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The Runt-related transcription factor (Runx) family has been suggested to play roles in stem cell regulation, tissue development, and oncogenesis in various tissues/organs. In this study, we investigated the possible functions of Runx1 and Runx3 in keratinocyte differentiation. Both Runx1 and Runx3 proteins were detected in primary cultures of mouse keratinocytes. Proteins were localized in the nuclei of undifferentiated keratinocytes but translocated to the cytoplasm of differentiated cells. The siRNA-mediated inhibition of Runx1 and Runx3 expression increased expression of keratin 1 and keratin 10, which are early differentiation markers of keratinocytes. In contrast, overexpression of Runx1 and Runx3 suppressed keratin 1 and keratin 10 expression. Endogenous Runx1 and Runx3 proteins were associated with the promoter sequences of keratin 1 and keratin 10 genes in undifferentiated but not differentiated keratinocytes. In mouse skin, the inhibition of Runx1 and Runx3 expression by keratinocyte-specific gene targeting increased the ratios of keratin 1- and keratin 10-positive cells in the basal layer of the epidermis. On the other hand, inhibition of Runx1 and Runx3 expression did not alter the proliferation capacity of cultured or epidermal keratinocytes. These results suggest that Runx1 and Runx3 likely function to directly inhibit differentiation-induced expression of keratin 1 and keratin 10 genes but are not involved in the regulation of keratinocyte proliferation.

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

Psoriasis is a chronic immune‐mediated inflammatory dermatosis. Recently, ozone therapy has been applicated to psoriasis treatment; however, the mechanism by which ozone therapy improves psoriasis remains unclear. The excessive proliferation and the differentiation of basal keratinocytes have been considered critical issues during pathological psoriasis process, in which keratin 6 (KRT6) and KRT10 might be involved. In the present study, KRT6, IL‐17 and IL‐22 protein within psoriasis lesions was decreased, while KRT10 and Tp63 protein in psoriasis lesions was increased by ozone treatment in both patient and IMQ mice psoriatic tissues. In the meantime, ozone treatment down‐regulated KRT6 mRNA and protein expression while up‐regulated KRT10 mRNA and protein expression within IL‐22 treated primary KCs; the cell viability of KCs was suppressed by ozone treatment. Moreover, Tp63 bound to KRT10 promoter region to activate its transcription in basal keratinocytes; the promotive effects of ozone on Tp63 and KRT10 were significantly reversed by Tp63 silence. Both TP63 and KRT10 mRNA expression were significantly increased by ozone treatment in psoriasis lesions; there was a positive correlation between Tp63 and KRT10 expression within tissue samples, suggesting that ozone induces the expression of Tp63 to enhance the expression of KRT10 and the differentiation of keratinocytes, therefore improving the psoriasis. In conclusion, the application of ozonated oil could be an efficient and safe treatment for psoriasis; ozone promotes the differentiation of keratinocytes via increasing Tp63‐mediated transcription of KRT10, therefore improving psoriasis.

Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes in vitro allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown. We show for the first time that PR and SLP themselves suppress expression of differentiation-specific keratins K1, K10 and K2 in normal human epidermal keratinocytes (NHEK) and two important cell lines, HaCaT and N/TERT-1. Removal of SLP increased expression of K1, K10 and K2 in 2D and 3D cultures, which was further enhanced in the absence of PR. The effect was reversed for K1 and K10 by adding all-trans retinoic acid (ATRA) but increased for K2 in the absence of PR. Furthermore, retinoid regulation of differentiation-specific keratins involves post-transcriptional mechanisms as we show KRT2 mRNA is stabilised whilst KRT1 and KRT10 mRNAs are destabilised in the presence of ATRA. Taken together, our results indicate that the presence of PR and SLP in cell culture media may significantly impact in vitro studies of keratinocyte differentiation.

Background Epidermolytic acanthoma (EA) is a rare acquired lesion demonstrating a characteristic histopathological pattern of epidermal degeneration referred to as epidermolytic hyperkeratosis (EHK). On histopathological analysis, EA appears nearly identical to inherited EHK-associated dermatoses such as epidermolytic ichthyosis and ichthyosis bullosa of Siemens. While it has been speculated that EA is caused by mutations in KRT10, KRT1, or KRT2 found in these inherited dermatoses, none have yet been identified. Herein, we aim to identify the contributions of keratin mutations to EA. Methods Using genomic DNA extracted from paraffin-embedded samples from departmental archives, we evaluated a discovery cohort using whole-exome sequencing (WES) and assessed remaining samples using Sanger sequencing screening and restriction fragment length polymorphism (RFLP) analysis. Results DNA from 16/20 cases in our sample was of sufficient quality for polymerase chain reaction amplification. WES of genomic DNA from lesional tissue revealed KRT10 c.466C > T, p.Arg156Cys mutations in 2/3 samples submitted for examination. RFLP analysis of these samples as well as eight additional samples confirmed the mutations identified via WES and identified four additional cases with Arg156 mutations. In sum, 6/11 screened cases showed hotspot mutation in KRT10. Conclusions Hotspot mutations in the Arg156 position of KRT10, known to cause epidermolytic ichthyosis, also underlie EA.

