Deactivation of TSH receptor signaling in filter-cultured pig
thyroid epithelial cells
Lars E.
Ericson and
Mikael
Nilsson
Institute of Anatomy and Cell Biology, Göteborg
University, SE 405 30 Goteborg, Sweden
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ABSTRACT |
Thyrotropin
[thyroid-stimulating hormone (TSH)] receptor on-off
signaling was studied in polarized monolayers of pig thyrocytes cultured on permeable support. Transepithelial resistance (R) and
potential difference (PD) were used as parameters to
monitor the effect of altered TSH concentrations on vectorial
electrolyte transport. TSH induced rapid but long-lasting changes in
R (decrease) and PD (increase) that were cAMP-dependent and related
to enhanced transcellular conductance of sodium and chloride.
Withdrawal of TSH from cultures prestimulated with TSH (0.1 mU/ml) for
48 h resulted in restitution of R to control level within 30 min.
Such deactivation was markedly accelerated by mild trypsinization, which degraded receptor-bound ligand without affecting TSH receptor responsiveness or ion transporting capacity. Small alterations in the
TSH concentration (0.01-0.1 mU/ml) were followed almost instantaneously by adjustments of R. In contrast, the reversal of
R after acute TSH stimulation (30 min) and subsequent TSH washout was delayed for several hours independently of cell surface
trypsinization. The observations indicate that, during continuous
exposure to physiological concentrations, TSH exerts a close
minute-to-minute surveillance of thyroid function and the rate-limiting
step of deactivation is the dissociation of ligand from the TSH
receptor at the cell surface. TSH-deprived cells briefly exposed to TSH are refractory to rapid deactivation, probably because of altered metabolism downstream of TSH receptor signal transduction.
cell polarity; vectorial ion transport; transepithelial potential
difference; transepithelial resistance
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INTRODUCTION |
THE STIMULATORY ACTION OF thyrotropin
[thyroid-stimulating hormone (TSH)] on the thyroid follicle
cell (thyrocyte) is mediated mainly by the cAMP pathway (12). After
addition of TSH, the intracellular concentration of cAMP is elevated
almost instantaneously, and stimulation of different thyroid functions,
e.g., iodide efflux (27, 28), exocytosis of thyroglobulin (4), and ion
transport (1, 2, 15), can be measured within a few minutes.
Conversely, withdrawal of TSH from cultured thyrocytes or inhibition of
pituitary release of TSH eventually leads to a downregulation, albeit
at different rates, of cellular functions involved in thyroid
hormone synthesis and release (5, 38). However, whereas the kinetics of
TSH receptor activation and downstream intracellular signaling have
been studied extensively (12), much less is known about the reverse
deactivation process. Knowledge in this respect is of significance to
thyroid physiology. For instance, TSH secretion is circadian and
pulsatile (7, 19), and thyroid morphology varies during a 24-h period
as a sign of changing TSH activity (19, 37, 43, 44).
In the present study, we have examined the kinetics of TSH-regulated
electrolyte transport and how this function is deactivated after
removal of TSH or modulated when the TSH concentration is altered
sequentially. Experiments were performed on primary cultures of pig
thyrocytes grown on filter in Transwell bicameral chambers [Transwell no. 3413, Costar, Cambridge, MA (27, 32)].
Confluent cells organize into a polarized monolayer with the apical
plasma membrane facing the upper culture chamber and the basolateral membrane that harbors the TSH receptor directed toward the filter and
the lower chamber (13, 26-29, 32). The culture system is thus a
model of an open follicle in which the apical and basal compartments,
separated by the cell layer, can be considered to represent the
follicular lumen and the extrafollicular space, respectively. The
epithelium develops a high transepithelial resistance (R) and a
significant potential difference (PD), both reflecting the magnitude of
vectorial ion transport. It is also known that TSH rapidly stimulates,
via cAMP, the transepithelial transport of Na+ and
Cl
(1, 2, 15, 32) and that the resulting increase in
membrane conductance corresponds to a reduced R and an increased PD
(29, 32). These parameters were used here to investigate the mechanism of TSH receptor deactivation.
