Osteopontin and protein kinase C regulate PDLIM2 activation and STAT1 ubiquitination in LPS-treated murine macrophages.

Abstract

The molecular pathways regulating signal transducer and activator of transcription 1 (STAT1) levels in states of inflammation are incompletely understood. The suppressor of cytokine signaling, protein inhibitor of STAT, and SHP-1/2 tyrosine phosphatases ultimately regulate activity of STAT molecules. However, these mechanisms do not degrade STAT proteins. In this regard, using a murine macrophage model of LPS stimulation, we previously demonstrated that osteopontin (OPN) increased STAT1 ubiquitination and 26 S proteasome degradation via the ubiquitin E3 ligase, PDLIM2. In this study, we further characterize OPN-dependent activation of PDLIM2 in a model of LPS-stimulated RAW264.7 murine macrophages. We identify serine 137 as a protein kinase C-phosphorylation site in PDLIM2 that is required for ubiquitination of STAT1. PDLIM2 phosphorylation requires OPN expression. Using phospho-mutants and phospho-mimetic constructs of PDLIM2, our in vivo and in vitro ubiquitination studies confirm the role of PDLIM2 in formation and degradation of Ub-STAT1. The functional consequences of PDLIM2-mediated STAT1 degradation were confirmed using an IFN-γ-regulated transcription factor STAT1α reporter construct and chromatin immunoprecipitation assay for the inducible nitric-oxide synthase promoter. In a murine cecal ligation and puncture model of sepsis in wild-type and OPN (-/-) animals, OPN was necessary for PDLIM2 serine phosphorylation and STAT1 ubiquitination in bone marrow macrophages. We conclude that OPN and PDLIM2 are important regulators of STAT1-mediated inflammatory responses.

• Authors: Hongtao Guo, Zhiyong Mi, Dawn E Bowles, Syamal D Bhattacharya, Paul C Kuo
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 285 Issue 48 Pg. 37787-96 (Nov 26 2010) ISSN: 1083-351X [Electronic] United States
• PMID: 20889505 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Retracted Publication)

Chemical References

• Adaptor Proteins, Signal Transducing
• LIM Domain Proteins
• Lipopolysaccharides
• Pdlim2 protein, mouse
• STAT1 Transcription Factor
• Stat1 protein, mouse
• Osteopontin
• Ubiquitin-Protein Ligases
• Protein Kinase C

Topics

• Adaptor Proteins, Signal Transducing (genetics, metabolism)
• Animals
• Cell Line
• Cells, Cultured
• Enzyme Activation
• LIM Domain Proteins
• Lipopolysaccharides (metabolism)
• Macrophages (enzymology, metabolism)
• Male
• Mice
• Mice, Knockout
• Osteopontin (genetics, metabolism)
• Phosphorylation
• Protein Kinase C (genetics, metabolism)
• STAT1 Transcription Factor (genetics, metabolism)
• Ubiquitin-Protein Ligases (genetics, metabolism)
• Ubiquitination