Cell differentiation in the embryonic periderm and in scaffolding epithelia of skin appendages.

Terminal differentiation of epithelial cells is critical for the barrier function of the skin, the growth of skin appendages, such as hair and nails, and the development of the skin of amniotes. Here, we present the hypothesis that the differentiation of cells in embryonic periderm shares characteristic features with the differentiation of epithelial cells that support the morphogenesis of cornified skin appendages during postnatal life. The periderm prevents aberrant fusion of adjacent epithelial sites during early skin development. It is shed off when keratinocytes of the epidermis form the cornified layer, the stratum corneum. A similar role is played by epithelia that ensheath cornifying skin appendages until they disintegrate to allow the separation of the mature part of the skin appendage from the adjacent tissue. These epithelia, exemplified by the inner root sheath of hair follicles and the epithelia close to the free edge of nails or claws, are referred to as scaffolding epithelia. The periderm and scaffolding epithelia are similar with regard to their transient functions in separating tissues and the conserved expression of trichohyalin and trichohyalin-like genes in mammals and birds. Thus, we propose that parts of the peridermal differentiation program were coopted to a new postnatal function during the evolution of cornified skin appendages in amniotes.

The Evolution of Transglutaminases Underlies the Origin and Loss of Cornified Skin Appendages in Vertebrates.

Transglutaminases (TGMs) cross-link proteins by introducing covalent bonds between glutamine and lysine residues. These cross-links are essential for epithelial cornification which enables tetrapods to live on land. Here, we investigated which evolutionary adaptations of vertebrates were associated with specific changes in the family of TGM genes. We determined the catalog of TGMs in the main clades of vertebrates, performed a comprehensive phylogenetic analysis of TGMs, and localized the distribution of selected TGMs in tissues. Our data suggest that TGM1 is the phylogenetically oldest epithelial TGM, with orthologs being expressed in the cornified teeth of the lamprey, a basal vertebrate. Gene duplications led to the origin of TGM10 in stem vertebrates, the origin of TGM2 in jawed vertebrates, and an increasing number of epithelium-associated TGM genes in the lineage leading to terrestrial vertebrates. TGM9 is expressed in the epithelial egg tooth, and its evolutionary origin in stem amniotes coincided with the evolution of embryonic development in eggs that are surrounded by a protective shell. Conversely, viviparous mammals have lost both the epithelial egg tooth and TGM9. TGM3 and TGM6 evolved as regulators of cornification in hair follicles and underwent pseudogenization upon the evolutionary loss of hair in cetaceans. Taken together, this study reveals the gain and loss of vertebrate TGM genes in association with the evolution of cornified skin appendages and suggests an important role of TGM9 in the evolution of amniotes.

Caspase-5: Structure, Pro-Inflammatory Activity and Evolution.

Caspase-5 is a protease that induces inflammation in response to lipopolysaccharide (LPS), a component of the cell envelope of Gram-negative bacteria. The expression level of the CASP5 gene is very low in the basal state, but strongly increases in the presence of LPS. Intracellular LPS binds to the caspase activation and recruitment domain (CARD) of caspase-5, leading to the formation of a non-canonical inflammasome. Subsequently, the catalytic domain of caspase-5 cleaves gasdermin D and thereby facilitates the formation of cell membrane pores through which pro-inflammatory cytokines of the interleukin-1 family are released. Caspase-4 is also able to form a non-canonical inflammasome upon binding to LPS, but its expression is less dependent on LPS than the expression of caspase-5. Caspase-4 and caspase-5 have evolved via the duplication of a single ancestral gene in a subclade of primates, including humans. Notably, the main biomedical model species, the mouse, has only one ortholog, namely caspase-11. Here, we review the structural features and the mechanisms of regulation that are important for the pro-inflammatory roles of caspase-5. We summarize the interspecies differences and the evolution of pro-inflammatory caspases in mammals and discuss the potential roles of caspase-5 in the defense against Gram-negative bacteria and in sepsis.

Comparative genomics of sirenians reveals evolution of filaggrin and caspase-14 upon adaptation of the epidermis to aquatic life.

