| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLE |
Institute of Interdisciplinary Research, Université Libre de Bruxelles, Campus Erasme, B-1070 Brussels, Belgium
Address all correspondence and requests for reprints to: F. Vandeput, Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Campus Erasme, Building C, Route de Lennik 808, 1070 Brussels, Belgium. E-mail: fvdeput{at}ulb.ac.be.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We found that TSH and forskolin are without effect on the phosphorylation and activity of the different classes of MAPKs. In contrast, all the cAMP-independent growth factors, whereas without effect on the phosphorylation and activity of JNKs and p38 MAPK, stimulated the ERKs. This effect was strong and sustained in response to HGF, epidermal growth factor and 12-myristate 13-acetate but weak and transient in response to insulin. Moreover, whereas in stimulated cells DNA synthesis was inhibited by PD 098059, an inhibitor of MAPK kinase 1 and consequently of ERKs, it was not modified by SB 203580, an inhibitor of p38 MAPK.
Taken together, these data 1) exclude a role of JNKs and p38 MAPK in the proliferation of dog and human thyrocytes; 2) suggest that the mitogenic action of the cAMP-independent agents requires a strong and sustained activation of both ERKs and phosphatidylinositol 3-kinase/protein kinase B as realized by HGF alone or by the other agents together with insulin; and 3) show that TSH and cAMP do not activate ERKs but that the weak activation of ERKs by insulin is nevertheless necessary for DNA synthesis to occur.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our ultimate goal is the understanding of the regulation of mitogenesis in the human thyroid cell. For availability reasons, dog thyroid cells are used as our main experimental system as, from what is known at present, it is in this species that the regulation of the thyrocytes presents the closest similitude with their human counterpart (12). Results are then extended to the human cell. In both cases, primary cultures were chosen as they are closer to physiology than the immortalized cell lines which are generally used.
In dog and human thyroid cells, three types of signals trigger DNA synthesis: EGF acting through a receptor tyrosine kinase, phorbol 12-myristate 13-acetate (PMA), a stimulator of protein kinase C, and TSH acting through an increase in the intracellular concentration of cAMP (13, 14). The mitogenic effect of these agents requires IGF-1 receptor stimulation by IGF-1 or by high concentrations of insulin (14, 15). By themselves, IGF-1 and insulin have little mitogenic action. As the real physiological stimulator of thyroid cells is TSH, the role of insulin/IGF-1 is described as permissive for TSH and, by extension, for EGF and PMA action. Hepatocyte growth factor (HGF) is the only growth factor so far that can induce DNA synthesis and proliferation in dog but not human thyrocytes cultured without insulin/IGF-1, thus acting as a full mitogenic factor (12). Although activation of ERKs has been proposed to be a common step of all mitogenic cascades (16), we have shown, in a previous work, that in the dog thyrocyte an increase in the phosphorylation of these kinases was not involved in the mitogenic action of TSH (17). However, TSH and cAMP activation of ERKs have been demonstrated in the Fischer rat thyroid (FRTL-5) cell line (18). More recently, we have also shown that TSH was without effect on the phosphatidylinositol (PI) 3-kinase/protein kinase B (PKB) pathway (19) which is also believed to play a major role in the induction of cellular proliferation (20).
With the hope to elucidate the mechanism by which TSH, through cAMP, is a signal for growth, the present study examined its effect, in the dog and human thyrocytes, on the phosphorylation and activity of the different classes of MAPKs, i.e. ERKs, JNKs, and p38 MAPK. This has been compared with the effect of HGF, EGF, PMA and several stress agents, i.e. sorbitol 400 mM, Na arsenite, and anisomycin. Moreover, the influence of PD 098059, an inhibitor of MEK1 and consequently of ERKs activation, and of SB 203580, an inhibitor of p38 MAPK have been assessed on the induction of DNA synthesis. In addition, to unravel the mechanism(s) of cooperation between insulin/IGF-1 and different growth factors leading to cell proliferation, we have also studied the effect of insulin/IGF-1 on the phosphorylation and activity of the different subfamilies of MAPKs. In a previous work, we had already shown that, in dog thyrocytes, insulin strongly stimulates the PI 3-kinase/PKB pathway, and we had suggested that this effect might account for the permissive action of insulin in thyrocytes proliferation (19).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
32P]ATP, and [3H]thymidine were obtained from Amersham International (Little Chalfont, Buckinghamshire, UK).
