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.

DNA hypomethylation leads to cGAS-induced autoinflammation in the epidermis.

DNA methylation is a fundamental epigenetic modification, important across biological processes. The maintenance methyltransferase DNMT1 is essential for lineage differentiation during development, but its functions in tissue homeostasis are incompletely understood. We show that epidermis-specific DNMT1 deletion severely disrupts epidermal structure and homeostasis, initiating a massive innate immune response and infiltration of immune cells. Mechanistically, DNA hypomethylation in keratinocytes triggered transposon derepression, mitotic defects, and formation of micronuclei. DNA release into the cytosol of DNMT1-deficient keratinocytes activated signaling through cGAS and STING, thus triggering inflammation. Our findings show that disruption of a key epigenetic mark directly impacts immune and tissue homeostasis, and potentially impacts our understanding of autoinflammatory diseases and cancer immunotherapy.

Pangolins Lack IFIH1/MDA5, a Cytoplasmic RNA Sensor That Initiates Innate Immune Defense Upon Coronavirus Infection.

Zoonotic infections are an imminent threat to human health. Pangolins were recently identified as carriers and intermediate hosts of coronaviruses. Previous research has shown that infection with coronaviruses activates an innate immune response upon sensing of viral RNA by interferon-induced with helicase C domain 1 (IFIH1), also known as MDA5. Here, we performed a comparative genomics study of RNA sensor genes in three species of pangolins. DDX58/RIG-I, a sensor of cytoplasmic viral RNA and toll-like receptors (TLR) 3, 7, and 8, which bind RNA in endosomes, are conserved in pangolins. By contrast, IFIH1 a sensor of intracellular double-stranded RNA, has been inactivated by mutations in pangolins. Likewise, Z-DNA-binding protein (ZBP1), which senses both Z-DNA and Z-RNA, has been lost during the evolution of pangolins. These results suggest that the innate immune response to viruses differs significantly between pangolins and other mammals, including humans. We put forward the hypothesis that loss of IFIH1 and ZBP1 provided an evolutionary advantage by reducing inflammation-induced damage to host tissues and thereby contributed to a switch from resistance to tolerance of viral infections in pangolins.

Cytosolic DNA sensing through cGAS and STING is inactivated by gene mutations in pangolins.

The release of DNA into the cytoplasm upon damage to the nucleus or during viral infection triggers an interferon-mediated defense response, inflammation and cell death. In human cells cytoplasmic DNA is sensed by cyclic GMP-AMP Synthase (cGAS) and Absent In Melanoma 2 (AIM2). Here, we report the identification of a "natural knockout" model of cGAS. Comparative genomics of phylogenetically diverse mammalian species showed that cGAS and its interaction partner Stimulator of Interferon Genes (STING) have been inactivated by mutations in the Malayan pangolin whereas other mammals retained intact copies of these genes. The coding sequences of CGAS and STING1 are also disrupted by premature stop codons and frame-shift mutations in Chinese and tree pangolins, suggesting that expression of these genes was lost in a common ancestor of all pangolins that lived more than 20 million years ago. AIM2 is retained in a functional form in pangolins whereas it is inactivated by mutations in carnivorans, the phylogenetic sister group of pangolins. The deficiency of cGAS and STING points to the existence of alternative mechanisms of controlling cytoplasmic DNA-associated cell damage and viral infections in pangolins.

Differential Evolution of the Epidermal Keratin Cytoskeleton in Terrestrial and Aquatic Mammals.

Keratins are the main intermediate filament proteins of epithelial cells. In keratinocytes of the mammalian epidermis they form a cytoskeleton that resists mechanical stress and thereby are essential for the function of the skin as a barrier against the environment. Here, we performed a comparative genomics study of epidermal keratin genes in terrestrial and fully aquatic mammals to determine adaptations of the epidermal keratin cytoskeleton to different environments. We show that keratins K5 and K14 of the innermost (basal), proliferation-competent layer of the epidermis are conserved in all mammals investigated. In contrast, K1 and K10, which form the main part of the cytoskeleton in the outer (suprabasal) layers of the epidermis of terrestrial mammals, have been lost in whales and dolphins (cetaceans) and in the manatee. Whereas in terrestrial mammalian epidermis K6 and K17 are expressed only upon stress-induced epidermal thickening, high levels of K6 and K17 are consistently present in dolphin skin, indicating constitutive expression and substitution of K1 and K10. K2 and K9, which are expressed in a body site-restricted manner in human and mouse suprabasal epidermis, have been lost not only in cetaceans and manatee but also in some terrestrial mammals. The evolution of alternative splicing of K10 and differentiation-dependent upregulation of K23 have increased the complexity of keratin expression in the epidermis of terrestrial mammals. Taken together, these results reveal evolutionary diversification of the epidermal cytoskeleton in mammals and suggest a complete replacement of the quantitatively predominant epidermal proteins of terrestrial mammals by originally stress-inducible keratins in cetaceans.

Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation of DNA in lingual cornifying keratinocytes without activating inflammatory responses.

The cornification of keratinocytes on the surface of skin and oral epithelia is associated with the degradation of nuclear DNA. The endonuclease DNase1L2 and the exonuclease Trex2 are expressed specifically in cornifying keratinocytes. Deletion of DNase1L2 causes retention of nuclear DNA in the tongue epithelium but not in the skin. Here we report that lack of Trex2 results in the accumulation of DNA fragments in the cytoplasm of cornifying lingual keratinocytes and co-deletion of DNase1L2 and Trex2 causes massive accumulation of DNA fragments throughout the cornified layers of the tongue epithelium. By contrast, cornification-associated DNA breakdown was not compromised in the epidermis. Aberrant retention of DNA in the tongue epithelium was associated neither with enhanced expression of DNA-driven response genes, such as Ifnb, Irf7 and Cxcl10, nor with inflammation. Of note, the expression of Tlr9, Aim2 and Tmem173, key DNA sensor genes, was markedly lower in keratinocytes and keratinocyte-built tissues than in macrophages and immune tissues, and DNA-driven response genes were not induced by introduction of DNA in keratinocytes. Altogether, our results indicate that DNase1L2 and Trex2 cooperate in the breakdown and degradation of DNA during cornification of lingual keratinocytes and aberrant DNA retention is tolerated in the oral epithelium.

Inactivation of DNase1L2 and DNase2 in keratinocytes suppresses DNA degradation during epidermal cornification and results in constitutive parakeratosis.

The stratum corneum of the epidermis constitutes the mammalian skin barrier to the environment. It is formed by cornification of keratinocytes, a process which involves the removal of nuclear DNA. Here, we investigated the mechanism of cornification-associated DNA degradation by generating mouse models deficient of candidate DNA-degrading enzymes and characterizing their epidermal phenotypes. In contrast to Dnase1l2 -/- mice and keratinocyte-specific DNase2 knockout mice (Dnase2 Δep ), Dnase1l2 -/- Dnase2 Δep mice aberrantly retained nuclear DNA in the stratum corneum, a phenomenon commonly referred to as parakeratosis. The DNA within DNase1L2/DNase2-deficient corneocytes was partially degraded in a DNase1-independent manner. Isolation of corneocytes, i.e. the cornified cell components of the stratum corneum, and labelling of DNA demonstrated that corneocytes of Dnase1l2 -/- Dnase2 Δep mice contained DNA in a nucleus-shaped compartment that also contained nucleosomal histones but lacked the nuclear intermediate filament protein lamin A/C. Parakeratosis was not associated with altered corneocyte resistance to mechanical stress, changes in transepidermal water loss, or inflammatory infiltrates in Dnase1l2 -/- Dnase2 Δep mice. The results of this study suggest that cornification of epidermal keratinocytes depends on the cooperation of DNase1L2 and DNase2 and indicate that parakeratosis per se does not suffice to cause skin pathologies.

The caspase-1 inhibitor CARD18 is specifically expressed during late differentiation of keratinocytes and its expression is lost in lichen planus.

BACKGROUND: CARD18 contains a caspase recruitment domain (CARD) via which it binds to caspase-1 and thereby inhibits caspase-1-mediated activation of the pro-inflammatory cytokine interleukin (IL)-1ß. OBJECTIVES: To determine the expression profile and the role of CARD18 during differentiation of keratinocytes and to compare the expression of CARD18 in normal skin and in inflammatory skin diseases. METHODS: Human keratinocytes were induced to differentiate in monolayer and in 3D skin equivalent cultures. In some experiments, CARD18-specific siRNAs were used to knock down expression of CARD18. CARD18 mRNA levels were determined by quantitative real-time PCR, and CARD18 protein was detected by Western blot and immunofluorescence analyses. In situ expression was analyzed in skin biopsies obtained from healthy donors and patients with psoriasis and lichen planus. RESULTS: CARD18 mRNA was expressed in the epidermis at more than 100-fold higher levels than in any other human tissue. Within the epidermis, CARD18 was specifically expressed in the granular layer. In vitro CARD18 was strongly upregulated at both mRNA and protein levels in keratinocytes undergoing terminal differentiation. In skin equivalent cultures the expression of CARD18 was efficiently suppressed by siRNAs without impairing stratum corneum formation. Epidermal expression of CARD18 was increased after ultraviolet (UV)B irradiation of skin explants. In skin biopsies of patients with psoriasis no consistent regulation of CARD18 expression was observed, however, in lesional epidermis of patients with lichen planus, CARD18 expression was either greatly diminished or entirely absent whereas in non-lesional areas expression was comparable to normal skin. CONCLUSIONS: Our results identify CARD18 as a differentiation-associated keratinocyte protein that is altered in abundance by UV stress. Its downregulation in lichen planus indicates a potential role in inflammatory reactions of the epidermis in this disease.

