Structural and functional consequences of loricrin mutations in human loricrin keratoderma (Vohwinkel syndrome with ichthyosis)

Although loricrin is the predominant protein of the cornified envelope (CE) in keratinocytes, loss or gain of loricrin function in mouse models produces only modest skin phenotypes. In contrast, insertional mutations resulting in a frameshift in the C-terminal domain of loricrin produce the characteristic ichthyosis of loricrin keratoderma in mouse and man. To ascertain the basis for the loricrin keratoderma phenotype, we assessed epidermal structure and stratum corneum (SC) function in a previously genotyped human loricrin keratoderma kindred. Our studies revealed abnormal corneocyte fragility and basal permeability barrier function, but accelerated repair kinetics. Despite fragility, increased water loss occurred predominantly via extracellular domains, which correlated with disorganized lamellar bilayers that were linked spatially to discontinuities of the CE. Accelerated barrier recovery was explicable by amplified lamellar body secretion, while partial normalization of the CE in the outer SC correlated with persistence of abundant calcium in the extracellular spaces (positioned to activate transglutaminase-1). These results show that the barrier abnormality in loricrin keratoderma is linked to a defective CE scaffold, resulting in increased extracellular permeability, as shown previously for another "scaffold disorder", lamellar ichthyosis. But in contrast to lamellar ichthyosis, the CE scaffold partially normalizes in the outer SC in loricrin keratoderma.     

Impaired cutaneous permeability barrier function, skin hydration, and sphingomyelinase activity in keratin 10 deficient mice

Point mutations in the suprabasal cytokeratins 1 (K1) or 10 (K10) in humans have been shown to be the cause of the congenital ichthyosis epidermolytic hyperkeratosis. Recently, a K10 deficient mouse model was established serving as a model for epidermolytic hyperkeratosis. Homozygotes suffered from severe skin fragility and died shortly after birth. Heterozygotes developed hyperkeratosis with age. To see whether phenotypic abnormalities in the mouse model were associated with changes in skin barrier function and skin water content we studied basal transepidermal water loss and capacity for barrier repair after experimental barrier disruption as well as stratum corneum hydration. Also, we determined the activities of acid and neutral sphingomyelinase key enzymes of the tumor necrosis factor and interleukin-1 signal transduction pathways generating the ceramides most important for epidermal permeability barrier homeostasis. Neonatal homozygotes showed an 8-fold increase in basal transepidermal water loss compared with wild type controls. Adult heterozygotes exhibited delayed barrier repair after experimental barrier disruption. Stratum corneum hydration was reduced in homozygous and heterozygous mice. Acid sphingomyelinase activity, which is localized in the epidermal lamellar bodies and generates ceramides for extracellular lipid lamellae in the stratum corneum permeability barrier, was reduced in homozygous as well as heterozygous animals. Neutral sphingomyelinase activity, which has a different location and generates ceramides involved in cell signaling, was increased. The reduction in acid sphingomyelinase activity may explain the recently described decreased ratio of ceramides to total lipids in K10 deficient mice. In summary, our results demonstrate the crucial role of the keratin filament for permeability barrier function and stratum corneum hydration.     

Basis for the permeability barrier abnormality in lamellar ichthyosis

The basis for the permeability barrier abnormality in lamellar ichthyosis (LI) is not known. LI is caused by mutations in the gene that encodes the enzyme, transglutaminase 1 (TGI), which is responsible for assembly of the cornified envelope (CE). TG1 also has been suggested recently to catalyze the covalent attachment of omega-hydroxyceramides (omega-OHCer) to the CE, forming the corneocyte-lipid envelope (CLE). We first assessed the barrier function and the permeability pathway of the water-soluble tracer, colloidal lanthanum, across the stratum corneum (SC) in patients with LI with absent (n = 4) or low (n = 2) TG1 activity/protein. Increased movement of tracer through the SC correlated with increased transcutaneous water loss, and tracer remained restricted to the SC interstices. Enhanced extracellular permeability, in turn, was explicable by truncation and fragmentation of extracellular lamellar membrane arrays. The resultant clefts in the SC interstices represent the likely pathway for increased water permeability. Moreover, tracer movement remained restricted to the interstices, despite the demonstration of increased corneocyte fragility associated with widespread variations in CE structure. Regardless of variability in CE structure, however, CLE structure and bound omega-OHCer content were normal. The normal CLE in LI may explain both the restriction of tracer to the SC interstices, as well as the presence of foreshortened membrane arrays with near-normal interlamellar dimensions. Finally, the demonstration of a normal CLE in LI also raises questions about the putative role of TG1 in forming the CLE. These results demonstrate: (1) the extracellular nature of increased permeability in LI; (2) discontinuities in extracellular membrane structures that account for the enhanced permeability in LI; (3) that these membrane abnormalities are both associated with and explained by abnormalities in the subjacent CE scaffold; and (4) an intact CLE is present in LI, despite abnormalities in the CE, which may restrict water movement to the SC interstices in LI.     

