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

초록

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 inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expressions in Raw 264.7 cells. We found that the expressions of PLDs were increased when we stimulated Raw 264.7 cells with LPS. By blocking of PLD activity using 1-butanol, NO synthesis and expressions of iNOS and COX-2 were decreased. To confirm the role of PLD in NO synthesis in macrophages, we transfected by PLD1 and PLD2, and their dominant negative forms, repectively. Interestingly, we found that only PLD2 overexpression, not PLD1, increased NO synthesis and expressions of iNOS and COX-2. Overexpression of dominant negative form of PLD2 (DN-PLD2) completely blocked LPS-induced NO production. While DN-PLD1 did not effect on LPS-induced NO synthesis. Moreover, LPS induced NO synthesis and expression of iNOS and COX-2 blocked by transfection of PLD2 siRNA, suggesting that LPS upregulate NO synthesis through PLD2. Furthermore, We monitored the phosphorylation of two downstream effectors of mTOR (p70 S6kinase and 4EBP1) in response to stimulation by LPS. To investigate the involvement of mTOR pathway in LPS-induced NO synthesis, we used mTOR inhibitor, rapamycin. Pretreatment with rapamycin decreased NO synthesis and expression of iNOS and COX-2, also phosphorylation of p70 S6kinase and 4EBP1 were inhibited. We have further explored the effects of 1-butanol and PLD2 siRNA on PLD/mTOR signaling and have obtained that phosphorylation of p70 S6kinase was decreased but 4EBP was not decreased. Taken together, the present study suggest that NO synthesis is regulated by PLD-dependent manner with involvement of mTOR pathway, and among PLD isozymes, PLD2 acts as an important regulator in NO production signal in Raw 264.7 cells.