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Cannabinoids Protect Astrocytes from Ceramide-induced Apoptosis through the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway*

Open AccessPublished:July 19, 2002DOI:https://doi.org/10.1074/jbc.M205797200
      Cannabinoids, the active components of marijuana and their endogenous counterparts, exert many of their actions on the central nervous system by binding to the CB1cannabinoid receptor. Different studies have shown that cannabinoids can protect neural cells from different insults. However, those studies have been performed in neurons, whereas no attention has been focused on glial cells. Here we used the pro-apoptotic lipid ceramide to induce apoptosis in astrocytes, and we studied the protective effect exerted by cannabinoids. Results show the following: (i) cannabinoids rescue primary astrocytes from C2-ceramide-induced apoptosis in a dose- and time-dependent manner; (ii) triggering of this anti-apoptotic signal depends on the phosphatidylinositol 3-kinase/protein kinase B pathway; (iii) ERK and its downstream target p90 ribosomal S6 kinase might be also involved in the protective effect of cannabinoids; and (iv) cannabinoids protect astrocytes from the cytotoxic effects of focal C2-ceramide administration in vivo. In summary, results show that cannabinoids protect astrocytes from ceramide-induced apoptosis via stimulation of the phosphatidylinositol 3-kinase/protein kinase B pathway. These findings constitute the first evidence for an “astroprotective” role of cannabinoids.
      THC
      Δ9-tetrahydrocannabinol
      ERK
      extracellular signal-regulated kinase
      GFAP
      glial-fibrillary acidic protein
      PI3K
      phosphatidylinositol 3-kinase
      PKB
      protein kinase B
      RSK
      p90 ribosomal S6 kinase
      TUNEL
      terminal dUTP nick-end labeling
      PBS
      phosphate-buffered saline
      HA
      hemagglutinin
      The effects exerted by marijuana and their derivatives through Δ9-tetrahydrocannabinol (THC)1 and other cannabinoid constituents have been known for many centuries. However, the molecular basis of these actions were not understood until the discovery of an endogenous cannabinoid system comprising two plasma membrane Gi/o-coupled cannabinoid receptors (CB1 (
      • Matsuda L.A.
      • Lolait S.J.
      • Brownstein M.
      • Young A.
      • Bonner T.I.
      ) and CB2 (
      • Munro S.
      • Thomas K.L.
      • Abu-Shaar M.
      )) and a family of endogenous ligands for those receptors (
      • Devane W.A.
      • Hanus L.
      • Breuer A.
      • Pertwee R.G.
      • Stevenson L.A.
      • Griffin G.
      • Gibson D.
      • Mandelbaum A.
      • Etinger A.
      • Mechoulam R.
      ,
      • Mechoulam R.
      • Ben Shabat S.
      • Hanus L.
      • Ligumsky M.
      • Kaminski N.E.
      • Schatz A.R.
      • Gopher A.
      • Almog S.
      • Martin B.R.
      • Compton D.R.
      • Pertwee R.G.
      • Griffin G.
      • Bayewitch M.
      • Barg J.
      • Vogel Z.
      ). Cannabinoid receptors mediate cannabinoid effects by coupling to different signaling pathways. Both the CB1 and the CB2 receptor signal inhibition of adenylyl cyclase (
      • Howlett A.C.
      ) and stimulation of extracellular signal-regulated kinase (ERK) (
      • Bouaboula M.
      • Poinot Chazel C.
      • Bourrie B.
      • Canat X.
      • Calandra B.
      • Rinaldi-Carmona M., Le
      • Fur G.
      • Casellas P.
      ), whereas the CB1 receptor is also coupled to modulation of Ca2+ and K+ channels (
      • Pertwee R.G.
      ), stimulation of the stress-activated p38 and c-Jun N-terminal kinases (
      • Rueda D.
      • Galve-Roperh I.
      • Haro A.
      • Guzmán M.
      ), stimulation of the focal adhesion kinase (
      • Derkinderen P.
      • Toutant M.
      • Kadaré G.
      • Ledent C.
      • Parmentier M.
      • Girault J.-A.
      ), hydrolysis of sphingomyelin (
      • Sánchez C.
      • Rueda D.
      • Ségui B.
      • Galve-Roperh I.
      • Levade T.
      • Guzmán M.
      ), and stimulation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) (
      • Gómez del Pulgar T.
      • Velasco G.
      • Guzmán M.
      ).
      The study of the potential therapeutic applications of cannabinoids has become one of the most exciting areas in the field. Ongoing research is determining whether cannabinoid ligands may be effective agents in the treatment of pain, glaucoma, and the wasting and emesis associated with acquired immunodeficiency syndrome and cancer chemotherapy (
      • Pertwee R.G.
      ,
      • Piomelli D.
      • Giuffrida A.
      • Calignano A.
      • Rodrı́guez de Fonseca F.
      ). In addition, cannabinoids are being investigated as potential antitumoral drugs (
      • Galve-Roperh I.
      • Sánchez C.
      • Cortés M.
      • Gómez del Pulgar T.
      • Izquiedo M.
      • Guzmán M.
      ,
      • Sánchez C.
      • de Ceballos M.L.
      • Gómez del Pulgar T.
      • Rueda D.
      • Corbacho C.
      • Velasco G.
      • Galve-Roperh I.
      • Huffman J.W.H.
      • Ramón y Cajal S.
      • Guzmán M.
      ,
      • Bifulco M.
      • Laezza C.
      • Portella G.
      • Vitale M.
      • Orlando P., De
      • Petrocellis L.
      • Di Marzo V.
      ) and therapeutic agents for neurological and neurodegenerative disorders (
      • Baker D.
      • Pryce G.
      • Croxford J.L.
      • Brown P.
      • Pertwee R.G.
      • Huffman J.W.
      • Layward L.
      ,
      • Mechoulam R.
      • Panikashvili D.
      • Sholami E.
      ). Neuroprotection by cannabinoids has been related to the CB1-mediated inhibition of voltage-sensitive Ca2+ channels to reduce Ca2+influx, glutamate release and excitotoxicity (
      • Piomelli D.
      • Giuffrida A.
      • Calignano A.
      • Rodrı́guez de Fonseca F.
      ,
      • Shen M.
      • Thayer S.A.
      ), and to the ability of cannabinoids to act as antioxidants (
      • Hampson A.J.
      • Grimaldi M.
      • Axelrod J.
      • Wink D.
      ,
      • Marsicano G.
      • Moosmann H.
      • Lutz B.
      • Bel C.
      ). However, nothing is known about the possible protective effect of cannabinoids on the major cell population of the central nervous system, namely the astrocytes, despite the pivotal role played by these cells in brain homeostasis. In addition, although the CB1 receptor is coupled to PI3K/PKB (
      • Gómez del Pulgar T.
      • Velasco G.
      • Guzmán M.
      ) and ERK activation (
      • Bouaboula M.
      • Poinot Chazel C.
      • Bourrie B.
      • Canat X.
      • Calandra B.
      • Rinaldi-Carmona M., Le
      • Fur G.
      • Casellas P.
      ), and both signaling routes are essential for neural cell survival (
      • Yuan J.
      • Yankner B.A.
      ), their possible involvement in the protection of neural cells by cannabinoids is as yet unknown.
      Ceramide, a sphingosine-based lipid, regulates a variety of cellular processes including differentiation, proliferation, and apoptosis (
      • Kolesnick R.N.
      • Krönke M.
      ). Interestingly, the pro-apoptotic effect of ceramide may be due, at least partially, to its ability to inhibit PKB (
      • Salinas M.
      • López-Valdaliso R.
      • Martı́n D.
      • Álvarez A.
      • Cuadrado A.
      ,
      • Schubert K.M.
      • Scheid M.P.
      • Duronio V.
      ). In addition, it has been shown that accumulation of ceramide in astrocytes leads to apoptosis (
      • Blázquez C.
      • Galve-Roperh I.
      • Guzmán M.
      ). Here we employed a cell-permeable analog of ceramide to induce apoptosis in astrocytes, and we studied (i) the protective role of cannabinoids and (ii) the involvement of PI3K/PKB and ERK pathways in such effect.

      DISCUSSION

      During the last few years, a number of reports have indicated that cannabinoids protect nervous cells from different insults (reviewed in Refs.
      • Piomelli D.
      • Giuffrida A.
      • Calignano A.
      • Rodrı́guez de Fonseca F.
      and
      • Mechoulam R.
      • Panikashvili D.
      • Sholami E.
      ). In line with those observations, data presented here show that cannabinoids, via activation of the CB1 receptor, protect astrocytes from ceramide-induced apoptosis in vitroand in vivo. Astrocytes have been traditionally considered as secondary players in the central nervous system scenario, and therefore all the previous studies on the protective role of cannabinoids on neural cells have involved neurons (see Refs.
      • Shen M.
      • Thayer S.A.
      and
      • Nagayama T.
