Skin Barrier Formation Research Articles

Transglutaminase 1: Emerging Functions beyond Skin.

Transglutaminase enzymes catalyze Ca2+- and thiol-dependent posttranslational modifications of glutamine-residues that include esterification, hydrolysis and transamidation, which results in covalent protein-protein crosslinking. Among the eight transglutaminase family members in mammals, transglutaminase 1 (TG1) plays a crucial role in skin barrier formation via crosslinking and insolubilizing proteins in keratinocytes. Despite this established function in skin, novel functions have begun merging in normal tissue homeostasis as well as in pathologies. This review summarizes our current understanding of the structure, activation, expression and activity patterns of TG1 and discusses its putative novel role in other tissues, such as in vascular integrity, and in diseases, such as cancer and fibrosis.

A cellular disease model toward gene therapy of TGM1-dependent lamellar ichthyosis

Lamellar Ichthyosis (LI) is a chronic disease, mostly caused by mutations in TGM1 gene, marked by impaired skin barrier formation. No definitive therapies are available and current treatments aim at symptomatic relief. LI mouse models often fail to faithfully replicate the clinical and histopathological features of human skin conditions. To develop advanced therapeutic approaches, as combined ex vivo cell and gene therapy, we established a human cellular model of LI by efficient CRISPR-Cas9-mediated gene ablation of the TGM1 gene in human primary clonogenic keratinocytes. Gene edited cells showed complete absence of Transglutaminase-1 (TG1) expression and recapitulated a hyperkeratotic phenotype with most of the molecular hallmarks of LI in vitro. Using a SINγ-RV vector expressing transgenic TGM1 under the control of its own promoter, we tested an ex vivo gene therapy approach and validate the model of LI as a platform for pre-clinical evaluation studies. Gene-corrected TGM1-null keratinocytes displayed proper TG1 expression, enzymatic activity and cornified envelopes formation, hence restored proper epidermal architecture. Single cell multiomic analysis demonstrated proviral integrations in holoclone-forming epidermal stem cells, which are crucial to epidermal regeneration. This study serves as a proof-of-concept for assessing the potential of this therapeutic approach in treating TGM1 dependent LI.

A gain-of-function variant in SREBF1 causes generalized skin hyperpigmentation with congenital cataracts.

Lipid metabolism has essential roles in skin barrier formation and the regulation of skin inflammation. Lipid homeostasis regulates skin melanogenesis, although the underlying mechanism remains largely unknown. Sterol regulatory element binding protein 1 (SREBP-1) is a key transcription factor essential for cellular lipid metabolism. Loss-of-function variants in SREBF1 are responsible for autosomal-dominant ichthyosis follicularis, alopecia and photophobia syndrome, emphasizing the significance of lipid homeostasis in skin keratinization. To identify the genetic basis of a new entity featuring diffuse skin hyperpigmentation with congenital cataracts, and to unravel the underlying mechanism for the pathogenesis of the SREBF1 variant. Whole-exome sequencing was performed to identify underlying genetic variants. Quantitative polymerase chain reaction, Western blot and immunofluorescence staining were used to assess the expression and the subcellular localization of the SREBF1 variant. The transcriptional activity of mutant SREBP-1 was determined by a luciferase reporter assay. A transgenic zebrafish model was constructed. Two unrelated patients presented with generalized skin hyperpigmentation with skin xerosis, congenital cataracts and extracutaneous symptoms. We identified a de novo nonsense variant c.1289C>A (p.Ser430*) in SREBF1 in both patients. The variant encoded a truncated protein that showed preferential nucleus localization, in contrast to wild-type SREBP-1 which - in sterol-sufficient conditions - is mainly localized in the cytoplasm. The luciferase reporter assay revealed that the p.Ser430* mutant exhibited enhanced transcriptional activity. Cultured patient primary melanocytes showed increased melanin synthesis vs. those from healthy controls. At 35 days postfertilization, the p.Ser430* transgenic zebrafish model exhibited more black spots, along with upregulated expression of melanogenic genes. We demonstrated that a gain-of-function variant of SREBF1 causes a previously undescribed disorder characterized by generalized skin hyperpigmentation and congenital cataracts. Our study reveals the involvement of SREBP-1 in melanogenesis and lens development, and paves the way for the development of novel therapeutic targets for skin dyspigmentation or cataracts.

