Abrogation of Nerve Growth Factor-induced Terminal Differentiation by ret Oncogene Involves Perturbation of Nuclear Translocation of ERK

Abstract

Oncogenic variants of the receptor tyrosine kinase, Ret, cause formation of tumors of neuroendocrine derivation in the multiple endocrine neoplasia type 2 and, thus, likely interfere with antiproliferative and/or differentiative extracellular signals. Here we took advantage of two rat pheochromocytoma-derived cell lines (PC12/MEN2A and PC12/MEN2B) to investigate whether Ret-induced nerve growth factor (NGF) unresponsiveness might involve impairment of ERK signaling. In fact, these cells, stably transfected with distinct forms of the active ret oncogene, fail to block proliferation, even upon NGF stimulation. In these cells we show the presence of both chronic ERKs activity and high expression levels of MKP-3, an ERK-specific phosphatase. Despite the presence of MKP-3, ERK activity can be further stimulated by NGF, but it fails to translocate into the nucleus and consequently to induce immediate-early gene transcription. Because of the presence of MKP-3, our results suggest the existence of a negative regulatory feedback acting on ERKs as a mechanism responsible for the abrogation of NGF-induced terminal differentiation. Indeed, MKP-3 seems to be implicated in the persistence of ERKs in cell cytoplasm. This interpretation is further supported by the observation that in ret-transfected cells, forced expression of an active form of MEK-1 may overcome this block; it restores transcription from the c-fos promoter, induces translocation of ERKs into the nucleus, and inhibits cell proliferation.Ret is a receptor tyrosine kinase whose expression is restricted to neuronal cells of the central and peripheral nervous system. Recently four ligands have been described for this receptor as follows: glial cell-derived neurotrophic factor, neurturin, artemin, and persephin (1).Germ line mutations of the receptor tyrosine kinase, Ret, are responsible for the multiple endocrine neoplasia (MEN) 1 type 2A and 2B syndrome and for the familial medullary thyroid carcinoma (2-5). MEN-2A and MEN-2B are distinct hereditary neoplastic syndromes both characterized by the presence of medullary thyroid carcinomas and pheochromocytomas (6, 7). Missense mutations at one of five cysteine residues (Cys-609, -611, -618, -620, and -634) clustered in the extra cytoplasmic domain of ret are the most frequent causative genetic events of familial medullary thyroid carcinoma and the MEN-2A syndrome (8). A single point mutation, which results in a Thr for Met substitution at codon 918 within the Ret catalytic domain, is responsible for the MEN-2B syndrome (8). These mutations convert ret into a dominant transforming gene and cause constitutive activation of its intrinsic tyrosine kinase activity, although their mechanism of activation differs (8 -10). In MEN-2 syndromes, the molecular mechanisms by which the mutated Ret contribute to the development of neuroendocrine neoplasms remain largely unknown. Indeed, the inheritance of mutated ret alleles implicates them in the pathogenesis of a generalized hyperplasia of the entire population of thyroid C-cells...

Asbestos-Induced MKP-3 Expression Augments TNF-α Gene Expression in Human Monocytes

“…This explanation does not necessarily explain the mechanism by which MKP-3 augments TNF-a gene expression unless MKP-3 was constitutively active. One study has shown, however, that a catalytically inactive mutant MKP-3 can bind to ERK and retain it in the cytoplasm, thus preventing ERK translocation to the nucleus (46). Although MKP-3 can be present in nonstimulated cells, we do not believe this to be the case in human monocytes because our data demonstrate that MKP-3 expression increases in a time-dependent manner after exposure to asbestos.…”

“…Cells expressing the wildtype MKP-3 expression vector had a significant decrease in luciferase activity, while cells expressing the mutant MKP-3 (C293S) had a partial recovery of the ELK-1 luciferase activity that remained below baseline ( Figure 4D). The fact that the mutant MKP-3 did not salvage ERK activation is not surprising, considering the fact that the amino-terminal, noncatalytic domain binds to the ERK and retains it in the cytoplasm regardless of whether ERK is phosphorylated (21,46,47). In particular, one study has demonstrated that overexpression of a catalytic mutant MKP-3 vector prevents downstream ERKmediated gene expression (21).…”

An undigested gliadin peptide activates innate immunity and proliferative signaling in enterocytes: the role in celiac disease

“…Complementary DNAs were generated from total RNA by using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) (25). The resulting cDNA samples were subjected to 10 cycles of polymerase chain reaction (PCR) amplification followed by real-time PCR using the TaqMan PreAmp Master Mix Kit Protocol (PN 4366127; Applied Biosystems).…”

Multiple endocrine neoplasia type 2 and RET: from neoplasia to neurogenesis

“…GRB7 or 10 binding (Y905) has also been linked to activation of this pathway 82. In some cell types, RET mediated activation of RAS dependent pathways can result in cellular differentiation,81 while in other cell types, RET activation may actually block RAS induced differentiation by preventing nuclear translocation of ERK and induction of immediate early gene transcription 83…”