help button home button Am J Pathol International Conference on Pathology of Chest Diseases
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Watanabe, T.
Right arrow Articles by Takahashi, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Watanabe, T.
Right arrow Articles by Takahashi, M.
(American Journal of Pathology. 2002;161:249-256.)
© 2002 American Society for Investigative Pathology

Regular Articles

Characterization of Gene Expression Induced by RET with MEN2A or MEN2B Mutation

Tsuyoshi Watanabe*{dagger}, Masatoshi Ichihara*, Mizuo Hashimoto*, Keiko Shimono{dagger}, Yoshie Shimoyama*, Tetsuro Nagasaka{ddagger}, Yoshiki Murakumo*, Hideki Murakami*, Hideshi Sugiura{dagger}, Hisashi Iwata{dagger}, Naoki Ishiguro{dagger} and Masahide Takahashi*

From the Departments of Pathology,*Orthopedic Surgery,{dagger}and Laboratory Medicine,{ddagger}Nagoya University Graduate School of Medicine, Nagoya, Japan


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Germ-line point mutations of the RET gene are responsible for multiple endocrine neoplasia (MEN) type 2A and 2B that develop medullary thyroid carcinoma and pheochromocytoma. We performed a differential display analysis of gene expression using NIH 3T3 cells expressing the RET-MEN2A or RET-MEN2B mutant proteins. As a consequence, we identified 10 genes induced by both mutant proteins and eight genes repressed by them. The inducible genes include cyclin D1, cathepsins B and L, and cofilin genes that are known to be involved in cell growth, tumor progression, and invasion. In contrast, the repressed genes include type I collagen, lysyl oxidase, annexin I, and tissue inhibitor of matrix metalloproteinase 3 (TIMP3) genes that have been implicated in tumor suppression. In addition, six RET-MEN2A- and five RET-MEN2B-inducible genes were identified. Among 21 genes induced by RET-MEN2A and/or RET-MEN2B, six genes including cyclin D1, cathepsin B, cofilin, ring finger protein 11 (RNF11), integrin-{alpha}6, and stanniocalcin 1 (STC1) genes were also induced in TGW human neuroblastoma cells in response to glial cell line-derived neurotrophic factor stimulation. Because the STC1 gene was found to be highly induced by both RET-MEN2B and glial cell line-derived neurotrophic factor stimulation, and the expression of its product was detected in medullary thyroid carcinoma with the MEN2B mutation by immunohistochemistry, this may suggest a possible role for STC1 in the development of MEN 2B phenotype.

The RET proto-oncogene encodes a receptor tyrosine kinase with a cadherin-related motif and a cysteine-rich domain in the extracellular domain and is located on chromosome 10q11.2.1,2 It has been demonstrated that RET is a functional receptor for four related neurotrophic factors including glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin, and persephin. These factors are known to require glycosylphosphatidylinositol-anchored co-receptors, GFR-{alpha}s, as ligand-binding components and to promote the survival of various central and peripheral neurons in culture.1,2 In addition, gene knockout studies revealed that the GDNF/RET signaling plays a crucial role in the development of the enteric nervous system and the kidney.3-6

Germline mutations of the RET gene cause dominant inherited cancer syndromes; multiple endocrine neoplasia (MEN) type 2A and 2B.7-10 MEN 2A is characterized by the development of medullary thyroid carcinoma (MTC), pheochromocytoma, and parathyroid hyperplasia. MEN 2B shows a more complex phenotype with association of MTC, pheochromocytoma, and developmental abnormalities such as mucosal neuroma, hyperganglionosis of the intestinal tract, and marfanoid skeletal changes. The MEN2A mutations were identified in cysteine residues of the RET extracellular domain, leading to ligand-independent RET dimerization.11,12 The MEN2B mutations were detected in methionine at codon 918 or in alanine at codon 883 in the tyrosine kinase domain and appear to activate RET without dimerization.12,13

A variety of signaling molecules were shown to be activated by GDNF or RET with MEN2 mutations.1,2 These include extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), AKT, c-Jun amino-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38MAPK), and phosholipase-C{gamma} (PLC-{gamma}). Intriguingly, it turned out that several major intracellular signaling pathways such as RAS/ERK. PI3-K/AKT, JNK, p38MAPK, and ERK5 pathways are activated mainly through phosphorylated tyrosine 1062 present in the carboxy-terminal region of RET.14-17 Consistent with this finding, we showed that the transforming activity of all MEN 2 mutant forms of RET was markedly impaired by a mutation at tyrosine 1062, indicating the importance of tyrosine 1062 on signal transduction for oncogenesis.18,19

To further elucidate the mechanisms of development of MEN 2A or MEN 2B phenotype, it is important to know which genes are induced by RET-MEN2A or RET-MEN2B mutant proteins. We performed a screening analysis of differential gene expression using a defined in vitro model of NIH 3T3 cells expressing RET-MEN2A and RET-MEN2B. In this study, we identified a number of genes induced downstream of RET signals and suggest that the stanniocalcin1 (STC1) gene expression may play a role in the MEN 2B phenotype.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture

NIH 3T3 cells and transfectants expressing RET with MEN2A mutation (cysteine 634 -> arginine) or RET with MEN2B mutation (methionine 918 -> threonine) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 8% calf serum (Hyclone, Logan, UT).