MCPIP1 contributes to the inflammatory response of UVB-treated keratinocytes.

BACKGROUND: Monocyte chemoattractant protein-1-induced protein-1 (MCPIP1), also known as regnase-1, negatively regulates many cellular processes including the cellular response to inflammatory agents, differentiation, viability, and proliferation. It possesses a PilT N-terminus (PIN) domain that is directly involved in regulating the stability of transcripts and miRNAs by recognizing stem loop structures and degrading them by endonucleolytic cleavage. OBJECTIVE: We investigated the role of MCPIP1 in the response of human primary keratinocytes to UVB stress. METHODS: Keratinocytes were treated with UVB, siRNA against MCPIP1, pharmacological inhibitors of signaling pathways, or subjected to control treatments. The mRNA and protein levels of MCPIP1 and MCPIP1-dependent changes gene expression were analyzed by quantitative (Q)-RT-PCRs and Western blots. Secretion of TNFα and IL-8 was determined by ELISA. RESULTS: UVB treatment of keratinocytes induced upregulation of MCPIP1 at the mRNA level after 4-8h and at the protein level after 8-16h. MCPIP1 abundance depended on NF-κB activity. Using an siRNA strategy, we found that diminished MCPIP1 resulted in an up-regulation of transcripts coding for IL-8, TNFα, COX-2, and BCL-2, as well as an enhanced release of IL-8. Moreover, decreased phosphorylation of NF-κB and p38 signaling pathways were observed in addition to a slight up-regulation of ERK1/2 directly after UVB treatment. Twenty-four hours later, decreased phosphorylation was observed only for NF-κB and p38. Furthermore, in MCPIP1-suppressed cells, the levels of pro-apoptotic Puma, the phosphorylated form of p53 and the abundance of its target p21 as well as the activity of caspase 3 decreased, while the level of cyclin D1 increased. CONCLUSION: MCPIP1 contributes to the UVB response of keratinocytes by altering metabolic and apoptotic processes and the release of inflammatory mediators.

Holocrine Secretion of Sebum Is a Unique DNase2-Dependent Mode of Programmed Cell Death.

Sebaceous glands produce sebum via holocrine secretion, a largely uncharacterized mode of programmed cell death that contributes to the homeostasis and barrier function of the skin. To determine the mechanism of DNA degradation during sebocyte cell death, we have inactivated candidate DNA-degrading enzymes by targeted gene deletions in mice. DNase1 and DNase1-like 2 were dispensable for nuclear DNA degradation in sebocytes. By contrast, epithelial cell-specific deletion of lysosomal DNase2 blocked DNA degradation in these cells. DNA breakdown during sebocyte differentiation coincided with the loss of LAMP1 and was accelerated by the abrogation of autophagy, the central cellular program of lysosome-dependent catabolism. Suppression of DNA degradation by the deletion of DNase2 resulted in aberrantly increased concentrations of residual DNA and decreased amounts of the DNA metabolite uric acid in secreted sebum. These results define holocrine secretion as a DNase2-mediated form of programmed cell death and suggest that autophagy-dependent metabolism, DNA degradation, and the molecular composition of sebum are mechanistically linked.

Localisation of keratin K78 in the basal layer and first suprabasal layers of stratified epithelia completes expression catalogue of type II keratins and provides new insights into sequential keratin expression.

Among the 26 human type II keratins, K78 is the only one that has not yet been explored with regard to its expression characteristics. Here, we show that, at both the transcriptional and translational levels, K78 is strongly expressed in the basal and parabasal cell layers with decreasing intensity in the lower suprabasal cells of keratinising and non-keratinising squamous epithelia and keratinocyte cultures. The same pattern has been detected at the transcriptional level in the corresponding mouse epithelia. Murine K78 protein, which contains an extraordinary large extension of its tail domain, which is unique among all known keratins, is not detectable by the antibody used. Concomitant studies in human epithelia have confirmed K78 co-expression with the classical basal keratins K5 and K14. Similarly, K78 co-expression with the differentiation-related type I keratins K10 (epidermis) and K13 (non-keratinising epithelia) occurs in the parabasal cell layer, whereas that of the corresponding type II keratins K1 (epidermis) and K4 (non-keratinising epithelia) unequivocally starts subsequent to the respective type I keratins. Our data concerning K78 expression modify the classical concept of keratin pair K5/K14 representing the basal compartment and keratin pairs K1/K10 or K4/K13 defining the differentiating compartment of stratified epithelia. Moreover, the K78 expression pattern and the decoupled K1/K10 and K4/K13 expression define the existence of a hitherto unperceived early differentiation stage in the parabasal layer characterized by K78/K10 or K78/K13 expression.

Keratins K2 and K10 are essential for the epidermal integrity of plantar skin.

BACKGROUND: K1 and K2 are the main type II keratins in the suprabasal epidermis where each of them heterodimerizes with the type I keratin K10 to form intermediate filaments. In regions of the ears, tail, and soles of the mouse, only K2 is co-expressed with K10, suggesting that these keratins suffice to form a mechanically resilient cytoskeleton. OBJECTIVE: To determine the effects of the suppression of both main keratins, K2 and K10, in the suprabasal plantar epidermis of the mouse. METHODS: Krt2(-/-) Krt10(-/-) mice were generated by crossing Krt2(-/-) and Krt10(-/-) mice. Epidermal morphology of soles of hind-paws was examined macroscopically and histologically. Immunofluorescence analysis and quantitative PCR analysis were performed to analyze the expression of keratins in sole skin of wildtype and Krt2(-/-) Krt10(-/-) mice. Highly abundant proteins of the sole stratum corneum were determined by electrophoretic and chromatographic separation and subsequent mass spectrometry. RESULTS: K2 and K10 are the most prominent suprabasal keratins in normal mouse soles with the exception of the footpads where K1, K9 and K10 predominate. Mice lacking both K2 and K10 were viable and developed epidermal acanthosis and hyperkeratosis in inter-footpad epidermis of the soles. The expression of keratins K1, K9 and K16 was massively increased at the RNA and protein levels in the soles of Krt2(-/-) Krt10(-/-) mice. CONCLUSIONS: This study demonstrates that the loss of the main cytoskeletal components of plantar epidermis, i.e. K2 and K10, can be only partly compensated by the upregulation of other keratins. The thickening of the epidermis in the soles of Krt2(-/-) Krt10(-/-) mice may serve as a model for pathomechanistic aspects of palmoplantar keratoderma.

Multifaceted role of TREX2 in the skin defense against UV-induced skin carcinogenesis.

TREX2 is a 3'-DNA exonuclease specifically expressed in keratinocytes. Here, we investigated the relevance and mechanisms of TREX2 in ultraviolet (UV)-induced skin carcinogenesis. TREX2 expression was up-regulated by chronic UV exposure whereas it was de-regulated or lost in human squamous cell carcinomas (SCCs). Moreover, we identified SNPs in the TREX2 gene that were more frequent in patients with head and neck SCCs than in healthy individuals. In mice, TREX2 deficiency led to enhanced susceptibility to UVB-induced skin carcinogenesis which was preceded by aberrant DNA damage removal and degradation as well as reduced inflammation. Specifically, TREX2 loss diminished the up-regulation of IL12 and IFNγ, key cytokines related to DNA repair and antitumor immunity. In UV-treated keratinocytes, TREX2 promoted DNA repair and passage to late apoptotic stages. Notably, TREX2 was recruited to low-density nuclear chromatin and micronuclei, where it interacted with phosphorylated H2AX histone, which is a critical player in both DNA repair and cell death. Altogether, our data provide new insights in the molecular mechanisms of TREX2 activity and establish cell autonomous and non-cell autonomous functions of TREX2 in the UVB-induced skin response.

Comparative genomics reveals conservation of filaggrin and loss of caspase-14 in dolphins.

