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Inhibition of Phosphatidylinositol 3-Kinase Induces Nitric-oxide Synthase in Lipopolysaccharide- or Cytokine-stimulated C6 Glial Cells*

Open AccessPublished:March 12, 1999DOI:https://doi.org/10.1074/jbc.274.11.7528
      Nitric oxide (NO) produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of demyelinating and neurodegenerative diseases. The present study underlines the importance of phosphatidylinositol 3-kinase (PI 3-kinase) in the expression of iNOS in C6 glial cells and rat primary astrocytes. Bacterial lipopolysaccharide (LPS) or interleukin-1β (IL-1β) was unable to induce the expression of iNOS and the production of NO in rat C6 glial cells. Similarly, wortmannin and LY294002, compounds that inhibit PI 3-kinase, were also unable to induce the expression of iNOS and the production of NO. However, a combination of wortmannin or LY294002 with LPS or IL-1β induced the expression of iNOS and the production of NO in C6 glial cells. Consistent with the induction of iNOS, wortmannin also induced iNOS promoter-derived chloramphenicol acetyltransferase activity in LPS- or IL-1β-treated C6 glial cells. The expression of iNOS by LPS in C6 glial cells expressing a dominant-negative mutant of p85α, the regulatory subunit of PI 3-kinase, further supports the conclusion that inhibition of PI 3-kinase provides a necessary signal for the induction of iNOS. Next we examined the effect of wortmannin on the activation of mitogen-activated protein (MAP) kinase and nuclear factor NF-κB in LPS- or IL-1β-stimulated C6 glial cells. In contrast to the inability of LPS and IL-1β alone to induce the expression of iNOS, both LPS and IL-1β individually stimulated MAP kinase activity and induced DNA binding and transcriptional activity of NF-κB. Wortmannin alone was unable to activate MAP kinase and NF-κB. Moreover, wortmannin had no effect on LPS- or IL-1β-mediated activation of MAP kinase and NF-κB, suggesting that wortmannin induced the expression of iNOS in LPS- or IL-1β-stimulated C6 glial cells without modulating the activation of MAP kinase and NF-κB. Similar to C6 glial cells, wortmannin also stimulated LPS-mediated expression of iNOS and production of NO in astrocytes without affecting the LPS-mediated activation of NF-κB. Taken together, the results from specific chemical inhibitors and dominant-negative mutant expression studies demonstrate that apart from the activation of NF-κB, inhibition of PI 3-kinase is also necessary for the expression of iNOS and production of NO.
      Nitric oxide (NO),
      The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase(s); iNOS, inducible NOS; LPS, lipopolysaccharide; IL-1β, interleukin-1β; IFN-γ, interferon-γ; NF-κB, nuclear factor κB; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; PI 3-kinase, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; Erk, extracellular signal-regulated kinase; l-NMA, l-N-methylarginine
      1The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase(s); iNOS, inducible NOS; LPS, lipopolysaccharide; IL-1β, interleukin-1β; IFN-γ, interferon-γ; NF-κB, nuclear factor κB; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; PI 3-kinase, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; Erk, extracellular signal-regulated kinase; l-NMA, l-N-methylarginine
      a vascular and neuronal messenger and a cytotoxic and cytostatic agent, is enzymatically formed from l-arginine by the enzyme nitric-oxide synthase (NOS). The NOS are basically divided into two forms. One constitutive form present in neurons (nNOS) and endothelial cells (eNOS) is a calcium-dependent enzyme, and the inducible form (iNOS) present in macrophages and astrocytes is regulated at the transcriptional level in response to stimuli (e.g. cytokine/lipopolysaccharide (LPS)) and does not require calcium for its activity (
      • Jaffrey S.R.
      • Snyder S.H.
      ,
      • Nathan C.
      ). Although the NO produced by iNOS accounts for the bactericidal and tumoricidal properties of macrophages, it is also of particular importance in the pathophysiologies of inflammatory neurological diseases including demyelinating disorders (e.g. multiple sclerosis, experimental allergic encephalopathy, X-adrenoleukodystrophy) and in ischemia and traumatic injuries associated with infiltrating macrophages and the production of proinflammatory cytokines (
      • Mitrovic B.
      • Ignarro L.J.
      • Montestruque S.
      • Smoll A.
      • Merril J.E.
      ,
      • Bo L.
      • Dawson T.M.
      • Wesselingh S.
      • Mork S.
      • Choi S.
      • Kong P.A.
      • Hanley D.
      • Trapp B.D.
      ,
      • Merrill J.E.
      • Ignarro L.J.
      • Sherman M.P.
      • Melinek J.
      • Lane T.E.
      ,
      • Koprowski H.
      • Zheng Y.M.
      • Heber-Katz E.
      • Fraser N.
      • Rorke L.
      • Fu Z.F.
      • Hanlon C.
      • Dietzshold B.
      ,
      • Cross A.H.
      • Misko T.P.
      • Lin R.F.
      • Hickey W.F.
      • Trotter J.L.
      • Tilton R.G.
      ,
      • Hooper D.C.
      • Bagsra O.
      • Marini J.C.
      • Zborek A.
      • Ohnishi S.T.
      • Kean R.
      • Champion J.M.
      • Sarker A.B.
      • Bobroski L.
      • Farber J.L.
      • Akaike T.
      • Maeda H.
      • Koprowski H.
      ). It is now increasingly clear that glial cells in the central nervous system also produce NO in response to the induction of iNOS by bacterial LPS and a series of cytokines including interleukin-1β (IL-1β), tumor necrosis factor-α, and interferon-γ (IFN-γ). Astrocytes in the healthy brain do not express iNOS; however, after ischemic, traumatic, neurotoxic, or inflammatory damage the reactive astrocytes express iNOS in the mouse, rat, and human (
      • Hu S.X.
      • Sheng W.S.
      • Peterson P.K.
      • Chao C.C.
      ,
      • Galea E.
      • Feinstein D.L.
      • Reis D.J.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ). NO derived from both astrocytes and macrophages is assumed to contribute to oligodendrocyte degeneration in demyelinating diseases and neuronal death during ischemia and trauma (
      • Mitrovic B.
      • Ignarro L.J.
      • Montestruque S.
      • Smoll A.
      • Merril J.E.
      ,
      • Bo L.
      • Dawson T.M.
      • Wesselingh S.
      • Mork S.
      • Choi S.
      • Kong P.A.
      • Hanley D.
      • Trapp B.D.
      ,
      • Merrill J.E.
      • Ignarro L.J.
      • Sherman M.P.
      • Melinek J.
      • Lane T.E.
      ).
      Characterization of the intracellular pathways required to transduce the signal from the cell surface to the nucleus for the induction of iNOS is an active area of investigation. Identification of the DNA binding site for nuclear factor (NF)-κB in the promoter region of iNOS (
      • Xie Q.-W.
      • Kashiwabara Y.
      • Nathan C.
      ) and inhibition of iNOS induction by inhibitors of NF-κB activation have established an essential role of NF-κB activation in the induction of iNOS (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Kwon G.
      • Corbett J.A.
      • Rodi C.P.
      • Sullivan P.
      • McDaniel M.L.
      ). Suppression of NF-κB and inhibition of iNOS expression (
      • Feinstein D.L.
      • Galea E.
      • Cermak J.
      • Chugh P.
      • Lyandvert L.
      • Reis D.J.
      ,
      • Nishiya T.
      • Uehara T.
      • Nomura Y.
      ) by inhibitors of tyrosine kinase in different cell types suggest the possible involvement of tyrosine phosphorylation in the activation of NF-κB and the induction of iNOS. Inhibition of LPS- and cytokine-induced activation of NF-κB and induction of iNOS by inhibitors of the mevalonate pathway and Ras farnesyl protein transferase also indicate that Ras may be involved in the activation of NF-κB and the induction of iNOS (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ). Again, increasing cAMP and protein kinase A activity has been shown to inhibit the activation of NF-κB and the induction of iNOS possibly because of the inhibition of Raf-1 (
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ). Recently we have also observed that PD98059, an inhibitor of mitogen-activated protein (MAP) kinase kinase (MEK), the kinase responsible for the activation of MAP kinase, inhibits the LPS-induced activation of NF-κB and the induction of iNOS in astrocytes, suggesting the possible involvement of the MAP kinase pathway in the LPS- and proinflammatory cytokine-mediated induction of iNOS (
      • Pahan K.
      • Sheikh F.G.
      • Khan M.
      • Namboodiri A.M.S.
      • Singh I.
      ). Taken together, these studies suggest that any alteration of the Ras-Raf-MEK-MAP kinase signal transduction pathway alters the activation of NF-κB and so the induction of iNOS in astrocytes and C6 glial cells.
      In this paper we present evidence that the signal mediated by inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) induces/stimulates the expression of iNOS in LPS- or cytokine-stimulated C6 glial cells and rat primary astrocytes and that the signal is not mediated via MAP kinase and NF-κB. Specific inhibitors of PI 3-kinase (wortmannin and LY294002) and expression of the dominant-negative mutant of p85α, the regulatory subunit of PI 3-kinase, induced the expression of iNOS in LPS- or cytokine-stimulated C6 glial cells or stimulated the expression of iNOS in rat primary astrocytes without modulating the LPS- or cytokine-mediated activation of MAP kinase and NF-κB, suggesting that apart from the activation of NF-κB by LPS or cytokines, the inhibition of PI 3-kinase also provides an essential signal for the expression of iNOS and production of NO in C6 glial cells and astrocytes.

      DISCUSSION

      The signaling events transduced by proinflammatory cytokines and LPS for the induction of iNOS are poorly understood. A complete understanding of the cellular signaling mechanisms involved in the induction of iNOS should identify novel targets for the therapeutic intervention in NO-mediated neuroinflammatory diseases. Recently PI 3-kinase-associated signaling events have been shown to prevent apoptosis in a number of cell types including cerebellar granule neurons (
      • Okada T.
      • Kawano Y.
      • Sakakakibara T.
      • Hazeki M.
      • Ui M.
      ) and hematopoietic cells (
      • Powis G.
      • Bonjouklian R.
      • Berggren M.M.
      • Gallegos A.
      • Abraham R.
      • Ashendel C.
      • Zalkow L.
      • Matter W.F.
      • Dodge J.
      • Grindey G.
      • Vlahos C.J.
      ). Several lines of evidence presented in this study support the conclusion that the inhibition of PI 3-kinase activity, independent of the activation of MAP kinase and NF-κB, induces/stimulates the expression of iNOS in C6glial cells and astrocytes. Our conclusion is based on the following observations. First, LPS or IL-1β alone induced the activation of MAP kinase and NF-κB, but they were ineffective in the modulation of the activity of PI 3-kinase and in the induction of the expression of iNOS. However, the combinations of LPS and IL-1β or LPS and IFN-γ induced the activation of MAP kinase and NF-κB, caused a transient inhibition of PI 3-kinase, and induced the expression of iNOS and production of NO. Second, the compounds (wortmannin and LY294002) that inhibit PI 3-kinase had no effect on the degree of activation of MAP kinase and activation of NF-κB and the expression of iNOS in C6glial cells. However, these inhibitors induced the expression of iNOS and the production of NO in LPS- or IL-1β-treated C6glial cells. In addition, LPS was able to induce the expression of iNOS in C6 cells transfected with a dominant-negative mutant of p85α but not in cells transfected with the empty vector. Consistent with this observation the inhibitors of PI 3-kinase also induced iNOS promoter-derived expression of CAT in LPS- or IL-1β-treated C6 glial cells. On the other hand, these inhibitors of PI 3-kinase had no effect on LPS- or IL-1β-mediated activation of MAP kinase and NF-κB. These observations indicate that in addition to the activation of NF-κB, inhibition of PI 3-kinase is also necessary for the expression of iNOS in C6 glial cells. Our hypothetical model depicting the signals for the biosynthesis of iNOS in C6 glial cells is summarized in Fig. 11. Consistent with the apoptotic activity of NO (
      • Liu Q.
      • Schacher D.
      • Hurth C.
      • Freund G.G.
      • Dantzer R.
      • Kelly K.W.
      ,
      • Minshall C.
      • Arkins S.
      • Freund G.G.
      • Kelly K.W.
      ) and the antiapoptotic activity of activated PI 3-kinase (
      • Okada T.
      • Kawano Y.
      • Sakakakibara T.
      • Hazeki M.
      • Ui M.
      ,
      • Powis G.
      • Bonjouklian R.
      • Berggren M.M.
      • Gallegos A.
      • Abraham R.
      • Ashendel C.
      • Zalkow L.
      • Matter W.F.
      • Dodge J.
      • Grindey G.
      • Vlahos C.J.
      ), the observed up-regulation of LPS- or cytokine-induced expression of iNOS and production of NO in both C6 glial cells and rat primary astrocytes by inhibitors of PI 3-kinase indicates that PI 3-kinase may function as a negative regulator in the induction of iNOS and that this property of PI 3-kinase may contribute to its antiapoptotic activity.
      Figure thumbnail gr11
      Figure 11Hypothetical model describing the signaling pathways for the expression of iNOS in C6 glial cells. MAPK, MAP kinase.
      Proinflammatory cytokines (tumor necrosis factor-α, IL-1β, or IFN-γ) and LPS bind to their respective receptors and induce iNOS expression via activation of NF-κB (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Xie Q.-W.
      • Kashiwabara Y.
      • Nathan C.
      ,
      • Kwon G.
      • Corbett J.A.
      • Rodi C.P.
      • Sullivan P.
      • McDaniel M.L.
      ,
      • Leist M.
      • Fava E.
      • Montecucco C.
      • Nicotera P.
      ,
      • Xie K.
      • Wang Y.
      • Huang S.
      • Xu L.
      • Bielenberg D.
      • Salas T.
      • McConkey D.J.
      • Jiang W.
      • Fidler I.J.
      ). The presence of a consensus sequence in the promoter region of iNOS for the binding of NF-κB (
      • Xie Q.-W.
      • Kashiwabara Y.
      • Nathan C.
      ) and the inhibition of iNOS expression with the inhibition of NF-κB activation establishes an essential role of NF-κB activation in the induction of iNOS (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Kwon G.
      • Corbett J.A.
      • Rodi C.P.
      • Sullivan P.
      • McDaniel M.L.
      ). Activation of NF-κB by various cellular stimuli involves the proteolytic degradation of IκBα and the concomitant nuclear translocation of the liberated NF-κB heterodimer (
      • Stefanova I.
      • Corcoran M.L.
      • Horak E.M.
      • Wahl L.M.
      • Bolen J.B.
      • Horak I.D.
      ,
      • Salkowski C.A.
      • Detore G.
      • McNally R.
      • van Rooijen N.
      • Vogel S.N.
      ). Although the biochemical mechanism underlying the degradation of IκBα remains unclear, it appears that degradation of IκBα induced by various mitogens and cytokines occurs in association with the transient phosphorylation of IκBα on serines 32 and 36 (
      • Beg A.A.
      • Ruben S.M.
      • Scheinman R.I.
      • Haskil S.
      • Rosen C.A.
      • Baldwin Jr., A.S.
      ). Upon phosphorylation, IκBα that is still bound to NF-κB apparently becomes a high affinity substrate for an ubiquitin-conjugating enzyme (
      • Sun S.-C.
      • Ganchi P.A.
      • Ballard D.W.
      • Greene W.C.
      ). After phosphorylation-controlled ubiquitination, the IκBα is rapidly and completely degraded by the 20 S or 26 S proteosome, and the NF-κB heterodimer is targeted to the nucleus (
      • Brown K.
      • Gerstberger S.
      • Carlson L.
      • Franzoso G.
      • Siebenlist U.
      ). Recently, it has been reported that 90-kDa ribosomal S6 kinase (p90 RSK), a downstream candidate of the well characterized Ras-Raf-MEK-MAP kinase pathway, phosphorylates the NH2-terminal regulatory domain of IκBα on serine 32 (
      • Ghoda L.
      • Lin X.
      • Greene W.C.
      ), suggesting the possible involvement of the MAP kinase pathway in the phosphorylation of IκBα and in the activation of NF-κB. Consistent with this observation, we also found that PD98059, an inhibitor of MEK, inhibited the LPS-induced activation of NF-κB in rat primary astrocytes (
      • Pahan K.
      • Sheikh F.G.
      • Khan M.
      • Namboodiri A.M.S.
      • Singh I.
      ).
      Earlier, we have also observed that cAMP derivatives that activate protein kinase A, mevalonate inhibitors that inhibit the p21ras, or antioxidants likeN-acetylcysteine inhibit the expression of iNOS by inhibiting the activation of NF-κB (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ). On the other hand, cell-permeable ceramides analogs and inhibitors of protein phosphate 1/2A stimulate the expression of iNOS in rat primary astrocytes by stimulating the activation of NF-κB (
      • Pahan K.
      • Sheikh F.G.
      • Khan M.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ). Here also we have observed that activation of NF-κB is an essential, but not sufficient, signal for the induction of iNOS. First, IFN-γ did not induce the activation of NF-κB, therefore wortmannin was unable to induce the expression of iNOS and the production of NO in C6 cells. Second, pyrrolidine dithiocarbamate, an inhibitor of NF-κB activation, blocked the activation of NF-κB and thereby inhibited the expression of iNOS, induced by the combination of LPS and wortmannin, suggesting that LPS- and wortmannin-induced expression of iNOS is dependent on the activation of NF-κB. However, LPS- or IL-1β-induced activation of NF-κB was not sufficient to induce the expression of iNOS in the absence of inhibition of PI 3-kinase.
      In summary, studies reported in this manuscript underscore the necessity of inhibition of PI 3-kinase in the LPS- or cytokine-mediated induction of iNOS. Moreover, the signal induced by inhibition of PI 3-kinase for the induction of iNOS is not mediated by MAP kinase or NF-κB.

      Acknowledgments

      We thank Dr. Avtar K. Singh for a review of the manuscript and helpful suggestions and Jan Ashcraft for technical help.

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