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-CONH(2) (WKYMVm)) to its specific receptor, FPRL1, in RBL-2H3 cells. Among the novel peptides, Trp-Arg-Trp-Trp-Trp-Trp-CONH(2) (WRWWWW (WRW(4))) showed the most potent activity in terms of inhibiting WKYMVm binding to FPRL1. We also found that WRW(4) 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 WRW(4) to the FPR family, we observed that WRW(4) specifically inhibit the increase in intracellular calcium by the FPRL1 agonists MMK-1, amyloid beta42 (Abeta42) peptide, and F peptide, but not by the FPR agonist, fMLF. To investigate the effect of WRW(4) on endogenous FPRL1 ligand-induced cellular responses, we examined its effect on Abeta42 peptide in human neutrophils. Abeta42 peptide-induced superoxide generation and chemotactic migration of neutrophils were inhibited by WRW(4), which also completely inhibited the internalization of Abeta42 peptide in human macrophages. WRW(4) 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.     

The neuropeptide pituitary adenylate cyclase activating polypeptide modulates Ca2+ and pro-inflammatory functions in human monocytes through the G protein-coupled receptors VPAC-1 and formyl peptide receptor-like 1

In human neutrophils, the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) acting via the G protein-coupled receptors vasoactive intestinal peptide/PACAP receptor 1 (VPAC-1) and formyl peptide receptor-like 1 (FPRL1) modulates Ca2+ and pro-inflammatory activities. We evaluated in human monocytes the importance of the Ca2+ signal and the participation of FPRL1 in PACAP-associated signaling pathways and pro-inflammatory activities. PACAP-evoked Ca2+ transient involved both Ca2+ influx and intracytoplasmic Ca2+ mobilisation. This was pertussis toxin, protein kinase A and adenylate cyclase dependent indicating the participation of Galphai and Galphas with mobilisation of both InsP3 sensitive and insensitive stores. Intra- or extracellular Ca2+ depletion resulted in the inhibition of PACAP-induced, Akt, ERK, p38 and NF-kappaB activations as well as a decrease in PACAP-associated reactive oxygen species (ROS) production and integrin CD11b membrane upregulation. The FPRL1 antagonist, Trp-Arg-Trp-Trp-Trp (WRW4), decreased PACAP-evoked Ca2+ signal, Akt, ERK phosphorylation, ROS and CD11b upregulation without affecting p38 phosphorylation. NF-kappaB inhibitors prevented PACAP-induced Ca2+ mobilisation. Monocytes pre-treatment with fMLP but not with LPS desensitised cells to the pro-inflammatory effects of PACAP. Thus, both intra- and extracellular Ca2+ play a role in controlling pro-inflammatory functions stimulated by PACAP which acts through a VPAC-1, FPRL1/Galphai/PI3K/ERK pathway and a VPAC-1/Galphas/PKA/p38 pathway to fully activate monocytes.     

Trp-Arg-Trp-Trp-Trp-Trp antagonizes formyl peptide receptor like 2-mediated signaling

Although formyl peptide receptor like 2 (FPRL2) has been regarded as an important classical chemoattractant receptor, its functional role and signaling pathway have not been fully investigated, because of the lack of its specific ligand. Recently F2L, a heme-binding protein fragment peptide, has been reported as an FPRL2-selective endogenous agonist. In the present study, we examined the effect of Trp-Arg-Trp-Trp-Trp-Trp-CONH2 (WRWWWW, WRW4), on F2L-induced cell signaling. WRW4 inhibited the activation of FPRL2 by F2L, resulting in the complete inhibition of intracellular calcium increase and chemotactic migration induced by F2L. WRW4 also completely inhibited F2L-induced NF-kappaB activation in FPRL2-transfected HEK293 cells. WRW4 specifically inhibited F2L-induced intracellular calcium increase and chemotactic migration in mature monocyte-derived dendritic cells, which express FPRL2 but not the other FPR family. Taken together, WRW4 is the first FPRL2 antagonist and is expected to be useful in the study of FPRL2 signaling and in development of drugs against FPRL2-related cellular responses.     

