Multidirectional activity of bakuchiol against cellular mechanisms of facial ageing - Experimental evidence for a holistic treatment approach.

OBJECTIVE: Skin ageing is a multifactorial process involving formation of reactive oxygen species, consecutive inflammation with reduced epidermal and dermal cell viability and resulting damage to the extracellular matrix. Effective dermocosmetic treatment modalities should ideally address these hallmarks in a holistic approach. Here, we determined the corresponding activity profile of bakuchiol, a plant-derived meroterpene, in an array of in vitro, ex vivo and in vivo studies and compared it to retinol, currently considered as gold standard in topical antiageing cosmetics. METHODS: The antioxidative capacity and power of bakuchiol and retinol were analysed by measuring 2,2'-diphenyl-1-picrylhydrazyl (DPPH) reduction via its absorption decay and electron spin resonance spectroscopy, respectively. Effects on prostaglandin E2 (PGE2), macrophage migration inhibitory factor (MIF), fibroblast growth factor 7 (FGF7), collagen type I and VII (COL1A1, COL7A1), fibronectin (FN) levels as well as the metabolization of water-soluble tetrazolium 1 (WST-1) were determined in human dermal fibroblasts. Epidermal regeneration was assessed utilizing an in vitro wound healing model. FN protein levels were analysed ex vivo after treatment with a formulation containing bakuchiol, retinol or vehicle using suction blister fluid. Skin condition improvement was determined in vivo in a split-face comparison study after application of bakuchiol or vehicle. RESULTS: In contrast to retinol, bakuchiol demonstrated high antioxidative efficacy. Levels of PGE2 and MIF were significantly decreased by both bakuchiol and retinol. Bakuchiol but not retinol significantly increased FGF7 protein levels. WST-1 metabolization levels were significantly augmented by bakuchiol and retinol. Bakuchiol and retinol application led to a significant augmentation of COL1A1, COL7A1 and FN protein levels. Wounds supplemented with bakuchiol but not retinol displayed a significant increase in epidermis regeneration. Clinically, areas treated with a bakuchiol-containing formulation showed a statistically significant increase in FN protein values after a 4-week application compared to untreated areas and areas treated with vehicle. CONCLUSION: These data provide evidence for the multidirectional efficacy of bakuchiol against cellular hallmarks of skin ageing. Its activity profile shares some common features with retinol but demonstrates several hitherto unknown biopositive effects in our studies, namely stimulation of the critical extracellular matrix component FN, and accelerated epidermal regeneration and wound healing.

OBJECTIF: le vieillissement de la peau est un processus multifactoriel impliquant la formation de dérivés réactifs de l'oxygène, une inflammation consécutive qui entraîne une viabilité réduite des cellules du derme et de l'épiderme, et endommage la matrice extracellulaire. Pour être efficaces, les traitements dermocosmétiques devraient dans l'idéal traiter ces caractéristiques selon une approche holistique. Ici, nous avons déterminé le profil d'activité correspondant du bakuchiol, un méroterpène d'origine végétale, dans une série d'études in vitro, ex vivo et in vivo, et l'avons comparé au rétinol, qui est aujourd'hui considéré comme la référence parmi les cosmétiques anti-âge topiques. MÉTHODES: la capacité antioxydante et la puissance du bakuchiol et du rétinol ont été analysées en mesurant la réduction du 2,2-diphényl-1-picrylhydrazyl (DPPH) selon sa décroissance par absorption et à l'aide d'une spectroscopie par résonance magnétique électronique, respectivement. Les effets sur la prostaglandine E2 (PGE2), le facteur inhibiteur de la migration (FIM) des macrophages, le facteur de croissance des fibroblastes 7 (FGF7), le collagène de type I et VII (COL1A1, COL7A1), les taux de fibronectine (FN), ainsi que la métabolisation du tétrazolium 1 soluble dans l'eau (WST-1) ont été déterminés dans des fibroblastes dermiques humains. La régénération épidermique a été évaluée à l'aide d'un modèle de cicatrisation des plaies in vitro. Les taux de fibronectine ont été analysés ex vivo après un traitement avec une formulation contenant du bakuchiol, du rétinol ou un excipient à l'aide d'un liquide d'aspiration sous forme de vésicules. L'amélioration de l'état de la peau a été déterminée in vivo dans une étude de comparaison en hémiface après l'application de bakuchiol ou d'un excipient. RÉSULTATS: Contrairement au rétinol, le bakuchiol s'est avéré présenter une efficacité antioxydante élevée. Les taux de PGE2 et de FIM ont significativement diminué avec le bakuchiol et le rétinol. L'application de bakuchiol s'est accompagnée d'une augmentation significative des taux de protéine FGF7, mais pas celle de rétinol. Les taux de métabolisation du WST-1 ont augmenté de façon significative avec le bakuchiol et le rétinol. L'application de bakuchiol et de rétinol a entraîné une augmentation significative des taux de protéines COL1A1, COL7A1 et fibronectine. Les plaies supplémentées en bakuchiol, mais pas en rétinol, ont montré une augmentation significative de la régénération épidermique. Sur le plan clinique, les zones traitées avec une formulation contenant du bakuchiol ont montré une augmentation statistiquement significative des taux de fibronectine après une application de 4 semaines par rapport aux zones non traitées et aux zones traitées avec un excipient. CONCLUSION: ces données fournissent des preuves de l'efficacité multidirectionnelle du bakuchiol contre les caractéristiques cellulaires du vieillissement de la peau. Son profil d'activité partage certaines caractéristiques communes avec le rétinol, mais démontre plusieurs effets biopositifs jusqu'alors inconnus dans nos études : la stimulation de la fibronectine, composante essentielle de la matrice extracellulaire, et une régénération épidermique et une cicatrisation accélérée des plaies.

Direct assessment of individual skin barrier components by electrical impedance spectroscopy.

BACKGROUND: Expression of the tight junction proteins Cldn1 and 4 is altered in skin diseases such as atopic dermatitis, and Cldn1 deficiency affects skin barrier formation. Impedance spectroscopy (IS) has been proven to allow detection of alterations in the skin barrier but is currently unable to separate effects on viable epidermis (VE) and stratum corneum (SC). METHODS: Effects of siRNA-mediated Cldn1 and 4 knockdown in reconstructed human epidermis (RHE) on VE and SC barrier function were investigated with Ussing chamber-based IS. Barrier components were sequentially altered, employing iron oxide nanoparticles and EGTA, to identify their contribution to the impedance spectrum. Resistance changes due to apically applied hyperosmolar electrolyte were used to identify barrier defects non-invasively. RESULTS: IS of RHE yielded two relaxation frequencies, representing the barrier properties of the SC (~1000 Hz) and VE (~100 Hz). As proof of concept, it was shown that the Cldn1 knockdown-induced resistance drop arises from the impairment of both SC and VE, indicated by a shift of both relaxation frequencies. Hyperosmolar electrolyte penetration allowed non-invasive detection of Cldn1 knockdown via time-dependent frequency shifts. The absence of Cldn4 knockdown-induced changes revealed the weaknesses of transepithelial electrical resistance analysis. CONCLUSION: In conclusion, the present technique allows to separately measure the barrier properties of SC and VE and further evaluate the Cldn1 and 4 knockdown impact on the skin barrier. As the measurement with agarose-embedded electrolyte allowed non-invasive identification of the Cldn1 knockdown, this opens the way to detailed in vivo skin barrier assessment.

Melanoma Associated Chitinase 3-Like 1 Promoted Endothelial Cell Activation and Immune Cell Recruitment.

Chitinase 3-like 1 (CHI3L1) is an enzymatically inactive mammalian chitinase that is associated with tumor inflammation. Previous research indicated that CHI3L1 is able to interact with different extracellular matrix components, such as heparan sulfate. In the present work, we investigated whether the interaction of CHI3L1 with the extracellular matrix of melanoma cells can trigger an inflammatory activation of endothelial cells. The analysis of the melanoma cell secretome indicated that CHI3L1 increases the abundance of various cytokines, such as CC-chemokine ligand 2 (CCL2), and growth factors, such as vascular endothelial growth factor A (VEGF-A). Using a solid-phase binding assay, we found that heparan sulfate-bound VEGF-A and CCL2 were displaced by recombinant CHI3L1 in a dose-dependent manner. Microfluidic experiments indicated that the CHI3L1 altered melanoma cell secretome promoted immune cell recruitment to the vascular endothelium. In line with the elevated VEGF-A levels, CHI3L1 was also able to promote angiogenesis through the release of extracellular matrix-bound pro-angiogenic factors. In conclusion, we showed that CHI3L1 is able to affect the tumor cell secretome, which in turn can regulate immune cell recruitment and blood vessel formation. Accordingly, our data suggest that the molecular targeting of CHI3L1 in the course of cancer immunotherapies can tune patients' response and antitumoral inflammation.

Use of Modified Clostridium perfringens Enterotoxin Fragments for Claudin Targeting in Liver and Skin Cells.

Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic in vitro studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.

Tight Junction Proteins Claudin-1 and Occludin Are Important for Cutaneous Wound Healing.

Tight junction (TJ) proteins are known to be involved in proliferation and differentiation. These processes are essential for normal skin wound healing. Here, we investigated the TJ proteins claudin-1 and occludin in ex vivo skin wound healing models and tissue samples of acute and chronic human wounds and observed major differences in localization/expression of these proteins, with chronic wounds often showing a loss of the proteins at the wound margins and/or in the regenerating epidermis. Knockdown experiments in primary human keratinocytes showed that decreased claudin-1 expression resulted in significantly impaired scratch wound healing, with delayed migration and reduced proliferation. Activation of AKT pathway was significantly attenuated after claudin-1 knockdown, and protein levels of extracellular signal-related kinase 1/2 were reduced. For occludin, down-regulation had no impact on wound healing in normal scratch assays, but after subjecting the cells to mechanical stress, which is normally present in wounds, wound healing was impaired. For both proteins we show that most of these actions are independent from the formation of barrier-forming TJ structures, thus demonstrating nonbarrier-related functions of TJ proteins in the skin. However, for claudin-1 effects on scratch wound healing were more pronounced when TJs could form. Together, our findings provide evidence for a role of claudin-1 and occludin in epidermal regeneration with potential clinical importance.

3D skin models for 3R research: The potential of 3D reconstructed skin models to study skin barrier function.

The skin barrier is an important shield regulating the outside-in as well as inside-out penetration of water, nutrients, ions and environmental stimuli. We can distinguish four different barrier compartments: the physical, chemical, immunological and microbial skin barrier. Well-functioning of those is needed to protect our body from the environment. To better understand the function and the contribution of barrier dysfunction in skin diseases, 3D skin or epidermal models are a valuable tool for in vitro studies. In this review, we summarize the development and application of different skin models in skin barrier research. During the last years, enormous effort was made on optimizing these models to better mimic the in vivo composition of the skin, by fine-tuning cell culture media, culture conditions and including additional cells and tissue components. Thereby, in vitro barrier formation and function has been improved significantly. Moreover, in this review we point towards changes and chances for in vitro 3D skin models to be used for skin barrier research in the nearby future.

Tight junctions in skin inflammation.

Inflammation of the skin is found after various external stimuli, e.g., UV radiation, allergen uptake, microbial challenge, or contact with irritants, as well as due to intrinsic, not always well-defined, stimuli, e.g., in autoimmune responses. Often, it is also triggered by a combination of both. The specific processes, which mean the kind of cytokines and immune cells involved and the extent of the reaction, depend not only on the trigger but also on the predisposition of the individual. Tight junctions (TJs) in the skin have been shown to form a barrier in the granular cell layer of the epidermis. Furthermore, TJ proteins were found in several additional epidermal layers. Besides barrier function, TJ proteins have been shown to be involved in proliferation, differentiation, cell-cell adhesion, and apoptosis in keratinocytes. In inflamed skin, TJ proteins are often affected. We summarize here the impact of skin inflammation on TJs, e.g., in various forms of dermatitis including atopic dermatitis, in skin infection, and in UV-irradiated skin, and discuss the role of TJs in these inflammatory processes.

Current knowledge on biomarkers for contact sensitization and allergic contact dermatitis.

Contact sensitization is common and affects up to 20% of the general population. The clinical manifestation of contact sensitization is allergic contact dermatitis. This is a clinical expression that is sometimes difficult to distinguish from other types of dermatitis, for example irritant and atopic dermatitis. Several studies have examined the pathogenesis and severity of allergic contact dermatitis by measuring the absence or presence of various biomarkers. In this review, we provide a non-systematic overview of biomarkers that have been studied in allergic contact dermatitis. These include genetic variations and mutations, inflammatory mediators, alarmins, proteases, immunoproteomics, lipids, natural moisturizing factors, tight junctions, and antimicrobial peptides. We conclude that, despite the enormous amount of data, convincing specific biomarkers for allergic contact dermatitis are yet to be described.

Diverse regulation of claudin-1 and claudin-4 in atopic dermatitis.

Tight junctions are important for skin barrier function. The tight junction protein claudin 1 (Cldn-1) has been reported to be down-regulated in nonlesional skin of atopic dermatitis (AD) patients. In contrast, we did not observe a significant down-regulation of Cldn-1 in nonlesional skin of the AD cohort used in this study. However, for the first time, a significant down-regulation of Cldn-1 in the upper and lower epidermal layers of lesional skin was detected. In addition, there was a significant up-regulation of Cldn-4 in nonlesional, but not lesional, AD skin. For occludin, no significant alterations were observed. In an AD-like allergic dermatitis mouse model, Cldn-1 down-regulation in eczema was significantly influenced by dermal inflammation, and significantly correlated with hallmarks of eczema (ie, increased keratinocyte proliferation, altered keratinocyte differentiation, increased epidermal thickness, and impaired barrier function). In human epidermal equivalents, the addition of IL-4, IL-13, and IL-31 resulted in a down-regulation of Cldn-1, and Cldn1 knockdown in keratinocytes resulted in abnormal differentiation. In summary, we provide the first evidence that Cldn-1 and Cldn-4 are differentially involved in AD pathogenesis. Our data suggest a role of Cldn-1 in AD eczema formation triggered by inflammation.

Sebaceous gland, hair shaft, and epidermal barrier abnormalities in keratosis pilaris with and without filaggrin deficiency.

Although keratosis pilaris (KP) is common, its etiopathogenesis remains unknown. KP is associated clinically with ichthyosis vulgaris and atopic dermatitis and molecular genetically with filaggrin-null mutations. In 20 KP patients and 20 matched controls, we assessed the filaggrin and claudin 1 genotypes, the phenotypes by dermatoscopy, and the morphology by light and transmission electron microscopy. Thirty-five percent of KP patients displayed filaggrin mutations, demonstrating that filaggrin mutations only partially account for the KP phenotype. Major histologic and dermatoscopic findings of KP were hyperkeratosis, hypergranulosis, mild T helper cell type 1-dominant lymphocytic inflammation, plugging of follicular orifices, striking absence of sebaceous glands, and hair shaft abnormalities in KP lesions but not in unaffected skin sites. Changes in barrier function and abnormal paracellular permeability were found in both interfollicular and follicular stratum corneum of lesional KP, which correlated ultrastructurally with impaired extracellular lamellar bilayer maturation and organization. All these features were independent of filaggrin genotype. Moreover, ultrastructure of corneodesmosomes and tight junctions appeared normal, immunohistochemistry for claudin 1 showed no reduction in protein amounts, and molecular analysis of claudin 1 was unremarkable. Our findings suggest that absence of sebaceous glands is an early step in KP pathogenesis, resulting in downstream hair shaft and epithelial barrier abnormalities.

Influence of Birch Bark Triterpenes on Keratinocytes and Fibroblasts from Diabetic and Nondiabetic Donors.

Impaired wound healing is one of the main risk factors associated with diabetes mellitus. Few options are available to treat diabetic wounds, and therefore efficient remedies are urgently needed. An interesting option might be an extract of birch bark (TE) that has been clinically proven to accelerate acute wound healing. We investigated the effects of TE and its main components betulin and lupeol in cultured normal keratinocytes and dermal fibroblasts from diabetic and nondiabetic donors. These in vitro models can provide insights into possible beneficial effects in wound healing. TE and betulin treatment led to increased mRNA levels of chemokines, pro-inflammatory cytokines, and mediators important in wound healing, e.g., IL-6, TNFα, IL-8, and RANTES. We observed a pronounced upregulation of MIF, IL-8, and RANTES on the protein level. Furthermore, a shape change of the actin cytoskeleton was seen in keratinocytes and fibroblasts, and the Rho-GTPases and p38-MAPK were found to be activated in keratinocytes. On the basis of our results, TE is worthy of further study as a potential option to influence wound-healing processes under diabetic conditions. These first insights need to be confirmed by clinical studies with diabetic patients.

Hereditary barrier-related diseases involving the tight junction: lessons from skin and intestine.

The tight junction (TJ) regulates paracellular barrier properties. TJs are composed of transmembrane proteins, i.e., claudins, occludin, tricellulin and junctional adhesion molecules as well as TJ plaque proteins. Their relative abundance and composition determines epithelial tightness. TJs undergo rapid regulation by various signalling pathways, either directly addressing TJ transmembrane proteins or via plaque proteins and the cytoskeleton. In the skin, TJs exert predominantly barrier functions, while in the intestine they also mediate paracellular ion and water transport. In diseases, TJs can either be primarily affected (hereditary TJ defects) or changes can result from secondary regulatory inputs as, e.g., in inflammatory diseases (secondary TJ defects). Secondary TJ defects can maintain disease activity, e.g., by enhanced antigen leak. This review discusses TJ composition, function and regulation as well as primary and secondary tight junction defects in a comparative manner in skin and intestine in order to elucidate similarities and differences.

Accelerated reepithelialization by triterpenes: proof of concept in the healing of surgical skin lesions.

The acceleration of wound healing is a major surgical concern. A triterpene extract from birch bark (Betulae cortex) experimentally enhances keratinocyte differentiation in vitro and accelerates wound healing ex vivo. We conducted an open, blind-evaluated, controlled, prospective, randomized (1:1) phase II clinical trial in patients requiring split-thickness skin graft transplantation at two university hospitals in Germany. Donor sites on the upper legs were covered with a moist silicone-coated dressing. Oleogel-S10 ointment containing 10% birch bark extract was randomly applied to the distal or proximal half of the wound, with the other half serving as an intraindividual control, for 14 days after the skin graft surgery. The primary efficacy variable was faster reepithelialization as determined from macrophotographs by independent, blinded experts. Twenty-four patients were randomized and completed the trial. After the 14-day test period, the planned interim analysis revealed a highly significant (p < 0.0001) superiority of Oleogel-S10 in the primary efficacy variable and the trial was terminated early due to ethical concerns. The treatment side was also better reepithelialized and more similar to normal skin after 3 months. In conclusion, Oleogel-S10 significantly accelerated reepithelialization at split-thickness skin graft donor sites. Treatment with Oleogel-S10 was safe and well tolerated.

Immunohistochemical analyses point to epidermal origin of human Merkel cells.

Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, ß1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. ß1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors.

Merkel cell polyomavirus detection in Merkel cell cancer tumors in Northern Germany using PCR and protein expression.

Merkel cell carcinoma is a highly malignant skin cancer which predominantly occurs in elderly and immunocompromised persons. The identification of the Merkel cell polyomavirus (MCPyV) has inaugurated a new understanding of Merkel cell carcinoma pathogenesis. The frequent detection of the virus in Merkel cell carcinoma tissue (70-90%), its monoclonal integration in the tumor cells and the expression of viral oncogenes highly suggest that MCPyV is causally linked to the pathogenesis of the majority of Merkel cell cancer (MCC) cases. Using qualitative and quantitative PCR together with immunohistochemical staining this study aimed at characterizing the presence of MCPyV sequences and viral early gene expression in a cohort of MCC cases (n = 32) selected in Northern Germany. 40-57% of the cases were identified as MCPyV positive with 40.6% of the cases positive by immunohistochemical staining and 51.6-57.6% positive by PCR. Interestingly, in the majority (64%) of LT-Antigen positive tumors only 25-50% of tumor cells express LT-Antigen. These data are in accord with published studies describing heterogeneity in MCPyV viral loads and suggest that detection of MCPyV in Merkel cell carcinoma by PCR should be undertaken using multiple primer pairs.

The connexin mimetic peptide Gap27 increases human dermal fibroblast migration in hyperglycemic and hyperinsulinemic conditions in vitro.

Significant increases in skin wound healing rates occur by reducing connexin-mediated communication (CMC). Gap27, a connexin (Cx) mimetic peptide targeted to the second extracellular loop of Cx43, which inhibits CMC, increases migration of human keratinocytes and dermal fibroblasts. To examine the efficacy of Gap27 in a hyperglycemic and hyperinsulinemic in vitro environment, cell migration, gap junction, and Cx hemichannel functionality and cell-substrate adhesion assays were performed on human dermal fibroblasts and diabetic fibroblast and keratinocytes. To investigate fibroblast genes involved in these processes, extra-cellular matrix (ECM) and adhesion gene expression was determined with a PCR array. Gap27 increased fibroblast migration in both euglycemia/euinsulinemia and hyperglycemia/hyperinsulinemia, and influenced migration in diabetic keratinocytes. Hyperglycemia/hyperinsulinemia reduced gap junction coupling in fibroblasts and Gap27 reduced CMC and cell adhesion to substrata in fibroblasts cultured in high glucose. Migrating dermal fibroblast ECM and cell adhesion genes were found to be differentially regulated by Gap27 in euglycemia and hyperglycemia. The PCR array showed that Gap27 upregulated 34 genes and downregulated 1 gene in euglycemic migrating fibroblasts. By contrast in hyperglycemia, Gap27 upregulated 1 gene and downregulated 9 genes. In euglycemic conditions, Gap27 induced upregulation of genes associated with ECM remodeling, whereas in hyperglycemia, ECM component genes were downregulated by Gap27. Thus, Gap27 improves cell migration during scrape-wound repair in hyperglycemia/hyperinsulinemia conditions in vitro, although migration of diabetic cells is less influenced. Our results suggest that this increase in motility may occur by decreasing gap junction and hemichannel activity and altering gene expression in the adhesion and ECM pathway.

Filaggrin genotype in ichthyosis vulgaris predicts abnormalities in epidermal structure and function.

Although it is widely accepted that filaggrin (FLG) deficiency contributes to an abnormal barrier function in ichthyosis vulgaris and atopic dermatitis, the pathomechanism of how FLG deficiency provokes a barrier abnormality in humans is unknown. We report here that the presence of FLG mutations in Caucasians predicts dose-dependent alterations in epidermal permeability barrier function. Although FLG is an intracellular protein, the barrier abnormality occurred solely via a paracellular route in affected stratum corneum. Abnormal barrier function correlated with alterations in keratin filament organization (perinuclear retraction), impaired loading of lamellar body contents, followed by nonuniform extracellular distribution of secreted organelle contents, and abnormalities in lamellar bilayer architecture. In addition, we observed reductions in corneodesmosome density and tight junction protein expression. Thus, FLG deficiency provokes alterations in keratinocyte architecture that influence epidermal functions localizing to the extracellular matrix. These results clarify how FLG mutations impair epidermal permeability barrier function.

Tight junctions and differentiation--a chicken or the egg question?

Skin barrier function is indispensable to prevent the uncontrolled loss of water and solutes and to protect the body from external assaults. To fulfil this function, keratinocytes undergo a complex pathway of differentiation that terminates in the formation of the stratum corneum. Additionally, tight junctions (TJs), which are cell-cell junctions localized in the stratum granulosum, are involved in the barrier function of the skin. Important biological and clinical roles of TJs are strongly suggested by altered TJ protein levels and distribution in skin diseases like psoriasis, ichthyosis and atopic dermatitis. Because these skin diseases show alterations in differentiation and TJs, it was suggested that changes in TJs might simply be a consequence of altered differentiation. However, in this viewpoint, we like to argue that the situation is not as simple and depends on the specific microenvironment. We discuss three hypotheses regarding the interplay between TJs/TJ proteins and differentiation: (1) TJs/TJ proteins are influenced by differentiation, (2) differentiation is influenced by TJs/TJ proteins, and (3) TJs/TJ proteins and differentiation are independent of each other. In addition, the concept is introduced that both processes are going on at the same time, which means that while one specific TJ protein/barrier component might be influenced by differentiation, the other may influence differentiation.