Epidermolytic palmoplantar keratoderma (EPPK) is characterized by diffuse hyperkeratosis affecting palms and soles with suprabasal epidermolysis or vacuolar degeneration histopathologically. The disorder is caused by heterozygous mutations in KRT9 or KRT1. Dominant-negative mutations in KRT1 could also result in epidermolytic ichthyosis with EPPK, a more severe entity affecting the entire body.To investigate the genetic basis and pathogenesis of two unrelated patients with EPPK and knuckle pads, both of whom were born to consanguineous parents of Chinese origin.Next-generation sequencing was applied to the two patients using genomic DNA extracted from peripheral blood. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining and Western blot (WB) were employed to evaluate mRNA and protein expression level. Ultrastructural changes of skin lesion were analysed using transmission electron microscopy.Two novel homozygous mutations, c.457CT (p.Gln153*) and c.33CG (p.Tyr11*) in KRT1, were identified in patients 1 and 2 respectively. The nonsense mutations were predicted to result in nonsense-mediated mRNA decay and absence of keratin 1, which was confirmed in the skin lesions from patient 1. Upregulated keratin 2 was detected both in the affected and unaffected skin samples from patient 1, while the protein abundance and distribution pattern of keratin 10 remained unchanged. An aberrant and clumped staining pattern of keratin 9 was noted in the palmar skin of patient 1.Homozygous 'knockout' mutations in KRT1 resulted in EPPK with knuckle pads rather than epidermolytic ichthyosis. We speculated that sparing of non-acral skin might be due to compensatory effect of keratin 2 upregulation by forming heterodimer with keratin 10.

Ichthyosis with confetti (IWC) is a genodermatosis associated with dominant‐negative variants in keratin 10 (KRT10) or keratin 1 (KRT1). These frameshift variants result in extended aberrant proteins, localized to the nucleus rather than the cytoplasm. This mislocalization is thought to occur as a result of the altered carboxy (C)‐terminus, from poly‐glycine to either a poly‐arginine or ‐alanine tail. Previous studies on the type of C‐terminus and subcellular localization of the respective mutant protein are divergent. In order to fully elucidate the pathomechanism of IWC, a greater understanding is critical. This study aimed to establish the consequences for localization and intermediate filament formation of altered keratin 10 (K10) C‐termini. To achieve this, plasmids expressing distinct KRT10 variants were generated. Sequences encoded all possible reading frames of the K10 C‐terminus as well as a nonsense variant. A keratinocyte line was transfected with these plasmids. Additionally, gene editing was utilized to introduce frameshift variants in exon 6 and exon 7 at the endogenous KRT10 locus. Cellular localization of aberrant K10 was observed via immunofluorescence using various antibodies. In each setting, immunofluorescence analysis demonstrated aberrant nuclear localization of K10 featuring an arginine‐rich C‐terminus. However, this was not observed with K10 featuring an alanine‐rich C‐terminus. Instead, the protein displayed cytoplasmic localization, consistent with wild‐type and truncated forms of K10. This study demonstrates that, of the various 3′ frameshift variants of KRT10, exclusively arginine‐rich C‐termini lead to nuclear localization of K10.

The red maple and sugar maple (Acer rubrum and A. saccharum, respectively) contain acertannins (ginnalins and maplexins), galloylated derivatives of 1,5-anhydro- d -glucitol (1,5-AG, 1). These compounds have a variety of potential medicinal properties and we have shown that some of them promote the expression of ceramide synthase 3. We now report on the beneficial effects of ginnalin B, (6-O-galloyl-1,5-AG, 5), leading to acceleration of skin metabolism and reduction of the turnover time. Ginnalin B dose-dependently increased the relative amount of keratin 10, keratin 1, and filaggrin gene, with maximal increase of 1.7-, 2.9, and 5.2-fold at 100 μM, respectively. The validation study showed that it had superior capacity to induce multiple stages of keratinocyte differentiation and significantly elevated the immunostaining site of keratin 10 and filaggrin in a 3-dimensional cultured human skin model, by 1.2 and 2.8-fold, respectively. Furthermore, ginnalin B caused the arrest of proliferation at the G0/G1 phase but it did not induce apoptotic cell death in normal human keratinocytes. Molecular studies revealed that ginnalin B up-regulated the levels of NOTCH1 and a concomitant increase p21 expression. Ginnalin B, therefore, represents a new class of promising functional and medical cosmetic compound and it could contribute to the maintenance of homeostasis of the epidermis.

Objectives To identify cytokeratins (CK) of significant correlations with clinical and histopathologic prognostic parameters in oral and oropharyngeal squamous cell carcinoma (SCC). Design The sample consisted of 100 cases retrieved from the archives of the Pathology Department/ King Hussein Cancer Center/Amman/ Jordan. Recorded data included: age, gender, location, grade, depth of invasion, the presence of epithelial dysplasia, tumor size, lymph node metastasis, number of positive lymph nodes, distant metastases, clinical stage, local recurrence, treatment modalities and 5-year survival rate. Immunohistochemical staining of 7 cytokeratins: 8, 10, 13, 14, 16, 18, and 19 was performed using standard protocols. Stained sections were digitized and analyzed using ImageJ-color deconvolution to identify the percentage of cytokeratin-positive area (score). Statistical tests used were: student t-test, analysis of variance, bivariate analysis and logistic regression. Results Lower CK8,18, 19 scores correlated with lower 5-year survival rate. Higher CK19 and lower CK 10, 14, 16 scores were associated with distant metastasis. Increased CK8, 18, 19 scores correlated with higher stage and with higher depth of invasion. Increased CK18 scores correlated with increased local recurrence. Higher CK10, 13, 16 scores correlated with well-differentiated grade. Higher CK19 and lower CK16 scores were associated with adjacent epithelial dysplasia. Regression analysis showed that better 5-year survival rate was significantly correlated with increased CK16, decreased CK18 and 19 scores. Conclusion Expression scores of a panel of cytokeratin are potential prognostic indicators for 5-year survival and correlates with other prognostic parameters.

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.

This pilot study was aimed at comparing TLR7/TLR9 expression, cytoskeletal arrangement, and cell proliferation by indirect immunofluorescence in parallel lesional and non lesional skin samples of guttate psoriasis (PG) and psoriasis vulgaris (PV) in five male patients for each group (n=10). TLR7 expression was detected throughout all the epidermal compartment in PV samples, while in PG skin was restricted to the granular layer. TLR9 was present in the granular layer of non lesional skin and in the suprabasal layers of PV/PG lesional skin. Cell proliferation was localized in all the epidermal layers in lesional PG and PV, consistently with the immunopositivity for the “psoriatic keratin” K16. In the suprabasal layers of lesional PG and PV skin, a similar K17 expression was detected and K10 exhibited a patchy distribution. The present results suggest that TLR7 expression can be considered an intrinsic and differential histomorphological feature of PV.

Despite the progress in chronic wound treatment, antibacterial cutaneous scaffold with high efficiency in wound healing is still the hot spot in the field. In present study, a functionalized silk fibroin (SF) cutaneous scaffold incorporated with natural medicine usnic acid (UA) is investigated, in which UA is used as an antibacterial and wound-healing reagent. Via electrospinning, UA-SF mixture is fabricated into UA-SF composite scaffold (USCS), which is composed of uniform nanofibers with average diameters of around 360 ± 10 nm. The interwoven nanofibers form mesh structure providing sufficient moisture permeability for scaffold. With methanol treatment, USCS presents improved mechanical properties and stability to protease XIV. In the presence of USCS, the growth rate of both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Streptococci pyogenes, Escherichia coli, and Pseudomonas aeruginosa, is significantly inhibited in plate culture and suspension assays. In a cutaneous excisional mouse wound model, USCS presents a significant increase of wound closure rate, compared with pure SF scaffold and commercial dressing, Tegaderm Hydrocolloid 3M . The histological assessments further prove that USCS can enhance re-epithelialization, vascularization, and collagen deposition in wound site to promote the wound-healing process, which indicates the potential application of USCS in chronic wound treatment.

Atopic Dermatitis is an inflammatory skin disease associated with broad defects in skin barrier function caused by increased levels of type-2 cytokines (IL-4 and IL-13) that repress keratinocyte (KC) differentiation. Although crucial in mediating allergic disease, the mechanisms for gene repression induced by type-2 cytokines remain unclear. In this study, we determined that gene repression requires the master regulator of the epidermal differentiation program, p63. We found that type-2 cytokine–mediated inhibition of the expression of genes involved in early KC differentiation, including keratin 1, keratin 10, and DSC-1, is reversed by p63 blockade. Type-2 cytokines, through p63, also regulate additional genes involved in KC differentiation, including CHAC-1, STC2, and CALML5. The regulation of the expression of these genes is ablated by p63 small interfering RNA as well. In addition, we found that IL-4 and IL-13 and Staphylococcus aureus lipoteichoic acid work in combination through p63 to further suppress the early KC differentiation program. Finally, we found that IL-4 and IL-13 also inhibit the activity of Notch, a transcription factor required to induce early KC differentiation. In conclusion, type-2 cytokine–mediated gene repression and blockade of KC differentiation are multifactorial, involving pathways that converge on transcription factors critical for epidermal development, p63 and Notch.

Purpose To investigate the effects and mechanisms of the peroxisome proliferator-activated receptor alpha (PPAR-α) agonist fenofibrate on the formation of ocular surface squamous metaplasia induced by topical benzalkonium chloride (BAC) in a mouse model. Methods Ocular surface squamous metaplasia was induced in 16 days by topical BAC application in mice. During the period of induction, mice were divided into four groups: no additional treatment (BAC+UT), topical vehicle (BAC+Vehicle), topical fenofibrate (BAC+Feno), or topical fenofibrate plus intraperitoneal injection of MK886 (BAC+Feno+MK886). The parameters of tear film were evaluated on day 16, and eye specimens were collected. Histologic investigation; PAS assays; immunostaining for cytokeratin 10 (K10), Ki67, and F4/80; and PCR assays for TNF-α and IL-6 were performed. Cell Counting Kit 8 (CCK-8) assays were performed to evaluate the inhibitory effects of fenofibrate on RAW264.7 cells. Results Fenofibrate suppressed the formation of BAC-induced instable tear film. In the BAC+Feno group, the expression of K10 and Ki67 was lower than in the other three groups. The number of goblet cells was reduced in eyes of the BAC+UT and BAC+Vehicle groups but was maintained in eyes of the BAC+Feno group. The number of F4/80-positive cells and the levels of TNF-α and IL-6 mRNA were significantly reduced in the cornea of the BAC+Feno group. These effects of fenofibrate could be attenuated by MK886. The cell viability of RAW264.7 cells could be significantly inhibited by fenofibrate in a dose-dependent pattern. Conclusions Topical application of fenofibrate suppressed the formation of ocular surface squamous metaplasia, which might be mediated through the PPAR-α signaling pathway.