Omega-hydroxyceramides are required for corneocyte lipid envelope (CLE) formation and normal epidermal permeability barrier function

Omega-hydroxyceramides (omega-OHCer) are the predominant lipid species of the corneocyte lipid envelope in the epidermis. Moreover, their omega-esterified-derivatives (acylCer) are major components of the stratum corneum extracellular lamellae, which regulate cutaneous permeability barrier function. Because epidermal omega-OHCer appear to be generated by a cytochrome P450-dependent process, we determined the effects of a mechanism-based inhibitor of omega-hydroxylation, aminobenzotriazole (ABT), on epidermal omega-OH Cer formation and barrier function. We first ascertained that ABT, but not hydroxybenzotriazole (OHBT), a chemical relative with no P450 inhibitory activity, inhibited the incorporation of [14C]-acetate into the omega-OH-containing Cer species in cultured human keratinocytes (68.1% +/- 6.9% inhibition versus vehicle-treated controls; p < 0.001), without altering the synthesis of other Cer and fatty acid species. In addition, ABT significantly inhibited the omega-hydroxylation of very long-chain fatty acids in cultured human keratinocytes. Topical application of ABT, but not OHBT, when applied to the skin of hairless mice following acute barrier disruption by tape-stripping, resulted in a significant delay in barrier recovery (e.g., 38.3% delay at 6 h versus vehicle-treated animals), assessed as increased transepidermal water loss. The ABT-induced barrier abnormality was associated with: (i) a significant decrease in the quantities of omega-OHCer in both the unbound and the covalently bound Cer pools; (ii) marked alterations of lamellar body structure and contents; and (iii) abnormal stratum corneum extracellular lamellar membrane structures, with no signs of cellular toxicity. Furthermore, pyridine-extraction of ABT- versus vehicle-treated skin, which removes all of the extracellular lamellae, leaving the covalently attached lipids, showed numerous foci with absent corneocyte lipid envelope in ABT- versus vehicle-treated stratum corneum. These results provide the first direct evidence for the importance of omega-OHCer for epidermal permeability function, and suggest further that acylCer and/or corneocyte lipid envelope are required elements in permeability barrier homeostasis.     

Normal ultrastructure, but altered stratum corneum lipid and protein composition in a mouse model for epidermolytic hyperkeratosis.

Recently, we established keratin 10-deficient mice, serving as a model for the hyperkeratotic skin disorder epidermolytic hyperkeratosis. The considerable ichthyosis in these mice suggested alterations in terminal differentiation and in the formation of a functional epidermal barrier. Here, we report on the ultrastructural organization and composition of the stratum corneum lipids and on the expression of two major cornified envelope proteins. Electron microscopy of ruthenium tetroxide postfixed skin samples demonstrated a normal extrusion and morphology of lamellar bodies as well as the formation of bona fide lamellar layers in neonatal keratin 10-deficient mice. When we studied the composition of the major stratum corneum lipids, however, we found significant changes. Most importantly, the analysis of ceramide subpopulations revealed that the total amount of ceramide 2 was elevated in keratin 10-deficient mice, whereas ceramides 1, 3, 4, and 5 were decreased among total stratum corneum lipids. The amount of the ceramide precursors sphingomyelin and glucosylceramide was reduced in the stratum corneum without accompanying changes in the mRNA coding for acid sphingomyelinase. Notably, we found an increased mRNA and protein content for involucrin in neonatal keratin 10-deficient mice, whereas the expression of loricrin was not changed. Our data demonstrate that, although the formation of lipid layers in the stratum corneum appeared to be normal, its lipid composition is significantly altered in keratin 10-deficient mice.     

