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S100 fused-type proteins (SFTPs) such as filaggrin, trichohyalin, and cornulin are differentially expressed in cornifying keratinocytes of the epidermis and various skin appendages. To determine evolutionarily conserved, and thus presumably important, features of SFTPs, we characterized nonmammalian SFTPs and compared their amino acid sequences and expression patterns with those of mammalian SFTPs. We identified an ortholog of cornulin and a previously unknown SFTP, termed scaffoldin, in reptiles and birds, whereas filaggrin was confined to mammals. In contrast to mammalian SFTPs, both cornulin and scaffoldin of the chicken are expressed in the embryonic periderm. However, scaffoldin resembles mammalian trichohyalin with regard to its expression in the filiform papillae of the tongue and in the epithelium underneath the forming tips of the claws. Furthermore, scaffoldin is expressed in the epithelial sheath around growing feathers, reminiscent of trichohyalin expression in the inner root sheath of hair. The results of this study show that SFTP-positive epithelia function as scaffolds for the growth of diverse skin appendages such as claws, nails, hair, and feathers, indicating a common evolutionary origin.

Urocanic acid (UCA) is produced by the enzyme histidase and accumulates in the stratum corneum of the epidermis. In this study, we investigated the photoprotective role of endogenous UCA in the murine skin using histidinemic mice, in which the gene encoding histidase is mutated. Histidase was detected by immunohistochemistry in the stratum granulosum and stratum corneum of the normal murine skin but not in the histidinemic skin. The UCA content of the stratum corneum and the UVB absorption capacity of aqueous extracts from the stratum corneum were significantly reduced in histidinemic mice as compared with wild-type mice. When the shaved back skin of adult mice was irradiated with 250mJcm-2 UVB, histidinemic mice accumulated significantly more DNA damage in the form of cyclobutane pyrimidine dimers than did wild-type mice. Furthermore, UVB irradiation induced significantly higher levels of markers of apoptosis in the epidermis of histidinemic mice. Topical application of UCA reversed the UVB-photosensitive phenotype of histidinemic mice and increased UVB photoprotection of wild-type mice. Taken together, these results provide strong evidence for an important contribution of endogenous UCA to the protection of the epidermis against the damaging effects of UVB radiation.

Degradation of nuclear DNA is a hallmark of programmed cell death. Epidermal keratinocytes die in the course of cornification to function as the dead building blocks of the cornified layer of the epidermis, nails, and hair. Here, we investigated the mechanism and physiological function of DNA degradation during cornification in vivo. Targeted deletion of the keratinocyte-specific endonuclease DNase1-like 2 (DNase1L2) in the mouse resulted in the aberrant retention of DNA in hair and nails, as well as in epithelia of the tongue and the esophagus. In contrast to our previous studies in human keratinocytes, ablation of DNase1L2 did not compromise the cornified layer of the epidermis. Quantitative PCRs showed that the amount of nuclear DNA was dramatically increased in both hair and nails, and that mitochondrial DNA was increased in the nails of DNase1L2-deficient mice. The presence of nuclear DNA disturbed the normal arrangement of structural proteins in hair corneocytes and caused a significant decrease in the resistance of hair to mechanical stress. These data identify DNase1L2 as an essential and specific regulator of programmed cell death in skin appendages, and demonstrate that the breakdown of nuclear DNA is crucial for establishing the full mechanical stability of hair.

Activator protein 1 (AP-1) proteins play key roles in the regulation of cell proliferation and differentiation. In this study we investigated the expression of Fos and Jun proteins in different models of terminal differentiation of human keratinocytes and in skin from psoriasis patients. All Jun and Fos proteins, with the exception of FosB, were efficiently expressed in keratinocytes in monolayer cultures. In contrast, in normal epidermis as well as in organotypic epidermal cultures, the expression pattern of AP-1 proteins was dependent on the differentiation stage. Fos proteins were readily detected in nuclei of keratinocytes of basal and suprabasal layers. JunB and JunD were expressed in all layers of normal epidermis. Interestingly, expression of c-Jun started suprabasally, then disappeared and became detectable again in distinct cells of the outermost granular layer directly at the transition zone to the stratum corneum. In psoriatic epidermis, c-Jun expression was prominent in both hyperproliferating basal and suprabasal keratinocytes, whereas c-Fos expression was unchanged. These data indicate that AP-1 proteins are expressed in a highly specific manner during terminal differentiation of keratinocytes and that the enhanced expression of c-Jun in basal and suprabasal keratinocytes might contribute to the pathogenesis of psoriasis.

Keratin K2 is one of the most abundant structural proteins of the epidermis; however, its biological significance has remained elusive. Here we show that suprabasal type II keratins, K1 and K2, are expressed in a mutually exclusive manner at different body sites of the mouse, with K2 being confined to the ear, sole, and tail skin. Deletion of K2 caused acanthosis and hyperkeratosis of the ear and the tail epidermis, corneocyte fragility, increased transepidermal water loss, and local inflammation in the ear skin. The loss of K2 was partially compensated by upregulation of K1 expression. However, a significant portion of K2-deficient suprabasal keratinocytes lacked a regular cytoskeleton and developed massive aggregates of the type I keratin, K10. Aggregate formation, but not hyperkeratosis, was suppressed by the deletion of both K2 and K10, whereas deletion of K10 alone caused clumping of K2 in ear skin. Taken together, this study demonstrates that K2 is a necessary and sufficient binding partner of K10 at distinct body sites of the mouse and that unbalanced expression of these keratins results in aggregate formation.

