Epidermal Differentiation Markers Research Articles

138 Expression of epidermal differentiation markers is modulated by AD-microbiota

Atopic Dermatitis (AD) is the most common chronic inflammatory skin disease, which is caused by an exaggerated T helper 2 cell-mediated immune responses and impairment of the skin barrier function. Single loss of function (LOF) mutations of filaggrin (FLG) are the strongest known genetic risk factor for AD. In Europeans an allele frequency of approximately 10% for a single LOF mutation of FLG can be found, but does not necessarily lead to AD-manifestation. FLG is essential for the skin barrier function. Mutations can thus lead to a disrupted skin barrier. It is known that atopic and healthy skin differ in bacterial diversity and that AD-skin is associated with dysbiosis. It is also known that healthy individuals with a single LOF mutation of FLG have a skin microbiota which is in part similar to that of AD patients. However, the mechanism through which the microbiota contributes in the pathogenesis of AD need further investigation and it is still unclear to what extent the AD microbiota is a driver of the disease. To investigate this question further, we stimulated 3D skin equivalents (3DSE) with collected microbiota of localization-, gender- and age-matched probands (AD- and healthy). Defined skin areas were rinsed with a saline/detergent solution to harvest the microbiota. Subsequently, the rinsing solution containing the microbiota was sequentially centrifuged to remove host mediators/cellular debris. The microbiota samples were then applied to the surface of 3DSE and the expression of skin differentiation markers like FLG were analyzed by real-time PCR. Our findings indicate that the treatment of the 3DSE with the AD-derived microbiota induce a decreased expression of FLG compared to 3DSE treated with the control-microbiota. These results support the hypothesis, that the AD-microbiota maintain the disrupted skin barrier and provide further evidence that the dysbiosis of the AD microbiota is linked to the manifestation/ development and course of this skin disease.

A Lipid Mixture Enriched by Ceramide NP with Fatty Acids of Diverse Chain Lengths Contributes to Restore the Skin Barrier Function Impaired by Topical Corticosteroid

Introduction: The stratum corneum (SC) is a skin barrier that consists of corneocytes, intercellular lipids, and corneodesmosomes. Ceramides are composed of sphingoid bases linked with various types of fatty acids (FAs), and they are an essential constituent of SC intercellular lipids. Among their subtypes, ceramide NP with a phytosphingosine base is especially important. Most of the previous studies on barrier recovery have focused on a specific ceramide with a single chain FA, not with diverse chain lengths. Skin barrier function is impaired by various factors, including topical corticosteroid. Objective: We evaluated whether a lipid mixture enriched by ceramide NP with FAs of diverse chain lengths (CER [NP]*) can restore the skin barrier function impaired by topical corticosteroid. Methods: Twenty-seven healthy adult male volunteers were recruited. Topical corticosteroid was applied on both volar forearms of volunteers. Then, the test cream containing a lipid mixture with CER (NP)* was applied on the left forearm, and a vehicle cream without a lipid mixture was applied on the right forearm of each subject. The functional parameters of the skin barrier were compared before and after the treatment. Epidermal differentiation markers, hyaluronic acid synthase 3 (HAS3), cytokine levels, and the lipid profiles in the SC were analyzed. Results: The functional parameters of the skin barrier, such as barrier recovery rate, SC integrity, and SC hydration were significantly improved in the test cream-applied site compared to the vehicle cream-applied sites. Filaggrin and HAS3 levels were significantly higher in the sites applied with the test cream. Interleukin (IL)-1α levels were also significantly increased in these sites. IL-2, IL-6, IL-10, and IL-13 levels were significantly decreased in the test cream-applied sites. Lipid analyses showed that C18, C20, and total ceramide NP levels significantly increased in the sites where the test cream was applied. Also, C16, C18, C20, C24, and total ceramide NP levels were significantly elevated in the test cream-applied sites after acute barrier disruption. Conclusion: Our results demonstrate that a lipid mixture enriched by CER (NP)* could recover the barrier function impaired by topical corticosteroid.

Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin.

