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converting enzyme activation through stimulation of 5-HT2B and 1D autoreceptors in neuronal cells
* Institut André Lwoff-Institut Pasteur, CNRS UPR 1983, Laboratoire de Différenciation Cellulaire et Prions, Villejuif Cedex, France;
Pharma Research Department, F. Hoffmann-La-Roche Ltd., Basel, Switzerland; and
Service de Biochimie, EA3621 Hôpital Lariboisière, Faculté de Pharmacie, Université Paris V, Paris, France
1 Correspondence: Différenciation Cellulaire et Prions, Institut André Lwoff, CNRS UPR 1983, 7 rue Guy Môquet, 94801 Villejuif Cedex, France. E-mail: bschneid{at}vjf.cnrs.fr
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ABSTRACT |
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1D adrenoceptors are functionally coupled to reactive oxygen species (ROS) synthesis through NADPH oxidase activation in 1C115-HT and 1C11NE cells. This observation constitutes the prime evidence that bioaminergic autoreceptors take part in the control of the cellular redox equilibrium in a neuronal context. Second, our data identify TACE (TNF- Converting Enzyme), a member of a disintegrin and metalloproteinase (ADAM) family, as a downstream target of the 5-HT2B and 1D receptor-NADPH oxidase signaling pathways. Upon 5-HT2B or 1D receptor stimulation, ROS fully govern TNF- shedding in the surrounding milieu of 1C115-HT or 1C11NE cells. Third, 5-HT2B and 1Dreceptor couplings to the NADPH oxidase-TACE cascade are strictly restricted to 1C11-derived progenies that have implemented a complete serotonergic or noradrenergic phenotype. Overall, these observations suggest that 5-HT2B and 1D autoreceptors may play a role in the maintenance of neuron- and neurotransmitter-associated functions. Eventually, our study may have implications regarding the origin of oxidative stress as well as up-regulated expression of proinflammatory cytokines in neurodegenerative disorders, which may relate to the deviation of normal signaling pathways.—Pietri, M., Schneider, B., Mouillet-Richard, S., Ermonval, M., Mutel, V., Launay, J.-M., Kellermann, O. Reactive oxygen species-dependent TNF- converting enzyme activation through stimulation of 5-HT2B and 1D autoreceptors in neuronal cells.
Key Words: bioaminergic autoreceptors • ROS signaling • metalloproteinase • neuronal differentiation • neuronal homeostasis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For instance, alterations of serotonin (5-hydroxytrypamine, 5-HT)- or norepinephrine (NE) -dependent signaling pathways have been reported in Alzheimer’s disease (1
, 2)
. Oxidative stress is also widely suspected to be a major cause of neuronal cells dysfunction or death in these diseases, since postmortem brain tissues from affected patients clearly show enhanced indices of reactive oxygen species (ROS) (3
, 4)
. A crucial unanswered question, however, is whether oxidative stress originates from deviation of ROS-coupled signaling pathways that normally take part in the control of neuronal functions.
Over the past few years, ROS have emerged as effectors of intracellular signaling cascades (5)
recruited upon stimulation of a wide variety of receptors, including receptors for cytokines, receptors tyrosine kinase, G-protein coupled receptors (GPCRs), or ion channel-linked receptors (for a review, see ref 6
). Recently, ROS have been suspected to mediate 5-HT2 receptor- as well as 1 adrenoceptor-dependent hypertrophy of smooth muscle cells (7
8
9)
or cardiomyocytes (10
, 11)
.
By innervating most parts of the brain, the 5-HT and NE monoaminergic systems control a wide range of behavioral and physiological processes, including sleep, cognition, and mood. As many as 14 5-HT receptors (12
, 13)
and 9 adrenoceptors (14)
, most of which are GPCRs, transduce 5-HT- and NE-associated signals. Assessing how each receptor contributes to 5-HT or NE physiological functions is greatly hindered both by the diversity of receptor subtypes and the lack of selective pharmacological tools for each subtype.