Background: Mast cells (MC) and basophils are effector cells of allergic reactions. Growth and function of these cells are regulated by a network of cytokines, other ligands, and respective cell surface membrane receptors. Methods: In the present study, we examined the expression of novel CD antigens on human lung MC, skin MC, blood basophils, the MC line HMC-1, and the basophil cell line KU-812. Expression of surface antigens was analyzed by monoclonal antibodies (mAbs) and indirect immunofluorescence staining techniques. Results: Primary MC were found to react with mAbs against KIT (CD117), the signal regulatory protein SIRP-α (CD172a), and the ectoenzyme E-NPP3 (CD203c). Human basophils were found to express large amounts of E-NPP3 and lower levels of Siglec-5 (CD170), CXCR4 (CD184) and SIRP-α. The HMC-1 cell line was recognized by mAbs against SIRP-α, CXCR4, endothelial protein C receptor (CD201) and E-NPP3. KU-812 cells were found to react with mAbs against E-NPP3, CXCR4 and glycophorin C (CD236R), but did not react with mAb against endothelial protein C receptor. Most of the other CD antigens tested disclosed negative results. Conclusions: In summary, our data provide further evidence that MC and basophils express a unique composition of surface antigens. The use of novel CD antibodies may help to isolate MC and basophils and to study their functional properties.

The removal of keratinocyte (KC) nuclear DNA by deoxyribonucleases (DNases) is an important step in the formation of normal stratum corneum (SC). However, the molecular identity of the DNA-degrading enzymes has so far remained elusive. Here we show that the endonuclease DNase1-like 2 (DNase1L2) is preferentially expressed in the epidermis and that its expression correlates with terminal differentiation of KC in vitro and in vivo. In biopsies of normal skin, DNase1L2 mRNA was regularly found in suprabasal KC and DNase1L2 protein was highly abundant in the stratum granulosum. In contrast to normal skin, DNase1L2 expression was downregulated in parakeratotic epidermis such as in psoriatic lesions. When DNase1L2 gene expression was knocked down by small interfering RNA in a human skin equivalent model, nuclei were maintained through all layers of the SC. Taken together, our data demonstrate that DNase1L2 plays an essential role in DNA degradation during terminal differentiation of epidermal KC.

The antimicrobial defense of the skin is partially mediated by RNase 7, an abundant ribonuclease of the stratum corneum (SC). Here, we investigated the expression and regulation of members of the RNase A family and of the endogenous RNase inhibitor (RI) protein in epidermal keratinocytes (KCs). Reverse transcription-PCR screening revealed that KCs expressed not only RNase 7 but also RNase 5, which was shown earlier to kill the yeast Candida albicans, as well as RNase 1, RNase 4, and RI. The mRNA and protein levels of RNase 5, RNase 7, and RI increased during KC differentiation. When RNase 5 and RNase 7 were incubated with RI in vitro, not only their ribonucleolytic activities but also their antimicrobial activities were strongly suppressed. Immunochemical analyses revealed that SC contains RNase 5, whereas RI was not detectable. Unlike recombinant RNase 5, recombinant RI was degraded when exposed to SC extract. The addition of aprotinin prevented the degradation of RI, indicating that serine proteases of the SC cleave RI. Taken together, this study adds RNase 5 to the list of antimicrobial factors present in the SC and suggests that proteases contribute indirectly to the defense function of the SC by releasing the RI-mediated inhibition of RNase 5 and RNase 7.

Mast cells (MCs) are multifunctional hematopoietic effector cells that produce and release an array of biologically active mediator substances. Growth and functions of MCs are regulated by cytokines, other extracellular factors, surface and cytoplasmic receptors, oncogene products, and a complex network of signal transduction cascades. Key regulators of differentiation of MCs appear to be stem cell factor (SCF) and its tyrosine kinase receptor KIT (c-kit proto-oncogene product=CD117), downstream-acting elements, and the mi transcription factor (MITF). Signaling through KIT is negatively regulated by the signal regulatory protein (SIRP)-alpha (CD172a)-SHP-1-pathway that is disrupted in neoplastic MCs in MC proliferative disorders. Both KIT and FcepsilonRI are involved in MC activation and mediator release. Activation of MCs through FcepsilonRI is associated with increased expression of activation-linked membrane antigens as well as with signaling events involving Lyn and Syk kinases, the phosphatidylinositol-3-kinase-pathway, Ras pathway, and the phospholipase C-protein kinase C pathway. A similar network of signaling is found in SCF-activated MCs. The current article gives an overview on signal transduction-associated and activation-linked antigens expressed in human MCs. Wherever possible the functional implication of signaling pathways and antigen expression are discussed.