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Age-related thinning and reduced cell proliferation in the human epidermis are associated with the accumulation of senescent cells and decreases in the number and function of epidermal stem cells. This study examined the expression of INHBA/Activin-A in human epidermis and expression differences with age, and the effect of Activin-A on epidermal stem/progenitor cells. Immunohistochemical staining was used to analyze age-related changes in the expression of INHBA/Activin-A in the epidermal tissue of young and old subjects. Epidermal INHBA/Activin-A expression levels, epidermal morphology, and the number of epidermal stem/progenitor cells or proliferating cells were investigated using older abdominal skin samples. The effects of Activin-A on the development of a three-dimensional (3D) reconstructed epidermis and cell proliferation were also assessed. INHBA/Activin-A expression levels in the human epidermis increased with age, although they varied among individuals. In the epidermis of older abdominal skin samples, INHBA/Activin-A expression levels negatively correlated with epidermal thickness, the rete ridge depth and the interdigitation index. The proportion of epidermal stem/progenitor cells and proliferating cells decreased with increases in INHBA/Activin-A expression levels. Activin-A had no effect on the differentiation of keratinocytes in the 3D-reconstructed epidermis; however, thinning of the 3D epidermis was noted. Moreover, the addition of Activin-A inhibited the proliferation of epidermal stem/progenitor cells in a concentration-dependent manner. Age-related increased in INHBA/Activin-A expression levels were observed in the human epidermis, and may contribute to epidermal thinning and decreases in the number of epidermal stem/progenitor cells and proliferative activity. • The expression of INHBA/Activin-A in human epidermis increased with age. • Reduced epidermal stem/progenitor cells in human epidermis highly expressing INHBA. • Reduced proliferating cells in human epidermis highly expressing INHBA. • Activin-A suppresses the proliferation of epidermal stem/progenitor cells. [ABSTRACT FROM AUTHOR]

• Claudin-7 expression was regulated by HMG-CoA reductase in the epidermis of psoriasis. • Claudin-7 was localized in the keratohyalin granules of psoriatic lesions. • Claudin-7 might be involved in psoriasis epidermal lipid metabolism. 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. In this study, we created a gene expression profile to investigate the effect of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors on epidermal homeostasis. 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. 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. 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. [ABSTRACT FROM AUTHOR]

• Melanosomes are concentrated in the basal keratinocytes for localized protection. • Regional distribution of melanosomes correlates with their selective transfer. • Regional distribution of melanosomes correlates with their differential degradation. Human skin is a highly efficient self-renewing barrier that is critical to withstanding environmental insults. Undifferentiated keratinocyte stem cells reside in the basal layer of the epidermis and in hair follicles that continuously give rise to progenies ensuring epidermal turnover and renewal. Ultraviolet (UV) radiation is a proven cause of skin keratinocyte cancers, which preferentially occur at sun-exposed areas of the skin. Fortunately, melanocytes produce melanin that is packaged in specific organelles (termed melanosomes) that are then delivered to nearby keratinocytes, endowing the recipient cells with photoprotection. It has long been thought that melanosome transfer takes place stochastically from melanocytes to keratinocytes via an as-yet-unrecognized manner. However, recent studies have indicated that melanosomes are distributed regionally in the basal layer of the skin, affording localized intensive photoprotection for progenitor keratinocytes and stem cells that reside in the microenvironment of the basal epidermis. In this review, we summarize current knowledge about molecular and cellular mechanisms that are responsible for the selective transfer and exclusive degradation of melanosomes in the epidermis, emphasizing implications for skin carcinogenesis. [ABSTRACT FROM AUTHOR]

• Rosacea epidermis show high HSD11B1 expression. • TLR3 agonist Poly(I:C) increases HSD11B1 expression in human epidermal keratinocytes. • Differentiated keratinocytes augments glucocorticoid synthetases induction by Poly(I:C). • The knockdown of TLR3 suppress the by Poly(I:C) stimuli. • TLR3 transduction augments HSD11B1 induction by Poly(I:C) in keratinocytes. While most skin diseases benefit from topical steroids, rosacea symptoms are exacerbated by topical steroids. In the rosacea pathogenesis, abnormal innate immune mechanisms including overexpression of the Toll-like receptor (TLR) have been proposed. However, the links between glucocorticoid metabolism and innate immunity in the epidermis have not been elucidated. In order to understand the pathology by which rosacea symptoms are exacerbated by steroids and environment stimuli, we examined the molecular links between the innate immune system and glucocorticoid synthesis in epidermis. We examined the expression of glucocorticoid-synthetic enzymes in rosacea skin. We stimulated epidermal keratinocytes by TLR ligands and examined the regulation of glucocorticoid-synthetic enzymes. We also employed siRNA and adenovirus vectors to knockdown and transduce TLR molecules, respectively. Rosacea epidermis showed high HSD11B1 in the granular layer. Among TLR ligands, TLR3 ligand Poly(I:C) enhanced the expression of multiple glucocorticoid-synthetic enzymes including HSD11B1 and CYP11A1, and increased cortisol in the cultured media. Induction of HSD11B1 by Poly(I:C) was abolished by pretreatment with TLR3 siRNA. Transfection with an adenoviral vector incorporating TLR3 enhanced HSD11B1 and CYP11A1 protein expression by Poly(I:C). In addition, cell staining revealed increased expression of HSD11B1 and CYP11A1 proteins in the group transfected with TLR3 under the same conditions. TLR3-stimulated epidermal keratinocytes and rosacea epidermis enhance the expression of glucocorticoid-synthetic enzymes, which would promote cortisol activation in the epidermis. The innate immunity modulates glucocorticoid-synthetic enzymes expression via the TLR3 pathway in epidermal keratinocytes. [ABSTRACT FROM AUTHOR]

