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Phosphatidylinositol 3-Kinase-dependent Extracellular Calcium Influx Is Essential for CX3CR1-mediated Activation of the Mitogen-activated Protein Kinase Cascade*

Open AccessPublished:August 24, 2001DOI:https://doi.org/10.1074/jbc.M009374200
      Fractalkine, the first member of the CX3C chemokine family, induces leukocyte chemotaxis through activation of its high affinity receptor, CX3CR1. Like other chemokine receptors, CX3CR1 is coupled to a pertussis toxin-sensitive heterotrimeric Gi protein, which is necessary for rapid rise in the concentration of intracellular calcium. Using a Chinese hamster ovary cell line stably transfected with the CX3CR1 receptor, we show that the source of calcium mobilized by fractalkine stimulation is the extracellular pool. Calcium influx is blocked by extracellular calcium chelators, as well as by divalent heavy metals such as Ni2+, Co2+, and Cd2+ without affecting the integrity of intracellular stores. Remarkably, selective phosphoinositide 3-kinase (PI3K) inhibitors, wortmannin and LY294002, abolish the wave extracellular calcium, suggesting that an active PI3K is necessary for this event. The influx of extracellular calcium is in turn required to trigger the activation of the p42/44 mitogen-activated protein/extracellular signal-regulated kinase pathway, but is not necessary for other signals downstream to PI3K, such as phosphorylation of Akt. The potential role of this signaling cascade in fractalkine-mediated chemotaxis is discussed.
      GPCR
      G protein-coupled receptor
      MAPK
      mitogen-activated protein kinase
      PI3K
      phosphoinositide 3-kinase
      CHO
      Chinese hamster ovary
      PBS
      phosphate-buffered saline
      BAPTA
      1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
      TTX
      tetrodotoxin
      HIV
      human immunodeficiency virus
      PCR
      polymerase chain reaction
      RT
      reverse transcription
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      Chemokines are small secreted proteins that stimulate the directional migration of leukocytes, playing a key role in the inflammatory response and infectious diseases (
      • Murphy P.M.
      ). Chemokines have been classified into four groups depending on the number and spacing of the first two conserved cysteine residues: CXC, CX3C, CC, and C (
      • Mennicken F.
      • Maki R.
      • de Souza E.B.
      • Quirion R.
      ). Fractalkine, also referred to as neurotactin, is a novel chemokine of the CX3C chemokine family (
      • Pan Y.
      • Lloyd C.
      • Zhou H.
      • Dolich S.
      • Deeds J.
      • Gonzalo J.A.
      • Vath J.
      • Gosselin M.
      • Ma J.
      • Dussault B.
      • Woolf E.
      • Alperin G.
      • Culpepper J.
      • Gutierrez-Ramos J.C.
      • Gearing D.
      ,
      • Combadiere C.
      • Salzwedel K.
      • Smith E.D.
      • Tiffany H.L.
      • Berger E.A.
      • Murphy P.M.
      ). Fractalkine is different from typical chemokines because of its molecular size and membrane-associated structure. The fractalkine molecule consists of a 373-amino acid polypeptide chain, which carries the 76-amino acid chemokine domain at the N terminus followed by a mucin-like stalk and a transmembrane domain (
      • Pan Y.
      • Lloyd C.
      • Zhou H.
      • Dolich S.
      • Deeds J.
      • Gonzalo J.A.
      • Vath J.
      • Gosselin M.
      • Ma J.
      • Dussault B.
      • Woolf E.
      • Alperin G.
      • Culpepper J.
      • Gutierrez-Ramos J.C.
      • Gearing D.
      ). The receptor for fractalkine was identified as CX3CR1 (previously V28), and, like other chemokine receptors, it belongs to the super family of G protein-coupled receptors (GPCRs)1 (
      • Combadiere C.
      • Salzwedel K.
      • Smith E.D.
      • Tiffany H.L.
      • Berger E.A.
      • Murphy P.M.
      ,
      • Imai T.
      • Hieshima K.
      • Haskell C.
      • Baba M.
      • Nagira M.
      • Nishimura M.
      • Kakizaki M.
      • Takagi S.
      • Nomiyama H.
      • Schall T.J.
      • Yoshie O.
      ).
      The physiological roles of fractalkine are only beginning to be understood. Fractalkine plays a central role in the trafficking of leukocytes in tissues with high blood flow, like the glomerular circuit (
      • Chen S.
      • Bacon K.B.
      • Li L.
      • Garcia G.E.
      • Xia Y.
      • Lo D.
      • Thompson D.A.
      • Siani M.A.
      • Yamamoto T.
      • Harrison J.K.
      • Feng L.
      ,
      • Feng L.
      • Chen S.
      • Garcia G.E.
      • Xia Y.
      • Siani M.A.
      • Botti P.
      • Wilson C.B.
      • Harrison J.K.
      • Bacon K.B.
      ). This function is attributed to its unique membrane bound structure, which enables it to form strong adhesive bonds with leukocytes expressing the CX3CR1 receptor in this high shear environment (
      • Fong A.M.
      • Robinson L.A.
      • Steeber D.A.
      • Tedder T.F.
      • Yoshie O.
      • Imai T.
      • Patel D.D.
      ,
      • Fong A.M.
      • Erickson H.P.
      • Zachariah J.P.
      • Poon S.
      • Schamberg N.J.
      • Imai T.
      • Patel D.D.
      ). These adhesive interactions between the membrane-bound fractalkine and cells expressing the fractalkine receptor are independent of CX3CR1 signal transduction or integrin function (
      • Haskell C.A.
      • Cleary M.D.
      • Charo I.F.
      ). However, fractalkine can also act as a typical chemokine in cell culture assays and in vivo since soluble forms of fractalkine are able to induce the migration of different types of T and natural killer cells through endothelial cells (
      • Pan Y.
      • Lloyd C.
      • Zhou H.
      • Dolich S.
      • Deeds J.
      • Gonzalo J.A.
      • Vath J.
      • Gosselin M.
      • Ma J.
      • Dussault B.
      • Woolf E.
      • Alperin G.
      • Culpepper J.
      • Gutierrez-Ramos J.C.
      • Gearing D.
      ,
      • Imai T.
      • Hieshima K.
      • Haskell C.
      • Baba M.
      • Nagira M.
      • Nishimura M.
      • Kakizaki M.
      • Takagi S.
      • Nomiyama H.
      • Schall T.J.
      • Yoshie O.
      ). A recent study showed that fractalkine is cleaved from membranes of neurons in culture in response to an excitotoxic stimulus (
      • Chapman G.A.
      • Moores K.
      • Harrison D.
      • Campbell C.A.
      • Stewart B.R.
      • Strijbos P.J.
      ). In the central nervous system, neuronally derived fractalkine mediates interactions between neurons and microglia upon nerve injury and promotes neuronal cell survival (
      • Boehme S.A.
      • Lio F.M.
      • Maciejewski-Lenoir D.
      • Bacon K.B.
      • Conlon P.J.
      ,
      • Harrison J.K.
      • Jiang Y.
      • Chen S.
      • Xia Y.
      • Maciejewski D.
      • McNamara R.K.
      • Streit W.J.
      • Salafranca M.N.
      • Adhikari S.
      • Thompson D.A.
      • Botti P.
      • Bacon K.B.
      • Feng L.
      ,
      • Meucci O.
      • Fatatis A.
      • Simen A.A.
      • Miller R.J.
      ). CX3CR1, in addition to the CCR5 and CXCR4 receptors, serves as a major co-receptor (along with the CD4) involved in the entry of dual cell tropic HIV-1 strain into target cells (
      • Faure S.
      • Meyer L.
      • Costagliola D.
      • Vaneensberghe C.
      • Genin E.
      • Autran B.
      • Delfraissy J.F.
      • McDermott D.H.
      • Murphy P.M.
      • Debre P.
      • Theodorou I.
      • Combadiere C.
      ,
      • Locati M.
      • Murphy P.M.
      ). In agreement with this fact, fractalkine was recently found to inhibit CX3CR1 receptor-mediated HIV-1 infection in vitro (
      • Faure S.
      • Meyer L.
      • Costagliola D.
      • Vaneensberghe C.
      • Genin E.
      • Autran B.
      • Delfraissy J.F.
      • McDermott D.H.
      • Murphy P.M.
      • Debre P.
      • Theodorou I.
      • Combadiere C.
      ).
      Despite its prominent role in inflammation and HIV infection, the signaling pathways triggered by CX3CR1 are poorly understood. As with other chemokine receptors, a major signaling consequence of CX3CR1 stimulation is a rapid mobilization of calcium. It is not clear, however, how this event is regulated and how elevated intracellular calcium is related to fractalkine-mediated cell migration. Recent studies have pointed at the importance of Gβγ/PI3Kγ-dependent signaling cascades in the process of neutrophil, macrophage, and lymphocyte cell migration (
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ,
      • Li Z.
      • Jiang H.
      • Xie W.
      • Zhang Z.
      • Smrcka A.V.
      • Wu D.
      ,
      • Sasaki T.
      • Irie-Sasaki J.
      • Jones R.G.
      • Oliveira-dos-Santos A.J.
      • Stanford W.L.
      • Bolon B.
      • Wakeham A.
      • Itie A.
      • Bouchard D.
      • Kozieradzki I.
      • Joza N.
      • Mak T.W.
      • Ohashi P.S.
      • Suzuki A.
      • Penninger J.M.
      ). However, the importance of these pathways in CX3CR1 signaling and biological functions has not been addressed. Finally, fractalkine stimulation of CX3CR1 has been shown to induce phosphorylation of p42/44 MAPK (
      • Maciejewski-Lenoir D.
      • Chen S.
      • Feng L.
      • Maki R.
      • Bacon K.B.
      ,
      • Meucci O.
      • Fatatis A.
      • Simen A.A.
      • Bushell T.J.
      • Gray P.W.
      • Miller R.J.
      ), but the architecture of this signaling cascade has yet to be determined. In this study, we have identified a PI3K-dependent signaling cascade, which upon CX3CR1 activation modulates extracellular calcium influx and MAPK activation.

      DISCUSSION

      The effects of chemoattractants such as formylmethionyleucylphenylalanine, interleukin 8, and C5a on neutrophil migration as well as those of regulated on activation normal T cell expressed and secreted, macrophage inflammatory protein-5, macrophage-derived chemokine, and stromal cell-derived factor-1 on macrophage migration have underscored the importance of Gβγ/PI3Kγ-dependent cascades in the process of chemotaxis (
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ,
      • Li Z.
      • Jiang H.
      • Xie W.
      • Zhang Z.
      • Smrcka A.V.
      • Wu D.
      ,
      • Sasaki T.
      • Irie-Sasaki J.
      • Jones R.G.
      • Oliveira-dos-Santos A.J.
      • Stanford W.L.
      • Bolon B.
      • Wakeham A.
      • Itie A.
      • Bouchard D.
      • Kozieradzki I.
      • Joza N.
      • Mak T.W.
      • Ohashi P.S.
      • Suzuki A.
      • Penninger J.M.
      ). We thus hypothesized that signaling cascades emanating from Gβγ and PI3K could also play an essential role in CX3CR1-mediated chemotaxis. The increase in cytosolic calcium concentration is a typical signaling readout of chemokine receptor activation. However, the significance of fast calcium mobilization with respect to cell migration is still being debated. To begin analyzing these questions in the context of CX3CR1-mediated signaling cascades, we established a CHO cell line expressing the CX3CR1 receptor (Fig. 1). Stimulation with 10 nm fractalkine induced robust signaling responses such as fast phosphorylation of MAPK and Akt (Fig.1 c), as well as a fast increase in cytosolic calcium concentrations (Fig. 1 b). The increase in [Ca2+]i could be the result of Gβγ activation, and subsequent phospholipase Cβ-mediated release of calcium from intracellular stores. Alternatively, the increase in [Ca2+]i could be caused by influx of extracellular calcium through plasma membrane channels (
      • Sozzani S.
      • Molino M.
      • Locati M.
      • Luini W.
      • Cerletti C.
      • Vecchi A.
      • Mantovani A.
      ). Our results demonstrate that fractalkine-mediated elevation of [Ca2+]i is caused by a potent influx of extracellular calcium (Figs. 2 and 3). This conclusion is based on the observations that the rise [Ca2+]i is: (a) completely dependent on the presence of extracellular calcium (Fig.2 b); (b) blocked by non-permeable calcium chelators such as BAPTA (20 µm) and EGTA (5 mm) (Fig. 2, c and d), under conditions that do not affect the integrity of the intracellular stores; (c) inhibited by general Ca2+ channel blockers such as Ni2+, Cd2+, and Co2+ cations (all 100 µm) (Fig. 3,c–e); and (d) not affected by inhibition of phospholipase C with U71322 (5 µm) (Fig. 3 b). Extracellular calcium mobilization in response to fractalkine is thus mediated directly by plasma membrane Ca2+ channels, and does not seem to depend on prior release of Ca2+ from intracellular stores.
      The calcium influx generated by activation of CX3CR1 is blocked by Pertussis toxin (Ref.
      • Imai T.
      • Hieshima K.
      • Haskell C.
      • Baba M.
      • Nagira M.
      • Nishimura M.
      • Kakizaki M.
      • Takagi S.
      • Nomiyama H.
      • Schall T.J.
      • Yoshie O.
      , and data not shown), which indicates the requirement of Go/i coupling. Gβγ subunits can bind directly and inhibit N-, P-, and Q-type voltage-gated channels predominantly present in excitable cells (
      • Herlitze S.
      • Garcia D.E.
      • Mackie K.
      • Hille B.
      • Scheuer T.
      • Catterall W.A.
      ,
      • Ikeda S.R.
      ,
      • Ruiz-Velasco V.
      • Ikeda S.R.
      ), but typically absent in non-excitable cells such as fibroblasts and lymphocytes. Gβγ dimers can also activate receptor-operated channels and voltage-operated L-type channels (
      • Barritt G.J.
      ,
      • Viard P.
      • Exner T.
      • Maier U.
      • Mironneau J.
      • Nurnberg B.
      • Macrez N.
      ). For example, GPCR agonists such as angiotensin II, noradrenalin, and GnRH can induce the opening of l-type channels (
      • Viard P.
      • Exner T.
      • Maier U.
      • Mironneau J.
      • Nurnberg B.
      • Macrez N.
      ,
      • Mulvaney J.M.
      • Zhang T.
      • Fewtrell C.
      • Roberson M.S.
      ). However, two different 1,4-dihydropiridine type/L-type channel inhibitors, nimodipine and nifedipine, did not affect fractalkine-induced calcium influx at high concentrations (Fig. 4). In contrast, the inhibitors flunarizine and amiloride significantly reduced the CX3CR1-mediated Ca2+ influx (Fig. 4), when used at concentrations considered to be specific for T-type channels (
      • Bijlenga P.
      • Liu J.H.
      • Espinos E.
      • Haenggeli C.A.
      • Fischer-Lougheed J.
      • Bader C.R.
      • Bernheim L.
      ,
      • Tang C.M.
      • Presser F.
      • Morad M.
      ,
      • Tytgat J.
      • Vereecke J.
      • Carmeliet E.
      ). However, two lines of evidence stand against the notion that these effects are truly mediated by T-type channels. First, depolarization with 30 mm KCl did not induce calcium influx (Fig. 5c), suggesting the absence of voltage-gated channels in the CHO-CX3CR1 cells; and second, we could not detect expression of α1G, α1H, or α1I subtype T-type channel mRNA in CHO-CX3CR1 cells by RT-PCR. Thus, the identity of the channel mediating fractalkine calcium influx remains to be determined. Our current hypothesis is that the rapid rise in [Ca2+]i is mediated by yet uncharacterized receptor-operated channels with unreported sensitivity to those compounds. The use of flunarizine and amiloride could actually help in identifying these channels. It is intriguing in this context that mibefradil, another relatively selective T-type channel blocker, can produce potent immunosuppression by inhibiting transmigration of CD4+ and CD8+ T cells through allogeneic endothelium (
      • Blaheta R.A.
      • Hailer N.P.
      • Brude N.
      • Wittig B.
      • Oppermann E.
      • Leckel K.
      • Harder S.
      • Scholz M.
      • Weber S.
      • Encke A.
      • Markus B.H.
      ). It would be also interesting to extend the analysis of the mechanisms of fractalkine-induced Ca2+ influx to excitable neurons, where CX3CR1 is abundantly expressed and plays a role in fractalkine-mediated neuronal survival (
      • Boehme S.A.
      • Lio F.M.
      • Maciejewski-Lenoir D.
      • Bacon K.B.
      • Conlon P.J.
      ,
      • Meucci O.
      • Fatatis A.
      • Simen A.A.
      • Miller R.J.
      ).
      Earlier reports have indicated a regulatory role for PI3K signals on calcium channels, particularly of the L- and N-types (
      • Blair L.A.
      • Marshall J.
      ,
      • Viard P.
      • Exner T.
      • Maier U.
      • Mironneau J.
      • Nurnberg B.
      • Macrez N.
      ). This prompted us to test whether fractalkine-mediated Ca2+influx was also mediated by PI3K. Indeed, two chemically different and selective PI3K inhibitors, wortmannin and LY294002, effectively reduced Ca2+ influx, whereas the specific MEK inhibitor PD98059 had no effect (Fig. 6). Both PI3K inhibitors also blocked fractalkine-induced activation of MAPK (Fig. 7), indicating that PI3K lies upstream of both Ca2+ and MAPK signaling events. In turn, inhibition of Ca2+ signaling by the extracellular Ca2+ chelators BAPTA and EGTA, and by Ca2+channel-blocking cations Ni2+ and Cd2+inhibited MAPK phosphorylation (Fig. 7 b). Similar to the effects on Ca2+ influx, flunarizine also reduced MAPK phosphorylation, but this effect was not observed with the L-type antagonists, nimodipine and nifedipine (Fig. 7 b). Attenuation of Ca2+ entry, however, had no effect on the phosphorylation of Akt at Ser473, which also requires activation of PI3K (Fig. 7 b). Consistently, the rise in [Ca2+]i by depletion of intracellular stores did not stimulate Akt phosphorylation (Fig.7 c). Taken together, these results delineate a signaling cascade involving the activation of PI3K. PI3K-derived signals then bifurcate in two separate effector pathways, one regulating the influx of extracellular calcium through a plasma membrane channel, and the other activating Akt and possibly other downstream effectors in a Ca2+-independent manner. Elevation of [Ca2+]i triggered by fractalkine stimulation is in turn required for MAPK pathway activation. Current efforts are under way to dissect more thoroughly the Ca2+-activated MAPK pathway. This important cascade involves three common sequential steps, namely activation of PI3K, elevated [Ca2+]i, and activation of MAPK.
      Fractalkine is thus a remarkable dual action molecule, regulating cell-cell interactions (
      • Haskell C.A.
      • Cleary M.D.
      • Charo I.F.
      ) and cell migration. Our preliminary studies suggest that a similar signaling cascade involving PI3K, [Ca2+]i, and MAPK is essential for fractalkine-induced chemotaxis of Jurkat T cells. Thus, signaling pathways diverging downstream of PI3K appear to play multiple and important roles in CX3CR1 function such as chemotaxis and cell survival (our preliminary results and Refs.
      • Boehme S.A.
      • Lio F.M.
      • Maciejewski-Lenoir D.
      • Bacon K.B.
      • Conlon P.J.
      and
      • Meucci O.
      • Fatatis A.
      • Simen A.A.
      • Miller R.J.
      , respectively). As PI3K and Akt may work to promote the polarization of signaling proteins required for triggering chemotaxis (
      • Servant G.
      • Weiner O.D.
      • Herzmark P.
      • Balla T.
      • Sedat J.W.
      • Bourne H.R.
      ), the question of how PI3K signals regulate [Ca2+]i, and consequently MAPK deserves further investigation. Interestingly, a recent report suggests that phosphatidylinositol 3,4,5-trisphosphate, the main product of PI3K, induces calcium entry through T cell plasma membrane channels by a novel uncharacterized mechanism (
      • Hsu A.L.
      • Ching T.T.
      • Sen G.
      • Wang D.S.
      • Bondada S.
      • Authi K.S.
      • Chen C.S.
      ). The Ca2+-mediated activation of the MAPK cascade may in turn play a role in the remodeling of the cytoskeleton required for CX3CR1-induced cell movement, perhaps by enhancing myosin light chain kinase activity and the phosphorylation of downstream targets involved in cell motility (
      • Klemke R.L.
      • Cai S.
      • Giannini A.L.
      • Gallagher P.J.
      • de Lanerolle P.
      • Cheresh D.A.
      ). Future studies designed to uncover the different components of this pathway could shed more light on the molecular events triggering the chemotactic response to fractalkine during inflammation.

      Acknowledgments

      We thank Sandra Schieferl and Kevin D'Arcy for technical assistance. We also thank Drs. Tim Hales and Peter Hornbeck for critical reading of the manuscript. We especially thank Dr. Michael Comb for encouragement and helpful discussions.

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