©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of Mitogen-activated Protein Kinase by Thyrotropin in Primary Cultured Human Thyroid Follicles (*)

(Received for publication, October 12, 1994; and in revised form, December 14, 1994)

Bertrand Saunier (§) Cathy Tournier Claude Jacquemin Michel Pierre

From the From Unité 96, INSERM, 94276 Le Kremlin-Bicêtre, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the thyroid, thyrotropin (TSH) stimulates both growth and function, and stimulates the production of cAMP which reproduces most of the effects of TSH. Here, we report evidence that TSH stimulates the mitogen-activated protein (MAP) kinase cascade through a cAMP-independent pathway, in human thyroid. TSH stimulated MAP kinase activity (4-9-fold the basal level) measured in the cytosolic fractions of primary cultured thyroid follicles. Maximal activity was reached after 20 min and remained sustained for 1-3 h, TSH being as potent as EGF; EC was 1.5 nM TSH. Only a single isoform of MAP kinase (p42) was detected in the follicles. p42 was phosphorylated on tyrosine residues and showed a reduced electrophoretic mobility in follicles stimulated by TSH. All these effects on MAP kinase were decreased by preincubation of the follicles with human anti-TSH receptor antibodies. The stimulation of MAP kinase by TSH was neither blocked by pertussis toxin nor reproduced by forskolin, cholera toxin, or 8-bromo-cAMP. In conclusion, in human thyroid cells, in contrast with previous observations on dog thyroid cells, TSH stimulates strongly MAP kinase through a pertussis toxin-insensitive and cAMP-independent pathway.


INTRODUCTION

Mitogen-activated protein kinase (MAP (^1)kinase or Erk) is an essential component of a signaling cascade, rapidly activated by MAP kinase/Erk kinase, which phosphorylates it on both a threonine and a tyrosine residue(1, 2) . This cascade can be stimulated in various cell types (3) not only by a range of growth factors, but also by hormones acting through receptors that are coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) (4, 5, 6) . TSH is well known to play an important physiological role in the function and growth of the thyroid gland(7) . The TSH receptor belongs to the family of seven transmembrane domain receptors, and is coupled, through heterotrimeric G proteins, to the stimulation of adenylyl cyclase (8) and, in certain species, to phospholipases C or A(2)(9, 10, 11, 12) . In dog thyroid cells, TSH induces cell proliferation but does not stimulate MAP kinase. This cell proliferation is most probably mediated by an elevation in intracellular cAMP, since TSH stimulates adenylyl cyclase in these cells, and forskolin is able to fully reproduce the TSH effect on cell division(13) . However, in WRT cells, a cell line derived from rat thyroid, TSH-induced mitogenicity is dependent not only on cAMP but also on Ras via a cAMP-independent pathway(14) . As Ras has been shown previously to stimulate MAP kinase activity in a number of cell types (15) , the question of whether MAP kinase is subject to regulation by TSH remains unanswered. Our present results demonstrate that TSH stimulates MAP kinase in human thyroid follicles.


EXPERIMENTAL PROCEDURES

Materials

Collagenase type I, protease type IX, antibiotics-antimycotics, human transferrin, vitamin C, bovine insulin, protein kinase A inhibitor, 8-bromo-cAMP, and 8-(4-chlorophenylthio)-cAMP were from Sigma; tissue culture dishes were from Falcon; Dulbecco's modified Eagle's medium (DMEM), DMEM/Ham's F-12 culture medium, and fetal calf serum from Life Technologies, Inc.; myelin basic protein peptide substrate of MAP kinase and anti-phosphotyrosine monoclonal antibodies were from UBI; [-P]ATP (1600 Ci/mmol), chemiluminescence reaction kit assay (ECL), and autoradiography films (Hyperfilm MP) were from Amersham Corp.; phosphocellulose filter paper (P-81) from Whatman; nitrocellulose membrane (BA 83) was from Schleicher & Schuell; anti-MAP kinase monoclonal antibodies were from Zymed Laboratories; anti-mouse immunoglobulin G (IgG) antibodies coupled to peroxidase were from Biosys; and protein G-Sepharose was from Boehringer Mannheim.

Bovine TSH (approx25 units/mg) was a generous gift from NIH or was purchased from UCB Bioproducts, Brussels, Belgium (approx40 units/mg) where identified; EGF was from Sigma, and bFGF from Genzyme.

Cell Culture and Preparation of Cell Extracts

The human thyroid tissues were obtained at surgery from patients with non-cancer thyroid diseases. Thyroid follicles were prepared as described (16) and modified as follows; the minced tissues were incubated in DMEM for 90 min at 37 °C in the presence of 0.4 mg/ml collagenase type I and 0.9 mg/ml protease type IX. Thyroid follicle sedimentation was more rapid than the other cells present, thus allowing a purification of the thyrocytes. The purification of thyrocytes was verified by electronic microscopy, as described in a previous study(17) . The isolated follicles were seeded in 6-cm tissue culture dishes and cultured for 2 days in DMEM/Ham's F-12 (1:1) medium supplemented with antibiotics-antimycotics, 1.25 µg/ml human transferrin, 40 µg/ml vitamin C, 1% fetal calf serum, and 5 µg/ml bovine insulin. Prior to use in experiments, the follicles were cultured overnight in DMEM/Ham's F-12 medium supplemented only with antibiotics-antimycotics and 40 µg/ml vitamin C.

After stimulation in serum-free media, the culture medium was removed, and the thyrocytes were washed twice with buffer A: 80 mM beta-glycerophosphate, pH 7.4, containing 20 mM EGTA and 15 mM MgCl(2). The cells were then harvested, sonicated for 3 s on ice in buffer A containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 4 µg/ml leupeptin, 10 µg/ml antipain, 1 mM trypsin inhibitor, 1 mM benzamidine, and 10 µg/ml pepstatin) and 1 mM phosphatase inhibitor orthovanadate (Na(3)VO(4)). Homogenates were centrifuged at 4 °C at 105,000 times g for 30 min. The supernatants were aliquoted and stored at -80 °C until use.

MAP Kinase Assay

The MAP kinase activity was measured by incubation of 2-5 µg of cytosolic proteins for 10 min at 30 °C with 1 mM substrate peptide containing the sequence of myelin basic protein phosphorylated by MAP kinase (18) (APRTPGGRR) in the following buffer: 12.5 mM MOPS and 12.5 mM beta-glycerophosphate, pH 7.2, containing 5 mM MgCl(2), 2 mM dithiothreitol, 0.5 mM EGTA, 1 mM orthovanadate, 10 µg/ml protein kinase A inhibitor, and 50 µM [-P]ATP (1-3 µCi/nmol). The reaction was stopped by 3% trichloroacetic acid for 30 min on ice. After centrifugation at 10,000 times g, the sample supernatants were then spotted onto 1-cm^2 phosphocellulose filter paper(19) , washed once overnight at 4 °C in 30% acetic acid containing 3 mM ATP (10 ml/filter), washed three times in 15% trichloroacetic acid, and dried with ether-alcohol. The radioactivity bound to the filters was quantified by liquid scintillation counting.

Immunoblotting Studies

Protein samples were prepared in denaturing conditions and were separated by electrophoresis in 8-15% SDS-PAGE, as described(20) , then blotted (X-blot) onto nitrocellulose membrane. Immunodetection was performed by incubating the membrane for 3 h with either 1/2,000 diluted anti-phosphotyrosine monoclonal antibodies or 1/2,000 diluted anti-MAP kinase monoclonal antibodies. After washing of the membrane in buffer B: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20, detection of antigen-antibody complexes was performed by further incubation of the membrane with 1/30,000 diluted anti-mouse IgG antibodies coupled to peroxidase for 1 h. After washing once in buffer B, the revelation was performed with the chemiluminescence reaction and autoradiography.

Preparation of Antibodies

IgG were purified by affinity chromatography on protein G-Sepharose from either control serum, or serum containing anti-TSH receptor antibodies either (i) stimulating adenylyl cyclase activity (TSAb)(21) , or (ii) blocking the stimulation by TSH of cAMP production (TBkAb) (22) in cultured thyroid cells. After concentration, quantification of the IgG was performed by Ouchterlony's radial immunodiffusion technique. The ability of the purified IgG to recognize the TSH receptor was verified by inhibition of the binding of I-TSH to purified thyroid membranes.


RESULTS

In order to test a possible effect of TSH on MAP kinase in human thyroid, we first performed a kinetic study of MAP kinase activation in primary cultured human thyroid follicles (Fig. 1), prepared as described under ``Experimental Procedures.'' The stimulation of follicles by 10 nM TSH was followed by an increase in MAP kinase activity, measured as described under ``Experimental Procedures,'' that reached a maximum of 4-9-fold the basal level after a 20-min stimulation period, depending on the available thyroid gland. This activity then decreased after 60 min to a level, which was approximately half of the maximal activation, and was sustained for at least 3 h. MAP kinase has previously been shown to require tyrosine phosphorylation for its activation(2) ; hence, we performed immunoblotting studies of cytosolic proteins with monoclonal anti-phosphotyrosine antibodies (Fig. 1B, upper panel). The treatment of thyroid follicles with 10 nM TSH induced the phosphorylation of a tyrosine residue of a protein with a molecular mass of 42 kDa (pp42), the apparent molecular mass reported for Erk2. Anti-MAP kinase monoclonal antibodies identified only one isoform of MAP kinase with a molecular mass of 42 kDa in cultured human thyroid cells (Fig. 1B, lowerpanel). It has been reported previously that the electrophoretic mobility of MAP kinase was decreased after its phosphorylation and activation(23) . After treatment of thyroid follicles with 10 nM TSH, a significant proportion of the 42-kDa MAP kinase presented the typical shift in mobility following electrophoresis (Fig. 1B, lower panel), the amount of which was correlated with both intensity of tyrosine phosphorylation of pp42 and MAP kinase activity. Moreover, when the immunoblotting studies with anti-phosphotyrosine and anti-MAP kinase antibodies were successively performed on the same membrane, the apparent molecular mass of the more slowly migrating MAP kinase was exactly the same as that of pp42. Hence, it would appear that TSH stimulated the activity of p42 MAP kinase in primary cultured human thyroid follicles.


Figure 1: Kinetic analysis of MAP kinase activation by TSH in cultured human thyroid follicles. Human thyroid follicles were isolated and cultured as described under ``Experimental Procedures'' and treated with 10 nM TSH, and cytosolic fractions were prepared. A, MAP kinase activity was measured by incubation of proteins from cytosolic fraction with peptide substrate and [-P]ATP. The peptide was spotted onto chromatographic paper, and the incorporated radioactivity was quantified by liquid scintillation. B, proteins (50 µg) of the same cytosolic fractions were prepared and subjected to SDS-PAGE (8-15%) and immunoblotting with either monoclonal anti-phosphotyrosine (alphaP-Tyr) or anti-MAP kinase (alphaMAP-K) antibodies. Antigen-antibody complexes were detected by further incubation with peroxidase-coupled antibodies, followed by a chemiluminescent reaction and autoradiography.



Maximal stimulation of p42 MAP kinase activity in response to 10 nM TSH was obtained after 20 min of stimulation. Hence, we performed dose-response assays to TSH at this time point. The maximal MAP kinase activity was obtained with 5 nM TSH (Fig. 2), with a calculated EC approx 1.5 nM. This corresponds to a considerably greater TSH concentration (approximately 3-fold more) than that required to produce the EC for the stimulation of cAMP production, and the apparent K(d) for the high affinity TSH binding site, in human thyroid(8) . These results correlated with those obtained when the cytosolic extracts were submitted to anti-phosphotyrosine and to anti-MAP kinase immunoblotting. To complement the results obtained in the previous experiments with bovine TSH (from NIH), we used another highly purified batch of TSH (from UCB Bioproducts), which elicited a similar stimulation of MAP kinase (not shown).