• S100 fused-type proteins have distinct surface chemistries at target binding site • Inter-EF-hand linker residues help S100 fused-type proteins anchor substrate • Profilaggrin B domain works with A domain to bind and stabilize protein targets • Profilaggrin AB complex binds coiled-coil region of keratin intermediate filaments • Annexin 2 domains I and II are positioned to interact with profilaggrin B domain Profilaggrin belongs to the S100 fused-type protein family expressed in keratinocytes and is important for skin barrier integrity. Its N-terminus contains an S100 ("A") domain and a unique "B" domain with a nuclear localization sequence. To determine whether profilaggrin B domain cooperates with the S100 domain to bind macromolecules. To characterize the biochemical and structural properties of the profilaggrin N-terminal "AB" domain and compare it to other S100 fused-type proteins. We used biochemical (protease protection, light scattering, fluorescence spectroscopy, pull-down assays) and computational techniques (sequence analysis, molecular modeling with crystallographic structures) to examine human profilaggrin and S100 fused-type proteins. Comparing profilaggrin S100 crystal structure with models of the other S100 fused-type proteins demonstrated each has a unique chemical composition of solvent accessible surface around the hydrophobic binding pocket. S100 fused-type proteins exhibit higher pocket hydrophobicity than soluble S100 proteins. The inter-EF-hand linker in S100 fused-type proteins contains conserved hydrophobic residues involved in binding substrates. Profilaggrin B domain cooperates with the S100 domain to bind annexin II and keratin intermediate filaments in a calcium-dependent manner using exposed cationic surface. Using molecular modeling we demonstrate profilaggrin B domain likely interacts with annexin II domains I and II. Steric clash analysis shows annexin II N-terminal peptide is favored to bind profilaggrin among S100 fused-type proteins. The N-terminal S100 and B domains of profilaggrin cooperate to bind substrate molecules in granular layer keratinocytes to provide epidermal barrier functions. [ABSTRACT FROM AUTHOR]

• IFN-γ acts as an inducer of tryptophan conversion to kynurenine preferentially in undifferentiated keratinocytes. • Following IFN-γ stimulation of reconstructed human epidermis expression of IDO-1 is only present in the basal layer of the epidermis. • Our study establishes the importance of using differentiated keratinocytes in a 3D model for investigating the keratinocyte response in vitro. Interferon-gamma (IFN-γ) represents a potent inducer for keratinocyte inflammatory and immune activation in vitro. Since tryptophan (trp) conversion to kynurenine (kyn) is involved in inflammation, the topical kyn/trp ratio may serve as a biomarker of skin inflammation. However, the trp metabolism in keratinocytes exposed to IFN-γ is not yet fully understood. The aim of this study was to establish a human epidermis model in order to quantify cytokine and kyn/trp secretion from IFN-γ stimulated cells and tissues. Moreover, to compare the cell response of 2D-cultured keratinocytes and the 3D epidermis model. Polycarbonate filters were used on which primary keratinocytes could attach and stratify in order to form the typical layers of reconstructed human epidermis (RHE). After IFN-γ treatment, secretion of kyn/trp was measured by high performance liquid chromatography. Gene and protein expression of indoleamine 2,3-dioxygenase 1 (IDO) was analyzed with real-time PCR and immunohistochemistry. The secretion of cytokines was quantified with ELISA. Trp catabolism to kyn was significantly increased (P < 0.01) in the 2D culture in response to IFN-γ treatment. Before kyn secretion, IDO was strongly upregulated (P < 0.001). IFN-γ treatment also increased the secretion of IL-6 and IL-8 from the keratinocytes. In the RHE, IDO was upregulated by IFN-γ, and kyn secretion could be detected. Interestingly, IDO expression was only present in the basal cells of the RHE. Our results suggest that IFN-γ acts as an inducer of trp degradation preferentially in undifferentiated keratinocytes, indicated by the IDO expression in the basal layer of the RHE. [ABSTRACT FROM AUTHOR]

• We analyzed epidermis-specific Drp1 knockout (EKO) mice. • Epidermal development in these mice was found to be normal. • Loss of Drp1 did not alter the UVB-induced apoptosis of keratinocytes. • Drp1 may become a novel therapeutic target for malignant keratinocytic skin lesions. Mitochondrial morphology is controlled by fission and fusion. Dynamin-related protein 1 (Drp1, dynamin-1-like protein (Dnml1)) regulates mitochondrial fission, which is associated with cell division and apoptosis. We previously reported that DRP1 is indispensable for cell growth in cutaneous squamous cell carcinoma. However, little is known about Drp1 in normal epidermis/keratinocytes. We investigated the function of Drp1 in normal epidermis/keratinocytes. Epidermis-specific Drp1 knockout (EKO) mice were analyzed. Epidermal development in the EKO mice were indistinguishable from those in the wild-type (WT) mice. Ultrastructural analysis and immunohistochemistry revealed that the mitochondria of keratinocytes in the EKO mice were neither elongated nor constricted. Drp1 knockdown did not diminish the cell growth of normal human keratinocytes. Both in vivo and in vitro , UVB-induced apoptosis in the EKO epidermis and keratinocytes did not differ from that in the WT mice. In chronic UVB-irradiation, the loss of Drp1 sensitized the epidermis to the development of skin tumors. Clinically, DRP1 is expressed more highly in sun-exposed skin than in non-exposed skin in individuals under age 40, but not in those over age 60. EKO mice demonstrate that Drp1 is dispensable for the development and apoptosis of the epidermis. Drp1 plays critical roles in malignant tumors; thus, the molecular machinery of mitochondrial dynamics involving Drp1 could be a novel therapeutic target for malignant keratinocytic lesions. On the other hand, the anti-tumorigenic role of Drp1 in chronic UVB-induced carcinogenesis need to be further investigated. [ABSTRACT FROM AUTHOR]

• Zinc deficiency induces epidermal malformation with dysregulation of acetylation process in epidermis. • Implication of the zinc-epigenetic axis in epidermal homeostasis • Possible involvement of zinc transporter ZIP10 in epidermal acetylation [ABSTRACT FROM AUTHOR]