Differential Display Analysis

Total RNAs were isolated from NIH 3T3 cells and transfectants expressing RET-MEN2A or RET-MEN2B mutant proteins using Trizol reagent (Gibco, Tokyo, Japan). After treating with RNase-free DNase I to eliminate contaminated chromosomal DNA, differential display-polymerase chain reaction (PCR) was performed using the TaKaRa rhodamine fluorescence differential display system (TaKaRa, Kyoto, Japan). The fluorescence products were resolved by electrophoresis on denaturing urea-4% polyacrylamide gels. Differentially expressed bands were identified using FM-BIO II (TaKaRa).

Northern Blot Analysis

Total RNA (10 µg) was separated on 1% agarose-formamide gels with formaldehyde and transferred onto Hybond-XL nylon membranes (Amersham Biosciences, Uppsala, Sweden). DNA fragments identified by the differential display method were labeled with [{alpha}-32P] dCTP (3000 Ci/mmol, Amersham Biosciences) using the High Prime DNA-labeling system (Roche Diagnostics, Mannheim, Germany) and used as probes for Northern hybridization at 68°C for 3 hours in QuikHyb Solution (Stratagene, Austin, TX). Signals were detected on X-ray films (RX-U) after exposure for appropriate time. To confirm equal loading of RNA, the membranes were also hybridized with [{alpha}-32P] dCTP-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA. Densitometric analysis was performed by scanning the imaging plate with the BAS-2000 system (Fujifilm, Tokyo, Japan).

SYBR Green-Based Real-Time Quantitative Reverse Transcriptase-PCR

Total RNAs were isolated from TGW human neuroblastoma cells stimulated with GDNF (100 ng/ml) for 0.5, 1, 2, 4, 8, 16, 24, and 48 hours, and used for real-time quantitative reverse transcriptase-PCR studies. cDNAs produced from total RNAs were added to 12.5 µl of PCR reaction mixture plus 12.5 µl of 2x SYBR Green master mix (Applied Biosystems, Foster City, CA) with 900 nmol/L of gene-specific primers and assays were performed according to the manufacturer’s instructions. All data were analyzed with the Applied Biosystems model 7700 and were normalized by the expression level of GAPDH mRNA as an internal control.

Western Blot Analysis

Anti-STC1 rabbit polyclonal antibody was generated against the carboxy-terminal 19 amino acids of mouse STC1. Cells were lysed in sodium dodecyl sulfate sample buffer (50 mmol/L Tris-HCl, pH 6.8, 5 mmol/L ethylenediaminetetraacetic acid, 2% sodium dodecyl sulfate, 10% sucrose, 20 µg/ml bromophenol blue) containing 80 mmol/L of dithiothreitol and protein concentrations in the lysates were determined by using the DC Protein Assay kit (Bio-Rad, Hercules, CA). Twenty µg of the proteins were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA). The membranes were blocked with 3% ovalbumin in TPBS (Tris-buffered saline with 0.5% Tween 20), and probed with the antibody. The blots were washed and probed with the swine anti-rabbit horseradish peroxidase-conjugated secondary antibody (DAKO, Glostrup, Denmark) before being visualized with the standard chemiluminescent technique (ECL, Amersham Biosciences).

Tet-Off System

MEF3T3 Tet-Off cells (Clontech, Palo Alto, CA) were grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% Tet-Off system-approved fetal bovine serum (Clontech) containing 50 UI/ml penicillin, 50 µg/ml streptomycin, 2 mmol/L L-glutamine, and 100 µg/ml G418 (Roche Diagnostics), and maintained at 37°C under a 5% CO2 atmosphere. The MEF3T3 Tet-Off cells grown on 60-minute tissue culture dishes were co-transfected with 10 µg of pTRE/RET-MEN2A or pTRE/RET-MEN2B plasmid and 0.5 µg of pTK-Hygr plasmid (Clontech) by Lipofectamine 2000 (Invitrogen, Tokyo, Japan). The cells were detached with trypsin 3 days after transfection and plated in the presence of doxycycline hydrochloride (0.01 to 1 µg/ml) (Sigma-Aldrich Japan K.K., Tokyo, Japan) and hygromycin B (200 µg/ml; Roche Diagnostics). The resulting hygromycin-resistant colonies were expanded and maintained in media supplemented with 100 µg/ml of G418, 100 µg/ml of hygromycin, and 200 ng/ml of doxycycline. To induce the expression of RET-MEN2A and RET-MEN2B mutant proteins, the cells were cultured in the absence of doxycycline for 72 hours.

Immunohistochemical Analysis

Human MTC tissues were obtained from eight patients who underwent operations at Nagoya University Hospital. Sections were deparaffinized with xylene, rehydrated, and preincubated with blocking buffer [10% normal goat serum in phosphate-buffered saline (PBS)] for 15 minutes at room temperature. Then they were reacted with the anti-RET or anti-STC1 antibody overnight at 4°C. After washing with PBS, they were treated with the secondary dextran peroxidase-conjugated goat anti-rabbit antibody (DAKO Envision system) and diaminobenzidine was used to visualize immune complexes.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Gene Expression Induced or Suppressed by RET with MEN2 Mutations

To identify differentially expressed genes in NIH 3T3 cells and NIH 3T3 cells expressing RET-MEN2A or RET-MEN2B mutant proteins [designated NIH-RET(MEN2A) or NIH-RET(MEN2B) cells], we performed a differential display analysis using their RNAs. We detected a total of 336 cDNA bands whose intensities showed clear differences (more than three times) among the samples from these RNAs (data not shown). After further separated by hemagglutinin-yellow and HA-red splitting techniques (TaKaRa), the cDNAs derived from the isolated bands were inserted into the TA cloning vector and sequenced. As a result, 130 known genes and 13 previously unidentified sequences were found (data not shown).

