Tumor Necrosis Factor-
, Sphingomyelinase, and Ceramide Inhibit
Store-operated Calcium Entry in Thyroid FRTL-5 Cells*
Kid
Törnquist
§¶,
Anna-Maria
Malm§
,
Michael
Pasternack**,
Robert
Kronqvist
,
Sonja
Björklund
,
Raimo
Tuominen§§, and
J. Peter
Slotte
From the
Department of Biology and the

Department of Biochemistry and Pharmacy,
Åbo Akademi University, BioCity, 20520 Turku, Finland and the
§§ Department of Pharmacology and Toxicology,
Institute of Biomedicine, the
Department of Biosciences,
Division of Animal Physiology, and the ** Institute of Biotechnology,
§ University of Helsinki, and the Minerva Foundation
Institute for Medical Research, 00250 Helsinki, Finland
 |
ABSTRACT |
Tumor necrosis factor
(TNF-
) is a potent
inhibitor of proliferation in several cell types, including thyroid
FRTL-5 cells. As intracellular free calcium
([Ca2+]i) is a major signal in activating
proliferation, we investigated the effect of TNF-
on calcium fluxes
in FRTL-5 cells. TNF-
per se did not modulate resting
[Ca2+]i. However, preincubation (10 min) of the
cells with 1-100 ng/ml TNF-
decreased the thapsigargin (Tg)-evoked
store-operated calcium entry in a concentration-dependent
manner. TNF-
did not inhibit the mobilization of sequestered
calcium. To investigate whether the effect of TNF-
on calcium entry
was mediated via the sphingomyelinase pathway, the cells were
pretreated with sphingomyelinase (SMase) prior to stimulation
with Tg. SMase inhibited the Tg-evoked calcium entry in a
concentration-dependent manner. Furthermore, an inhibition of
calcium entry was obtained after preincubation of the cells with the
membrane-permeable C2-ceramide and C6-ceramide analogues. The inactive ceramides dihydro-C2 and
dihydro-C6 showed only marginal effects. Neither SMase,
C2-ceramide, nor C6-ceramide affected the
release of sequestered calcium. C2- and
C6-ceramide also decreased the ATP-evoked calcium entry,
without affecting the release of sequestered calcium. The effect of
TNF-
and SMase was inhibited by the kinase inhibitor staurosporin
and by the protein kinase C (PKC) inhibitor calphostin C but not by
down-regulation of PKC. However, we were unable to measure a
significant activation of PKC using TNF-
or C6-ceramide.
The effect of TNF-
was not mediated via activation of either c-Jun
N-terminal kinase or p38 kinase. We were unable to detect an increase
in the ceramide (or sphingosine) content of the cells after stimulation
with TNF-
for up to 30 min. Thus, one mechanism of action of
TNF-
, SMase, and ceramide on thyroid FRTL-5 cells is to inhibit
calcium entry.
 |
INTRODUCTION |
An abundance of reports has shown that the cytokine tumor necrosis
factor-
(TNF-
)1 has
diverse effects upon several cell systems. TNF-
also potently modulates thyroid function, especially growth and differentiation. In
humans, the injection of TNF-
decreases serum triiodothyronine and
TSH levels (1), whereas in rats and mice TNF-
decreases both serum
triiodothyronine and thyroxine levels and serum TSH levels (2, 3). In
human thyroid cells in culture, TNF-
decreases the TSH-evoked
incorporation of 125I and the secretion of triiodothyronine
and thyroxine (4). TNF-
attenuates also the production of
thyroglobulin and cAMP in these cells (5). In rat FRTL-5 cells, TNF-
inhibits the TSH-evoked uptake of iodide and inhibits mitogen-evoked
cell proliferation (6-8). Furthermore, TNF-
inhibits the
TSH-evoked type I 5'-deiodinase activity, the expression of both the
thyroid peroxidase gene and the thyroglobulin gene (9-11), and
TSH-evoked hydrogen peroxide production (12).
TNF-
binds to two membrane receptors, a 55- and a 75-kDa receptor.
Of these two forms, the 55-kDa receptor apparently is the most
important (13, 14). Binding of TNF-
to FRTL-5 cells has also been
reported (3), although the receptor types have not been characterized.