Ichthyosis in Sjögren–Larsson syndrome reflects defective barrier function due to abnormal lamellar body structure and secretion

Sjögren–Larsson syndrome is a genetic disease characterized by ichthyosis, mental retardation, spasticity and mutations in the ALDH3A2 gene coding for fatty aldehyde dehydrogenase, an enzyme necessary for oxidation of fatty aldehydes and fatty alcohols. We investigated the cutaneous abnormalities in 9 patients with Sjögren–Larsson syndrome to better understand how the enzymatic deficiency results in epidermal dysfunction. Histochemical staining for aldehyde oxidizing activity was profoundly reduced in the epidermis. Colloidal lanthanum perfusion studies showed abnormal movement of tracer into the extracellular spaces of the stratum corneum consistent with a leaky water barrier. The barrier defect could be attributed to the presence of abnormal lamellar bodies, many with disrupted limiting membranes or lacking lamellar contents. Entombed lamellar bodies were present in the cytoplasm of corneocytes suggesting blockade of lamellar body secretion. At the stratum granulosum–stratum corneum interface, non-lamellar material displaced or replaced secreted lamellar membranes, and in the stratum corneum, the number of lamellar bilayers declined and lamellar membrane organization was disrupted by foci of lamellar/non-lamellar phase separation. These studies demonstrate the presence of a permeability barrier abnormality in Sjögren–Larsson syndrome, which localizes to the stratum corneum interstices and can be attributed to abnormalities in lamellar body formation and secretion.     

Structural basis for the barrier abnormality following inhibition of HMG CoA reductase in murine epidermis.

Recent studies have shown that increased epidermal 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase activity is crucial for the barrier recovery response that follows solvent-induced barrier perturbation. Upregulation of this enzyme leads to increased cholesterologenesis, formation and secretion of cholesterol-enriched lamellar bodies, and barrier repair. Topical lovastatin-induced inhibition of HMG CoA reductase activity both delays the acute barrier-repair response, as well as leading to a chronic barrier abnormality when applied repeatedly to intact skin. Presently, we assessed the effects of repeated topical applications of two different specific inhibitors of HMG CoA reductase on barrier function, the lamellar body-secretory system, and stratum corneum intercellular domains, with functional and morphologic parameters. Once-daily applications of lovastatin or fluindostatin (XU62-320; Sandoz) for 4-8 d to intact hairless mouse epidermis produced a progressive abnormality in barrier function (transepidermal water loss greater than 2.0-5.0 in treated versus less than 0.25 mg/cm2/h for weakly active analogues or vehicle controls). The barrier defect was preceded by alterations in lamellar body internal structure and a partial failure of lamellar body secretion into the stratum corneum interstices, further confirmed by enzyme cytochemistry. Moreover, the deposition of abnormal lamellar body contents resulted in the formation of clefts in the intercellular spaces at the stratum granulosum-stratum corneum interface, resulting in increased permeability through these domains shown by lanthanum perfusion. Applications of irritants, even when producing a barrier abnormality, did not alter the lamellar body secretory system. Co-applications of cholesterol with the inhibitors reversed both the barrier abnormality and the abnormalities in the lamellar body secretory system that occur with the inhibitor alone. Finally, membrane bilayer structures in the mid-to-outer stratum corneum of inhibitor-treated specimens appeared normal, but the intercellular domains displayed enormously expanded lacunae. However, because similar dilatations also occurred in vehicle-treated samples, they can be attributed to the vehicle alone. These studies provide further evidence that the inhibitor-induced defect in barrier function a) is initiated by inhibition of HMG CoA reductase; b) can be attributed to defects in both lamellar body structure and deposition with resultant abnormalities in intercellular membrane domains in the lower stratum corneum; and c) is further enhanced by permissive effects of the vehicle on the permeability of the outer stratum corneum.     

