RET expression and neuron-like differentiation of pheochromocytoma and normal chromaffin cells.

Receptor tyrosine kinase RET is normally expressed at low levels in chromaffin cells and high levels in sympathetic neurons. Paradoxically, it is overexpressed in subsets of pheochromocytomas. The overexpressed protein is usually wild-type, except in multiple endocrine neoplasia type 2. Possible explanations for overexpression include tumor origin from RET-expressing sympathoadrenal progenitors that escape developmental culling during embryogenesis, or reactivation of signaling pathways related to neuronal differentiation. Normal adult chromaffin and pheochromocytoma cells can undergo neuron-like differentiation in cell culture. In this investigation, cultured cells from two normal human adrenal medullas, two of three human pheochromocytomas, and one extra-adrenal paraganglioma showed RET induction corresponding with extensive nerve growth factor-induced outgrowth of neurite-like processes, while one pheochromocytoma showed neither processes nor RET induction. RET was similarly upregulated in parallel with process outgrowth in cultures of normal rat chromaffin cells and PC12 rat pheochromocytoma cells. In contrast, mouse pheochromocytoma cells that constitutively express high levels of wild-type RET together with other neuronal progenitor markers showed no further RET increase after cyclic AMP-induced process outgrowth. The RET-activating ligand GDNF was anti-apoptotic for mouse pheochromocytoma but not for PC12 cells. The findings suggest that overexpression of RET in pheochromocytomas could result either from a secondary event that activates signaling pathways mediating adult chromaffin cell plasticity or as a component of a persistent sympathoadrenal progenitor phenotype. Whether wild-type RET contributes to tumor development or is merely a lineage marker for cells at various stages of neuronal differentiation may vary, with other tumor characteristics.

Pheochromocytomas in Nf1 knockout mice express a neural progenitor gene expression profile.

Pheochromocytomas are adrenal medullary tumors that typically occur in adult patients, with increased frequency in multiple endocrine neoplasia type 2, von Hippel-Lindau disease, familial paraganglioma syndromes and neurofibromatosis type 1 (NF1). Pheochromocytomas arise in adult mice with a heterozygous knockout mutation of exon 31 of the murine Nf1 gene, providing a mouse model for pheochromocytoma development in NF1. We performed a microarray-based gene expression profiling study comparing mouse pheochromocytoma tissue to normal adult mouse adrenal medulla to develop a basis for studying the pathobiology of these tumors. The findings demonstrate that pheochromocytomas from adult neurofibromatosis knockout mice express multiple developmentally regulated genes involved in early development of both the CNS and peripheral nervous system. One of the most highly overexpressed genes is receptor tyrosine kinase Ret, which is known to be transiently expressed in the developing adrenal gland, down-regulated in adult adrenals and often overexpressed in human pheochromocytomas. Real-time polymerase chain reaction validated the microarray results and immunoblots confirmed the overexpression of Ret protein. Other highly expressed validated genes include Sox9, which is a neural crest determinant, and Hey 1, which helps to maintain the progenitor status of neural precursors. The findings are consistent with the recently proposed concept that persistent neural progenitors might give rise to pheochromocytomas in adult mouse adrenals and suggest that events predisposing to tumor development might occur before formation of the adrenal medulla or migration of cells from the neural crest. However, the competing possibility that developmentally regulated neural genes arise secondarily to neoplastic transformation cannot be ruled out. In either case, the unique profile of gene expression opens the mouse pheochromocytoma model to new applications pertinent to neural stem cells and suggests potential new targets for treatment of pheochromocytomas or eradication of their precursors.

Up-regulation of ret by reserpine in the adult rat adrenal medulla.