• TLR3 agonist Poly(I:C) increases uptake of melanosome-rich globules, melanosomes, and melanocores by keratinocytes. • Poly(I:C) increases protease-activated receptor-2 (PAR-2) expression. • The knockdown of PAR-2 does not completely suppress endo/phagocytosis of melanin by Poly(I:C) stimuli. • Poly(I:C) increases the expressions and activation of RHOA and CDC42. • The knockdown of RHOA and CDC42 suppress endo/phagocytosis of melanin by Poly(I:C) stimuli. Keratinocytes and melanocytes in human epidermis express Toll-like receptors (TLR) and induce immune responses. We previously reported that TLR3 stimulation increases melanosome transport from perinuclear to cell membrane in melanocytes and enhanced release of melanosome from melanocytes, which were followed by increase in melanosome uptake into keratinocytes. In this study, we investigated whether TLR3 stimuli directly affect keratinocytes to enhance melanosome uptake. To observe keratinocyte's melanosome uptake ability precisely without melanocytes influences, we isolated melanosomes from human melanocytes and applied isolated melanosomes to keratinocytes stimulated by Poly(I:C). Poly(I:C)-stimulated keratinocytes enhanced uptake of isolated melanosome-rich globules five-times as much as control. Poly(I:C) increases the RNA and protein expressions of RHOA and CDC42, which are small GTP-binding proteins inducing the endocytosis. Pull-down assay showed that Poly(I:C) increased the GTP-binding RHOA and CDC42, suggesting TLR3 stimulation activated RHOA and CDC42. The knockdown of TLR3 suppressed RHOA and CDC42 induction by Poly(I:C). Consistently, the knockdown of RHOA and CDC42 significantly suppressed the melanosome-rich globules uptake by Poly(I:C)-stimulated keratinocytes. Because RHOA and CDC42 activation induces endocytosis by modification of actin stress fiber and filopodia formation, respectively, these results suggested that TLR3 stimulation enhances melanosome uptake into keratinocytes through endocytosis mechanisms. Combining with the data of our previous publications, TLR3, which signal is activated by sensing viral molecules, enhance pigmentation by controlling both melanin transport system by RAB GTPases induction in melanocytes and uptake system by RHOA and CDC42 in keratinocytes. [ABSTRACT FROM AUTHOR]

• Neurofibroma highly expresses polydom. • Polydom promotes TGFβ production via ITGB8-induction. • TGFβ develops neurofibroma by producing PDGFB. Neurofibromas in von Recklinghausen's disease (vRD) can develop in the dermis. Therefore, we hypothesized that a dermal niche exists that promotes the development of these neurofibromas in subjects with vRD. The purpose of this study is to examine the function of polydom, known as a ligand for integrin, mediating cell adhesion, and expressed in mouse nerve tissue, in promotion of neurofibroma. Molecular, transcriptome and immunohistochemical analysis were performed to investigate the association between polydom expression and neurofibroma development. Polydom mRNA levels were significantly higher in neurofibroma tissue than in control tissue. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of RNA purified from primary cultured dermal neurofibroma cells demonstrated significantly higher polydom mRNA expression in cells derived from the surrounding dermis of neurofibromas compared to those from normal human dermal fibroblasts. RNA sequencing was used to compare gene expression between cultured cells derived from dermal neurofibroma-surrounding tissue with or without polydom knockdown. Subsequent gene ontology assays revealed that expression of integrinβ8 (ITGB8) , a factor that releases transforming growth factor-β (TGF-β) from pro-TGF-β, was downregulated following polydom knockdown, suggesting upregulation of polydom-mediated TGF-β production. Furthermore, we observed a strong association between polydom expression and the increase in platelet-derived growth factor B (PDGFB) expression in primary cultured cells from the surrounding dermis of neurofibromas exposed to TGF-β1. Our results suggest that increased polydom expression in the dermis surrounding neurofibromas may promote dermal neurofibroma development by activating the TGF-β signaling pathway. [ABSTRACT FROM AUTHOR]

• KLKs, mainly KLK5 and KLK7, play crucial roles in epidermal barrier formation. • Many KLKs seem to be involved in the pathophysiology of inflammatory skin diseases. • Elucidating the role of epidermal KLKs will contribute to developing new treatment. Identifying the function of kallikrein-related peptidases (KLKs) in the epidermis has elicited great interest over recent decades. KLKs comprise 15 serine proteases, and their activities are regulated by complex and fine-tuned mechanisms involving the proteolytic activation cascade, endogenous inhibitors, and environmental factors. When the balance is disrupted, excessive or insufficient protease activity can impair epidermal barrier homeostasis. KLKs are involved in various events, such as skin inflammation, wound healing, pruritus, anti-bacterial activity, and viral susceptibility. One of the primary roles of KLKs, mainly KLK5 and KLK7, is physiological desquamation. Both proteases are also involved in the development of inflammatory skin diseases with barrier abnormalities, e.g., Netherton syndrome and atopic dermatitis (AD). In Netherton syndrome, unrestricted activity of KLK5 due to loss of the major endogenous inhibitor, lymphoepithelial Kazal-type-related inhibitor (LEKTI), destroys the component molecules of corneodesmosome, leading to Th2 and Th17 inflammation. Meanwhile, the increased activity of KLK7 in the hyperkeratotic lesions of chronic AD is suppressed by upregulated LEKTI. The functions and implications of other KLKs including KLK6 and KLK8 in healthy and diseased skin such as psoriasis represent an exciting but relatively unexplored area. Clarifying the function of epidermal KLKs will enable development of disease-specific biomarkers and new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background Centrosomes contain two centrioles: a pre-existing mature centriole and a newly formed immature centriole. Each centriole is duplicated once within a cell cycle, which is crucial for proper centrosome duplication and cell division. Objective To describe the centrosome duplication cycle in human epidermis, Bowen’s disease (BD), and squamous cell carcinoma (SCC). Methods Immunofluorescent staining of centriolar proteins and Ki-67 was used to evaluate cell cycles and the number of centrioles. Centrobin and Outer dense fiber of sperm tails 2 (ODF2) were used as markers for immature and mature centrioles, respectively. Results Normal human primary epidermal keratinocytes in a monolayered culture have one centrobin + centriole (CTRB 1+ cells) supposed in G0/G1 phases or have two centrobin + centrioles (CTRB 2+ cells) supposed in S−G2 phase. In a three-dimensional culture and in vivo human epidermis, the majority of suprabasal cells were CTRB 2+ cells, in spite of their non-proliferative Ki-67 − nature. The tumor mass of BD and SCC contained CTRB 1+ cells and Ki-67 + proliferating and Ki-67 − non-proliferative CTRB 2+ cells. Clumping cells in BD had increased numbers of centrioles, with an approximate 1:1 to 2:1 ratio of centrobin + to ODF2 + centrioles. Conclusions The cell cycle arrest of suprabasal cells is distinct from the G0 arrest of monolayered epithelial cells. Tumor mass of BD and SCC contained non-proliferative cells with the characteristics of the suprabasal cells of normal epidermis. A constant ratio of the number of centrobin + to ODF2 + centrioles indicates that multiple centrioles were induced by cell division failure rather than centriole overduplication in clumping cells. [ABSTRACT FROM AUTHOR]

