Richard D. Ye

International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the Formyl Peptide Receptor (FPR) Family

Formyl peptide receptors (FPRs) are a small group of seven-transmembrane domain, G protein-coupled receptors that are expressed mainly by mammalian phagocytic leukocytes and are known to be important in host defense and inflammation. The three human FPRs (FPR1, FPR2/ALX, and FPR3) share significant sequence homology and are encoded by clustered genes. Collectively, these receptors bind an extraordinarily numerous and structurally diverse group of agonistic ligands, including N-formyl and nonformyl peptides of different composition, that chemoattract and activate phagocytes. N-formyl peptides, which are encoded in nature only by bacterial and mitochondrial genes and result from obligatory initiation of bacterial and mitochondrial protein synthesis with N-formylmethionine, is the only ligand class common to all three human receptors. Surprisingly, the endogenous anti-inflammatory peptide annexin 1 and its N-terminal fragments also bind human FPR1 and FPR2/ALX, and the anti-inflammatory eicosanoid lipoxin A4 is an agonist at FPR2/ALX. In comparison, fewer agonists have been identified for FPR3, the third member in this receptor family. Structural and functional studies of the FPRs have produced important information for understanding the general pharmacological principles governing all leukocyte chemoattractant receptors. This article aims to provide an overview of the discovery and pharmacological characterization of FPRs, to introduce an International Union of Basic and Clinical Pharmacology (IUPHAR)-recommended nomenclature, and to discuss unmet challenges, including the mechanisms used by these receptors to bind diverse ligands and mediate different biological functions.

A Stimulatory Role for cGMP-Dependent Protein Kinase in Platelet Activation

It is currently accepted that cGMP-dependent protein kinase (PKG) inhibits platelet activation. Here, we show that PKG plays an important stimulatory role in platelet activation. Expression of recombinant PKG in a reconstituted cell model enhanced von Willebrand factor (vWF)-induced activation of the platelet integrin alpha(IIb)beta(3). PKG knockout mice showed impaired platelet responses to vWF or low doses of thrombin and prolonged bleeding time. Human platelet aggregation induced by vWF or low-dose thrombin was inhibited by PKG inhibitors but enhanced by cGMP. Furthermore, a cGMP-enhancing agent, sildenafil, promoted vWF- or thrombin-induced platelet aggregation. The cGMP-stimulated platelet responses are biphasic, consisting of an initial transient stimulatory response that promotes platelet aggregation and a subsequent inhibitory response that limits the size of thrombi.

Lipopolysaccharide Stimulates Platelet Secretion and Potentiates Platelet Aggregation via TLR4/MyD88 and the cGMP-Dependent Protein Kinase Pathway

Bacterial LPS induces rapid thrombocytopenia, hypotension, and sepsis. Although growing evidence suggests that platelet activation plays a critical role in LPS-induced thrombocytopenia and tissue damage, the mechanism of LPS-mediated platelet activation is unclear. In this study, we show that LPS stimulates platelet secretion of dense and α granules as indicated by ATP release and P-selectin expression, and thus enhances platelet activation induced by low concentrations of platelet agonists. Platelets express components of the LPS receptor-signaling complex, including TLR (TLR4), CD14, MD2, and MyD88, and the effect of LPS on platelet activation was abolished by an anti-TLR4-blocking Ab or TLR4 knockout, suggesting that the effect of LPS on platelet aggregation requires the TLR4 pathway. Furthermore, LPS-potentiated thrombin- and collagen-induced platelet aggregation and FeCl3-induced thrombus formation were abolished in MyD88 knockout mice. LPS also induced cGMP elevation and the stimulatory effect of LPS on platelet aggregation was abolished by inhibitors of NO synthase and the cGMP-dependent protein kinase (PKG). LPS-induced cGMP elevation was inhibited by an anti-TLR4 Ab or by TLR4 deficiency, suggesting that activation of the cGMP/protein kinase G pathway by LPS involves the TLR4 pathway. Taken together, our data indicate that LPS stimulates platelet secretion and potentiates platelet aggregation through a TLR4/MyD88- and cGMP/PKG-dependent pathway.

Cutting Edge: TLR2 Is a Functional Receptor for Acute-Phase Serum Amyloid A

Induced secretion of acute-phase serum amyloid A (SAA) is a host response to danger signals and a clinical indication of inflammation. The biological functions of SAA in inflammation have not been fully defined, although recent reports indicate that SAA induces proinflammatory cytokine expression. We now show that TLR2 is a functional receptor for SAA. HeLa cells expressing TLR2 responded to SAA with potent activation of NF-κB, which was enhanced by TLR1 expression and blocked by the Toll/IL-1 receptor/resistance (TIR) deletion mutants of TLR1, TLR2, and TLR6. SAA stimulation led to increased phosphorylation of MAPKs and accelerated IκBα degradation in TLR2-HeLa cells, and results from a solid-phase binding assay showed SAA interaction with the ectodomain of TLR2. Selective reduction of SAA-induced gene expression was observed in tlr2−/− mouse macrophages compared with wild-type cells. These results suggest a potential role for SAA in inflammatory diseases through activation of TLR2.

Protein Kinase C-δ Regulates Thrombin-Induced ICAM-1 Gene Expression in Endothelial Cells via Activation of p38 Mitogen-Activated Protein Kinase

ABSTRACT The procoagulant thrombin promotes the adhesion of polymorphonuclear leukocytes to endothelial cells by a mechanism involving expression of intercellular adhesion molecule 1 (ICAM-1) via an NF-κB-dependent pathway. We now provide evidence that protein kinase C-δ (PKC-δ) and the p38 mitogen-activated protein (MAP) kinase pathway play a critical role in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. We observed the phosphorylation of PKC-δ and p38 MAP kinase within 1 min after thrombin challenge of human umbilical vein endothelial cells. Pretreatment of these cells with the PKC-δ inhibitor rottlerin prevented the thrombin-induced phosphorylation of p38 MAP kinase, suggesting that p38 MAP kinase signals downstream of PKC-δ. Inhibition of PKC-δ or p38 MAP kinase by pharmacological and genetic approaches markedly decreased the thrombin-induced NF-κB activity and resultant ICAM-1 expression. The effects of PKC-δ inhibition were secondary to inhibition of IKKβ activation and of subsequent NF-κB binding to the ICAM-1 promoter. The effects of p38 MAP kinase inhibition occurred downstream of IκBα degradation without affecting the DNA binding function of nuclear NF-κB. Thus, PKC-δ signals thrombin-induced ICAM-1 gene transcription by a dual mechanism involving activation of IKKβ, which mediates NF-κB binding to the ICAM-1 promoter, and p38 MAP kinase, which enhances transactivation potential of the bound NF-κB p65 (RelA).

Identification of Peptides That Antagonize Formyl Peptide Receptor-Like 1-Mediated Signaling

Formyl peptide receptor-like 1 (FPRL1) is an important classical chemoattractant receptor that is expressed in phagocytic cells in the peripheral blood and brain. Recently, various novel agonists have been identified from several origins, such as host-derived molecules. Activation of FPRL1 is closely related to inflammatory responses in the host defense mechanism and neurodegenerative disorders. In the present study we identified several novel peptides by screening hexapeptide libraries that inhibit the binding of one of FPRL1’s agonists (Trp-Lys-Tyr-Met-Val-d-Met-CONH2 (WKYMVm)) to its specific receptor, FPRL1, in RBL-2H3 cells. Among the novel peptides, Trp-Arg-Trp-Trp-Trp-Trp-CONH2 (WRWWWW (WRW4)) showed the most potent activity in terms of inhibiting WKYMVm binding to FPRL1. We also found that WRW4 inhibited the activation of FPRL1 by WKYMVm, resulting in the complete inhibition of the intracellular calcium increase, extracellular signal-regulated kinase activation, and chemotactic migration of cells toward WKYMVm. For the receptor specificity of WRW4 to the FPR family, we observed that WRW4 specifically inhibit the increase in intracellular calcium by the FPRL1 agonists MMK-1, amyloid β42 (Aβ42) peptide, and F peptide, but not by the FPR agonist, fMLF. To investigate the effect of WRW4 on endogenous FPRL1 ligand-induced cellular responses, we examined its effect on Aβ42 peptide in human neutrophils. Aβ42 peptide-induced superoxide generation and chemotactic migration of neutrophils were inhibited by WRW4, which also completely inhibited the internalization of Aβ42 peptide in human macrophages. WRW4 is the first specific FPRL1 antagonist and is expected to be useful in the study of FPRL1 signaling and in the development of drugs against FPRL1-related diseases.

Activation of NF-κB by bradykinin through a Gα(q)- and Gβγ-dependent pathway that involves phosphoinositide 3-kinase and Akt

Recent work has suggested a role for the serine/threonine kinase Akt and IκB kinases (IKKs) in nuclear factor (NF)-κB activation. In this study, the involvement of these components in NF-κB activation through a G protein-coupled pathway was examined using transfected HeLa cells that express the B2-type bradykinin (BK) receptor. The function of IKK2, and to a lesser extent, IKK1, was suggested by BK-induced activation of their kinase activities and by the ability of their dominant negative mutants to inhibit BK-induced NF-κB activation. BK-induced NF-κB activation and IKK2 activity were markedly inhibited by RGS3T, a regulator of G protein signaling that inhibits Gαq, and by two Gβγ scavengers. Co-expression of Gαq potentiated BK-induced NF-κB activation, whereas co-expression of either an activated Gαq(Q209L) or Gβ1γ2 induced IKK2 activity and NF-κB activation without BK stimulation. BK-induced NF-κB activation was partially blocked by LY294002 and by a dominant negative mutant of phosphoinositide 3-kinase (PI3K), suggesting that PI3K is a downstream effector of Gαq and Gβ1γ2 for NF-κB activation. Furthermore, BK could activate the PI3K downstream kinase Akt, whereas a catalytically inactive mutant of Akt inhibited BK-induced NF-κB activation. Taken together, these findings suggest that BK utilizes a signaling pathway that involves Gαq, Gβ1γ2, PI3K, Akt, and IKK for NF-κB activation.

Regulation of nuclear factor κB activation by G-protein-coupled receptors

Accumulating evidence indicates that G‐protein‐coupled receptors (GPCRs) play an active role in transcriptional regulation. In leukocytes, activation of receptors for several chemokines and classic chemoattractants has been associated with enhanced expression of proinflammatory cytokines and chemokines. GPCRs in endothelial and epithelial cells also regulate transcription and contribute to the expression of cytokines, adhesion molecules, and growth factors that are essential for extravasation of leukocytes and tissue repair. Nuclear factor (NF) κB is one of the most important transcription factors responsible for the expression of these proinflammatory genes. Recent studies have shown that GPCRs utilize several different pathways to activate NF‐κB. These pathways differ from the ones induced by classic cytokines in that they are initiated by heterotrimeric G‐proteins, but they converge to IκB phosphorylation and nuclear translocation/modification of the NF‐κB proteins. GPCR‐induced NF‐κB activation provides an effective means for local expression of cytokine and growth factor genes due to the wide distribution of these receptors. Chemokine‐induced, GPCR‐mediated production of chemokines constitutes an autocrine regulatory mechanism for the growth of certain malignant tumors and enhances the recruitment of leukocytes to sites of inflammation.

Gαq and Gβγ Regulate PAR-1 Signaling of Thrombin-Induced NF-κB Activation and ICAM-1 Transcription in Endothelial Cells

As thrombin binding to the G protein-coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, Galpha(q), and the Gbetagamma dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits Galpha(q), or alpha-transducin (Galpha(t)), a scavenger of the Gbetagamma, markedly decreased NF-kappaB activity induced by PAR-1 activation. We determined the downstream signaling targets activated by Galpha(q) and Gbetagamma that mediate NF-kappaB activation. Expression of the kinase-defective protein kinase C (PKC)-delta mutant inhibited NF-kappaB activation induced by the constitutively active Galpha(q) mutant, but had no effect on NF-kappaB activity induced by Gbeta(1)gamma(2). In related experiments, NF-kappaB as well as ICAM-1 promoter activation induced by Gbeta(1)gamma(2) were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated Galpha(q). Cotransfection of the catalytically inactive Akt mutant inhibited the NF-kappaB activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of Galpha(q) and PKC-delta. These results support a model in which ligation of PAR-1 induces NF-kappaB activation and ICAM-1 transcription by the engagement of parallel Galphaq/PKC-delta and Gbetagamma/PI3-kinase pathways that converge at Akt.

The redox-sensitive cation channel TRPM2 modulates phagocyte ROS production and inflammation

The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a nonselective and redox-sensitive cation channel, inhibited ROS production in phagocytic cells and prevented endotoxin-induced lung inflammation in mice. TRPM2-deficient mice challenged with endotoxin (lipopolysaccharide) had an enhanced inflammatory response and diminished survival relative to that of wild-type mice challenged with endotoxin. TRPM2 functioned by dampening NADPH oxidase–mediated ROS production through depolarization of the plasma membrane in phagocytes. As ROS also activate TRPM2, our findings establish a negative feedback mechanism for the inactivation of ROS production through inhibition of the membrane potential–sensitive NADPH oxidase.