We analyzed the differential expression of 130 known genes in NIH 3T3, NIH-RET(MEN2A), and NIH-RET(MEN2B) cells by Northern blotting. Twenty-nine genes were confirmed to be differentially expressed in these cells (Figure 1) . Based on their expression patterns, they were classified into four types: 1) 10 genes induced by both RET-MEN2A and RET-MEN2B mutant proteins (type I); 2) six genes induced predominantly by RET-MEN2A (type II); 3) five genes induced predominantly by RET-MEN2B (type III); and 4) eight genes repressed by RET-MEN2A and RET-MEN2B (type IV) (Figure 1) . Type I includes cyclin D1, cofilin, and cathepsin L and B genes that are known to be involved in cell growth, tumor progression, and invasion whereas type IV includes type I collagen, lysyl oxidase, annexin I, and tissue inhibitor of matrix metalloproteinase 3 (TIMP3) genes that have been implicated in tumor suppression. Type II and type III include various genes associated with different physiological functions.



View larger version (57K):
[in this window]
[in a new window]
  		Figure 1. Characterization of differentially expressed genes by Northern blot analysis. A portion (10µg) of total RNA extracted from NIH 3T3, NIH-RET(MEN2A), or NIH-RET(MEN2B) cells were analyzed by Northern blotting with cDNA probes of the designated mouse genes. Type I, the genes induced by both RET-MEN2A and RET-MEN2B mutant proteins; type II, the genes induced predominantly by RET-MEN2A; type III, the genes induced predominantly by RET-MEN2B; type IV, the genes repressed by both RET-MEN2A and RET-MEN2B. RNF11, ring finger protein 11; IFN-ß, interferon-ß; TB-2-like 1, TB-2-like 1 protein; HSC73, heat shock protein 73; EMK2, ELKL motif kinase 2; ITGA6, integrin-{alpha}6; PKA-RI, protein kinase A regulatory subunit I; TACC3, transforming acidic coiled coil-containing gene family 3; PRNPA, prion-related protein A; MPT, mitochondrial phosphate transporter; STC1, stanniocalcin 1; Phex, phosphate-regulating gene with homology to endopeptidases on the X chromosome; EIF4G3, eukaryotic translation initiation factor 4G3; neuropsin, serine protease 19; PLOD2, procollagen-lysine,2-oxyglutarate,5-dioxygenase 2; COLIA1, type I collagen {alpha}1 chain; COLIA2, type I collagen {alpha}2 chain; TIMP3, tissue inhibitor of metalloproteinases 3; SDF1{alpha}, stromal cell-derived factor-1{alpha}; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

 
Identification of GDNF-Inducible Genes

We next investigated whether RET-MEN2A- and/or RET-MEN2B-inducible genes were induced by GDNF stimulation. TGW human neuroblastoma cells that endogenously express RET and GFR-{alpha}1 were treated with GDNF for 0.5, 1, 2, 6, 12, 24, and 48 hours, and their total RNAs were extracted for Northern blotting. As shown in Figure 2A , six of 21 differentially expressed genes were also induced by GDNF, although the time course of their induction was different depending on the genes. They included cyclin D1, cathepsin B, cofilin, ring finger protein 11 (RNF11), integrin-{alpha}6 (ITGA6), and stanniocalcin 1 (STC1) genes. Cyclin D1, cathepsin B, cofilin, and RNF11 belonged to the type I gene group, and ITGA6 and STC1 belonged to type II and type III gene groups, respectively. These results were confirmed by real-time quantitative reverse transcriptase-PCR using RNAs from GDNF-treated TGW cells that were separately extracted (Figure 2B) .



View larger version (65K):
[in this window]
[in a new window]
  		Figure 2. Identification of GDNF-inducible genes. A: Total RNAs isolated from TGW neuroblastoma cells treated with GDNF (100 ng/ml) for 0, 0.5, 1, 2, 6, 12, 24, or 48 hours were analyzed by Northern blotting with cDNA probes of the designated human genes. The membranes were also probed with GAPDH cDNA as a control. B: Total RNAs separately isolated from TGW cells treated with GDNF were analyzed by real-time quantitative reverse transcriptase-PCR as described in Materials and Methods. The results show averages from at least three independent experiments. The bars indicate standard errors.

 
STC1 Expression in Cells Expressing RET-MEN2B Mutant Proteins

Because only STC1 was found to be induced by both RET-MEN2B and GDNF, and was suggestive of a role in early skeletal development20 that is affected in MEN 2B, we further investigated the expression of the STC1 protein in the transfectants.

We developed a rabbit polyclonal antibody against the carboxy-terminal 19 amino acids of mouse STC1. To show the specificity of this antibody, we transiently transfected Flag-tagged mouse STC1 cDNA into COS7 cells and their cell lysates were analyzed by Western blotting with anti-Flag and anti-STC1 antibodies. Both antibodies specifically detected a 32-kd band that is consistent with the predicted molecular mass of STC1 (Figure 3A) . As expected from the result of Northern blot analysis (Figure 1) , the 32-kd band was detected more strongly in the lysate from NIH-RET(MEN2B) cells than in the lysate from NIH-RET(MEN2A) cells (Figure 3B) .



