Akt-dependent Pp2a activity is required for epidermal barrier formation during late embryonic development

Acquisition of epidermal barrier function occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. We investigated the molecular pathways active during epidermal barrier formation. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55α or B55α), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. Our data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. We therefore describe a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development

Interplay of Staphylococcal and Host Proteases Promotes Skin Barrier Disruption in Netherton Syndrome.

Netherton syndrome (NS) is a monogenic skin disease resulting from loss of function of lymphoepithelial Kazal-type-related protease inhibitor (LEKTI-1). In this study we examine if bacteria residing on the skin are influenced by the loss of LEKTI-1 and if interaction between this human gene and resident bacteria contributes to skin disease. Shotgun sequencing of the skin microbiome demonstrates that lesional skin of NS subjects is dominated by Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). Isolates of either species from NS subjects are able to induce skin inflammation and barrier damage on mice. These microbes promote skin inflammation in the setting of LEKTI-1 deficiency due to excess proteolytic activity promoted by S. aureus phenol-soluble modulin α as well as increased bacterial proteases staphopain A and B from S. aureus or EcpA from S. epidermidis. These findings demonstrate the critical need for maintaining homeostasis of host and microbial proteases to prevent a human skin disease

Water permeation through stratum corneum lipid bilayers from atomistic simulations

Stratum corneum, the outermost layer of skin, consists of keratin filled rigid non-viable corneocyte cells surrounded by multilayers of lipids. The lipid layer is responsible for the barrier properties of the skin. We calculate the excess chemical potential and diffusivity of water as a function of depth in lipid bilayers with compositions representative of the stratum corneum using atomistic molecular dynamics simulations. The maximum in the excess free energy of water inside the lipid bilayers is found to be twice that of water in phospholipid bilayers at the same temperature. Permeability, which decreases exponentially with the free energy barrier, is reduced by several orders of magnitude as compared to with phospholipid bilayers. The average time it takes for a water molecule to cross the bilayer is calculated by solving the Smoluchowski equation in presence of the free energy barrier. For a bilayer composed of a 2:2:1 molar ratio of ceramide NS 24:0, cholesterol and free fatty acid 24:0 at 300K, we estimate the permeability P=3.7e-9 cm/s and the average crossing time \tau_{av}=0.69 ms. The permeability is about 30 times smaller than existing experimental results on mammalian skin sections.Comment: latex, 8 pages, 6 figure

Molecular organization and in situ assembly of the human skin barrier

A deficient skin barrier function is a characteristic feature of skin diseases such as eczema, psoriasis and the ichtyoses. A malformation of the lipid matrix might be a major factor in barrier deficient skin disease. To better understand barrier function impairments in the future we studied the in situ assembly and molecular organization of the healthy skin barrier. Presently, two different models for the lipid secretion system, lamellar body system, have been proposed – the Landmann model and the membrane-folding model. The Landmann model proposes lipid secretion into the extracellular space between viable and cornified epidermis via a diffusion/fusion based process of multiple discrete lipid containing vesicles, while the membrane folding model proposes lipid secretion via a single and coherent continuous tuboloreticular system associating the cytoplasmic transGolgi with the extracellular space. In this thesis the continuity versus discreteness of the lamellar body system was addressed with three complementary 3D electron microscopy methodologies - tomography of vitreous sections (TOVIS), freeze-substitution serial section electron tomography (FS-SET) and focused ion beam scanning electron microscopy (FIB-SEM) tomography, using cryo-electron microscopy of vitreous sections (CEMOVIS) as a high-resolution 2D reference. We show that lamellar bodies are not discrete vesicles but part of a tubuloreticular membrane network filling out a large portion of the cytoplasm and being continuous with the plasma membrane of stratum granulosum cells. The next step in skin barrier formation is the lipid molecular assembly. We found five different characteristic high-resolution CEMOVIS patterns that correspond to five different steps in the lipid reorganization process. Using a novel approach combining very-high magnification cryo-electron microscopy of vitreous skin section (CEMOVIS) defocus-series, molecular modelling and electron microscopy simulation we determined the molecular organisation of the lipid matrix in each of the five steps. We conclude that the human skin barrier is formed by a cubic-to-lamellar lipid bilayer phase transition followed by a rearrangement of the ceramide hydrocarbon chains from a hairpin-like to a fully splayed conformation with a concomitant displacement of cholesterol from symmetric to asymmetric distribution between lipid layers. Finally we show with the same novel approach that when the skin barrier formation is complete, the lipid matrix is organized as asymmetrical stacked bilayers of fully extended ceramides with cholesterol associated to the ceramide sphingoid moiety. This rationalizes the skin’s low permeability towards both water and towards hydrophilic and lipophilic substances, as well as the skin barrier’s robustness towards hydration and dehydration, environmental temperature and pressure changes, stretching, compression, bending and shearing. The near-native high resolution molecular description of skin barrier and its formation presented in this thesis opens the door for molecular dynamics modelling as well as in-vitro modelling, of barrier function and its deficiency in normal and diseased skin

The microbiome in patients with atopic dermatitis.