IntroductionThe skin is comprised of various kinds of cells and has three layers, the epidermis, dermis and subcutaneous adipose tissue. Stem cells in each tissue duplicate themselves and differentiate to supply new cells that function in the tissue, and thereby maintain the tissue homeostasis. In contrast, senescent cells accumulate with age and secrete senescence-associated secretory phenotype (SASP) factors that impair surrounding cells and tissues, which lowers the capacity to maintain homeostasis in each tissue. Previously, we found Gremlin 2 (GREM2) as a novel SASP factor in the skin and reported that GREM2 suppressed the differentiation of adipose-derived stromal/stem cells. In the present study, we investigated the effects of GREM2 on stem cells in the epidermis and dermis.MethodsTo examine whether GREM2 expression and the differentiation levels in the epidermis and dermis are correlated, the expressions of GREM2, stem cell markers, an epidermal differentiation marker Keratin 10 (KRT10) and a dermal differentiation marker type 3 procollagen were examined in the skin samples (n = 14) randomly chosen from the elderly where GREM2 expression level is high and the individual differences of its expression are prominent. Next, to test whether GREM2 affects the differentiation of skin stem cells, cells from two established lines (an epidermal and a dermal stem/progenitor cell model) were cultured and induced to differentiate, and recombinant GREM2 protein was added.ResultsIn the human skin, the expression levels of GREM2 varied among individuals both in the epidermis and dermis. The expression level of GREM2 was not correlated with the number of stem cells, but negatively correlated with those of both an epidermal and a dermal differentiation markers. The expression levels of epidermal differentiation markers were significantly suppressed by the addition of GREM2 in the three-dimensional (3D) epidermis generated with an epidermal stem/progenitor cell model. In addition, by differentiation induction, the expressions of dermal differentiation markers were induced in cells from a dermal stem/progenitor cell model, and the addition of GREM2 significantly suppressed the expressions of the dermal differentiation markers.ConclusionsGREM2 expression level did not affect the numbers of stem cells in the epidermis and dermis but affects the differentiation and maturation levels of the tissues, and GREM2 suppressed the differentiation of stem/progenitor cells in vitro. These findings suggest that GREM2 may contribute to the age-related reduction in the capacity to maintain skin homeostasis by suppressing the differentiation of epidermal and dermal stem/progenitor cells.

DECODING THE GENE EXPRESSION PROGRAM EVOLVED IN ODONTOGENIC KERATOCYST BY THE COMBINATION OF DEEP TRANSCRIPTOMICS ANALYSES AND COMPUTATIONAL BIOLOGY TOOLS

<h3>Background</h3> <i>Transcriptomics</i> analysis of odontogenic keratocysts (OKCs) has been described in 3 microarray-based studies utilizing fresh-frozen or formalin-fixed paraffin-embedded samples. To date, RNA-sequencing (RNA-seq) technology has been applied only for the transcriptomics analysis of OKC-derived fibroblasts. <h3>Objective</h3> RNA-seq analysis of the entire OKC tissue will enable the identification of the gene expression profile that accompanies OKC development. <h3>Methods</h3> Total RNA was extracted from formalin-fixed paraffin-embedded samples of 3 OKCs and 3 dental follicles of third molars. RNA library preparation and 150-bp paired-end sequencing were implemented using an Illumina Hiseq4000 Sequencer followed by application of advanced computational biology tools; for example, edgeR software package and Galaxy platform. <h3>Results</h3> RNA-sequencing resulted in ∼15 to 40 million paired-end reads per sample. A total of 1335 differentially expressed genes (DEGs) between OKCs and dental follicles (fold change ≥2) were captured. Gene ontology classification analysis identified markers of epidermal cell development and differentiation (e.g., DSP, TFAP2A, FOXM1) among the top upregulated DEGs, markers of extracellular matrix organization and nervous system regulation (e.g., ADAMTS14, COL3A1, NAV3), among the top downregulated DEGs, and various transcription factors in both categories exemplified by the upregulation of the epidermal master regulator TP63 in OKCs. <h3>Conclusions</h3> This is the first RNA-seq-based study of the entire OKC. The preliminarily results show significant upregulation of genes commuting to epidermis development/differentiation, a finding consistent with OKC histopathologic phenotype. The identification of novel DEGs is critical to access unknown elements of the molecular mechanism underlying OKC pathogenesis and biologic behavior, with potential for future therapeutic applications.

Effects of Aloe vera Flower Extract and Its Active Constituent Isoorientin on Skin Moisturization via Regulating Involucrin Expression: In Vitro and Molecular Docking Studies.

Skin moisturization is very crucial for maintaining the flexibility, viscoelasticity, and differentiation of the epidermis and its deprivation causes several diseases from dry skin to dermatitis. Aloe vera, a miracle plant having diverse medicinal properties including skin moisturization effects. This study investigated for the first time the molecular mechanism targeting skin moisturization effects of the Aloe vera flower and its major active constituent. By treating human epidermal keratinocytes (HaCaT cells) with Aloe vera flower water extract (AFWE), we found that AFWE upregulated epidermal involucrin by activating the expression of protein kinase C, p38, and ERK 1/2. Additionally, it modulated filaggrin, increased aquaporin expression, and hyaluronan synthesis via a balanced regulation of HAS1 and HYAL1 protein. Similarly, it was able to protect UVB-induced photodamage. Western blot analysis, ELISA, and qRT- PCR were performed to evaluate various epidermal differentiation markers and moisturization-related factors on human epidermal keratinocytes (HaCaT cells). TLC and HPLC were used to detect and analyze the chemical constituents. Among them, we found that an active component of Aloe vera flower, isoorientin (IO) has a high binding affinity to all of its targeted proteins such as involucrin, PKC, P38, etc. through molecular docking assay. This study indicated that the Aloe vera flower and its active constituent, IO can be used as a prominent ingredient to enhance skin barrier function and improve its related pathologies.

Substrate membrane bearing close-packed array of micron-level pillars incrassates air-exposed three-dimensional epidermal equivalent model.

We showed previously that a thick three-dimensional epidermal equivalent can be constructed with passaged keratinocytes on a patterned surface. We first carried out computer simulations of a three-dimensional epidermal equivalent model built on close-packed arrays of 10µm, 15µm, 20µm, 30µm, and 60µm diameter pillars. Based on these predictions, we evaluated epidermal equivalents built on a series of porous plastic membranes bearing arrays of pillars 15µm, 20µm, 25µm, 30µm, and 50µm in diameter. The simulations predicted that a model having near-physiological thickness would be formed on 15~30µm pillars. In the results of in vitro study, the thickest epidermal equivalent was obtained on the 20µm pillars. Epidermal differentiation markers, filaggrin and loricrin, were expressed at the upper layer of the epidermal equivalent model, and tight-junction proteins, claudin-1 and ZO-1, were expressed on the cell membranes. BrdU-positive cells were observed at the base and also at the top of the pillars. The results of the study suggested that mathematical modeling might be a useful tool to guide biological studies.

SMOC1 and IL-4 and IL-13 Cytokines Interfere with Ca2+ Mobilization in Primary Human Keratinocytes

Immunoregulatory effects of IL-4 and IL-13 and alterations of keratinocyte (KC) differentiation are important factors in the pathogenesis of atopic dermatitis. This study investigated the role of IL-4 and IL-13 in KC responses to changes in extracellular calcium (Ca2+) and analyzed differentiation signals elicited via a Ca2+ sensor, SMOC1. Real-time dynamics of transmembrane Ca2+ influx were assessed in live KCs by flow cytometry and microscopy. Exposure of KCs to a high Ca2+ environment (1.3 mM) triggered a rapid intracellular Ca2+ influx, whereas IL-4- and IL-13-treated cells exhibited a significant decrease in the peak amplitude of Ca2+ influx (P < 0.01). IL-17A and IL-22 did not elicit such responses. Evaluation of intracellular Ca2+ dynamics by microscopy confirmed these observations and revealed heterogeneity of individual KC responses. IL-4 and IL-13 significantly inhibited the expression of Ca2+-binding protein SMOC1 (P < 0.001). Inhibition of epidermal differentiation markers were also observed in SMOC1 small interfering RNA-transfected KCs. Concurrently, the deletion of SMOC1 increased the amplitude of Ca2+ peak response (P < 0.05). In conclusion, our results provide innovative data that IL-4 and IL-13 regulate KC sensitivity to microenvironmental Ca2+ changes and inhibit Ca2+-induced KC differentiation signals. SMOC1 inhibition by IL-4 and IL-13 alters Ca2+ transport in KCs and inhibits differentiation, suggesting a new target for treatment of atopic dermatitis.

