Differential Involvement of the Actin Cytoskeleton in Differentiation and Mitogenesis of Thyroid Cells: Inactivation of Rho Proteins Contributes to Cy
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《内分泌学杂志》
Institute of Interdisciplinary Research (IRIBHM) (N.F., S.B., J.E.D., C.M., P.P.R., S.D.), Universite Libre de Bruxelles, Campus Erasme, B-1070 Brussels, Belgium
Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie (K.A.), Albert-Ludwigs-Universitat Freiburg, D-79104 Freiburg, Germany
Abstract
In thyroid epithelial cells, TSH via cAMP induces a rounding up of the cells associated with actin stress fiber disruption, expression of differentiation genes and cell cycle progression. Here we have evaluated the role of small G proteins of the Rho family and their impact on the actin cytoskeleton in these different processes in primary cultures of canine thyrocytes. TSH and forskolin, but not growth factors, rapidly inactivated RhoA, Rac1, and Cdc42, as assayed by detection of GTP-bound forms. Using toxins that inactivate Rho proteins (toxin B, C3 exoenzyme) or activate them [cytotoxic necrotizing factor 1 (CNF1)], in comparison with disruption of the actin cytoskeleton by dihydrocytochalasin B (DCB) or latrunculin, two unexpected conclusions were reached: 1) inactivation of Rho proteins by cAMP, by disorganizing actin microfilaments and inducing cell retraction, could be necessary and sufficient to mediate at least part of the cAMP-dependent induction of thyroglobulin and thyroid oxidases, but only partly necessary for the induction of Na+/I– symporter and thyroperoxidase; 2) as indicated by the effect of their inhibition by toxin B and C3, some residual activity of Rho proteins could be required for the induction by cAMP-dependent or -independent mitogenic cascades of DNA synthesis and retinoblastoma protein (pRb) phosphorylation, through mechanisms targeting the activity, but not the stimulated assembly, of cyclin D3-cyclin-dependent kinase 4 complexes. However, at variance with current concepts mostly derived from fibroblast models, DNA synthesis induction and cyclin D3-cyclin-dependent kinase 4 activation were resistant to actin depolymerization by dihydrocytochalasin B in canine thyrocytes, which provides a first such example in a normal adherent cell.
Introduction
THE SMALL G PROTEINS Rho, Rac, and Cdc42 are mainly involved in the control of the cytoskeleton architecture and cell movement (1), but they also participate in several other important cellular activities including the control of cell proliferation and specific gene expression (2). Classically, whereas activation of Rho induces actin polymerization and microfilament assembly, activation of Rac and Cdc42 cause lamellipodia and filopodia formation, respectively (3). These proteins are active when they bind GTP and are inactivated by the hydrolysis of GTP to GDP. Both the binding and hydrolysis are controlled by specific guanosine nucleotide exchange factors (GEFs) and GTPase-activating proteins respectively (4). One best-characterized effector pathway of RhoA-GTP is the activation of the Rho kinases (ROCK), which phosphorylate and activate the LIM domain-containing protein kinase (LIM kinase) to phosphorylate and inhibit the actin-depolymerizing protein cofilin, thus leading to microfilament assembly and stress fiber formation (5).
Experiments mainly performed in fibroblast systems have indicated that integrin-mediated cell adhesion to extracellular matrix, the resulting RhoA-dependent actin stress fiber formation and focal adhesion formation, and subsequent cell spreading are strictly required for induction of normal cell cycle progression by growth factors (6, 7). Demonstrated Rho/ROCK-dependent mechanisms include sustained activation by growth factors of the Ras/Raf/ERK cascade, G1 phase induction of cyclin D1 and down-regulation of the cyclin-dependent kinase (CDK) 2 inhibitors p21cip1 and p27kip1, leading to CDK4 and CDK2 activations, phosphorylation of the retinoblastoma protein (pRb) and entry into S phase (8). In addition to being needed for proliferation in nontransformed cells, Rho family proteins are necessary for oncogenic Ras-mediated transformation by reducing p21cip1 levels (9). The fundamental role of Rho proteins in mitogenic signaling by both growth factor and oncogenic Ras-dependent pathways, through cyclin D1 up-regulation and p21cip1 or p27kip1 down-regulation, has been extended to some epithelial cell lines (10, 11, 12).
The physiologically relevant model of canine thyroid epithelial cells in primary culture (13, 14) displays several interesting characteristics to reevaluate the relationships and proposed mechanisms between Rho family proteins, actin microfilament organization, cell function, expression of differentiation genes, and cell cycle progression. In these cells, a unique association of actin stress fiber disruption, expression of differentiation genes and cell cycle progression is concomitantly observed in response to TSH, through a cAMP increase. Via cAMP elevation and cAMP-dependent protein kinase [protein kinase A (PKA)] activation, TSH acutely induces phagocytosis (15) [the in vitro manifestation of the macropinocytosis of thyroglobulin (Tg) involved in stimulated thyroid hormone secretion (16)], which is associated to microfilament depolymerization and stress fiber disruption accompanied by dephosphorylation of cofilin (17) and myosin light chains (15). This precedes long-term morphological changes associated with modulation of the synthesis of various cytoskeleton proteins (18, 19, 20) and parallels the induction by TSH/cAMP of thyroid differentiation genes (21). Both the cAMP-dependent morphological changes and differentiation expression are inhibited by growth factors such as epidermal growth factor (EGF) (22) and hepatocyte growth factor (HGF) (23), which induce cell proliferation and cell cycle progression through a classical Ras/Raf/Erk/c-jun pathway leading to D-type cyclin induction (24, 25, 26, 27, 28). By contrast, in fully differentiated dog thyrocytes stimulated by TSH, the distinct (29) positive control of G0-S phase progression and DNA synthesis initiation by sustained cAMP elevations does not activate Ras- or phosphatidylinositol 3 kinase-dependent signaling cascades (26, 27, 28, 30) and does not up-regulate D-type cyclins (25). It nevertheless requires the activity of cyclin D3-CDK4 complexes (25), which are assembled (25), and subsequently activated depending on a continuous stimulation (31). Also unlike growth factors, TSH and cAMP paradoxically enhance the expression of p27kip1, which plays a positive role in the nuclear import and activity of cyclin D3-CDK4 complexes (32, 33).
In this work, we define the effects of thyroid signal transduction cascades on RhoA, Rac1, and Cdc42 activity, and through the use of specific Rho modulating toxins compared with actin depolymerization by dihydrocytochalasin B (DCB) or latrunculin B, their role in thyroid-specific gene expression and cell cycle regulation. Our data point to an important contribution of the cAMP-dependent inhibition of Rho-dependent stress fibers in the elusive cAMP-dependent regulation of some major thyroid differentiation genes, and provide evidence of a new cell cycle control target that depends on the activity of Rho proteins but not on actin polymerization, at variance with current concepts (7, 34).
Materials and Methods
Cell cultures
Dog thyroid epithelial cells (obtained according to a protocol approved by the institutional animal use committee) were cultured in monolayer (2 x 104 cells/cm2) in a medium that included DMEM, Ham’s F12 medium, and MCDB 104 medium (2:1:1, vol/vol) supplemented by ascorbic acid (40 μg/ml), and antibiotics (14). As indicated, bovine insulin (5 μg/ml) (Sigma, St. Louis, MO) was added or not to this medium since the seeding. Four or 5 d after seeding, quiescent cells were treated as indicated in figure legends. The various agents used in this study were: forskolin (Calbiochem, La Jolla, CA), fetal bovine serum (FBS) (Life Technologies, Inc., Paisley, Scotland, UK), and bovine TSH, murine EGF, TPA (12-O-tetradecanoylphorbol-13-acetate), and DCB, all purchased from Sigma. Latrunculin B was purchased from A.G. Scientific (San Diego, CA). Toxin B (35), the fusion toxin C2IN-C3 + C2II (36), and glutathione S-transferase (GST)-CNF1 (37) were prepared at the Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie (Freiburg, Germany). Human dermal fibroblasts (a cell strain derived from fetal skin) were cultured in DMEM + 10% FBS and made quiescent by a 3-d serum deprivation.
DNA synthesis
Cells in 3.5-cm Petri dishes were stimulated for 48 h by various mitogenic agents. Bromodeoxyuridine (BrdUrd) was added for the last 24 h. BrdUrd incorporation was detected by immunofluorescence, and BrdUrd-labeled nuclei were counted (1000/dish) as described (29).
Indirect immunofluorescence detection of actin (20)
Cells in Petri dishes were fixed with methanol for 10 min at –20 C, permeabilized with 0.1% Triton X-100 in PBD (pH 7.5), at room temperature and blocked for 20 min with normal sheep serum (5% in PBS containing 0.05% BSA [PBS/BSA]). They were then incubated for 2 h at room temperature or overnight at 4 C with a rabbit antiactin antibody (1/50, Sigma) diluted in PBS/BSA. After washing, the cells were then incubated for 2 h with fluorescein conjugated antirabbit IgG.
Western blotting detections of proteins
They were performed as described (33) using the following antibodies: p27kip1 (C-19) from Santa Cruz Biotechnology (Santa Cruz, CA), pRb (C-15) (Santa Cruz), CDK4 (Santa Cruz), cyclin D3 (DCS-22) and -tubulin (clone DM1A) from NeoMarkers (Fremont, CA), cyclin D1 (DCS-6) from Dr. J. Bartek (Danish Cancer Society, Copenhagen, Denmark). Our antibodies against Tg (22) and thyroid oxidase (ThOX1) (38) were characterized previously.
