Contribution of MKP-1 regulation of p38 to endotoxin tolerance.
Endotoxin tolerance has been characterized as diminished TNF-alpha expression after a second LPS stimulus and is dependent on new protein synthesis. LPS-induced expression of TNF-alpha is partly regulated by the p38 mitogen-activated protein (MAP) kinase, which post-transcriptionally stabilizes TNF-alpha mRNA. The dual-specific phosphatase, MKP-1, has been shown to negatively regulate p38 via dephosphorylation. We hypothesized that MKP-1 expression induced during tolerance regulates TNF-alpha expression by inhibiting p38 activity. To test this hypothesis, tolerance was induced in THP-1 cells, and naive or tolerized cells were rechallenged 18 h later with LPS (1 microg/mL) and TNF-alpha production was measured. Under similar conditions, nuclear proteins were isolated after LPS stimulation and were analyzed for phospho-p38 and MKP-1 by Western blot. Transient overexpression of MKP-1 was achieved using an adenoviral expression strategy and infected cells subsequently treated with LPS for TNF-alpha production and p38 activation. Results showed that LPS tolerance was induced as reflected by decreased TNF-alpha. Induction of LPS hyporesponsiveness could be mimicked by overexpression of MKP-1 but not beta-gal. MKP-1 expression was noted only in LPS-tolerized or Ad-MKP-1 infected cells. In the canonical and Ad-MKP-1-mediated tolerance models, decreased phospho-p38 activity was observed. MKP-1s role in mediating endotoxin tolerance was further confirmed by demonstrating the inability to fully tolerize peritoneal macrophages isolated from MKP-1 null mutant (vs. wild type) mice (24% vs. 72% reductions, respectively). These data demonstrate that the dual specific phosphatase MKP-1 is an important mediator of endotoxin tolerance via p38 regulation.
Nimah
Marianne
M
Division of Critical Care Medicine, Cincinnati Children's Hospital Research Foundation, University of Cincinnati Medical School, Cincinnati, Ohio 45229-3039, USA.
Zhao
Bin
B
Denenberg
Alvin G
AG
Bueno
Orlando
O
Molkentin
Jeffery
J
Wong
Hector R
HR
Shanley
Thomas P
TP
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D054638:Dual Specificity Phosphatase 1; D004731:Endotoxins / Q000378:metabolism*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism / Q000502:physiology*; D047468:Immunoprecipitation; D008070:Lipopolysaccharides / Q000378:metabolism; D008264:Macrophages / Q000166:cytology; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D010749:Phosphoprotein Phosphatases / Q000378:metabolism / Q000502:physiology*; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism / Q000502:physiology*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004731:Endotoxins; D017874:Immediate-Early Proteins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; C517020:Dusp1 protein, mouse; D017027:Protein Tyrosine Phosphatases