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. 2013 Sep 27;8(9):e77027.
doi: 10.1371/journal.pone.0077027. eCollection 2013.

Reactive oxygen species are required for 5-HT-induced transactivation of neuronal platelet-derived growth factor and TrkB receptors, but not for ERK1/2 activation

Jeff S Kruk  1 , Maryam S Vasefi, John J Heikkila, Michael A Beazely
Affiliations

Reactive oxygen species are required for 5-HT-induced transactivation of neuronal platelet-derived growth factor and TrkB receptors, but not for ERK1/2 activation

Jeff S Kruk et al. PLoS One. .

Abstract

High concentrations of reactive oxygen species (ROS) induce cellular damage, however at lower concentrations ROS act as intracellular second messengers. In this study, we demonstrate that serotonin (5-HT) transactivates the platelet-derived growth factor (PDGF) type β receptor as well as the TrkB receptor in neuronal cultures and SH-SY5Y cells, and that the transactivation of both receptors is ROS-dependent. Exogenous application of H₂O₂ induced the phosphorylation of these receptors in a dose-dependent fashion, similar to that observed with 5-HT. However the same concentrations of H₂O₂ failed to increase ERK1/2 phosphorylation. Yet, the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin blocked both 5-HT-induced PDGFβ receptor phosphorylation and ERK1/2 phosphorylation. The increases in PDGFβ receptor and ERK1/2 phosphorylation were also dependent on protein kinase C activity, likely acting upstream of NADPH oxidase. Additionally, although the ROS scavenger N-acetyl-l-cysteine abrogated 5-HT-induced PDGFβ and TrkB receptor transactivation, it was unable to prevent 5-HT-induced ERK1/2 phosphorylation. Thus, the divergence point for 5-HT-induced receptor tyrosine kinase (RTK) transactivation and ERK1/2 phosphorylation occurs at the level of NADPH oxidase in this system. The ability of 5-HT to induce the production of ROS resulting in transactivation of both PDGFβ and TrkB receptors may suggest that instead of a single GPCR to single RTK pathway, a less selective, more global RTK response to GPCR activation is occurring.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. H2O2 increases PDGFβ receptor phosphorylation in SH-SY5Y cells and primary neuron cultures.
(A) SH-SY5Y cells were treated with vehicle (VEH) or 0.01 to 100 µM H2O2 for 5 min. Following drug treatments, cell lysates were evaluated by Western blot analysis as described in Materials and Methods. Data were normalized to total PDGFRβ protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-1021 immunoreactivity compared to vehicle-treated cells. Representative blots for phospho-PDGFRβ 1021 (pY1021) and PDGFRβ at 180 kDa are shown. (B) Primary mouse cortical neuron cultures were treated with 0.1 µM H2O2 for 5 min. Lysates were evaluated for phospho-Y1021 as described in “A”. (C) SH-SY5Y cell cultures were pretreated with vehicle or 1000 µM of the ROS scavenger N-acetyl-l-cysteine (NAC) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. (Data are representative of 4-6 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test, or Student’s t-test).
Figure 2
Figure 2. H2O2 concentrations sufficient for inducing PDGFβ receptor phosphorylation do not result in cell death.
SH-SY5Y cells were treated with 0, 0.1, 1, 10, 100, or 1000 µM H2O2 for (A) 30 min, or (B) overnight. Following treatment with MTT reagents and lysis, cell viability was measured and compared to control (VEH) values. (Data are representative of 4 independent experiments. * = p < 0.05 compared to vehicle-treated cells, one-way ANOVA, Tukey post-test).
Figure 3
Figure 3. 5-HT-induced PDGFβ receptor transactivation requires PKC and NADPH oxidase.
