Proliferation Of Human Keratinocytes Research Articles

Self-Assembled Fibroblast Growth Factor Nanoparticles as a Therapeutic for Oxidant-Induced Neuronal and Skin Cell Injury.

Traumatic brain injury (TBI) and spinal cord injury (SCI) are neurological conditions that result from immediate mechanical injury, as well as delayed injury caused by local inflammation. Furthermore, TBI and SCI often lead to secondary complications, including pressure wounds of the skin, which can heal slowly and are prone to infection. Pressure wounds are localized areas of damaged tissue caused by prolonged pressure on the skin due to immobility and loss of neurological sensation. With the aim to ameliorate these symptoms, we investigated whether fibroblast growth factors 2 (FGF-2) could contribute to recovery. FGF-2 plays a significant role in both neurogenesis and skin wound healing. We developed a recombinant fusion protein containing FGF-2 linked to elastin-like polypeptides (FGF-ELP) that spontaneously self-assembles into nanoparticles at around 33 °C. The nanoparticle's size was ranging between 220 and 250 nm in diameter at 2 μM. We tested this construct for its ability to address neuronal and skin cell injuries. Hydrogen peroxide was used to induce oxidant-mediated injury on cultured neuronal cells to mimic the impact of reactive oxidants released during the inflammatory response in vivo. We found that FGF-ELP nanoparticles protected against hydrogen peroxide-mediated injury and promoted neurite outgrowth. In the skin cell models, cells were depleted from serum to mimic the reduced levels of nutrients and growth factors in chronic skin wounds. FGF-ELP increased the proliferation and migration of human keratinocytes, fibroblasts, and endothelial cells. FGF-ELP is, therefore, a potentially useful agent to provide both neuroprotection and promotion of cellular processes involved in skin wound healing.

Type 17 immune response promotes oral epithelial cell proliferation in periodontitis

ObjectivesThis study aims to investigate the effects of type 17 immune response on the proliferation of oral epithelial cells in periodontitis. DesignA time-dependent ligature induced periodontitis mouse model was utilized to explore gingival hyperplasia and the infiltration of interleukin 17A (IL-17A) positive cells. Immunohistochemistry and flow cytometry were employed to determine the localization and expression of IL-17A in the ligature induced periodontitis model. A pre-existing single-cell RNA sequencing dataset, comparing individuals affected by periodontitis with healthy counterparts, was reanalyzed to evaluate IL-17A expression levels. We examined proliferation markers, including proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription (STAT3), Yes-associated protein (YAP), and c-JUN, in the gingival and tongue epithelium of the periodontitis model. An anti-IL-17A agent was administered daily to observe proliferative changes in the oral mucosa within the periodontitis model. Cell number quantification, immunofluorescence, and western blot analyses were performed to assess the proliferative responses of human normal oral keratinocytes to IL-17A treatment in vitro. ResultsThe ligature induced periodontitis model exhibited a marked infiltration of IL-17A-positive cells, alongside significant increase in thickness of the gingival and tongue epithelium. IL-17A triggers the proliferation of human normal oral keratinocytes, accompanied by upregulation of PCNA, STAT3, YAP, and c-JUN. The administration of an anti-IL-17A agent attenuated the proliferation in oral mucosa. ConclusionsThese findings indicate that type 17 immune response, in response to periodontitis, facilitates the proliferation of oral epithelial cells, thus highlighting its crucial role in maintaining the oral epithelial barrier.

LncRNA MIR181A2HG negatively regulates human keratinocytes proliferation by binding SRSF1.

The online version contains supplementary material available at 10.1007/s10616-024-00621-6.

Phloroglucinol Enhances Anagen Signaling and Alleviates H2O2-Induced Oxidative Stress in Human Dermal Papilla Cells.

Phloroglucinol (PG) is one of the abundant isomeric benzenetriols in brown algae. Due to its polyphenolic structure, PG exhibits various biological activities. However, the impact of PG on anagen signaling and oxidative stress in human dermal papilla cells (HDPCs) is unknown. In this study, we investigated the therapeutic potential of PG for improving hair loss. A non-cytotoxic concentration of PG increased anagen-inductive genes and transcriptional activities of β-Catenin. Since several anagen-inductive genes are regulated by β-Catenin, further experiments were performed to elucidate the molecular mechanism by which PG upregulates anagen signaling. Various biochemical analyses revealed that PG upregulated β-Catenin signaling without affecting the expression of Wnt. In particular, PG elevated the phosphorylation of protein kinase B (AKT), leading to an increase in the inhibitory phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at serine 9. Treatment with the selective phosphoinositide 3-kinase/AKT inhibitor, LY294002, restored the increased AKT/GSK3β/β-Catenin signaling and anagen-inductive proteins induced by PG. Moreover, conditioned medium from PG-treated HDPCs promoted the proliferation and migration of human epidermal keratinocytes via the AKT signaling pathway. Subsequently, we assessed the antioxidant activities of PG. PG ameliorated the elevated oxidative stress markers and improved the decreased anagen signaling in hydrogen peroxide (H2O2)-induced HDPCs. The senescence-associated β-galactosidase staining assay also demonstrated that the antioxidant abilities of PG effectively mitigated H2O2-induced senescence. Overall, these results indicate that PG potentially enhances anagen signaling and improves oxidative stress-induced cellular damage in HDPCs. Therefore, PG can be employed as a novel therapeutic component to ameliorate hair loss symptoms.

Inflammation Can Be a High-Risk Factor for Mucosal Nonunion of MRONJ by Regulating SIRT1 Signaling When Treated with an Oncologic Dose of Zoledronate.

Zoledronate (ZA) stands as a highly effective antiresorptive agent known to trigger medication-related osteonecrosis of the jaw (MRONJ). Its clinical dosages primarily encompass those used for oncologic and osteoporosis treatments. While inflammation is recognized as a potential disruptor of mucosal healing processes associated with ZA, prior research has overlooked the influence of varying ZA dosages on tissue adaptability. Therefore, a deeper understanding of the specific mechanisms by which inflammation exacerbates ZA-induced MRONJ, particularly when inflammation acts as a risk factor, remains crucial. Cell proliferation and migration of human oral keratinocytes (HOK) was analyzed after treatment with different doses of ZA and/or lipopolysaccharide (LPS) to assess their possible effect on mucosal healing of extraction wounds. Mouse periodontitis models were established using LPS, and histological changes in extraction wounds were observed after the administration of oncologic dose ZA. Hematoxylin and eosin (HE) staining and immunofluorescence were used to evaluate mucosal healing. In vitro, LPS did not exacerbate the effects of osteoporosis therapeutic dose of ZA on the proliferation and migration of HOK cells, while aggravated these with the oncologic dose of ZA treatment by inducing mitochondrial dysfunction and oxidative stress via regulating SIRT1 expression. Furthermore, SIRT1 overexpression can alleviate this process. In vivo, local injection of LPS increased the nonunion of mucous membranes in MRONJ and decreased the expression of SIRT1, PGC-1α, and MnSOD. Inflammation aggravates oncologic dose of ZA-induced mitochondrial dysfunction and oxidative stress via a SIRT1-dependent pathway, enhancing the risk of impaired mucosal healing in MRONJ. Our study implies that inflammation becomes a critical risk factor for MRONJ development at higher ZA concentrations. Elucidating the mechanisms of inflammation as a risk factor for mucosal non-healing in MRONJ could inform the development of SIRT1-targeted therapies.

Assessment of Some Unsymmetrical Porphyrins as Promising Molecules for Photodynamic Therapy of Cutaneous Disorders.

In order to select for further development novel photosensitizers for photodynamic therapy in cutaneous disorders, three unsymmetrical porphyrins, namely 5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl) porphyrin (P2.2), 5-(2-hydroxy-5-methoxyphenyl)-10,15,20-tris-(4-carboxymethylphenyl) porphyrin (P3.2), and 5-(2,4-dihydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl) porphyrin (P4.2), along with their fully symmetrical counterparts 5,10,15,20-tetrakis-(4-acetoxy-3-methoxyphenyl) porphyrin (P2.1) and 5,10,15,20-tetrakis-(4-carboxymethylphenyl) porphyrin (P3.1) were comparatively evaluated. The absorption and fluorescence properties, as well as atomic force microscopy measurements were performed to evaluate the photophysical characteristics as well as morphological and textural properties of the mentioned porphyrins. The cellular uptake of compounds and the effect of photodynamic therapy on the viability, proliferation, and necrosis of human HaCaT keratinocytes, human Hs27 skin fibroblasts, human skin SCL II squamous cell carcinoma, and B16F10 melanoma cells were assessed in vitro, in correlation with the structural and photophysical properties of the investigated porphyrins, and with the predictions regarding diffusion through cell membranes and ADMET properties. All samples were found to be isotropic and self-similar, with slightly different degrees of aggregability, had a relatively low predicted toxicity (class V), and a predicted long half-life after systemic administration. The in vitro study performed on non-malignant and malignant skin-relevant cells highlighted that the asymmetric P2.2 porphyrin qualified among the five investigated porphyrins to be a promising photosensitizer candidate for PDT in skin disorders. P2.2 was shown to accumulate well within cells, and induced by PDT a massive decrease in the number of metabolically active skin cells, partly due to cell death by necrosis. P2.2 had in this respect a better behavior than the symmetric P.2.1 compound and the related asymmetric compound P4.2. The strong action of P2.2-mediated PDT on normal skin cells might be an important drawback for further development of this compound. Meanwhile, the P3.1 and P3.2 compounds were not able to accumulate well in skin cells, and did not elicit significant PDT in vitro. Taken together, our experiments suggest that P2.2 can be a promising candidate for the development of novel photosensitizers for PDT in skin disorders.

Neutrophil-Derived MicroRNA-1290 Promotes Keratinocyte Proliferation in Psoriasis

The pathological hallmark of psoriasis is the infiltration of neutrophils into the skin. Some neutrophil-derived microRNAs (miRNAs) serve as biomarkers for various diseases, but none have been reported for psoriasis. In this study, we investigated the involvement of miRNAs released from neutrophils in psoriasis pathogenesis. We compared the expression of miRNAs in the sera of patients with psoriasis with that in healthy individuals and found that the expression of 2 miRNAs-miR-223 and miR-1290-was significantly upregulated in the sera of patients with psoriasis. The serum levels of these miRNAs positively correlated with the PASI and CRP levels. We used all-trans retinoic acid to induce the differentiation of human promyelocytic leukemia HL-60 cells into neutrophil-like cells and found that the release of both miRNAs increased during differentiation. Furthermore, the release of miR-1290 was increased by TNF-α in neutrophil-like cells and human neutrophils. Treatment with the miR-1290 precursor promoted the proliferation of human keratinocytes, increased the proportion of S-phase cells, and upregulated the phosphorylation of extracellular signal-regulated kinase 1/2. These results suggest that miR-1290 plays a vital role in regulating neutrophil differentiation and keratinocyte proliferation and could be a serum marker of psoriasis severity.

MiR-96-5p Suppresses Progression of Arsenite-Induced Human Keratinocyte Proliferation and Malignant Transformation by Targeting Denticleless E3 Ubiquitin Protein Ligase Homolog.

Long-term exposure to arsenic has been linked to a variety of cancers, among which skin cancer is the most prevalent form. However, the mechanism underlying arsenic carcinogenesis is unclear, and there is still limited information on the role of miRNAs in arsenic-induced skin cancer. This study aims to explore the role of miR-96-5p in the arsenite-induced proliferation and malignant transformation of human HaCaT keratinocytes. The GEO database (accession numbers GSE97303, GSE97305, and GSE97306) was used to extract mRNA and miRNA expression profiles of HaCaT cells treated with or without 0.1 μmol/L sodium arsenite for 3 and 7 weeks. In this paper, according to the CCK8 assay result, HaCaT cells exposed to 0.1 μmol/L sodium arsenite for 48 h were finalized. CCK8, MTT, EdU incorporation, and colony formation assays were used to determine the viability and proliferation of HaCaT cells and transformed HaCaT (T-HaCaT) cells. The subcellular localization and relative expression levels of DTL, as well as miR-96-5p in HaCaT cells induced by arsenite, were determined via immunofluorescence, RT-qPCR, and Western blot. Dual-luciferase reporter assay was performed to identify miR-96-5p bound directly to DTL. Transfection of miR-96-5p mimics or DTL siRNA was conducted to verify the arsenite-induced viability of HaCaT cells and T-HaCaT cells. T-HaCaT cells and nude mice were used to construct arsenite-induced malignant transformation and an in vivo xenograft model to demonstrate the over-expressed effect of miR-96-5p. The results showed that DTL was the target gene of miR-96-5p. Meanwhile, we also found that 0.1 μmol/L sodium arsenite upregulated DTL by decreasing the miR-96-5p level, leading to the proliferation and malignant transformation of HaCaT cells. MiR-96-5p agomir treatment slowed the growth of transplanted HaCaT cells transformed by arsenite in a manner associated with DTL downregulation in the nude mice xenograft model. Taken together, we confirmed that miR-96-5p, as a potent regulator of DTL, suppressed arsenite-induced HaCaT cell proliferation and malignant transformation, which might provide a novel therapeutic target for the treatment of arsenic-induced skin cancer.

Phytochemical and Nutraceutical Screening of Ethanol and Ethyl Acetate Phases of Romanian Galium verum Herba (Rubiaceae)

Galium species are used worldwide for their antioxidant, antibacterial, antifungal, and antiparasitic properties. Although this plant has demonstrated its antitumor properties on various types of cancer, its biological activity on cutaneous melanoma has not been established so far. Therefore, the present study was designed to investigate the phytochemical profile of two extracts of G. verum L. herba (ethanolic and ethyl acetate) as well as the biological profile (antioxidant, antimicrobial, and antitumor effects) on human skin cancer. The extracts showed similar FT-IR phenolic profiles (high chlorogenic acid, isoquercitrin, quercitrin, and rutin), with high antioxidant capacity (EC50 of ethyl acetate phase (0.074 ± 0.01 mg/mL) > ethanol phase (0.136 ± 0.03 mg/mL)). Both extracts showed antimicrobial activity, especially against Gram-positive Streptococcus pyogenes and Staphylococcus aureus bacilli strains, the ethyl acetate phase being more active. Regarding the in vitro antitumor test, the results revealed a dose-dependent cytotoxic effect against A375 melanoma cell lines, more pronounced in the case of the ethyl acetate phase. In addition, the ethyl acetate phase stimulated the proliferation of human keratinocytes (HaCaT), while this effect was not evident in the case of the ethanolic phase at 24 h post-stimulation. Consequently, G. verum l. could be considered a promising phytocompound for the antitumor approach of cutaneous melanoma.

Exosomes derived from uMSCs promote hair regrowth in alopecia areata through accelerating human hair follicular keratinocyte proliferation and migration.

Alopecia areata (AA) is a complex genetic disease that results in hair loss due to an autoimmune-mediated attack on the hair follicle. Mesenchymal stem cells (MSCs) have great potential to induce hair regeneration due to their strong secretion ability and multidirectional differentiation. Recent studies have revealed that the therapeutic potential of MSCs comes from their secretion ability, which can produce large amounts of bioactive substances and regulate the key physiological functions of subjects. The secretion products of MSCs, such as vesicles, exosomes, and conditioned media, have significant advantages in preparing of biological products derived from stem cells. Human umbilical cord mesenchymal stem cells (uMSCs) are the best choice for exosome production. uMSCs are obtained from the human umbilical cord. The umbilical cord is easy to obtain, and the efficiency of uMSCs isolation and culture higher than that of obtaining MSCs from bone marrow or adipose tissue. In this study, we investigated the effects of exosomes released from uMSCs in AA mice. In summary, due to easy isolation and cultivation, simple preparation, and convenient storage, it is possible to obtain uMSCs, or uMSCs exosomes for research and clinical treatment.

WTAP-Mediated N6-Methyladenosine of RNAs Facilitate the Pathophysiology of Atopic Dermatitis

N6-methyladenosine (m6A) is the most abundant dynamic and reversible internal chemical modification of RNA in eukaryotic cells and is essential in multiple pathophysiological processes. However, it has not been reported in atopic dermatitis (AD). We used Arraystar m6A-mRNA epitranscriptomic microarray to screen for differentially expressed genes (DEGs) and their m6A levels, and m6A-related enzymes in patients with AD. We confirmed that the m6A RNA methyltransferase Wilms tumor 1-associated protein (WTAP) and two candidate DEGs (S100 calcium-binding protein A9 [S100A9] and serpin peptidase inhibitor, clade B (ovalbumin), member 3 [SERPINB3] were significantly upregulated in keratinocytes in public data and epidermal lesions of patients with AD. In vitro cell experiments confirmed that WTAP influenced the expression of the two candidate DEGs and promoted primary human epidermal keratinocytes (HEKs) proliferation while inhibiting HEKs differentiation. Furthermore, we showed that WTAP, S100A9, and SERPINB3 expression correlated with AD severity. Our findings revealed that WTAP-mediated m6A modification promoted the expression of S100A9 and SERPINB3 to aggravate HEK proliferation and dysdifferentiation contributing to the pathophysiological development of AD.

Effects of an Adipose Mesenchymal Stem Cell-Derived Conditioned medium and TGF-β1 on Human Keratinocytes In Vitro.

Human keratinocytes play a crucial role during skin wound healing and in skin replacement therapies. The secretome of adipose-derived stem cells (ASCs) has been shown to secrete pro-healing factors, among which include TGF-β1, which is essential for keratinocyte migration and the re-epithelialization of cutaneous wounds during skin wound healing. The benefits of an ASC conditioned medium (ASC-CM) are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in keratinocytes. Here, we evaluated the composition and the innate characteristics of the ASC secretome and its biological effects on keratinocyte maturation and wound healing in vitro. In particular, we detected high levels of different growth factors, such as HGF, FGFb, and VEGF, and other factors, such as TIMP1 and 4, IL8, PAI-1, uPA, and IGFBP-3, in the ASC-CM. Further, we investigated, using immunofluorescence and flow cytometry, the distinct effects of a human ASC-CM and/or synthetic TGF-β1 on human keratinocyte proliferation, migration, and cell apoptosis suppression. We demonstrated that the ASC-CM increased keratinocyte proliferation as compared to TGF-β1 treatment. Further, we found that the ASC-CM exerted cell cycle progression in keratinocytes via regulating the phases G1, S, and G2/M. In particular, cells subjected to the ASC-CM demonstrated increased DNA synthesis (S phase) compared to the TGF-β1-treated KCs, which showed a pronounced G0/G1 phase. Furthermore, both the ASC-CM and TGF-β1 conditions resulted in a decreased expression of the late differentiation marker CK10 in human keratinocytes in vitro, whereas both treatments enhanced transglutaminase 3 and loricrin expression. Interestingly, the ASC-CM promoted significantly increased numbers of keratinocytes expressing epidermal basal keratinocyte markers, such DLL1 and Jagged2 Notch ligands, whereas those ligands were significantly decreased in TGF-β1-treated keratinocytes. In conclusion, our findings suggest that the ASC-CM is a potent stimulator of human keratinocyte proliferation in vitro, particularly supporting basal keratinocytes, which are crucial for a successful skin coverage after transplantation. In contrast, TGF-β1 treatment decreased keratinocyte proliferation and specifically increased the expression of differentiation markers in vitro.

Effects of Schumann resonance on the proliferation and migration of normal human epidermal keratinocytes and the expression of DEFB1 and SIRT1.

When the skin is damaged and its barrier function is disrupted, the proliferation and migration of epidermal keratinocytes are vital for repairing the damaged area. The Schumann resonance at 7.8 Hz has been reported to protect rat cardiomyocytes against oxidative stress and inhibit the proliferation of B16 mouse melanoma cells. However, its effect on the skin is unknown. In this study, we applied 7.8-Hz electromagnetic waves to normal human epidermal keratinocytes (NHEKs) and investigated its effects on cell proliferation and migration, β-defensin (DEFB1) and sirtuin 1 (SIRT1) expression. We performed cell proliferation assay, cell migrationassay and gene expression analysis of DEFB1 and SIRT1. We found that the application of 7.8-Hz electromagnetic waves caused a 2.8-fold increase in NHEK proliferation, enhanced cell migration, and increased the expression of DEFB1 and SIRT1 by 2.4-fold and 4.9-fold, respectively. These results suggest that the application of 7.8-Hz electromagnetic waves may contribute to improving the skin barrier function and skin ulcer.

Inhibition of the CoREST Repressor Complex Promotes Wound Re-Epithelialization through the Regulation of Keratinocyte Migration

Wound healing is a complex process involving phases of hemostasis, inflammation, proliferation, and remodeling. The regenerative process in the skin requires coordination between many regulators, including signaling molecules, transcription factors, and the epigenetic machinery. In this study, we show that chromatin regulators HDAC1 and LSD1, key components of the CoREST repressor complex, are upregulated in the regenerating epidermis during wound repair. We also show that corin, a synthetic dual inhibitor of the CoREST complex and HDAC1/LSD1 activities, significantly accelerates wound closure through enhanced re-epithelialization in a mouse tail wound model. Acetylated H3K9 (methylation of histone H3 at lysine 9) expression, a histone modification targeted by HDAC1, is increased in keratinocytes after topical treatment with 100 nM and 1 μM of corin. Invitro experiments demonstrate that corin promotes migration and inhibits the proliferation of human keratinocytes. Furthermore, expression levels of genes promoting keratinocyte migration, such as AREG, CD24, EPHB2, ITGAX, PTGS, SCT1, SERPINB2, SERPINE1, SLPI, SNAI2, and TWIST, increased in keratinocytes treated with corin. These data demonstrate that dual inhibition of class I histone deacetylases and LSD1 by corin may serve as a new approach for promoting wound re-epithelialization and provide a platform for further applications of corin for the treatment of chronic wounds.

Combination of Pinocembrin and Epidermal Growth Factor Enhances the Proliferation and Survival of Human Keratinocytes.

Re-epithelialization is delayed in aged skin due to a slow rate of keratinocyte proliferation, and this may cause complications. Thus, there has been development of new therapies that increase treatment efficacy for skin wounds. Epidermal growth factor (EGF) has been clinically used, but this agent is expensive, and its activity is less stable. Therefore, a stable compound possessing EGF-like properties may be an effective therapy, especially when combined with EGF. The current study discovered that pinocembrin (PC) effectively synergized with EGF in increasing keratinocyte viability. The combination of PC and EGF significantly enhanced the proliferation and wound closure rate of the keratinocyte monolayer through activating the phosphorylation of ERK and Akt. Although these effects of PC were like those of EGF, we clearly proved that PC did not transactivate EGFR. Recent data from a previous study revealed that PC activates G-protein-coupled receptor 120 which further activates ERK1/2 and Akt phosphorylation. Therefore, this clearly indicates that PC possesses a unique property to stimulate the growth and survival of keratinocytes through activating a different receptor, which subsequently conveys the signal to cross-talk with the effector kinases downstream of the EGFR, suggesting that PC is a potential compound to be combined with EGF.

LncRNA MIR31HG Regulates Proliferation and Migration by Targeting Matrix Gla Protein in Nonsyndromic Cleft Lip With or Without Cleft Palate.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common craniofacial birth defect with complex etiologies. Recently, the dysregulation of long noncoding RNAs (lncRNAs) has been implicated in many developmental diseases, including NSCL/P. However, the functions and mechanisms of lncRNAs in NSCL/P have not been fully elucidated. In this study, we found that lncRNA MIR31HG in NSCL/P patients was significantly downregulated than that in healthy individuals (GSE42589, GSE183527). In addition, single nucleotide polymorphism rs58751040 in MIR31HG was nominally associated with NSCL/P susceptibility (odds ratio: 1.29, 95% confidence interval: 1.03-1.54, p = 4.93 × 10-2) through a case-control study (504 NSCL/P cases and 455 controls). Luciferase activity assay showed that the C allele of rs58751040 revealed a decreased transcription activity of MIR31HG than the G allele. Moreover, knockdown of MIR31HG promoted cell proliferation and migration in human oral keratinocytes and human embryonic palate mesenchyme. Bioinformatic analysis and cellular studies suggested that MIR31HG may confer susceptibility to risk of NSCL/P through matrix Gla protein (MGP) signaling. In summary, we identified a novel lncRNA involved in the development of NSCL/P.

Defective cathepsin Z affects EGFR expression and causes autosomal dominant palmoplantar keratoderma.

The abnormal function of epidermal growth factor receptor (EGFR) has recently been shown to underlie various disorders of cornification. To delineate the genetic basis of a novel dominant form of palmoplantar keratoderma (PPK). Whole-exome (WES) and direct sequencing, quantitative real-time polymerase chain reaction, protein modelling, confocal immunofluorescence microscopy, immunoblotting, three-dimensional skin equivalents and an enzyme activity assay were used to delineate the genetic basis of a novel dominant form of PPK. WES revealed heterozygous variants (c.274T > C and c.305C > T) in CTSZ (encoding cathepsin Z) in four individuals (belonging to three unrelated families) with focal PPK. Bioinformatics and protein modelling predicted the variants to be pathogenic. Previous studies have suggested that EGFR expression may be subject to cathepsin regulation. Immunofluorescence revealed reduced cathepsin Z expression in the upper epidermal layers and concomitant increased epidermal EGFR expression in patients harbouring CTSZ variants. Accordingly, human keratinocytes transfected with constructs expressing PPK-causing variants in CTSZ displayed reduced cathepsin Z enzymatic activity, as well as increased EGFR expression. In line with the role played by EGFR in the regulation of keratinocyte proliferation, human keratinocytes transfected with the PPK-causing variants showed significantly increased proliferation that was abolished upon exposure to erlotinib, an EGFR inhibitor. Similarly, downregulation of CTSZ resulted in increased EGFR expression and increased proliferation in human keratinocytes, suggestive of a loss-of-function effect of the pathogenic variants. Finally, three-dimensional organotypic skin equivalents grown from CTSZ-downregulated cells showed increased epidermal thickness and EGFR expression as seen in patient skin; here, too, erlotinib was found to rescue the abnormal phenotype. Taken collectively, these observations attribute to cathepsin Z a hitherto unrecognized function in epidermal differentiation.

Designing a robust biocompatible porous polymeric membrane using Laponite and graphene oxide for versatile and selective adsorption of water contaminants

For reclaiming contaminated water, nanoparticle-incorporated porous polymeric membranes provide the requisite functionality for efficient and selective adsorption of toxic molecules. To this end, a simple yet innovative material selection strategy was adopted to prepare a robust and high-performance membrane system by the phase-inversion method using thermoplastic polyurethane (TPU), Laponite, graphene oxide (GO) and a hybrid derived from their aqueous “co-dispersion”, viz., Laponite/GO (LGO). The addition of 12 wt% LGO substantially improved the hydrophilicity, porosity, pure water flux (PWF), and mechanical properties of TPU membranes. Notably, the above membrane showed a striking 20-fold increase in PWF (from ∼27 to ∼652 Lm-2h−1), a 43-fold increase in elastic modulus, a 10-fold increase in tensile strength and a 3-fold increase in toughness and displayed synergistic improvement in mechanical properties. At 12 wt% LGO, the rejection efficiency of > 98%, >95%, and ∼86%, and the adsorption capacity of ∼47 mg/g, ∼49.3 mg/g and ∼16.8 mg/g for methylene blue (MB), crystal violet (CV) and rhodamine B (RhB) dyes, respectively were observed. The dye adsorption efficiency was comparable with that of poly(vinylidene fluoride) (PVDF)-LGO membranes. Owing to the anionic functional groups in LGO, the nanocomposite membranes displayed excellent selectivity of adsorption. The dye adsorption by the nanofiller-TPU membranes showed a pseudo-2nd-order kinetic behavior, indicating chemisorption; while the pristine TPU showed marginal physisorption of dyes. The LGO-filled membranes also showed efficient adsorption of Pb2+ and As5+ ions from water, with efficiency increasing with LGO content, showing > 90% efficiency with 15 wt% LGO. The nanocomposite membranes showed no cytotoxicity effect and supported the proliferation of human epidermal keratinocytes (HaCaT) cells. On these accounts, the LGO-TPU membranes promise the development of high-performance and cost-effective filtration membranes.

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.

IL17A Blockade with Ixekizumab Suppresses MuvB Signaling in Clinical Psoriasis

Unbiased informatics approaches have the potential to generate insights into uncharacterized signaling pathways in human disease. In this study, we generated longitudinal transcriptomic profiles of plaque psoriasis lesions from patients enrolled in a clinical trial of the anti-IL17A antibody ixekizumab (IXE). This dataset was then computed against a curated matrix of over 700 million data points derived from published psoriasis and signaling node perturbation transcriptomic and chromatin immunoprecipitation-sequencing datasets. We observed substantive enrichment within both psoriasis-induced and IXE-repressed gene sets of transcriptional targets of members of the MuvB complex, a master regulator of the mitotic cell cycle. These gene sets were similarly enriched for pathways involved in the regulation of the G2/M transition of the cell cycle. Moreover, transcriptional targets for MuvB nodes were strongly enriched within IXE-repressed genes whose expression levels correlated strongly with the extent and severity of the psoriatic disease. In models of human keratinocyte proliferation, genes encoding MuvB nodes were transcriptionally repressed by IXE, and depletion of MuvB nodes reduced cell proliferation. Finally, we made the expression and regulatory networks that supported this study available as a freely accessible, cloud-based hypothesis generation platform. Our study positions inhibition of MuvB signaling as an important determinant of the therapeutic impact of IXE in psoriasis.