Differential calcium regulation of proinflammatory activities in human neutrophils exposed to the neuropeptide pituitary adenylate cyclase-activating protein

The neuropeptide pituitary adenylate cyclase-activating protein (PACAP) acts via the G protein-coupled receptor vasoactive intestinal peptide/PACAP receptor-1 to induce phospholipase C/calcium and MAPK-dependent proinflammatory activities in human polymorphonuclear neutrophils (PMNs). In this study, we evaluate other mechanisms that regulate PACAP-evoked calcium transients, the nature of the calcium sources, and the role of calcium in proinflammatory activities. Reduction in the activity of PMNs to respond to PACAP was observed after cell exposure to inhibitors of the cAMP/protein kinase A, protein kinase C, and PI3K pathways, to pertussis toxin, genistein, and after chelation of intracellular calcium or after extracellular calcium depletion. Mobilization of intracellular calcium stores was based on the fact that PACAP-associated calcium transient was decreased after exposure to 1) thapsigargin, 2) Xestospongin C, and 3) the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenyl hydrazone; inhibition of calcium increase by calcium channel blockers, by nifedipine and verapamil, indicated that PACAP was also acting on calcium influx. Such mobilization was not dependent on a functional actin cytoskeleton. Homologous desensitization with nanomoles of PACAP concentration and heterologous receptors desensibilization by G protein-coupled receptor agonists were observed. Intracellular calcium depletion modulated PACAP-associated ERK but not p38 phosphorylation; in contrast, extracellular calcium depletion modulated PACAP-associated p38 but not ERK phosphorylation. In PACAP-treated PMNs, reactive oxygen species production and CD11b membrane up-regulation in contrast to lactoferrin release were dependent on both intra- and extracellular calcium, whereas matrix metalloproteinase-9 release was unaffected by extracellular calcium depletion. These data indicate that both extracellular and intracellular calcium play key roles in PACAP proinflammatory activities.     

Regulation of human polymorphonuclear leukocytes functions by the neuropeptide pituitary adenylate cyclase-activating polypeptide after activation of MAPKs

Anti-inflammatory activities of pituitary adenylate cyclase-activating protein (PACAP) are mediated in part through specific effects on lymphocytes and macrophages. This study shows that in human polymorphonuclear neutrophils (PMNs), PACAP acts as a proinflammatory molecule. In PMNs, vaso-intestinal peptide/PACAP receptor 1 (VPAC-1) was the only receptor found to be expressed by RT-PCR. Using VPAC-1 Ab, we found that VPAC-1 mRNA was translated into proteins. In PMNs, PACAP increases cAMP, inositol triphosphate metabolites, and calcium. It activates two of the three members of the MAPK superfamily, the ERK and the stress-activated MAPK p38. U73122, an inhibitor of phospholipase C (PLC), inhibits PACAP-induced ERK activation, whereas p38 MAPK phosphorylation was unaffected. Using specific pharmalogical inhibitors of ERK (PD098059) and p38 MAPK (SB203580), we found that PACAP-mediated calcium increase was ERK and PLC dependent and p38 independent. PACAP primes fMLP-associated calcium increase; it also primes fMLP activation of the respiratory burst as well as elastase release, these last two processes being ERK and PLC dependent and p38 MAPK independent. PACAP also increases membrane expression of CD11b and release of lactoferrin and metallo proteinase-9 (MMP-9). These effects were PLC dependent (CD 11b, lactoferrin, MMP-9), ERK dependent (CD 11b, lactoferrin, MMP-9), and p38 dependent (CD11b, lactoferrin). We conclude that PACAP is a direct PMN activator as well as an effective PMN priming agent that requires PLC, ERK, and p38 MAPK activities.     

Mechanism of the protective effect of intraperitoneally administered agonists for formyl peptide receptors against chemotherapy-induced alopecia

Previously, we found that an intraperitoneally administered chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), and MMK-1, a selective agonist of formyl peptide receptor-like 1 (FPRL1) receptor, the low affinity subtype of the fMLP receptor, prevented the alopecia in neonatal rats induced by the anticancer agent etoposide. The anti-alopecia effect of fMLP was not inhibited at all by Boc-FLFLF, a selective antagonist of formylpeptide receptor (FPR), which is the high affinity subtype of the fMLP receptor, but it was partly inhibited by Trp-Arg-Trp-Trp-Trp-Trp-NH(2) (WRW(4)), an antagonist of FPRL1 receptor. On the other hand, the anti-alopecia effect of MMK-1 was completely abolished by WRW(4). The anti-alopecia effects of fMLP and MMK-1 were also inhibited by Lys-D-Pro-Thr (K(D)PT) and pyrrolidine dithiocarbamate, which are inhibitors of interleukin-1 (IL-1) and nuclear factor-kappaB (NF-kappaB) respectively. Hence, we suggest that the anti-alopecia mechanisms of intraperitoneally administered fMLP and MMK-1 include activation of NF-kappaB via IL-1 release downstream of the FPRL1 receptor homolog in rats.     

