A network map of GDNF/RET signaling pathway in physiological and pathological conditions

Abstract

Glial cell line-derived neurotrophic factor (GDNF) signals through a multi-component receptor system predominantly consisting of glycosyl-phosphatidylinositol-anchored GDNF family receptor alpha-1 (GFRα1) and the Rearranged during transfection (RET) receptor tyrosine kinase. GDNF/RET signaling is vital to the central and peripheral nervous system, kidney morphogenesis, and spermatogenesis. In addition, the dysregulation of the GDNF/RET signaling has been implicated in the pathogenesis of cancers. Despite the extensive research on GDNF/RET signaling, a molecular network of reactions induced by GDNF reported across the published literature. However, a comprehensive GDNF/RET pathway resource is currently unavailable. We describe an integrated signaling pathway reaction map of GDNF/RET consisting of 1151 molecular reactions. These include information pertaining to 52 molecular association events, 70 enzyme catalysis events, 36 activation/inhibition events, 22 translocation events, 856 gene regulation events, and 115 protein-level expression events induced by GDNF in diverse cell types. We developed a comprehensive GDNF/RET signaling network map based on these molecular reactions. The pathway map was made accessible through WikiPathways database (https://www.wikipathways.org/index.php/Pathway:WP5143).

Graphical abstract

Biocuration and development of gene regulatory network map of GDNF/RET signaling pathway

Supplementary Information

The online version contains supplementary material available at 10.1007/s12079-023-00726-1.

Introduction

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for the midbrain dopaminergic (DA) neurons (Lin et al. 1993). GDNF was first discovered in 1991 by researchers at Synergen, a biotechnology-based company in Boulder, Colorado, USA (Vastag 2010). It is a prominent member of the GDNF family of ligands (GFLs) that also comprises artemin (ARTN), neurturin (NRTN), and persephin (PSPN) (Zihlmann et al. 2005). A wide variety of cell types synthesize and secrete GDNF, including glial cells such as astrocytes, oligodendrocytes, Schwann cells, motor neurons (MNs), and skeletal muscles. GDNF is first synthesized as a precursor protein pro-GDNF. After a series of protein cleavage and processing, the 211 amino acid pro-GDNF is finally converted into the active and mature form of GDNF (Cintron-Colon et al. 2020).

The GDNF signals several intracellular pathways through receptors, including a unique multi-component receptor system, preferably the glycosyl-phosphatidylinositol (GPI)-linked cell surface co-receptor, GFRα1, and the signal transducer RET (REarrangement during Transfection) receptor tyrosine kinase. Besides GFRα1 and RET, GDNF also signals through neural cell adhesion molecule-1 (NCAM1), syndecan-3 (SDC3), and integrin-β1 (ITGB1), independent of RET. In mammals, two major alternate splice isoforms of RET that differ in their C-terminal tails (RET9 and RET51) are considered to exhibit different signaling properties in vivo (de Graaff et al. 2001). Cells that co-express both RET and GFRα1 receptors mediate cis signaling. The signaling mediated by soluble or immobilized GFRα1 undergoes ‘trans’-signaling, where RET is activated outside the lipid rafts by GFRα1 (Paratcha et al. 2001).

The GDNF/RET signaling is primary to the central and peripheral nervous system as a survival factor for multiple neuron types and a neuroprotective factor during many neurodegenerative disorders (Takahashi 2001). The RET signaling pathway is essential for the growth and formation of kidneys, as well as the maturation of spermatogonia, which are the precursors of sperm cells (Schuchardt et al. 1996; Meng et al. 2000). It is known that RET is an oncogenic driver in several cancers, including papillary thyroid carcinomas (PTCs), non-small cell lung cancers (NSCLCs), breast, ovarian, and head and neck tumors (Adashek et al. 2021; Thein et al. 2021; Regua et al. 2022). In addition, mutations in the RET gene have been associated with thyroid melanoma and multiple endocrine neoplasia (MEN) syndromes-2A and -2B (Adashek et al. 2021). Therefore, GDNF/RET signaling systems are being targeted with multiple therapeutic approaches in the treatment of diverse diseases, including neurological conditions and cancers (Plaza-Menacho et al. 2014; Mahato and Sidorova 2020).

