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Molecular Endocrinology, doi:10.1210/me.2003-0173
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Molecular Endocrinology 18 (4): 1004-1017
Copyright © 2004 by The Endocrine Society

Biological Effects of the Dual Phenotypic Janus Mutation of ret Cosegregating with Both Multiple Endocrine Neoplasia Type 2 and Hirschsprung’s Disease

Elena Arighi, Anna Popsueva, Debora Degl’Innocenti, Maria Grazia Borrello, Cristiana Carniti, Nina M. Perälä, Marco A. Pierotti and Hannu Sariola

Developmental Biology (E.A., A.P., N.M.P., H.S.), Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland FIN-00014; Department of Experimental Oncology (E.A., D.D., M.G.B., C.C., M.A.P.), Istituto Nazionale Tumori, 20133 Milan, Italy; and Helsinki University Central Hospital (HUCH) Laboratory Diagnostics (H.S.), Paediatric Pathology, HUCH, Helsinki, Finland FIN-00029

Address all correspondence and requests for reprints to: Hannu Sariola, Developmental Biology, Institute of Biomedicine, Biomedicum Helsinki, P.O. Box 63, University of Helsinki, Helsinki, Finland FIN-00014. E-mail: Hannu.Sariola{at}helsinki.fi.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gain-of-function mutations of ret receptor tyrosine kinase, the signaling receptor for glial cell line-derived neurotrophic factor, cause sporadic thyroid and adrenal malignancies as well as endocrine cancer syndromes, such as multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma. Loss-of-function mutations of ret cause Hirschsprung’s disease (HSCR) or colonic aganglionosis. In 20–30% of families with a mutation at residues 609, 611, 618, or 620 of RET, MEN 2A and familial medullary thyroid carcinoma cosegregate with HSCR. These mutations constitutively activate RET due to aberrant disulfide homodimerization and diminish the level of RET at the plasma membrane. It is not known how these mutations simultaneously lead to both gain- and loss-of-function RET-associated diseases. We provide an explanation for the dual phenotypic Janus mutation at Cys620 of RET. In Madin-Darby canine kidney (MDCK) cells, the Janus mutation impairs the glial cell line-derived neurotrophic factor-induced effects of RET on cell migration, differentiation, and survival but simultaneously promotes rapid cell proliferation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ret GENE encodes a transmembrane receptor tyrosine kinase with two main isoforms of 1072 (short form) or 1114 (long form) amino acids. Gain-of-function mutations of ret in germ line cause three types of endocrine cancer syndromes. Multiple endocrine neoplasia type 2A (MEN 2A) is a familial cancer syndrome with an autosomal dominant trait and high penetrance. It is characterized by bilateral and multicentric medullary thyroid carcinomas (MTCs) arising from calcitonin-secreting thyroid C cells, pheochromocytoma, and, less frequently, hyperplasia of the parathyroid glands. Multiple endocrine neoplasia type 2B (MEN 2B) includes MTC, pheochromocytoma, oral neuromas, ganglioneuromatosis of the digestive tract, and skeletal abnormalities. Familial MTC (FMTC) is defined by the sole occurrence of MTC (reviewed in Refs.1, 2, 3). Different allelic mutations in ret are responsible for these three cancer syndromes, and a correlation between the position of the mutations and the clinical phenotypes has been established (4). The vast majority of MEN 2A and FMTC mutations occur in a cluster of six highly conserved cysteines (Cys 609, 611, 618, 620 in exon 10; 630 and 634 in exon 11). Missense mutations affecting noncysteine residues within the tyrosine kinase domain have been found in FMTC families. Most MEN 2B cases are caused by a unique mutation (M918T), which lies in the catalytic domain of RET. The nature of the MEN 2 mutations dictates the molecular mechanisms of RET activation. The MEN 2B mutation modifies the substrate specificity of the tyrosine kinase. The MEN 2A and FMTC mutations involving cysteines result in constitutive activation of the tyrosine kinase through the formation of intermolecular disulfide-bonded RET homodimers (reviewed in Ref.5).

HSCR or colonic aganglionosis is the main cause for congenital constipation with an incidence of 1/5000 live births. This developmental disorder is characterized by the absence of the enteric ganglia along variable lengths of colon. It is a multigenic disorder with low penetrance and variable expression. To date, eight genes have been associated with HSCR. The major susceptibility gene is ret, in which mutations have been identified in 50% of familial and 15–35% of sporadic HSCR cases (reviewed in Ref.6). The range of ret mutations including insertions, deletions, nonsense, missense, and splicing mutations is suggestive of a loss of RET function in HSCR. Most HSCR-associated mutations indeed disable the activation or expression of RET (7). However, a simple activating vs. inactivating model of gene action is not sufficient to explain the cosegregation of MEN 2A/FMTC with HSCR in patients with a point single mutation at residues 609, 611, 618, or 620.

RET receptor tyrosine kinase and glial cell line-derived neurotrophic factor (GDNF) family receptor {alpha}1 (GFR{alpha}1) form the signaling receptor complex for GDNF. It first forms a high-affinity complex with glycosylphosphatidylinositol-anchored or soluble GFR{alpha}1, which then activates RET (reviewed in Ref.8). In the presence of soluble GFR{alpha}1, ret-transfected MDCK cells respond to GDNF like parental MDCK cells respond to hepatocyte growth factor (HGF) (9). HGF, the ligand for Met receptor tyrosine kinase (10), induces scattering, chemotactic movements, and tubule formation in these cells (11). In GFR{alpha}1-expressing cells, GDNF activates Met via Src, and this signaling pathway is RET independent (12).

In this study, we analyzed the biological effects of classical loss-of-function (HSCR-associated) and gain-of-function (MEN 2 causing) mutations in ret, and compared these mutations with the double-faced Janus mutation at Cys620 of RET. Like the two mutually incompatible faces of the Roman god Janus, this mutation causes both HSCR and MEN2 in a sizeable fraction of families. The Janus-RET-expressing MDCK cells are incapable of responding chemotactically to GDNF. The cells also fail to migrate, form branching tubules, and be protected from apoptosis in response to GDNF. All these features are shared by HSCR mutations and the Janus Cys620 mutation of RET. On the other hand, the MDCK cells expressing the Janus-C620R protein proliferate rapidly, at the same rate as the MEN 2A-C634R-expressing cells, whereas the proliferation rate is very low in the cells transfected with ret bearing HSCR mutations. Our results explain how a single mutation in ret is associated with loss- and gain-of-function effects and can lead to opposing disease phenotypes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Activity of RET Mutations in HSCR, MEN 2A, or FMTC
To analyze the biological effects of loss-of-function and gain-of-function mutations of the ret gene, we introduced mutations identified in patients with HSCR, MEN 2A, or FMTC to the coding sequence of the short isoform of ret by site-directed mutagenesis. The residues Cys620 (Janus-C620R), Cys634 (MEN2A-C634R), Ser765 (HSCR-S765P), Ser767 (HSCR-S767R), or Glu768 (FMTC-E768D) were replaced. The positions of these mutations in the RET protein are represented in Fig. 1Go. All constructs were sequenced and cloned into the pcDNA3 eukaryotic expression vector. The synthesis of the correctly sized proteins by the constructs was verified by transient expression in 293T human embryonic kidney (HEK) cells (Fig. 2AGo, bottom panel). The 140-kDa product corresponds to an incompletely glycosylated precursor of RET present in the endoplasmic reticulum whereas the 160-kDa band is the mature RET protein, fully glycosylated and expressed at the cell surface (13).



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  		Fig. 1. RET Receptor Tyrosine Kinase with a Signal Peptide (SP), Cadherin-Like (CAD), Cysteine-Rich (CYS), Transmembrane (TM), and Tyrosine Kinase (TK) Domains

The disease-associated mutations analyzed in this study are marked.

 


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  		Fig. 2. Expression and Activation of the RET Proteins and PLC{gamma}

A, In vitro immunokinase assays of the RET proteins. Wild-type RET (RET), RET-K758R, Janus-C620R, MEN2A-C634R, HSCR-S765P, HSCR-S767R, and FMTC-E768D proteins were transiently expressed in HEK 293T cells, immunoprecipitated with anti-RET antiserum ({alpha}RET), and subjected to autokinase assay and immunokinase assay against the myelin basic protein (MBP). The kinase-deficient RET-K758R (K758R) protein was used as negative control. Equal amounts of the immunoprecipitates were subjected to Western blotting using anti-RET antibodies ({alpha}RET). The numbers below the lanes indicate the fold increase relative to wild-type RET as quantified by ImageQuant software (MediaCybernetics, Inc., Silver Spring, MD). IP, Immunoprecipitation; WB, Western blotting. B, HSCR-mutant proteins fail to phosphorylate PLC{gamma}. The lysates from mock-transfected, wild-type RET, Janus-C620R, MEN 2A-C634R, HSCR-S765P, HSCR-S767R, and FMTC-E768D-expressing COS cells were immunoprecipitated with anti-PLC antibody ({alpha}PLC{gamma}), followed by immunoblotting with phosphotyrosine antibody ({alpha}Ptyr). The same filter was reprobed with {alpha}PLC{gamma} antibody. The lower panels show total cell lysates subjected to immunoblotting with {alpha}Ptyr, {alpha}RET, or {alpha}PLC{gamma} antibodies. A and B, Results are representative of three independent experiments.

 
We have previously demonstrated that the MEN 2A-C634R protein is constitutively phosphorylated, and its autokinase and kinase activities toward the exogenous substrate myelin basic protein (MBP) are higher than those of wild-type RET (14). The mutation Janus-C620R also leads to constitutive RET activity (15). Both MEN 2A-C634R and Janus-C620R mutations activate RET by causing ligand-independent disulfide-bridged homodimerization (15, 16, 17, 18). We now tested the effects of other disease-associated mutations on the RET kinase activity. The HSCR-S765P mutation caused a 100-fold decrease in the autokinase activity of RET and the phosphorylation of MBP (Fig. 2AGo). Autokinase activity of the HSCR-S767R protein as well as phosphorylation of the exogenous substrate were lower than in the wild-type RET. The gain-of-function FMTC-E768D mutation (19) did not affect the RET autokinase activity, but its ability to phosphorylate MBP was 5-fold higher than that of wild-type RET.

We next examined the phosphorylation of phospholipase C {gamma} (PLC{gamma}) in COS cells transiently transfected with the different ret mutant constructs. The PLC{gamma} pathway is impaired by HSCR-associated mutations of RET (20). Phosphorylation of PLC{gamma} was induced in cells expressing RET, Janus-C620R, MEN2A-C634R, or FMTC-E768D proteins, whereas it was undetectable in cells expressing the HSCR-S765P or HSCR-767R proteins and in mock-transfected cells (Fig. 2BGo). Because PLC{gamma} activation is correlated to its phosphorylation (21), the results indicate that PLC{gamma} is activated by wild-type RET and gain-of-function RET mutant proteins. Complete or partial loss of RET kinase activity and disruption of PLC{gamma} signaling are functional consequences of the S765P- and S767R-HSCR mutations, in accordance with previous studies (7, 20).