The present study exploits a neuroectodermal murine progenitor, 1C11, endowed with the capacity to differentiate into serotonergic (1C115-HT) or noradrenergic (1C11NE) neuronal cells (15)
to probe the possible involvement of 5-HT or NE in regulating the cellular redox state in neurons. Upon induction by Bt2cAMP, almost 100% of 1C11 cells acquire, within 4 days, a complete serotonergic phenotype (1C115-HT d4) including 5-HT synthesis, storage, and uptake. 1C115-HT cells respond to external 5-HT from day 2 via 5-HT2B and 5-HT1B/1D receptors (16)
. At day 4, implementation of 5-HT2A receptors extends the repertoire of neurotransmitter receptors present on 1C115-HT cells. Under combined addition of Bt2cAMP and DMSO, 100% of 1C11 cells are converted within 12 days into fully functional noradrenergic neurons (1C11NE d12) able to synthesize, store, and take up NE. 1C11NE cells transduce NE signals through a single 1D adrenoceptor induced at day 8 (15)
. Along either differentiation program, the bioaminergic receptors act as autoreceptors and mediate the effects of 5-HT or NE in the coordination and/or onset of all neurotransmitter-associated functions (15)
.
Here, we show that NADPH oxidase-dependent ROS synthesis is elicited upon stimulation of 5-HT2B receptors or 1D adrenoceptors in 1C115-HT or 1C11NE bioaminergic neurons. The ROS response imparted by each receptor is restricted to fully differentiated 1C11-derived progenies. In search for downstream targets of the 5-HT2B or 1D receptor-NADPH oxidase signaling pathways, we identified TNF- converting enzyme (TACE), a member of a disintegrin and metalloproteinase (ADAM) family, as being activated by 5-HT or NE in 1C115-HT or 1C11NE cells, respectively.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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18
19
. LY 266097 was synthesized at Hoffman-La Roche AG (Basel, Switzerland) according to ref 20
. Antagonists (indoramin, 5-methylurapidil, WB-4101, terazosin, alfuzosin, doxazosin, prazosin, albanoquil) of adrenoceptor subtypes were chosen according to ref 15
.
Antibodies
C-20, a goat polyclonal antibody against the p47PHOX NADPH oxidase subunit, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). N-19, a goat polyclonal antibody against the p67PHOX subunit, was from Santa Cruz Biotechnology. C-14, a rabbit polyclonal antibody targeting the Rac1 p21 GDP/GTP binding protein was from Santa Cruz Biotechnology.
Cell culture and enzyme inhibition
1C11 cells were grown and induced to differentiate along the serotonergic (1C115-HT) or catecholaminergic (1C11NE) pathway as described previously (15)
. NADPH oxidase activity was inhibited with diphenyleneiodonium (DPI Sigma) by pretreating cells at 37°C for 45 min in culture medium containing the inhibitor. TACE was switched off by pretreating cells with TAPI-2 (TNF- processing inhibitor-2, Peptides International, Louisville, KY, USA), following the same procedure as for DPI experiments.
Reactive oxygen species detection
Extracellular release of ROS from 1C11 precursor cells and their neuronal progenies (1C115-HT and 1C11NE) was followed using the fluorogenic reagent OxyBurstR Green H2HFF BSA (Molecular Probes, Breda, The Netherlands) (21)
. In a typical assay, cells grown in 96-well microculture plate were washed twice with PBS supplemented with 1 mM Ca2+ and Mg2+ and preincubated for 2 min at 37°C in the presence of the fluorogenic reagent (10 µg·mL–1) in serotonin-free culture medium without phenol red to avoid fluorescence interference. For stimulation of 1C115-HT or 1C11NE cells with 5-HT2 agonists or norepinephrine (NE), respectively, fluorescence was continuously recorded at 
= 528 nm (slit width=10 nm) with excitation at = 488 nm (slit width=10 nm) using a Cary Eclipse fluorometer (Varian Inc., Palo Alto, CA, USA).
Membrane fraction preparation and immunoprecipitation of 32P-labeled p47PHOX and p67PHOX NADPH oxidase subunits
1C11 precursor, 1C115-HT, or 1C11NE cells were washed in PBS supplemented with 1 mM Ca2+ and Mg2+ and incubated in phosphate-free DMEM containing 50 µCi [32P]PO43– (NEN-Life Science) per milliliter per 106 cells for 1 h at 37°C. After receptor stimulation with agonists or NE, 32P-labeled cells were washed twice with ice-cold PBS, then scraped off into PBS containing a cocktail of protease inhibitors. After centrifugation, the supernatant was removed and the pellet was frozen at –80°C. Plasma membranes were prepared according to ref 16
in the presence of 100 mM Na3VO4 to avoid protein dephosphorylation. p47PHOX and p67PHOX NADPH oxidase subunits were immunoprecipitated from the plasma membrane fraction overnight at 4°C under gentle mixing using G Sepharose beads (Amersham Pharmacia, Piscataway, NJ, USA) coupled to C-20 and N-19 antibodies, respectively. Immunocomplexes were resolved by 10% SDS-PAGE and transferred to Immobilon membranes. 32P-labeled p47PHOX and p67PHOX were detected using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA).
Small G-protein Rac1 activation assay
Rac1 GDP/GTP binding protein activation in 1C11 precursor cells and their neuronal progenies, 1C115-HT and 1C11NE, was assessed upon binding of [35S]GTP
S (NEN-Life Science), a nonhydrolyzable analog of GTP. Plasma membranes of 1C11, 1C115-HT, or 1C11NE cells (16)
were loaded in assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, and 3 mM MgCl2) at a final protein concentration of 0.5 mg/mL. Test compounds (agonists of 5-HT2B receptors, NE or PMA) were added to the membrane fraction along with 5 µM GDP and 0.05 nM [35S]GTPS and incubated at 37°C for 15 min. The small G-protein Rac1 protein was immunoprecipitated overnight at 4°C under gentle mixing using A Sepharose beads (Amersham Pharmacia) coupled to C-14 antibody. Immunocomplexes were resolved by 10% SDS-PAGE and transferred to Immobilon membranes. Bound [35S]GTPS to Rac1 was detected using a PhosphorImager (Molecular Dynamics).
Tumor necrosis factor- (TNF-) quantification
TNF- from culture media was measured with ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Cytosolic extracts of 1C115-HT and 1C11NE neuronal cells were prepared by incubating cells for 30 min at 4°C in NET lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 100 mM Na3VO4, and a cocktail of protease inhibitors). Extracts were centrifuged at 14,000 x g for 15 min. Protein concentrations in the supernatants were measured by using the bicinchoninic acid method (Pierce, Rockford, IL, USA). To standardize the results, induced TNF- shedding was expressed in TNF- units per milligram of cytosolic protein.
Radioligand binding
Binding experiments were performed on intact cells using 1C115-HT, 1C11NE cells or mouse LMTK– fibroblasts stably transfected with cDNAs encoding mouse 5-HT2A, 5-HT2B receptors, or 1D adrenoceptors (as described in refs 15
, 16
, 22
).
Data analysis
Binding data were analyzed by the iterative nonlinear fitting software Prism (GraphPad, San Diego, CA, USA). This allowed the calculation of dissociation equilibrium constants (KD) for saturation experiments, as well as inhibition constants (KI) for displacement studies. Statistical analysis on small groups used the Student test from the Kaleidagraph software (Abelbeck, Reading, PA, USA). The chosen significance criterion was P < 0.001. All values are given as means ± SE mean or 95% confidence intervals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). With 1C115-HT d4 cells that express all three 5-HT receptor subtypes, 5-CT stimulation still failed to induce ROS release (Fig. 1A
). By contrast, ROS synthesis became detectable as soon as 10 min after DOI addition, and reached a plateau after 30 min stimulation (Fig. 1B
, inset). The intensity of ROS release increased with agonist concentration in a dose-dependent manner (Fig. 1B
). The maximal response (40-fold over basal level) was reached at 20 nM DOI, allowing us to define an associated EC50 value of 8.9 ± 0.8 nM DOI (n=5). These data highlight that, in fully differentiated 1C115-HT d4 cells, stimulation of 5-HT2 receptors elicits ROS release.
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The 5-HT-induced ROS production in 1C115-HT fully differentiated serotonergic cells is mediated by the 5-HT2B receptor subtype
Since DOI targets both 5-HT2A and 5-HT2B receptor subtypes, we selected several agonists with distinct binding properties toward each receptor to determine their relative contributions to the DOI-induced ROS release. Stimulation of 1C115-HT d4 cells with 1 µM 5-HT triggered the greatest ROS response observed in this study (Fig. 1C
). BW723C86 (40 nM) and Ro 60.0175 (5 nM), two selective agonists of 5-HT2B receptors, promoted ROS generation with an efficacy comparable to that initiated by 5-HT (95% and 92%, respectively; Fig. 1C
). The DOI (40 nM) response represented 73% of the 5-HT-mediated ROS synthesis (Fig. 1C
). The agonist MK212 (1.5 µM), which preferentially binds 5-HT2A receptors but also recognizes 5-HT2B receptors, elicited a ROS production equivalent to only 50% of that induced by 5-HT (Fig. 1C
). These data suggest that the ROS production recorded with 1C115-HT d4 cells relates to the 5-HT2B receptor subtype.
By selecting a panel of agonists and antagonists that allows us to discriminate between 5-HT2B and 5-HT2A receptor subtypes and to specify the 5-HT2B receptor (17
18
19)
, we sought to confirm that the DOI-induced ROS production was imparted by 5-HT2B receptors. The pharmacological profile of the 5-HT2B receptor in 1C115-HT d4 cells was determined by measuring the binding affinities (pKI) of each drug used as a competitor of DOI binding. The dose-response effects of agonists on ROS release were then directly followed to determine pEC50 values. In the case of antagonists, their capacity to inhibit the DOI-dependent ROS synthesis was monitored to calculate pKB values. A highly significant correlation (rs=0.865, n=19, P<0.0001) was obtained between the agonists and antagonists binding constants (pKI) measured with 1C115-HT d4 cells and the apparent equilibrium constants (pEC50 and pKB) deduced from the drug effects on ROS production (Fig. 1D
). Because the set of selected drugs specifies the 5-HT2B receptor (17
18
19)
, these pharmacological data demonstrate that ROS production in 1C115-HT d4 cells is strictly related to 5-HT2B receptor activity.
The absence of ROS response upon 5-HT2B receptor stimulation at day 2 of differentiation cannot be accounted for by changes in the number of 5-HT2B receptors (2500 sites/cell at day 2 as well as day 4) or in the pharmacological profile of the receptor (15
, 23)
. We may instead propose that the onset of the 5-HT2B-dependent ROS coupling in 1C115-HT cells relates to the implementation of the overall serotonergic functions per se.
NADPH oxidase controls 5-HT2B receptor-dependent ROS production
Because NADPH oxidase is a powerful ROS generator, we probed an involvement of this enzyme in the 5-HT2B receptor-mediated ROS response. NADPH oxidase is a multicomponent protein whose activation relies on phosphorylation of the cytosolic subunits p47PHOX and p67PHOX, their translocation to the plasma membrane, and recruitment of the small GTPases Rac (24
, 25)
. Exposure of 1C115-HT d4 cells to 40 nM DOI or BW723C86 led to a significant increase in the phosphorylation level of both p47PHOX and p67PHOX subunits associated with the plasma membrane fraction as early as 5 min after agonist stimulation (Fig. 2
A). Agonist treatment of 1C115-HT d4 cells strongly activated the small GTPase Rac1 as monitored through its capacity to bind [35S]GTPS (Fig. 2A
). We definitively assessed the engagement of NADPH oxidase in 5-HT2B receptor-dependent signaling pathways, using DPI, a selective inhibitor of NADPH oxidase. Exposure of 1C115-HT d4 cells to 100 µM DPI prior to DOI addition fully cancelled the ROS production (Fig. 2B
). These overall results identify NADPH oxidase-dependent ROS production as a novel feature of 5-HT2B receptor signaling in 1C11-derived serotonergic neurons.
