The role of glial cell line‐derived neurotrophic factor family member artemin in neurological disorders and cancers

Abstract Artemin (ARTN) is a member of the glial cell line‐derived neurotrophic factor (GDNF) family ligands (GFLs), which encompasses family members, GDNF, neurturin (NRTN) and persephin (PSPN). ARTN is also referred to as Enovin or Neublastin, and bears structural characteristics of the TGF‐β superfamily. ARTN contains a dibasic cleavage site (RXXR) that is predicted to be cleaved by furin to yield a carboxy‐terminal 113 amino acid mature form. ARTN binds preferentially to receptor GFRα3, coupled to a receptor tyrosine kinase RET, forming a signalling complex for the regulation of intracellular pathways that affect diverse outcomes of nervous system development and homoeostasis. Standard signalling cascades activated by GFLs via RET include the phosphorylation of mitogen‐activated protein kinase or MAPK (p‐ERK, p‐p38 and p‐JNK), PI3K‐AKT and Src. Neural cell adhesion molecule (NCAM) is an alternative signalling receptor for ARTN in the presence of GFRα1, leading to activation of Fyn and FAK. Further, ARTN also interacts with heparan sulphate proteoglycan syndecan‐3 and mediates non‐RET signalling via activation of Src kinases. This review discusses the role of ARTN in spinal cord injury, neuropathic pain and other neurological disorders. Additionally, ARTN plays a role in non‐neuron tissues, such as the formation of Peyer's patch‐like structures in the lymphoid tissue of the gut. The emerging role of ARTN in cancers and therapeutic resistance to cancers is also explored. Further research is necessary to determine the function of ARTN in a tissue‐specific manner, including its signalling mechanisms, in order to improve the therapeutic potential of ARTN in human diseases.

characteristics as a dimeric and cysteine-knot motif like molecule, which resembles distant members of the TGF-β superfamily. 2,[6][7][8] Under physiological conditions, ARTN promotes sensory neuron survival and peripheral nerve homoeostasis, 9 and the cell survival of dopaminergic neurons of the ventral mesencephalon in the brain. 10 ARTN knockout mice revealed abnormalities in the sympathetic nervous system (SNS), with defective migration and axonal projection pattern of SNS. 11 In pathological nerve injury, ARTN plays a distinct role in neuropathic pain and morphological alterations of nerves. 12 In addition to its role in neural tissue tropism, ARTN acts as an attractant of intestinal hematopoietic cells and participates in the formation of Peyer's patch-like structures in the gut. 4,9 This review discusses the role of ARTN in spinal cord injury repair, neuropathic pain and other neurological disorders. An overview of the molecular structure, signalling pathways, and gene expression of ARTN is presented. In addition, the emerging role of ARTN in various types of cancers is surveyed. Further understanding of the role of ARTN in a tissue-specific manner and its underlying signalling mechanisms will help us to develop ARTN as a therapeutic target for neurological diseases and cancers.

| MOLECUL AR S TRUC TURE AND E XPRE SS I ON OF ARTN
Multiple sequence analyses indicate that human ARTN shares considerable (approximately 60%-65%) amino acid sequence identity to rat, mouse, pig, pantry and rhesus macaque ARTN homologs, particularly at their carboxyl termini (approximately 85%-90%), indicating that ARTN is a well-conserved protein among mammalian species ( Figure 1A,B).
At the molecular structural level, human ARTN contains an amino-terminal signal sequence and is expressed as a preproprotein, F I G U R E 1 ARTN amino acid sequence analysis. A, Multiple sequence analysis reveals that human ARTN shares considerable amino acid sequence identity to rat, mouse, chimpanzee, rhesus macaque and pig ARTN homologs, particularly in the mature form at the C terminus, indicating that ARTN is a well-conserved protein among mammalian species. B, Family tree of ARTN in various species which undergoes further protein cleavage at the dibasic motif (RXXR) predicted to be cleaved by furin to yield a carboxy-terminal 113 amino acid mature form of functional ARTN with a disulphide-linked homodimer of 28 kDa protein ( Figure 2A). 4,5,9 ARTN bears sequence similarity with GDNF, NRTN and PSPN which also contain putative furin cleavage site of RXXR at amino acid residues 77, 95 and 60, respectively ( Figure 2A). 9 Recombinant ARTN proteins were expressed and purified from bacterial hosts, and exhibited biological activity associated with disulphide structural folding. 13,14 Three dimensional structure of ARTN is known, and it resembles that of GDNF and NRTN, and has some homology with TGF-beta family members ( Figure 2B). 15 At the transcriptional level, ARTN mRNAs were reported to be expressed in developing nerve roots, 9 Schwann cells 6 and embryonic vascular smooth muscle cells. 11 The expression of ARTN was regulated by the master activator protein 1 (AP-1) transcription factor, c-Jun, in Schwann cells. 16 Upregulation of ARTN was also identified in mesenchymal stem cells (MSCs) transplanted onto the cortex of brain injured rats, indicating a potential role of ARTN in central neural tissue regeneration. 17

