Osteopontin Induces Ubiquitin-Dependent Degradation of STAT1 in RAW264.7 Murine Macrophages

Abstract

In systemic inflammation induced by endotoxin (LPS), the macrophage produces the majority of the circulating NO metabolites. However, while the molecular pathways which up-regulate iNOS expression have been extensively studied in the macrophage, little is known of the parallel counterregulatory pathways which repress or inhibit macrophage iNOS expression. Using both in vivo and in vitro murine models of endotoxin (LPS) stimulation, we have previously demonstrated that NO feedback inhibits its own synthesis by increasing transcription of osteopontin (OPN), a potent transrepressor of inducible NO synthase expression. In this current study, using a system of LPS-treated RAW264.7 macrophages, we go on to demonstrate that OPN increases STAT1 ubiquitination and subsequent 26s proteasome-mediated degradation to inhibit STAT1 dependent iNOS promoter activity, transcription, and protein expression. In addition, we identify STAT-interacting LIM protein as the critical STAT ubiquitin E3 ligase critical for STAT1 degradation in this setting. OPN has not been linked previously to STAT1 degradation. This regulation of STAT1 degradation underlies OPN′s effect as an inhibitor of iNOS gene transcription. These are novel findings and define OPN as a unique and as yet, poorly characterized, transactivator of STAT1 degradation by the ubiquitin-proteasome system.

Osteopontin and Protein Kinase C Regulate PDLIM2 Activation and STAT1 Ubiquitination in LPS-treated Murine Macrophages

“…Tanaka et al (8) were the first to identify PDLIM2 protein (previously known as SLIM or mystique) as a STAT ubiquitin E3 ligase and demonstrate a central role for ubiquitination in regulation of the IFN-STAT signaling pathway. Previously, using a murine macrophage model of LPS stimulation, we found that OPN increased STAT1 ubiquitination and degradation through the ubiquitin E3 ligase, PDLIM2 (9). In this study, we build upon our former observations to demonstrate that LPS-and OPNdependent activation of PDLIM2 and subsequent STAT1 ubiquitination require protein kinase C (PKC)-regulated phosphorylation of PDLIM2 Ser-137.…”

“…In Vitro Ubiquitination Assay-The in vitro ubiquitination assay was described previously (9). Recombinant wild-type PDLIM2, mutant His-tagged PDLIM2, or empty His vector was expressed in RAW264.7 cells.…”

Smurf1 Protein Negatively Regulates Interferon-γ Signaling through Promoting STAT1 Protein Ubiquitination and Degradation

“…However, the E3 ligases responsible for the STAT1 ubiquitination are not defined. In this regard, PDLIM2 has been identified as an E3 ligase for STAT1 ubiquitination (15,17). PDLIM2, as a nuclear protein, was shown to specifically interact with the tyrosine-phosphorylated STAT1 and mediates the degradation of active STAT1.…”

“…Plasmid Constructs and Transfection-Mammalian expression plasmids for FLAG-tagged STAT1 and luciferase reporter constructs bearing the mouse iNOS promoter have been described previously (17). Myc-tagged STAT1 and Smurf1 were obtained by PCR and cloned into the pCMV-Myc plasmids (Promega).…”

Efeitos in vitro de extratos naturais sobre Enterococcus faecalis, Staphylococcus aureus, Escherichia coli e endotoxinas em canais radiculares

“…Para avaliar os efeitos citotóxicos do conteúdo coletado dos canais radiculares, foi utilizada a linhagem de macrófagos de camundongo RAW 264.7, pois estes macrófagos são freqüentemente utilizados em pesquisa para avaliar os efeitos de LPS e produção de citocinas e outros mediadores da resposta inflamatória (Jiang et al, 2003, Refai et al, 2004Gao et al, 2007;Lee et al, 2007;Xu et al, 2007). Para quantificar as citocinas (IL-1 e TNF-), foram utilizados os kits para reação de ELISA da R & D, pois vários estudos publicados em periódicos internacionais com seletiva política editorial utilizam os kits do fabricante R & D para dosar citocinas (Hong et al, 2004;Bostanci et al, 2007;Lee et al, 2008;Youn et al, 2008 (Koo et al, 2000;Ferreira et al, 2007;Cardoso et al, 2009).…”