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MATERIALS AND METHODS |
Chemicals.
Bovine TSH, IBMX, amiloride, forskolin, furosemide, trypsin (type III,
from bovine pancreas), and trypsin inhibitor were purchased from Sigma
Chemical, St Louis, MO.
Cell culture.
The methods of follicle isolation and culture have been described
previously (27). Briefly, pig thyroid glands were minced and repeatedly
digested with collagenase and disintegrated by pipetting. The follicle
fragments thus obtained were rinsed from connective tissue elements and
single cells by filtrations and washings and then seeded on microporous
filters of bicameral chambers (pore size 0.4 µm) coated with collagen
type I (0.3 mg/ml). Culture was carried out in MEM, supplemented with
5% fetal calf serum, penicillin (200 U/ml), streptomycin (200 µg/ml), and fungizone (2.5 µg/ml) at 37°C in a CO2
(5%) incubator (all medium components from GIBCO, Paisley, UK). The
same medium was used in both chamber compartments (200 µl apically
and 500 µl basally). After 4-5 days of culture, the cells were
organized into a confluent polarized monolayer on the filter. Growth to
confluence occurred in the absence of TSH. Long-term TSH stimulation,
as applied in the experiments presented in this paper, has no
growth-stimulating effect on confluent pig thyrocytes, which thus stay
in monolayer formation on the filter (13).
Experimental design.
From previous studies (26-29, 32), we know that filter-cultured
pig thyrocytes form a very tight epithelial barrier designated by a
high transepithelial R. In such a tight epithelium, the paracellular flux of ions and molecules is negligible unless the epithelial barrier
is impaired, e.g., after removal of extracellular Ca2+
(26). Under normal conditions, therefore, R reflects mainly the
transcellular transfer of ions; a high resistance (or low conductance)
indicates a low transfer, and a low resistance (or high conductance)
depends on a high transfer of ions across the monolayer. An altered
transcellular conductance is also reflected by changes in
transepithelial PD (29, 32).
All experiments reported here were performed during the 2nd wk after
seeding and only on cultures in which the tightness (R >6,000
· cm2) was ascertained before TSH
stimulation. "TSH-deprived" and "prestimulated" cultures,
respectively, were kept for 48 h in serum-supplemented medium with or
without 0.1 mU/ml of TSH present in the basal chamber. R and
PD were measured with a Millicell ERS ohmmeter (Millipore, Bedford, MA)
under sterile conditions outside the incubator, and the cultures were
generally kept in the incubator between measurements. The details of
each type of experiment are described in the text. Addition or removal
of substances was performed by exchanging the basal (in a few instances
the apical) medium. If not otherwise indicated, experimental
observations were made on triplicate cultures, and a particular type of
experiment was performed in at least three different seedings. Data
from a single representative experiment are presented in the graphs.
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RESULTS |
Effects of TSH and TSH withdrawal on R and PD.
Pig thyrocytes grown to confluence on filter in the absence of TSH
established a high transepithelial R, often exceeding the detection limit of the ohmmeter (20 k
/filter or 6.7 k
· cm2 for the type of
inserts used), and a PD of ~20 mV (apical side negative) across the
cell layer. TSH (0.1 mU/ml) added to the basal medium induced a rapid
fall in R to about 1 k
· cm2 and a
gradual increase in PD to >30 mV (Fig.
1). This effect of TSH is known to be
mimicked by forskolin (30), indicating involvement of cAMP as second
messenger, and it depends on increased transcellular ion transport (26,
29, 32) with maintained function of the tight junctions
(30). In the continued presence of 0.1 mU/ml TSH, both R and PD were
sustained at the new steady-state levels for
48 h without signs of
desensitization (Fig. 1). A higher TSH dose (1 mU/ml) was only slightly
more effective in depressing R and elevating PD, whereas a smaller dose
(0.01 mU/ml) caused only minor changes compared with untreated cultures
after 48 h (not shown). The response to 0.1 mU/ml was thus close to maximal and therefore chosen for acute and long-term stimulation (the
latter referred to as "prestimulated cultures") throughout the
experiments unless otherwise stated.

