Osteopontin and protein kinase C regulate PDLIM2 activation and STAT1 ubiquitination in LPS-treated murine macrophages.
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.
Guo
Hongtao
H
Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.
Mi
Zhiyong
Z
Bowles
Dawn E
DE
Bhattacharya
Syamal D
SD
Kuo
Paul C
PC
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004789:Enzyme Activation; D060588:LIM Domain Proteins; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D048868:Adaptor Proteins, Signal Transducing; D060588:LIM Domain Proteins; D008070:Lipopolysaccharides; C508508:Pdlim2 protein, mouse; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D053495:Osteopontin; D044767:Ubiquitin-Protein Ligases; D011493:Protein Kinase C