Molecular cloning of mouse epidermal cystatin A and detection of regulated expression in differentiation and tumorigenesis

Abstract

A gamma gt 11 cDNA expression library representing mouse epidermis mRNA was screened with polyclonal rabbit antiserum directed against 10-13 kDa epidermal antigens that had previously been shown to be regulated during epidermal differentiation. A cDNA clone was detected and isolated and its identity as the coding sequence for one of the antigens was confirmed by translation of hybrid-selected mRNA from mouse epidermis. The cDNA sequence predicted a peptide homologous to the reported sequence of rat epidermis cystatin A, a thiol proteinase inhibitor. This identification was confirmed by cross-reactivity of the gamma clone fusion protein with authentic antiserum to rat epidermis cystatin A. Southern gel analysis showed that mouse DNA may contain several closely related genes homologous to the cystatin probe. An mRNA of about 0.6 kb from epidermis and cultured mouse epidermal cells hybridized with the cystatin probe on northern analysis. The abundance of the message was high in cultured basal cells and was selectively diminished by inducing terminal differentiation in culture with an elevated Ca2+ concentration in the medium. Cystatin message was abundant in chemically induced mouse skin papillomas but reduced in carcinomas. In epidermis, mRNA was localized to the less differentiated basal and lower spinous layers by in situ hybridization. Regulation of expression of cystatin A in epidermis and tumors suggests that it may be important in the control of normal keratinocyte proliferation and differentiation and in malignant conversion.