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Endocrinology Vol. 142, No. 7 3152-3162
Copyright © 2001 by The Endocrine Society

ARTICLES

Dystroglycan Is Present in Rat Thyroid and Rat Thyroid Cells and Responds to Thyrotropin1

Barbara J. Collins, Gleb Gorelick and Arthur B. Schneider

Section of Endocrinology and Metabolism, University of Illinois, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Dr. Barbara J. Collins, Section of Endocrinology and Metabolism, MC 640, University of Illinois, 1819 West Polk Street, Chicago, Illinois 60612. E-mail: bcollins{at}uic.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dystroglycan is a high affinity laminin-binding glycoprotein originally described as a member of the dystrophin-associated glycoprotein complex in muscle. We have demonstrated the presence of dystroglycan in the thyroid using immunocytochemistry, immunoblots, ligand binding assays, and relative quantitative RT-PCR. In intact rat thyroid glands, antibodies against the {alpha} (extracellular, laminin-binding subunit) and ß (cytoplasmic/membrane bound) portions of the dystroglycan protein reacted at basolateral membranes where they colocalized with laminin. Western-blotted protein from the Fischer rat thyroid cell line FRTL-5 reacted with both the {alpha}- and ß-dystroglycan antibodies. The {alpha}-dystroglycan-reactive band colocalized with laminin-binding activity, and the protein and binding activity were decreased by TSH. In contrast, in the culture medium of these cells, {alpha}-dystroglycan was increased by TSH. The ß-dystroglycan antibody recognized the full-length 43-kDa band and an approximately 30-kDa truncated form. The truncated form was reduced in cells cultured with TSH, whereas the full-length form was not significantly diminished by TSH. Immunofluorescence of FRTL-5 cells in the absence of TSH showed a colocalization of dystroglycan and laminin. This was disrupted by the addition of TSH and was correlated to morphological changes. PCR amplification of complementary DNA with primer pairs from {alpha}- and ß-dystroglycan produced appropriately sized bands, whose sequence had identical protein-coding sequences and more than 96% nucleotide homology to mouse dystroglycan sequences. Relative quantitative RT-PCR of ß-dystroglycan messenger RNA showed reduced expression in cells cultured with TSH. We conclude that dystroglycan is present in rat thyroid and in FRTL5 rat thyroid cells and that TSH reduces its expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DYSTROGLYCAN is a transmembrane glycoprotein originally characterized as a member of the dystrophin-associated glycoprotein (DAG) complex in muscle sarcolemma. At its extracellular face it binds laminin with high affinity, and at its intracellular terminus it binds dystrophin or utrophin (1, 2, 3). Comprised of a highly glycosylated extracellular portion ({alpha}-dystroglycan) noncovalently linked to a smaller membrane-bound portion (ß-dystroglycan), dystroglycan is encoded by a single gene on human chromosome 3p21 (4). It has been proposed that dystroglycan forms a continuous link from the extracellular matrix to the actin cytoskeleton, providing structural integrity and perhaps transducing signals, in a manner similar to the integrins (5, 6). Dystroglycan, originally described in brain as cranin (7), has also been found in peripheral nerve, kidney, lung, and salivary gland tissues and in a variety of cell lines (4, 8, 9, 10, 11, 12, 13). Unlike other proteins of the DAG complex, mutations in the dystroglycan gene have not been implicated as a cause of myopathy, and indeed, dystroglycan knockout mice manifest lethal defects early in embryogenesis (embryonic days 6.5–7.5), before myogenesis has begun (14). This critical role in development along with its wide distribution in many cell types suggest a pivotal role for dystroglycan, which is further substantiated by evidence of its requirement for basement membrane assembly (15). Dystroglycan messenger RNA (mRNA) and protein have not been documented previously in thyroid. Here we describe the presence of dystroglycan in rat thyroid tissue and in the Fischer rat thyroid cell line FRTL-5 using immunocytochemical techniques, immunoblots, ligand binding, and RT-PCR. We show that incubation of FRTL-5 cells with TSH causes time-dependent changes in dystroglycan levels and characteristics.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
FRTL-5 cells were purchased from American Type Culture Collection (Manassas, VA). The cells were maintained in Coon’s modification of F-12 medium supplemented with 5% calf serum plus insulin, somatostatin, transferrin, hydrocortisone, and His-Gly-Asp peptide (basal medium) (16). For long-term TSH stimulation, bovine TSH at 1.0 mU/ml was used. Withdrawal of TSH from FRTL-5 cells slowed cell division. Therefore, to equalize the number of FRTL-5 cells tested with and without TSH, FRTL-5 cells destined for the basal (no TSH) condition were plated with TSH at twice the density of cells destined for chronic TSH, grown with TSH for 2–4 days, and then switched to TSH-free medium for 5–7 days before use. Cells acutely stimulated with TSH received TSH at 4.0 mU/ml added to the basal medium for 2–18 h before cell harvesting. Cells were fed twice weekly and maintained in 5% CO2 in a humidified incubator at 37 C. For protein and RNA isolation, cells were grown in Falcon 75-cm2 tissue culture flasks, and for immunocytochemistry they were plated in Falcon four-chamber CultureSlides (7). In some experiments incubation medium from the final 3 days was collected to assess secreted protein. All reagents were from Sigma (St. Louis, MO) unless otherwise specified.

Rat thyroids
The thyroid was obtained from an adult female rat after transcardial perfusion with PBS and 3% paraformaldehyde in phosphate buffer, under an institutionally approved protocol. The thyroid, attached to the trachea, was postfixed for 2 h and cryoprotected in 20% sucrose in phosphate buffer, and 20-µm frozen sections were thaw-mounted and processed for immunofluorescence by a modification of the protocol used for cells, with primary and secondary antibody incubations carried out overnight at 4 C.

Antibodies
The antibody against {alpha}-dystroglycan used for blotting and immunofluorescence was a mouse monoclonal IgG raised against rabbit skeletal muscle membranes (mAb VIA4–1, Upstate Biotechnology, Inc., Lake Placid, NY). It was used for immunofluorescence at 1:100 dilution (10 µg/ml) and for Western blotting at 1:500 dilution. Monoclonal antibody 6C1 (available through Chemicon, Temecula, CA) against cranin ({alpha}-dystroglycan) was a gift from Dr. Neil Smalheiser. This IgM class antibody against a peptide corresponding to amino acids 572–604 of the human sequence (7, 17) was provided as hybridoma tissue culture medium and was used undiluted. The ß-dystroglycan antibody (clone 43DAG/8D5, Novocastra Laboratories, Newcastle, UK) was raised against a synthetic peptide containing 15 of the last 16 amino acids in the human ß-dystroglycan C-terminal region, supplied as tissue culture supernatant, and used at a concentration of 5–10 ng/ml (dilution, 1:5–10) for immunofluorescence and at a dilution of 1:200 for Western blotting. The antibody against thyroglobulin (Sigma) was used at a 1:1000 dilution. The anti-laminin antibody (Sigma) was used at a 1:200 dilution for immunofluorescence and a 1:5000 dilution for ligand binding blots. Mouse and rabbit IgG at a concentration of 10 µg/ml or mouse antihuman ß-1 integrin (Chemicon) or rabbit anti-ret (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used as IgG-negative controls. An IgM antibody, 1A5, prepared at the same time as the 6C1 antibody that did not recognize the 6C1 antigen by enzyme-linked immunosorbent assay was used as a negative control for the 6C1 antibody (17). Fluorescein isothiocyanate- and Texas Red-conjugated secondary antibodies used for immunofluorescence were obtained from Vector Laboratories, Inc. (Burlingame, CA). Peroxidase-conjugated anti-IgG secondary antibodies for Western blotting were purchased from Sigma.

Immunocytochemistry
Samples used for differential interference contrast (DIC) microscopy were cells reacted with monoclonal antibody 6C1 against {alpha}DG and visualized by peroxidase-diaminobenzidine hydrochloride (DAB) staining for brightfield photography. These cells were fixed in glutaraldehyde (0.5%) and reacted by an avidin-biotin peroxidase method with DAB visualization. The slides, which had been mounted in Permount (Fisher Scientific, Pittsburgh, PA) for brightfield photography, were recovered for DIC by removing the mounting medium with xylene followed by rehydration in graded ethanol. They were remounted in Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA) with 4,6-diamino-2-phenylindole (DAPI) to localize nuclei and visualized with Nomarski optics on a Carl Zeiss LSM 510 laser scanning confocal microscope (New York, NY) with a UV laser to visualize DAPI-stained nuclei. Using this method, the DAB-stained antigen is revealed as a black precipitate on a phase contrast image.

Cells were processed for immunofluorescence by the method of Bacallao et al. (18) using the pH shift paraformaldehyde method, in which cells were first fixed in 3% paraformaldehyde in K+-PIPES buffer with CaCl2 and MgCl2, pH 6.8, followed by 3% paraformaldehyde in sodium borate buffer, pH 11.0. After treatment with NaBH4 in PBS, the cells were blocked in 0.2% fish gelatin (Amersham Pharmacia Biotech, Arlington Heights, IL) in PBS with 0.1% Triton X-100, incubated in primary antibodies, and reacted with fluorescein-conjugated antimouse IgG (rat-adsorbed) and Texas Red-conjugated antirabbit IgG. The slides were mounted in Vectashield with DAPI and visualized with the Carl Zeiss LSM 510 laser scanning confocal microscope using an argon/krypton laser. Both single plane and stacked horizontal Z-sections at 1-µm intervals were obtained using the LSM software. Orthogonal images perpendicular to the xy-plane were electronically reconstructed from the stacked Z-section images.

Laminin binding and Western blotting
Membrane proteins from FRTL-5 cells cultured in flasks were extracted by a modification of the method of Smalheiser and Kim (17). Cells were scraped into cold Tris-EDTA-NaCl buffer (pH 7.6) containing phenylmethylsulfonylfluoride and N-ethylmaleimide, and the cell suspension was homogenized on ice in a Dounce homogenizer (Kontes Co., Vineland, NJ). A portion of the crude homogenate was retained for total protein. Centrifugation of the remainder at 12,000 x g yielded a supernatant of soluble cytosolic proteins and a pellet containing the crude membrane fraction. The pellet was solubilized for 2 h on ice in triethanolamine buffer (pH 7.4) containing NaCl, CaCl2, and MgCl2 (hereafter referred to as TEA buffer) with 10% glycerol and 2% Triton X-100. The detergent-insoluble proteins were removed by recentrifugation and protein concentration of the total homogenate, soluble cytosolic proteins and solubilized membrane proteins were determined with the Pierce Chemical Co. bicinchoninic acid protein assay of methanol-precipitated aliquots and standards. Protein fractions were precipitated in 5 vol methanol at -20 C, recovered by centrifugation, and resuspended at a concentration of 5 mg/ml in 2x denaturing PAGE buffer containing dithiothreitol. The proteins were electrophoresed on a 6.5% or 7.5% SDS-PAGE gel and transferred to Hybond P membranes (Amersham Pharmacia Biotech) using a Tris-acetate-EDTA transfer buffer. The amount of protein loaded onto the gels varied by fraction to provide comparable signals: 50 µg total homogenate, 25 µg membrane proteins, and 100 µg soluble cytosolic proteins. The transferred blots were blocked in 1% nonfat dry milk (blotting grade, Bio-Rad Laboratories, Inc., Hercules, CA) in PBS for Western blots, or TEA buffer for laminin binding. The blots were reacted for laminin binding following the method of Smalheiser and Kim (17). Blots reacted with antibodies without ligand binding were processed similarly, except PBS was used in place of TEA buffer. The signal was developed by chemiluminescence with ECL Plus (Amersham Pharmacia Biotech) (7), and multiple film exposures for different lengths of time were made to establish a linear range. Film images were scanned by a ScanJet 4C/T with DeskScan software (Hewlett-Packard Co., Palo Alto, CA) and quantified with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA) with background corrections made for each lane.