Alteration of epidermal lipid composition as a result of deficiency in the magnesium transporter Nipal4

Lipids in the stratum corneum play an important role in the formation of the skin permeability barrier. The causative gene for congenital ichthyosis, NIPAL4, encodes a Mg2+ transporter and is involved in increases in intracellular Mg2+ concentrations that depend on keratinocyte differentiation. However, the role of this increased Mg2+ concentration in skin barrier formation and its effect on the lipid composition of the stratum corneum has remained largely unknown. Therefore, in the present study, we performed a detailed analysis of epidermal lipids in Nipal4 KO mice via TLC and MS. Compared with WT mice, the Nipal4 KO mice showed compositional changes in many ceramide classes (including decreases in ω-O-acylceramides and increases in ω-hydroxy ceramides), together with increases in ω-hydroxy glucosylceramides, triglycerides, and free fatty acids and decreases in ω-O-acyl hydroxy fatty acids containing a linoleic acid. We also found increases in unusual ω-O-acylceramides containing oleic acid or palmitic acid in the KO mice. However, there was little change in levels of cholesterol or protein-bound ceramides. The TLC analysis showed that some unidentified lipids were increased, and the MS analysis showed that these were special ceramides called 1-O-acylceramides. These results suggest that elevated Mg2+ concentrations in differentiated keratinocytes affect the production of various lipids, resulting in the lipid composition necessary for skin barrier formation.

The WD Domain of Atg16l1 Crucial for LC3-Associated Phagocytosis Is Not Required for Preserving Skin Barrier Function in Mice

The skin is a multifunctional organ, forming a barrier between the external and internal environment, thereby functioning as a safeguard against extrinsic factors. Autophagy has been implicated in epidermal differentiation and in preserving skin homeostasis. LC3-associated phagocytosis (LAP) uses some but not all components of autophagy. The Atg16l1 ( WD) mouse model lacks the WD40 domain required for LAP and has been widely used to study the effects of LAP deficiency and autophagy on tissue homeostasis and response to infection. In this study, the WD model was used to study the relationship between LAP and skin homeostasis by determining whether LAP-deficient mice display a cutaneous phenotype. Skin histology of wild-type and WD mice aged 1 year revealed minor morphological differences in the tail skin dermal layer. RT-qPCR and western blot analysis showed no differences in key keratin expression between genotypes. Skin barrier formation, assessed by dye permeation assays, demonstrated full and proper formation of the skin barrier at embryonic day 18.5 in both genotypes. Biomechanical analysis of the skin showed decreased skin elasticity in aged WD but not wild-type mice. In summary, the LAP-deficient WD mice displayed subtle alterations in dermal histology and age-related biomechanical changes.

Clinical effectiveness of emollients with 30% urea and antiseptic Dorogov's stimulator in liposomal form in the complex therapy of chronic dermatoses

One of the modern trends in the study of the protective barrier of the skin is the study of the dynamics of the skin process. As a result of the active inflammatory process, a decrease in moisture levels occurs, a violation of the hydrolipid mantle of the skin and a decrease in natural moisturizing factors. The formation and maintenance of a protective skin barrier is an important pathogenetic tool in the treatment of inflammatory dermatoses. Chronic dermatoses often require regular topical therapy, so safe, convenient and effective topical regimens with good cosmetic properties can be very beneficial for this group of patients. Moisturizing and barrier-restoring formulations are clinically recognized as valuable topical treatments for patients with chronic dermatoses.

A modified protocol for studying filaggrin degradation using a reconstructed human epidermis model under low and high humidity.

