Sp1 elements regulate transcriptional activity within the murine Toll-like receptor 4 promoter.
OBJECTIVE
Toll-like receptor 4 (TLR4) mediates the innate immune response to lipopolysaccharide (LPS) by a complex intracellular signaling pathway that activates the transcription factor nuclear factor (NF)-kappaB, with subsequent production of inflammatory cytokines. The precise mechanisms involved have not been delineated. We hypothesized that specific regulatory elements exist within the TLR4 promoter.


METHODS
We cloned regions of the murine TLR4 promoter (0.25 kb to 2 kb; -2000 bp to +244 bp) into the pGL3-Basic plasmid containing the firefly luciferase gene and used the resulting constructs to transfect HEK293 or RAW 264.7 cells. After 24 h, we used LPS to stimulate RAW 264.7 cells. We quantified relative light units (RLUs) of cell lysates and secreted tumor necrosis factor (TNF)-alpha. For pairwise comparison, we used Student's t-test. Sequence analysis of the promoter revealed several putative Sp1 sites. We used two constructs showing increased promoter activity, TLR4-750/-1 and TLR4-500/-1, to transfect cells in the presence of a specific Sp1 inhibitor, mithramycin A.


RESULTS
Of the six promoter constructs, four showed greater transcriptional activity (p < 0.05 vs. pGL3-Basic), as measured by luciferase activity. However, we did not observe differences in transcriptional activity of the promoter in five of those six constructs when we stimulated transfected RAW 264.7 cells with 10 ng of LPS. This finding suggests that transcriptional regulation of the promoter is unaffected by cellular changes caused by LPS. Both TLR4-750/-1 and TLR4-500/-1 showed dose-dependent reductions in transcriptional activity in the presence of increasing concentrations of the specific Sp1 inhibitor mithramycin A in both HEK293 and RAW 264.7 cells (p < 0.05 vs. no mithramycin A).


CONCLUSIONS
At least one Sp1 transcriptional regulatory element is present within the murine TLR4 promoter (range -750 bp to -250 bp). This finding holds promise for manipulating this fundamental inflammatory response.
Wasiluk
Karen R
KR
Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455, USA. wasil002@umn.edu
McCulloch
Karen A
KA
Banton
Kaysie L
KL
Dunn
David L
DL
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D008070:Lipopolysaccharides; D016329:Sp1 Transcription Factor; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4