Molecularand Cellular Endocrinologv 76 (1991) 135-148 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50

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MOLCEL 02468

Intermediate filaments in normal thyrocytes: modulation of vimentin expression in primary cultures Joelle Coclet, Franpise

Lamy, Fabienne Rickaert, Jacques E. Dumont and Pierre P. Roger

Instituteof InterdisciplinaryResearch Schal of Medicine, Free University of Brussels,B-1070 Brussels,Belgium (Received 15 October 1990; accepted 19 December1990)

Key woruk Thyroid; Vimentin; Cytokeratin; Morphology; Thyrotropin; Epidermalgrowth factor; (Dog)

In dog thyrocyte primary cultures, the antagonistic effects of thyrotropin (TSH) and epidermal growth factor (EGF) on differentiation expression were accompagnied by distinct long-term morphological changes: TSH-treated cells showed an epitheloid morphology; EGF reversibly induced a fusiform shape. Using indirect immunofluorescence microscopy and two-dimensional gel electrophoresis, we studied the modifications in the distribution and synthesis of the intermediate filament proteins of the cytoskeleton in response to TSH and EGF. These factors had little effect on the expressionof cytokeratins 8 and 18, which were expressed in 98% of cells. However, ‘I’SH induced a profound redistribution of cytokeratins (and actin) with the appearance of a marked staining of cell junctions. Vimentin was coexpressed with cytokeratins in about 40% of Cells from normal thyroid follicles freshly isolated by collagenase. During culture, immunostained vimentin network progressively developed in 90%of control and EGF-treated cells simultaneously with vimentin synthesis. In contrast, only 20%of TSH-treated cells reacted with vimentin antibody and we observed a marked decreasein vimentin synthesis in response to TSH. Therefore, vimentin synthesis, which should occur in at least some normal thyroid follicles in vivo, was inhibited in vitro by TSH which promotes differentiation expression. However, EGF-treated cells thereafter cultured with TSH regained an epitheloid morphology and differentiation in spite of the persistency of a complete network of vimentin.

Introduction Cell growth and differentiation are particularly dependent on cell shape, direct cell-cell interaction, and mechanical as well as chemical proper-

Address for correspondence: Pierre Roger, Institute of Interdisciplinary Research, School of Medicine, Free University of Brussels, Campus Erasme, route de Lennik 808, B-1070 Brussels, Belgium.

ties of cell substratum (Ben Ze’ev, 1987; Watt et al., 1988; Ingber and Folkman, 1989). Gene regulation by environmental conditions that affect cellular organization could occur in part through the cytoskeleton which provides a protein continuum between cell membrane and nucleus (Bissell and Barcellos-Hof,1987).Extracellular matrix proteins such as fibronectin and laminin bind to transmembrane receptors (integrins) that are coupled with the cytoskeleton via talin and vinculin (Carraway and Carothers-Carraway, 1989). Cyto-

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skeletal association with polyribosomes affects mRNA stability and rates of protein synthesis (Farmer et al., 1983), while its interaction with the nuclear matrix could affect mRNA processing and, possibly, rates of transcription (Bissel and Barcellos-Hof, 1987). On the other hand, cell contacts and morphogenesis are largely determined by the organ~tion and composition of the cytoskeletal networks, which are themselves dependent on changes in cell configuration. Thus, there is a 'dynamic reciprocity' (Bissel and Barcellos-Hof, 1987; Ben Ze'ev, 1989) where modifications of cytoskeleton influence cell morphology, which in turn, via the cytoskeleton~ could regulate the expression of genes related to growth, morphogenesis and differentiation. The cytoskeleton in higher eukaryotic cells is composed of three major classes of protein polymer networks: microtubules, actin micro;ilaments and intermediate filaments. Intermediate filaments comprise a diverse and heterogeneous family of subunit proteins which are expressed in tissuespecific and developmentally regulated patterns (Steinert, 1988). Among them, the various cytokeratins specify the different epithelial cell types and their filaments determine the mechanical strength of epithelia by their interaction with the desmosomal plaque proteins in intercellular junctions. Vimentin is the only intermediate filament protein expressed in mesenchymal cells. It interacts with lamin B of the nuclear lamina through the nuclear pores (Oeorgatos and Blobel, 1987). It has even been proposed that vimentin and the other intermediate filament protein subtypes could each regulate a gene set that may be different in different cell types (Chan et al., 1989). The first descriptions of coexpression of vimentin and cytokeratins in epithelial cells were from cancer cells or from in vitro cultured cells, and were interpreted as indicating their dedifferentiation (Lazarides, 1982; Ramaekers et al., 1983; Domagala et al., 1989). According to Miettinen et al. (1984), the coexpression of vimentin and cytokeratins can be used as a marker of aggress~'ee thyroid carcinomas. Greenburg and Hay (1988) reported that a striking transformation of rat thyrocytes into fibroblast-like cells is associated with a progressive replacement of cytokeratins by a vimentin net-