Calcipotriol/Betamethasone Dipropionate Foam Inhibits Th17 Cytokine Secretion and Improves Epidermal Barrier Markers in a Human Th17 Skin Inflammation Model.

IntroductionT-helper 17 (Th17) cytokines play a key role in the pathophysiology of psoriasis by driving inflammatory responses that lead to epidermal alterations. Markers of epidermal differentiation, including the proteins loricrin (LOR), filaggrin (FLG) and involucrin (IVL), are dysregulated in psoriatic skin. The fixed-dose combination of calcipotriol/betamethasone dipropionate (Cal/BD) foam and clobetasol propionate (CP) are widely used, effective topical treatments for psoriasis. In this study, we investigated the effects of Cal/BD foam and CP cream on Th17 cytokine secretion and epidermal differentiation using a human Th17 skin inflammation model (InflammaSkin®).MethodsThe fixed-dose combination Cal/BD foam and the CP cream were applied once and twice daily, respectively, onto the air-exposed epidermal surface of InflammaSkin cultures for 7 days. Th17 cytokine levels were measured in culture supernatants, and gene expression analysis and immunohistochemical staining for LOR, FLG and IVL were performed on the skin samples.ResultsTopical treatment with Cal/BD foam almost completely inhibited Th17 cytokine secretion and upregulated LOR and IVL expression, but not FLG expression, at the mRNA and protein levels. Topical treatment with CP cream significantly reduced Th17 cytokine levels, but to a lesser extent than Cal/BD foam, and did not improve expression of any of the epidermal differentiation markers.ConclusionCompared with CP treatment, the fixed-dose combination Cal/BD foam showed a greater suppression of Th17 cytokine secretion and improved epidermal differentiation, resulting in an overall higher degree of improvement of the skin. These results support our understanding of the mechanisms behind the clinical efficacy observed for Cal/BD foam and of its use for long-term proactive treatment of psoriasis vulgaris.

Suprabasin-derived bioactive peptides identified by plasma peptidomics

Identification of low-abundance, low-molecular-weight native peptides using non-tryptic plasma has long remained an unmet challenge, leaving potential bioactive/biomarker peptides undiscovered. We have succeeded in efficiently removing high-abundance plasma proteins to enrich and comprehensively identify low-molecular-weight native peptides using mass spectrometry. Native peptide sequences were chemically synthesized and subsequent functional analyses resulted in the discovery of three novel bioactive polypeptides derived from an epidermal differentiation marker protein, suprabasin. SBSN_HUMAN[279–295] potently suppressed food/water intake and induced locomotor activity when injected intraperitoneally, while SBSN_HUMAN[225–237] and SBSN_HUMAN[243–259] stimulated the expression of proinflammatory cytokines via activation of NF-κB signaling in vascular cells. SBSN_HUMAN[225–237] and SBSN_HUMAN[279–295] immunoreactivities were present in almost all human organs analyzed, while immunoreactive SBSN_HUMAN[243–259] was abundant in the liver and pancreas. Human macrophages expressed the three suprabasin-derived peptides. This study illustrates a new approach for discovering unknown bioactive peptides in plasma via the generation of peptide libraries using a novel peptidomic strategy.

