Targeted disruption of the pemphigus vulgaris antigen (desmoglein 3) gene in mice causes loss of keratinocyte cell adhesion with a phenotype similar to pemphigus vulgaris

Abstract

In patients with pemphigus vulgaris (PV), autoantibodies against desmoglein 3 (Dsg3) cause loss of cell-cell adhesion of keratinocytes in the basal and immediate suprabasal layers of stratified squamous epithelia. The pathology, at least partially, may depend on protease release from keratinocytes, but might also result from antibodies interfering with an adhesion function of Dsg3. However, a direct role of desmogleins in cell adhesion has not been shown. To test whether Dsg3 mediates adhesion, we genetically engineered mice with a targeted disruption of the DSG3 gene. DSG3 -/- mice had no DSG3 mRNA by RNase protection assay and no Dsg3 protein by immunofluorescence (IF) and immunoblots. These mice were normal at birth, but by 8-10 d weighed less than DSG3 +/- or +/+ littermates, and at around day 18 were grossly runted. We speculated that oral lesions (typical in PV patients) might be inhibiting food intake, causing this runting. Indeed, oropharyngeal biopsies showed erosions with histology typical of PV, including suprabasilar acantholysis and "tombstoning" of basal cells. EM showed separation of desmosomes. Traumatized skin also had crusting and suprabasilar acantholysis. Runted mice showed hair loss at weaning. The runting and hair loss phenotype of DSG3 -/- mice is identical to that of a previously reported mouse mutant, balding (bal). Breeding indicated that bal is coallelic with the targeted mutation. We also showed that bal mice lack Dsg3 by IF, have typical PV oral lesions, and have a DSG3 gene mutation. These results demonstrate the critical importance of Dsg3 for adhesion in deep stratified squamous epithelia and suggest that pemphigus autoantibodies might interfere directly with such a function.

Figure 1

Targeting strategy and Southern blot confirmation of targeted alleles. (A) Intron–exon organization of the mouse DSG3 gene. The exons (vertical blocks) and introns (horizontal lines) are drawn to scale, with the exception of intron 1 that is >6 kb in size. (B) Targeting strategy. Vertical box indicates exon 1 (lighter shading indicates part of exon 1 that is deleted in targeting strategy). neo, neomycin-resistance cassette; tk, herpes thymidine kinase cassette; B, BamHI sites; NotI, restriction site used to linearize vector; (bold horizontal lines) portions of the DSG3 gene that were used in targeting vector; (thickest horizontal line) pUC vector sequences. Probe indicated was used in Southern blots described below. (C) Southern blot of DNA from wild type (+/+) and targeted (+/−) ES cell clones. DNA was digested with BamHI. Targeted allele shows a 2.7-kb band; wild-type allele shows a 9-kb band. (D) Southern blot of tail DNA from pups of DSG3 +/− × DSG3 +/− mating. −/−, both DSG3 alleles are targeted, resulting in only a 2.7-kb band. +/− animals show both a recombinant (2.7 kb) and wild-type (9 kb) allele, whereas +/+ animals only show a wild-type allele. Lane C shows control ES cell DNA.

Figure 2

Lack of Dsg3 RNA and protein in homozygous targeted mice. (A) RNase protection assay of tongue lysates from wild type (+/+) and targeted (−/−) mice. (B) Immunofluorescence of tongue from wild-type and targeted mice shows absence of Dsg3 from −/− mice, but presence of Dsg 1 and 2 (Dsg1/2, identified by mAb DG3.10), and plakoglobin. The submucosa of −/− mice shows increased nonspecific fluorescence probably as a result of inflammation. (C) Western blot of tongue lysates. Dsg3 is absent in −/− mice (arrowhead), but Dsg1 and 2 are present (arrow) to the same degree as in +/+ mice. Bar, 25 μm.

Figure 3

DSG3 −/− mice are runts and have skin erosions and eye lesions. (A) DSG3 −/− mice are runts. Upper mouse is DSG3 −/−; lower mouse is a +/+ littermate. (B) Weight graph shows that −/− mice (open circles), compared with +/+ and +/− littermates (filled circles), are born with equal weight but by about day 8–10 are lagging in weight gain. Weight loss is seen about day 20, approximately the time of weaning and start of solid food. (C) Nipple erosions in a DSG3 −/− nursing mother. (D) Snout erosion and conjunctivitis in a DSG3 −/− mouse.

Figure 3

DSG3 −/− mice are runts and have skin erosions and eye lesions. (A) DSG3 −/− mice are runts. Upper mouse is DSG3 −/−; lower mouse is a +/+ littermate. (B) Weight graph shows that −/− mice (open circles), compared with +/+ and +/− littermates (filled circles), are born with equal weight but by about day 8–10 are lagging in weight gain. Weight loss is seen about day 20, approximately the time of weaning and start of solid food. (C) Nipple erosions in a DSG3 −/− nursing mother. (D) Snout erosion and conjunctivitis in a DSG3 −/− mouse.

Figure 4

Histology of oral mucous membranes and skin in DSG3 −/− mice and human PV patients. (A) DSG3 −/−: inflammatory oral erosion of the tongue. (B) Human PV: inflammatory oral erosion. (C) DSG3 −/−: oral lesion shows basal cells are separated from each other and the suprabasilar epithelium is lost. This is a characteristic histology called a “row of tombstones.” (D) Human PV: oral lesion shows a “row of tombstones.” (E) DSG3 −/−: early oral lesion shows suprabasilar acantholysis with intact suprabasilar epithelium separated from basal cells. (F) DSG3 −/−: early skin lesion on dorsum of foot near where skin was traumatized by cutting. (G) Human PV: skin lesion shows typical suprabasilar acantholysis. Bar: (A and B) 160 μm; (C–G) 40 μm.

