A tandem GC box motif is necessary for lipopolysaccharide-induced transcription of the type II TNF receptor gene.

Abstract

LPS induces the expression of the gene encoding the type II TNF-alpha receptor in mononuclear phagocytes. To elucidate the nuclear signaling mechanisms involved in this response, a 772-bp fragment of the TNFRII gene promoter was analyzed by deletion and site-specific mutagenesis following transient transfection in the macrophage-like cell line RAW264.7. A region located between -100 and -75 relative to the transcription start site was found to be essential for LPS sensitivity. This contained a GC-rich region composed of two tandemly arrayed Sp-1 sites. While mutation of this region confirmed that it was essential for LPS sensitivity, the sequence was not able to confer LPS sensitivity upon a heterologous promoter. Internal deletion and site-specific mutagenesis of the 100-bp fragment identified regions immediately flanking an initiator region (Inr) site that were also necessary for sensitivity to LPS. Electrophoretic mobility shift assays demonstrated that the GC box bound Sp-1 and Sp-3, although the level of binding activity did not vary with LPS stimulation. An oligonucleotide probe containing nucleotide positions -45 to -18 also bound several protein complexes, but these were not enhanced by LPS. These findings indicate that the tandem GC box is necessary, but not sufficient, for LPS-mediated transcription of the TNFRII gene. A second apparently novel motif, located within 15 nucleotides of the Inr, is also necessary.