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[Cancer Research 65, 1343-1351, February 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Cytoplasmic Domain of proEGF Affects Distribution and Post-Translational Modification of Microtubuli and Increases Microtubule-Associated Proteins 1b and 2 Production in Human Thyroid Carcinoma Cells

Janette Pyka1, Aleksandra Glogowska2, Henning Dralle1, Cuong Hoang-Vu1 and Thomas Klonisch2

1 Clinics of Surgery, Medical Faculty, Martin-Luther-University Halle-Wittenberg, Halle, Germany and 2 Department of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

Requests for reprints: Thomas Klonisch, Department of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada. Phone: 204-789-3893; Fax: 204-789-3920; E-mail: klonisch{at}cc.umanitoba.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We studied the distribution of transcripts encoding the cytoplasmic domain of the membrane-anchored precursor epidermal growth factor (proEGFcyt) and a novel cytoplasmic proEGF splice isoform with a deleted exon 23 and an out-of-frame fusion of exon 24 (proEGFdel23) in human normal and neoplastic thyroid tissues. In papillary thyroid carcinoma (PTC), coexpression of transcripts encoding for both proEGFcyt and proEGFdel23 correlated with poor differentiation of PTC. To determine potential roles of the cytoplasmic proEGF domain in human thyroid cells, we generated stable transfectants of the human follicular thyroid carcinoma cell line FTC-133 overexpressing the normal cytoplasmic domain proEGFcyt, a truncated proEGFcyt composed of the peptide sequence encoded by exons 22 and 23 (proEGF22.23) and proEGFdel23. The proEGFcyt and proEGF22.23 transfectants displayed significantly reduced proliferation rates, an enlarged cellular phenotype, and alterations in the distribution and post-translational modification of the microtubular system. These transfectants also displayed increased production of microtubule-associated proteins 1b and 2c, which was absent in FTC-133-proEGFdel23 or FTC-133-empty plasmid transfectants. This is the first evidence of an involvement of proEGF cytoplasmic domain in microtubular stability in the human thyroid carcinoma cell line FTC-133 and may suggest a specific role for the cytoplasmic domain of membrane-anchored proEGF, particularly exon 23, in thyroid carcinoma. The up-regulation of proEGFdel23 in poorly differentiated PTC and the exclusive detection of both proEGF isoforms in undifferentiated thyroid carcinoma may indicate an involvement of this novel truncated proEGFdel23 cytoplasmic domain during dedifferentiation processes of human thyroid cells.

Key Words: proEGF • cytoplasmic domain • thyroid • carcinoma • human


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The epidermal growth factor (EGF) is the founding member of a family of structurally related EGF receptor (EGFR) ligands, which also include transforming growth factor-{alpha} (TGF-{alpha}), heparin-binding EGF-like growth factor, amphiregulin, epiregulin, and the neuregulins (1). Binding of the EGF-like ligands to transmembrane receptor tyrosine kinase EGFRs of the ErbB family (ErbB1-4) results in increased cellular proliferation and changes in differentiation (2). The human normal and neoplastic thyroid gland has been identified as a source of EGF (3). Both EGF and TGF-{alpha} are potent in vitro and in vivo mitogens for normal and neoplastic thyroid follicular cells, and activation of the EGFR by EGF or TGF-{alpha} enhances cell migration and promotes dedifferentiation of thyrocytes (4). Coexpression of EGF or TGF-{alpha} and EGFR results in more aggressive disease in thyroid cancers (5). A high degree of coexpression of EGF/TGF-{alpha} and EGFR is observed in metastasizing thyroid carcinoma and is predictive of a poor prognosis (6). Like all EGF family members, EGF is synthesized as a transmembrane precursor proform (proEGF) that undergoes ectodomain cleavage, primarily mediated by members of the ADAM family, to release soluble and biologically active EGF (7). In the distal tubules of kidney and in keratinocytes, membrane-bound proforms of EGF are predominantly present (8). proEGF-like ligands are biologically active in a juxtacrine manner by binding to and activating EGFR on neighboring cells (9). Pro–heparin-binding EGF-like growth factor is the receptor for diphtheria toxin and acts as an antiapoptotic factor in kidney epithelial cells (10). The intracellular and transmembrane domains of proEGF, pro-amphiregulin, and proTGF-{alpha} are important in determining their cellular localization and proteolytic processing (11–14). Membrane anchorage and correct processing of TGF-{alpha} depend on the presence of the COOH-terminal valine in the cytoplasmic proTGF-{alpha} domain (14). Furthermore, the cytoplasmic domain of some proEGF-like ligands seems to be a site of protein interactions. The cytoplasmic domain of proTGF-{alpha} was shown to associate with a 86- and 106-kDa protein, with the 106-kDa component possessing kinase activity (15). The cytoplasmic domain of pro–heparin-binding EGF-like growth factor was shown to interact with a promyelocytic leukemia zinc finger protein, a transcriptional repressor localized in the nucleus, resulting in altered mitogenic cellular signaling (16, 17). Released from the membrane-anchored proform of neuregulin-1 (Nrg-1) on EGFR binding, the intracellular domain of Nrg-1 (Nrg-1-ICD) enters the nucleus and represses expression of several regulators of apoptosis (18). Nrg-1-ICD also associates with the member of protein serine/threonine kinases LIM kinase and promyelocytic leukemia zinc finger protein (18).

Information is lacking on the potential cellular role of the cytoplasmic domain of proEGF (proEGFcyt). We have determined the expression of proEGF and a novel proEGF isoform (proEGFdel23) in normal and neoplastic human thyroid tissues and thyroid carcinoma cell lines. Stable transfectants of the human follicular thyroid carcinoma (FTC) cell line FTC-133 overexpressing proEGFcyt or its truncated exon 22.23 version (proEGF22.23) revealed altered proliferation and marked changes in microtubular organization. This is the first report on novel functions of the cytoplasmic proEGF domain in human thyroid carcinoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human Thyroid Tissues. Thyroid tissue samples were obtained from patients undergoing surgery for clinical indications (goiter: 46; thyroid carcinoma: 40 papillary, 22 follicular, and 14 anaplastic; Table 1). This study was approved by the ethical committee of the Faculty of Medicine, Martin-Luther-University Halle-Wittenberg, and the informed consent of the patients was obtained before tissue collection. Tissues were either cryopreserved or fixed in formalin and embedded in paraffin. For the extraction of total RNA, tissues were frozen in liquid nitrogen and stored at –80°C until used.


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  		Table 1. List of PTC, FTC, and UTC tissues employed in this study as classified by pTNM status

 
Cell Culture. The human thyroid papillary carcinoma cell line BCPAP, the follicular carcinoma cell lines FTC-133, FTC-236, FTC-238, and the dedifferentiated carcinoma cell lines Hth-74, C-643, 8305C, and 8505C were propagated in DMEM F-12 medium (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% FCS in a 5% CO2 atmosphere at 37°C. Medium was changed every second day, and cells were routinely passaged every 5 to 7 days.

RNA Processing, Reverse Transcription-PCR, and Cloning of Eukaryotic Expression Constructs. Total RNA of the human thyroid cell lines and the human thyroid tissues was isolated with Trizol reagent (Invitrogen, Karlsruhe, Germany). The amount of RNA isolated was spectrophotometrically determined at 260 and 280 nm (19). Total RNA (1 µg) obtained from the thyroid carcinoma cell lines and thyroid tissues was used for first-strand cDNA synthesis employing the SuperScript reverse transcriptase kit and 500 ng/mL oligo(dT) primer (both Life Technologies, Heidelberg, Germany). All PCR reactions were carried out in 25 µL solution containing 1 µL cDNA, 2.5 µL of 10x Advantage cDNA polymerase mix buffer, 100 µmol/L deoxynucleotide triphosphate, 10 pmol/L each of forward and reverse primers, and 2.5 units Advantage cDNA polymerase mix (BD Biosciences Clontech, Heidelberg, Germany). For amplification of proEGFcyt/del23 transcripts from tissues or cell lines, the PCR cycles consisted of an initial denaturation for 3 minutes at 95°C followed by 40 cycles of a 1-minute denaturation step at 95°C, annealing for 1 minute at 65°C, and a 2-minute elongation step at 72°C employing the PCR primer pairs (forward) 5'-GGCCATGGAGACTCAGAAGCTGCTATCGAAAAACCC-3' and (reverse) 5'-ACGGATCCTCACTGAGTCAGCTCCATTTGGTG-3'. For cloning of the cytoplasmic domain of human proEGF, the human proEGF expression plasmid phEGF101 (generously provided by Prof. G. Bell, University of Chicago, Chicago, IL) was employed (20); the proEGFdel23 isoform was amplified from cDNA of the human thyroid carcinoma cell line FTC-238. Amplification of proEGFcyt, proEGF22.23, and proEGFdel23 was done for seven cycles with denaturation at 95°C for 1 minute, annealing at 65°C for 1 minute, and elongation at 72°C for 2 minutes followed by 30 cycles with denaturation at 95°C for 1 minute and annealing and elongation at 72°C for 3 minutes employing the PCR primers EcoRI-proEGF (forward) 5'-GGAATTCACTCAGAAGCTATCGAAAAACCC-3' and NotI-proEGF (reverse) 5'-GGCGGCCGCTCACTGAGTCAGCTCCATTTGGTG-3' for amplification of proEGFcyt or proEGFdel23 or NotI-proEGF22.23 (reverse) 5'-GGCGGCCGCTCACCCATCTGCTGCCTGGCCATC-3' for amplification of proEGF22.23. All amplicons were separated in a 1% agarose gel in TAE buffer and visualized by ethidium bromide staining. Amplicons were purified by Wizard PCR Preps DNA purification resin, cloned into the pGEM-T vector (both Promega, Heidelberg, Germany), and sequenced in both directions employing the Thermo Sequenase Dye Terminator Cycle Sequencing Pre-Mix kit (Amersham Biosciences, Piscataway, NJ) and T7 or SP6 sequencing primers. Verified EcoRI/NotI proEGFcyt, proEGF22.23, and proEGFdel23 constructs were cloned into eukaryotic expression vector pcDNA4/HisMaxC (Invitrogen).

Stable proEGFcyt, proEGF22.23, and proEGFdel23 FTC-133 Transfectants. One day before stable transfection of cells with 1 µg of the proEGFcyt, proEGF22.23, or proEGFdel23 construct in pcDNA4/HisMaxC or pcDNA4/HisMaxC empty plasmid vector itself, human FTC-133 were seeded into six-well plates and cultured overnight to reach 80% confluence. Cells were stably transfected using the Metafectene Transfection kit (Biontex Laboratories, Munich, Germany) according to the manufacturer's instructions. Selection was initiated 48 hours after transfection with Zeozin at 25 µg/mL (Invitrogen), and individual FTC-133 clones were picked twice and expanded. All experiments were carried out on at least three different clones per stable transfection.

Proliferation Assay. The assays were carried out on two different clones per transfection and repeated twice each. To determine proliferation rates, stable FTC-133 transfectants were seeded into six-well plates at a total of 3 x 104 cells per well and cultured for 4 days with a daily change of medium. Cells were harvested in their exponential phase at a confluence of 70% using 500 µL cell detaching reagent (Accutase II, PAA Laboratories) per well and the number of transfectants was recalculated in a Neubauer chamber. BrdU proliferation assays were done according to the manufacturer's instructions using the colorimetric BrdU cell proliferation ELISA kit (Roche Diagnostics GmbH, Penzberg, Germany). Stable FTC-133 transfectants were seeded into 96-well plates with the total amount of 2,000 cells per well and cultured overnight. Reaction was stopped with 1 mol/L H2SO4 and absorbance was measured in an ELISA reader at 450 and 620 nm.

Taxol and Nocodazole Treatment. Incubations with the microtubule-stabilizing drug taxol and the microtubule-depolymerizing drug nocodazole (both Calbiochem, Darmstadt, Germany) occurred for 1 hour at a concentration of 25 µmol/L on cells at 80% confluence before the experiment.

Immunodetection on Cell Transfectants. Immunofluorescent detection of actin was done with a phalloidin-FITC conjugate (1:100, Sigma, Deisenhofen, Germany) and monoclonal antibodies toward acetylated tubulin (clone 6-11B-1), polyglutamylated tubulin (both 1:200, all Sigma), {alpha}-tubulin (1:250, Invitrogen, Karlsruhe, Germany), human cytokeratin (clone MNF116, 1:200, DAKO, Hamburg, Germany), and human microtubule-associated proteins (MAP) 1b (clone AA6) and 2 (clone HM-2, both 1:200, Sigma) were employed. FTC-133 transfectants were plated onto glass slides and cultured to reach 80% confluence. Cells were fixed in 4% paraformaldehyde in PBS. Membrane permeabilization was done with 0.003% Triton X-100 in PBS for 7 minutes at room temperature (actin), with 1% Triton X-100 in 5 mmol/L PIPES (pH 6.7) plus 2 mmol/L EGTA for 40 seconds at 4°C (acetylated and polyglutamylated tubulin), or with 1 mg/mL trypsin (PAA Laboratories) for 10 minutes at 37°C (cytokeratin). No separate permeabilization step occurred for the immunodetection of MAP1b and MAP2. Blocking of unspecific binding with 10% normal goat serum in PBS plus 2% bovine serum albumin and 0.05% saponin for 30 minutes at room temperature, incubations with primary antibody dilutions in blocking buffer occurred for 1 hour at room temperature (for actin staining) or overnight at 4°C. After incubation with FITC- or TRITC-conjugated secondary donkey anti-mouse antibody (1:400, both Jackson ImmunoResearch, Hamburg, Germany) for 1 hour at room temperature, nuclear staining was done with 0.01 mg/mL Hoechst stain (Sigma) or 10 µg/mL 7-amino-actinomycin D (Molecular Probes). Cells were mounted in fluoroguard antifade reagent (Bio-Rad, Munich, Germany) and viewed with the Axioplan fluorescent microscope (Zeiss, Jena, Germany). Images were captured and edited with an AxioCam camera and Axiovision software (Zeiss), respectively.

Coimmunoprecipitation. Protein A-Sepharose bead (60 µL, 50%, Roche Diagnostics) solutions were preincubated with either 8 µg anti-Xpress antibody (Invitrogen) or IgG1 (Dianova, Hamburg, Germany) for 1 hour at room temperature. After washing with buffer containing 50 mmol/L HEPES-KOH, 60 mmol/L potassium acetate, 5 mmol/L magnesium acetate, 0,1% Triton X-100, 10% glycerol, 1 mmol/L NaF, 1 mmol/L DTT, and 1 mmol/L DMFS, beads were incubated with 5 mg/mL bovine serum albumin diluted in the washing buffer for 30 minutes at room temperature. Cells were harvested at 80% confluence and lysed in lysis buffer containing 50 mmol/L Tris-HCl, 50 mmol/L NaCl, 0,2% Triton X-100, 10% glycerol, 1 mmol/L DTT, and 1 mmol/L phenylmethylsulfonyl fluoride for 30 minutes at 4°C. After centrifugation at 13,000 rpm at 4°C for 15 minutes, supernatants were added to the Sepharose beads and incubated for 2 hours at 4°C under gentle agitation. Beads were washed thrice with buffer A, and specifically bound proteins were eluted from Sepharose beads by adding washing buffer containing 10% SDS.

Western Blot Analysis. Proteins were extracted in lysis buffer containing 62,5 mmol/L Tris, 2% SDS, and 10% saccharose for 30 minutes at 4°C. The amount of protein was determined by Bradford method (Bio-Rad). Cellular proteins were extracted with 1x SDS gel loading buffer (19), boiled for 5 minutes at 90°C, and centrifuged at 13,000 rpm for 5 minutes at 4°C, and supernatants were separated by SDS-PAGE and blotted onto a Hybond nitrocellulose membrane (Amersham, Braunschweig, Germany). Membranes were blocked at room temperature for 1 hour in TBS plus 0.05% Tween 20 or PBS plus Tween 20 containing 3% bovine serum albumin or 5% skimmed milk for the detection of the Xpress tag, tubulins, or MAP1b and MAP2, respectively. A monoclonal antibody directed against the Xpress tag (1:5,000, Invitrogen) at the NH2 terminus of the proEGFcyt, proEGF22.23, and proEGFdel23 fusion proteins was used to select for stable FTC-133 transfectants. For the detection of human MAP1b, MAP2, and tubulins, membranes were incubated overnight at 4°C with the antibodies (all 1:1,000) followed by a 1-hour incubation with a horseradish peroxidase–conjugated goat anti-mouse Ig (1:20,000, Dianova). Specific immunobinding was detected with the enhanced chemiluminescence kit (Amersham).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of a Novel Isoform of the Cytoplasmic Domain of proEGF in Human Thyroid Tissues and Human Thyroid Carcinoma Cell Lines. Reverse transcription-PCR analysis of human thyroid tissues (Table 1) and human thyroid carcinoma cell lines was done with a primer pair specifically amplifying the cytoplasmic domain of human proEGF, which is encoded by exons 22 to 24. Two different amplicons at 371 and 450 bp were detected (Fig. 1). Sequence analysis confirmed the 450-bp amplicon to encode the complete proEGF cytoplasmic domain (proEGFcyt). The 371-bp amplicon encoded a novel alternative splice variant of the cytoplasmic domain of proEGF lacking complete exon 23 followed by an out-of-frame insertion of exon 24 and generation of a preliminary stop codon (proEGFdel23; Fig. 2). The COOH-terminally encoded 13 amino acids of the truncated proEGFdel23 distinctly differed from the amino acid sequence encoded by exon 24 in proEGFcyt (Fig. 2).



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  		Figure 1. Reverse transcription-PCR analysis of cDNA preparations from human goiter and neoplastic PTC, FTC, and UTC thyroid carcinoma as well as human thyroid carcinoma cell lines employing oligonucleotide primers specific for the cytoplasmic domain of human EGF precursor. A 450-bp amplicon (1 and 2) encoding the full-length nucleic acid sequence of proEGF cytoplasmic domain (proEGFcyt) and an additional 371-bp amplicon (proEGFdel23) were detected (1). No PCR amplicons were observed when template was omitted in PCR reaction (3). Amplification of the housekeeping gene GAPDH served as an internal control.

 


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  		Figure 2. Nucleic acid and deduced amino acid sequences of proEGFcyt and proEGFdel23 cloned from cDNA of the human thyroid carcinoma cell line FTC-238. Three independent PCR amplifications were sequenced in both directions to exclude sequencing errors, and identical sequencing results were obtained in all cases. Alignments of the nucleotide sequences of proEGFcyt and proEGFdel23 revealed a deletion of the complete exon 23 followed by a 1-bp frameshift resulting in a premature stop codon in the former exon 24 (20). The nucleic acid sequence encoded by frameshifted exon 24 encoded a 13–amino acid peptide in proEGFdel23 with no homology to the amino acid sequence encoded by the in-frame exon24 of proEGFcyt (*identical nucleotide; * identical amino acid).

 
The human follicular and dedifferentiated thyroid carcinoma cell lines FTC-133 and 8305C, respectively, showed no expression of either of the two proEGF isoforms. In normal and neoplastic human thyroid tissues and the human thyroid carcinoma cell lines FTC-236, FTC-238, Hth-74, C-643, and 8505C, the proEGFdel23 isoform was always coexpressed with proEGFcyt. The latter was also expressed in the absence of proEGFdel23 (Table 2). More than 75% of all goiter and FTC tissues and 90% of papillary thyroid carcinoma (PTC) tissues expressed transcripts for either of the proEGF isoforms. By contrast, ~64% of human undifferentiated thyroid carcinoma (UTC) tissues either were devoid of proEGF isoforms or exclusively displayed coexpression of both proEGF isoforms (34%) but not proEGFcyt alone (Table 2). In addition, in PTC tissues displaying coexpression of both proEGF isoforms, 84% of these tissues were staged in pT3/T4, whereas only 16% staged pT1/T2. No significant correlation could be found between the expression of either of the proEGF isoforms and the age or gender of the patients (Table 1). Immunoreactive EGF was detected in the cytoplasm of thyrocytes of all PTC, FTC, and UTC tissues investigated. However, EGF was not an exclusive product of human thyrocytes but was also detected in interstitial cells of human thyroid carcinoma (data not shown). This suggests that EGF, although not a marker for human thyrocytes, is present and potentially active within the human thyroid gland.


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  		Table 2. Expression pattern of proEGFcyt and proEGFdel23 in human thyroid tissues (% of investigated tissues)

 
Proliferation Rates Are Decreased in FTC-133 Overexpressing proEGFcyt and proEGF22.23. To study the functional role of proEGFcyt and proEGFdel23 in human thyroid cells, stable transfectants overexpressing these proEGF isoforms, as well as stable transfectants expressing the engineered proEGF domain encoded by exons 22 and 23 (proEGF22.23), were generated in FTC-133, which were devoid of endogenous proEGF expression (Fig. 3A). The proEGF isoforms were expressed as fusion proteins containing a NH2-terminal Xpress tag, which allowed specific Western blot detection of the immunoreactive proEGF proteins of the expected sizes in clonal FTC-133 cell lines (Fig. 3B). FTC-133 stably transfected with the pcDNA4/HisMaxC empty plasmid vector served as negative control. Overexpression of proEGFcyt and proEGF22.23 resulted in proliferation rates 3-fold lower in cell counts (Fig. 3C) or ~2-fold lower in BrdU proliferation assays (Fig. 3D) compared with the FTC-133-proEGFdel23 or empty plasmid control transfectants.



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  		Figure 3. Schematic diagram of the cytoplasmic proEGF constructs employed for establishing stable FTC-133 transfectants. Nucleotide and amino acid sequences of exons 22 to 24 as well as the 13–amino acid sequence of proEGFdel23 (13 amino acids) refer to the sequences shown in Fig. 2 (A). Employing the anti-Xpress antibody, Western blot analysis of total cell lysates of stable FTC-133 transfectants revealed immunobands at the expected sizes of 20.7, 14.4, and 12.6 kDa for immunoreactive proEGFcyt, proEGF22.23, and proEGFdel23 fusion proteins containing a NH2-terminal Xpress tag, respectively. FTC-133 stably transfected with pcDNA4/HisMaxC empty vector, not revealing immunoreactive bands, were used as negative control (B). Cell counting (C) and BrdU cell proliferation assays (D) were employed to determine the proliferation rate of the stable transfectants FTC-133-proEGFcyt, proEGF22.23, proEGFdel23, and FTC-133 empty plasmid control. FTC-133-proEGFcyt and proEGF22.23 stable transfectants showed a significant decrease in proliferation, whereas in FTC-133-proEGFdel23 and the empty plasmid control transfectants proliferation rates were unchanged.

 
Microtubular System Is Altered in FTC-133-proEGFcyt Transfectants. Stable FTC-133 transfectants overexpressing proEGFcyt or proEGF22.23 displayed an enlarged cellular body size (Fig. 4A, C, E, G, J, K, and M) and the presence of multiple nuclei (Fig. 4A, C, and E) when compared with stable transfectants of FTC-133-proEGFdel23 or empty plasmid control. Overexpression of proEGF22.23 revealed a phenotype similar to that of proEGFcyt, whereas the phenotype of proEGFdel23 was similar to that of the empty plasmid control. The structure and composition of the cytoskeleton determine the cell shape; thus, we investigated the distribution of different cytoskeletal components in those FTC-133 transfectants. No differences were observed in the distribution of actin filaments among the stable FTC-133 transfectants, indicating that the phenotypical changes seen in FTC-133-proEGFcyt or proEGF22.23 transfectants were caused by cytoskeletal components other than actin (Fig. 4A and B). In addition, we did not observe changes in the distribution of cytokeratins among these FTC-133 transfectants (data not shown).



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  		Figure 4. Immunofluorescence on paraformaldehyde-fixed stable FTC-133-proEGFcyt and proEGFdel23 transfectants for the detection of cytoskeletal components. Immunofluorescent staining of FTC-133-proEGF22.23 revealed a phenotype similar to that of FTC-133-proEGFcyt, whereas the phenotype of FTC-133-proEGFdel23 was similar to that of the empty plasmid control (FTC-133-proEGFcyt and proEGFdel23). FITC-conjugated phalloidin was used to detect actin (A and B, green). Immunoreactive detection of {alpha}-tubulin (C and D, green), acetylated {alpha}-tubulin (E-H, red), MAP1b (I and J, red), MAP2 (K and L, red), or polyglutamylated tubulin (M and N, green) was done with monoclonal antibodies and FITC- or TRITC-conjugated secondary antibodies. For nuclear staining, either 7-amino-actinomycin D stain (A and B, red) or Hoechst stain (D-N, blue) were used. No changes were observed in the distribution and structure of actin among stable transfectants overexpressing proEGFcyt (A) and empty plasmid controls (B). Immunoreactive staining of {alpha}-tubulin revealed a broad ramified microtubular distribution in FTC-133-proEGFcyt transfectants (C). This phenotype was not observed in empty plasmid control transfectants (D), which displayed a more condensed and aggregated arrangement of microtubules. The high level of {alpha}-tubulin acetylation in FTC-133-proEGFcyt (E) decreased on treatment with the tubulin depolymerizing drug nocodazole (G). By contrast, the low acetylation level of {alpha}-tubulin in empty plasmid control (F) was significantly increased on treatment with the tubulin stabilizer taxol (H). No further increase in acetylation was observed in FTC-133-proEGFcyt transfectants on taxol treatment (data not shown), indicating maximal levels of acetylation in those transfectants. Immunofluorescent staining of MAPs showed an up-regulation of MAP1b and MAP2 protein in FTC-133-proEGFcyt (I and K) compared with empty plasmid control transfectants (J and L), whereas no differences were observed in their cytoplasmic distribution (I-L). Immunofluorescent detection of polyglutamylated tubulin in FTC-133-proEGFcyt showed a generalized cytoplasmic distribution (M). By contrast, in FTC-133-proEGFdel23 and empty plasmid controls, polyglutamylated tubulin was concentrated around the nucleus (N).

 
Both distribution and composition of components of the microtubular cytoskeletal system were distinctly different in FTC-133-proEGFcyt or proEGF22.23 compared with FTC-133-proEGFdel23 and empty plasmid transfectants. Immunofluorescence revealed a broad ramified cytoplasmic distribution of {alpha}-tubulin in proEGFcyt and proEGF22.23 transfectants (Fig. 4C). This contrasted with the condensed microtubular aggregates observed in FTC-133-proEGFdel23 and empty plasmid transfectants (Fig. 4D). In contrast to the latter transfectants (Figs. 4F and 5A-C and K-N), overexpression of proEGFcyt or proEGF22.23 in FTC-133 led to a significant increase in immunoreactive acetylated {alpha}tubulin as determined by immunofluorescence (Fig. 4E) and Western blot analysis (Fig. 5D-G and O; H-J), suggesting decreased dynamics in microtubular turnover and high microtubular stability in FTC-133-proEGFcyt transfectants. To provide further evidence for a link between tubulin acetylation and microtubular turnover, the level of acetylation of {alpha}-tubulin in the FTC-133 transfectants was assessed on exposure to the microtubule-stabilizing drug taxol and the microtubule depolymerizer nocodazole. Treatment with nocodazole caused a decrease in {alpha}-tubulin acetylation in FTC-133-proEGFcyt or FTC-133-proEGF22.23 as determined by immunofluorescence (Fig. 4G) and Western blot analysis (Fig. 5D-G and O; H-J). By contrast, taxol treatment did not result in a further detectable increase in tubulin acetylation in FTC-133-proEGFcyt or FTC-133-proEGF22.23 but caused increased tubulin acetylation in FTC-133-proEGFdel23 and empty plasmid transfectants (Fig. 4H). Coimmunoprecipitation experiments revealed no direct interaction of proEGFcyt with tubulin (data not shown). These results suggest enhanced microtubular stability of FTC-133 transfectants overexpressing proEGFcyt or proEGF22.23 as a result of increased acetylation of {alpha}-tubulins.



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  		Figure 5. Western blot analysis on protein extracts from different transfectants for acetylated {alpha}-tubulin showed a significant increase of acetylated {alpha}-tubulin in FTC-133-proEGFcyt (D-G and O) and FTC-133-proEGF22.23 (H-J) compared with FTC-133-proEGFdel23 (K-N) and FTC-133 empty plasmid control (A-C). Treatment with 25 µmol/L nocodazole for 1 hour resulted in decreased acetylation levels in FTC-133-proEGFcyt (D-G and O) and FTC-133-proEGF22.23 (H-J), whereas no changes in acetylation were observed in FTC-133-proEGFdel23 (K-N) or empty plasmid controls (A-C). Western blot detection of immunoreactive human MAP1b and MAP2 showed increased expression of MAP1b and MAP2c protein in FTC133-proEGFcyt (D-G and O) and FTC-133-proEGF22.23 (H-J) compared with proEGFdel23 (K-N) or control cells (A-C). No changes where seen in the amount of polyglutamylated tubulin present among the transfectants. Western blot detection of ß-actin in the total cell lysates was used to verify equal protein loading.

 
Localization of Polyglutamylated Tubulin Is Altered and MAP1b and MAP2 Are Increased in FTC-133-proEGFcyt. FTC-133-proEGFcyt and FTC-133-proEGF22.23 transfectants revealed a spotty distribution of polyglutamylated tubulin across the cytoplasm (Fig. 4M). By contrast, FTC-133 overexpressing proEGFdel23 or FTC-133-negative controls displayed perinuclear localization of polyglutamylated tubulin (Fig. 4N). Despite these differences in the distribution of polyglutamylated tubulin, neither of the stable FTC-133 transfectants produced significantly increased amounts of immunoreactive polyglutamylated tubulin as determined by Western blot analysis (Fig. 5A-O). Overexpression of immunoreactive MAP1b and the 75-kDa MAP2 isoform MAP2c was observed in FTC-133-proEGFcyt and FTC-133-proEGF22.23 transfectants by immunofluorescence (Fig. 4G and J) or Western blot analysis (Fig. 5D-G and O; H-J). By contrast, thyroid carcinoma cell transfectants overexpressing proEGFdel23 and cells harboring the empty plasmid control displayed only weak MAP1b and MAP2c production (Figs. 4J and L and 5A-C and K-N).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Immunoreactive EGF has been reported in the cytosolic and nuclear compartment of thyroid follicular cells and seems to play a multifunctional role in human thyroid tissues (3, 21, 22). In normal and neoplastic human thyroid tissues, EGF acts as a dedifferentiating factor for thyrocytes (23–27). There is controversial evidence on the potential contribution of EGF for neoplastic transformation of thyrocytes. Although higher expression levels of EGF and TGF-{alpha} had been reported in malignant thyroid carcinoma (28, 22), no correlation between EGF expression and thyroid pathologies was observed by others (29). Our transcriptional analysis of transmembrane proEGF in human thyroid carcinoma tissues and cell lines revealed the presence of two proEGF isoforms showing marked differences in their cytoplasmic domains. Alternative splicing of precursor mRNAs can generate precursor EGF family member isoforms of functionally diverse properties as shown recently by the transmembrane NTAK/Nrg-2, a neural- and thymus-derived activator for ErbB kinases (30). In human goiter, PTC and FTC tissues, proEGF, but not proEGFdel23, was detected as an individual transcript, whereas proEGFdel23 was always found to be coexpressed with proEGF. Of the human thyroid tissues investigated, the detection of proEGFdel23 transcripts in the majority of advanced tumor stages (pT3/T4) of PTC and in UTC suggests an as-yet unidentified role for this truncated proEGF isoform during dedifferentiation in human thyroid carcinoma cells.

To further elucidate the role of the cytosolic domain of transmembrane proEGF and its splice version in human thyroid carcinoma cells, we generated stable FTC-133 transfectants. A significant decrease in proliferation and an enlarged cellular size with multiple nuclei was exclusively observed in FTC-133-proEGFcyt overexpressing the native proEGF cytoplasmic domain but not in FTC-133-proEGFdel23 or plasmid control stable transfectants. Interestingly, a similar change in cellular shape was observed in FTC-133-proEGF22.23 transfectants, suggesting that the 30–amino acid sequence encoded by exon 23, but not the peptides encoded by either exon 22 or exon 24 at both ends of the proEGFcyt domain, may be involved in mediating these morphologic changes. The intracellular and transmembrane domains of proEGF, pro-amphiregulin, and proTGF-{alpha} were shown to be important in determining the cellular localization and proteolytic processing of these proEGF members (11–14). In fact, the presence of a single residue in the cytoplasmic domain of proTGF-{alpha}, the COOH-terminal valine, was shown to be essential for membrane anchorage and correct processing of TGF-{alpha} (14). The cytoplasmic domain of proTGF-{alpha} was also shown to associate with a 86- and 106-kDa protein, with the 106-kDa component possessing kinase activity (15). Interaction of the intracellular domain of pro–heparin-binding EGF-like growth factor with promyelocytic leukemia zinc finger protein, a transcriptional repressor localized in the nucleus, resulted in altered mitogenic cellular signaling (16, 17). Both promyelocytic leukemia zinc finger protein and LIM kinase have been shown recently to associate with the intracellular domain of proNrg-1 (Nrg-1-ICD), and when released from the membrane-anchored proform on EGFR binding, Nrg-1-ICD can enter the nucleus and repress expression of several regulators of apoptosis (18). The changes in cellular shape observed in FTC-133-proEGFcyt and FTC-133-proEGF22.23 stable transfectants provide first evidence for a novel and unique role of proEGFcyt among transmembrane proEGF-like members and implicated the exon 23–encoded peptide of proEGFcyt as a suitable target region for future structure-function analysis. The search for peptide motifs within the exon 23–encoded peptide only revealed a single myristoylation site. The morphologic changes in FTC-133-proEGFcyt transfectants coincided with alterations in the level of post-translational acetylation of {alpha}-tubulin. The effects of proEGFcyt on the acetylation of {alpha}-tubulin were in large part reversible by the actions of the microtubule-targeting drugs and known microtubular stabilizer and destabilizer taxol and nocodazole, respectively. This provided evidence for an involvement of tubulin acetylation in the cellular phenotype of FTC-133-proEGFcyt and indicated a functional system of tubulin (de)acetylases in all FTC-133 transfectants (32–35). Enhanced levels of acetylated tubulin stabilize microtubuli and have been associated with decreased microtubular dynamics and cell motility (36–40). The level of tubulin acetylation in the FTC-133-proEGFcyt transfectants was maximal as taxol treatment did not result in a further detectable increase in acetylation of {alpha}-tubulin and indicates an indirect action of proEGFcyt on the (de)acetylase system (41). Thus, being a novel, highly efficient natural tubulin acetylating agent, overexpression of the cytoplasmic proEGF domain may be of therapeutic value particularly in less differentiated thyroid carcinoma lacking proEGFcyt (42–46).

The specific and exclusive transcriptional up-regulation and increased production of immunoreactive MAP1b and MAP2c in FTC-133-proEGFcyt seemed to be an additional mechanism by which proEGFcyt affects microtubular dynamics. MAPs mediate the interaction of actin filaments and microtubules, thus contributing to cell shape, motility, and plasticity (47–49). Both MAP1b and MAP2c stabilize microtubules and promote the formation of long microtubule bundles (50–53). Polyglutamylation of {alpha}-tubulin is a post-transcriptional modification, which modulates the affinity of tubulin for MAPs (54). Although the amount of polyglutamylated tubulin produced among the transfectants was similar, their distinctly different distribution pattern in FTC-133-proEGFcyt could potentially have an indirect effect on the local kinetics of MAP-tubulin interaction in these transfectants (55). Polyglutamylation of {alpha}-tubulin is also required for centriole formation, an organelle with the most stable microtubules (56). Alterations in both distribution of polyglutamylated tubulin and MAP-tubulin interactions are likely to affect centriole formation/stability and, in addition to hyperacetylated tubulin, could contribute to the decrease in proliferation and incomplete mitosis resulting in multinuclear FTC-133-proEGFcyt transfectants.

In conclusion, this study has provided first insights into the unique role of the native cytoplasmic domain of proEGF as a novel and versatile modulator of microtubular dynamics in human thyroid carcinoma cells.


   Acknowledgments
 
Grant support: German Cancer Research (Deutsche Krebshilfe).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Katrin Hammje, Christine Fröhlich, and Anja Winkler for their excellent technical support.

Received 6/ 8/04. Revised 11/ 4/04. Accepted 12/ 1/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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