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Fig. 1.
Short- and long-term effects of thyrotropin (TSH) on
transepithelial resistance (R, top) and potential difference (PD,
bottom) in filter-cultured pig thyrocytes. TSH (0.1 mU/ml) was
added to basal medium (arrow), and R and PD were measured
repeatedly during culture for 48 h. Open symbols, control cultures;
filled symbols, TSH-treated cultures. Data shown are means ± SD,
n = 3.
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We next addressed the question of how fast the TSH-induced changes of R
and PD were reversed after removal of TSH from the culture medium.
Washout of TSH after 30 min of stimulation in previously TSH-deprived
cultures had no immediate effect on R, and full restitution was not
observed until after several hours in TSH-free medium (Fig.
2). In contrast, washout of TSH from prestimulated cultures (exposed to TSH for 48 h) caused a much more
rapid recovery of R, which generally was completed within 30 min (Figs.
2 and 3). TSH withdrawal also induced a fall in PD (Fig.
3).

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Fig. 2.
Effect of TSH withdrawal ("washout") on transepithelial R after
acute and long-term TSH stimulation. Cultures were exposed to TSH (0.1 mU/ml) for 30 min (squares) or 48 h (circles) and then exposed to
TSH-free medium (filled symbols) or continuously cultured in presence
of TSH (open symbols). Arrows indicate acute addition (1) and washout
(2) of TSH. Note different levels of R obtained after acute and
long-term TSH stimulation, respectively. Data shown are means ± SD,
n = 3.
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Fig. 3.
Effect of washout of TSH on transepithelial R ( ) and PD ( ) in
cultures prestimulated with TSH (0.1 mU/ml) for 48 h. Data shown are
means ± SD, n = 3.
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Effects of amiloride and furosemide on R and PD.
Because the TSH-induced changes in R and PD are due to stimulation of
transepithelial ion transport (29, 32), we examined whether the effect
of omitting TSH was mimicked by ion transport inhibitors. Amiloride and
furosemide were added to the apical and basal mediums, respectively. In
TSH-deprived cultures, these agents caused a decrease in PD to about
the same extent (Fig. 4), although the
effect of furosemide was slightly delayed. At the same time, R remained
at the highest detection limit (not shown), indicating that the
reduction of PD was indeed due to inhibited ion transport and not
associated with a loss of barrier function. The ion transport blockers
might in fact be expected to increase R further, but this was not
possible to register due to limitations of the ohmmeter.

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Fig. 4.
Effects of amiloride and furosemide on transepithelial PD in
TSH-deprived cultures. According to previous findings on site of action
of drugs (3, 19), amiloride (0.1 mM) was added to apical medium, and
furosemide (0.1 mM) was added to basal medium (arrow).
Untreated, amiloride, furosemide. Transepithelial
R was maintained at highest detectable level (20 k /filter) during
treatments (not shown). Data shown are means ± SD, n = 3.
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In cultures prestimulated with TSH for 48 h and still exposed to the
ligand, furosemide induced a gradual increase in R (Fig. 5A) and a rapid fall in PD (Fig.
5B). In contrast, amiloride had only marginal effects on R and
PD (Fig. 5, A and B). Thus inhibition of a
furosemide-sensitive effector mechanism at the basolateral cell surface
reproduced, albeit at a slower rate, the response of R and PD to TSH
withdrawal in prestimulated cultures.

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Fig. 5.
Effects of amiloride and furosemide on transepithelial R
(A) and transepithelial PD (B) in cultures
prestimulated with TSH for 48 h. Amiloride (0.1 mM, ) and furosemide
(0.1 mM, ) were added to apical and basal medium, respectively
(arrows). Gradual changes in R and PD in TSH-stimulated cultures not
exposed to inhibitors ( ) are generally observed during repeated
transfer of wells in and out of CO2 incubator. Data shown
are means ± SD, n = 3.
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Effects of IBMX, forskolin, and trypsinization on R and PD.
As shown in Fig. 2, the deactivation of TSH-induced ion transport was
much more rapid in prestimulated cultures than after acute TSH
stimulation. In principle, the rate-limiting step of deactivation might
be the effector mechanism itself, i.e., the ion transporter proteins in
the plasma membrane or at any level of the upstream TSH receptor
signaling pathway. However, the rapid rise of R observed after removal
of TSH from prestimulated cultures was completely prevented by the
phosphodiesterase inhibitor IBMX (Fig. 6),
indicating that deactivation involved breakdown of cAMP. Interestingly,
IBMX was able to decrease R in TSH-deprived cultures (Fig. 6),
supporting the notion that the TSH receptor has an intrinsic stimulatory effect on adenylate cyclase leading to the formation of
cAMP also in the absence of agonist (42).

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Fig. 6.
Effect of IBMX on transepithelial R with or without TSH prestimulation
(0.1 mU/ml) for 48 h. IBMX (0.5 mM) was added to basal medium (arrow);
prestimulated cultures were simultaneously exchanged for TSH-free
medium. Untreated cultures, IBMX, washout of TSH,
washout of TSH in presence of IBMX. Data shown are means ± SD,
n = 3.
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To study further the deactivation mechanism, trypsin was used as a tool
to rapidly eliminate TSH, and possibly the extracellular portion also
of its receptor, from the cell surface. In prestimulated cultures, the
recovery of R after washout of TSH was markedly accelerated by trypsin
being added to the basal medium (Fig. 7). This occurred without impairment of the epithelial barrier, unless a
high trypsin concentration (1% and in some experiments 0.1%) was
applied (Fig. 7). Similar results were obtained when trypsinization was
allowed in the presence of TSH (not shown). In contrast, trypsin did
not influence the lack of rapid restitution of R when TSH was removed
after acute stimulation (Fig. 8). Thus
deactivation of TSH receptor signaling by trypsin required that the
cells be long-term stimulated with TSH.

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Fig. 7.
Effect of cell surface trypsinization on transepithelial R in cultures
prestimulated with TSH (0.1 mU/ml) for 48 h. Basal medium was switched
to TSH-free solution without ( ) or with trypsin at different
concentrations: 0.001% ( ), 0.01% ( ), and 0.1% ( ).
Corresponding end-point levels of PD were 48 mV (washout of TSH), 23 mV
(0.001% trypsin), 14 mV (0.01% trypsin), 0 mV (0.1% trypsin). Note
decrease of R after treatment with 0.1% trypsin to level below that
recorded in cultures continuously exposed to TSH in absence of trypsin
( ). Data shown are means ± SD, n = 3. See text for further
comments.
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Fig. 8.
Effect of trypsin on restitution of transepithelial R after short- and
long-term TSH stimulation. Cultures were exposed to TSH (0.1 mU/ml) for 30 min (circles; first arrow, TSH addition) or 48 h
(squares) and then transferred to TSH-free medium (second arrow) with
or without addition of 0.001% trypsin. Open symbols, control;
filled symbols, trypsin treatment. Data shown are means ± SD,
n = 3.
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Treatment of prestimulated cultures with IBMX (Fig.
9A) or forskolin (Fig. 9B)
abolished the effect of trypsin on R. This indicates that trypsin
affected the ligand-receptor complex at the cell surface rather than
affecting postreceptor events along the signaling pathway or ion
transport molecules in the plasma membrane. To further elucidate the
main target of trypsinization, prestimulated cultures were first
exposed to trypsin during a short washout period and then stimulated
with TSH again. As shown in Fig. 10,
readdition of TSH caused a rapid decrease of R to almost the same level
as that induced when trypsin was omitted; thus the TSH receptor was
still capable of binding TSH and transmitting the signal into the cell.
On the other hand, the bioactivity of TSH was lost when TSH-containing
medium was trypsinized before being added to the cells (Fig.
9C). Together these observations indicate that trypsin affected
mainly the ligand rather than the receptor.

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Fig. 9.
Effect of trypsin on action of IBMX (A), forskolin (B),
and TSH (C) on transepithelial R. A: prestimulated
cultures (0.1 mU/ml TSH for 48 h) were switched to TSH-free medium
(bars 1 and 2) or incubated with 0.001% trypsin along
with prestimulatory dose of TSH (bars 3 and 4) in
absence (bars 1 and 3) or presence (bars 2 and
4) of 0.5 mM IBMX. B: TSH-deprived cultures were
exposed to 0.1 mU/ml TSH (bars 2 and 3) or 50 µM
forskolin (bars 4 and 5) in absence (bars 2 and
4) or presence (bars 3 and 5) of 0.1% trypsin.
C: untreated cultures were exposed to 0.1 mU/ml TSH (bar
2) or to TSH-containing medium that had been preincubated with
0.01% trypsin 15 min before use (bar 3); trypsin action on
cells was prevented by simultaneously adding trypsin inhibitor (0.1 mg/ml). Open bars (1), R recorded in control cultures for each
experiment; hatched and solid bars, treatments without and with
trypsin, respectively, in all graphs. Data shown are means ± SD,
n = 3, of end-point values of R obtained after 30 (A
and B) or 4 (C) min of incubation, respectively. See
text for further comments.
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Fig. 10.
Effect of TSH on transepithelial R after short-term trypsinization in
TSH-prestimulated cultures. Basal medium was switched to TSH-free
medium without ( ) or with ( ) addition of 0.001% trypsin (first
arrow). After incubation for 20 min, trypsin was replaced by medium
containing trypsin inhibitor (0.1 mg/ml). TSH (0.1 mU/ml) was readded
(second arrow) 30 min after beginning of washout period. Note that
response to TSH after trypsinization is slightly less pronounced than
it is after washout in absence of trypsin. Data shown are means ± SD,
n = 3.
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Effect of stepwise changes of TSH concentration on R.
Readdition of TSH to prestimulated cultures after washout caused again
a rapid decrease in R (Figs. 10 and 11). Moreover, this effect could be
repeated in several cycles of exchange between TSH-free and
TSH-containing media (Fig. 11).
Reciprocal alterations, albeit less prominent, were observed for PD
after the same treatment (not shown). With respect to the fairly high
TSH concentration (0.1 mU/ml) used in these experiments, the response
to TSH at levels closer to the physiological range was examined. As
shown in Fig. 12, stepwise changes of TSH
in the interval 0.01-0.1 mU/ml were followed almost immediately by
adjustments of R. The close relationship between R and the TSH level
was also evident when an intermediate concentration of TSH (0.025 mU/ml) was applied to prestimulated cultures, either directly when R
was still low or after a washout period when R had recovered (Fig.
13), irrespective of the starting point
at which a similar intermediate steady-state level of R was eventually
reached.

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Fig. 11.
Effect of alternating removal and readdition of TSH on transepithelial
R in long-term (48 h) prestimulated cultures. Arrows indicate exchange
for TSH-free (1) and TSH-containing (2) medium; corresponding R values
are indicated by and . TSH doses used for prestimulation and
restimulation after washout were the same (0.1 mU/ml). Data are means ± SD, n = 3.
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Fig. 12.
Dose-dependent effect of TSH on transepithelial R after long-term TSH
prestimulation. Different concentrations of TSH (0.125-0.1 mU/ml)
were added sequentially (arrows) after short periods of TSH withdrawal
(bars). * Transient reversal of R regularly occurring during
restitution after TSH washout. Data shown are from single culture
recording. See text for further comments.