Ichthyosis: Mechanisms of Disease

The disorders of cornification (ichthyoses) comprise acquired and inherited disorders characterized clinically by generalized scaling and histologically by hyperkeratosis. They may arise through defects in the production or maintenance of a normal cornified cell compartment, or both. The stratum corneum is composed of protein-enriched and lipid-depleted corneocytes ("bricks") surrounded by an intercellular domain ("mortar") composed of hydrophobic, lipid-enriched membrane bilayers, and containing desmosomes and a limited array of hydrolytic enzymes. Mechanisms whereby a genetic defect involving either the bricks or the mortar may result in abnormal stratum corneum retention are discussed using ichthyosis vulgaris and recessive X-linked ichthyosis as examples. In addition, epidermal hyperproliferation, which floods the cornified cell compartment with incompletely formed units, results in hyperkeratosis. To date, no primary disorders of epidermal hyperproliferation have been defined. Recent work, however, demonstrates that stratum corneum barrier function regulates epidermal DNA synthesis. For example, in essential fatty acid deficiency, barrier dysfunction is responsible at least in part ror the epidermal hyperproliferation. Defective barrier function due to defective lamellar body secretion may also underlie the phenotypic changes after birth in harlequin ichthyosis; that is, from the massive, constrictive hyperkeratosis of the newborn to an exfoliative erythroderma in survivors. The mechanisms whereby specific defects in cornification result in generalized scaling disease are only beginning to be defined. Yet, even at this early stage, the view of the stratum corneum as a tightly organized structure whose function is highly regulated is emerging. Hence, the disorders of cornification should provide important insights into stratum corneum structure and function.     

Short-term glucocorticoid treatment compromises both permeability barrier homeostasis and stratum corneum integrity: inhibition of epidermal lipid synthesis accounts for functional abnormalities

Prolonged exposure of human epidermis to excess endogenous or exogenous glucocorticoids can result in well-recognized cutaneous abnormalities. Here, we determined whether short-term glucocorticoid treatment would also display adverse effects, specifically on two key epidermal functions, permeability barrier homeostasis and stratum corneum integrity and cohesion, and the basis for such changes. In humans 3 d of treatment with a potent, commonly employed topical glucocorticoid (clobetasol), applied topically, produced a deterioration in barrier homeostasis, characterized by delayed barrier recovery and abnormal stratum corneum integrity (rate of barrier disruption with tape strippings) and stratum corneum cohesion (microg protein removed per stripping). Short-term systemic and topical glucocorticoid produced similar functional defects in mice, where the basis for these abnormalities was explored further. Both the production and secretion of lamellar bodies were profoundly decreased in topical glucocorticoid-treated mice resulting in decreased extracellular lamellar bilayers. These structural changes, in turn, were attributable to a profound global inhibition of lipid synthesis, demonstrated both in epidermis and in cultured human keratinocytes. The basis for the abnormality in stratum corneum integrity and cohesion was a diminution in the density of corneodesmosomes in the lower stratum corneum. We next performed topical replacement studies to determine whether lipid deficiency accounts for the glucocorticoid-induced functional abnormalities. The abnormalities in both permeability barrier homeostasis and stratum corneum integrity were corrected by topical applications of an equimolar distribution of free fatty acids, cholesterol, and ceramides, indicating that glucocorticoid-induced inhibition of epidermal lipid synthesis accounts for the derangements in both cutaneous barrier function and stratum corneum integrity/cohesion. These studies indicate that even short-term exposure to potent glucocorticosteroids can exert profound negative effects on cutaneous structure and function. Finally, topical replenishment with epidermal physiologic lipids could represent a potential method to reduce the adverse cutaneous effects of both topical glucocorticoid treatment and Cushing's syndrome.     

Localization of ceramide and glucosylceramide in human epidermis by immunogold electron microscopy.