IL-33, a chromatin-associated multifunctional cytokine, is implicated in the pathogenesis of atopic dermatitis (AD), an inflammatory skin disorder characterized by skin barrier dysfunction. IL-33 accumulates in the nuclei of epidermal keratinocytes (KCs) in AD lesions. However, it is unclear whether nuclear IL-33 directly contributes to the pathogenesis of AD. IL-31, a pruritogenic cytokine primarily produced by T helper type 2 cells, is elevated in AD lesions and promotes AD development by suppressing KC differentiation and inducing itching. In this study, we investigated the involvement of nuclear IL-33 in IL-31‒mediated suppression of KC differentiation. In monolayer cultures and living skin equivalent, IL-31 increased the expression of full-length IL-33 and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in the nuclei of human KCs, which in turn downregulated the expression of differentiation markers. We found that IL-31 and IL-4/IL-13 use very similar mechanisms to inhibit KC differentiation: nuclear IL-33 combines with phosphorylated STAT3 and functions as a STAT3 transcription cofactor, promoting phosphorylated STAT3 binding to the FLG promoter to inhibit its transcription; moreover, the nuclear IL-33/phosphorylated STAT3 complex drives the downregulation of keratin 1 and keratin 10 by reducing the availability of the transcription factor RunX1. Therefore, nuclear IL-33 plays an important role in IL-31‒mediated differentiation suppression by regulating STAT3 activation in human KCs.

Retinol, a derivative of vitamin A, is a ubiquitous compound used to treat acne, reduce wrinkles and protect against conditions like psoriasis and ichthyosis. While retinol is used as the primary active ingredient (AI) in many skin care formulations, its efficacy is often limited by an extreme sensitivity to degrade and toxicity at high concentrations. While microencapsulation is an appealing method to help overcome these issues, few microencapsulation strategies have made a major translational impact due to challenges with complexity, cost, limited protection of the AI and poor control of the release of the AI. We have developed a class of silicone particles that addresses these challenges for the encapsulation, protection and controlled release of retinol and other hydrophobic compounds. The particles are prepared by the sol-gel polymerization of silane monomers, which enables their rapid and facile synthesis at scale while maintaining a narrow size distribution (i.e., CV 85%), (ii) protect retinol from degradation (yielding a half-life 9× greater than unencapsulated retinol) and (iii) slowly release retinol over several hours (at rates from 0.14 to 0.67 μg cm−2 s−1/2). To demonstrate that the controlled release of retinol from the particles can reduce irritation, we performed a double blind study on human subjects and found that formulations containing our particles were 12–23% less irritating than identical formulations containing Microsponge® particles (an industry standard by Amcol, Inc.). To show that the silicone particles can elicit a favorable biological response, similar to the Microsponge® particles, we applied both formulations to reconstructed human epidermal tissues and found an upregulation of keratin 19 (K19) and a downregulation of K10, indicating that the reduced irritation observed in the human study was not caused by reduced activity. We also found a decrease in the production of interleukin-1α (IL-1α) compared to formulations containing the Microsponge particles, suggesting lower irritation levels and supporting the findings from the human study. Finally, we show that the silicone particles can encapsulate other AIs, including betamethasone, N, N-diethyl-meta-toluamide (DEET), homosalate and ingenol mebutate, establishing these particles as a true platform technology.

Background The development of an alternative medicine to treat atopic dermatitis (AD) from natural sources is necessary. Aims To improve skin barrier dysfunction by enhancing the differentiation of human keratinocytes with the fermented Scutellaria baicalensis. Methods Scutellaria baicalensis was fermented with Lactobacillus plantarum and extracted with 70% ethanol (FE). Antioxidant activities and the regulation of the gene expression related to keratinocyte differentiation were measured as well as its proliferation. Result This work first proved that the FE had multiple activities, both increasing keratinocyte differentiation and proliferation: The FE greatly up-regulated expression of the genes of keratinocyte differentiation such as involucrin, keratin 10, and transglutaminase-1 (TG-1) up to 4.06-fold, which was 3 times higher than the 2 other extracts. The effect of baicalein on keratinocyte differentiation was also first found; however, its efficacy was lower than that of the fermented extract. The FE proved to effectively accelerate keratinocyte differentiation, rather than to initiate the differentiation, and also showed an ability of stimulating keratinocyte proliferation up to 2.8 × 106 viable cells/mL as well as 70.24 ng/mL of collagen production in fibroblasts. High efficacy of the FE was confirmed by synergistic effects of large amounts of various bioactive substances in the extracts as baicalein alone did not show remarkable effects and even positive controls had not much better activities than the FE. Conclusion The fermented extract was able to improve skin barrier dysfunction, and the ointment with 1%-5% (v/v) of the extract be directly used for skin clinical trials to treat AD.

