Differentiation Of Human Keratinocytes Research Articles

GSDMD suppresses keratinocyte differentiation by inhibiting FLG expression and attenuating KCTD6-mediated HDAC1 degradation in atopic dermatitis.

Recent studies have shown that activated pyroptosis in atopic dermatitis (AD) switches inflammatory processes and causes abnormal cornification and epidermal barrier dysfunction. Little research has focused on the interaction mechanism between pyroptosis-related genes and human keratinocyte differentiation. The AD dataset from the Gene Expression Omnibus (GEO) was used to identify differently expressed pyroptosis-related genes (DEPRGs). Hub genes were identified and an enrichment analysis was performed to select epithelial development-related genes. Lesions of AD patients were detected via immunohistochemistry (IHC) to verify the hub gene. Human keratinocytes cell lines, gasdermin D (GSDMD) overexpression, Caspase1 siRNA, Histone Deacetylase1 (HDAC1) siRNA, and HDAC1 overexpression vectors were used for gain-and-loss-of-function experiments. Regulation of cornification protein was determined by qPCR, western blot (WB), immunofluorescence (IF), dual-luciferase reporter assay, co-immunoprecipitation (Co-IP), and chromatin immunoprecipitation (ChIP). A total of 27 DEPRGs were identified between either atopic dermatitis non-lesional skin (ANL) and healthy control (HC) or atopic dermatitis lesional skin (AL) and HC. The enrichment analysis showed that these DEPRGs were primarily enriched in the inflammatory response and keratinocytes differentiation. Of the 10 hub genes identified via the protein-protein interaction network, only GSDMD was statistically and negatively associated with the expression of epithelial tight junction core genes. Furthermore, GSDMD was upregulated in AD lesions and inhibited human keratinocyte differentiation by reducing filaggrin (FLG) expression. Mechanistically, GSDMD activated by Caspase1 reduced FLG expression via HDAC1. HDAC1 decreased FLG expression by reducing histone acetylation at the FLG promoter. In addition, GSDMD blocked the interaction of Potassium Channel Tetramerization Domain Containing 6 (KCTD6) and HDAC1 to prohibit HDAC1 degradation. This study revealed that GSDMD was upregulated in AD lesions and that GSDMD regulated keratinocytes via epigenetic modification, which might provide potential therapeutic targets for AD.

13 UV filters regulate epidermal differentiation and psoriasis-like inflammation in human keratinocytes

13 UV filters regulate epidermal differentiation and psoriasis-like inflammation in human keratinocytes

Protein Kinase CK2 Promotes Proliferation, Abnormal Differentiation, and Proinflammatory Cytokine Production of Keratinocytes via Regulation of STAT3 and Akt Pathways in Psoriasis

Protein kinase CK2 is a constitutively active and ubiquitously expressed serine/threonine kinase that is closely associated with various types of cancers, autoimmune disorders, and inflammation. However, the role of CK2 in psoriasis remains unknown. Herein, the study indicated elevated expression of CK2 in skin lesions from patients with psoriasis and from psoriasis-like mice. In the psoriasis-like mouse model, the CK2-specific inhibitor CX-4945 ameliorated imiquimod-induced psoriasis symptoms with reduced proliferation, abnormal differentiation, inflammatory cytokine production (especially IL-17A) of keratinocytes, and infiltration of γδ T cells. In invitro studies, exogenous CK2 promoted hyperproliferation and abnormal differentiation of human keratinocytes, which were reversed by the suppression of CK2 with CX-4945 or siRNA. Furthermore, knockdown of CK2 reduced IL-17A expression and abolished IL-17A-induced proliferation and inflammatory cytokine expression in keratinocytes. Interestingly, IL-17A increased the expression of CK2 in keratinocytes, thereby establishing a positive feedback loop. In addition, suppression of CK2 inhibited the activation of STAT3 and Akt signaling pathways in human keratinocytes and imiquimod-induced psoriatic lesions of mice. These findings indicate that a highly expressed CK2 level in the skin lesions is required in the development of psoriasis by promoting epidermal hyperplasia, abnormal differentiation, and inflammatory response via regulation of the STAT3 and Akt signaling pathways. CK2 may be a target for the treatment of psoriasis.

