The current study was conducted to elucidate the mechanism through which TNF-alpha stimulates expression of MUC1, a membrane-tethered mucin. A549 human lung alveolar cells treated with TNF-alpha exhibited significantly higher MUC1 protein levels in detergent lysates compared with cells treated with vehicle alone. Increased MUC1 protein levels were correlated with significantly higher levels of MUC1 mRNA in TNF-alpha-treated cells compared with controls. However, TNF-alpha did not alter MUC1 transcript stability, implying increased de novo transcription induced by the cytokine. TNF-alpha increased MUC1 gene promoter activity in A549 cells transfected with a promoter-luciferase reporter plasmid. Both U0126, an inhibitor of MEK1/2, and dominant negative ERK1 prevented TNF-alpha-induced MUC1 promoter activation, and anti-TNFR1 antibody blocked TNF-alpha-stimulated ERK1/2 activation. MUC1 promoter activation by TNF-alpha also was blocked by mithramycin A, an inhibitor of Sp1, as well as either deletion or mutation of a putative Sp1 binding site in the MUC1 promoter located between nucleotides -99 and -90. TNF-alpha-stimulated binding of Sp1 to the MUC1 promoter in intact cells was demonstrated by chromatin immunoprecipitation assay. We conclude that TNF-alpha induces MUC1 gene transcription through a TNFR1 --> MEK1/2 --> ERK1 --> Sp1 pathway.