Glucosylceramide fed mice skin before SDS treatment (0d), after SDS treatment (2d)
ABSTRACT: Dietary glucosylceramide (GC) improves skin barrier function. To elucidate the molecular mechanisms involved, we used a microarray system to analyze mRNA expression in SDS-treated dorsal skin of hairless mouse. Transepidermal water loss of mouse skin was increased by SDS treatment and the increase was significantly reduced by prior oral administration of glucosylceramides. Microarray-evaluated mRNA expression ratios showed statistically significant increase of expression of genes related to cornified envelope and tight junction formation versus all genes in glucosylceramide-fed/SDS-treated mouse skin. We then examined the contribution of glucosylceramide metabolites to tight junction formation of cultured keratinocytes. SDS treatment of cultured keratinocytes significantly decreased the transepidermal electrical resistance, and the decrease was significantly ameliorated in the presence of sphingosine or phytosphingosine, the major metabolites of glucosylceramide. These results suggest that oral administration of glucosylceramide improves skin barrier function by upregulating genes associated with both cornified envelope and tight junction formation. Two-condition experiment, effect of oral intake of GC on mice skin before SDS treatment (0d), and after SDS treatment(2d), Biological replicates: 3 replicates for 0d, 4 replicates for 2d.
Project description:Dietary glucosylceramide (GC) improves skin barrier function. To elucidate the molecular mechanisms involved, we used a microarray system to analyze mRNA expression in SDS-treated dorsal skin of hairless mouse. Transepidermal water loss of mouse skin was increased by SDS treatment and the increase was significantly reduced by prior oral administration of glucosylceramides. Microarray-evaluated mRNA expression ratios showed statistically significant increase of expression of genes related to cornified envelope and tight junction formation versus all genes in glucosylceramide-fed/SDS-treated mouse skin. We then examined the contribution of glucosylceramide metabolites to tight junction formation of cultured keratinocytes. SDS treatment of cultured keratinocytes significantly decreased the transepidermal electrical resistance, and the decrease was significantly ameliorated in the presence of sphingosine or phytosphingosine, the major metabolites of glucosylceramide. These results suggest that oral administration of glucosylceramide improves skin barrier function by upregulating genes associated with both cornified envelope and tight junction formation. Overall design: Two-condition experiment, effect of oral intake of GC on mice skin before SDS treatment (0d), and after SDS treatment(2d), Biological replicates: 3 replicates for 0d, 4 replicates for 2d.
Project description:Keratinocyte terminal differentiation is the process that ultimately forms the epidermal barrier that is essential for mammalian survival. This process is controlled, in part, by signal transduction and gene expression mechanisms, and the epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Using microarray analysis of a confluent cell density-induced model of keratinocyte differentiation, we identified 2,676 genes that are regulated by epidermal growth factor (EGF), a ligand of the EGFR. We further discovered, and separately confirmed by functional assays, that EGFR activation abrogates all of the known essential processes of keratinocyte differentiation by 1) decreasing the expression of lipid matrix biosynthetic enzymes, 2) regulating numerous genes forming the cornified envelope, and 3) suppressing the expression of tight junction proteins. In organotypic cultures of skin, EGF acted to impair epidermal barrier integrity, as shown by increased transepidermal water loss. As defective epidermal differentiation and disruption of barrier function are primary features of many human skin diseases, we used bioinformatic analyses to identify genes that are known to be associated with skin diseases. Compared with non-EGF-regulated genes, EGF-regulated genes were significantly enriched for skin disease genes. These results provide a systems-level understanding of the actions of EGFR signaling to inhibit keratinocyte differentiation, providing new insight into the role of EGFR imbalance in skin pathogenesis.
Project description:Transglutaminase 1 (TGase 1) is one of the genes implicated in autosomal recessive congenital ichthyosis. Skin from TGase 1(-/-) mice, which die as neonates, lacks the normal insoluble cornified envelope and has impaired barrier function. Characterization of in situ dye permeability and transepidermal water loss revealed defects in the development of the skin permeability barrier in TGase 1(-/-) mice. In the stratum corneum of the skin, tongue, and forestomach, intercellular lipid lamellae were disorganized, and the corneocyte lipid envelope and cornified envelope were lacking. Neonatal TGase 1(-/-) mouse skin was taut and erythrodermic, but transplanted TGase 1(-/-) mouse skin resembled that seen in severe ichthyosis, with epidermal hyperplasia and marked hyperkeratosis. Abnormalities in those barrier structures remained, but transepidermal water loss was improved to control levels in the ichthyosiform skin. From these results, we conclude that TGase 1 is essential to the assembly and organization of the barrier structures in stratified squamous epithelia. We suggest that the ichthyosiform skin phenotype in TGase 1 deficiency develops the massive hyperkeratosis as a physical compensation for the defective cutaneous permeability barrier required for survival in a terrestrial environment.
Project description:In chronic wounds, biofilm infects host tissue for extended periods of time. This work establishes the first chronic preclinical model of wound biofilm infection aimed at addressing the long-term host response. Although biofilm-infected wounds did not show marked differences in wound closure, the repaired skin demonstrated compromised barrier function. This observation is clinically significant, because it leads to the notion that even if a biofilm infected wound is closed, as observed visually, it may be complicated by the presence of failed skin, which is likely to be infected and/or further complicated postclosure. Study of the underlying mechanisms recognized for the first time biofilm-inducible miR-146a and miR-106b in the host skin wound-edge tissue. These miRs silenced ZO-1 and ZO-2 to compromise tight junction function, resulting in leaky skin as measured by transepidermal water loss (TEWL). Intervention strategies aimed at inhibiting biofilm-inducible miRNAs may be productive in restoring the barrier function of host skin.
Project description:Atopic dermatitis (AD) is characterized by epidermal tight junction (TJ) defects and a propensity for Staphylococcus aureus skin infections. S. aureus is sensed by many pattern recognition receptors, including Toll-like receptor 2 (TLR2). We hypothesized that an effective innate immune response will include skin barrier repair, and that this response is impaired in AD subjects. S. aureus-derived peptidoglycan (PGN) and synthetic TLR2 agonists enhanced TJ barrier and increased expression of TJ proteins, claudin-1 (CLDN1), claudin-23 (CLDN23), occludin, and Zonulae occludens 1 (ZO-1) in primary human keratinocytes. A TLR2 agonist enhanced skin barrier recovery in human epidermis wounded by tape stripping. Tlr2(-/-) mice had a delayed and incomplete barrier recovery following tape stripping. AD subjects had reduced epidermal TLR2 expression as compared with nonatopic subjects, which inversely correlated (r=-0.654, P=0.0004) with transepidermal water loss (TEWL). These observations indicate that TLR2 activation enhances skin barrier in murine and human skin and is an important part of a wound repair response. Reduced epidermal TLR2 expression observed in AD patients may have a role in their incompetent skin barrier.
Project description:Diabetes causes skin complications, including xerosis and foot ulcers. Ulcers complicated by infections exacerbate skin conditions, and in severe cases, limb/toe amputations are required to prevent the development of sepsis. Here, we hypothesize that hyperglycemia induces skin barrier dysfunction with alterations of epidermal integrity. The effects of hyperglycemia on the epidermis were examined in streptozotocin-induced diabetic mice with/without insulin therapy. The results showed that dye leakages were prominent, and transepidermal water loss after tape stripping was exacerbated in diabetic mice. These data indicate that hyperglycemia impaired skin barrier functions. Additionally, the distribution of the protein associated with the tight junction structure, tight junction protein-1 (ZO-1), was characterized by diffuse and significantly wider expression in the diabetic mice compared to that in the control mice. In turn, epidermal cell number was significantly reduced and basal cells were irregularly aligned with ultrastructural alterations in diabetic mice. In contrast, the number of corneocytes, namely, denucleated and terminally differentiated keratinocytes significantly increased, while their sensitivity to mechanical stress was enhanced in the diabetic mice. We found that cell proliferation was significantly decreased, while apoptotic cells were comparable in the skin of diabetic mice, compared to those in the control mice. In the epidermis, Keratin 5 and keratin 14 expressions were reduced, while keratin 10 and loricrin were ectopically induced in diabetic mice. These data suggest that hyperglycemia altered keratinocyte proliferation/differentiation. Finally, these phenotypes observed in diabetic mice were mitigated by insulin treatment. Reduction in basal cell number and perturbation of the proliferation/differentiation process could be the underlying mechanisms for impaired skin barrier functions in diabetic mice.
Project description:Combining genome-wide microarray and functional analyses, we found that EGFR activation abrogates barrier function, increasing transepidermal water loss (TEWL) and transepithelial permeability of water-soluble ions and higher molecular weight dextrans, in part by disrupting the expression of tight junction proteins. EGF decreases certain lipid matrix free fatty acids and ceramides by its actions to repress the expression of specific biosynthetic enzymes. Activation of EGFR inhibits cornified envelope formation by regulating the expression of 59 percent of the known contributing genes. EGF-responsiveness enriches more than 100 genes known to be associated with skin diseases. These data are used to obtain 2,676 density-dependent genes that are differentially expressed in response to EGF treatment. The 16 microarrays were preprocessed using the 5th percentile of region method in dChip. Genes with at least a 1.5 fold difference between the untrated samples at 50% and 100% confluent cell density were exported for further analysis. Two-way ANOVA was used to identify differentially expressed genes by either density or treatment factors using JMP Genomics 4.1 (SAS). Multiple hypothesis testing was corrected by Benjamini-Hochberg false discovery rate control at the 0.05 level. Pair-wise comparisons were performed using the Tukey’s Honestly Significant Difference test.
Project description:Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes. Using C57BL/6 wild-type and Toll-like receptor 4 (TLR4)-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier, and immune cell recruitment. Our results show that papain affects the skin barrier by increasing transepidermal water loss, degrading TJ proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells, and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was TLR4 independent and, in spite of its capacity to degrade epidermal TJ proteins, does not rely on its enzymatic function. From our data, we conclude that papain has all features to act as a strong allergen via the skin.
Project description:Hailey-Hailey disease (HHD) and Darier's disease (DD) are caused by mutations in Ca(2+)-ATPases with the end result of desmosomal disruption and suprabasal acantholysis. Tight junctions (TJ) are located in the granular cell layer in normal skin and contribute to the epidermal barrier. Aberrations in the epidermal differentiation, such as in psoriasis, have been shown to lead to changes in the expression of TJ components. Our aim was to elucidate the expression and dynamics of the TJ proteins during the disruption of desmosomes in HHD and DD lesions. Indirect immunofluorescence and avidin-biotin labeling for TJ, desmosomal and adherens junction proteins, and subsequent analyses with the confocal laser scanning microscope were carried out on 14 HHD and 14 DD skin samples. Transepidermal water loss (TEWL) was measured in normal and lesional epidermis of nine HHD and eight DD patients to evaluate the function of the epidermal barrier in HHD and DD skin. The localization of TJ proteins claudin-1, claudin-4, ZO-1, and occludin in perilesional HHD and DD epidermis was similar to that previously described in normal skin. In HHD lesions the tissue distribution of ZO-1 expanded to the acantholytic spinous cells. In agreement with previous findings, desmoplakin was localized intracellularly. In contrast claudin-1 and ZO-1 persisted in the cell-cell contact sites of acantholytic cells. TEWL was increased in the lesional skin. The current results suggest that TJ components follow different dynamics in acantholysis of HHD and DD compared to desmosomal and adherens junction proteins.