Ceramides and glucosylceramides are pivotal molecules in multiple biologic processes such as apoptosis, signal transduction, and mitogenesis. In addition, ceramides are major structural components of the epidermal permeability barrier. The barrier ceramides derive mainly from the enzymatic hydrolysis of glucosylceramides. Recently, anti-ceramide and anti-glucosylceramide anti-sera have become available that react specifically with several epidermal ceramides and glucosylceramides, respectively. Here we demonstrate the detection of two epidermal covalently bound omega-hydroxy ceramides and one covalently bound omega-hydroxy glucosylceramide species by thin-layer chromatography immunostaining. Moreover, we show the ultrastructural distribution of ceramides and glucosylceramides in human epidermis by immunoelectron microscopy on cryoprocessed skin samples. In basal epidermal cells and dermal fibroblasts ceramide was found: (i) at the nuclear envelope; (ii) at the inner and outer mitochondrial membrane; (iii) at the Golgi apparatus and the endoplasmic reticulum; and (iv) at the plasma membrane. The labeling density was highest in mitochondria and at the inner nuclear membrane, suggesting an important role for ceramides at these sites. In the upper epidermis, ceramides were localized: (i) in lamellar bodies; (ii) in trans-Golgi network-like structures; (iii) at the cornified envelope; and (viii) within the intercellular space of the stratum corneum, which is in line with the known analytical data. Glucosylceramides were detected within lamellar bodies and in trans-Golgi network-like structures of the stratum granulosum. The localization of glucosylceramides at the cornified envelope of the first corneocyte layer provides further proof for the existence of covalently bound glucosylceramides in normal human epidermis.     

Epidermal-specific defect of GPI anchor in Pig-a null mice results in Harlequin ichthyosis-like features

We previously demonstrated that the epidermal-specific glycosylphosphatidylinositol (GPI)-anchor-deficient mice, generated by Pig-a gene disruption (Pig-a null mice), exhibited wrinkled and dry skin with hyperkeratosis and abnormal differentiation, and they died within a few days after birth. Here, we investigated the basis for the early demise of these animals, and the potential role of epidermal structural and biochemical abnormalities. The rapid demise of these animals was associated with both diminished epidermal permeability barrier function and decreased stratum corneum (SC) water content. The barrier abnormality could be attributed abnormal internal contents of lamellar bodies, with a downstream failure to generate normal extracellular lamellar bilayers in the SC. Moreover, processing profilaggrin to its monomeric form was impaired in Pig-a null mouse epidermis, while levels of the differentiation-specific proteins, involucrin, loricrin and profilaggrin were normal. Failure of filaggrin processing was accompanied by decreased activity of protein phosphatase 2A, an enzyme involved in profilaggrin to filaggrin processing. Thus, these studies demonstrate a critical role for GPI anchor and GPI-anchored proteins in divergent arms of epidermal terminal differentiation. While the permeability barrier abnormality can be attributed to defects in the lamellar body secretory system, the hydration abnormality is, in part, due to lack of availability of filaggrin-derived proteolytic products. Finally, since the dual abnormalities in the lamellar body secretory system and filaggrin processing resemble two key features of human Harlequin ichthyosis, Pig-a null mice could provide an appropriate analog for further studies of this disease.     

Structural and biochemical basis for the UVB-induced alterations in epidermal barrier function

Ultraviolet light (UVR) induces a myriad of cutaneous changes, including delayed disruption of the permeability barrier with higher doses. To investigate the basis for the UVB-induced barrier alteration, we assessed the epidermal lamellar body secretory system at various time points before and after barrier disruption with a single high dose of UVB (7.5 MED) to murine epidermis. Morphological data were correlated with changes in epidermal proliferation and lipid synthesis, indicative of lamellar body generation. Twenty-four hours following UVB, the stratum corneum (SC) is normal, but a layer of abnormal, vacuolated, and lamellar body (LB)-deficient cells is present, immediately beneath the stratum granulosum (SG)/SC interface. Immediately subjacent to this band of damaged cells, normal keratinocytes that contain intact LBs are present. By 72 h, concomitant with the appearance of a barrier abnormality, extensively damaged cells persist at the SC/SG interface, and abnormal lamellar membrane structures appear in the lower SC. Upper stratum spinosum (SS) and lower SG cells appear normal, with increased numbers of LBs. A barrier abnormality is still present at 96 h, in association with membrane abnormalities in the lower SC interstices, but up to four normal-appearing, subjacent SG cell layers are present. By 120 h, accelerated LB formation and precocious LB extrusion occur throughout the thickened SG; normal lamellar membranes are present in the lower SC; and barrier recovery is almost complete. Whereas, epidermal synthesis of the major barrier lipid species (i.e., cholesterol, fatty acids, and ceramides, including acylceramides) is reduced or unchanged at 24 and 48 h, it increases significantly 72 h after exposure to UVB. Therefore, the delayed disruption of the permeability barrier following acute UVB exposure results from the arrival of a band of lamellar body-incompetent (i.e., damaged) cells at the SG/SC interface. The subsequent, rapid recovery of the barrier, in turn, results from compensatory hyperplasia of subjacent, undamaged SS/SG cells, generating increased numbers and contents of LB. These results underscore the critical role of the stratum compactum in mediating barrier function, and suggest that beneficial therapeutic effects of UV exposure may be due to enhanced lipid production and barrier regeneration.     

Harlequin ichthyosis and other autosomal recessive congenital ichthyoses: the underlying genetic defects and pathomechanisms

Autosomal recessive congenital ichthyoses (ARCI) include several severe subtypes including harlequin ichthyosis (HI), lamellar ichthyosis and non-bullous congenital ichthyosiform erythroderma. Patients with these severe types of ichthyoses frequently show severe hyperkeratosis and scales over a large part of the body surface form birth and their quality of life is often severely affected. Recently, research into the pathomechanisms of these severe congenital ichthyoses have advanced dramatically and led to the identification of several causative genes and molecules underlying the genetic defects. To date, seven loci have been identified that are associated with ARCI and, among them, five causative genes and molecules have been detected. The five genes are transglutaminase 1 gene (TGM1), ABCA12, two lipoxygenase genes, ALOXE3 and ALOX12B and ichthyin. One of these components, ABCA12, has recently been shown to be a keratinocyte lipid transporter associated with lipid transport in lamellar granules and loss of ABCA12 function leads to a defective lipid barrier in the stratum corneum, resulting in the HI phenotype. Transglutaminse 1 deficiency was reported to cause a malformed cornified cell envelope leading to a defect in the intercellular lipid layers in the stratum corneum and defective stratum corneum barrier function resulting in an ichthyosis phenotype. Thus, defective intercellular lipid layers are major findings in autosomal recessive congenital ichthyoses. Information concerning ARCI genetic defects and disease pathomechanisms are beneficial for providing better treatments and genetic counseling including prenatal diagnosis for families affect by ichthyoses.     

Barrier function of the skin: "la raison d'être" of the epidermis

The primary function of the epidermis is to produce the protective, semi-permeable stratum corneum that permits terrestrial life. The barrier function of the stratum corneum is provided by patterned lipid lamellae localized to the extracellular spaces between corneocytes. Anucleate corneocytes contain keratin filaments bound to a peripheral cornified envelope composed of cross-linked proteins. The many layers of these specialized cells in the stratum corneum provide a tough and resilient framework for the intercellular lipid lamellae. The lamellae are derived from disk-like lipid membranes extruded from lamellar granules into the intercellular spaces of the upper granular layer. Lysosomal and other enzymes present in the extracellular compartment are responsible for the lipid remodeling required to generate the barrier lamellae as well as for the reactions that result in desquamation. Lamellar granules likely originate from the Golgi apparatus and are currently thought to be elements of the tubulo-vesicular trans-Golgi network. The regulation of barrier lipid synthesis has been studied in a variety of models, with induction of several enzymes demonstrated during fetal development and keratinocyte differentiation, but an understanding of this process at the molecular genetic level awaits further study. Certain genetic defects in lipid metabolism or in the protein components of the stratum corneum produce scaly or ichthyotic skin with abnormal barrier lipid structure and function. The inflammatory skin diseases psoriasis and atopic dermatitis also show decreased barrier function, but the underlying mechanisms remain under investigation. Topically applied "moisturizers" work by acting as humectants or by providing an artificial barrier to trans-epidermal water loss; current work has focused on developing a more physiologic mix of lipids for topical application to skin. Recent studies in genetically engineered mice have suggested an unexpected role for tight junctions in epidermal barrier function and further developments in this area are expected. Ultimately, more sophisticated understanding of epidermal barrier function will lead to more rational therapy of a host of skin conditions in which the barrier is impaired.     

