Phospholipase D2 acts as an important regulator in Nitric oxide synthesis in Raw 264.7 cells

Abstract

The purpose of this study was to identify the role of phospholipase D2 (PLD2) in lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis. LPS enhanced NO synthesis and expressions of inducible nitric oxide synthase (iNOS) in macrophage cell line, Raw 264.7 cells. We found that the expressions of PLDs were increased when we stimulated Raw 264.7 cells with LPS. To confirm the role of PLD in NO synthesis, we transfected each of PLD1, PLD2, and their dominant negative forms to Raw 264.7 cells, respectively. Interestingly, we found that only PLD2 overexpression, not PLD1, increased NO synthesis and iNOS expression. Moreover, LPS-induced NO synthesis and iNOS expression were blocked by PLD2 siRNA, suggesting that LPS upregulates iNOS expression and NO synthesis through PLD2. Next, we investigated the p70S6K1-ERK1/2-STAT3 signaling pathway in LPS-induced NO synthesis mechanism. To examine the involvement of p70S6K1 pathway, we used p70S6 kinase inhibtor, rapamycin. Inhibition of p70S6 kinase decreased NO synthesis as well as iNOS expression. Also, we found that activation of ERK1/2-STAT3 is involved in LPS-induced p70S6 kinase pathway. Moreover, Knockdown of PLD2 with siRNA also decreased phosphorylation of p70S6 kinase, ERK1/2 and STAT3. Taken together, the present study suggests that NO synthesis is regulated by PLD2-dependent manner with involvement of p70S6kinase- ERK1/2-STAT3 pathway in Raw 264.7 cells.