PNPLA1 defects in patients with autosomal recessive congenital ichthyosis and KO mice sustain PNPLA1 irreplaceable function in epidermal omega-O-acylceramide synthesis and skin permeability barrier.

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of monogenic genodermatoses that encompasses non-syndromic disorders of keratinization. The pathophysiology of ARCI has been linked to a disturbance in epidermal lipid metabolism that impaired the stratum corneum function, leading to permeability barrier defects. Functional characterization of some genes involved in ARCI contributed to the identification of molecular actors involved in epidermal lipid synthesis, transport or processing. Recently, PNPLA1 has been identified as a gene causing ARCI. While other members of PNPLA family are key elements in lipid metabolism, the function of PNPLA1 remained unclear. We identified 5 novel PNPLA1 mutations in ARCI patients, mainly localized in the putative active enzymatic domain of PNPLA1. To investigate Pnpla1 biological role, we analysed Pnpla1-deficient mice. KO mice died soon after birth from severe epidermal permeability defects. Pnpla1-deficient skin presented an important impairment in the composition and organization of the epidermal lipids. Quantification of epidermal ceramide species highlighted a blockade in the production of ω-O-acylceramides with a concomitant accumulation of their precursors in the KO. The virtually loss of ω-O-acylceramides in the stratum corneum was linked to a defective lipid coverage of the resistant pericellular shell encapsulating corneocytes, the so-called cornified envelope, and most probably disorganized the extracellular lipid matrix. Finally, these defects in ω-O-acylceramides synthesis and cornified envelope formation were also evidenced in the stratum corneum from PNPLA1-mutated patients. Overall, our data support that PNPLA1/Pnpla1 is a key player in the formation of ω-O-acylceramide, a crucial process for the epidermal permeability barrier function.

Activography reveals aberrant proteolysis in desquamating diseases of differing backgrounds.

The role of epidermal proteolysis in overdesquamation was revealed in Netherton syndrome, a rare ichthyosis due to genetic deficiency of the LEKTI inhibitor of serine proteases. Recently, we developed activography, a new histochemical method, to spatially localize and semiquantitatively assess proteolytic activities using activity-based probes. Activography provides specificity and versatility compared to in situ zymography, the only available method to determine enzymatic activities in tissue biopsies. Here, activography was validated in skin biopsies obtained from an array of distinct disorders and compared with in situ zymography. Activography provides a methodological advancement due to its simplicity and specificity and can be readily adapted as a routine diagnostic assay. Interestingly, the levels of epidermal proteolysis correlated with the degree of desquamation independent of skin pathology. Thus, deregulated epidermal proteolysis likely represents a universal mechanism underlying aberrant desquamation.

Counterregulation between thymic stromal lymphopoietin- and IL-23-driven immune axes shapes skin inflammation in mice with epidermal barrier defects.

BACKGROUND: Epidermal barrier dysfunction has been recognized as a critical factor in the initiation and exacerbation of skin inflammation, particularly in patients with atopic dermatitis (AD) and AD-like congenital disorders, including peeling skin syndrome type B. However, inflammatory responses developed in barrier-defective skin, as well as the underlying mechanisms, remained incompletely understood. OBJECTIVE: We aimed to decipher inflammatory axes and the cytokine network in mouse skin on breakdown of epidermal stratum corneum barrier. METHODS: We generated Cdsn(iep-/-) mice with corneodesmosin ablation in keratinocytes selectively in an inducible manner. We characterized inflammatory responses and cytokine expression by using histology, immunohistochemistry, ELISA, and quantitative PCR. We combined mouse genetic tools, antibody-mediated neutralization, signal-blocking reagents, and topical antibiotic treatment to explore the inflammatory axes. RESULTS: We show that on breakdown of the epidermal stratum corneum barrier, type 2 and type 17 inflammatory responses are developed simultaneously, driven by thymic stromal lymphopoietin (TSLP) and IL-23, respectively. Importantly, we reveal a counterregulation between these 2 inflammatory axes. Furthermore, we show that protease-activated receptor 2 signaling is involved in mediating the TSLP/type 2 axis, whereas skin bacteria are engaged in induction of the IL-23/type 17 axis. Moreover, we find that IL-1ß is induced in skin of Cdsn(iep-/-) mice and that blockade of IL-1 signaling suppresses both TSLP and IL-23 expression and ameliorates skin inflammation. CONCLUSION: The inflammatory phenotype in barrier-defective skin is shaped by counterregulation between the TSLP/type 2 and IL-23/type 17 axes. Targeting IL-1 signaling could be a promising therapeutic option for controlling skin inflammation in patients with peeling skin syndrome type B and other diseases related to epidermal barrier dysfunction, including AD.