The expression of filaggrin and its stepwise proteolytic degradation are critical events in the terminal differentiation of epidermal keratinocytes and in the formation of the skin barrier to the environment. Here, we investigated whether the evolutionary transition from a terrestrial to a fully aquatic lifestyle of cetaceans, that is dolphins and whales, has been associated with changes in genes encoding filaggrin and proteins involved in the processing of filaggrin. We used comparative genomics, PCRs and re-sequencing of gene segments to screen for the presence and integrity of genes coding for filaggrin and proteases implicated in the maturation of (pro)filaggrin. Filaggrin has been conserved in dolphins (bottlenose dolphin, orca and baiji) but has been lost in whales (sperm whale and minke whale). All other S100 fused-type genes have been lost in cetaceans. Among filaggrin-processing proteases, aspartic peptidase retroviral-like 1 (ASPRV1), also known as saspase, has been conserved, whereas caspase-14 has been lost in all cetaceans investigated. In conclusion, our results suggest that filaggrin is dispensable for the acquisition of fully aquatic lifestyles of whales, whereas it appears to confer an evolutionary advantage to dolphins. The discordant evolution of filaggrin, saspase and caspase-14 in cetaceans indicates that the biological roles of these proteins are not strictly interdependent.

Loss of keratin K2 expression causes aberrant aggregation of K10, hyperkeratosis, and inflammation.

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.

Mechanisms and emerging functions of DNA degradation in the epidermis.

Degradation of DNA is involved in key processes maintaining the integrity of the epidermis such as the cornification of keratinocytes, the removal of damaged cells and the defense against potentially harmful microorganisms. Recent studies have characterized some of the molecular mechanisms and physiological functions of DNA degradation in the epidermis. Deoxyribonuclease (DNase)1L2 and TREX2 were identified as epidermis-specific DNases and DNase 2 was shown to be the predominant DNA-degrading enzyme on the surface of the skin. Here we review the latest insights into the DNA catabolism in the skin and discuss open questions pertaining to the molecular biology of epidermal DNA breakdown.

A comparative proteomic study of human skin suction blister fluid from healthy individuals using immunodepletion and iTRAQ labeling.

Aberrations in skin morphology and functionality can cause acute and chronic skin-related diseases that are the focus of dermatological research. Mechanically induced skin suction blister fluid may serve as a potential, alternative human body fluid for quantitative mass spectrometry (MS)-based proteomics in order to assist in the understanding of the mechanisms and causes underlying skin-related diseases. The combination of abundant-protein removal with iTRAQ technology and multidimensional fractionation techniques improved the number of identified protein groups. A relative comparison of a cohort of 8 healthy volunteers was thus recruited in order to assess the net variability encountered in a healthy scenario. The technology enabled the identification, to date, of the highest number of reported protein groups (739) with concomitant relative quantitative data for over 90% of all proteins with high reproducibility and accuracy. The use of iTRAQ 8-plex resulted in a 66% decrease in protein identifications but, despite this, provided valuable insight into interindividual differences of the healthy control samples. The geometric mean ratio was close to 1 with 95% of all ratios ranging between 0.45 and 2.05 and a calculated mean coefficient of variation of 15.8%, indicating a lower biological variance than that reported for plasma or urine. By applying a multistep sample processing, the obtained sensitivity and accuracy of quantitative MS analysis demonstrates the prospective value of the approach in future research into skin diseases.

In situ labeling of DNA reveals interindividual variation in nuclear DNA breakdown in hair and may be useful to predict success of forensic genotyping of hair.

Hair fibers are formed by keratinocytes of the hair follicle in a process that involves the breakdown of the nucleus including DNA. Accordingly, DNA can be isolated with high yield from the hair bulb which contains living keratinocytes, whereas it is difficult to prepare from the distal portions of hair fibers and from shed hair. Nevertheless, forensic investigations are successful in a fraction of shed hair samples found at crime scenes. Here, we report that interindividual differences in the completeness of DNA removal from hair corneocytes are major determinants of DNA content and success rates of forensic investigations of hair. Distal hair samples were permeabilized with ammonia and incubated with the DNA-specific dye Hoechst 33258 to label DNA in situ. Residual nuclear DNA was visualized under the fluorescence microscope. Hair from some donors did not contain any stainable nuclei, whereas hair of other donors contained a variable number of DNA-positive nuclear remnants. The number of DNA-containing nuclear remnants per millimeter of hair correlated with the amount of DNA that could be extracted and amplified by quantitative PCR. When individual hairs were investigated, only hairs in which DNA could be labeled in situ gave positive results in short tandem repeat typing. This study reveals that the completeness of DNA degradation during cornification of the hair is a polymorphic trait. Furthermore, our results suggest that in situ labeling of DNA in hair may be useful for predicting the probability of success of forensic analysis of nuclear DNA in shed hair.

DNase 2 is the main DNA-degrading enzyme of the stratum corneum.

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.

Essential role of the keratinocyte-specific endonuclease DNase1L2 in the removal of nuclear DNA from hair and nails.

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.