Keratinized mucosa is of fundamental importance to maintain healthy gingival tissue, and understanding the mechanisms of oral mucosa keratinization is crucial to successfully manage healthy gingiva. Previous studies have shown a strong involvement of the basement membrane in the proliferation and differentiation of epithelial cells. Therefore, first, to identify the keratinized mucosa-specific basement membrane components, immunohistochemical analysis for the six alpha chains of type IV collagen was performed in 8-week-old mice. No difference in the expression pattern of type IV collagen α1(IV) and α2(IV) chains was observed in the keratinized and non-keratinized mucosa. Interestingly, however, type IV collagen α5(IV) and α6(IV) chains specifically were strongly detected in the keratinized mucosa. To analyze the functional roles of the type IV collagen isoform α6(IV) in oral mucosa keratinization, we analyzed Col4a6-knockout mice. Epithelial developmental delay and low levels of KRT10 were observed in new-born Col4a6-knockout mice. Additionally, in vitro experiments with loss-of function analysis using human gingival epithelial cells confirmed the important role of α6(IV) chain in epithelial keratinization. These findings indicate that α112:α556 (IV) network, which is the only network that includes the α6(IV) chain, is one regulator of KRT10 expression in keratinization of oral mucosal epithelium.

N-3 polyunsaturated fatty acids (n-3 PUFAs), and in particular docosahexaenoic acid (DHA), have many beneficial metabolic effects, including reducing epidermal thickness in patients with psoriasis. The positive impacts of DHA in psoriasis could be mediated by its interactions with the PPAR signaling pathway, as well as by its secretion of anti-inflammatory bioactive metabolites, but the detailed metabolism is still not understood. In the present study, we evaluated the influence of DHA on the main features of psoriasis and its effects on the PPAR signaling pathway, in a psoriatic in vitro skin model. Healthy and psoriatic skin substitutes were produced according to the tissue-engineered self-assembly method, using culture media supplemented with 10 μM of DHA. The presence of DHA led to a reduction in the abnormal cell differentiation of psoriatic keratinocytes, seen in the increased expression of filaggrin and keratin 10. DHA was incorporated into the membrane phospholipids of the epidermis and transformed principally into eicosapentaenoic acid (EPA). Furthermore, the addition of DHA into the culture medium led to a decrease in the levels of lipid mediators derived from n-6 PUFAs, mainly prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE). Finally, DHA supplementation rebalanced the expression of PPAR receptors and caused a decrease in the secretion of TNF-α. Altogether, our results show that DHA possesses the ability to attenuate the psoriatic characteristics of psoriatic skin substitutes, mostly by restoring epidermal cell differentiation and proliferation, as well as by reducing inflammation.

IL-33 is a chromatin-associated multifunctional cytokine implicated in the pathogenesis of atopic dermatitis (AD), an inflammatory skin disorder characterized by skin barrier dysfunction. The previous reports show that IL-33 is highly detected in the nucleus of epidermal keratinocytes in AD lesions compared with that in unaffected or normal skin. However, it is unclear whether intracellular IL-33 directly contributes to the pathogenesis of AD. T helper type 2 cytokines IL-4 and IL-13 that are elevated in AD lesions suppress keratinocyte differentiation to impair skin barrier function. We investigated whether intracellular IL-33 is involved in IL-4 and IL-13 function. In monolayer culture and living skin equivalent analyses, IL-4 and IL-13 increased the expression of full-length IL-33 in the nucleus of keratinocytes by activating the MAPK/extracellular signal‒regulated kinase kinase/extracellular signal‒regulated kinase signaling pathway, which is necessary for the inhibition of differentiation markers FLG, LOR, keratin 1, and keratin 10. The nuclear IL-33 functions as a transcription cofactor of signal transducer and activator of transcription 3, increasing the binding of phosphorylated signal transducer and activator of transcription 3 to FLG promoter, thereby inhibiting its transcription, and it inhibits the expression of transcription factor RUNX1 by signal transducer and activator of transcription 3 and signal transducer and activator of transcription 6, thereby downregulating LOR, keratin 1, and keratin 10. Thus, the elevated nuclear IL-33 in the epidermis of AD lesions may be involved in the pathogenesis of AD by inhibiting keratinocyte differentiation and skin barrier function.

Background:The epidermis is a continuously regenerating tissue maintained by a balance between proliferation and differentiation, with imbalances resulting in skin disease. We have previously found that in mouse keratinocytes, the lipid-metabolizing enzyme phospholipase D2 (PLD2) is associated with the aquaglyceroporin, aquaporin 3 (AQP3), an efficient transporter of glycerol. Our results also show that the functional interaction of AQP3 and PLD2 results in increased levels of phosphatidylglycerol (PG) in response to an elevated extracellular calcium level, which triggers keratinocyte differentiation. Indeed, we showed that directly applying PG can promote keratinocyte differentiation.Objective:We hypothesized that the differentiative effects of this PLD2/AQP3/PG signaling cascade, in which AQP3 mediates the transport of glycerol into keratinocytes followed by its PLD2-catalyzed conversion to PG, are mediated by protein kinase CβII (PKCβII), which contains a PG-binding domain in its carboxy-terminus. Method: To test this hypothesis we used quantitative RT-PCR, western blotting and immunocytochemistry.Results:We first verified the presence of PKCβII mRNA and protein in mouse keratinocytes. Next, we found that autophosphorylated (activated) PKCβII was redistributed upon treatment of keratinocytes with PG. In the unstimulated state phosphoPKCβII was found in the cytosol and perinuclear area; treatment with PG resulted in enhanced phosphoPKCβII localization in the perinuclear area. PG also induced translocation of phosphoPKCβII to the plasma membrane. In addition, we observed that overexpression of PKCβII enhanced calcium- and PG-induced keratinocyte differentiation without affecting calcium-inhibited keratinocyte proliferation.Conclusion:These results suggest that the PG produced by the PLD2/AQP3 signaling module may function by activating PKCβII.

Epidermolytic ichthyosis (EI) is a rare disorder of cornification caused by mutations in KRT1 and KRT10, encoding two suprabasal epidermal keratins. Because of the variable clinical features and severity of the disease, histopathology is often required to correctly direct the molecular analysis. EI is characterized by hyperkeratosis and vacuolar degeneration of the upper epidermis, also known as epidermolytic hyperkeratosis, hence the name of the disease. In the current report, the authors describe members of 2 families presenting with clinical features consistent with EI. The patients were shown to carry classical mutations in KRT1 or KRT10, but did not display epidermolytic changes on histology. These observations underscore the need to remain aware of the limitations of pathological features when considering a diagnosis of EI.

Skin is the largest organ of the human body. Thus far, tissue engineering of skin has developed rapidly and has used many types of growth factors and nanofibrous scaffolds. In this study, we differentiated neonate keratinocytes for epithelialization on the polycaprolactone-Platelet gel (PCL-PG) scaffold. Fabricated PCL nanofibers prepared by electrospinning technology and coated by platelet gel. Subsequently, the structure of the scaffold was evaluated by SEM, FTIR-ATR, contact angle and tensile test assays. After seeding the neonate keratinocytes on neat PCL and PCL-PG scaffolds, the epidermal maturation was tested by detecting cytokeratin 10 and loricrin determinants by immunocytochemistry; moreover, keratinocyte genes such as keratin 14, keratin 10, and Involucrin were investigated by real-time PCR. The results of MTT assay indicated an increase in cell viability and cell proliferation of neonate keratinocytes on PCL-PG nanofiber scaffolds compared with PCL. RT-PCR and immunocytochemical analysis showed better cell differentiation on the PCL-PG scaffolds than neat PCL. Furthermore, SEM microscopy images demonstrated that neo-keratinocytes enhance adhesion and proliferation on PCL-PG nanofiber scaffolds. We found that PG increases biocompatibility and wettability of scaffold, cell adhesion, and expression of keratinocyte markers. Overall, this procedure is recommended to be employed in skin tissue engineering and wounds healing.

Objective Psoriasis is an immune-mediated inflammatory disease. Despite advances in the study of its pathogenesis, the exact development mechanism of psoriasis remains to be fully elucidated. Hyperproliferative epidermis plays a crucial role in psoriasis. This study aimed to investigate the effects of interleukin-36

Psoriasis is one of the most common chronic inflammatory skin diseases, it is characterized by hyperproliferation of keratinocytes and infiltration of inflammatory cells. Several in vitro studies have reported that interleukin (IL)-22 is involved in excessive proliferation and abnormal differentiation of human keratinocytes. However, the association between IL-22 and CCAAT enhancer binding protein α (C/EBPα) in the pathogenesis of psoriasis remains unclear. Therefore, the present study aimed to investigate the association between IL-22 and C/EBPα, and the effects of IL-22 on the proliferation and differentiation of keratinocytes. Keratinocytes were treated with different concentrations of IL-22 (30, 60 and 90 ng/ml) and subsequently cells were collected at different time intervals. The expression levels of the key molecules of the mitogen-activated protein kinase (MAPK) signaling pathway were detected using western blot analysis. In addition, the effect of IL-22 on the proliferation rate of keratinocytes and the mRNA expression levels of C/EBPα were determined using a Cell Counting Kit-8 assay and reverse transcription-quantitative PCR, respectively. Furthermore, keratinocytes were transfected with C/EBPα small interfering (si)RNA or control using Lipofectamine® 2000. The results revealed that IL-22 significantly induced the proliferation of keratinocytes and the expression of phosphorylated (p)-JNK, p-ERK and p-p38 (P

To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild‐type human keratin‐1/keratin‐10 helix 1B heterotetramer at 3.0 A resolution. It revealed biochemical determinants for the A 11 mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10‐1B heterodimer dictated tetramer assembly: the N‐terminal hydrophobic pocket (defined by L227 K1 , Y230 K1 , F231 K1 , and F234 K1 ), the K10 hydrophobic stripe, K1 interaction residues, and the C‐terminal anchoring knob (formed by F314 K1 and L318 K1 ). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full‐length filament assembly. Individual knob residue mutant F314A K1 , but not L318A K1 , abolished 1B tetramer formation. The K1‐1B knob/pocket mechanism is conserved across keratins and many non‐keratin intermediate filaments. To demonstrate how pathogenic mutations cause skin disease by altering filament assembly, we additionally determined the 2.39 A structure of K1/10‐1B containing a S233L K1 mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1 S233L /K10‐1B in solution without affecting secondary structure. The K1 S233L /K10‐1B octamer structure revealed S233L K1 causes aberrant hydrophobic interactions between 1B tetramers.

Background: As non-cellulosic &beta, d-glucans are known to exert wound-healing activity by triggering keratinocytes into cellular differentiation, the functionality of a semisynthetic lichenan-based polysaccharide on skin cell physiology was investigated. Methods: &gamma, Propoxy-sulfo-lichenan (&gamma, PSL, molecular weight 52 kDa, &beta, 1,3/1,4-p-d-Glucose, degree of substitution 0.7) was prepared from lichenan. Differentiation of primary human keratinocytes was assayed by the protein analysis of differentiation specific markers and by gene expression analysis (qPCR). The gene array gave insight into the cell signaling induced by the polysaccharide. Results: &gamma, PSL (1 to 100 &mu, g/mL) triggered keratinocytes, in a concentration-dependent manner, into the terminal differentiation, as shown by the increased protein expression of cytokeratin 1 (KRT1). Time-dependent gene expression analysis proved differentiation-inducing effects, indicating strong and fast KRT1 gene expression, while KRT10 expression showed a maximum after 12 to 24 h, followed by downregulation to the basal level. Involucrin gene expression was only changed to a minor extent, which was similar to loricrin and transglutaminase. Gene array indicated the influence of &gamma, PSL on MAP kinase and TGF-&beta, mediated signaling towards keratinocyte differentiation. Conclusion: The propoxylated lichenan may improve wound healing by topical application to promote the terminal barrier formation of keratinocytes.

// Santasree Banerjee 1,2 , Qian Wu 1 , Yuyi Ying 1 , Yanni Li 1 , Matsuyuki Shirota 3 , Dante Neculai 1 and Chen Li 1 1 Department of Cell Biology and Medical Genetics, School of Medicine, Zhejiang University, Hangzhou, China 2 BGI-Shenzhen, Shenzhen, China 3 Department of Applied Information Sciences, Graduate School of Information Sciences, Tohoku University, Sendai, Japan Correspondence to: Chen Li, email: // Keywords : genodermatoses, missense mutations, coiled-coil heterodimer, in silico analysis, Pathology Section Received : March 30, 2016 Accepted : May 17, 2016 Published : July 13, 2016 Abstract The K1 and K10 associated genodermatoses are characterized by clinical symptoms of mild to severe redness, blistering and hypertrophy of the skin. In this paper, we set out to computationally investigate the structural and functional effects of missense mutations on the 2B domain of K1/K10 heterodimer and its consequences in disease phenotype. We modeled the structure of the K1/K10 heterodimer based on crystal structures for the human homolog K5/K14 heterodimer, and identified that the missense mutations exert their effects on stability and assembly competence of the heterodimer by altering physico-chemical properties, interatomic interactions, and inter-residue atomic contacts. Comparative structural analysis between all the missense mutations and SNPs showed that the location and physico-chemical properties of the substituted amino acid are significantly correlated with phenotypic variations. In particular, we find evidence that a particular SNP ( K10 , p.E443K) is a pathogenic nsSNP which disrupts formation of the hydrophobic core and destabilizes the heterodimer through the loss of interatomic interactions. Our study is the first comprehensive report analyzing the mutations located on 2B domain of K1/K10 heterodimeric coiled-coil complex.

Epidermolytic ichthyosis (EI) due to KRT10 mutations is a rare, typically autosomal dominant, disorder characterized by generalized erythema and cutaneous blistering at birth followed by hyperkeratosis and less frequent blistering later in life. We identified two KRT10 mutations p.Q434del and p.R441P in subjects presenting with a mild EI phenotype. Both occur within the mutational “hot spot” of the keratin 10 (K10) 2B rod domain, adjacent to severe EI-associated mutations. p.Q434del and p.R441P formed collapsed K10 fibers rather than aggregates characteristic of severe EI KRT10 mutations such as p.R156C. Upon differentiation, keratinocytes from p.Q434del showed significantly lower apoptosis (P-value

During keratinocyte stratification and wound healing, keratinocytes undergo a switch between differentiation and motility. However, limited knowledge exists on the mechanisms of the switch. We have previously demonstrated that the expression of CD9 was changed in different wound stages and involved in the regulation of keratinocyte migration. In this study, we showed that CD9 expression was increased in both human and mouse keratinocytes undergoing differentiation. CD9 overexpression in keratinocytes stimulated terminal differentiation and reduced cell motility. CD9 silencing inhibited calcium-induced keratinocyte differentiation and increased cell motility. Furthermore, CD9 overexpression recruited E-cadherin to the plasma membrane and subsequently activated PI3K/Akt signaling, while CD9 knockdown inhibited the recruitment of E-cadherin to the plasma membrane and PI3K/Akt activation. Importantly, silencing E-cadherin expression or inhibiting PI3K/Akt signaling reversed CD9 overexpression-induced differentiation and -reduced motility. These results demonstrate that CD9 acts as an important node that regulates keratinocyte differentiation and motility. The recruitment of E-cadherin to the plasma membrane and activation of the PI3K/Akt signaling pathway mediated by CD9 play an important role in these processes.

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The gaseous mediators nitric oxide (NO), carbon monoxide (CO) and lately also hydrogen sulfide (H2S) have been described to contribute to the interplay of protein type- and lipid mediators in the regulation of wound healing. In particular, the recently reported role of H2S in skin repair remains largely unresolved. Therefore we assessed the expressional kinetics of potential H2S-producing enzymes during undisturbed skin repair: the cystathionine-γ-lyase (CSE), the cystathionine-β-synthase (CBS) and the 3-mercaptopyruvate sulfurtransferase (MPST). All three enzymes were not transcriptionally induced upon wounding and remained silent through the acute inflammatory and proliferative phase of skin repair. By contrast, CSE expression started to increase significantly at the later stages of healing, when cellular proliferation ceases within the granulation tissue and neoepidermis. The importance of H2S production in late healing phases was supported by a strong induction of otherwise not-induced CBS to complement the loss of CSE function in CSE-deficient mice. Immunohistochemistry revealed hair follicle keratinocytes and basal keratinocytes of the neo-epidermis covering the wound area as sources of CSE expression. Subsequent in vitro studies implicated a role of CSE-derived H2S for keratinocyte differentiation: the H2S-donor GYY4137 markedly increased the Ca2+-triggered expression of the early keratinocyte differentiation markers cytokeratin 10 (CK10) and involucrin (IVN) in cultured human keratinocytes. Here, GYY4137-derived H2S strongly enhanced CK10 expression by increasing the binding of RNA polymerase II to the CK10 promoter.

Varicella zoster virus (VZV) is a skin-tropic virus that infects epidermal keratinocytes and causes chickenpox. Although common, VZV infection can be life-threatening, particularly in the immunocompromized. Therefore, understanding VZV-keratinocyte interactions is important to find new treatments beyond vaccination and antiviral drugs. In VZV-infected skin, kallikrein 6 and the ubiquitin ligase MDM2 are upregulated concomitant with keratin 10 (KRT10) downregulation. MDM2 binds to KRT10, targeting it for degradation via the ubiquitin-proteasome pathway. Preventing KRT10 degradation reduced VZV propagation in culture and prevented epidermal disruption in skin explants. KRT10 knockdown induced expression of NR4A1 and enhanced viral propagation in culture. NR4A1 knockdown prevented viral propagation in culture, reduced LC3 levels, and increased LAMP2 expression. We therefore describe a drug-able pathway whereby MDM2 ubiquitinates and degrades KRT10, increasing NR4A1 expression and allowing VZV replication and propagation.

Conventional approaches can repair minor skin injuries; however, severe burn injuries require innovative approaches for efficient and better wound repair. Recent studies indicate that stem cell-based regenerative therapies can restore severe damaged skin both structurally and functionally. The current study aims to evaluate the wound healing potential of skin substitute derived from human neonatal stem cells (hNSCs) using a severe burn injury rat model. Amniotic epithelial cells (AECs) and mesenchymal stem cells (MSCs) were isolated from placenta (a source of neonatal stem cells) by explant culture method. After characterization, AECs and umbilical cord-MSCs were differentiated into keratinocyte and fibroblasts, respectively. Morphological changes, and expression of corresponding keratinocyte and fibroblast specific markers were used to verify differentiation into respective lineage. A skin substitute was developed by mixing hNSCs-derived skin cells (hNSCs-SCs) in plasma for transplantation in a rat model of severe burn injury. Results indicated that placenta-derived AECs and MSCs were efficiently differentiated into skin cells, that is, keratinocytes and fibroblasts, respectively, as indicated by morphological changes, immunostaining, and polymerase chain reaction analysis. Further, transplantation of hNSCs-SCs seeded in plasma significantly improved basic skin architecture, re-epithelization rate, and wound healing concurrent with reduced apoptosis. In conclusion, neonatal stem cell-derived skin substitute efficiently improved severe burn wounds in a rat model of burn injury. Unique properties of placenta-derived stem cells make them superlative candidates for the development of "off-the-shelf" artificial skin substitutes for future use.

Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions. Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period. Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology.

To explore the genetic cause and clinical features of a neonate with bullous congenital ichthyosiform erythroderma.The patient was examined thoroughly. Following the extraction of genomic DNA, next generation sequencing was performed to analyze the genetic cause.The patient manifested generalized erythema, blistering, and extensive exfoliation of the skin. A heterozygous missence 482TG mutation was found in the first exon of KRT10 gene, which led to a p.L161W alteration in its protein product.The de novo mutation of the KRT10 gene probably underlies the disease in the child.