TGF-β1 enhances degradation of IFN-γ-induced iNOS protein via proteasomes in RAW 264.7 cells

TGF-β1 is a well-known immunosuppressive cytokine that inhibits inducible nitric oxide synthase (iNOS) gene expression in various cells including macrophages. In this study, we investigated the suppressive mechanisms of TGF-β1 on IFN-γ-induced iNOS gene expression using the murine macrophage-like cell line RAW 264.7. TGF-β1 decreased iNOS protein amount through enhanced degradation, although TGF-β1 did not affect IFN-γ-induced iNOS mRNA level or stability. In addition, the enhancement of iNOS protein degradation by TGF-β1 treatment was almost completely blocked by MG132, a proteasome inhibitor. Furthermore, TGF-β1 enhanced the trypsin-like activity of proteasomes in the presence of IFN-γ, although did not enhance the peptidylglutamyl-peptide hydrolyzing and chymotrypsin-like activities of proteasomes. The level of ubiquitinated iNOS protein was not significantly altered by IFN-γ or IFN-γ plus TGF-β1 treatment. Because MG132 inhibited iNOS protein degradation and IFN-γ plus TGF-β1 treatment increased the trypsin-like activity of proteasomes, we hypothesized that TGF-β1 might enhance iNOS protein degradation via the ubiquitin-proteasome pathway in the presence of IFN-γ. We propose that these mechanisms of TGF-β1 in the posttranslational regulation of iNOS gene expression may contribute to suppression of excess nitric oxide during inflammatory processes.