However, the molecular events associated with GDNF/RET signaling are scattered across literature. They have not been assembled in a form that can be used for multi-omics level approaches. Hence, we report the assembly and development of a topology-oriented comprehensive GDNF/RET signaling network map. This GDNF/RET signaling pathway map forms part of the neurological system pathways such as BDNF/p75NTR (Sandhya et al. 2013), oxytocin-oxytocin receptor signaling (Chatterjee et al. 2016), galanin-galanin receptor (Gopalakrishnan et al. 2021), serotonin (Sahu et al. 2018), and corticotropin-releasing hormone (Subbannayya et al. 2013) developed by our group and submitted to NetPath (Kandasamy et al. 2010) and WikiPathways (Kutmon et al. 2016). Here, we describe the generation of an integrated pathway map of GDNF/RET signaling by manual curation. This comprehensive signaling pathway map can be used for knowledge-based analysis of multi-omics datasets for improvement of our understanding of fundamental biological processes associated with GDNF signaling.

Methodology

A literature survey was carried out in PubMed to gather the articles associated with GDNF signaling using the search term ‘("GDNF" OR "glial cell-derived neurotrophic factor" OR "ATF" OR "ATF1" OR "ATF2" OR "astrocyte-derived trophic factor") AND ("signaling" OR "signalling" OR "pathway")’. A manual screening of these articles containing relevant information of signaling events reported to be induced by GDNF was performed. Later, manual curation of the signaling events was carried out from the screened articles based on NetPath annotation criteria (Kandasamy et al. 2010). These events were categorized into five groups such as (a) molecular association (protein–protein interactions (PPIs)), (b) enzyme-catalysis (enzyme–substrate reactions and also the post-translational modifications), (c) activation/inhibition events, (d) protein translocation events, and e) gene regulation events at mRNA/protein level (Kandasamy et al. 2010). The curated signaling events were merged into a single pathway map and each curated reaction was hyperlinked to the PubMed identifier of the research article from which the reactions have been curated. The PathVisio tool was used for the representation and visualization of the network map (Kutmon et al. 2015). The GDNF pathway map was made freely accessible through WikiPathways database.

Results and discussion

From 3,259 articles that were manually screened, 1061 articles which had relevant information pertaining to GDNF signaling were retrieved. A total of 1151 signaling events associated with GDNF stimulation were curated from these shortlisted articles (Supplementary Data S1). These events included categories of 52 molecular associations, 70 enzyme catalysis, 36 activation/inhibition reactions, and 22 translocation events. Furthermore, we also cataloged 115 protein and 856 mRNA expression regulation events modulated by GDNF stimulation in various mammalian cells/cell lines. A topology-oriented GDNF/RET signaling network map was manually drawn using PathVisio software and made freely accessible through WikiPathways database (https://www.wikipathways.org/index.php/Pathway:WP5143) (Fig. 1).

Fig. 1.

Schematic representation of GDNF signaling pathway. Schematic representation of GDNF-induced signaling reactions. The signaling pathway map represents molecules involved in ligand-receptor interactions and GDNF-activated downstream molecular events including molecular association, enzyme catalysis, translocation, and gene regulation events. Information regarding the post-translational modification site and the residue is also shown in the pathway

The signaling pathways via GDNF/GFRα1/RET complex

The GDNF/RET signaling pathway is a significant pathway to explore for its critical role in the development and maintenance of the nervous system (Takahashi 2001). It is also involved in the development of other organs, such as the kidneys (Costantini and Shakya 2006) and the pancreas (Lucini et al. 2008). Most of the GDNF effects are mediated by the receptor tyrosine kinase RET and GFRα1. The binding of GDNF to GFRα1/RET leads to auto-phosphorylation of tyrosine residues of RET including Tyr⁹⁰⁵, Tyr¹⁰¹⁵, Tyr¹⁰⁶², Tyr¹⁰⁹⁶, Tyr⁶⁸⁷, Tyr⁹⁸¹, Ser⁶⁹⁶_, and Thr⁶⁷⁵. The former 4 residues are only specific to the RET51 isoform and eventually become the docking sites for adaptor proteins such as Grb7/Grb10, PLCγ, Shc/Enigma, and Grb2, respectively, leading to the activation of various signaling modules (Airaksinen et al. 1999; Takahashi 2001).

For instance, the phosphorylation of RET at Tyr¹⁰⁶² is found to be critical for the function of RET51, which is required for the activation of RAS/MAPK, p38MAPK, JNK and PI3K/AKT pathway (Besset et al. 2000; Segouffin-Cariou and Billaud 2000). Further, these pathways are vital for enteric neuroblasts differentiation and survival, and the branching morphogenesis of the ureteric bud (Fisher et al. 2001). Similarly, phosphorylated Tyr¹⁰¹⁵ residue activates PLCγ pathway, which regulates Ca²⁺ influx from the endoplasmic reticulum and thereby induces calcium signaling (Kawai and Takahashi 2020).

GDNF/RET signaling in cell-specific functions

The GDNF/RET signaling promote cell survival and/or neurite outgrowth in several neuronal subpopulations such as in Neuro2a cells via the activation of PI3K/AKT1 pathway (Encinas et al. 2001) and in sympathetic neurons via MAPK1/3 activation (Coulpier and Ibanez 2004). However, GDNF/SDC3 signaling is implicated in the regulation of neurite outgrowth in hippocampal neurons (Bespalov et al. 2011). Further, GDNF/GFRα1/NCAM1 signaling is reported to promote dendrite growth and spine formation via the activation of FAK, SRC and MAPK1/3 in the hippocampus (Irala et al. 2016). In TGW cell lines, the activated RET by Tyr¹⁰⁶² phosphorylation leads to the association between RET with GAB1 and SHC, which induces the activation of CREB and NF-kB through RAS/MAPK and PI3K/AKT pathways, respectively, which are involved in neuron survival (Hayashi et al. 2000). In adult rat substantia nigra, GDNF stimulates PI3K/AKT downstream signaling resulting in the translocation of NF-kB to nucleus and regulates the higher expression of Calbindin-D28K thereby protecting the neurons from degeneration, the main pathological change associated with Parkinson's disease (PD) (Howland and Wang 2008). In neuronal cell line RN33B, which is RET independent, GDNF promotes cell survival by activation of SRC-like kinase, phosphorylation of CREB, and protein up-regulation of FOS rather than activating RAS/MAPK pathway. In MN1 cells, GDNF stimulates the activation of RAS/MAPK and PI3K/AKT1 pathways, PLCγ phosphorylation, which results in the up-regulation of CREB and FOS at mRNA and protein level, respectively, which are essential for cell survival (Trupp et al. 1999).

The GDNF also plays an important role in spermatogonial stem cell (SSC) functions. In C18-4 cells, a mouse spermatogonial stem cell line, GDNF stimulation induces the phosphorylation and association of RET between SHC1 and GRB2 which in turn induces the phosphorylation of CREB1, ATF1, and TNF via the RAS/MAPK1/3 pathway that promotes the transcription of c-FOS, which leads to SSC proliferation (He et al. 2008). Similarly, GDNF/ GFRα1/RET induces spermatogenesis in mice spermatogonial progenitor cells (SPCs) by inducing the phosphorylation of RPS6 and RPTOR via MAPK1/3 kinase signaling, an important step in self-renewal and mitotic proliferation of SPCs (Wang et al. 2017).

GDNF/RET signaling in cancer

The GDNF/RET signaling is implicated in the development and metastasis of several cancers (Mulligan 2018; Subbiah and Cote 2020). In in vitro cell and animal models of breast cancer, GDNF/RET signaling induces MAPK and JNK activation that leads to tumor cell proliferation and survival suggesting the potential role of RET as a novel therapeutic target in breast cancer (Boulay et al. 2008). GDNF stimulates GFRα1 and induces the activation of NFKB1 which promotes the invasive potential in human pancreatic cancer cells (Takahashi et al. 2004). GDNF stimulates GFRα1 and induces the activation of AKT1, MAPK1, MAPK8, PIK3CA, AKT1, RHOA, NRAS, RAC1, and RNASE9, which are involved in tumor migration and invasion in pancreatic cancer PANC-1 cells (Veit et al. 2004). The exposure of cytotoxic agents to prostate cancer cells promotes the increased expression of GDNF which stimulates GFRα1 and induces the activation of SRC and MAPK1, which enhances prostate cancer resistance to genotoxic chemotherapy (Huber et al. 2015).

The GDNF secretion by nerve cells increases the PDL1 expression through JAK2-STAT1 signaling pathway contributing to immune surveillance escape in head and neck squamous cell carcinoma (HNSCC) (Lin et al. 2017). Activation of GDNF/RET/AKT signaling in neuroblastoma (NB) cells inhibits the GSK3β activity, resulting in increases in PTCH1, GLI2 and GLI1 and decreases in GLI3, which activates hedgehog signaling pathway and thereby promotes the proliferation of NB cells and tumor growth (Ruan et al. 2016). In breast cancer cells, GDNF stimulates GFRα1 and induces the phosphorylation of RET (Tyr 905), ESR1 (Ser 167 and 118), AKT1 (Ser 473), JUN (Ser 73), RPS6KB1 (Thr 389), MAPK1/3 (Tyr204/Thr202), MAPK8/9 (Thr183/Tyr185) and upregulation of PGR, TFF1, TOP2A at gene level in MCF7 cells which promoted the survival of aromatase inhibitor-resistant cells (Morandi et al. 2013). In human glioblastoma cells, GDNF stimulates GFRα1 and induces the upregulation of VEGFA and FMOD at mRNA and protein levels and subsequently, the transport of VEGFA from extracellular vesicle to cytoplasm which induces angiogenesis (Chen et al. 2018). A recent study has shown that RET and MEN2A molecular activities can be inhibited by the dephosphorylation of protein tyrosine phosphatase receptor-type-A (PTPRA) which in turn regulates GDNF-dependent RET-RAS-MAPK growth signaling and the oncogenic activity of RET (Yadav et al. 2020). Unlike the other RET-induced malignancies, which are caused due to RET gene mutation, papillary thyroid carcinoma (PTC), the most common form of thyroid cancer is mediated as a result of chromosomal translocation of RET gene (Santoro and Carlomagno 2013; Prescott and Zeiger 2015). The application of small molecule inhibitors capable of inhibiting RET protein kinase activity has been an effective therapeutic approach for PTCs (Prescott and Zeiger 2015).

Conclusions

We developed a comprehensive GDNF/RET signaling pathway map based on the information derived from the literature related to GDNF signaling. The diverse signaling modules and the interactions specific to the phosphosites in RET and also their information pertaining to distinct RET variants are distinguished in this pathway map. We believe that our map provide effective visualization of the pathway to derive novel insights into GDNF/RET signaling mechanisms by gene-set enrichment analysis approaches using future multiomic datasets. Information on the GDNF-mediated cellular responses in this map will facilitate effective integration and updation of the map with evolving information on GDNF/RET signaling as a platform for reference and analysis of GDNF associated physiological processes and pathological conditions.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

GDNF

Glial-cell-line-derived neurotrophic factor

GFRα1

GDNF family receptor alpha-1

GFRAs

GDNF family receptor alpha members

GFLs

GDNF family ligands

GPI

Glycosyl-phosphatidylinositol

RET

Rearranged during transfection

NCAM1

Neural cell adhesion molecule-1

CNS

Central nervous system

MAPK

Mitogen-activated protein kinase

PLC

Phospholipase-C

SDC3

Syndecan-3

ITGB1

Integrin-β1