Janus-C620R Dimers Are Unresponsive to GDNF
The ability of the gain-of-function mutations to activate and loss-of-function mutations to inactivate RET is apparently the molecular basis of MEN 2 and HSCR, respectively, but this model does not explain why HSCR and MEN 2 cosegregate, albeit with low penetrance, in a sizeable fraction of the families whose affected members carry the Cys620 mutation of RET (22, 23). To address this issue, we established stable RET-MDCK cell lines expressing the above-mentioned RET constructs. Each cell line expressed similar amounts of RET proteins (Fig. 3AGo, bottom panel). To verify that the level of RET receptor present at the cell membrane was also comparable, proteins expressed at the surface of the RET-MDCK cells were biotinylated in vivo. Biotin-labeled proteins were precipitated with anti-RET antibody, and the resulting complexes were subjected to SDS-PAGE and Western blotting. Probing of the filters with the streptavidin reagent revealed that similar levels of the 160-kDa mature form of RET were present at the surface of the RET-MDCK cell lines (Fig. 3AGo). In addition to the 160-kDa form, also the 140-kDa precursor slightly reacted with the streptavidin-biotin peroxidase complexes, probably because the 140-kDa protein is the predominant form in RET-MDCK cells and it is present in excess compared with the mature 160-kDa form.



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  		Fig. 3. Cell Surface Expression and Phosphorylation of RET by GDNF

A, Biotinylation of RET proteins. MDCK cells stably expressing wild-type RET or the following mutant proteins, Janus-C620R, MEN 2A-C634R, HSCR-S765P, HSCR-S767R, and FMTC-E768D, were labeled with biotin and immunoprecipitated with anti-RET ({alpha}RET) antibody. The samples were separated on 6% SDS-PAGE and reacted with streptavidin-biotin peroxidase complex. Half of the immunoprecipitated samples were also reacted with anti-RET antibody. B, Phosphorylation of RET by GDNF. After 2 h starvation in serum-free medium, the MDCK cells expressing wild-type RET were stimulated with 100 ng/ml GDNF together with 100 ng/ml sGFR{alpha}1. The cell lysates were immunoprecipitated with anti-RET antibody ({alpha}RET), followed by immunodetection with antiphosphotyrosine antibody ({alpha}PTyr). The lower panel shows the reprobing of the same filter with anti-RET antibody. C, RET phosphorylation induced by 2 h stimulation with GDNF/sGFR{alpha}1 in MDCK cells expressing wild-type RET or the following mutants: Janus-C620R, MEN 2A-C634R, HSCR-S765P, HSCR-S767R, and FMTC-E768D. The same filter was reprobed with anti-RET antibody. A–C, Results are representative of three independent experiments. WB, Western blot; IP, immunoprecipitation.

 
The GDNF responsiveness of wild-type and mutant RET proteins was analyzed by phosphorylation of RET when exposed to soluble GFR{alpha}1 (sGFR{alpha}1) together with GDNF (9). RET phosphorylation was observed after 2 h of GDNF/sGFR{alpha}1 treatment (Fig. 3BGo) and persisted up to 15 h (data not shown). This is in accord with the previously reported extended lag period in the activation of RET by GDNF and sGFR{alpha}1 (9, 24). The delay is most probably due to the kinetics of GDNF/sGFR{alpha}1 association in solution. Nevertheless, the phosphorylation pattern of individual tyrosine residues of RET is the same in both cis and trans stimulation (24). The phosphorylation of the Janus-C620R protein either in the absence or in the presence of GDNF/sGFR{alpha}1 was low, suggesting that the covalent dimers formed by Janus-C620R were unresponsive to GDNF (Fig. 3CGo). The faint phosphorylation of both forms, the immature 140-kDa precursor present in the endoplasmic reticulum and the mature glycosylated 160-kDa protein present at the plasma membrane, was likely due to the constitutive ligand-independent kinase activity and to the cross-phosphorylation due to the crowd of the exogenously expressed Janus-C620R protein. In contrast, the MEN 2A-C634R dimers were sensitive to GDNF. They were highly autoactive, and exhibited an increase in the level of tyrosine phosphorylation upon GDNF/sGFR{alpha}1 treatment. As expected, both HSCR-RET proteins failed to respond to GDNF, although HSCR-S767R showed a significant level of autophosphorylation. The gain-of-function FMTC-E768D protein was highly responsive to ligand stimulation (Fig. 3CGo).

Both Janus-C620R and HSCR Mutations Impair Motility and Migration of GDNF-Stimulated RET-Expressing Cells
RET-expressing MDCK cell lines exhibit increased motility, dissociation, and directed migration in response to GDNF plus sGFR{alpha}1 (9). We now studied the motility of mutant RET-expressing cells in the presence of GDNF plus sGFR{alpha}1, and the chemotactic migration of cells toward GDNF by the Boyden chamber chemotaxis assay. The motility and directed migration were fully abolished in MDCK cells expressing Janus-C620R and HSCR proteins (Fig. 4Go). In contrast, MEN 2A-C634R-expressing cells migrated through the filter even without stimulation by the ligand. Consistent with the increase of tyrosine phosphorylation after ligand stimulation, GDNF plus sGFR{alpha}1 further increased the motility and migration of MEN 2A-C634R-expressing cells. In the cells expressing the FMTC-E768D protein motility and migration were completely ligand dependent.



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  		Fig. 4. Both Janus-C620R and HSCR Proteins Impair Cellular Motility and Migration in Response to GDNF

In Boyden dual-chamber assay, mock-transfected, wild-type RET, Janus-C620R, MEN 2A-C634R, HSCR-S765P, HSCR-S767R, and FMTC-E768D expressing cells were stimulated by GDNF (100 ng/ml), added to both the bottom and top chambers to assay chemokinesis and only to the bottom chamber to assay chemotaxis. Culture media contained 100 ng/ml of sGFR{alpha}1. Analysis was performed with at least two different clones of each RET mutation. Mock-transfected MDCK cells were used as a control. Membranes with the migrated cells were fixed, stained with Giemsa solution, and photographed. One representative experiment of three experiments is shown. The number of migrated cells was counted as described in Materials and Methods. The results shown represent the means ± SEM (n = 3); *, P < 0.05; **, P < 0.01; ***, P < 0.005.

 
Janus-C620R- and HSCR-Expressing Cells Do Not Branch in Response to GDNF
To further analyze the effects of the Janus-C620R in MDCK cells, we examined their ability to form branching tubules in response to GDNF in three-dimensional collagen gel. This property of MDCK cells may be considered as an in vitro epithelial tubulogenesis model, which resembles branching of developing ureteric buds (9, 11, 25). The Janus-C620R mutation allowed only poor branching of RET-MDCK cells that was not increased by GDNF/sGFR{alpha}1 (Fig. 5Go). The two HSCR-associated mutations were entirely incapable of promoting branching of RET-MDCK cells. The MEN 2A-C634R mutation promoted in the cells a constitutive ligand-independent branching activity, which was further increased by GDNF plus sGFR{alpha}1. The FMTC-E768D mutation is a gain-of-function mutation with a poor oncogenic potential and transforming activity in fibroblasts in the absence of GDNF (26, 27). In accordance, the FMTC-E768D protein was unable to induce branching and migration of MDCK cells in the absence of GDNF.



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  		Fig. 5. Janus-C620R and HSCR Mutant Proteins Are Unable to Mediate Branching in Response to GDNF in Three-Dimensional Collagen Culture

MDCK cells expressing the wild-type RET, Janus-C620R, MEN 2A-C634R, HSCR-S765P, HSCR-S767R, and FMTC-E768D were treated with GDNF (100 ng/ml) alone, sGFR{alpha}1 (100 ng/ml) alone, or GDNF (100 ng/ml) together with sGFR{alpha}1 (100 ng/ml). GDNF or sGFR{alpha}1 alone was applied to exclude any possible RET-independent effect. At least two different clones of each RET mutation were used for the analyses, and no discrepancies were found between clones. Mock-transfected MDCK cells were used as a control. The morphology of RET-expressing MDCK cells grown for 3 d in collagen culture with sGFR{alpha}1 alone or GDNF plus sGFR{alpha}1 is shown (magnification, x40). Only the tubules longer than two-cell body diameters were counted. The results are representative of at least three independent experiments. Means ± SEM of four to six independent repeats are shown in the graph. **, P < 0.01; ***, P < 0.005.

 
GDNF Does Not Protect Janus-C620R- and HSCR-Expressing Cells from Apoptosis
Apoptosis in the absence of cell-matrix interactions (also called anoikis) was analyzed to determine whether the Janus-C620R mutation affects the sensitivity to cell death. Normal cell-matrix interactions were disrupted by plating MDCK cells on culture dishes coated with poly-hydroxyethylmethacrylate (polyHEMA) to prevent cell attachment (28), and apoptotic cells were identified by terminal deoxynucleotide transferase (TdT)-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) after 8 h of culture. Anoikis was significantly alleviated in RET-expressing MDCK cells by GDNF plus sGFR{alpha}1 (Fig. 6Go). The cells expressing the Janus-C620R and HSCR proteins were not protected from apoptosis by the RET ligand. In contrast, GDNF rescued MDCK cells expressing MEN 2A-C634R and FMTC-E768D from apoptosis. In accordance with the high autoactivity, the MEN 2A-C634R diminished apoptosis also in the absence of ligand as compared with wild-type RET.



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  		Fig. 6. GDNF Does Not Protect Janus-C620R- and HSCR-RET-Expressing Cells from Apoptosis

Mock-transfected and wild-type RET-, Janus-C620R-, MEN 2A-C634R-, HSCR-S765P-, HSCR-S767R-, and FMTC-E768D-expressing MDCK cells were pretreated with 100 ng/ml GDNF/sGFR{alpha}1 or left unstimulated for 12 h in medium containing 0.5% FCS and then plated onto polyHEMA-coated dishes. After 8 h in the presence or absence of GDNF/sGFR{alpha}1, cells were harvested, and the percentages of cells undergoing apoptosis were determined by TUNEL reactivity against the total number of cells calculated using Hoechst staining. The results shown are representative of three independent experiments. Means ± SEM of at least five fields of view (2 x 103 cells per field) are shown. *, P < 0.05.

 
Janus-C620R and MEN 2A-C634R Promote Rapid Cell Proliferation
GDNF-induced proliferation of the cells expressing wild-type and mutated RET proteins was studied by thiazolyl blue tetrazolium bromide (MTT) (Fig. 7AGo) and bromodeoxyuridine (BrdU) (Fig. 7BGo) cell proliferation assays. GDNF plus sGFR{alpha}1 induced cell proliferation in RET-MDCK cells as much as HGF in mock-transfected MDCK cells. Janus-C620R-expressing cells also showed ligand-independent high proliferative activity that was not further increased by GDNF/sGFR{alpha}1 or HGF in colorimetric assay (Fig. 7AGo). BrdU incorporation confirmed the high proliferative rate and inability of Janus-C620R-MDCK cells to respond to GDNF by proliferation (Fig. 7BGo). Similar ligand-independent high proliferative activity was observed in MEN 2A-C634R-expressing cells. The cells expressing FMTC-E768D increased their growth rate only in the presence of ligand. No increase in the growth rate was seen by GDNF plus sGFR{alpha}1 in HSCR-RET-expressing cells (Fig. 7Go, A and B, and data not shown).



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  		Fig. 7. Growth Rate of RET-MDCK Cells in Response to GDNF

A, Proliferation of the cells was estimated using MTT cell proliferation assays. Cells were serum starved in 0.5% FCS for 2 d, treated with 100 ng/ml GDNF/sGFR{alpha}1 or 50 ng/ml HGF or left untreated for 24, 48, and 72 h, and then assayed for MTT dye conversion. Only results relative to 72-h treatment are shown. Data relative to other time points are published as supplemental data available on The Endocrine Society’s Journals Online web site at http://med.endojournals.org. Results are presented as average ± SEM of three independent experiments each measured in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.005. B, Proliferation of the RET-MDCK cells was verified by BrdU cell proliferation assays. Plates were cultured for 24 h and 48 h. The results shown are relative to 48-h treatment and are representative of three independent experiments Data relative to other time points are published as supplemental data on The Endocrine Society’s Journals Online web site at http://med.endojournals.org. Means ± SEM of five independent repeats are shown. *, P < 0.05; **, P < 0.01.

 
Janus-C620R-Expressing Cells Form Colonies in Soft Agar but Less Efficiently than MEN2A-C634R-Expressing Cells
MDCK cells are untransformed epithelial cells that exhibit certain parallels to the process of tumorigenesis, when disruption of cell-cell contacts, increased cell motility, and ability to grow in soft agar are induced, leading to the transition from an epithelial to a mesenchymal phenotype (29). To determine whether the Janus-C620R protein transformed the cells, a soft-agar assay was performed by comparing the colony-forming efficiency of Janus-C620R- and MEN2A-C634R-expressing cells to that of wild-type RET-expressing cells. The soft agar assay was also performed on RET-expressing NIH 3T3 cells. NIH-MEN2A-C634R-expressing cells showed a high clonogenic efficiency and gave rise to colonies in about 10 d. In contrast, NIH 3T3-Janus-C620R cells formed colonies, but with a much lower efficiency and longer latency (3 wk). Wild-type RET cells did not form colonies in soft agar (Fig. 8AGo). The ability of MDCK-MEN2A-C634R cells to form colonies in soft agar was clearly reduced compared with NIH 3T3-RET-C634R cells (Fig. 8BGo). We did not see colonies formed by MDCK-RET-C620R cells.



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  		Fig. 8. Clonogenic Activity in Soft Agar of NIH 3T3 and MDCK Cells Expressing Wild-Type RET, MEN2A-C634R, or Janus-C620R

A, Colony formation was tested in soft agar containing NIH 3T3 cells (104 cells) stably expressing RET, MEN2A-C634R, or Janus-C620R. Colonies were stained and photographed 2 and 3 wk after plating. Results were confirmed in three independent assays. B, MDCK cells (7.5 x 104 cells) stably expressing RET, MEN2A-C634R, or Janus-C620R were plated in soft agar. Colonies were stained and photographed 2 and 3 wk after plating. These results are representative of three independent assays.

 
The results of the biological effects of the mutant RET proteins analyzed in this study are summarized in Table 1Go.


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  		Table 1. Summary of Biological Effects of Mutant RET Proteins Segregating with MEN 2, HSCR, or Both

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The functional and biological consequences of different mutant RET proteins segregating with either MEN 2 or HSCR, or both, were approached. Like HSCR-associated mutations of RET, the Janus-C620R-expressing cells are unresponsive to GDNF and are unable to migrate, differentiate, and be protected from apoptosis in response to GDNF. On the other hand, the covalent dimerization of Janus-RET confers to the cells ligand-independent rapid proliferation activity, which is at the same rate as in the MEN 2A-C634R-expressing cells, and anchorage-independent growth (Table 1Go). These ligand-independent activities may be responsible for C cell hyperplasia, followed by tumor development in FMTC. Our results may also explain why the MEN 2A-C634R mutation does not associate with HSCR.

We used the MDCK cell model to study the biological effects of different mutations in the short form of RET. Although the two isoforms of RET behave similarly in a number of in vitro assays, several observations have suggested that they may have different tissue-specific effects on embryogenesis and tumorigenesis. Studies with monoisoformic RET-deficient mice indicate that the short form of RET is sufficient to support normal embryogenesis, whereas mice expressing only the long isoform have severe defects in the innervation of the gut and renal development (30). In particular, we focused on the mutation of Cys620 to reconcile why it cosegregates with HSCR and MEN 2A/FMTC. Cys620 mutation decreases the cell surface expression of RET (18, 31) and activates its kinase domain (15, 18), similar to the MEN 2A-C634R mutation, a known gain-of-function mutation (17). Different amino acid substitutions of the same cysteine result in similar transforming activity, indicating that the degree of the autoactivity depends on the position of cysteine mutations rather than the substituting amino acids (31).

