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,Michael A Beazely,Maryam S Vasefi,John J Heikkila,Shilpa J Buch

doi:10.1371/journal.pone.0077027

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.

Highlights

Serotonin (5-HT) is a tryptophan-derived signaling molecule best known for its role as a neurotransmitter [1]
Based on transactivation pathways described in other cell types [11,24], we postulated that reactive oxygen species (ROS) are involved in the 5-HT-induced transactivation of neuronal platelet-derived growth factor type β (PDGFβ) receptors
To determine if 5-HT-induced transactivation of PDGFβ receptors involved the generation of endogenous ROS, we pretreated the cells with the ROS scavenger, N-acetyl-Lcysteine, followed by 100 nM 5-HT for 5 min (Figure 1C)

Summary

Introduction

Serotonin (5-HT) is a tryptophan-derived signaling molecule best known for its role as a neurotransmitter [1]. The platelet-derived growth factor type β (PDGFβ) receptor is an important receptor tyrosine kinase (RTK) for the development of the CNS [7,8]. Ligand binding results in the dimerization and activation of the receptor, which triggers intracellular kinase domain-mediated transautophosphorylation of several tyrosine residues [7]. Multiple intracellular signaling pathways are initiated that result primarily in the promotion of cell growth [7], the roles of PDGF signaling in the developed CNS have not been fully elucidated. The magnitude of activation of the PDGFβ receptor during transactivation (as measured by tyrosine phosphorylation) is typically much less than ligand-induced activation [10]. This may explain why ligand-induced activation results in rapid down-regulation of RTKs such as the PDGFβ receptor [9], whereas down-regulation of transactivated PDGFβ receptors has not been observed [10,17]

Methods

Results

Conclusion