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TRIM28/TIF1β and Fli-1 negatively regulate peroxynitrite generation via DUOX2 to decrease the shedding of
Rui Yamaguchi1, Misa Haraguchi1, Reona Yamaguchi2
1Graduate School of Medical Science, Kumamoto Health Science University, Kitaku Izumi-machi 325 Kumamoto 861-5598, Japan.
Abstract:
The chemokine fractalkine is synthesized as a membrane-bound protein, but studies have shown that serum levels of soluble fractalkine are elevated in inflammatory and autoimmune diseases. Patients with autoimmune diseases also have increased serum levels of neuropeptide substance P (SP). The shedding activity of the ADAM family is induced by peroxynitrite, but that of SP is unclear. Treatment of human macrophages with SP upregulated levels of membrane-bound fractalkine. Interestingly, small interfering RNA (siRNA) for DUOX2 further increased membrane-bound fractalkine but decreased soluble fractalkine compared with cells treated with SP alone. SP induced nitric oxide 2/inducible nitric oxide synthase (NOS2/iNOS) mRNA and increased levels of nitrotyrosine, a biomarker of peroxynitrite, whereas transfection with DUOX2 siRNA blunted upregulation of nitrotyrosine. Most importantly, N(ω)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) decreased protein levels of nitrotyrosine and concomitantly increased expression of membrane-bound fractalkine after exposure to SP. As for the signaling pathway of TGFβ1 (an inhibitor of iNOS mRNA expression), silencing of RNA for TAK-1 upregulated membrane-bound fractalkine, but silencing of RNA for the Smad family did not. Interfering RNA of transcription factor specificity protein 1 (Sp1) upregulated protein levels of TGFβ1/LAP. Most importantly, double transfection with siRNA for Sp1 and TRIM28/TIF1βor Fli-1 led to a significant increase in TGFβ1/LAP levels and a corresponding reduction of NOS2/iNOS, which inhibited the shedding of membrane-bound fractalkine. In conclusion, TRIM28/TIF1β and Fli-1 negatively regulate TGFβ1 expression to upregulate the generation of peroxynitrite, leading to increased shedding of membrane-bound fractalkine induced by SP.
Insights
Neuropeptide substance P (SP) induces fractalkine shedding via peroxynitrite generation, a process regulated by TRIM28/TIF1β and Fli-1. These factors inhibit TGFβ1, promoting peroxynitrite and fractalkine release in autoimmune diseases.
Area of Science:
- Immunology
- Molecular Biology
- Cellular Signaling
Background:
- Soluble fractalkine levels are elevated in inflammatory and autoimmune diseases.
- Neuropeptide substance P (SP) levels are also increased in autoimmune diseases.
- The mechanism of SP-induced fractalkine shedding remains unclear.
Purpose of the Study:
- To investigate the role of SP in fractalkine shedding.
- To elucidate the signaling pathways involved in SP-induced fractalkine release.
- To identify regulatory factors controlling fractalkine shedding in the context of autoimmune conditions.
Main Methods:
- Human macrophages were treated with SP and subjected to siRNA-mediated gene silencing (DUOX2, TAK-1, Smad family, Sp1, TRIM28/TIF1β, Fli-1).
- Levels of membrane-bound and soluble fractalkine were measured.
- Nitric oxide 2/inducible nitric oxide synthase (NOS2/iNOS) mRNA, nitrotyrosine, and TGFβ1/LAP protein levels were assessed.
- Inhibitors such as N(ω)-nitro-L-arginine methyl ester (L-NAME) were used.
Main Results:
- SP treatment upregulated membrane-bound fractalkine and induced NOS2/iNOS and nitrotyrosine.
- DUOX2 siRNA increased membrane-bound fractalkine but decreased soluble fractalkine and nitrotyrosine.
- L-NAME inhibited nitrotyrosine and increased membrane-bound fractalkine after SP exposure.
- Sp1 interference upregulated TGFβ1/LAP; combined siRNA for Sp1 and TRIM28/TIF1β or Fli-1 increased TGFβ1/LAP, reduced NOS2/iNOS, and inhibited fractalkine shedding.
Conclusions:
- TRIM28/TIF1β and Fli-1 negatively regulate TGFβ1 expression.
- This regulation upregulates peroxynitrite generation, leading to SP-induced shedding of membrane-bound fractalkine.
- The findings reveal a novel pathway contributing to fractalkine dysregulation in autoimmune diseases.