The occurrence of HSCR in many MEN 2A/FMTC pedigrees is difficult to explain with a gain-of-function mutation in RET, which is typical for the MEN 2 mutations. Proposed explanations have been a decreased cell surface expression of RET (18, 31, 32), a kinase activity under a threshold required for cell survival (33), and an inability to respond to GDNF and protect RET-expressing cells from apoptosis (34). The Janus mutation promotes high proliferation of the cells similar to the MEN 2A-C634R, apparently accounting for oncogenic capacity associated with the mutation. GDNF no longer increases the proliferation rate of Janus-C620R-MDCK cells, and they are completely unable to migrate and branch in response to GDNF. Insensitivity to GDNF renders cells more prone to apoptosis. These features are shared by HSCR-associated mutations of RET, which are considered as loss-of-function mutations.

Our data may also explain why the MEN 2A-C634R mutation is not associating with HSCR. Unlike the Janus-C620R dimers, MEN 2A-C634R dimers are responsive to GDNF and promote migration, branching, and protection from apoptosis. The ability of MEN 2A-C634R cells to respond to GDNF might allow MEN 2A-C634R-expressing enteric neuron system progenitors to undergo GDNF-induced migration toward and within the gut wall. In accordance, MEN 2A-C634R dimers in neuroectodermal cells are also inducible by GDNF (34).

The findings suggest that the autokinase and kinase activity of RET and its full glycosylation and ability to reach the cell membrane, as well as its responsiveness to GDNF, all affect the functional consequences of RET mutations. The results support a model in which the different MEN 2 phenotypes are determined by constitutive RET activation at a different intensity, associated with the availability of GDNF in different tissues. The complete MEN 2A phenotype is associated with a high activation of RET, which can be still enhanced by binding of the ligand. The cells expressing the MEN2A-C634R protein were constitutively active in all biological assays, and GDNF further potentiated them. Mutations in noncysteine domains of RET causing FMTC are associated with a much lower level of RET activation, and the presence of GDNF is critical for an oncogenic capacity, at least for mutation at residue 768 (E768D). Hyperplasia of the thyroid C cells may be due to active RET signaling during development of the thyroid gland, which expresses GDNF. On the other hand, the absence of pheochromocytomas and hyperparathyroidism in FMTC might be because the ligand is absent from parathyroid and adrenal medulla. Noteworthy, among alterations at Cys634, the mutation that changes cysteine to arginine (C634R) has not been found among families with FMTC (4). The speed of malignant transformation apparently correlates with level of RET activation. The MEN 2A-C634R mutation accounts for the very early onset of MTC, and the FMTC-E768D mutation results in an attenuated form with late onset (35). The features of the Janus-C620R-RET support the hypothesis in which the degree of RET signaling during development may explain the coexistence of MEN 2 with HSCR (5). The low autophosphorylation level of Janus-C620R compared with MEN2A-C634R dimers and the lack of GDNF-induced activities such as chemotactic migration, differentiation, and protection from apoptosis, are characteristics of HSCR-associated mutations, whereas anchorage-independent growth of NIH 3T3 cells and rapid cell proliferation of MDCK cells are features associated with oncogenic mutations of RET. Thus, one may speculate that high RET activity during embryogenesis may be sufficient to initiate C cell hyperplasia, but without augmentation by the ligand, it is insufficient on some genetic backgrounds to drive the development of enteric neuroblasts and rescue them from apoptotic death.

Recently, an association between renal abnormalities and the Janus mutation in ret has been established (36). During embryogenesis, RET is expressed in the embryonic kidney by the tips of the ureteric bud. GDNF is produced by the metanephric blastema and promotes the development of the ureteric bud. The mice lacking gdnf, ret, or gfr{alpha}1 show severe defects in enteric innervation and renal differentiation (reviewed in Ref.37). The inability of Janus-C620R-expressing kidney epithelial cells to form tubules in response to GDNF fits well with the clinical observation that patients with FMTC/MEN 2A carrying the Janus-C620R mutation occasionally exhibit renal malformations. Interestingly, also a few families with HSCR have developmental abnormalities of the kidneys (38).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Transfections
Early-passage MDCK cells were provided by Dr. Eero Lehtonen (Haartman Institute, University of Helsinki). Cells were cultured in MEM, containing 10% fetal calf serum (FCS). To generate stable lines, subconfluent cells in 10-cm dishes were transfected with pcDNA3, pcDNA3-RET, pcDNA3-RET-C620R, pcDNA3-RET-C634R, pcDNA3-RET-K758R, pcDNA3-RET-S765P, pcDNA3-RET-S767R, pcDNA3-RET-E768D, using Fugene 6 (Roche Diagnostics, Indianapolis, IN). Stable clones were selected in 400 ng/ml G418 (Life Technologies, Gaithersburg, MD). HEK 293T and COS7 cells were grown in DMEM supplemented with 10% FCS, transiently transfected by calcium phosphate precipitation and diethylaminoethyl-dextran method, respectively. NIH 3T3 cells were cultured in DMEM containing 10% FCS. NIH 3T3 (2 x 105 per 10-cm diameter dish) cells were stably transfected by calcium phosphate coprecipitation using 200 ng plasmid DNA and 40 µg mouse carrier DNA. G418-resistant colonies were selected in DMEM plus 10% calf serum and G418 antibiotic (0.5 µg/ml).