Background The boundaries between actinic keratosis (AK), Bowen’s disease (BD), and cutaneous squamous cell carcinoma (cSCC) are sometimes not clear. Large-scale proteomic profiling studies of these lesions are also non-existent. Objective To evaluate proteomic changes between normal epidermis, AK, BD and cSCC that could support a molecular classification and improve our understanding of disease progression. Methods Microdissected formalin-fixed paraffin embedded samples of normal epidermis (n = 4, pooled), AK (n = 10), BD (n = 10) and cSCC (n = 10) were analyzed by mass spectrometry. Following normalization and multiple testing adjustments, differential abundance analysis was performed using Linear Models for Microarray data. Proteins were filtered for significance (adjusted p-value ≤ 0.05) and fold change of at least ±1.5. Comparative bioinformatics analysis was performed using Ingenuity Pathway Analysis (IPA) software. Proteomic findings were subsequently substantiated using immunohistochemistry. Results 2073 unique proteins were identified. cSCC had the highest number of differentially abundant proteins (63 proteins) followed by BD (58 proteins) and AK (46 proteins). Six proteins (APOA1, ALB, SERPINA1, HLA-B, HP and TXNDC5) were differentially abundant in cSCC compared to AK. Immunohistochemical analysis corroborated changes in MIF, RPL37A and TXNDC5. IPA analysis predicted that cell proliferation, angiogenesis and inflammatory reactions were significantly activated in cSCC compared to BD and AK. Cell death and DNA damage were predicted to be inhibited in BD. Conclusion Our study supports the concept that AK and BD are precursors of cSCC. The identification of proteome changes indicates disruption of repair, pro-apoptotic, and tumor promoting pathways. Our findings will help select targets for classification and treatment. [ABSTRACT FROM AUTHOR]

Background A variety of human skin disorders is characterized by defects in the epidermal barrier, leading to dehydration, itchiness, and rashes. Previously published literature suggests that microtubule stabilization at the cortex of differentiating keratinocytes is necessary for the formation of the epidermal barrier. Objectives We tested whether stabilization of microtubules with paclitaxel or epothilone B can repair barrier defects that were experimentally induced in three-dimensional culture models of epidermis. Methods We established two models of defective epidermis in vitro, using three-dimensional cultures of primary human keratinocytes on filter supports: immature reconstructed human epidermis (RHE), and RHE that was compromised by treatment with inflammatory cytokines, the latter mimicking defects seen in atopic dermatitis. Results Both paclitaxel and epothilone B promoted keratinocyte differentiation, accumulation of junctional proteins at the cell cortex, and the early appearance of lamellar bodies in immature RHE, whereas destabilization of microtubules by nocodazole had the reverse effect. Moreover, stabilization of microtubules rescued the barrier after cytokine treatment. The rescued barrier function correlated with the restoration of filaggrin and loricrin protein levels, the cortical accumulation of junctional proteins (E-cadherin, β-catenin, and claudin-1), and with the secretion of lamellar bodies. Conclusions Our data suggest that the microtubule network is important for the formation of the epidermis, and that stabilization of microtubules promotes barrier formation. Microtubule stabilization may support regeneration of damaged skin, by restoring or improving the barrier. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD) is a chronic inflammatory skin disease causing a strong impact on quality of life. Its pathophysiology is the result of complex interactions involving immunological, genetic and environmental factors. Although there are several published in vitro three-dimensional models mimicking AD, none of them have taken all these pathophysiological features into account; thus, finding the right model may be complicated. This paper reviews the literature on the different reconstructed epidermis models of AD as well as their relevance. We focused our attention on both the defect of the epidermal barrier and the inflammation linked to the immune system. [ABSTRACT FROM AUTHOR]

Research on the genetic abnormalities and pathogenetic processes of ichthyoses has progressed remarkably, and many causative genes and molecules have been identified in ichthyoses and ichthyosis syndromes. Most of the genes/molecules causative of ichthyosis are associated with the barrier function of the stratum corneum, and defects in the skin barrier play important roles in the pathogenesis of various ichthyosis phenotypes. It has been elucidated that, of the ichthyosis-causative genes, ABCA12 , ALOXE3, ALOX12B , CYP4F22, CERS3, ABHD5 , PNPLA1 and ELOVL4 work in the formation of the corneocyte lipid envelope (CLE), a structure that is essential to sound skin barrier function. The CLE mostly consists of ultra-long-chain (ULC) ceramides derived from ULC-acylceramide (EOS; a combination of esterified ω-hydroxy fatty acids and sphingosines). In this review, I shed light on the synthesis, metabolism and transport of epidermal ceramides, especially on ULC-acylceramide and the processes of CLE formation. In addition, I summarize the pathogeneses of various ichthyoses and ichthyosis syndromes from the aspects of abnormal synthesis of ULC-acylceramide and malformation of the CLE. Investigations on the pathomechanisms of ichthyoses have provided novel knowledge on the synthesis and metabolism of ceramides in the epidermis. Conversely, research on the dynamics of epidermal ceramides has contributed to the elucidation of the pathogenesis of ichthyoses. Advances in our understanding of the biology of epidermal lipids and the disease pathogeneses of ichthyoses and ichthyosis syndromes promise to provide clues for the development of effective therapies for ichthyosis patients in the near future. [ABSTRACT FROM AUTHOR]