The mammalian epidermis has evolved to protect the body in a dry environment. Genes of the epidermal differentiation complex (EDC), such as FLG (filaggrin), are implicated in the barrier function of the epidermis. Here, we investigated the molecular evolution of the EDC in sirenians (manatees and dugong), which have adapted to fully aquatic life, in comparison to the EDC of terrestrial mammals and aquatic mammals of the clade Cetacea (whales and dolphins). We show that the main subtypes of EDC genes are conserved or even duplicated, like late cornified envelope (LCE) genes of the dugong, whereas specific EDC genes have undergone inactivating mutations in sirenians. FLG contains premature stop codons in the dugong, and the ortholog of human CASP14 (caspase-14), which proteolytically processes filaggrin, is pseudogenized in the same species. As FLG and CASP14 have also been lost in whales, these mutations represent convergent evolution of skin barrier genes in different lineages of aquatic mammals. In contrast to the dugong, the manatee has retained functional FLG and CASP14 genes. FLG2 (filaggrin 2) is truncated in both species of sirenians investigated. We conclude that the land-to-water transition of sirenians was associated with modifications of the epidermal barrier at the molecular level.

Evolutionary origin of Hoxc13-dependent skin appendages in amphibians.

Cornified skin appendages, such as hair and nails, are major evolutionary innovations of terrestrial vertebrates. Human hair and nails consist largely of special intermediate filament proteins, known as hair keratins, which are expressed under the control of the transcription factor Hoxc13. Here, we show that the cornified claws of Xenopus frogs contain homologs of hair keratins and the genes encoding these keratins are flanked by promoters in which binding sites of Hoxc13 are conserved. Furthermore, these keratins and Hoxc13 are co-expressed in the claw-forming epithelium of frog toe tips. Upon deletion of hoxc13, the expression of hair keratin homologs is abolished and the development of cornified claws is abrogated in X. tropicalis. These results indicate that Hoxc13-dependent expression of hair keratin homologs evolved already in stem tetrapods, presumably as a mechanism for protecting toe tips, and that this ancestral genetic program was coopted to the growth of hair in mammals.

Comparative genomics of monotremes provides insights into the early evolution of mammalian epidermal differentiation genes.

The function of the skin as a barrier against the environment depends on the differentiation of epidermal keratinocytes into highly resilient corneocytes that form the outermost skin layer. Many genes encoding structural components of corneocytes are clustered in the epidermal differentiation complex (EDC), which has been described in placental and marsupial mammals as well as non-mammalian tetrapods. Here, we analyzed the genomes of the platypus (Ornithorhynchus anatinus) and the echidna (Tachyglossus aculeatus) to determine the gene composition of the EDC in the basal clade of mammals, the monotremes. We report that mammal-specific subfamilies of EDC genes encoding small proline-rich proteins (SPRRs) and late cornified envelope proteins as well as single-copy EDC genes such as involucrin are conserved in monotremes, suggesting that they have originated in stem mammals. Monotremes have at least one gene homologous to the group of filaggrin (FLG), FLG2 and hornerin (HRNR) in placental mammals, but no clear one-to-one pairwise ortholog of either FLG, FLG2 or HRNR. Caspase-14, a keratinocyte differentiation-associated protease implicated in the processing of filaggrin, is encoded by at least 3 gene copies in the echidna. Our results reveal evolutionarily conserved and clade-specific features of the genetic regulation of epidermal differentiation in monotremes.

Development-Associated Genes of the Epidermal Differentiation Complex (EDC).

The epidermal differentiation complex (EDC) is a cluster of genes that encode protein components of the outermost layers of the epidermis in mammals, reptiles and birds. The development of the stratified epidermis from a single-layered ectoderm involves an embryo-specific superficial cell layer, the periderm. An additional layer, the subperiderm, develops in crocodilians and over scutate scales of birds. Here, we review the expression of EDC genes during embryonic development. Several EDC genes are expressed predominantly or exclusively in embryo-specific cell layers, whereas others are confined to the epidermal layers that are maintained in postnatal skin. The S100 fused-type proteins scaffoldin and trichohyalin are expressed in the avian and mammalian periderm, respectively. Scaffoldin forms the so-called periderm granules, which are histological markers of the periderm in birds. Epidermal differentiation cysteine-rich protein (EDCRP) and epidermal differentiation protein containing DPCC motifs (EDDM) are expressed in the avian subperiderm where they are supposed to undergo cross-linking via disulfide bonds. Furthermore, a histidine-rich epidermal differentiation protein and feather-type corneous beta-proteins, also known as beta-keratins, are expressed in the subperiderm. The accumulating evidence for roles of EDC genes in the development of the epidermis has implications on the evolutionary diversification of the skin in amniotes.

