Caspase-14 protects against epidermal UVB photodamage and water loss

Abstract

Caspase-14 belongs to a conserved family of aspartate-specific proteinases. Its expression is restricted almost exclusively to the suprabasal layers of the epidermis and the hair follicles1,2,3,4. Moreover, the proteolytic activation of caspase-14 is associated with stratum corneum formation, implicating caspase-14 in terminal keratinocyte differentiation and cornification5,6. Here, we show that the skin of caspase-14-deficient mice was shiny and lichenified, indicating an altered stratum-corneum composition. Caspase-14-deficient epidermis contained significantly more alveolar keratohyalin F-granules, the profilaggrin stores. Accordingly, caspase-14-deficient epidermis is characterized by an altered profilaggrin processing pattern and we show that recombinant caspase-14 can directly cleave profilaggrin in vitro. Caspase-14-deficient epidermis is characterized by reduced skin-hydration levels and increased water loss. In view of the important role of filaggrin in the structure and moisturization of the skin, the knockout phenotype could be explained by an aberrant processing of filaggrin. Importantly, the skin of caspase-14-deficient mice was highly sensitive to the formation of cyclobutane pyrimidine dimers after UVB irradiation, leading to increased levels of UVB-induced apoptosis. Removal of the stratum corneum indicate that caspase-14 controls the UVB scavenging capacity of the stratum corneum.

Main

The formation of corneocytes is a specialized form of cell death that does not involve the classical apoptotic caspases2,7. To analyse the role of caspase-14 during terminal keratinocyte differentiation, caspase-14-deficient mice were generated by homologous recombination (see Supplementary Information, Fig. S1a). Heterozygous caspase-14+/− mice were intercrossed to obtain homozygous caspase-14+/+ and caspase-14−/− mice. The genotype was confirmed by Southern blotting, and the absence of caspase-14 protein by western blotting (see Supplementary Information, Fig. S1b, c). In total epidermal extracts from wild-type mice approximately 50% of caspase-14 is proteolytically activated during epidermal differentiation, as indicated by the presence of the typical p20 and p10 subunits. It was determined that caspase-14 preferentially cleaves the WEHD tetrapeptide motif8, similarly to caspase-1. Epidermal extracts from caspase-14-deficient mice, in contrast with extracts from wild-type mice, did not show WEHDamc cleavage activity (Fig. 1a). This indicates that in homeostatic conditions all detectable proteolytic activity on WEHD in the epidermis is due to caspase-14. The expression levels of apoptotic executioner caspases, such as caspase-3 and −7, were not affected. In contrast with caspase-14, caspase-3 and -7 remained unprocessed during terminal keratinocyte differentiation (see Supplementary Information, Fig. S1c), as also indicated by the absence of DEVDase activity in the epidermis (Fig. 1a). This confirms that the apoptotic proteolytic cascade is not activated during homeostatic programmed cell death in the epidermis2. Extensive immunohistochemical analysis of different tissues confirmed that caspase-14 is expressed only in cornifying epithelia, such as the epidermis and Hassall's bodies9. Moreover, caspase-14 was also expressed in the forestomach of mice, which is cornified like epidermis. Caspase-14 expression was ablated in all these tissues in caspase-14−/− mice (see Supplementary Information, Fig. S1d). However, no gross histological abnormalities were observed in caspase-14-deficient mice. Our results, using caspase-14−/− mice as a control, indicate that the previously reported immunostaining in the epithelium of the choroid plexus and the pigmented retina layer9, was non-specific (see Supplementary Information, Fig. S2a, b).

Figure 1: Histomorphological characterization of caspase-14-deficient epidermis.
figure1

(a) Measurement of WEHDamc and DEVDamc cleavage activity in epidermal extracts of wild-type and caspase-14−/− mice. The error bars represent the mean ± s.d. of three mice. (b) Macroscopic observation of wild-type and caspase-14−/− neonate mice. (c) Eosin–haematoxylin staining of skin sections of caspase-14+/+ and caspase-14−/− neonatal mice. (d) Scanning electron microscopy of epidermis of caspase-14+/+ and caspase-14−/− neonatal mice. (e) Transmission electron microscopy of caspase-14+/+ and caspase-14−/− neonate epidermis. Alveolar mottled keratohyalin F-granules are indicated by arrows. Neonates were 3.5–6.5 days old. (f) Confocal microscopy analysis with anti-filaggrin antibody on skin sections of 5.5-day-old mice. The scale bars represent 1 mm in b, 40 μm in c, 100 μm in d, 1 μm in e and 10 μm in f.

Caspase-14−/− mice were born at the expected Mendelian ratio, were fertile, and their long-term survival was indistinguishable from that of wild-type mice (up to 20 months). The skin of newborn caspase-14−/− mice was shinier and more lichenified than that of wild-type mice (Fig. 1b). Lichenification is characterized by more pronounced skin lines. This phenotype became more prominent from day 2–3 and lasted throughout adulthood. Both wild-type and caspase-14-deficient mice showed normal hair growth, and caspase-14−/− mice did not suffer from abnormal hair loss during hair cycling. Light microscopic examination of haematoxylin–eosin-stained sections of newborn and adult caspase-14-deficient epidermis revealed no abnormalities in the stratum basale, stratum spinosum, stratum granulosum or hair follicles (Fig. 1c). The stratum corneum, where caspase-14 is proteolytically activated1,3,4, also showed no histological abnormalities. However, scanning microscopic analysis showed that caspase-14−/− mice had larger scales than caspase-14+/+ animals (Fig. 1d), indicating at least partial involvement of caspase-14 in the desquamation process. Indeed, some caspase-14 protein was found associated with corneodesmosomes10. The ultrastructure of hair shafts was similar in wild-type and caspase-14-deficient mice (see Supplementary Information, Fig. S2c). In addition, comparison of wild-type and caspase-14−/− epidermis by transmission electron microscopy (Fig. 1e), showed that a significantly higher fraction (75%) of alveolar, swollen keratohyalin F-granules was present in the transition layer of caspase-14-deficient epidermis than in wild-type epidermis (8%). These swollen F-granules are characterized by a mottled appearance, the biochemical nature of which is not known. Such structures were reported earlier in transglutaminase-1-deficient mice11. Because caspase-14 is activated only during cornification, differences between wild-type and caspase-14-deficient epidermis are expected mainly in the transition and cornified layers. F-granules are stores of profilaggrin, a major epidermal histidine-rich protein. Profilaggrin (relative molecular mass (Mr) 500,000)contains an amino-terminal sequence comprising an A domain that binds calcium and a B domain (the function of which is not clear), followed by tandem repeats of filaggrin and a unique carboxy-terminal sequence. During terminal differentiation of keratinocytes, profilaggrin is processed into mature filaggrin units that assist the formation of macrofibril keratin bundles12,13. Later, these monomers are degraded to free amino acids that contribute to the so-called natural moisturizing factors maintaining epidermal hydration14. Confocal microscopy indicated that filaggrin accumulated in the upper layers of the stratum corneum in caspase-14−/− epidermis (Fig. 1f). In contrast, the distribution and expression level of K5 and K14 (early differentiation markers), and K1, K10, loricrin and involucrin (late differentiation markers), were not different in epidermal, thymus and forestomach sections of the two genotypes (see Supplementary Information, Fig. S3a and data not shown). Filaggrin is also an important component of cornified envelopes that replace the plasma membrane in corneocytes5. Therefore, cornified envelopes were isolated from newborn mice: the cornified envelopes from caspase-14-deficient skin did not differ in shape or size from wild type when examined by phase contrast microscopy (data not shown).

Based on the occurrence of altered F-granules and filaggrin distribution in caspase-14−/− epidermis, the profilaggrin processing pattern was compared in wild-type and caspase-14-deficient mice. Western blots showed that the 32K filaggrin monomer band was equally well formed in both caspase-14+/+ and caspase-14−/− epidermis, but in contrast with wild-type tissue, filaggrin fragments of 15–25K accumulated in caspase-14-deficient epidermis (Fig. 2a). These fragments are derived from the filaggrin monomer as the antibody used in this study was raised against a specific peptide present in this monomer. In addition, proteins were eluted from the 15–25K range of an SDS gel on which wild-type and caspase-14−/− epidermal extracts were separated. Using mass spectrometry analysis, sixfold more peptide spectra derived from the filaggrin monomer were identified in caspase-14−/− extracts compared with wild-type extracts (data not shown). This confirmed the nature of the accumulating filaggrin fragments observed in the western blot experiment. A similar aberration in filaggrin processing was observed in the cornified epithelium of the forestomach (data not shown). These data provide in vivo proof that deficiency in caspase-14 affects the correct degradation of (pro)filaggrin. Interestingly, flaky tail mice, which express an abnormal profilaggrin protein that is not processed to filaggrin monomers, have large scales similar to those of caspase-14-deficient mice13. In contrast to caspase-14−/− mice, flaky tail mice exhibit marked attenuation of the epidermal granular layer and orthokeratotic hyperkeratosis. This is probably due to the lack of mature filaggrin monomers in the flaky tail mice.

