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Desmoglein isoform distribution affects stratum corneum structure and function.

Elias PM, Matsuyoshi N, Wu H, Lin C, Wang ZH, Brown BE, Stanley JR - J. Cell Biol. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of California San Francisco, San Francisco, California 94143, USA.

ABSTRACT
Desmogleins are desmosomal cadherins that mediate cell-cell 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.

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Genotyping of involucrin-Dsg 3 transgenic mice. (A) Transgenic construct. Inv, involucrin; mDsg3, mouse Dsg 3; FLAG, indicates nucleotides encoding the FLAG peptide epitope; polyA, polyadenylation signal. NotI shows restriction sites used to clone the mDsg 3-FLAG cDNA into the pH3700-pL2 parental vector. (B) PCR strategy to differentiate the transgene from the genomic mouse Dsg 3 DNA. (Arrows) Position of primers, (gray rectangles) exons, (black line) intron. Tg, transgene. (C) PCR analysis of transgenic mouse (+) compared with nontransgenic (−) littermate. (Arrowhead) 671-bp genomic PCR product, (arrow) 213-bp transgene product. (D) Southern blot detects transgene (arrow) and genomic DNA (arrowhead).
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Figure 1: Genotyping of involucrin-Dsg 3 transgenic mice. (A) Transgenic construct. Inv, involucrin; mDsg3, mouse Dsg 3; FLAG, indicates nucleotides encoding the FLAG peptide epitope; polyA, polyadenylation signal. NotI shows restriction sites used to clone the mDsg 3-FLAG cDNA into the pH3700-pL2 parental vector. (B) PCR strategy to differentiate the transgene from the genomic mouse Dsg 3 DNA. (Arrows) Position of primers, (gray rectangles) exons, (black line) intron. Tg, transgene. (C) PCR analysis of transgenic mouse (+) compared with nontransgenic (−) littermate. (Arrowhead) 671-bp genomic PCR product, (arrow) 213-bp transgene product. (D) Southern blot detects transgene (arrow) and genomic DNA (arrowhead).

Mentions: 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-dUTP–labeled 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 peroxidase–conjugated antidigoxygenin antibody and developed with a chemiluminescence substrate (CSPD; Boehringer).


Desmoglein isoform distribution affects stratum corneum structure and function.

Elias PM, Matsuyoshi N, Wu H, Lin C, Wang ZH, Brown BE, Stanley JR - J. Cell Biol. (2001)

Genotyping of involucrin-Dsg 3 transgenic mice. (A) Transgenic construct. Inv, involucrin; mDsg3, mouse Dsg 3; FLAG, indicates nucleotides encoding the FLAG peptide epitope; polyA, polyadenylation signal. NotI shows restriction sites used to clone the mDsg 3-FLAG cDNA into the pH3700-pL2 parental vector. (B) PCR strategy to differentiate the transgene from the genomic mouse Dsg 3 DNA. (Arrows) Position of primers, (gray rectangles) exons, (black line) intron. Tg, transgene. (C) PCR analysis of transgenic mouse (+) compared with nontransgenic (−) littermate. (Arrowhead) 671-bp genomic PCR product, (arrow) 213-bp transgene product. (D) Southern blot detects transgene (arrow) and genomic DNA (arrowhead).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169464&req=5

Figure 1: Genotyping of involucrin-Dsg 3 transgenic mice. (A) Transgenic construct. Inv, involucrin; mDsg3, mouse Dsg 3; FLAG, indicates nucleotides encoding the FLAG peptide epitope; polyA, polyadenylation signal. NotI shows restriction sites used to clone the mDsg 3-FLAG cDNA into the pH3700-pL2 parental vector. (B) PCR strategy to differentiate the transgene from the genomic mouse Dsg 3 DNA. (Arrows) Position of primers, (gray rectangles) exons, (black line) intron. Tg, transgene. (C) PCR analysis of transgenic mouse (+) compared with nontransgenic (−) littermate. (Arrowhead) 671-bp genomic PCR product, (arrow) 213-bp transgene product. (D) Southern blot detects transgene (arrow) and genomic DNA (arrowhead).
Mentions: 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-dUTP–labeled 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 peroxidase–conjugated antidigoxygenin antibody and developed with a chemiluminescence substrate (CSPD; Boehringer).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of California San Francisco, San Francisco, California 94143, USA.

ABSTRACT
Desmogleins are desmosomal cadherins that mediate cell-cell 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.

Show MeSH
Related in: MedlinePlus