Epidermal Barrier Development via Corneoptosis: A Unique Form of Cell Death in Stratum Granulosum Cells.

Epidermal development is responsible for the formation of the outermost layer of the skin, the epidermis. The establishment of the epidermal barrier is a critical aspect of mammalian development. Proper formation of the epidermis, which is composed of stratified squamous epithelial cells, is essential for the survival of terrestrial vertebrates because it acts as a crucial protective barrier against external threats such as pathogens, toxins, and physical trauma. In mammals, epidermal development begins from the embryonic surface ectoderm, which gives rise to the basal layer of the epidermis. This layer undergoes a series of complex processes that lead to the formation of subsequent layers, including the stratum intermedium, stratum spinosum, stratum granulosum, and stratum corneum. The stratum corneum, which is the topmost layer of the epidermis, is formed by corneoptosis, a specialized form of cell death. This process involves the transformation of epidermal keratinocytes in the granular layer into flattened dead cells, which constitute the protective barrier. In this review, we focus on the intricate mechanisms that drive the development and establishment of the mammalian epidermis to gain insight into the complex processes that govern this vital biological system.

Type 2 Inflammation Contributes to Skin Barrier Dysfunction in Atopic Dermatitis.

Skin barrier dysfunction, a defining feature of atopic dermatitis (AD), arises from multiple interacting systems. In AD, skin inflammation is caused by host-environment interactions involving keratinocytes as well as tissue-resident immune cells such as type 2 innate lymphoid cells, basophils, mast cells, and T helper type 2 cells, which produce type 2 cytokines, including IL-4, IL-5, IL-13, and IL-31. Type 2 inflammation broadly impacts the expression of genes relevant for barrier function, such as intracellular structural proteins, extracellular lipids, and junctional proteins, and enhances Staphylococcus aureus skin colonization. Systemic anti‒type 2 inflammation therapies may improve dysfunctional skin barrier in AD.

Homoharringtonine is a transdermal granular permeation enhancer.

Although modulation of claudin-1-based tight junction (TJ) in stratum granulosum is an option for transdermal absorption of drugs, granular permeation enhancers have never been developed. We previously found that homoharringtonine (HHT), a natural alkanoid, weakened intestinal epithelial barrier with changing expression and cellular localization of TJ components such as claudin-1 and claudin-4. In the present study, we investigated whether HHT is an epidermal granular permeation enhancer. Treatment of normal human epidermal keratinocytes (NHEK) cells with HHT decreased claudin-1 and claudin-4 but not zonula occludens-1 and E-cadherin. HHT lowered TJ-integrity in NHEK cells, accompanied by permeation-enhancement of dextran (4 kDa) in a dose-dependent manner. Transdermal treatment of mice with HHT weakened epidermal barrier. HHT treatment enhanced transdermal absorption of dextran with a molecular mass of up to 10 kDa. Together, HHT may be a transdermal absorption enhancer.

Loricrin and NRF2 Coordinate Cornification.

Cornification involves cytoskeletal cross-linkages in corneocytes (the brick) and the secretion of lipids/adhesion structures to the interstitial space (the mortar). Because the assembly of lipid envelopes precedes corneocyte maturation, loricrin is supposed to be dispensable for the protection against desiccation. Although the phenotypes of Lor knockout (LKO) mice are obscure, the antioxidative response on the KEAP1/NRF2 signaling pathway compensates for the structural defect in utero. In this study, we asked how the compensatory response is evoked after the defects are repaired. To this end, the postnatal phenotypes of LKO mice were analyzed with particular attention to the permeability barrier function primarily maintained by the mortar. Ultrastructural analysis revealed substantially thinner cornified cell envelopes and increased numbers of lamellar granules in LKO mice. Superficial epidermal damages triggered the adaptive repairing responses that evoke the NRF2-dependent upregulation of genes associated with lamellar granule secretion in LKO mice. We also found that corneodesmosomes are less degraded in LKO mice. The observation suggests that loricrin and NRF2 are important effectors of cornification, in which proteins need to be secreted, cross-linked, and degraded in a coordinated manner.

