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.
Keywords: ADAM family; DUOX2; Fractalkine; Peroxynitrite; Shedding.
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