Activation of platelet-activating factor receptor-coupled G alpha q leads to stimulation of Src and focal adhesion kinase via two separate pathways in human umbilical vein endothelial cells

Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G alpha(q) protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G alpha(q) protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activation of G alpha(q) protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC beta 3 through its G alpha(q) protein-coupled receptor, whereas activation of phospholipase C gamma 1 (PLC gamma 1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130(Cas), Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.     

Basic fibroblast growth factor stimulates fibronectin expression through phospholipase C gamma, protein kinase C alpha, c-Src, NF-kappaB, and p300 pathway in osteoblasts

Fibronectin (Fn) is involved in early stages of bone formation and basic fibroblast growth factor (bFGF) is an important factor regulating osteogenesis. bFGF increased Fn expression, which was attenuated by phosphatidylinositol phospholipase inhibitor (U73122), protein kinase C inhibitor (GF109203X), Src inhibitor (PP2), NF-kappaB inhibitor (PDTC), IkappaBalpha phosphorylation inhibitor (Bay 117082), or IkappaB protease inhibitor (TPCK). bFGF-induced increase of Fn-luciferase activity was antagonized by cells transfected with Fn construct without NF-kappaB regulatory site. Stimulation of osteoblasts with bFGF activated IkappaB kinase alpha/beta (IKK alpha/beta) and increased IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-kappaB-specific DNA-protein complex and kappaB-luciferase activity. bFGF-mediated an increase of IKKalpha/beta activity and DNA-binding activity was inhibited by U73122, GF109203X, or PP2. The binding of p65 to the NF-kappaB element, as well as the recruitment of p300 and the enhancement of p50 acetylation on the Fn promoter was enhanced by bFGF. Overexpression of constitutively active FGF receptor 2 (FGFR2) increased Fn-luciferase activity, which was inhibited by co-transfection with dominant negative (DN) mutants of PLCgamma2, PKCalpha, c-Src, IKKalpha, or IKKbeta. Our results suggest that bFGF increased Fn expression in rat osteoblasts via the FGFR2/PLCgamma2/PKCalpha/c-Src/NF-kappaB signaling pathway.     

Activation and regulation of platelet-activating factor receptor: role of G(i) and G(q) in receptor-mediated chemotactic, cytotoxic, and cross-regulatory signals

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role(s) of G(i) and G(q) in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing G alpha(i3) (PAFR-G alpha(i3)) or G alpha(q) (PAFR-G alpha(q)) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-G alpha(i3), or PAFR-G alpha(q) was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca2+ mobilization, phosphoinositide (PI) hydrolysis, and secretion of beta-hexosaminidase. PAFR-G alpha(i3) and PAFR-G alpha(q) mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-G alpha(i3), but not PAFR-G alpha(q), mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated beta arrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca2+ mobilization to PAF by all the receptors but only PAFR-G alpha(i3) activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that G(i) exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both G(i) and G(q) activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by G(i), the data suggest that differential activation of both G(i) and G(q) by PAFR likely mediate specific as well as redundant signaling pathways.     

Inverse agonist-induced signaling and down-regulation of the platelet-activating factor receptor

Platelet-activating factor (PAF) is a potent phospholipid mediator involved in several diseases such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G-protein-coupled receptor family. Following stimulation, PAFR becomes rapidly desensitized; this refractory state is dependent on PAFR phosphorylation, internalization and down-regulation. In this report, we show that the PAFR inverse agonist, WEB2086, can induce phosphorylation and down-regulation of PAFR. Using selective inhibitors, we determined that the agonist, PAF, and WEB2086 could induce phosphorylation of PAFR by PKC. Moreover, dominant-negative (DN) mutant of PKC isoforms beta inhibited WEB2086-stimulated PAFR phosphorylation, whereas PAF-stimulated phosphorylation was inhibited by DN PKCalpha and delta. WEB2086 also induced PAFR down-regulation which could be blocked by PKC inhibitors and by DN PKCbeta. WEB2086-induced down-regulation was dynamin-dependent but arrestin-independent. Unlike PAF, WEB2086-stimulated intracellular trafficking of PAFR was independent of Rab5. Specific inhibitors of lysosomal proteases and of proteasomes were both effective in reducing WEB2086-induced PAFR down-regulation, indicating the importance of receptor targeting to both lysosomes and proteasomes in long-term cell desensitization to WEB2086. These results indicate that although both agonists and inverse agonists induce receptor PAFR down-regulation, this may be accomplished through different signal transduction and trafficking pathways.     

Janus kinase 2 activation by the platelet-activating factor receptor (PAFR): roles of Tyk2 and PAFR C terminus

Platelet-activating factor (PAF) is a phospholipid with multiple physiological and pathological actions. The PAF receptor (PAFR) belongs to the G protein-coupled, heptahelical receptor superfamily. Recently, we have shown that PAF signals through the Janus kinase (Jak)/STAT pathway and that Tyk2 plays an essential role in PAF-induced PAFR promoter 1 activation. In the present study we found that PAF stimulated Jak2 tyrosine phosphorylation in the monocytic cell line MonoMac-1 as well as in COS-7 cells transfected with PAFR and Jak2 cDNAs. The use of a G protein-uncoupled PAFR (D289A) mutant indicated that Jak2 activation was G protein independent. Interestingly, following PAF stimulation, Jak2 coimmunoprecipitated with PAFR in the presence of active Tyk2, but not with a kinase-inactive Tyk2 mutant, K930I. Moreover, Tyk2-K930I completely blocked PAF-stimulated Jak2 phosphorylation. Gradual deletion of C-terminal residues of the PAFR resulted in progressively decreased Jak2 activation. Deletion of 12 C-terminal residues in mutant V330Stop diminished Jak2 tyrosine phosphorylation by 17%. Further deletions of 25-37 residues from the PAFR C-tail (C317Stop, M311Stop, and T305Stop) resulted in a 50% decrease in Jak2 phosphorylation compared with the wild-type receptor. Complete removal of the C tail resulted in a mutant (K298Stop) that failed to activate Jak2, suggesting that the receptor C-terminal region contains important domains for Jak2 activation. Finally, the coexpression of a minigene encoding the C terminus of PAFR partially inhibited PAF-induced kinase activation. Taken together, our results indicate that PAF activates Jak2 and that Tyk2 and the C-terminal tail of PAFR are of critical importance for PAF-induced Jak2 activation.     

PAF enhances MMP-2 production in rat aortic VSMCs via a β-arrestin2-dependent ERK signaling pathway

Platelet-activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a potent phospholipid mediator and has been reported to be localized in atherosclerotic plaque. However, its role in the progression of atherosclerosis remains unclear. In the present study, we investigated the role of PAF in the production of matrix metalloproteinase (MMP) in primary vascular smooth muscle cells (VSMCs). When rat aortic primary VSMCs were stimulated with PAF (1 nmol/l), the expressions of MMP-2 mRNA and protein, but not of MMP-9, were significantly increased, and these upregulations were markedly attenuated by inhibiting extracellular signal-regulated kinases (ERKs) using molecular and pharmacological inhibitors, but not by using inhibitors of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. Likewise, ERK phosphorylation was markedly enhanced in PAF-stimulated VSMCs, and this was attenuated by WEB2086, but not by EGF receptor inhibitor, demonstrating the specificity of PAF receptor (PAFR) in PAF-induced ERK phosphorylation. In immunofluorescence studies, β-arrestin2 in PAF-stimulated VSMCs colocalized with PAFR and phosphorylated ERK (P-ERK). Coimmunoprecipitation results suggest that β-arrestin2-bound PAFRs existed as a complex with P-ERK. In addition, PAF-induced ERK phosphorylation and MMP-2 production were significantly attenuated by β-arrestin2 depletion. Taken together, the study shows that PAF enhances MMP-2 production in VSMCs via a β-arrestin2-dependent ERK signaling pathway.     