Differential activation of formyl peptide receptor-like 1 by peptide ligands

Formyl peptide receptor-like 1 (FPRL1) plays a key role in the regulation of immune responses. The activation of FPRL1 induces a complicated pattern of cellular signaling, which results in the regulation of several immune responses, such as chemotactic migration and the production of reactive oxygen species (ROS). Because some of these cellular responses are not beneficial to the host, ligands that selectively modulate these cellular responses are useful. His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that binds to FPRL1. In this study, we generated various HFYLPM analogues and examined their effects on cellular responses via FPRL1 in FPRL1-expressing rat basophilic leukemia-2H3 cells or in primary human neutrophils. Among the HXYLPM analogues, His-Arg-Tyr-Leu-Pro-Met (HRYLPM) activated a broad spectrum of cellular signaling events, including an intracellular Ca(2+) concentration increase, phosphoinositide 3-kinase, extracellular signal-regulated kinase, and Akt activation, however, His-Glu-Tyr-Leu-Pro-Met (HEYLPM) activated only intracellular Ca(2+) concentration and Akt but did not increase Ca(2+). In addition, HRYLPM was found to stimulate chemotaxis and ROS generation via phosphoinositide 3-kinase and an intracellular Ca(2+) concentration increase, respectively, whereas HEYLPM stimulated chemotaxis but not ROS generation. With respect to the molecular mechanisms involved in the differential action of HRYLPM and HEYLPM, we found that HRYLPM but not HEYLPM competitively inhibited the binding of (125)I-labeled Trp-Lys-Tyr-Met-Val-D-Met-NH(2) (WKYMVm, a FPRL1 ligand) to FPRL1. This study demonstrates that the important chemoattractant receptor, FPRL1, may be differentially modulated by distinct peptide ligands. We also suggest that HRYLPM and HEYLPM may be used to selectively modulate FPRL1.     

The neuropeptide pituitary adenylate cyclase activating protein stimulates human monocytes by transactivation of the Trk/NGF pathway

Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.     

The role of beta-arrestins in the formyl peptide receptor-like 1 internalization and signaling

The N-formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor (GPCR) that transmits intracellular signals in response to a variety of agonists, many of them being clearly implicated in human pathology. beta-arrestins are adaptor proteins that uncouple GPCRs from G protein and regulate receptor internalization. They can also function as signal transducers through the scaffolding of signaling molecules, such as components of the extracellular signal-regulated kinase (ERK) cascade. We investigated the role of beta-arrestins in ligand-induced FPRL1 internalization and signaling. In HEK293 cells expressing FPRL1, fluorescence microscopy revealed that agonist-stimulated FPRL1 remained co-localized with beta-arrestins during endocytosis. Internalization of FPRL1, expressed in a mouse embryonic fibroblast (MEF) cell line lacking endogenous beta-arrestins, was highly compromised. This distinguishes FPRL1 from the prototypical formyl peptide receptor FPR that is efficiently internalized in the absence of beta-arrestins. In both HEK293 and MEF cells, FPRL1-mediated ERK1/2 activation was a rapid and transient event. The kinetics and extent of ERK1/2 activation were not significantly modified by beta-arrestin overexpression. The pattern of FPRL1-mediated ERK1/2 activation was similar whether cells express or not beta-arrestins. Furthermore, treatment of the FPRL1 expressing cells with pertussis toxin inhibited ERK1/2 activation in MEF and in HEK293 cells. These results led us to conclude that activation of ERK1/2 mediated by FPRL1 occurs primarily through G protein signaling. Since beta-arrestin-mediated signaling has been observed essentially for receptors coupled to G proteins other than G(i), this may be a characteristic of G(i) protein-coupled chemoattractant receptors.     