      • Sinor A.D.
      • Simon R.P.
      • Chen J.
      • Graham S.H.
      • Jin K.
      • Greenberg D.A.
      ,
      • Sinor A.D.
      • Irvin S.M.
      • Greenberg D.A.
      ,
      • Panikashvili D.
      • Simeonidou C.
      • Ben-Shabat S.
      • Hanus L.
      • Breuer A.
      • Mechoulam R.
      • Shohami E.
      ,
      • Van der Stelt M.
      • Veldhuis W.B.
      • Bär P.R.
      • Veldink G.A.
      • Vliegenthart J.F.G.
      • Nicolay K.
      ,
      • Van der Stelt M.
      • Veldhuis W.B.
      • van Haaften G.W.
      • Fezza F.
      • Bisogno T.
      • Bär P.R.
      • Veldink G.A.
      • Vliegenthart J.F.G., Di
      • Marzo V.
      • Nicolay K.
      , for example). However, it is currently well established that astrocytes, the most abundant cells of the mammalian brain, are involved in numerous functions such as supply of nutrients to neurons (
      • Tsacopoulos M.
      • Magistretti P.J.
      ), establishment of synapses (
      • Ullian E.M.
      • Sapperstein S.K.
      • Christopherson K.S.
      • Barres B.A.
      ), and generation of neurons (
      • Doetsch F.
      • Caillé I.
      • Lim D.A.
      • Garcı́a-Verdugo J.M.
      • Álvarez-Buylla A.
      ). In addition, in the context of the present study astrocytes are known to take up (
      • Di Marzo V.
      • Fontana A.
      • Cadas H.
      • Schinelli S.
      • Cimino G.
      • Schwartz J.C.
      • Piomelli D.
      ) and produce (
      • Walter L.
      • Franklin A.
      • Witting A.
      • Moller T.
      • Stella N.
      ) endocannabinoids. Thus, most likely the complex mechanisms underlying defense against brain injury (and in particular the mechanisms mediated by cannabinoids) also involve protection of astrocytes.
      Several observations presented in this report indicate that cannabinoids protect primary astrocytes from ceramide-induced apoptosis via CB1 receptor-mediated stimulation of the PI3K/PKB pathway. (i) Blockade of the CB1 receptor or inhibition of PI3K abolishes the protective effect of cannabinoids. (ii) Cannabinoid treatment leads to reactivation of PKB in parallel to prevention of apoptosis. (iii) Overexpression of a dominant-negative form of PKB abrogates the protective effect of cannabinoids. It is well established that challenge with ceramide leads to apoptosis in several experimental models, and this may be at least partially due to dephosphorylation and inactivation of PKB by a ceramide-activated phosphatase (
      • Salinas M.
      • López-Valdaliso R.
      • Martı́n D.
      • Álvarez A.
      • Cuadrado A.
      ,
      • Schubert K.M.
      • Scheid M.P.
      • Duronio V.
      ). Our results suggest that cannabinoids (via activation of the PI3K pathway) and ceramide (via phosphatase activation) may compete for the modulation of PKB activity in astrocytes. Supporting this notion, overexpression of ceramide-sensitive wild-type PKB abrogated the apoptotic effect of ceramide. Because activation of PKB triggers the phosphorylation of different targets involved in preventing apoptosis, including Bad, forkhead transcription factors, IκB kinase, and caspase 9 (
      • Vanhaesebroeck B.
      • Alessi D.R.
      ), ceramide inhibition of PKB could lead to suppression of the survival signal, whereas cannabinoid-dependent reactivation of the pathway would restore it.
      Expression of a dominant-negative form of PKB abolishes the protective effect of cannabinoids but does not induce apoptosis by itself, indicating that the apoptotic effect of ceramide and therefore the generation of a survival signal may also depend on the modulation of additional pathways. Thus, several data suggest that the ERK pathway may participate together with PKB activation in the anti-apoptotic effect of cannabinoids as follows: (i) inhibition of the ERK pathway also prevents the protective effect of cannabinoids, and (ii) astrocyte challenge with cannabinoids leads to activation of both ERK and RSK. One of the mechanisms whereby ERK prevents apoptosis in neural cells involves activation of its downstream kinase RSK as this kinase phosphorylates Bad and the transcription factor cAMP-response element-binding protein (
      • Yuan J.
      • Yankner B.A.
      ). Thus RSK may act synergistically with PKB to prevent apoptosis (
      • Nebreda A.R.
      • Gavin A.-C.
      ). In our model, triggering of the survival signal is accompanied by a consistent activation of ERK and RSK. Nevertheless, incubation with ceramide leads to apoptosis and activation of ERK and RSK, although to a lower extent than with cannabinoid co-treatment. Interestingly, blockade of PI3K prevents the effect of cannabinoids on ERK and RSK but not ceramide-induced activation of these kinases. These data are in line with recent results of our group
      I. Galve-Roperh, D. Rueda, T. Gómez del Pulgar, G. Velasco, and M. Guzmán, submitted for publication.
      showing that stimulation of ERK by cannabinoids depends on PI3K and suggest that the latter may be involved in the pro-survival effect of cannabinoids also via activation of the ERK/RSK pathway. It is worth noting that RSK activation also depends on phosphorylation by 3-phosphoinositide-dependent kinase 1 on its N-terminal domain (
      • Williams M.R.
      • Arthur J.S.C.
      • Balendran A.
      • Van der Kaay J.
      • Poli V.
      • Cohen P.
      • Alessi D.R.
      ). Although that phosphorylation site has been suggested to be constitutive (
      • Williams M.R.
      • Arthur J.S.C.
      • Balendran A.
      • Van der Kaay J.
      • Poli V.
      • Cohen P.
      • Alessi D.R.
      ), it cannot be ruled out that under certain circumstances PI3K activation could lead to 3-phosphoinositide-dependent kinase 1-dependent phosphorylation and activation of RSK (
      • Park J.
      • Hill M.M.
      • Hess D.
      • Brazil D.P.
      • Hofsteenge J.
      • Hemmings B.A.
      ).
      In short, data presented here indicate that cannabinoids protect primary astrocytes from ceramide-induced apoptosis via activation of the PI3K/PKB pathway. Our data also suggest that cannabinoids are involved in protecting astrocytes in vivo. Although the mechanisms of ceramide generation in astrocytes in vivo are still unknown, it is possible that exposure to proinflammatory cytokines (
      • Singh I.
      • Pahan K.
      • Khan M.
      • Singh A.K.
      ) or to saturated fatty acids (
      • Blázquez C.
      • Galve-Roperh I.
      • Guzmán M.
      ) may increase ceramide production in astrocytes during situations of brain injury. It is curious that, unlike this protective effect on astrocytes, cannabinoids induce apoptosis of glioma cells (
      • Galve-Roperh I.
      • Sánchez C.
      • Cortés M.
      • Gómez del Pulgar T.
      • Izquiedo M.
      • Guzmán M.
      ,
      • Sánchez C.
      • de Ceballos M.L.
      • Gómez del Pulgar T.
      • Rueda D.
      • Corbacho C.
      • Velasco G.
      • Galve-Roperh I.
      • Huffman J.W.H.
      • Ramón y Cajal S.
      • Guzmán M.
      ,
      • Gómez del Pulgar T.
      • Velasco G.
      • Sánchez C.
      • Haro A.
      • Guzmán M.
      ). This difference between transformed (glioma) and non-transformed cells (astrocytes) could be due to their ability to synthesize ceramide in response to cannabinoids. Thus, cannabinoids induce apoptosis on glioma cells via stimulation of ceramide synthesis de novo(
      • Gómez del Pulgar T.
      • Velasco G.
      • Sánchez C.
      • Haro A.
      • Guzmán M.
      ), whereas challenge to cannabinoids does not induce ceramide synthesis de novo in astrocytes.
      T. Gómez del Pulgar, G. Velasco, and M. Guzmán, unpublished results.
      Taken together, these data suggest that cannabinoid receptors are coupled to different pathways and therefore lead to different responses in glioma cells and astrocytes. Accordingly, cannabinoids are being tested as potential antitumoral drugs in the treatment of malignant gliomas and, given the crucial role of astrocytes in brain homeostasis and neuroprotection, our results raise the suggestive although still speculative idea of their usage as therapeutic agents for the management of neurodegenerative disorders.

      Acknowledgments

      We are grateful to Dr. D. Alessi, Dr. W. Ogawa, Dr. C. Sutherland, Dr. R. Mechoulam, and Sanofi Synthelabo for the kind donation of reagents; Dr. J. Lizcano and Dr. I. Galve-Roperh for helpful suggestions on the signaling experiments; Dr. L. López- Mascaraque and Dr. L. M. Garcı́a-Segura for helpful suggestions on the in vivo experiments; and A. Carracedo, Dr. C. Blázquez, Dr. D. Rueda, and M. E. Fernández de Molina for technical assistance.

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