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Psoriasis is a kind of severe immune-mediated systemic skin disorder, becoming a worldwide public health concern. Daturataturin A (DTA), a withanolide compound, exerts excellent anti-inflammatory and anti-proliferative properties. The objective of this study is to elucidate the effect of DTA on psoriasis and its potential mechanism. We established psoriasis-like keratinocytes model by stimulating HaCaT cells with M5 cocktail cytokines including Interleukin (IL)-17A, IL-22, oncostatin M, IL-1α, and tumor necrosis factor-α (TNF-α), followed by intervention with DTA. The potential effects and mechanisms of DTA on psoriasis were evaluated in vitro. DTA was found to be able to inhibit hyperproliferation, promote apoptosis, decrease the release of pro-inflammatory cytokines, downregulate keratin expression, and improve lipid metabolism via regulating the peroxisome proliferator-activated receptor (PPAR) signaling pathway by M5 cocktail cytokines stimulation in HaCaT cells. DTA ameliorated lipid metabolism of psoriasis and exerted the potential anti-psoriasis effects by regulating PPAR pathway in vitro, suggesting that DTA may act as a new therapeutic agent for psoriasis. [ABSTRACT FROM AUTHOR]

Senescent cells are typically characterized by a stable proliferation arrested in dividing cells accompanied with a senescence‐associated secretory phenotype (SASP). Skin cellular senescence is the primary cause of skin aging, whereas the lack of identified skin senescence markers limits our understanding of the mechanisms involved in skin aging. Recent studies have revealed that intracellular calcium signaling has emerged as a key player in regulating cellular senescence and aging. However, the implication and roles of calcium signaling in skin keratinocyte senescence remain only partially understood. In this study, we developed a model for skin keratinocyte senescence using ionizing radiation (I/R) stimulation and found that the calcium‐associated gene transglutaminase 2 (TGM2) was significantly induced compared with normal control. Interestingly, inhibition of TGM2 was found to delay skin keratinocyte senescence by suppressing I/R‐promoted intracellular calcium signaling, accumulation of reactive oxygen species (ROS), DNA damage, as well as NF‐κB‐mediated SASP secretion. Taken together, our findings demonstrate that inhibition of TGM2 contributes to bypassing I/R‐induced skin keratinocyte senescence and sheds light on novel strategies against skin stresses caused by I/R. [ABSTRACT FROM AUTHOR]

• Integrins are transmembrane receptors for extracellular matrix adhesion. • Keratinocyte integrins regulate factors such as matrix proteins, proteases, growth factors, and cytokines that are secreted into the microenvironment (i.e., the secretome). • Integrin α3β1 is emerging as a regulator of the secretome in keratinocytes. • Integrin-regulated secreted factors mediate crosstalk to the microenvironment by: (1) promoting matrix generation through the regulation of ECM/BM proteins, (2) promoting matrix remodeling through the regulation of proteases, or (3) supporting stromal cells through the regulation of cytokines and/or growth factors. • Secreted factors regulated by certain integrins can promote paracrine crosstalk to distinct cells such as immune cells, endothelial cells, and fibroblasts to control both pathological (i.e., tumor growth and progression) and repair (i.e., wound healing) processes. Integrins are cellular transmembrane receptors that physically connect the cytoskeleton with the extracellular matrix. As such, they are positioned to mediate cellular responses to microenvironmental cues. Importantly, integrins also regulate their own microenvironment through secreted factors, also known as the integrin-mediated secretome. Epidermal integrins, or integrins expressed by keratinocytes of the skin epidermis, regulate the cutaneous microenvironment through the contribution of matrix components, via proteolytic matrix remodeling, or by mediating factors like cytokines and growth factors that can promote support for nearby but distinct cells of the stroma, such as immune cells, endothelial cells, and fibroblasts. This role for integrins is enhanced during both pathological and repair tissue remodeling processes, such as tumor growth and progression and wound healing. This review will discuss examples of how the epithelial integrin-mediated secretome can regulate the tissue microenvironment. Although different epithelial integrins in various contexts will be explored, emphasis will be given to epidermal integrins that regulate the secretome during wound healing and cutaneous tumor progression. Epidermal integrin α3β1 is of particular focus as well, since this integrin has been revealed as a key regulator of the keratinocyte secretome. [ABSTRACT FROM AUTHOR]

Objective: Human skin is the first line of defence from environmental factors such as solar radiation and is susceptible to premature ageing, including a disruption in epidermal differentiation and homeostasis. We evaluated the impact of a Galactomyces Ferment Filtrate (GFF) on epidermal differentiation and response to oxidative stress. Methods: We used transcriptomics, both spatial and traditional, to assess the impact of GFF on epidermal biology and homeostasis in keratinocytes (primary or immortalized) and in ex vivo skin explant tissue. The effect of GFF on cell adhesion rates, cellular ATP levels and proliferation rates were quantitated. Oxidative phosphorylation and glycolytic rates were measured under normal and stress‐induced conditions. Results: Transcriptomics from keratinocytes and ex vivo skin explants from multiple donors show GFF induces keratinocyte differentiation, skin barrier development and cell adhesion while simultaneously repressing cellular stress and inflammatory related processes. Spatial transcriptomics profiling of ex vivo skin indicated basal keratinocytes at the epidermal‐dermal junction and cornifying keratinocytes in the top layer of the epidermis as the primary cell types influenced by GFF treatment. Additionally, GFF significantly increases crosstalk between suprabasal and basal keratinocytes. To support these findings, we show that GFF can significantly increase cell adhesion and proliferation in keratinocytes. GFF also protected overall cellular bioenergetics under metabolic or oxidative stress conditions. Conclusion: Our findings provide novel insights into cellular differences and epidermal spatial localization in response to GFF, supporting previous findings that this filtrate has a significant impact on epidermal biology and homeostasis, particularly on spatially defined crosstalk. We propose that GFF can help maintain epidermal health by enhancing keratinocyte crosstalk and differentiation/proliferation balance as well as promoting an enhanced response to stress. [ABSTRACT FROM AUTHOR]

Background: Psoriatic patients tend to develop metabolic syndrome (MS). MS accelerates psoriasis, but the exact molecular mechanisms are poorly understood. Objectives: We aim to investigate the impact of leptin on keratinocyte insulin sensitivity and explore its underlying molecular mechanism, which might play a role in the pathogenesis of this disease. Methods: ELISA and immunohistochemistry were applied respectively to detect the level of leptin in serum and in lesion of psoriatic patients with and without MS. The HaCaT cell line was cultured and western-blot assay was performed to assess the change of insulin sensibility. q-PCR and western-blot assay were applied to detect the SOCS3 expressions. Knockdown of SOCS3 were generated in HaCaT cell line by siRNA. Leptin and insulin were treated for 6days and K10 expression was evaluated by western-blot assay. Results: Patients with MS had higher level of leptin in serum and lesions than their counterparts without MS. Serum levels of leptin was negatively correlated to PASI decline index in psoriatic patients. Long-term treatment of leptin induced insulin resistance in HaCaT cell line, as indicated by elevated expression of p-IRS-1 (ser636) and lower p-PKB (ser473). Leptin treatment up-regulated the mRNA and protein expression of SOCS3. Knockdown of SOCS3 blocked the effect of leptin-induced insulin resistance. Leptin treatment attenuated insulin-elicited K10 expression. Conclusions: Leptin induces insulin resistance by upregulating SOCS3 and give rise to differentiation disorder of keratinocyte. Insulin resistance may serve as a target for anti-psoriatic therapies. [ABSTRACT FROM AUTHOR]

Background: The aim of This study is to investigate the effects of Keratinocyte differentiation factor 1 (KDF1) on cervical cancer cells and the underlying mechanisms. Methods: The Gene Expression Profiling Interactive Analysis database was used to analyse KDF1 expression in cervical cancer and paracancerous tissue samples. The correlation between the expression of KDF1 and clinicopathological features was also analysed. Cervical cancer cells (HeLa cells) with KDF1 overexpression or knockdown were constructed. Reverse transcription polymerase chain reaction was used to detect the mRNA expression of KDF1 in cervical cancer tissues and cells. In different treatment groups of cervical cancer cells, protein expression of KDF1, cell viability, invasion, and migration were subsequently confirmed by western blotting, CCK-8 assay, transwell assay, and wound healing assay, respectively. A PI3K inhibitor (LY294002) was used to detect the effect of KDF1 on the phosphoinositide 3-kinase (PI3K)/Protein Kinase B (AKT) pathway. Results: KDF1 was highly expressed in cervical cancer tissues and cell lines (p < 0.01), and was significantly associated with poor prognosis (p< 0.05). Knockdown of KDF1 in HeLa cells resulted in a significant decrease in cell proliferation, migration, and invasion, as well as phosphorylated PI3K (P-PI3K) and p-AKT levels (p< 0.01). However, KDF1 overexpression activated the PI3K/AKT pathway and significantly enhanced the malignant biological behaviour of cervical cancer cells (p< 0.01). Additionally, the PI3K inhibitor reduced the proliferation, invasion, and migration of HeLa cells overexpressing KDF1 (p < 0.01). Conclusion: KDF1 enhances cervical cancer viability and migration by activating the PI3K/AKT pathway, and may serve as a therapeutic target for patients with cervical cancer. [ABSTRACT FROM AUTHOR]