Mice deficient for the epidermal dermokine ß and γ isoforms display transient cornification defects.

Expression of the human dermokine gene (DMKN) leads to the production of four dermokine isoform families. The secreted α, ß and γ isoforms have an epidermis-restricted expression pattern, with Dmkn ß and γ being specifically expressed by the granular keratinocytes. The δ isoforms are intracellular and ubiquitous. Here, we performed an in-depth characterization of Dmkn expression in mouse skin and found an expression pattern that was less complex than in humans. In particular, mRNA coding for the δ family were absent. Homozygous mice null for the Dmkn ß and γ isoforms had no obvious phenotype but only a temporary scaly skin during the first week of life. The pups null for the Dmkn ß and γ isoforms had smaller keratohyalin granules and their cornified envelopes were more sensitive to mechanical stress. At the molecular level, amounts of profilaggrin and filaggrin monomers were reduced whereas amino acid components of the natural moisturizing factor were increased. In addition, the electrophoretic mobility of involucrin was modified, suggesting post-translational modifications. Finally, the mice null for the Dmkn ß and γ isoforms strongly overexpressed Dmkn α. These data are evocative of compensatory mechanisms relevant to the temporary phenotype. Overall, we improved the knowledge of Dmkn expression in mouse and highlighted a role for Dmkn ß and γ in cornification.

Expanding the Clinical and Genetic Spectrum of KRT1, KRT2 and KRT10 Mutations in Keratinopathic Ichthyosis.

Twenty-six families with keratinopathic ichthyoses (epidermolytic ichthyosis, superficial epidermolytic ichthyosis or congenital reticular ichthyosiform erythroderma) were studied. Epidermolytic ichthyosis is caused by mutations in the genes KRT1 or KRT10, mutations in the gene KRT2 lead to superficial epidermolytic ichthyosis, and congenital reticular ichthyosiform erythroderma is caused by frameshift mutations in the genes KRT10 or KRT1, which lead to the phenomenon of revertant mosaicism. In this study mutations were found in KRT1, KRT2 and KRT10, including 8 mutations that are novel pathogenic variants. We report here the first case of a patient with congenital reticular ichthyosiform erythroderma carrying a mutation in KRT10 that does not lead to an arginine-rich reading frame. Novel clinical features found in patients with congenital reticular ichthyosiform erythroderma are described, such as mental retardation, spasticity, facial dysmorphisms, symblepharon and malposition of the 4th toe.

Analysis of protein-protein interaction between late cornified envelope proteins and corneodesmosin.

Deletion of two members of the late cornified envelope (LCE) family, LCE3B and LCE3C (LCE3C_LCE3B-del), has been identified as risk factor for psoriasis with a possible role in skin barrier function. Moreover, genetic interaction between LCE3C_LCE3B-del and HLA-C*06, located in the psoriasis susceptibility regions 4 and 1 (PSORS4 and 1), has been reported in several populations. Because of high linkage disequilibrium between the PSORS1 genes HLA-C*06 and corneodesmosin (CDSN), both genes are potentially involved in psoriasis. As corneodesmosin and LCE proteins are both constituents of the stratum corneum, we investigated potential direct protein-protein interactions between six LCE proteins and two corneodesmosin sequence variants. Partial colocalization of LCE2 and CDSN was observed in normal and psoriasis skin using immunofluorescence microscopy. Co-expression of eCFP-LCE and mRFP-CDSN proteins in COS-1 cells and human adult keratinocytes, and GST pull-down results did not provide evidence for direct interactions between LCE proteins and CDSN variants.

Hypotrichosis simplex of the scalp is associated with nonsense mutations in CDSN encoding corneodesmosin.

We have identified nonsense mutations in the gene CDSN (encoding corneodesmosin) in three families suffering from hypotrichosis simplex of the scalp (HSS; OMIM 146520). CDSN, a glycoprotein expressed in the epidermis and inner root sheath (IRS) of hair follicles, is a keratinocyte adhesion molecule. Truncated CDSN aggregates were detected in the superficial dermis and at the periphery of hair follicles. Our findings suggest that CDSN is important in normal scalp hair physiology.

Epidermal 1-O-acylceramides appear with the establishment of the water permeability barrier in mice and are produced by maturating keratinocytes.

1-O-Acylceramides (1-OACs) have a fatty acid esterified to the 1-hydroxyl of the sphingosine head group of the ceramide, and recently we identified these lipids as natural components of human and mouse epidermis. Here we show epidermal 1-OACs arise shortly before birth during the establishment of the water permeability barrier in mice. Fractionation of human epidermis indicates 1-OACs concentrate in the stratum corneum. During in vitro maturation into reconstructed human epidermis, human keratinocytes dramatically increase 1-OAC levels indicating they are one source of epidermal 1-OACs. In search of potential enzymes responsible for 1-OAC synthesis in vivo, we analyzed mutant mice with deficiencies of ceramide synthases (Cers2, Cers3, or Cers4), diacylglycerol acyltransferases (Dgat1 or Dgat2), elongase of very long fatty acids 3 (Elovl3), lecithin cholesterol acyltransferase (Lcat), stearoyl-CoA desaturase 1 (Scd1), or acidic ceramidase (Asah1). Overall levels of 1-OACs did not decrease in any mouse model. In Cers3 and Dgat2-deficient epidermis they even increased in correlation with deficient skin barrier function. Dagt2 deficiency reshapes 1-OAC synthesis with an increase in 1-OACs with N-linked non-hydroxylated fatty acids and a 60% decrease compared to control in levels of 1-OACs with N-linked hydroxylated palmitate. As none of the single enzyme deficiencies we examined resulted in a lack of 1-OACs, we conclude that either there is functional redundancy in forming 1-OAC and more than one enzyme is involved, and/or an unknown acyltransferase of the epidermis performs the final step of 1-OAC synthesis, the implications of which are discussed.

A large-scale multi-technique approach identifies forty-nine new players of keratinocyte terminal differentiation in human epidermis.

At the latest stage of terminal differentiation in the epidermis, granular keratinocytes (GKs) undergo cornification, a programmed cell death required for the establishment of a functional skin barrier. A complex genetic regulatory network orchestrates the underlying biochemical modifications, but very few transcription factors specific to this programme have been identified to date. Here, we describe a large-scale, multi-technique approach performed on cells purified from normal human epidermis, primarily focusing on the identification of regulators. We combined data from microarray analysis of cell fractions enriched in GKs or basal keratinocytes, from an expressed sequence tag (EST) library built from GKs and from an in silico promoter analysis of 52 differentiation-associated genes. Among 3576 genes potentially expressed in GK, 298 candidates were selected, and half were directly profiled for the first time in the different layers of the epidermis by quantitative real-time PCR. Forty-nine genes upregulated during terminal differentiation, associated with numerous function of GK including lipid synthesis and secretion, were identified. Of 94 transcription factors detected, 37 were found to be either positively or negatively regulated, suggesting their involvement as regulators of gene expression in the GKs. These results largely extend the number of genes known as involved in the latest step of the terminal differentiation of human epidermis as well as the number of transcription factors known to control the expression of these genes.

Corneodesmosomes and corneodesmosin: from the stratum corneum cohesion to the pathophysiology of genodermatoses.

Corneodesmosin (CDSN) was identified 20 years ago by raising monoclonal antibodies against human plantar stratum corneum. The protein is specific to corneodesmosomes, cell-junction structures that, in humans, are found in the epidermis, the hard palate epithelium, and the inner root sheath of the hair follicles. Synthesized by the granular keratinocytes and secreted via the lamellar bodies, CDSN is incorporated into the desmoglea of the desmosomes, shortly before their transformation into corneodesmosomes during cornification. CDSN displays adhesive properties, mostly attributable to its N-terminal glycine-rich domain, and is sequentially proteolyzed as corneocytes migrate towards the skin surface prior to desquamation. The recent inactivation of Cdsn in mice induced a lethal epidermal barrier disruption and hair follicle degeneration, related to corneodesmosome dysfunction. That confirmed the essential role of the protein in maintaining integrity of the epidermis and the hair follicle. The CDSN gene is located in PSORS1, the major psoriasis susceptibility locus on the chromosome 6, but to date its involvement in the disease pathophysiology is not clear. By contrast, two different monogenic diseases associated with nonsense mutations in CDSN, were recently identified. First, hypotrichosis simplex of the scalp in which mutated CDSN accumulates in the dermis and forms amyloid deposits; then, peeling skin disease in which the genetic defect induces dyscohesion of the stratum corneum, responsible for abnormal desquamation and increased skin penetration of allergens.

Fatal Neonatal DOLK-CDG as a Rare Form of Syndromic Ichthyosis.

Neonatal collodion baby or ichthyosis can pose a diagnostic challenge, and in many cases, only additional organ involvement or the course of the disease will help differentiate between non-syndromic and syndromic forms. Skin abnormalities are described in about 20% of the congenital disorders of glycosylation (CDG). Among those, some rare CDG forms constitute a special group among the syndromic ichthyoses and can initially misdirect the diagnosis towards non-syndromic genodermatosis. DOLK-CDG is such a rare subtype, resulting from a defect in dolichol kinase, in which the congenital skin phenotype (often ichthyosis) is later associated with variable extracutaneous features such as dilatative cardiomyopathy, epilepsy, microcephaly, visual impairment, and hypoglycemia and may lead to a fatal course. We report two neonatal cases of lethal ichthyosis from the same family, with distal digital constrictions and a progressive course leading to multi-organ failure and death. Postmortem trio whole-exome sequencing revealed the compound heterozygous variants NM_014908.3: c.1342G>A, p.(Gly448Arg) and NM_014908.3: c.1558A>G, p.(Thr520Ala) in the DOLK gene in the first affected child, which were confirmed in the affected sibling. Reduced staining with anti-α-Dystroglycan antibody was observed in frozen heart tissue of the second child as an expression of reduced O-mannosylation due to the dolichol kinase deficiency. In addition to the detailed dermatopathological changes, both cases presented hepatic and extrahepatic hemosiderosis on histological examination. Our patients represent an early and fatal form of DOLK-CDG with a striking presentation at birth resembling severe collodion baby. Both cases emphasize the phenotypic variability of glycosylation disorders and the importance to broaden the differential diagnosis of ichthyosis and to actively search for organ involvement in neonates with ichthyosis.

A 4.2 kb upstream region of the human corneodesmosin gene directs site-specific expression in hair follicles and hyperkeratotic epidermis of transgenic mice.

Corneodesmosin (CDSN) is a desmosomal protein expressed in the epidermis during the late stages of differentiation and in the inner root sheath of hair follicles. The homophilic adhesive properties of the protein suggest that it reinforces keratinocyte cohesion in the upper layers of the epidermis (stratum granulosum and stratum corneum). In this study, we analyzed the expression of the CDSN gene in 16 human tissues. We confirmed the closely restricted expression pattern of CSDN. Indeed, apart from the skin, the mRNA was significantly detected only in the placenta and the thymus. As a step in elucidating the mechanisms of tissue-specific expression, transgenic mice bearing a 4.2 kb fragment of the human CSDN gene promoter linked to the LacZ gene were generated. The reporter-gene expression was detected in special areas of the inner root sheath of the hair follicles and the hair medulla but not in the epidermis. Induction of epidermis hyperproliferation however either by pharmacological agents or by wounding led to strong expression of the reporter gene in the keratinocytes of the stratum granulosum and the parakeratotic corneocytes of the stratum corneum. The data suggest that the genomic sequences and/or regulating factors responsible for the cell-specific expression of the human CDSN gene in the normal hair follicle as well as in the hyperproliferative epidermis are different from those necessary for expression in the normal epidermis.