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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2003 Nov;163(5):1771–1779. doi: 10.1016/S0002-9440(10)63537-7

Recessive Epidermolysis Bullosa Simplex Phenotype Reproduced in Vitro

Ablation of Keratin 14 Is Partially Compensated by Keratin 17

Abdoelwaheb El Ghalbzouri *, Marcel Jonkman ‡, Johanna Kempenaar *, Maria Ponec *
PMCID: PMC1892449  PMID: 14578178

Abstract

Recessive epidermolysis bullosa simplex (REBS) is characterized by generalized cutaneous blistering in response to mechanical trauma. This results from fragility of the basal keratinocytes that lack keratin tonofilaments because of homozygote null mutation in the keratin 14 gene. REBS patients display in addition focal dyskeratotic skin lesions with histology of epidermolytic hyperkeratosis (EHK) and tonofilament clumping in the suprabasal layers of the epidermis. In this study we examined whether it is possible to mimic in vitro the bullous and dyskeratotic cellular phenotype. For this purpose, fibroblasts from nondyskeratotic (K14−/−) and dyskeratotic (K14−/−) skin of a REBS patient and fibroblasts from a healthy donor (K14+/+) were isolated and incorporated into collagen matrices. Subsequently, fresh biopsies originating from the nondyskeratotic and dyskeratotic skin of the patient and from a healthy donor were placed onto the collagen matrices and cultured at the air-liquid interface. Epidermal morphogenesis was evaluated on the basis of tissue morphology and the expression of a series of keratins. The results of the present study indicate that basal cell vacuolization in REBS can be mimicked in vitro but not the EHK. Fibroblasts seem to play an important regulatory role in establishing the REBS phenotype. These findings suggest that wild-type fibroblasts may enhance the stability of K14−/− keratinocytes in vitro.

In stratified squamous epithelia morphogenesis and formation of distinct cell compartments is associated with specific expression and distribution of various proteins among which are the keratins. Keratins are heteropolymeric proteins that form the intermediate filament cytoskeleton in epithelial cells. More then 20 keratins have been identified in various epithelial tissues, and they can be grouped in either type I, or acidic (K9 to K20), and type II, or basic (K1 to K8), keratins. 1 Mutations in several keratin genes have been found to cause a variety of human diseases affecting the epidermis and other epithelial structures. Epidermolysis bullosa simplex (EBS) was the first mechanobullous disease for which the underlying genetic lesion was found 2-4 to be caused by dominant-negative mutations in the K5 and K14 genes. More recently, a recessive form of EBS (REBS) has been described in which the keratin intermediate filaments are absent because of homozygous null mutation in the K14 gene. 5-10

REBS is characterized by generalized cutaneous blistering in response to mechanical trauma, resulting from fragility of the basal keratinocytes. In addition, the program of epidermal differentiation is focally disturbed resulting in exfoliative dyskeratotic plaques with histological appearance of epidermolytic hyperkeratosis (EHK) and suprabasal keratin clumping. 7

In the past a few in vitro studies have been performed to examine the performance of epidermolysis bullosa fibroblasts in fibroblast-induced collagen contraction. Fibroblasts derived from patients with recessive dystrophic epidermolysis bullosa cannot elongate and spread out when incorporated into a collagen matrix. They are therefore poor at contracting that collagen lattice in comparison to healthy fibroblasts. 11 Eisen and colleagues 12 demonstrated that fibroblasts from patients with recessive dystrophic epidermolysis bullosa, dominant dystrophic epidermolysis bullosa, and dominant epidermolysis bullosa simplex, show an extremely broad range of contractility (normal, poor, and hypercontraction). 12-14 However, until now little information is available on the possible role of keratinocyte-fibroblast interaction on the manifestation of this skin disorder. The reason for this is the limited size of the available skin samples derived from patients. Enzymatic treatment is a commonly used approach for keratinocyte isolation from the epidermis. However, use of a skin biopsy as starting material would yield an insufficient number of keratinocytes required forthe generation of human skin equivalents (HSEs). Such HSEs are mostly constructed by seeding a relatively high number of human keratinocytes (∼1 × 105/cm2) on top of a dermal matrix (eg, a fibroblast-populated collagen matrix) and subsequent culturing at the air-liquid interface. Various studies have demonstrated that HSEs formed under these conditions show high similarity with the native tissue. It consists of a fully differentiated epidermis with a basal, spinous, granular, and cornified layers. 15-20 The presence of fibroblasts in the underlying dermal equivalent facilitates the formation of the basement membrane, the presence of which is required for the maintenance of skin integrity. 20-22 To overcome the limited availability of skin samples derived from patients suffering with various skin disorders one can start an organotypic culture by placing a skin punch biopsy onto a dermal matrix and subsequent culture at the air-liquid interface. 23,24 A similar approach has been used in the present study with skin biopsies derived from the nondyskeratotic and dyskeratotic skin of the patient and from a healthy donor. To assess the effect of fibroblasts on epidermal morphogenesis, fibroblasts from nondyskeratotic (K14−/−) and dyskeratotic (K14−/−) skin of a REBS patient and fibroblasts from a healthy donor (K14+/+) were isolated and incorporated into collagen matrices. Subsequently, fresh biopsies originating from nondyskeratotic, dyskeratotic, or healthy skin were placed onto the collagen matrices and cultured for 32 days at the air-liquid interface. Hereafter, epidermal morphogenesis was evaluated on the basis of tissue morphology and the expression of a number of structural proteins (K5, K6, K10, K14, K15, K16, and K17). The results of the present study indicate that basal cell vacuolization in REBS can be mimicked in vitro but not the EHK.

Materials and Methods

Keratinocyte Cultures

Keratinocytes were isolated from the skin biopsies and cultured in serum-free medium (BioWhittaker, Verviers, Belgium) as described previously. 7

Fibroblast Cultures

To establish fibroblast cultures, 4-mm biopsies were taken from dyskeratotic and nondyskeratotic REBS patient skin. In addition, fibroblasts from a healthy donor have been isolated. The culture was established as described earlier. 18 The fibroblasts were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 5% fetal calf serum (Gibco, Invitrogen, Breda, The Netherlands), penicillin (100 IU/ml), and streptomycin (100 μg/ml) (ICN Biomedicals, Inc., Costa Mesa, CA). Passages two to five were used for the experiments.

Dermal Equivalents

Collagen

Hydrated collagen gels were prepared as described by Smola, 25 using a 4-mg/ml collagen solution isolated from rat tails. Briefly, the collagen solution was used directly or mixed at 4°C with fibroblast suspension to reach a final density of 1 × 105 cells/ml. Subsequently, 2.5 ml of collagen mixture was pipetted onto a filter insert (6-well plate; Costar). After gel polymerization, culture medium was added and the collagen matrices were subsequently incubated overnight.

Dermal equivalents (D) were prepared 24 hours before initiation of the organotypic culture and populated with fibroblasts from either intact skin (nEHK) and dyskeratotic skin with EHK of a REBS patient, or from healthy control skin (C), D(nEHK), D(EHK), and D(C), respectively.