Immunoprecipitation and pRb-kinase assay
Cyclin D3-CDK4 complexes were immunoprecipitated using a monoclonal antibody against cyclin D3 (DCS-28; NeoMarkers, Fremont, CA) and their pRb-kinase activity was assayed exactly as described (31, 32). Immune complexes were incubated in the presence of 2 mM ATP and 0.5 μg of a 56-kDa pRb fragment (amino acids 379–928) (QED Bioscience, San Diego, CA). After SDS-PAGE separation and transfer to polyvinylidene difluoride membranes of proteins of the reaction mixture, the phosphorylation on Ser780 of the pRb fragment (specifically ascribed to CDK4) was detected using the phosphospecific-pRb (Ser780) antibody from Cell Signaling Technology (Beverly, MA). Membranes were then reprobed using antibodies for CDK4 (DCS-156 from Cell Signaling Technology) and cyclin D3.
Rho protein activation assays
The amount of GTP-bound Rho proteins was assessed by GST pull-down assay, using the Cdc42 and Rac1-interactive binding (CRIB) domain from human PAK1B (for the study of Rac 1 and Cdc42) or the Rho binding domain from murine Rhotekin (for the study of RhoA) fused to the GST protein. Plasmids coding for these proteins were gifts from Dr. J. G. Collard (The Netherlands Cancer Institute, Amsterdam, The Netherlands). Proteins were expressed as described (39). Small G protein activation was determined as follows: stimulated cells (from a 9- or 6-cm dish) were rapidly washed with ice-cold PBS, lysed and scraped in 800 μl of GST-fish buffer [10% glycerol, 50 mM Tris (pH 7.4), 100 mM NaCl, 1% Nonidet P-40, 2 mM MgCl2 supplemented with protease inhibitors]. The lysate was clarified by centrifugation. Supernatants (650 μl) were incubated for 30 min at 4 C with glutathione-agarose beads (Sigma) preloaded with the GST fusion protein. After incubation, beads were washed three times in GST-fish buffer and then resuspended in Laemmli buffer. Samples were analyzed by SDS-PAGE (15%) followed by transfer on polyvinylidene difluoride membranes. Immunodetections were performed using antibodies for Rac1 (Transduction Laboratories, Lexington, KY), Cdc42 (P1; Santa Cruz) or RhoA (26C4; Santa Cruz). Aliquots of whole-cell lysate were also resolved to ascertain that the same total amount of Rho proteins was assayed.
Northern blotting mRNA analyses
Subconfluent cell monolayers (from a 9-cm dish) were rapidly scraped in 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was extracted using the RNeasy Mini kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. Eight micrograms of total RNA were denatured in glyoxal and fractioned by electrophoresis on a 1% agarose gel. RNA quality was verified by acridine orange staining. RNAs were transferred to a nylon membrane (pall Biodyne A) using 20x saline sodium citrate. After baking, the blots were prehybridized and hybridized as described (24). The same filter was probed successively with different probes: dog thyroperoxidase (TPO) cDNA 2-kb BamHI, Tg cDNA 1.1-kb PstI, rat Na+/I– symporter (NIS) cDNA 2.4-kb EcoRI/XmnI (40), and human ThOX1 cDNA 4.6-kb EcoRI/XbaI fragments (38).
All the experiments were reproduced in independent cultures at least twice and in general three or four times with similar results.
Results
Disruption of actin stress fibers by TSH and cAMP but not by growth factors
Dog thyrocytes cultured for 4 d in basal control medium exhibited a dense network of stress fibers as shown by indirect immunofluorescence labeling of actin (Fig. 1). This was not modified by a treatment of 20 min with insulin (5 μg/ml), EGF (25 or 100 ng/ml), HGF (100 ng/ml) (Fig. 1) or 10% FBS (not shown). On the other hand, as previously described (20), TSH (1 mU/ml) and the general adenylyl cyclase activator forskolin (10–5 M) completely disrupted this microfilament network and induced membrane ruffling (Fig. 1). Previous studies have associated these acute processes to dephosphorylation of both myosin light chains and cofilin (15, 17). Within a few hours, TSH and forskolin then progressively promoted rounding up and cytoplasmic arborization of the cells that remain associated by dendrite-like extensions (see Fig. 3D) (20, 41). These morphological changes would be compatible with an involvement of Rac1 in the ruffling induced by the cAMP cascade, whereas reorganization of the actin cytoskeleton could be explained by RhoA inactivation in these conditions.
cAMP-dependent cascade but not growth factors inactivates Rho proteins RhoA, Rac1, and Cdc42
The effects of the different thyroid signal transduction cascades on the activation of Rac1, Cdc42, and RhoA were tested by the detection of the GTP-loaded forms of these proteins. As shown in Fig. 2, EGF (100 ng/ml) had no effect on Rac and Cdc42, but activated RhoA, after 1–30 min of stimulation. In parallel experiments using the same assay, carbachol did activate Rac1 (42). On the other hand, TSH (1 mU/ml) and forskolin (10–5 M) inactivated not only RhoA but also Rac1 and Cdc42 (Fig. 2). The same results were obtained in cells previously cultured for 4 d in the presence of insulin (not shown). The ruffling induced by cAMP cascade activating agents was thus not a result of Rac1 activation in dog thyrocytes. The disappearance of RhoA-GTP induced by TSH and forskolin was consistent with the hypothesis that the disruption of actin stress fibers is mediated by RhoA inactivation.
Rho protein inactivation is involved in the cAMP-dependent morphological changes
To determine whether the inhibition by cAMP of the three Rho family proteins (Rho, Rac, and Cdc42) could play a role in the cAMP-dependent functions of dog thyrocytes, we have incubated the cells with the Clostridium difficile toxin B. Depending on internalization via receptor-mediated endocytosis and possibly proteolytic release of its active enzyme domain, toxin B monoglucosylates Rho subfamily proteins Rho, Rac, and Cdc42 at Thr35/37 (43). This glucosylation inactivates the Rho GTPases by inhibiting their effector coupling (44). Exposure of cells to 100 ng of toxin B/ml (but not 500 pg/ml) for 24 h efficiently inhibited binding of Rac1 and RhoA to their effectors (Fig. 3A). These inhibitory effects persisted at least for 3 d (not shown). Moreover, the presence of total Rac1 and Cdc42 proteins, but not RhoA, was strongly reduced by toxin B (Fig. 3A), as observed by others, likely because these glucosylated proteins are more degraded by the proteasome (45, 46). Treatment of dog thyrocytes with toxin B (100 ng/ml, 24 h), much like forskolin, disrupted the actin cytoskeleton and potently induced cell rounding-up and arborization (Fig. 3D). Preliminary experiments had shown that such a long treatment with toxin B was required to induce these effects in the denser parts of the cell monolayer, as described in other polarized epithelial cells where toxin B receptors might be more concentrated on the basolateral side (47).
To study the involvement of Rho apart from Rac/Cdc42, thyrocytes were also incubated with the C3 exoenzyme, which selectively inactivates RhoA, RhoB and RhoC by catalyzing their ADP-ribosylation at Asn41 (48). Because C3 and related C3-like transferases do not readily enter the cells, we used the fusion toxin C2IN–C3, comprising the catalytic domain of C3 transferase of C. limosum and part of the C2I toxin of C. botulinum, which together with the activated binding component C2II of the C2 toxin allows efficient uptake into the cells (36). Such a C3 treatment also induced the disorganization of the actin cytoskeleton and cell retraction associated with even more conspicuous dendrite-like extensions of some cells (Fig. 3D).
On the other hand, to activate Rho, Rac, and Cdc42 and thus prevent their inhibition by cAMP, we have also treated the cells with CNF1. This toxin, produced by uropathogenic Escherichia coli, deamidates the glutamine 61/63 of Rho, Rac, and Cdc42, which blocks the intrinsic or the GTPase-activating protein-induced hydrolysis of GTP, leading to the permanent activation of these G proteins (49). As shown in Fig. 3B, Rac1 and Cdc42 were strongly activated by treatment of the cells for 24 h with the CNF1 toxin fused to the GST protein (GST-CNF1). Although the amount of Rac1-GTP was increased by GST-CNF1, the total amount of Rac1 was decreased. This decrease, also observed by others (50, 51), could result from a proteasome-mediated degradation of deamidated Rac1. RhoA activation by GST-CNF1 was reflected in SDS-PAGE by the upward 1-kDa shift characteristic of CNF1-induced deamidation of RhoA (52) (Fig. 3C). Preincubation of the cells for 8 h with GST-CNF1 prevented the actin stress fiber disruption induced by forskolin (Fig. 3D).
Collectively, these observations suggested that the cAMP-induced inactivation of Rho proteins, particularly RhoA, is both necessary and sufficient to mediate the cAMP-dependent actin reorganization in dog thyrocytes.
Rho-mediated actin cytoskeleton reorganization plays a crucial role in the cAMP-dependent expression of thyroid differentiation genes
To investigate whether the modulation of Rho proteins could be involved in the expression of some differentiation characteristics of the thyroid, we have assessed the effect of toxin B and CNF1 on the level of Tg and ThOX proteins assayed by western blotting, as well as on the accumulation of the mRNAs of these genes and of the NIS and TPO genes by Northern blotting. To evaluate the role of actin organization, the effects of toxin B were compared with those of DCB, a membrane permeant inhibitor of actin polymerization, which unlike cytochalasin B has little effect on sugar transport. Disruption of the actin cytoskeleton was seen 1 h min after DCB administration (Fig. 3D), culminating in complete cell retraction by 5 h (Fig. 3D), and these effects persisted for at least 48 h (not shown). Before treatment with these various agents, thyrocytes were cultured for 3 d in the presence of EGF (25 ng/ml) to repress Tg expression (22). Cells were then transferred in basal medium without EGF, pretreated for 24 h with toxin B (100 ng/ml), or for 8 h with either DCB (10 μM) or GST-CNF1 (1.5 μg/ml), and then stimulated or not for 48 h with forskolin in the presence of these different drugs. As previously shown (21, 22, 38, 40), forskolin strongly induced the accumulation of Tg, TPO, NIS, and ThOXs proteins and/or mRNAs (Fig. 4). Toxin B or DCB did not affect the induction by forskolin of Tg and ThOXs proteins (Fig. 4A). Surprisingly, toxin B or DCB used alone induced an accumulation of Tg and ThOXs proteins and mRNAs (Fig. 4, A and B), but as previously shown in the case of forskolin stimulation (19), they reduced the presence of -tubulin (Fig. 4A). The same result was obtained with latrunculin B (0.1–10 μM), another actin polymerization inhibitor (53) (not shown). These drugs did not increase cellular cAMP levels (not shown). On the other hand, GST-CNF1, which activates Rho proteins, decreased the induction by forskolin of not only Tg and ThOXs but also of NIS and TPO mRNAs (Fig. 4). Nevertheless, on the contrary of what we observed for Tg and ThOXs, toxin B and DCB did not induce the accumulation of TPO and NIS mRNAs (Fig. 4B).
