Matriptase-1 expression is lost in psoriatic skin lesions and is downregulated by TNFα in vitro.

BACKGROUND AND OBJECTIVES: Matriptase-1 participates in terminal keratinocyte (KC) differentiation. Knockdown of matriptase-1 in skin equivalent cultures leads to impaired KC differentiation and retention of nuclei in the stratum corneum. Here, we investigated the expression and regulation of matriptase-1 in psoriatic skin and in KC in vitro. PATIENTS AND METHODS: Matriptase-1 expression in healthy and psoriatic skin and its regulation in skin equivalents were analyzed by Western blotting, immunofluorescence staining, qRT-PCR, and activity assays. Involvement of the nuclear factor kappa B (NFκB) signaling pathway was investigated by adenoviral overexpression of a dominant-negative form of IKK2. RESULTS: Matriptase-1 expression was detected in the stratum granulosum of healthy human skin and in skin equivalent cultures. Its expression and activity was strongly reduced in lesional skin of patients with psoriasis. Addition of TNFα to skin equivalent cultures resulted in complete loss of matriptase-1 expression accompanied by disturbed KC differentiation. Mechanistically, we were able to show that TNFα-induced downregulation of matriptase-1 was inhibited by blocking the IKK2/NFκB signaling pathway. CONCLUSIONS: Given that matriptase-1 participates in terminal KC differentiation, its absence in psoriatic skin lesions indicates that this contributes to the barrier disturbances in this disease. Our data suggests that blocking the IKK2/NFκB-pathway represents a potential target for the treatment of psoriasis.

Identification of reptilian genes encoding hair keratin-like proteins suggests a new scenario for the evolutionary origin of hair.

The appearance of hair is one of the main evolutionary innovations in the amniote lineage leading to mammals. The main components of mammalian hair are cysteine-rich type I and type II keratins, also known as hard alpha-keratins or "hair keratins." To determine the evolutionary history of these important structural proteins, we compared the genomic loci of the human hair keratin genes with the homologous loci of the chicken and of the green anole lizard Anolis carolinenis. The genome of the chicken contained one type II hair keratin-like gene, and the lizard genome contained two type I and four type II hair keratin-like genes. Orthology of the latter genes and mammalian hair keratins was supported by gene locus synteny, conserved exon-intron organization, and amino acid sequence similarity of the encoded proteins. The lizard hair keratin-like genes were expressed most strongly in the digits, indicating a role in claw formation. In addition, we identified a novel group of reptilian cysteine-rich type I keratins that lack homologues in mammals. Our data show that cysteine-rich alpha-keratins are not restricted to mammals and suggest that the evolution of mammalian hair involved the co-option of pre-existing structural proteins.

Identification of novel mammalian caspases reveals an important role of gene loss in shaping the human caspase repertoire.

Proteases of the caspase family play central roles in apoptosis and inflammation. Recently, we have described a new gene encoding caspase-15 that has been inactivated independently in different mammalian lineages. To determine the dynamics of gene duplication and loss in the entire caspase gene family, we performed a comprehensive evolutionary analysis of mammalian caspases. By comparative genomics and reverse transcriptase-polymerase chain reaction analyses, we identified 3 novel mammalian caspase genes, which we tentatively named caspases-16 through -18. Caspase-16, which is most similar in sequence to caspase-14, has been conserved in marsupials and placental mammals, including humans. Caspase-17, which is most similar to caspase-3, has been conserved among fish, frog, chicken, lizard, and the platypus but is absent from marsupials and placental mammals. Caspase-18, which is most similar to caspase-8, has been conserved among chicken, platypus, and opossum but is absent from placental mammals. These gene distribution patterns suggest that, in the evolutionary lineage leading to humans, caspase-17 was lost after the split of protherian and therian mammals and caspase-18 was lost after the split of marsupials and placental mammals. In the canine genome, the number of caspases has been reduced by the fusion of the neighboring genes caspases-1 and -4, resulting in a single coding region. Further lineage-specific gene inactivations were found for caspase-10 in murine rodents and caspase-12 in humans, rabbit, and cow. Lineage-specific gene duplications were found for caspases-1, -3, and -12 in opossum and caspase-4 in primates. Other caspases were generally conserved in all mammalian species investigated. Using the positions of introns as stable characters during recent vertebrate evolution, we define 3 phylogenetic clades of caspase genes: caspases-1/-2/-4/-5/-9/-12/-14/-15/-16 (clade I), caspases-3/-6/-7/-17 (clade II), and caspases-8/-10/-18/CFLAR (clade III). We conclude that gene inactivations have occurred in each of the 3 caspase clades and that gene loss has been as critical as gene duplication in the evolution of the human repertoire of caspases.

