TGF-beta1 enhances degradation of IFN-gamma-induced iNOS protein via proteasomes in RAW 264.7 cells.
TGF-beta1 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-beta1 on IFN-gamma-induced iNOS gene expression using the murine macrophage-like cell line RAW 264.7. TGF-beta1 decreased iNOS protein amount through enhanced degradation, although TGF-beta1 did not affect IFN-gamma-induced iNOS mRNA level or stability. In addition, the enhancement of iNOS protein degradation by TGF-beta1 treatment was almost completely blocked by MG132, a proteasome inhibitor. Furthermore, TGF-beta1 enhanced the trypsin-like activity of proteasomes in the presence of IFN-gamma, 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-gamma or IFN-gamma plus TGF-beta1 treatment. Because MG132 inhibited iNOS protein degradation and IFN-gamma plus TGF-beta1 treatment increased the trypsin-like activity of proteasomes, we hypothesized that TGF-beta1 might enhance iNOS protein degradation via the ubiquitin-proteasome pathway in the presence of IFN-gamma. We propose that these mechanisms of TGF-beta1 in the posttranslational regulation of iNOS gene expression may contribute to suppression of excess nitric oxide during inflammatory processes.
Mitani
Toshifumi
T
Department of Biochemistry and Molecular Medicine, Shimane University Faculty of Medicine, Izumo 693-8501, Japan.
Terashima
Masaharu
M
Yoshimura
Hitoshi
H
Nariai
Yuko
Y
Tanigawa
Yoshinori
Y
D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007371:Interferon-gamma / Q000494:pharmacology*; D007976:Leupeptins / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009097:Multienzyme Complexes; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D025801:Ubiquitin / Q000378:metabolism
D007976:Leupeptins; D009097:Multienzyme Complexes; D012333:RNA, Messenger; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D025801:Ubiquitin; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde