The inhibitory effect of raloxifene on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells is mediated through a ROS/p38 MAPK/CREB pathway to the up-regulation of heme oxygenase-1 independent of estrogen receptor.
In this study, we demonstrate that raloxifene, a selective estrogen receptor modulator, is a potent inducer of the anti-inflammatory enzyme heme oxygenase-1 (HO-1). In RAW264.7 macrophages, raloxifene induced HO-1 mRNA and protein expression. Pretreatment of ICI182780, an estrogen receptor (ER) antagonist or knock-down of endogenous ERα or ERβ gene by RNA interference failed to reverse raloxifene-mediated HO-1 induction, indicating an estrogen receptor-independent mechanism. Interestingly, the raloxifene-induced HO-1 expression was suppressed by reactive oxygen species (ROS) scavengers, including glutathione, TEMPO, Me(2)SO, 1,10-phenanthroline, or allopurinol. In addition, buthionine sulfoximine, an inhibitor of reduced glutathione synthesis, or Fe(2+)/Cu(2+) ions enhanced the positive effect of raloxifene on HO-1 expression. Consistent with these findings, raloxifene induced production of intracellular ROS and increased xanthine oxidase activity in vitro. Additional experiments revealed the involvement of mitogen-activated protein kinase (MAPK) kinase6 and p38 MAPK in the up-regulation of HO-1 by raloxifene and identified p38 MAPK as a downstream effector of ROS. Furthermore, the ROS-p38 MAPK cascade targeted the transcription factor cAMP-responsive element-binding protein (CREB). Finally, the functional significance of HO-1 induction was revealed by raloxifene-mediated inhibition of inducible nitric oxide synthase expression and nitric oxide production, a response reversed by the inhibition of HO-1 protein synthesis or blockade of p38 MAPK or xanthine oxidase activity. Therefore, identification of ROS-p38 MAPK-CREB-linked cascade as cellular relays in raloxifene-mediated HO-1 expression defines the signaling events that could participate in raloxifene-mediated anti-inflammatory response.
Lee
Sin-Ae
SA
Department of Biochemistry, College of Natural Sciences, Kangwon National University, 192-1 Hyoja-2-dong, Chuncheon 200-701, South Korea.
Kim
Eun-Ye
EY
Jeon
Woo-Kwang
WK
Woo
Chang-Hoon
CH
Choe
Jongseon
J
Han
Sanghwa
S
Kim
Byung-Chul
BC
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D051547:Heme Oxygenase-1 / Q000096:biosynthesis / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D020849:Raloxifene Hydrochloride / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D015854:Up-Regulation / Q000187:drug effects*; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D017362:Cyclic AMP Response Element-Binding Protein; C045076:Fenton's reagent; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D011960:Receptors, Estrogen; D020845:Selective Estrogen Receptor Modulators; D019820:Xanthine; D009569:Nitric Oxide; D020849:Raloxifene Hydrochloride; D006861:Hydrogen Peroxide; D007501:Iron; D052247:Nitric Oxide Synthase Type II; D051547:Heme Oxygenase-1; D014969:Xanthine Oxidase; D048051:p38 Mitogen-Activated Protein Kinases