Primary culture
Cells were obtained from dog and human thyroid tissue. Dog thyroids were taken out of euthanized animals that had been used for physiological experiments. Normal human thyroid tissue was obtained from patients undergoing surgery for partial thyroidectomies following the rules of the University Hospital Ethical Committee. The normal histological appearance of the tissue was checked by a pathologist. Follicles were isolated by mild digestion of fresh tissue with collagenase (50 mg/ml) and, in the case of human tissue, with collagenase (50 mg/ml) and dispase (100 µg/ml). The follicles were cultured in monolayer (2 x 104 cells/cm2) in the following medium: DMEM/Ham’s F-12 medium/MCDB 104 medium (2:1:1, by vol) supplemented with 2 mM sodium pyruvate, 40 µg/ml ascorbic acid, 100 U penicillin/ml, 100 µg streptomycin/ml, fungizone 2.5 µg/ml. Human thyrocytes were cultured for the first 24 h with 1% serum to ensure optimal spreading of the follicles. After culture for 4 d, the cells were stimulated for various periods of time with different agents: bovine TSH (1 mU/ml), forskolin (10-5 M), human recombinant HGF (50 ng/ml), bovine insulin (5 µg/ml), PMA (10 ng/ml), EGF (25 ng/ml), sorbitol (400 mM), anisomycin (25 ng/ml), or sodium arsenite (100 µM). In some cases, cells were treated with PD 098059 (10–50 µM) or with SB 203580 (10 µM) for 30 min before activation.
DNA synthesis
After 4 d of culture, dog thyroid cells were incubated for 48 h with the mitogenic agents. Bromodeoxyuridine (BrdU) was added for the last 24 h. The number of cells entering into DNA synthesis was estimated by the frequency of BrdU-labeled nuclei, as revealed by immunofluorescence. In each triplicate dish, at least 1000 nuclei chosen at random in different fields were counted in blind (21).
Gel electrophoresis and immunodetection of proteins
At the end of the culture period, total cellular proteins were separated according to molecular mass by SDS-PAGE (10% gel) and immunodetected after Western blotting as described previously (22) with antibodies against phospho-ERKs, -JNKs, and -p38 MAPK. Immunoblots were detected by chemiluminescence using the ECL kit (Amersham International). After stripping, the membrane was reprobed with anti ERK 2 or anti p38 MAPK antibodies, which served as gel loading controls.