Figure 2: Caspase-14 mediates profilaggrin processing, and controls transepidermal water loss and stratum corneum hydration.
figure2

(a) Western blot analysis of profilaggrin processing in epidermal lysates of 5.5-day-old wild-type mice and caspase-14−/− mice. Accumulating filaggrin bands are indicated with an arrowhead. (b) Epidermal lysate from caspase-14-deficient mice was incubated with 400 ng wild-type recombinant caspase-14 (hc14) with or without zVADfmk, or 400 ng recombinant catalytically dead caspase-14 mutant (hc14C132A). These lysates were analysed by western blotting using an anti-filaggrin antibody. An uncropped image of the scan is shown in the Supplementary Information, Fig. S5. (c, d) An in vitro translated N-terminal human or mouse profilaggrin fragment (hFG1 or mFG2, respectively; see schematic representation: A, A domain; B, B domain; FG, partial filaggrin monomer) was incubated with wild-type recombinant caspase-14 with or without zVADfmk, or with caspase-14C132A mutant. The profilaggrin fragments were visualized by autoradiography. Note that when hFG1 is translated, two protein bands appear (arrows) due to internal initiation at a downstream methionine26. Cleaved profilaggrin products are indicated with asterisks. (e, f) Transepidermal water loss (TEWL) and hydration levels of adult and 4.5–6.5-day-old neonates were measured using a TEWAmeter (Courage and Khazaka, TM210) or a Corneometer (Courage and Khazaka, CM825), respectively. The number of animals tested is indicated. Asterisks indicate P <0.0001, unless otherwise indicated (unpaired Student's t-test on mean values ± s.d. using Graphpad software).

To determine whether the defect in filaggrin degradation could be complemented by caspase-14, caspase-14-deficient epidermal extracts were treated with enzymatically active recombinant caspase-14, which could restore degradation of the filaggrin fragments (Fig. 2b). In addition, we in vitro translated cDNA constructs encoding N-terminal fragments of human (Fig. 2c) or mouse (Fig. 2d) profilaggrin and treated them with recombinant caspase-14 (ref. 15), which resulted in processing at multiple sites. In both experiments (Fig. 2b–d), addition of the pan-caspase inhibitor zVADfmk to recombinant caspase-14 prevented (pro)filaggrin processing. Moreover, the enzymatically dead C132A caspase-14 mutant (caspase-14C132A) could not cleave (pro)filaggrin (Fig. 2b–d). Thus, the altered (pro)filaggrin processing pattern observed in caspase-14−/− mice can be explained by the absence of direct cleavage of (pro)filaggrin by caspase-14.

Genetic studies recently established that filaggrin has an important role in epidermal barrier formation12,16. The formation of a functional barrier during embryonic development occurs from day E16.5–17.5 and coincides with stratum corneum formation, profilaggrin processing and caspase-14 activation17,18. To examine whether epidermal barrier formation was affected during embryonic development in caspase-14-deficient mice, an in situ toluidine blue penetration assay was performed. This assay evaluates differences in the outside–in epidermal barrier. The formation of the skin barrier was essentially the same in wild-type and caspase-14-knockout embryos; on day E16.5 the barrier started to form at the dorsal part and spread around the embryo to the ventral part (see Supplementary Information, Fig. S3b). Formation of the barrier on the dorsal side was not uniform for all embryos on day E16.5, as observed by others18. On day E17.5, the embryonic skin barrier was completely formed in wild-type, as well as in caspase-14−/− embryos (see Supplementary Information, Fig. S3b). After birth the cornified layer also acts as a physical inside–out barrier. Therefore, transepidermal water loss (TEWL) and hydration was measured to determine whether the water barrier was affected in caspase-14-deficient mice. TEWL was 20% higher in caspase-14-deficient mice (Fig. 2e). Consequently, the stratum corneum hydration level was consistently approximately 20% lower in newborn and adult caspase-14-deficient mice (Fig. 2f). To determine whether a defect in the tight junction performance was the underlying cause for increased TEWL, dermal injections of a biotin tracer were performed. In both wild-type and caspase-14-deficient mice, the diffusion of the tracer to the skin surface was prevented by functional tight junctions (data not shown). Our findings demonstrate that the specialized cell death process leading to cornification is affected in caspase-14−/− mice, resulting in a less efficient skin barrier. Lower hydration levels could be explained by incomplete degradation of filaggrin fragments in caspase-14−/− mice (Fig. 2a), leading to lower levels of free amino acids that have an important role as natural moisturizing factors of the stratum corneum14.

One of the most important functions of the skin is to protect against environmental stress, of which protection against UVB is of major importance. The back skin of newborn wild-type and knockout mice was irradiated with 250 mJ cm−2 or 750 mJ cm−2 of UVB. Intriguingly, haematoxylin–eosin-stained sections of caspase-14-deficient skin displayed significantly more epidermal damage at the 250 mJ cm−2 dose than wild-type skin, and exhibited massive ballooning of cells in the stratum spinosum and granulosum when exposed to 750 mJ cm−2 (Fig. 3a). Accordingly, caspase-14-deficient skin displayed a large number of sunburnt cells with apoptotic hallmarks, such as pyknotic nuclei, caspase-3 activation and internucleosomal DNA degradation (Fig. 3). Caspase-3-positive cells were at least 2–3 times more numerous in UVB-irradiated epidermis from caspase-14-deficient mice than from wild-type mice (Fig. 3b, c). Similarly, quantification of the epidermal TUNEL-positive cells, demonstrating internucleosomal DNA degradation, indicated that more TUNEL-positive cells were present in caspase-14−/− than in caspase-14+/+ epidermis (Fig. 3d, e). These experiments were performed in neonatal mice (5.5 days after birth) and confirmed in adult mice (see Supplementary Information, Fig. S4a), indicating the persistence of the observed phenotype. UVB-induced apoptosis in the skin is p53 dependent19. To evaluate whether alternative p53-independent apoptotic pathways were activated in the highly UVB sensitive caspase-14−/− mice, we crossed our mice with p53-deficient mice20. Ablation of p53 resulted in similar inhibition of apoptosis in the caspase-14+/+ and caspase-14−/− background (see Supplementary Information, Fig. S4b). Taken together, our results demonstrate that caspase-14 is involved in the biochemical processes required to protect the skin against UVB-induced apoptosis.

Figure 3: Caspase-14-deficient mice are highly sensitive to UVB-induced apoptosis.
figure3

(a) Haematoxylin–eosin-stained sections of epidermis of non-irradiated and UVB-irradiated 5.5-day-old mice. Samples were taken 24 h after irradiation. Arrows indicate cells with pyknotic nuclei. (b) Immunohistochemical staining of epidermal sections (as in a) with an antibody against active caspase-3. Arrows indicate cells containing active caspase-3. (c) Quantification of active caspase-3-positive cells. All cells in the epidermis staining with the antibody against active caspase-3 were counted over a linear distance of 1.3 mm. The error bars represent the mean ± s.d. of three mice from a representative experiment. (d) TUNEL staining of sections as in a. Arrows indicate TUNEL-positive cells. (e) Quantification of TUNEL-positive cells in the epidermis. TUNEL-positive cells of the interfollicular epidermis were counted over a linear distance of 1.3 mm. The results represent an average from two mice from a representative experiment. The scale bars represent 40 μm in a, 20 μm in b and 7 μm in d.

There are several possibilities why the epidermis of caspase-14-deficient mice is more sensitive to apoptosis induced by UVB: first, keratinocytes of caspase-14-deficient mice may be more sensitive to UVB-induced apoptosis; and second, in view of the observed alterations in the terminal differentiation program of keratinocytes in caspase-14−/− epidermis, the UVB scavenging capacity of the stratum corneum may be diminished. To distinguish between the two possibilities, the sensitivity of freshly prepared keratinocytes derived from caspase-14−/− and wild-type mice to UVB-induced apoptosis was compared. In addition, we tested several other DNA-damaging apoptotic stimuli and found that their apoptotic responses to UVB irradiation, doxorubicin, etoposide or cisplatin were similar (Fig. 4a). UVB induces direct formation of cyclobutane pyrimidine dimers (CPDs), which are crucial for triggering UVB-induced apoptosis21. There was no difference in the observed CPD levels of UVB-irradiated wild-type and caspase-14−/− primary keratinocyte cultures (Fig. 4b). This demonstrates that there are no cell autonomous differences in DNA-damage sensitivity between wild-type and caspase-14-deficient keratinocytes. To explore the second possibility, we reasoned that if the UVB-filtering capacity of the stratum corneum is reduced in caspase-14−/− mice, more cyclobutane pyrimidine dimers (CPDs) would be expected immediately after UVB irradiation. Almost no CPD staining was observed 15 min after irradiation of caspase-14+/+ mice with 250 mJ cm−2, and only a minor CPD staining when 750 mJ cm−2 was used (Fig. 4c). This UVB-induced DNA damage was completely repaired after 24 h. In contrast, when caspase-14−/− mice were irradiated with similar doses, a very strong CPD staining was observed throughout the epidermis immediately after irradiation, and remained for at least 24 h. The increased DNA damage in caspase-14−/− epidermis was then confirmed by Southwestern dot blots (Fig. 4d). These CPD-positive cells eventually undergo apoptosis, corresponding to the increased number of apoptotic cells detected in caspase-14−/− epidermis after UVB irradiation (Fig. 3).

Figure 4: The UVB-filtering capacity of the stratum corneum is reduced in caspase-14-deficient mice.
figure4

(a) Freshly isolated primary keratinocytes derived from 1-day-old caspase-14+/+ and caspase-14−/− mice were cultured and irradiated with different doses of UVB or treated with the DNA-damage inducing agents doxorubicin (1 μg ml−1), etoposide (50 μM), cisplatin (100 μM) or DMSO control. Cell death was measured with an MTT assay27. The error bars represent the mean ± s.d. of four independent experiments. (b) Southwestern dot-blot analysis of caspase-14+/+ and caspase-14−/− primary keratinocytes that were irradiated with different doses of UVB. Genomic DNA was extracted 30 min after UVB irradiation, dot blotted (1 μg) and revealed with an anti-CPD antibody. Results show one out of four independent experiments. (c) Anti-CPD staining of non-irradiated and UVB-irradiated epidermis of 5.5-day-old caspase-14+/+ and caspase-14−/− mice. Samples were taken 15 min or 24 h after irradiation with different doses of UVB. Results show one out of three independent experiments. (d) Southwestern dot-blot analysis, using an anti-CPD antibody, of genomic DNA extracted from the epidermis of 5.5-day-old caspase-14+/+ and caspase-14−/− mice that were irradiated with different doses of UVB. Genomic DNA was extracted at different times after UVB irradiation (15 min, 6 and 24 h). Data shown are from one of three independent experiments. To compile the image, electronic cuts were introduced after all samples were blotted and scanned on a single membrane. (e) Anti-CPD staining of tape-stripped mice. The stratum corneum of caspase-14+/+ and caspase-14−/− mice was removed from the back of 5.5-old-mice by tape-stripping. Control and tape-stripped mice were irradiated with 750 mJ cm−2 UVB. Epidermal samples were taken 15 min after exposure and stained with an anti-CPD antibody. The scale bars represent 10 μm in c and e.

Because the increase in CPD staining was observed immediately after UVB irradiation (15 min), the UVB-filtering capacity of caspase-14−/− corneum must have decreased dramatically. To substantiate this hypothesis, caspase-14+/+ and caspase-14−/− mice were tape-stripped to remove the stratum corneum and then irradiated with UVB. Tape-stripped epidermis of caspase-14+/+ mice became at least as sensitive to UVB-induced CPD formation as non-tape-stripped caspase-14−/− mice (Fig. 4e). In contrast, removal of the stratum corneum in caspase-14-deficient mice only mildy affected the CPD levels after UVB irradiation. In agreement with the observed CPD levels, the difference in UVB scavenging capacity of the stratum corneum in caspase-14+/+ and caspase-14−/− was also reflected in the subsequent apoptotic response of control and tape-stripped mice (see Supplementary Information, Fig. S4c). These findings also confirm that the difference in UVB sensitivity is not due to a cell autonomous defect in the keratinocytes. We want to stress that the basal and stratum spinosum cells that also lack enzymatically active caspase-14 in wild-type mice1,2,3, specifically undergo more UVB-induced DNA damage and apoptosis in the caspase-14−/− epidermis. The latter observation again points to a stratum corneum defect in caspase-14 knockout mice. In addition, there was no difference in UV light reflection between caspase-14+/+ and caspase-14−/− skin (data not shown). Our experiments on caspase-14-deficient mice indicate that the absence of caspase-14 during cornification severely alters the biochemistry of this process during terminal differentiation of keratinocytes, reducing the efficacy of the barrier against UVB irradiation and water loss.

In spite of the clear association of the expression and proteolytic activation of caspase-14 with the formation of cornified envelopes, the role of caspase-14 in this process has been enigmatic. Here, we show that absence of caspase-14 severely affects the functional outcome of epidermal cornification. Although embryonic outside–in barrier formation during development is not affected or delayed, epidermal hydration is reduced in neonate and adult caspase-14−/− mice because of increased trans-epidermal water loss. The altered processing pattern of profilaggrin indicates that it could be involved in this phenotype. It has been demonstrated that profilaggrin processing has a crucial role in hydration22. Recently, the importance of filaggrin has been underscored by demonstrating that loss-of-function mutations in the profilaggrin gene underlie the skin disease ichthyosis vulgaris, and that they strongly predispose to atopic dermatitis and asthma12,16. The latter may be due to an increased entry of allergens and infectious agents in these patients. The structure of caspase-14-deficient skin shows hyperlinearity and lower hydration, characteristics of filaggrin-deficient human skin12. Importantly, the UVB protective properties of the stratum corneum are severely reduced in caspase-14-deficient mice. The non-pigmentary photoprotection by the epidermis, such as observed in vitiligo in which patches of skin lose their pigmentation, is not yet understood. Caspase-14-deficient mice may serve as a model for studying the mechanism of stratum corneum hydration and photoprotection against UVB irradiation. Identification of the molecules that are involved in caspase-14-dependent skin-barrier formation will be of clinical relevance.

Methods

Western blotting.

The epidermis of 5.5-day-old neonatal mice was isolated. Skin was incubated in PBS with 10 mM EDTA at 56 °C for 10 min, after which epidermis was mechanically separated from dermis. Epidermis was lysed in NP-40 buffer (10 mM Tris–HCl at pH 7, 200 mM NaCl, 5 mM EDTA, 10% glycerol, 1% NP-40, 100 nM PMSF, 0.15 nM aprotinin, 2.1 nM leupeptin) and freeze-thawed ten times in liquid nitrogen. Samples were centrifuged, then 20 μg protein was separated on gel and transferred to a nitrocellulose membrane. Membranes were incubated with primary antibodies and appropriate HRP-labelled secondary antibodies (GE Healthcare, Diegem, Belgium). Detection was performed with the Western LightningT chemiluminescence reagent plus kit (PerkinElmer, Waltham, MA).

Caspase-activity assay.

Epidermal extracts from wild-type and knockout mice were prepared in grinding buffer (100 mM HEPES buffer at pH 7.5 containing 200 mM NaCl) and tested for caspase activity by incubating 50 μg of extract in caspase-14 assay buffer (10 mM PIPES at pH 7.5, 10% sucrose, 100 mM NaCl, 1.3 M sodium citrate, 0.1 mM EDTA, 10 mM DTT) and 50 μM WEHDamc or DEVDamc (Peptide Institute, Barnet, UK). Fluorescence was monitored for 50 min with a Cytofluor Multiwell Reader series 4000 at 37 °C (Perseptive Biosystems, Cambridge, MA).

Antibodies.

Primary antibodies used were directed against mouse caspase-3 and -14 (ref. 2), mouse Keratin 1 (K1), K5, K10, filaggrin, loricrin and involucrin (all purchased from Covance, Berkeley, CA), active caspase-3 (Bio-Connect, Huissen, The Netherlands) and CPDs (Kamiya Biomedical Company, Seattle, WA).

Histological and immunohistochemical analysis.

Tissues were fixed for 2 h in 4% paraformaldehyde and embedded in paraffin. Sections of 5 μm were stained with haematoxylin–eosin or processed for immunohistochemical analysis. Primary antibodies were detected with the appropriate HRP (DAB) kit (Dako EnVision System, Glastrup, Denmark).

Electron microscopy.

For scanning electron microscopy, back skins of 4.5-day-old neonatal mice were fixed in Karnovsky buffer for 2 days at 4 °C and rinsed twice in sodium-cacodylate buffer. Fixed tissues were dehydrated in graded ethanol solutions (25, 35, 50, 75, 85, 95 and 100%). Dehydrated skins were rinsed three times with liquid CO2, critical-point dried, mounted on stubs with glue (Pattex, Henkel, Germany) and coated with gold. They were examined with a Jeol JSM-840 scanning electron microscope. For transmission electron microscopy, skin biopsies from the backs of 6.5-day-old mice were treated as previously described23. Postfixation was performed in 1% OsO4 sodium-cacodylate buffer containing K3Fe(CN)6 at 4 °C for 12 h. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a Jeol 1200 EXII electron microscope at 80 kV.

UVB irradiation.

TL20W/12RS Philips UV lamps were used (peak emission at 310 nm), and a plastic lid was used to block UVC emission. The UVB dose was monitored with an IL1700 radiometer equipped with a SED005/TLS312/W detector (International Light, Purmerend, The Netherlands). The backs of wild-type and caspase-14−/− mice were irradiated at a dose rate of 0.7 mJ cm−2 s−1.

TUNEL assay.

TUNEL assay was performed on 5 μm paraffin sections from skin with the in situ cell death detection kit, TMR red (Roche, Vilvoorde, Belgium), according to the manufacturers instructions. Stained sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Houson, TX).

Southwestern dot-blot analysis.

Epidermis was isolated as described for western blotting. Genomic DNA was isolated from epidermis and 0.5 μg was used for Southwestern dot-blot analysis using the anti-CPD antibody24.

Isolation of primary newborn keratinocytes.

Primary keratinocytes were isolated as previously described25. Whole skins of 1-day-old mice were floated on 2.5 U ml−1 Dispase II (Roche) in defined keratinocyte-SFM (K-SFM; Gibco, Merelbeke, Belgium) overnight at 4 °C. Epidermis was separated form dermis, incubated in 5× trypsin–EDTA solution for 10 min at 37 °C. Cells were seeded in K-SFM supplemented with keratinocyte growth supplement (10784-015, Gibco), insulin, hydrocortisone and hEGF (Clonetics, New Jersey, NJ), 1 ng ml−1 Cholera toxin (Sigma, Bornem, Belgium), gentamycin (50 μg ml−1), 100 μg ml−1 penicillin–streptomycin and 10% LPS-free FCS in a humidified incubator at 34 °C with 7.5% CO2. Next day cells were refreshed with complete K-SFM without FCS.

In vitro cleavage assay.

Isolated mouse epidermis from 5.5-day-old neonates was homogenized by sonication in caspase-14 assay buffer (supplemented with 1 μg ml−1 leupeptin, 1 μg ml−1 aprotinin and 0.5 mM PMSF), freeze-thawed ten times, centrifuged at 20,000g (20 min at 4 °C), and supernatant was collected. Epidermal lysate was incubated with recombinant human (h) caspase-14 wild type with or without 20 μM zVADfmk or hcaspase-14C132A mutant in caspase-14 assay buffer for 3 h at 37 °C. Filaggrin profiles were analysed by immunoblotting.

The sequence for the N-terminal 467 amino acids of human profilaggrin was cloned in the pCDNA3 vector (hFG1)15 and the N-terminal 283 amino acids of mouse profilaggrin were cloned in the GBKT7 (Clontech, Mountain View, CA) expression vector (mFG2). 35S-methionine-labelled profilaggrin fragments were prepared with the coupled transcription–translation assay TNT kit (Promega, Leiden, The Netherlands). Translation reactions were incubated as mentioned above. Cleavage products were separated by 15% SDS–PAGE; reactions had to be diluted 1:5 because of high salt concentration. Gels were vacuum dried and the resulting labelled cleavage products were analysed by autoradiography.

Note: Supplementary Information is available on the Nature Cell Biology website.

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Acknowledgements

We thank A. Bredan for editing the manuscript and E. van Damme, A. Meeus and W. Deckers for technical assistance. This work was supported in part by the Interuniversitaire Attractiepolen V, the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, the Epistem 6th framework EC-RTD grant and Ghent University GOA project. G.D. is a postdoctoral fellow at the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, E.H. has an Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie (IWT) predoctoral grant and P.O. had an Emmanuel Verscheuren and an IWT predoctoral grant. R.P. was supported by R01 AR49183 from the National Institutes of Health.

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Denecker, G., Hoste, E., Gilbert, B. et al. Caspase-14 protects against epidermal UVB photodamage and water loss. Nat Cell Biol 9, 666–674 (2007). https://doi.org/10.1038/ncb1597

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