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

J Biol Chem. 2010 Nov 26;285(48):37787-96. doi: 10.1074/jbc.M110.161869. Epub 2010 Oct 1.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Retracted Publication

MeSH terms

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

Substances

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