Organotypic Cultures

To start organotypic cultures, 8-mm biopsies (B) taken from intact skin (nEHK), and dyskeratotic skin (EHK) of an REBS patient, and from normal control skin (C) were used within 24 hours thereafter. The biopsies were divided into four fragments and each fragment was placed onto a dermal equivalents D(nEHK), D(EHK), and D(C), respectively. This resulted in generation of the following organotypic cultures: with biopsy from intact nondyskeratotic REBS skin: B(nEHK)/D(nEHK), B(nEHK)/D(EHK), B(nEHK)/D(C); with biopsy from dyskeratotic REBS skin: B(EHK)/D(EHK), B(EHK)/D(nEHK), B(EHK)/D(C); with biopsy from healthy control skin: B(C)/D(nEHK), B(C)/D(EHK), B(C)/D(C). The cultures were immediately grown for 5 days at the air-liquid (A/L) interface using keratinocyte medium supplemented with 5% serum, followed by a 4-day culture in medium supplemented with 1% serum, 1 × 10−5 mol/L l-carnitine (Sigma), 1 × 10−2 mol/L l-serine (Sigma), 1 μmol/L dl-α-tocopherol-acetate and a lipid supplement containing 25 μmol/L palmitic acid, 15 μmol/L linoleic acid, 7 μmol/L arachidonic acid, and 2.4 × 10−5 mol/L bovine serum albumin (Sigma). 18 Thereafter, the same medium has been used except that serum was omitted, the concentration of linoleic acid was increased to 30 μmol/L and 50 μmol/L/ml ascorbic acid (Sigma) was added. 18 The cultures were grown for 32 days at the A/L interface. Culture medium was renewed twice a week.

Passaging of Organotypic Cultures

After 32 days of culture at the A/L interface, fragments of the outgrowing part (periphery of the culture) from each type of organotypic culture was harvested and subsequently transferred onto dermal equivalents populated with various types of fibroblasts (nEHK, EHK, C), using the same combinations as described above. The cultures were kept under the same conditions as described earlier.

Immunohistochemistry and Immunofluorescence Microscopy

Immunohistochemical analysis was performed using 5-μm frozen sections, which after sectioning at −20°C, were air-dried overnight and fixed in acetone for 10 minutes. The primary antibodies used in the present study are listed in Table 1 ▶ . After incubation with primary antibodies, sections were stained with avidin-biotin-peroxidase complex system (streptABcomplex/HRP; DAKO, Glostrup, Denmark), as described by the suppliers with the following minor modifications: Phosphate-buffered saline was used instead of Tris-buffered saline. All sections were counterstained with hematoxylin.

Table 1.

Primary Antibodies Used for Immunohistochemical and Immunofluorescence Staining of Tissue Sections

Sections Antibody designation Source*
Frozen K14 (LL001) Dr. I. M. Leigh, London, England
K14 (LL002) Dr. I. M. Leigh, London, England
K14 (RCK107) Monosan, Uden, The Netherlands
K10 (DE-K10) ICN Biomedicals Inc, Ohio
K5&8 (RCK102) Monosan, Uden, The Netherlands
K15 (LHK15) Neomarkers, Fremont, CA
Keratin 6 (Ks6.KA12) Sanbio B. V. Uden, the Netherlands
Keratin 16 (LL0025) Dr. I. M. Leigh, London, England
Keratin 17 (CK-E3) Sigma, Saint Louis, MO
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*Antibodies not purchased from indicated sources were personal gifts from the investigator named.

For immunofluorescence, the harvested cultures were processed as previously described using the biotin-streptavidin system. 7 Digital fluorescence microscopy was performed according to Bruins and colleagues 26 allowing detection of low levels of fluorescence. Immunofluorescence on cultured keratinocytes was previously described. 27 In brief, cells were grown on glass coverslips, washed with phosphate-buffered saline (PBS), fixed with 1% formaldehyde in PBS and permeabilized by Triton X-100. Hereafter they were blocked with 1% (w/v) bovine serum albumin in PBS. After rinsing the cells three times, they were incubated with Alexa 488-labeld goat anti-mouse IgG for 30 minutes at room temperature. Finally the nuclei were stained blue with bisbenzamide.

Results

Cellular Phenotype

Histological appearance showed morphological differences between EHK and nEHK skin areas of the K14 (−/−) REBS patient. The epidermis in dyskeratotic EHK plaques was hyperproliferative consisting of 4 to 11 cell layers (Figure 1A) ▶ , whereas the epidermis of nondyskeratotic nEHK skin consists only of 5 to 6 cell layers (Figure 1B) ▶ .

Figure 1.

Figure 1.

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Histological appearance of a dyskeratotic (A) and nondyskeratotic (B) skin. Shown are hematoxylin-stained cross sections. The epidermis in dyskeratotic EHK plaques was hyperproliferative consisting of 4 to 11 cell layers, whereas the epidermis of nondyskeratotic nEHK skin consists only of five to six cell layers. Original magnifications, ×200.

Construction of HSEs with Biopsies Obtained from a REBS Patient

To determine whether the skin phenotype can be reconstituted in vitro, biopsies originating from the nondyskeratotic and dyskeratotic skin of the REBS patient and from the healthy volunteer were divided each into four different fragments. Individual fragments were subsequently transferred onto a dermal equivalent populated with either type of fibroblasts (nEHK, EHK, or C, respectively). During the 32-day culture at the A/L interface, lateral expansion of the epidermis was noticed (Figure 2) ▶ . For establishment of the secondary culture, a small fragment was harvested from the periphery of the primary culture and transferred onto a freshly prepared dermal equivalent populated with the same type of fibroblasts as used in primary cultures (Figure 2) ▶ .

Figure 2.

Figure 2.

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Macroscopic view of the passaging procedure. Generation of a HSE with a biopsy obtained from a REBS patient. Primary culture is established by placing a skin biopsy onto fibroblast-populated collagen matrix and culturing for 32 days at the air-liquid interface. After this culture period the collagen matrix was fully covered with keratinocytes originating from the biopsy. To establish a secondary culture, a fragment from the primary HSE is transferred onto fibroblast-populated collagen matrix.

Irrespective of the skin donor used, a fully differentiated epidermis was formed after 32-days of culture at the A/L interface (Figure 3A) ▶ . However, differences in epidermal architecture have been noticed. When a biopsy from nondyskeratotic or dyskeratotic skin was cultured on a dermal matrix containing fibroblasts derived either from nondyskeratotic or dyskeratotic skin, vacuolization of the basal cells was observed. In contrast to this, when dermal equivalents were populated with fibroblasts originating from a healthy (C) donor no such morphological changes have been noticed (Figure 3A) ▶ . The observed changes in epidermal architecture persisted after passaging of the primary cultures (Figure 3B) ▶ . The epidermal morphology in organotypic cultures established with a biopsy derived from the healthy donor was similar irrespective the type of fibroblasts used and did not show any abnormalities (data not shown).

Figure 3.

Figure 3.

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The REBS phenotype is reproduced under the in vitro conditions. Histological appearance of reconstructed epidermis established on collagen matrices populated with dyskeratotic (EHK), nondyskeratotic (nEHK), and healthy fibroblasts (C) after 32-day culture at the air-liquid interface. Cross sections of primary (A) and secondary (B) cultures are shown. In both primary and secondary cultures, a fully differentiated epidermis was formed irrespective of the skin donor used. In HSEs generated with a biopsy originating from nondyskeratotic or dyskeratotic skin on a dermal matrix containing fibroblasts derived either from nondyskeratotic or dyskeratotic skin, vacuolization of the basal cells was observed in both primary and secondary cultures. H&E-stained paraffin cross sections are shown. Original magnifications, ×800.

Differential Expression of K14 Protein in REBS-HSE Constructs

For examination of keratin expression antibodies directed against K5, K10, K14, K15, K16, and K17 have been used. As shown in Figure 4 ▶ , the immunohistochemical staining of the native epidermis originating from a healthy donor with various K14 antibodies resulted in positive staining of the basal and first suprabasal layer with RCK107 antibody; with LL001 and LL002 antibodies the entire epidermis was positively stained. K5 was uniformly decorating the basal layer and locally also some cells in the first suprabasal layer. In contrast to K14 and K5, only local expression of K15 in basal and first suprabasal layer was noticed. K10 was expressed in all suprabasal layers. A similar staining pattern was seen in organotypic cultures established with biopsies originating from a healthy donor ▶ ▶ (Figures 4 and 5B) ▶ . Inspection of the K14-staining pattern revealed a weak local staining in B(nEHK)/D(nEHK) and B(EHK)/D(nEHK) cultures in basal and suprabasal cells when RCK107 antibody was used. In contrast to this, with LL001 and LL002 antibodies no positively stained cells were detected. Similar observations have been made with biopsies taken from the patient (Figure 5A) ▶ . As shown in Figure 5B ▶ , K5 was generally present. Weak suprabasal K5 staining was also noticed in B(nEHK)/D(nEHK) and B(EHK)/D(nEHK) cultures. Local expression of K15 was observed in all cultures examined.

Figure 4.

Figure 4.

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Keratin expression in native skin. Shown are paraffin cross sections of skin stained for K14 (using antibodies RCK107, LLOO1, and LLOO2), K5, K15, and K10. Positively stained cells in the basal and first suprabasal epidermal layer were seen with RCK107 antibody, with both L001 and L002 antibodies the entire viable epidermis was stained. K5 decorated continuously and K15 locally the basal and first suprabasal epidermal layer. K10 expression was observed in all suprabasal cell layers. Original magnifications, ×200.

Figure 5.

Figure 5.

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Differential expression of K14 protein in REBS-HSE constructs. Keratin expression in HSEs generated with fibroblasts and keratinocytes originating from a healthy donor; in the skin of the REBS patient; and in HSEs generated with biopsies originating from nondyskeratotic (nEHK), dyskeratotic (EHK) skin on collagen matrices populated with fibroblasts isolated from EHK, nEHK, and healthy fibroblasts (C). Shown are paraffin cross-sections stained for K14 using different antibodies: RCK107, LLOO1, and LLOO2 (A); for K5, K15, K10 (B); and K6, K16, K17 (C). Note: RCK107 antibody showed a weak local staining in B(nEHK)/D(nEHK) and B(EHK)/D(nEHK) cultures; with LL001 and LL002 antibodies no positively stained cells were detected. A similar observation has been made in biopsies taken from the patient. Strong K17 staining was observed in basal cell layer in B(nEHK)/D(nEHK) and B(EHK)/D(EHK) cultures with K17 deposition concentrated in the vacuoles. Original magnifications, ×200.

Figure 5A.

Figure 5A.

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Continued

Expression of Specific Proteins Related to Keratinocyte Cell Activity

To establish whether the expression of various keratins (K6, K16, and K17), usually expressed in keratinocyte hyperproliferative phenotype, was examined in all skin reconstructs. Using immunohistochemistry, we observed that K6 was expressed in all viable cell layers irrespective of the type of fibroblast or keratinocyte used (Figure 5C) ▶ . The K16 expression in cultures generated with skin biopsies originating from the healthy donor was lower as compared to the cultures generated with the patient’s skin biopsies and K17 was absent. In cultures generated with biopsies originating from a healthy donor (C) and in B(nEHK)/D(C) cultures, K16 was very weakly expressed in all of the suprabasal cell layers. However, its expression was much higher in B(nEHK)/D(nEHK), B(EHK)/D(EHK) (Figure 5C) ▶ , and B(EHK)/D(C) (data not shown). A high K17 expression was observed in the basal cell layer in B(nEHK)/D(nEHK) and B(EHK)/D(EHK) cultures with K17 deposition concentrated in cells with vacuoles (Figure 5C) ▶ . The expression of these keratins in B(nEHK)/D(C) cultures was similar to that observed in cultures established with skin derived from a healthy donor.

K14 Is Not Expressed in Proliferating Keratinocytes

When keratinocytes derived from the healthy donor or a REBS patient were grown under proliferating (low calcium) conditions, K14 staining was detectable with both LL001 and RCK107 antibodies in cultures established with healthy donor keratinocytes (Figure 6) ▶ . In cultures established with REBS keratinocytes, positive staining was observed with RCK107 antibody whereas with LL001 all cells stained negatively also when both antibodies were used simultaneously.

Figure 6.

Figure 6.

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RCK107 is expressed in keratinocyte cultures established from REBS skin whereas LL001 is not. Keratinocytes originating from a healthy donor or REBS patient were cultured under submerged conditions in low-calcium medium. Shown is immunofluorescence staining with RCK107 and LLOO1 and double staining with both antibodies. Note the positive staining with RCK107 in REBS keratinocytes. Original magnifications, ×1000.

Discussion

The objective of the present study was to examine the possibility to reproduce the REBS phenotype in vitro. To accomplish this, organotypic co-cultures have been established with biopsies originating from nondyskeratotic (nEHK), and dyskeratotic (EHK) REBS skin and from healthy donor (C) on collagen lattices populated with fibroblasts originating from either source. Irrespective of the biopsy source used, a fully differentiated epidermis was formed after a 32-day culture period. A mild vesicular phenotype was reproduced under our organotypic culture conditions. This was mainly manifested by extensive vacuolization of the basal cells and expression of K17. In REBS patients the keratin filament aggregation is the earliest recognized morphological abnormality and precedes the other distinct morphological changes such as cellular vacuolization. 28-30 In vivo, vacuolization of basal keratinocytes is not present in intact unrubbed skin of a REBS patient (Figure 1B) ▶ . 7 The presence of vacuolization in K14−/− basal keratinocytes in organotypic co-cultures indicates that the cytoskeleton is weaker or the mechanical stress is stronger under our culture conditions. K14(−/−) cells cultured in a monolayer, however, did not show vacuolization; thus we conclude that the cells experience more mechanical stress in organotypic cultures than in monolayer cultures or in skin.

Interestingly, fibroblasts seem to play an important regulatory role in epidermal morphogenesis because the vacuolar basal cell phenotype was reverted to nonvacuolated in organotypic cultures when the REBS biopsy was cultured on a collagen matrix populated with normal control fibroblasts. These effects persisted also in secondary cultures. Various studies have documented the importance of mesenchymal cells for keratinocyte growth and differentiation in culture. 25,31-36 Recent studies from our laboratory showed that for the generation of reconstructed epidermis closely mimicking the native tissue appropriate culture conditions are required. 20,37 When grown in serum-free media and in the absence of growth factors, the number and the functional state of fibroblasts incorporated into the collagen matrix is crucial for the normalization of the epidermal differentiation program and of the deposition of various basement membrane (BM) proteins and hemidesmosomal (HD) components. 20,37 A double-paracrine mechanism has been suggested tocontrol keratinocyte proliferation and differentiation. 35,36 Through release of interleukin (IL)-1, keratinocyte can enhance release of growth factors such as KGF, GM-CSF, IL-6, or IL-8 in dermal cells, which in turn stimulate keratinocyte proliferation. 36,38,39 The IL-1-induced KGF and GM-CSF has been reported to be regulated through c-jun and junB transcription factors. 40 Also parathyroid hormone-related peptide induced KGF expression in dermal fibroblasts. 41

The disorder of cornification is another feature associated with REBS phenotype. 7,42,43 However, the EHK seen in the REBS patient could not be reproduced in vitro. The lack of EHK under the in vitro conditions may be caused by limited culture time (4 weeks), high relative humidity, buffered condition, and/or the absence of mechanical shear forces.

The intermediate filaments in the basal layer areformed by combination of type I and II keratins (K5/K14/K15). In the basal layer of the normal skin the K5 and K14 are ubiquitously present, whereas K15 shows a patchy pattern. A similar expression pattern was detected in the present study in HSEs established with cells originating from a healthy donor. The discontinuous expression of K15 in basal human skin keratinocytes was also reported by Waseem and colleagues, 44 in contrast to other human stratified tissues, such as esophagus, where continuous expression was detected. In vivo, K14 is absent in the REBS patient and the lack of K14 is compensated by up-regulation of the alternative type I keratin K15. 7 Similar up-regulation at both mRNA and protein level in hair follicle from a patient suffering from REBS has been reported by Waseem and colleagues. 44 The expression of type II keratin K5 in REBS patients is similar to that seen in normal skin. 7 The K14 ablation could also be reproduced in vitro in organotypic cultures established with REBS biopsies independently of fibroblasts used for population of the dermal compartment. These observations were made with both the LL001 and LL002 antibodies, both recognizing the C-terminal portion of the K14 protein. Remarkably, when RCK107 antibody directed against K14 was used, 45 weak patchy staining was observed in REBS-HSE cultures. This finding is suggestive that this antibody recognizes another protein in addition to K14. It should be noted that in cultures established with K14-ablated keratinocytes, K5 and K15 were expressed in all HSEs irrespective of the origin of the skin and fibroblasts used for skin reconstruction. Culture conditions are probably very important for K15 expression in organotypic cultures, as in cultures generated on de-epidermized dermis by Waseem and colleagues, 44 high expression of K6 and K16 and the absence of K15 was demonstrated. This can be ascribed to the hyperproliferative culture conditions used because expression of K15 mRNA and protein were found to be down-regulated in psoriatic epidermis and in hypertrophic scar. 44 Namely, the presence of fibroblasts and the use of serum-free media is most probably crucial for normalization of the terminal differentiation program. 37

Under our in vitro conditions K15 expression did not seem to be up-regulated. A similar observation has been made by Troy and Turksen, 46 who found similar K15 expression in cultures of K14−/− and K14+/+ murine keratinocytes. Furthermore, Lloyd and colleagues 47 demonstrated that neonatal mutant mice with an ablated K14 gene K15 did not compensate for the loss of K14.

It has been suggested that the presence of a K5/K14 cytoskeleton is a prerequisite for the formation of a normal K1/K10 network. 48 However, similar to the in vivo situation, the suprabasal expression of the early differentiation marker K10 was observed in all types of HSEs established in the present study. Our study therefore does not give an explanation for the disturbance of epidermal differentiation in K14−/− skin, as manifested by the expression of K17 in basal keratinocytes with vacuoles.

Keratins K6, K16, and K17 are absent in native tissue. In wound healing or in hyperproliferative disorders, the keratin pair K6 and K16 is transiently expressed in the suprabasal cell layers. 49 The expression of these keratins under the in vitro conditions is regulated by the fibroblasts present in the dermal compartment. They are always expressed in laterally expanding epidermis, where the keratinocytes are migrating, proliferating, and the terminal differentiation program is still not complete. 37 In all REBS-HSEs tested, the presence of K16 and K17 was noticed. Remarkable is the expression of K17 in the vacuolated basal keratinocyte. This deposition was only detected in B(nEHK)/D(nEHK), B(I/D(EHK), and B(EHK)/D(C), but not in healthy donor [(B(C)/D(C)] and B(nEHK)/D(C). We hypothesize that up-regulation of K17 is the result of disturbance of epidermal differentiation, where the expression of this protein in the basal cells containing vacuoles was observed.

A quite remarkable observation was the normalizing effect on epidermal morphogenesis by control fibroblasts, which stabilize REBS cells by an unknown factor, without the expression of K14. A recent report suggests that keratin synthesis is not restricted only to cells of epithelial origin. Namely, Katagata and colleagues 50 have recently demonstrated the presence of K14, K5, and K16 polypeptides in cultured dermal fibroblasts. The possibility that wild-type fibroblasts may be capable to compensate for the K14 deficiency in REBS keratinocytes by production and transfer of K14 is in our opinion unlikely.

In conclusion, the results of the present study clearly show that the effects of K14 ablation in REBS can be reproduced in vitro but the EHK not. Furthermore, the possibility to generate HSEs with skin biopsies of patients suffering with genetic disorders offer an attractive approach for in vitro studies focusing on the mechanism of various pathological conditions.

Footnotes

Address reprint requests to Dr. Maria Ponec, Department of Dermatology, Leiden University Medical Center, Sylvius Laboratory, P.O. Box 9503, 2300 RA Leiden, The Netherlands. E-mail: m.h.ponec-waelsch@lumc.nl.

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