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Fig. 13.
Dose-dependent effect of TSH on transepithelial R after prestimulation
with TSH (0.1 mU/ml) for 48 h. Cultures, either kept in prestimulation
medium ( ) or briefly incubated in TSH-free medium ( ; first arrow,
start of washout) were switched to medium containing intermediary
concentration (0.025 mU/ml) of TSH (second arrow, filled symbols). Data
shown are means ± SD, n = 3.
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DISCUSSION |
Deactivation of TSH-regulated vectorial electrolyte transport.
A major theme of the present study was to examine the effect of TSH
withdrawal on ion transport evaluated by measurements of
transepithelial R and PD in filter-cultured pig thyrocytes. The
ultimate purpose was to elucidate the mechanism by which TSH-regulated thyroid functions may be turned off in response to decreasing levels of
ligand and a corresponding silencing of TSH receptor signaling.
Deactivation was investigated in two situations, 1) after acute
stimulation (for 30 min) with a moderate TSH concentration (0.1 mU/ml)
in previously TSH-deprived cells, and 2) after prolonged exposure (for 48 h) to the same dose of TSH (referred to as
prestimulation). As previously described (2, 29, 32) and confirmed in
the present study, TSH stimulates Na+ and
Cl
conductance as reflected by a rapid (within
minutes) but long-lasting (for days) decrease in R and an increase in
PD. We found that withdrawal of TSH in acutely stimulated cells
resulted in a very slow recovery of the bioelectrical parameters, which
did not reach control levels (as in untreated cultures) until after
several hours. In contrast, in prestimulated cultures, full restitution of R was observed within 30 min after the switch to the TSH-free medium. Based on the effects of amiloride, an inhibitor of the apical
Na+ channel (3), and furosemide, an inhibitor of the
basolateral NaKCl2 symporter (22), the prevailing
electrolyte transport mechanisms were also found to differ between
acutely and chronically TSH-stimulated cells. The rapid changes of R
and PD occurring in response to acute TSH have been previously known to
be largely due to enhanced Na+ influx through an
amiloride-sensitive channel in the apical plasma membrane (15). In
contrast, the present results collectively indicated that
Cl
cotransport contributed more to the R and PD
levels measured in prestimulated cells. In fact, furosemide added to
the basal medium mimicked a TSH washout response (increasing R and
decreasing PD), even in the presence of TSH, whereas amiloride
administered apically did not. Altogether, these observations indicate
that deactivation of TSH-regulated electrolyte transport as a result of
TSH withdrawal is accelerated in prestimulated thyrocytes and that this
is predominantly an effect of turning off the basal-to-apical transport
of Cl
.
Rate-limiting step of deactivation after long-term TSH stimulation.
Deactivation of TSH-regulated thyroid functions certainly involves many
steps along the TSH receptor signaling pathway, e.g., interrupted
transduction of the G protein-coupled signal to adenylate cyclase,
diminished adenylate cyclase activity, degradation of intracellular
cAMP, and dephosphorylation of downstream signaling intermediate and
effector molecules. To elucidate the rate-limiting step, we
investigated whether deactivation was influenced by a mild cell surface
trypsinization that did not challenge the barrier function of the
epithelium. In TSH-prestimulated cultures, such treatment caused an
almost instantaneous reversal of R to high levels. Because the
trypsin-induced rise in R was not observed in cultures simultaneously
exposed to forskolin or the phosphodiesterase inhibitor IBMX, it is
likely that neither the generation of cAMP nor the ion transporter(s)
in the plasma membrane was affected. Moreover, pretreatment with
trypsin reduced the ability of TSH to reactivate ion transport only
marginally, supporting previous notions (39) that both ligand binding
and signal transduction of the TSH receptor are largely resistant to
trypsin. In contrast, the bioactivity of soluble TSH was rapidly
abolished by trypsin. Together, these findings strongly suggest that
TSH receptor deactivation is enhanced by proteolytic removal of
receptor-bound TSH. This in turn implies that the TSH receptor ceases
to signal, or at least that the signaling is markedly reduced, shortly
after being dissociated from its cognate ligand. After TSH is removed
from the cell surface, the downstream intracellular signaling cascade appears to be almost instantaneously interrupted. Although not measured, this probably comprises a rapid degradation of cAMP and
dephosphorylation of protein kinase A (PKA)-regulated target proteins.