In vitro kinase assays
After stimulation, the cells were solubilized in Lysis buffer [120 mM NaCl, 25 mM Tris/HCl (pH 7.6), 1 mM benzamidine, 1 mM EDTA, 2 mM EGTA, 50 mM NaF, 15 mM sodium pyrophosphate, 1.5 mM MgCl2, 20 mM ß-glycerophosphate, 0.5% sodium deoxycholate, 1% (vol/vol) Nonidet P-40, 60 µg/ml Pefabloc, 1 mM Na3VO4, and 10 µg/ml each of leupeptin and pepstatin]. Protein samples were subjected to immunoprecipitation using specific antibodies coupled to protein A-Sepharose. Samples containing 200 µg protein or 30 µg protein were subjected to immunoprecipitation using polyclonal antibodies to p46 JNK, p38 MAPK, or ERK2, respectively. After washing the immunoprecipitates twice with Lysis buffer and twice with kinase buffer [25 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM ß-glycerophosphate, 20 mM tetra-sodium diphosphate, 1 mM Na3VO4, 2 mM dithiothreitol], kinase activity was assayed in a final volume of 30 µl kinase buffer containing 20 µM ATP, 3 µCi [32P]ATP, and 2 µg GST-c-Jun, 2 µg ATF-2, or 5 µg myelin basic protein for p46 JNK, p38 MAPK, or ERK2, respectively. The phosphorylation reaction was allowed to proceed for 15 min at 30 C. Reaction mixtures were resolved by SDS-PAGE and the amount of 32P incorporated into the substrate was quantified using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Inositol phosphate (IP) determinations
For IP determinations, after 3 d of culture in the presence of insulin (5 µg/ml), cells were incubated with 10 µCi/ml [3H]inositol. The next day, dishes were washed three times with Krebs-Ringer-HEPES buffer (KRH), preincubated in KRH plus LiCl (10 mM) for 30 min, and then incubated for 15 min in the previous medium supplemented with different concentrations of TSH, 10-5 M forskolin or 100 µM ATP, the two last steps being performed at 37 C. The incubation was stopped by the addition of ice-cold 3% HClO4, and 3H-labeled IPs (inositol mono-, bis-, and trisphosphates) were isolated and assayed by stepwise chromatography on AG1 x 8 resin (23). The cell debris in the bottom of the dishes was dissolved in 1 M NaOH and counted as PIs. Results are expressed as the percent radioactivity incorporated in IPs over the sum of radioactivity in IPs and PIs.
cAMP determinations
After 4 d of culture, cells were washed with KRH, preincubated in this medium for 30 min at 37 C, and then incubated for 15 min at the same temperature in the absence or in the presence of the different agents. At the end of the incubation, the medium was aspirated and replaced by 0.1 M HCl. The cell extracts were dried in a vacuum concentrator, and cAMP determination was performed by competitive binding assay, as described previously (24). Results are expressed as picomoles of cAMP per dish.
Statistical methods
One-way ANOVAs were performed followed by the multiple comparison test of Tukey.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). DNA synthesis was evaluated as the percentage of BrdU-labeled nuclei. The role of JNKs was not assessed in this way as, until now, no specific inhibitor of this enzyme has been described. Table 1 shows that PD 098059 (50 µM) but not SB 203580 (10 µM) strongly inhibits DNA synthesis in cells stimulated by TSH, EGF, or PMA in the presence of insulin, or by HGF alone. Figure 1 shows, in a representative experiment, that in cells stimulated by TSH, forskolin, EGF, and PMA in the presence of insulin or by HGF alone, the inhibition, by PD 098059, of DNA synthesis and of ERKs activity is concentration dependent. As activation of ERKs occurs through phosphorylation of threonine and tyrosine at the Thr-Glu-Tyr motif, an antibody raised against this phosphorylated motif was used to detect activated ERKs after separation of total cellular proteins by SDS-PAGE and Western blotting. A significant inhibition of ERKs phosphorylation and of DNA synthesis was already observed in the presence of 10 µM PD 098059 for all conditions tested except in cells incubated with PMA plus insulin, a condition that resulted in the strongest stimulation of ERKs. In this case, 20 µM PD 098059 was required to decrease significantly both ERKs phosphorylation and DNA synthesis. It is worth noting that even at the highest concentration of PD 098059 tested there was no evidence of nonspecific toxicity, i.e. no decrease in cell number or increase in the low number of floating cells was observed. The lack of effect of SB 203580 on DNA synthesis shown in Table 1 was confirmed in additional experiments in which 3.3, 5, or 10 µM SB 203580 were added every 8 h (three times) during the prereplicative phase of the cell cycle. The persisting potency of SB 203580 after 8 h of incubation was first verified on p38 MAPK phosphorylation. This was realized as follows: after 8 h of preincubation with or without SB 203580, dog thyrocytes were stimulated for 15 min with 400 mM sorbitol. Anti-phospho-p38 MAPK immunoblots of whole cell extracts revealed a strong decrease of p38 MAPK phosphorylation in 3.3, 5, or 10 µM SB 203580 treated cells (data not shown).
|
|
|
MAPKs phosphorylation after dog thyrocytes stimulation by growth factors and stress agents
After 4 d of culture in basal medium, dog thyrocytes were spread and quiescent. At that time, they were stimulated by the different agonists for 15 min except for Na arsenite, which remained present for 60 min. These periods of cellular activation were chosen as they were shown to lead to optimal stimulation of MAPKs in our model (17) or in other systems (26, 27, 28, 29). Immunodetections by anti-phosphorylated MAPKs (Fig. 2) showed that treatment of the cells with TSH or forskolin was not followed by an increase in the phosphorylation of any group of MAPKs, although a marked phosphorylation of ERKs in response to the other growth factors (EGF, insulin, PMA, and HGF) and a marked phosphorylation of p46 JNK and of p38 MAPK in response to several stress agents could be observed. Interestingly, among the stress agents, only 400 mM sorbitol led to an increase in the phosphorylation of ERKs. In this representative experiment, a small increase in the phosphorylation of p38 MAPK in response to PMA and HGF was also noticed. This effect was not reproduced in all the experiments performed. The two nitrocellulose membranes were reprobed after stripping respectively with an antibody for p38 MAPK and for ERK2. In both cases, a signal of the same intensity was seen in each lane showing that the same amount of cellular proteins was loaded per lane.
|
) confirms the lack of effect of TSH and forskolin on any subgroup of MAPKs. They show that, although the strongest stimulation of ERKs was achieved by PMA (9.7x) and the weakest one by insulin (3.2x), EGF, HGF, and 400 mM sorbitol had a similar effect on this activity (5.8, 5.5, and 5.1, respectively). These results also confirm the lack of any significant effect of HGF, EGF, insulin, and PMA on p46 JNK and p38 MAPK activities, although these kinases were stimulated from three to eight times by the different stress agents.
Kinetic analysis of MAPKs phosphorylation and activity after dog thyrocytes stimulation by the different mitogens and by insulin
Figure 3 shows that, in TSH-treated cells, the level of phosphorylation of the different MAPKs was indistinguishable from the control level at all time points studied from 5 min to 4 h. In the same representative experiment, a stimulation of the cells for 15 min with PMA and 400 mM sorbitol led to a robust increase in the phosphorylation of ERKs and of the stress-activated kinases (JNKs and p38 MAPK), respectively. The same results were obtained when the cells were stimulated by forskolin instead of TSH (data not shown).
|
shows a quantitative evaluation of ERKs activity after stimulation of dog thyrocytes by TSH, insulin, EGF, HGF, and PMA for different periods of time from 5 min to 6 h. Stimulation of ERKs activity by insulin, EGF, HGF, and PMA was rapid, as it was already noticed at the first time point studied (5 min). Maximal activation of ERKs by these agents was noticed at the same time point (15 min). This response was the strongest for PMA (about 10-fold), of the same order of magnitude for HGF and EGF (about 6-fold) and much weaker for insulin (less than 3-fold). Whereas a decrease of ERK activation to about half of its maximal value was already observed after 1 h of action of EGF and insulin, it was only observed after 6 h of action of HGF and PMA. However, a significant activation of ERKs by EGF was observed for at least 4 h. Figure 4 also shows the lack of effect of TSH on ERKs activity at any time point studied from 5 min to 6 h, which is in agreement with its lack of effect on ERKs phosphorylation shown in Fig 3. To exclude an earlier and transient effect of TSH on ERKs activity as the one reported by Iacovelli et al. (18) in FRTL-5 cells, additional experiments were performed in which thyrocytes were stimulated with TSH for 2, 4, and 8 min, with the same result (data not shown).