As an interface with the environment, the skin is a complex ecosystem colonized by many microorganisms that coexist in an established balance. The cutaneous microbiome inhibits colonization with pathogens, such as Staphylococcus aureus, and is a crucial component for function of the epidermal barrier. Moreover, crosstalk between commensals and the immune system is now recognized because microorganisms can modulate both innate and adaptive immune responses. Host-commensal interactions also have an effect on the developing immune system in infants and, subsequently, the occurrence of diseases, such as asthma and atopic dermatitis (AD). Later in life, the cutaneous microbiome contributes to the development and course of skin disease. Accordingly, in patients with AD, a decrease in microbiome diversity correlates with disease severity and increased colonization with pathogenic bacteria, such as S aureus. Early clinical studies suggest that topical application of commensal organisms (eg, Staphylococcus hominis or Roseomonas mucosa) reduces AD severity, which supports an important role for commensals in decreasing S aureus colonization in patients with AD. Advancing knowledge of the cutaneous microbiome and its function in modulating the course of skin disorders, such as AD, might result in novel therapeutic strategies

Skin cleansing and emolliating for older people: A quasi-experimental pilot study.

AIMS: The aims of this study were to (i) assess the effect of low-cost hygiene and emollient regimens on the skin barrier function (SBF) of people aged >65 year with xerosis (dry skin) on their lower legs; (ii) to assess the utility of portable measures of skin barrier function in terms of stratum corneum hydration (SCH) and transepidermal water loss (TEWL) in community settings; and iii) to provide evidence for a randomised controlled trial on the treatment of adults in a resource-poor country with dry skin on their lower legs which causes and exacerbates the skin disease podoconiosis (non-filarial elephantiasis). BACKGROUND: Age increases the risk of impaired skin barrier function which can precipitate skin breakdown. Older skin is frequently characterised by troublesome xerosis and pruritus (itching). Hygiene and emollient practices are central to maintaining skin integrity but are currently under-researched. METHOD: A quasi-experimental pilot study of five combinations of cleansing and emollient interventions was applied to the xerotic lower legs of ten participants with no skin disease for five consecutive days. Stratum corneum hydration and transepidermal water loss were measured at baseline and day six. Products were chosen because of effectiveness, low cost and availability in a poor-resource country. RESULTS: The greatest difference in transepidermal water loss pre-intervention-postintervention was indicated by the regimen of soapy water, 2% glycerine soak and Vaseline(™) (mean 1.14, SD 1.27). This regimen also indicated the greatest difference in stratum corneum hydration (mean 7.92, SD 3.93). The improvement in stratum corneum hydration was significantly greater than for the control (p = .011), soap (p = .050) or water soak (p = .011). CONCLUSION: A regimen of washing skin with soapy water, soaking in 2% glycerine for 30 min and applying Vaseline(™) has a beneficial effect on the skin barrier function in older people. The study supports previous findings on the positive effects of glycerine on skin barrier function. IMPLICATIONS FOR PRACTICE AND RESEARCH: Skin barrier function in older people can be improved using a regimen of washing, soaking in 2% glycerine and applying Vaseline(™)

By protecting against cutaneous inflammation, epidermal pigmentation provided an additional advantage for ancestral humans.

Pigmentation evolved in ancestral humans to protect against toxic, ultraviolet B irradiation, but the question remains: "what is being protected?" Because humans with dark pigmentation display a suite of superior epidermal functions in comparison with their more lightly pigmented counterparts, we hypothesized and provided evidence that dark pigmentation evolved in Africa to support cutaneous function. Because our prior clinical studies also showed that a restoration of a competent barrier dampens cutaneous inflammation, we hypothesized that resistance to inflammation could have provided pigmented hominins with yet another, important evolutionary benefit. We addressed this issue here in two closely related strains of hairless mice, endowed with either moderate (Skh2/J) or absent (Skh1) pigmentation. In these models, we showed that (a) pigmented mice display a markedly reduced propensity to develop inflammation after challenges with either a topical irritant or allergen in comparison with their nonpigmented counterparts; (b) visible and histologic evidence of inflammation was paralleled by reduced levels of pro-inflammatory cytokines (i.e., IL-1α and INFα); (c) because depigmentation of Skh2/J mouse skin enhanced both visible inflammation and pro-inflammatory cytokine levels after comparable pro-inflammatory challenges, the reduced propensity to develop inflammation was directly linked to the presence of pigmentation; and (d) furthermore, in accordance with our prior work showing that pigment production endows benefits by reducing the surface pH of skin, acidification of albino (Skh1) mouse skin also protected against inflammation, and equalized cytokine levels to those found in pigmented skin. In summary, pigmentation yields a reduced propensity to develop inflammation, consistent with our hypothesis that dark pigmentation evolved in ancestral humans to provide a suite of barrier-linked benefits that now include resistance to inflammation

The epidthelial sodium channel ENaC and its regulators in the epidermal permeability barrier function

The highly amiloride-sensitive epithelial sodium channel ENaC is well known to be involved in controlling whole body sodium homeostasis and lung liquid clearance. ENaC expression has also been detected in the skin of amphibians and mammals. Mice lacking ENaC expression lose rapidly weight associated with an epidermal barrier defect that develops following birth. This dehydration is accompanied with a highly abnormal lipid matrix composition and an impaired skin surface acidification. This strongly suggests a role of ENaC in the maturation of barrier function rather than in the prenatal generation of the barrier, and may be as such an important modulator for skin hydration. In parallel, gene targeting experiments of regulators of ENaC activity, membrane serine proteases, also termed channel activating proteases, like CAP1/Prss8 and matriptase/MT-SP1 by themselves have been shown to be crucial for the epidermal barrier function. In our review, we mainly focus on the role of ENaC and its regulators in the skin and discuss their importance in the epidermal permeability barrier function

Penetration enhancing effect of phytoceramides

Ceramides are essential components in the stratum corneum barrier function. Different classes of ceramides are present in human skin, differing in the nature of sphingosine and acyl moieties with respect to chain length, degree of saturation and the presence of an OH group [1]. Ceramides with a saturated sphingosine base containing a hydroxyl function at C4 are known as phytoceramides. A few studies demonstrated the penetration enhancing properties of ceramides [2-5], however, systematic studies using phytoceramides are lacking. This led us to assess the penetration enhancing effect of phytosphingosine and a series of nine phytoceramides via transdermal experiments using in vitro Franz diffusion cells. As transdermal model compounds, testosterone, caffeine and ibuprofen were tested in a 50:50 (V/V) EtOH:H2O dose formulation [6]. Results showed that the penetration enhancing effect of the phytoceramides depends on the used model compound. Selected phytoceramides exhibited a penetration enhancing ratio of more than two. References [1] Janůšová, B.; Zbytovská, J.; Lorenc, P.; Vavrysová, H.; Palát K.; Hrabálek, A.; Vávrová, K. (2011). Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes. Biochimica et Biophysica, 1811, 129–137. [2] Vávrová, K.; Hrabálek, A.; Dolezal, P.; Holas, T.; Zbytovská, J. (2003). L-serine and glycine based ceramide analogues as transdermal permeation enhancers: polar head size and hydrogen bonding. Bioorganic & medicinal chemistry letters, 13, 2351-2353. [3] Vávrová, K.; Zbytovská, J.; Hrabálek, A. (2005). Amphiphilic transdermal permeation enhancers: structure-activity relationships. Current Medicinal Chemistry, 12, 2273-2291. [4] Novotý, J.; Janůšová, B.; Novotý, M.; Hrabálek, A.; Vávrová, K. (2009). Short-chain ceramides decrease skin barrier properties. Skin pharmacology and Physiology, 22, 22-30. [5] Sinko, B.; Kökösi, J.; Avdeef, A.; Takács-Novák, K. (2009). A PAMPA study of the permeability-enhancing effect of new ceramide analogues. Chemistry & Biodiversity, 6, 1867-1874. [6] Baert, B.; Deconinck, E.; Van Gele, M.; Slodicka, M.; Stoppie, P.; Bode, S.; Slegers, G.; Vander Heyden, Y.; Lambert, J.; Beetens, J.; De Spiegeleer, B. (2007). Transdermal penetration behaviour of drugs: CART-clustering, QSPR and selection of model compounds. Bioorganic & medicinal chemistry, 15(22), 6943-6955