Stable Expression of Transfected Human Involucrin Gene in Various Cell Types: Evidence for In Situ Cross-Linking by Type I and Type II Transglutaminase

Abstract

Involucrin is a 68-kd precursor of the cornified envelope in keratinocytes. During terminal differentiation, involucrin becomes covalently cross-linked to other proteins by a membrane-anchored calcium-activated transglutaminase (TG) to form part of the cornified envelope. To better understand this process, we have used vector-mediated gene transfer to express human involucrin in a variety of cell types. Authentic involucrin protein was expressed in Chinese hamster ovarian (CHO) cells (fibroblasts), PtK2 rat kangaroo kidney cells (simple epithelial), and rat epidermal keratinocytes (stratifying squamous epithelial). The expression vector included an independent transcription unit encoding the aminoglycoside phosphotransferase gene (neo-r) allowing selection of stable involucrin-positive, G418-resistant clonal cell lines. In each cell type, involucrin levels were comparable to the endogenous expression observed in SCC-13 cells. Immunofluorescence localization studies revealed a uniform involucrin distribution throughout the cytoplasm in each cell type. Elevation of intracellular calcium resulted in cross-linking of involucrin in both CHO cells and rat keratinocytes, a process that was inhibited by EDTA and cystamine. In contrast, no cross-linking was observed in PtK2 cells or long-term passaged rat keratinocytes. Transglutaminase assays showed that rat keratinocytes contain predominantly a particulate (type I) form, whereas CHO cells contain only soluble TG (type II). PtK2 cells and long-term passaged keratinocytes, which were unable to cross-link involucrin, contained barely detectable TG activity. These results indicate 1) that involucrin is an efficient in situ substrate for both type I and type II transglutaminase and 2) that involucrin cross-linking is strictly TG dependent.