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Requirement of Phosphatidylinositol 3-Kinase Activation and Calcium Influx for Leukotriene B4-induced Enzyme Release*

Open AccessPublished:September 19, 2002DOI:https://doi.org/10.1074/jbc.M208051200
      Leukotriene B4(LTB4) is a potent lipid mediator involved in host defense and inflammatory responses. It causes chemotaxis, generation of reactive oxygen species, and degranulation. However, only little is known of the molecular mechanisms by which LTB4 induces these biological activities. To analyze the intracellular signaling pathways to mediate lysosomal enzyme release through the cloned LTB4 receptor (BLT1), we transfected BLT1 to rat basophilic leukemia cells (RBL-2H3). LTB4 dose-dependently released β-hexosaminidase, and the release was mostly inhibited when the cells were pretreated with pertussis toxin, indicating that the degranulation is mediated by Gi proteins. LTB4activated phosphatidylinositol 3-kinase (PI3-K) through Gi, and inhibition of PI3-K by wortmannin or LY290042 inhibited degranulation. Granulocytes from PI3-Kγ-deficient mice showed reduced LTB4-induced degranulation, suggesting that this isozyme of PI3-K is involved in the degranulation. LTB4 also caused calcium release from intracellular stores and calcium influx from the outside milieu through Gi, but only the calcium influx is critical for the lysosomal enzyme release. Calcium influx and PI3-K activation are both downstream events of Gi, since they were inhibited by pertussis toxin. These two events are in essence independent each other, because calcium depletion did not affect PI3-K, and inhibition of PI3-K did not attenuate calcium influx significantly. Thus, our results have clearly shown that LTB4 binds BLT1 and activates Gi-like protein, and both PI3-Kγ activation and a sustained calcium elevation by calcium influx are necessary for enzyme release in these cells.
      Leukotreine B4 (LTB4),
      The abbreviations used are: LTB4, leukotriene B4(5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid); PI3-K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-triphosphate; BAPTA/AM, 1,2-bis-(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; BSA, bovine serum albumin; PTX, pertussis toxin; PMA, phorbol 12-myristate 13-acetate; fMLP, formylmethionylleucylphenylalanine; MPO, myeloperoxidase.
      1The abbreviations used are: LTB4, leukotriene B4(5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid); PI3-K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-triphosphate; BAPTA/AM, 1,2-bis-(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; BSA, bovine serum albumin; PTX, pertussis toxin; PMA, phorbol 12-myristate 13-acetate; fMLP, formylmethionylleucylphenylalanine; MPO, myeloperoxidase.
      a metabolite of arachidonic acid, is one of the most potent chemoattractants for neutrophils. LTB4activates leukocytes, leading to chemotaxis, release of lysosomal enzyme, and production of superoxide anions, thus playing important roles in host defense (
      • Ford-Hutchinson A.W.
      • Bray M.A.
      • Doig M.V.
      • Shipley M.E.
      • Smith M.J.
      ,
      • Samuelsson B.
      • Dahlen S.E.
      • Lindgren J.A.
      • Rouzer C.A.
      • Serhan C.N.
      ). Overproduction of LTB4 is involved in various inflammatory diseases including psoriasis (
      • Iversen L.
      • Kragballe K.
      • Ziboh V.A.
      ), bronchial asthma (
      • Turner C.R.
      • Breslow R.
      • Conklyn M.J.
      • Andresen C.J.
      • Patterson D.K.
      • Lopez-Anaya A.
      • Owens B.
      • Lee P.
      • Watson J.W.
      • Showell H.J.
      ), rheumatoid arthritis (
      • Griffiths R.J.
      • Pettipher E.R.
      • Koch K.
      • Farrell C.A.
      • Breslow R.
      • Conklyn M.J.
      • Smith M.A.
      • Hackman B.C.
      • Wimberly D.J.
      • Milici A.J.
      • et al.
      ), inflammatory bowel diseases (
      • Sharon P.
      • Stenson W.F.
      ), and ischemic renal failure (
      • Noiri E.
      • Yokomizo T.
      • Nakao A.
      • Izumi T.
      • Fujita T.
      • Kimura S.
      • Shimizu T.
      ). LTB4 binds to a specific G protein-coupled receptor named BLT1, which was cloned in our laboratory (
      • Yokomizo T.
      • Izumi T.
      • Chang K.
      • Takuwa Y.
      • Shimizu T.
      ). Signaling to activate leukocytes through BLT1 is poorly understood, in part because most of the previous studies have been done using peripheral leukocytes, especially granulocytes. The lifetime of these cells is short (several days), which makes it difficult to perform studies using various drugs or gene transfection. We have previously analyzed signaling pathway from BLT1 using Chinese hamster ovary cells stably expressing human BLT1 (
      • Yokomizo T.
      • Izumi T.
      • Chang K.
      • Takuwa Y.
      • Shimizu T.
      ). In these cells, LTB4 increases intracellular IP3 levels by Gq-like protein(s), inhibits adenylyl cyclase activity, and activates chemotaxis through Gi-like protein(s). In hematopoietic cells, however, the intracellular events leading to release of lysosomal enzymes and generation of oxygen species are poorly understood. We generated rat basophilic leukemia (RBL-2H3) cells overexpressing BLT1 and examined the pathway to release lysosomal enzyme through BLT1.
      The major pathway of activating lysosomal enzyme release in mast cell involves the aggregation of high affinity receptors (FcεRI) for IgE by corresponding antigens (
      • Segal D.M.
      • Taurog J.D.
      • Metzger H.
      ,
      • Ujike A.
      • Ishikawa Y.
      • Ono M.
      • Yuasa T.
      • Yoshino T.
      • Fukumoto M.
      • Ravetch J.V.
      • Takai T.
      ). This activation of FcεRI induces histamine release following the activation of tyrosine kinases (Lyn, Syk, Btk, etc.), phosphatidylinositol 3-kinase (PI3-K) (
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ,
      • Hata D.
      • Kawakami Y.
      • Inagaki N.
      • Lantz C.S.
      • Kitamura T.
      • Khan W.N.
      • Maeda-Yamamoto M.
      • Miura T.
      • Han W.
      • Hartman S.E.
      • Yao L.
      • Nagai H.
      • Goldfeld A.E.
      • Alt F.W.
      • Galli S.J.
      • Witte O.N.
      • Kawakami T.
      ,
      • Kawakami Y.
      • Yao L.
      • Tashiro M.
      • Gibson S.
      • Mills G.B.
      • Kawakami T.
      ,
      • Beaven M.A.
      • Baumgartner R.A.
      ), and calcium increase (
      • Kim T.D.
      • Eddlestone G.T.
      • Mahmoud S.F.
      • Kuchtey J.
      • Fewtrell C.
      ). Although G protein-coupled receptor-mediated activation of PI3-K was also found in fMLP-stimulated human neutrophils and thrombin-stimulated human platelets (
      • Kucera G.L.
      • Rittenhouse S.E.
      ,
      • Traynor-Kaplan A.E.
      • Thompson B.L.
      • Harris A.L.
      • Taylor P.
      • Omann G.M.
      • Sklar L.A.
      ,
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Kelly K.L.
      • Ruderman N.B.
      • Chen K.S.
      ), the physiological role of PI3-K in these cells and its relation to calcium increase are somewhat uncertain. Here we demonstrate that LTB4-induced enzyme release requires a PI3-Kγ activation and calcium influx by the interaction of BLT1 and Gi.

      DISCUSSION

      Enzyme release is one of the most important biological responses of inflammatory cells by LTB4 stimulation, leading to inflammation and host defense against infection (
      • Samuelsson B.
      • Dahlen S.E.
      • Lindgren J.A.
      • Rouzer C.A.
      • Serhan C.N.
      ). To study signaling for the enzyme release, we used rat basophilic leukemia (RBL) cells, which contain the same functional G proteins reported in human neutrophils (
      • Hide M.
      • Ali H.
      • Price S.R.
      • Moss J.
      • Beaven M.A.
      ). Expression of LTB4 receptor in RBL cells allowed us to examine the signaling pathway to LTB4-dependent enzyme release.
      LTB4 plays its functions via specific G protein-coupled receptors named BLT1 and BLT2 (
      • Yokomizo T.
      • Izumi T.
      • Chang K.
      • Takuwa Y.
      • Shimizu T.
      ,
      • Yokomizo T.
      • Kato K.
      • Terawaki K.
      • Izumi T.
      • Shimizu T.
      ), and we transfected BLT1, a high affinity and blood cell-specific LTB4 receptor to RBL cells. LTB4 is reported to activate the Gi and G16 classes of G proteins (
      • Gaudreau R.
      • Le Gouill C.
      • Metaoui S.
      • Lemire S.
      • Stankova J.
      • Rola-Pleszczynski M.
      ) to inhibit adenylate cyclase and to activate phospholipase C, respectively. Saito et al.(
      • Saito H.
      • Okajima F.
      • Molski T.F.
      • Sha'afi R.I.
      • Ui M.
      • Ishizaka T.
      ) reported that PTX-sensitive G protein mediates thrombin- and compound 48/80-induced histamine release in mast cells and basophils. In this study, we showed that a series of signaling including Gi, calcium influx, and PIP3 production are necessary for the ultimate enzyme release through LTB4-BLT1 interaction.
      First, as shown in Fig. 2 A, the degranulation is totally sensitive to PTX treatment, suggesting that Gi-like molecules are related to enzyme release. Next, we examined the effects of various inhibitors and toxins on LTB4-dependent enzyme release. We found that LTB4-triggered enzyme release was inhibited by wortmannin and LY294002 (Figs. 2 B and 3), suggesting that PI3-K is deeply involved in degranulation. We then examined whether LTB4 can indeed activate PI3-K and found that PIP3 production was enhanced to 2–3-fold upon LTB4 stimulation (Fig. 4, A–D). Pretreatment of the cells with PTX completely abolished LTB4-dependent PIP3 production, suggesting that Gi-like molecules are required for LTB4-dependent PI3-K activation (Fig.4 E). Pretreatment of the cells with various kinase inhibitors did not affect LTB4-induced enzyme releases (Fig. 2 B). Depletion of conventional protein kinase C by overnight PMA treatment did not inhibit enzyme releases (Fig.2 A). GF109203X at higher concentrations showed inhibitory effects on the enzyme release (data not shown). GF109203X is an inhibitor for both conventional protein kinase C and atypical protein kinase C (
      • Hofmann J.
      ). Therefore, it is possible that atypical protein kinase C is involved in either step for degranulation (
      • Ozawa K.
      • Szallasi Z.
      • Kazanietz M.G.
      • Blumberg P.M.
      • Mischak H.
      • Mushinski J.F.
      • Beaven M.A.
      ,
      • Ozawa K.
      • Yamada K.
      • Kazanietz M.G.
      • Blumberg P.M.
      • Beaven M.A.
      ). Haribabuet al. (
      • Haribabu B.
      • Zhelev D.V.
      • Pridgen B.C.
      • Richardson R.M.
      • Ali H.
      • Snyderman R.
      ) reported that the chemotaxis was blocked by PTX, showing that BLT1 couples to PTX-sensitive Gi-like proteins in RBL-BLT1 cells. They also showed that BLT1-mediated calcium increase and enzyme release were almost insensitive to PTX or wortmannin treatment. In our study, BLT1-mediated calcium increase, enzyme release, and PIP3 production were sensitive to PTX (Figs.2 A, 4 E, and 5). We do not have clear explanations for this discrepancy, but the experimental conditions and stable cell lines with different expression levels of BLT1 can cause the apparent difference.
      Calcium mobilization by FcεRI cross-linking is essential to antigen-induced enzyme release in RBL cells (
      • Kim T.D.
      • Eddlestone G.T.
      • Mahmoud S.F.
      • Kuchtey J.
      • Fewtrell C.
      ). Thus, we examined the relationship between calcium mobilization and enzyme release. Calcium mobilization from internal stores and subsequent calcium entry from the extracellular space are the two major components of calcium increase following activation of cell surface receptors (
      • Penner R.
      • Fasolato C.
      • Hoth M.
      ). In our experiments, EGTA or SKF96365, a calcium channel blocker, inhibited LTB4-induced enzyme release, showing the critical role of calcium influx in enzyme release. Calcium depletion in these cells by BAPTA/AM loading resulted in the delayed increase of intracellular calcium (Fig. 7 B). BAPTA/AM treatment caused the similar delay in LTB4-induced enzyme release (Fig. 6 B). The local high calcium concentrations by calcium influx or a sustained calcium elevation with a critical duration might be necessary for the enzyme release.
      Finally, we have performed a series of experiments to identify the relationship between calcium influx and PI3-K activation. Neither EGTA nor BAPTA/AM treatment affected LTB4-depended PIP3 production (Fig. 4 E), showing that PI3-K activation does not require calcium increase. Conversely, we determined whether inhibition of PI3-K affects calcium mobilization or calcium influx, since several investigators have reported that PI3-K regulates phospholipase Cγ in RBL cells with FcεRI activation (
      • Ching T.T.
      • Hsu A.L.
      • Johnson A.J.
      • Chen C.S.
      ,
      • Smith A.J.
      • Surviladze Z.
      • Gaudet E.A.
      • Backer J.M.
      • Mitchell C.A.
      • Wilson B.S.
      ). We found that treatment of cells with wortmannin or LY294002 did not significantly change the calcium response by LTB4 stimulation (Fig. 8). In three independent lines of PI3-Kγ null mice, calcium mobilization in neutrophils was examined using various chemoattractants. fMLP (
      • 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.
      ,
      • Li Z.
      • Jiang H.
      • Xie W.
      • Zhang Z.
      • Smrcka A.V.
      • Wu D.
      ), C5a, platelet-activating factor, or IL-8 (
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ) induced similar calcium increase in neutrophils from wild-type and PI3-Kγ null mice. We also confirmed that LTB4-induced calcium mobilization was not reduced in PI3-Kγ null granulocytes (Fig. 9 C).
      It is an important future issue how Gi activates PI3-K in RBL-2H3 cells. The role of the βγ subunit of G protein might be the most important, since PI3-Kγ activated by the βγ subunit of G proteins induces respiratory burst in hematopoietic cells (
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      ,
      • Stephens L.R.
      • Eguinoa A.
      • Erdjument-Bromage H.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.S.
      • Thelen M.
      • Cadwallader K.
      • Tempst P.
      • Hawkins P.T.
      ). Neutrophils of PI3-Kγ null mice impaired chemoattractant-induced respiratory burst and chemotaxis (
      • 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.
      ,
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ). Our present data (Fig. 9) showed clearly that PI3-Kγ is the most important PI3-K isozyme in LTB4-dependent degranulation. Signals to calcium influx from Gi molecules are another concern for future studies. They are either due to direct activation of calcium channels or due to the capacitance calcium entry as proposed (
      • Parekh A.B.
      • Penner R.
      ,
      • Putney Jr., J.W.
      ,
      • Barritt G.J.
      ).
      In summary, using RBL-2H3 cells expressing BLT1, we found that PTX-sensitive Gi molecules are important for the degranulation process. Gi then activates PI3-K and calcium influx, and both are required for enzyme release in inflammatory cells. We further demonstrated that calcium influx and PI3-K might be processes that are independent from each other, although both are downstream events of Gi activation. Although these two signals are required for degranulation, other downstream molecules of Gi can also play roles in the enzyme release.

      Acknowledgments

      We thank Dr. N. Nakatani, Dr. K. Hirose (University of Tokyo) and M. Murakami (Showa University) for discussion.

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