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Involvement of Sp1 in lipopolysaccharide-induced expression of HDC mRNA in RAW 264 cells
Noriyasu Hirasawa1, Masashi Torigoe, Kuniyuki Kano
1Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan. hirasawa@mail.pharm.tohoku.ac.jp
Abstract:
The involvement of Sp1 in the lipopolysaccharide (LPS)-induced transcription of HDC mRNA in the mouse macrophage-like cell line RAW 264 was analyzed. LPS increased the levels of HDC mRNA 4 h after the stimulation in a concentration-dependent manner. Mithramycin A, an inhibitor of the binding of the Sp family to the GC box, reduced the LPS-induced increase in the levels of HDC mRNA at 4 h and HDC protein at 8 h in a concentration-dependent manner. By conducting electrophoretic mobility shift assays, we found that one of the transcription factors binding to the DNA probe containing the GC box sequence of the mouse HDC gene promoter region was Sp1, and that levels of Sp1-DNA probe complexes were increased by stimulation with LPS although the protein levels of Sp1 were not changed. These results suggested that Sp1 is one of the transcription factors that regulate the LPS-induced expression of HDC in RAW 264 cells.
Insights
Lipopolysaccharide (LPS) increases histamine decarboxylase (HDC) mRNA in RAW 264 cells. The transcription factor Sp1 plays a key role in this LPS-induced HDC expression, as shown by inhibitor studies and binding assays.
Area of Science:
- Immunology and Molecular Biology
- Gene Regulation and Transcription Factor Function
Background:
- Lipopolysaccharide (LPS) is a potent immune activator.
- Histamine decarboxylase (HDC) is involved in histamine synthesis.
- Understanding gene regulation during immune responses is crucial.
Purpose of the Study:
- To investigate the role of the transcription factor Sp1 in LPS-induced HDC gene expression.
- To analyze the mechanism of HDC mRNA and protein regulation by LPS in RAW 264 cells.
Main Methods:
- RAW 264 macrophage cell line stimulation with LPS.
- Quantitative analysis of HDC mRNA levels.
- Inhibition studies using Mithramycin A (Sp1 inhibitor).
- Electrophoretic mobility shift assays (EMSA) to assess Sp1-DNA binding.
Main Results:
- LPS significantly increased HDC mRNA levels in a dose-dependent manner.
- Mithramycin A reduced LPS-induced HDC mRNA and protein expression.
- EMSA confirmed Sp1 binding to the HDC gene promoter's GC box.
- LPS stimulation enhanced Sp1 binding to the GC box without altering Sp1 protein levels.
Conclusions:
- Sp1 is a critical transcription factor regulating LPS-induced HDC expression in RAW 264 cells.
- The study elucidates a specific molecular mechanism in LPS-mediated immune cell activation.
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