皮肤屏障功能研究进展

皮肤屏障主要由角化包膜和脂质膜、中间丝聚合蛋白、角蛋白、角化桥粒、板层小体和角质层角质形成细胞问质、紧密连接等组成,防止水分的丢失及阻止外界的侵害,维持机体内稳态.基因突变、变应原、微生物、紫外线等因素共同作用下,能导致皮肤屏障结构、代谢及功能的异常,引起鱼鳞病、特应性皮炎等皮肤病.阐明皮肤病与屏障结构及功能异常联系,指导开发有效的诊疗和预防措施.     

Impaired sphingomyelinase activity and epidermal differentiation in atopic dermatitis

A defective permeability barrier leads to the penetration of environmental allergens into the skin and initiates immunological reactions and inflammation crucially involved in the pathogenesis of atopic dermatitis (AD). Decreased stratum corneum ceramide content may cause the defect in permeability barrier function consistently found in AD. Acid and neutral sphingomyelinase (A- and N-SMase) generate ceramides with structural and signal transduction functions in epidermal proliferation and differentiation. We determined epidermal SMase activities, DNA synthesis, involucrin, loricrin, filaggrin, and keratin expression in lesional and non-lesional skin of AD patients. We found decreased epidermal A-SMase activity in lesional and non-lesional skin, correlating with reduced stratum corneum ceramide content and disturbed barrier function. N-SMase activity was reduced in non-lesional skin and more significantly reduced in lesional skin, correlating with impaired expression of cornified envelope proteins and keratins, important for skin barrier function. Changes in involucrin, loricrin, filaggrin, keratin K 5 (basal) and K 16 (proliferation associated) were noticed in non-lesional and lesional skin, whereas changes in K 10 (suprabasal), K 6 (proliferation associated), and K 17 (inflammation associated) were found only in lesional skin. In summary, reduction in SMase-generating ceramides and impaired differentiation are involved in the defective barrier function found in AD.     

An update on molecular aspects of the non-syndromic ichthyoses

Abstract:  Ichthyosis includes a number of subtypes from congenital severe forms, such as harlequin ichthyosis (HI), to mild non-congenital forms, such as ichthyosis vulgaris. Recently, research into the pathomechanisms of ichthyoses has dramatically advanced and led to the identification of several causative genes and molecules underlying the genetic defects. In most types of ichthyosis, pathogenic mechanisms are associated with defects in skin barrier function. Three major components of the stratum corneum barrier are (i) intercellular lipid layers, (ii) cornified cell envelope and (iii) keratin-filaggrin degradation products. The causative molecules underlying ichthyosis subtypes include ABCA12, lipoxygenase-3, 12 R -lipoxygenase, CYP4F2 homolog, ichthyin and steroid sulphatase and all these are thought to be related to the intercellular lipid layers. Transglutaminase 1 has a function in cornified cell envelope formation. Keratins 1, 10 and 2 are involved in the keratin network of suprabasal keratinocytes and filaggrin are essential for formation of keratohyalin granules. In fact, loss of ABCA12 function leads to a defective lipid barrier in the stratum corneum, resulting in the HI phenotype and ABCA12 is a known keratinocyte lipid transporter associated with lipid transport in lamellar granules. Filaggrin gene mutations in ichthyosis vulgaris cause keratohyalin granule deficiency. Information concerning genetic defects and ichthyotic disease pathomechanisms are beneficial to develop effective therapy and provide information for genetic counselling including prenatal diagnosis for families affected by ichthyotic disease.     