It has been previously reported that the half-life of intracellular
cAMP is only ~2 min in dog thyroid cells (42) and that
phosphodiesterase activity is upregulated by TSH in pig thyrocytes
(36). Thus, in the chronically stimulated pig thyroid epithelium
sensing a sudden decrease in TSH concentration, dissociation of the
ligand receptor complex at the cell surface is rate limiting for the
rapid deactivation of TSH-regulated electrolyte transport.
Rate-limiting step of deactivation after acute TSH stimulation.
As discussed above, the reversal of R after withdrawal of TSH from
cultures that had been acutely stimulated for only 30 min was markedly
delayed compared with that observed in prestimulated cultures. This
difference was also apparent during trypsin treatment, which, unlike
after long-term TSH stimulation, did not enhance deactivation. Provided
that the effect of trypsin on the ligand receptor complex is equivalent
regardless of the duration of TSH exposure, these observations in
acutely stimulated cells suggest that intracellular signaling continues
when the signal input from the TSH receptor at the cell surface is
interrupted. A likely reason for this is that intracellular cAMP
persists at a level high enough to allow a continuous stimulation of
electrolyte transport, designated by a low R, even after
ligand-receptor dissociation. Because TSH increases the synthesis of
phosphodiesterase in pig thyrocytes (36), it is possible that this
enzyme is downregulated in TSH-deprived cultures and that this will
modulate the responsiveness to TSH. Another factor that might influence
the magnitude and duration of cAMP formation is the number of TSH
receptors available for ligand binding at the cell surface. TSH has
been found to regulate negatively the TSH receptor expression in FRTL-5
cells (33). On the other hand, the receptor level seems rather
resistant to changes in the TSH concentration in dog thyrocytes (23). Whether TSH influences the expression of TSH receptor in pig thyrocytes is unknown.
Possible mechanisms of TSH receptor signal deactivation.
The precise mechanism by which the TSH receptor signal is rapidly
deactivated when TSH is withdrawn from prestimulated cultures cannot be
determined with certainty. However, it is known that the association
and dissociation constants for TSH binding to the receptor are of the
same order (8, 40), suggesting that TSH bound to the receptor might be
in dynamic equilibrium with soluble TSH in the extracellular fluid.
Thus one possibility is that a release of receptor-bound ligand
directly alters the properties of the receptor, presumably by a
conformational change, which then ends signal transduction. This is
also supported by the data obtained in the trypsin experiments. Another
possibility is that the ligand-receptor complex, similarly to that of
other polypeptide hormones and their receptors, is cleared from the
cell surface by endocytosis and afterward by dissociation of the
complex in an endosomal compartment and that internalization is
important for deactivation. In response to agonist, many G
protein-coupled receptors are rapidly desensitized by
phosphorylation-dependent binding to arrestins, which mediate
uncoupling from the G proteins and endocytosis of the receptor (14,
21). Subsequent resensitization to the preligand-exposed state is
believed to involve endosomal dephosphorylation and recycling of the
receptor to the cell surface. Evidence for ligand-induced
internalization of the TSH receptor has been obtained in nonthyroid
cells overexpressing the transfected receptor (17, 18). Moreover,
desensitization of the TSH receptor might also be arrestin dependent
(20, 24, 25). However, TSH-induced desensitization is generally
recognized after acute stimulation, like the present experiments on
TSH-deprived cultures in which deactivation was markedly delayed.
