Ultrastructural Localization of Caspase-14 in Human Epidermis
Dipartimento di Biologia Evoluzionistica Sperimentale, University of Bologna, Bologna, Italy (LA); Department of Dermatology, Medical University of Vienna, Vienna, Austria (MD,CR,ET,LE); and Centre de Recherches et d'Investigations Epidermiques et Sensorielles (CE.R.I.E.S.), Neuilly, France (ET)
Correspondence to: Lorenzo Alibardi, Dept. of Biology, University of Bologna, via Selmi 3, 40126 Bologna, Italy. E-mail: Alibardi{at}biblio.cib.unibo.it
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Summary |
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(J Histochem Cytochem 52:15611574, 2004)
Key Words: human epidermis cornification caspase-14 ultrastructural immunocytochemistry
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Introduction |
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Profilaggrin, a histidin-rich protein that aggregates keratins, is present in F-granules of composite keratohyalin (Fukuyama and Epstein 1986; Holbrook 1989
; Steven et al. 1990
; Manabe et al. 1991
). F-granules have been described for both rodents and human keratohyalin (Yoneda et al. 1992
; Ishida-Yamamoto et al. 1993
). During terminal keratinocyte differentiation they are processed into mature filaggrin units and later degraded to form the so-called natural moisturizing factor of the stratum corneum, whereby only a small amount remains chemically associated with keratins 1 and 10 and corneous cell proteins (Resing and Dale 1991
; Steinert and Marekov 1995
; Ishida-Yamamoto et al. 1999
,2000
).
Loricrin, the main sulfur-rich protein of keratohyalin, is instead localized in round or L-granules in rodents (especially mouse) keratohyalin (Steven et al. 1990; Hardman et al. 1998
), or in amorphous material often associated with F-granules of the composite human keratohyalin (Yoneda et al. 1992
; Ishida-Yamamoto et al. 1993
,1996
,2000
). Another major protein of terminal differentiating keratinocytes, involucrin, is both cytoplasmic and associated with the amorphous material of composite keratohyalin granules (Warhol et al. 1985
; Ishida-Yamamoto and Iizuka 1994
). Proteins such as involucrin, small proline-rich proteins, sciellin, elafin, and eventually loricrin are deposited against the cytoplasmic side of the plasma membrane and desmosomes of terminally differentiating keratinocytes, while ceramides and waxes are deposited mainly on the external surface of the membrane (Kalinin et al. 2002
; Menon and Norlen 2002
).
Most transformation processes during keratinocyte cornification are mediated by specific enzymes. For example, the formation of the insoluble cornified cell envelope is achieved by transglutaminases and by sulfhydryl oxidase, which crosslink proteins via isopeptide and disulfide bonds, respectively (Polakowska and Goldsmith 1991; Hashimoto et al. 2000
).
Because the transition of keratinocytes of the granular layer into enucleate corneocytes represents a form of cell death, we recently screened the caspase family of pro-apoptotic proteases for members specifically expressed in these cells. One caspase, caspase-14, was found to be almost exclusively present in differentiating keratinocytes (Eckhart et al. 2000a,b
; Lippens et al. 2003
). Like other caspases, caspase-14 is expressed as a proenzyme that is activated by cleavage into a large and a small subunit.
The protein substrate of caspase-14 is unknown at present. Activation of caspase-14 was found to be associated with the formation of the stratum corneum in epidermal equivalent models in vitro, and processed caspase-14 was detected in the stratum corneum of human skin (Eckhart et al. 2000a). Immunohistochemical (IHC) analysis revealed that caspase-14 is also present in the upper layers of the epidermis, in sebaceous glands and hair follicles. Immunoreactivity was found primarily in the cytosol but occasionally also in the nuclei of keratinocytes (Eckhart et al. 2000a
; Lippens et al. 2003
). Knowledge of the detailed cytological localization of casp-14 is desirable because this may be a valuable guide in the search for the protein substrate. Therefore, we determined the subcellular localization of caspase-14 in human epidermis by immunoelectron microscopy.
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Materials and Methods |
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Skin Samples
For Western blot analysis, skin samples were obtained from mammary reduction plastic surgery and stratum corneum squames were collected from psoriasis patients after obtaining their informed consent. For immunolocalization of caspase-14, samples of normal human skin (hospital biopsies), collected from the arm and finger of three individuals, were used. Samples (2 x 4 mm) were collected under ethical and legal approval.
Electrophoresis and Immunoblotting
Epidermal proteins and proteins from stratum corneum squames were prepared as described previously and subjected to Western blotting analysis according to a published protocol (Eckhart et al. 2000a). The antibody raised against human caspase-14 was used at a dilution of 1:1000. As control, the anti-caspase-14 antibody was preincubated with 3 µg/µl recombinant human caspase-14 for 2 hr at RT.
Tissue Preparation
The tissues were immediately fixed at 04C for 46 hr with in a modified Carnoy's fluid (9 parts 90% ethanol and 1 part acetic acid). In addition, some pieces from the last sample were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. Tissues were dehydrated at 04C and embedded in Bioacryl resin under UV polymerization at 04C (Scala et al. 1992). Using an ultramicrotome, tissues were sectioned at 12-µm thickness for light microscopic observations. Parallel sections were collected over chromoalum/albumin-coated slides for the following immunocytochemical reaction. From tissues fixed in 4% paraformaldehyde, thin sections at 4080-nm thickness were collected on nickel grids for ultrastructural immunogold cytochemistry.
IHC and Electron Microscopy
The dilution was 1:5001000 for the anti-human caspase-14 or 1:1500 for the mouse caspase-14 antibodies in 0.05 M Tris-HCl at pH 7.6 with 2% BSA for light immunocytochemistry, or with 1% coldwater fish gelatin for ultrastructural immunocytochemistry. In controls the primary antibody was preabsorbed with the antigen by mixing the antibody solution with a solution of recombinant human caspase-14 (2 µg/µl) at a ratio of 1:1 for 1 hr at RT. In other negative controls the primary antibody was omitted from the incubating solution. Light immunocytochemistry was performed using a secondary HRP-conjugated anti-rabbit IgG antibody (Biorad, Richmond, CA, or Sigma, St Louis, MO) at a dilution of 1:50, which was revealed by a diaminobenzidine (DAB) reaction. For transmission electron microscopy (TEM) immunocytochemistry the secondary (anti-rabbit IgG) antibody was conjugated to 10-nm gold (Sigma). To check for co-localization of involucrin and caspase-14 in the epidermis, a double-labeling immunostaining was performed with a monoclonal anti-human involucrin antibody (clone SY5; Sigma) that is specific for human epidermis (Alibardi and Maderson 2003) and with the anti-murine caspase-14 antibody. The secondary antibodies were a 10-nm gold-conjugated goat anti-mouse IgG (for involucrin) and a 20-nm gold-conjugated goat anti-rabbit IgG (for caspase-14). Thin sections were lightly stained in 4% uranyl acetate. The ultrastructural immunocytochemical observation was done using a Philips CM-100 electron microscope operating at 80 kV.
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Results |
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Caspase-14 immunoreactivity was restricted to the granular and mainly to the transitional and corneous layers of human epidermis (Figures 2 and 3). Basal and spinous layers were only weakly stained or were immunonegative. Labeling was seen over keratohyalin granules of the upper granular layer, as detailed below in the ultrastructural description. Negative controls performed by preabsorption of the primary antibody with recombinant antigen or by omitting the primary antibody yielded greatly reduced or no labeling (Figure 4).
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In cells of the upper granular and transitional layers, diffuse but consistent labeling was seen among keratin bundles and over keratohyalin composite granules but not along the plasma membrane (Figure 6). In controls, either negative (omitting the primary anti-human or anti-mouse antibodies), no, or little labeling was seen, including the labeling over the corneous layer that was almost completely abolished (Figure 7).
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Very strong immunolabeling was found in partially or completely mature corneocytes of the lowermost corneous layer, both in the corneous cytoplasm and, at highest intensity, in areas occupied by seemingly nuclear remnants (Figures 5 and 16). Some gold particles were consistently observed near the cornified envelope or were more clearly associated with corneodesmosomes present along the irregular surface and interlocking spines of mature corneocytes (Figures 17 and 18). The labeling was intracellular and was nearly absent extracellularly among corneocytes.
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Discussion |
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By high-resolution electron microscopy caspase-14 was found to be mainly cytosolic, but distinct labeling was also detected in the nucleus and at desmosomes (see below). The cytosolic labeling increased from the lower to the upper layer of the stratum granulosum, where it was preferentially detected on the electron-pale areas of composite keratohyalin. Double-labeling experiments revealed that caspase-14 and involucrin are present at the same sites in these cells. Because previous reports have shown that loricrin and other minor proteins destined to form the cornified envelope co-localize with involucrin (Warhol et al. 1985; Steven et al. 1990
; Manabe et al. 1991
; Ishida-Yamamoto et al. 1996
,2000
; Presland et al. 1997
; Ishida-Yamamoto and Iizuka 1998
), our findings suggest that all these components of keratohyalin are potential substrates for caspase-14. By contrast, the weak labeling intensity of F-granules suggests that filaggrin and its binding partners are less likely candidates. Interestingly, co-localization of caspase-14 and involucrin was not detectable in the stratum corneum. Whereas caspase-14 reactivity increased and remained at least partially cytosolic, the involucrin labeling decreased towards the mature stratum corneum. The low involucrin labeling is probably caused by crosslinking through transglutaminase in the course of its integration into the cornified cell envelope (Ishida-Yamamoto and Iizuka 1998
; Ishida-Yamamoto et al. 2000
; Kalinin et al. 2002
). Caspase-14 seemed to remain soluble, making it plausible that caspase-14 plays a role in the mature corneocyte. Consistent with such a possible role, preparations of caspase-14 from the stratum corneum contained caspase-14 fragments of the size of mature subunits, suggesting that the enzyme is in its active state (Eckhart et al. 2000a
).
