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.

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.

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.

Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma.

Leukemic cutaneous T-cell lymphomas (L-CTCL) are lymphoproliferative disorders of skin-homing mature T-cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L-CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L-CTCL, which correlated with the increased clonal T-cell count in the blood. Dual inhibition of STAT3/5 using small-molecule degraders and multi-kinase blockers abolished L-CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L-CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti-leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L-CTCL.

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.

A centric view of JAK/STAT5 in intestinal homeostasis, infection, and inflammation.

Cytokines, growth factors or hormones take action through the JAK/STAT5 signaling pathway, which plays a critical role in regulating the intestinal response to infection and inflammation. However, the way in which STAT5 regulates intestinal epithelial compartment is largely ignored due to the lack of genetic tools for proper exploration and because the two STAT5 transcription factors (STAT5A and STAT5B) have some redundant but also distinct functions. In this review article, by focusing on STAT5 functions in the intestinal undifferentiated and differentiated epithelia, we discuss major advances of the growth factor/cytokine-JAK/STAT5 research in view of intestinal mucosal inflammation and immunity. We highlight the gap in the research of the intestinal STAT5 signaling to anticipate the gastrointestinal explorative insights. Furthermore, we address the critical questions to illuminate how STAT5 signaling influences intestinal epithelial cell differentiation and stem cell regeneration during homeostasis and injury. Overall, our article provides a centric view of the relevance of the relationship between chronic inflammatory diseases and JAK/STAT5 pathway and it also gives an example of how chronic infection and inflammation pirate STAT5 signaling to worsen intestinal injuries. Importantly, our review suggests how to protect a wound healing from gastrointestinal diseases by modulating intestinal STAT5.