Johanna M. Brandner

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.

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.

Up-Regulated Expression of Zonula Occludens Protein-1 in Human Melanoma Associates with N-Cadherin and Contributes to Invasion and Adhesion

During the process of malignant transformation, nascent melanoma cells escape keratinocyte control through down-regulation of E-cadherin and instead communicate among themselves and with fibroblasts via N-cadherin-based cell-cell contacts. The zonula occludens (ZO) protein-1 is a membrane-associated component of both the tight and adherens junctions found at sites of cell-cell contact. In most cancers, levels of ZO-1 are typically down-regulated, leading to increased motility. Here we report the novel observation that ZO-1 expression is up-regulated in melanoma cells and is located at adherens junctions between melanoma cells and fibroblasts. Immunofluorescence and co-immunoprecipitation studies showed co-localization of ZO-1 with N-cadherin. Down-regulation of ZO-1 in melanoma cells through RNA interference produced marked changes in cell morphology--leading to a less-dendritic, more rounded phenotype. Consistent with a role in N-cadherin-based adhesion, RNAi-treated melanoma cells were less adherent and invasive when grown in a collagen gel. These data provide the first evidence that increased ZO-1 expression in melanoma contributes to the oncogenic behavior of this tumor and further illustrate that protein products of genes, such as ZO-1, can function in either a pro- or anti-oncogenic manner when expressed in different cellular contexts.

Alteration of Tight Junction Proteins Is an Early Event in Psoriasis: Putative Involvement of Proinflammatory Cytokines

Psoriasis is an inflammatory skin disease characterized by hyperproliferation of keratinocytes, impaired barrier function, and pronounced infiltration of inflammatory cells. Tight junctions (TJs) are cell-cell junctions that form paracellular barriers for solutes and inflammatory cells. Altered localization of TJ proteins in the epidermis was described in plaque-type psoriasis. Here we show that localization of TJ proteins is already altered in early-stage psoriasis. Occludin, ZO-1, and claudin-4 are found in more layers than in normal epidermis, and claudin-1 and -7 are down-regulated in the basal and in the uppermost layers. In plaque-type psoriasis, the staining patterns of occludin and ZO-1 do not change, whereas the claudins are further down-regulated. Near transmigrating granulocytes, all TJ proteins except for junctional adhesion molecule-A are down-regulated. Treatment of cultured keratinocytes with interleukin-1beta and tumor necrosis factor-alpha, which are present at elevated levels in psoriatic skin, results in an increase of transepithelial resistance at early time points and a decrease at later time points. Injection of interleukin-1beta into an ex vivo skin model leads to an up-regulation of occludin and ZO-1, resembling TJ protein alteration in early psoriasis. Our results show for the first time that alteration of TJ proteins is an early event in psoriasis and is not the consequence of the more profound changes found in plaque-type psoriasis. Our data indicate that cytokines are involved in alterations of TJ proteins observed in psoriasis.

Tight junctions form a barrier in human epidermis.

Tight junctions (TJ) are cell-cell junctions that have proved to form a paracellular barrier for solutes and water between cells of epithelia, including the stratum granulosum of the stratified epithelium of the epidermis of newborn mice. In mice lacking claudin-1, a major barrier-forming TJ component, this barrier was abolished. However, the role of TJ in human skin is controversially discussed as unambiguous data were missing so far. Here, we investigated TJ barrier function in healthy human skin as well as in skin samples from psoriatic lesions which are characterized by an altered localization of TJ proteins. We demonstrate for human skin that occludin- and claudin-1-positive sites in the stratum granulosum form a barrier for extracellular biotin-SH (557Da) and that in psoriatic skin the localization of the barrier and the TJ proteins are altered in parallel.

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells

During early wound healing (WH) events Connexin 43 (Cx43) is down‐regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down‐regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non‐diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non‐diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non‐diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non‐diabetic cells. The cause of the discrepancy between diabetic and non‐diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368‐phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.

Tight Junction Proteins in the Skin

It has long been accepted that tight junctions (TJ) are crucial for the formation and maintenance of the paracellular barrier and for cell polarity in simple epithelia and endothelia. Moreover, it is long known that they play a role in barrier function of amphibian skin. However, only in recent years were TJ and TJ proteins identified in the epidermis of men and mice. Their involvement in the barrier function of mammalian skin has been shown. This review summarizes our current knowledge about TJ and TJ proteins in mammalian skin.

Barriers and more: functions of tight junction proteins in the skin

Although the existence of tight junction (TJ) structures (or a secondary epidermal barrier) was postulated for a long time, the first description of TJ proteins in the epidermis (occludin, ZO‐1, and ZO‐2) was only fairly recent. Since then, a wealth of new insights concerning TJs and TJ proteins, including their functional role in the skin, have been gathered. Of special interest is that the epidermis as a multilayered epithelium exhibits a very complex localization pattern of TJ proteins, which results in different compositions of TJ protein complexes in different layers. In this review, we summarize our current knowledge about the role of TJ proteins in the epidermis in barrier function, cell polarity, vesicle trafficking, differentiation, and proliferation. We hypothesize that TJ proteins fulfill TJ structure‐dependent and structure‐independent functions and that the specific function of a TJ protein may depend on the epidermal layer where it is expressed.

Contribution of Tight Junction Proteins to Ion, Macromolecule, and Water Barrier in Keratinocytes

Tight junctions (TJs) form a selective barrier for ions, water, and macromolecules in simple epithelia. In keratinocytes and epidermis, TJs were shown to be involved in individual barrier functions. The absence of the TJ protein claudin-1 (Cldn1) in mice results in a skin-barrier defect characterized by lethal water loss. However, detailed molecular analyses of the various TJ barriers in keratinocytes and the contribution of distinct TJ proteins are missing. Herein, we discriminate TJ-dependent paracellular resistance from transcellular resistance in cultured keratinocytes using the two-path impedance spectroscopy. We demonstrate that keratinocyte TJs form a barrier for Na(+), Cl(-), and Ca(2+), and contribute to barrier function for water and larger molecules of different size. In addition, knockdown of Cldn1, Cldn4, occludin, and zonula occludens-1 increased paracellular permeabilities for ions and larger molecules, demonstrating that all of these TJ proteins contribute to barrier formation. Remarkably, Cldn1 and Cldn4 are not critical for TJ barrier function for water in submerged keratinocyte cultures. However, Cldn1 influences stratum corneum (SC) proteins important for SC water barrier function, and is crucial for TJ barrier formation for allergen-sized macromolecules.

Expression and localization of tight junction-associated proteins in human hair follicles

Tight junctions (TJ) are barrier-forming intercellular junctions selectively sealing cells and controlling the paracellular pathway. They have been well-characterized in simple epithelia and endothelia but have only recently been described in stratified epithelia such as epidermis, oesophagus and oral mucosa. Various epithelial layers which are partly in morphogenic continuity with the epidermis and develop therefrom during early fetal life, build the human hair follicle. The barrier function of these epithelial layers seems to be important for the universal continuity of the barrier represented by the skin. We show the presence of the TJ proteins ZO-1, occludin, and various claudins in the hair follicle and demonstrate their impressive heterogeneous distribution pattern within a given stratum as well as within its different epithelial layers. Coexpression of the various TJ proteins, arguing for typical TJ structures, can be observed especially in cell layers facing the hair shaft and the stratum corneum, and in addition at the border between the outer and inner root sheaths. Usually they are found in close proximity to desmosomal and adherens junction proteins. The morphological and biological importance of these findings and the possible roles of TJ in hair follicles, e.g. in follicular penetration, are discussed.