(A) SH-SY5Y cell cultures were pretreated with vehicle or 0.1, 1 or 10 µM of the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. Following drug treatments, cell lysates were evaluated by immunoblot analysis as described in Materials and Methods. Data were normalized to total PDGFRβ protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-1021 immunoreactivity compared to vehicle-treated cells. Representative blots for phospho-PDGFRβ 1021 (pY1021) and PDGFRβ at 180 kDa are shown. (B) Cell cultures were pretreated with vehicle or 1, 10 or 100 µM of the NADPH oxidase inhibitor apocynin for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min, and results were analyzed for phospho-Y1021 as described in “A”. (C) Cultures were pretreated with vehicle or 0.1 µM of the PKC inhibitor Go 6983 for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min, and results were analyzed for phospho-Y1021 as described in “A”. (Data are representative of 3-5 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test).
Figure 4
Figure 4. 5-HT can transactivate TrkB receptors via ROS.
(A) SH-SY5Y cells were treated with vehicle (VEH) or 0.01 to 10 µM H2O2 for 5 min. Following drug treatments, cell lysates were evaluated by Western blot analysis as described in Materials and Methods. Data were normalized to total TrkB protein expression and are expressed as the fold change (average ± S.E.M.) in TrkB phospho-816 immunoreactivity compared to vehicle-treated cells. Representative blots for phospho-TrkB Y816 (pY816) and TrkB at 145 kDa are shown. (B) Cell cultures were incubated with 0.1 µM 5-HT for 0, 1, 2, 5, 10, or 15 min, and fold change in TrkB Y816 phosphorylation was measured with respect to vehicle. (C) Cultures were pretreated with vehicle or 1000 µM of the ROS scavenger N-acetyl-l-cysteine (NAC) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. Normalized data was analyzed for phospho-TrkB Y816. (D) Cells were incubated overnight with 0.01 or 0.1 µg/mL pertussis toxin (Ptx) followed by 5 min treatment with 0.1 µM 5-HT. (E) Cell cultures were pretreated with vehicle or 1 or 10 µM of the PDGF receptor kinase inhibitor AG 1296 for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. Western blots were evaluated for changes in phospho-TrkB Y816. (Data are representative of 5-6 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test).
Figure 5
Figure 5. 5-HT induced ERK1/2 phosphorylation diverges from the transactivation pathway at or after NADPH oxidase.
(A) SH-SY5Y cells were treated with 0.01 to 100 µM H2O2 for 5 min. Following drug treatments, cell lysates were evaluated by Western blot analysis as described in Materials and Methods. Data were normalized to total ERK1/2 protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-ERK immunoreactivity compared to vehicle-treated cells. (B) SH-SY5Y cell cultures were pretreated with vehicle or 10, 100 or 1000 µM of the ROS scavenger N-acetyl-l-cysteine (NAC) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min and lysates were evaluated as in “A”. Cell cultures were also pretreated with vehicle or the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) (C) or apocynin (D) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min, and results were analyzed for phospho-ERK1/2 as described in “A”. (E) Cultures were pretreated with vehicle or 0.1 µM of the PKC inhibitor Go 6983 for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min, and results were analyzed for phospho-ERK1/2 as described above. Representative blots of phospho-ERK1/2 and total ERK1/2 at 42 and 44 kDa are shown. (Data are representative of 4-8 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test).
Figure 6
Figure 6. Mechanism of PDGFβ and TrkB receptor transactivation.
Gαi-coupled GPCRs such as 5-HT1A initiate transactivation signaling, which gets relayed through Gα or Gβγ subunits. PLC activation results in intracellular calcium release and activation of PKC. The NADPH oxidase subunits subsequently assemble and produce ROS. Active NADPH oxidase is required for both 5-HT-induced RTK and ERK1/2 phosphorylation but only endogenous ROS (or exogenous H2O2) is involved in RTK transactivation.
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Grants and funding

This research was funded through generous start-up funding from the University of Waterloo, Faculty of Science, by the National Science and Engineering Research Council of Canada, and by the Queen Elizabeth II Graduate Scholarship in Science and Technology awarded to JSK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.