TNF-
activates different sphingomyelinases in cells, resulting in
the hydrolysis of sphingomyelin to ceramide and stimulation of the
mitogen-activated protein kinase cascade, or the Jun kinase 1 (JNK-1)
cascade (15). TNF-
may also activate protein kinase C (PKC) (13).
The ceramide-evoked activation of NF
B is probably important in
linking the TNF-
-evoked stimulus to transcriptional activity in the
nucleus (16, 17). In human papillary thyroid carcinoma cells TNF-
has been shown to activate NF
B (18), indicating that this signaling
pathway also is present in thyroid cells. The type of SMase activated
upon stimulation is apparently crucial for the ultimate fate of the
cells, as the SMase-evoked production of ceramide may lead to
activation of apoptosis (via JNK-1), stimulate proliferation (via
mitogen-activated protein kinase), or protection against cytotoxicity
(15, 19). Of the reported effects of TNF-
on FRTL-5 cells, the
inhibition of proliferation (20), the inhibition of type I 5'deodinase activity (11), and the inhibition of TSH-evoked production of hydrogen
peroxidase (12) have also been induced by exogenous ceramide,
suggesting that these events are the result of the TNF-
-evoked activation of a sphingomyelinase and the hydrolysis of sphingomyelin to ceramide.
Other sphingomyelin breakdown products, like sphingosine (SP) and
sphingosine 1-phosphate (SPP), potently stimulate proliferation and
mobilize sequestered calcium in several cell types (21). These effects
of SP and SPP have also been observed in thyroid FRTL-5 cells (22-24).
Recent reports also show that SP attenuated store-operated calcium
entry (25, 26). Sphingosines and ceramides seem to have mostly opposite
effects on cellular proliferation. As both SP and SPP mobilize
sequestered calcium and stimulate calcium entry in FRTL-5 cells, two
important events in the initiation of proliferation, we thought that it
would be of interest to investigate whether ceramides could have any
effect on the regulation of calcium fluxes in these cells. Our results
showed that, in FRTL-5 cells, TNF-
, SMase, and ceramides potently
attenuated calcium entry. Thus, one mechanism of action of TNF-
on
thyroid cells is an inhibition of calcium entry.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Culture medium, serum, and hormones needed for
the cell culture was purchased from Life Technologies, Inc., Biological
Industries (Beth Haemek, Israel), and Sigma. Culture dishes were
obtained from Falcon Plastics (Oxnard, CA) or from Greiner (Germany).
GF109203X, N-acetylsphingosine (C2-ceramide),
and N-hexanoylsphingosine (C6-ceramide) and the
inactive forms N-acetylsphinganine (dihydro-C2)
and N-hexanoylsphinganine (dihydro-C6) were
purchased from Biomol (Plymouth Meeting, PA). Phenylmethylsulfonyl
fluoride, 1,2-sn-dioctanoyl-acylglycerol (DAG),
phosphatidylserine (PS), Triton X-100, staurosporin, phorbol 12-myristate 13-acetate (PMA), and sphingomyelinase were all purchased from Sigma. Human recombinant tumor necrosis factor-
, calphostin C,
and D609 were from Alexis Corp. (Laufelfingen, Switzerland). The
anti-active c-Jun N-terminal kinase was from Promega (Madison, WI). The
p38 kinase inhibitor SB203580 was from Calbiochem. Fura 2-AM and
bisoxonol were purchased from Molecular Probes, Inc. (Eugene, OR).
Thapsigargin was from LC Services Corp. (Woburn, MA). Lipid standards
(bovine brain sphingomyelinase, ceramide, and sphingosine) were
obtained from Sigma. All other chemicals used were of reagent grade.
Whatman P81 phosphocellulose paper was purchased from Whatman (UK).
[
-32P]ATP is a product of Amersham Corp. (UK). Bovine
TSH was a generous gift from Dr. A. F. Parlow (NHPP, NIDDK, National
Institutes of Health).
Cell Culture--
Rat thyroid FRTL-5 cells were a generous gift
of Dr. Egil Haug (Akers Hospital, Oslo, Norway). The cells were grown
in Coon's modified Ham's F-12 medium, supplemented with 5% calf
serum, and six hormones (27) (insulin, 10 µg/ml; transferrin, 5 µg/ml; hydrocortisone, 10 nM; the tripeptide
Gly-L-His-L-Lys, 10 ng/ml; TSH, 0.3 milliunits/ml; somatostatin, 10 ng/ml) in a water-saturated atmosphere
of 5% CO2 and 95% air at 37 °C. Before an experiment, cells from one donor culture dish were harvested with a 0.2% trypsin solution and plated onto plastic 100- or 35-mm culture dishes. The
cells were grown for 7-8 days before an experiment, with 2-3 changes
of the culture medium. Fresh medium was always added 24 h prior to
an experiment. In the current-clamp experiments, the cells were grown
on round coverslips in 24-well culture dishes.
Measurement of [Ca2+]i--
The medium was
aspirated, and the cells were harvested with HEPES-buffered saline
solution (HBSS, in millimolar concentrations: NaCl, 118; KCl, 4.6;
glucose, 10; CaCl2, 1.0; HEPES, 20; pH 7.2) lacking
Ca2+ but containing 0.02% EDTA and 0.1% trypsin. After
washing the cells three times by pelleting, the cells were incubated
with 1 µM Fura 2-AM for 30 min at 37 °C. Following the
loading period, the cells were washed twice with HBSS buffer and
incubated for at least 10 min at room temperature and washed once
again. Fluorescence was measured with a Hitachi F2000 fluorimeter. The
excitation wavelengths were 340 and 380 nm, and emission was measured
at 510 nm. The signal was calibrated by addition of 1 mM
CaCl2 and Triton X-100 to obtain maximal fluorescence.
Chelating extracellular Ca2+ with 5 mM EGTA and
the addition of Tris-base was used to elevate pH above 8.3 to obtain
minimal fluorescence. [Ca2+]i was calculated as
described by Gryenkiewicz et al. (28), using a computer
program designed for the fluorimeter with a Kd value
of 224 nM for Fura 2.
Measurement of Ceramide and Sphingosine Production--
Cells
grown on 35-mm dishes were labeled with
L-[3-3H]serine (5 µCi/ml) for 48 h in
6H medium. The plates were then washed twice with PBS (in millimolar
concentrations; NaCl, 137; KCl, 2.7; Na2HPO4,
8, KH2PO4, 1.5; pH 7.4) and incubated for
1 h in serum-free Ham's F-12 medium in a water bath at 37 °C.
Then TNF-
(final concentration 100 ng/ml) was added, and the plates
were incubated for 3-30 min. In some experiments SMase (final
concentration 100 milliunits/ml diluted in Ham's F-12) was added to
the plates, and the plates were incubated for 30 min. After the
incubation, the plates were rapidly washed with ice-cold PBS and then
frozen. Lipids were extracted by two 20-min incubations in 2 ml of
hexane:propanol (3:2, v/v) on a shaker at room temperature. For protein
measurements, the proteins were hydrolyzed in 1 ml of 0.1 M
NaOH and determined according to Lowry et al. (29). The
lipid extracts were pipetted to glass tubes and dried in a gentle
stream of air. The dried lipids were dissolved in 70 µl of
hexane:2-propanol. Sphingomyelin was determined by application of the
lipid extract to plastic-backed TLC plates (Whatman). The lipids were
separated using chloroform:methanol:concentrated acetic acid:water
(50:30:8:3) (30). After detection using iodine vapor, the appropriate
bands were cut, and radioactivity was measured in a scintillation
counter. Ceramide and sphingosine were separated using high performance
TLC plates (Merck), and the lipids were separated by two elutions using
chloroform:methanol:2 N NH4OH (40:10:1) (31).
The plates were allowed to dry between the separate runs. The lipids
were detected using iodine vapor, and the appropriate bands were
scraped into scintillation vials, and the radioactivity was determined.