View larger version (29K):
[in this window]
[in a new window]
  		Figure 3. Detection of STC1 protein by Western blot analysis. A: COS-7 cells were transfected with the expression vector carrying Flag-tagged full-length mouse STC1 cDNA. The cell lysates from original COS-7 cells (lanes 2, 4, and 6) or transfectant (lanes 1, 3, and 5) were analyzed by Western blotting with normal rabbit IgG (lanes 1 and 2), anti-STC1 antibody (lanes 3 and 4), or anti-Flag antibody (lanes 5 and 6). A 32-kd STC1 band is indicated. B: Total cell lysates from NIH 3T3, NIH-RET(MEN2A), or NIH-RET(MEN2B) cells were analyzed by Western blotting with anti-RET, anti-phosphotyrosine (pY), anti-ERK2, and anti-STC1 antibodies. C: MEF3T3 Tet-Off cells were transfected with pTRE expression vector (Clontech) carrying RET-MEN2A or RET-ME2B cDNA. Cell lysates from NIH-RET(MEN2B) or MEF3T3 Tet-Off transfectants incubated with or without doxycycline (100 ng/ml) were analyzed by Western blotting with anti-RET, anti-pY, anti-ERK2, anti-phophoERK1/2, and anti-STC1 antibodies. Tet, tetracycline (doxycycline).

 
To confirm that the STC1 expression is highly induced by the RET-MEN2B mutant protein, we used MEF 3T3 Tet-Off cells in which the gene expression was repressed by addition of tetracycline (doxycycline). The cells were co-transfected with the pTRE (tetracycline-responsive element)-2 vector containing RET-MEN2A or RET-MEN2B cDNA and the pTK-Hyg selection vector. When tetracycline was removed from media in the transfectants, the expression of RET-MEN2A or RET-MEN2B mutant proteins were clearly induced, accompanying the increase of ERK1/2 phosphorylation (Figure 3C) . Under the same condition, the STC1 expression was induced more strongly in the cells expressing RET-MEN2B than in the cells expressing RET-MEN2A (Figure 3C) .

STC1 Expression in Medullary Thyroid Carcinoma

Finally, we examined the STC1 expression in specimens of human MTC by immunohistochemistry. We stained two sections of sporadic MTCs, four sections of MEN2A-MTCs, and two sections of MEN2B-MTCs. No RET mutations were detected in two sporadic MTCs. Interestingly, two specimens of MEN2B-MTC were strongly stained with anti-STC1 antibody (Figure 4F) . Two MEN2A-MTCs were weakly stained with the antibody (Figure 4E) , and two sporadic MTCs and two MEN2A-MTCs were almost unstained (Figure 4D and data not shown). RET expression was detected in all specimens of both MEN2A MTCs and MEN2B MTCs to variable degrees (Figure 4, B and C) .



View larger version (80K):
[in this window]
[in a new window]
  		Figure 4. Expression of STC1 in human MTC carrying the MEN2B mutation. Bright-field micrographs of histological paraffin sections from sporadic MTC (A and D), MEN2A-MTC (B and E), and MEN2B-MTC (C and F) were stained with anti-RET (A–C) or anti-STC1 (D–F) antibody. Diaminobenzidine was used to visualize immune complexes. They were counterstained by hematoxylin. When immunohistochemical analyses were performed without the first antibody as a negative control, no staining was observed in these cases. Original magnifications, x200.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
MEN 2A and MEN 2B are dominantly inherited cancer syndromes characterized by the development of MTC and pheochromocytoma. In addition to these tumors, 10 to 30% patients of MEN 2A develop parathyroid hyperplasia whereas MEN 2B is characterized by a variety of developmental anomalies including mucosal neuroma, intestinal ganglioneuroma, marfanoid skeletal changes, and a male reproductive defect. The MEN2A mutations were detected mainly in one of six cysteine residues of the RET extracellular domain, leading to disulfide-linked RET dimerization.11,12 The MEN2B mutations were found at methionine 918 or alanine 883 in the kinase domain and activated RET without dimerization.12,13 Although we and other investigators reported the differences in signal transduction activated by RET-MEN2A or RET-MEN2B mutant proteins,21-24 which signalings are critical for the development of disease phenotypes still remains unknown. To elucidate further the mechanisms of development of MEN 2A and MEN 2B phenotypes, it is interesting to characterize the differential gene expression induced by RET-MEN2A and RET-MEN2B.

In the current study, we performed a differential display analysis of gene expression using RNAs from NIH 3T3, NIH-RET(MEN2A), and NIH-RET(MEN2B) cells. We identified a total of 29 genes whose differential expression was confirmed by Northern blotting. Although we identified 101 other genes by differential display, the differences in the levels of their mRNA expression were less than two times among three cell lines in Northern blot analyses (data not shown). Thus, the interpretation of the results obtained by sensitive techniques such as differential display and DNA microarray should be cautious without confirmation by different techniques.

Some of RET-MEN2A and RET-MEN2B-inducible genes (type I genes) including cyclin D1, cofilin, and cathepsin B and L genes were reported to be involved in the cell growth, cell motility, and cancer invasion,25-28 suggesting that the increased expression of these genes contributes to malignant properties of RET-MEN2A- or RET-MEN2B-expressing cells. However, the roles of other genes such as decorin and interferon-ß are controversial because they were reported to suppress tumor growth.29,30 In addition, the functions of RNF11, foocen, and TB2-like 1 genes belonging to type I group are currently unknown.31,32 On the other hand, we identified eight genes suppressed by both RET-MEN2A and RET-MEN2B mutant proteins (type IV genes). Several genes such as type I collagen, lysyl oxidase, TIMP3, and annexin I genes are down-regulated in cancer cells and have been implicated in tumor suppression.33-35 Thus, it seems likely that the changes of expression of certain type I and type IV genes directly correlate to tumor development or progression.