&amp;lt;i&amp;gt;Rehmannia&amp;lt;/i&amp;gt; Root Improves Extracellular Matrix Production and Epidermal Differentiation by Upregulation of the Estrogen Receptor

Estrogen is essential for the skin to maintain its physiological function. The binding of estrogen to the estrogen receptor (ER) activates gene transcription, which has biological effects on the target tissue. Estrogen levels and ER expression are known to decrease with aging and exposure to ultraviolet light (UV); therefore, increased estrogen levels and ER expression may improve age-related changes in the skin. Rehmannia root has been reported to have blood circulation-promoting and anti-inflammatory effects; however, few studies have reported the effects of Rehmannia root on skin. In this study, we examined the effects of Rehmannia glutinosa Libosch. var. purpurea Makino root extract (RE) on ER expression, and estrogen, RE, or their related ingredients increased ER expression in human epidermal keratinocytes, human dermal fibroblasts, and skin models. Moreover, RE increased the production of basic fibroblast growth factor, transforming growth factor β1, and epidermal growth factor. The mixture of estrogen and RE improved extracellular matrix (ECM) production to a greater degree than estrogen and RE independently. Although high population doubling levels (PDL) and UV irradiation downregulated ER expression, RE upregulated ER expression in high PDL cells and UV irradiated cells. In addition, RE increased the expression of epidermal differentiation marker proteins compared to their expression levels in the absence of RE. The collective findings suggest that RE aids in the prevention of skin aging by upregulating the ER expression that has been decreased by aging and UV and promoting estrogen activity, ECM production, and epidermal differentiation.

The ceramide [NP]/[NS] ratio in the stratum corneum is a potential marker for skin properties and epidermal differentiation

BackgroundSpecific species of ceramides (Cer), major constituents of lipids in the stratum corneum (SC), are decreased and are correlated with SC barrier and water-holding functions in the skin of patients with atopic dermatitis (AD) or psoriasis (Pso). However, possible correlations between Cer subclass ratios and skin properties in barrier-disrupted skin and in healthy skin remain unclear. The objective of this study was to identify a new marker to evaluate skin properties and epidermal differentiation in SC not only in barrier-disrupted skin but also in healthy skin.MethodsThe Cer subclass ratios in the SC of healthy control subjects and in patients with AD or Pso were evaluated. Correlations with candidate markers and facial skin features of healthy Japanese females (20–74 years old, n = 210) were investigated. Variations of markers during epidermal differentiation were studied in human epidermis and in cultured keratinocytes.ResultsThe ratios of Cer [NP]/[NS], Cer [NH]/[NS], Cer [NP]/[AS], Cer [NH]/[NS], Cer [NDS]/[AS], Cer [AH]/[AS] and Cer [EOP]/[AS] showed significant differences between non-lesional skin of AD patients and normal skin of healthy control subjects, as well as Pso patients and their healthy control subjects. The Cer [NP]/[NS] ratio was correlated with SC functional parameters (transepidermal water loss and capacitance) and with skin appearance (texture, scaling and color) even in the cheek skin of healthy female subjects. The Cer [NP]/[NS] ratio in the SC was approximately 18-times higher than in living keratinocytes, and it increased as they differentiated.ConclusionsThe Cer [NP]/[NS] ratio in the SC is a potential marker for skin properties and epidermal differentiation in barrier-disrupted skin as well as in healthy skin.

The Expression Pattern of Epidermal Differentiation Marker Keratin 10 in the Normal Human Breast and Breast Cancer Cells.

Cells of the human breast gland express an array of keratins, of which some are used for characterizing both normal and neoplastic breast tissue. However, the expression pattern of certain keratins has yet to be detailed. Here, the expression of a differentiation marker of epidermal epithelium, keratin 10 (K10), was investigated in the human breast gland. While in normal breast tissue generally less than 1% of luminal epithelial cells expressed K10, in women >30 years of age glandular structures with K10-positive (K10pos) cells were found at higher frequency than in younger women. K10pos cells belong to a mature luminal compartment as they were negative for cKIT, positive for Ks20.8, and mostly non-cycling. In breast cancer, around 16% of primary breast carcinomas tested were positive for K10 by immunohistochemistry. Interestingly, K10pos tumor cells generally exhibit features of differentiation similar to their normal counterparts. Although this suggests that K10 is a marker of tumor differentiation, data based on gene expression analysis imply that high levels of K10 dictate a worse outcome for breast cancer patients. These findings can form the basis of future studies that should unravel which role K10 may play as a marker of breast cancer.

Atopic dermatitis displays stable and dynamic skin transcriptome signatures

Skin transcriptome studies in atopic dermatitis (AD) showed broad dysregulation as well as "improvement" under therapy. These observations were mainly made in trials and based on microarray data. Our aim was to explore the skin transcriptome and the impact of systemic treatment in patients of the TREATgermany registry. Biopsy specimens from 59 patients with moderate-to-severe AD before and 30 patients 12 weeks after start of systemic treatment (dupilumab [n= 22] or cyclosporine [n= 8]) and from 31 healthy controls were subjected to mRNA sequencing. Differential expression, pathway enrichment, correlation, and coexpression network analysis were conducted. Both lesional and nonlesional skin showed a stable "core" signature characterized by disturbed epidermal differentiation and activation of IL-31/IL-1 signaling. Asecond dynamic signature showed progressive enrichment for type 2 inflammation, TH17 signaling, and natural killer cell function. Markers correlated with disease activity have functions in epidermal barrier properties and immune modulation. IL4RA was among the top 3 central dysregulated genes. Cyclosporine led to a more pronounced global transcriptome reversion and normalized TH17 cell/IL23 signaling, whereas dupilumab led to a stronger increase in level of epidermal differentiation markers. Both treatments strongly decreased levels of type 2 markers, but overall the residual profile was still profoundly different from that of healthy skin. Lower levels of IL4RA and IL13 and high IL36A expression were related to a stronger clinical response to dupilumab. The AD core signature is characterized by dysregulation of genes related to keratinocyte differentiation and itch signaling. Adynamic signature reflects progressive immune responses dominated by type 2 cytokines with an additional role of TH17 and natural killer cell signaling.

212 Regulated mitochondrial fission via NIX and DRP1 in keratinocytes drives epidermal differentiation

Epidermal keratinocytes initiate wholesale organelle degradation during cornification, but the mechanisms driving this process are poorly understood. Using live confocal microscopy, we imaged single organelle dynamics in organotypic human epidermis focusing on mitochondria, which are known to regulate keratinocyte differentiation. Mitochondria in the lower epidermis underwent cyclic fission and fusion, but in the upper layers, they became stably fragmented, depolarized, and acidified, leading us to investigate if they were targeted for autophagic degradation. We found differentiating keratinocytes up-regulate the autophagy receptor NIX, which is concentrated on granular layer mitochondria in both organotypic epidermis and human skin biopsies. Thus, we hypothesized NIX could serve as an initiation signal for mitochondrial degradation during cornification. Accordingly, ectopic expression of NIX in undifferentiated keratinocytes was sufficient to induce mitochondrial fragmentation and led to premature differentiation of epidermal cultures. To test the requirement for NIX in epidermal morphogenesis, we used CRISPR/Cas9 gene editing to generate NIX-deficient human organotypic epidermis. NIX-targeted cultures exhibited impaired expression of epidermal differentiation markers and aberrant mitochondrial degradation seen by electron microscopy. Delineating the underlying mechanism, we showed that NIX enhanced mitochondrial localization of FIS1, a receptor for the membrane fission GTPase DRP1. Further, over-expression of FIS1 induced fragmentation of keratinocyte mitochondria similar to NIX. Finally, we showed that compromising DRP1 function directly using a dominant-negative mutant blocked mitochondrial fragmentation in the upper epidermal layers, resulting in abnormal morphology and differentiation of suprabasal keratinocytes. In sum, we report a novel pathway in differentiating keratinocytes driving break-down of mitochondria via the autophagy receptor NIX and DRP1 in order to support proper epidermal morphogenesis.

CYP11A1-derived vitamin D3 products protect against UVB-induced inflammation and promote keratinocytes differentiation

UVB radiation mediates inflammatory responses causing skin damage and defects in epidermal differentiation. 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) interacts with the vitamin D3 receptor (VDR) to regulate inflammatory responses. Additionally, 1,25(OH)2D3/VDR signaling represents a potential therapeutic target in the treatment of skin disorders associated with inflammation and poor differentiation of keratinocytes. Since the protective effect of 1,25(OH)2D3 against UVB-induced skin damage and inflammation is recognized, CYP11A1-derived vitamin D3-hydroxyderivatives including 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3 and 1,20,23(OH)3D3 were tested for their anti-inflammatory and skin protection properties in UVB-irradiated human epidermal keratinocytes (HEKn). HEKn were treated with secosteroids for 24 h pre- and post-UVB (50 mJ/cm2) irradiation. Secosteroids modulated the expression of the inflammatory response genes (IL-17, NF-κB p65, and IκB-α), reducing nuclear-NF-κB-p65 activity and increasing cytosolic-IκB-α expression as well as that of pro-inflammatory mediators, IL-17, TNF-α, and IFN-γ. They stimulated the expression of involucrin (IVL) and cytokeratin 10 (CK10), the major markers of epidermal differentiation, in UVB-irradiated cells. We conclude that CYP11A1-derived hydroxyderivatives inhibit UVB-induced epidermal inflammatory responses through activation of IκB-α expression and suppression of NF-kB-p65 activity and its downstream signaling cytokines, TNF-α, and IFN-γ, as well as by inhibiting IL-17 production and activating epidermal differentiation.

Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα.

Progenitor cells at the basal layer of skin epidermis play an essential role in maintaining tissue homeostasis and enhancing wound repair in skin. The proliferation, differentiation, and cell death of epidermal progenitor cells have to be delicately regulated, as deregulation of this process can lead to many skin diseases, including skin cancers. However, the underlying molecular mechanisms involved in skin homeostasis remain poorly defined. In this study, with quantitative proteomics approach, we identified an important interaction between KDF1 (keratinocyte differentiation factor 1) and IKKα (IκB kinase α) in differentiating skin keratinocytes. Ablation of either KDF1 or IKKα in mice leads to similar but striking abnormalities in skin development, particularly in skin epidermal differentiation. With biochemical and mouse genetics approach, we further demonstrate that the interaction of IKKα and KDF1 is essential for epidermal differentiation. To probe deeper into the mechanisms, we find that KDF1 associates with a deubiquitinating protease USP7 (ubiquitin-specific peptidase 7), and KDF1 can regulate skin differentiation through deubiquitination and stabilization of IKKα. Taken together, our study unravels an important molecular mechanism underlying epidermal differentiation and skin tissue homeostasis.

Re-epithelialization and immune cell behaviour in an ex vivo human skin model

A large body of literature is available on wound healing in humans. Nonetheless, a standardized ex vivo wound model without disruption of the dermal compartment has not been put forward with compelling justification. Here, we present a novel wound model based on application of negative pressure and its effects for epidermal regeneration and immune cell behaviour. Importantly, the basement membrane remained intact after blister roof removal and keratinocytes were absent in the wounded area. Upon six days of culture, the wound was covered with one to three-cell thick K14+Ki67+ keratinocyte layers, indicating that proliferation and migration were involved in wound closure. After eight to twelve days, a multi-layered epidermis was formed expressing epidermal differentiation markers (K10, filaggrin, DSG-1, CDSN). Investigations about immune cell-specific manners revealed more T cells in the blister roof epidermis compared to normal epidermis. We identified several cell populations in blister roof epidermis and suction blister fluid that are absent in normal epidermis which correlated with their decrease in the dermis, indicating a dermal efflux upon negative pressure. Together, our model recapitulates the main features of epithelial wound regeneration, and can be applied for testing wound healing therapies and investigating underlying mechanisms.

Rab25 Deficiency Perturbs Epidermal Differentiation and Skin Barrier Function in Mice.

Rab25, a member of the Rab11 small GTPase family, is central to achieving cellular polarity in epithelial tissues. Rab25 is highly expressed in epithelial cells of various tissues including breast, vagina, cervix, the gastrointestinal tract, and skin. Rab25 plays key roles in tumorigenesis, mainly by regulating epithelial differentiation and proliferation. However, its role in skin physiology is relatively unknown. In this study, we demonstrated that Rab25 knock-out (KO) mice show a skin barrier dysfunction with high trans-epidermal water loss and low cutaneous hydration. To examine this observation, we investigated the histology and epidermal differentiation markers of the skin in Rab25 KO mice. Rab25 KO increased cell proliferation at the basal layer of epidermis, whereas the supra-basal layer remained unaffected. Ceramide, which is a critical lipid component for skin barrier function, was not altered by Rab25 KO in its distribution or amount, as determined by immunohistochemistry. Notably, levels of epidermal differentiation markers, including loricrin, involucrin, and keratins (5, 14, 1, and 10) increased prominently in Rab25 KO mice. In line with this, depletion of Rab25 with single hairpin RNA increased the expression of differentiation markers in a human keratinocyte cell line, HaCaT. Transcriptomic analysis of the skin revealed increased expression of genes associated with skin development, epidermal development, and keratinocyte differentiation in Rab25 KO mice. Collectively, these results suggested that Rab25 is involved in the regulation of epidermal differentiation and proliferation.

BMP2-mediated PTEN enhancement promotes differentiation of hair follicle stem cells by inducing autophagy

The proliferation and differentiation of hair follicle stem cells (HFSCs) is regulated by several signaling pathways, including BMP and PTEN. Therefore, this study intended to clarify the potential effects of two such regulators, BMP2 and PTEN, on HFSC differentiation. HFSCs were subjected to BMP2, noggin (BMP2 ligand inhibitor), rapamycin (Rapa, autophagy inducer), 3-methyladenine (3-MA, autophagy inhibitor), or shRNA against PTEN. The differentiation of HFSCs was evaluated using oil red O staining and autophagy was assessed using the transmission electron microscope. Then expression of epidermal differentiation marker (K10 and involucrin), adipogenic markers (PPAR-γ2, aP2, perilipin2, and Adipoq), keratinocyte-specific marker (K15), proliferation-related markers (PCNA and Ki67) and autophagy-related factors (Atg5, Atg7, Atg12, Beclin-1 and LC3-II/LC3-I) was examined by RT-qPCR and Western blot analysis. Next, HFSCs were treated with 3-MA, or shRNA against Atg5 or Atg7 to verify the effect of autophagy on differentiation of BMP2-treated HFSCs. Finally, the effect of BMP2 on HFSC differentiation was verified by a mouse wound model. HFSCs overexpressing BMP2 exhibited elevated expression of epidermal differentiation marker, adipogenic markers and autophagy-related factors but inhibited expression of keratinocyte-specific marker and proliferation-related markers. Furthermore, we found that PTEN promoted the differentiation of BMP2-treated HFSCs by inducing autophagy. In vivo experiments further confirmed the roles of BMP2/PTEN on differentiation of HFSCs. Taken together, BMP2 up-regulated PTEN and consequently induced autophagy to facilitate HFSC differentiation.

IL-1β–Primed Mesenchymal Stromal Cells Improve Epidermal Substitute Engraftment and Wound Healing via Matrix Metalloproteinases and Transforming Growth Factor-β1

Since the 1980s, deep and extensive skin wounds and burns are treated with autologous split-thickness skin grafts, or cultured epidermal autografts, when donor sites are limited. However, the clinical use of cultured epidermal autografts often remains unsatisfactory because of poor engraftment rates, altered wound healing, and reduced skin functionality. In the past few decades, mesenchymal stromal cells (MSCs) have raised much attention because of their anti-inflammatory, protrophic, and pro-remodeling capacities. More specifically, gingival MSCs have been shown to possess enhanced wound healing properties compared with other tissue sources. Growing evidence also indicates that MSC priming could potentiate therapeutic effects in diverse invitro and invivo models of skin trauma. In this study, we found that IL-1β-primed gingival MSCs promoted cell migration, dermal-epidermal junction formation, and inflammation reduction invitro, as well as improved epidermal substitute engraftment invivo. IL-1β-primed gingival MSCs had different secretory profiles from naive gingival MSCs, characterized by an overexpression of transforming growth factor-β and matrix metalloproteinase (MMP) pathway agonists. Eventually, MMP-1, MMP-9, and transforming growth factor-β1 appeared to be critically involved in IL-1β-primed gingival MSC mechanisms of action.