Duplication of the caspase-12 prodomain and inactivation of NLRC4/IPAF in the dog.

Caspases-1, 4, 5, and 12 and other proteins containing caspase recruitment domains (CARDs) play crucial roles in the induction of inflammatory processes. Recently, hybrid caspase-1/4 mRNAs encoding proteases with two CARDs were identified in cat and dog, indicating that the molecular machinery of caspase-dependent inflammation has an unconventional composition in members of the order Carnivora. Here we extended these studies and identified, both in cat and dog, splice variants of caspase-12, which also contained two CARDs. Comparative genomics analysis of the repertoire of canine CARD proteins revealed that the gene encoding NLRC4/IPAF, which is implicated in the inflammatory response to cytosolic flagellin, was inactivated by deleterious mutations in the dog. Our results demonstrate that the repertoires of CARD proteins in cat and dog differ significantly from that of humans and suggest the existence of uncharacterized pathways of inflammasome-mediated signaling in Carnivora.

Transcription of the caspase-14 gene in human epidermal keratinocytes requires AP-1 and NFkappaB.

Caspase-14, a protease involved in skin barrier formation, is specifically expressed in epidermal keratinocytes (KCs). Here, we mapped three start sites of transcription of the human caspase-14 gene and analyzed the upstream chromosomal region for promoter activity. Reporter gene assays identified a core promoter region proximal to the first exon and a distal regulatory region which differentially suppressed promoter activity in KC and other cells. Sequence elements in the proximal promoter were bound by the transcription factors AP-1 (JunB, c-Jun, JunD, Fra-1 and Fra-2) and NFkappaB (p50 and RelB). Our data reveal the basic organization of the human caspase-14 promoter and suggest an important role of AP-1 and NFkappaB in the transcriptional control of caspase-14.

Histidase expression in human epidermal keratinocytes: regulation by differentiation status and all-trans retinoic acid.

BACKGROUND: Histidase (histidine ammonia lyase) converts histidine into urocanic acid, the main ultraviolet (UV) light absorption factor of the stratum corneum. It is unknown if and how histidase is regulated in the epidermis. OBJECTIVE: We have investigated the transcriptional regulation of histidase expression in epidermal keratinocytes. METHODS: Human epidermal keratinocytes were cultured in vitro and exposed to UV irradiation, a number of cytokines and all-trans retinoic acid (ATRA) (1 microM). Keratinocyte differentiation was triggered by maintaining confluent cells in monolayer culture and by establishing three-dimensional skin equivalents. The mRNA expression level of histidase in keratinoytes as well as in the epidermis and other tissues was determined by quantitative real-time PCR. Protein expression was determined by Western blot analysis. RESULTS: Human epidermis contained higher levels of histidase transcripts than all other tissues investigated. Expression of histidase strongly increased at the mRNA and protein levels during differentiation of primary keratinocytes in vitro. Treatment of keratinocytes with UVA and UVB did not significantly change the expression level of histidase. By contrast, ATRA suppressed histidase expression almost completely. CONCLUSIONS: Our results show that histidase is upregulated during keratinocyte differentiation and that ATRA but not UV irradiation modulates the expression level of histidase. Suppression of histidase-mediated production of urocanic acid may contribute to the increase in UV sensitivity that is caused by treatment with retinoids.

Retinoic acid increases the expression of p53 and proapoptotic caspases and sensitizes keratinocytes to apoptosis: a possible explanation for tumor preventive action of retinoids.

Retinoids influence growth and differentiation of keratinocytes (KCs) and are widely used for the management of skin diseases and for prevention of nonmelanoma skin cancer (NMSC) in predisposed patients. Here we investigated the effect of all-trans-retinoic acid (ATRA) on KC apoptosis. When KCs were cultured in confluent monolayers for several days, they acquired resistance against UVB-induced apoptosis. In contrast, when the cells were treated with 1 micromol/L ATRA for 6 days and subsequently irradiated with different doses of UVB, they underwent massive apoptosis as assessed by morphology, expression of activated caspase-3, and DNA fragmentation. The same effect was observed when doxorubicin was used instead of UVB. Analysis by real-time PCR and Western blot revealed that ATRA treatment strongly increased the mRNA and protein expression of p53 and caspase-3, -6, -7, and -9, which are key regulators of apoptosis. UVB irradiation of ATRA-treated cells but not of control cells led to the accumulation of p53 protein and of its target gene Noxa. Inhibition of p53 and caspases with alpha-pifithrin and z-Val-Ala-Asp-fluoromethyl ketone, respectively, blocked UVB- and doxorubicin-induced apoptosis in ATRA-treated KCs. Analogous to the observed ATRA effects in monolayer cultures, in vitro-generated organotypic skin cultures reacted with up-regulation of p53 and proapoptotic caspases and displayed increased sensitivity to UVB-induced apoptosis. The ability of retinoic acid to regulate the expression of proapoptotic genes and to sensitize KCs to apoptosis may play a role in their prevention of NMSC in transplant patients and patients with DNA-repair deficiencies.

Knockdown of filaggrin impairs diffusion barrier function and increases UV sensitivity in a human skin model.

Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.

Caspase-15 is autoprocessed at two sites that contain an aspartate residue in the P1' position.

Our recent characterization of porcine caspase-15 suggested that the amino terminus of the small catalytic subunit is formed by proteolytic processing between the consecutive aspartate residues D277 and D278. Since a charged residue (D278) is highly unusual in the P1' position of a caspase cleavage site, we further characterized the mechanism of caspase-15 autoproteolysis. Amino acid sequence alignments showed that D277 and D278 as well as another pair of aspartates, D270 and D271, were evolutionarily conserved among species of the mammalian clade Laurasiatheria. Site-directed mutations of these four residues and analysis of recombinant proteins showed that D270 was crucial for autoproteolysis whereas the three other aspartates were dispensable for separation of the catalytic subunits. Mutation of D270 prevented catalytic activation and abolished subsequent processing at D277. Together with previous reports, our results show that caspase-15, unlike all other caspases, efficiently cleaves sites with an aspartate in the P1' position.

Gene silencing in a human organotypic skin model.

Here we present a simple and highly reproducible method which allows the study of the effects of a single gene knockdown in an organotypic skin model. Human keratinocytes (KC) were transfected with backbone-modified short interfering RNAs (siRNAs) specific for vascular endothelial growth factor (VEGF) and matriptase-1. Twenty-four hours later the transfected cells were seeded onto fibroblast collagen suspensions and allowed to build up a multilayered epidermis by culture at the air/medium interface for 7 days. Protein expression of both targeted genes remained down-regulated by more than 80% up to 8 days after transfection. As expected, VEGF knockdown by siRNA did not alter epidermis formation in our organotypic skin model. By contrast ablation of matriptase-1 led to aberrant KC differentiation and impaired filaggrin processing and resulted in an epidermal phenotype closely resembling that of matriptase-1 deficient mouse skin. Our results suggest that siRNA-mediated gene silencing is highly efficient in an organotypic skin model and readily allows the assessment of the roles of individual genes during terminal KC differentiation.

Identification and characterization of a novel mammalian caspase with proapoptotic activity.

Caspases are essential proteases in programmed cell death and inflammation. Studies in murine and human cells have led to the characterization of 14 members of this enzyme family. Here we report the identification of caspase-15, a novel caspase that is expressed in various mammalian species including pig, dog, and cattle. The caspase-15 protein contains a catalytic domain with all amino acid residues critical for caspase activity and a prodomain that is predicted to fold into a pyrin domain structure, which is a unique feature among mammalian caspases. Recombinant porcine caspase-15 underwent autocatalytic processing into its subunits and cleaved both tetrapeptide caspase substrates and the apoptosis regulator protein Bid in vitro. Overexpression of caspase-15 in mammalian cells induced proenzyme maturation, cleavage of Bid, activation of caspase-3, and eventually cell death. Both the proteolytic and the pro-apoptotic activity of caspase-15 were abolished by mutation of the active site cysteine. Since a homolog of caspase-15 is absent in the human and the mouse genome, our results reveal an unexpected variability in the molecular apoptotic machinery of mammals.