The Value of Type IV Collagen Immunohistochemical Staining in the Differential Diagnosis of Autoimmune Subepidermal Bullous Diseases.

Autoimmune subepidermal bullous diseases (AISBDs) exhibit various clinical presentations, histological appearances, prognoses, and responses to treatment. Many diagnostic techniques, such as direct immunofluorescence (IF), indirect salt-split skin IF, and enzyme-linked immunosorbent assays, are used in the differential diagnoses of AISBDs. However, these techniques require fresh frozen tissue, expensive laboratory equipment, and sophisticated laboratory techniques. The purpose of this study was to evaluate the value of type IV collagen immunohistochemical (IHC) staining for the differential diagnosis of AISBDs. Paraffin-embedded blocks of skin biopsies were selected from 28 patients with autoimmune subepidermal bullous diseases. Among these 28 cases, 24 patients exhibited bullous pemphigoid (BP), 2 exhibited epidermolysis bullosa acquisita (EBA), 1 exhibited linear immunoglobulin A dermatosis (LAD), and 1 exhibited bullous systemic lupus erythematosus (BSLE). Sections were stained for type IV collagen and examined to determine the location of type IV collagen in the subepidermal blister. Type IV collagen positivity was observed on the base of the subepidermal blister in patients with BP (24 of 24 cases) and LAD (1 of 1 case). Staining was observed on the roof of the blister in patients with EBA (2 of 2 cases) and BSLE (1 of 1 case), and irregular staining was also observed on the base in patients with EBA. In conclusion, type IV collagen IHC staining is a simple and useful diagnostic technique for the differential diagnosis of AISBDs.

Suprabasal Dsg2 expression in transgenic mouse skin confers a hyperproliferative and apoptosis-resistant phenotype to keratinocytes.

Desmoglein 2 (Dsg2), a component of the desmosomal cell-cell adhesion structure, has been linked to invasion and metastasis in squamous cell carcinomas. However, it is unknown whether--and if so how--Dsg2 contributes to the malignant phenotype of keratinocytes. In this study, we addressed the consequences of Dsg2 overexpression under control of the involucrin promoter (Inv-Dsg2) in the epidermis of transgenic mice. These mice exhibited epidermal hyperkeratosis with slightly disrupted early and late differentiation markers, but intact epidermal barrier function. However, Inv-Dsg2 transgene expression was associated with extensive epidermal hyperplasia and increased keratinocyte proliferation in basal and suprabasal epidermal strata. Cultured Inv-Dsg2 keratinocytes showed enhanced cell survival in the anchorage-independent state that was critically dependent on EGF receptor activation and NF-kappaB activity. Consistent with the hyperproliferative and apoptosis-resistant phenotype of Inv-Dsg2 transgenic keratinocytes, we observed enhanced activation of multiple growth and survival pathways, including PI 3-kinase/AKT, MEK-MAPK, STAT3 and NF-kappaB, in the transgenic skin in situ. Finally, Inv-Dsg2 transgenic mice developed intraepidermal skin lesions resembling precancerous papillomas and were more susceptible to chemically induced carcinogenesis. In summary, overexpression of Dsg2 in epidermal keratinocytes deregulates multiple signaling pathways associated with increased growth rate, anchorage-independent cell survival, and the development of skin tumors in vivo.

Loss of cell adhesion in Dsg3bal-Pas mice with homozygous deletion mutation (2079del14) in the desmoglein 3 gene.

Pemphigus encompasses a group of autoimmune blistering diseases with circulating pathogenic autoantibodies recognizing several proteins, including the desmosomal cadherin, desmoglein 3. Targeted disruption of the Dsg3 gene by homologous recombination (Dsg3tm1stan) in mouse results in fragility of the skin and oral mucous membranes, analogous to the human disease. In addition, the Dsg3tm1stan mice develop phenotypic runting and hair loss, identical to that of the mouse mutant, Dsg3bal-2J. The Dsg3bal-2J mice are homozygous for a 1 bp insertion (2275insT) in the Dsg3 gene resulting in a nonfunctional Dsg3 mRNA. In this study, we characterized an allelic mutation, Dsg3bal-Pas, with clinical features similar to those in Dsg3bal-2J. We have identified a 14 bp deletion in exon 13 of the Dsg3 gene resulting in a frameshift and premature termination codon 7 bp downstream from the site of the deletion and causing a truncation of the desmoglein 3 polypeptide by 199 amino acids, eliminating virtually all of the intracellular domain. We demonstrate that, although a Dsg3 mRNA transcript was detectable in Dsg3bal-Pas skin, the corresponding protein for desmoglein 3 was completely absent in the oral mucosal epithelium of homozygous Dsg3bal-Pas compared with that of +/Dsg3bal-Pas mice. No significant changes in the expression of desmogleins 1 and 2 were detected. To elucidate a potential mechanism causing loss of cell adhesion in the Dsg3bal-Pas mice, we generated a myc-tagged truncated Dsg3bal-Pas desmoglein 3 protein and expressed it in keratinocytes. The myc-tagged truncated Dsg3bal-Pas desmoglein 3 protein was found predominantly in the cytoplasm possibly due to increased proteolytic degradation. Cell surface staining was also detected but was jagged, not linear along the cell-cell border like that observed for the full-length desmoglein 3. The expression of the myc-tagged truncated Dsg3bal-Pas desmoglein 3 protein resulted in a reduction in staining of other desmosomal proteins, including desmoglein 1 and 2, plakophilin 2, and plakoglobin. In addition, the cells expressing myc-tagged truncated Dsg3bal-Pas desmoglein 3 protein underwent dramatic changes in cell morphology and exhibited striking extensive filopodia. Collectively, these data showed that the perturbation of desmoglein 3 found in the Dsg3bal-Pas mice resulted in disadhesion of keratinocytes manifested with blistering phenotype.

Metastasis-associated protein (MTA)1 enhances migration, invasion, and anchorage-independent survival of immortalized human keratinocytes.

The human metastasis-associated gene (MTA1), a member of the nucleosome remodeling complex with histone deacetylase activity, is frequently overexpressed in biologically aggressive epithelial neoplasms. Here, we extend this observation to squamous carcinoma cells, which express high levels of MTA1 relative to normal or immortalized keratinocytes. To address functional aspects of MTA1 expression, we established variants of human immortalized keratinocytes (HaCaT cells) by expressing MTA1 cDNA in both the sense and antisense orientations. We demonstrate that (1) forced MTA1 expression enhances migration and invasion of immortalized keratinocytes; (2) MTA1 expression is necessary but not sufficient for cell survival in the anchorage independent state; (3) MTA1 contributes to expression of the anti-apoptotic Bcl-2 family member Bcl-x(L); (4) MTA1 expression in immortalized keratinocytes depends, in part, on activation of the epidermal growth factor receptor (EGFR). These results establish that, in keratinocytes, MTA1 expression contributes to several aspects of the metastatic phenotype including survival in the anchorage independent state, migration, and invasion.