Correspondence to: John R. Stanley, Department of Dermatology, University of Pennsylvania, 211 CRB, 415 Curie Blvd., Philadelphia, PA 19104. Tel:(215) 898-3240 Fax:(215) 573-2033 E-mail:jrstan{at}mail.med.upenn.edu.
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Abstract |
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Desmogleins are desmosomal cadherins that mediate cellcell adhesion. In stratified squamous epithelia there are two major isoforms of desmoglein, 1 and 3, with different distributions in epidermis and mucous membrane. Since either desmoglein isoform alone can mediate adhesion, the reason for their differential distribution is not known. To address this issue, we engineered transgenic mice with desmoglein 3 under the control of the involucrin promoter. These mice expressed desmoglein 3 with the same distribution in epidermis as found in normal oral mucous membranes, while expression of other major differentiation molecules was unchanged. Although the nucleated epidermis appeared normal, the epidermal stratum corneum was abnormal with gross scaling, and a lamellar histology resembling that of normal mucous membrane. The mice died shortly after birth with severe dehydration, suggesting excessive transepidermal water loss, which was confirmed by in vitro and in vivo measurement. Ultrastructure of the stratum corneum showed premature loss of cohesion of corneocytes. This dysadhesion of corneocytes and its contribution to increased transepidermal water loss was confirmed by tape stripping. These data demonstrate that differential expression of desmoglein isoforms affects the major function of epidermis, the permeability barrier, by altering the structure of the stratum corneum.
Key Words: cadherins, cell adhesion molecules, desmosome, epidermis, skin
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Introduction |
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Desmogleins and desmocollins are transmembrane glycoproteins of the desmosome, a cellcell adhesive structure prominent in epithelial tissues (
Several lines of evidence suggest that these glycoproteins are critical for the cellcell adhesion function of the desmosome. First, desmogleins and desmocollins are members of the cadherin gene superfamily, of which the prototypic members (e.g., E-cadherin) mediate adhesion. Second, in some systems, transfection of desmosomal cadherins with plakoglobin, an intracellular plaque protein of the desmosome, confers adhesive properties to cells (
Although both desmogleins and desmocollins may be needed for adequate cell adhesion, why there are different isoforms of each in different layers of the same tissue is not clear. For example, desmoglein (Dsg)1 1 is expressed throughout all the nucleated cell layers of the epidermis, but Dsg 3 is found only in the deep epidermis, whereas in oral mucous membrane Dsg 3, like Dsg 1, is found throughout all the nucleated cell layers (
One explanation for the variable distribution of desmoglein isoforms in different epithelia might be to provide adhesion that is appropriate for the specific types of stress to which these epithelia are subjected. However, another possibility is that there are functions of desmogleins beyond simple adhesion. Accordingly, we hypothesized that the differential distribution of desmoglein isoforms might influence the differentiation and/or function of these epithelia. To test this hypothesis, we constructed a transgenic mouse in which the distribution of Dsg 3 in epidermis was similar to that in oral mucous membranes. In these mice, the epidermal stratum corneum displayed some properties that resembled those of the stratum corneum in mucosal epithelia, including histological features, decreased cohesion, and increased transepidermal water loss that often resulted in lethality during the first week of life due to dehydration. These results demonstrate that the distribution of desmoglein isoforms in stratified squamous epithelia regulates both the structure and function of these epithelia.
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Materials and Methods |
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Construction and Genotyping of Involucrin-Dsg 3 Transgenic Mouse
The involucrin promoter vector (pH3700-pL2), which also contained the first involucrin intron, an SV40 intron, a ß-galactosidase gene, and an SV40 polyadenylation site, was a gift from Dr. Lorne Taichman (State University of New York at Stony Brook, Stony Brook, NY;
Mouse tail DNA, extracted with Puregene Genomic DNA Isolation Kit (Gentra Systems), was used for PCR and Southern blotting to establish genotypes. PCR primers from desmoglein 3 exon 7 (5'-AACTTCCCAGTGCTCAGAGAGTCTC-3') and exon 8 (5'-TTAACCACCTTCAGGATGCCTTC-3') were used to identify genomic DNA (671-bp product) or transgenic cDNA (213-bp product) (see Fig 1 B). Southern blotting was performed with a digoxigenin-dUTPlabeled probe generated by PCR of mouse Dsg 3 cDNA with primer sets starting at nucleotide positions 976 and 1497 (5'-GAAGGCATCCTGAAGGTGGT-3' and 5'-CTTCTCGAGGACAACTGACG-3'), according to the PCR DIG probe synthesis kit (Boehringer). Genomic DNA was digested with HindIII, electrophoresed, and transferred to MagnaGraph nylon membranes (Osmonics), and then, after hybridization and washing, was incubated with horseradish peroxidaseconjugated antidigoxygenin antibody and developed with a chemiluminescence substrate (CSPD; Boehringer).
