Sp1-associated activation of macrophage inflammatory protein-2 promoter by CpG-oligodeoxynucleotide and lipopolysaccharide

Abstract

Macrophage inflammatory protein-2 (MIP-2) is a C-X-C chemokine that is important in recruiting neutrophils to inflammatory sites. Our previous reports demonstrated that lipopolysaccharide (LPS) or CpG-oligode-oxynucleotide (CpG-ODN) rapidly induce MIP-2 gene expression in the macrophage cell line, RAW 264.7. Here, we show that the DNA sequence of the MIP-2 promoter between -114 and +14 is sufficient for strong promoter activity in LPS- or CpG-ODN-stimulated RAW 264.7 cells. Importantly, comprehensive mutant analysis reveals that an Sp1 element in the promoter region between -114 and -94 is essential for synergistic MIP-2 promoter activation by NF-kappaB and c-Jun regardless of the presence of an AP-1 site. By combining deletion or site-specific mutant analysis with immunocomplex assays, we also confirmed that Sp1 mediates the recruitment of transcription factors NF- kappaB and c-Jun in LPS- or CpG-ODN-treated RAW 264.7 cells. Several lines of experimental evidence imply that the Sp1-binding element is an important determinant of MIP-2 promoter activity, and that NF-kappaB, c-Jun and Sp1 can functionally cooperate to elicit maximal activation of the promoter.

Inhibitory effects of flavonoids on TNF-α-induced IL-8 gene expression in HEK 293 cells

“…We performed SDS-PAGE and Western blot analysis as described elsewhere (25). Briefly, HEK 293 cells were preincubated with flavonoids (kaempferol, quercetin, fisetin, or chrysin) at a concentration of 40 μM for 1 h prior to TNF-α treatment for the indicated time periods.…”

“…The effects of flavonoids on TNF-α-induced NF-κB p65 nuclear localization were detected by an indirect immunofluorescence assay using confocal microscopy as described previously (25). Briefly, HEK 293 cells (1 × 10 5 cells/well) were cultured in 24-well plates and then preincubated with flavonoids (kaempferol, quercetin, fisetin, or chrysin) at a concentration of 40 μM for 1 h prior to TNF-α treatment for 30 min.…”

Direct role of NF‐κB activation in Toll‐like receptor‐triggered HLA‐DRA expression

“…We then ligated this fragment into KpnI and HindIII sites of the luciferase reporter plasmid pGL3-Basic vector (Promega, Madison, WI), which yielded the reporter construct pHLA-DRA-Luc. To introduce a site-specific mutation in NF-jB-binding site, we abrogated the transcription factor recognition site and replaced with non-functional sequence by two-step PCR mutagenesis method [38]. The method uses 5 0 -primer DRA(-323) and 3 0 -primer DRA(+43), along with primers that encode the following sequences in sense or antisense orientation: mNF-jB, -312 GCTCTCACTTTAccTGTTTggATTGATTCTATTC -279 .…”

“…We performed SDS-PAGE and Western blot analysis as described elsewhere [38]. After treating the cells with IFN-c (200 U/mL), CpG-DNA (3 lM) or LPS (100 ng/mL) for the indicated periods, we harvested the cells and lysed them in 10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and Complete Protease Inhibitor Cocktail (Roche).…”

“…Nuclear extracts of the cells treated with IFN-c, CpG-DNA, or LPS for 30 min were prepared as described elsewhere [38]. We purchased the NF-jB consensus oligonucleotide from Santa Cruz Biotechnology as a probe, and synthesized HLA-DRA promoter fragments that encompassed -323 to -282 (the HLA-DRA NF-jB) by the customer service of GenoTech.…”