work. This phenomenon presents analogies with the shift from cytokeratin to vimentin expression in mesothelial cells changing from a cuboidal to a fusiform shape during rapid growth in culture (Conneil and Rheinwald, 1983) or in mammary epithelial cells that spontaneously give rise to fibroblast-like cells (Dulbecxo et al., 1981). Profound changes in morphology are also observed in canine thyrocytes in primary culture in association with changes in differentiation expression (Roger and Dumont, 1984; Roger et al., 1985; Pohl et al., 1990). Thus, canine thyrocytes that proliferate in response to thyrotropin (TSH) acting through cAMP adopt an epithelial cuboidal morphology and express thyroid differentiation at high levels, while epidermal growth factor (EGF) inhibits the expression of differentiation and induces these cells to grow with a fusiform, fibroblast-like pattern. In the present study, we characterize the changes in intermediate filament protein synthesis and distribution which underline the reversible and hormone-dependent changes in morpholosy and differentiation expression that occur during the culture of canine thyrocytes. Material and methods

Primary cultures of dog thyroidfollicular cells The cells were obtained from dog thyroid as detailed previously (Roger et al., 1982). Briefly, the tissue was digested by collagenase (type I, 150 U/ml; Worthington Biochemical Corp., Freehold, N J, U.S.A.) so that the resulting suspension consisted mainly of fragmented as well as intact follicles. These follicles were seeded ( + 2 × 104 ceUs/cm2) in 35 mm tissue culture-treated Petri dishes; the attached follicles developed in 1-2 days as a monolayer. The cells were cultured in the following mixture that constituted the control medium (Roger and Dumont, 1984; Roger et al., 1987): Dulbecco's modified Eagle's medium (DMEM) + Ham's F12 medium (Flow Laboratories, Irvine, U.K.)+ MCDB 104 medium (Gibco Laboratories, Paisley, U.K.) (2:1 : 1, v/v/v) supplemented with 2 mM glutamine (Flow Laboratories), ascorbic acid (40/~g/ml), insulin (5/~g/ml) (Sigma Chemical Co., St. Louis, MO, U.S.A.), 1% fetal bovine serum (Gibco Laboratories), antibio-

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tics (100 U penicillin/nd, 100/tg streptomycin/ml and 2.5 pg amphotericin B/ml, Flow Laboratories). The cells were kept in a water-saturated incubator at 37°C in an atmosphere of 5% CO2 in air. The medium was renewed every other day. After one day of culture (day 1), bovine TSH (Armour Pharmaceutical Co., Chicago, IL, U.S.A.) or routine EGF (Sigma Chemical Co.) were added to some dishes and their presence maintained throughout the culture period.

Indirect immunofluorescence staining The following antibodies and dilutions were used: monoclonal anti-actin (1/250; Amersham, U.K.), monoclonal anti-vimentin (1/5; RPN 1102, .4anersham), monoclonal antibodies against specific simple epithelia cytokeratins (8 and 18; RPN 1166 and RPN 1160 respectively, Amersham), polyclonal broad-spectrum rabbit anti-cytokeratin antibody (1/100; Dakopatts Z622), fluoresceinconjugated sheep anti-mouse immunoglobulin G (1/50; Boehringer, F.R.G.), Texas Red-conjugated swine anti-rabbit immunoglobulins (1/100; Dakopatts). Cells in the Petri dishes were fixed in methanol for 7 rain at -20°C, ri~lsed in TBS (tris(hydroxymethyl)aminomethane 10 raM, NaCI 155 raM, pH 8.2) and permeabilized with Triton X-100 (0.15%), 10 rain, at room temperature. Subsequent rinses were followed by addition, for 30 rain, of normal sheep serum (1/20 in TBS containing 0.1 ~ bovine serum albumin) and the cells were incubated with the diluted primary antibody overnight at 4°C. Then cells were washed and incubated with the fluorochrome conjugated secondary antibody for 1 h. After further thorough washings, cells were mounted in Gelvatol (polyvinylalcohol, Monsanto) containing 100 mg/ml 1,4-diazobicyclo[2,2,2]octane in order to delay the fluorescence fading. Negative controls were replacement of the primary antibody by either anti-NSE (neuron-specific enolase) or by nz~mal serum. Cells were viewed on a Zeiss epifluorescence microscope and were photographed with 400 iso Fuji films. For double vimentin/cytokeratin immunofluorescence labeling, mouse monoclonal antivimentin and rabbit polyclonal anti-cytokeratins were simultaneously applied and revealed using the combination of fluorescent antibodies against

mouse and rabbit immunoglobulins. Positive control of double vimentin/keratin labeling was done on HeLa cells.

Intermediate filament extraction and two.dimensional (21)) gel electrophoresis [35S]Methionine (50 pCi/ml) was added during the last 12 h of the cell culture. At the end of the culture, the cells were rapidly rinsed at room temperature in 0.9~o NaCI. A Triton X-100 and high salt-insoluble fraction was prepared according to Ben-Ze'ev et al. (1986) by scraping and vigorously mixing the cells in a buffer containing 0.6 M KCI, 0.5~ Triton X-100, 14 mM mercaptoethanol, 2.5 mM EGTA, 5 mM MgCI 2, 1 mM PMSF (phenylmethyl-sulfonyl fluoride) and 10 mM Hepes (N2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid) pH 7.4. The intermediate filament-rich fraction was sedimented by centrifugation in an Eppendorf centrifuge for 3-5 rain at 4°C, solubilized in O'Farrel's lysis buffer (1975) and analyzed by 2D gel electrophoresis as described previously (Lamy et al., 1986). Proteins were separated by isoelectric focusing on cylindrical gels in the first dimension with 3.2~ Servalytes pH 5-7 plus 0.8~ Servalytes pH 2-11 and according to molecular mass on sodium dodecyl sulfate, linear gradient (6-165) polyacrylamide slab gels in the second dimension. Fluorography was carried out with the autoradiographic enhancer Enhance, NEN. Gels were dried and exposed to Amersham hyper film-MP. They were kept at - 8 0 ° C during the required exposure time. Results from indirect immunofluorescence and electrophoretic analysis of intermediate filaments were reproduced in at least five independent primary cultures in duplicate dishes. Blotting and immunodetection of proteins After separation by 2D gel electrophoresis, proteins were transferred to a nitrocellulose membrane pH 79 (Schleicher & Schuel, Dassel, F.R.G.) for 16 h at 60 V and 4°C. Blotting was performed in a Trans Blot cell as described in the operating instructions from Bio-Rad (Richmond, CA, U.S.A.). Mouse monoclonal anti-vimentin (IgG) antibody (RPN 1102, Amersham, U.K.) or anticytokeratin 8 antibody (RPN 1166, Amersham~

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were used at a 1/5 dilution. 125I-labeled antimouse immunoglobulin (Amersham, U.K.) was used as a second antibody for development by autoradiography. Autoradiography before immunoreaction was done by direct contact of the nitrocellulose sheet with Amersham hyper film~max, which allows detection of ass-labeled proreins. After immunoreaction, an exposed and processed film was placed between the nitrocellulose sheet and an Amersham hyper film-MP in order to prevent the formation of spots by 35Slabeled proteins but also to allow the recording of

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spots due to 125I-anti-mouse immunoglobulin complexes. An intensifying screen (Siemens Special) further increased the intensity of the spots due to 125i. Results

Morphology of dog thyrocytes A continuous treatment of dog thyrocytes in primary culture with TSH or E G F profoundly affects their morphology. As previously shown (Roger and Dumont, 1984), while cells cultured

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Fig. 1. Morphology of dog thyroid cells. Phase contrast microscopy of living cells (× 100). Characteristic fields with confluent monolayer are shown. (A) Control cells cultured for 9 days. (B) Cells cultured for 9 days in the continuous presence of TSH (1 mU/ml) added at day 1. (C) Cells cultured for 9 days in the continuous presence of EGF (25 ng/ml) added at day 1. (D) The cells were cultured for 6 days in the pre~ence of EGF. After elimination of EGF, they were then cultured with TSH (1 mU/ml) until day 9.

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for 9 days in control conditions displayed a very spread, moderately elongated morphology (Fig. 1A), cells continuously treated with TSH presented a characteristic cuboidal epitheloid morphology and occasionally reached a very high local

density (Fig. 1B). EGF-treated cells, though also reaching a quite high density, were characterized by a quite different morphology: cells progressively became fusiform, very elongated, in fact fibrobl~t-like (Fig. 1C). This morphological effect

Fig. 2. Anti-actin indirect immunofluorescence of dog thyroid cells (× 600). (A) Control cells cultured for 9 days presenting a very abundant actin stress fiber network. (B) Cells cultured for 9 days with EGF (25 ng/ml) added at day 1. (C) Cells cultured for 9 days with TSH (1 mU/ml) added at day 1. Notice the absence of actin stress fiber, the punctiform staining and the labeling of intercellular limits in TSH-treated cells.

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Organization of actin microfilaments The long-term effects of TSH and EGF on actin microfilarnent distribution are shown in Fig. 2. Indirect immunofluorescent labeling of actin showed that the very spread unstimulated cells contained a very abundant network of microfilament bundles (stress fibers), which spanned the whole cell length (Fig. 2A). A similar organization of actin was observed in the fusiform EGF-treated cells (Fig. 2B). By contrast, cells continuously treated with TSH displayed a diffuse cytoplasmic anti-actin immunoreactivity with fine granulations. Very few stress fibers were seen, but the cell limits were stained (Fig. 2C).

Distribution of cytokeratin intermediate filaments

Fig. 3. Anti-cytokeratin 8 indirect immunofluorescence of dog thyroid cells ( x 600). (A) Control cells cultured for 9 days. (B) Cells cultured for 9 days with TSH (1 mU/mi) added at day 1. Notice the redistribution of keratin filaments and the high labeling of intercellular limits in TSH-treated cells.

of EGF was reversible. After eliminatiol~ of EGF and addition of TSH, the cells regained the characteristic cuboidal shape (Fig. 1D).

Dog thyrocytes in primary culture as human thyroid follicular cells presented a marked reactivity for monoclonal antibodies directed against keratins 8 (type I basic) (Fig. 3) and 18 (type II acidic) (not shown). These keratins are specific of simple epithelia (Moll et al., 1982). Their expression was maintained in vitro, even in the fusiform 'fibroblast-like' dog thyrocytes treated with EGF (not shown). The cytokeratin networks revealed either with keratin 8, 18 monoclonal antibodies, or with a wide-spectrum keratin polyclonal antibody were undistinguishable (not shown). Unstimulated (control) cells presented a very filamentous cytokeratin network (Fig. 3A), while in EGF-treated cells this network presented a more diffuse cytoplasmic distribution (not shown). As shown in Fig. 3B, TSH-treated cells were characterized by a completely different pattern of the cytokeratin network. Keratin fibers radiated from a nuclear location toward the cell periphery. A bright, continuous labeling of the cell limits was observed in TSH-treated cells (Fig. 3B), but not in control (Fig. 3A) or in EGF-treated cells (not

Fig. 4. Double-immunofluorescence labeling of cytokeratins (A, C, E, and G) and vimentin (B, D, F, and H) in cultured dog thyroid cells (× 200). Exactly the same microscopical fields were viewed for red fluorescence (cytokeratins; A, C, E, and G) and green fuorescence (vimentin; B, D, F, and H). Pictures were from the experiment shown in Fig. 1. (A and B) Nine-day-old control cells. (C and D) Cells continuously treated with EGF (25 ng/ml) until day 9. (E and F) Cells continuously treated with TSH (1 mU/mi) until day 9. (G and H) Cells cultured with EGF for 6 days and then treated for 3 days with TSH (1 mU/ml) after elimination of EGF. Notice the coexpression of cytokeratin and vimentin in the majority of cells in all the situations, except in cells continuously stimulated with TSH (E and F), which lacked vimentin immunoreactivity. The field in E and F was selected to show the comparison with one contaminating fibroblast, which was positive for vimentin, but negative for cytokeratin immunostaining. Arrows in d and B indicate that a minority of cytokeratin-positive control cells lacked vimentin immunoreactivity.

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Coexpression of cytokeratins and vimentin in cultured thyrocytes Dog thyrocytes cultured for 9 days in the presence of either TSH or EGF were compared to control unstimulated cells. The intermediate filaments were doubly labeled using a vimentin monoclonal antibody revealed with a fluoresceinconjugated antibody to mouse immunoglobulins and ~, rabbit antiserum to keratin which was revealed with a secondary Texas Red-conjugated antibody. The morphology of these cells had been viewed by phase-contrast microscopy and is shown in Fig, 1. Fig. 4 shows that control (Fig. 4A and B) and EGF-treated cells (Fig. 4C and D) conrained both a keratin and a vimentin network. While more than 98% of cells expressed cytokeratins, about 90~ of cells coexpressed vimentin. Thus, a small proportion of keratin-positive cells did not express a vimcntin network (indicated by arrowheads on Fig. 4A and B). These cells were dispersed among the cell monolayer and were not distinguished by any particular morphology. By contrast, the majority (80~ in the experiment shown) of keratin-positive TSH-treated cells did not contain vimentin (Fig. 4F). When found, the coexpression of vimentin and keratin was more frequent in the most spread cells of TSH-treated cultures (not shown). Very rare (less than 1~) spindle-shape fibroblast-like cells were positive for vimehtin but negative for keratin immunostaining (Fig. 4F). The cuboidal shape that c~aracterized TSHtreated cells was restored by a 3-day stimulation with TSH of cells which had been cultured for 6 days with EGF (Fig. 1D). This morphological effect of TSH occurred despite the persistence of a vimentin network in the EGF- and then TSHtreated cells (Fig. 4H). The intercellular heterogeneity of keratin immunoreactivity was particularly apparent after this latter cell treatment (Fig. 4G). It was not correlated to the vimentin immunoreactivity (Fig. 4H). The coexpression of cytokeratins and vimentin in cultured thyrocytes was confirmed by the analysis by 2D gel electrophoresis of an intermediate

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Fig. 5. Demonstration of vimentin in primary cultares of dog thyroid cells by 2D gel electrophoresis and immunoblotting. Dog thyrocytes were cultured for 9 days in the control medium containing 1~ serum and labeled with [35S]methionine for the last 12 h of the culture. The Triton X-100-high salt-insoluble fraction which is enriched in intermediate filaments was separated by 2D gel electrophoresis and transferred to a nitrocellulose membrane as described in Material and Methods. (A) Autoradiograph o f the blot before immunodetection. (B) Autoradio~,aph of ~hc ~ m e blot after specific immunoreaction against vimentin. On the left part of the autoradiograph, delimited by a dotted line, only 1251radiation was recorded by using a screen that stops 35S radiation as described in Material and Methods. Localization of vimentin and vimentin degradation products on autoradiograph A was done by superposition of autoradiograph B. a, residual actin; v, vimentin; the arrowheads indicate the position of the major degradation products of vimentin.

filament (IF)-enriched fraction extracted from these cells. Fig. 5 shows the immunodetection of vimentin in the IF-enriched fraction isolated from [35S]methionine-labeled control thyrocytes after 9 days of culture and separated by 2D gel electrophoresis. The monoclonal antibody used also recognized the characteristic degradation products of vimentin that form a 'staircase' pattern toward the more acidic part of the gel (Ben-Ze'ev et al., 1986). A parallel but less intense 'staircase' pattern of vimentin degradation products was also revealed by this anti-vimentin antibody. Ben Ze'ev et al. (1986) have suggested that this second set of vimentin degradation products were obtained from a more basic cleavage product of vimentin with a slightly lower Mr. Keratin 8 was similarly demonstrated by the immunoblotting procedure (not shown). Keratins 18 and 19 were tentatively identified by their characteristic electrophoretic motilities (Fig. 6,4) according to Moll's classification of human keratins (Moll et al., 1982).

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Fig. 6. Differential regulation of vimentin synthesis in dog thyroid cells in response to TSH and EGF. Dog thyrocytes were cultured for 9 days in the presence of 1~ serum. (A) Control, (B) with TSH (1 mU/ml), (C) with EGF (25 ng/ml), (D) with EGF (25 ng/ml) for 6 days and then with TSH alone (1 mU/ml) for 3 days. Twelve hours before the end of the culture 50 /tCi/ml [35S]methionine was added to the medium. The IF-enriched fractions were isolated from samples containing equal amounts of total trichloroacetic acid-precipitable [35S]methionine counts and separated by 2D-gel electrophoresis as described in Material and Methods. Autoradiographs of the 2D gels are shown, a, residual actin; v, vimentin; the arrowheads indicate the position of the major degradation products of vimenti=~,; 8, 18, 19, cytokeratins according to the nomenclature of Moil. This figure illustrates the same experiment as Figs. 1 and 4.

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Fig. 7. Influence of TSH and EGF on the vimentin content of dog thyroid cells evaluated by silver staining. Dog thyrocytes were cultured for 9 days in the presence of 1~ serum, (A) Control, (B) with TSH (1 mU/ml), (C) with EGF (25 ng/ml), (D) with EGF (25 ng/ml) for 6 days and then with TSH alone (1 mU/ml) for 3 days. The IF-enriched fractions were isolated from samples containing equal amounts of total cell proteins and separated by 2D-gel electrophoresis as described in Material and Methods. a; residual actin; v, vimentin; the arrows indicate the position of major degradation products of vimentin; 8, 18, 19, cytokeratins according to the nomenclature of Moll. This figure illustrates the same experiment as Figs. 1, 4, and 6.

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Fig. 6 shows that a continuous treatment of dog thyrocytes with TSH for 9 days had strongly repressed the synthesis of vimentin (Fig. 6B) while a similar treatment of the ce~Js for 9 days with E G F had no obvious effect on the synthesis of this protein (Fig. 6C). A 3-day treatment with TSH of cells previously cultured for 6 days with EGF had a weaker inhibitory effect on vimentin synthesis

(Fig. 6D) than the one observed in cells continuously stimulated with TSH. Fig. ?B shows that TSH-treated thyrocytes had only a very low content of vimentin, as compared to control thyrocytes (Fig. 7,4). This certainly results from the potent repression of vLmentin synthesis by this hormone. By contrast, cells treated with E G F for 6 days and then with TSH

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C Fig. 8. Coexpressionof cytokeratins and vimentinin some follicles that were freshly isolated by collagenasedigestionof a dog thyroid gland. Double-immunofluorescencelabelingof cytokeratins(A) and vimentin(B) (x 200). (C) Viment~nidentified in freshly isolated follicles by silver staining of W-enriched fraction separated by 2D-gel electrophoresis, v, vimentin; 8, 18, 19, cytokeratins according to the nomenclatureof Moll.

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for 3 days (Fig. 7/)) had conserved the same content of vimentin as cells continuously treated with EGF for 9 days (Fig. 7C), despite the moderate inhibition of vimentin synthesis observed in the same conditions (Fig. 6D). "Ibis is probably due to the long half-life of vimentin (Connell and Rheinwald, 1983). Thus cells continuously cultured with TSH (9 days) (Fig. 7B) or treated with TSH for only 3 days after being cultured with EGF for 6 days (Fig. 7D) are distinguished by a very different vimentin content. No obvious or reproducible changes were observed for the synthesis or amount of cytokeratin 8 and the peptide tentatively identified as cytokeratin 18 while the synthesis and amount of the peptide whose migration in 2D gel corresponds to that of cytokeratin 19 seem to be decreased in TSHtreated cells (Figs. 6 and 7). The same results were observed when total cellular proteins instead of IF-enriched fractions were separated by 2D gel electrophoresis (data not shown). These data correlate well with the qualitative evaluation of vimentin and cytokeratins realized by indirect immunofluorescence microscopy (Fig. 4).

Coexpression of vimentin and cytokeratins in some freshly isolated thyroid follicles Coexpression of vimentin and cytokeratin intermediate filaments is still considered an exceptional feature of normal epithelial tissues in vivo, while it is frequently observed in epithelial cell cultures in vitro. We have thus assessed the presence of vimentin in thyroid follicles freshly released by collagenase digestion of the dog thyroid tissue. Surprisingly, double-labeling immunofluorescence experiments showed that while some follicles presented only a cytokeratin immunoreactivity, others were completely immunoreactive for both vimentin and cytokeratins (Fig. 8,4 and B). In some follicles, only few cells contained both vimentin and cytokeratins. The presence of vimentin in freshly isolated follicles was confirmed by 2D gel electrophoresis (Fig. 8C). In different experiments, the proportion of cells which coexpress vimentin and cytokeratin in freshly isolated follicles was ranging from 30 to 6070. This could not be due to a rapid neosynthesis of vimentin during follicle isolation since exactly the same figures were obtained when dissecting and

digesting the thyroid gland in the presence of cycloheximide (10 /~g/ml), which blocks protein synthesis by 9770 (not shown). The coexpression of vimentin and cytokeratin rapidly generalized during the course of the primary culture and the development of the cell monolayer. After day 4 of the primary culture in control conditions, only few cells (10-2070) remained negative for vimentin. They were dispersed in the cell monolayer, but preferentially localized in area with high local cell density (not shown). The generalization of the coexpression of vimentin and cytokeratins did not seem to be due to a selection of vimentin-cytokeratin-positive follicles (the follicles were put into culture with a high yield (7570)), nor to the in vitro proliferation since vimentin appeared even in unstimulated serum-free cultures (not shown) where the majority of cells are quiescent (Roger et a!., 1987). Discussion

Dog thyroid epithelial cells developing as a monolayer in primary culture expressed the simple epithelium cytokeratins 8, 18 and possibly 19 that are found in the human thyroid follicular cells in vivo (Dockhorn-Dworniczak et al., 1987; HenzenLockmans et al., 1987). In addition, they were found to express vimentin, the intermediate filament protein of mesenchymal cells which, as classically considered, can also be expressed in epithelial cells, but only in abnormal situations such as neoplastic transformation or in vitro cell culture (Lazarides, 1982; Steinert, 1988). Our immunological and electrophoretical demonstration of vimentin presence and synthesis in dog thyrocytes in primary culture contradicts a previous statement that dog thyrocytes only contained cytokeratin intermediate filaments (Nielsen et al., 1985). The coexpression of vimentin and cytokeratins in dog thyrocytes (which was also observed in monolayer cultures of bovine and human thyrocytes; unpublished results) was not purely a 'culture artefact' that could be ascribed to in vitro dedifferentiation: we also demonstrated it in about 40% of cells in dog thyroid follicles that were freshly isolated in conditions that prevented protein neosynthesis. This implies that many normal thyroid follicular cells should contain vimentin in

146

vivo and suggests an important heterogeneity in thyroid tissue with regard to the presence of this cytoskeleton network. Whether this could be related to the known intercellular and interfollicular heterogeneity of morphological and functional responses in thyroid gland (Dumont, 1971) is not known. Previous studies on the composition of intermediate filaments in thyroid gland were quite controversial. Although all the authors agree on the coexpression of vimentin and cytokeratins in various thyroid carcinomas (Miettinen et al., 1984; Buley, 1987; Dockhorn-Dworniczak et al., 1987; Henzen-Logmans et al., 1987), in normal human tissue Miettinen et al. found only cytokeratin immunoreactivities while the other grOUl:S reported the coexpression of vimentin and cytokeratin in a fraction of follicular cells ranging from 5% (Henzen-Logmans et al., 1987) to a great majority (Dockhorn-Dworniczak et al., 1987"L Also contrasting with our results, rat thyroid cells in primary culture on collagen gels were reported not to express vimentin (Greenburg and Hay, 1988). The reasons for these discrepancies are unclear, but the claim of vimentin absence was not supported by data from electrophoretic techniques. The presence of vimentin in normal thyroid follicular cells precludes its use as a marker of thyroid tumors. Recently, coexpression of vimentin and cytokeratins has also been reported in several normal epithelia in vivo (La Rocca and Rheinwald, 1984; Czernobilsky et al., 1985; Paranko et al., 1986; Kasper and Stosiek, 1989; Kasper et al., 1989). We observed a progressive generalization of the coexpression of vimentin and cytokeratins in unstimulated and EGF-treated dog thyroid primary cultures. This was also seen in cells that remain quiescent in the absence of serum (data not shown), indicating that vimentin synthesis was not related to in vitro growth stimulation as reported in other systems (Connell and Rheinwald, 1983; Ferrari et al., 1986). According to Ben-Ze'ev (1987), the appearance of vimentin could be related to the spreading of cells as a monolayer and inversely correlated with confluence density. Indeed, vimentin-negative cells were more frequently found in areas where cells are less spread due to higher density. However, at any time of the cul-

ture, a minority of well-spread cells that were dispersed in the cell monolayer, remained devoid of vimentin immunoreactivity. This suggests that a vimentin network is not necessary for spreading of a cytokeratin containing cell. In other epithelial cell systems, including rat thyroid follicular cells, a striking transformation into fusiform mesenchymal-like cells has been associated with cessation of cytokeratin expression and appearance of vimentin (Dulbecco et al., 1981; Connell and Rheinwald, 1983; Greenburg and Hay, 1988). This illustrated quite well the assumption that the expression of particular intermediate filament subtypes might be important for determination of a cell type-specific morphology and the execution of a normal cell differentiation program. We observed here that a continuous treatment of dog thyrocytes with TSH, which maintains a characteristic epithelial morphology and stimulates differentiation expression (Roger and Dumont, 1984; Roger et al., 1985; Pohl et al., 1990), also strongly repressed the synthesis and accumulation of vimentin in a majority of cells. However, TSH had no appreciable effect on the expression of cytokeratin 8 and 18, and EGF, which reversibly inhibits differentiation expression and induces a fusiform, fibroblast-like morphology, did not inhibit cytokeratin synthesis, nor did it further increase the vimentin synthesis, at least on a 12-day treatment. Therefore, the transition from the differentiation-expressing epitheloid cells treated with TSH to the apparently dedifferentiated fusiform EGF-treated cells does not represent a 'mesenchymal transformation' as reported by Greenburg and Hay (1988), but rather illustrates the high versatility of shape in epithelial cells. The importance of the potent inhibition of vimentin expression for the morphological and differentiation effects that TSH exerts via cAMP remains unclear. When cells cultured for 6 days with EGF were then stimulated with TSH for 3 days, a weaker inhibition of vimentin synthesis was achieved, which did not lead to a detectable decrease in the cell content of vimentin. In this case, the reinduction by TSH of the epithelial morphology and of differentiation expression in the whole cell population (Pohl et al., 1990) occurs despite the persistence of a complete vimentin network in the great majority of cells.

147

The long-term morphological changes induced by TSH are better correlated with dramatic redistributions of both microfilaments and cytokeratin intermediate filaments. The effect on actin filaments could be the consequence of the disruption of stress fibers that TSH acutely induces via protein ldnase A activation (Roger et al., 1988) and, likely, changes in myosin light chain phosphorylation (Ikeda et al., 1986), and of the inhibition of the synthesis of actin (Passareiro et al., 1985) and high molecular weight isoforms of tropomyosin (Roger et al., 1989). The mechanism by which the distribution of cytokeratin filaments might be modified by TSH is obscure. However, the redistribution of both cytokeratin and actin immunostaining in a continuous labeling of intercellular limits suggests that TSH induced the formation of junctional complexes, such as desmosomes, which interact with cytokeratins (reviewed by Carraways and Carothers-Carraways, 1989). This might profoundly affect the intercellular communication in these monolayer cultures. It is possible that the TSH-induced formation of these tight cell contacts might indirectly contribute to repress the synthesis of vimentin. To conclude, this study has shown that vimentin synthesis, which seems also to occur in normal thyroid epithelial cells in vivo, can be modulated by TSH, at least in in vitro cell cultures. Although vimentin has been shown to provide a protein continuum between cell membrane and nucleus, which was proposed to transmit signals affecting gene expression, we were unable to find a close correlation between vimentin presence and either a particular morphology or modifications of differentiation expression. Acknowledgments This work was made possible thanks to grants from the Ministrre de la Politique Scientifique (Science de la Vie), F.R.S.M. (Fonds de la Recherche Scientifique Mrdicale), the Cancer Research Funds of the Caisse Grnrrale d'Epargne et de Retraite, the 'Association contre le Cancer' and thanks to Euratom Contract B10-C-360-81-B. The authors thank Mrs C. Doudelet for excellent technical assistance. Pierre P. Roger is a senior research assistant of the National Fund for Scien-

tific Research (Belgium). They also thank Prof. J.J. Vanderhaegen and Mrs. M. Authelet (Laboratoire d'Anatomie Pathologique et de Microscopie Electronique, Free University of Brussels) for access to epifluorescence microscopes and for technical advise. References Ben-Ze'ev, A. (1987) J. Cell. Sci. Suppl. 8, 293-312. Ben-Ze'ev, A. (1989) in Cell Shape: Determinants, Regulation and Regulatory Role, pp. 95-119, Academic Press, New York. Bissel, M.J. and Barcellos-Hoff, M.M. (1987) J. Cell Sci. Suppl. 8, 327-343. Buley, I.D., Gatter, K.C., Heryet, A. and Mason, D.Y. (1987) J. Clin. Pathol. 40, 136-1a2. Carraway, K.L. and Carothers-earraway, C.A. (1989) Biochim. Biophys. Acta 988, 147-171. Chart, D., Goate, A. and Puck, T.T. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2747-2751. Connell, N.D. and Rheinwald, J.G. (1983) Cell 34, 245-253. Czernobilsky, B., Moll, R., Levy, R. and Franke, W.W. (1985) Eur. J. Cell Biol. 37, 175-190. Dockhorn-Dwomiczak, B., Franke~ W.W., Schr~kler, S., Czernobilsky, B., Gould, V.E. and Bt~cker, W. (1987) Differentiation 35, 53-71. Domagala, W., Lasota, J., Chosia, M., Szadowska, A., Weber, K. and Osborn, M. (1989) Cancer 63, 504-517. Dulbecco, R., Henahan, M., Bowman, M., Okada, S., Battifora, H. and Unger, M. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2345-2349. Dumont, J.E. (1971) Vitam. Horm. 29, 287. Farmer, S.R., Wan, K.M., Ben-Ze'ev, A. and Penman, S. (1983) Mol. Cell. Biol. 3, 182-189. Ferrari, S., Battini, R., Kacjmarek, L., Rittling, S., Calabretta, B., de Riel, J.K., Philiponis, V., Wet, J.F. and Baserga, R. (1986) Mol. Cell. Biol. 6, 3614-3620. Georg'~tos, S.D. and Blobel, G. (1987) J. Cell Biol. 105, 105115; 117-125. Greenburg, G. and Hay, E.D. (1988) Development 102, 605622. Henzen-Logmans, S.C., Mullink, H., Ramaekers, F.C.S., Tadema, T. and Meijer, C.J.L.M. (1987) Virchows Arch. Abt. A Pathol. Anat. Histopathol. 410, 347-354. Ikeda, M., Deery, W.J., Nielsen, T.B., Ferdows, M.S. and Field, J.B. (1986) Endocrinology 119, 591-599. Ingber, D.E. and Folkman, J. (1989) in Cell Shape: Determinants, Regulation and Regulatory Role, pp. 3-31, Academic Press, New York. Kasper, M. and Stosiek, P. (1989) Cell Tissue Res. 257, 661664. Kasper, M., Stosiek, P., van Muijen, G.N.P. and Moll, R. (1989) Histochemistry 93, 93-103. Lamy, F., Roger, P.P., Lecocq, R. and Dumont, J.E. (1986) Eur. J. Biochem. 155, 265-272.

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