Filaggrin (FLG) is an essential protein that plays a vital role in maintaining skin barrier function and moisture levels, allowing the skin to adapt to dry environments. However, the precise temporal dynamics of FLG metabolism in the human epidermis remain poorly understood, and suitable tools to study these time-dependent effects are currently lacking. To investigate the molecular mechanisms and time course of FLG metabolism and skin barrier function under high- and low-humidity conditions, utilizing a reconstructed epidermis model. EpiSkin specimens cultured under humid or dry conditions for varying durations (2-48 h) were compared by assessing FLG degradation and skin barrier formation using immunofluorescence staining and western blotting. Under conditions of low humidity, the proteolysis of FLG in EpiSkin increased between 4 and 12 h and was accompanied by elevated levels of cysteine-aspartic protease (caspase)-14. The expression of peptidyl arginine deiminase 1 and calpain 1 also increased at 4 h. However, after 24 h, the expression of these three FLG-degrading proteins significantly decreased. Conversely, the levels of pyrrolidone-5-carboxylic acid and urocanic acid initially decreased at 2 h and then increased between 12 and 24 h. Additionally, the expression of skin barrier proteins, such as FLG, transglutaminase 5, loricrin and zonula occludens-1, decreased starting from 12 h. Notably, epidermal cell viability and activity were also inhibited. We propose a reliable and ethical model to study the temporal dynamics of FLG metabolism and its role in skin barrier function. Using a commercially reconstructed epidermis to mimic dry skin formation obviates the need for animal and human testing.

Oat (Avena sativa L.) Sprouts Restore Skin Barrier Function by Modulating the Expression of the Epidermal Differentiation Complex in Models of Skin Irritation.

Oats (Avena sativa L.) are used as therapeutic plants, particularly in dermatology. Despite numerous studies on their skin moisturization, anti-inflammation, and antioxidation effects, the precise molecular mechanisms of these effects are only partially understood. In this study, the efficacy of oat sprouts in the treatment of allergic contact dermatitis (ACD) was investigated, and their specific phytoconstituents and exact mechanisms of action were identified. In the in vivo ACD model, by stimulating the mitogen-activated protein kinase signaling pathway, oat sprouts increased the expression levels of proteins associated with skin barrier formation, which are produced during the differentiation of keratinocytes. In addition, in a lipopolysaccharide-induced skin irritation model using HaCaT, steroidal saponins (avenacoside B and 26-deglucoavenacoside B) and a flavonoid (isovitexin-2-o-arabinoside) of oat sprouts regulated the genetic expression of the same proteins located on the adjacent locus of human chromosomes known as the epidermal differentiation complex (EDC). Furthermore, oat sprouts showed immunomodulatory functions. These findings suggest the potential for expanding the use of oat sprouts as a treatment option for various diseases characterized by skin barrier disruption.

Spontaneous Transition of Spherical Coacervate to Vesicle-Like Compartment.

Numerous biological systems contain vesicle-like biomolecular compartments without membranes, which contribute to diverse functions including gene regulation, stress response, signaling, and skin barrier formation. Coacervation, as a form of liquid-liquid phase separation (LLPS), is recognized as a representative precursor to the formation and assembly of membrane-less vesicle-like structures, although their formation mechanism remains unclear. In this study, a coacervation-driven membrane-less vesicle-like structure is constructed using two proteins, GG1234 (an anionic intrinsically disordered protein) and bhBMP-2 (a bioengineered human bone morphogenetic protein 2). GG1234 formed both simple coacervates by itself and complex coacervates with the relatively cationic bhBMP-2 under acidic conditions. Upon addition of dissolved bhBMP-2 to the simple coacervates of GG1234, a phase transition from spherical simple coacervates to vesicular condensates occurred via the interactions between GG1234 and bhBMP-2 on the surface of the highly viscoelastic GG1234 simple coacervates. Furthermore, the shell structure in the outer region of the GG1234/bhBMP-2 vesicular condensates exhibited gel-like properties, leading to the formation of multiphasic vesicle-like compartments. A potential mechanism is proposed for the formation of the membrane-less GG1234/bhBMP-2 vesicle-like compartments. This study provides a dynamic process underlying the formation of biomolecular multiphasic condensates, thereby enhancing the understanding of these biomolecular structures.

Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1

Transglutaminase 1 (TGM1) plays an essential role in skin barrier formation by cross-linking proteins in differentiated keratinocytes. Here, we established a protocol for the antibody-dependent detection of TGM1 protein and the parallel detection of TGM activity. TGM1 immunoreactivity initially increased and co-localized with membrane-associated TGM activity during keratinocyte differentiation. TGM activity persisted upon further differentiation of keratinocytes, whereas TGM1 immunoreactivity was lost under standard assay conditions. Pretreatment of tissue sections with the proteases trypsin or proteinase K enabled immunodetection of TGM1 in cornified keratinocytes, indicating that removal of other proteins was a prerequisite for TGM1 immunolabeling after cornification. The increase of TGM activity and subsequent loss of TGM1 immunoreactivity could be replicated in HEK293T cells transfected with TGM1, suggesting that protein cross-linking mediated by TGM1 itself may lead to reduced recognition of TGM1 by antibodies. To screen for proteins potentially regulating TGM1, we performed Virotrap experiments and identified the CAPNS1 subunit of calpain as an interaction partner of TGM1. Treatment of keratinocytes and TGM1-transfected HEK293T cells with chemical inhibitors of calpain suppressed transglutamination. Our findings suggest that calpain contributes to the control of TGM1-mediated transglutamination and proteins cross-linked by transglutamination mask epitopes of TGM1.

Inhibition of cutaneous heat-sensitive Ca2+ -permeable transient receptor potential vanilloid 3 channels alleviates UVB-induced skin lesions in mice.

Ultraviolet B (UVB) radiation causes skin injury by trigging excessive calcium influx and signaling cascades in the skin keratinocytes. The heat-sensitive Ca2+ -permeable transient receptor potential vanilloid 3 (TRPV3) channels robustly expressed in the keratinocytes play an important role in skin barrier formation and wound healing. Here, we report that inhibition of cutaneous TRPV3 alleviates UVB radiation-induced skin lesions. In mouse models of ear swelling and dorsal skin injury induced by a single exposure of weak UVB radiation, TRPV3 genes and proteins were upregulated in quantitative real-time PCR and Western blot assays. In accompany with TRPV3 upregulations, the expressions of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were also increased. Knockout of the TRPV3 gene alleviates UVB-induced ear swelling and dorsal skin inflammation. Furthermore, topical applications of two selective TRPV3 inhibitors, osthole and verbascoside, resulted in a dose-dependent attenuation of skin inflammation and lesions. Taken together, our findings demonstrate the causative role of overactive TRPV3 channel function in the development of UVB-induced skin injury. Therefore, topical inhibition of TRPV3 may hold potential therapy or prevention of UVB radiation-induced skin injury.

Harlequin Ichthyosis: A Rare Skin Disorder

Harlequin ichthyosis, an exceptionally rare and severe genetic skin disorder, presents a unique intersection of genetics, skin biology, and clinical manifestation. This congenital condition, a severe form of ichthyosis, has captivated the medical community due to its striking clinical features. It arises from genetic mutations in the ABCA12 gene, disrupting skin barrier formation. The clinical presentation is characterized by diamond-shaped scales, facial distortions, and medical challenges, particularly in neonates. Historical accounts have contributed to our evolving understanding, culminating in recent genetic advancements. While no cure exists, treatment involves a combination of medical interventions and supportive care. The psychosocial impact on affected individuals and families is profound. Despite challenges, the future is promising, driven by genomics, precision medicine, novel therapies, and collaborative research. Harlequin ichthyosis represents a remarkable example of progress at the intersection of science and compassion.

Introduction of a carboxylic acid group into pyrazolylpyridine derivatives increased selectivity for inhibition of the 20-HETE synthase CYP4A11/4F2