MEN2A- or MEN2B-inducible genes (type II or type III genes) showed a wide diversity in their possible physiological functions. They include protein kinases (EMK2, PKA-RI),36,37 cell adhesion molecule (ITGA6),38,39 microtubule-associated protein (TACC3),40 protease (neuropsin),41 and translation initiation factor (EIF4G3).42 Of 11 type II or type III genes identified, we focused on the STC1 gene because it was reported that it may be involved in the early skeletal development20 that is affected in MEN 2B patients.

STC was originally identified as a hormone that is synthesized and secreted by the corpuscles of Stannius, unique endocrine glands associated with kidney and embryologically derived from nephric ducts.43 Human STC1 was cloned in a study aimed at identifying genes involved in the control of cellular proliferation, using a simian virus 40 early region-transfected human fibroblast culture.44 Bacterial or Chinese hamster ovary cell-synthesized recombinant human STC was found to reduce renal phosphate excretion in rats, and to reduce calcium absorption and increase phosphate absorption in the rat and pig duodenum.45 These findings suggested that mammalian STC acts as a regulator of calcium and phosphate homeostasis in an autocrine/paracrine manner. In addition, the expression pattern of STC1 in mouse embryos was indicative of a role in early skeletal patterning and joint formation.20,46-48

Our current studies using the stable transfectants and Tet-Off cells demonstrated that STC1 was highly induced by the RET-MEN2B mutant protein. The high level of its expression was also observed in MTC from MEN 2B patients. Although no RET mutations were found in two sporadic MTCs examined in this study, the investigation of STC1 expression in sporadic MTCs with the MEN2B mutation could be important to show the correlation between its expression and the MEN2B mutation. In addition, given that mammalian STC is involved in early skeletal development, it is possible that high levels of STC1 expression during embryogenesis may be associated with marfanoid skeletal changes developed in MEN 2B. However, to elucidate this relation, it will be necessary to investigate which tissues and cells during human embryogenesis express STC1 whose expression level is influenced by the MEN2B mutation. STC1 was also reported to be expressed in a variety of tumors including lung cancer, pheochromocytoma, neuroblastoma, osteosarcoma, and fibrosarcoma and to be useful as a molecular marker for their micrometastasis.49-53 Thus, a high level of STC1 expression in MTC carrying the MEN2B mutation may affect biological properties of tumor cells. Further investigation of STC1 expression and functions in human tissues may provide a new insight into the development of MEN 2B phenotype.

In addition to the skeletal development, several physiological roles for STC1 were suggested. We showed that STC1 was induced in neuronal cells in response to GDNF stimulation, suggesting that it plays a role in the GDNF/RET signaling pathway. Because STC1 is expressed in brain neurons and functions as a molecular guard of neurons during cerebral ischemia as observed for GDNF,54-56 this may imply a physiological link between STC1 and GDNF/RET signaling in the nervous system. Moreover, the fact that the high STC1 expression was detected in ureteric bud/collecting duct cells of E14.5-18.5 mouse metanephric kidney and overlapped with RET expression at this stage suggests the possibility that STC1 expression and GDNF/RET signaling cooperatively function in kidney development.3-6,57-59 Future studies will clarify the interesting roles of STC1 in the GDNF/RET signaling pathway as well as in tumor biology.


   Acknowledgements
 
We thank K. Imaizumi, K. Uchiyama, and M. Kozuka for their technical assistance.


   Footnotes
 
Address reprint requests to Masahide Takahashi, Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: mtakaha{at}med.nagoya-u.ac.jp

Supported by a grant-in-aid for Center of Excellence Research from the Ministry of Education, Science, Sports, and Culture of Japan.

