Sylvain G. Bourgoin

V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway.

The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.

ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPγS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.

A role for phosphatidic acid in COPI vesicle fission yields insights into Golgi maintenance

Proteins essential for vesicle formation by the Coat Protein I (COPI) complex are being identified, but less is known about the role of specific lipids. Brefeldin-A ADP-ribosylated substrate (BARS) functions in the fission step of COPI vesicle formation. Here, we show that BARS induces membrane curvature in cooperation with phosphatidic acid. This finding has allowed us to further delineate COPI vesicle fission into two sub-stages: 1) an earlier stage of bud-neck constriction, in which BARS and other COPI components are required, and 2) a later stage of bud-neck scission, in which phosphatidic acid generated by phospholipase D2 (PLD2) is also required. Moreover, in contrast to the disruption of the Golgi seen on perturbing the core COPI components (such as coatomer), inhibition of PLD2 causes milder disruptions, suggesting that such COPI components have additional roles in maintaining Golgi structure other than through COPI vesicle formation.

Expression and activity of prostaglandin endoperoxide synthase-2 in agonist-activated human neutrophils

Proinflammatory agents were assessed for their capacity to stimulate the expression of the inducible cyclooxygenase isoform (COX‐2) in human neutrophils. A number of agents, including PMA, opsonized bacteria and zymosan, LPS, GM‐CSF, TNF‐α, and fMLP, induced COX‐2 protein expression through signaling pathways involving transcription and protein synthesis events. Northern blots showed that freshly isolated neutrophils expressed low levels of COX‐2 mRNA, which rapidly increased after incubation with inflammatory agents. A characterization of the signal transduction pathways leading to COX‐2 protein expression was initiated. In LPS‐treated neutrophils, efficient induction of COX‐2 required the presence of serum and involved ligand binding to the CD14 surface antigen. The specific inhibitor of p38 mitogen‐activated protein kinase (p38 MAPK), SB 203580, had little effect on the induction of COX‐2 expression in neutrophils, in contrast to what had been previously observed with other inflammatory cell types. Depending on the agonist present, ethanol differentially blocked the stimulated expression of COX‐2, raising the possibility that phospholipase D activation might take part in the process of COX‐2 induction. Major COX‐2‐derived prostanoids synthesized by inflammatory neutrophils were identified by liquid‐chromatography and tandem mass‐spectrometry as TXA2 and PGE2. The agonist‐induced synthesis of TXA2 and PGE2 was effectively blocked by cycloheximide and by the specific COX‐2 inhibitor NS‐398. These results show that COX‐2 can be induced in an active state by different classes of inflammatory mediators in the neutrophil. They support the concept that, in these cells, the COX‐2 isoform is preeminent over COX‐1 for the stimulated‐production of prostanoids, and also suggest that neutrophil COX‐2 displays a distinct profile of expression among circulatory cells.—Pouliot, M., Gilbert, C., Borgeat, P., Poubelle, P. E., Bourgoin, S., Créminon, C., Maclouf, J., McColl, S. R., Naccache, P. H. Expression and activity of prostaglandin endoperoxide synthase‐2 in agonist‐activated human neutrophils. FASEB J. 612, 1109–1123 (1998)

REGULATION OF PHOSPHOLIPASE D BY PHOSPHORYLATION-DEPENDENT MECHANISMS

The rapid production of phosphatidic acid following receptor stimulation has been demonstrated in a wide range of mammalian cells. Virtually every cell uses phosphatidylcholine as substrate to produce phosphatidic acid in a controlled reaction catalyzed by specific PLD isoforms. Considerable effort has been directed at studying the regulation of PLD activities and subsequent work has characterized a family of proteins including PLD1 and PLD2. Whereas both PLD enzymes are dependent on phosphatidylinositol 4, 5-bisphosphate for activity only the PLD1 isoform was strongly stimulated by the small GTPases ARF and RhoA and by protein kinase Calpha as well. A role for tyrosine kinase activities in the membrane recruitment of small GTPases, in the synthesis of phosphatidylinositol 4,5-bisphosphate and tyrosine phosphorylation of PLD1 and PLD2 has been uncovered. However, it still not clear exactly how tyrosine phosphorylation of proteins contributes to PLD activation in cells. Here we review the data linking tyrosine phosphorylation of proteins to the activation of PLD and describe recent finding on the sites and possible mechanisms of action of tyrosine kinases in receptor-mediated PLD activation. Finally, a model illustrating the potential complex interplay linking these signaling events with the activation of PLD is presented.

Crystal-induced neutrophil activation VI. Involvement of FcγRIIIB (CD16) and CD11b in response to inflammatory microcrystals

The inflammatory reaction associated with the deposition of monosodium urate (MSU) crystals in synovial spaces is known to be due to interactions with polymorphonuclear neutrophils mediated by presently unidentified surface structures. In this study, we have observed that antibodies directed against CD16 (VIFcRIII) and CD11b (VIM12) selectively and potently inhibit the activation of neutrophils by MSU crystals. The responses affected include the stimulation of tyrosine phosphorylation, activation of the tyrosine kinase syk, tyrosine phosphorylation of the proto‐oncogene Cbl, mobilization of calcium, and stimulation of the activity of phospholipase D and of the production of superoxide anions. Tyrosine phosphorylation responses to MSU crystals develop during the Me2SO4‐induced differentiation of HL‐60 cells in parallel with the surface expression of CD16. These data strongly support the hypothesis that inflammatory microcrystals interact opportunistically with CD16 initially, and that the signal transduction pathways activated thereby depend on CD11b. An examination of the relevance of the hypothesis that an uncontrolled activation of CD16/CD11b may play a role in inflammatory reactions associated with a dysregulation of neutrophil function (other than crystal arthropathies) appears warranted on the basis of the present results.—Barabé, F., Gilbert, C., Liao, N., Bourgoin, S. G., Naccache, P. H. Crystal‐induced neutrophil activation: involvement of FcγRIIIB (CD16) in response to inflammatory microcrystals. FASEB J. 12, 209–220 (1998)

Cross-Talk between CD14 and Complement Receptor 3 Promotes Phagocytosis of Mycobacteria: Regulation by Phosphatidylinositol 3-Kinase and Cytohesin-1

The glycosylphosphatidyl anchored molecule CD14 to the monocyte membrane plays a prominent role in innate immunity, and the paradigms for CD14 selective signaling are beginning to be elucidated. In this study, transfected human monocytic cell line THP-1 and Chinese hamster ovary (CHO) fibroblastic cells were used to examine phagocytosis of Mycobacterium bovis bacillus Calmette-Guérin (BCG). Flow cytometry was combined with molecular and biochemical approaches to demonstrate a dual mechanism for BCG internalization involving either CD14 alone or a CD14-regulated complement receptor (CR)3-dependent pathway. Phagocytosis by CD14-positive THP-1 cells was attenuated by phosphatidylinositol-3 inhibitors LY294002 and wortmannin and experiments using transfected CHO cells showed substantial accumulation of phosphatidylinositol-3,4,5-trisphosphate at the BCG attachment site in CHO cells expressing CD14 and TLR2 suggesting that bacteria bind to CD14 and use TLR2 to initiate a PI3K signaling pathway. Additional experiments using blocking Abs showed that anti-TLR2 Abs inhibit phagocytosis of BCG by THP-1 cells. Furthermore, knockdown of cytohesin-1, a PI3K-regulated adaptor molecule for β2 integrin activation, specifically abrogated CD14-regulated CR3 ingestion of BCG consistent with the observation of physical association between CR3 and cytohesin-1 in cells stimulated with mycobacterial surface components. These findings reveal that mycobacteria promote their uptake through a process of “inside-out” signaling involving CD14, TLR2, PI3K, and cytohesin-1. This converts low avidity CR3 into an active receptor leading to increased bacterial internalization.

Phospholipases D1 and D2 Coordinately Regulate Macrophage Phagocytosis

Phagocytosis is a fundamental feature of the innate immune system, required for antimicrobial defense, resolution of inflammation, and tissue remodeling. Furthermore, phagocytosis is coupled to a diverse range of cytotoxic effector mechanisms, including the respiratory burst, secretion of inflammatory mediators and Ag presentation. Phospholipase D (PLD) has been linked to the regulation of phagocytosis and subsequent effector responses, but the identity of the PLD isoform(s) involved and the molecular mechanisms of activation are unknown. We used primary human macrophages and human THP-1 promonocytes to characterize the role of PLD in phagocytosis. Macrophages, THP-1 cells, and other human myelomonocytic cells expressed both PLD1 and PLD2 proteins. Phagocytosis of complement-opsonized zymosan was associated with stimulation of the activity of both PLD1 and PLD2, as demonstrated by a novel immunoprecipitation-in vitro PLD assay. Transfection of dominant-negative PLD1 or PLD2 each inhibited the extent of phagocytosis (by 55–65%), and their combined effects were additive (reduction of 91%). PLD1 and PLD2 exhibited distinct localizations in resting macrophages and those undergoing phagocytosis, and only PLD1 localized to the phagosome membrane. The COS-7 monkey fibroblast cell line, which has been used as a heterologous system for the analysis of receptor-mediated phagocytosis, expressed PLD2 but not PLD1. These data support a model in which macrophage phagocytosis is coordinately regulated by both PLD1 and PLD2, with isoform-specific localization. Human myelomonocytic cell lines accurately model PLD-dependent signal transduction events required for phagocytosis, but the heterologous COS cell system does not.

Chemotactic Factor-Induced Recruitment and Activation of Tec Family Kinases in Human Neutrophils. II. Effects of LFM-A13, a Specific Btk Inhibitor

Tyrosine phosphorylation events play major roles in the initiation and regulation of several functional responses of human neutrophils stimulated by chemotactic factors such as the bacterially derived tripeptide formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). However, the links between the G protein-coupled receptors, the activation of the tyrosine kinases, and the initiation of neutrophil functional responses remain unclear. In the present study we assessed the effects of a Btk inhibitor, leflunomide metabolite analog (LFM-A13), on neutrophils. LFM-A13 decreased the tyrosine phosphorylation induced by fMet-Leu-Phe and inhibited the production of superoxide anions and the stimulation of adhesion, chemotaxis, and phospholipase D activity. We observed a decreased accumulation of phosphatidylinositol-3,4,5-trisphosphate in response to fMet-Leu-Phe in LFM-A13-pretreated cells even though the inhibitor had no direct effect on the lipid kinase activity of the p110γ or p85/p110 phosphatidylinositol 3-kinases or on the activation of p110γ by fMet-Leu-Phe. The phosphorylation of Akt and of extracellular signal-regulated kinases 1/2 and p38 were similarly inhibited by LFM-A13. LFM-A13 also negatively affected the translocation of Rac-2, RhoA, ADP ribosylation factor-1, Tec, Bmx, and Btk induced by fMet-Leu-Phe. The results of this study provide evidence for an involvement of Btk and possibly other Tec kinase family members in the regulation of the functional responsiveness of human neutrophils and link these events, in part at least, to the modulation of levels of phosphatidylinositol-3,4,5-trisphosphate.

Regulation of lysophosphatidic acid receptor expression and function in human synoviocytes: Implications for rheumatoid arthritis?

Lysophosphatidic acid (LPA), via interaction with its G-protein coupled receptors, is involved in various pathological conditions. Extracellular LPA is mainly produced by the enzyme autotaxin (ATX). Using fibroblast-like synoviocytes (FLS) isolated from synovial tissues of patients with rheumatoid arthritis (RA), we studied the expression profile of LPA receptors, LPA-induced cell migration, and interleukin (IL)-8 and IL-6 production. We report that FLS express LPA receptors LPA1-3. Moreover, exogenously applied LPA induces FLS migration and secretion of IL-8/IL-6, whereas the LPA3 agonist l-sn-1-O-oleoyl-2-methyl-glyceryl-3-phosphothionate (2S-OMPT) stimulates cytokine synthesis but not cell motility. The LPA-induced FLS motility and cytokine production are suppressed by LPA1/3 receptor antagonists diacylglycerol pyrophosphate and (S)-phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl) ester (VPC32183). Signal transduction through p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and Rho kinase is involved in LPA-mediated cytokine secretion, whereas LPA-induced cell motility requires p38 MAPK and Rho kinase but not p42/44 MAPK. Treatment of FLS with tumor necrosis factor-α (TNF-α) increases LPA3 mRNA expression and correlates with enhanced LPA- or OMPT-induced cytokine production. LPA-mediated superproduction of cytokines by TNF-α-primed FLS is abolished by LPA1/3 receptor antagonists. We also report the presence of ATX in synovial fluid of patients with RA. LPA1/3 receptor antagonists and ATX inhibitors reduce the synovial fluid-induced cell motility. Together the data suggest that LPA1 and LPA3 may contribute to the pathogenesis of RA through the modulation of FLS migration and cytokine production. The above results provide novel insights into the relevance of LPA receptors in FLS biology and as potential therapeutic targets for the treatment of RA.