Figure 5

Ultrastructure of lesional posterior lingual epithelium in DSG3 −/− mice. (A) An edge of a blister cavity, denoted by “C” in a DSG3 −/− mouse. The base is formed by a single layer of basal keratinocytes with a characteristic “tombstone” cytoarchitecture. The roof consists of intact suprabasal epithelium with occasional associated acantholytic keratinocytes (*). (B) Blister in a DSG3 −/− mouse shows separation of desmosomes which form half-desmosomes (double arrows) with tonofilaments still attached. (C) Higher magnification of a half-desmosome shows an intact intracytoplasmic dense plaque and its associated tonofilaments. There is residual flocculent material (arrowheads) along the cell membrane. (D) Desmosome in DSG3 +/+ littermate. (E) Desmosome in DSG3 −/− mouse has normal appearance. (F) Hemidesmosome in DSG3 +/+ littermate. (G) Hemidesmosome in DSG3 −/− mouse shows normal appearance. Bars: (A) 10 μm; (B) 0.5 μm; (C–G) 50 nm.

Figure 6

Analysis of balding mice compared with DSG3 −/− mice. (A) Balding phenotype of a 1-mo-old DSG3 −/− mouse compared with a normal littermate (above). (B) Histology of an oral mucosal blister in a bal/bal mouse shows suprabasilar acantholysis. (C) Immunofluorescence of bal/bal tongue shows no Dsg3 compared with control littermate. (D) RNase protection assay of tongue lysates demonstrates that bal/bal mice synthesize DSG3 mRNA, as compared with DSG3 −/− mice that do not. Note that DSG3 +/− mice demonstrate about half of the mRNA of wild-type mice.

Figure 6

Analysis of balding mice compared with DSG3 −/− mice. (A) Balding phenotype of a 1-mo-old DSG3 −/− mouse compared with a normal littermate (above). (B) Histology of an oral mucosal blister in a bal/bal mouse shows suprabasilar acantholysis. (C) Immunofluorescence of bal/bal tongue shows no Dsg3 compared with control littermate. (D) RNase protection assay of tongue lysates demonstrates that bal/bal mice synthesize DSG3 mRNA, as compared with DSG3 −/− mice that do not. Note that DSG3 +/− mice demonstrate about half of the mRNA of wild-type mice.

Figure 6

Analysis of balding mice compared with DSG3 −/− mice. (A) Balding phenotype of a 1-mo-old DSG3 −/− mouse compared with a normal littermate (above). (B) Histology of an oral mucosal blister in a bal/bal mouse shows suprabasilar acantholysis. (C) Immunofluorescence of bal/bal tongue shows no Dsg3 compared with control littermate. (D) RNase protection assay of tongue lysates demonstrates that bal/bal mice synthesize DSG3 mRNA, as compared with DSG3 −/− mice that do not. Note that DSG3 +/− mice demonstrate about half of the mRNA of wild-type mice.

Figure 7

Analysis of the DSG3 mutation in balding mice. (A) Heteroduplex analysis of a 400-bp segment of PCR-amplified mouse DSG3, containing exon 14 and flanking intronic sequences, revealed heteroduplex bands in case of a heterozygous bal/+ mouse (lane 2), while control mouse (lane 1) and a homozygous bal/bal (lane 3) showed a homoduplex band only. (B) Direct nucleotide sequencing of DNA shown on lane 2 in A revealed a heterozygous 1-bp insertion, 2275insT, which resulted in frame-shift (upper panel), in comparison with normal sequence in DNA obtained from a control mouse shown on lane 1 in A (lower panel). Sequencing of DNA on lane 3 revealed that bal/bal mouse was homozygous for the 2275insT mutation (middle panel). (C) Allele-specific oligonucleotide hybridization (ASO) was used to verify the presence of the mutation in balding mice. Specifically, PCR-amplified DNA from the control mouse hybridized with the wild-type oligomer (WT) only (lane 1), while homozygous bal/bal mouse hybridized with the mutant (M) probe only (lane 3). DNA from bal/+ mouse hybridized with both the WT and M probes, indicating that these mice were heterozygous for the mutation (lane 2).

Figure 7

Analysis of the DSG3 mutation in balding mice. (A) Heteroduplex analysis of a 400-bp segment of PCR-amplified mouse DSG3, containing exon 14 and flanking intronic sequences, revealed heteroduplex bands in case of a heterozygous bal/+ mouse (lane 2), while control mouse (lane 1) and a homozygous bal/bal (lane 3) showed a homoduplex band only. (B) Direct nucleotide sequencing of DNA shown on lane 2 in A revealed a heterozygous 1-bp insertion, 2275insT, which resulted in frame-shift (upper panel), in comparison with normal sequence in DNA obtained from a control mouse shown on lane 1 in A (lower panel). Sequencing of DNA on lane 3 revealed that bal/bal mouse was homozygous for the 2275insT mutation (middle panel). (C) Allele-specific oligonucleotide hybridization (ASO) was used to verify the presence of the mutation in balding mice. Specifically, PCR-amplified DNA from the control mouse hybridized with the wild-type oligomer (WT) only (lane 1), while homozygous bal/bal mouse hybridized with the mutant (M) probe only (lane 3). DNA from bal/+ mouse hybridized with both the WT and M probes, indicating that these mice were heterozygous for the mutation (lane 2).

Figure 8

Histology of bald back skin from a 24-d-old DSG3 −/− mouse. Note dilated telogen hair follicle containing a clump of pigment but no hair shaft. Bar, 35 μm.