Accepted for publication April 15, 2002.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Takahashi M: The GDNF/RET signaling pathway and human diseases. Cytokine Growth Factor Rev 2001, 12:361-373[Medline]
  2. Manie S, Santoro M, Fusco A, Billaud M: The RET receptor: function in development and dysfunction in congenital malformation. Trends Genet 2001, 17:580-589[Medline]
  3. Schuchardt A, D’Agati V, Larsson-Blomberg L, Costantini F, Pachnis V: Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 1994, 367:380-383[Medline]
  4. Moore MW, Klein RD, Farinas I, Sauer H, Armanini M, Phillips H, Reichardt LF, Ryan AM, Carver-Moore K, Rosenthal A: Renal and neuronal abnormalities in mice lacking GDNF. Nature 1996, 382:76-79[Medline]
  5. Pichel JG, Shen L, Sheng HZ, Granholm AC, Drago J, Grinberg A, Lee EJ, Huang SP, Saarma M, Hoffer BJ, Sariola H, Westphal H: Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 1996, 382:73-76[Medline]
  6. Sanchez MP, Silos-Santiago I, Frisen J, He B, Lira SA, Barbacid M: Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 1996, 382:70-73[Medline]
  7. Mulligan LM, Kwok JB, Healey CS, Elsdon MJ, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L: Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993, 363:458-460[Medline]
  8. Donis-Keller H, Dou S, Chi D, Carlson KM, Toshima K, Lairmore TC, Howe JR, Moley JF, Goodfellow P, Wells SA, Jr: Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet 1993, 2:851-856[Abstract/Free Full Text]
  9. Hofstra RM, Landsvater RM, Ceccherini I, Stulp RP, Stelwagen T, Luo Y, Pasini B, Hoppener JW, van Amstel HK, Romeo G: A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature 1994, 367:375-376[Medline]
  10. Carlson KM, Dou S, Chi D, Scavarda N, Toshima K, Jackson CE, Wells SA, Jr, Goodfellow PJ, Donis-Keller H: Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B. Proc Natl Acad Sci USA 1994, 91:1579-1583[Abstract/Free Full Text]
  11. Asai N, Iwashita T, Matsuyama M, Takahashi M: Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations. Mol Cell Biol 1995, 15:1613-1619[Abstract]
  12. Santoro M, Carlomagno F, Romano A, Bottaro DP, Dathan NA, Grieco M, Fusco A, Vecchio G, Matoskova B, Kraus MH: Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science 1995, 267:381-383[Abstract/Free Full Text]
  13. Iwashita T, Asai N, Murakami H, Matsuyama M, Takahashi M: Identification of tyrosine residues that are essential for transforming activity of the ret proto-oncogene with MEN2A or MEN2B mutation. Oncogene 1996, 12:481-487[Medline]
  14. Segouffin-Cariou C, Billaud M: Transforming ability of MEN2A-RET requires activation of the phosphatidylinositol 3-kinase/AKT signaling pathway. J Biol Chem 2000, 275:3568-3576[Abstract/Free Full Text]
  15. Hayashi H, Ichihara M, Iwashita T, Murakami H, Shimono Y, Kawai K, Kurokawa K, Murakumo Y, Imai T, Funahashi H, Nakao A, Takahashi M: Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor. Oncogene 2000, 19:4469-4475[Medline]
  16. Besset V, Scott RP, Ibanez CF: Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase. J Biol Chem 2000, 275:39159-39166[Abstract/Free Full Text]
  17. Hayashi Y, Iwashita T, Murakami H, Kato Y, Kawai K, Kurokawa K, Tohnai I, Ueda M, Takahashi M: Activation of BMK1 via tyrosine 1062 in RET by GDNF and MEN2A mutation. Biochem Biophys Res Commun 2001, 281:682-689[Medline]
  18. Asai N, Murakami H, Iwashita T, Takahashi M: A mutation at tyrosine 1062 in MEN2A-Ret and MEN2B-Ret impairs their transforming activity and association with shc adaptor proteins. J Biol Chem 1996, 271:17644-17649[Abstract/Free Full Text]
  19. Iwashita T, Kato M, Murakami H, Asai N, Ishiguro Y, Ito S, Iwata Y, Kawai K, Asai M, Kurokawa K, Kajita H, Takahashi M: Biological and biochemical properties of Ret with kinase domain mutations identified in multiple endocrine neoplasia type 2B and familial medullary thyroid carcinoma. Oncogene 1999, 18:3919-3922[Medline]
  20. Stasko SE, Wagner GF: Possible roles for stanniocalcin during early skeletal patterning and joint formation in the mouse. J Endocrinol 2001, 171:237-248[Abstract]
  21. Murakami H, Iwashita T, Asai N, Shimono Y, Iwata Y, Kawai K, Takahashi M: Enhanced phosphatidylinositol 3-kinase activity and high phosphorylation state of its downstream signalling molecules mediated by ret with the MEN 2B mutation. Biochem Biophys Res Commun 1999, 262:68-75[Medline]
  22. Bocciardi R, Mograbi B, Pasini B, Borrello MG, Pierotti MA, Bourget I, Fischer S, Romeo G, Rossi B: The multiple endocrine neoplasia type 2B point mutation switches the specificity of the Ret tyrosine kinase towards cellular substrates that are susceptible to interact with Crk and Nck. Oncogene 1997, 15:2257-2265[Medline]
  23. Zhou S, Carraway KL, III, Eck MJ, Harrison SC, Feldman RA, Mohammadi M, Schlessinger J, Hubbard SR, Smith DP, Eng C: Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature 1995, 373:536-539[Medline]
  24. Salvatore D, Melillo RM, Monaco C, Visconti R, Fenzi G, Vecchio G, Fusco A, Santoro M: Increased in vivo phosphorylation of ret tyrosine 1062 is a potential pathogenetic mechanism of multiple endocrine neoplasia type 2B. Cancer Res 2001, 61:1426-1431[Abstract/Free Full Text]
  25. Jang TJ, Kang MS, Kim H, Kim DH, Lee JI, Kim JR: Increased expression of cyclin D1, cyclin E and p21(Cip1) associated with decreased expression of p27(Kip1) in chemically induced rat mammary carcinogenesis. Jpn J Cancer Res 2000, 91:1222-1232[Medline]
  26. Lah TT, Kos J: Cysteine proteinases in cancer progression and their clinical relevance for prognosis. Biol Chem 1998, 379:125-130[Medline]
  27. Yan S, Sameni M, Sloane BF: Cathepsin B and human tumor progression. Biol Chem 1998, 379:113-123[Medline]
  28. Aizawa H, Fukui Y, Yahara I: Live dynamics of Dictyostelium cofilin suggests a role in remodeling actin latticework into bundles. J Cell Sci 1997, 110:2333-2344[Abstract]
  29. Kresse H, Schonherr E: Proteoglycans of the extracellular matrix and growth control. J Cell Physiol 2001, 189:266-274[Medline]
  30. Vannucchi S, Percario ZA, Chiantore MV, Matarrese P, Chelbi-Alix MK, Fagioli M, Pelicci PG, Malorni W, Fiorucci G, Romeo G, Affabris E: Interferon-beta induces S phase slowing via up-regulated expression of PML in squamous carcinoma cells. Oncogene 2000, 19:5041-5053[Medline]
  31. Seki N, Hattori A, Hayashi A, Kozuma S, Sasaki M, Suzuki Y, Sugano S, Muramatsu MA, Saito T: Cloning and expression profile of mouse and human genes, Rnf11/RNF11, encoding a novel RING-H2 finger protein. Biochim Biophys Acta 1999, 1489:421-427[Medline]
  32. Calero M, Whittaker GR, Collins RN: Yop1p, the yeast homolog of the polyposis locus protein 1, interacts with Yip1p and negatively regulates cell growth. J Biol Chem 2001, 276:12100-12112[Abstract/Free Full Text]
  33. Andreu T, Beckers T, Thoenes E, Hilgard P, von Melchner H: Gene trapping identifies inhibitors of oncogenic transformation. The tissue inhibitor of metalloproteinases-3 (TIMP3) and collagen type I alpha2 (COL1A2) are epidermal growth factor-regulated growth repressors. J Biol Chem 1998, 273:13848-13854[Abstract/Free Full Text]
  34. Csiszar K: Lysyl oxidases: a novel multifunctional amine oxidase family. Prog Nucleic Acid Res Mol Biol 2001, 70:1-32[Medline]
  35. Kang JS, Calvo BF, Maygarden SJ, Caskey LS, Mohler JL, Ornstein DK: Dysregulation of annexin I protein expression in high-grade prostatic intraepithelial neoplasia and prostate cancer. Clin Cancer Res 2002, 8:117-123[Abstract/Free Full Text]
  36. Hurov JB, Stappenbeck TS, Zmasek CM, White LS, Ranganath SH, Russell JH, Chan AC, Murphy KM, Piwnica-Worms H: Immune system dysfunction and autoimmune disease in mice lacking Emk (Par-1) protein kinase. Mol Cell Biol 2001, 21:3206-3219[Abstract/Free Full Text]
  37. Goodwin SF, Del Vecchio M, Velinzon K, Hogel C, Russell SR, Tully T, Kaiser K: Defective learning in mutants of the Drosophila gene for a regulatory subunit of cAMP-dependent protein kinase. J Neurosci 1997, 17:8817-8827[Abstract/Free Full Text]
  38. Ashton GH, Sorelli P, Mellerio JE, Keane FM, Eady RA, McGrath JA: Alpha 6 beta 4 integrin abnormalities in junctional epidermolysis bullosa with pyloric atresia. Br J Dermatol 2001, 144:408-414[Medline]
  39. Georges-Labouesse E, Mark M, Messaddeq N, Gansmuller A: Essential role of alpha 6 integrins in cortical and retinal lamination. Curr Biol 1998, 8:983-986[Medline]
  40. Gergely F, Karlsson C, Still I, Cowell J, Kilmartin J, Raff JW: The TACC domain identifies a family of centrosomal proteins that can interact with microtubules. Proc Natl Acad Sci USA 2000, 97:14352-14357[Abstract/Free Full Text]
  41. He XP, Shiosaka S, Yoshida S: Expression of neuropsin in oligodendrocytes after injury to the CNS. Neurosci Res 2001, 39:455-462[Medline]
  42. McKendrick L, Morley SJ, Pain VM, Jagus R, Joshi B: Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo. Eur J Biochem 2001, 268:5375-5385[Medline]
  43. Wagner GF, Milliken C, Friesen HG, Copp DH: Studies on the regulation and characterization of plasma stanniocalcin in rainbow trout. Mol Cell Endocrinol 1991, 79:129-138[Medline]
  44. Chang AC, Janosi J, Hulsbeek M, de Jong D, Jeffrey KJ, Noble JR, Reddel RR: A novel human cDNA highly homologous to the fish hormone stanniocalcin. Mol Cell Endocrinol 1995, 112:241-247[Medline]
  45. Madsen KL, Tavernini MM, Yachimec C, Mendrick DL, Alfonso PJ, Buergin M, Olsen HS, Antonaccio MJ, Thomson AB, Fedorak RN: Stanniocalcin: a novel protein regulating calcium and phosphate transport across mammalian intestine. Am J Physiol 1998, 274:96-102
  46. Jiang WQ, Chang AC, Satoh M, Furuichi Y, Tam PP, Reddel RR: The distribution of stanniocalcin 1 protein in fetal mouse tissues suggests a role in bone and muscle development. J Endocrinol 2000, 165:457-466[Abstract]
  47. Yoshiko Y, Kosugi T, Koide Y: Effects of a synthetic N-terminal fragment of stanniocalcin on the metabolism of mammalian bone in vitro. Biochim Biophys Acta 1996, 1311:143-149[Medline]
  48. Yoshiko Y, Son A, Maeda S, Igarashi A, Takano S, Hu J, Maeda N: Evidence for stanniocalcin gene expression in mammalian bone. Endocrinology 1999, 140:1869-1874[Abstract/Free Full Text]
  49. Jellinek DA, Chang AC, Larsen MR, Wang X, Robinson PJ, Reddel RR: Stanniocalcin 1 and 2 are secreted as phosphoproteins from human fibrosarcoma cells. Biochem J 2000, 350:453-461
