Dysregulated Cell Signaling in LPS‐stimulated Macrophages by Myeloid‐specific Conditional Knock Out of the Catalytic Subunit of Protein Phosphatase 2A

Activation of the innate immune response triggered by pathogen-associated molecular patterns forms the pathophysiologic basis for many of the acute, systemic inflammatory diseases such as sepsis and acute respiratory distress syndrome. While cell activation is mediated by protein kinases, protein phosphatases play counterbalancing roles. Previously we demonstrated that protein phosphatase 2A (PP2A) inhibited LPS-induced gene transcription of TNFα by negatively modulating JNK pathway. We also showed that PP2A negatively regulates TNFα mRNA stability in a manner dependent on p38. These studies were performed with incompletely selective pharmacologic inhibitors raising uncertainty as to the precise role of PP2A in cell signaling. To overcome these problems and to study PP2A regulation in vivo, we established conditional knock out PP2A by inserting loxP sites at both 5' and 3' ends of a 1.0-kb exon1 region on the genome of the catalytic subunit of PP2A (PP2ACα). After breeding with LysM-Cre mice (Jackson laboratory), a myeloid-specific gene deletion of PP2ACα strain (PP2ACαlox/lox;LyZ-Cre ) was established. While conditional knockout animals appeared phenotypically normal, bone marrow derived macrophages showed diminished PP2ACα expression. After LPS stimulation, TNFα secretion was dramatically increased. Western blot analysis showed highly upregulated phosphorylation of p38,p44/42,IKK/IκB and p65. Phospho-JNK/c-Jun was upregulated to a modest level. A gene array analysis is being conducted to identify the global changes of gene expression. Furthermore, we are also using this conditional knock out model to investigate the roles of PP2A in sepsis-induced endotoxin tolerance.