Fig. 4. Identification of the transcription factors mediating OPN transcription in HMGB1-stimulated VSMCs.
(A) Three lengths of the OPN promoter were individually constructed in a luciferase-based reporter vector to produce pGL3-OPN-2284 (full-length), pGL3-OPN-538 and pGL3-OPN-234 promoter constructs. The VSMCs were transiently transfected with these promoter constructs and an empty luciferase vector (pGL3) for 24 h, and then stimulated with HMGB1 (100 ng/ml) for 1 h. The differences in promoter activity between the control and HMGB1-stimulated cells were presented as the means ± SEMs of 6 independent experiments. **p < 0.01 vs. non-treated control. (B) Nucleotide sequence of the -538 ~ -234 region of the OPN promoter. The transcription factor binding sites were identified using TF Search software. The sequences of the potential binding sites for AP-1 and C/EBPβ in pLuc-OPN-538 were underlined. (C) The binding activity of AP-1 on the OPN promoter segment in HMGB1-treated VSMCs was assessed using a ChIP assay. IgG was used as the negative control. The relative binding intensities to the input signals were quantified and presented as the means ± SEMs of 6 independent experiments. **p < 0.01 vs. the value at 0 h. OPN, osteopontin; VSMCs, vascular smooth muscle cells; HMGB1, high-mobility group box 1; AP-1, activator protein 1; ChIP, chromatin immunoprecipitation.
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