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Pemphigus vulgaris antigen is a cadherin-like desmosomal cell adhesion molecule expressed primarily in suprabasal keratinocytes within the epidermis. Previously characterized structural features have defined this molecule as a desmoglein, DSG3. In this study, we have cloned the human DSG3 gene and examined the transcriptional regulation of its expression. The total gene consisted of 15 exons and was estimated to span >23 kilobases. Comparison of exon-intron organization of DSG3 with bovine DSG1 and several classical cadherin genes revealed striking conservation of the structure. Up to 2.8 kilobases of the upstream genomic sequences were sequenced and found to contain several putative cis-regulatory elements. The promoter region was GC-rich and TATA-less, similar to previously characterized mammalian cadherin promoters. The putative promoter region was subcloned into a vector containing chloramphenicol acetyl transferase reporter gene. Transient transfections with a series of deletion clones indicated that the DSG3 promoter demonstrated keratinocyte-specific expression, as compared with dermal fibroblasts examined in parallel, and fine mapping identified a 30-base pair segment at -200 to -170 capable of conferring epidermal specific expression. The results provide evidence for the transcriptional regulation of the pemphigus vulgaris antigen gene, potentially critical for development of the epidermis and physiologic terminal differentiation of keratinocytes.

Interferon-gamma (IFN-gamma) has been shown to regulate epidermal keratinocyte growth and differentiation. In this study, we examined the effects of recombinant human IFN-gamma on the expression of the gene encoding the 230-kDa bullous pemphigoid antigen (BPAG1), a marker of the mitotic basal cell phenotype in the epidermis. Northern analysis revealed a dose- and time-dependent suppression of BPAG1 expression by IFN-gamma in cultured human keratinocytes from several different donors, and incubation of the cells with IFN-gamma in the presence of cycloheximide demonstrated that this effect required ongoing protein synthesis. The inhibition of BPAG1 gene expression was also demonstrated at the protein level by indirect immunofluorescence using a monoclonal antibody recognizing the human 230-kDa bullous pemphigoid antigen. Transient transfections of cultured keratinocytes with BPAG1 promoter-chloramphenicol acetyltransferase reporter gene plasmids indicated marked suppression of the promoter activity by IFN-gamma, and deletion constructs were able to identify a defined region containing the responsive element (IFN-gamma inhibitory element). Reduced transcription of the BPAG1 gene by IFN-gamma was also demonstrated by in vitro nuclear run-on assays. These data, which indicate inactivation of transcription of a basal keratinocyte-specific gene of transcription of a basal keratinocyte-specific gene (BPAG1) by IFN-gamma, provide novel insight into the mechanisms of IFN-gamma-mediated keratinocyte gene regulation and epidermal differentiation in inflammatory diseases.

The 230-kDa bullous pemphigoid antigen gene (BPAG1) is expressed exclusively in basal keratinocytes of epidermis. In this study, we have identified a novel cis-element, keratinocyte responsive element 3 (KRE3), at position -216 to -197 of the human BPAG1 gene. A promoter-CAT construct containing this element had approximately 50-fold higher expression than a similar construct devoid of this sequence when tested in transient transfections of cultured human keratinocytes. However, there was no effect on the low base-line level of expression in cultured skin fibroblasts. KRE3 contains a palindromic sequence 5'-CAAATATTTG-3', and mutations in this sequence significantly reduced the promoter activity. Gel mobility shift assays with an oligomer containing KRE3 sequence demonstrated binding activity with nuclear proteins isolated from keratinocytes. One of the DNA/protein complexes was clearly specific, since competition with > 12.5-fold excess of the unlabeled oligomer resulted in disappearance of this band. No specific binding activity was noted with nuclear proteins extracted from fibroblasts. Thus, KRE3 appears to serve as the binding site for keratinocyte-specific trans-activating factor(s), and KRE3 may thus confer the tissue-specific expression to the BPAG1 gene.