The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-α–induced priming of the NADPH oxidase in human neutrophils

Abstract

Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-␣ (TNF-␣), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-␣ primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-␣-induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-␣. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-␣-induced priming of neutrophil ROS production induced by N-formylmethionyl-leucyl-phenylalanine peptide (fMLF). TNF-␣ enhanced fMLF-induced IntroductionNeutrophils play an important role in host defense against invading pathogens and in inflammation. In response to stimulating agents, such as the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF), neutrophils release large amounts of superoxide anions and other reactive oxygen species (ROS) in a phenomenon called the respiratory burst. ROS produced by the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play a key role in host defenses, 1-3 but excessive ROS production can damage healthy bystander tissues, thereby contributing to inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel diseases, and acute respiratory distress syndrome. 4,5 Neutrophil ROS production is mediated by the phagocyte NADPH oxidase, also called NOX2. NADPH oxidase is a multicomponent enzyme system that catalyzes NADPH-dependent reduction of oxygen to superoxide anion. 6,7 In resting cells, the NADPH oxidase is inactive and its components are distributed between the cytosol and membranes. When cells are activated, the cytosolic components (p47phox, p67phox, p40phox, and Rac2) migrate to the membranes, where they associate with the membranebound components (p22phox and gp91phox/NOX2, which form the flavocytochrome b558) to assemble the catalytically active oxidase. 7,8 During NADPH oxidase activation, p47phox, p67phox, p40phox, p22phox, and gp91phox/NOX2 become phosphorylated. 9-13 p47phox phosphorylation on several serines plays a pivotal role in oxidase activation in intact cells. 14,15 Neutrophil ROS production is enhanced or primed by a variety of mediators, including proinflammatory cytokines, such as tumor necrosis factor-␣ (TNF-␣). In vitro, TNF-␣ induces a very weak oxidative response by neutrophils but strongly enhances ROS release on exposure to a secondary stimulus, such as the bacterial peptide fMLF. [16][17][18] This "priming" of neutrophil ROS production plays a detrimental role in a variety of human inflammatory diseases, in which ROS hyperp...

Pin1 Protein Regulates Smad Protein Signaling and Pulmonary Fibrosis

“…S12). These data suggest that in cells, Smad6 phosphorylation on one or more of the four Ser-Pro sites enhanced the interaction with Pin1 (30). Despite these data, using generic anti-phosphoantibodies, we were unable to show increased Smad6 Ser (supplemental Fig.…”

Cooperation of p40 with p47 for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis

“…In unstimulated phagocytes, the oxidase is dissociated and inactive; the flavocytochrome b 558 is stored on the membranes of intracellular granules (4), and the other Phox proteins associate in a separate cytoplasmic ternary complex (p47 phox -p67 phox -p40 phox ) (5) in a dephosphorylated state (6 -8). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse to newly forming phago-somes, and the cytoplasmic ternary complex binds to these membranes; p47 phox is phosphorylated (9,10), thereby inducing conformational changes that promote interaction of the ternary complex with p22 phox (11), and p40 phox also undergoes conformational changes by disruption of the intramolecular PX-PB1 domain interaction to enable the ternary complex to bind through the p40 phox PX domain to PI(3)P (12,13), which is enriched in phagosomes (14 -16).…”

The TLR7/8 Agonist CL097 Primes N-Formyl-Methionyl-Leucyl-Phenylalanine–Stimulated NADPH Oxidase Activation in Human Neutrophils: Critical Role of p47phox Phosphorylation and the Proline Isomerase Pin1

“…Circulating neutrophils were isolated from healthy volunteers by Polymorphprep (Axis Shield, Dundee, Scotland) gradient centrifugation (26,27). The neutrophil band was collected, and the cells were washed in PBS and counted.…”

“…Polyclonal anti-TLR7 and anti-TLR8 Abs were from Abcam (Cambridge, U.K.). The rabbit polyclonal Abs against phospho-sites p47phox and p47phox have been described elsewhere (27).…”

“…This priming effect at the infection site could facilitate the rapid elimination of the pathogen; however, excessive and uncontrolled priming could participate to the inflammatory reaction and thus be self-destructive (22,23). The priming effect has been shown to be controlled by the partial phosphorylation of p47phox (24)(25)(26), the activation of the proline isomerase 1 (Pin1) (27), and the translocation of the flavocytochrome b588 to the plasma membranes (28,29).…”