| Abstract |
The 230-kDa bullous pemphigoid antigen gene is expressed primarily, if not exclusively, in basal keratinocytes of the epidermis. Keratinocyte responsive element 3, a cis-element at position -216 to -197 of the human 230-kDa bullous pemphigoid antigen gene promoter, confers tissue-specific expression to this gene (Tamai et al: J Biol Chem 270:7609-7614, 1995). In this study, we investigated the functional characteristics of keratinocyte responsive element 3 on the 230-kDa bullous pemphigoid antigen gene core promoter by transient transfections of cultured normal human keratinocytes and normal human fibroblasts, as well as of lung carcinoma (A549), osteosarcoma (OST), and gastric adenocarcinoma (GT3TKB) cell lines. A 230-kDa bullous pemphigoid antigen gene core promoter/luciferase reporter gene plasmid construct, pBPL, was modified to develop a series of constructs (pKBPL-p4KBPL), which have insertions of one, two, three, or four tandem repeats of keratinocyte responsive element 3, and these plasmids were used in transient transfections of the cultured cells. The promoter activities of pKBPL-p4KBPL constructs, relative to pBPL, in normal human keratinocytes were 7.6-, 15.5-, 4.6-, and 2.7-fold higher, respectively, whereas no upregulatory effect by keratinocyte responsive element 3 insertion was observed in other cell lines tested. prKBPL, a plasmid constructed with keratinocyte responsive element 3 in reverse orientation, showed essentially no activity in normal human keratinocytes. Insertion of a random 20 bp sequence between keratinocyte responsive element 3 and the 230-kDa bullous pemphigoid antigen gene core promoter resulted in about 40% reduction of luciferase activity in normal human keratinocytes. These data suggest that keratinocyte responsive element 3 functions as a position-, copy number-, and orientation-dependent cis-element contributing to tissue-specific regulation of the 230-kDa bullous pemphigoid antigen gene.
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