20-Hydroxyeicosatetraenoic acid (20-HETE) is a lipid mediator and one of the major arachidonic acid metabolites whose formation is mainly catalyzed by the enzymes cytochrome P450 (CYP) 4F2 and CYP4A11. Several studies have suggested that 20-HETE is involved in the pathogenesis of renal diseases, including diabetic nephropathy and autosomal dominant polycystic kidney disease, and we previously reported compound 1 as a dual inhibitor of CYP4A11/4F2 with therapeutic potential against renal fibrosis. Subsequent studies revealed that compound 1, the dual CYP4A11/4F2 inhibitor, however, exhibited low selectivity over another CYP4F subtype, CYP4F22, which catalyzes ω-hydroxylation of ultra-long-chain fatty acids (ULCFAs); ULCFAs are important for the formation of acylceramides, which play a role in skin barrier formation. Therefore, we sought to develop a CYP4A11/4F2 inhibitor that would show greater CYP4A11/4F2 selectivity against CYP4F22, to avoid potential dermatological side effects. We re-evaluated a series of compounds from our 20-HETE program and identified pyrazolylpyridine derivatives containing a carboxylic acid group showing only weak CYP4F22 inhibition. Subsequent optimization studies from these derivatives led to identification of compound 15, which showed CYP4A11/4F2 inhibition with improved selectivity against CYP4F22. Compound 15 inhibited 20-HETE production in both human and rat renal microsomes and did not inhibit ω-hydroxylation of ULCFAs in human keratinocytes. Compound 15 also significantly inhibited renal 20-HETE production after oral administration.

Determining the structure of protein-bound ceramides, essential lipids for skin barrier function

Protein-bound ceramides, specialized ceramides covalently bound to corneocyte surface proteins, are essential for skin permeability barrier function. However, their exact structure and target amino acid residues are unknown. Here, we found that epoxy-enone (EE) ceramides, precursors of protein-bound ceramides, as well as their synthetic analog, formed stable conjugates only with Cys among nucleophilic amino acids. NMR spectroscopy revealed that the β-carbon of the enone was attached by the thiol group of Cys via a Michael addition reaction. We confirmed the presence of Cys-bound EE ceramides in mouse epidermis by mass spectrometry analysis of protease-digested epidermis samples. EE ceramides were reversibly released from protein-bound ceramides via sulfoxide elimination. We found that protein-bound ceramides with reversible release properties accounted for approximately 60% of total protein-bound ceramides, indicating that Cys-bound EE ceramides are the predominant protein-bound ceramides. Our findings provide clues to the molecular mechanism of skin barrier formation by protein-bound ceramides.

Efficacy and safety of oral palmitoleic acid supplementation for skin barrier improvement: A 12-week, randomized, double-blinded, placebo-controlled study

BackgroundPalmitoleic acid (omega-7) has been reported to be effective primarily for metabolic disorders. Recently, it has been reported to help improve quality of life (QoL) by improving skin symptoms. ObjectiveThe aim of this randomized, double-blinded, placebo-controlled clinical study is to evaluate the efficacy and safety of oral palmitoleic acid in improving skin barrier, elasticity, and wrinkle formation in adult women. MethodsIn this randomized, double-blind, placebo-controlled clinical study, 90 healthy participants were enrolled and received 500 mg/day palmitoleic acid (intervention) or corn oil without palmitoleic acid (control) for 12 weeks. Skin hydration and transepidermal water loss and skin elasticity, surface roughness, eye wrinkle volume, and wrinkle severity were measured at 6-week intervals to assess the skin barrier function and efficacy in wrinkle improvement, respectively. ResultsAfter 12 weeks, skin hydration and transepidermal water loss significantly improved in the intervention group compared to the control group. Skin elasticity, surface roughness, eye wrinkle volume, wrinkle severity, and participant-assessed clinical improvement score did not significantly improve compared with the control group. ConclusionOral palmitoleic acid effectively improves the skin barrier function improvement, which may enhance QoL in aging adults.

1397 LSD1 is critical for epidermal development and skin barrier formation

1397 LSD1 is critical for epidermal development and skin barrier formation

771 Striatin-interacting protein 1 (STRIP1) is important for skin barrier formation

771 Striatin-interacting protein 1 (STRIP1) is important for skin barrier formation

Neryl acetate, the major component of Corsican Helichrysum italicum essential oil, mediates its biological activities on skin barrier.

Corsican Helichrysum italicum essential oil (HIEO) is characterized by high concentrations of neryl acetate, and we previously demonstrated that Corsican HIEO increases the expression of genes that are part of the differentiation complex (involucrin, small proline rich proteins, late cornified envelope, S100 protein family). The biological activities of HIEO and neryl acetate (NA) were compared to identify how NA contributes to HIEO activity on human skin. NA, as a part component of HIEO, was tested on skin explant models for 24 hours and 5 days in comparison with HIEO. We analyzed the biological regulations in the skin explant by transcriptomic analysis, skin barrier protein immunofluorescence, lipid staining and ceramide analysis by liquid chromatography-mass spectrometry. Transcriptomic analysis revealed that 41.5% of HIEO-modulated genes were also regulated by NA and a selected panel of genes were confirmed by qquantitative reverse transcription PCR analysis. Those genes are involved in epidermal differentiation, skin barrier formation and ceramide synthesis. Involucrin (IVL), involved in formation of the cornified envelope (CE), was upregulated at both gene and protein levels after 24 hours and 5 days respectively. After 5 days of treatment, total lipids and ceramides were also increased. Our results demonstrate that NA mediates a large part of Corsican HIEO activity on skin barrier formation.

High-speed AFM reveals a rare structural state for TRPV3.

TRPV3 is a cation selective ion channel belonging to the vanilloid-subfamily of temperature sensitive TRP channels. TRPV3 function is implicated in cutaneous pain, itch, and temperature-sensation, as well as in skin barrier formation, wound healing, and hair growth, while its dysfunction is associated with many diseases such as the Olmsted syndrome, atopic dermatitis, and rosacea. TRPV3 is activated and sensitized upon repeated stimulation by warm temperatures as well as by numerous chemicals, including plant extracts, lipid metabolites, and synthetic small molecules such as 2-aminoethoxydiphenyl borate (2-APB) and diphenylboronic anhydride (DPBA). TrpV3, like all other TRP channels, displays a homo-tetrameric structure, and possesses a similar architecture to those of the other thermo-sensitive TRPV channels. Multiple Cryo-EM structures of TRPV3 have been solved, revealing various conformations assigned as open, closed, and sensitized states. However, much information is still lacking regarding the full molecular-level mechanism of TrpV3 sensitization and gating, and it appears that more structural states have yet to be determined. Using high-speed atomic force microscopy (HS-AFM), which allows the direct imaging of membrane proteins at the single-molecule level in membrane and in buffer solution at physiological temperature, we reveal a hitherto uncharacterized and rare structural state. Interestingly, we demonstrate that the prevalence of this state is significantly increased upon the addition of DPBA, which has been shown in the past to lead to a secondary and enhanced TRPV3 current. With the ability of HS-AFM to visualize dynamic processes in real-time, we demonstrate the transience and reversibility of this state, providing an explanation as to why this state has so far eluded structural determination by cryo-EM. Overall, our results provide crucial structural information regarding TRPV3, and enable a more complete understanding of its full structural gating mechanism.

Dysregulated ceramide metabolism in mouse progressive dermatitis resulting from constitutive activation of Jak1

Coordinated lipid metabolism contributes to maintaining skin homeostasis by regulating skin barrier formation, immune reactions, thermogenesis, and perception. Several reports have documented the changes in lipid composition in dermatitis, including in atopic dermatitis (AD); however, the specific mechanism by which these lipid profiles are altered during AD pathogenesis remains unknown. Here, we performed untargeted and targeted lipidomic analyses of an AD-like dermatitis model resulting from constitutive activation of Janus kinase 1 (Spade mice) to capture the comprehensive lipidome profile during dermatitis onset and progression. We successfully annotated over 700 skin lipids, including glycerophospholipids, ceramides, neutral lipids, and fatty acids, many of which were found to be present at significantly changed levels after dermatitis onset, as determined by the pruritus and erythema. Among them, we found the levels of ceramides composed of nonhydroxy fatty acid and dihydrosphingosine containing very long-chain (C22 or more) fatty acids were significantly downregulated before AD onset. Furthermore, in vitro enzyme assays using the skin of Spade mice demonstrated the enhancement of ceramide desaturation. Finally, we revealed topical application of ceramides composed of nonhydroxy fatty acid and dihydrosphingosine before AD onset effectively ameliorated the progression of AD symptoms in Spade mice. Our results suggest that the disruption in epidermal ceramide composition is caused by boosting ceramide desaturation in the initiation phase of AD, which regulates AD pathogenesis.