| ARTN RECEP TOR AND S I G NALLING PATHWAYS
GFLs, including ARTN, signal by the formation of a complex receptor system consisting of a ligand-specific non-signalling receptor subunit GFRα, coupled to a receptor tyrosine kinase, RET ( Figure 3). [18][19][20] Four GFRα receptors have been identified (GFRα1-4), and ARTN first binds to GFRα3 which is attached to the membrane via glycosylphosphatidylinositol (GPI) anchor. 9,21 And then the ARTN-GFRα3 complex binds to and activates receptor tyrosine kinase RET by triggering phosphorylation of RET intracellular tyrosine residues, 21,22 and this pathway is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. 23 GFRα3 was found to be strongly expressed in dorsal root ganglia cells and Schwann cells, 24 and in the developing brain, as well as in developing and adult peripheral nerves. 25 Consistently, RET mRNA was strongly expressed in motor neurons and dorsal root ganglion neurons. 24 Several cascades activated by GFLs via RET include the phosphorylation of MAPK (p-ERK, p-p38 and p-JNK), PI3-AKT and Src ( Figure 3). [26][27][28][29] GFL signalling is diverse, and ARTN could also signal independently of RET in combination with other receptors. 2,7,9,30 The GFRα1 has been suggested to be a receptor for ARTN, 9,31 but displays much weaker binding to ARTN than GDNF 32 and lack of functional role in ARTN-mediated cell activity. 30,33,34 In addition, GDNF signalling was identified via the neural cell adhesion molecule F I G U R E 2 Molecular structure of ARTN. A, Human homolog of ARTN contains an N terminal signal sequence and is expressed as a preproprotein, which contains a putative furin cleavage site of RXXR at amino acid residue 107, and is further processed as a mature form of a 113 amino acid functional ARTN with a putative glycosylation site, and disulphide-linked homodimer of 28 kDa peptide. ARTN shares a similar structure with GDNF, NRTN and PSPN which also contain putative furin cleavage site of RXXR at amino acid residues 77, 95 and 60, respectively. B, Tertiary structure analysis showing that ARTN shares typical features of TGF-β superfamily as predicted by EBI-based bioinformatics (https://www.ebi.ac.uk/pdbe/entry / pdb/2ASK) It was observed that intracellular cyclic adenosine monophosphate (cAMP) elevation leads to the activation of protein kinase A (PKA) in neuronal cells. 37 Serine 696 in RET was identified as a putative phosphorylation site by upstream PKA, whereas phosphorylation of tyrosine 1062 in RET is crucial for the downstream activation of phosphatidylinositol 3-kinase (PI3K). 37 Consistently, using Ret-null Wolffian ducts in a budding experiment, it was shown that PKA is involved in GDNF-dependent effect. 38 (Figure 3). On the other hand, PKA might also act as a downstream regulator of RET. Using by primary dorsal root ganglion neurons in culture, ARTN was found to support the extension of neurites through activation of PKA to phosphorylate cAMP-response element binding protein (CREB) (Figure 3). 39 The utility of ARTN signalling is exemplified by regulating the expression of genes involved in various biological processes, including arginase I in spinal cord neurons, 39 TRPV1 in cutaneous sensory neurons, 40 TRPA1 in trigeminal afferents, 41 VEGF-A in human microvascular endothelial cells, 42 and TRIO and F-actin binding protein (TRIOBP) and integrin, beta 5 (ITGβ5) in SMMC-7721 cells. 43 However, the transcription regulations by ARTN-mediated signalling are incompletely defined and require further investigation.

| THE ROLE OF ARTN IN S PINAL CORD INJ URY REPAIR
Spinal cord injury (SCI) is debilitating and often has a poor prognosis, significantly due to our lack of understanding of its complex pathogenesis. Functional recovery following SCI requires axon regrowth into the site of injury with synaptic connectivity to target tissues. 44 Increasing evidence shows that ARTN plays an important role in the functional recovery after spinal cord injury. 39,[45][46][47] Further, ARTN was found to enhance neurite extension in vitro by overcoming myelin inhibition and may have the potential to improve motor recovery following SCI in a rodent model. 39 Consistently, ARTN has been shown to preserve small types of nerve fibres, and type C fibre conduction velocity in the dorsal horn after SCI in a rodent model, 30 and administration of ARTN led to the formation of sensory fibres reaching to the site of injury, and facilitated the restoration of sensory function following SCI. 48 ARTN also modulates neurite initiation and neurite elongation, and the branching of unmyelinated sensory neurons in the spinal cord dorsal root ganglion (DRG) of mature rats, 29,49 as well as the regeneration of myelinated axons. 50 Further, in DRG injury rats, the expressions of GFRα1 and GFRα3 were increased whereas the expression of GFRα2 was unchanged. 51 ARTN was found to promote neurite outgrowth and actin polymerization in mature DRG by affecting the transcription of many target genes 49 and stimulate the topographically correct regeneration of DRG axons in rodent dorsal root crush models. 52 ARTN also induced and the regeneration of large, myelinated sensory afferents, 46 and the peripheral nerve regeneration, and functional restoration of nerve fibres in partial lesions distal to DRG, 53 which may be partially attributed to decreased caspase 3 and activating transcription factor 3 (ATF3) gene expression. 53 F I G U R E 3 ARTN signalling. ARTN mediates the activation of the GFRα3/RET receptor complex. ARTN binds to GFRα3 which is attached to the membrane via GPI anchor. The GFRα1/RET complex has also been suggested to be a putative receptor for ARTN. Several cascades activated by GFLs via RET include the phosphorylation of MAPK (p-ERK, p-p38 and p-JNK), PI3K-AKT and Src. In addition, NCAM is an alternative signalling receptor for ARTN only in the presence of GFRα1, leading to activation of Fyn and FAK. Further, ARTN also interacts with heparan sulphate proteoglycan syndecan-3 and mediates non-RET signalling via activation of Src-type kinases. Interestingly, PKA might act as regulator of upstream of RET via cAMP, or as a downstream regulator of RET

| THE ROLE OF ARTN IN NEUROPATHI C PA I N
ARTN and fellow GDNF family members also regulate the sensitivity of thermal nociceptors and hyperalgesia induced by inflammation, 54 which appears to be mediated by ARTN-GFRα3 interactions. 55 Systemically administered ARTN was able to restore nociceptive and sensorimotor functions following injury. 48 Further, peripherally derived ARTN appeared to play an important role in inflammatory and neuropathic pain through the regulation of TRPV1/A1 (members of the TRP family of cation channels, that are activated by noxious thermal and chemical stimuli) expression in primary afferent neurons in DRG. 56 ARTN also modulates heat hyperalgesia and cold responses via TRPM8-associated signalling in mice, 57 which might involve the regulation of nicotinic acetylcholine receptor (nAChR) gene expression in thermal hypersensitivity. 58 In the light of the important role of ARTN in neuropathic pain, ARTN might serve as a potential target for pain therapeutics. 59 For example, small molecule BT13 that mimics the effect of ARTN has been shown to selectively activate RET signalling and to support neurite growth in a similar way to ARTN, providing additional indication of the potential of ARTN for developing medications to treat neuropathic pain. 60 In particular, ARTN could be delivered selectively to neurons that are responsible for cold pain, 61 potentially through modulation of the expression of TRPV1 (a polymodal calcium-permeable cation channel activated by heat and inflammatory stimuli) and TRPA1 (an ionotropic channel responsive to cold and chemical stimuli) in cutaneous sensory neurons. 40 Recently, ARTN (BG00010) was trialled clinically for pain relief of unilateral sciatica. 62,63 Results of the trial supported the further development of ARTN (BG00010) for the treatment of neuropathic pain. 62 Further, randomized, double-blinded, placebo-controlled phase 2 trial showed evidence of pain relief ARTN (BG00010) with adverse event of headache, feeling hot and pruritus. 64 Consistently, ARTN appears to mediate hypersensitivity and itch to warmth, leading to abnormal peripheral innervation, thermal hyperalgesia and provoked itch sensation in pruritic skin disorders, such as atopic dermatitis (AD). 65 More recently, ARTN is involved in bone pain behaviour in a model of inflammatory bone via the activation and sensitization of bone afferent neurons. 66

| THE ROLE OF ARTN IN OTHER NEUROLOG IC AL DISORDER S
In additional to SCI, increasingly data have implicated a role of ARTN in other neurological disorders. For instance, ARTN was found to induce neurite outgrowth from sympathetic neurons in the early stages of embryos 67 and exert distinct effects on the generation, survival and growth of sympathetic neurones in vivo. 68 ARTN could also enhance the regeneration of sensory axons in the brainstem. 46 ARTN was detected expressed in blood vessels during periods of early sympathetic differentiation, where is it considered to be a guidance factor, possibly by chemoattractive activity, for the growth of sympathetic fibres. 11 Using in vitro isolated mouse embryonic motor neurons, ARTN was shown to act as a survival factor for parasympathetic preganglionic motor neurons through GFRα3/Syndecan-3 activation. 69,70 Further, ARTN was found to improve functional outcome after sciatic nerve injuries in rats. 70 In that study, the sciatic nerve was transected and treated with a fibrin sealant containing ARTN, and the results revealed that ARTN increased the number of regenerating motor neurons. 70

| THE ROLE OF ARTN IN NON -NEUROLOG IC AL DISORDER S
In addition to neural tissues, ARTN might affect non-neural organs.
For instance, ARTN is a strong attractant of gut haematopoietic cells, inducing the formation of ectopic Peyer's patch-like structures, which is suggestive of a role in intestine organogenesis. 84 It was revealed that haematopoietic cells in the gut exhibit a random pattern of motility before forming the unique primordia structure of Peyer's patches via RET signalling. Knockout mice study showed that GFRa3 deficiency exhibited impaired Peyer's patch development, suggesting that ARTN/ GFRa3/RET mediates this process. 84 Overexpression of ARTN in chronic pancreatitis disturbs tissue homoeostasis, leading to pancreatic fibrosis. 85 Using quantitative PCR (polymerase chain reaction) and Western blot analyses, it was found that ARTN and GFRα3 were significantly overexpressed in chronic pancreatitis, and positively correlated with the severity of fibrosis.
Further, transforming growth factor beta1 (TGF-β1) upregulated the expression of ARTN in human pancreatic stellate cells (hPSCs) which might contribute to this pathogenesis. 85

| THE EMERG ING ROLE OF ARTN IN C ANCERS
Recently, a role of ARTN in tumorigenesis, tumour metastasis and drug resistance is emerging. 86  In non-small-cell lung carcinoma (NSCLC), ARTN was identified to arbitrate the progression of human NSCLC, as determined by clinical and laboratory findings. 92 NSCLC tissues showed increased expression of ARTN and advanced lymph node metastasis, which was accompanied by increased migration and invasion of NSCLC cells via upregulation of BCL2 transcription. 92 Overexpression of ARTN was also found to promote the proliferation and invasiveness of lung cancer cells in vitro. 93 Similarly, the prognostic significance of ARTN expression in laryngeal squamous cell carcinoma (LSCC) may serve as predictors of LSCC progression and outcome in patients with LSCC. 94 In pancreatic adenocarcinoma (PCa), ARTN appears to participate in the generation of pancreatic neuropathy 95 and to stimulate the invasion and neurotrophic function of PCa in vivo and in vitro. 96 Consistently, ARTN and CXC chemokine receptor 4 (CXCR4) were found to be overexpressed in pancreatic cancer tissues, and the migration and invasion of pancreatic cancer cells appears to be promoted by Akt and ERK 1/2/NF-κB signalling and the stromal cell-derived factor 1α (SDF-1α)/CXCR4 axis. 97 In hepatocellular carcinoma (HCC), ARTN was found to promote the growth and progression of HCC based on increased clinical tissue expression, increased tumour size, increased relapse and shorter survival time. 98 Further laboratory investigation found that HCC cells overexpressing ARTN demonstrated reduced apoptosis, increased proliferation and EMT, and increased motility via hypoxia-induced factor 1-α (HIF-1α) regulated AKT signalling, indicating that ARTN may function in a hypoxic environment and promote HCC. 98 In endometrial carcinoma (EC), the expression levels of ARTN proteins were found to be positively correlated with the stage of EC and lymphatic metastasis. 99 Additionally, ARTN is implicated as a pathogenic factor for certain acute myeloid leukaemia (AML) patients, which warrants further investigation. 100 Activation of ARTN/GFRα3mediated RET signalling in AML cells requires further clinical investigation. 100 Further, downstream RET-mTORC1 signalling is found to promote AML cell growth through the suppression of autophagy and stabilization of leukaemia-genic drivers, indicating the potential of RET as a therapeutic target in subgroups of AML patients. 100 More recently, it was found that in the enlarged spleen of hosts bearing advanced tumours, erythroblast-like cells are enriched and boost tumour progression via producing ARTN into the blood. 43 Using hepatocellular carcinoma (HCC) tissues from in HCC patients, the protein levels of GFRa3 and phosphorylated RET were examined by immunohistochemistry (IHC) with automated cell acquisition. It was revealed that higher GFRa3 mRNA expression and RET phosphorylation in HCC tissues were correlated with the reduced disease-free survival of HCC patients. Further, the higher levels of serum ARTN were correlated with the higher levels of GFRa3 expression or higher RET phosphorylation in HCC tissues in patients with shorter disease-free survival. 43 Consistently, in vivo depletion or deficiency of ARTN inhibits the growth of HCC and abolishes tumour-promoting ability of erythroblast-like cells. 43 Bioinformatics gene analyses indicate that the ARTN transcripts are expressed in human and mouse cancers, most abundantly in Ewing's sarcoma in human and in melanoma in mouse (Figure 4). 101 However, the mechanism by which ARTN is differentially expressed and involved in various types of cancers warrants further investigation.  Xu and Jiake Xu supervised the study and revised the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.