  50. Groves TC, Wagner GF, DiMattia GE: cAMP signaling can antagonize potent glucocorticoids post-transcriptional inhibition of stanniocalcin gene expression. J Endocrinol 2001, 171:499-516[Abstract]
  51. Miura W, Mizunashi K, Kimura N, Koide Y, Noshiro T, Miura Y, Furukawa Y, Nagura H: Expression of stanniocalcin in zona glomerulosa and medulla of normal human adrenal glands, and some adrenal tumors and cell lines. Apmis 2000, 108:367-372[Medline]
  52. Lal A, Peters H, St Croix B, Haroon ZA, Dewhirst MW, Strausberg RL, Kaanders JH, van der Kogel AJ, Riggins GJ: Transcriptional response to hypoxia in human tumors. J Natl Cancer Inst 2001, 93:1337-1343[Abstract/Free Full Text]
  53. Fujiwara Y, Sugita Y, Nakamori S, Miyamoto A, Shiozaki K, Nagano H, Sakon M, Monden M: Assessment of stanniocalcin-1 mRNA as a molecular marker for micrometastases of various human cancers. Int J Oncol 2000, 16:799-804[Medline]
  54. Zhang KZ, Westberg JA, Paetau A, von Boguslawsky K, Lindsberg P, Erlander M, Guo H, Su J, Olsen HS, Andersson LC: High expression of stanniocalcin in differentiated brain neurons. Am J Pathol 1998, 153:439-445[Abstract/Free Full Text]
  55. Zhang K, Lindsberg PJ, Tatlisumak T, Kaste M, Olsen HS, Andersson LC: Stanniocalcin: a molecular guard of neurons during cerebral ischemia. Proc Natl Acad Sci USA 2000, 97:3637-3642[Abstract/Free Full Text]
  56. Wang Y, Lin SZ, Chiou AL, Williams LR, Hoffer BJ: Glial cell line-derived neurotrophic factor protects against ischemia-induced injury in the cerebral cortex. J Neurosci 1997, 17:4341-4348[Abstract/Free Full Text]
  57. Stasko SE, Wagner GF: Stanniocalcin gene expression during mouse urogenital development: a possible role in mesenchymal-epithelial signalling. Dev Dyn 2001, 220:49-59[Medline]
  58. Meng X, Lindahl M, Hyvonen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, Saarma M, Sariola H: Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000, 287:1489-1493[Abstract/Free Full Text]
  59. Tsuzuki T, Takahashi M, Asai N, Iwashita T, Matsuyama M, Asai J: Spatial and temporal expression of the ret proto-oncogene product in embryonic, infant and adult rat tissues. Oncogene 1995, 10:191-198[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
T. S. Gujral, W. van Veelen, D. S. Richardson, S. M. Myers, J. A. Meens, D. S. Acton, M. Dunach, B. E. Elliott, J. W.M. Hoppener, and L. M. Mulligan
A Novel RET Kinase-{beta}-Catenin Signaling Pathway Contributes to Tumorigenesis in Thyroid Carcinoma
Cancer Res., March 1, 2008; 68(5): 1338 - 1346.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Surg. Oncol.Home page
C. J. Wray, T. A. Rich, S. G. Waguespack, J. E. Lee, N. D. Perrier, and D. B. Evans
Failure to Recognize Multiple Endocrine Neoplasia 2B: More Common Than We Think?
Ann. Surg. Oncol., January 1, 2008; 15(1): 293 - 301.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
P. J. Pollard, M. El-Bahrawy, R. Poulsom, G. Elia, P. Killick, G. Kelly, T. Hunt, R. Jeffery, P. Seedhar, J. Barwell, et al.
Expression of HIF-1{alpha}, HIF-2{alpha} (EPAS1), and Their Target Genes in Paraganglioma and Pheochromocytoma with VHL and SDH Mutations
J. Clin. Endocrinol. Metab., November 1, 2006; 91(11): 4593 - 4598.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
J. W. B. de Groot, T. P. Links, J. T. M. Plukker, C. J. M. Lips, and R. M. W. Hofstra
RET as a Diagnostic and Therapeutic Target in Sporadic and Hereditary Endocrine Tumors
Endocr. Rev., August 1, 2006; 27(5): 535 - 560.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
H. Y. Yeung, K. P. Lai, H. Y. Chan, N. K. Mak, G. F. Wagner, and C. K. C. Wong
Hypoxia-Inducible Factor-1-Mediated Activation of Stanniocalcin-1 in Human Cancer Cells
Endocrinology, November 1, 2005; 146(11): 4951 - 4960.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
M. Drosten, G. Hilken, M. Bockmann, F. Rodicker, N. Mise, A. N. Cranston, U. Dahmen, B. A. J. Ponder, and B. M. Putzer
Role of MEN2A-Derived RET in Maintenance and Proliferation of Medullary Thyroid Carcinoma
J Natl Cancer Inst, August 18, 2004; 96(16): 1231 - 1239.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. M. Myers and L. M. Mulligan
The RET Receptor Is Linked to Stress Response Pathways
Cancer Res., July 1, 2004; 64(13): 4453 - 4463.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Jain, M. A. Watson, M. K. DeBenedetti, Y. Hiraki, J. F. Moley, and J. Milbrandt
Expression Profiles Provide Insights into Early Malignant Potential and Skeletal Abnormalities in Multiple Endocrine Neoplasia Type 2B Syndrome Tumors
Cancer Res., June 1, 2004; 64(11): 3907 - 3913.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Fukuda, M. Ichihara, T. Morinaga, K. Kawai, H. Hayashi, Y. Murakumo, S. Matsuo, and M. Takahashi
Identification of a Novel Glial Cell Line-derived Neurotrophic Factor-inducible Gene Required for Renal Branching Morphogenesis
J. Biol. Chem., December 12, 2003; 278(50): 50386 - 50392.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Watanabe, T.
Right arrow Articles by Takahashi, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Watanabe, T.
Right arrow Articles by Takahashi, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS