The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-alpha-induced priming of the NADPH oxidase in human neutrophils - PubMed (original) (raw)

. 2010 Dec 23;116(26):5795-802.

doi: 10.1182/blood-2010-03-273094. Epub 2010 Oct 18.

Marie-Anne Gougerot-Pocidalo, Gilles Hayem, Silvia Ciappelloni, Houssam Raad, Riad Arabi Derkawi, Odile Bournier, Yolande Kroviarski, Xiao Zhen Zhou, James S Malter, Ping K Lu, Aghleb Bartegi, Pham My-Chan Dang, Jamel El-Benna

Affiliations

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

Tarek Boussetta et al. Blood. 2010.

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-α (TNF-α), 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-α primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-α-induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-α. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-α-induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-α enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-α-induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy.

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Figures

Figure 1

Figure 1

Pin1 is expressed in human neutrophils and is activated by TNF-α and fMLF. (A) Recombinant Pin1 (0.5 μg) and a resting human neutrophil (PMN) lysate (2 × 106 cells) were analyzed by SDS-PAGE and Western blot with an anti-Pin1 antibody. (B) Resting human neutrophils were lysed by nitrogen cavitation, and fractions were isolated on a Percoll gradient. Proteins were analyzed by SDS-PAGE and Western blot with an anti-Pin1 antibody. IB indicates immunoblot. (C) Recombinant Pin1 (0.25 nmol) was used to measure activity by recording the absorbance (at 390 nm) of free p_-nitroaniline (pNA) cleaved by trypsin from Trp-Phe-Tyr-Ser(PO3H2)-Pro-Arg-pNA in the absence and presence of juglone (250nM). (D) Neutrophils were incubated in the absence and presence of juglone (250nM for 30 minutes) and then treated with TNF-α (20 ng/mL), fMLF (10−7M), or TNF-α, followed by fMLF (TNF-α + fMLF), before lysis. Pin1 activity was determined by measuring the absorbance of free pNA cleaved from Trp-Phe-Tyr-Ser(PO3H2)-Pro-Arg-pNA. Data are mean plus or minus SEM of 6 experiments. *P < .01 compared with resting neutrophils. §_P < .01 TNF-α + fMLF compared with fMLF. #P < .001 with versus without juglone.

Figure 2

Figure 2

Pin1 is required for TNF-α–induced priming of ROS production. (A) Neutrophils were incubated in Hanks buffer containing juglone (250nM) for 30 minutes; then TNF-α (20 ng/mL) was added for 20 minutes before stimulation with fMLF (10−7M). ROS production was measured with a luminol-amplified chemiluminescence technique. (B) Total chemiluminescence in each experimental condition is expressed as mean plus or minus SEM of 6 experiments. (C) PPIn (50 μM) was tested in the same conditions as juglone. (D) Data are mean plus or minus SEM of 6 experiments. (E) Neutrophils were incubated with juglone or PPIn and then stimulated with PMA (100 ng/mL) before measuring ROS production with a luminol-amplified chemiluminescence technique (one experiment representative of 3). (F) Total chemiluminescence in each experimental condition is expressed as mean plus or minus SEM of 3 experiments. *P < .01 compared with inhibitor-free conditions.

Figure 3

Figure 3

TNF-α enhances fMLF-induced Pin1 and p47phox translocation to the membranes in human neutrophils and the effect of juglone. (A) Neutrophils were treated with TNF-α (20 ng/mL), fMLF (10−7M), or TNF-α followed by fMLF (TNF-α + fMLF) and then lysed by nitrogen cavitation. Membranes and cytosols were separated by ultracentrifugation on a sucrose gradient. Proteins were analyzed by SDS-PAGE and Western blotting with anti-Pin1, anti-p47phox, and anti-pSer345 antibodies. (B) Neutrophils were treated with TNF-α, fMLF, or TNF-α + fMLF in the absence or presence of 250nM juglone and then lysed. Membranes were separated by ultracentrifugation on a sucrose gradient, and proteins were analyzed by SDS-PAGE and immunoblotting (IB). (representative of 7 experiments).

Figure 4

Figure 4

Pin1 interacts with p47phox via phosphorylated Ser345. (A) Neutrophils were treated with TNF-α alone, fMLF alone, or TNF-α followed by fMLF (TNF-α + fMLF), and then lysed and incubated with GST-Pin1 in the presence of glutathione beads. The beads were then washed, Pin1 was released by thrombin cleavage, and proteins were analyzed by SDS-PAGE and Western blot (IB). (B) Recombinant p47phox was phosphorylated with p38MAPK and then repurified and incubated with GST-Pin1 and glutathione-agarose beads. The beads were washed 3 times, and proteins were analyzed by SDS-PAGE and immunoblotting (IB). (C) p47phox peptides containing phosphorylated or nonphosphorylated Ser345 were coupled to ovalbumin, spotted on nitrocellulose membranes, and overlaid with recombinant Pin1. Pin1 was detected with a specific antibody. Experiments are representative of 3.

Figure 5

Figure 5

Pin1 induces conformational changes of p47phox via binding to phosphorylated Ser345. (A) p47phox was phosphorylated with p38MAPK, incubated with Pin1 in the presence or absence of juglone, and subjected to trypsin cleavage; peptides were analyzed with Tris-tricine gels and immunoblotting with an antibody directed against the whole recombinant p47phox protein. (B) Gels were scanned and peptides were quantified with Scion image Beta 4.03 for Windows 95 to XP software from the National Institutes of Health. Experiments are representative of 3.

Figure 6

Figure 6

Pin1 facilitates phosphorylation of p47phox by PKC, both in vitro and in intact neutrophils. (A) p47phox was phosphorylated with p38MAPK and then incubated with or without Pin1 preincubated with or without juglone. Where indicated, PKC was added in the presence of 32P-γ-ATP. Proteins were analyzed by SDS-PAGE, autoradiography, and immunoblotting (IB). (B) Phosphorylated p47phox peptides containing phospho-Ser315 (p-Ser315), p-Ser320, p-Ser328, or p-Ser345 were subjected to 16% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and revealed with anti p-Ser315, p-Ser320, p-Ser328, or p-Ser345 antibodies. (C) Neutrophils were incubated with or without GF109203X (GFX) (5μM) for 15 minutes, stimulated with PMA, and proteins were analyzed with SDS-PAGE and Western blotting using anti–phospho-Ser315, anti–phospho-Ser320, anti–phospho-Ser328, and anti-p47phox antibodies. (D) Neutrophils were treated with TNF-α and fMLF, alone or together, in the absence or presence of juglone. Neutrophils were then lyzed, and proteins were analyzed with SDS-PAGE and Western blotting with anti–phospho-Ser315, anti–phospho-Ser320, anti–phospho-Ser328, anti–phospho-Ser345, and anti-p47phox antibodies. All experiments are representative of 3.

Figure 7

Figure 7

Models of the different conformations of p47phox in resting, primed, and activated neutrophils: role of Pin1 and phosphorylation. In resting cells, p47phox is not phosphorylated and has a constrained conformation because of the tight interaction between SH3 domains and the autoinhibitory region (AIR). (1) During priming, p47phox is first phosphorylated by a MAPKinase (ERK1/2 or p38MAPK) on Ser345, and (2) activated Pin1 then binds to this site, (3) inducing the first conformational changes that allow PKC isoforms to phosphorylate p47phox on other sites during activation. (4) Phosphorylation of p47phox on several sites at its C-terminal tail prevents the SH3/AIR interaction, allowing the cryptic SH3 domains to bind to the proline-rich region (PRR) of p22phox (5) and NADPH oxidase hyperactivation.

Comment in

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