Thrombin induces inducible nitric oxide synthase expression via the MAPK, MSK1, and NF-κB signaling pathways in alveolar macrophages

In this study, we investigated the roles of mitogen activated protein kinase (MAPK), mitogen stress-activated protein kinase 1 (MSK1), and nuclear factor-κB (NF-κB) signaling pathways in thrombin-induced inducible nitric oxide synthase (iNOS) expression in alveolar macrophages (NR8383). Treatment of NR8383 cells with thrombin caused an increase in iNOS expression in a concentration- and time-dependent manner. Treatment of NR8383 cells with SB203580 (4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole, a p38 MAPK inhibitor), PD98059 (2′-amino-3′-methoxyflavone, a MAPK kinase (MEK) inhibitor), and SP600125 (anthra[1-9-cd]pyrazol-6(2H)-one, a JNK inhibitor) all inhibited thrombin-induced iNOS expression. Stimulation of cells with thrombin caused an increase in p38 MAPK, ERK, and JNK phosphorylation. Treatment of cells with Ro 31-8220 (an MSK1 inhibitor) and MSK1 small interfering RNA (MSK1 siRNA) both inhibited thrombin-induced iNOS expression. Thrombin caused time-dependent activation of MSK1 Ser531 phosphorylation, which was inhibited by SB203580 and PD98059, but not by SP600125. Treatment of cells with pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) inhibited thrombin-induced iNOS expression in a concentration-dependent manner. Treatment of NR8383 cells with thrombin induced κB-luciferase activity and p65 Ser276 phosphorylation. Thrombin-induced increases in p65 Ser276 phosphorylation and κB-luciferase activity were inhibited by SB203580, PD98059, Ro 31-8220, and MSK1 siRNA. Taken together, these results suggest that the signaling pathways of MAPK, MSK1, and NF-κB play important roles in thrombin-induced iNOS expression in alveolar macrophages.