(Received for publication, October 12, 1994; and in revised form, December 14, 1994)
From the
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.
Mitogen-activated protein kinase (MAP ()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
(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.
Bovine TSH (25 units/mg) was a generous gift from NIH or was
purchased from UCB Bioproducts, Brussels, Belgium (
40 units/mg)
where identified; EGF was from Sigma, and bFGF from Genzyme.
After
stimulation in serum-free media, the culture medium was removed, and
the thyrocytes were washed twice with buffer A: 80 mM -glycerophosphate, pH 7.4, containing 20 mM EGTA and
15 mM MgCl
. 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
VO
). Homogenates were centrifuged at 4
°C at 105,000
g for 30 min. The supernatants were
aliquoted and stored at -80 °C until use.
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 (
P-Tyr) or anti-MAP kinase (
MAP-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
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
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
(P-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 (200% 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
, 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.
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)
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
-PLC. This
hypothesis is reinforced by the fact that, in these cells, carbachol
which stimulates PIP
-PLC(31) , also stimulates MAP
kinase(13) . Although the stimulation of PIP
-PLC by
TSH involving
-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
-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
-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-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(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
-subunits (38) which may not be specific to the
-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.