724 Loss of pinin induces differentiation of human epidermal keratinocytes

724 Loss of pinin induces differentiation of human epidermal keratinocytes

Effect of Hypertrophic Scar Fibroblast-Derived Exosomes on Keratinocytes of Normal Human Skin

Epidermal keratinocytes are highly activated, hyper-proliferated, and abnormally differentiated in the post-burn hypertrophic scar (HTS); however, the effects of scar fibroblasts (SFs) on keratinocytes through cell-cell interaction in HTS remain unknown. Here, we investigated the effects of HTSF-derived exosomes on the proliferation and differentiation of normal human keratinocytes (NHKs) compared with normal fibroblasts (NFs) and their possible mechanism to provide a reference for clinical intervention of HTS. Fibroblasts were isolated and cultured from HTS and normal skin. Both HTSF-exosomes and NF-exosomes were extracted via a column-based method from the cell culture supernatant. NHKs were treated for 24 or 48 h with 100 μg/mL of cell-derived exosomes. The expression of proliferation markers (Ki-67 and keratin 14), activation markers (keratins 6, 16, and 17), differentiation markers (keratins 1 and 10), apoptosis factors (Bax, Bcl2, caspase 14, and ASK1), proliferation/differentiation regulators (p21 and p27), and epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and vimentin) was investigated. Compared with NF-exosomes, HTSF-exosomes altered the molecular pattern of proliferation, activation, differentiation, and apoptosis, proliferation/differentiation regulators of NHKs, and EMT markers differently. In conclusion, our findings indicate that HTSF-derived exosomes may play a role in the epidermal pathological development of HTS.

Heme Oxygenase-1 Is Upregulated during Differentiation of Keratinocytes but Its Expression Is Dispensable for Cornification of Murine Epidermis.

The epidermal barrier of mammals is initially formed during embryonic development and continuously regenerated by the differentiation and cornification of keratinocytes in postnatal life. Cornification is associated with the breakdown of organelles and other cell components by mechanisms which are only incompletely understood. Here, we investigated whether heme oxygenase 1 (HO-1), which converts heme into biliverdin, ferrous iron and carbon monoxide, is required for normal cornification of epidermal keratinocytes. We show that HO-1 is transcriptionally upregulated during the terminal differentiation of human keratinocytes in vitro and in vivo. Immunohistochemistry demonstrated expression of HO-1 in the granular layer of the epidermis where keratinocytes undergo cornification. Next, we deleted the Hmox1 gene, which encodes HO-1, by crossing Hmox1-floxed and K14-Cre mice. The epidermis and isolated keratinocytes of the resulting Hmox1f/f K14-Cre mice lacked HO-1 expression. The genetic inactivation of HO-1 did not impair the expression of keratinocyte differentiation markers, loricrin and filaggrin. Likewise, the transglutaminase activity and formation of the stratum corneum were not altered in Hmox1f/f K14-Cre mice, suggesting that HO-1 is dispensable for epidermal cornification. The genetically modified mice generated in this study may be useful for future investigations of the potential roles of epidermal HO-1 in iron metabolism and responses to oxidative stress.

203 Constitutive expression of heme oxygenase-1 in differentiated keratinocytes is abolished in a new mouse model for the study of epidermal iron metabolism and redox balance

Cornification of epidermal keratinocytes is associated with the breakdown of organelles and other cell components. The degradation of hemoproteins, such as cytochrome c and cytochrome P450 family members, releases heme, a potent source of oxidative stress. Heme oxygenase 1 (HO-1) converts heme into ferrous iron, carbon monoxide and biliverdin, a strong antioxidant. Here, we show by single cell transcriptomics and immunohistochemistry that HO-1 is upregulated during terminal differentiation of human keratinocytes, leading to the accumulation of HO-1 mRNA and protein in the granular layer of the epidermis.

High Glucose Induces Late Differentiation and Death of Human Oral Keratinocytes.

Keratinocytes are essential cells for wound repair. Impaired oral wound healing is common in diabetic patients with periodontal disease. High glucose, or hyperglycemia, impairs the cellular function of different cell types. However, it is unknown whether high glucose has a detrimental effect on the functions of oral keratinocytes. In the current study, a human gingival keratinocyte cell line, telomerase immortalized gingival keratinocytes (TIGK), was treated with high glucose (24 and 48 mM) for up to 120 h. Proliferation, migration, cell viability, and production of markers of differentiation, growth factors and enzymatic antioxidants were assessed after high glucose treatment. The results showed that high glucose significantly inhibited TIGK proliferation and migration. High glucose also induced significant cell death through apoptosis and necrosis as determined by flow cytometry, especially at 120 h after high glucose treatment. Necrosis was the dominant form of cell death induced. Real-time PCR showed that high glucose treatment upregulated mRNA expression of late keratinocyte differentiation makers, such as keratin 1, 10, 13 and loricrin, and downregulated enzymatic antioxidants, including superoxide dismutase 1, catalase, nuclear factor erythroid 2 -related factor 2, heme oxygenase 1. In conclusion, high glucose impairs the proliferation and migration of oral keratinocytes and likely induces cell death through the promotion of late cell differentiation and down-regulation of enzymatic antioxidants.

Differentiation-related epigenomic changes define clinically distinct keratinocyte cancer subclasses.

Keratinocyte cancers (KC) are the most prevalent malignancies in fair‐skinned populations, posing a significant medical and economic burden to health systems. KC originate in the epidermis and mainly comprise basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). Here, we combined single‐cell multi‐omics, transcriptomics, and methylomics to investigate the epigenomic dynamics during epidermal differentiation. We identified ~3,800 differentially accessible regions between undifferentiated and differentiated keratinocytes, corresponding to regulatory regions associated with key transcription factors. DNA methylation at these regions defined AK/cSCC subtypes with epidermal stem cell‐ or keratinocyte‐like features. Using cell‐type deconvolution tools and integration of bulk and single‐cell methylomes, we demonstrate that these subclasses are consistent with distinct cells‐of‐origin. Further characterization of the phenotypic traits of the subclasses and the study of additional unstratified KC entities uncovered distinct clinical features for the subclasses, linking invasive and metastatic KC cases with undifferentiated cells‐of‐origin. Our study provides a thorough characterization of the epigenomic dynamics underlying human keratinocyte differentiation and uncovers novel links between KC cells‐of‐origin and their prognosis.

Epigenetic remodeling of downstream enhancer regions is linked to selective expression of the IL1F10 gene in differentiated human keratinocytes

Interleukin (IL)-38, encoded by the IL1F10 gene, is a member of the IL-1 family of cytokines. IL-38 is constitutively expressed in epithelia in healthy humans, and in particular in epidermal keratinocytes in the skin. IL-38 expression is closely correlated with keratinocyte differentiation. The aim of this study was to further characterize the regulation of IL1F10 expression and the mechanisms involved in its selective induction in differentiated human keratinocytes. We observed coordinated expression of two IL1F10 transcripts, transcribed from two different promoters, upon differentiation of primary human keratinocytes. Using ENCODE datasets and ChIP-qPCR on ex vivo isolated normal human epidermis, we identified regulatory regions located downstream of the IL1F10 gene, which displayed features of differentiated keratinocyte-specific enhancers. Expression of the IL1F10 gene was linked to changes in the epigenetic landscape at these downstream enhancer regions in human epidermis. Overexpression of the transcription factors KLF4 and TAp63β in an immortalized normal human keratinocyte (iNHK) cell line promoted the expression of mRNA encoding the differentiation markers keratin 10 and involucrin, and of IL1F10. ChIP-qPCR experiments indicated that KLF4 and TAp63β overexpression also modified the chromatin state of the proximal downstream enhancer region, suggesting a role for KLF4 and TAp63β in directly or indirectly regulating IL1F10 transcription.In conclusion, expression of the IL1F10 gene in differentiated keratinocytes in normal human epidermis involves coordinated transcription from two promoters and is linked to epigenetic remodeling of enhancer regions located downstream of the gene.

IL-38 orchestrates proliferation and differentiation in human keratinocytes.

Interleukin (IL)-38 is a member of the IL-1 cytokine family with reported anti-inflammatory activity. The highest constitutive IL-38 expression is detected in the skin, where it is mainly produced by differentiating keratinocytes. However, little data are available regarding its biological functions. In this study, we investigated the role of IL-38 in skin physiology. We demonstrate here that dermal fibroblasts and epithelial cells of skin appendages, such as eccrine sweat glands and sebaceous glands, also express IL-38. Next, using two- and three-dimensional cell cultures, we show that endogenous expression of IL-38 correlates with keratinocyte differentiation and its ectopic overexpression inhibits keratinocyte proliferation and enhances differentiation. Accordingly, immunohistochemical analysis revealed downregulation of IL-38 in skin pathologies characterized by keratinocyte hyperproliferation, such as psoriasis and basal or squamous cell carcinoma. Finally, intracellular IL-38 can shuttle between the nucleus and the cytoplasm and its overexpression modulates the activity of the transcription regulators YAP and ID1. Our results indicate that IL-38 can act independently from immune system activation and suggest that it may affect the epidermis directly by decreasing proliferation and promoting differentiation of keratinocytes. These data suggest an important role of keratinocyte-derived IL-38 in skin homeostasis and pathologies characterized by epidermal alterations.

441 Super-resolution imaging for nuclear pore quantification in human keratinocyte differentiation

Nuclear Pore Complexes (NPCs) serve as essential gateways between the nucleus and cytoplasm. Dysregulation of NPC function is associated with aging and cancer, yet how NPC is implicated in epidermal homeostasis and skin disorders remains largely unexplored. Here, we show that the 74% of genes encoding nucleoporins (NUPs), the building blocks constituting the NPC, are significantly downregulated (p<0.05) in primary human keratinocyte differentiation. NUP93, an essential NUP for determining the protein diffusion size limit across the nuclear pores, is among the most drastically downregulated NUPs.

In vitro characterization of iridoid and phenylethanoid glycosides from Cistanche phelypaea for nutraceutical and pharmacological applications.

"Desert hyacinths" are a remarkable group of parasitic plants belonging to genus Cistanche, including more than 20 accepted species typically occurring in deserts or coastal dunes parasitizing roots of shrubs. Several Cistanche species have long been a source of traditional herbal medicine or food, being C.deserticola and C.tubulosa the most used in China. This manuscript reports the isolation and identification of some phenylethanoid and iridoid glycosides, obtained from the hydroalcoholic extract of C.phelypaea collected in Spain. The present study aims to characterize the antioxidant activity of C.phelypaea metabolites in the light of their application in nutraceutical and cosmeceutical industries and the effect of acetoside, the most abundant metabolite in C.phelypaea extract, on human keratinocyte and pluripotent stem cell proliferation and differentiation. Our study demonstrated that acetoside, besides its strong antioxidant potential, can preserve the proliferative potential of human basal keratinocytes and the stemness of mesenchymal progenitors necessary for tissue morphogenesis and renewal. Therefore, acetoside can be of practical relevance for the clinical application of human stem cell cultures in tissue engineering and regenerative medicine.

Micromotor-Assisted Keratinocytes Migration in a Floating Paper Chip.

In vitro epidermis models are important to evaluate and study disease progression and possible dermal drug delivery. An in vitro epidermis model using floating paper chips as a scaffold for proliferation and differentiation of primary human keratinocytes is reported. The formation of the four main layers of the epidermis (i.e., basal, spinosum, granulose, and cornified layers) is confirmed. The development of a cornified layer and the tight junction formation are evaluated as well as the alterations of organelles during the differentiation process. Further, this in vitro model is used to assess keratinocyte migration. Finally, magnetic micromotors are assembled, and their ability to aid cell migration on paper chips is confirmed when a static magnetic field is present. Taken together, this attempt to combine bottom-up synthetic biology with dermatology offers interesting opportunities for studying skin disease pathologies and evaluate possible treatments.

Sphingosine 1-Phosphate Signaling at the Skin Barrier Interface.

Simple SummarySphingosine 1-phosphate is a bioactive phospholipid derived from cell membranes. It is biosynthesized and metabolized within the cell and released extracellularly to trigger various cellular responses via sphingosine 1-phosphate receptors. Keratinocyte and mast cell functions are responsible for skin barrier acquired and innate immunity, and express sphingosine 1-phosphate receptors. These receptors regulate not only cell differentiation and proliferation, but also immune responses such as cell migration and cytokine secretion, through G-protein-mediated signaling pathways. Sphingosine 1-phosphate and its receptor signaling pathways are also involved in several skin diseases along with various receptor agonists and are being investigated as potential therapeutic agents. Here, we focus on the association of sphingosine 1-phosphate and its receptors within the skin barrier interface.Sphingosine 1-phosphate (S1P) is a product of membrane sphingolipid metabolism. S1P is secreted and acts via G-protein-coupled receptors, S1PR1-5, and is involved in diverse cellular functions, including cell proliferation, immune suppression, and cardiovascular functions. Recent studies have shown that the effects of S1P signaling are extended further by coupling the different S1P receptors and their respective downstream signaling pathways. Our group has recently reported that S1P inhibits cell proliferation and induces differentiation in human keratinocytes. There is a growing understanding of the connection between S1P signaling, skin barrier function, and skin diseases. For example, the activation of S1PR1 and S1PR2 during bacterial invasion regulates the synthesis of inflammatory cytokines in human keratinocytes. Moreover, S1P-S1PR2 signaling is involved in the production of inflammatory cytokines and can be triggered by epidermal mechanical stress and bacterial invasion. This review highlights how S1P affects human keratinocyte proliferation, differentiation, immunoreaction, and mast cell immune response, in addition to its effects on the skin barrier interface. Finally, studies targeting S1P-S1PR signaling involved in inflammatory skin diseases are also presented.

Role of CD99 in regulating homeostasis and differentiation in normal human epidermal keratinocytes

CD99 is a glycoprotein primarily expressed in immune cells. Physiologically, it is involved in the adhesion, migration, and development of immune cells. The presence of CD99 in the skin was first reported in 2016 and its function is yet to be determined. In this study, we aimed to understand the role of CD99 in the skin using normal human epidermal keratinocytes (NHEK). CD99 expression increased with the confluency of NHEK, while the CD99-high expressing NHEK lost their stem cell properties and played a role in barrier function. We characterized CD99-expressing NHEK as cells committed to early differentiation because they expressed early differentiation markers. However, the deficiency of CD99 in NHEK disrupted homeostasis and caused aberrant differentiation, as evidenced by larger cells with lesser Ki67 staining and higher expression of terminal differentiation markers. Hence, we propose that CD99 is involved in maintaining homeostasis and initiating early differentiation in the skin.

Retinoic acid induces hyaluronic acid production through the klotho-mediated EGFR signaling pathway in human epidermal keratinocytes

All-trans retinoic acid (RA) is an effective anti-aging chemical substance widely used in skin-care products. RA compromises epidermal differentiation and induces keratinocyte proliferation, causing hyaluronic acid production through mechanisms that are not completely understood. Klotho protein causes the differentiation of human epidermal keratinocytes. Klotho gene expression is mediated by epidermal growth factor (EGF), which inhibits cell apoptosis in aging-related diseases. The klotho gene causes human aging syndrome, including short lifespan, skin atrophy, and osteoporosis. We investigated the relationship between RA and klotho in epidermal keratinocytes for the first time. In human epidermal keratinocytes, RA induced klotho gene expression. Treatment with both RA and recombinant klotho induced hyaluronic acid production in human epidermal keratinocytes. However, in klotho small interfering RNA (siRNA)-transfected keratinocytes, RA produced less hyaluronic acid than in the control group, indicating that RA may partially regulate hyaluronic acid production through a klotho-dependent pathway. Knockdown of klotho gene expression inactivated the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway, which is involved in hyaluronic acid production. We concluded that the effect of RA on hyaluronic acid production is partly regulated through the klotho-mediated EGFR signaling pathway in human epidermal keratinocytes.