The receptor tyrosine kinase, ret, is activated by glial cell line-derived neurotrophic factor, neurturin and related ligands that bind to glycosylphosphatidylinositol-tailed receptors GFRalpha1-4. Ret expression is developmentally regulated and detectable only at very low levels in adult adrenal medulla. However, mutations of ret that cause constitutive activation or alter signal transduction give rise to adrenal medullary hyperplasia and pheochromocytomas in humans with hereditary multiple endocrine neoplasia (MEN) syndromes 2A and 2B and in animal models. These discordant observations pose the conundrum of how a molecule barely detectable in the adult adrenal can contribute to development of adrenal medullary pathology that typically occurs in adults. We recently reported that depolarization and phorbol esters that activate protein kinase C act synergistically with neurturin to up-regulate ret protein and mRNA expression in adult rat chromaffin cell cultures. Those findings suggested that ret expression in vivo is not static and might be regulated in part by neurally derived signals. We show here that the anti-hypertensive agent reserpine, which is known to cause a reflex increase in trans-synaptic stimulation of chromaffin cells, increases expression of ret mRNA and protein in adult rat adrenal medullary tissue in vivo. Elevated ret protein levels are detectable both by immunoblots and immunohistochemistry, which shows immunoreactive ret in chromaffin cells and neurons after reserpine administration. The finding that ret expression is subject to up-regulation by environmental signals in vivo suggests that epigenetic factors might influence the development of adrenal medullary disease by affecting the expression of ret. It is known that long-term administration of reserpine leads to the development of adrenal medullary hyperplasia and pheochromocytomas in rats. Our findings suggest potential utility of the rat model for studying the roles of ret in the adrenal medulla and the mechanisms of its involvement in MEN 2 and other pheochromocytoma syndromes.

Animal models of pheochromocytoma.

Pheochromocytomas are neuroendocrine tumors of adrenal chromaffin cells. They are rare in all species except rats but occur with increased frequency in several human familial tumor syndromes. Concurrence of pheochromocytoma with other tumors sometimes parallels these human syndromes in rats, bovines, horses and dogs but a shared genetic basis for human and spontaneously occurring animal pheochromocytomas has thus far not been established. Pheochromocytomas are inducible in rats by a variety of non-genotoxic substances that may act indirectly by stimulating chromaffin cell proliferation. They are not known to be similarly inducible in other species but arise with increased frequency in transgenic and knockout mice that to varying degrees recapitulate human tumor syndromes. Preliminary evidence suggests that homologous somatic genetic changes might contribute to pheochromocytoma development in humans and some mouse models. The nerve growth factor-responsive PC12 cell line, established from a rat pheochromocytoma, has for almost 30 years served as a research tool for many aspects of neurobiology involving normal and neoplastic conditions. Recently developed pheochromocytoma cell lines from neurofibromatosis knockout mice supplement the PC12 line and have generated additional applications. Advantages of the mouse lines include expression of substantial levels of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase and expression of high levels of the receptor tyrosine kinase, Ret, which is characteristic of sporadic and familial human pheochromocytomas but not of PC12 cells. Disadvantages include an apparently less stable phenotype. It is difficult to establish pheochromocytoma cell lines from any species, although the tumor cells persist in culture for many months. Understanding of factors that permit pheochromocytoma cells to proliferate might itself provide important insights for tumor biology.

Potentiation of mitogenesis in adult rat chromaffin cell cultures by immunosuppressive agent FK506.

The immunosuppressive drugs FK506 and cyclosporin inhibit T- and B-lymphocyte proliferation and exert neuritogenic and/or cytoprotective effects on several types of neurons. While the immunosuppressive actions of both drugs are mediated in large part by inhibition of the Ca(2+)-dependent phosphatase, calcineurin, FK506 is known to exert additional effects. In the present study, FK506 is shown to potentiate mitogenic effects of the neurotrophic factor, neurturin, on normal adult rat adrenal chromaffin cells in culture. The effect is not seen with cyclosporin or with a non-immunosuppressive analog of FK506, GPI-1046. The finding of increased mitogenesis in response to FK506 may have applications to the study of normal and neoplastic neuroendocrine cells and to understanding the development of some types of tumors in transplant patients.