In addition to the cytoplasm, caspase-14 was also detected in the nucleus of granular layer cells as well as in nuclear remnants within cells of the transitional layer and corneus layer. It is therefore possible that caspase-14 is involved in regulation of the breakdown of the nucleus in terminally differentiating keratinocytes (Fukuyama et al. 1984; Karasek 1988
; Holbrook 1989
). A closely related protease, caspase-3, is known to cleave ICAD, the inhibitor caspase-activated DNase (CAD) during regular apoptosis (Enari et al. 1998
). Whether caspase-14 initiates an analogous degradation pathway in transitional layer keratinocytes remains to be determined by molecular biological studies. Alternatively, it is conceivable that caspase-14 targets structural proteins, such as periphilin, which localize to the nucleus before being transferred to the cell surface (Kazerounian and Aho 2003
). Strikingly, periphilin was found to be susceptible to processing by caspase-5 in vitro. We will investigate whether this potential substrate can also be cleaved by caspase-14.
The finding that some immunolabeling for caspase-14 was associated with desmosomes and the cornified envelope may support the hypothesis that proteins eventually constituting these structures are substrates for this enzyme (Warhol et al. 1985; Steven et al. 1990
; Ishida-Yamamoto and Iizuka 1994
,1998
). The detection of caspase-14 near corneodesmosomes may also suggest that this enzyme intervenes with desquamation of corneocytes.
The following technical issues must be considered when the data presented here are interpreted. First, it must be noted that our investigation of Bioacryl-embedded epidermis did not permit analysis of lamellar bodies in the granular layers. These organelles can be revealed only by standard osmium staining or by specific staining with ruthenium tetroxide, whereby both techniques destroy the immunoreactivity of typical antigens (Menon and Norlen 2002). Although lamellar bodies contain a large number of hydrolytic enzymes (Holbrook 1989
; Menon and Norlen 2002
), they are considered more likely to be involved in the lipid metabolism of corneocytes than in protein degradation during cornification. Second, the precise localization of involucrin along the cornified envelope can be revealed only by demasking agents such as proteases, which were not used in our study. Our interpretation that caspase-14 and involucrin do not co-localize in the stratum corneum is based on published studies on involucrin in which this methodology was used (Ishida-Yamamoto and Iizuka 1994
,1998
; Ishida-Yamamoto et al. 1996
,2000
). Third, when tissues are embedded in Bioacryl it is difficult to differentiate between F-keratohyalin granules and fragments of nuclear material that may even be part of composite keratohyalin. In our investigations, some components of composite keratohyalin granules (F-granules) appeared as reticular organelles made up of coarse filaments of
10-nm diameter. These data are in agreement with earlier reports on the ultrastructure of keratohyalin in mammalian epidermis (Fukuyama and Epstein 1986
; Holbrook 1989
; Steven et al. 1990
; Manabe et al. 1991
).
Because the available anti-caspase-14 antibodies do not discriminate between proenzyme and catalytically active subunits in IHC, our data on the intracellular distribution of caspase-14 do not reveal the site of initial procaspase-14 activation. Previously we have shown by Western blotting analysis that caspase-14 subunits appear concomitant with cornification and that almost all caspase-14 proenzyme is converted to the subunits in mature stratum corneum (Eckhart et al. 2000a). Ongoing studies in our laboratories are aimed at the identification of caspase-14 protein species in preparations of cytoplasmic and nuclear proteins of terminally differentiating keratinocytes. The specific degradative substrates (Chien et al. 2002
; Kuechle et al. 2001
) of caspase-14 will be identified through biochemical and molecular biological studies. With the information provided here, the screening of substrates can be focused on a subset of proteins localizing to certain compartments of terminally differentiated keratinocytes.
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Acknowledgments |
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We thank Martin Stichenwirth, Heinz Fischer, Daniela Burtscher, and Andreas Kapetanopoulos for technical support and helpful discussions, and Wim Declercq (Flanders Interuniversity Institute for Biotechnology and Ghent University; Ghent, Belgium) for providing the antibody against murine caspase-14.
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Footnotes |
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Literature Cited |
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