Platelet-activating factor-induced chemokine gene expression requires NF-kappaB activation and Ca2+/calcineurin signaling pathways. Inhibition by receptor phosphorylation and beta-arrestin recruitment

Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing carboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-kappaB activation and CCL2 production. PAF caused translocation of beta-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (DeltaST-PAFR). Interestingly, PAF induced significantly higher NF-kappaB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing DeltaST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-kappaB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-kappaB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and beta-arrestin recruitment inhibit CCL2 production by blocking both NF-kappaB activation and Ca2+/calcineurin-dependent signaling pathways.     

Trafficking, ubiquitination, and down-regulation of the human platelet-activating factor receptor

Platelet-activating factor (PAF) is a potent phospholipid mediator involved in various disease states such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G protein-coupled receptor family. Following PAF stimulation, cells become rapidly desensitized; this refractory state can be maintained for hours and is dependent on PAFR phosphorylation, internalization, and down-regulation. In this report, we characterized ligand-induced, long term PAFR desensitization, and pathways leading to its degradation. Some GPCRs are known to be targeted to proteasomes for degradation while others traffic via the early/late endosomes toward lysosomes. Specific inhibitors of lysosomal proteases and inhibitors of the proteasome were effective in reducing the ligand-induced PAFR down-regulation by 40 and 25%, respectively, indicating the importance of receptor targeting to both lysosomes and proteasomes in long term cell desensitization to PAF. The effects of the proteasome and lysosomal protease inhibitors were additive and, together, completely blocked ligand-induced degradation of PAFR. Using dominant-negative Rab5 and 7 and colocalization of the PAFR with the early endosome autoantigen I (EEAI) or transferrin, we confirmed that ligand-induced PAFR down-regulation was Rab5/7-dependent and involved lysosomal degradation. In addition, we also demonstrated that PAFR was ubiquitinated in an agonist-independent manner. However, a dominant negative ubiquitin ligase (NCbl) reduced PAFR ubiquitination and inhibited ligand-induced but not basal receptor degradation. Our results indicate that PAFR degradation can occur via both the proteasome and lysosomal pathways and ligand-stimulated degradation is ubiquitin-dependent.     

Activation of ERK1/2 by platelet-activating factor receptor is independent of receptor internalisation and G-protein activation

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator involved in a broad range of physiological and pathophysiological processes. The receptor of PAF (PAFR) is a heptahelical G-protein-coupled receptor. We have shown previously that upon agonist stimulation, PAFR internalised through clathrin-coated vesicles in an arrestin-dependent, but G-protein-coupling-independent manner. In the current report, we demonstrate that PAF stimulates Erk1/2 phosphorylation and: (1). dominant negative mutants of arrestins and dynamin do not influence Erk1/2 activation, (2). hypertonic conditions do not decrease the extent of Erk1/2 phosphorylation, (3). internalisation-deficient and/or G-protein-coupling-deficient mutants of PAFR activate Erk1/2 as efficiently as the wild-type PAFR, and (4). inhibition of epidermal growth factor receptor (EGFR) does not block Erk1/2 activation. Taken together, our results suggest that PAFR-mediated activation of mitogen-activated protein kinases Erk1/2 does not require receptor endocytosis, receptor tyrosine kinase transactivation or G-protein activation. In addition, our studies reveal that PAFR-mediated signals of G-protein activation, receptor internalisation and MAPK activation are differentially regulated by receptor structure and/or conformation.     

Distinct roles of receptor phosphorylation, G protein usage, and mitogen-activated protein kinase activation on platelet activating factor-induced leukotriene C(4) generation and chemokine production

Platelet activating factor (PAF) interacts with cell surface G protein-coupled receptors on leukocytes to induce degranulation, leukotriene C(4) (LTC(4)) generation, and chemokine CCL2 production. Using a basophilic leukemia RBL-2H3 cell line expressing wild-type PAF receptor (PAFR) and a phosphorylation-deficient mutant (mPAFR), we have previously demonstrated that receptor phosphorylation mediates desensitization of PAF-induced degranulation. Here, we sought to determine the role of receptor phosphorylation on PAF-induced LTC(4) generation and CCL2 production. We found that PAF caused a significantly enhanced LTC(4) generation in cells expressing mPAFR when compared with PAFR cells. In contrast, PAF-induced CCL2 production was greatly reduced in mPAFR cells. Pertussis toxin and U0126, which inhibit G(i) and p44/42 mitogen-activated protein kinase (ERK) activation, respectively, caused very little inhibition of PAF-induced CCL2 production (approximately 20% inhibition). In contrast, these inhibitors almost completely blocked both PAF-induced ERK phosphorylation and LTC(4) generation in PAFR cells. However, in mPAFR cells pertussis toxin only partially inhibited PAF-induced ERK phosphorylation. A Ca(2+)/calmodulin inhibitor had no effect on PAF-induced ERK phosphorylation in PAFR cells but completely blocked the response in mPAFR cells. These data demonstrate that receptor phosphorylation, which serves to desensitize PAF-induced LTC(4) generation, is required for chemokine CCL2 production. They also indicate a previously unrecognized selectivity in G protein usage and ERK activation for PAF-induced responses. Whereas PAF-induced CCL2 production is, in large part, mediated independently of G(i) activation or ERK phosphorylation, LTC(4) generation requires ERK phosphorylation, which is mediated by different G proteins depending on the phosphorylation status of the receptor.     

Platelet activating factor-induced neuronal apoptosis is initiated independently of its G-protein coupled PAF receptor and is inhibited by the benzoate orsellinic acid

The bioactive lipid mediator platelet activating factor (PAF) is recognized as a key effecter of neuronal apoptosis, yet it is not clear whether its G-protein coupled receptor (PAFR) initiates or prevents PAF neurotoxicity. Using PAFR-/- and congenic wild-type mice, we show that PAF triggers caspase-3/7 activity and neuronal death in PAFR-/- but not PAFR+/+ cerebellar granule neurons. Restoring receptor expression by recombinant adenoviral infection protected cells from PAF challenge. Neuronal death was not mediated by nitric oxide or N-methyl-d-aspartate receptor signaling given that N-nitro-l-arginine methyl ester and MK-801 did not inhibit PAF-induced neuronal loss in PAFR-/- neurons. To intervene in PAFR-independent neurotoxicity, the anti-apoptotic actions of three structurally distinct PAF antagonists were compared to a panel of plant and fungal benzoic acid derivatives. We found that the PAF antagonist BN 52021 but not FR 49175 or CV 3988 inhibited PAFR-independent neurotoxicity. Orsellinic acid, a fungal-derived benzoic acid, blocked PAF-mediated neuronal apoptosis without affecting PAFR-mediated neuroprotection. These findings demonstrate that PAF can transduce apoptotic death in primary neurons independently of its G-protein coupled receptor, that PAFR activation is neuroprotective, and that orsellinic acid effectively attenuates PAFR-independent neuronal apoptosis.     

Reduced pain behaviors and extracellular signal-related protein kinase activation in primary sensory neurons by peripheral tissue injury in mice lacking platelet-activating factor receptor

Peripheral tissue injury causes the release of various mediators from damaged and inflammatory cells, which in turn activates and sensitizes primary sensory neurons and thereby produces persistent pain. The present study investigated the role of platelet-activating factor (PAF), a phospholipid mediator, in pain signaling using mice lacking PAF receptor (pafr-/- mice). Here we show that pafr-/- mice displayed almost normal responses to thermal and mechanical stimuli but exhibit attenuated persistent pain behaviors resulting from tissue injury by locally injecting formalin at the periphery as well as capsaicin pain and visceral inflammatory pain without any alteration in cytoarchitectural or neurochemical properties in dorsal root ganglion (DRG) neurons and a defect in motor function. However, pafr-/- mice showed no alterations in spinal pain behaviors caused by intrathecally administering agonists for N-methyl-d-aspartate (NMDA) and neurokinin(1) receptors. A PAFR agonist evoked an intracellular Ca(2+) response predominantly in capsaicin-sensitive DRG neurons, an effect was not observed in pafr-/- mice. By contrast, the PAFR agonist did not affect C- or Adelta-evoked excitatory post-synaptic currents in substantia gelatinosa neurons in the dorsal horn. Interestingly, mice lacking PAFR showed reduced phosphorylation of extracellular signal-related protein kinase (ERK), an important kinase for the sensitization of primary sensory neurons, in their DRG neurons after formalin injection. Furthermore, U0126, a specific inhibitor of the ERK pathway suppressed the persistent pain by formalin. Thus, PAFR may play an important role in both persistent pain and the sensitization of primary sensory neurons after tissue injury.     

RGS4 inhibits platelet-activating factor receptor phosphorylation and cellular responses

To define the role of regulators of G-protein signaling (RGS) in chemoattractant-mediated responses, RGS4 and the receptors for platelet-activating factor (PAFR), formylated peptides (FR), or interleukin-8 (CXCR1) were stably coexpressed in a rat basophilic leukemia (RBL-2H3) cell line. The data demonstrate that RGS4 inhibited responses by PAFR (i.e., phosphoinositide (PI) hydrolysis, Ca2+ mobilization) but not by FR or CXCR1. An N-terminal 33 amino acid deletion mutant of RGS4 (DeltaRGS4), deficient in GAP (GTPase activating protein) activity and plasma membrane localization, had no effect on either PAFR, FR, or CXCR1. RGS4, but not DeltaRGS4, also blocked phosphorylation of PAFR by platelet-activating factor (PAF) and, unexpectedly, by phorbol 12-myristate 13-acetate (PMA); it also blocked cross-phosphorylation by formylmethionylleucylphenylalanine (fMLP). A point mutant of RGS4 (N88S), deficient in GAP activity but not membrane localization, partially blocked PAFR phosphorylation but had no effect on PAFR-mediated PI hydrolysis and Ca2+ mobilization. Truncation of the cytoplasmic tail of PAFR (mPAFR) resulted in a loss of its susceptibility to inhibition by RGS4. Taken together, the data indicate that of the receptors studied, RGS4 selectively inhibited responses to PAFR, which preferentially couples to Gq. At the level of expression studied, RGS4 did not inhibit FR or CXCR1 which activates Gi to transduce cellular signals. Since the tail-deleted mutant of PAFR was not affected by RGS4, and RGS4 blocked homologous as well as heterologous phosphorylation of this receptor, it is possible that RGS4 interferes sterically with the cytoplasmic tail of PAFR. Thus, in addition to stimulating the GTPase activity of Galpha, RGS4 prevents G protein activation by PAFR and the homologous and heterologous phosphorylation of this receptor.     

Extracellular oxidation by taurine chloramine activates ERK via the epidermal growth factor receptor

Taurine is present in high concentrations in neutrophils, and when the cells are stimulated taurine can react with hypochlorous acid (HOCl) to form taurine-chloramine (Tau-Cl). This compound retains oxidant activity and can affect the neutrophil itself or surrounding tissue cells. We have investigated the effects of Tau-Cl on MAPK signaling in human umbilical vein endothelial cells (HUVEC). Tau-Cl caused no loss in intracellular glutathione or inactivation of the thiol-sensitive enzyme glyceraldehyde-3-phosphate dehydrogenase, indicating that it had not entered the cells. However, stimulation of HUVEC with Tau-Cl (20-100 microM) induced the rapid activation of ERK within 10 min. This activation was abolished by inhibition of MEK by U0126, indicating that it was not because of direct oxidation of ERK. No activation of p38 was detected. These results suggest that Tau-Cl reacts with a cell membrane target that results in intracellular ERK activation. Tau-Cl over the same concentration range and time scale stimulated epidermal growth factor (EGF) receptor tyrosine phosphorylation in A431 cells and HUVEC. The EGF receptor inhibitor PD158780 significantly attenuated Tau-Cl-induced phosphorylation of both the EGF receptor and ERK. This implicates the EGF receptor in the upstream activation of ERK. The Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine had no effect on Tau-Cl-induced EGF receptor or ERK activation. We propose that Tau-Cl acts on an oxidant-sensitive target on the cell surface, this being either the EGF receptor itself or another target that can interact with the EGF receptor, with consequential activation of ERK.     

Heterogeneity in the sn-1 carbon chain of platelet-activating factor glycerophospholipids determines pro- or anti-apoptotic signaling in primary neurons

The platelet-activating factor (PAF) family of glycerophospholipids accumulates in damaged brain tissue following injury. Little is known about the role of individual isoforms in regulating neuronal survival. Here, we compared the neurotoxic and neuroprotective activities of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C(16)-PAF) and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C(18)-PAF) in cerebellar granule neurons. We find that both C(16)-PAF and C(18)-PAF cause PAF receptor-independent death but signal through different pathways. C(16)-PAF activates caspase-7, whereas C(18)-PAF triggers caspase-independent death in PAF receptor-deficient neurons. We further show that PAF receptor signaling is either pro- or anti-apoptotic, depending upon the identity of the sn-1 fatty acid of the PAF ligand. Activation of the PAF G-protein-coupled receptor (PAFR) by C(16)-PAF stimulation is anti-apoptotic and inhibits caspase-dependent death. Activation of PAFR by C(18)-PAF is pro-apoptotic. These results demonstrate the importance of the long-chain sn-1 fatty acid in regulating PAF-induced caspase-dependent apoptosis, caspase-independent neurodegeneration, and neuroprotection in the presence or absence of the PAF receptor.     

Platelet-activating factor receptor-deficient mice are protected from experimental sleep apnea-induced learning deficits

Intermittent hypoxia (IH) during sleep, a hallmark of sleep apnea, is associated with neurobehavioral impairments, regional neurodegeneration and increased oxidative stress and inflammation in rodents. Platelet-activating factor (PAF) is an important mediator of both normal neural plasticity and brain injury. We report that mice deficient in the cell surface receptor for PAF (PAFR-/-), a bioactive mediator of oxidative stress and inflammation, are protected from the spatial reference learning deficits associated with IH. Furthermore, PAFR-/- exhibit attenuated elevations in inflammatory signaling (cyclo-oxygenase-2 and inducible nitric oxide synthase activities), degradation of the ubiquitin-proteasome pathway and apoptosis observed in wild-type littermates (PAFR+/+) exposed to IH. Collectively, these findings indicate that inflammatory signaling and neurobehavioral impairments induced by IH are mediated through PAF receptors.     

Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication

The hepatitis C virus (HCV) causes chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. We have previously shown that HCV nonstructural proteins induce activation of STAT-3 via oxidative stress and Ca2+ signaling (G. Gong, G. Waris, R. Tanveer, and A. Siddiqui, Proc. Natl. Acad. Sci. USA 98:9599-9604, 2001). In this study, we focus on the signaling pathway leading to STAT-3 activation in response to oxidative stress induced by HCV translation and replication activities. Here, we demonstrate the constitutive activation of STAT-3 in HCV replicon-expressing cells. The HCV-induced STAT-3 activation was inhibited in the presence of antioxidant (pyrrolidine dithiocarbamate) and Ca2+ chelators (BAPTA-AM and TMB-8). Previous studies have shown that maximum STAT-3 transactivation requires Ser727 phosphorylation in addition to tyrosine phosphorylation. Using a series of inhibitors and dominant negative mutants, we show that HCV-induced activation of STAT-3 is mediated by oxidative stress and influenced by the activation of cellular kinases, including p38 mitogen-activated protein kinase, JNK, JAK-2, and Src. Our results also suggest a potential role of STAT-3 in HCV RNA replication. We also observed the constitutive activation of STAT-3 in the liver biopsy of an HCV-infected patient. These studies provide an insight into the mechanisms by which HCV induces intracellular events relevant to liver pathogenesis associated with the viral infection.