Steroid sulfatase deficiency causes cellular senescence and abnormal differentiation by inducing Yippee-like 3 expression in human keratinocytes

Human steroid sulfatase (STS) is an enzyme that catalyzes the hydrolysis of dehydroepiandrosterone sulfate (DHEAS), estrone sulfate (E1S), and cholesterol sulfate. Abnormal expression of STS causes several diseases including colorectal, breast, and prostate cancer and refractory skin disease. In particular, accumulation of intracellular cholesterol sulfate by STS deficiency leads to a skin disorder with abnormal keratinization called X-linked ichthyosis (XLI). To determine the detailed mechanisms of XLI, we performed RNA sequencing (RNA-seq) analysis using human keratinocyte HaCaT cells treated with cholesterol and cholesterol sulfate. Of the genes with expression changes greater than 1.5-fold, Yippee-like 3 (YPEL3), a factor expected to affect cell differentiation, was found. Induction of YPEL3 causes permanent growth arrest, cellular senescence, and inhibition of metastasis in normal and tumor cells. In this study, we demonstrate that YPEL3 expression was induced by STS deficiency and, using the CRISPR/Cas9 system, a partial knock-out (STS+/−) cell line was constructed to establish a disease model for XLI studies. Furthermore, we show that increased expression of YPEL3 in STS-deficient cell lines promoted cellular senescence and expression of keratinization-related proteins such as involucrin and loricrin. Our results suggest that upregulation of YPEL3 expression by STS deficiency may play a crucial role in inducing cellular senescence and abnormal differentiation in human keratinocytes.

107 Single-cell transcriptomics of avian skin reveals evolutionarily ancient keratinocyte differentiation genes

The genetic program of epidermal keratinocyte differentiation has evolved by adaptation to a dry terrestrial environment. The dynamics of epidermal gene expression is largely conserved between human and mouse, the standard model of biomedical research. However, only little is known about keratinocyte differentiation in non-mammalian vertebrates. Here we performed a single-cell transcriptome study of keratinocyte differentiation in avian skin. Keratinocytes and other skin cells from the leg skin of 3 chicks were analyzed by single-cell RNA-sequencing. We identified two distinct populations of differentiated keratinocytes corresponding to hard scales and soft inter- scale epidermis. The scale keratinocytes contained cysteine-rich keratins, one of which could be immunolocalized in the tissue using a newly raised antibody. By contrast, interscale keratinocytes contained KRT9 and KRT78-like keratins with low cysteine contents. The transcriptome of the latter type of keratinocytes showed many similarities to the transcriptome of keratinocytes in the granular layer of human epidermis, including expression of established differentiation markers, such as loricrin and claudin 1, and as yet incompletely characterized genes, such as POF1B and HOPX. Furthermore, genes involved in lipid metabolism and transport, such as ELOVL4, ABCA12, FABP5 and GLTP were upregulated during both chicken and human keratinocyte differentiation. In conclusion, comparative analysis of chicken and human skin at the single-cell level identifies regulators of epidermal differentiation that have evolved prior to divergence of the phylogenetic lineages leading to birds and mammals.

Nano- and Micro-Porous Chitosan Membranes for Human Epidermal Stratification and Differentiation.

The creation of partial or complete human epidermis represents a critical aspect and the major challenge of skin tissue engineering. This work was aimed at investigating the effect of nano- and micro-structured CHT membranes on human keratinocyte stratification and differentiation. To this end, nanoporous and microporous membranes of chitosan (CHT) were prepared by phase inversion technique tailoring the operational parameters in order to obtain nano- and micro-structured flat membranes with specific surface properties. Microporous structures with different mean pore diameters were created by adding and dissolving, in the polymeric solution, polyethylene glycol (PEG Mw 10,000 Da) as porogen, with a different CHT/PEG ratio. The developed membranes were characterized and assessed for epidermal construction by culturing human keratinocytes on them for up to 21 days. The overall results demonstrated that the membrane surface properties strongly affect the stratification and terminal differentiation of human keratinocytes. In particular, human keratinocytes adhered on nanoporous CHT membranes, developing the structure of the corneum epidermal top layer, characterized by low thickness and low cell proliferation. On the microporous CHT membrane, keratinocytes formed an epidermal basal lamina, with high proliferating cells that stratified and differentiated over time, migrating along the z axis and forming a multilayered epidermis. This strategy represents an attractive tissue engineering approach for the creation of specific human epidermal strata for testing the effects and toxicity of drugs, cosmetics and pollutants.