Chromaffin cell mitogenesis by neurturin and glial cell line-derived neurotrophic factor.

Neurturin and glial cell line-derived neurotrophic factor are novel mitogens for normal adult rat chromaffin cells in vitro. These neurotrophic factors differ from the previously described adult chromaffin cell mitogens, nerve growth factor and basic fibroblast growth factor, in that their effects are potentiated by depolarization and activation of protein kinase C. Neurturin and glial cell line-derived neurotrophic factor signal via the receptor tyrosine kinase, ret, but may also act independently of ret. Both depolarization and phorbol esters act synergistically with neurturin to up-regulate ret protein expression in chromaffin cell cultures, suggesting a mechanism for potentiation of mitogenesis. However, a direct role for ret in mitogenesis has not been established. Stimulation by neurturin causes increased phosphorylation of extracellular signal-regulated kinases 1 and 2 in cultured chromaffin cells, and mitogenesis is prevented by inhibitors of their phosphorylation. Inhibitors of phosphatidylinositol 3-kinase also prevent mitogenesis. The present findings suggest the hypothesis that neurotrophic factors and neurally derived signals might cooperatively regulate chromaffin cell proliferation in vivo in the rat. In addition, trans-synaptic stimulation might provide a route by which epigenetic factors could influence the development of adrenal medullary hyperplasia in humans with hereditary multiple endocrine neoplasia syndromes 2A and 2B by affecting expression and/or activation of ret.

Mitogenic signaling by cyclic adenosine monophosphate in chromaffin cells involves phosphatidylinositol 3-kinase activation.

Increase of intracellular cyclic adenosine monophosphate by the permeant cyclic adenosine monophosphate analog, 8-(4-chlorophenylthio)-adenosine 3':5'- cyclic monophosphate, is mitogenic for normal adult rat chromaffin cells. The mitogenic effect is blocked by the phosphatidylinositol 3-kinase inhibitor, LY294002, and is associated with accumulation of phosphorylated Akt and p70S6 kinase, suggesting that cyclic adenosine monophosphate activates Type l phosphatidylinositol 3-kinase. The mechanism of activation was examined in PC12 pheochromocytoma cells, which are neoplastic chromaffin cells that exhibit many of the biochemical characteristics of their normal counterparts. Incubation of PC12 cells with 8-(4-chlorophenylthio)-adenosine 3':5'- cyclic monophosphate led to a significant increase in total phosphatidylinositol 3-kinase activity that was sensitive to low concentrations of LY294002. The increase was maximal at 1 h and returned to basal levels within six hours. Immunoprecipitation studies showed no increase in phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immune complexes from PC12 cells stimulated by 8-(4-chlorophenylthio)-adenosine 3':5'- cyclic monophosphate, in contrast to cells stimulated by nerve growth factor. Instead, activity was demonstrated in association with p110gamma and p85. These findings suggest that cyclic adenosine monophosphate causes activation of Types IA and IB phosphatidylinositol 3-kinase by a novel mechanism in chromaffin and pheochromocytoma cells. That activation may contribute to chromaffin cell proliferation and to the development and progression of pheochromocytomas. J. Cell. Biochem. Suppl. 36: 89-98, 2001.

C-cell hyperplasia, pheochromocytoma and sympathoadrenal malformation in a mouse model of multiple endocrine neoplasia type 2B.

Dominantly inherited multiple endocrine neoplasia type 2B (MEN2B) is characterized by tumors of the thyroid C-cells and adrenal chromaffin cells, together with ganglioneuromas of the gastrointestinal tract and other developmental abnormalities. Most cases are caused by substitution of threonine for Met918 in the RET receptor tyrosine kinase, which is believed to convert the RET gene to an oncogene by altering the enzyme's substrate specificity. We report the production of a mouse model of MEN2B by introduction of the corresponding mutation into the ret gene. Mutant mice displayed C-cell hyperplasia and chromaffin cell hyperplasia progressing to pheochromocytoma. Homozygotes did not develop gastrointestinal ganglioneuromas, but displayed ganglioneuromas of the adrenal medulla, enlargement of the associated sympathetic ganglia and a male reproductive defect. Surprisingly, homozygotes did not display any developmental defects attributable to a loss-of-function mutation. Thus, while our results support the conclusion that the Met918Thr substitution is responsible for MEN2B, they suggest that the substrate specificity of the RET kinase does not interfere with its normal role in the development of the kidneys and enteric nervous system.

Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice.

Transplantable tumors and cell lines have been developed from pheochromocytomas arising in mice with a heterozygous knockout mutation of the neurofibromatosis gene, Nf1. Nf1 encodes a ras-GTPase-activating protein, neurofibromin, and mouse pheochromocytoma (MPC) cells in primary cultures typically show extensive spontaneous neuronal differentiation that may result from the loss of the remaining wild-type allele and defective regulation of ras signaling. However, all MPC cell lines express neurofibromin, suggesting that preservation of the wild-type allele may be required to permit the propagation of MPC cells in vitro. MPC lines differ from PC12 cells in that they express both endogenous phenylethanolamine N-methyltransferase (PNMT) and full-length PNMT reporter constructs. PNMT expression is increased by dexamethasone and by cell-cell contact in suspension cultures. Mouse pheochromocytomas are a new tool for studying genes and signaling pathways that regulate cell growth and differentiation in adrenal medullary neoplasms and are a unique model for studying the regulation of PNMT expression.

Vitamin D3-induced proliferative lesions in the rat adrenal medulla.

Adrenal medullary hyperplasia and pheochromocytomas are induced in rats by a variety of non-genotoxic agents, and we have hypothesized that these agents induce lesions indirectly by stimulating chromaffin cell proliferation. Vitamin D3, which has not been previously associated with adrenal medullary proliferative lesions, is the most potent in vivo stimulus to chromaffin cell proliferation yet identified. The present investigation utilized the vitamin D3 model to prospectively test the relationship between mitogenicity and focal proliferative lesions in the adrenal medulla and to determine early events in the pathogenesis of these lesions. Charles River Crl:CD BR rats were treated with 0; 5000; 10,000; or 20,000 IU/kg/day of vitamin D3 in corn oil (5 ml/kg) by oral intubation. Rats were killed after 4, 8, 12, or 26 weeks of treatment, following a final week of labeling with bromodeoxyuridine (BrdU) using a mini-pump. Adrenal sections were double-stained for BrdU and phenylethanolamine-N-methyl transferase (PNMT) to discriminate epinephrine (E) from norepinephrine (NE) cells or for vesicular acetylcholine transporter (VAchT) to identify cholinergic nerve endings. Vitamin D3 caused a 4-5-fold increase in BrdU labeling at week 4, diminishing to a 2-fold increase by week 26. An initial preponderance of labeled E cells gave way to a preponderance of labeled NE cells. By week 26, 17/19 (89%) animals receiving the 2 highest doses of vitamin D3 had focal adrenal medullary proliferative lesions, in contrast to an absence of lesions in control rats. The lesions encompassed a spectrum including BrdU-labeled "hot spots" not readily visible on H and E sections, hyperplastic nodules, and pheochromocytomas. Lesions were usually multicentric, bilateral, and peripheral in location, and almost all were PNMT-negative. The lesions were not cholinergically innervated, suggesting autonomous proliferation. Hot spots, hyperplastic nodules, and pheochromocytomas appear to represent a continuum rather than separate entities. Their development might involve selective responses of chromaffin cell subsets to mitogenic signals, influenced by both innervation and corticomedullary interactions. A number of non-genotoxic compounds that induce pheochromocytomas in rats are known to affect calcium homeostasis. The results of this study provide further evidence to support the hypothesis that altered calcium homeostasis is indirectly involved in the pathogenesis of pheochromocytomas, via effects on chromaffin cell proliferation.