Phosphoinositide-3-kinase and mitogen activated protein kinase signaling pathways mediate acute NGF sensitization of TRPV1

Abstract

Nerve growth factor (NGF) induces an acute sensitization of nociceptive DRG neurons, in part, through sensitization of the capsaicin receptor TRPV1 via the high affinity trkA receptor. The mechanisms linking trkA and TRPV1 remain controversial with several candidate signaling pathways proposed. Utilizing adult rat and mouse DRG neurons and CHO cells co-expressing trkA and TRPV1, we have investigated the signaling events underlying acute TRPV1 sensitization by NGF combining biochemical, electrophysiological, pharmacological, mutational and genetic knockout approaches. Pharmacological interference with p42/p44 mitogen activated protein kinase (MAPK) or phosphoinositide-3-kinase (PI3K), but not PLC abrogated sensitization of capsaicin responses. Co-expression of TRPV1 with wild-type or Y785F (PLC signal deficient) mutant human trkA reconstituted NGF sensitization. In contrast, TRPV1 co-expressed with MAPK signaling deficient Y490A or PI3K signaling deficient Y751F trkA mutants exhibited weaker sensitization. Biochemical analysis of p42/p44 and Akt phosphorylation confirmed the specificity of pharmacological agents and trkA mutants. Finally, NGF sensitization of capsaicin responses was greatly reduced in neurons from p85α (regulatory subunit of PI3K) null mice. These data strongly suggest that PI3K and MAPK pathways, but not the PLC pathway underlie the acute sensitization of TRPV1 by NGF.

Introduction

The transient receptor potential vanilloid 1 (TRPV1) receptor, a member of the transient receptor potential superfamily of cation channels, was expression cloned as the capsaicin receptor (Caterina et al., 1997). TRPV1 is highly expressed in small diameter sensory neurons including those in dorsal root ganglia (DRG) and trigeminal ganglia (TG). Its activation by different noxious stimuli including heat, protons and chemicals such as capsaicin and anandamide, as well as the striking similarities between the behavior of the cloned TRPV1 and the native receptor expressed in DRG or TG neurons (Tominaga et al., 1998), have established it as a molecular marker of nociceptive sensory neurons. Several molecules released during inflammation or injury, including histamine, protons, bradykinin, prostaglandins and nerve growth factor (NGF), can induce hyperalgesia or allodynia, primarily through sensitization of primary nociceptors. Evidence has accumulated that several of these factors including bradykinin and prostaglandins can trigger this sensitizing effect on sensory neurons through protein kinase C (PKC) or cAMP-dependent protein kinase (PKA)-mediated phosphorylation of TRPV1 (Premkumar and Ahern, 2000, Chuang et al., 2001, Sugiuar et al., 2004, Lopshire and Nicol, 1998, Bhave et al., 2002, Bhave et al., 2003, Mohapatra and Nau, 2003). The details of the mechanisms by which other agents including NGF induce sensitization remain unclear and controversial (Zhang and McNaughton, 2006).

It is well documented that NGF plays a critical role in the development and survival of primary nociceptors through transcriptional mechanisms. However, recent studies have shown that NGF is also an important factor in inflammatory or injury-induced hyperalgesia. NGF can promote maintenance of hyperalgesic states through p38 MAP-kinase-mediated non-transcriptional increases in TRPV1 expression in skin (Ji et al., 2002). In addition to such long-term affects, acute sensitization of sensory neurons by NGF has been observed. In cultured rat DRG neurons NGF can increase TTX-resistant sodium currents and reduce voltage-gated potassium currents, effects attributed to NGF signaling through the low affinity p75 receptor (Zhang et al., 2002). Shu and Mendell, 1999a, Shu and Mendell, 1999b first reported an acute sensitization of capsaicin responses by NGF in acutely dissociated rat DRG neurons, a response that can be recapitulated by co-expression of the high affinity trkA receptor and TRPV1 in heterologous systems (Chuang et al., 2001, Zhu and Oxford, 2003, Zhu et al., 2004). Moreover, there is a developmental switch in acute NGF sensitization of TRPV1 in postnatal rat DRG neurons (Zhu et al., 2004), which likely reflects plasticity in the signaling pathway linking the two receptors rather than changes in expression of either receptor. TrkA, as a receptor tyrosine kinase, traditionally signals through activation of one of three major biochemical pathways: phospholipase C (PLCγ), p42/p44 mitogen-activated protein kinase (ERK) or phosphoinositide-3-kinase (PI3K). Although several studies have attempted to elucidate the molecular mechanisms through which NGF sensitizes TRPV1, a consensus has not yet been achieved as published studies have utilized different cell types, different functional endpoints, and have not routinely verified biochemical specificity of signaling interventions.

Using the Xenopus oocyte expression system, the Julius laboratory first presented evidence suggesting that NGF-TrkA activation of PLCγ and subsequent hydrolysis of phosphatidylinositol-4,5-biphosphate (PIP₂) relieves a tonic inhibition of TRPV1 by PIP₂ (Chuang et al., 2001). A subsequent study identified a C-terminal domain in TRPV1 between amino acids 777 and 820 critical for the NGF sensitization (Prescott and Julius, 2003). PLCγ was also suggested to play a role in the NGF-induced sensitization of heat responses in adult DRG neurons (Galoyan et al., 2003). However, a recent study using calcium imaging of cultured mouse DRG neurons has suggested the sensitizing effect of NGF involves the PI3K pathway, but not the ERK or PLCγ pathways (Bonnington and McNaughton, 2003). An additional study in primary adult rat DRG neurons has suggested that both PI3K and ERK mediate inflammatory heat hyperalgesia through TRPV1 sensitization (Zhuang et al., 2004). Similarly our preliminary investigations in both adult rat DRG neurons and in Chinese Hamster Ovary (CHO) cells co-expressing TRPV1 and TrkA support the involvement of both PI3K and ERK pathways, but not the PLCγ pathway, in acute sensitization of TRPV1 by NGF (Zhu and Oxford, 2003). Furthermore, it has been recently reported that PIP₂ resynthesis is necessary for recovery of functional TRPV1 following desensitization (Liu et al., 2005), an observation seemingly opposite to the prediction of an inhibitory role for PIP₂. Finally, two recent reports suggest that the critical endpoint of acute trkA signaling in DRG neurons is enhanced trafficking of TRPV1 to the plasma membrane perhaps involving phosphorylation of TRPV1 by src kinase (Zhang et al., 2005, Stein et al., 2006).

To further elucidate the complicated signaling mechanisms between trkA receptors and TRPV1 sensitization, we have combined biochemical, mutagenesis and electrophysiological approaches in both rat and mouse neurons and in mammalian expression systems. Our results indicate that NGF sensitizes TRPV1 independent of extracellular calcium, that this sensitizing effect is mediated by TrkA and requires its downstream PI3K and ERK pathways, possibly in part through activation of ERK by PI3K. While it appears unlikely that PLCγ hydrolysis of PIP₂ is directly involved in this phenomenon, a related signaling pathway through protein kinase C (PKC) modulates NGF-induced TRPV1 sensitization. Finally, src kinase appears to be only one of the final effectors involved but cannot account for TRPV1 sensitization in its entirety.