|
Since the link between 5-HT2B receptors and ROS is restricted to fully differentiated 1C115-HT d4 cells, we wondered whether the expression of NADPH oxidase varied between day 2 and day 4 of the serotonergic program. Irrespective of the stage of differentiation, the phorbol ester PMA (1 µM), a potent activator of NADPH oxidase through protein kinase C (PKC) (26)
, elicited comparable ROS responses (Fig. 2C
). Membrane-associated phosphorylated p47PHOX and p67PHOX components were detected as early as 1 min after PMA stimulation with 1C11, 1C115-HT d2, and 1C115-HT d4 cells (Fig. 2D
). Whatever the differentiation state of the cells, exposure to PMA promoted Rac1 activation (Fig. 2D
). Finally, at any time of the serotonergic program, the PMA-induced ROS response was abrogated by addition of 100 µM DPI (Fig. 2C
), demonstrating the functionality of NADPH oxidase throughout 1C11 serotonergic differentiation. Our overall findings emphasize that NADPH oxidase becomes a target of 5-HT2B receptors signaling upon implementation of a complete serotonergic phenotype.
1D Adrenoceptor stimulation elicits NADPH oxidase-dependent ROS production in fully differentiated noradrenergic 1C11NE d12 cells
Along the noradrenergic differentiation program, a single 1D adrenoceptor is induced at day 8. The number of 1D sites (2200 sites/cell) and the pharmacological profile of the receptor do not change from day 8 to day 12, when differentiation is completed (15)
. 1C11NE cells were exposed to NE (1–100 nM) to test whether 1D receptors could instigate ROS release. At day 8 of differentiation, NE failed to induce any ROS production (Fig. 3
A, inset). ROS release became measurable at day 12 in cells harboring a fully functional noradrenergic phenotype (1C11NE d12). The ROS response was detectable 10 min after 1D receptor stimulation and reached a plateau after 30 min (Fig. 3A
, inset). It increased with NE concentration in a dose-dependent manner (Fig. 3A
). A maximal response (10-fold over basal level) was obtained with 50 nM NE, corresponding to an associated EC50 value of 18.2 ± 0.1 nM NE (n=5).
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A set of selective antagonists was further used to confirm that the NE-induced ROS synthesis was controlled by 1D adrenoceptors. A highly significant correlation (rs=0.994, n=8, P<0.0001) was obtained between the antagonists binding constants (pKI) measured with 1C11NE d12 cells and the apparent equilibrium constants (pKB) deduced from the inhibitory effects of antagonists on NE-induced ROS production (Fig. 3B
). Thus, we conclude that the adreno 1D receptor is functionally linked to ROS synthesis in noradrenergic cells.
Finally, NADPH oxidase appears to be an intracellular target of 1D adrenoceptor signaling activity. Indeed, exposure of 1C11NE d12 cells to 100 nM NEstimulated phosphorylation and plasma membrane translocation of p47PHOX and p67PHOX subunits concomitant with Rac1 activation (Fig. 3C
). Besides, treatment of 1C11NE d12 cells with DPI abrogated the NE-induced ROS production (Fig. 3D
). As observed for the serotonergic pathway, NADPH oxidase is functional throughout noradrenergic differentiation, as evidenced by PMA effects on ROS release (Fig. 3C, D
).
Our data demonstrate a functional coupling of adreno 1D autoreceptors to NADPH oxidase-dependent ROS synthesis in 1C11NE d12 cells. Again, the onset of all neurotransmitter-associated functions of noradrenergic neurons at day 12 of the differentiation program appears to be a prerequisite for NE-dependent ROS production.
The coupling of 5-HT2B receptors to NADPH oxidase instructs TACE activation
ROS, as signaling transducers, may regulate transcription factors or promote kinase activation or ectodomain shedding of membrane-bound proteins (5
, 6
, 27)
. The ectodomain shedding of many cell surface proteins is carried out by enzymes of the ADAM (a disintegrin and metalloproteinase) family. One of the most well-known ADAM members is TACE (tumor necrosis factor- converting enzyme), also called ADAM 17, which primarily governs the shedding of TNF- (28)
. In view of a recent report describing a redox-mediated activation of TACE in monocytic cells (27)
, we wondered whether TACE could represent a downstream target of the 5-HT2B receptor-NADPH oxidase signaling cascade in serotonergic neurons.
Release of TNF- (17 kDa), which results from the cleavage of the 26 kDa transmembrane pro-TNF- protein, was used as a sensitive marker to assess TACE-mediated ectodomain shedding. 1C115-HT cells were exposed to either 40 nM DOI or 40 nM BW723C86 for 5 min up to 240 min. At day 2 of differentiation, agonists failed to induce any TNF- shedding (data not shown). With 1C115-HT d4 cells, TNF- release became detectable in the culture medium as soon as 10 min after receptor stimulation. Maximum levels were reached after 30 min of exposure to agonists (Fig. 4
A). Comparable TNF- responses were monitored with DOI or BW723C86 (Fig. 4A
). Agonist-induced TNF- release was totally abolished upon addition of 50 nM ritanserin, an inverse agonist of 5-HT2 receptor subtypes, or 5 nM RS 127.445, a selective antagonist of 5-HT2B receptors (Fig. 4A
). Exposure of 1C115-HT d4 cells to BW723C86 (40 nM) in combination with 100 µM TAPI-2, a TACE inhibitor, failed to induce any TNF- shedding (Fig. 4B
). These data demonstrate that the 5-HT2B receptor instigates TNF- shedding in fully differentiated 1C115-HT d4 cells and introduce TACE as a new downstream target of 5-HT2B receptors.
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We next examined whether the TACE-mediated TNF- shedding observed in response to 5-HT2B receptor stimulation would depend on NADPH oxidase activation. Exposure of 1C115-HT d4 cells to 100 µM DPI switched off the BW723C86-induced TNF- release (Fig. 4C
). The PMA-dependent TNF- release was also quenched upon DPI treatment (Fig. 4C
). These data highlight that, in 1C115-HT d4 cells, TACE activation is strictly ROS dependent. They draw a functional link between the 5-HT2B receptor and TNF- shedding through a NADPH oxidase-mediated signaling cascade.
The 1D autoreceptor-dependent ROS coupling controls TNF- shedding in fully differentiated noradrenergic 1C11NE d12 cells
We assessed whether, in the 1C11 cell system, 1D adrenoceptors exposed to NE induced TACE-dependent TNF- shedding. Though 1D receptors are implemented as early as day 8 of 1C11 noradrenergic differentiation, TNF- release could not be monitored before day 12 (data not shown). In the presence of 100 nM NE, TNF- release by 1C11NE d12 cells reached its maximum at 30 min (Fig. 4D
). TNF- shedding was quenched upon addition of 100 µM TAPI-2 or 100 µM DPI (Fig. 4D
). We therefore conclude that in fully differentiated 1C11NE noradrenergic cells, 1D adrenoceptors instruct TACE activation through NADPH oxidase-induced ROS signaling.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 29
, 30)
, on the one hand, and that of 1D adrenoceptors on noradrenergic neurons of the locus coeruleus (31
32
33)
, on the other, define these bioaminergic receptors as candidate protagonists participating in the regulation of serotonergic- or noradrenergic-associated functions. That these receptors may act in vivo as autoreceptors is strongly suggested by the detection of their corresponding transcripts or binding sites in serotonergic (12)
and noradrenergic (31)
neurons. In support of this hypothesis is the observation that 1 adrenoceptors modulate 2 adrenoceptor-dependent NE firing within an autocrine loop (32)
. However, uncovering the couplings that link such autoreceptors to their specific cellular targets is hindered by the variety of signals and potential cross-talks involved in cellular responses in intact tissues. To perhaps overcome this limitation, we used the bipotential neuroectodermal 1C11 progenitor able to convert into serotonergic (1C115-HT) or noradrenergic (1C11NE) neurons expressing defined subsets of receptors (15
, 16)
.
The present work establishes that 5-HT2B or 1D receptor stimulation causes NADPH oxidase-dependent ROS generation in 1C115-HT and 1C11NE cells, respectively. Functional couplings between 5-HT2B or 1D receptors and NADPH oxidase are demonstrated by measuring highly significant correlations between the binding constants of agonists and antagonists on each receptor, on one side, and the dose-response effects of the same drugs on NADPH oxidase activity on the other. In corollary, our data provide the prime evidence that neurotransmitter receptors take part in the regulation of the redox state in neuronal cells.
The ability of 5-HT2B or 1D receptors to recruit NADPH oxidase is restricted to fully differentiated 1C11-derived serotonergic (1C115-HT d4) or noradrenergic (1C11NE d12) progenies. A delay in the onset of the functional NADPH oxidase multicomponent enzymatic system cannot be put forward to account for this neuronal-specific response. Indeed, NADPH oxidase-dependent ROS production can be induced after PMA treatment in the 1C11 cell system whatever the state of differentiation, as monitored in ref 21
. It is difficult to understand why the 5-HT2B- and 1D receptor-dependent NADPH signalings are specific to 1C11 cells harboring a complete neuronal phenotype whereas functional couplings of the same receptors to other effectors (PLC, NOS, and PLA2 for the 5-HT2B receptor, PLC for the 1D receptor) occur from day 2 of the serotonergic differentiation or day 8 of the noradrenergic program (15
, 22
, 23
, 34)
. Over the past few years, sphingolipid- and cholesterol-rich membrane microdomains ("lipid raft") have emerged as preferential transduction "centers" (35
36
37)
. Notably, the dynamic assembly and enzymatic function of the NADPH oxidase complex has been reported to be associated with rafts (38)
. Considering the PMA-induced NADPH oxidase activation, it is likely that irrespective of the stage of differentiation, rafts in which operational signaling events may occur are present in 1C11 cells. To account for the lack of functional coupling between 5-HT2B or 1D receptors and NADPH oxidase at intermediate stages of differentiation, we may assume that these proteins are each localized in distinct raft microdomains. At the final stages of differentiation, maturation of the membrane properties of 1C115-HT or 1C11NE cells would be associated with a spatial redistribution of signaling partners, thereby enlarging the repertoire of the 5-HT2B or 1D receptor downstream pathways. The ability of these bioaminergic receptors to recruit NADPH oxidase would hence depend strictly on the availability of the latter protein in the vicinity of the receptors. Alternatively, we may propose that the 5-HT2B- or 1D receptor-associated NADPH oxidase signaling cascades involve yet-to-be-identified intermediate molecules specifically expressed in fully differentiated cells. In any case, restriction to mature bioaminergic neurons of the receptor couplings to NADPH oxidase argues against the idea that these signaling cascades instruct early neuronal differentiation processes. Rather, by taking part in the control of the cellular redox balance, the bioaminergic receptors may contribute to the regulation and/or maintenance of neuronal- or neurotransmitter-associated functions.
Another major finding is the identification of the TACE metalloproteinase as a novel downstream target of the 5-HT2B- and 1D receptor-associated signaling pathways in 1C11-derived neuronal cells. TACE activation has been reported to result from a post-translational processing in which ROS oxidation of a crucial cysteine thiol group leads to full enzymic activity (27)
. In line with these data, we further demonstrate that agonist-induced ROS responses fully govern 5-HT2B or 1D receptor-mediated TACE activity. Therefore, in 5-HT2B and 1D receptor-TACE cascades, NADPH oxidase-dependent ROS act as second message signals and control the biochemical activation of TACE. In turn, TACE catalyzes the shedding of TNF- in the extracellular environment of mature 1C115-HT or 1C11NE cells.
TNF- exerts its action through two membrane receptors, TNFR1 and TNFR2, which may subsequently mobilize promiscuous pathways (39
, 40)
. The large spectrum of TNF- downstream intracellular effectors accounts for the wide array of cellular responses regulated by this cytokine, including apoptosis, cell survival, proliferation (41)
, and/or differentiation (42)
. In the CNS, TNF- may contribute to neuroprotection and neurosurvival mechanisms (43)
. With fully differentiated 1C115-HT and 1C11NE cells, we did not observe any cell death upon agonist-induced TNF- shedding (result not shown). We therefore favor a hypothesis where the coupling between 5-HT2B or 1D receptors and TACE-dependent TNF- release takes part in the maintenance of the neuronal phenotype. Several observations are in line with this idea. First, neuronal-associated TNF- down-regulates NE release from noradrenergic nerve terminals in the CNS (44)
. In addition, brain administration of exogenous TNF- enhances 5-HT catabolism (45)
. Besides, TNF- regulates GPCRs signaling function through modulation of heterotrimeric G-protein expression levels and activities (40
, 46)
. Finally, TACE has been reported to control the level of active TNF- by catalyzing TNF- shedding, on the one hand, and by regulating the cleavage of the membrane-expressed receptor TNFR2 into a soluble form that can sequestrate TNF- on the other (47)
. As a whole, these reports strongly suggest that TNF- plays an important role in the regulatory loops that control bioamine metabolism. By identifying new signal transduction events linking two bioaminergic receptors to TACE-dependent TNF- shedding, our study unveils a scenario within which 5-HT or NE take part in the fine-tuning of their own integrated metabolisms.
Dysfunctions of the 5-HT or NE systems appear to be at the root of several neurological diseases. The occurrence of defective bioaminergic signaling in various neurodegenerative disorders such as Alzheimer’s disease is now widely documented (1)
. In these affections, neuronal injury or death associated with aberrant protein aggregation may originate from interference with normal signal transduction pathways. Oxidative stress (3
, 4)
and up-regulated expression of proinflammatory cytokines such as TNF- (48
49
50
51)
, which both play a central role in neuronal cell demise, are common hallmarks of these pathologies. By linking 5-HT2B and adreno 1D autoreceptors to NADPH oxidase and TACE, the present work gives some clues as to how alterations of 5-HT or NE normal signaling pathways, in relation to abnormal protein aggregation, may cause imbalances in ROS and TNF- levels. Targeting these normal pathways to alleviate loss of neuronal homeostasis may represent new therapeutic prospects in the field of neurodegenerative disorders.
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ACKNOWLEDGMENTS |
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Received for publication February 2, 2005. Accepted for publication March 8, 2005.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
cytokine production by ventricular fibroblasts. Circulation 110,969-974-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes. J. Mol. Cell. Cardiol. 33,131-139[CrossRef][Medline]
(1)-adrenoceptor signaling in adult rat cardiac myocytes. Am. J. Physiol. 282,C926-C934
-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate. FASEB J. 15,303-305 converting enzyme. Int. J. Biochem. Cell Biol. 34,1-5[CrossRef][Medline]
(1)-adrenoceptors that suppress the GIRK conductance in adult rat locus coeruleus neurons. Br. J. Pharmacol. 135,226-232[CrossRef][Medline]
, interleukin 1 and related cytokines in brain development: normal and pathological. Dev. Neurosci. 14,1-10[Medline]
): its role in noradrenergic functioning and modification of its expression following antidepressant drug administration. J. Neuroimmunol. 79,84-90[CrossRef][Medline]
: neuroendocrine, central monoamine, and behavioral variations. J. Neurosci. 19,5654-5665 (i2) subunits in human polymorphonuclear leukocytes. J. Immunol. 158,913-918[Abstract]
, interleukin 1 , and glial fibrillary acidic protein in experimental Creutzfeldt-Jakob disease in mice. Proc. Natl. Acad. Sci. USA 93,9754-9758This article has been cited by other articles:
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  L. Monassier, M.-A. Laplante, F. Jaffre, P. Bousquet, L. Maroteaux, and J. de Champlain Serotonin 5-HT2B Receptor Blockade Prevents Reactive Oxygen Species-Induced Cardiac Hypertrophy in Mice Hypertension, August 1, 2008; 52(2): 301 - 307. [Abstract] [Full Text] [PDF]
|
|
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|
 S. Thifault, S. Ondrej, Y. Sun, A. Fortin, E. Skamene, R. Lalonde, J. Tremblay, and P. Hamet Genetic determinants of emotionality and stress response in AcB/BcA recombinant congenic mice and in silico evidence of convergence with cardiovascular candidate genes Hum. Mol. Genet., February 1, 2008; 17(3): 331 - 344. [Abstract] [Full Text] [PDF]
|
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 Y.-J. Peng, G. Yuan, F. J. Jacono, G. K. Kumar, and N. R. Prabhakar 5-HT evokes sensory long-term facilitation of rodent carotid body via activation of NADPH oxidase J. Physiol., October 1, 2006; 576(1): 289 - 295. [Abstract] [Full Text] [PDF]
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 H. Ohtsu, P. J. Dempsey, and S. Eguchi ADAMs as mediators of EGF receptor transact |