Comparative immunohistochemical analysis suggests a conserved role of EPS8L1 in epidermal and hair follicle barriers of mammals.

The mammalian skin and its appendages depend on tightly coordinated differentiation of epithelial cells. Epidermal growth factor receptor (EGFR) pathway substrate 8 (EPS8) like 1 (EPS8L1) is enriched in the epidermis among human tissues and has also been detected in the epidermis of lizards. Here, we show by the analysis of single-cell RNA-sequencing data that EPS8L1 mRNA is co-expressed with filaggrin and loricrin in terminally differentiated human epidermal keratinocytes. Comparative genomics indicated that EPS8L1 is conserved in all main clades of mammals, whereas the orthologous gene has been lost in birds. Using a polyclonal antibody against EPS8L1, we performed an immunohistochemical screening of skin from diverse mammalian species and immuno-electron microscopy of human skin. EPS8L1 was detected predominantly in the granular layer of the epidermis in monotremes, marsupial, and placental mammals. The labeling was partly associated with cell membranes, and it was evident along the perimeter of keratinocytes at the transition with the cornified layer of the epidermis, similar to involucrin distribution. Basal, spinous, and the fully mature cornified layers lacked immunolabeling of EPS8L1. In addition to the epidermis, the hair follicle inner root sheath (IRS) was immunolabeled. Both epidermal granular layer and IRS contribute to the barrier function of the skin, suggesting that EPS8L1 is involved in the regulation of these barriers.

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.

Iron Metabolism of the Skin: Recycling versus Release.

The skin protects the body against exogenous stressors. Its function is partially achieved by the permanent regeneration of the epidermis, which requires high metabolic activity and the shedding of superficial cells, leading to the loss of metabolites. Iron is involved in a plethora of important epidermal processes, including cellular respiration and detoxification of xenobiotics. Likewise, microorganisms on the surface of the skin depend on iron, which is supplied by the turnover of epithelial cells. Here, we review the metabolism of iron in the skin with a particular focus on the fate of iron in epidermal keratinocytes. The iron metabolism of the epidermis is controlled by genes that are differentially expressed in the inner and outer layers of the epidermis, establishing a system that supports the recycling of iron and counteracts the release of iron from the skin surface. Heme oxygenase-1 (HMOX1), ferroportin (SLC40A1) and hephaestin-like 1 (HEPHL1) are constitutively expressed in terminally differentiated keratinocytes and allow the recycling of iron from heme prior to the cornification of keratinocytes. We discuss the evidence for changes in the epidermal iron metabolism in diseases and explore promising topics of future studies of iron-dependent processes in the skin.

Heme Oxygenase-1 Is Upregulated during Differentiation of Keratinocytes but Its Expression Is Dispensable for Cornification of Murine Epidermis.

The epidermal barrier of mammals is initially formed during embryonic development and continuously regenerated by the differentiation and cornification of keratinocytes in postnatal life. Cornification is associated with the breakdown of organelles and other cell components by mechanisms which are only incompletely understood. Here, we investigated whether heme oxygenase 1 (HO-1), which converts heme into biliverdin, ferrous iron and carbon monoxide, is required for normal cornification of epidermal keratinocytes. We show that HO-1 is transcriptionally upregulated during the terminal differentiation of human keratinocytes in vitro and in vivo. Immunohistochemistry demonstrated expression of HO-1 in the granular layer of the epidermis where keratinocytes undergo cornification. Next, we deleted the Hmox1 gene, which encodes HO-1, by crossing Hmox1-floxed and K14-Cre mice. The epidermis and isolated keratinocytes of the resulting Hmox1f/f K14-Cre mice lacked HO-1 expression. The genetic inactivation of HO-1 did not impair the expression of keratinocyte differentiation markers, loricrin and filaggrin. Likewise, the transglutaminase activity and formation of the stratum corneum were not altered in Hmox1f/f K14-Cre mice, suggesting that HO-1 is dispensable for epidermal cornification. The genetically modified mice generated in this study may be useful for future investigations of the potential roles of epidermal HO-1 in iron metabolism and responses to oxidative stress.

Differential Loss of OAS Genes Indicates Diversification of Antiviral Immunity in Mammals.

One of the main mechanisms of inducing an antiviral response depends on 2'-5'-oligoadenylate synthetases (OAS), which sense double-stranded RNA in the cytoplasm and activate RNase L. Mutations leading to the loss of functional OAS1 and OAS2 genes have been identified as important modifiers of the human immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we performed comparative genomics to search for inactivating mutations of OAS genes in other species of mammals and to establish a model for the diversifying evolution of the OAS gene family. We found that a recombination of the OAS and OAS-like (OASL) loci has led to the loss of OAS2 in camelids, which also lack OAS3. Both paralogs of OASL and OAS3 are absent in Asian pangolins. An evolutionarily ancient OAS paralog, which we tentatively name OAS4, has been lost in pangolins, bats and humans. A previously unknown OAS gene, tentatively named OAS5, is present in Yangochiroptera, a suborder of bats. These differences in the OAS gene repertoire may affect innate immune responses to coronaviruses and other RNA viruses.

Transglutaminase Activity Is Conserved in Stratified Epithelia and Skin Appendages of Mammals and Birds.

The cross-linking of structural proteins is critical for establishing the mechanical stability of the epithelial compartments of the skin and skin appendages. The introduction of isopeptide bonds between glutamine and lysine residues depends on catalysis by transglutaminases and represents the main protein cross-linking mechanism besides the formation of disulfide bonds. Here, we used a fluorescent labeling protocol to localize the activity of transglutaminases on thin sections of the integument and its appendages in mammals and birds. In human tissues, transglutaminase activity was detected in the granular layer of the epidermis, suprabasal layers of the gingival epithelium, the duct of sweat glands, hair follicles and the nail matrix. In the skin appendages of chickens, transglutaminase activity was present in the claw matrix, the feather follicle sheath, the feather sheath and in differentiating keratinocytes of feather barb ridges. During chicken embryogenesis, active transglutaminase was found in the cornifying epidermis, the periderm and the subperiderm. Transglutaminase activity was also detected in the filiform papillae on the tongue of mice and in conical papillae on the tongue of chickens. In summary, our study reveals that transglutaminase activities are widely distributed in integumentary structures and suggests that transglutamination contributes to the cornification of hard skin appendages such as nails and feathers.

Inactivation of Autophagy in Keratinocytes Reduces Tumor Growth in Mouse Models of Epithelial Skin Cancer.

Autophagy is a ubiquitous degradation mechanism, which plays a critical role in cellular homeostasis. To test whether autophagy suppresses or supports the growth of tumors in the epidermis of the skin, we inactivated the essential autophagy gene Atg7 specifically in the epidermal keratinocytes of mice (Atg7∆ep) and subjected such mutant mice and fully autophagy-competent mice to tumorigenesis. The lack of epithelial Atg7 did not prevent tumor formation in response to 7, 12-dimethylbenz(a)anthracene (DMBA) as the initiator and 12-O tetradecanoylphorbol-13-acetate (TPA) as the promoter of tumor growth. However, the number of tumors per mouse was reduced in mice with epithelial Atg7 deficiency. In the K5-SOS EGFRwa2/wa2 mouse model, epithelial tumors were initiated by Son of sevenless (SOS) in response to wounding. Within 12 weeks after tumor initiation, 60% of the autophagy-competent K5-SOS EGFRwa2/wa2 mice had tumors of 1 cm diameter and had to be sacrificed, whereas none of the Atg7∆ep K5-SOS EGFRwa2/wa2 mice formed tumors of this size. In summary, the deletion of Atg7 reduced the growth of epithelial tumors in these two mouse models of skin cancer. Thus, our data show that the inhibition of autophagy limits the growth of epithelial skin tumors.

Evolutionary diversification of epidermal barrier genes in amphibians.

The epidermal differentiation complex (EDC) is a cluster of genes encoding components of the skin barrier in terrestrial vertebrates. EDC genes can be categorized as S100 fused-type protein (SFTP) genes such as filaggrin, which contain two coding exons, and single-coding-exon EDC (SEDC) genes such as loricrin. SFTPs are known to be present in amniotes (mammals, reptiles and birds) and amphibians, whereas SEDCs have not yet been reported in amphibians. Here, we show that caecilians (Amphibia: Gymnophiona) have both SFTP and SEDC genes. Two to four SEDC genes were identified in the genomes of Rhinatrema bivittatum, Microcaecilia unicolor and Geotrypetes seraphini. Comparative analysis of tissue transcriptomes indicated predominant expression of SEDC genes in the skin of caecilians. The proteins encoded by caecilian SEDC genes resemble human SEDC proteins, such as involucrin and small proline-rich proteins, with regard to low sequence complexity and high contents of proline, glutamine and lysine. Our data reveal diversification of EDC genes in amphibians and suggest that SEDC-type skin barrier genes have originated either in a common ancestor of tetrapods followed by loss in Batrachia (frogs and salamanders) or, by convergent evolution, in caecilians and amniotes.

Identification of New Biological Pathways Involved in Skin Aging From the Analysis of French Women Genome-Wide Data.

Skin aging is an ineluctable process leading to the progressive loss of tissue integrity and is characterized by various outcomes such as wrinkling and sagging. Researchers have identified impacting environmental factors (sun exposure, smoking, etc.) and several molecular mechanisms leading to skin aging. We have previously performed genome-wide association studies (GWAS) in 502 very-well characterized French women, looking for associations with four major outcomes of skin aging, namely, photoaging, solar lentigines, wrinkling, and sagging, and this has led to new insights into the molecular mechanisms of skin aging. Since individual SNP associations in GWAS explain only a small fraction of the genetic impact in complex polygenic phenotypes, we have made the integration of these genotypes into the reference Kegg biological pathways and looked for associations by the gene set enrichment analysis (GSEA) approach. 106 pathways were tested for association with the four outcomes of skin aging. This biological pathway analysis revealed new relevant pathways and genes, some likely specific of skin aging such as the WNT7B and PRKCA genes in the "melanogenesis" pathway and some likely involved in global aging such as the DDB1 gene in the "nucleotide excision repair" pathway, not picked up in the previously published GWAS. Overall, our results suggest that the four outcomes of skin aging possess specific molecular mechanisms such as the "proteasome" and "mTOR signaling pathway" but may also share common molecular mechanisms such as "nucleotide excision repair."

Comparative genomics reveals evolutionary loss of epiplakin in cetaceans.

The adaptation of vertebrates to different environments was associated with changes in the molecular composition and regulation of epithelia. Whales and dolphins, together forming the clade cetaceans, have lost multiple epithelial keratins during or after their evolutionary transition from life on land to life in water. It is unknown whether the changes in keratins were accompanied by gain or loss of cytoskeletal adapter proteins of the plakin family. Here we investigated whether plakin proteins are conserved in cetaceans and other vertebrates. Comparative analysis of genome sequences showed conservation of dystonin, microtubule actin crosslinking factor 1 (MACF1), plectin, desmoplakin, periplakin and envoplakin in cetaceans. By contrast, EPPK1 (epiplakin) was disrupted by inactivating mutations in all cetaceans investigated. Orthologs of EPPK1 are present in bony and cartilaginous fishes and tetrapods, indicating an evolutionary origin of EPPK1 in a common ancestor of jawed vertebrates (Gnathostomes). In many vertebrates, EPPK1 is flanked by an as-yet uncharacterized gene that encodes protein domains homologous to the carboxy-terminal segment of MACF1. We conclude that epiplakin, unlike other plakins, was lost in cetaceans.