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The Sprouty (SPRY) family of proteins includes important regulators of downstream signaling initiated by receptor tyrosine kinases. In the present study, we investigated the role of SPRY proteins in intracellular signaling via the RET receptor tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF). Expression of SPRY1, SPRY2, SPRY3 and SPRY4 in HEK293T cells transfected with RET and GDNF receptor family α1 (GFRα1) genes significantly reduced sustained ERK activation as well as ELK-1 activation. Because expression of SPRY2 was efficiently induced by GDNF in TGW human neuroblastoma cells expressing RET and GFRα1, we further investigated the role of SPRY2 in the growth and differentiation of TGW cells. Expression of wild-type SPRY2 (WT-SPRY2) decreased the growth of TGW cells. In contrast, expression of a dominant negative form of SPRY2 (MT-SPRY2, with a mutated tyrosine residue) enhanced cell proliferation. In addition, expression of WT-SPRY2 reduced GDNF-dependent neurite outgrowth of TGW cells, whereas expression of MT-SPRY2 enhanced it. Taken together, our results suggest that SPRY2 regulates GDNF-dependent proliferation and differentiation of TGW neuroblastoma cells mediated by RET tyrosine kinase. (Cancer Sci 2007; 98: 815–821)
The RET receptor tyrosine kinase (RTK) regulates a variety of cellular processes, including proliferation, survival, differentiation, migration, chemotaxis, branching morphogenesis and synaptic plasticity.(1–5) RET signaling is activated by the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GDNF, neurtuin, artemin and persephin). Unlike other RTK, however, the ligands do not bind to RET directly, but require glycosylphosphatidylinositol-anchored cell surface proteins called GDNF family receptor α 1–4 (GFRα1–4) as ligand-binding components.(1) GDNF, neurtuin, artemin and persephin use GFRα1, GFRα2, GFRα3 and GFRα4, respectively, as their preferred receptors. Complex formation results in the activation of several signaling pathways, including the RAS/ERK, phosphatidylinositol-3 kinase/Akt, p38 MAPK, phospholipase Cγ, and RAC/c-Jun amino terminal kinase (JNK) pathways.(6–12) Gene knockout studies have demonstrated that the GDNF/RET signaling pathway is crucial for development of the enteric nervous system and the kidneys, as well as spermatogenesis.(13–17)
RET mutations are responsible for the development of several human diseases, including papillary thyroid carcinoma, multiple endocrine neoplasia (MEN) types 2A and 2B, familial medullary thyroid carcinoma and Hirschsprung's disease.(5,18) MEN 2A and 2B share the clinical features of medullary thyroid carcinoma and pheochromocytoma, whereas familial medullary thyroid carcinoma is characterized by the development of medullary thyroid carcinoma alone. In addition, 10–30% of MEN 2A patients develop parathyroid hyperplasia, whereas MEN 2B patients show a more complex phenotype that includes ganglioneuromatosis of the gastrointestinal tract, mucosal neuroma and marfanoid habitus. Papillary thyroid carcinoma is caused by somatic rearrangement of RET, and MEN 2A, MEN 2B and familial medullary thyroid carcinoma are caused by its germ-line point mutations. These mutations lead to RET gain-of-function. However, loss-of-function mutations of RET lead to the development of Hirschsprung's disease, which is a congenital malformation associated with the absence of enteric neurons.
Sprouty (SPRY) proteins have been found to antagonize fibroblast growth factor (FGF) signaling during tracheal branching in Drosophila.(19) Drosophila SPRY proteins are conserved as inhibitors of RTK signaling.(20) They specifically suppress RAS/ERK signaling activated by RTK, leaving the phosphatidylinositol-3 kinase/Akt and other MAPK pathways unaffected. The mechanism by which SPRY blocks ERK activation remains unclear, and may depend on the cellular context or the specific RTK.(21) The mouse and human genomes each contain four SPRY genes (SPRY1–4) encoding proteins that have molecular masses of 32–34 kDa. It has been reported that SPRY1, SPRY2 and SPRY4 inhibit FGF- and vascular endothelial growth factor (VEGF)-induced ERK activation, but do not affect epidermal growth factor (EGF)-induced ERK activation.(22,23) These findings suggest the possibility that SPRY proteins are rather selective inhibitors of RTK.
A recent report revealed that Spry2-deficient mice develop hyperganglionosis in the enteric nervous system, probably resulting from enhancement of ERK activation in the GDNF/RET signaling pathway.(24) In the present study, we investigated the role of SPRY in GDNF/RET signaling in cell growth and differentiation using TGW human neuroblastoma cells.
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RET receptor tyrosine kinase controls a variety of cellular processes including proliferation, differentiation and migration.(1–3) Its activity is tightly controlled through the coordinated action of both positive and negative regulators that function at multiple levels of the signal transduction cascade, and at different time points within the GDNF-induced response. When this process goes awry, developmental defects and malignancy may result. It has been demonstrated that SPRY expression is positively regulated by the ERK pathway (that it antagonizes), yielding a negative feedback loop.(29) In agreement with this model, GDNF treatment induced apparent upregulation of SPRY2 expression in TGW neuroblastoma cells, although upregulation of SPRY4 expression in TGW cells by GDNF was weak, and expression of SPRY1 and SPRY3 was not apparent. Thus, individual SPRY genes may be regulated by specific combinations of factors to allow optimal control of signaling and function in a tissue-specific manner.(30)
Overexpression of SPRY1–4 proteins inhibited sustained ERK activation by GDNF, whereas overexpression did not appear to affect transient ERK activation. This is in agreement with the finding that activation of exogenous SPRY2 was low 5 min after GDNF stimulation (Fig. 1d). In contrast, expression of dominant-negative MT-SPRY2 enhanced transient ERK activation as well as sustained ERK activation. This result suggests that MT-SPRY2 expression may significantly inhibit the activity of endogenous SPRY2 even 5 min after GDNF stimulation.
It is well established that GDNF/RET signaling plays a crucial role in regulating the proliferation, differentiation and migration of enteric neural crest cells.(31,32) Thus, defects in signaling result in the development of Hirschsprung's disease, a congenital malformation of the enteric nervous system.(1–5,18) In addition, it was reported that GDNF/RET signaling is involved in proliferation and differentiation of neuroblastoma cells.(33) In the present study, we investigated the effect of SPRY2 expression on proliferation or differentiation of TGW neuroblastoma cells. Consistent with the finding that SPRY2 expression inhibits ERK activation in TGW cells, GDNF-dependent proliferation of TGW cells was significantly impaired by expression of WT-SPRY2. In contrast, expression of the dominant-negative MT-SPRY2 (in which tyrosine 55 was replaced by alanine) enhanced cell proliferation. This finding suggests that SPRY2 regulates the growth of neuroblastoma cells by modulating ERK activity downstream of RET tyrosine kinase.
GDNF treatment can induce neurite outgrowth of TGW cells.(28) Neurite outgrowth is an established marker of neuronal differentiation that requires concerted intracellular signaling cascades.(34) Our recent study revealed that GDNF-dependent neurite outgrowth in TGW neuroblastoma cells was accelerated by sustained ERK activation.(28) We observed that expression of WT-SPRY2 significantly reduced sustained ERK activation by GDNF whereas expression of MT-SPRY2 enhanced it. Consistent with this finding, TGW cells expressing WT-SPRY2 produced shorter neurites in the presence of GDNF than did control TGW cells. In contrast, GDNF treatment of TGW cells expressing MT-SPRY2 developed longer neurites, indicating that SPRY2 also regulates neuronal differentiation.
In mice, Spry2 deficiency leads to ganglionic megacolon.(24) That report suggests that the hyperresponsiveness of enteric neurons to GDNF leads to enteric nervous system (ENS) hyperganglionosis in these mice. This finding suggests that Spry negatively regulates proliferation of enteric neural crest cells during embryogenesis. In addition, Spry1-deficient mice show kidney abnormalities resulting from hyperresponsiveness of the Wolffian duct to GDNF.(35,36) Thus, the SPRY family of proteins plays critical roles in normal development of both the enteric nervous system and the kidney by regulating ERK signaling downstream of RET tyrosine kinase.
SPRY1 and/or SPRY2 are downregulated in several types of cancers, including breast, prostate and liver cancers,(37) suggesting that the change in SPRY expression may be associated with the malignant phenotypes of these cancers. Conversely, SPRY2 expression was found to be upregulated in melanoma with B-RAF mutations. For example, SPRY2 protein binds to wild-type B-RAF but not to mutant B-RAF (V599E mutation), resulting in decreased inhibition of ERK signaling by mutant B-RAF.(38) Complete understanding of the role of these proteins in carcinogenesis will require analysis of the mechanisms by which each protein in the SPRY family inhibits RTK signaling.