Functional importance of an Sp1- and an NFkB-related nuclear protein in a keratinocyte-specific promoter of rabbit K3 keratin gene.

We have shown previously that a 300-base pair (bp) 5' upstream sequence of rabbit keratin K3 gene (RK3) can function as a keratinocyte-specific promoter in transient transfection assays. Electrophoretic mobility shift assays using various overlapping and mutated oligonucleotides established that corneal keratinocyte nuclear proteins bound in vitro to two sites (B and E). Immunosupershift and UV cross-linking established that the keratinocyte nuclear binding protein of site B (5'-GGGGCTTTCC-3', -262 to -253 bp) was NFkB consisting of the p65 and p50 subunits. The E site contained an unusual GC-rich motif (5'-CCGCCCCCTG-3', -203 to -194 bp) whose sequence deviated from the Sp1 consensus in 4 out of 10 positions; this site bound an Sp 1-related keratinocyte nuclear protein. Mutagenesis of the NFkB, GC motif, and both sites abolished 20, 50, and 75%, respectively, of the promoter activity in transfected keratinocytes. The NFkB-like keratinocyte nuclear protein was barely detectable in kidney epithelial cells, HeLa, and fibroblasts. The Sp1-related nuclear protein was abundant in keratinocytes and simple epithelial cells, but was much less abundant in fibroblasts. These results indicate that NFkB is present in significant quantities in keratinocyte nuclei and that the tissue restriction of the NFkB- and Sp1-related proteins, in combination with other factors, may contribute to the keratinocyte specificity of RK3 promoter.