Involvement of Phospholipase D 1 and 2 in the subcellular localization and activity of formyl-peptide-receptors in the human colonic cell line HT29

Epithelial cells of the alimentary tract play a central role in the mucosal host defence against pathogens and in the recognition of agonists that interact with mucosal surfaces. In particular, the formyl peptide receptor (FPR) family and their three human subtypes: FPR, formyl-peptide-receptor-like-1 (FPRL1) and FPRL2, are involved in the host defence against pathogens that mediate epithelial responses thus upregulating inflammation. To elucidate the mechanisms by which FPR function, we examined the influence of phospholipase D (PLD) 1 and 2 on the activity and signal transduction of human enterocytes cell line HT29. PLD is a key enzyme involved in secretion, endocytosis and receptor signalling. We inhibited PLD1 and 2 by small interference RNA (siRNA) and determined the activity of formyl peptide receptors using Western blotting and cAMP level measurements. We then analyzed the distribution of formyl peptide receptors FPR, FPRL1 and FPRL2 compared to a control. In this study, we demonstrated that the depletion of PLD1 and 2 resulted in a marked reduction of formyl peptide receptor activity due to inhibited extracellular-signal regulated kinases 1/2 (ERK1/2), phosphorylation and cAMP level reduction. In addition, we observed an intracellular accumulation of FPR, FPRL1 and FPRL2 as a result of receptor recycling inhibition using fluorescence microscopy. The constitutive internalization rate was unaffected. Our results support the importance of PLD1 and 2 in formyl peptide receptor function and the role of endocytosis, receptor recycling and reactivation for receptor activity.     

F2L, a peptide derived from heme-binding protein, inhibits formyl peptide receptor-mediated signaling

F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein. Very recently, F2L was identified as an endogenous chemoattractant peptide acting specifically through formyl peptide receptor-like (FPRL)2. In the present study, we report that F2L stimulates chemotactic migration in human neutrophils. However, F2L inhibits formyl peptide receptor (FPR) and FPRL1 activities, resulting in the complete inhibition of intracellular calcium increases, and superoxide generation induced by N-formyl-Met-Leu-Phe, MMK-1, or Trp-Lys-Tyr-Met-Val-d-Met (WKYMVm) in human neutrophils. In terms of the inhibitory role of F2L on FPR- and FPRL-mediated signaling, we found that F2L competitively inhibits the binding of (125)I-WKYMVm to its specific receptors, FPR and FPRL1. F2L is the first endogenous molecule that inhibits FPR- and FPRL1-mediated signaling, and is expected to be useful in the study of FPR and FPRL1 signaling and in the development of drugs to treat diseases involving the FPR family of receptors.     

Contrasting effects of PACAP and carbachol on [Ca]i and inositol phosphates in human neuroblastoma NB-OK-1 cells

The effects of PACAPs on [Ca²⁺]_i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1–27) and PACAP(1–38) increased [Ca²⁺]_i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca²⁺]_i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca²⁺. Carbachol also increased [Ca²⁺]_i in a biphasic manner, but it mobilized intracellular Ca²⁺ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca²⁺ entry, this being accompanied by a more marked and prolonged elevation of IP₃ and IP₄ than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca²⁺ handling through PACAP receptors than with muscarinic M₁ receptors.     

GSK3beta modulates PACAP-induced neuritogenesis in PC12 cells by acting downstream of Rap1 in a caveolae-dependent manner

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic peptide. Here, we show that PACAP recruits Rap1 into caveolin-enriched membrane subdomains in PC12 cells and activates Rap1, nuclear ERK1/2, Elk-1 and CREB in a caveolae-dependent manner. We reveal that GSK3beta is a novel modulator in PACAP signalling. PACAP induces phosphorylation of serine 9 in GSK3beta, which is inhibited by silencing Rap1. Lithium and valproate promote but wortmannin and LY294002 attenuate PACAP-induced phosphorylation of both GSK3beta and ERK1/2, whereas MEK inhibitor PD98059 inhibits nerve growth factor- but not PACAP-induced phosphorylation of GSK3beta, suggesting that GSK3beta operates downstream of Rapt 1 but upstream of ERK1/2 in PACAP signalling. Inhibition or stimulation of GSK3beta results in a 2-fold increase and 6-fold decrease in PACAP-induced neurite outgrowth, respectively. These results reveal an important role of caveolae in the signal transduction of PACAP and that GSK3beta is a critical regulator in PACAP-induced neuronal differentiation.     

Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1beta.

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1beta. IL-1beta induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1beta for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1beta. IL-1beta primed neutrophils for enhanced release of superoxide (O(2)(-)) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1beta also induced O(2)(-) release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1beta and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1beta induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1beta and activation of this cascade mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.     