Figure 2: Concentration dependence of the stimulation by TSH of MAP kinase in human thyroid follicles. Cultured human thyroid follicles were treated with TSH for 20 min. The MAP kinase activity was assayed in cytosolic fractions, as in Fig. 1A. Proteins from the same fractions were subjected to immunoblot analysis with anti-phosphotyrosine antibodies (alphaP-Tyr), as in Fig. 1B.



To further characterize the mechanism of TSH-mediated MAP kinase activity, we employed two specific antisera, TSAb and TBkAb, which contain antibodies able to specifically stimulate and inhibit, respectively, the TSH receptor. When the stimulation of MAP kinase was evaluated by the shift of the activated p42 in SDS-PAGE, the preincubation of thyroid follicles with 0.3 mg/ml control IgG, TBkAb, or TSAb was not followed by stimulation of MAP kinase (Fig. 3). However, a slight increase in MAP kinase activity (less than 2-fold the control), measured by phosphorylation of substrate peptide, was observed when the follicles were incubated at least 1 h in the presence of TSAb, but not in the presence of TBkAb, as compared to the control (not shown). Although the stimulation of MAP kinase elicited following 20 min of TSH (5 nM) addition was not affected by preincubation of thyroid follicles with control antibodies, it was decreased when preincubation was performed with anti-TSH receptor antibodies (Fig. 3). Corresponding to this result, the stimulation by 5 nM TSH for 20 min of MAP kinase activity was decreased by more than 50% by the preincubation of follicles with anti-TSH receptor antibodies, but not with control antibodies (not shown). The incomplete inhibition of the TSH-mediated stimulation of MAP kinase may be because the titer of the anti-TSH receptor antibodies is too low in comparison with the TSH concentration used for these experiments. Unfortunately, it was not possible to greatly increase the antibody concentration in these experiments, because of the appearance of nonspecific effects.


Figure 3: Inhibition by anti-TSH receptor antibodies of the TSH-induced stimulation of MAP kinase in human thyroid follicles. Thyroid follicles were preincubated for 1 h with 0.3 mg/ml IgG purified from normal serum (Control) or from serum containing anti-thyrotropin receptor blocking (TBkAb) or stimulating (TSAb) antibodies. After stimulation of the follicles by 5 nM TSH for 20 min, MAP kinase immunoblots were performed on cytosolic fractions, as in Fig. 1B.



EGF is a growth factor well known to be a mitogenic stimulus to thyroid cells(24, 25) . The stimulation of MAP kinase activity after a 30-min addition of 5 nM TSH was of the same order of magnitude as that observed after a 10-min addition of 10 nM EGF (Fig. 4). The MAP kinase response to EGF was not decreased by preincubation of thyroid follicles with anti-TSH receptor antibodies (not shown). Moreover, bFGF, known to a major MAP kinase-stimulating factor in a range of cells, and which has been found to be a contaminant in some TSH preparations(26) , had no stimulatory effect on MAP kinase activity in human thyroid follicles (Fig. 4).


Figure 4: MAP kinase activity after stimulation of human thyroid follicles by TSH, cAMP, or growth factors. Cytosolic fractions were prepared from human thyroid follicles, unstimulated (Cont.), or stimulated by 5 nM TSH for 30 min, 1 mM 8-bromo-cAMP for 30 min, 10 nM EGF for 10 min, or 1 nM bFGF for 10 min. MAP kinase assays were performed on the cytosolic fractions, as described in Fig. 1A.



Several groups have shown that cAMP can stimulate MAP kinase activity (27, 28) . As TSH stimulates cAMP production in thyrocytes, we studied whether an increase in cAMP concentration could modulate the MAP kinase activity of cultured follicles. In conditions where TSH strongly stimulated MAP kinase activity, the treatment of follicles for 30 min with 8-bromo-cAMP, a permeant analogue of cAMP that gives a similar stimulation of protein kinase A to that elicited by TSH, had only a slight (approx200% of control) stimulating effect (Fig. 4). Similar results were obtained with 8-(4-chlorophenylthio)-cAMP, another permeant analogue of cAMP. The maximal effect was reached 20 min after its addition. At this time, the MAP kinase activity increased with the concentration of 8-(4-chlorophenylthio)-cAMP and reached a plateau (300% of the control) at 1 mM. Neither the treatment of follicles for 15 or 30 min with 10 µM forskolin, a diterpene that directly stimulates adenylyl cyclase, and increased cAMP production to the same extent as TSH, nor the treatment for 1 h with 0.3 or 3 µg/ml cholera toxin, which ADP-ribosylates and inhibits the GTPase activity of G(s), leading to permanent activation of adenylyl cyclase, reproduced the TSH-mediated simulation of MAP kinase activity (not shown).

Hormones, which activate MAP kinase through heterotrimeric G protein-coupled receptors, may do so via pertussis toxin-sensitive G proteins(5, 29, 30) . Thyroid follicles were pretreated by 0.2 µg/ml pertussis toxin for 3.5 h, which completely ADP-ribosylates the cellular complement of pertussis toxin-sensitive G proteins (not shown). As shown in Fig. 5, pertussis toxin, which by itself was able to stimulate the activity of MAP kinase in the absence of agonist by approximately 2-fold, did not inhibit the stimulation by TSH of MAP kinase.


Figure 5: Pertussis toxin fails to inhibit TSH-induced stimulation of MAP kinase in human thyroid follicles. Thyroid follicles were pretreated or not with 0.2 mg/ml pertussis toxin for 3 h 30 min. Then, the follicles were stimulated by 5 nM TSH for 20 min. MAP kinase activity and corresponding MAP kinase immunoblot were performed as in Fig. 1.




DISCUSSION

TSH is well known to play a major role not only in the control of thyroid function, but also in the stimulation of thyroid cell proliferation(7) . We show that MAP kinase was stimulated by TSH in cultured human thyroid follicles, since it enhanced with a similar time course and in a correlated manner: (i) the cytosolic activity that phosphorylates a peptide substrate of MAP kinase and (ii) the tyrosine phosphorylation of a 42-kDa protein corresponding to the activated MAP kinase, which presented a typical reduction in electrophoretic mobility (23) . Furthermore, anti-TSH receptor antibodies, but not control antibodies, decreased the response of MAP kinase to TSH.

In contrast to our results, in dog thyroid gland, TSH did not stimulate MAP kinase, unlike other mitogens such as EGF or 12-O-tetradecanoylphorbol-13-acetate(13) . This might be due to a difference in the proximal coupling of the TSH receptor. Indeed, contrary to what was reported in human thyroid(9) , it was demonstrated in canine thyroid that the TSH receptor was not coupled to phosphatidylinositol bisphosphate (PIP(2)) hydrolysis by a PLC(31) . The simplest explanation of the absence of stimulation of MAP kinase by TSH in dog thyroid cells would be the TSH receptor being uncoupled from PIP(2)-PLC. This hypothesis is reinforced by the fact that, in these cells, carbachol which stimulates PIP(2)-PLC(31) , also stimulates MAP kinase(13) . Although the stimulation of PIP(2)-PLC by TSH involving alpha-subunits of G proteins has been reported in human thyroid(32) , we have as yet no evidence of its implication in the stimulation of MAP kinase by TSH. Indeed, oncogenic G(q) alpha-subunits mutants, which lack GTPase activity, do not stimulate the MAP kinase cascade when expressed in COS cells(33, 34) , whereas they stimulate efficiently PIP(2)-PLC(35) .

Another explanation of the discrepant results between canine and human thyroid could be the existence of a different effect of cAMP on the MAP kinase cascade. In some cell types, cAMP has been found to inhibit the stimulation of MAP kinase by growth factors(36) . It was not reported by Dumont and collaborators whether cAMP could have negative effect on the MAP kinase cascade in canine thyroid. This is probably not the case in human thyroid, since TSH stimulates both MAP kinase and cAMP production. In other cell types cAMP stimulates the MAP kinase cascade (27, 28) . In dog thyroid cells, forskolin and TSH, which are both cAMP-elevating agents, did not stimulate MAP kinase(13) . In human thyroid follicles, we found that, in conditions where TSH produced a robust activation of MAP kinase, neither forskolin nor cholera toxin, which increase cAMP production, nor permeant analogues of cAMP, could stimulate MAP kinase activity at the same extent as TSH. These results suggest that TSH stimulates MAP kinase by a pathway largely independent from that of intracellular cAMP elevation. Furthermore, we have recently reported the expression of TSH receptors and the existence of TSH effects in cultured astroglial cells(37) . In these cells, TSH stimulated MAP kinase(42) , but neither InsP(2)-PLC nor cAMP production(37) , suggesting another signaling pathway could be involved in the pathway from the TSH receptor to the MAP kinase cascade.

The mitogenic effect of TSH in the thyroid gland is attributed to the stimulation of cAMP production, via G(s)(7) . However, in slices of human thyroid gland, TSH-mediated DNA synthesis is only in part explained by an elevation in intracellular cAMP(16) . Kupperman and co-workers (14) have shown in WRT, a cell line derived from rat thyroid, that the cAMP-independent portion of DNA synthesis stimulated by TSH was inhibited by microinjection of dominant negative Ras. The Ras protein is located on the signaling pathway of mitogens which stimulate the MAP kinase cascade(15) . Hormones stimulating MAP kinase have been shown to activate Ras, via pertussis toxin-sensitive G proteins(5, 29, 30) . In human thyroid follicles, pertussis toxin did not affect the stimulation of MAP kinase by TSH (Fig. 5), suggesting G proteins were not implicated in this TSH effect. However, it has been shown that Ras-Raf might be activated through heterotrimeric G proteins beta-subunits (38) which may not be specific to the alpha-subunits they are associated with. Moreover, MAP kinase/Erk kinase kinases have recently been cloned, which are different from Raf and show homologies with yeast MAP kinase kinase kinases(39) , which are stimulated by homologs of the mammalian heterotrimeric G proteins. This leaves open the possibility for TSH to stimulate MAP kinase through another pathway distinct to that employing Raf.

In M cells, another cell line derived from rat thyroid, TSH was unable to stimulate proliferation while it increased the intracellular concentration of cAMP(40) . In a human thyroid carcinoma cell line transfected with the human TSH receptor cDNA, TSH led to the stimulation of the cAMP signaling pathway, but was growth-inhibitory (41) . These examples suggest that mitogenic effects of TSH on the thyroid may also depend on the connection of more distal targets of the TSH signaling pathways. We clearly demonstrate that TSH stimulates MAP kinase in cultured human thyroid follicles, it remains to be determined to what extent this enzyme could be implicated in the transduction of the mitogenic signal induced by TSH.


FOOTNOTES

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

§
To whom correspondence should be addressed: U96, INSERM, 80, rue du Général Leclerc, 94276 Le Kremlin-Bicêtre Cedex, France. Fax: 33-1-49-59-85-40.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; TSH, thyrotropin; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; PAGE, polyacrylamide gel electrophoresis; PIP(2), phosphatidylinositol bisphosphate; PLC, phospholipase C.


ACKNOWLEDGEMENTS

We are indebted to F. Gayral and J. Orgiazzi for providing human thyroid glands and anti-TSH receptor antibodies, respectively. TSH was generously provided by the NIDDK (NHPP, University of Maryland, College Park, MD). We warmly thank J. Pouysségur and F. McKenzie for helpful discussion and having read the manuscript carefully.


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