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Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection
Published on: September 2, 2014
The regulation of EN-RAGE (S100A12) gene expression in human THP-1 macrophages
Takamasa Hasegawa1, Atsushi Kosaki, Tatsuji Kimura
1Department of Medicine II, Kansai Medical University, 10-15 Fumizono-cho, Osaka 570-8507, Moriguchi, Japan.
Abstract:
EN-RAGE is a ligand for the receptor for advanced glycation end products (RAGE) and may be involved in the development of diabetic macro- and micro-angiopathy. This study is designed to investigate the regulation of EN-RAGE gene expression in human macrophages. The amounts of EN-RAGE mRNA were measured in cultured human THP-1 macrophages after treatment with various stimuli known to modulate atherosclerosis. First, interleukin-6 (IL-6), a proinflammatory cytokine, increased the level of EN-RAGE mRNA by approximately 2-fold in a time- and a dose-dependent fashion. EN-RAGE protein was detected in the cultured medium and increased significantly by the addition of IL-6. The induction was abolished by pretreatment with the JAK kinase inhibitor and cycloheximide, but not with the MEK kinase inhibitor. Second, pioglitazone (PIO), a thiazolidinedione, decreased the level of EN-RAGE mRNA by approximately 25% of the basal in a time- and a dose-dependent fashion. Pioglitazone also inhibited the induction of EN-RAGE mRNA by IL-6. These results indicate the production of EN-RAGE is induced by IL-6 through de novo protein synthesis via the JAK-STAT kinase pathway and inhibited by the activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) in human macrophages.
Insights
Interleukin-6 (IL-6) increases EN-RAGE in macrophages via JAK-STAT signaling. Pioglitazone (PIO) inhibits EN-RAGE production, suggesting a role in diabetic vascular complications.
Area of Science:
- Endocrinology
- Immunology
- Molecular Biology
Background:
- EN-RAGE, a ligand for the receptor for advanced glycation end products (RAGE), is implicated in diabetic vascular complications.
- Understanding EN-RAGE gene regulation in macrophages is crucial for addressing atherosclerosis.
Purpose of the Study:
- To investigate the regulatory mechanisms of EN-RAGE gene expression in human macrophages.
- To determine the effects of key inflammatory and metabolic modulators on EN-RAGE production.
Main Methods:
- Cultured human THP-1 macrophages were treated with interleukin-6 (IL-6) or pioglitazone (PIO).
- EN-RAGE mRNA levels were quantified using quantitative assays.
- Protein levels were assessed, and pathway inhibitors (JAK, MEK, cycloheximide) were employed.
Main Results:
- IL-6 significantly increased EN-RAGE mRNA and protein in a time- and dose-dependent manner, mediated by JAK-STAT pathway and de novo protein synthesis.
- Pioglitazone (PIO) dose- and time-dependently decreased basal EN-RAGE mRNA levels and inhibited IL-6-induced EN-RAGE production.
- The JAK kinase inhibitor and cycloheximide abolished IL-6 induction, while the MEK kinase inhibitor did not.
Conclusions:
- Macrophage EN-RAGE production is upregulated by IL-6 via JAK-STAT signaling and de novo protein synthesis.
- Peroxisome proliferator-activated receptor-gamma (PPARgamma) activation by PIO inhibits EN-RAGE expression.
- These findings highlight potential therapeutic targets for diabetic angiopathy.