Site-Directed Mutagenesis and Plasmid Construction
The RET mutants, C620R, K758R, S765P, S767R, and E768D, were generated by site-specific mutagenesis, using the GeneEditor (Promega Corp., Madison, WI) kit and the oligonucleotides 5'-AAG TGC TTC CGC GAG CCC GAA GAC-3', 5'-ACG GTG GCC GTG AGG ATG CTG AAA-3', 5'-GAG AAC GCC CCC CCG AGT GAG CTT-3', 5'-AAC GCC TCC CCG AGG GAG CTT CGA GAC-3', 5'-GCC TCC CCG AGT GAT CTT CGA GAC CTG-3' as primers, respectively. The cDNA insert was cloned into the mammalian expression vector pcDNA3 carrying the geneticin (G418) resistance gene. The cloning of wild-type RET and RET-C634R in pCEP9ß has been previously reported (14). Fragments were excised from the pCEP9ß vector with HindIII and NotI and cloned into pcDNA3.

Immunoprecipitation, Western Blotting, and in Vitro Kinase Assays
To analyze RET phosphorylation, subconfluent stably transfected MDCK cell lines were starved for 2 h before stimulation in serum-free MEM. After indicated time periods at 37 C with 100 ng/ml GDNF (Cephalon, Inc., West Chester, PA) together with 100 ng/ml soluble recombinant GFR{alpha}1 (R&D Systems, Minneapolis, MN) cells were lysed in lysis buffer (10 mM sodium phosphate, pH 7, 100 mM NaCl, 1% Triton X-100, 5 mM EDTA) supplemented with a protease inhibitors cocktail (Roche Diagnostics) and phosphatase inhibitor 1 mM Na3VO4. Equal amounts of cell lysates were incubated with protein A-Sepharose (Amersham Biosciences, Buckinghamshire, UK) precoated with anti-RET antibodies (C-19 rabbit polyclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

COS cells were lysed in PLCLB Buffer (50 mM HEPES, pH 7.4; 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM Na4P2O7, 10 mM NaF) supplemented with a protease inhibitors cocktail (Roche Diagnostics) and with 1 mM Na3VO4. PLCLB extracts were immunoprecipitated with anti-PLC-{gamma} antibody (PLC-{gamma}1, Santa Cruz Biotechnology, Inc.). For total cell lysates, cells were lysed in sodium dodecyl sulfate (SDS) lysis buffer (62.5 mM Tris-HCl, pH 6.8; 2% SDS).

For Western blotting, total extracts and immunoprecipitates were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. The filters were blocked with 5% milk or 2% BSA, visualized by anti-RET, antiphosphotyrosine (anti-PTyr, Upstate Biotechnology, Inc., Lake Placid, NY) and anti-PLC-{gamma} primary antibodies as indicated and horseradish peroxidase-conjugated secondary antibodies (DAKO, Glostrup, Denmark) and detected by the enhanced chemiluminescence (ECL) method (Amersham Biosciences).

For the RET in vitro immunokinase assays, the anti-RET immunoprecipitates were washed twice with lysis buffer, once with incubation buffer (50 mM HEPES, pH 7.2; 20 mM MnCl2, 5 mM phenylmethylsulfonyl fluoride), and incubated for 15 min at +4 C in 20 µl of the same buffer containing 0.5 mM dithiothreitol, 4 µCi {gamma}-ATP diluted with unlabeled ATP to the final concentration of 26 pmol ATP per sample, and 50 µM MBP. The reactions were stopped by an equal volume of 2x reducing Laemmli buffer. Proteins were eluted and separated in 6% or 15% SDS-PAGE. 32P-labeled bands were visualized by autoradiography. The intensity of the bands corresponding to phosphorylated MBP was quantified by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and was expressed as the fold increase relative to unstimulated wild-type RET, which was given the arbitrary value of 1.

Biotin Labeling of Cell Surface Proteins
Biotinylation was performed with ECL Protein Biotinylation Module (Amersham Biosciences) according to the manufacturer’s instructions. Briefly, MDCK cells (5 x 106) stably expressing mutant RET proteins were washed twice with cold PBS. Biotinylation was performed with 40 µl biotinylation reagent in 40 mM bicarbonate buffer (2 ml/10-cm diameter dish); the plates were incubated 15 min on ice on an orbital shaker. The biotinylation buffer was removed, and cells were washed twice with cold PBS containing 50 mM glycine. Cells were lysed in RIPA buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40). Cell extracts were immunoprecipitated with anti-RET antibodies (C-19 rabbit polyclonal, Santa Cruz Biotechnology), resolved by SDS-PAGE, and transferred onto nitrocellulose membranes. The filters were blocked with 5% membrane blocking agent (Amersham Biosciences), visualized by streptavidin-horseradish peroxidase conjugate (Amersham Biosciences), and revealed with the ECL system.

Cell Motility and Migration Assays
Cells (105) were suspended in 300 µl MEM containing 10% FCS and seeded into 24-well cell culture inserts with filters (Boyden chambers) (pore size, 8 µm; Falcon). GDNF/sGFR{alpha}1, GDNF alone, or sGFR{alpha}1 alone were added to both the bottom and top chambers at 100 ng/ml for the motility assay, and to the bottom chamber only for the migration assay. After 48 h, cells were fixed and stained as described (9). Pictures of the cells migrated through the filter were taken using a Zeiss microscope equipped with a charge-coupled device camera (AxioCam, Carl Zeiss, Thornwood, NY) and the cells in four fields of each membrane were counted using the ImagePro3 program. Each condition was repeated in at least three independent experiments. The average number per field and SEM were calculated. The statistical significance of the means was estimated by Student’s t test.

Branching Morphogenesis Assays
Trypsinized cells were mixed 1:3 with collagen type I solution and plated. MEM with 10% FCS was overlaid on the gels with both 100 ng/ml GDNF and sGFR{alpha}1, 100 ng/ml GDNF alone, or 100 ng/ml sGFR{alpha}1 alone. Cells in collagen were cultured for 3 d, fixed by 2.5% glutaraldehyde in PBS, and counted under a light microscope. Each experiment was repeated at least three times. The average number and SEM were calculated. Statistical significance was determined by Student’s t test.

Anoikis, Cell Apoptosis Assays, and Hoechst Staining
Cell lines were pretreated with 100 ng/ml GDNF/sGFR{alpha}1 or left unstimulated for 12 h in medium containing 0.5% FCS before being trypsinized and plated onto dishes coated with poly-hydroxyethylmethacrylate (polyHEMA, Sigma Chemical Co., St. Louis, MO) in the presence or absence of GDNF/sGFR{alpha}1. Plates were prepared as described previously (28). After 8 h, cells were collected and cytospin preparations were fixed with 4% PFA (paraformaldehyde) and permeabilized with ethanol-acetic acid (2:1). The total number of cells was estimated by Hoechst staining. Apoptotic cells were detected by TUNEL histochemistry using the ApopTag fluorescein in situ apoptosis detection kit (Intergen Co., Purchase, NY), according to the manufacturer’s instructions. TUNEL and Hoechst reactivity were examined and photographed with a fluorescence microscope (Carl Zeiss) equipped with a charge-coupled device camera (AxioCam, Zeiss). Cells were counted using the ImagePro3 program. The average number and SEM were calculated. Significance of the differences between means was estimated by Student’s t test.

MTT Cell Proliferation Assays
Cell proliferation was evaluated using the cell proliferation kit I (MTT, Roche Diagnostics). This assay is designed for the spectrophotometric quantification of cell growth and viability. Briefly, 103 cells per well were seeded on 96-well plates in 10% FCS MEM. After 24 h, cells were serum starved in MEM containing 0.5% FCS for 2 d and then treated with 100 ng/ml GDNF/sGFR{alpha}1 or 50 ng/ml HGF or left untreated. Separated plates were cultured for 24, 48, and 72 h. After incubation periods, 10 µl of the MTT labeling reagent were added to each well, and the plates were kept for 4 h at 37 C. The reactions were terminated by adding lysis buffer (10% SDS in 0.01 M HCl). Absorbance readings were taken at 540 nm using a Multiscan microtiter plate reader (Labsystems, Inc., Marlboro, MA). Each experiment was performed in triplicate and repeated at least three times. The average number and SEM were calculated.

BrdU Cell Proliferation Assays
Cells (104/well) were seeded on coverslips in full-serum MEM. Cells were starved for 24 h in 0.5% FCS MEM. Next day, 100 ng/ml GDNF/sGFR{alpha}1 or 100 ng/ml sGFR{alpha}1 were added to the media. Separated plates were cultured for 24 h and 48 h. One hour before the fixation BrdU (1:1000, Amersham Biosciences) was added. Cells were fixed with 70% ethanol and washed with PBS, and immunocytochemistry was done using the Anti-Bromodeoxyuridine + Nuclease kit (Amersham Biosciences). Coverslips were mounted on the slides, three images from each slide were taken with a fluorescent microscope, and the percentage of proliferating cells was counted. The average number and SEM were calculated.

Soft Agar Assay
The anchorage-independent growth assay was carried out as follows. A bottom layer of agar was prepared in 60-mm dishes using 3 ml of 0.5% noble agar (Agarose type VII, Sigma) in growth medium. Next, 1.5 ml of 0.33% noble agar in growth medium containing NIH 3T3 (104 cells) or MDCK (7.5 x 104 cells) stably expressing wild-type RET, MEN2A-C634R, or Janus-C620R were added on top of the solidified bottom layer. The colonies were dyed 2 and 3 wk later with p-iodonitrotetrazolium violet (Sigma) for 24 h at 37 C and then photographed.


   ACKNOWLEDGMENTS
 
We thank Dr. Eero Lehtonen (Haartman Institute, University of Helsinki) for MDCK cells; Maria G. Rizzetti, Piera Mondellini, Maria T. Radice, and Agnes Viherä for technical assistance; and Dr. Kirmo Wartiovaara, Dr. Vijay Kumar, and Dr. Italia Bongarzone for helpful discussion.


   FOOTNOTES
 
This work was supported by the Italian Association for Cancer Research (AIRC), the Academy of Finland, Centre for International Mobility (CIMO) scholarship, National Technology Agency of Finland (TEKES) Drug 2000 Program, Helsinki University Central Hospital Research Funding, Center of Excellence funding by the Academy of Finland, and Sigrid Juselius grants to H.S. (Biocentrum Helsinki fellow).

E.A. and A.P. contributed equally to this work.

The laboratories of M.A.P. and H.S. contributed equally to this work.

Abbreviations: BrdU, Bromodeoxyuridine; FCS, fetal calf serum; FMTC, familial medullary thyroid carcinoma; GDNF, glial cell line-derived neurotrophic factor; GFR{alpha}1, GDNF family receptor {alpha}1; HEK, human embryonic kidney; HGF, hepatocyte growth factor; HSCR, Hirschsprung’s disease; MBP, myelin basic protein; MDCK, Madin-Darby canine kidney; MEN, multiple endocrine neoplasia; MTC, medullary thyroid carcinoma; MTT, thiazolyl blue tetrazolium bromide; PLC, phospholipase C; poly-HEMA, polyhydroxyethylmethacrylate; SDS, sodium dodecyl sulfate; sGFR, soluble GFR; TUNEL, terminal deoxynucleotide transferase (TdT)-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling.

Received for publication May 12, 2003. Accepted for publication December 31, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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