Here, we investigated the potential interaction between bromodomain-containing protein 4 (BRD4), an established epigenetic modulator and transcriptional coactivator, and p63, a member of the p53 transcription factor family, essential for epithelial development and skin homeostasis. Our protein–protein interaction assays demonstrated a strong and conserved physical interaction between BRD4 and the p53 family members—p63, p73, and p53—suggesting a shared binding region among these proteins. While the role of BRD4 in cancer development through its interaction with p53 has been explored, the effects of BRD4 and Bromodomain and Extra Terminal (BET) inhibitors in non-transformed cells, such as keratinocytes, remain largely unknown. Our functional analyses revealed changes in cellular proliferation and differentiation in keratinocytes depleted of either p63 or BRD4, which were further supported by using the BRD4 inhibitor JQ1. Transcriptomic analyses, chromatin immunoprecipitation, and RT-qPCR indicated a synergistic mechanism between p63 and BRD4 in regulating the transcription of keratinocyte-specific p63 target genes, including HK2, FOXM1, and EVPL. This study not only highlights the complex relationship between BRD4 and p53 family members but also suggests a role for BRD4 in maintaining keratinocyte functions. Our findings pave the way for further exploration of potential therapeutic applications of BRD4 inhibitors in treating skin disorders. [ABSTRACT FROM AUTHOR]

Senescent cells secrete a senescence‐associated secretory phenotype (SASP), which can induce senescence in neighboring cells. Human dermal papilla (DP) cells lose their original hair inductive properties when expanded in vitro, and rapidly accumulate senescent cells in culture. Protein and RNA‐seq analysis revealed an accumulation of DP‐specific SASP factors including IL‐6, IL‐8, MCP‐1, and TIMP‐2. We found that combined senolytic treatment of dasatinib and quercetin depleted senescent cells, and reversed SASP accumulation and SASP‐mediated repressive interactions in human DP culture, resulting in an increased Wnt‐active cell population. In hair reconstitution assays, senolytic‐depleted DP cells exhibited restored hair inductive properties by regenerating de novo hair follicles (HFs) compared to untreated DP cells. In 3D skin constructs, senolytic‐depleted DP cells enhanced inductive potential and hair lineage specific differentiation of keratinocytes. These data revealed that senolytic treatment of cultured human DP cells markedly increased their inductive potency in HF regeneration, providing a new rationale for clinical applications of senolytic treatment in combination with cell‐based therapies. [ABSTRACT FROM AUTHOR]

Key points Pannexin (PANX) channels are present in skin and facilitate the movement of signalling molecules during cellular communication. PANX1 and PANX3 function in skin homeostasis and keratinocyte differentiation but were previously reduced in a small cohort of human cutaneous squamous cell carcinoma (cSCC) tumours compared to normal epidermis. In our study we used SCC‐13 cells, limited publicly available RNA‐seq data and a larger cohort of cSCC patient‐matched samples to analyse PANX1 and PANX3 expression and determine the association between their dysregulation and the malignant properties of cSCC. In a bioinformatics analysis, PANX1 transcripts were increased in cSCC and head and neck SCC tumours compared to normal tissues, but PANX3 mRNA showed no differences. However, in our own cohort PANX3 transcripts were decreased in cSCC compared to patient‐matched aged skin, whereas PANX1 protein was upregulated in cSCC. PANX1 localized to all regions within the cSCC tumour microenvironment, and increased levels were associated with larger tumour dimensions. To investigate PANX1 function in SCC‐13 cells, we deleted PANX1 via CRISPR/Cas9 and treated with PANX1 inhibitors, which markedly reduced cell growth and migration. To assess PANX3 function in cutaneous carcinogenesis, we employed the 7,12‐dimethylbenz(a)anthracene/12‐otetradecanoylphorbol‐13‐acetate (DMBA/TPA) model using our global Panx3 knockout (KO) mice, where 60% of wild‐type and 100% of KO mice formed precancerous papillomas. Average papilloma volumes at endpoint were significantly increased in KO mice and showed moderate evidence of increases in KO mice over time. Collectively, these findings suggest PANX1 and PANX3 dysregulation may have potential tumour‐promoting and tumour‐suppressive effects for keratinocyte transformation, respectively. Pannexin 1 (PANX1) and pannexin 3 (PANX3) are channel‐forming proteins which are critical in the normal maintenance and function of keratinocytes in the skin but may become altered in cutaneous squamous cell carcinoma (cSCC) tumours. In this study we used a combination of culture models, mouse models and patient‐derived tissues. We found PANX1 levels are increased in cSCC tumours and present in all tumour regions, functioning to promote cSCC cell growth and migration. Conversely, PANX3 levels are decreased in cSCC tumours, and this protein reduces the incidence and growth of precancerous lesions. Taken together our data indicate that in cSCC these pannexin family members seem to have opposite effects, in either promoting or restricting cancer cell properties. These results help us to better understand the mechanisms of malignant transformation of keratinocytes and offer a new potential therapeutic target for the treatment of advanced cSCC. [ABSTRACT FROM AUTHOR]

Background: Tissue engineering techniques offer new strategies to understand complex processes in a controlled and reproducible system. In this study, we generated bilayered human tissue substitutes consisting of a cellular connective tissue with a suprajacent epithelium (full-thickness stromal-epithelial substitutes or SESS) and human tissue substitutes with an epithelial layer generated on top of an acellular biomaterial (epithelial substitutes or ESS). Both types of artificial tissues were studied at sequential time periods to analyze the maturation process of the extracellular matrix. Results: Regarding epithelial layer, ESS cells showed active proliferation, positive expression of cytokeratin 5, and low expression of differentiation markers, whereas SESS epithelium showed higher differentiation levels, with a progressive positive expression of cytokeratin 10 and claudin. Stromal cells in SESS tended to accumulate and actively synthetize extracellular matrix components such as collagens and proteoglycans in the stromal area in direct contact with the epithelium (zone 1), whereas these components were very scarce in ESS. Regarding the basement membrane, ESS showed a partially differentiated structure containing fibronectin-1 and perlecan. However, SESS showed higher basement membrane differentiation, with positive expression of fibronectin 1, perlecan, nidogen 1, chondroitin-6-sulfate proteoglycans, agrin, and collagens types IV and VII, although this structure was negative for lumican. Finally, both ESS and SESS proved to be useful tools for studying metabolic pathway regulation, revealing differential activation and upregulation of the transforming growth factor-β pathway in ESS and SESS. Conclusions: These results confirm the relevance of epithelial-stromal interaction for extracellular matrix development and differentiation, especially regarding basement membrane components, and suggest the usefulness of bilayered artificial tissue substitutes to reproduce ex vivo the extracellular matrix maturation and development process of human tissues. Highlights: 1- Artificial bilayered substitutes developed by tissue engineering allow the spatiotemporal study of extracellular matrix maturation and development. 2- Epithelial tissue substitutes generated on the surface of acellular biomaterials display limited differentiation levels, with limited expression of relevant extracellular matrix components. 3- Full-thickness stromal-epithelial substitutes with a cellular stroma showed significantly higher levels of maturation and differentiation, with higher expression of epithelial (cytokeratin 10, claudin) and stromal markers (collagens, proteoglycans). 4- For the formation and development of the basement membrane, an interaction process between the epithelial and the stromal cells is necessary for the synthesis of most fibrillar and non-fibrillar components of this structure. 5- Our results confirm the intrinsic capability of epithelial cells to synthesize extracellular matrix components and the positive inductive effect of paracrine factors released by stromal cells. [ABSTRACT FROM AUTHOR]

The epidermis, the outer layer of the skin, relies on a delicate balance of cell growth and keratinocyte differentiation for its function and renewal. Recent research has shed light on exosomes' role in facilitating skin communication by transferring molecules like miRNAs, which regulate gene expression post-transcriptionally. Additionally, these factors lead to skin aging through oxidative stress caused by reactive oxygen species (ROS). In this research project, experiments were conducted to study the impact of Sun2000 solar simulator irradiation on exosomal miRNA profiles in HEKa cells. We hypothesized that acute oxidative stress induced by solar simulator irradiation would alter the expression profile of exosomal miRNAs in HEKa cells. The cells were exposed to different durations of irradiation to induce oxidative stress, and the levels of reactive ROS were measured using the CellROX Deep Red flow cytometry assay kit. Exosomes were isolated from both control and irradiated cells, characterized using DLS and SEM techniques, and their miRNAs were extracted and analyzed using qPCR. Solar simulator irradiation led to a time-dependent increase in intracellular ROS and a decrease in cell viability. Exosomal size increased in irradiated cells. Fifty-nine exosomal miRNAs were differentially expressed in irradiated HEKa cells, including hsa-miR-425-5p, hsa-miR-181b-5p, hsa-miR-196b-5p, hsa-miR-376c-3p, and hsa-miR-15a-5p. This study highlights the significant impact of solar radiation on exosomal miRNA expression in keratinocytes, suggesting their potential role in the cellular response to oxidative stress. [ABSTRACT FROM AUTHOR]

For several decades, there has been growing interest in the influence of low-frequency magnetic fields (LFMFs) and red LED light on the healing process. Keratinocytes are cells that play a significant role in the process of wound healing and tissue regeneration. A human keratinocyte cell line (HaCaT) was exposed to an LFMF with low induction (180–195 Hz; 60 µT, magnetostimulation), red LED light (630 nm; 300 mW, LED therapy), and their combined action (magneto-LED therapy) in in vitro culture conditions. On day 4 and 8 of the experiment, the following parameters were determined: adhesion/proliferation, adenylate kinase (AK), nitric oxide (NO), cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF-α), metalloproteinases (MMP-2, MMP-9), and collagen IV. It was shown that magnetostimulation caused an increase in keratinocyte adhesion/proliferation and IL-8 secretion and a decrease in IL-12 secretion. The LED therapy resulted in a transient increase in the secretion of NO and cytokines IL-1, IL-12, and IL-6 in keratinocytes. The use of magneto-LED therapy resulted in an increase in keratinocyte adhesion/proliferation, the secretion of cytokines IL-6 and IL-8, and NO with a simultaneous decrease in MMP-9 secretion. The results of our studies showed that the action of an LFMF with low-induction and LED light on keratinocytes can modulate the biological activity of keratinocytes towards improving the skin healing process. [ABSTRACT FROM AUTHOR]

Anal Squamous Cell Carcinoma (SCCA) is a rare Human Papillomavirus type 16 (HPV16)-associated carcinoma whose pathogenesis is still poorly understood. Recent studies based on biopsy and Next Generation Sequencing (NGS) approaches have linked the viral episomal status to aggressive SCCA phenotypes, suggesting a potential role of the 16E5 oncoprotein in tumor development. Our previous findings indicated that 16E5 induces Fibroblast Growth Factor Receptor 2 (FGFR2) isoform switching, aberrant mesenchymal FGFR2c expression, Epithelial Mesenchymal Transition (EMT), and cell invasion in various in vitro human keratinocyte models, as well as in the in vivo context of cervical Low-grade Squamous Intraepithelial Lesions (LSILs). To further explore the role of 16E5 in epithelial carcinogenesis, this study aims to investigate the molecular profile in HPV-related anal lesions. The results showed a significant positive correlation between 16E5 and FGFR2c, as well as 16E5 or FGFR2c and key EMT-related transcription factors, particularly in the group of HPV16 positive anal samples not containing without high grade lesions. Additionally, by coupling the molecular analysis with an interactome investigation, we hypothesized a potential functional interplay between the Ca2+ channel Transient Receptor Potential Ankyrin 1 (TRPA1) and FGFR2c, mediated by 16E5 during the establishment of the oncogenic signaling. These findings will help to elucidate the actual relevance of 16E5 in the early progression of anal lesions and contribute to determine its potential as target for future preventive approaches for HPV16-positive SCCA. [ABSTRACT FROM AUTHOR]

Background: The physiological relevance of cell-to-cell communication mediated by small extracellular vesicle-encapsulated microRNAs (sEV-miRNAs) remains debated because of the limiting representativity of specific miRNAs within the extracellular pool. We hypothesize that sEV-miRNA non-canonical function consisting of the stimulation of Toll-like receptor 7 (TLR7) may rely on a global shift of the sEV cargo rather than on the induction of one or few specific miRNAs. Psoriasis represents an ideal model to test such hypothesis as it is driven by overt activation of TLR7-expressing plasmacytoid dendritic cells (pDCs) following keratinocyte damage. Methods: To mimic the onset of psoriasis, keratinocytes were treated with a cocktail of psoriatic cytokines or UV-irradiated. SmallRNA sequencing was performed on sEVs released by healthy and UV-treated keratinocytes. sEV-miRNAs were analyzed for nucleotide composition as well as for the presence of putative TLR7-binding triplets. Primary human pDCs where stimulated with sEVs +/- inhibitors of TLR7 (Enpatoran), of sEV release (GW4869 + manumycin) and of TLR7-mediated pDC activation (anti-BDCA-2 antibody). Secretion of type I IFNs and activation of CD8+T cells were used as readouts. qPCR on psoriatic and healthy skin biopsies was conducted to identify induced miRNAs. Results: sEV-miRNAs released by damaged keratinocytes revealed a significantly higher content of TLR7-activating sequences than healthy cells. As expected, differential expression analysis confirmed the presence of miRNAs upregulated in psoriatic skin, including miR203a. More importantly, 76.5% of induced miRNAs possessed TLR7-binding features and among these we could detect several previously demonstrated TLR7 ligands. In accordance with this in silico analysis, sEVs from damaged keratinocytes recapitulated key events of psoriatic pathogenesis by triggering pDCs to release type I interferon and activate cytotoxic CD8+T cells in a TLR7- and sEV-dependent manner. Discussion: Our results demonstrate that miR203a is just one paradigmatic TLR7-activating miRNA among the hundreds released by UV-irradiated keratinocytes, which altogether trigger pDC activation in psoriatic conditions. This represents the first evidence that cell damage shifts the miRNA content of sEVs towards a TLR7-activating cargo capable to propagate inflammation and immunity, offering strong support to the physiological role of systemic miRNA-based cell-to-cell communication. [ABSTRACT FROM AUTHOR]

The demand for skin models as alternatives to animal testing has grown due to ethical concerns and the need for accurate substance evaluation. These alternatives, known as New Approach Methodologies (NAMs), are increasingly used for regulatory decisions. Current skin models from primary human cells often rely on bovine collagen, raising ethical issues. This study explores self-assembled skin models (SASM) as a new method, utilizing hair follicle-derived keratinocytes reprogrammed into induced pluripotent stem cells (iPSC) and differentiated into fibroblasts and keratinocytes. The model relies on the ability of fibroblasts to secrete collagen to produce a xeno-free dermal layer and on the differentiation of keratinocytes to create a functional epidermal layer. These layers exhibited confirmed metabolic activity and the capability to withstand test substances. The successful development of SASM underscores the significance of accurate alternatives in dermatological research, providing an ethical and reliable option for substance evaluation and regulatory testing. [ABSTRACT FROM AUTHOR]

Z-DNA-binding protein 1 (ZBP1) is an interferon-inducible sensor of Z-DNA and Z-RNA, which has emerged as a critical regulator of cell death and inflammation. ZBP1 binds Z-DNA and Z-RNA via its Zα domains, and signals by engaging RIPK3 and RIPK1 via its RIP homotypic interaction motifs (RHIMs). Here, we show that mice express an alternatively-spliced shorter ZBP1 isoform (ZBP1-S), which harbours the Zα domains but lacks the RHIMs, and acts as an endogenous inhibitor of the full-length protein (ZBP1-L). Mice and cells expressing only ZBP1-S are resistant to ZBP1-mediated cell death and inflammation. In contrast, cells lacking ZBP1-S show increased ZBP1-L-induced death compared to cells expressing both isoforms. Moreover, loss of the short isoform accelerates and exacerbates skin inflammation induced by ZBP1-mediated necroptosis of RIPK1-deficient keratinocytes, revealing an important physiological role of ZBP1-S. Mechanistically, ZBP1-S suppresses ZBP1-L-mediated cell death by binding to Z-nucleic acids via its Zα domains. Therefore, ZBP1-S acts as an endogenous inhibitor that competes with full-length ZBP1-L for binding Z-nucleic acid ligands to fine-tune ZBP1-mediated cell death and inflammation. Synopsis: Mice express full-length protein ZBP1-L as well as an alternatively-spliced shorter isoform (ZBP1-S) containing only its two Zα domains but lacking the three RHIM motifs. This study shows that ZBP1-S acts as an endogenous inhibitor that suppresses activation of ZBP1-L-mediated necroptosis and inflammation by endogenous Z-RNA ligands. Both ZBP1-S and ZBP1-L are induced by interferons and interact with endogenous Z-RNAs via their Zα domains. ZBP1-S suppresses ZBP1-L activation in a Zα domain-dependent manner. Loss of ZBP1-S results in increased ZBP1-L-mediated cell death. Specific ablation of ZBP1-S causes exaggerated ZBP1-L-induced necroptosis and inflammation in mice lacking RIPK1 in keratinocytes. Interferons induce a ZBP1-S isoform that competitively interacts with endogenous Z-RNAs and thereby suppresses ZBP1-induced necroptosis and inflammation. [ABSTRACT FROM AUTHOR]

Background: Streptococcus canis is a commensal bacterium in companion animals. This microorganism can infect humans who have been in deep contact with or bitten by pet dogs, suggesting that the skin/soft tissue is one of infection entry sites. To understand pathological process in human cells, we aimed to determine S. canis transcriptomic changes in invasive environments of human keratinocytes. Methods: We selected one isolate from candidates with whole-genome sequences, based on re-obtained cell invasion ability (CIA) data into human keratinocytes along with bacterial cytotoxicity. RNA-sequencing was conducted for the samples at baselines and 2 h/5 hr post-inoculation using NovaSeq 6000. Global/differential gene expression analyses [principal component analysis (PCA)/k-means clustering analysis/differentially expressed gene (DEG) analyses] were performed. We classified DEGs into their functional categories. To validate transcriptomic results, we did quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Results: FU1 isolate was selected from seven candidates, based on re-obtained CIA data with less cytotoxicity. Total read bases of 6.17–9.02 Gbp were obtained by RNA-sequencing. PCA and k-means clustering analysis indicated clustering according to their inoculation times. Volcano plots and Venn diagrams revealed that S. canis invasion into keratinocytes produced altered distributions of many genes. Gene ontology enrichment analysis showed most of the gene expressions were downregulated. DEG functional analysis showed the downregulated DEGs belonging to energy production and conversion/carbohydrate transport and metabolism/amino acid transport and metabolism/nucleotide transport and metabolism, with the upregulated DEGs belonging to transcription. qRT-PCR assays for downregulated/upregulated expressions of four genes (pgk–slo/opuAA–kdpB) validated transcriptomic results. Conclusion: Our observations suggest that S. canis can downregulate its metabolism-associated gene expressions in human keratinocyte environments. The observed gene expression changes can imply the latent infection in human cells. Further investigation is needed to elucidate the underlying mechanisms for the latent infection. [ABSTRACT FROM AUTHOR]

Incontinence‐associated dermatitis (IAD) is one of the most serious complications in older people with incontinence. Controlling urine property in absorbent pads could be effective for preventing IAD caused by bacterial urine. However, no animal model has been established to evaluate their effectiveness. This study aimed to induce IAD‐like skin changes using absorbent pads containing bacterial urine and to confirm their pathophysiology in rats. Hairless Wistar Yagi rats were divided into the bacteria‐containing urine (BU) and the bacteria‐free urine (U) groups. A 10‐h‐attachment of absorbent pads containing artificial urine with/without bacteria to the skin pretreated with sodium lauryl sulfate was performed repeatedly for 5 days. Macroscopic findings and skin barrier function were examined every day, and histological changes, inflammatory responses and bacterial quantification in tissue samples were examined on Day 5. The BU group exhibited significant skin redness from Day 3, significant elevation of transepidermal water loss from Day 1, and histological changes, including significantly thickened epidermis, abnormal keratinocyte differentiation and erythrocyte leakage. Inflammation, confirmed by higher myeloperoxidase‐positive cells, elevated tumour necrosis factor‐α expression, and vascular endothelial damage, indicated by CD31 and pentraxin 3‐positive cells, were observed in the BU group. The bacterial quantification showed no significant difference between the groups. IAD‐like skin changes including histological changes and inflammation were suggested to be caused by urine properties altered by bacteria. This study proposed a new animal model for evaluating the effectiveness of absorbent pads in controlling the urine properties of bacterial urine on preventing IAD. [ABSTRACT FROM AUTHOR]

The NLRP1 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-1) inflammasome is the most important inflammasome in human keratinocytes. It plays a crucial role in regulating innate immunity in the skin. This study aimed to evaluate NLRP1 inflammasome activation and the corresponding levels of detection in different keratinocyte cell lines to identify a suitable in vitro model for analyzing inflammasome activation in keratinocytes. We compared NLRP1 inflammasome activation, expression, and cell death among primary keratinocytes and immortalized keratinocyte cell lines HaCaT, HaSKpw, and SVTERT upon stimulation with ultraviolet B (UVB) irradiation or talabostat. The effects of both NLRP1 inducers on cell death and the modification of NLRP1 molecules were examined using fluorescence-activated cell sorting analysis, Western blotting, and an enzyme-linked immunosorbent assay. The key inflammasome components had varied expression levels among the keratinocyte cell models, with the highest expression observed in primary keratinocytes. Moreover, our data showed that both UVB and talabostat triggered cell death, and NLRP1 inflammasome activation was readily detected in primary keratinocytes but not in the analyzed immortalized keratinocyte cell lines. Therefore, we do not recommend the use of the immortalized keratinocyte cell lines HaCaT, HaSKpw, and SVTERT for analyzing inflammasome activation in keratinocytes; we strongly recommend the use of primary keratinocytes for these studies. [ABSTRACT FROM AUTHOR]

Bio-sourced insect repellents are becoming more popular due to their safer applications. Known for its strong fly-repellent property, Cis, trans-para-menthane-3,8-diol (PMD) is the main component of the lemon eucalyptus essential oil and is synthesized from citronellal. In April 2005, US Centers for Disease Control approved two fly repellents that do not contain N,N-diethyl-meta-toluamide (DEET), including PMD. Due to the intentional and pervasive human exposure caused by DEET as insect repellent, concerns have been raised about its toxicological profile and potential harm to people. We hypothesized PMD would have a different toxicological profile than DEET. We synthesized PMD from Eucalyptus citriodora using green chemistry methods and analyzed its structures by 1H-NMR,13C-NMR, and GC/MS spectral methods. We used MTS assay to determine the percentage inhibition of PMD and DEET on keratinocyte (human epidermal keratinocyte [HaCaT]) cells. The xCelligence system was used and followed at real time. Effects of PMD and DEET on zebrafish embryo development were monitored and levels of lipid peroxidation, glutathione-S-transferase (GST), superoxide dismutase (SOD), and acetylcholinesterase (AchE) were evaluated at 72 h post-fertilization using spectrophotometric methods. Our results showed that while DEET inhibited human keratinocyte cell growth, while imporved cell viability and proliferation was exposed in PMD exposed group. In zebrafish embryos, PMD was less toxic in terms of development, oxidant-antioxidant status, and AChE activities than DEET. Based on these results we suggest an efficient method using green chemistry for the synthesis of PMD, which is found to be less toxic in zebrafish embryos and human keratinocyte cells. [ABSTRACT FROM AUTHOR]

There are comorbidities and complications in atopic dermatitis and psoriasis that often occur after the appearance of skin inflammation. Statistically, data show that patients with psoriasis and atopic dermatitis have a shorter life expectancy than patients without psoriatic dermatitis, due to the occurrence of arteriosclerosis, myocardial infarction, and cerebral infarction. Many types of skin inflammation are treated with various antibody preparations, and marked improvement in patients' quality of life can be achieved. The next theme is to understand the pathogenesis of arteriosclerosis, myocardial infarction, stroke, and other complications associated with dermatitis and to find treatments and drugs to reduce their occurrence. The skin, a crucial immune organ, generates large amounts of inflammatory cytokines in response to various stimuli, leading to systemic inflammation and potential damage to internal organs. The link between inflammatory skin conditions like psoriasis and atopic dermatitis with serious health complications such as vascular disorders and systemic amyloidosis has been increasingly recognized. In psoriasis, biological treatments targeting Interleukin (IL)-17A, a key cytokine, have shown promise in reducing cardiovascular risks. Recent developments include treatments that target both IL-17A and IL-17F in the psoriasis field, though each cytokine's impact on internal organ damage is still under debate. Among visceral complications secondary to dermatitis, systemic amyloidosis and atherosclerosis have been reported to be controlled by suppressing IL-17 in the early stages of dermatitis. Still, it remains unclear whether suppressing IL-17 prevents organ damage in the late stages of persistent severe dermatitis. A study using a long-lasting dermatitis mouse model that overexpressed human caspase-1 in keratinocytes (Kcasp1Tg) investigated the effects of deleting IL-17A and IL-17F on visceral complications. Cross-mating Kcasp1Tg with IL-17A-, IL-17F-, and IL-17AF-deficient mice assessed the skin and visceral organs histologically, and RT-PCR analysis of aortic sclerosis markers was performed. Despite less improvement in dermatitis, deletion of IL-17A in Kcasp1Tg mice showed promising results in reducing multiple organ amyloidosis. On the other hand, the effect was observed in both IL-17A and IL-17F deleted mice for aortic sclerosis. The inhibition of IL-17A and IL-17F was suggested to reduce the risk of developing comorbidities in internal organs. IL-17A and IL-17F were found to act similarly or produce very different results, depending on the organ. [ABSTRACT FROM AUTHOR]

Urothelial carcinoma (UC) is prevalent, especially in elderly males. The high rate of recurrence, treatment regime, and follow-up monitoring make UC a global health and economic burden. Arsenic is a ubiquitous toxicant that can be found in drinking water, and it is known that exposure to arsenic is associated with UC development. Around 25% of diagnosed UC cases are muscle-invasive (MIUC) which have poor prognosis and develop chemoresistance, especially if tumors are associated with squamous differentiation (SD). The immortalized UROtsa cell line is derived from normal human urothelium and our lab has malignantly transformed these cells using arsenite (As3+). These cells represent a basal subtype model of MIUC and the tumors derived from the As3+-transformed cells histologically and molecularly resemble clinical cases of the basal subtype of MIUC that have focal areas SD and expression of the basal keratins (KRT1, 5, 6, 14, and 16). Our previous data demonstrate that KRT6 protein expression correlates to areas of SD within the tumors. For this study, we performed a lentiviral knockdown of KRT6 in As3+-transformed UROtsa cells to evaluate the effects on morphology, gene/protein expression, growth, colony formation, and cisplatin sensitivity. The knockdown of KRT6 resulted in decreased expression of the basal keratins, decreased growth, decreased colony formation, and increased sensitivity to cisplatin, the standard treatment for MIUC. The results of this study suggest that KRT6 plays a role in UC cell growth and is an exploitable target to increase cisplatin sensitivity for MIUC tumors that may have developed resistance to cisplatin treatment. [ABSTRACT FROM AUTHOR]

Psoriasis is a chronic skin disease characterized by the hyperproliferation of keratinocytes and an overactive autoimmune response. Photodynamic therapy (PDT) has been established as a promising intervention for alleviating psoriasis. However, the current transdermal delivery of photosensitizers is inefficient and imprecise. In this study, we developed a foamed microemulsion nanodroplets system containing chlorin e6 (Ce6 FM), exhibiting precise epidermal targeting and retention, which targeted the aberrantly proliferating epidermal cells at psoriatic skin lesions and avoided the damage to the normal cutaneous cells. Upon application in a psoriatic mouse model, Ce6 FM efficiently induced keratinocyte apoptosis by generating reactive oxygen species under laser. Furthermore, Ce6 FM-based PDT activated the cyclooxygenase-2-induced immunosuppressive pathway in keratinocytes, resulting in the amelioration of the autoimmune microenvironment in psoriatic skin. Additionally, Ce6 FM-based PDT did not induce skin damage or atrophy associated with non-targeted halometasone treatment. Overall, Ce6 FM-based PDT holds promise as an effective, safe and compliant strategy for psoriasis treatment. [ABSTRACT FROM AUTHOR]

Psoriasis is an inflammatory skin disease that relapses frequently. Keratinocyte apoptosis dysregulation plays a crucial role in the pathological mechanisms of psoriasis. PANoptosis is a process with intermolecular interaction among pyroptosis, apoptosis, and necroptosis. The mechanism of PANoptosis in the occurrence and development of psoriasis is still unclear. Here we present a novel approach by identifying PANoptosis-related signatures (PANoptosis-sig) from skin tissue of psoriasis patients and healthy controls on transcriptional and protein levels. Five PANoptosis-sig (TYMP, S100A8, S100A9, NAMPT, LCN2) were identified. Enrichment analysis showed they were mainly enriched in response to leukocyte aggregation, leukocyte migration, chronic inflammatory response and IL−17 signaling pathway. Single cell transcriptome analysis showed TYMP and NAMPT were expressed in almost all cell populations, while LCN2, S100A8 and S100A9 were significantly highly expressed in keratinocyte. We then constructed predictive and diagnostic models with the PANoptosis-sig and evaluated their performance. Finally, unsupervised consensus clustering analysis was conducted to ascertain psoriasis molecular subtypes by the PANoptosis-sig. The psoriasis cohort was divided into two distinct subtypes. Immune landscape showed that the stromal score of cluster 1 was significantly higher than cluster 2, while the immune and estimate scores of cluster 2 were expressively higher than cluster 1. Cluster 1 exhibited high expression of Plasma cells, Tregs and Mast cells resting, while cluster 2 showed high expression of T cells, Macrophages M1, Dendritic cells activated, and Neutrophils in immune infiltration analysis. And cluster 2 was more sensitive to immune checkpoints. In conclusion, our findings revealed potential biomarkers and therapeutic targets for the prevention, diagnosis, and treatment of psoriasis, enhancing our understanding of the molecular mechanisms underlying PANoptosis. [ABSTRACT FROM AUTHOR]

Introduction: The resolution of the skin's inflammatory response is only possible if its barrier function is restored. TRPV1 channel activation plays an important role during inflammation but the effect of this activation on the skin barrier under inflammatory conditions has not been clarified. We hypothesize that it could potentially aid the keratinocyte barrier by reducing inflammatory cytokine release and promoting tight junction development. Methods: To explore the role of TRPV1 activation in inflammation, we designed and optimized an in vitro model of keratinocytes with basal epidermal layer characteristics using HaCaT cells and TNFα to induce inflammation. Results: TNFα increased the gene expression of tight junction protein claudin 1 (CLDN1) by at least 2.60 ± 0.16-fold, in a concentration-dependent manner, over a 48 h period. The administration of a capsaicin pre-treatment reduced the CLDN1 expression to 1.51 ± 0.16-fold during the first 6 h after TNFα induction, whereas IL-8 cytokine release was reduced 0.64 ± 0.17-fold. After 48 h, CLDN1 protein levels increased by a factor of 6.57 ± 1.39 compared to cells only treated with TNFα. Discussion: These results suggest that activation of TRPV1 by capsaicin can potentiate the increase in CLDN1 expression and CLDN1 protein synthesis induced by TNFα in cultured keratinocytes, while reducing the release of IL-8. [ABSTRACT FROM AUTHOR]

The transcription factor NRF2 plays an important role in many biological processes and is a promising therapeutic target for many disease states. NRF2 is highly expressed in the skin and is known to play a critical role in diabetic wound healing, a serious disease process for which treatment options are limited. However, many existing NRF2 activators display off-target effects due to their electrophilic mechanism, underscoring the need for alternative approaches. In this work, we investigated two recently described non-electrophilic NRF2 activators, ADJ-310 and PRL-295, and demonstrated their efficacy in vitro and in vivo in human keratinocytes and Leprdb/db diabetic mice. We also compared the downstream targets of PRL-295 to those of the widely used electrophilic NRF2 activator CDDO-Me by RNA sequencing. Both ADJ-310 and PRL-295 maintained human keratinocyte cell viability at increasing concentrations and maintained or improved cell proliferation over time. Both compounds also increased cell migration, improving in vitro wound closure. ADJ-310 and PRL-295 enhanced the oxidative stress response in vitro, and RNA-sequencing data showed that PRL-295 activated NRF2 with a narrower transcriptomic effect than CDDO-Me. In vivo, both ADJ-310 and PRL-295 improved wound healing in Leprdb/db diabetic mice and upregulated known downstream NRF2 target genes in treated tissue. These results highlight the non-electrophilic compounds ADJ-310 and PRL-295 as effective, innovative tools for investigating the function of NRF2. These compounds directly address the need for alternative NRF2 activators and offer a new approach to studying the role of NRF2 in human disease and its potential as a therapeutic across multiple disease states. [ABSTRACT FROM AUTHOR]

Psoriasis is a chronic skin disease characterized by abnormal proliferation and inflammation of epidermal keratinocytes. Fibroblast growth factor 12 (FGF12) is implicated in the regulation of diverse cellular signals; however, its precise mechanism in psoriasis requires further investigation. In this study, high expression of FGF12 is observed in the epidermis of skin lesion in psoriasis patients and imiquimod (IMQ)‐induced psoriasis like‐dermatitis. Moreover, specific loss of FGF12 in keratinocytes in IMQ‐induced psoriasis model alleviates psoriasis‐like symptoms and reduces proliferation. In vitro RNA sequencing demonstrates that knockdown of FGF12 effectively arrests the cell cycle, inhibits cell proliferation, and predominantly regulates the p53 signaling pathway. Mechanistically, FGF12 is selectively bound to the RING domain of MDM2, thus partially inhibiting the binding of β‐Trcp to MDM2. This interaction inhibits β‐Trcp‐induced‐K48 ubiquitination degradation of MDM2, thereby suppressing the activity of the p53 signaling pathway, which results in excessive cell proliferation. Last, the alleviatory effect of FGF12 deficiency on psoriasis progression is reversed by p53 knockdown. In summary, these findings provide valuable insights into the mechanisms by which FGF12 suppresses p53 signaling in keratinocytes, exacerbating the development of psoriasis. This positive regulatory loop highlights the potential of FGF12 as a therapeutic target to manage psoriasis. [ABSTRACT FROM AUTHOR]

Non‐healing diabetic wounds and ulcer complications, with persistent cell dysfunction and obstructed cellular processes, are leading causes of disability and death in patients with diabetes. Currently, there is a lack of guideline‐recommended hypoglycemic drugs in clinical practice, likely due to limited research and unclear mechanisms. In this study, it is demonstrated that liraglutide significantly accelerates wound closure in diabetic mouse models (db/db mice and streptozotocin‐induced mice) by improving re‐epithelialization, collagen deposition, and extracellular matrix remodeling, and enhancing the proliferation, migration, and adhesion functions of keratinocytes. However, these effects of improved healing by liraglutide are abrogated in dedicator of cytokinesis 5 (Dock5) keratinocyte‐specific knockout mice. Mechanistically, liraglutide induces cellular function through stabilization of unconventional myosin 1c (Myo1c). Liraglutide directly binds to Myo1c at arginine 93, enhancing the Myo1c/Dock5 interaction by targeting Dock5 promoter and thus promoting the proliferation, migration, and adhesion of keratinocytes. Therefore, this study provides insights into liraglutide biology and suggests it may be an effective treatment for diabetic patients with wound‐healing pathologies. [ABSTRACT FROM AUTHOR]

Keratin intermediate filaments confer structural stability to epithelial tissues, but the reason this simple mechanical function requires a protein family with 54 isoforms is not understood. During skin wound healing, a shift in keratin isoform expression alters the composition of keratin filaments. If and how this change modulates cellular functions that support epidermal remodeling remains unclear. We report an unexpected effect of keratin isoform variation on kinase signal transduction. Increased expression of wound-associated keratin 6A, but not of steady-state keratin 5, potentiated keratinocyte migration and wound closure without compromising mechanical stability by activating myosin motors to increase contractile force generation. These results substantially expand the functional repertoire of intermediate filaments from their canonical role as mechanical scaffolds to include roles as isoform-tuned signaling scaffolds that organize signal transduction cascades in space and time to influence epithelial cell state. [Display omitted] • During skin wound healing, the isoform composition of keratin filaments changes • Wound-associated keratin isoforms organize cellular signals to activate myosin • Keratin isoform-dependent signaling is separable from canonical mechanical function Nanes et al. explore why the seemingly simple mechanical function of keratin intermediate filaments requires a protein family with 54 isoforms. They find that skin wound healing triggers a change in keratin isoform expression not to alter force resistance but to organize signals activating myosin and promoting cell motility. [ABSTRACT FROM AUTHOR]

The article delves into the function of periostin in atopic dermatitis (AD) by examining cell interactions in skin biopsy samples from healthy individuals, AD patients, and psoriasis patients. It reveals that POSTN and its receptors are mainly expressed in keratinocytes and fibroblasts, with higher POSTN levels in AD compared to healthy skin and psoriasis. The study suggests a T cell-fibroblast-keratinocyte axis in AD, with periostin playing a crucial role in linking Th2-type responses to keratinocyte activation. The research underscores the significance of periostin as a key molecule in AD, hinting at the potential benefits of targeting periostin signaling for AD treatment. [Extracted from the article]

Background: ABO blood group antigens (ABH antigens) are carbohydrate chains glycosylated on epithelial and red blood cells. Recent findings suggest reduced ABH expression in psoriasis and atopic dermatitis, a chronic inflammatory skin disease with retained scale. H antigen, a precursor for A and B antigens, is synthesized by fucosyltransferase 1 (FUT1). Desmosomes, critical for skin integrity, are known to require N-glycosylation for stability. We investigate the impact of H antigens, a specific type of glycosylation, on desmosomes in keratinocytes. Method: Primary human keratinocytes were transfected with FUT1 siRNA or recombinant adenovirus for FUT1 overexpression. Cell adhesion and desmosome characteristics and their underlying mechanisms were analyzed. Result: The knockdown of FUT1, responsible for H2 antigen expression in the skin, increased cell-cell adhesive strength and desmosome size in primary cultured keratinocytes without altering the overall desmosome structure. Desmosomal proteins, including desmogleins or plakophilin, were upregulated, suggesting enhanced desmosome assembly. Reduced H2 antigen expression via FUT1 knockdown led to increased keratinocyte differentiation, evidenced by elevated expression of differentiation markers. Epidermal growth factor receptor (EGFR) has been described to be associated with FUT1 and promotes cell migration and differentiation. The effects of FUT1 knockdown were recapitulated by an EGFR inhibitor concerning desmosomal proteins and cellular differentiation. Further investigation demonstrated that the FUT1 knockdown reduced EGFR signaling by lowering the levels of EGF ligands rather than directly regulating EGFR activity. Moreover, FUT1 overexpression reversed the effects observed in FUT1 knockdown, resulting in the downregulation of desmosomal proteins and differentiation markers while increasing both mRNA and protein levels of EGFR ligands. Conclusion: The expression level of FUT1 in the epidermis appears to influence cell-cell adhesion and keratinocyte differentiation status, at least partly through regulation of H2 antigen and EGFR ligand expression. These observations imply that the fucosylation of the H2 antigen by FUT1 could play a significant role in maintaining the molecular composition and regulation of desmosomes and suggest a possible involvement of the altered H2 antigen expression in skin diseases, such as psoriasis and atopic dermatitis. [ABSTRACT FROM AUTHOR]

The epidermis of the skin and skin appendages, such as nails, hair and sebaceous glands, depend on a balance of cell proliferation and terminal differentiation in order to fulfill their functions at the interface of the body and the environment. The differentiation of epithelial cells of the skin, commonly referred to as keratinocytes, involves major remodeling processes that generate metabolically inactive cell remnants serving as building blocks of the epidermal stratum corneum, nail plates and hair shafts. Only sebaceous gland differentiation results in cell disintegration and holocrine secretion. A series of studies performed in the past decade have revealed that the lysosome-dependent intracellular degradation mechanism of autophagy is active during keratinocyte differentiation, and the blockade of autophagy significantly alters the properties of the differentiation products. Here, we present a model for the autophagy-mediated degradation of organelles and cytosolic proteins as an important contributor to cellular remodeling in keratinocyte differentiation. The roles of autophagy are discussed in comparison to alternative intracellular degradation mechanisms and in the context of programmed cell death as an integral end point of epithelial differentiation. [ABSTRACT FROM AUTHOR]

We genetically manipulated HaCaT cells, a spontaneously immortalised normal keratinocyte cell line, to stably express two different coloured luciferase reporter genes, driven by interleukin 8 (IL‐8) and ubiquitin‐C (UBC) promoters, respectively. Subsequently, we generated a three‐dimensional (3D) skin‐like in vitro composite (SLIC) utilising these cells, with the objective of monitoring bioluminescence emitted from the SLIC. This SLIC was generated on non‐woven silica fibre membranes in differentiation medium. Immunohistochemical analyses of skin differentiation markers in the SLIC revealed the expression of keratins 2 and 10, filaggrin, and involucrin, indicating mature skin characteristics. This engineered SLIC was employed for real‐time bioluminescence monitoring, allowing the assessment of time‐ and dose‐dependent responses to UV stress, as well as to hydrophilic and hydrophobic chemical loads. Notably, evaluation of responses to hydrophobic substances has been challenging with conventional 2D cell culture methods, suggesting the need for a new approach, which this technology could address. Our observations suggest that engineered SLIC with constitutively expressing reporters driven by selected promoters which are tailored to specific objectives, significantly facilitates assays exploring the physiological functions of skin cells based on genetic response mechanisms. It also highlights new avenues for evaluating the physiological impacts of various compounds designed for topical application to human skin. [ABSTRACT FROM AUTHOR]

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterised by epidermal virus replication in skin and mucosa and the formation of blisters. We have previously shown that VZV infection has a profound effect on keratinocyte differentiation, altering the normal pattern of epidermal gene expression. In particular, VZV infection reduces expression of suprabasal keratins 1 and 10 and desmosomal proteins, disrupting epidermal structure to promote expression of a blistering phenotype. Here, we extend these findings to show that VZV infection upregulates the expression of keratin 15 (KRT15), a marker expressed by basal epidermal keratinocytes and hair follicles stem cells. We demonstrate that KRT15 is essential for VZV replication in the skin, since downregulation of KRT15 inhibits VZV replication in keratinocytes, while KRT15 exogenous overexpression supports viral replication. Importantly, our data show that VZV upregulation of KRT15 depends on the expression of the VZV immediate early gene ORF62. ORF62 is the only regulatory gene that is mutated in the live attenuated VZV vaccine and contains four of the five fixed mutations present in the VZV Oka vaccine. Our data indicate that the mutated vaccine ORF62 is not capable of upregulating KRT15, suggesting that this may contribute to the vaccine attenuation in skin. Taken together our data present a novel association between VZV and KRT15, which may open a new therapeutic window for a topical targeting of VZV replication in the skin via modulation of KRT15. [ABSTRACT FROM AUTHOR]

Increased prevalence of skin ageing is a growing concern due to an ageing global population and has both sociological and psychological implications. The use of more clinically predictive in vitro methods for dermatological research is becoming commonplace due to initiatives and the cost of clinical testing. In this study, we utilise a well‐defined and characterised bioengineered skin construct as a tool to investigate the cellular and molecular dynamics involved in skin ageing from a dermal perspective. Through incorporation of ageing fibroblasts into the dermal compartment we demonstrate the significant impact of dermal‐epidermal crosstalk on the overlying epidermal epithelium. We characterise the paracrine nature of dermal‐epidermal communication and the impact this has during skin ageing. Soluble factors, such as inflammatory cytokines released as a consequence of senescence associated secretory phenotype (SASP) from ageing fibroblasts, are known to play a pivotal role in skin ageing. Here, we demonstrate their effect on epidermal morphology and thickness, but not keratinocyte differentiation or tissue structure. Through a novel in vitro strategy utilising bioengineered tissue constructs, this study offers a unique reductionist approach to study epidermal and dermal compartments in isolation and tandem. [ABSTRACT FROM AUTHOR]

The study published in the Journal of Diabetes explores the relationship between stratum corneum hydration levels and HbA1c levels in elderly Chinese individuals. The research indicates a negative correlation between stratum corneum hydration and HbA1c levels, with individuals with type 2 diabetes showing lower hydration levels. The findings suggest that improving stratum corneum hydration could be an alternative approach in managing type 2 diabetes, as low hydration levels may increase proinflammatory cytokines linked to the disease's pathogenesis. Further studies are needed to understand the interaction between stratum corneum hydration and type 2 diabetes. [Extracted from the article]

The epidermis, the most superficial layer of the human skin, serves a critical barrier function, protecting the body from external pathogens and allergens. Dysregulation of epidermal differentiation contributes to barrier dysfunction and has been implicated in the pathology of various dermatological diseases, including atopic dermatitis (AD). Mucopolysaccharide polysulphate (MPS) is a moisturising agent used to treat xerosis in patients with AD. However, its mechanism of action on keratinocytes, the main constituents of the epidermis, remains unclear. In this study, we investigated the effect of MPS on keratinocytes by subjecting adult human epidermal and three‐dimensional cultured keratinocytes to MPS treatment, followed by transcriptome analysis. The analysis revealed that MPS treatment enhances keratinocyte differentiation and suppresses proliferation. We focused on amphiregulin (AREG), a membrane protein that belongs to the epidermal growth factor (EGF) family and possesses a heparin‐binding domain, as a significant target among the genes altered by MPS. MPS exerted an inhibitory effect directly on AREG, rather than on EGF receptors or other members of the EGF family. Furthermore, AREG leads to a reduction in epidermal barrier function, whereas MPS contributes to barrier enhancement via AREG inhibition. Collectively, these findings suggest that MPS modulates barrier function through AREG inhibition, offering insights into potential therapeutic strategies for skin barrier restoration. [ABSTRACT FROM AUTHOR]

Currently, there are no specific antiviral therapeutic approaches targeting Human papillomaviruses (HPVs), which cause around 5% of all human cancers. Specific antiviral reagents are particularly needed for HPV-related oropharyngeal cancers (HPV+OPCs) whose incidence is increasing and for which there are no early diagnostic tools available. We and others have demonstrated that the estrogen receptor alpha (ERα) is overexpressed in HPV+OPCs, compared to HPV-negative cancers in this region, and that these elevated levels are associated with an improved disease outcome. Utilizing this HPV+-specific overexpression profile, we previously demonstrated that estrogen attenuates the growth and cell viability of HPV+ keratinocytes and HPV+ cancer cells in vitro. Expansion of this work in vivo failed to replicate this sensitization. The role of stromal support from the tumor microenvironment (TME) has previously been tied to both the HPV lifecycle and in vivo therapeutic responses. Our investigations revealed that in vitro co-culture with fibroblasts attenuated HPV+-specific estrogen growth responses. Continuing to monopolize on the HPV+-specific overexpression of ERα, our co-culture models then assessed the suitability of the selective estrogen receptor modulators (SERMs), raloxifene and tamoxifen, and showed growth attenuation in a variety of our models to one or both of these drugs in vitro. Utilization of these SERMs in vivo closely resembled the sensitization predicted by our co-culture models. Therefore, the in vitro fibroblast co-culture model better predicts in vivo responses. We propose that utilization of our co-culture in vitro model can accelerate cancer therapeutic drug discovery. [ABSTRACT FROM AUTHOR]