Moreover, we did not observe any signs of desensitization on the
long-term action of TSH on R and PD at the given dose. It thus remains
an open question whether TSH receptor internalization influences the
responsiveness of prestimulated thyrocytes to reduced ligand
concentrations. Regardless of this, the almost instantaneous
deactivation observed after clearance of soluble and surface-bound TSH
by trypsin treatment strongly suggests that internalization is not a
prerequisite for turning off TSH receptor signaling. An alternative,
albeit speculative, mechanism for deactivation might be removal of
ligand-bound receptor by proteolysis. Indeed, shedding of the
ectodomain of the TSH receptor as a result of matrix metalloprotease
and protein disulfide isomerase activities has been identified (9, 39).
On-off signaling of the TSH receptor: possible physiological
implications.
Cultures prestimulated with TSH are conceivably a more adequate model
for studying the normal regulation of thyroid function than cultures
deprived of TSH. There is reason to believe, therefore, that the
dynamic deactivation of TSH receptor signaling and effector function as
has been observed in prestimulated cultures represents a mechanism of
physiological significance. This is further supported by the findings
that the steady-state level of R was rapidly adjusted to small
variations in the TSH concentration within or close to the
physiological range (12) and that such adjustments were possible to
reproduce in on-line recordings of the same culture during a
considerable period of time. Depending on the concentration of ligand,
it thus seems that the TSH receptor cycles between on-off signaling and
that this is transduced to corresponding modulation of the effector
mechanisms that govern plasma membrane conductivity. Altogether, the
present data indicate that TSH exerts a close surveillance on thyroid
activity. The influence of TSH is tonic but can be adjusted rapidly,
almost on a minute-to-minute basis, by means of an altered
concentration of the ligand. This in turn supports the notion that the
pulsatile and circadian release of TSH from the pituitary (7, 19) is
likely transmitted to rapid changes in thyroid structure and function
(19, 37, 43, 44). In terms of altered electrolyte transport, there are
several indications pointing to the possibility that this may have
important physiological implications. For instance, in rats with
inhibited TSH secretion caused by hypophysectomy or thyroxin treatment, the viscosity of the colloid in the follicle lumen is very high, and
the diffusion of newly iodinated thyroglobulin is consequently slow
(31, 34). However, shortly after injection of TSH, the iodinated
molecules are spread out evenly in the colloid as a sign of
unrestricted diffusion (16). Such altered properties of the colloid,
which are probably caused by changes in the ion and water content, may
facilitate iodination and endocytosis of thyroglobulin and may
ultimately enhance the release of thyroid hormone to the bloodstream
(35). Transepithelial ion transport identified in thyrocytes is
bidirectional with respect to the apical-basal polarity of the
epithelium (2). Na+ flux occurs preferentially in the
apical-to-basal direction via apical sodium channels and the
Na+-K+-ATPase located in the basolateral plasma
membrane (32). In contrast, Cl
is transported mainly
in the opposite direction through a basolateral NaKCl2
symporter and anion channels present in the apical plasma membrane (1,
6, 11). TSH positively regulates both transporting mechanisms via the
cAMP-PKA pathway, but, as suggested by the present data, the relative
importance may vary depending on the duration of TSH stimulation.
Activation of tyrosine kinase receptors and protein kinase C has also
been found to induce distinct changes in transepithelial R and PD
in filter-cultured thyrocytes (13, 29), further indicating that these
parameters are closely related to the functional status of the thyroid epithelium.
 |
ACKNOWLEDGEMENTS |
We are grateful to Therese Carlsson for superb technical assistance.
 |
FOOTNOTES |
This work was supported by grants from the Swedish Medical Research
Council (12X-537), Assar Gabrielsson Foundation, and the Harald
Jeansson and Harald and Greta Jeansson Foundations.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: LE Ericson,
Institute of Anatomy and Cell Biology, Göteborg University, PO
Box 420, SE 405 30 Göteborg, Sweden (E-mail:
Lars.E.Ericson{at}anatcell.gu.se).
Received 27 April 1999; accepted in final form 28 October 1999.
 |
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