During their turnover from the basal to the upper-most epidermal layers primary keratinocytes (KC) differentiate into corneocytes, thereby forming the tight barrier that protects the organism against transepidermal water loss and from external damaging influences (Haake and Holbrook, 1999; Latkowski and Freedberg, 1999). During the transition to corneocytes, commonly referred to as terminal differentiation, KC die in a tightly controlled fashion (for reviewGandarillas, 2000; Melino et al, 1998). This type of cell death is in some aspects, such as the loss of the nuclei, reminiscent of programmed cell death in other cells; however, the fact that the end-point of this process is not complete disintegration of cells, but formation of cellular ghosts that are resistant to biochemical and biophysical degradation, i.e., corneocytes, sets it apart from conventional apoptosis, which aims at the complete removal of cells without leaving a trace.
Caspases are the key enzymes of apoptosis. They cleave their substrates specifically at 4 amino-acid motifs leading to the inactivation and degradation of a large number of regulatory and structural proteins (Chang and Yang, 2000). In addition, caspases can cleave and activate each other thereby regulating and amplifying apoptotic signals. Caspase activity was detected in epidermal preparations (Takahashi et al, 1998) and activation of caspase-3 was localized to cells at the transition zone between the granular and cornified layers of the epidermis (Weil et al, 1999) and related to the formation of the skin barrier. Recently, we and others have reported that caspase-14, the latest member of the caspase family (Ahmad et al, 1998; Hu et al, 1998; Van de Craen et al, 1998) is almost exclusively expressed in the epidermis (Eckhart et al, 2000b; Lippens et al, 2000). We could show that the activation of human caspase-14 occurs during the terminal differentiation of KC in vitro (Eckhart et al, 2000) and we have suggested that it plays a part in the terminal differentiation of KC but not in conventional apoptosis.
To expand our knowledge of the role of caspases in the terminal differentiation of KC we have analyzed here the regulation of caspase-14, -3, and -8 expression by retinoids, which are potent modulators of KC differentiation (Fisher and Voorhees, 1996). Our finding that caspase-14, but not caspase-3 and -8, is regulated by retinoids in a differentiation-dependent fashion, supports the role of caspase-14 in the terminal differentiation of KC and sets it further apart from the classical pathways of apoptosis.
MATERIALS AND METHODS
Cell culture
Human neonatal foreskin-derived KC were obtained from Clonetics (San Diego, CA) and cultured as described by the manufacturer under low Ca2+ conditions (0.15 mM) in serum-free keratinocyte growth medium (KGM), i.e., modified MCDB 153 medium containing 0.1 ng human recombinant epidermal growth factor per ml, 5 
g insulin per ml, 0.5 g hydrocortisone per ml, 0.4% bovine pituitary extract, 50 g gentamycin per ml, and 50 ng amphotericin B per ml. Second to fourth passage KC were used for all experiments. For differentiation-promoting confluence assays KC were seeded at low density in six-well plates, grown to confluence (designated as "day 0") and cultured for up to 5 d postconfluence (designated as "day 5"). Retinoids or dimethyl sulfoxide were added to fresh KGM at indicated days and medium changes were performed daily.
Human dermal fibroblasts were obtained by explant cultures of de-epidermized skin specimen derived from routine breast reduction surgery. Fibroblasts were subcultured in Dulbecco's modified Eagle medium (Gibco, BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum and were used between passages 3 and 10.
Skin equivalents
In vitro reconstructed skin equivalents were generated essentially as described byStark et al (1999) with modifications. Briefly, 2.5 ml of a collagen solution containing 2.4 mg per ml bovine collagen type I (Vitrogen 100, Collagen Corporation, CA), 1
Hank's buffered saline solution (Gibco), 10% fetal bovine serum and 1105 fibroblasts per ml, adjusted to pH 7.4 with 2 M NaOH, were poured into 24 mm cell culture inserts (Falcon, Becton Dickinson, Schwechat, Austria), which were placed in special deep six-well trays (Falcon). After gelation at 37°C in a humidified atmosphere for 2 h in the absence of CO2, gels were equilibrated in 16 ml prewarmed KGM (2 ml inside, 14 ml outside the insert) for 2 h at 37°C in a 5% CO2/95% air environment in a humidified incubator. The medium inside the insert was then removed and replaced by 2 ml KGM containing 3105 KC per cm2 (
1.3106 cells). After submerged culture overnight, the medium was changed to 10 ml serum-free keratinocyte defined medium (SKDM) outside the insert, and the cultures were kept at the air–liquid interface from that time point onwards. SKDM is a high Ca2+ medium (1.3 mM) consisting of KGM except for the omission of bovine pituitary extract and containing 10 g transferrin per ml (Clonetics), 0.1% bovine serum albumin (Sigma, St Louis, MO) and 50 g L-ascorbic acid per ml. Culture medium for skin equivalents was replaced by fresh prewarmed SKDM every 2–3 d and culture was continued for up to 6 d. At indicated days samples were taken by punch biopsy, formalin-fixed and processed for either hematoxylin and eosin (H&E) staining or immunofluorescence. Simultaneously, the remaining part of the skin equivalents or additional, equally treated skin equivalents were lyzed for Western blot and Northern blot analysis.
Retinoids
All-trans retinoic acid (RA) and all-trans retinol (Sigma) were dissolved in dimethylsulfoxide and stored as 10-2M aliquots at -20°C protected from light. At the time of use, retinoids were diluted in KGM with the addition of 0.1% bovine serum albumin (Sigma) to stabilize the retinoids or in SKDM for skin equivalents (Hodam and Creek, 1996). Dimethyl sulfoxide at 0.01% or below served as control.
Immunostaining
Paraffin sections of skin equivalents were immuno-stained according to a previously published protocol (Weninger et al, 1999). Briefly, dewaxed sections were boiled in 0.01 M citrate buffer (pH 6.0) in a microwave oven at 500 W for 10 min, cooled to room temperature and incubated with 2% bovine serum albumin (Sigma)/10% goat serum (DAKO, Glostrup, Denmark) for 30 min to block nonspecific binding of the secondary antibody. The primary antibodies were applied for 1 h at room temperature followed by 30 min incubation with 4 ng per ml Alexa 546 (or 488) goat anti-rabbit or goat anti-mouse anti-sera (Molecular Probes, Leiden, the Netherlands). Primary antibodies were directed against caspase-14 (rabbit anti-serum;Lippens et al, 2000), filaggrin (mouse IgG1, Biomedical Technologies Inc., Stoughton, MA), and keratin 19 (mouse IgG1, DAKO). For control purposes the caspase-14 anti-serum was preincubated with 10 g recombinant caspase-14 per ml for 1 h at room temperature. In situ detection of active caspase-3 was obtained by immunofluorescence staining with two different anti-sera specifically recognizing the activated form of caspase-3 only (rabbit anti-serum, R&D Systems, Minneapolis, MN; rabbit monoclonal, Research Diagnostics Inc., Flanders, NJ). A similar protocol as described above was used for anti-caspase-3 staining except that cryosections were used without special pretreatment.
In situ detection of apoptosis
Apoptotic cells were identified on H&E-stained sections by the characteristics of apoptosis, i.e., cell shrinkage with condensed, pyknotic nuclei and eosinophilic cytoplasm. Detection of apoptotic cells was confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling with the "in situ cell death detection kit" (Boehringer Mannheim, Vienna, Austria) according to the manufacturer's instructions.
Western blot analysis
For Western blot analysis all samples were lyzed in sodium dodecyl sulfate lysis buffer (62.5 mM Tris–HCl, pH 6.8, 6 M urea, 2% sodium dodecyl sulfate, 0.00125% bromophenol blue, 5%
-mercaptoethanol). After ultrasonication and removal of insoluble cell debris, 40 g of protein were electrophoresed through an 8–18% gradient polyacrylamide gel and blotted on to a nitrocellulose membrane (Bio-Rad, Hercules, CA). Blots were blocked for 2 h in blocking buffer (phosphate-buffered saline with 7.5% nonfat dry milk, 2% bovine serum albumin, 0.1% Tween) and incubated with caspase-14 anti-serum (1: 1000) (Lippens et al, 2000), caspase-8 anti-serum (1: 1000, kind gift from Peter Krammer (Scaffidi et al, 1997), caspase-3 (1: 1000, Pharmingen, San Diego, CA), and filaggrin (1: 200, Biomedical Technologies) antibodies in blocking buffer overnight at 4°C. Subsequently, membranes were washed in phosphate-buffered saline with 5% nonfat dry milk, 0.1% Tween, incubated with peroxidase-conjugated goat anti-rabbit IgG Fc antibody (Pierce, Rockford, IL 1: 10,000 in blocking buffer) for 1 h at room temperature, washed in phosphate-buffered saline and developed using the ECL chemiluminescence detection system (Amersham Buckinghamshire, UK).
Northern blot analysis
Northern blot analysis was performed as previously described (Eckhart et al, 2000b). The probe for caspase-14 was generated by 32P-deoxycytidine triphosphate random labeling of the polymerase chain reaction product amplified from human KC cDNA with the primers 5'-ATATGATATGTCAGGTGCCCG-3' (sense) and 5'-CTTTGGTGACACACAGTATTAG-3' (anti-sense). The probes for -actin were generated analogously, using the primers 5'-TTCCCCT CCATCGTGGGGCGCCCCAGGCACCAGGGC-3' (sense) and 5'-GGC-GACGTAGCACAGCTTCTC-3' (anti-sense).
RESULTS
When primary KC were allowed to differentiate by culturing them at confluence (Lee et al, 1998), expression of the differentiation-associated marker profilaggrin was induced and increased substantially over the following days (Figure 1a). The addition of 10-6M RA, a potent inhibitor of KC differentiation (Fisher and Voorhees, 1996), nearly completely inhibited this induction (Figure 1a) (Asselineau and Darmon, 1995). Similarly, the induction of pro-caspase-14 (p28), which as we have recently shown is induced in differentiating conditions (Eckhart et al, 2000b), was inhibited by RA treatment (Figure 1a). In contrast to caspase-14 protein levels, which after RA treatment stayed steady throughout the culture period, caspase-14 mRNA was reduced by RA treatment to nearly undetectable levels within the first 24 h, indicating its downregulation at the transcriptional level (Figure 1a). Treatment with different concentrations of RA demonstrated the concentration dependence of the effect on pro-caspase-14 protein expression (Figure 1b). Similar inhibition of pro-caspase-14 expression was observed with retinol (not shown), the physiologic precursor that is converted to active RA after uptake into cells (Fisher and Voorhees, 1996), as well as with the all-trans RA isomers 13-cis RA and 9-cis RA (not shown).
Figure 1.
Retinoids downregulate caspase-14 expression in primary keratinocyte cultures. (a) Primary KC were differentiated by culturing them at confluence and differentiation was inhibited by addition of all-trans RA starting at day 1 after confluence for up to 4 d. The induction of both the epidermal differentiation marker profilaggrin (1st panel) as well as of pro-caspase-14 (2nd panel) was strongly reduced. Northern blot analysis (3rd panel) revealed downregulation of caspase-14 at the mRNA level. Equal loading of RNA was confirmed by ethidium bromide staining of the gel (4th panel). (b) Primary KC were cultured for 4 d in the presence of different concentrations of RA starting at day 1 after confluence. Western blot analysis demonstrated the concentration dependence of the downregulation of pro-caspase-14 by RA.
Full figure and legend (94K)In analogy to the absence of profilaggrin processing (Figure 1a), no cleavage of pro-caspase-14 was detected in confluent KC cultures (Figure 1a) (Eckhart et al, 2000b). This is most likely due to the fact that in submerged cultures KC differentiate only incompletely (Asselineau et al, 1989). In contrast to KC in monolayer cultures, KC in skin equivalents (skin equivalents) form a stratified, regularly differentiated epidermis under the stimulus of air exposure (Stark et al, 1999). Under these conditions profilaggrin is processed (Asselineau and Darmon, 1995) and also pro-caspase-14 must be activated, as cleavage products become detectable (Figure 2a) (Eckhart et al, 2000b). In skin equivalents retinoids prevent regular epidermal differentiation (Asselineau and Darmon, 1995). We could confirm that RA treatment of skin equivalents for 4 and 6 d inhibited both profilaggrin induction and processing (Figure 2a), and we found that the expression of pro-caspase-14 (p28) was strongly reduced by RA treatment (Figure 2a) without the appearance of cleavage products, which would be indicative of its activation. Analogous to KC in monolayer culture, Northern blot analysis revealed a strong inhibition of caspase-14 mRNA expression (Figure 2b).
Figure 2.
Caspase-14 expression and activation is inhibited by retinoids in organotypic skin cultures. KC were differentiated in three-dimensional in vitro reconstructed skin equivalents cultured at the air–liquid interface and differentiation was inhibited by RA treatment. (a) Western blot analysis showed expression of both the differentiation marker profilaggrin and of pro-caspase-14, as well as their activation by cleavage (filaggrin: 38 kDa, caspase-28: 17, 11 kDa) in this improved in vivo-like culture conditions. RA treatment strongly inhibited expression and activation of both proteins. (b) Northern blot analysis demonstrated that the regulation of caspase-14 occurred at the level of mRNA expression.
Full figure and legend (76K)To understand better the regulation of caspase-14 expression and its temporal and spatial association with the inhibition of KC differentiation by RA, we studied skin equivalents by H&E staining and immunofluorescence analysis of the in situ expression of caspase-14 and KC differentiation markers. At 4 and 6 d of culture, control skin equivalents showed all features of regular epidermal differentiation (Figure 3a,c), including strong expression of filaggrin (Figure 3i,k) and caspase-14 (Figure 3e,g). Treatment with 10-6M RA for 4 d prevented the formation of both granular and cornified KC layers (Figure 3b). After 6 d, epidermal thickness had strongly increased, stratification was disturbed, KC appeared to lose their orientation, and no cornified layer was formed (Figure 3d). Both caspase-14 and filaggrin expression were strongly reduced after 4 d (Figure 3f,j) and only found in scattered cells after 6 d (Figure 3h,l). The possibility that filaggrin and caspase-14 downregulation was due to an overall reduction of protein synthesis was excluded by the demonstration that de novo expression of the "wet epithelial" keratin 19 was strongly induced by RA treatment (Figure 3m–p) (Asselineau and Darmon, 1995).
Figure 3.
Inhibition of keratinocyte differentiation by retinoic acid in organotypic epidermal cultures is paralleled by downregulation of caspase-14. In vitro differentiated skin equivalents (skin equivalents) were generated by culture at the air–liquid interface for 4 and 6 d and their differentiation was inhibited by treatment with RA. The association of caspase-14 regulation by RA with the inhibition of differentiation was analyzed by H&E (a–d) and immunofluorescence stainings for caspase-14 (e–h), filaggrin (i–l), and keratin 19 (m–p). As compared with control (a), treatment with 10-6M RA for 4 d led to loss of the granular and cornified epidermal layers and KC retained their nuclei in the uppermost layers (arrows). After 6 d of RA treatment, in addition, the epidermal thickness was increased, regular stratification was disturbed, and up to 20% of KC showed "sunburn-cell" morphology characteristic for apoptosis (d, arrowheads). Simultaneously, as compared with untreated control samples (e,g), caspase-14 expression was nearly completely inhibited after 4 d (f, arrows), and after 6 d only scattered KC stained weakly positive (h, arrows). Downregulation of the differentiation marker filaggrin served as a control for the inhibitory effects of RA treatment on epidermal differentiation (i–l). By contrast, keratin 19, characteristic for "wet epithelia", was expressed after 4 d (n) and 6 d (p) of RA treatment, and was absent in control samples (m,o). Bars=50 m.
As activation of caspase-3 has been suggested to be involved in the transition of viable KC into corneocytes (Weil et al, 1999) and caspase-8 has been shown to associate with caspase-14 (Hu et al, 1998) we also analyzed the expression, regulation, and activation of these caspases during differentiation and its inhibition by RA. In KC monolayer culture we found that, in contrast to caspase-14, both caspase-3 and -8 were strongly expressed at the time of culture initiation and that the expression levels during confluence slightly decreased and did not change, respectively (Figure 4a). As opposed to the potent downregulation of caspase-14 by RA treatment, no regulation of caspase-8 was observed and caspase-3 levels slightly increased after addition of RA (Figure 4a). The results on caspase-3 and -8 expression obtained in monolayer culture were comparable with those found in skin equivalents: both caspases were strongly expressed and caspase-3 pro-enzyme levels were strongly increased by RA treatment, whereas those of caspase-8 remained unchanged (Figure 4b). In contrast to the activation of caspase-14 in regularly differentiated skin equivalents, no cleavage of caspase-3 or -8 could be detected (Figure 4b) under control conditions; however, RA treatment of skin equivalents for 6 d, led to caspase-3 activation as determined by the occurrence of the 17 kDa large subunit in western blot analysis, indicative of ongoing apoptosis (Figure 4b). By immunofluorescence staining we could demonstrate that caspase-3 activation was confined to individual cells in suprabasal layers (Figure 5b), which in H&E staining were characterized as apoptotic cells comprising 10–20% of all KC (Figure 3d, arrowheads). These immunostaining data were confirmed by using a second anti-serum (not shown) and by blocking experiments with human recombinant active caspase-3 (not shown). In contrast to an earlier report (Weil et al, 1999), we could not detect caspase-3 activation in cells at the transition zone between the granular and cornified layer in untreated skin equivalents. Our data demonstrate that, as opposed to caspase-14, the inducer and effector caspases 8 and 3, respectively, are not regulated in a differentiation-dependent manner in KC, and that RA treatment can lead to caspase-3 activation in KC.
Figure 4.
Caspase-3 and -8 are not regulated in a differentiation dependent manner in primary KC and differentiating skin equivalents. Primary KC were differentiated by culturing at confluence (a) or in stratified skin equivalents (b) and differentiation was inhibited by addition of RA. Expression of caspase-3 and -8 was analyzed by Western blot. (a) Unlike caspase-14, both caspase-3 and -8 expression were neither up-regulated during differentiation nor inhibited by RA treatment. Rather, caspase-3 pro-enzyme levels slightly decreased after extended culture at confluence and slightly increased by additional RA treatment. Expression levels of pro-caspase-8 were unchanged under any conditions tested. (b) No differentiation-associated regulation of caspase-3 and -8 by RA treatment was detectable. Moreover, no cleavage cloud be detected in regularly differentiating SE, indicating its dispensability for regular epidermal differentiation. However, caspase-3 activation, as demonstrated by the appearance of the cleaved 17 kDa large subunit, was readily detectable after RA treatment.
Full figure and legend (93K)Figure 5.
Apoptosis of KC in skin equivalents after RA-mediated abrogation of differentiation involves activation of caspase-3. Skin equivalents were cultured at the air–liquid interface for 6 d and their differentiation was inhibited by treatment with RA. In situ analysis of caspase-3 activation by immunofluorescence with an anti-serum specific for activated caspase-3 was performed. (a) In control samples, caspase-3 activation was detected only in few scattered apoptotic cells (arrows) but not in the terminal differentiating layers. (b) In addition to inhibition of differentiation, RA treatment strongly increased the number of active caspase-3-positive cells indicative of apoptosis. Bar=50 m.