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Antibodies
A rabbit affinity-purified antibody against extracellular domain 5 of mouse Dsg 3 (
Rabbit antisera against extracellular domain 5 of Dsg 3 and Dsg 1 were used for immunoblotting, as was a mouse monoclonal antiFLAG antibody (Eastman Kodak Co.). The rabbit antimouse Dsg 3 antiserum against extracellular domain 5 of Dsg 3 was prepared as described (
Rabbit antibodies against loricrin, involucrin, filaggrin, and keratin 10 were obtained from Babco.
Immunofluorescence and Immunoblotting
Indirect immunofluorescence of formalin-fixed mouse skin with rabbit antibodies was performed as previously described (
For immunoblotting, mouse back skin or mucous membrane (tongue) was homogenized on dry ice, and then extracted with Laemmli sample buffer. Samples with equal protein amounts (Protein Assay Kit; Bio-Rad Laboratories), were electrophoresed on 6% Tris-glycine polyacrylamide gels (Novex), and then transferred to nitrocellulose sheets (Trans-Blot; Bio-Rad Laboratories). The sheets were incubated for 1 h in blocking buffer [5% fat-free milk powder in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl (TBS)]. The first antibody, diluted in blocking buffer, was then applied for 1 h at room temperature. After two washes with 0.1% Tween-20 in TBS (antiFLAG) or 0.5% Triton X-100, 0.5 M NaCl in TBS (antiDsg), the sheets were incubated for 2 h with horseradish peroxidaseconjugated antirabbit or antimouse IgG (Bio-Rad Laboratories) diluted 1:1,000 in blocking buffer. After washing with 0.1% Tween-20 in TBS, the signals were detected with chemiluminescence (ECL; Amersham Pharmacia Biotech). Signals were quantitated by densitometry with ImageQuant software (Molecular Dynamics).
Transepidermal Water-loss Measurement
6-mm punch biopsies, taken from the flank skin of 38-d-old transgenic pups and normal littermates, were placed in silicone high vacuum grease (Dow Corning) dermal side down on parafilm, and then weighed every 30 min (
We also used an electrolytic water analyzer (Meeco) to measure transepidermal water loss of 6-d-old pups from three sites (both shoulders and left hip), as previously described (
Electron Microscopy
Skin samples, fixed in Karnovsky's fixative, were analyzed as previously described (
Corneocyte Tape Stripping
D-Squame disks (CuDerm Corp.) were used to strip stratum corneum cells as previously described (
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Results |
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Genetic and Expression Analysis of Involucrin-Dsg 3 Transgenic Mice
To determine whether different distributions of desmoglein isoforms affect the structure or function of epidermis, we engineered a transgenic mouse expressing mouse Dsg 3 in epidermis in the pattern normally found only in mucous membranes. The transgene placed mouse Dsg 3 cDNA under the control of the involucrin promoter (Fig 1 A). Nucleotides encoding the FLAG peptide sequence, which can be detected with specific antibodies, were added to the 3' end of the coding sequence. Since involucrin is expressed normally in the suprabasilar layers of epidermis (
Analysis by PCR with primers in exons 7 and 8 of mouse Dsg 3 DNA identified a germline band of 671 bp, compared with a band from the transgene cDNA of 213 bp, clearly differentiating the transgenic mouse from the nontransgenic littermate (Fig 1B and Fig C). This genotyping was confirmed by Southern blotting (Fig 1 D), which showed a copy number for the transgene of 47 and 51, respectively, in the first and second founders' lines.
Expression of the transgene was detected by Western blotting for the FLAG-tagged mouse Dsg 3 of extracted skin from transgenic neonatal mice (Fig 2 A). Distribution of expression of the transgenic mouse Dsg 3-FLAG was detected by immunofluorescence with antiFLAG antibodies of mouse skin, which showed cell-surface staining in the suprabasilar layers of the epidermis (Fig 2 B). The distribution of both transgenic and nontransgenic Dsg 3 in mouse skin was detected by immunofluorescence with an antimouse Dsg 3 antibody. Whereas nontransgenic littermates showed Dsg 3 only in the deep epidermis (Fig 2 C), transgenic animals showed Dsg 3 throughout the entire epidermis (Fig 2 D), in a pattern identical to that described in oral mucous membrane (
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To determine the approximate ratios of Dsg 3 to Dsg 1 in transgenic skin compared with normal mucous membrane, we quantitated the band intensities of Western blots (Fig 2 E). These data showed that the ratio of Dsg 3 to Dsg 1 in transgenic skin of founder 1 was approximately that seen in normal oral mucous membrane (i.e., tongue), but higher than that seen in normal epidermis. The ratio of Dsg 3 to Dsg 1 in the transgenic skin of founder 2 (2.3) was higher than in founder 1 (1.7), although both showed similar scaling phenotypes. These data show that the Dsg 3 to Dsg 1 ratio in transgenic skin more closely resembles that found normal tongue mucosa than that found in normal epidermis.
