Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin

Caspase-14 is a protease that is mainly expressed in suprabasal epidermal layers and activated during keratinocyte cornification. Caspase-14-deficient mice display reduced epidermal barrier function and increased sensitivity to UVB radiation. In these mice, profilaggrin, a protein with a pivotal role in skin barrier function, is processed correctly to its functional filaggrin (FLG) repeat unit, but proteolytic FLG fragments accumulate in the epidermis. In wild-type stratum corneum, FLG is degraded into free amino acids, some of which contribute to generation of the natural moisturizing factors (NMFs) that maintain epidermal hydration. We found that caspase-14 cleaves the FLG repeat unit and identified two caspase-14 cleavage sites. These results indicate that accumulation of FLG fragments in caspase-14(-/-) mice is due to a defect in the terminal FLG degradation pathway. Consequently, we show that the defective FLG degradation in caspase-14-deficient skin results in substantial reduction in the amount of NMFs, such as urocanic acid and pyrrolidone carboxylic acid. Taken together, we identified caspase-14 as a crucial protease in FLG catabolism.     

IL‐6 Stimulates but is not essential for stratum corneum formation and permeability barrier development during gestation

Please cite this paper as: IL‐6 Stimulates but is not essential for stratum corneum formation and permeability barrier development during gestation. Experimental Dermatology 2010; 19 : e31–e36. Abstract: The regulation of epidermal ontogenesis is a complex process. Previous studies have shown that cytokines (IL‐1, TNFα and IL‐6) regulate permeability barrier homeostasis in adult mice. Recently, we reported that IL‐1 and TNFα accelerate stratum corneum (SC) formation and permeability barrier development in foetal rodents. Here, we determined whether IL‐6 also regulates SC formation and permeability barrier development during late gestation. Using a rat skin explant model, we demonstrated that IL‐6 accelerates permeability barrier formation in a time‐ and dose‐dependent fashion. This acceleration of barrier formation is attributable to (a) accelerated lamellar membrane maturation, (b) formation of a multi‐layer SC and (c) enhanced expression of epidermal differentiation markers. When comparing epidermis of IL‐6‐deficient (knockout mice) and wild‐type foetal mice at days 16–18, we could not detect any abnormalities in either SC formation or the expression of differentiation markers in knockout (KO) mice. In parallel, the basal expression levels of IL‐6 mRNA in epidermis and IL‐6 protein in amniotic fluid were very low, with only a minimal change in IL‐6 receptor mRNA levels in epidermis of days 16–22 foetal mice. These low IL‐6 levels may account, at least in part, for the absence of epidermal abnormalities in IL‐6 KO mice. In conclusion, exogenous IL‐6 accelerates epidermal ontogenesis, but it is not essential for normal epidermal maturation.     

Exogenous origin of n-alkanes in pathologic scale.

BACKGROUND--Although n-alkanes accumulate in some disorders of cornification, recent studies using radioactive carbon 14 content by accelerator mass spectrometry point to an exogenous origin for alkanes in normal stratum corneum, and their derivation in congenital ichthyosiform erythroderma remains controversial. DESIGN AND RESULTS--Using 14C content to measure sample age, the n-alkane fractions from two patients with congenital ichthyosiform erythroderma contained no detectable contemporary materials. By electron microscopy, alkane-enriched emollients (petrolatum [Vaseline]) permeated to all levels of stratum corneum of hairless mice, expanding the intercellular domains and distorting membrane bilayers. Similar ultrastructural changes were also observed in the stratum corneum of patients with congenital ichthyosiform erythroderma. When alkanes were excluded, no differences in lipid content were evident between two forms of autosomal recessive ichthyosis. CONCLUSIONS--These data demonstrate that scale n-alkanes in disorders of cornification derive from environmental sources and indicate the pervasiveness of petroleum-based emollients in skin. Therefore, epidermal lipid analyses must be interpreted with caution. However, these studies do not rule out an important therapeutic and/or pathogenic role for exogenous n-alkanes in skin.     

Z‐Ligustilide inhibits benzo(a)pyrene‐induced CYP1A1 upregulation in cultured human keratinocytes via ROS‐dependent Nrf2 activation

Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is an environmental contaminant that can induce cytochrome P4501A1 (CYP1A1) upregulation via aryl hydrocarbon receptor (AhR) activation and provoke inflammation. Here, we investigated the effect of Z‐Ligustilide, an active ingredient isolated from the medicinal plants Cnidium officinale and Angelica acutiloba, on BaP‐induced CYP1A1 upregulation in normal human epidermal keratinocytes (NHEKs) as well as its underlying mechanisms. Z‐Ligustilide significantly inhibited BaP‐induced CYP1A1 upregulation in NHEKs. Treatment of NHEKs with Z‐Ligustilide induced Nuclear factor‐E2‐related factor 2 (Nrf2) nuclear translocation and expression of the Nrf2‐regulated genes for haeme oxygenase‐1 (HO‐1) and NAD(P)H:quinine oxidoreductase‐1 (NQO1). AhR silencing, SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor) and LY294002 (a PI3K inhibitor) did not suppress Z‐Ligustilide‐induced Nrf2 activation. Moreover, treatment of NHEKs with Z‐Ligustilide increased reactive oxygen species (ROS) and L‐N‐acetylcysteine (L‐NAC, an antioxidant) attenuated Z‐ligustilide‐induced Nrf2 nuclear translocation and HO‐1 expression. L‐NAC or knock‐down of Nrf2 significantly attenuated the inhibitory effects of Z‐Ligustilide on BaP‐induced CYP1A1 upregulation in NHEKs. Taken together, these findings suggest that Z‐Ligustilide can suppress BaP‐induced CYP1A1 upregulation through ROS‐dependent Nrf2 pathway activation and may be beneficial in preventing or treating BaP‐induced skin damage.     

TINCR is not a non-coding RNA but encodes a protein component of cornified epidermal keratinocytes

Long non-coding RNAs have been implicated in the regulation of a plethora of biological processes, yet it has been challenging to verify that they are truly not coding for proteins. Terminal differentiation-induced non-coding RNA (TINCR) is a 3.7-kilobase mRNA that is highly abundant in epidermal keratinocytes prior to cornification. Here, we report the presence of an evolutionarily conserved open reading frame in TINCR and the identification of peptides derived from this open reading frame in the proteome of human stratum corneum. Our results demonstrate that TINCR is a protein-coding RNA and suggest that the TINCR-encoded protein is involved in keratinocyte cornification.     

Modulation of HMGB1 translocation and RAGE/NFκB cascade by quercetin treatment mitigates atopic dermatitis in NC/Nga transgenic mice

Quercetin, glycosylated form of flavonoid compound, has potent antioxidant and anti‐inflammatory properties. In this study, we have investigated the effects of quercetin on skin lesion, high‐mobility group box (HMGB)1 cascade signalling and inflammation in atopic dermatitis (AD) mouse model. AD‐like lesion was induced by the application of house dust mite extract to the dorsal skin of NC/Nga transgenic mouse. After AD induction, quercetin (50 mg/kg, p.o) was administered daily for 2 weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for HMGB1, receptor for advanced glycation end products (RAGE), toll‐like receptor (TLR)4, nuclear factor (NF)κB, nuclear factor erythroid‐2‐related factor (Nrf)2, kelch‐like ECH‐associated protein (Keap)1, extracellular signal‐regulated kinase (ERK)1/2, cyclooxygenase (COX)2, tumor necrosis factor (TNF)α, interleukin (IL)‐1β, IL‐2Rα and other inflammatory markers in the skin of AD mice. In addition, serum levels of T helper (Th) cytokines (interferon (IFN)γ, IL‐4) were measured by enzyme‐linked immunosorbent assay. Quercetin treatment attenuated the development of AD‐like skin lesions. Histological analysis showed that quercetin inhibited hyperkeratosis, parakeratosis, acanthosis, mast cells and infiltration of inflammatory cells. Furthermore, quercetin treatment downregulated cytoplasmic HMGB1, RAGE, nuclear p‐NFκB, p‐ERK1/2, COX2, TNFα, IL‐1β, IL‐2Rα, IFNγ and IL‐4 and upregulated nuclear Nrf2. Our data demonstrated that the HMGB1/RAGE/NFκB signalling might play an important role in skin inflammation, and quercetin treatment could be a promising agent for AD by modulating the HMGB1/RAGE/NFκB signalling and induction of Nrf2 protein.     

Saprophytic bacteria and fungi colonize stearoyl coenzyme-A desaturase-1 knockout skin

Lipids synthesized on the skin are critical to the antimicrobial barrier. Skin lipids also facilitate survival of lipophilic skin commensals in an otherwise dry and acidic ecological landscape. Thus, skin-specific stearoyl-coenzyme A desaturase 1 knockout mice (Scd1^ΔK14) with sebocyte atrophy and decreased synthesis of monounsaturated fatty acids, triglycerides and wax diesters have dry, inflamed skin. Here, we used 16S rRNA (V1-V2 and V1-V9) and internal transcribed spacer 1 (ITS1) amplicon sequencing to compare bacterial and fungal skin microbiomes between Scd1^ΔK14 mice and wildtype control mice (Scd1^fl/fl) in a barrier facility. Saprophytic bacteria including Sporosarcina spp. and Staphylococcus lentus and saprophytic fungi including Alternaria infectoria were found in higher relative abundance in the Scd1^ΔK14 group (ANCOM). Analysis of community diversity (Shannon index) revealed greater fungal alpha diversity in the Scd1^ΔK14 group (p = 0.009, Kruskal–Wallis). Principal coordinates analysis (Bray–Curtis dissimilarity) showed that both bacterial (p = 0.002, PERMANOVA) and fungal communities (p = 0.006, PERMANOVA) of the Scd1^ΔK14 group were unique from the wildtype group. Altogether, these results suggest that sebaceous gland-derived lipids normally restrict the skin microbiome, and in the absence of these lipids, a greater diversity of opportunistic organisms are able to colonize the surface of skin.     

Modulating T cell functions does not alleviate chronic inflammatory skin lesions in K5.TGFβ1 transgenic mice

Please cite this paper as: Modulating T cell functions does not alleviate chronic inflammatory skin lesions in K5.TGFβ1 transgenic mice. Experimental Dermatology 2009; 19: 406–415. Abstract: To use mice with chronic hyperproliferative skin inflammation as psoriasis models, their thorough phenotypic and functional characterization is indispensable. Mice with keratin 5 promoter‐controlled overexpression of latent human Transforming Growth Factor (TGF)β1 within the basal epidermis (K5.TGFβ1 mice) show a psoriasiform phenotype, but the underlying pathogenic mechanisms are not entirely clear. To elucidate the contribution of T lymphocytes to the pathogenesis in K5.TGFβ1 mice, we used three complementary approaches: first, peripheral T cells were eradicated via systemic treatment with CD3‐ or CD4‐depleting antibodies. However, this elimination did not alleviate the chronic inflammatory disorder. Second, bone marrow transplantation from transgenic mice into wildtype recipients and vice versa resulted in the expected reconstitution of both adaptive and innate immune system but had little effect on the cutaneous phenotype both in wildtype and transgenic chimeras. Third, based on the hypothesis that the disease course could be modulated by regulatory T cells (Tregs), we expanded Tregs in vivo using a superagonistic anti‐CD28 antibody. While this treatment achieved a threefold increase in Foxp3‐expressing Tregs, there was little, if any, effect on the chronic skin inflammation. We conclude from our findings that T cells play little, if any, role in the skin lesions of K5.TGFβ1 mice.     

Expression of epidermal<Emphasis Type="Italic"> N</Emphasis>-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate receptors (NMDAR1) depends on formation of the granular layer—analysis in diseases with parakeratotic cornification

Ionotrope glutamate receptors of the N-methyl-d-aspartate (NMDA) receptor type are expressed on keratinocytes and influence the intracellular calcium concentration. The importance of NMDA receptors in pathophysiological processes in the skin is, however, still unclear. Epidermal distribution patterns of NMDA receptors were investigated in dermatoses with parakeratotic cornification (psoriasis vulgaris and verrucae vulgares) and compared to the expression of filaggrin. The expression of NMDA receptors (R1 component) in paraffin-embedded normal epidermis (n=22), psoriasis vulgaris (n=21) and verrucae vulgares (n=23) was examined and evaluated by means of digital image analysis. For quantitative characterization of the distribution patterns, a quotient was formed of the expression in the stratum granulosum and stratum basale (NMDA ratio). The distribution of NMDAR1 was compared to the immunohistochemical expression of filaggrin. Additionally the expression of filaggrin was investigated in HaCaT cells after treatment with the NMDA receptor antagonist MK-801. NMDA receptors were demonstrated in the epidermis of all preparations. In healthy skin, the highest receptor density was found in the stratum granulosum. This distribution pattern was basically also present in the dermatoses examined. Thus, the occurrence of parakeratosis in psoriasis vulgaris, but not in verrucae vulgares, was characterized by a significant reduction in the NMDA ratio (reduced expression of NMDAR1 in the upper epidermis). The immunohistochemical distribution of filaggrin was similar to that of NMDAR1. In HaCaT cells MK-801 suppressed the expression of filaggrin. NMDA receptors are expressed in human epidermis under physiological conditions especially in the stratum granulosum. Their reduced expression within parakeratotic epidermis in psoriasis vulgaris may be evidence of impaired intracellular calcium influx in this disease.     

Barrier lipid composition and response to plasma lipids: A direct comparison of mouse dorsal back and ear skin

The skin of the ear and the back are frequently selected sites in skin research using mouse models. However, distinct responses to treatment have been described between these two sites in several studies. Despite the crucial role of the stratum corneum (SC) in the skin barrier function of both dorsal back and ear skin, it remains unclear whether differences in lipid composition might underlie altered responses. Here, we compared the skin morphology and the barrier lipid composition of the ear with the back skin of wild-type mice. The ear contained more corneocyte layers in the SC and its barrier lipid composition was enriched with sphingosine ceramide subclasses, especially the short ones with a total chain length of 33-34 carbons. The free fatty acid (FFA) profile in the ear skin shifted towards shorter chains, significantly reducing the mean chain length to 23.3 vs 24.7 carbons in the back skin. In line, FFA species in the ear displayed a twofold increase in unsaturation index (P < .001). Gene expression in the ear skin revealed low expression of genes involved in lipid synthesis and uptake, indicating a reduced metabolic activity. Finally, the effects of hypercholesterolaemia on SC FFA composition was compared in ear and back skin of apolipoprotein E knockout (APOE^−/−) mice. Interestingly, the FFA profile in APOE^−/− ear skin was minimally affected, while the FFA composition in the back skin was markedly changed in response to hypercholesterolaemia. In conclusion, ear and back skin have distinct barrier lipids and respond differently to elevated plasma cholesterol.     

Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.

There is evidence that the "acid mantle" of the stratum corneum is important for both permeability barrier formation and cutaneous antimicrobial defense. The origin of the acidic pH of the stratum corneum remains conjectural, however. Both passive (e.g., eccrine/sebaceous secretions, proteolytic) and active (e.g., proton pumps) mechanisms have been proposed. We assessed here whether the free fatty acid pool, which is derived from phospholipase-mediated hydrolysis of phospholipids during cornification, contributes to stratum corneum acidification and function. Topical applications of two chemically unrelated secretory phospholipase sPLA2 inhibitors, bromphenacylbromide and 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol, for 3 d produced an increase in the pH of murine skin surface that was paralleled not only by a permeability barrier abnormality but also altered stratum corneum integrity (number of strippings required to break the barrier) and decreased stratum corneum cohesion (protein weight removed per stripping). Not only stratum corneum pH but also all of the functional abnormalities normalized when either palmitic, stearic, or linoleic acids were coapplied with the inhibitors. Moreover, exposure of intact murine stratum corneum to a neutral pH for as little as 3 h produced comparable abnormalities in stratum corneum integrity and cohesion, and further amplified the inhibitor-induced functional alterations. Furthermore, short-term applications of an acidic pH buffer to inhibitor-treated skin also reversed the abnormalities in stratum corneum integrity and cohesion, despite the ongoing decrease in free fatty acid levels. Finally, the secretory-phospholipase-inhibitor-induced alterations in integrity/cohesion were in accordance with premature dissolution of desmosomes, demonstrated both by electron microscopy and by reduced desmoglein 1 levels in the stratum corneum (shown by immunofluorescence staining and visualized by confocal microscopy). Together, these results demonstrate: (i) the importance of phospholipid-to-free-fatty-acid processing for normal stratum corneum acidification; and (ii) the potentially important role of this pathway not only for barrier homeostasis but also for the dual functions of stratum corneum integrity and cohesion.     

Licochalcone A activates Nrf2 in vitro and contributes to licorice extract‐induced lowered cutaneous oxidative stress in vivo

The retrochalcone licochalcone A (LicA) has previously been shown to possess antimicrobial and anti‐inflammatory properties. In this study, we focused on pathways responsible for the antioxidative properties of LicA. In vitro, LicA protected from oxidative stress mediated by reactive oxygen species (ROS) by activating the expression of cytoprotective phase II enzymes. LicA induced nuclear translocation of NF‐E2‐related factor 2 (Nrf2) in primary human fibroblasts and elevated the expression of the cytoprotective and anti‐inflammatory enzymes heme oxygenase 1 and glutamate–cysteine ligase modifier subunit. LicA‐treated cells displayed a higher ratio of reduced to oxidized glutathione and decreased concentrations of ROS in UVA‐irradiated human dermal fibroblasts, as well as in activated neutrophils. In vivo, ultraweak photon emission analysis of skin treated with LicA‐rich licorice extract revealed a significantly lowered UVA‐induced luminescence, indicative for a decrease in oxidative processes. We conclude from these data that topical application of licorice extract is a promising approach to induce Nrf2‐dependent cytoprotection in human skin.     

Delphinidin,a dietary antioxidant,induces human epidermal keratinocyte differentiation but not apoptosis: studies in submerged and three‐dimensional epidermal equivalent models

Delphinidin (Del), [3,5,7,3'‐,4'‐,5'‐hexahydroxyflavylium], an anthocyanidin and a potent antioxidant abundantly found in pigmented fruits and vegetables exhibits proapoptotic effects in many cancer cells. Here, we determined the effect of Del on growth, apoptosis and differentiation of normal human epidermal keratinocytes (NHEKs) in vitro in submerged cultures and examined its effects in a three‐dimensional (3D) epidermal equivalent (EE) model that permits complete differentiation reminiscent of in vivo skin. Treatment of NHEKs with Del (10–40 μm ; 24–48 h) significantly enhanced keratinocyte differentiation. In Del‐treated cells, there was marked increase in human involucrin (hINV) promoter activity with simultaneous increase in the mRNA and protein expressions of involucrin and other epidermal differentiation markers including procaspase‐14 and transglutaminase‐1 (TGM1), but without any effect on TGM2. Del treatment of NHEKs was associated with minimal decrease in cell viability, which was not associated with apoptosis as evident by lack of modulation of caspases, apoptosis‐related proteins including Bcl‐2 family of proteins and poly(ADP‐ribose) polymerase cleavage. To establish the in vivo relevance of our observations in submerged cultures, we then validated these effects in a 3D EE model, where Del was found to significantly enhance cornification and increase the protein expression of cornification markers including caspase‐14 and keratin 1. For the first time, we show that Del induces epidermal differentiation using an experimental system that closely mimics in vivo human skin. These observations suggest that Del could be a useful agent for dermatoses associated with epidermal barrier defects including aberrant keratinization, hyperproliferation or inflammation observed in skin diseases like psoriasis and ichthyoses.