Presence of Pituitary Adenylate Cyclase-Activating Polypeptide Receptors in Y-79 Human Retinoblastoma Cells

Abstract: Cytochemical analysis demonstrated that a high percentage of human Y-79 retinoblastoma cells displayed a specific labeling by the biotinyl derivative of pituitary adenylate cyclase-activating polypeptide (PACAP), a novel neuropeptide of the secretin-vasoactive intestinal peptide (VIP) family of peptides. In cell membranes, the two molecular forms of PACAP, the one with 38 (PACAP 38) and the other with 27 (PACAP 27) amino acids, displaced the binding of ¹²⁵I-PACAP 27 with IC₅₀ values in the picomolar range and increased adenylyl cyclase activity by 100-fold with EC₅₀ values of 27 and 180 p M , respectively. VIP, human peptide histidine-isoleucine, glucagon, and secretin were much less effective and potent in both receptor assays. The PACAP receptor antagonists PACAP 6–27 and PACAP 6–38 and an antiserum directed against the stimulatory G protein G_s inhibited the PACAP stimulation of adenylyl cyclase. In intact cells, both PACAPs and VIP failed to stimulate the phosphoinositide hydrolysis, whereas in cell membranes PACAP 38, but not the other peptides, produced a modest increase (40%) of inositol phosphate formation with an EC₅₀ value of 22 n M . However, this effect was not antagonized by either PACAP 6–38 or PACAP 6–27. These data demonstrate the presence in human Y-79 retinoblastoma cells of specific PACAP receptors and provide further evidence that PACAP may act as a neurotransmitter/neuromodulator in mammalian retina.     

Inhibition of protein kinase C and anti-CD3-induced Ca2+ influx in Jurkat T cells by a synthetic peptide with sequence identity to HIV-1 gp41

We have previously shown that a synthetic peptide containing env residues 581-597 from HIV-1 inhibits lymphoproliferation of human PBMC. We have investigated the molecular mechanism(s) by which this HIV-1-derived peptide inhibits CD3-mediated signal transduction. We show that the peptide containing residues 581-597 from the HIV-1 transmembrane protein gp41 specifically inhibited the intracellular Ca2+ influx in Jurkat cells stimulated by the mAb OKT3 whereas it had no effect on the production of inositol triphosphate. In addition, the peptide inhibited protein kinase C (pkC)-mediated phosphorylation of the CD3 gamma-chain in intact cells and directly inhibited partially purified pkC. The inhibition was noncompetitive with respect to the substrates histone and ATP and independent of the regulatory domain of the enzyme. Furthermore, the peptide required internalization for inhibitory activity because no inhibition of lymphoproliferation was observed when cells were treated with peptide at 4 degrees C. Based on these results obtained with the peptide aa581-597, we postulate that the transmembrane protein gp41 of HIV-1 may inhibit pkC activity and thus block pkC-dependent immune function contributing to the immunosuppression of HIV-1-infected individuals.     

The neuropeptide pituitary adenylate cyclase activating protein is a physiological activator of human monocytes

Pituitary adenylate cyclase activating protein (PACAP) and its structurally related vasointestinal peptide (VIP) bind to three G-protein-coupled receptors named VPAC1 and VPAC2 for VIP/PACAP receptors and PAC1 for PACAP preferred receptors. We report that in freshly isolated human monocytes PACAP acts as a pro-inflammatory molecule. By RT-PCR, VPAC1 mRNA was the only receptor found to be expressed; VPAC1 protein was detected by Western blotting and visualized by immunohistochemistry. Signaling pathways activated by PACAP include the extracellular regulated kinase (ERK), the stress-activated MAPK p38, the focal adhesion kinase, Pyk2 and its associated cytoskeleton protein paxillin and the phosphatidylinositol 3-kinase (PI-3K). PACAP induces a transient peak in cytoplasmic calcium associated with an increase in reactive oxygen species production and upregulation in membrane expression of the integrin CD11b as well as the complement receptor 1. Control of the different pathways and functions stimulated by PACAP were evaluated using Phospholipase C (PLC), PI-3K, ERK and p38 MAPK inhibitors and led to the conclusion that PLC and to a lesser degree PI-3K activation are upstream events occurring in VPAC1 mediated PACAP stimulation of monocytes and are in contrast to ERK and p38 mandatory for the initiation of other cellular events associated with monocytes activation.     

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC1HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways: Akt ACTIVATION THROUGH PHOSPHATIDYLINOSITOL 3-KINASE �� AND VESICLE ENDOCYTOSIS FOR NEURONAL SURVIVAL*

MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC₁ receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC₁HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC₁HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC₁HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on Gα_q/phosphatidylinositol 3-kinase γ activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase γ-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC₁HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways.