Stat proteins play a role in tumor necrosis factor alpha gene expression

Trauma produces dysfunction in immunity, which appears to be partially related to alterations in the cytokine response. Signal transducer and activator of transcription proteins (STATs) mediate activation of several cytokine genes. However, the effect of STAT proteins on tumor necrosis factor-alpha (TNFα) activation is not fully defined. We identified binding sites for STAT 3 and STAT 5/6 within the promoter region of TNFα and hypothesize that alterations in these sites would affect TNFα expression. The TNFα promoter was inserted into the luciferase reporter vector, and binding sites for STAT 3, STAT 5/6, and activator protein-1 (AP-1) were mutated using site-directed mutagenesis. Murine macrophages were transfected with the resultant plasmids, then incubated with and without lipopolysaccharide (LPS) or IFNα. Gene expression was measured by dual luciferase assay. Mutation of the STAT 3 binding site was associated with decreased LPS-inducible activity. Mutation of the AP-1 and STAT 5/6 consensus binding sites alone had no effect on TNFα expression. However, combined mutation of both STAT 5/6 and AP-1 was associated with increased LPS-inducible activity. Mutations of the STAT binding sites in the promoter region of TNFα affect TNFα gene expression. These results suggest a regulatory role for STATs in TNF gene transcription.