Marek Haftek, MD, PhD, DR. CNRS, Laboratoire de Recherche Dermatologique, EA4169, Faculté de Médecine et de Pharmacie, 8, Av. Rockefeller, F-69373 Lyon, France, Tel./fax: +33 (0)478 777 140, e-mail: firstname.lastname@example.org
Abstract: Several tight junction (TJ) proteins were detected in the living layers of adult human epidermis, and TJ-like membrane ridges were observed at the top of the stratum granulosum (SG) in freeze-fracture studies. We applied standard and immunoelectron microscopy to look for TJ-derived structures in the stratum corneum (SC) of human adult epidermis and in cornified envelopes purified from the plantar SC. Besides confirming claudin-1 labelling in the proximity of SG desmosomes, we also observed immunolocalization near corneodesmosomes in the lower SC. In addition, TJ proteins were consistently detected in the purified cornified envelopes. Lateral but not horizontal walls of the corneocytes showed frequent points of molecular fusion between lipid envelopes. These structural associations were very frequently localized at the top of the lateral corneocyte membranes, thus sealing the extremities of lateral intercorneocyte spaces. We propose that TJ-like structures persist in the SC and contribute to the reinforcement of lateral contacts and to the formation of membrane interdigitations between corneocytes. Their presence could contribute to subdivision of the extracellular spaces of SC into consecutive individualized compartments. Intercellular lipids, enzymes and other (glyco)protein content could thus evolve in the keratinized epidermal layer at different paces, as preprogrammed in the underlying living cells and influenced by the environment, e.g. humidity. Such situation might explain differences in the degradation rates between the ‘peripheral’ and the ‘non-peripheral’ corneodesmosomes observed during physiological desquamation, as previously suggested by us and others.
Human stratum corneum (SC) is the final product of epidermal differentiation that provides barrier function to the skin. Composed of cornified epithelial cells, called corneocytes, and sealed with the lipid-rich extracellular matrix, this thin epidermal layer is relatively impermeable to water and water-soluble substances. Like the entire epidermis, SC is constantly recycled in a highly regulated and interactive process in which superficial cell losses by desquamation at the skin surface are readily compensated by the conversion of the uppermost living keratinocytes into corneocytes (1,2). Such a programmed cell death is associated with several important changes in the cell shape and structure leading to the accumulation of flattened, laterally interdigitated, cornified keratinocytes forming the lower part of the SC, the SC compactum. During cornification, phospholipids of the keratinocyte plasma membrane are replaced by a single layer of ceramides. The latter are covalently bound by transglutaminase 1 to the protein envelope that is also cross-linked at the cell periphery by the same enzyme (3,4). The proper functioning of the SC barrier depends largely on its cohesion. Therefore, it is important to note that desmosomes, the principal interkeratinocyte junctions contributing to the mechanical strength of the epidermal tissue, also become integrated into the cornified envelopes and persist within the SC (5,6). Corneodesmosomes, the modified desmosomes of SC, are evenly distributed around the corneocytes of SC compactum. Following a complex interplay between pH-sensitive hydrolases and their natural inhibitors in hydrophilic and hydrophobic compartments of the SC intercellular spaces, corneodesmosomes undergo proteolytic cleavage resulting in the formation of SC disjunctum and, finally, in desquamation (7–9). Interestingly, degradation of corneodesmosomes does not occur at the same rate all around the cells. The flat, upper and lower surfaces of corneocytes forming successive cell layers become deprived of the junctions earlier than the lateral, interdigitated corneocyte edges. This results in a characteristic layered appearance of the upper SC, histologically described as ‘basket weave’ pattern (7,10). However, the mechanism of this spatial disparity in corneodesmosome degradation is largely unknown.
In this context, we were alerted when discovering that tight junction (TJ)-like discrete regions of contact were not only found in the stratum granulosum (SG) but also persisted in the horny layer.
We proposed that these structures, which are distributed at the periphery of flattening cells, contributed to the reinforcement of lateral contacts and to the formation of lateral plasma membrane interdigitations. Persistence of these TJ-like structures, immobilized through the cross-linking of cornified envelopes, may result in an increased lateral cohesion between corneocytes in the SC compactum. This could explain differences in the degradation rates between the ‘peripheral’ and the ‘non-peripheral’ corneodesmosomes observed during physiological desquamation. Moreover, fusion between the lateral corneocyte walls could contribute to the physical and functional subdivision of the extracellular space of the SC into horizontal compartments. In such individualized extracellular volumes situated at various depths, molecular interactions between lipid and protein components could progress at different paces, influenced both by the input from the granular layer keratinocytes at the base and by the environmental conditions at the skin surface. Preliminary findings leading to this concept have been first presented during the post-International Investigative Dermatology satellite joint meeting of the Society for Skin Structure Research (Japan) and the Society for Cutaneous Ultrastructure Research (Europe) May 2008, Otsu, Japan (Haftek M., invited speaker) and then developed during several scientific conventions (11–14). Very recently, this viewpoint has been supported by an independent study (15).
Discrete points of contact reminiscent of TJ structures and co-localizing with claudin-1 labelling are detected between the granular layer keratinocytes in normal adult human epidermis
Immunogold labelling of ultra-thin sections of Lowicryl K4M-embedded skin with an antibody to the TJ protein claudin-1, used here as a marker, confirmed its expression at the surface of keratinocytes (see Data S1 for methods and the additional Fig. S1). In the stratum spinosum (SS), the labelling was abundant and distributed along the plasma membranes but was not associated with any discernable junction structures (Fig. 1a). In the upper SS, claudin-1 labelling was frequently found flanking desmosomes (Fig. 1d).
In the granular layer, peripheral distribution of claudin-1 became even less uniform and plasma membrane alignments characteristic of TJ could occasionally be detected at the sites of immunogold labelling (Fig. 1b). The labelling of cell surfaces persisted in the lowermost horny layers (Fig. 1c). Here, claudin-1 was frequently encountered at the periphery of corneodesmosomes, consistently with its distribution close to desmosomes in the living layers, but typical bi-lamellar TJ structures were no more observed in the vicinity of the labelling.
Cornified cell envelopes isolated from human plantar SC show presence of TJ-characteristic proteins
To investigate the localization of TJ proteins in the SC in more detail, we studied human cornified cell envelopes. Focal reactivity of antibodies to claudin-1 and, to a lesser degree, to occludin was observed on sections of Lowicryl-embedded purified cell envelopes (Fig. 2). Immunogold labelling was specifically distributed over the envelope structures with no apparent background. Double labelling using an antibody to desmoglein-1 confirmed the anatomical proximity of expression of TJ proteins and corneodesmosomes already observed on skin sections. Interestingly, sites of attachment between cornified envelopes belonging to two different cells were occasionally observed within the preparation. These points of persistent adhesion showed labelling with antibodies to claudin-1 and occludin. Ultrastructure of these attachment points suggested fusion between the lipid layers covering isolated cross-linked envelopes.
Accessibility of TJ proteins to specific antibodies may be compromised on native or plastic-embedded sections because of a compact organization of the junction components and their cross-linking in the upper keratinocyte layers, hence the necessity for the use of the antigen retrieval methods like sodium metaperiodate treatment (this study) or trypsin digestion (15). In the case of enzymatic digestion, the antigen localization is revealed often at the price of a lesser quality of the morphological preservation.
Regions of apparent fusion between lipid envelopes can be observed between the lateral corneocyte walls but not between the successive cell strata
Close examination of standard electron microscopy samples of the human SC allows observation of peculiar structures situated at the lateral faces of corneocytes (Fig. 3). In the SC compactum, the cells evolving at the same level frequently showed close apposition between portions of their lateral walls. Morphometric approach indicated that the total thickness of lipid membranes forming these points was equal or slightly inferior to the sum of thicknesses of two individual membranes, suggesting molecular fusion within the structures (Fig. S1). Such regions of apparent fusion were frequently intercalated between the corneodesmosomes attaching side-to-side cells but were not observed between the flat surfaces of corneocytes facing overlying cell layers. Typically, corneodesmosomes were also less frequent in this latter localization. Most importantly from the mechanistic point of view, the points of lateral association between cell envelopes were quite often observed in the upper parts of the lateral intercorneocyte spaces, thereby isolating them from the higher-level compartments.
What are the functions of TJ-like structures in the horny layer?
The various roles of TJs and TJ proteins in human epidermis are not yet fully understood (16–19). Classically, this type of junction divides the extracellular spaces situated beneath the TJ belts from those located above them. At the same time, strands of transmembrane TJ proteins separate portions of cell membranes situated on each side of the junction, thus polarizing cells and segregating pole-specific membrane molecules. To fulfil such functions, TJs must form a continuous band circumventing entire cell perimeter, contiguous with similar structures expressed on the neighbouring cells. Such is the situation in several simple epithelia where, e.g. TJs located around the apical poles of enterocytes exert their fencing role and seal the intercellular spaces against the lumen of the digestive tract (20–22).
TJs in adult human epidermis, at the steady state, do not seem to occur in the shape of complete belts but, according to early freeze-fracture studies, rather take on the appearance of more or less developed spots (23,24). Only on the surface of the granular layer keratinocytes, at the interface with SC, abundant TJ-like protein strands, similar but not identical to those present in simple epithelia, have been observed with the use of freeze-fracturing technique (25). They form an alveolar network, frequently surrounding the desmosomes and interconnecting them.
This discontinuous distribution could partially explain why TJ-like structures have only very recently been admitted to exist and play a role in adult human epidermis, based on transmission electron microscopy studies (25–29). In tissue cross-sections, ephemeral points of membrane bridging located in the high part of the lateral spaces, between the last keratinocytes of the granular layer, have proved amenable to labelling using TJ-specific anti-occludin antibodies. This very limited localization of the labelled structures remains in contrast to the wide distribution of typical TJ proteins, such as claudin-1.
Apparently, complete TJ belts have been described in the organotypic cultures of human epidermal keratinocytes (30,31), and TJ structures are constantly encountered in the apical position on sections of human foetal epidermis (25,27,28). However, both situations differ from the normal adult epidermis exposed to dry environment, where apically located TJ are harder to immunolocalise (15,26). Despite the problems with ultrastructural visualization of TJ belts in normal human epidermis, recent publications point to the existence of a functional TJ barrier, effective at least for small molecules of approximately 550 Da (32,33). Also, deregulation of TJs in pathological conditions, paralleling an impaired function of the epidermal barrier, supports the effective role of these junctions in this respect (18,26,34–36).
In murine epidermis, TJs encircling granular layer keratinocytes are additionally reinforced by expression of tricellulin – at the points of contact between three adjacent TJ belts – to provide an efficient barrier, which is complementary to that of the SC (37,38). The pioneering studies by Furuse et al. (39) have demonstrated that in newborn mice, TJs play an important function in controlling transepidermal water flux, because animals with the invalidated claudin-1 gene were unable to survive after birth, as they suffered from a rapid severe dehydration. Additionally, these studies have underscored the essential role of claudin-1 in the TJ function. Indeed, in the absence of claudin-1, the TJ system expressing occludin proved leaky, indicating by the way insufficient functional compensation by the other members of the family of claudins. The situation in humans seems to diverge in a significant way from that observed in mice. Naturally occurring human mutants that fail to produce claudin-1 develop NISCH syndrome [neonatal ichthyosis associated with sclerosing cholangitis, recently termed ichthyosis hypotrichosis and sclerosing cholangitis (IHSC) syndrome according to the new consensus nomenclature of ichthyoses (40)] but survive after birth, and their premature death is because of dysfunction of their liver and renal simple epithelia (41,42). A better protection from the epidermal water loss in claudin-1-deficient patients with IHSC syndrome could be because of a putative higher redundancy of the TJ protein system in humans. Also, in human development, there is much more time to compensate a barrier problem than in mice. Thickening of the SC observed in IHSC syndrome may be interpreted as the tissue’s successful attempt to compensate for the compromised TJ function. It remains to be studied whether slowing down of the desquamation process leading to hyperkeratosis in patients with IHSC syndrome is linked to an eventual over-expression of TJ structures lacking claudin-1. The role of TJs and the involvement of claudin-1 in hair development and structure are not yet well understood. Therefore, the biological mechanism leading to scalp hypotrichosis and scarring alopecia observed in IHSC syndrome have no straightforward explication so far (41,43). As understanding of life processes increasingly relies on systems biology, the answers to these puzzling questions should be brought about by a larger-scale ‘-omics’ science (44).
Our immunocytochemical studies of normal human epidermis in adults have confirmed previous transmission electron microscopy and freeze-fracture observations, suggesting the presence of TJ structures at the top of the SG (24–26,45). Typical membrane associations coinciding with the claudin-1 immunolabelling proved infrequent on vertical sections of normal adult epidermis, despite a relatively abundant expression of TJ proteins by keratinocytes. Therefore, we suppose that fully structured, multi-strand TJs are not a constant characteristics of this tissue at the steady state. Nevertheless, the shear presence of TJs in the granular layer suggests that the junctions’ proteins may get entrapped within the cornified envelopes during the process of transglutaminase-1-mediated cross-linking. Such a fate is common for another type of intercellular junction, the desmosome (3–5,8). Indeed, we could detect claudin-1 and occludin on cornified envelopes isolated from plantar SC. Focal distribution of these transmembrane TJ proteins, their frequent association with portions of cornified envelopes expressing desmoglein-1 and their presence at points of persisting contacts between structures originally belonging to two different cells are compatible with our observations of the epidermal sections. Furthermore, close examination of the lowermost layers of the SC compactum resulted in the detection of sites of peripheral fusion between lipid envelopes of corneocytes. Distribution of these sites along the lateral walls of corneocytes and, particularly, their presence in the upper parts of the lateral intercorneocyte spaces were highly suggestive of their TJ origin. In fact, fine TJ-like structures were previously visualized in this anatomical localization in the most superficial layer of the SG in normal human skin (24,45). TJ-characteristic proteins could be immunolocalized to such sites in foetal (25) and adult human epidermis (15,26).
It is likely that TJ-like points of adhesion distributed at the rim of the flattened corneocytes contribute to the reinforcement of lateral cohesion and to the formation of plasma membrane interdigitations at this localization. Persistence of these structures, immobilized through the cross-linking of cornified envelopes, may result in additional lateral riveting between corneocytes in the SC compactum. This could explain the differences in degradation rates between the ‘peripheral’ and ‘non-peripheral’ corneodesmosomes observed during physiological desquamation (10,11,13–15). It may also contribute to subdivision of the extracellular space of the SC into consecutive horizontal compartments, separately evolving their pH-regulated interactions between hydrolases, their endogenous inhibitors and their substrates (7, 12 14, 46). Indeed, the points of firm interaction between corneocyte cell envelopes may hinder diffusion of the extracellular proteases and their interaction with structural protein substrates present in corneodesmosomes. Direct contribution of TJs to the ‘cohesive force’ of SC cannot be excluded either but remains to be studied. Indeed, both corneodesmosome degradation and direct sticking of corneocyte lipid envelopes after experimental removal of intercellular lipids have been incriminated as causes of the observed modifications of the cohesive force measured in the SC (47,48). The lateral points of fusion between lipid cell envelopes observed in the horny layer, and putatively resulting from the persistence of TJ structures, could thus directly add to the increased lateral intercorneocyte cohesion.
This work was carried out within the following collaborative networks: European Epidermal Barrier Research Network (E2BRN; http://www.e2brn.com) and COST SkinBAD action (BM0903) supported by the European Union RTD Framework Programme. The study was performed according to the guidelines of the local ethics committee and to the Declaration of Helsinki principles. Electron microscopy samples have been observed at the Centre Technologique des Microscopies (CTμ) of University Lyon1, Villeurbanne, France.
MH, SC, performed the research; MH, VJ, designed the research study; YS, PP, FD, contributed essential reagents or tools; MH, VJ, KP, FF, FP analysed the data; MH wrote the paper.