Ultraviolet B-induced alterations of the skin barrier and epidermal calcium gradient

Ultraviolet irradiation induces a variety of cutaneous changes, including epidermal permeability barrier disruption. In the present study, we assessed the effects of ultraviolet B (UVB) irradiation in epidermal barrier function and calcium distribution in murine epidermis. Adult hairless mice were exposed to a single dose of UVB (0.15 J/cm(2)). Barrier function was evaluated by transepidermal water loss (TEWL), lanthanum perfusion. The morphological alterations were examined by histology, immunohistochemistry and electron microscopy using ruthenium tetroxide (RuO(4)) postfixation. For evaluation of the effect on epidermal calcium distribution, the ion-capture cytochemistry was employed. UVB irradiation caused a significant increase in TEWL, which peaked at day 4. In parallel, the increased number of sunburn cells and the changes in epidermal hyperplasia and proliferation were observed. Electron microscopic observation demonstrated that the water-soluble lanthanum tracer was present in the extracellular stratum corneum domains, and the increased intercellular permeability was correlated with defective organization of the extracellular lipid lamellar bilayers of the stratum corneum. Moreover, UVB irradiation also caused an appearance of calcium precipitates in the stratum corneum and transitional cell layers as well as the increased cytosolic calcium in the lower epidermis, reflecting the alterations of the epidermal calcium gradient. These results suggest that the changes of the epidermal calcium distribution pattern may correlate with the perturbation of the epidermal barrier induced by UVB irradiation.     

Glycerol regulates stratum corneum hydration in sebaceous gland deficient (asebia) mice

The only known function of human sebaceous glands is the provocation of acne. We assessed here whether sebum influences stratum corneum hydration or permeability barrier function in asebia J1 and 2 J mice, with profound sebaceous gland hypoplasia. Asebia J1 mice showed normal permeability barrier homeostasis and extracellular lamellar membrane structures, but they displayed epidermal hyperplasia, inflammation, and decreased (>50%) stratum corneum hydration, associated with a reduction in sebaceous gland lipids (wax diesters/monoesters, sterol esters). The triglyceride content of both asebia and control stratum corneum was low, consistent with high rates of triglyceride hydrolysis within the normal pilosebaceous apparatus, despite high rates of triglyceride synthesis. Although a mixture of synthetic, sebum-like lipids (sterol/wax esters, triglycerides) did not restore normal stratum corneum hydration to asebia skin, topical glycerol, the putative product of triglyceride hydrolysis in sebaceous glands, normalized stratum corneum hydration, and the glycerol content of asebia stratum corneum was 85% lower than in normal stratum corneum. In contrast, another potent endogenous humectant (urea) did not correct the abnormality. The importance of glycerol generation from triglyceride in sebaceous glands for stratum corneum hydration was demonstrated further by (i) the absence of sebaceous-gland-associated lipase activity in asebia mice, whereas abundant enzyme activity was present in the glands of control mice; and (ii) the inability of high concentrations of topical triglyceride to correct the hydration abnormality, despite the presence of abundant lipase activity in asebia stratum corneum. These results show that sebaceous-gland-derived glycerol is a major contributor to stratum corneum hydration.     

The skin: an indispensable barrier

Abstract: The skin forms an effective barrier between the organism and the environment preventing invasion of pathogens and fending off chemical and physical assaults, as well as the unregulated loss of water and solutes. In this review we provide an overview of several components of the physical barrier, explaining how barrier function is regulated and altered in dermatoses. The physical barrier is mainly localized in the stratum corneum (SC) and consists of protein‐enriched cells (corneocytes with cornified envelope and cytoskeletal elements, as well as corneodesmosomes) and lipid‐enriched intercellular domains. The nucleated epidermis also contributes to the barrier through tight, gap and adherens junctions, as well as through desmosomes and cytoskeletal elements. During epidermal differentiation lipids are synthesized in the keratinocytes and extruded into the extracellular domains, where they form extracellular lipid‐enriched layers. The cornified cell envelope, a tough protein/lipid polymer structure, resides below the cytoplasmic membrane on the exterior of the corneocytes. Ceramides A and B are covalently bound to cornified envelope proteins and form the backbone for the subsequent addition of free ceramides, free fatty acids and cholesterol in the SC. Filaggrin is cross‐linked to the cornified envelope and aggregates keratin filaments into macrofibrils. Formation and maintenance of barrier function is influenced by cytokines, 3′,5′‐cyclic adenosine monophosphate and calcium. Changes in epidermal differentiation and lipid composition lead to a disturbed skin barrier, which allows the entry of environmental allergens, immunological reaction and inflammation in atopic dermatitis. A disturbed skin barrier is important for the pathogenesis of contact dermatitis, ichthyosis, psoriasis and atopic dermatitis.