|
Figure 5 shows that, as in dog thyrocytes, TSH, forskolin, and insulin were without effect on the phosphorylation of JNKs and of p38 MAPK. Forskolin was also without effect on the phosphorylation of ERKs. Surprisingly, TSH provoked an increase in the phosphorylation of ERKs, comparable to the one observed in response to insulin. The simultaneous addition of both agents did not seem to lead to any further increase in this phosphorylation. This activation of ERKs by TSH but not by forskolin was confirmed by the quantitative evaluation of ERKs activity after treatment of the cells by these agents. The effect of insulin and of PMA on this activity has also been measured (Table 3). As in the human cell, TSH, at high concentrations stimulates phospholipase C through Gq (a G protein subtype demonstrated to allow receptor coupling to phospholipases of the ß subclass in a pertussis-toxin-insensitive manner; Ref. 30), the accumulation of IPs was measured. As shown in Table 4 and as previously shown (30), at the concentration of 1 mU/ml that enhanced ERKs activity, TSH did not increase the formation of these compounds, excluding an effect of this hormone on ERKs through Gq. As preincubation of TSH with TSH neutralizing antibodies prevented its effect on the accumulation of cAMP (Table 5) but not on the phosphorylation of ERKs (Fig. 5), it was concluded that this phosphorylation was due to a contaminant capable of stimulating the ERKs pathway in the human but not in the dog thyrocyte.
|
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this work, we show, by immunoblotting and immuno-complex kinase assays that, in primary cultures of dog and human thyrocytes, the cAMP-dependent agents, TSH and forskolin, are without effect on the phosphorylation and activity of the different subgroups of MAPKs, i.e. ERKs, JNKs, and p38 MAPK although this investigation was carried out over a time range from 2 min to 4 h. We also show that among these different subgroups of MAPKs, only ERKs are activated by the cAMP-independent growth factors i.e. HGF, EGF, and insulin and by PMA.
It is interesting to note that Hara et al. (31) also observed that, in human thyroid cells, TSH, at a concentration comparable to the one we used (10-9 M), was without effect on JNK activity. On the other hand, it has been reported that in CHO cells stably transfected with the human TSH receptor, TSH (1 mU/ml), and forskolin (10-5 M) caused an increase in the phosphorylation and activity of p38 MAPK (32). As this experimental system has no relation to thyroid cells, this result only indicates that signaling pathways implicating cAMP are potentially able to stimulate p38 MAPK in these cells, which had been established for other models. In rat epididymal fat cells, for example, the ß-adrenergic agonist isoproterenol and the cell permeable cAMP analog, chlorophenylthio-cAMP strongly increase the activity of p38 MAPK (29). Also, in PC12 cells, forskolin was shown to activate p38 MAPK (33).
The lack of effect of the cAMP-independent agents (HGF, EGF, insulin, PMA) on the activity of JNKs and p38 MAPK that we observed in thyrocytes was no surprise as it is now clear that the activation of these kinases depends not only on the stimulus but also on the cell type. For example, insulin can stimulate p38 MAPK in 3T3-L1 adipocytes (34) but down-regulates this activity in chick forebrain neuron cells (35). The fact that we did not observe any significant modification of the phosphorylation and activity of the JNKs and p38 MAPK after stimulation of the thyrocytes by any of the growth factors studied strongly precludes a role for these kinases in the pathways leading to thyroid cell proliferation. This is supported by the fact that treatment of dog thyrocytes with SB 203580, an inhibitor of p38 MAPK, was without effect on DNA synthesis in cells stimulated by these agents. Similar results were recently obtained in human osteoblasts (36), where it was shown that although an activation of ERKs was required for extracellular calcium stimulation of proliferation, p38 MAPK, and JNK were not activated by calcium in these cells and that proliferation was not affected by SB 203580. It is important to stress here that, although JNKs and p38 MAPK can be activated by several growth factors at least in certain cell types, their involvement in cell proliferation is far from being demonstrated, even in these cells (2, 3).
We also show, in this work, that in dog thyrocytes as in other cell types the cAMP-independent agents (HGF, EGF, insulin, PMA) stimulated ERKs phosphorylation and activity. PD 098059, an inhibitor of MEK, inhibited this activity and DNA synthesis in stimulated cells in a concentration-dependent manner, showing that ERK activation by these growth factors was necessary for the induction of DNA synthesis. The activation of ERKs by these agents was rapid, reaching its maximal value in about 15 min, and then declined differently depending on the stimulating agent. PMA was the strongest effector as it stimulated ERKs to about 10-fold over basal, whereas EGF and HGF had about the same potency (
6-fold over basal), and insulin was the weakest effector (3-fold over basal). Interestingly, whereas HGF and PMA promoted a sustained activation of ERKs (more than 6 h), the one promoted by insulin and EGF was already strongly diminished after 1 h. As a whole, these results are in agreement with those obtained in other systems such as Swiss 3T3 fibroblasts (37), PC12 cells (38), and vascular smooth muscle cells (39), but it is worth noting that, in thyrocytes as in smooth muscle cells (39), EGF action although strongly diminished after 1 h, persisted for at least 4 h. Our work also shows that, even a strong and sustained activation of ERKs is not sufficient to promote DNA synthesis in thyrocytes as PMA when acting alone, contrary to HGF, is not a mitogen for these cells.
What differentiates the action of HGF from that of PMA in dog thyrocytes is that, whereas both agents induced a strong and sustained activation of the MEK/ERKs pathway, only HGF stimulated the PI 3-kinase/PKB pathway, the inhibition of which also prevents DNA synthesis to occur (19). Interestingly, we have shown, in our previous work (19), that the activation of the PI 3-kinase/PKB pathway was strong and sustained after both HGF and insulin action but was much weaker and transient in response to EGF. This shows that a strong and sustained activation of the PI 3-kinase/PKB pathway per se is not sufficient to trigger DNA synthesis as insulin is not a mitogen for dog and human thyrocytes. The fact that HGF, the only mitogenic agent in thyrocytes that does not require insulin for its action, activates both MEK/ERKs and PI 3-kinase/PKB pathways strongly and in a sustained way and that PD 098059 as well as wortmannin inhibit HGF induced DNA synthesis suggests that a robust and sustained activation of both pathways could be necessary and even sufficient to induce DNA synthesis. The fact that both MEK/ERKs and PI3K/PKB pathways must be stimulated strongly and in a sustained way to achieve the mitogenic action of growth factors is further supported by the requirement of insulin in addition to EGF or PMA for this action. It is interesting to note here that, in fibroblasts, a sustained activation of ERKs (40) and also an activation of PI 3-kinase (41) have independently been claimed to be required for the accumulation of endogenous cyclin D1 and for S phase entry. Moreover, it has been suggested that this might only be achieved through a participation of both and possibly other pathways (41, 42).
In dog thyrocytes, TSH and forskolin were without effect on ERKs phosphorylation and activity. This lack of effect of cAMP on ERKs was also observed in the Wistar rat thyroid cell line (43) and in Swiss 3T3 fibroblasts where, as in thyrocytes, cAMP is a mitogenic signal (44). This is at variance with the FRTL-5 cell line (18) and a few other cell types such as PC12 cells (45, 46) and brown adipocytes (47), where it has been shown that cAMP can also directly activate ERKs (48). Activation of ERKs by TSH and cAMP in the FRTL-5 cell line has been reported to be due to a protein kinase A (PKA)-independent activation of the newly identified cAMP-Epac-Rap1-B Raf cascade (18). Although in dog thyrocytes (49) and in the Wistar rat thyroid cell line (50) Rap1 is also activated by TSH and forskolin, in these cells, it does not trigger the activation of the B Raf-MAPK pathway. Of course, in most cell types, cAMP antagonizes the activation of ERKs and of mitogenesis by growth factors. The fact that, in dog thyrocytes pretreated with PD098059, DNA synthesis elicited by TSH and forskolin in the presence of insulin was decreased in a concentration-dependent manner suggests that although TSH and cAMP do not activate the ERKs, the small activation of these kinases by insulin is nevertheless necessary for DNA synthesis to occur.
In human thyrocytes, we have clearly established that the weak effect of TSH on ERKs phosphorylation, which was not reproduced by forskolin, was not due to TSH per se but to the presence of nonidentified contaminant(s) in the TSH preparation. Indeed, we showed that, in agreement with previously published work (30), at the concentration used (1 mU/ml) TSH did not provoke the formation of IPs, thus excluding an activation of ERKs through Gq. Moreover, we showed that TSH-neutralizing antibodies, although preventing the effect of this hormone on cAMP formation, did not prevent its stimulation of ERKs phosphorylation. Our results obtained in primary cultures of human thyrocytes are in agreement with and give physiological relevance to those of Corrèze et al. (51) obtained with CHO cells expressing the human TSH receptor, an experimental system unrelated to the thyroid. The presence of contaminants in TSH preparations could also explain the stimulation of ERKs in response to TSH but not to forskolin observed by Saunier et al. (52) in cultured human thyroid follicles. It should be noted that the available commercial TSH is purified from bovine pituitaries that are notoriously rich in various growth factors. Moreover, the fact that our group as the group of Corrèze (51) obtained the same results with rhTSH as with bovine TSH, notably an increase in ERKs phosphorylation in wild-type CHO, shows that not only commercial bovine TSH preparations but also commercial rhTSH preparations contain contaminant(s) capable of stimulating the ERKs pathway by a TSH receptor-independent mechanism. With regard to the mitogenic regulation of thyroid cell proliferation, there is therefore no discrepancy between TSH and cAMP actions.
The mechanism by which cAMP triggers mitogenesis in dog and human thyrocytes thus remains enigmatic. It requires PKA activity (53). However the activation of PKA is not sufficient to reproduce the cAMP-dependent mitogenic activity (53). Thus the role of other cAMP-dependent but PKA-independent pathways remains to be defined. What has already been demonstrated is that in the cooperation between TSH and insulin to achieve DNA synthesis, the accumulation of cyclin D3 promoted by insulin/IGF-1 is necessary for the further assembly and activation of cyclin D3-cdk4 complexes elicited by TSH (21).
Thus, the present work further demonstrates the high degree of similarity of the dog and human thyroid cell culture with regard to mitogenic control, the complete reproduction of the TSH effects by forskolin, i.e. the mediation of these TSH effects by cAMP. It shows again the generation of artifacts caused by the use of high TSH concentrations or the use of experimental models unrelated to thyroid cells. As ERKs are involved in the mitogenic action of cAMP in other experimental models, this emphasizes again that this action of cAMP is a characteristic of the differentiation of each cell type and therefore might be expected to differ from one cell type to another.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
1 F.V. and S.P. made equal contributions to this study. 
Abbreviations: ATF-2, Activating transcription factor 2; BrdU, bromodeoxyuridine; CHO, Chinese hamster ovary; EGF, epidermal growth factor; FRTL-5, Fischer rat thyroid cell line; GST, glutathione-S-transferase; HGF, hepatocyte growth factor; IP, inositol phosphate; JNK, c-Jun N-terminal kinases; KRH, Krebs-Ringer-HEPES; MEK, MAPK kinase; PI, phosphatidylinositol; PMA, phorbol 12-myristate 13-acetate; PKA, protein kinase A; PKB, protein kinase B; rhTSH, recombinant human TSH.
Received November 20, 2001.
Accepted for publication December 31, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1-adrenergic Erk1/2 activation is Src- but not Gi-mediated in Brown adipocytes. J Biol Chem 275:22670–22677This article has been cited by other articles:
 |
|
 |
|
  S. Tsui, V. Naik, N. Hoa, C. J. Hwang, N. F. Afifiyan, A. Sinha Hikim, A. G. Gianoukakis, R. S. Douglas, and T. J. Smith Evidence for an Association between Thyroid-Stimulating Hormone and Insulin-Like Growth Factor 1 Receptors: A Tale of Two Antigens Implicated in Graves' Disease J. Immunol., September 15, 2008; 181(6): 4397 - 4405. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. R. H. Buch, H. Biebermann, H. Kalwa, O. Pinkenburg, D. Hager, H. Barth, K. Aktories, A. Breit, and T. Gudermann G13-dependent Activation of MAPK by Thyrotropin J. Biol. Chem., July 18, 2008; 283(29): 20330 - 20341. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Dremier, M. Milenkovic, S. Blancquaert, J. E. Dumont, S. O. Doskeland, C. Maenhaut, and P. P. Roger Cyclic Adenosine 3',5'-Monophosphate (cAMP)-Dependent Protein Kinases, But Not Exchange Proteins Directly Activated by cAMP (Epac), Mediate Thyrotropin/cAMP-Dependent Regulation of Thyroid Cells Endocrinology, October 1, 2007; 148(10): 4612 - 4622. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Costa, M. Senou, F. Van Rode, J. Ruf, M. Capello, D. Dequanter, P. Lothaire, C. Dessy, J. E. Dumont, M.-C. Many, et al. Reciprocal Negative Regulation between Thyrotropin/3',5'-Cyclic Adenosine Monophosphate-Mediated Proliferation and Caveolin-1 Expression in Human and Murine Thyrocytes Mol. Endocrinol., April 1, 2007; 21(4): 921 - 932. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Paternot, J. E. Dumont, and P. P. Roger Differential Utilization of Cyclin D1 and Cyclin D3 in the Distinct Mitogenic Stimulations by Growth Factors and TSH of Human Thyrocytes in Primary Culture Mol. Endocrinol., December 1, 2006; 20(12): 3279 - 3292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T Kogai, K Taki, and G A Brent Enhancement of sodium/iodide symporter expression in thyroid and breast cancer. Endocr. Relat. Cancer, September 1, 2006; 13(3): 797 - 826. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Gilbert, A. G. Gianoukakis, S. Salehi, J. Moorhead, P. V. Rao, M. Z. Khan, A. M. McGregor, T. J. Smith, and J. P. Banga Monoclonal pathogenic antibodies to the thyroid-stimulating hormone receptor in Graves' disease with potent thyroid-stimulating activity but differential blocking activity activate multiple signaling pathways. J. Immunol., April 15, 2006; 176(8): 5084 - 5092. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Calipel, F. Mouriaux, A.-L. Glotin, F. Malecaze, A.-M. Faussat, and F. Mascarelli Extracellular Signal-regulated Kinase-dependent Proliferation Is Mediated through the Protein Kinase A/B-Raf Pathway in Human Uveal Melanoma Cells J. Biol. Chem., April 7, 2006; 281(14): 9238 - 9250. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Fortemaison, S. Blancquaert, J. E. Dumont, C. Maenhaut, K. Aktories, P. P. Roger, and S. Dremier Differential Involvement of the Actin Cytoskeleton in Differentiation and Mitogenesis of Thyroid Cells: Inactivation of Rho Proteins Contributes to Cyclic Adenosine Monophosphate-Dependent Gene Expression but Prevents Mitogenesis Endocrinology, December 1, 2005; 146(12): 5485 - 5495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C Correze, J-P Blondeau, and M Pomerance p38 mitogen-activated protein kinase contributes to cell cycle regulation by cAMP in FRTL-5 thyroid cells Eur. J. Endocrinol., July 1, 2005; 153(1): 123 - 133. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
