Hornerin is a component of the epidermal cornified cell envelopes.

A single-nucleotide polymorphism within the gene encoding hornerin (HRNR) has recently been linked with atopic dermatitis (AD) susceptibility. HRNR shares features with filaggrin, a key protein for keratinocyte differentiation, but conflicting reports have been published concerning its expression in the epidermis, and its role is still unknown. To analyze HRNR expression and function in the epidermis, anti-HRNR antibodies were produced and used in Western blot analysis and immunohistochemical, confocal, and immunoelectron microscopy analyses of human skin and of cornified cell envelopes purified from plantar stratum corneum. We also tested whether HRNR was a substrate of transglutaminases. In the epidermis, HRNR was detected at the periphery of keratohyalin granules in the upper granular layer and at the corneocyte periphery in the whole cornified layer. Detected in Western blot analysis as numerous bands, HRNR was relatively insoluble and only extracted from epidermis with urea and/or reducing agents. The presence of HRNR in the purified envelopes was confirmed by immunoelectron microscopy and by Western blot analysis after V8-protease digestion. HRNR was shown to be a substrate of transglutaminase 3. These data demonstrate that HRNR is a component of cornified cell envelopes of human epidermis. Its reduced expression in AD may contribute to the epidermal barrier defect observed in the disease.

KazrinE is a desmosome-associated liprin that colocalises with acetylated microtubules.

Kazrin is a widely expressed, evolutionarily conserved cytoplasmic protein that binds the cytolinker protein periplakin. Multiple functions of kazrin have been reported, including regulation of desmosome assembly, embryonic tissue morphogenesis and epidermal differentiation. Here, we identify kazrinE as a kazrin isoform that contains a liprin-homology domain (LHD) and forms complexes with kazrinA, kazrinB and kazrinC. As predicted from the presence of the LHD, kazrinE can associate with the leukocyte common antigen-related (LAR) protein tyrosine phosphatase in a phosphorylation-dependent manner. When overexpressed in epidermal keratinocytes, kazrinE induces changes in cell shape and stimulates terminal differentiation. Like the other kazrin isoforms, kazrinE localises to the nucleus and desmosomes. However, in addition, kazrinE associates with stabilised microtubules via its LHD. During terminal differentiation, the keratinocyte microtubule network undergoes extensive reorganisation; in differentiating keratinocytes, endogenous kazrinE colocalises with microtubules, but periplakin does not. We speculate that the kazrinE-microtubule interaction contributes to the mechanism by which kazrin regulates desmosome formation and epidermal differentiation.

Deimination and expression of peptidylarginine deiminases during cutaneous wound healing in mice.

Deimination, the conversion of protein-bound arginines into citrullines, is a post-translational modification catalyzed by a peptidylarginine deiminase (Pad). In the epidermis, three Pads are expressed, namely Pad1, 2 and 3, and the major deiminated protein is filaggrin. Deimination of fibrin has been observed in various pathological inflammatory conditions. Here, we analyzed the expression of Pads and citrullination of proteins during cutaneous wound healing, i.e. in a physiological inflammatory condition. Full-thickness punches were performed on adult mouse back skin, and wound recovery was analyzed over 10 days by immunohistology and western blotting. Pad1 was immunodetected in all the neo-epidermis. Pad3, normally expressed in the stratum granulosum, was not detected in the hyperproliferative tongue of the neo-epidermis, but was shown to be co-localized with (pro)filaggrin in a large number of keratinocyte layers in its differentiating part. Deiminated proteins were detected in the stratum corneum of the neo-epidermis in the late phase of re-epithelialization, and in the clot and the clot-derived scab. In the clot where we only detected Pad4, one of the deiminated proteins was shown to be fibrin. Deimination of the clot proteins, and more generally wound healing and keratinocyte differentiation, seemed to be Pad2-independent, as shown using Padi2(-/-) mice.

[Deimination or citrullination, a post-translational modification with many physiological and pathophysiological facets]. / La désimination ou citrullination: une modification post-traductionnelle aux multiples facettes.

Deimination or citrullination, is a post-translational modification with many facets. It is involved in several basic cellular processes, including gene regulation, embryonic development and terminal differentiation, and also in various pathophysiological mechanisms linked to severe human diseases such as multiple sclerosis and rheumatoid arthritis. Deimination, the calcium-dependent enzymatic conversion of peptidyl-arginine to peptidyl-citrulline, induces a decrease in the charge of the modified proteins with major consequences on their conformation, stability and/or interactions, and therefore on their functions. Five isotypes of peptidylarginine deiminases (1-4 and 6), exist in humans with a variable tissue expression. These highly conserved enzymes are closely regulated at transcriptional and post-transcriptional levels, probably including auto-deimination.

Peptidylarginine deiminases and deimination in biology and pathology: relevance to skin homeostasis.

Deimination corresponds to the transformation of arginine residues within a peptide sequence into citrulline residues. Catalyzed by peptidylarginine deiminases, it decreases the net positive charge of proteins, alters intra and intermolecular ionic interactions and probably the folding of target proteins. Deimination has recently been implicated in several physiological and pathological processes. Here, we describe the enzymes involved in this post-translational modification, focusing on their expression, location and roles in skin, as well as their known protein substrates in the epidermis and hair follicles. We discuss also the potential involvement of deimination in human diseases including cutaneous disorders.

Peptidylarginine deiminase isoforms 1-3 are expressed in the epidermis and involved in the deimination of K1 and filaggrin.

Post-translational conversion of arginine to citrulline residues is catalyzed by peptidylarginine deiminases (PAD). Although the existence of five isoforms of PAD has been reported in rodents and humans, their tissue distribution, substrate specificity, and physiological function have yet to be explored. In the epidermis, deimination of filaggrin and keratins is involved in maintaining hydration of the stratum corneum (SC), and hence the cutaneous barrier function. Here, RT-PCR, western blotting, and confocal microscopy analyses with anti-peptide antibodies highly specific for each of the PAD1-4 demonstrated that only PAD1-3 are expressed in mouse and human epidermis. PAD1 was detected in all layers, including the SC, and PAD2 in all the living layers, whereas PAD3 expression was shown to be restricted to the granular layer and lower SC. Moreover, PAD1 and 3 were observed to co-localize with (pro)filaggrin, and PAD2 to be located at the keratinocyte periphery in the stratum granulosum. We also detected PAD1 in extracts of superficial SC, where K1 is deiminated. Moreover, we showed that PAD1 and 3 are able to modify filaggrin in vitro. These data strongly suggest that each enzyme exerts a specific role in the course of epidermis differentiation.

Peptidylarginine deiminase isoforms are differentially expressed in the anagen hair follicles and other human skin appendages.

Peptidylarginine deiminases (PAD) catalyze the conversion of arginine residues to citrullines. Five isoforms are known that present distinct tissue locations. In the epidermis, like in the skin, only PAD1, 2, and 3 are expressed. Their pattern of expression in skin appendages is not known. Here, confocal microscopy analysis using highly specific antibodies demonstrated that PAD1 and 3 are expressed in human anagen hair follicles, PAD1 and 2, in arrector pili muscles and sweat glands, whereas no PAD were detected in sebaceous glands. PAD1 was detected in the cuticle and the Huxley layer of the inner root sheath (IRS), and in the companion layer. PAD3 was localized in the medulla, and in the three layers of the IRS. Using anti-modified citrulline antibodies, we also showed that deiminated proteins appeared in the lower part of the IRS, first in the Henle layer, then in the cuticle, and finally in the Huxley layer. Our data demonstrate that PAD3 is the enzyme that deiminates trichohyalin in the medulla and the Henle layer, indicate that PAD1 and 3 are involved in the hair follicle program of differentiation, and suggest a role for PAD1 and 2 in the physiology of sweat glands and arrector pili muscles.

Comparative analysis of the mouse and human peptidylarginine deiminase gene clusters reveals highly conserved non-coding segments and a new human gene, PADI6.

Peptidylarginine deiminases (PADs) convert arginine residues in proteins into citrullines. They are suspected to be involved in multiple sclerosis and rheumatoid arthritis pathophysiology, and they play a role in epidermis homeostasis and possibly in regulation of gene expression through histone modification. In humans, four isoforms encoded by the genes PADI1-4 are known so far. We here report the characterization and comparative analysis of the human (355 kb) and mouse (240 kb) PAD gene clusters on chromosomes 1p35-36 and 4E1, respectively. We characterized an as yet unknown human PADI6 gene, and cloned the corresponding cDNA encoding a 694-amino-acid protein. RT-PCR analysis showed a rather restricted pattern of tissue-specific expression, mainly in ovary, testis and peripheral blood leukocytes. Nucleotide substitution rates suggest that PADI genes are under purifying selection. Comparative analysis of the human and mouse sequences identified 251 conserved non-coding segments predominantly clustered within the promoter regions, the large (>10 kb) first intron of each of the genes PADI1-3, and an 8 kb PADI1-2 intergenic region. The presence of numerous transcription factor binding sites suggests the segments are putative regulatory elements. This study is the first description of the human PADI6 gene and encoded protein, and the first step towards a better understanding of the coordinated regulation of PADI gene expression.

Knockdown of filaggrin in a three-dimensional reconstructed human epidermis impairs keratinocyte differentiation.

Atopic dermatitis is a chronic inflammatory skin disorder characterized by defects in the epidermal barrier and keratinocyte differentiation. The expression of filaggrin, a protein thought to have a major role in the function of the epidermis, is downregulated. However, the impact of this deficiency on keratinocytes is not really known. This was investigated using lentivirus-mediated small-hairpin RNA interference in a three-dimensional reconstructed human epidermis (RHE) model, in the absence of other cell types than keratinocytes. Similar to what is known for atopic skin, the experimental filaggrin downregulation resulted in hypogranulosis, a disturbed corneocyte intracellular matrix, reduced amounts of natural moisturizing factor components, increased permeability and UV-B sensitivity of the RHE, and impaired keratinocyte differentiation at the messenger RNA and protein levels. In particular, the amounts of two filaggrin-related proteins and one protease involved in the degradation of filaggrin, bleomycin hydrolase, were lower. In addition, caspase-14 activation was reduced. These results demonstrate the importance of filaggrin for the stratum corneum properties/functions. They indicate that filaggrin downregulation in the epidermis of atopic patients, either acquired or innate, may be directly responsible for some of the disease-related alterations in the epidermal differentiation program and epidermal barrier function.

Exons 5-15 of kazrin are dispensable for murine epidermal morphogenesis and homeostasis.

Kazrin binds to periplakin and ARVCF catenin, and regulates adhesion and differentiation of cultured human keratinocytes. To explore kazrin function in vivo, we generated a kazrin gene-trap mouse in which only exons 1-4 were expressed, fused to ß-galactosidase. On transient transfection, the protein encoded by exons 1-4 did not enter the nucleus, but did cause keratinocyte shape changes. The mice had no obvious defects in skin development or homeostasis, and periplakin and desmoplakin localization was normal. Expression of the kazrin-ß-galactosidase fusion protein faithfully reported endogenous kazrin expression. Kazrin was not expressed in embryonic epidermis and was first detected at postnatal day 1. In adult mice, epidermal kazrin expression was less widespread than in humans and Xenopus, being confined to the bulb of anagen hair follicles, the infundibulum, and parakeratotic tail epidermis. In anagen bulbs, kazrin was expressed by a band of cells with elongated morphology and low desmoplakin levels, suggesting a role in morphogenetic cell movements. We conclude that exons 5-15 of kazrin, encoding the nuclear localization signal and C-terminal domain, are not required for epidermal development and function. The previously reported role of kazrin in regulating cell shape appears to reside within the N-terminal coiled-coil domain encoded by exons 1-4.

An intronic enhancer driven by NF-κB contributes to transcriptional regulation of peptidylarginine deiminase type I gene in human keratinocytes.

Peptidylarginine deiminases (PADs) catalyze the conversion of protein-bound arginine to citrulline residues. In human epidermis, where filaggrin is the main deiminated protein, three PADs are detected with specific patterns of expression depending on the keratinocyte (KC) differentiation state. Previous characterizations of the PAD-encoding gene promoters have shown that proximal regulation alone is not sufficient to explain this specificity of expression. In this work, we describe an evolutionarily highly conserved nucleotide segment located in the first intron of the PAD1 gene (PADI1). Luciferase reporter assays showed that it enhances the activity of the PADI1 promoter, in a calcium- and orientation-independent manner. Mutation of a putative NF-κB cis-element markedly reduced its enhancer activity, which also confirmed its potential regulatory function. Chromatin immunoprecipitation assays evidenced the binding of both p65 and p50 NF-κB subunits to the cis-element, and RNA interference inhibition assays confirmed that NF-κB contributes to the PADI1 transcriptional control. Furthermore, the intronic enhancer and promoter of PADI1 potentially interact through chromatin looping, as indicated by chromosome conformation capture assays. Our findings provide evidence that an NF-κB-mediated signaling pathway is involved in PADI1 regulation in human epidermal KCs.

Kazrin regulates keratinocyte cytoskeletal networks, intercellular junctions and differentiation.

Kazrin is an evolutionarily conserved protein that is upregulated during keratinocyte terminal differentiation. Kazrin localizes to desmosomes and binds the epidermal cornified envelope protein periplakin. Kazrin overexpression in human epidermal keratinocytes caused profound changes in cell shape, reduced filamentous actin, reorganized keratin filaments, and impaired assembly of intercellular junctions. These effects were attributable to decreased Rho activity in kazrin-overexpressing cells. Kazrin overexpression also stimulated terminal differentiation and reduced clonal growth in culture. Knockdown of kazrin decreased expression of differentiation markers and stimulated proliferation without changing total Rho activity. We conclude that kazrin is a dual regulator of intercellular adhesion and differentiation in keratinocytes and regulates these processes by Rho-dependent and -independent mechanisms.

Mice deficient in involucrin, envoplakin, and periplakin have a defective epidermal barrier.

The cornified envelope is assembled from transglutaminase cross-linked proteins and lipids in the outermost epidermal layers and is essential for skin barrier function. Involucrin, envoplakin, and periplakin form the protein scaffold on which the envelope assembles. To examine their combined function, we generated mice deficient in all three genes. The triple knockouts have delayed embryonic barrier formation and postnatal hyperkeratosis (abnormal accumulation of cornified cells) resulting from impaired desquamation. Cornified envelopes form but are ultrastructurally abnormal, with reduced lipid content and decreased mechanical integrity. Expression of proteases is reduced and the protease inhibitor, serpina1b, is highly upregulated, resulting in defective filaggrin processing and delayed degradation of desmoglein 1 and corneodesmosin. There is infiltration of CD4+ T cells and a reduction in resident gammadelta+ T cells, reminiscent of atopic dermatitis. Thus, combined loss of the cornified envelope proteins not only impairs the epidermal barrier, but also changes the composition of T cell subpopulations in the skin.

Peptidyl arginine deiminase type 2 (PAD-2) and PAD-4 but not PAD-1, PAD-3, and PAD-6 are expressed in rheumatoid arthritis synovium in close association with tissue inflammation.

OBJECTIVE: Autoantibodies to citrullinated proteins (ACPAs) are specific for rheumatoid arthritis (RA) and probably are involved in its pathophysiology. Citrullyl residues, posttranslationally generated by peptidyl arginine deiminase (PAD), are indispensable components of ACPA-targeted epitopes. The aim of this study was to identify which PAD isotypes are expressed in the synovial tissue (ST) of patients with RA and are involved in the citrullination of fibrin, the major synovial target of ACPAs. METHODS: Expression of all PAD isotypes, including the recently described PAD type 6 (PAD-6), was explored by reverse transcription-polymerase chain reaction and immunoblotting, first in blood-derived mononuclear leukocytes from healthy donors, then in ST samples from 16 patients with RA and 11 control patients (4 with other arthritides and 7 with osteoarthritis [OA]). In ST samples from patients with RA, PADs were localized by immunohistochemistry. RESULTS: In lymphocytic and monocytic cells and, similarly, in ST samples from patients with RA, the PAD-2, PAD-4, and PAD-6 genes were found to be transcribed, but only PAD-2 and PAD-4 enzymes were detected. PAD-2 was also expressed in ST from control patients, including those with OA, while PAD-4 was preferentially expressed in ST from patients with other arthritides. In RA, the expression levels of PAD-2 and PAD-4 were correlated with the intensity of inflammation (cell infiltration, hypervascularization, and synovial lining hyperplasia), and both enzymes were demonstrable within or in the vicinity of citrullinated fibrin deposits. CONCLUSION: PAD-2 and PAD-4 are the only PAD isotypes expressed in the ST of patients with RA and those with other arthritides. Inflammatory cells are a major source, but PAD-4 also comes from hyperplastic synoviocytes. Both isotypes are probably involved in the citrullination of fibrin.

cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I.

Peptidylarginine deiminases (PADs) catalyse a post-translational modification of proteins through the conversion of arginine residues into citrullines. The existence of four isoforms of PAD (types I, II, III and IV) encoded by four different genes, which are distinct in their substrate specificities and tissue-specific expression, was reported in rodents. In the present study, starting from epidermis polyadenylated RNA, we cloned by reverse transcriptase-PCR a full-length cDNA encoding human PAD type I. The cDNA was 2711 bp in length and encoded a 663-amino-acid sequence. The predicted protein shares 75% identity with the rat PAD type I sequence, but displays only 50-57% identity with the three other known human isoforms. We have described the organization of the human PAD type I gene on chromosome 1p36. A recombinant PAD type I was produced in Escherichia coli and shown to be enzymically active. Human PAD type I mRNAs were detected by reverse transcriptase-PCR not only in the epidermis, but also in various organs, including prostate, testis, placenta, spleen and thymus. In human epidermis extracts analysed by Western blotting, PAD type I was detected as a 70 kDa polypeptide, in agreement with its predicted molecular mass. As shown by immunohistochemistry, the enzyme was expressed in all the living layers of human epidermis, with the labelling being increased in the granular layer. This is the first description of the human PAD type I gene and the first demonstration of its expression in epidermis.