A gene-centric approach to biomarker discovery identifies transglutaminase 1 as an epidermal autoantigen

Autoantigen discovery is a critical challenge for the understanding and diagnosis of autoimmune diseases. While autoantibody markers in current clinical use have been identified through studies focused on individual disorders, we postulated that a reverse approach starting with a putative autoantigen to explore multiple disorders might hold promise. We here targeted the epidermal protein transglutaminase 1 (TGM1) as a member of a protein family prone to autoimmune attack. By screening sera from patients with various acquired skin disorders, we identified seropositive subjects with the blistering mucocutaneous disease paraneoplastic pemphigus. Validation in further subjects confirmed TGM1 autoantibodies as a 55% sensitive and 100% specific marker for paraneoplastic pemphigus. This gene-centric approach leverages the wealth of data available for human genes and may prove generally applicable for biomarker discovery in autoimmune diseases.

Identification of Chicken Transglutaminase 1 and In Situ Localization of Transglutaminase Activity in Avian Skin and Esophagus

Transglutaminase 1 (TGM1) is a membrane-anchored enzyme that cross-links proteins during terminal differentiation of epidermal and esophageal keratinocytes in mammals. The current genome assembly of the chicken, which is a major model for avian skin biology, does not include an annotated region corresponding to TGM1. To close this gap of knowledge about the genetic control of avian cornification, we analyzed RNA-sequencing reads from organotypic chicken skin and identified TGM1 mRNA. By RT-PCR, we demonstrated that TGM1 is expressed in the skin and esophagus of chickens. The cysteine-rich sequence motif required for palmitoylation and membrane anchorage is conserved in the chicken TGM1 protein, and differentiated chicken keratinocytes display membrane-associated transglutaminase activity. Expression of TGM1 and prominent transglutaminase activity in the esophageal epithelium was also demonstrated in the zebra finch. Altogether, the results of this study indicate that TGM1 is conserved among birds and suggest that chicken keratinocytes may be a useful model for the study of TGM1 in non-mammalian cornification.

Physiological Effects of Manganese Gluconate and Calcium Combination from Saint-Gervais Mont Blanc Spring Water for Human Keratinocytes Differentiation

Alterations of skin barrier function affect quality of life and there is a need to develop dermatological/cosmetic treatments to reinforce or restore it. Inspiring of Hailey-Hailey disease, in which barrier alteration is due to a mutation of a Calcium-transporting protein (ATP2C1), we focused on the role of minerals and more especially those contained in Saint-Gervais Mont Blanc (SGMB) spring water to reinforce barrier function. Objectives: Demonstrate the interest to enrich SGMB spring water with manganese to improve both keratinocytes differentiation and barrier function. Methods: Effects of treatments on the expression of ATP2C1 and on the expression of key markers in keratinocyte differentiation and barrier function were studied by gene expression analysis on keratinocytes monolayers and also by measuring the protein expression of transglutaminase 1 using in situ immunofluorescence and image analysis in keratinocytes monolayers. Results: SGMB spring water stimulates transcriptomic expression of key markers involved in keratinocytes differentiation and barrier function while manganese gluconate has no effect. Combination of both dramatically enhances keratinocytes differentiation, in a synergistic way, at both transcriptomic and protein level. None of treatments modulated ATP2C1 expression. Conclusion: These results highlight the interest to enrich SGMB spring water with manganese to boost keratinocytes differentiation and barrier function.

c-Myc Upregulated by High Glucose Inhibits HaCaT Differentiation by S100A6 Transcriptional Activation

Keratinocyte differentiation dysfunction in diabetic skin is closely related to impaired skin barrier functions. We investigated the effects of c-Myc and S100A6 on Human immortal keratinocyte line (HaCaT) or keratinocyte differentiation and potential mechanisms. The expression levels of differentiation makers such as transglutaminase 1 (TGM1), loricrin (LOR), and keratin 1 (K1) were significantly reduced, while the expression of c-Myc was significantly increased in HaCaT cells cultured in high glucose and wound margin keratinocytes from diabetic rats and human patients. Overexpression of c-Myc caused differentiation dysfunction of HaCaT, while knocking down c-Myc promoted differentiation. High glucose increased the expression of c-Myc and inhibited differentiation in HaCaT cells by activating the WNT/β-catenin pathway. Moreover, inhibition of c-Myc transcriptional activity alleviated the differentiation dysfunction caused by high glucose or overexpression of c-Myc. c-Myc binds to the S100A6 promoter to directly regulate S100A6 expression and high glucose promoted S100A6 transcription. The expression of S100A6 was increased in HaCaT cultured with high glucose and wound margin keratinocytes from diabetic rats and human patients. However, the expression of S100A6 was decreased during normal HaCaT differentiation. HaCaT cells treated with S100A6 recombinant protein showed differentiation dysfunction. The expressions of TGM1, LOR and K1 in knockdown S100A6 HaCaT cells were higher than those in the control group. Overexpression of c-Myc or high glucose caused differentiation dysfunction of HaCaT cells, and was rescued by knocking down S100A6. These findings illustrate a new mechanism by which c-Myc upregulated by high glucose inhibits HaCaT differentiation by directly activating S100A6 transcription. Thus, c-Myc and S100A6 may be potential targets for the treatment of chronic diabetic wounds.

Lipid Metabolic Events Underlying the Formation of the Corneocyte Lipid Envelope

Cornified cells of the stratum corneum have a monolayer of an unusual lipid covalently attached to the outer surface. This is referred to as the corneocyte lipid envelope (CLE). It consists of a monolayer of ω-hydroxyceramides covalently attached to the outer surface of the cornified envelope. The CLE is essential for proper barrier function of the skin and is derived from linoleate-rich acylglucosylceramides synthesized in the viable epidermis. Biosynthesis of acylglucosylceramide and its conversion to the cornified envelope is complex. Acylglucosylceramide in the bounding membrane of the lamellar granule is the precursor of the CLE. The acylglucosylceramide in the limiting membrane of the lamellar granule may be oriented with the glucosyl moiety on the inside. Conversion of the acylglucosylceramide to the CLE requires removal of the glucose by action of a glucocerebrosidase. The ester-linked fatty acid may be removed by an as yet unidentified esterase, and the resulting ω-hydroxyceramide may become ester linked to the outer surface of the cornified envelope through action of transglutaminase 1. Prior to removal of ester-linked fatty acids, linoleate is oxidized to an epoxy alcohol through action of 2 lipoxygenases. This can be further oxidized to an epoxy-enone, which can spontaneously attach to the cornified envelope through Schiff’s base formation. Mutations of genes coding for enzymes involved in biosynthesis of the CLE result in ichthyosis, often accompanied by neurologic dysfunction. The CLE is recognized as essential for barrier function of skin, but many questions about details of this essentiality remain. What are the relative roles of the 2 mechanisms of lipid attachment? What is the orientation of acylglucosylceramide in the bounding membrane of lamellar granules? Some evidence supports a role for CLE as a scaffold upon which intercellular lamellae unfold, but other evidence does not support this role. There is also controversial evidence for a role in stratum corneum cohesion. Evidence is presented to suggest that covalently bound ω-hydroxyceramides serve as a reservoir for free sphingosine that can serve in communicating with the viable epidermis and act as a potent broad-acting antimicrobial at the skin surface. Many questions remain.

Hydroxylumisterols, Photoproducts of Pre-Vitamin D3, Protect Human Keratinocytes against UVB-Induced Damage

Lumisterol (L3) is a stereoisomer of 7-dehydrocholesterol and is produced through the photochemical transformation of 7-dehydrocholesteol induced by high doses of UVB. L3 is enzymatically hydroxylated by CYP11A1, producing 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3. Hydroxylumisterols function as reverse agonists of the retinoic acid-related orphan receptors α and γ (RORα/γ) and can interact with the non-genomic binding site of the vitamin D receptor (VDR). These intracellular receptors are mediators of photoprotection and anti-inflammatory activity. In this study, we show that L3-hydroxyderivatives significantly increase the expression of VDR at the mRNA and protein levels in keratinocytes, both non-irradiated and after UVB irradiation. L3-hydroxyderivatives also altered mRNA and protein levels for RORα/γ in non-irradiated cells, while the expression was significantly decreased in UVB-irradiated cells. In UVB-irradiated keratinocytes, L3-hydroxyderivatives inhibited nuclear translocation of NFκB p65 by enhancing levels of IκBα in the cytosol. This anti-inflammatory activity mediated by L3-hydroxyderivatives through suppression of NFκB signaling resulted in the inhibition of the expression of UVB-induced inflammatory cytokines, including IL-17, IFN-γ, and TNF-α. The L3-hydroxyderivatives promoted differentiation of UVB-irradiated keratinocytes as determined from upregulation of the expression at the mRNA of involucrin (IVL), filaggrine (FLG), and keratin 14 (KRT14), downregulation of transglutaminase 1 (TGM1), keratins including KRT1, and KRT10, and stimulation of ILV expression at the protein level. We conclude that CYP11A1-derived hydroxylumisterols are promising photoprotective agents capable of suppressing UVB-induced inflammatory responses and restoring epidermal function through targeting the VDR and RORs.