keratohyalin granules within epidermal granular cells. During epidermal terminal differentiation, the approximately 400 kDa profilaggrin polyprotein is dephosphorylated and rapidly cleaved by serine proteases to form monomeric filaggrin (37 kDa), which binds to and condenses the keratin cytoskeleton and thereby contributes to the cell compaction process that is required for squame biogenesis. Within the squames, filaggrin is citrullinated, which promotes its unfolding and further degradation into hygroscopic amino acids, which constitute one element of natural moisturising factor. Loss of profilaggrin or filaggrin leads to a poorly formed stratum corneum (ichthyosis), which is also prone to water loss (xerosis). Recent human genetic studies strongly suggest that perturbation of skin barrier function as a result of reduction or complete loss of filaggrin expression leads to enhanced percutaneous transfer of allergens. Filaggrin is therefore in the frontline of defence, and protects the body from the entry of foreign environmental substances that can otherwise trigger aberrant immune responses.
Project description:Naturally occurring saponins have been reported to have anti-inflammatory and immunomodulatory effects. However, the effects of gracillin, a main saponin component of Dioscorea quinqueloba (D. quinqueloba), on atopic dermatitis (AD), have not been previously studied. The aim of this study was to determine whether gracillin isolated from D. quinqueloba has an anti-AD effect on 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin lesions in SKH-1 hairless mice. Topical co-treatment of gracillin and DNCB for two weeks markedly reduced symptoms typical of AD (redness, itching, swelling and skin lichenification), decreased transepidermal water loss (TEWL) and increased skin hydration. In addition, gracillin strongly inhibited PI-induced IL-4 expression in RBL-2H3 cells and in the skins of AD mice. Our results suggest gracillin is a potential candidate for the prevention and treatment of AD and other inflammatory skin disorders.
Project description:Flaky tail mice (FTM) with gene mutations to FLG and TMEM79 are known to spontaneously develop atopic dermatitis (AD)-like dermatitis with age. Interestingly, we have already reported that the spontaneous development of dermatitis exhibits site-specificity, i. e. , the face and neck develop spontaneous dermatitis, while the dorsal flank shows normal skin in old FTM. In fact, transepidermal water loss and stratum corneum (SC) pH increased, SC hydration decreased, and epidermis thickened in the neck, but not in the dorsal flank. The detailed pathogenic mechanisms of such site-specific emergence of dermatitis in FTM remain unclear. To clarify the pathogenesis of site-specificity, we analyzed skin of the neck and flank in old FTM using a microarray. Overall design: The gene expressions of the skin on neck and flank in FTM were analyzed.
Project description:Skin aspartic acid protease (SASPase) is believed to be a key enzyme involved in filaggrin processing during epidermal terminal differentiation. Since little is known about the regulation of SASPase function, the aim of this study was to identify involved protein partners in the process. Yeast two hybrid analyses using SASPase as bait against a human reconstructed skin library identified that the N-terminal domain of filaggrin 2 binds to the N-terminal fragment of SASPase. This interaction was confirmed in reciprocal yeast two hybrid screens and by Surface Plasmon Resonance analyses. Immunohistochemical studies in human skin, using specific antibodies to SASPase and the N-terminal domain of filaggrin 2, showed that the two proteins partially co-localized to the stratum granulosum. In vitro enzymatic assays showed that the N-terminal domain of filaggrin 2 enhanced the autoactivation of SASPase to its 14 kDa active form. Taken together, the data suggest that the N-terminal domain of filaggrin 2 regulates the activation of SASPase that may be a key event upstream of filaggrin processing to natural moisturizing factors in the human epidermis.
