Role of Phospholipase D2 in Nitric oxide synthesis through p70S6kinase-ERK1/2-STAT3 (Ser727) pathway of 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 expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) 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, we transfected 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. Moreover, LPS induced NO synthesis and expressions of iNOS and COX-2 were blocked by PLD2 siRNA, suggesting that LPS upregulates NO synthesis through PLD2. Next, we investigated the mTOR-p70S6kinase-ERK1/2-STAT3(Ser727) signaling pathway in LPS-induced NO induction mechanism. To examine the involvement of mTOR-p70S6kinase pathway, we used p70S6kinase inhibitor, rapamycin. Inhibition of p70S6kinase decreased NO synthesis as well as expressions of iNOS and COX-2. Also, we found that activation of ERK1/2-STAT3 (Ser727) is involved in LPS-induced mTOR-P70S6kinase pathway. Moreover, knockdown of PLD2 with siRNA also decreased phosphorylation of p70S6kinase, ERK1/2 and STAT3. Taken together, the present study suggests that NO synthesis is regulated by PLD2-dependent manner with involvement of mTOR pathway through activation of p70 S6kinase-ERK1/2-STAT3.