Background The epidermis shows a reverse iron gradient from the basal layer to the stratum corneum and consequently, little epidermal intracellular iron is lost by desquamation. Objective To clarify the underlying mechanism of iron salvage. Methods We first used immunohistochemistry and mRNA quantification to demonstrate the distinctive expression pattern of iron metabolism molecules. The obtained results were confirmed using normal human epidermal keratinocytes (NHEKs) during in vitro differentiation. We next examined the effects of reducing ferroportin expression in vitro by ferroportin-specific siRNAs or hepcidin on the intracellular iron content of cultured NHEKs. Finally, we compared epidermal and systemic iron metabolism between Fpn Epi-KO mice and control mice. Results The results of both mRNA and protein expression analysis showed that most molecules participating in iron import and storage were expressed in the lower epidermis, while those involved in iron release from heme or iron transport were expressed in the upper epidermis. Consistent with their expression, keratinocyte differentiation reduced intracellular iron content. We next demonstrated that reducing ferroportin expression in vitro by ferroportin-specific siRNAs or hepcidin significantly increased the intracellular iron content. Finally, we showed that the iron content of the epidermis and squames was significantly greater in Fpn Epi-KO mice than in control mice, and that Fpn Epi-KO exhibited a more rapid decrease in blood hemoglobin concentration than control mice on a low iron diet. Conclusion These studies demonstrated that the epidermis is equipped with a machinery by which intracellular iron in differentiated keratinocytes is excreted to the extracellular space before reaching the stratum corneum. [ABSTRACT FROM AUTHOR]

Background Recent studies have demonstrated podoplanin expression in several tumors, which has been associated with lymph node metastasis and poor prognosis. Podoplanin expression in peritumoral cells such as cancer-associated fibroblasts also correlates with tumor progression in several cancers. However, podoplanin expression and its association with extramammary Paget's disease (EMPD) remain unclear. Objective In this study, we examined whether the presence of podoplanin expression in tumor cells or peritumoral basal keratinocytes correlated with aggressive behavior in patients with EMPD and investigated the mechanisms of podoplanin-mediated tumor invasion in this disorder. Methods Skin samples of 37 patients with EMPD were investigated by immunohistochemical analysis. The functions of podoplanin in keratinocytes were examined in vitro by RT-PCR and with invadopodia gelatin-degradation assays using HaCaT cells. Results Podoplanin was not identified in tumor cells in all cases. Podoplanin expression in peritumoral basal keratinocytes was observed in 25 patients (67.6%). In in situ EMPD, 50% of cases (9 in 18) exhibited podoplanin-positive keratinocytes, whereas 84.2% (16 in 19) demonstrated positive staining in invasive EMPD ( P < 0.05). Podoplanin expression in peritumoral keratinocytes was also associated with tumor thickness ( P < 0.005). By immunohistochemical analysis, podoplanin-positive peritumoral keratinocytes were found to be negative for E-cadherin, one of the major adhesion molecules of keratinocytes, which might contribute to tumor invasion into the dermis through a crack in the basal cell layer induced by down-regulation of cell adhesion therein. We further found that podoplanin-positive keratinocytes exhibited invadopodia, which are thought to function in the migration of cancer cells through tissue barriers, indicating that podoplanin-positive peritumoral basal keratinocytes might assist tumor invasion by degrading the extracellular matrix. Conclusion The presence of podoplanin expression in peritumoral keratinocytes correlates with aggressive behavior in EMPD and might therefore serve as a useful prognostic marker for patients with EMPD. [ABSTRACT FROM AUTHOR]

Background Deimination (also known as citrullination), the conversion of arginine in a protein to citrulline, is catalyzed by a family of enzymes called peptidylarginine deiminases (PADs). Three PADs are expressed in the epidermis, one of their targets being filaggrin. Filaggrin plays a central role in atopic dermatitis and is a key protein for the epidermal barrier. It aggregates keratins and is cross-linked to cornified envelopes. Following its deimination, it is totally degraded to release free amino acids, contributing to the natural moisturizing factor (NMF). The mechanisms controlling this multistep catabolism in human are unknown. Objective To test whether external humidity plays a role, and investigate the molecular mechanisms involved. Methods Specimens of reconstructed human epidermis (RHEs) produced in humid or dry conditions (>95% or 30–50% relative humidity) were compared. Results RHEs produced in the dry condition presented structural changes, including a thicker stratum corneum and a larger amount of keratohyalin granules. The transepidermal water loss and the stratum corneum pH were decreased whereas the quantity of NMF was greater. This highly suggested that filaggrin proteolysis was up-regulated. The expression/activity of the proteases involved in filaggrin breakdown did not increase while PAD1 expression and the deimination rate of proteins, including filaggrin, were drastically enhanced. Partial inhibition of PADs with Cl-amidine reversed the effect of dryness on filaggrin breakdown. Conclusion These results demonstrate the importance of external humidity in the control of human filaggrin metabolism, and suggest that deimination plays a major role in this regulation. [ABSTRACT FROM AUTHOR]

Background Although many kinds of keratinocyte-derived factors are known to regulate the proliferation and differentiation of human melanocytes, it is not well defined whether dermis-derived factors work in a similar way. Objective The aim of this study is to clarify whether dermal factors are involved in regulating the proliferation and differentiation of human melanocytes. Methods Human epidermal melanoblasts were cultured serially in a serum-free growth medium. Platelet-derived growth factor-BB (PDGF-BB) was supplemented to the medium, and the effects on the proliferation of melanoblasts/melanocytes and the differentiation of melanocytes were studied. Results PDGF-BB stimulated the proliferation of melanoblasts cultured in melanoblast-proliferation medium, but inhibited the proliferation of melanocytes cultured in melanocyte-proliferation medium. By contrast, PDGF-BB stimulated the differentiation, dendritogenesis, and melanogenesis of melanocytes through the stimulation of tyrosinase activity and the expressions of tyrosinase and tyrosinase-related protein-1. Conclusion These results suggest that PDGF-BB regulates the proliferation and differentiation of human melanocytes in a differentiation-stage-specific manner. PDGF-BB seems to be one of the dermal factors that regulate the proliferation and differentiation of human melanocytes. [ABSTRACT FROM AUTHOR]

Background Skin aging is a process of structural and compositional remodeling that can be manifested as wrinkling and sagging. Remarkably, the dermis plays a dominant role in the process of skin aging. Recent studies suggest that microRNAs (miRNAs) may play a role in the regulation of gene expression in organism aging. However, studies about age-related miRNAs in human skin remain limited. Objective To obtain an overall view of miRNAs expression in human aged dermis by comparison of dermis samples between young and elderly, construct the miRNA-gene-network and reveal the pivotal miRNAs in the regulatory network. Methods Human dermis tissue was obtained from 12 donors, including 6 of young group and 6 of elderly one. The miRNA microarray and data analysis were performed. Target genes of differentially expressed miRNAs were predicted, followed by a gene ontology and pathway enrichment analysis. A miRNA-gene-network was then constructed, and the pivotal miRNAs in the network was revealed. Primary human dermal fibroblasts (HDFs) were isolated, and the cellular senescence was induced by serial passaging. Alteration in the expression of miRNAs between young and senescent fibroblasts was evaluated by real-time quantitative RT-PCR. MiR-34b-5p mimics were transfected into primary HDFs. Subsequent cell cycle analysis was performed and expression level of COL1A1, elastin and MMP-1 were evaluated. Results The expression of a total of 40 miRNAs (25 upregulated and 15 downregulated) was found to be significantly altered in aged dermis compared with young dermis. Real-time quantitative PCR results confirmed the differential expression of miR-34 family and miR-29 family between young and aged dermis. A computational approach demonstrated that predicted target genes of the miRNA profile were found to be mainly involved in processes including cell adhesion, collagen synthesis, positive or negative regulation of transcription, as well as pathways such as insulin signaling pathway, ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) signaling pathway and Focal adhesion pathway. The miRNA-Gene-Network revealed that miR-34 family, miR-29 family and miR-424 may play a dominant role in the regulatory network. A similar miRNA alteration was observed in senescent fibroblasts in vitro, and the age-related miRNA profile may interact with p16 pathway to regulate the fibroblasts’ senescence. Additionally, transfection of miR-34b-5p mimics induced cell cycle arrest in HDFs, decreased the expression of both COL1A1 and elastin and increased MMP-1 expression. Conclusion The miR-34 family and miR-29 family expressed differentially in young and aged dermis. MiR-34 in HDFs modulated the cell function and expression of MMP-1, COL1A1 and elastin. The miRNAs may play critical roles in affecting dermis aging. [ABSTRACT FROM AUTHOR]

The skin is known to be the largest organ in human organism creating interface with outer environment. The skin provides protective barrier against pathogens, physical and chemical insults, and against uncontrolled loss of water. The barrier function was primarily attributed to the stratum corneum (SC) but recent studies confirmed that epidermal tight junctions (TJs) also play important role in maintaining barrier properties of the skin. Independent observations indicate that barrier function and its recovery is impaired in aged skin. However, trans-epidermal water loss (TEWL) values remains rather unchanged in elderly population. UV radiation as major factor of photoageing impairs TJ proteins, but TJs have great self-regenerative potential. Since it may be possible that TJs can compensate TEWL in elderly due to its regenerative and compensatory capabilities, important question remains to be answered: how are TJs regulated during skin ageing? This review provides an insight into TJs functioning as epidermal barrier and summarizes current knowledge about the impact of ageing on the barrier function of the skin and epidermal TJs. [ABSTRACT FROM AUTHOR]

Background K1 and K2 are the main type II keratins in the suprabasal epidermis where each of them heterodimerizes with the type I keratin K10 to form intermediate filaments. In regions of the ears, tail, and soles of the mouse, only K2 is co-expressed with K10, suggesting that these keratins suffice to form a mechanically resilient cytoskeleton. Objective To determine the effects of the suppression of both main keratins, K2 and K10, in the suprabasal plantar epidermis of the mouse. Methods Krt2 −/− Krt10 −/− mice were generated by crossing Krt2 −/− and Krt10 −/− mice. Epidermal morphology of soles of hind-paws was examined macroscopically and histologically. Immunofluorescence analysis and quantitative PCR analysis were performed to analyze the expression of keratins in sole skin of wildtype and Krt2 −/− Krt10 −/− mice. Highly abundant proteins of the sole stratum corneum were determined by electrophoretic and chromatographic separation and subsequent mass spectrometry. Results K2 and K10 are the most prominent suprabasal keratins in normal mouse soles with the exception of the footpads where K1, K9 and K10 predominate. Mice lacking both K2 and K10 were viable and developed epidermal acanthosis and hyperkeratosis in inter-footpad epidermis of the soles. The expression of keratins K1, K9 and K16 was massively increased at the RNA and protein levels in the soles of Krt2 −/− Krt10 −/− mice. Conclusions This study demonstrates that the loss of the main cytoskeletal components of plantar epidermis, i.e. K2 and K10, can be only partly compensated by the upregulation of other keratins. The thickening of the epidermis in the soles of Krt2 −/− Krt10 −/− mice may serve as a model for pathomechanistic aspects of palmoplantar keratoderma. [ABSTRACT FROM AUTHOR]

Background Human amniotic epithelial cells (hAECs) is a desirable reserve of stem cells. Human de-epidermized dermis (DED) retains basic tissue structure and parts of the basement membrane (BM) components at the acelluIar dermal surface, and provides a potential tool for skin regeneration. Objective To evaluate the potential role of hAECs in skin regeneration, we used DED to perform organotypic culture of hAECs to develop organotypic skin. Methods HAECs were isolated and cultured. Biological characteristics of hAECs were determined by immunocytochemistry and flow cytometry. To prepare DED, the epidermis was removed and then repeated freeze-thaw cycles. HAECs and fibroblast were seeded onto DED to perform the submerged culture for 3 days and then to be maintained at the air-liquid interface for 14 days to form organotypic culture. To identify whether the obtained DED retain the BM structure and components, the histological characteristics of DED and the BM were detected by immunohistochemistry. To evaluate whether the organotypic skin has similar histological characteristics with normal human skin, the marks of epidermal proliferation and differentiation and basement membrane component were detected by immunohistochemistry. Moreover, cell ultrastructure, cell–cell contact and ultrastructure of BM were examined under the transmission electron microscopy. Results HAECs has stem-cell characteristics with strong pluripotent Oct-4 and embryonic marker SSEA-4 expression. DED has effectively cleansed the cell components and continuous distributions of laminin and collagen IV. The histological appearance of tissue-engineered skin in vitro has 4 to 9 continuous layers of stratified epithelium and is similar to normal human skin in morphology. Immunohistochemical studies revealed that proliferation and differentiation markers such as Ki67, CK19, CK14, CK10, filaggrin but not CK18 expressed similar pattern characteristics to normal human epidermis. In addition, Periodic acid-Schiff stain showed that a uniform red staining strip located at the epidermal-dermal junction. BM component proteins (type IV collagen and laminin) and cell adhesion protein (desmoglein) were detected by immunohistochemistry in organotypic skin. Ultrastructurally, desmosomes, hemidesmosomes and BM zone (BMZ) were observed in organotypic skin. Conclusions Our studies indicate that the hAECs is a promising stem cell source for tissue-engineered skin, and DED with hAECs is a potential application prospects in regenerative medicine. [ABSTRACT FROM AUTHOR]

Background The ceramide metabolite, sphingosine-1-phosphate (S1P), regulates multiple cellular functions in keratinocytes (KC). We recently discovered that production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), is stimulated via a NF-κB-dependent mechanism that is activated by S1P when S1P is generated by sphingosine kinase (SPHK) 1. Objective We investigated whether pharmacological modulation of SPHK1 activity, using a novel synthetic SPHK1 activator, (S)-methyl 2-(hexanamide)-3-(4-hydroxyphenyl) propanoate (MHP), stimulates CAMP expression. Methods MHP-mediated changes in both S1P and CAMP downstream mediators were analyzed in normal cultured human KC by q RT-PCR, Western immunoblot, ELISA, confocal microscopy for immunohistochemistry, HPLC and ESI–LC/MS/MS, and microbial pathogen invasion/colonization in a human epidermal organotypic model. Results Treatment with MHP directly activated SPHK1 and increased cellular S1P content in normal cultured human KC. Because MHP did not inhibit S1P lyase activity, which hydrolyses S1P, augumented S1P levels could be attributed to increased synthesis rather than blockade of S1P degradation. Next, we found that exogenous MHP significantly stimulated CAMP mRNA and protein production in KC, increases that were significantly suppressed by si RNA directed against SPHK1, but not by a scrambled control si RNA. NF-κB activation, assessed by nuclear translocation of NF-κB, occurred in cells following incubation with MHP. Conversely, pretreatment with a specific inhibitor of SPHK1 decreased MHP-induced nuclear translocation of NF-κB, and significantly attenuated the MHP-mediated increase in CAMP production. Finally, topical MHP significantly suppressed invasion of the virulent Staphylococcus aureus into murine skin explants. Conclusion MHP activation of SPHK1, a target enzyme of CAMP production, can stimulate innate immunity. [ABSTRACT FROM AUTHOR]

Background Collective motion of keratinocytes is involved in morphogenesis, homeostasis, and wound healing of the epidermis. Yet how the collective motion of keratinocytes emerges from the behavior of individual cells is still largely unknown. Objective The aim of this study was to find the cellular behavior that links single and collective motion of keratinocytes. Methods We investigated the behavior of two-cell colonies of HaCaT keratinocytes by a combination of time-lapse imaging and image processing. Results The two-cell colonies of HaCaT cells were formed as a contacted pair of keratinocyte clones. Image analysis and cell culture experiments revealed that the rotational speed of two-cell colonies was positively associated with their proliferative capacity. α6 integrin was required for the rotational motion of two-cell keratinocyte colonies. We also confirmed that two-cell colonies of keratinocytes predominantly exhibited the rotational, but not translational, motion, two modes of motion in a contact pair of rotating objects. Conclusion The rotational motion is the primary motion of two-cell keratinocyte colonies and its speed is positively associated with their proliferative capacity. This study suggests that the assembly of rotating keratinocytes generates the collective motion of proliferative keratinocytes during morphogenesis and wound healing of the epidermis. [ABSTRACT FROM AUTHOR]

Background The dermal papilla is composed of a small clump of mesenchymal cells, called dermal papilla cells (DPCs). DPCs closely interact with epidermal cells to give rise to hair follicles and shafts during hair follicle development and the hair cycle. DPCs are promising cell sources for hair regeneration therapy for alopecia patients. However, once DPCs are put into conventional two-dimensional culture conditions, they quickly lose their capability to produce hair follicles. Objective We aimed to expand a sufficiently large population of DPCs that retain their hair inductive activity. Methods Murine DPCs were cultured in the presence of platelet-derived growth factor-AA (PDGF-AA) and fibroblast growth factor 2 (FGF2). Expressions of follicular-related genes were analyzed by real time PCR and hair inductive activity was determined by patch assay and chamber assay in vivo. Results FGF2 significantly increased the expression of platelet-derived growth factor receptor alpha (PDGFRα) in cultured vibrissal DPCs. PDGF-AA, a ligand of PDGFRα, promoted proliferation of DPCs synergistically when utilized with FGF2 and enhanced the expression of several follicular-related genes in DPCs. Hair reconstitution assays revealed that DPCs treated with both PDGF-AA and FGF-2 were able to maintain their hair inductive activity better than those treated with FGF2 alone. Conclusion Both cell proliferation and hair inductive activity in murine DPCs are maintained by the synergistic effect of FGF2 and PDGF-AA. [ABSTRACT FROM AUTHOR]

Background Notch signaling controls a number of cellular processes, including cell fate decisions, proliferation, differentiation, and survival/apoptosis, in multiple tissues. In the epidermis, Notch1 functions as a molecular switch that controls the transition of cells from an undifferentiated state into a differentiated state. Objective To clarify the functions of Notch in the regenerated epidermis during wound healing. Methods Wounds on mouse skin were immunostained. To investigate the functions of Notch, Notch was inhibited in primary keratinocytes by treatment with a γ-secretase inhibitor and by small interfering RNA-mediated knockdown, and was activated by a recombinant adenovirus approach. Results Notch1 and Notch2 were down-regulated in the regenerated epidermis during wound healing. To clarify the significance of this down-regulation, we examined its effect on expression of the interleukin (IL)-1 family of proinflammatory cytokines because wounds are exposed to pathogens from the outside world. Among the IL-1 family, IL-36α expression was induced by Notch inhibition. This was consistent with the decreased IL-36α expression in Notch-overexpressing keratinocytes. Notch down-regulation in the regenerated epidermis may reinforce defense against stress from the outside world by inducing IL-36α expression. Next, we examined the effects of Notch down-regulation on keratinocyte growth and differentiation. Notch down-regulation did not alter keratinocyte proliferation. On the other hand, Notch1 down-regulation suppressed induction of spinous layer-specific keratins (keratin1 and keratin10) in keratinocytes, which was consistent with the decreased expression of these keratins in the regenerated epidermis. The reduced levels of these keratins would increase cellular flexibility. Conclusion Notch down-regulation in the epidermis appears to contribute to tissue regeneration during wound healing. [ABSTRACT FROM AUTHOR]

Background The barrier function of the epidermis is integral to personal well-being, and defects in the skin barrier are associated with several widespread diseases. Currently there is a limited understanding of system-level proteomic changes during epidermal stratification and barrier establishment. Objective Here we report the quantitative proteogenomic profile of an in vitro reconstituted epidermis at three time points of development in order to characterize protein changes during stratification. Methods The proteome was measured using data-dependent “shotgun” mass spectrometry and quantified with statistically validated label-free proteomic methods for 20 replicates at each of three time points during the course of epidermal development. Results Over 3600 proteins were identified in the reconstituted epidermis, with more than 1200 of these changing in abundance over the time course. We also collected and discuss matched transcriptomic data for the three time points, allowing alignment of this new dataset with previously published characterization of the reconstituted epidermis system. Conclusion These results represent the most comprehensive epidermal-specific proteome to date, and therefore reveal several aspects of barrier formation and skin composition. The limited correlation between transcript and protein abundance underscores the importance of proteomic analysis in developing a full understanding of epidermal maturation. [ABSTRACT FROM AUTHOR]

Background Previous studies have shown that human sebum may play a role in barrier function but with much debate. Objective To elucidate the effects of human sebum on skin barrier function. Methods We used hairless mouse skin to study the functional and morphological alternation of epidermis after the application of human sebum. Results The results showed a significant increase in transepidermal water loss and erythema value, and a decrease in skin hydration, accompanied by epidermal hyperplasia with parakeratosis following sebum application. Nile red staining together with electron microscopic examination confirmed the underlying mechanisms for sebum-induced barrier disruption are related directly to the interaction of sebum with the intracellular lipid lamellae of the SC, thereby leading to the increase in the fluidity of SC intracellular lipids as demonstrated by ATR-FTIR measurement. An inflammatory reaction characterized by an enhanced cytokine cascade, including up-regulation of TNF-α, IL-1α and IL-6, was also observed. On the other hand, there were insignificant expression of thymic stromal lymphopoietin and unchanged serum levels of IgE, suggesting non-immunogenic stimulation by sebum treatment. Conclusion It may be concluded that inflammation induced by excess amount of sebum is more likely an irritant contact dermatitis rather than an allergic one. Moreover, these findings implicated possible relationships between sebum, irritant contact dermatitis, and seborrheic dermatitis. [ABSTRACT FROM AUTHOR]

Background The tight junction (TJ) barrier is located in the granular layer of the epidermis. Filaggrin deficiency predisposes patients to atopic dermatitis (AD) by impairing stratum corneum (SC) barrier function. Altered TJ barrier function has been observed in the skin of patients with AD; however, it remains unclear whether TJ function is influenced by filaggrin deficiency directly or secondarily via skin inflammation. Objective To investigate the in vivo effects of filaggrin deficiency and skin inflammation on epidermal TJ function. Methods Morphological changes in the TJ were investigated in filaggrin knockout mice and mice with hapten-induced dermatitis using en face visualization of epidermal sheets, and functional changes in the TJ were assessed with an in vivo permeation assay using tracers of various sizes. Results In filaggrin knockout mice, there was no apparent change in the honeycomb morphology of the TJ, TJ component mRNA expression, or TJ barrier function in neonates and adults, indicating that filaggrin-deficiency had no direct effects on the TJ. By contrast, in mice with hapten-induced dermatitis, the mRNA expression of TJ components was decreased markedly and the TJ barrier function was size-dependently impaired: the TJ leaked small tracers (<5 kDa), but not large tracers (>30 kDa). Conclusion Filaggrin deficiency did not affect the epidermal TJ barrier directly, but once dermatitis occurred, the skin inflammation induced TJ dysfunction. Since TJ dysfunction induces the SC barrier impairment, skin inflammation will enhance skin permeability to external antigens and result in a vicious cycle of barrier dysfunction and skin inflammation. [ABSTRACT FROM AUTHOR]

Background The interactions between human epidermal melanocytes and their cellular microenvironment are important in the regulation of human melanocyte functions or in their malignant transformation into melanoma. Although the basement membrane extracellular matrix (BM-ECM) is one of major melanocyte microenvironments, the effects of BM-ECM on the human melanocyte functions are not fully explained at a molecular level. Objective This study was aimed to characterize the molecular and cellular interactions between normal human melanocytes (NHMs) and BM-ECM. Methods We investigated cell culture models of normal human melanocytes or melanoma cells on three-dimensional (3D) Matrigel to understand the roles of the basement membrane microenvironment in human melanocyte functions. Melanogenesis and melanobast biomarker expression in both primary human melanocytes and melanoma cells on 3D Matrigel were evaluated. Results We found that NHMs migrated and formed reversible paired box 3 (PAX3) expressing cell clusters on three-dimensional (3D) Matrigel. The melanogenesis was significantly decreased in the PAX3 expressing cell cluster. The expression profile of PAX3, SOX10, and MITF in the melanocyte cluster on 3D Matrigel was similar to that of melanoblasts. Interestingly, PAX3 and SOX10 showed an inverse expression profile in NHMs, whereas the inverse expression pattern of PAX3 and SOX10 was disrupted in melanoma MNT1 and WM266-4 cells. Conclusion The human melanocyte culture on 3D Matrigel provides an alternative model system to study functions of human melanoblasts. In addition, this system will contribute to the elucidation of PAX3-related tumorigenic mechanisms to understand human melanoma. [ABSTRACT FROM AUTHOR]