Therefore, inactivation of Rho proteins by cAMP appeared to be instrumental in the cAMP-dependent expression of these major thyroid differentiation genes. Furthermore, disruption of the actin cytoskeleton mediated by cAMP-dependent inactivation of Rho proteins was sufficient to mimic in part the cAMP-dependent induction of Tg and ThOXs, but not the induction of NIS and TPO.
Rho protein activity, but not actin cytoskeleton integrity, is required for DNA synthesis induction in dog thyrocytes
Quiescent dog thyroid cells cultured in the presence of insulin were pretreated for 24 h with various concentrations of toxin B and then stimulated with TSH, EGF, or TPA for 48 h in the presence of this drug. Toxin B at 10 and 100 ng/ml inhibited DNA synthesis induced by all these agents (Fig. 5A) and also by forskolin (10–5 M) or HGF (50 ng/ml) (not shown). Toxin B did not affect cell viability, as judged from trypan blue staining and Hoechst dye staining of nuclei (not shown). Rho could be an important target of the inhibition of DNA synthesis by toxin B. Indeed, the more selective toxin C3 (the C2IN-C3/C2II complex) also inhibited the stimulation of DNA synthesis by TSH, EGF, or TPA (Fig. 5B). On the other hand, simultaneous activation of Rho, Rac and Cdc42 proteins by a 48-h treatment with GST-CNF1 (1.5 μg/ml) in the presence of insulin was not sufficient to induce DNA synthesis (not shown), at variance with the induction of DNA synthesis reported for Swiss 3T3 cells (54). Therefore, some activity of the Rho proteins was necessary for the mitogenic stimulations of dog thyrocytes by both cAMP-dependent and cAMP-independent cascades, but the activation of these proteins was not sufficient to elicit such an effect.
In the same experiments, DCB (10 μM) for 1 or 5 h completely disrupted the actin stress fibers (Fig. 3D), but it did not prevent DNA synthesis induced by the subsequent administration of TSH, EGF, or TPA while maintaining DCB presence (Fig. 5A). In other experiments, DCB also did not prevent DNA synthesis stimulated by EGF + serum (10%) (not shown). This surprising observation sharply contrasted with the complete inhibition by the same treatment with DCB (10 μM, added 1 h before mitogenic stimulation) of serum-stimulated DNA synthesis in cultured human skin fibroblasts (Fig. 5C), as observed in various fibroblast or endothelial cell systems (55, 56, 57, 58). The inhibitory effects of toxin B and C3 on dog thyrocyte DNA synthesis were thus not mediated by actin cytoskeleton alterations.
Toxin B, but not dihydrocytochalasin B, inhibits pRb phosphorylation
Dog thyroid cells were pretreated with toxin B (24 h) or DCB (1 or 24 h) before stimulating them in the presence of these drugs for 20 and 32 h with TSH (maximum cAMP-dependent mitogenic stimulation) or with the combination of EGF and 10% FBS (ES; maximum cAMP-independent mitogenic stimulation). Hyperphosphorylation of pRb induced by both mitogenic stimulations was clearly inhibited by toxin B (Fig. 6A). In sharp contrast, DCB did not affect pRb phosphorylation induced by TSH or EGF + serum, irrespective of the timing of its addition (1 h or even 24 h before administration of mitogens) (Fig. 6A). pRb phosphorylation was thus a critical target of the actin-independent requirement for Rho protein activity in dog thyroid cell cycle progression.
Regulation of cell cycle regulatory proteins by Rho proteins and actin cytoskeleton integrity
To identify the molecular basis of the requirement for Rho protein activity in the pRb phosphorylation process, the expression of G1 phase cyclins, CDK4 and p27kip1 was analyzed in dog thyrocytes treated or not with toxin B. DCB treatment was also included in these experiments, but quantitative comparison between toxin B and DCB treatments is limited by the different timing of their respective administrations justified by their different modes and kinetics of action. As previously shown (25), the levels of cyclin D3 and of the less abundant cyclin D1 were moderately increased by a 20-h treatment with EGF + serum (ES) but not by TSH (Fig. 6B). Conversely, stimulation of dog thyroid cells for 20 h or 32 h with TSH, but not ES, increased the expression of p27kip1 (Fig. 6B) (33). Both Toxin B and DCB reduced the basal expression of cyclin D3 and they also inhibited the accumulation of cyclin D1 and cyclin D3 stimulated by ES (Fig. 6B). Toxin B increased the amount of p27kip1 in all the conditions (but no longer in the presence of TSH at 32 h) (Fig. 6B). This was partly reproduced by DCB only in the absence of the mitogenic factors (Fig. 6B). On the other hand, CDK4 concentration was not modified by toxin B and DCB (Fig. 6B). Therefore, as in other cell types D-type cyclin expression depended at least in part on Rho protein-dependent integrity of the actin cytoskeleton. By contrast, the accumulation of p27kip1 in response to Rho protein inactivation might be partly independent of the actin cytoskeleton.
The activity of Rho proteins, but not actin cytoskeleton integrity, is required for the activation of cyclin D3-CDK4
In dog thyrocytes, TSH and cAMP weakly affect the accumulation of D-type cyclins or CDK4 (as seen in Fig. 6B), but they induce the assembly of required complexes of cyclin D3-CDK4 (25). EGF + serum also stimulates the formation and activity of cyclin D3-CDK4 complexes. To understand the mechanism by which toxin B inhibits the phosphorylation of pRb, dog thyrocytes were pretreated for 24 h with toxin B (100 ng/ml) or 1 h with DCB (10 μM) and the formation and activity of cyclin D3-CDK4 complexes were analyzed 20 h after cell stimulation by TSH or EGF + serum (ES) in the presence of these drugs. Consistent with its inhibitory effect on pRb phosphorylation described above, toxin B strongly inhibited the pRb-kinase activity associated with cyclin D3 in cells stimulated by TSH or ES (Fig. 7). By contrast, interestingly, toxin B did not prevent the association of CDK4 with cyclin D3 induced by these mitogenic agents. On the other hand, as expected because it did not inhibit pRb phosphorylation, DCB weakly affected the assembly and activity of cyclin D3-CDK4 complexes (Fig. 7). Our observations thus suggested that the activity of Rho proteins, independently of their effect on the actin microfilaments, was required for the activation but not for the assembly of cyclin D3-CDK4 in response to different mitogenic stimulations. These results identify a new Rho-dependent mechanism that is limiting for pRb-phosphorylation and thus cell cycle progression independently of actin cytoskeleton organization.
Discussion
Rho protein inactivation and actin depolymerization contribute to the regulation of thyroid cell function and gene expression by TSH and cAMP
The present study in canine thyrocytes shows for the first time that the TSH-cAMP pathway inactivates the three small G proteins RhoA, Rac1 and Cdc42. A similar inhibition of RhoA activity by cAMP has been described in other cell types including melanocytes and endothelial, lymphoid and neuronal cells (59, 60, 61). By contrast, cAMP and PKA activate Rac1 or Cdc42 (61) in various cell systems (62, 63), although Rac inactivation by PKA has also been described in endothelial cells (64). The membrane ruffling induced by cAMP in dog thyrocytes is thus not mediated by activation of Rac1 but through alternative pathways, which is consistent with reports indicating that Rac inactivation does not eliminate membrane ruffling in some systems (65, 66).
As in other cell systems, Rho inactivation in dog thyrocytes appears both necessary and sufficient to mediate the reorganization of actin microfilaments and cell rounding up (41) induced by TSH through cAMP and PKA activation, inasmuch as they were mimicked not only by toxin B but also by the C3 exoenzyme and prevented by Rho protein activation by CNF1 toxin. The cAMP-dependent inactivation of RhoA could result from its phosphorylation at Ser188 by PKA (59, 60), which promotes formation of RhoA:RhoGDI complexes, thereby decreasing the membrane association of RhoA (59, 67) and the binding of RhoA to its effector Rho kinase (60). PKA could also inhibit Rho signaling by phosphorylating the recently identified AKAP-Lbc, an A-kinase anchoring protein (AKAP) with a Rho-GEF activity. This phosphorylation promotes the binding of this AKAP to a 14-3-3 protein, blocking its Rho-GEF activity (68). The best known RhoA-GTP downstream effectors are ROCK, which phosphorylate several proteins controlling actin organization, such as LIM kinases. The resulting inhibition of LIM kinase (5) probably causes the dephosphorylation and activation of cofilin previously demonstrated in dog thyrocytes (17), itself leading to actin filament network disruption. Because ROCK also catalyzes the inhibitory phosphorylation of myosin phosphatase (69), the cAMP-dependent inhibition of RhoA should similarly contribute to explain the inhibition of myosin light chains phosphorylation observed in dog thyrocytes stimulated by TSH and cAMP (15).
Presumably as a result of both these effects, TSH through cAMP increases the level of unpolymerized G-actin in thyroid cells (70). One functional consequence of such an effect in vivo is to facilitate endocytosis (71). For example, RhoA activation blocks through ROCK the engulfment of apoptotic bodies in a phagocytic Chinese hamster ovary cell line and a macrophage cell line (72). Indeed, the first major acute functional effect of the TSH-cAMP pathway in thyroid is to induce the macropinocytosis of Tg, and after the proteolysis of this protein, thyroid hormone secretion (16). In dog thyroid cell culture, this acute functional effect is reflected by an enhanced phagocytic activity (15). Inhibition of RhoA by TSH and cAMP could therefore play a crucial role in stimulated thyroid hormone secretion.