Involucrin-Dsg 3 Transgenic Mice Show an Abnormal Epidermal Stratum Corneum with Histological Similarities to Oral Mucous Membrane
Although transgenic mice appeared normal at birth, within 23 d they developed scaling and were noted to be smaller than control littermates (Fig 3 A), even though they had visible milk in their stomachs. The degree of scaling of transgenic mice was somewhat variable and correlated positively with the degree of transgene expression, as determined by Western blotting with antiFLAG antibody. For example, while mice heterozygous for the transgene from the first founder were nonscaling, homozygous mice were scaling and showed at least twice as much expression of Dsg 3-FLAG by Western blotting. Heterozygous mice from the second founder showed scaling at age 23 d, and Western blots for Dsg 3-FLAG showed equivalent amounts to that seen in homozygotes from the first founder. Homozygous mice from the first founder died by age 710 d. Most heterozygote mice from the second founder died by 10 d, but some survived and those that did were able to breed.
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Scaly, transgenic mice were killed and autopsied at 46 d. Other than the skin, the histology of all organs and tissues, including mucous membranes, was normal. The epidermis showed a strikingly altered stratum corneum (Fig 3 C). Compared with the normal stratum corneum of skin, which typically shows a "basket weave" pattern (Fig 3 B), the stratum corneum of the transgenic epidermis was thinner with a compact lamellar pattern, similar to that seen in normal oral mucous membrane (Fig 3 D).
Involucrin-Dsg 3 Transgenic Mice Demonstrate Abnormal Cutaneous Barrier Function
Other than those of the skin, the only abnormalities noted in the transgenic neonatal mice were a significantly increased hematocrit and serum protein concentration, as well as decreased weight. At age 67 d, the hematocrit of the scaly transgenic mice was 43 ± 1.2% (mean ± SEM, n = 7), compared with 32 ± 0.7% (n = 7) for normal littermates; the total serum protein of these transgenic mice was 4.1 ± 0.1 g/dl (mean ± SEM), compared with 2.3 ± 0.1 g/dl for normal littermates. The weight of these transgenics was 2.6 ± 0.09 g (mean ± SEM), compared with nontransgenic littermates of the same age that weighed 3.9 ± 0.16 g. This hemoconcentration, along with excess scale and altered histology of the stratum corneum, suggested that the transgenic mice might have a permeability barrier defect, reflected by increased transepidermal water loss leading to dehydration, decreased weight, and eventual death.
To test this hypothesis, we first isolated 7-d-old transgenic and nontransgenic littermates from their mothers and weighed them for 5 h (Fig 4 A). In all cases, transgenic mice lost weight faster than nontransgenic mice. Presumably this accelerated weight loss of transgenic mice could be ascribed to increased transepidermal water loss. However, it is possible that other fluid loss (e.g., urine) could have accounted for these differences, although no other evidence of fluid loss was apparent.
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To examine epidermal barrier function directly, we measured the rate of water loss from the surface of 6-mm punch biopsies from the skin of transgenic and nontransgenic 38-d-old mice. We performed paired comparisons for weight change, as a measure of surface water loss, over time of a transgenic and nontransgenic skin sample for each experiment. In each paired comparison (n = 8), the transgenic skin displayed higher rates of transcutaneous water loss than did the nontransgenic skin (Fig 4 B shows two-paired comparison and mean ± SEM of all data). We also measured rates of transepidermal water loss in vivo at the skin surface of 6-d-old pups with an electrolytic water analyzer. Transgenic mice displayed significantly higher rates of water loss, 43 ± 9 x 10-2 mg/cm2 per h (mean ± SEM, n = 4), in comparison with nontransgenic littermates, 18 ± 2 x 10-2 mg/cm2 per h (mean ± SEM, n = 3).
Analysis of Epidermal Ultrastructure of Involucrin-Dsg 3 Mice
The epidermal nucleated cell layers of the living epidermis showed comparable normal ultrastructure, including desmosomal substructure, in transgenic and nontransgenic littermates. However, the epidermal stratum corneum of transgenic animals differed strikingly from that of nontransgenic littermates. Normally, with the transition from the stratum granulosum to the stratum corneum, and then, again, in the superficial stratum corneum, the desmosome changes structure (
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Tape Stripping of Epidermis Results in Increased Rate of Transepidermal Water Loss in Involucrin-Dsg 3 Mice
For further evidence of decreased corneocyte adhesion, we measured protein obtained with serial tape stripping of the stratum corneum in transgenic mice and normal littermates, a standard measure that correlates with corneocyte adhesion (
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Expression of Dsg 3 in the Superficial Epidermis Does Not Affect Expression of Major Differentiation Markers
To rule out the possibility that the expression of Dsg 3 in the superficial epidermis might have major effects on multiple pathways of differentiation in the epidermis, we determined expression of loricrin, involucrin, filaggrin, and K10 in the transgenic and normal epidermis by Western blotting (Fig 8). There were no major changes seen in the expression of these differentiation molecules. These data show that the expression of Dsg 3 in the superficial epidermis does not cause a generalized perturbation in the overall differentiation of epidermis, although we cannot rule out more subtle changes.
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Discussion |
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Desmogleins play a major role in stabilizing cellcell adhesion in the living layers of the epidermis. This function is perhaps best illustrated in the disease pemphigus in which autoantibodies against these transmembrane glycoproteins of the desmosome cause blisters due to loss of keratinocyte adhesion. Although desmogleins provide this critical function, it is not clear why different desmoglein isoforms are expressed at different levels in various stratified squamous epithelia. To address this issue, we constructed transgenic mice that expressed Dsg 3 throughout the entire epidermis, as occurs in normal oral mucous membrane, in contrast to its normal expression only in the deep epidermis. These mice displayed a dramatic phenotype, characterized by scaling shortly after birth, dehydration (usually leading to lethality within the first 10 d of life), a histologically abnormal epidermal stratum corneum, decreased adhesion of corneocytes, and increased transepidermal water loss.
This phenotype was unlikely to be due to toxicity of transgene overexpression, because the ratio of Dsg 3 to Dsg 1 in transgenic epidermis approximated that in normal oral mucous membrane (i.e., tongue). Although this ratio was somewhat higher than in tongue, at least in founder 2, the transgenic tongue had Dsg 3 to Dsg 1 ratios even higher than those of normal mucous membrane and transgenic epidermis, yet did not show any gross abnormalities indicative of toxicity. In addition, the function and processing of the transgenic Dsg 3 seems normal. For example, we know that the transgenic Dsg 3 is expressed on the cell surface, does not accumulate in the cytoplasm (as it might in severe overexpression), and provides physiologic functional adhesion, because it compensates for loss of Dsg 1 adhesion in pemphigus foliaceus (
Interestingly, changing the Dsg 3 to Dsg 1 ratio in transgenic epidermis to more closely approximate that seen in mucous membranes results in an epidermal stratum corneum that histologically and ultrastructurally resembles the stratum corneum of mucosal epithelia. Furthermore, the transgenic epidermal stratum corneum, like mucous membrane (
These data demonstrate that the distribution of desmosomal isoforms within epidermis affects the structure and function of the stratum corneum. How this occurs is suggested by the ultrastructural studies of transgenic skin. In normal stratum corneum, there is an orderly processing of the desmosome, with gradual dissolution beginning about two to three cell layers above the stratum granulosumstratum corneum interface and continuing to the surface of the skin (
Although it may first appear paradoxical that the histology of transgenic epidermis, which shows premature dissolution of desmosomes, shows a compact lamellar stratum corneum compared with the typical normal basket weave pattern, there is no correlation between the latter pattern and desmosome function or lack thereof. The histology of stratum corneum is an artifact of processing (probably due to loss of lipids and inability to rehydrate those areas with routine processing), because fully rehydrated stratum corneum shows closely opposed cells without gaps (
Together, these observations suggest that the ratio of desmosome isoforms in the stratum corneum affects physiologic desmosomal processing, which in turn regulates the degree of corneocyte adhesion and barrier function. Exactly how the expression of Dsg 3 in the stratum corneum might affect desmosomal processing is not obvious, in part because the mechanisms of desquamation in the stratum corneum have not been well characterized. Perhaps the phenotype we observe is simply a consequence of inherently different adhesive functions of Dsg 3 and Dsg 1, such that incorporation of both does not lend mechanical stability to the junctions in the stratum corneum. However, how this instability would lead to degradation of the desmosome is not clear. There are proteolytic and hydrolytic enzymes that are likely involved in regulating desquamation in the stratum corneum, and at least some of these enzymes may concentrate around corneodesmosomes (
In summary, these studies not only demonstrate that the distribution of isoforms of a desmosomal cadherin can have dramatic effects on the major physiologic function of epidermis (i.e., permeability barrier formation), but also underscore the importance of desmosomes in regulating that function.
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Footnotes |
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1 Abbreviation used in this paper: Dsg, desmoglein.
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Acknowledgements |
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We thank Drs. Leena Pulkinen and Fushan Wang for providing the cDNA constructs used in these studies, and Michael Shapiro and Dr. Peter Koch for helping to produce and characterize antidesmoglein antibodies.
Submitted: 1 December 2000
Revised: 1 March 2001
Accepted: 1 March 2001
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References |
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