DISCUSSION
The late stages of epidermal differentiation comprise several well defined changes such as the reorganization of the cytoskeleton, the appearance of granules containing cornified envelope precursor proteins, the assembly of the cornified envelope, and the degradation of the nucleus (Haake and Holbrook, 1999; Latkowski and Freedberg, 1999). In recent years many investigators have sought to clarify the relationship between KC death occurring during these final steps of epidermal differentiation and conventional apoptosis (Gandarillas, 2000). Depending on the methods used and the cell culture conditions, some common and some distinguishing features of KC death in the course of their transition to corneocytes and apoptosis were identified (Melino et al, 1998; Gandarillas, 2000). In the chain of events leading to apoptosis caspases are key enzymes that are able to amplify both apoptotic signals and execute late steps of the cell death program by cleaving crucial structural and regulatory cellular target proteins (Chang and Yang, 2000). The involvement of caspases in the formation of the cornified layer was recently suggested byTakahashi et al (1998) andWeil et al (1999), who detected caspase activity in preparations of the stratum corneum and blocked epidermal differentiation in vitro using caspase inhibitors, respectively. We have recently demonstrated that a new caspase, i.e., caspase-14, which belongs to the group of executioner caspases (Chang and Yang, 2000), is almost exclusively expressed in KC of the epidermis, the hair follicles, and the sebaceous glands (Eckhart et al, 2000a, b; Lippens et al, 2000). In contrast to other executioner caspases it is dispensable for and not activated during conventional KC apoptosis; however, cultivation of KC under conditions leading to the formation of a cornified layer leads to activation of caspase-14 (Eckhart et al, 2000b), suggesting its participation in this process. Here we show that in contrast to caspase-3 and -8, which are involved in classical apoptotic pathways, caspase-14 is downregulated in KC by retinoids under conditions that prevented formation of a cornified layer. The finding that caspase-14 is regulated analogously to proteins involved in the formation of the skin barrier such as filaggrin and involucrin (Fisher and Voorhees, 1996) is an additional piece of evidence that caspase-14 also plays a part in this process. The most likely explanation regarding the mechanisms of RA-mediated downregulation of caspase-14 expression is transrepression of AP-1-mediated gene activation, as has been shown for several differentiation associated genes (Fisher and Voorhees, 1996). Preliminary data on the caspase-14 promoter show that it contains at least two potential AP-1 binding sites that could participate in transrepression (unpublished observations).
In contrast to caspase-14, caspase-3 was not downregulated in skin equivalents by RA treatment. In addition, whereas activation of caspase-3 was not detectable in untreated controls by western blot analysis, it became readily detectable after RA treatment. In situ staining with anti-sera specific for activated caspase-3 corroborated these data. Whereas only few scattered cells expressing activated caspase-3 were found in untreated skin equivalents, up to 20% of cells were positive after treatment of skin equivalents for 6 d. By light microscopy, a similar percentage of cells showed the typical "sunburn cell" morphology (Bayerl et al, 1995) of apoptotic KC. Our data on the RA-mediated KC apoptosis in skin equivalents are in accordance with what has been reported previously (Haake and Cooklis, 1997). As this KC apoptosis occurred in the virtual absence of caspase-14 our data provide further evidence that this caspase is dispensable for conventional KC apoptosis. On the other hand, our results on the presence of activated caspase-3 are in contrast to the observation of the activation of caspase-3 in terminal differentiating KC at the transition zone to the cornified layer (Weil et al, 1999). In agreement with these authors we detected a faint staining of single transitional cells in untreated skin equivalents (not shown); however, we found that this staining was nonspecific as it could not be blocked by preincubation of the serum with active recombinant human caspase-3 (not shown). Under the same conditions the strong staining for activated caspase-3 of cells with apoptotic morphology was completely blocked (not shown). AsWeil et al (1999) did not perform blocking experiments to confirm the specificity of their detection of activated caspase-3 we suggest that these authors might have detected nonspecific binding of their anti-caspase-3 anti-serum. This assumption is further supported by the fact that the caspase cleavage products detected by the authors in western blot analysis did not correspond to the size expected for the fragments of activated caspase-3. In this context it is also interesting that it was shown recently that the anti-serum used by Weil et al recognizes caspase-6 in addition to caspase-3 (Zheng et al, 2000). We therefore believe that the blockade of in vitro differentiation of KC by caspase inhibitors reported by Weil et al might have been due to the inhibition of caspases other than caspase-3.
In conclusion, the observed differences in RA-mediated regulation of the inducer and effector caspases, caspase-8 and caspase-3, and of caspase-14 suggest that regulation and activation of caspase-14 is distinct from that of the classical caspase cascade. Our finding that caspase-14 is regulated by retinoids analogously to proteins of terminal KC differentiation strongly suggest its involvement in skin barrier formation.
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