Whereas the mechanisms responsible for the cell specificity of expression of thyroid differentiation genes, including Tg, have been detailed in depth (73), the mechanisms underlying their transcriptional regulation by TSH and cAMP (21, 74) remain elusive despite intensive investigation (75, 76). Our study provides the first evidence of an important role of the inactivation by cAMP of Rho-mediated actin polymerization in the cAMP-dependent expression of some major thyroid differentiation genes, much like what has been described in the case of cAMP-induced melanoma cell differentiation (77). Indeed, activation of Rho proteins by the CNF1 toxin repressed the induction by forskolin of thyroid differentiation genes including Tg, thyroperoxidase, NIS and ThOXs. Furthermore, inactivation of Rho proteins by toxin B and even microfilament disruption by dihydrocytochalasin B or latrunculin B were sufficient to partially mimic the induction by TSH and cAMP of Tg and ThOX proteins and mRNAs in dog thyrocytes. Interestingly, the oncogenic dedifferentiating RET/PTC rearrangement of papillary thyroid carcinomas stimulates the formation of Rho-dependent stress fibers in PC Cl3 thyroid cell line (78), and constitutively activated RhoA represses the transcription of the Tg gene in part by interfering with the activity of the thyroid transcription factor TTF-1 in FRTL-5 cells (79). Further studies should thus evaluate in vivo the importance of the relationship between Rho activity and cell shape in the physiopathologic control of Tg synthesis during TSH stimulation or in the dedifferentiation associated with carcinogenic processes.
In the present study, a congruence of the effects of TSH/cAMP, toxin B and DCB was observed not only on the induction of Tg and ThOXs, but also on cell cycle regulatory proteins: partial repression of cyclin D3 [mostly demonstrated in the absence of insulin in the case of the TSH effect (80)] and accumulation of p27kip1. The reduction by TSH and cAMP of the synthesis of various cytoskeleton proteins in dog thyrocytes, including actin (19), high-molecular-weight tropomyosins isoforms that stabilize the actin stress fibers (20, 81), vimentin (18), and -tubulin (Fig. 4A) (19) could also depend on the acute reorganization of actin cytoskeleton mediated by Rho inactivation. In agreement with previous proposals (82, 83), we have suggested that this conclusion could be extended to a large fraction of modifications of gene expression by TSH and cAMP because their effects on the synthesis of most of 45 unidentified proteins are mimicked by culturing unstimulated human thyrocytes as dense aggregates instead of monolayers that also reduces cytoskeleton organization (81). Mechanisms involved in the control of gene expression by cell shape and cytoskeleton organization are complex and still poorly understood (83). Integrins, which connect the actin cytoskeleton to focal adhesion sites, appear to play the role of major receptors for mechanotransduction (7, 34). G actin itself directly controls gene expression (84, 85, 86) by interfering with some transcription coactivators (85, 87). Changes in the actin cytoskeleton also affect the stability and translation of some mRNAs (88, 89).
Thyroid cell S phase entry depends on Rho proteins but resists to cytochalasin
The inhibition of dog thyroid cell S-phase entry stimulated by TSH or growth factors by toxin B and exoenzyme C3 generalizes the requirement of an activity of Rho proteins for cell cycle progression, as in Swiss3T3 and NIH3T3 fibroblasts or rat aortic smooth muscle cells (90, 91, 92). Thus, even in the cAMP-dependent mitogenic stimulation associated with a reduction by cAMP of the activity of Rho proteins, a residual activity of at least one of these proteins must exert a crucial permissive action for DNA synthesis induction. This apparent paradox is by no means unique in the analysis of the cAMP-dependent mitogenesis of thyrocytes. For instance, unlike growth factors, cAMP strongly represses cyclin D3 in dog thyrocytes, but in the presence of insulin that partly overcomes this inhibition, cyclin D3 is specifically required in the mitogenic stimulation by cAMP, but not in the stimulation by growth factors (25, 80). Inactivation of RhoA by C3 and dominant-negative RhoA also induces G1 arrest in rat thyroid FRTL-5 cells (93), whereas in rat thyroid WRT cells a dominant-negative Rac1 impairs cAMP-stimulated DNA synthesis (94).
The required involvement of Rho proteins and actin cytoskeleton integrity for D-type cyclins up-regulation and p27kip1 down-regulation, as demonstrated in many cell systems (7, 11, 95), are apparently confirmed in the present experiments. In dog thyrocytes, toxin B and cytochalasin (DCB) partly inhibited the basal or EGF + serum-stimulated accumulation of cyclin D1 and cyclin D3, and treatment with toxin B (more potently than DCB) sufficed to up-regulate p27kip1. However, these effects are clearly insufficient to explain the strong inhibition by toxin B of pRb phosphorylation and G1/S transition stimulated by cAMP-dependent and cAMP-independent mitogenic pathways: 1) the partial reduction of cyclin D3 levels was insufficient to lead to a decrease of cyclin D3-CDK4 complexes in cells stimulated by TSH or EGF + serum; 2) in dog thyrocytes stimulated by TSH, up-regulated p27kip1 supports, rather than inhibits, the activity of cyclin D3-CDK4 complexes (32); 3) cyclin D1 and D3 levels were similarly reduced by toxin B and DCB, but DCB did not inhibit pRb phosphorylation and DNA synthesis, in sharp contrast with the situation found in fibroblasts and vascular cells.
A new mechanism should thus explain the present inhibition of pRb phosphorylation by Rho protein inactivation, which, at variance with the current concepts (6, 7, 34), is not mediated by actin depolymerization and stress fiber disruption. Strikingly, Rho protein inactivation by toxin B did not inhibit the mitogen-dependent assembly of cyclin D3-CDK4 complexes, the mechanism of which is still unknown in dog thyrocytes stimulated by TSH/cAMP (25, 33) as in fibroblasts stimulated by growth factors. Instead, we show here that toxin B, but not DCB, inhibits the pRb-kinase activity of the assembled cyclin D3-CDK4 complexes, which is thus demonstrated as a new target for cell cycle control by Rho proteins. This observation provides new evidence supporting the concept that the catalytic activity of cyclin D-CDK complexes critically depends on additional regulated mechanisms (31, 32), besides the induction of D-type cyclins and their association with CDK and p27kip1. The nuclear translocation of the cyclin D3-CDK4 complex (33) and the phosphorylation of CDK4 within this complex have been identified as regulated targets in dog thyrocytes (31, 32). Interestingly, the homologous Thr160-phosphorylation of CDK2 has been found to directly depend on pathways inhibited by statins (96), which prevent the membrane translocation and activation of RhoA (10).
Studies using drugs, such as cytochalasin D, that disrupt microfilaments have suggested that normal cells (in fact mostly fibroblasts and capillary endothelial cells) require an intact actin cytoskeleton to pass through the G1/S restriction point (34, 55, 56, 57, 58). For instance, kinetics analysis using cytochalasin pulses have defined a narrow time window of 3 h just before restriction point in which actin cytoskeleton integrity is critical for G1/S transition (58). Our unexpected finding that the G1/S transition elicited by a variety of mitogens in dog thyrocytes was resistant to sustained actin depolymerization by continuous presence of cytochalasin D and subsequent cell rounding-up, demonstrates that the actin cytoskeleton-sensitive cell cycle checkpoint(s) is a cell type-specific characteristic. To our knowledge, induction of DNA synthesis in the presence of cytochalasin has only been observed in some transformed cells (97), and in lymphocytes (98) that proliferate in suspension culture. The absence of an actin-dependent cell cycle checkpoint in thyroid epithelial cells explains that their proliferation is clearly compatible with a reduced polymerization of actin and disruption of stress fibers induced by TSH and cAMP or by phorbol esters (20). DNA synthesis responses to EGF (99) or to TSH/cAMP (100) have been demonstrated in suspension cultures of thyroid follicles, which do not form stress fibers (101). Except for endothelial cells and wound fibroblasts, actin stress fibers are not detected in most proliferating cell types in vivo (102). Therefore, the cell proliferation requirement for spreading on a solid substrate and associated formation of stress fibers may appear as a specialized characteristic of some cell types, such as fibroblasts and endothelial cells, in order for them to obey the specific spatial constraints of their proliferation in vivo. It is not a major determining factor for the proliferation of thyroid follicle epithelial cells.
Acknowledgments
We thank Dr. J. G. Collard and J. P. ten Klooster for kindly providing us the GST-PAK-CRIB and C21-GST constructions, and Dr. J. Bartek for cyclin D1 antibody. We are also grateful to C. Degraef for technical assistance, S. Paternot for technical advice and help in pRb-kinase assay and L. Giandon (Cytogenetic Laboratory, Erasme Hospital) for the fibroblasts culture.
Footnotes
This work was supported by grants from the Fonds National de la Recherche Scientifique (FNRS), Fonds de la Recherche Scientifique Medicale (FRSM), Operation Televie, Fonds pour la formation a la Recherche dans l’Industrie et l’Agriculture (FRIA), Fondation Rose et Jean Hoguet, Fondation Van Buuren, Ple d’Action Interuniversitaire (PAI), Federation Belge Contre le Cancer, and Actions de Recherche Concertees (ARC) de la Communaute Franaise de Belgique. S.B. is a fellow of the Televie. P.P.R. is a Research Associate of the FNRS.
First Published Online August 25, 2005
Abbreviations: AKAP, A-kinase anchoring protein; BrdUrd, bromodeoxyuridine; CDK, cyclin-dependent kinase; CNF1, cytotoxic necrotizing factor 1; DCB, dihydrocytochalasin B; EGF, epidermal growth factor; FBS, fetal bovine serum; GEF, guanosine nucleotide exchange factors; GST, glutathione S-transferase; HGF, hepatocyte growth factor; NIS, Na+/I– symporter; PKA, protein kinase A; pRb, retinoblastoma protein; ROCK, Rho kinases; Tg, thyroglobulin; ThOX, thyroid oxidase; TPO, thyroperoxidase.
Accepted for publication August 15, 2005.
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Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie (K.A.), Albert-Ludwigs-Universitat Freiburg, D-79104 Freiburg, Germany
Abstract
In thyroid epithelial cells, TSH via cAMP induces a rounding up of the cells associated with actin stress fiber disruption, expression of differentiation genes and cell cycle progression. Here we have evaluated the role of small G proteins of the Rho family and their impact on the actin cytoskeleton in these different processes in primary cultures of canine thyrocytes. TSH and forskolin, but not growth factors, rapidly inactivated RhoA, Rac1, and Cdc42, as assayed by detection of GTP-bound forms. Using toxins that inactivate Rho proteins (toxin B, C3 exoenzyme) or activate them [cytotoxic necrotizing factor 1 (CNF1)], in comparison with disruption of the actin cytoskeleton by dihydrocytochalasin B (DCB) or latrunculin, two unexpected conclusions were reached: 1) inactivation of Rho proteins by cAMP, by disorganizing actin microfilaments and inducing cell retraction, could be necessary and sufficient to mediate at least part of the cAMP-dependent induction of thyroglobulin and thyroid oxidases, but only partly necessary for the induction of Na+/I– symporter and thyroperoxidase; 2) as indicated by the effect of their inhibition by toxin B and C3, some residual activity of Rho proteins could be required for the induction by cAMP-dependent or -independent mitogenic cascades of DNA synthesis and retinoblastoma protein (pRb) phosphorylation, through mechanisms targeting the activity, but not the stimulated assembly, of cyclin D3-cyclin-dependent kinase 4 complexes. However, at variance with current concepts mostly derived from fibroblast models, DNA synthesis induction and cyclin D3-cyclin-dependent kinase 4 activation were resistant to actin depolymerization by dihydrocytochalasin B in canine thyrocytes, which provides a first such example in a normal adherent cell.
Introduction
THE SMALL G PROTEINS Rho, Rac, and Cdc42 are mainly involved in the control of the cytoskeleton architecture and cell movement (1), but they also participate in several other important cellular activities including the control of cell proliferation and specific gene expression (2). Classically, whereas activation of Rho induces actin polymerization and microfilament assembly, activation of Rac and Cdc42 cause lamellipodia and filopodia formation, respectively (3). These proteins are active when they bind GTP and are inactivated by the hydrolysis of GTP to GDP. Both the binding and hydrolysis are controlled by specific guanosine nucleotide exchange factors (GEFs) and GTPase-activating proteins respectively (4). One best-characterized effector pathway of RhoA-GTP is the activation of the Rho kinases (ROCK), which phosphorylate and activate the LIM domain-containing protein kinase (LIM kinase) to phosphorylate and inhibit the actin-depolymerizing protein cofilin, thus leading to microfilament assembly and stress fiber formation (5).
Experiments mainly performed in fibroblast systems have indicated that integrin-mediated cell adhesion to extracellular matrix, the resulting RhoA-dependent actin stress fiber formation and focal adhesion formation, and subsequent cell spreading are strictly required for induction of normal cell cycle progression by growth factors (6, 7). Demonstrated Rho/ROCK-dependent mechanisms include sustained activation by growth factors of the Ras/Raf/ERK cascade, G1 phase induction of cyclin D1 and down-regulation of the cyclin-dependent kinase (CDK) 2 inhibitors p21cip1 and p27kip1, leading to CDK4 and CDK2 activations, phosphorylation of the retinoblastoma protein (pRb) and entry into S phase (8). In addition to being needed for proliferation in nontransformed cells, Rho family proteins are necessary for oncogenic Ras-mediated transformation by reducing p21cip1 levels (9). The fundamental role of Rho proteins in mitogenic signaling by both growth factor and oncogenic Ras-dependent pathways, through cyclin D1 up-regulation and p21cip1 or p27kip1 down-regulation, has been extended to some epithelial cell lines (10, 11, 12).
The physiologically relevant model of canine thyroid epithelial cells in primary culture (13, 14) displays several interesting characteristics to reevaluate the relationships and proposed mechanisms between Rho family proteins, actin microfilament organization, cell function, expression of differentiation genes, and cell cycle progression. In these cells, a unique association of actin stress fiber disruption, expression of differentiation genes and cell cycle progression is concomitantly observed in response to TSH, through a cAMP increase. Via cAMP elevation and cAMP-dependent protein kinase [protein kinase A (PKA)] activation, TSH acutely induces phagocytosis (15) [the in vitro manifestation of the macropinocytosis of thyroglobulin (Tg) involved in stimulated thyroid hormone secretion (16)], which is associated to microfilament depolymerization and stress fiber disruption accompanied by dephosphorylation of cofilin (17) and myosin light chains (15). This precedes long-term morphological changes associated with modulation of the synthesis of various cytoskeleton proteins (18, 19, 20) and parallels the induction by TSH/cAMP of thyroid differentiation genes (21). Both the cAMP-dependent morphological changes and differentiation expression are inhibited by growth factors such as epidermal growth factor (EGF) (22) and hepatocyte growth factor (HGF) (23), which induce cell proliferation and cell cycle progression through a classical Ras/Raf/Erk/c-jun pathway leading to D-type cyclin induction (24, 25, 26, 27, 28). By contrast, in fully differentiated dog thyrocytes stimulated by TSH, the distinct (29) positive control of G0-S phase progression and DNA synthesis initiation by sustained cAMP elevations does not activate Ras- or phosphatidylinositol 3 kinase-dependent signaling cascades (26, 27, 28, 30) and does not up-regulate D-type cyclins (25). It nevertheless requires the activity of cyclin D3-CDK4 complexes (25), which are assembled (25), and subsequently activated depending on a continuous stimulation (31). Also unlike growth factors, TSH and cAMP paradoxically enhance the expression of p27kip1, which plays a positive role in the nuclear import and activity of cyclin D3-CDK4 complexes (32, 33).
In this work, we define the effects of thyroid signal transduction cascades on RhoA, Rac1, and Cdc42 activity, and through the use of specific Rho modulating toxins compared with actin depolymerization by dihydrocytochalasin B (DCB) or latrunculin B, their role in thyroid-specific gene expression and cell cycle regulation. Our data point to an important contribution of the cAMP-dependent inhibition of Rho-dependent stress fibers in the elusive cAMP-dependent regulation of some major thyroid differentiation genes, and provide evidence of a new cell cycle control target that depends on the activity of Rho proteins but not on actin polymerization, at variance with current concepts (7, 34).
Materials and Methods
Cell cultures
Dog thyroid epithelial cells (obtained according to a protocol approved by the institutional animal use committee) were cultured in monolayer (2 x 104 cells/cm2) in a medium that included DMEM, Ham’s F12 medium, and MCDB 104 medium (2:1:1, vol/vol) supplemented by ascorbic acid (40 μg/ml), and antibiotics (14). As indicated, bovine insulin (5 μg/ml) (Sigma, St. Louis, MO) was added or not to this medium since the seeding. Four or 5 d after seeding, quiescent cells were treated as indicated in figure legends. The various agents used in this study were: forskolin (Calbiochem, La Jolla, CA), fetal bovine serum (FBS) (Life Technologies, Inc., Paisley, Scotland, UK), and bovine TSH, murine EGF, TPA (12-O-tetradecanoylphorbol-13-acetate), and DCB, all purchased from Sigma. Latrunculin B was purchased from A.G. Scientific (San Diego, CA). Toxin B (35), the fusion toxin C2IN-C3 + C2II (36), and glutathione S-transferase (GST)-CNF1 (37) were prepared at the Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie (Freiburg, Germany). Human dermal fibroblasts (a cell strain derived from fetal skin) were cultured in DMEM + 10% FBS and made quiescent by a 3-d serum deprivation.
DNA synthesis
Cells in 3.5-cm Petri dishes were stimulated for 48 h by various mitogenic agents. Bromodeoxyuridine (BrdUrd) was added for the last 24 h. BrdUrd incorporation was detected by immunofluorescence, and BrdUrd-labeled nuclei were counted (1000/dish) as described (29).
Indirect immunofluorescence detection of actin (20)
Cells in Petri dishes were fixed with methanol for 10 min at –20 C, permeabilized with 0.1% Triton X-100 in PBD (pH 7.5), at room temperature and blocked for 20 min with normal sheep serum (5% in PBS containing 0.05% BSA [PBS/BSA]). They were then incubated for 2 h at room temperature or overnight at 4 C with a rabbit antiactin antibody (1/50, Sigma) diluted in PBS/BSA. After washing, the cells were then incubated for 2 h with fluorescein conjugated antirabbit IgG.
Western blotting detections of proteins
They were performed as described (33) using the following antibodies: p27kip1 (C-19) from Santa Cruz Biotechnology (Santa Cruz, CA), pRb (C-15) (Santa Cruz), CDK4 (Santa Cruz), cyclin D3 (DCS-22) and -tubulin (clone DM1A) from NeoMarkers (Fremont, CA), cyclin D1 (DCS-6) from Dr. J. Bartek (Danish Cancer Society, Copenhagen, Denmark). Our antibodies against Tg (22) and thyroid oxidase (ThOX1) (38) were characterized previously.
Immunoprecipitation and pRb-kinase assay
Cyclin D3-CDK4 complexes were immunoprecipitated using a monoclonal antibody against cyclin D3 (DCS-28; NeoMarkers, Fremont, CA) and their pRb-kinase activity was assayed exactly as described (31, 32). Immune complexes were incubated in the presence of 2 mM ATP and 0.5 μg of a 56-kDa pRb fragment (amino acids 379–928) (QED Bioscience, San Diego, CA). After SDS-PAGE separation and transfer to polyvinylidene difluoride membranes of proteins of the reaction mixture, the phosphorylation on Ser780 of the pRb fragment (specifically ascribed to CDK4) was detected using the phosphospecific-pRb (Ser780) antibody from Cell Signaling Technology (Beverly, MA). Membranes were then reprobed using antibodies for CDK4 (DCS-156 from Cell Signaling Technology) and cyclin D3.
Rho protein activation assays
The amount of GTP-bound Rho proteins was assessed by GST pull-down assay, using the Cdc42 and Rac1-interactive binding (CRIB) domain from human PAK1B (for the study of Rac 1 and Cdc42) or the Rho binding domain from murine Rhotekin (for the study of RhoA) fused to the GST protein. Plasmids coding for these proteins were gifts from Dr. J. G. Collard (The Netherlands Cancer Institute, Amsterdam, The Netherlands). Proteins were expressed as described (39). Small G protein activation was determined as follows: stimulated cells (from a 9- or 6-cm dish) were rapidly washed with ice-cold PBS, lysed and scraped in 800 μl of GST-fish buffer [10% glycerol, 50 mM Tris (pH 7.4), 100 mM NaCl, 1% Nonidet P-40, 2 mM MgCl2 supplemented with protease inhibitors]. The lysate was clarified by centrifugation. Supernatants (650 μl) were incubated for 30 min at 4 C with glutathione-agarose beads (Sigma) preloaded with the GST fusion protein. After incubation, beads were washed three times in GST-fish buffer and then resuspended in Laemmli buffer. Samples were analyzed by SDS-PAGE (15%) followed by transfer on polyvinylidene difluoride membranes. Immunodetections were performed using antibodies for Rac1 (Transduction Laboratories, Lexington, KY), Cdc42 (P1; Santa Cruz) or RhoA (26C4; Santa Cruz). Aliquots of whole-cell lysate were also resolved to ascertain that the same total amount of Rho proteins was assayed.
Northern blotting mRNA analyses
Subconfluent cell monolayers (from a 9-cm dish) were rapidly scraped in 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was extracted using the RNeasy Mini kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. Eight micrograms of total RNA were denatured in glyoxal and fractioned by electrophoresis on a 1% agarose gel. RNA quality was verified by acridine orange staining. RNAs were transferred to a nylon membrane (pall Biodyne A) using 20x saline sodium citrate. After baking, the blots were prehybridized and hybridized as described (24). The same filter was probed successively with different probes: dog thyroperoxidase (TPO) cDNA 2-kb BamHI, Tg cDNA 1.1-kb PstI, rat Na+/I– symporter (NIS) cDNA 2.4-kb EcoRI/XmnI (40), and human ThOX1 cDNA 4.6-kb EcoRI/XbaI fragments (38).
All the experiments were reproduced in independent cultures at least twice and in general three or four times with similar results.
Results
Disruption of actin stress fibers by TSH and cAMP but not by growth factors
Dog thyrocytes cultured for 4 d in basal control medium exhibited a dense network of stress fibers as shown by indirect immunofluorescence labeling of actin (Fig. 1). This was not modified by a treatment of 20 min with insulin (5 μg/ml), EGF (25 or 100 ng/ml), HGF (100 ng/ml) (Fig. 1) or 10% FBS (not shown). On the other hand, as previously described (20), TSH (1 mU/ml) and the general adenylyl cyclase activator forskolin (10–5 M) completely disrupted this microfilament network and induced membrane ruffling (Fig. 1). Previous studies have associated these acute processes to dephosphorylation of both myosin light chains and cofilin (15, 17). Within a few hours, TSH and forskolin then progressively promoted rounding up and cytoplasmic arborization of the cells that remain associated by dendrite-like extensions (see Fig. 3D) (20, 41). These morphological changes would be compatible with an involvement of Rac1 in the ruffling induced by the cAMP cascade, whereas reorganization of the actin cytoskeleton could be explained by RhoA inactivation in these conditions.
cAMP-dependent cascade but not growth factors inactivates Rho proteins RhoA, Rac1, and Cdc42
The effects of the different thyroid signal transduction cascades on the activation of Rac1, Cdc42, and RhoA were tested by the detection of the GTP-loaded forms of these proteins. As shown in Fig. 2, EGF (100 ng/ml) had no effect on Rac and Cdc42, but activated RhoA, after 1–30 min of stimulation. In parallel experiments using the same assay, carbachol did activate Rac1 (42). On the other hand, TSH (1 mU/ml) and forskolin (10–5 M) inactivated not only RhoA but also Rac1 and Cdc42 (Fig. 2). The same results were obtained in cells previously cultured for 4 d in the presence of insulin (not shown). The ruffling induced by cAMP cascade activating agents was thus not a result of Rac1 activation in dog thyrocytes. The disappearance of RhoA-GTP induced by TSH and forskolin was consistent with the hypothesis that the disruption of actin stress fibers is mediated by RhoA inactivation.
Rho protein inactivation is involved in the cAMP-dependent morphological changes
To determine whether the inhibition by cAMP of the three Rho family proteins (Rho, Rac, and Cdc42) could play a role in the cAMP-dependent functions of dog thyrocytes, we have incubated the cells with the Clostridium difficile toxin B. Depending on internalization via receptor-mediated endocytosis and possibly proteolytic release of its active enzyme domain, toxin B monoglucosylates Rho subfamily proteins Rho, Rac, and Cdc42 at Thr35/37 (43). This glucosylation inactivates the Rho GTPases by inhibiting their effector coupling (44). Exposure of cells to 100 ng of toxin B/ml (but not 500 pg/ml) for 24 h efficiently inhibited binding of Rac1 and RhoA to their effectors (Fig. 3A). These inhibitory effects persisted at least for 3 d (not shown). Moreover, the presence of total Rac1 and Cdc42 proteins, but not RhoA, was strongly reduced by toxin B (Fig. 3A), as observed by others, likely because these glucosylated proteins are more degraded by the proteasome (45, 46). Treatment of dog thyrocytes with toxin B (100 ng/ml, 24 h), much like forskolin, disrupted the actin cytoskeleton and potently induced cell rounding-up and arborization (Fig. 3D). Preliminary experiments had shown that such a long treatment with toxin B was required to induce these effects in the denser parts of the cell monolayer, as described in other polarized epithelial cells where toxin B receptors might be more concentrated on the basolateral side (47).
To study the involvement of Rho apart from Rac/Cdc42, thyrocytes were also incubated with the C3 exoenzyme, which selectively inactivates RhoA, RhoB and RhoC by catalyzing their ADP-ribosylation at Asn41 (48). Because C3 and related C3-like transferases do not readily enter the cells, we used the fusion toxin C2IN–C3, comprising the catalytic domain of C3 transferase of C. limosum and part of the C2I toxin of C. botulinum, which together with the activated binding component C2II of the C2 toxin allows efficient uptake into the cells (36). Such a C3 treatment also induced the disorganization of the actin cytoskeleton and cell retraction associated with even more conspicuous dendrite-like extensions of some cells (Fig. 3D).
On the other hand, to activate Rho, Rac, and Cdc42 and thus prevent their inhibition by cAMP, we have also treated the cells with CNF1. This toxin, produced by uropathogenic Escherichia coli, deamidates the glutamine 61/63 of Rho, Rac, and Cdc42, which blocks the intrinsic or the GTPase-activating protein-induced hydrolysis of GTP, leading to the permanent activation of these G proteins (49). As shown in Fig. 3B, Rac1 and Cdc42 were strongly activated by treatment of the cells for 24 h with the CNF1 toxin fused to the GST protein (GST-CNF1). Although the amount of Rac1-GTP was increased by GST-CNF1, the total amount of Rac1 was decreased. This decrease, also observed by others (50, 51), could result from a proteasome-mediated degradation of deamidated Rac1. RhoA activation by GST-CNF1 was reflected in SDS-PAGE by the upward 1-kDa shift characteristic of CNF1-induced deamidation of RhoA (52) (Fig. 3C). Preincubation of the cells for 8 h with GST-CNF1 prevented the actin stress fiber disruption induced by forskolin (Fig. 3D).
Collectively, these observations suggested that the cAMP-induced inactivation of Rho proteins, particularly RhoA, is both necessary and sufficient to mediate the cAMP-dependent actin reorganization in dog thyrocytes.
Rho-mediated actin cytoskeleton reorganization plays a crucial role in the cAMP-dependent expression of thyroid differentiation genes
To investigate whether the modulation of Rho proteins could be involved in the expression of some differentiation characteristics of the thyroid, we have assessed the effect of toxin B and CNF1 on the level of Tg and ThOX proteins assayed by western blotting, as well as on the accumulation of the mRNAs of these genes and of the NIS and TPO genes by Northern blotting. To evaluate the role of actin organization, the effects of toxin B were compared with those of DCB, a membrane permeant inhibitor of actin polymerization, which unlike cytochalasin B has little effect on sugar transport. Disruption of the actin cytoskeleton was seen 1 h min after DCB administration (Fig. 3D), culminating in complete cell retraction by 5 h (Fig. 3D), and these effects persisted for at least 48 h (not shown). Before treatment with these various agents, thyrocytes were cultured for 3 d in the presence of EGF (25 ng/ml) to repress Tg expression (22). Cells were then transferred in basal medium without EGF, pretreated for 24 h with toxin B (100 ng/ml), or for 8 h with either DCB (10 μM) or GST-CNF1 (1.5 μg/ml), and then stimulated or not for 48 h with forskolin in the presence of these different drugs. As previously shown (21, 22, 38, 40), forskolin strongly induced the accumulation of Tg, TPO, NIS, and ThOXs proteins and/or mRNAs (Fig. 4). Toxin B or DCB did not affect the induction by forskolin of Tg and ThOXs proteins (Fig. 4A). Surprisingly, toxin B or DCB used alone induced an accumulation of Tg and ThOXs proteins and mRNAs (Fig. 4, A and B), but as previously shown in the case of forskolin stimulation (19), they reduced the presence of -tubulin (Fig. 4A). The same result was obtained with latrunculin B (0.1–10 μM), another actin polymerization inhibitor (53) (not shown). These drugs did not increase cellular cAMP levels (not shown). On the other hand, GST-CNF1, which activates Rho proteins, decreased the induction by forskolin of not only Tg and ThOXs but also of NIS and TPO mRNAs (Fig. 4). Nevertheless, on the contrary of what we observed for Tg and ThOXs, toxin B and DCB did not induce the accumulation of TPO and NIS mRNAs (Fig. 4B).
Therefore, inactivation of Rho proteins by cAMP appeared to be instrumental in the cAMP-dependent expression of these major thyroid differentiation genes. Furthermore, disruption of the actin cytoskeleton mediated by cAMP-dependent inactivation of Rho proteins was sufficient to mimic in part the cAMP-dependent induction of Tg and ThOXs, but not the induction of NIS and TPO.
Rho protein activity, but not actin cytoskeleton integrity, is required for DNA synthesis induction in dog thyrocytes
Quiescent dog thyroid cells cultured in the presence of insulin were pretreated for 24 h with various concentrations of toxin B and then stimulated with TSH, EGF, or TPA for 48 h in the presence of this drug. Toxin B at 10 and 100 ng/ml inhibited DNA synthesis induced by all these agents (Fig. 5A) and also by forskolin (10–5 M) or HGF (50 ng/ml) (not shown). Toxin B did not affect cell viability, as judged from trypan blue staining and Hoechst dye staining of nuclei (not shown). Rho could be an important target of the inhibition of DNA synthesis by toxin B. Indeed, the more selective toxin C3 (the C2IN-C3/C2II complex) also inhibited the stimulation of DNA synthesis by TSH, EGF, or TPA (Fig. 5B). On the other hand, simultaneous activation of Rho, Rac and Cdc42 proteins by a 48-h treatment with GST-CNF1 (1.5 μg/ml) in the presence of insulin was not sufficient to induce DNA synthesis (not shown), at variance with the induction of DNA synthesis reported for Swiss 3T3 cells (54). Therefore, some activity of the Rho proteins was necessary for the mitogenic stimulations of dog thyrocytes by both cAMP-dependent and cAMP-independent cascades, but the activation of these proteins was not sufficient to elicit such an effect.
In the same experiments, DCB (10 μM) for 1 or 5 h completely disrupted the actin stress fibers (Fig. 3D), but it did not prevent DNA synthesis induced by the subsequent administration of TSH, EGF, or TPA while maintaining DCB presence (Fig. 5A). In other experiments, DCB also did not prevent DNA synthesis stimulated by EGF + serum (10%) (not shown). This surprising observation sharply contrasted with the complete inhibition by the same treatment with DCB (10 μM, added 1 h before mitogenic stimulation) of serum-stimulated DNA synthesis in cultured human skin fibroblasts (Fig. 5C), as observed in various fibroblast or endothelial cell systems (55, 56, 57, 58). The inhibitory effects of toxin B and C3 on dog thyrocyte DNA synthesis were thus not mediated by actin cytoskeleton alterations.
Toxin B, but not dihydrocytochalasin B, inhibits pRb phosphorylation
Dog thyroid cells were pretreated with toxin B (24 h) or DCB (1 or 24 h) before stimulating them in the presence of these drugs for 20 and 32 h with TSH (maximum cAMP-dependent mitogenic stimulation) or with the combination of EGF and 10% FBS (ES; maximum cAMP-independent mitogenic stimulation). Hyperphosphorylation of pRb induced by both mitogenic stimulations was clearly inhibited by toxin B (Fig. 6A). In sharp contrast, DCB did not affect pRb phosphorylation induced by TSH or EGF + serum, irrespective of the timing of its addition (1 h or even 24 h before administration of mitogens) (Fig. 6A). pRb phosphorylation was thus a critical target of the actin-independent requirement for Rho protein activity in dog thyroid cell cycle progression.
Regulation of cell cycle regulatory proteins by Rho proteins and actin cytoskeleton integrity
To identify the molecular basis of the requirement for Rho protein activity in the pRb phosphorylation process, the expression of G1 phase cyclins, CDK4 and p27kip1 was analyzed in dog thyrocytes treated or not with toxin B. DCB treatment was also included in these experiments, but quantitative comparison between toxin B and DCB treatments is limited by the different timing of their respective administrations justified by their different modes and kinetics of action. As previously shown (25), the levels of cyclin D3 and of the less abundant cyclin D1 were moderately increased by a 20-h treatment with EGF + serum (ES) but not by TSH (Fig. 6B). Conversely, stimulation of dog thyroid cells for 20 h or 32 h with TSH, but not ES, increased the expression of p27kip1 (Fig. 6B) (33). Both Toxin B and DCB reduced the basal expression of cyclin D3 and they also inhibited the accumulation of cyclin D1 and cyclin D3 stimulated by ES (Fig. 6B). Toxin B increased the amount of p27kip1 in all the conditions (but no longer in the presence of TSH at 32 h) (Fig. 6B). This was partly reproduced by DCB only in the absence of the mitogenic factors (Fig. 6B). On the other hand, CDK4 concentration was not modified by toxin B and DCB (Fig. 6B). Therefore, as in other cell types D-type cyclin expression depended at least in part on Rho protein-dependent integrity of the actin cytoskeleton. By contrast, the accumulation of p27kip1 in response to Rho protein inactivation might be partly independent of the actin cytoskeleton.
The activity of Rho proteins, but not actin cytoskeleton integrity, is required for the activation of cyclin D3-CDK4
In dog thyrocytes, TSH and cAMP weakly affect the accumulation of D-type cyclins or CDK4 (as seen in Fig. 6B), but they induce the assembly of required complexes of cyclin D3-CDK4 (25). EGF + serum also stimulates the formation and activity of cyclin D3-CDK4 complexes. To understand the mechanism by which toxin B inhibits the phosphorylation of pRb, dog thyrocytes were pretreated for 24 h with toxin B (100 ng/ml) or 1 h with DCB (10 μM) and the formation and activity of cyclin D3-CDK4 complexes were analyzed 20 h after cell stimulation by TSH or EGF + serum (ES) in the presence of these drugs. Consistent with its inhibitory effect on pRb phosphorylation described above, toxin B strongly inhibited the pRb-kinase activity associated with cyclin D3 in cells stimulated by TSH or ES (Fig. 7). By contrast, interestingly, toxin B did not prevent the association of CDK4 with cyclin D3 induced by these mitogenic agents. On the other hand, as expected because it did not inhibit pRb phosphorylation, DCB weakly affected the assembly and activity of cyclin D3-CDK4 complexes (Fig. 7). Our observations thus suggested that the activity of Rho proteins, independently of their effect on the actin microfilaments, was required for the activation but not for the assembly of cyclin D3-CDK4 in response to different mitogenic stimulations. These results identify a new Rho-dependent mechanism that is limiting for pRb-phosphorylation and thus cell cycle progression independently of actin cytoskeleton organization.
Discussion
Rho protein inactivation and actin depolymerization contribute to the regulation of thyroid cell function and gene expression by TSH and cAMP
The present study in canine thyrocytes shows for the first time that the TSH-cAMP pathway inactivates the three small G proteins RhoA, Rac1 and Cdc42. A similar inhibition of RhoA activity by cAMP has been described in other cell types including melanocytes and endothelial, lymphoid and neuronal cells (59, 60, 61). By contrast, cAMP and PKA activate Rac1 or Cdc42 (61) in various cell systems (62, 63), although Rac inactivation by PKA has also been described in endothelial cells (64). The membrane ruffling induced by cAMP in dog thyrocytes is thus not mediated by activation of Rac1 but through alternative pathways, which is consistent with reports indicating that Rac inactivation does not eliminate membrane ruffling in some systems (65, 66).
As in other cell systems, Rho inactivation in dog thyrocytes appears both necessary and sufficient to mediate the reorganization of actin microfilaments and cell rounding up (41) induced by TSH through cAMP and PKA activation, inasmuch as they were mimicked not only by toxin B but also by the C3 exoenzyme and prevented by Rho protein activation by CNF1 toxin. The cAMP-dependent inactivation of RhoA could result from its phosphorylation at Ser188 by PKA (59, 60), which promotes formation of RhoA:RhoGDI complexes, thereby decreasing the membrane association of RhoA (59, 67) and the binding of RhoA to its effector Rho kinase (60). PKA could also inhibit Rho signaling by phosphorylating the recently identified AKAP-Lbc, an A-kinase anchoring protein (AKAP) with a Rho-GEF activity. This phosphorylation promotes the binding of this AKAP to a 14-3-3 protein, blocking its Rho-GEF activity (68). The best known RhoA-GTP downstream effectors are ROCK, which phosphorylate several proteins controlling actin organization, such as LIM kinases. The resulting inhibition of LIM kinase (5) probably causes the dephosphorylation and activation of cofilin previously demonstrated in dog thyrocytes (17), itself leading to actin filament network disruption. Because ROCK also catalyzes the inhibitory phosphorylation of myosin phosphatase (69), the cAMP-dependent inhibition of RhoA should similarly contribute to explain the inhibition of myosin light chains phosphorylation observed in dog thyrocytes stimulated by TSH and cAMP (15).
Presumably as a result of both these effects, TSH through cAMP increases the level of unpolymerized G-actin in thyroid cells (70). One functional consequence of such an effect in vivo is to facilitate endocytosis (71). For example, RhoA activation blocks through ROCK the engulfment of apoptotic bodies in a phagocytic Chinese hamster ovary cell line and a macrophage cell line (72). Indeed, the first major acute functional effect of the TSH-cAMP pathway in thyroid is to induce the macropinocytosis of Tg, and after the proteolysis of this protein, thyroid hormone secretion (16). In dog thyroid cell culture, this acute functional effect is reflected by an enhanced phagocytic activity (15). Inhibition of RhoA by TSH and cAMP could therefore play a crucial role in stimulated thyroid hormone secretion.
Whereas the mechanisms responsible for the cell specificity of expression of thyroid differentiation genes, including Tg, have been detailed in depth (73), the mechanisms underlying their transcriptional regulation by TSH and cAMP (21, 74) remain elusive despite intensive investigation (75, 76). Our study provides the first evidence of an important role of the inactivation by cAMP of Rho-mediated actin polymerization in the cAMP-dependent expression of some major thyroid differentiation genes, much like what has been described in the case of cAMP-induced melanoma cell differentiation (77). Indeed, activation of Rho proteins by the CNF1 toxin repressed the induction by forskolin of thyroid differentiation genes including Tg, thyroperoxidase, NIS and ThOXs. Furthermore, inactivation of Rho proteins by toxin B and even microfilament disruption by dihydrocytochalasin B or latrunculin B were sufficient to partially mimic the induction by TSH and cAMP of Tg and ThOX proteins and mRNAs in dog thyrocytes. Interestingly, the oncogenic dedifferentiating RET/PTC rearrangement of papillary thyroid carcinomas stimulates the formation of Rho-dependent stress fibers in PC Cl3 thyroid cell line (78), and constitutively activated RhoA represses the transcription of the Tg gene in part by interfering with the activity of the thyroid transcription factor TTF-1 in FRTL-5 cells (79). Further studies should thus evaluate in vivo the importance of the relationship between Rho activity and cell shape in the physiopathologic control of Tg synthesis during TSH stimulation or in the dedifferentiation associated with carcinogenic processes.
In the present study, a congruence of the effects of TSH/cAMP, toxin B and DCB was observed not only on the induction of Tg and ThOXs, but also on cell cycle regulatory proteins: partial repression of cyclin D3 [mostly demonstrated in the absence of insulin in the case of the TSH effect (80)] and accumulation of p27kip1. The reduction by TSH and cAMP of the synthesis of various cytoskeleton proteins in dog thyrocytes, including actin (19), high-molecular-weight tropomyosins isoforms that stabilize the actin stress fibers (20, 81), vimentin (18), and -tubulin (Fig. 4A) (19) could also depend on the acute reorganization of actin cytoskeleton mediated by Rho inactivation. In agreement with previous proposals (82, 83), we have suggested that this conclusion could be extended to a large fraction of modifications of gene expression by TSH and cAMP because their effects on the synthesis of most of 45 unidentified proteins are mimicked by culturing unstimulated human thyrocytes as dense aggregates instead of monolayers that also reduces cytoskeleton organization (81). Mechanisms involved in the control of gene expression by cell shape and cytoskeleton organization are complex and still poorly understood (83). Integrins, which connect the actin cytoskeleton to focal adhesion sites, appear to play the role of major receptors for mechanotransduction (7, 34). G actin itself directly controls gene expression (84, 85, 86) by interfering with some transcription coactivators (85, 87). Changes in the actin cytoskeleton also affect the stability and translation of some mRNAs (88, 89).
Thyroid cell S phase entry depends on Rho proteins but resists to cytochalasin
The inhibition of dog thyroid cell S-phase entry stimulated by TSH or growth factors by toxin B and exoenzyme C3 generalizes the requirement of an activity of Rho proteins for cell cycle progression, as in Swiss3T3 and NIH3T3 fibroblasts or rat aortic smooth muscle cells (90, 91, 92). Thus, even in the cAMP-dependent mitogenic stimulation associated with a reduction by cAMP of the activity of Rho proteins, a residual activity of at least one of these proteins must exert a crucial permissive action for DNA synthesis induction. This apparent paradox is by no means unique in the analysis of the cAMP-dependent mitogenesis of thyrocytes. For instance, unlike growth factors, cAMP strongly represses cyclin D3 in dog thyrocytes, but in the presence of insulin that partly overcomes this inhibition, cyclin D3 is specifically required in the mitogenic stimulation by cAMP, but not in the stimulation by growth factors (25, 80). Inactivation of RhoA by C3 and dominant-negative RhoA also induces G1 arrest in rat thyroid FRTL-5 cells (93), whereas in rat thyroid WRT cells a dominant-negative Rac1 impairs cAMP-stimulated DNA synthesis (94).
The required involvement of Rho proteins and actin cytoskeleton integrity for D-type cyclins up-regulation and p27kip1 down-regulation, as demonstrated in many cell systems (7, 11, 95), are apparently confirmed in the present experiments. In dog thyrocytes, toxin B and cytochalasin (DCB) partly inhibited the basal or EGF + serum-stimulated accumulation of cyclin D1 and cyclin D3, and treatment with toxin B (more potently than DCB) sufficed to up-regulate p27kip1. However, these effects are clearly insufficient to explain the strong inhibition by toxin B of pRb phosphorylation and G1/S transition stimulated by cAMP-dependent and cAMP-independent mitogenic pathways: 1) the partial reduction of cyclin D3 levels was insufficient to lead to a decrease of cyclin D3-CDK4 complexes in cells stimulated by TSH or EGF + serum; 2) in dog thyrocytes stimulated by TSH, up-regulated p27kip1 supports, rather than inhibits, the activity of cyclin D3-CDK4 complexes (32); 3) cyclin D1 and D3 levels were similarly reduced by toxin B and DCB, but DCB did not inhibit pRb phosphorylation and DNA synthesis, in sharp contrast with the situation found in fibroblasts and vascular cells.
A new mechanism should thus explain the present inhibition of pRb phosphorylation by Rho protein inactivation, which, at variance with the current concepts (6, 7, 34), is not mediated by actin depolymerization and stress fiber disruption. Strikingly, Rho protein inactivation by toxin B did not inhibit the mitogen-dependent assembly of cyclin D3-CDK4 complexes, the mechanism of which is still unknown in dog thyrocytes stimulated by TSH/cAMP (25, 33) as in fibroblasts stimulated by growth factors. Instead, we show here that toxin B, but not DCB, inhibits the pRb-kinase activity of the assembled cyclin D3-CDK4 complexes, which is thus demonstrated as a new target for cell cycle control by Rho proteins. This observation provides new evidence supporting the concept that the catalytic activity of cyclin D-CDK complexes critically depends on additional regulated mechanisms (31, 32), besides the induction of D-type cyclins and their association with CDK and p27kip1. The nuclear translocation of the cyclin D3-CDK4 complex (33) and the phosphorylation of CDK4 within this complex have been identified as regulated targets in dog thyrocytes (31, 32). Interestingly, the homologous Thr160-phosphorylation of CDK2 has been found to directly depend on pathways inhibited by statins (96), which prevent the membrane translocation and activation of RhoA (10).
Studies using drugs, such as cytochalasin D, that disrupt microfilaments have suggested that normal cells (in fact mostly fibroblasts and capillary endothelial cells) require an intact actin cytoskeleton to pass through the G1/S restriction point (34, 55, 56, 57, 58). For instance, kinetics analysis using cytochalasin pulses have defined a narrow time window of 3 h just before restriction point in which actin cytoskeleton integrity is critical for G1/S transition (58). Our unexpected finding that the G1/S transition elicited by a variety of mitogens in dog thyrocytes was resistant to sustained actin depolymerization by continuous presence of cytochalasin D and subsequent cell rounding-up, demonstrates that the actin cytoskeleton-sensitive cell cycle checkpoint(s) is a cell type-specific characteristic. To our knowledge, induction of DNA synthesis in the presence of cytochalasin has only been observed in some transformed cells (97), and in lymphocytes (98) that proliferate in suspension culture. The absence of an actin-dependent cell cycle checkpoint in thyroid epithelial cells explains that their proliferation is clearly compatible with a reduced polymerization of actin and disruption of stress fibers induced by TSH and cAMP or by phorbol esters (20). DNA synthesis responses to EGF (99) or to TSH/cAMP (100) have been demonstrated in suspension cultures of thyroid follicles, which do not form stress fibers (101). Except for endothelial cells and wound fibroblasts, actin stress fibers are not detected in most proliferating cell types in vivo (102). Therefore, the cell proliferation requirement for spreading on a solid substrate and associated formation of stress fibers may appear as a specialized characteristic of some cell types, such as fibroblasts and endothelial cells, in order for them to obey the specific spatial constraints of their proliferation in vivo. It is not a major determining factor for the proliferation of thyroid follicle epithelial cells.
Acknowledgments
We thank Dr. J. G. Collard and J. P. ten Klooster for kindly providing us the GST-PAK-CRIB and C21-GST constructions, and Dr. J. Bartek for cyclin D1 antibody. We are also grateful to C. Degraef for technical assistance, S. Paternot for technical advice and help in pRb-kinase assay and L. Giandon (Cytogenetic Laboratory, Erasme Hospital) for the fibroblasts culture.
Footnotes
This work was supported by grants from the Fonds National de la Recherche Scientifique (FNRS), Fonds de la Recherche Scientifique Medicale (FRSM), Operation Televie, Fonds pour la formation a la Recherche dans l’Industrie et l’Agriculture (FRIA), Fondation Rose et Jean Hoguet, Fondation Van Buuren, Ple d’Action Interuniversitaire (PAI), Federation Belge Contre le Cancer, and Actions de Recherche Concertees (ARC) de la Communaute Franaise de Belgique. S.B. is a fellow of the Televie. P.P.R. is a Research Associate of the FNRS.
First Published Online August 25, 2005
Abbreviations: AKAP, A-kinase anchoring protein; BrdUrd, bromodeoxyuridine; CDK, cyclin-dependent kinase; CNF1, cytotoxic necrotizing factor 1; DCB, dihydrocytochalasin B; EGF, epidermal growth factor; FBS, fetal bovine serum; GEF, guanosine nucleotide exchange factors; GST, glutathione S-transferase; HGF, hepatocyte growth factor; NIS, Na+/I– symporter; PKA, protein kinase A; pRb, retinoblastoma protein; ROCK, Rho kinases; Tg, thyroglobulin; ThOX, thyroid oxidase; TPO, thyroperoxidase.
Accepted for publication August 15, 2005.
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