MC5R Contributes to Sensitivity to UVB Waves and Barrier Function in Mouse Epidermis.

MC5R is known for its role in the exocrine function of sebaceous glands, but other functions in the epidermis remain unclear. This study focused on the relationship between MC5R and homeostasis in the epidermis and examined the role of MC5R in mice whose skin was irradiated with UVB waves. UVB irradiation-induced skin ulcers and severe inflammation at lower doses in homozygotes of MC5R-deficient (i.e., MC5R -/- ) mice (150 mJ/cm2) than the doses in wild-type mice (500 mJ/cm2). Transepidermal water loss was increased (approximately 10-fold) in adult MC5R -/- mice compared with that in wild-type mice. In neonates, a dye exclusion assay showed no remarkable difference between MC5R -/- and wild-type mice. After UVB irradiation, compared with wild-type mice, MC5R -/- mice showed increased inflammatory cell infiltration in the dermis of the ulcerative region, significantly increased thickness of the epidermis in the nonulcerative region, significantly more prickle cells in the nonulcerative region, and increased serum IL-6 levels but decreased IL-10 levels. Transmission electron microscopy revealed fewer lamellar granules, less lipid secretion, and an expansion of the trans-Golgi network in the epidermis in MC5R -/- mice. This study elucidated the increased sensitivity to UVB irradiation and decreased barrier function in MC5R -/- mice.

Claudin-7 in keratinocytes is downregulated by the inhibition of HMG-CoA reductase and is highly expressed in the stratum granulosum of the psoriatic epidermis.

BACKGROUND: Cholesterol is de novo synthesized in the upper epidermis and plays an important role in maintaining the normality of skin. Studying the impact of the inhibition of cholesterol de novo synthesis in the epidermis may help understand how skin homeostasis is regulated. OBJECTIVE: In this study, we created a gene expression profile to investigate the effect of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors on epidermal homeostasis. METHODS: A microarray analysis was performed using normal keratinocytes with or without HMG-CoA reductase inhibitor (pitavastatin) treatment. Real-time PCR confirmed the reproducibility of genes with altered expression in keratinocytes treated with HMG-CoA reductase inhibitors. Among these genes, we focused on reduced expression of claudin 7 histologically confirmed by immunohistochemical staining, in situ hybridization, and immunoelectron microscopy. RESULTS: Claudin-7 was highly expressed in the stratum granulosum of psoriatic lesions but was not expressed in the normal epidermis. Immunoelectron microscopy revealed that claudin-7 was localized in the keratohyalin granules of psoriatic lesions. CONCLUSION: These results indicate that claudin-7 expression was regulated by HMG-CoA reductase in the epidermis and might play a pathogenic role in the keratohyalin granules found in the epidermal granular layer of psoriasis.

Extracting the elasticity of the human skin in microscale and in-vivo from atomic force microscopy experiments using viscoelastic models.

Detecting mechanical properties of the intact skin in-vivo leads to a novel quantitative method to diagnose skin diseases and to monitor skin conditions in clinical settings. Current research and clinical methods that detect skin mechanics have major limitations. The in-vitro experiments are done in non-physiological conditions and in-vivo clinical methods measurer unwanted mechanics of underneath fat and muscle tissues but report the measurement as skin mechanics. An ideal skin mechanics should be captured at skin scale (i.e., micron-scale) and in-vivo. However, extreme challenges of capturing the in-vivo skin mechanics in micron-scale including skin motion due to heart beep, breathing and movement of the subject, has hindered measurement of skin mechanics in-vivo.This study for the first time captures micro-scale mechanics (elasticity and viscoelasticity) of top layers of skin (i.e., the stratum corneum (SC) and stratum granulosum (SG)) in-vivo. In this study, the relevant literature is reviewed and Atomic Force Microscopy (AFM) was used to capture force-indentation curves on the fingertip skin of four human subjects at a high indentation speed of 40 µm/s. The skin of the same subject were tested in-vitro at 10 different indentation speeds ranging from 0.125 to 40 µm/s by AFM. This study extracts the in-vivo elasticity of SC and SG by detecting time-dependency of tested tissue using a fractional viscoelastic standard linear model developed for indentation. The in-vivo elasticity of SC and SG were smaller in females and in-vitro elasticity were higher than that of in-vivo results. The results were consistent with previous observations.