Isolation of dystroglycan from conditioned medium
Three-day culture medium from three large flasks each of TSH-free or chronic TSH-cultured cells (~75 ml) was carefully aspirated and centrifuged for 5 min at 1000 x g to remove cells. In a modification of the methods of Matsumura et al. (19) and Smalheiser and Kim (17), the supernatants were decanted and adjusted to 0.5 M NaCl before adding to 5 ml wheat germ agglutinin (WGA)-Sepharose beads (Vector Laboratories, Inc., Burlingame, CA) in 50-ml polypropylene centrifuge tubes. The WGA-Sepharose beads were incubated with medium by gentle rotation overnight at 4 C, washed twice with 50 mM Tris-HCl, pH 7.4, containing 0.5 M NaCl, and eluted with 45 ml 50 mM Tris-HCl containing 0.3 M N-acetylglucosamine (Sigma). The eluate was concentrated in a Centriprep-30 (Amicon, Inc., Beverly, MA) and reconstituted with TEA buffer. The concentration and reconstitution steps were repeated to reduce the sugar concentration to less than 0.01 M. The eluate, in 12 ml TEA buffer, was added to 2 ml conditioned laminin affinity beads (EHS laminin coupled to Bio-Rad Laboratories, Inc., Affigel beads by a hydrazide procedure) and incubated by gentle rotation overnight at 4 C. The beads were rinsed with TEA buffer and eluted with 12 ml high salt/chelator buffer containing 1 M NaCl, 10 mM EGTA, 10 mM EDTA, and 0.1% Triton X-100 in TEA buffer. The eluate was concentrated, repeatedly diluted with TEA, and reconcentrated. An aliquot of the final concentrate was taken for protein determination, and the protein sample was precipitated in 5 vol methanol for subsequent electrophoresis. A 20-µg aliquot of total protein was loaded onto SDS-polyacrylamide gels and processed as indicated above for {alpha}-dystroglycan and laminin binding.

RNA isolation, RT, and PCR amplification
Total RNA was isolated from cells grown in 75-cm2 flasks by Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH), and reverse transcribed using the Retroscript kit (Ambion, Inc., Austin, TX) or Advantage RT for PCR (CLONTECH Laboratories, Inc., Palo Alto, CA). RNA for sequencing purposes was deoxyribonuclease-treated before RT. PCR was performed (GeneAmp 9600 System, Perkin-Elmer Corp., Norwalk, CT) using Perkin-Elmer Corp. AmpliTaq Gold or Platinum Taq (Life Technologies, Inc., Gaithersburg, MD) and a hot start protocol with 40 cycles of amplification. Three sets of dystroglycan-specific primers from the mouse dystroglycan sequence were used: a 269-bp region at the N-terminus of {alpha}-dystroglycan within exon 1 (GGA TGT CTG TGG ACA ACT GGC; CCC ACT GGA GGC AAT TAA ATC), a 425-bp region of {alpha}-dystroglycan in the middle region (GAG CCC ACA GCC GTT ATT AC; TGA ACC CAC GAT TTC TCA CC), and a 346-bp region encompassing most of the cytoplasmic portion of ß-dystroglycan (GAG GAC CAG GCC ACC TTT ATT AAG; CAG GCG CTT GTG GGT TAA GG). A fourth primer pair that overlapped the last 132 bp of the 346-bp C-terminal amplified region (CCT AAC GCA CCT CCC TAT CAG C; CAG GCG CTT GTG GGT TAA GG) was used only for sequencing. The HPLC-purified primers were prepared by Operon Technologies, Inc. (Alameda CA).

Cloning and sequencing
The PCR-amplified products were purified with GeneClean (BIO 101, La Jolla, CA) and cloned into the Novagen (Madison, WI) pT7Blue-3 Perfectly Blunt cloning vector. Colonies, grown on agar with ampicillin or carbenicillin and tetracycline plus X-galactosidase and isopropyl- ß-D-thiogalactopyranoside, were selected by blue-white screening and replated. A portion of the colony lysate PCR product was sequenced by the Amersham Pharmacia Biotech T7 Sequenase method according to the manufacturer’s protocol, using [33P]deoxy-ATP. The products were run on a 6% polyacrylamide Tris-borate-EDTA-urea gel and visualized by autoradiography.

Relative quantitative RT-PCR
Relative expression of mRNA for ß-dystroglycan was assessed by use of the QuantumRNA (Ambion, Inc.) 18S ribosomal RNA internal standard primers and competimers along with primers amplifying the 346-bp region coding for the cytoplasmic portion of ß-dystroglycan. Following the manufacturer’s directions, complementary DNA (cDNA) was prepared by RT from total RNA isolated from TSH-free, chronic TSH, and 2-h acute TSH-treated cells. The cDNA was amplified by PCR with Platinum-Taq for 26 cycles with the ß-dystroglycan primer and the competing 18S ribosomal RNA primer:competimer pair at a ratio of 3:7. [{alpha}-32P]Deoxy-CTP incorporation into the PCR reaction enabled visualization of the product on a 6% denaturing polyacrylamide-urea-Tris-borate-EDTA gel using a PhosphorImager (Molecular Dynamics, Inc.). Quantification of the signal was accomplished with the ImageQuant software program included with the phosphorimaging program. The ratio of the signal obtained for the ß-dystroglycan-amplified product to that of the 18S signal was computed and used as the measure by which to compare expression between conditions. Results were normalized by expressing the results as a percentage of the basal (no TSH) value.

Statistical comparisons
Immunoblot and RT-PCR data were analyzed by two-tailed paired t tests, comparing each TSH-stimulated condition to the basal (no TSH) results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dystroglycan immunoreactivity in rat thyroid
In rat thyroid tissue, confocal microscopy of immunofluorescence signals revealed that {alpha}-dystroglycan immunoreactivity was localized to the basolateral surface of the follicular cells (Fig. 1Go, A, C, and D). At the basal surface, colocalization of the dystroglycan signal with that of laminin (Fig. 1Go, B–D) was apparent, but laterally dystroglycan reactivity was abundantly expressed without interacting with laminin. The antibody to ß-dystroglycan gave a similar pattern (Fig. 1EGo), whereas thyroglobulin reactivity was confined to the colloid (Fig. 1FGo). Mouse and rabbit IgG controls were negative (Fig. 1GGo).



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  		Figure 1. Fluorescence immunoreactivity of adult rat thyroid fixed frozen sections. In these cross-sections of thyroid follicles, the basal surface of each cell is at the outside of the follicle, and the apical surface faces the colloid (black in all panels except F, where it is green). The lateral surfaces form the cell-cell interfaces seen between the apical and basal surfaces. A–D are the same field, with {alpha}-dystroglycan in green (fluorescein isothiocyanate), laminin in red (Texas Red), and their colocalization in yellow (C and D). D shows a portion of the confocal image in C scanned at higher resolution and at twice the magnification to provide a better image of the lateral distribution of dystroglycan at the cell interfaces (green) and its interaction (yellow) at the basal surface with laminin (red). The bar in each panel represents 20 µm. E is the composite exposure of ß-dystroglycan in green with laminin in red. Most of the follicular cells here were captured in cross-section, showing the membrane-associated signal of ß-dystroglycan. Some longitudinally sectioned areas provide evidence for the basolateral distribution as seen for {alpha}- dystroglycan and colocalization with laminin (yellow). F shows thyroglobulin immunoreactivity in green, as a positive control, restricted to the colloid. The blue staining is of DAPI-reactive nuclei in the follicular cells. G is a negative control using mouse and rabbit IgG at 10 µg/ml (green and red, respectively), with DAPI used to show nuclei. E, F, and G are at the same magnification as A, B, and C.

 
Dystroglycan immunoreactivity in FRTL-5 cells
Figure 2Go (top row) shows DIC phase images of FRTL-5 cells reacted with mAb 6C1 against {alpha}-dystroglycan after culture in the absence of TSH, stimulated with TSH for 2 h, and stimulated with TSH chronically. Cells without TSH were flattened and spread, and the immunoreactivity of {alpha}- dystroglycan was diffusely spread throughout the cytoplasm. Acute application of TSH caused the cells to round up, withdrawing cell contact from the substrate surface and concentrating the mAb 6C1 antigen in the reduced cytoplasmic volume. In chronically exposed cultures, the cells tended to grow in small piles within which the mAb 6C1 antigen is clustered. The negative control IgM antibody 1A5 was negative (data not shown). Using a different monoclonal antibody against {alpha}-dystroglycan (VIA4) for immunofluorescent visualization by confocal microscopy, the morphological changes seen in the DIC images were duplicated. The different fixation conditions and antibody, and concurrent reaction with a laminin antibody, provide additional information about the localization of {alpha}-dystroglycan. As can be seen in the next three panels of Fig. 2Go, showing {alpha}-dystroglycan in green, laminin in red, and superimposition of the two in yellow, there was extensive colocalization of the laminin and {alpha}-dystroglycan signal in the TSH-free cells (left). This overlap was seen at cell-cell and cell-matrix interfaces. Also, there was a diffuse punctate distribution of {alpha}-dystroglycan immunoreactivity that did not overlap the laminin signal. With the application of TSH for 2 h (center column), the extensive laminin-bound meshwork began to break up, leaving many cells isolated from each other and unattached to laminin. In the chronic TSH panels (right column), clumps of cells can be seen expressing both laminin and {alpha}-dystroglycan, but with little colocalization. The laminin was expressed predominantly within and below the clumps of cells, but not between. This difference is seen better in the orthogonal views (ortho) of the no TSH and chronic TSH cells. The yellow spots in no TSH cells indicate colocalization with laminin, and green shows the rather diffuse signal of {alpha}-dystroglycan. This colocalization was absent in TSH-treated cells, where laminin was basally located separately from {alpha}-dystroglycan, here seen concentrated at the apical surface of these cells. Immunoreactivity to ß-dystroglycan antibody in TSH-free FRTL-5 cells (similar to the images in Fig. 2Go and shown in black and white in Fig. 3Go) produced a membrane-associated pattern that was highly colocalized with laminin.



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  		Figure 2. DIC images (top row) of FRTL-5 cells incubated without TSH (left), TSH for 2 h (middle), or chronic TSH (right) and reacted with antibody 6C1 against {alpha}-dystroglycan. Immunofluorescence of FRTL-5 cells incubated, as described above, without TSH, TSH for 2 h, and TSH chronically and reacted with antibody VIA-4 against {alpha}-dystroglycan (second row) or antilaminin antibody (third row), and superimposed (fourth row). A yellow signal indicates colocalization of the laminin and dystroglycan signals. The two narrow panels at the bottom labeled Ortho are orthogonal (Z-plane) images constructed from Z-scanned stacked images of the corresponding No TSH or Chronic TSH flat images.

 


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  		Figure 3. ß-Dystroglycan and laminin immunofluorescence in FRTL5 cells, cultured without TSH. The signal for ß-dystroglycan is predominantly colocalized with that of laminin and is strongest at cell-cell interfaces.

 
Immunoblotting and laminin binding
The immunocytochemical data indicated that TSH produced major changes in the morphology of FRTL-5 cells, in the distribution of dystroglycan, and in the relationship of dystroglycan to laminin. We investigated the effects of TSH on dystroglycan further by immunoblotting FRTL-5 cell protein fractions with dystroglycan antibodies and by laminin binding to blotted protein fractions. Total homogenate, soluble cytosolic, and membrane proteins were isolated from FRTL-5 cells cultured in basal medium without TSH, with chronic TSH, and with acute TSH for 2, 4, 8, 12, and 18 h and were processed for immunoblotting and laminin binding. Figure 4Go shows protein and binding levels, with representative dystroglycan immunoblots or laminin-binding blots below each graph. The {alpha}-dystroglycan reactive bands (Fig. 4AGo) colocalized with the laminin binding bands (Fig. 4BGo) between 120 and 150 kDa, agreeing with the range previously published for {alpha}-dystroglycan (20) from muscle and brain. Graphs of the immunoblot spot densities for {alpha}-dystroglycan and laminin binding over time in TSH (Fig. 4Go, A and B) show that total homogenate, soluble cytosolic proteins, and membrane-associated protein are all significantly reduced in chronic TSH. This effect reached significance after 4 h of exposure to TSH, corresponding to the time when the TSH-stimulated cells have completely changed their morphology from flattened, where dystroglycan is colocalized with laminin, to rounded up and detached from the laminin substratum. The data suggest that this morphological change is accompanied by coordinate decreases in {alpha}-dystroglycan and laminin binding.



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  		Figure 4. Data analyzed from immunoblots of {alpha}-dystroglycan (A), ß-dystroglycan (C and D), and ligand binding blots of laminin (B) and of protein isolated from FRTL-5 cells incubated without TSH (basal condition); with TSH for 2, 4, or 8–12-18 h (combined); or with chronic incubation with TSH. All results are presented as a percentage of the calculated density of the spot for basal levels (no TSH) in each blot, which was set at 100%. The bars represent the mean and SE of four to eight experiments. (An asterisk indicates P < 0.05.) Below each graph is a representative blot of the data analyzed. B, The lower band represents laminin binding to dystroglycan, and the upper band (marker) represents endogenous (~200 kDa) laminin. C and D, The pattern of ß-dystroglycan immunoreactivity in total and membrane fractions (the cytosolic fraction did not contain sufficient antigen to react with the antibody). The data for the 43-kDa and approximately 30-kDa subunits were taken from the same gels, but are graphed separately.

 
In blots reacted with the ß-dystroglycan antibody, the expected band at 43 kDa was recognized (Fig. 4CGo), but did not diminish significantly with time in TSH in either the total homogenate or the membrane-bound fraction. (The soluble cytosolic fraction did not contain enough ß-dystroglycan-reactive protein to be detectable). An additional ß-dystroglycan-reactive band at approximately 30 kDa was strongly detected in the TSH-free condition in both total homogenate and membrane-bound fractions (Fig. 4DGo), but decreased with time in TSH and, although still detectable in the total homogenate fraction, was virtually absent in the membrane fraction of cells maintained in chronic TSH.

The disparate expression of {alpha}- and ß-dystroglycan after TSH stimulation suggested that the two subunits were dissociating from each other. ß-Dystroglycan immunoreactivity was retained at the membrane in both TSH-free and TSH-cultured cells, and this perception was reinforced by the constancy of the 43-kDa band in immunoblotting. However, {alpha}-dystroglycan immunoreactivity was greatly reduced after TSH, suggesting that the {alpha}-subunit was being lost. To test this, 3-day incubation medium was collected from cells cultured with or without TSH, and dystroglycan from the medium was enriched by passage over WGA-Sepharose and laminin affinity beads. Figure 5Go is the result of a representative experiment, showing that in the medium, unlike the other fractions, {alpha}-dystroglycan was more than 2-fold more abundant in the presence of TSH than in its absence, and this difference was mirrored by laminin binding. Examination of the flow-through fraction from the laminin beads (data not shown) found no {alpha}-dystroglycan-reactive material in the TSH-free fraction, indicating that a modification of {alpha}- dystroglycan, rendering it incapable of binding laminin, cannot account for this difference. Some {alpha}-dystroglycan immunoreactivity was detected in the flow-through from the TSH-cultured cells, most likely indicating a saturation of the laminin-binding capacity of the beads, which would indicate that the difference between {alpha}-dystroglycan release in TSH-free and TSH-cultured cells may be greater than 2-fold.



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  		Figure 5. Blots of {alpha}-dystroglycan and laminin binding in the presence (+), indicating chronic TSH, or the absence (-) of TSH. The cytosolic and membrane fractions were isolated in the same manner as all other blotted proteins, but the Medium fraction was enriched on WGA-Sepharose and laminin affinity beads.

 
PCR amplification and relative quantitative RT-PCR with dystroglycan primers
The immunoreactivity of thyroid cell proteins to three dystroglycan antibodies and colocalization with laminin binding established the presence of dystroglycan in thyroid. Although immunoblotting indicated a reduction in dystroglycan with the addition of TSH to the cells, the question remained of whether there was a direct action of TSH to suppress transcription of dystroglycan mRNA or if there was only a redistribution of dystroglycan within the cell. To answer this question, we examined the expression of dystroglycan mRNA from FRTL-5 cells cultured with or without TSH. Three primer sets were used for PCR amplification of cDNA prepared from total RNA isolated from FRTL-5 cells. Fragments of the correct size were amplified, and no product was seen when RT was omitted (data not shown). The products from primers amplifying the N-terminal (exon 1) of {alpha}-dystroglycan and the cytoplasmic region of ß-dystroglycan (within exon 2) were cloned and sequenced (Fig. 6Go; GenBank accession no. AF357215 and AF357216). Of the 222 bp of cDNA that gave the sequence for 74 amino acids at the N-terminus of {alpha}-dystroglycan, only 7 bp were different from the mouse sequence (GenBank accession no. X86073), and all of these represented codon variants of the same amino acids. Within these 222 nucleotides, 27 were different from the human sequence (GenBank accession no. L19711), accounting for 11 amino acid changes. These were the same amino acid variations seen between mouse and human. The amplified region for the ß-dystroglycan cytoplasmic tail using two overlapping primer pairs gave 327 bp, of which 12 bp were different from the mouse sequence (GenBank accession no. U43512), and these changes represented only codon variants of the same amino acids, with one exception, where the rat coded for methionine (as in the human) instead of valine as in the mouse. In this region there were 30 bp differences between rat and human. These results confirm that Fischer rat thyroid cells contain dystroglycan mRNA and that the protein sequences for the regions studied are virtually identical to those of mouse dystroglycan, whereas the nucleotide sequences for these regions vary by less than 4%.



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  		Figure 6. Comparison of rat dystroglycan cDNA sequences to human and mouse sequences. Boldface letters indicate those bases in which the rat differs from either the human or the mouse. The amino acids coded by these sequences are listed above each triplet. When human and mouse or rat amino acid sequences are different, the human is listed first, and the mouse second. Top, The N-terminal 222 bp of {alpha}-dystroglycan of rat (GenBank accession no. AF357215), compared with that of human and mouse published sequences (GenBank accession no. L19711 and X86073, respectively). Bottom, The C-terminal region of ß-dystroglycan (GenBank accession no. AF357216), whose 327 bp encode for the last 109 amino acids of the protein. The mouse sequence is that of GenBank accession no. U43512.

 
Assessment of ß-dystroglycan mRNA expression by RT-PCR was calculated as a ratio of dystroglycan mRNA to 18S ribosomal RNA, as displayed on a denaturing polyacrylamide gel. The results were expressed as the percent difference from the ratio of the no TSH group, which gave the highest expression of the three groups tested. These data (Table 1Go) show that ß-dystroglycan mRNA expression in chronic TSH was reduced to 57.2% of that in TSH-free medium (P < 0.05). After only 2 h in TSH, expression was reduced by 27% (P < 0.03) relative to that in cells cultured without TSH. These results show that the reduction in the amount of dystroglycan protein after TSH is due to decreased production and not solely to its redistribution.


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  		Table 1. Relative expression of FRTL-5 cell ß-dystroglycan (C-terminal) mRNA by Ambion QuantumRNA relative quantitative RT-PCR

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Dystroglycan, originally described in muscle (1, 2, 3), has been found to be widely expressed (4, 7, 8, 10, 11, 12). This is the first report of dystroglycan in thyroid and the first observation of its control by TSH. The results point to a role for dystroglycan in the thyroid, where it may participate in the formation of the follicular architecture. They may also provide further insight into the regulation and processing of dystroglycan. {alpha}-Dystroglycan and laminin binding were more abundant in membrane and cytosolic proteins from FRTL-5 cells cultured in the absence of TSH, whereas TSH stimulation facilitated the release of {alpha}-dystroglycan into the medium. Membrane-associated ß-dystroglycan was relatively unchanged by TSH. However, a lower molecular mass (~30 kDa) band recognized by the ß-dystroglycan antibody in the absence of TSH did, like {alpha}-dystroglycan, decrease with time in TSH. Relative quantitative RT-PCR for dystroglycan confirmed a comparable suppression of mRNA levels in TSH-treated cells.

Dystroglycan is transcribed as a single mRNA from a highly conserved gene. The translated protein is composed of an {alpha}-subunit, which, because of glycosylation, varies in size from 120 kDa in brain to 156 kDa in muscle or 190 kDa in Torpedo electroplax organ (21), and a ß-subunit, which migrates at 43 kDa in all species. These two moieties are noncovalently linked, but their coordinate expression has not been universally established. Evidence to the contrary exists from several investigators who found, as we did, that {alpha}- dystroglycan may be recovered in medium of cultured cell lines (19), suggesting that it may be actively secreted. Our results in FRTL-5 cells show that this process is increased by TSH and is correlated with a morphological phenotype that exhibits reduced laminin binding and little colocalization of {alpha}-dystroglycan with laminin in situ. Conversely, in the absence of TSH, {alpha}-dystroglycan colocalizes with laminin, membrane-bound {alpha}-dystroglycan is increased, and release into the medium is decreased. These results indicate that attachment to laminin is inversely correlated with release of {alpha}-dystroglycan into the medium, suggesting that the noncovalent bond between the {alpha}- and ß-subunits is more likely to dissociate if the {alpha}-subunit is not bound to a ligand.

ß-Dystroglycan, normally seen on immunoblots at 43 kDa, was also recognized as an approximately 30-kDa form in TSH-deprived cells. This truncated form is reduced in intensity by half within 4 h of the addition of TSH and is virtually undetectable in the chronic presence of TSH, in contrast to the 43-kDa form, which remains essentially unchanged. Several other researchers have noted a lower molecular mass band in the vicinity of 25–30 kDa (12, 20, 22, 23). This band has been ascribed to proteolysis (12). The recent report by Losasso et al. (23) supports this and demonstrates the truncated form in a variety of carcinoma cell lines. Unlike our results, they found that the presence of the truncated ß-subunit was correlated with the absence of the {alpha}-subunit. We found that the truncated form of ß-dystroglycan was expressed coordinately with the {alpha}-subunit, especially in the absence of TSH, where {alpha}-dystroglycan’s colocalization with laminin is most apparent and when there is reduced shedding of the {alpha}-subunit into the medium. This suggests that the conformation of dystroglycan while bound to laminin may in some way protect the natural cleavage site from dissociation, exposing instead the juxtamembrane portion of the extracellular domain of ß-dystroglycan to proteolytic cleavage. This would leave the 30-kDa portion of ß-dystroglycan attached to the cell membrane, releasing the entire {alpha}dystroglycan portion along with the extracellular remnant of ß-dystroglycan. Our data indicate that when firmly bound to laminin, as seen most prominently in the absence of TSH, the shedding of the {alpha}-subunit into the medium is inhibited. In the absence of laminin binding, {alpha}-dystroglycan is either constitutively secreted into the medium or passively dissociated from the full-length ß-dystroglycan during collection of the medium. All of these various morphological and biochemical changes may be pointing to a mechanism by which the cell uses its dystroglycan-laminin interface to spread and move over the substratum, perhaps in conjunction with other molecular species, such as integrins, that interact with the basement membrane and extracellular matrix. However, even without the elucidation of such a mechanism, the data from our system indicate that {alpha}-dystroglycan in the culture medium is inversely related to the amount of laminin binding, whereas the amount of truncated ß-dystroglycan recoverable is directly proportional to laminin binding.

On the basis of our data it cannot be said with certainty that TSH is exerting a direct effect on dystroglycan. Although there is responsiveness of the dystroglycan gene to TSH, we do not know whether this is a direct effect of TSH signal transduction pathways on the dystroglycan gene or whether this is secondary to the morphological changes that are induced by TSH acting through other pathways. Dystroglycan is one link in a chain of proteins from actin to laminin. Therefore, it is possible that its abundance and location can be influenced by any event in the cell that affects one or more of these components. For example, in muscle the absence of intracellular dystrophin in Duchenne’s muscular dystrophy results in the down-regulation of dystroglycan expression (1). In our system FRTL-5 cell morphology is drastically reorganized by TSH. Whether dystroglycan contributes to this reorganization or is a passive element in the altered cell architecture, reacting to the changes in the cytoskeleton, cannot yet be answered definitively.

The pivotal role emerging for dystroglycan is based on its extremely high affinity for laminin, which is 1000-fold greater than that of the laminin receptors of the integrin family (8), and its affinity for proteoglycans such as agrin and perlecan (24, 25, 26, 27). We have demonstrated that dystroglycan is distributed basolaterally in intact rat thyroid and colocalizes in part with laminin at the basal surface of the follicular cells. In membrane extracts of FRTL-5 cells, dystroglycan and laminin binding are sensitive to TSH, suggesting that dystroglycan and its interaction with laminin may play a role in the formation or maintenance of the thyroid follicle. Other studies have shown that laminin or merosin, a variant form of laminin, is synthesized by primary cultures of thyroid cells and that a form of basal lamina is present at the basal surface of follicular structures when thyroid cells are cultured under appropriate conditions (28, 29, 30, 31, 32, 33). FRTL-5 cells are also reported to be capable of synthesizing proteins of the extracellular matrix that are focally organized at structures appearing as basement membranes (34). Proteoglycans have been described in porcine (35) and human thyroid (36, 37), and perlecan, in particular, has been reported to be expressed in human thyroid tissue (33). Whether the dystroglycan-binding proteoglycans perlecan and agrin also play a role with dystroglycan in the thyroid remains to be determined.

We have provided evidence that dystroglycan is present in rat thyroid tissue and in the rat thyroid cell line FRTL-5, where it is responsive to TSH. The control of the amount and nature of dystroglycan by TSH is evidence of a potentially important physiological role. Thyroid cell lines may offer a system to study the nature and significance of dystroglycan-laminin interactions in the thyroid, providing clues to its function in epithelial cells in general.


   Acknowledgments
 
We thank Dr. Neil Smalheiser (University of Illinois, Institute for Psychiatric Research, Chicago, IL) for the 6C1 and 1A5 antibodies, for help in developing the laminin binding assays, and for the laminin affinity beads. We are also grateful to Dr. Mei Ling Chen (Research Resources Center, University of Illinois, Chicago, IL) for assistance with confocal microscopy.


   Footnotes
 
1 This work was supported by NCI Grant CA 21518 (to A.B.S.). Portions of these results were presented at the 79th and 80th Annual Meetings of The Endocrine Society, 1997 and 1998. Back

Received January 26, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ervasti JM, Ohlendieck K, Kahl SD, Gaver MG, Campbell KP 1990 Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. Nature 345:315–319[CrossRef][Medline]
  2. Ibraghimov-Beskrovnaya O, Ervasti JM, Leveille CJ, Slaughter CA, Sernett SW, Campbell KP 1992 Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature 355:696–702[CrossRef][Medline]
  3. Durbeej M, Henry MD, Campbell KP 1998 Dystroglycan in development and disease. Curr Opin Cell Biol 10:594–601[CrossRef][Medline]
  4. Ibraghimov-Beskrovnaya O, Mialtovich A, Ozcelik T, Yang B, Koepnick K, Francke U, Campbell KP 1993 Human dystroglycan: skeletal muscle cDNA, genomic structure, origin of tissue specific isoforms and chromosomal localization. Hum Mol Genet 2:1651–1657[Abstract/Free Full Text]
  5. Ohlendieck K 1996 Towards an understanding of the dystrophin-glycoprotein complex: linkage betwen the extracellular matrix and the membrane cytoskeleton in muscle fibers. Eur J Cell Biol 69:1–10[Medline]
  6. Henry MD, Campbell KP 1999 Dystroglycan inside and out. Curr Opin Cell Biol 11:602–607[CrossRef][Medline]
  7. Smalheiser NR, Schwartz NB 1987 Cranin: a laminin-binding protein of cell membranes. Proc Natl Acad Sci USA 84:6457–6461[Abstract/Free Full Text]
  8. Gee SH, Blacker RW, Douville PJ, Provost PR, Yurchenco PD, Carbonetto S 1993 Laminin-binding protein 120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan, and binds with high affinity to the major heparin binding domain of laminin. J Biol Chem 268:14972–14980[Abstract/Free Full Text]
  9. Gorecki DC, Derry JMJ, Barnard EA 1994 Dystroglycan: brain localization and chromosome mapping in the mouse. Hum Mol Genet 3:1589–1597[Abstract/Free Full Text]
  10. Yamada H, Shimizu T, Tanaka T, Campbell KP, Matsumura K 1994 Dystroglycan is a binding protein of laminin and merosin in peripheral nerve. FEBS Lett 352:49–53[CrossRef][Medline]
  11. Durbeej M, Larsson E, Ibraghimov-Beskrovnaya O, Roberds S, Campbell KP, Ekblom P 1995 Non-muscle {alpha}-dystroglycan is involved in epithelial development. J Cell Biol 130:79–91[Abstract/Free Full Text]
  12. Durbeej M, Henry MD, Ferletta M, Campbell KP, Ekblom P 1998 Distribution of dystroglycan in normal adult mouse tissues. J Histochem Cytochem 46:449–457[Abstract/Free Full Text]
  13. James M, Nguyen thi Man, Wise CJ, Jones GE, Morris GE 1996 Utrophin-dystroglycan complex in membranes of adherent cultured cells. Cell Motil Cytoskel 33:163–174[CrossRef][Medline]
  14. Williamson RA, Henry MD, Daniels KJ, Hrstka RF, Lee JC, Sunada Y, Ibraghimov-Beskrovnaya O 1997 Dystroglycan is essential for early embryonic development: disruption of Reichert’s membrane in Dag1-null mice. Hum Mol Genet 6:831–841[Abstract/Free Full Text]
  15. Henry MD, Campbell KP 1998 A role for dystroglycan in basement membrane assembly. Cell 95:859–870[CrossRef][Medline]
  16. Ambesi-Impiombato FS, Parks LAM, Coon HG 1980 Culture of hormone-dependent functional epithelial cells from rat thyroids. Proc Natl Acad Sci USA 77:3455–3459[Abstract/Free Full Text]
  17. Smalheiser NR, Kim E 1995 Purification of cranin, a laminin binding membrane protein. J Biol Chem 270:15425–15433[Abstract/Free Full Text]
  18. Bacallao R, Kiai K, Jesaitis L 1995 Guiding principles of specimen preservation for confocal fluorescence microscopy. In: Pawley JB (ed) Handbook of Biological Confocal Microscopy. 2nd ed. Plenum Press, New York, pp 311–325
  19. Matsumura K, Chiba A, Yamada H, Fukuta-Ohi H, Fujita S, Endo T, Kobata A, Anderson LVB, Kanazawa I, Campbell KP 1997 A role of dystroglycan in schwannoma cell adhesion to laminin. J Biol Chem 272:13904–13910[Abstract/Free Full Text]
  20. Ervasti JM, Campbell KP 1991 Membrane organization of the dystrophin-glycoprotein complex. Cell 66:1121–1131[CrossRef][Medline]
  21. Henry MD, Campbell KP 1996 Dystroglycan: an extracellular matrix receptor linked to the cytoskeleton. Curr Opin Cell Biol 8:625–631[CrossRef][Medline]
  22. Mummery R, Sessay A, Lai FA, Beesley PW 1996 ß-Dystroglycan: subcellular localisation in rat brain and detection of a novel immunologically related, postsynaptic density-enriched protein. J Neurochem 66:2455–2459[Medline]
  23. Losasso C, Di Tomasso F, Sgambato A, Ardito R, Cittadini A, Giardina B, Petrucci TC, Brancaccio A 2000 Anomalous dystroglycan in carcinoma cell lines. FEBS Lett 484:194–198[CrossRef][Medline]
  24. Gee SH, Montanaro F, Lindenbaum MH, Carbonetto S 1994 Dystroglycan-{alpha}, a dystrophin-associated glycoprotein, is a functional agrin receptor. Cell 77:675–686[CrossRef][Medline]
  25. Yamada H, Denzer AJ, Hori H, Tanaka T, Anderson LVB, Fujita S, Fukuta-Ohi H, Shimizu T, Ruegg MA, Matsumura K 1996 Dystroglycan is a dual receptor for agrin and laminin-2 in Schwann cell membrane. J Biol Chem 271:23418–23423[Abstract/Free Full Text]
  26. Gesemann M, Brancaccio A, Schumaker B, Ruegg MA 1998 Agrin is a high-affinity binding protein of dystroglycan in non-muscle tissue. J Biol Chem 273:600–605[Abstract/Free Full Text]
  27. Peng HB, Ali AA, Daggett DF, Rauvala H, Hassell JR, Smalheiser NR 1998 The relationship between perlecan and dystroglycan and its implication in the formation of the neuromuscular junction. Cell Adhes Commun 5:475–489[Medline]
  28. Garbi C, Wollman SH 1982 Basal lamina formation on thyroid epithelia in separated follicles in suspension culture. J Cell Biol 94:489–492[Abstract/Free Full Text]
  29. Wadeleux P, Nusgens B, Foidart J, Lapiere C, Winand R 1985 Synthesis of basement membrane components by differentiated thyroid cells. Biochim Biophys Acta 846:257–264[Medline]
  30. Andre F, Fillipi P, Feracci H 1994 Merosin is synthesized by thyroid cells in primary culture irrespective of cellular organization. J Cell Sci 107:183–193[Abstract]
  31. Fröhlich E, Wahl R, Reutter K 1995 Basal lamina formation by porcine thyroid cells grown in collagen- and laminin-deficient medium. Histochem J 27:602–608[Medline]
  32. Yap AS, Stevenson BR, Keast JR, Manley SW 1995 Cadherin-mediated adhesion and apical membrane assembly define distinct steps during thyroid epithelial polarization and lumen formation. Endocrinology 136:4672–4680[Abstract]
  33. Bürgi-Saville ME, Gerber H, Peter H-J, Paulsson M, Aeschlimann D, Glaser C, Kaempf J, Ruchti C, Sidiropoulos I, Bürgi U 1997 Expression patterns of extracellular matrix components in native and cultured normal human thyroid tissue and in human toxic adenoma tissue. Thyroid 7:347–356[Medline]
  34. Garbi C, Zurzolo C, Bifulco M, Nitsch L 1988 Synthesis of extracellular matrix glycoproteins by a differentiated thyroid epithelial cell line. J Cell Physiol 135:39–46[CrossRef][Medline]
  35. Shishiba Y, Yanagishita M 1983 Presence of heparan sulfate proteoglycan in thyroid tissue. Endocrinol Jpn 30:637–641[Medline]
  36. Shishiba Y, Yanagishita M, Tanaka T, Ozawa T, Kadowaki N 1984 Abnormal accumulation of proteoglycan in human thyroid adenocarcinoma tissue. Endocrinol Jpn 31:501–507[Medline]
  37. Katoh R, Muramatsu A, Kawaoi A, Komiyama A, Suzuki A, Hemmi A, Katayama S 1993 Alteration of the basement membrane in human thyroid diseases: an immunohistochemical study of type IV collagen, laminin and heparan sulphate proteoglycan. Virchows Arch A Pathol Anat 423:417–424[CrossRef]



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