Background and Purpose: 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)-κB, 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)-α. 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).
Conclusion: 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.