Modulation of lipid fluidity likely contributes to the fructose/xylitol-induced acceleration of epidermal permeability barrier recovery.

We previously showed that topical application of hexoses such as fructose accelerates barrier recovery after disruption. We also showed that various hexoses and polyols interact with phospholipid and alter the phase transition temperature. Thus, we hypothesized that the improvement of barrier recovery by hexoses and polyols might be related to the interaction with phospholipid. Here, we tested this idea by examining the effects of xylitol (a component of some skin-care products) and fructose on lipid dynamics in an epidermal-equivalent model at the single-cell level by means of two-photon microscopy after staining with Laurdan, a fluorescent dye sensitive to the physical properties of its membrane environment. First, we confirmed that topical application of xylitol aqueous solution on tape-stripped human skin accelerated barrier recovery. Then, we examined changes of lipid fluidity in the epidermal-equivalent model after application of water or an aqueous solution of xylitol or fructose. Application of xylitol and/or fructose increased the lipid fluidity in the uppermost part of the stratum granulosum layer, compared to treatment with water alone, and accelerated the exocytosis of lamellar bodies to the intercellular domain between stratum corneum and stratum granulosum. Our results support the idea that the improvement of epidermal barrier homeostasis upon topical application of xylitol or fructose is due to increased lipid fluidity in the uppermost layer of the stratum granulosum, which enables accelerated release of lipid from the stratum granulosum, thereby improving the lamellar structure and accelerating epidermal permeability barrier recovery.

Morphological and Functional Analyses of the Tight Junction in the Palatal Epithelium of Mouse.

Tight junction (TJ) is one of the cell-cell junctions and known to have the barrier and fence functions between adjacent cells in both simple and stratified epithelia. We examined the distribution pattern, constitutive proteins, and permeability of TJ in the stratified squamous epithelium of the palatal mucosa of mice. Ultrastructural observations based on the ultrathin section and freeze-fracture methods revealed that poorly developed TJs are located at the upper layer of the stratum granulosum. The positive immunofluorescence of occludin (OCD), claudin (CLD)-1 and -4 were localized among the upper layer of the stratum granulosum showing a dot-like distribution pattern. And CLD-1 and -4 were localized among the stratum spinosum and the lower part of stratum granulosum additionally showed a positive reaction along the cell profiles. Western blotting of TJ constitutive proteins showed OCD, CLD-1, -2, -4, and -5 bands. The permeability test using biotin as a tracer revealed both the areas where biotin passed through beyond OCD positive points and the areas where biotin stopped at OCD positive points. These results show that poor TJs localize at the upper layer of the stratum granulosum of the palatal epithelium, and the TJs are leaky and include at least CLD-1 and -4.

Epidermal tight junctions in health and disease.

The skin, the largest organ of the body, is an essential barrier that under homeostatic conditions efficiently protects and/or minimizes damage from both environmental (e.g. microorganisms, physical trauma, ultraviolet radiation) and endogenous (e.g., cancers, inflammation) factors. This formidable barrier function resides mainly in the epidermis, a dynamic, highly-stratified epithelium. The epidermis has 2 major barrier structures: stratum corneum, the outmost layer and tight junctions, intercellular junctions that seal adjacent keratinocytes in the stratum granulosum, found below the stratum corneum. In recent years there have been significant advances in our understanding of tight junction function, composition and regulation. Herein we review what is known about tight junctions in healthy skin and keratinocyte culture systems and highlight the dynamic crosstalk observed between tight junctions and the cutaneous immune system. Finally we discuss the preliminary observations suggesting that tight junction function or protein expression may be relevant for the pathogenesis of a number of common cutaneous inflammatory and neoplastic conditions.

Expression of the Ly6/uPAR-domain proteins C4.4A and Haldisin in non-invasive and invasive skin lesions.

C4.4A and Haldisin belong to the Ly6/uPAR/α-neurotoxin protein domain family. They exhibit highly regulated expression profiles in normal epidermis, where they are confined to early (C4.4A) and late (Haldisin) squamous differentiation. We have now explored if dysregulated expressions occur in non-invasive and invasive skin lesions. In non-invasive lesions, their expression signatures were largely maintained as defined by that of normal epidermis. The scenario was, however, markedly different in the progression towards invasive squamous cell carcinomas. In its non-invasive stage (carcinoma in situ), a pronounced attenuation of C4.4A expression was observed, but upon transition to malignant invasive squamous cell carcinomas, the invasive fronts regained high expression of C4.4A. A similar progression was observed for the early stages of benign infiltrating keratoacanthomas. Interestingly, this transition was accompanied by a shift in the predominant association of C4.4A expression with CK1/10 in the normal epidermis to CK5/14 in the invasive lesions. In contrast, Haldisin expression maintained its confinement to the most-differentiated cells and was hardly expressed in the invasive lesions. Because this altered expression of C4.4A was seen in the invasive front of benign (keratoacanthomas) and malignant (squamous cell carcinomas) neoplasms, we propose that this transition of expression is primarily related to the invasive process.

Analysis of aquaporin 9 expression in human epidermis and cultured keratinocytes.

Aquaporin 9 (AQP9) is a member of the aquaglyceroporin family that transports glycerol, urea and other small solutes as well as water. Compared to the expression and function in epidermal keratinocytes of AQP3, another aquaglyceroporin, our knowledge of epidermal AQP9 remains elusive. In this study, we investigated the expression of AQP9 in the human epidermis and cultured keratinocytes. Immunofluorescence studies revealed that AQP9 expression is highly restricted to the stratum granulosum of the human epidermis, where occludin is also expressed at the tight junctions. Interestingly, the AQP3 staining decreased sharply below the cell layers in which AQP9 is expressed. In cultured normal human epidermal keratinocytes (NHEK), knock-down of AQP9 expression in the differentiated cells induced by RNA interference reduced glycerol uptake, which was not as pronounced as was the case with AQP3 knock-down cells. In contrast, similar reduction of urea uptake was detected in AQP9 and AQP3 knock-down cells. These findings suggested that AQP9 expression in NHEK facilitates at least the transport of glycerol and urea. Finally, we analyzed the effect of retinoic acid (RA), a potent stimulator of keratinocyte proliferation, on AQP3 and AQP9 mRNA expression in differentiated NHEK. Stimulation with RA at 1 µM for 24 h augmented AQP3 expression and down-regulated AQP9 expression. Collectively, these results indicate that AQP9 expression in epidermal keratinocytes is regulated in a different manner from that of AQP3.

A homozygous nonsense mutation in the gene for Tmem79, a component for the lamellar granule secretory system, produces spontaneous eczema in an experimental model of atopic dermatitis.

BACKGROUND: Flaky tail (ma/ma Flg(ft/ft)) mice have a frameshift mutation in the filaggrin (Flg(ft)) gene and are widely used as a model of human atopic dermatitis associated with FLG mutations. These mice possess another recessive hair mutation, matted (ma), and develop spontaneous dermatitis under specific pathogen-free conditions, whereas genetically engineered Flg(-/-) mice do not. OBJECTIVE: We identified and characterized the gene responsible for the matted hair and dermatitis phenotype in flaky tail mice. METHODS: We narrowed down the responsible region by backcrossing ma/ma mice with wild-type mice and identified the mutation using next-generation DNA sequencing. We attempted to rescue the matted phenotype by introducing the wild-type matted transgene. We characterized the responsible gene product by using whole-mount immunostaining of epidermal sheets. RESULTS: We demonstrated that ma, but not Flg(ft), was responsible for the dermatitis phenotype and corresponded to a Tmem79 gene nonsense mutation (c.840C>G, p.Y280*), which encoded a 5-transmembrane protein. Exogenous Tmem79 expression rescued the matted hair and dermatitis phenotype of Tmem79(ma/ma) mice. Tmem79 was mainly expressed in the trans-Golgi network in stratum granulosum cells in the epidermis in both mice and humans. The Tmem79(ma/ma) mutation impaired the lamellar granule secretory system, which resulted in altered stratum corneum formation and a subsequent spontaneous dermatitis phenotype. CONCLUSIONS: The Tmem79(ma/ma) mutation is responsible for the spontaneous dermatitis phenotype in matted mice, probably as a result of impaired lamellar granule secretory system and altered stratum corneum barrier function.

The urokinase receptor homolog Haldisin is a novel differentiation marker of stratum granulosum in squamous epithelia.

Several members of the Ly-6/uPAR (LU)-protein domain family are differentially expressed in human squamous epithelia. In some cases, they even play important roles in maintaining skin homeostasis, as exemplified by the secreted single domain member, SLURP-1, the deficiency of which is associated with the development of palmoplantar hyperkeratosis in the congenital skin disorder Mal de Meleda. In the present study, we have characterized a new member of the LU-protein domain family, which we find to be predominantly expressed in the stratum granulosum of human skin, thus resembling the expression of SLURP-1. In accordance with its expression pattern, we denote this protein product, which is encoded by the LYPD5 gene, as Haldisin (human antigen with LU-domains expressed in skin). Two of the five human glycolipid-anchored membrane proteins with multiple LU-domains characterized so far are predominantly confined to squamous epithelia (i.e., C4.4A), to stratum spinosum, and Haldisin to stratum granulosum under normal homeostatic conditions. Whether Haldisin is a prognostic biomarker for certain epithelial malignancies, like C4.4A and SLURP-1, remains to be explored.

Cell death by cornification.

Epidermal keratinocytes undergo a unique form of terminal differentiation and programmed cell death known as cornification. Cornification leads to the formation of the outermost skin barrier, i.e. the cornified layer, as well as to the formation of hair and nails. Different genes are expressed in coordinated waves to provide the structural and regulatory components of cornification. Differentiation-associated keratin intermediate filaments form a complex scaffold accumulating in the cytoplasm and, upon removal of cell organelles, fill the entire cell interior mainly to provide mechanical strength. In addition, a defined set of proteins is cross-linked by transglutamination in the cell periphery to form the so-called cornified envelope. Extracellular modifications include degradation of the tight linkages between corneocytes by excreted proteases, which allows corneocyte shedding by desquamation, and stacking and modification of the excreted lipids that fill the intercellular spaces between corneocytes to provide a water-repellant barrier. In hard skin appendages such as hair and nails these tight intercorneocyte connections remain permanent. Various lines of evidence exist for a role of organelle disintegration, proteases, nucleases, and transglutaminases contributing to the actual cell death event. However, many mechanistic aspects of kearatinocyte death during cornification remain elusive. Importantly, it has recently become clear that keratinocytes activate anti-apoptotic and anti-necroptotic pathways to prevent premature cell death during terminal differentiation. This review gives an overview of the current concept of cornification as a mode of programmed cell death and the anti-cell death mechanisms in the epidermis that secure epidermal homeostasis. This article is part of a Special Section entitled: Cell Death Pathways.

Giant sublingual epidermoid cyst resembling plunging ranula.

Epidermoid and dermoid cysts represent less than 0.01% of all oral cavity cysts. We describe a rare case of large epidermoid cyst in floor of mouth, with an oral as well as submental component resembling plunging ranula reported in the literature from India. We present a case of a 16-year-old girl with complaints of a mass in sublingual region, difficulty chewing, and dysphagia for about 5 months. Fine-needle aspiration cytology showed keratin flakes and proteinaceous material. Contrast-enhanced CT oral cavity was done and showed 7.0 × 5 × 4.5 cm well-circumscribed non-enhancing cystic mass extending into the floor of the mouth. On examination, a firm swelling was noticed in the submental area, extending down to the thyroid notch. The patient underwent surgical removal of the mass. On histopathology, acidophilic stratum corneum and basophilic dot like staining of stratum granulosum, which is the hallmark of an epidermoid cyst, were seen.