Project description:INTRODUCTION:Healthcare professionals tend to recommend emollients based primarily on patient/consumer preference and cost, with cheaper options assumed to be therapeutically equivalent. The aim of this study was therefore to compare the effects on skin hydration of two emollients prescribed in the UK, Doublebase Dayleve™ gel (DELP) and a cheaper alternative, Zerobase Emollient™ cream (ZBC). METHODS:This was a single-centre, randomised, double-blind, concurrent bi-lateral (within-patient) comparison in 18 females with atopic eczema and dry skin on their lower legs. DELP gel and ZBC cream were each applied to one lower leg twice daily for 4 days and on the morning only on day 5. The efficacy of both products was assessed by hydration measurements using a Corneometer CM825 probe (Courage-Khazaka Electronic). The measurements were made three times daily on days 1 to 5. The primary efficacy variable was the area under the curve (AUC) of the change from baseline corneometer readings over the 5 days. RESULTS:Skin hydration using DELP gel was significantly higher than using ZBC cream (p < 0.0001). The cumulative increase in skin hydration observed for DELP gel was substantial and long lasting. In contrast, for ZBC cream, there was no significant improvement of the cumulative skin hydration as measured by the AUC (p = 0.22). CONCLUSION:DELP gel achieved substantial, long-lasting and cumulative skin hydration, whilst ZBC cream achieved no measurable improvement in skin hydration compared to before treatment. Healthcare professionals should be aware that different emollients can perform differently. FUNDING:Dermal Laboratories Ltd. TRIAL REGISTRATION:EudraCT number:2014-001026-16.
Project description:The stratum corneum (SC), the outermost barrier of mammalian bodies, consists of layers of cornified keratinocytes with intercellular spaces sealed with lipids. The insolubility of the SC has hampered in-depth analysis, and the SC has been considered a homogeneous barrier. Here, we applied time-of-flight secondary ion mass spectrometry to demonstrate that the SC consists of three layers with distinct properties. Arginine, a major component of filaggrin-derived natural moisturizing factors, was concentrated in the middle layer, suggesting that this layer functions in skin hydration. Topical application of metal ions revealed that the outer layer allowed their passive influx and efflux, while the middle and lower layers exhibited distinct barrier properties, depending on the metal tested. Notably, filaggrin deficiency abrogated the lower layer barrier, allowing specific metal ions to permeate viable layers. These findings elucidate the multi-layered barrier function of the SC and its defects in filaggrin-deficient atopic disease patients.
Project description:INTRODUCTION: To evaluate the efficacy and safety of a pseudoceramide-containing moisturizer as maintenance therapy in patients with mild-to-moderate atopic dermatitis (AD). METHODS: This was a prospective, single-arm, open-label clinical trial of a twice-daily application of a pseudoceramide-containing moisturizer for 4 weeks as maintenance therapy in 40 patients with stable, mild-to-moderate AD in a tropical climate. Clinical and skin barrier assessment was done at week 0, week 2 and week 4. Any adverse effects were also recorded during the study period. RESULTS: The objective scoring atopic dermatitis decreased from 29.1 [interquartile range (IQR) 21.9-33.7] at week 0 to 22.0 (IQR 21.2-27.8) at week 4 (p < 0.001). There was no detectable difference in transepidermal water loss after 4 weeks; however, stratum corneum (SC) hydration was significantly increased from 39.7 (IQR 35.3-46.4) at week 0 to 49.2 (IQR 41.2-54.6) after 4 weeks (p < 0.001). Both Dermatology Life Quality Index and patient-oriented eczema measure showed significant improvement at week 4 (p < 0.001). The moisturizer was well tolerated with no serious adverse events recorded. CONCLUSION: After 4 weeks of barrier maintenance therapy with a pseudoceramide moisturizer, there was a significant improvement in disease severity, SC hydration and quality of life in both pediatric and adult patients with mild-to-moderate AD.
Project description:Flaky tail mice (FTM) with gene mutations to FLG and TMEM79 are known to spontaneously develop atopic dermatitis (AD)-like dermatitis with age. Interestingly, we have already reported that the spontaneous development of dermatitis exhibits site-specificity, i. e. , the face and neck develop spontaneous dermatitis, while the dorsal flank shows normal skin in old FTM. In fact, transepidermal water loss and stratum corneum (SC) pH increased, SC hydration decreased, and epidermis thickened in the neck, but not in the dorsal flank. The detailed pathogenic mechanisms of such site-specific emergence of dermatitis in AD remain unclear. To clarify the pathogenesis of site-specificity, we analyzed skin of the neck and flank in old FTM using a microarray. Overall design: The gene expressions of neck compared to that of flank in FTM were analyzed.