To clarify the mechanism underlying regulation of MUC6 expression, we isolated the 5' flanking region of the MUC6 gene (5'-MUC6). We determined the transcription start site of the MUC6 gene, and found a TATA box at -35 to -29 bp, a putative NF kappa B consensus sequence at -173 to -164 bp, and putative Sp family binding sites at -530 to -521 and -847 to -838 bp. The luciferase activity of 5'-MUC6 gradually decreased with deletion of these sites. NF kappa B inhibitory factor I kappa B decreased the luciferase activity, and forced expression of NF kappa B induced MUC6 transcription. An inhibitor of Sp family binding, mithramycin A, suppressed MUC6 transcripts, and Sp1 and Sp3 overexpression up-regulated them. Binding of Sp family members to their putative sites was confirmed by electrophoretic mobility shift assays. Our results suggest that MUC6 transcription is regulated by NF kappa B and Sp family members. (c) 2005 Elsevier Inc. All rights reserved.