Dermal papilla cells (DPCs) are located at the bottom of the hair follicle and play a critical role in hair growth, shape, and cycle. Epidermal growth factor receptor (EGFR) and Wnt/β-catenin signaling pathways are essential in promoting keratinocyte activation as well as hair follicle formation in DPCs. Piperonylic acid is a small molecule that induces EGFR activation in keratinocytes. However, the effects of piperonylic acid on DPCs in regard to the stimulation of hair growth have not been studied. In the present study, piperonylic acid was shown to activate the Wnt/β-catenin signaling pathway in addition to the EGFR signaling pathway in DPCs. Piperonylic acid suppressed DKK1 expression, which presumably promoted the accumulation of β-catenin in the nucleus. In addition, piperonylic acid promoted cyclin D upregulation and cell growth and increased the expression of alkaline phosphatase (ALP), a DPC marker. In a clinical study, the group that applied a formulation containing piperonylic acid had a significantly higher number of hairs per unit area than the placebo group. These results identify piperonylic acid as a promising new candidate for hair loss treatment. [ABSTRACT FROM AUTHOR]

The skin epidermis provides a barrier that is imperative for preventing transepidermal water loss (TEWL) and protecting against environmental stimuli. The underlying molecular mechanisms for regulating barrier functions and sustaining its integrity remain unclear. RORα is a nuclear receptor highly expressed in the epidermis of normal skin. Clinical studies showed that the epidermal RORα expression is significantly reduced in the lesions of multiple inflammatory skin diseases. In this study, we investigate the central roles of RORα in stabilizing skin barrier function using mice with an epidermis-specific Rora gene deletion (RoraEKO). While lacking spontaneous skin lesions or dermatitis, RoraEKO mice exhibited an elevated TEWL rate and skin characteristics of barrier dysfunction. Immunostaining and Western blot analysis revealed low levels of cornified envelope proteins in the RoraEKO epidermis, suggesting disturbed late epidermal differentiation. In addition, an RNA-seq analysis showed the altered expression of genes related to "keratinization" and "lipid metabolism" in RORα deficient epidermis. A lipidomic analysis further uncovered an aberrant ceramide composition in the RoraEKO epidermis. Importantly, epidermal Rora ablation greatly exaggerated percutaneous allergic inflammatory responses to oxazolone in an allergic contact dermatitis (ACD) mouse model. Our results substantiate the essence of epidermal RORα in maintaining late keratinocyte differentiation and normal barrier function while suppressing cutaneous inflammation. [ABSTRACT FROM AUTHOR]

Keratinocytes play an essential role in the inflammatory phase of wound regeneration. In addition to migrating and proliferating for tissue regeneration, they produce a large amount of cytokines that modulate the inflammatory process. Previous studies have shown that subthermal treatment with radiofrequency (RF) currents used in capacitive resistive electric transfer (CRET) therapy promotes the proliferation of HaCat keratinocytes and modulates their cytokine production. Although physical therapies have been shown to have anti-inflammatory effects in a variety of experimental models and in patients, knowledge of the biological basis of these effects is still limited. The aim of this study was to investigate the effect of CRET on keratinocyte proliferation, cytokine production (IL-8, MCP-1, RANTES, IL-6, IL-11), TNF-α secretion, and the expression of MMP9, MMP1, NF-κB, ERK1/2, and EGFR. Human keratinocytes (HaCat) were treated with an intermittent 448 kHz electric current (CRET signal) in subthermal conditions and for different periods of time. Cell proliferation was analyzed by XTT assay, cytokine and TNF-α production by ELISA, NF-κB expression and activation by immunofluorescence, and MMP9, MMP1, ERK1/2, and EGF receptor expression and activation by immunoblot. Compared to a control, CRET increases keratinocyte proliferation, increases the transient release of MCP-1, TNF-α, and IL-6 while decreasing IL-8. In addition, it modifies the expression of MMPs and activates EGFR, NF-κB, and ERK1/2 proteins. Our results indicate that CRET reasonably modifies cytokine production through the EGF receptor and the ERK1/2/NF-κB pathway, ultimately modulating the inflammatory response of human keratinocytes. [ABSTRACT FROM AUTHOR]

Introduction: Digital dermatitis (DD) in cattle appears with high prevalence; nevertheless, the knowledge on its pathogenesis is still limited. In this context, in vitro skin models represent a valuable tool to facilitate the study of DD. Methods: Two in vitro skin models were established using bovine distal limb skin: a skin explant model and an organotypic skin model. For the skin explant model, skin samples were cultured with an air-liquid interface for up to 7 days. Besides routine histopathological examination, readout parameters were Ki-67 and cleaved Caspase-3 stainings. For the organotypic model, primary keratinocytes were layered on top of a dermal equivalent containing mainly mitotically inactive fibroblasts and maintained for up to 21 days. At regular intervals (days 7, 14, and 21), cultured skin samples were taken for (immuno)histological analysis. Results: Both cultures could be maintained for the entire duration of the intended culture period. In the histopathological assessment, explant skin cultures showed ballooning degeneration of keratinocytes and segmental necrosis starting at day 5 of culturing. Initially, basal keratinocytes in the organotypic model differentiated as demonstrated by positive Keratin 14, Desmoglein-1, Loricrin, and Involucrin immunofluorescent stainings. Ki-67 was observed occasionally and suprabasally still after 21 days of culture. Conclusion: Both in vitro models proved dependable and constitute a viable option for replacing experiments on live animals, each with its own benefits. Whereas skin explants include all cell types available in vivo and can therefore reflect realistic cell-cell interactions and signaling pathways, the organotypic model offers a higher standardization and reproducibility. Depending on the focus of future studies, both models can be used for specific experimental purposes of bovine dermatological research in general or specialized questions concerning (infectious) claw diseases as, e.g., DD. [ABSTRACT FROM AUTHOR]

Background: Conspicuous facial pores are benign but represent a cosmetic concern for men and women. Recent works described dermal and epidermal impairments as clinical causes of enlarged pores. Morphological modifications of skin at the site of pores were associated with collagen density loss, possible alteration of extracellular matrix and abnormal differentiation of keratinocytes. Aims: A composition containing mannose‐6‐phosphate (Active Complex) was designed to address these different aspects of pore enlargement. In vitro and ex vivo evaluations were conducted in different models mimicking disturbance of dermal and epidermal functions. The pore refining activity of Active Complex was assessed in two clinical trials studying a Caucasian women cohort and an Asian men cohort. Results: At the dermal level, Active Complex upregulated collagen I and decorin synthesis, and genes encoding collagens I, III, V, VII, XVII; suggesting its ability to favor collagen fiber organization and anchorage. The downregulation of matrix metalloprotease, involved in extracellular matrix degradation, reinforced the protective effect of Active Complex in the dermis. Active Complex down modulated differentiation markers in keratinocytes as well as genes involved in cell renewal. Study of reconstructed human epidermis modeling keratinocyte hyperproliferation revealed that Active Complex mitigated two markers of this state: number of nuclei in the stratum corneum and involucrin expression. Clinical trials confirmed the pore refining activity of Active Complex on men and women of different ages and ethnicities; −24% total skin pore area after 56 days of application on women, and −30.2% on men after 7 days. Conclusions: This work demonstrates the interest to target dermal and epidermal modifications described in conspicuous pore area, especially dermis fiber organization, to address this cosmetic concern. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. The aim of this study was to evaluate transcriptional changes in the sole epidermis and dermis of bovine claws with septic sole ulceration of the lateral claw. Assessment included changes in transcripts orchestrating epidermal homeostatic processes, including epidermal proliferation, differentiation, inflammation, and cell signaling. Sole epidermis and dermis samples were removed from region 4 of lesion-bearing lateral and lesion-free medial claws of pelvic limbs in multiparous, lactating Holstein cows. Control sole epidermis and dermis samples were obtained from region 4 of lateral claws of normal pelvic limbs. Transcript abundances were evaluated by real-time PCR, and relative expression analyzed by ANOVA. Relative to normal lateral claws, sole epidermis and dermis in ulcer-bearing claws exhibited downregulation of genes associated with growth factors, growth factor receptors, activator protein 1 (AP-1) and proto-oncogene (CMYC) transcription components, cell cycle elements, lateral cell-to-cell signaling elements, and structures of early and late keratinocyte differentiation. These changes were accompanied by upregulation of proinflammatory transcripts interleukin 1 α (IL1A), interleukin1 β (IL1B), interleukin 1 receptor 1 (IL1R1), inducible nitric oxide synthase (NOS2), the inflammasome components NOD-like receptor protein 3 (NLRP3), pyrin and caspase recruitment domain (PYCARD), caspase-1 interleukin converting enzyme (CASPASE), the matrix metalloproteinases (MMP2 and MMP9), and the anti-inflammatory genes interleukin 1 receptor antagonist (IL1RN) and interleukin1 receptor 2 (IL1R2). Transcript abundance varied across epidermis and dermis from the ulcer center, margin, and epidermis and dermis adjacent to the lesion. Sole epidermis and dermis of lesion-free medial claws exhibited changes paralleling those in the adjacent lateral claws in an environment lacking inflammatory transcripts and downregulated IL1A , interleukin 18 (IL18), tumor necrosis factor α (TNFA), and NOS2. These data imply perturbations in signal pathways driving epidermal proliferation and differentiation are associated with, but not inevitably linked to epidermis and dermis inflammation. Further work is warranted to better define the role of crushing tissue injury, sepsis, metalloproteinase activity, and inflammation in sole ulceration. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Pododermatitis aseptica hemorrhagica circumscripta is associated with metalloproteinase 2 weakening of distal phalangeal suspensory structures and sinkage of the distal phalanx in the claw capsule. Pressure from the tuberculum flexorium on the sole epidermis and dermis produces hemorrhagic tissue injury and defective horn production appearing as yellow-red, softened claw horn in region 4 of the sole. A model of the MAPK/ERK signal cascade orchestrating epidermal-dermal homeostasis was employed to determine if sterile inflammatory responses are linked to disturbed signal transduction for epidermal homeostasis in sole epidermis and dermis. The objective was to assess shifts in target genes of inflammation, up- and downstream MAPK/ERK signal elements, and targeted genes supporting epidermal proliferation and differentiation. Sole epidermis and dermis were removed from lateral claws bearing lesions of PAHC, medial claws from the same limb and lateral claws from completely normal limbs of multiparous, lactating Holstein cows. The abundance levels of targeted transcripts were evaluated by real-time PCR. Lesion effects were assessed by ANOVA, and mean comparisons were performed with t -tests to assess variations between mean expression in ulcer-bearing or medial claw dermis and epidermis and completely normal lateral claw dermis and epidermis or between ulcer-bearing dermis and epidermis and medial claw dermis and epidermis. The lesions were sterile and showed losses across multiple growth factors, their receptors, several downstream AP1 transcription components, CMYC, multiple cell-cycle and terminal differentiation elements conducted by MAPK/ERK signals and β 4, α 6, and collagen 17A hemidesmosome components. These losses coincided with increased cytokeratin 6, β 1 integrin, proinflammatory metalloproteinases 2 and 9, IL1B and physiologic inhibitors of IL1B, the decoy receptor, and receptor antagonist. Medial claw epidermis and dermis from limbs with lateral claws bearing PAHC showed reductions in upstream MAPK/ERK signal elements and downstream targets that paralleled those in hemorrhagic lesions. Inhibitors of IL1B increased in the absence of real increases in inflammatory targets in the medial claw dermis and epidermis. Losses across multiple signal path elements and downstream targets were associated with negative effects on targeted transcripts supporting claw horn production and wound repair across lesion-bearing lateral claws and lesion-free medial claw dermis and epidermis. It was unclear if the sterile inflammation was causative or a consequence of these perturbations. [ABSTRACT FROM AUTHOR]

ABSTRACT: Pododermatitis aseptica hemorrhagica circumscripta is associated with metalloproteinase 2 weakening of distal phalangeal suspensory structures and sinkage of the distal phalanx in the claw capsule. Pressure from the tuberculum flexorium on the sole epidermis and dermis produces hemorrhagic tissue injury and defective horn production appearing as yellow-red, softened claw horn in region 4 of the sole. A model of the MAPK/ERK signal cascade orchestrating epidermal-dermal homeostasis was employed to determine if sterile inflammatory responses are linked to disturbed signal transduction for epidermal homeostasis in sole epidermis and dermis. The objective was to assess shifts in target genes of inflammation, up- and downstream MAPK/ERK signal elements, and targeted genes supporting epidermal proliferation and differentiation. Sole epidermis and dermis were removed from lateral claws bearing lesions of PAHC, medial claws from the same limb and lateral claws from completely normal limbs of multiparous, lactating Holstein cows. The abundance levels of targeted transcripts were evaluated by real-time PCR. Lesion effects were assessed by ANOVA, and mean comparisons were performed with t-tests to assess variations between mean expression in ulcer-bearing or medial claw dermis and epidermis and completely normal lateral claw dermis and epidermis or between ulcer-bearing dermis and epidermis and medial claw dermis and epidermis. The lesions were sterile and showed losses across multiple growth factors, their receptors, several downstream AP1 transcription components, CMYC, multiple cell-cycle and terminal differentiation elements conducted by MAPK/ERK signals and β 4, α 6, and collagen 17A hemidesmosome components. These losses coincided with increased cytokeratin 6, β 1 integrin, proinflammatory metalloproteinases 2 and 9, IL1B and physiologic inhibitors of IL1B, the decoy receptor, and receptor antagonist. Medial claw epidermis and dermis from limbs with lateral claws bearing PAHC showed reductions in upstream MAPK/ERK signal elements and downstream targets that paralleled those in hemorrhagic lesions. Inhibitors of IL1B increased in the absence of real increases in inflammatory targets in the medial claw dermis and epidermis. Losses across multiple signal path elements and downstream targets were associated with negative effects on targeted transcripts supporting claw horn production and wound repair across lesion-bearing lateral claws and lesion-free medial claw dermis and epidermis. It was unclear if the sterile inflammation was causative or a consequence of these perturbations.

Background and rationale: Ultraviolet-B (UVB) light induces dermal inflammation, although it is mostly absorbed in the epidermis. Recent reports suggest extracellular vesicles (EVs) act as a mediator of photodamage signaling. Melatonin is reported to be a protective factor against UV-induced damage. We hypothesized that EVs derived from UVB-irradiated keratinocytes might trigger proinflammatory responses in dermal cells and tested whether melatonin can ameliorate UVB-induced inflammation. Methods: We used UVB-irradiated HaCaT cells, primary keratinocytes and STING knock-out mice to model production of EVs under photodamaging conditions and performed immunoblotting and ELISA to measure their effect on dermal macrophages. Results: UVB-irradiated keratinocytes produce an increased number of EVs that contain higher concentrations of DNA and protein compared with controls. KC-derived EVs (KEVs) induced a STING- and inflammasome-mediated proinflammatory response in macrophages in vitro, and a pronounced inflammatory infiltrate in mouse dermis in vivo. Melatonin ameliorated KEVs inflammatory effect both in vitro and in vivo. Conclusions: This data suggests EVs are mediators in a crosstalk that takes place between keratinocytes and their neighboring cells as a result of photodamage. Further studies exploring EVs induced by damaging doses of UVB, and their impact on other cells will provide insight into photodamage and may help develop targeted therapeutic approaches. [ABSTRACT FROM AUTHOR]

Psoriasis is a chronic, relapsing, and refractory immune-mediated skin disease with the etiology and pharmaceutical targets remaining unsatisfactorily addressed. Topical herbal-derived compounds, such as tryptanthrin (Tryp), have been considered as an alternative therapy for psoriasis due to their lower costs and fewer side effects compared to other therapies. However, the effectiveness of topically administered drugs is substantially limited by the thickened pathological skin barrier and the low bioavailability of drugs in the deeper layers of the lesion. Ethosomes, being a novel phospholipid-based vesicle system with high content of ethanol, have been implicated in enhancing topical drug absorption and restoring psoriatic lesions. In this study, taking advantages of ethosomes as a soft and malleable drug carrier, we constructed the Tryp-loaded ethosome (Tryp-ES) through a one-step microfluidics-based technique. The optimal formulation of Tryp-ES was achieved by adding amino-acid-derived surfactant sodium lauroyl glutamate, and Tryp-ES exhibited homogeneous particle size and favorable stability at room temperature. In vitro evaluations showed that Tryp of Tryp-ES could be easily internalized into cells and accumulated in cell nuclei, hence inhibited the abnormally proliferated keratinocytes by inducing apoptosis. In vivo and in vitro assessment using psoritic skin of mice revealed that Tryp-ES had preferred skin retention and permeation of loaded drugs within the initial 1 h of topical administration, which could be attributed to transient disintegrations of cell membranes by ethosomes, thus improved cellular fluidity and permeability. Notably, a synergistic effect of ethosomes and Tryp was found in psoriatic mice. Tryp-ES-treated mice showed substantially ameliorated symptoms of psoriasis and reduced pathological alterations due to hyperplasia, inflammation and angiogenesis, without detectable local or systemic toxicities. Interestingly, lipidomics analysis confirmed that the supplementation of phospholipids, as in the form of ethosome vehicles, was an alterantive strategy to relieve psoriatic pathologies. Taken together, this study provides a novel impact for ethosomal topical delivery of Tryp and underlines their potential as an effective therapy for the management of psoriasis. [ABSTRACT FROM AUTHOR]

Skin aging has been associated with the onset of various skin issues, and recent studies have identified an increase in Cdc42 activity in naturally aging mice. While previous literature has suggested that CASIN, a specific inhibitor of Cdc42 activity, may possess anti‐aging properties, its specific effects on the epidermis and dermis, as well as the underlying mechanisms in naturally aging mice, remain unclear. Our study revealed that CASIN demonstrated the ability to increase epidermal and dermal thickness, enhance dermal‐epidermal junction, and stimulate collagen and elastic fiber synthesis in 9‐, 15‐, and 24‐month‐old C57BL/6 mice in vivo. Moreover, CASIN was found to enhance the proliferation, differentiation, and colony formation and restore the cytoskeletal morphology of primary keratinocytes in naturally aging skin in vitro. Furthermore, the anti‐aging properties of CASIN on primary fibroblasts in aging mice were mediated by the ribosomal protein RPL4 using proteomic sequencing, influencing collagen synthesis and cytoskeletal morphology both in vitro and in vivo. Meanwhile, both subcutaneous injection and topical application exhibited anti‐aging effects for a duration of 21 days. Additionally, CASIN exhibited anti‐inflammatory properties, while reduced expression of RPL4 was associated with increased inflammation in the skin of naturally aging mice. Taken together, our results unveil a novel function of RPL4 in skin aging, providing a foundational basis for future investigations into ribosomal proteins. And CASIN shows promise as a potential anti‐aging agent for naturally aging mouse skin, suggesting potential applications in the field. [ABSTRACT FROM AUTHOR]