Activation of Protein Kinase C--
Immediately before exposure
of the cells, the 6H medium was removed from the wells and the
treatment was started by adding 0H medium containing various
concentrations of the substances. Protein kinase C (PKC) activity was
measured by the method of Kikkawa et al. (32) and Roskoski
(33), with some modifications (34). The experiments were terminated by
removing the medium and washing the cells three times with an ice-cold
Ca2+-free salt solution (in millimolar concentrations:
NaCl, 145; KCl, 5.2; NaH2PO4, 1; glucose, 11.2;
HEPES, 15; pH 7.4). The cells were scraped from the plates and
homogenized by sonication (2 × 15 s) in an ice-cold lysis
buffer (containing in millimolar concentrations: EDTA, 2;
phenylmethylsulfonyl fluoride, 1; Tris-HCl, 20; pH 7.5, and 50 µg/ml
leupeptin). Homogenates were centrifuged for 60 min at 100,000 × g at 4 °C. The supernatant served as the soluble
fraction. The pellet was dispersed into the same buffer containing
0.1% Triton X-100, and the homogenate was incubated on ice for 60 min.
The mixture was centrifuged at 100,000 × g for 60 min
at 4 °C. This supernatant constituted the particulate PKC activity.
The protein content in both subcellular fractions was measured
according to Bradford (35). In the PKC assay, the final reaction
mixture (100 µl) contained in millimolar concentrations the
following: Tris-HCl, 35; pH 7.5; mM EGTA, 0.25; EDTA, 0.5; MgCl2, 6; phenylmethylsulfonyl fluoride, 0.25; PKC-specific
substrate peptide FKKSFKL-NH2, 34 nM (36);
CaCl2, 1; and 0.1 mM [32P]ATP
(100-200 cpm/pmol). The mixture also contained leupeptin (12.5 µg/ml), phosphatidylserine (PS, 40 µg/ml), and diacylglycerol (DAG,
8 µg/ml). PKC activity was calculated as the difference in the
activity in the presence and absence of CaCl2, PS, and DAG.
The activity in the absence of CaCl2, PS, and DAG was the same as that obtained when only PS and DAG were omitted. The reaction was started by adding protein (0.7-1.5 µg). The samples were
incubated for 5 min at 30 °C, and the reaction was stopped by
spotting 25 µl of each reaction mixture onto Whatman P81
phosphocellulose paper (1.5 × 1.5 cm). The papers were washed
three times in 75 mM phosphoric acid. After air-drying, the
radioactivity measured was determined. The results are expressed as
nanomoles of inorganic phosphate incorporated to substrate
peptide/mg of protein/min.
Immunoblotting of PKC Isoenzymes--
SDS-polyacrylamide gel
electrophoresis was run using a minigel apparatus (Midget
Electrophoresis Unit, Pharmacia, Sweden). Proteins (1 and 3 µg per
well for soluble and particulate proteins, respectively) were loaded
onto 8% polyacrylamide-SDS gels and separated according to molecular
weight. The proteins were electrophoretically transferred to
methylcellulose membranes. The membranes were incubated three times for
15 min at 45 °C in Tween/TBS (TTBS, containing in millimolar
concentrations: NaCl, 500; Tris-base, 20; pH 7.5, and 0.1% Tween 20)
containing 5% fat-free dry milk and 15 min in TTBS. Then the membranes
were incubated for 2 h with 1:4000-1:80,000 dilution of rabbit
polyclonal anti-rat PKC antibodies that recognize
,
1,
2,
,
,
, and
subtypes of PKC (37). A horseradish peroxidase-labeled goat anti-rabbit antibody (Bio-Rad) was used as the
secondary antibody, and the immunoreactive bands were visualized by
enhanced chemiluminescence (ECL, Amersham Corp., UK). The localization of immunoreactive proteins was compared with those of prestained molecular weight markers (Life Technologies, Inc.).
Electrophoresis and Western Blotting of c-Jun N-terminal
Kinase--
The cells were grown on 60- or 100-mm plates as described
above. The cells were harvested as above and were allowed to rest for
30 min at 37 °C in HBSS. After incubation with TNF-
for 1-30 min, the cells were centrifuged and extracted in ice-cold lysis buffer
(containing in mM concentrations: NaCl, 100;
Na3VO4, 2; EDTA, 2; Tris-base, 20 mM, pH 8.0; and 3% Nonidet P-40). A sample of the extract
was mixed with an equal volume of boiling SDS buffer (glycerol, 20%;
2-mercaptoethanol, 10%; SDS, 4%; bromphenol blue, 0.02%; Tris-base,
0.125 mM, pH 6.8). Equal amounts of protein were separated
by SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels.
The proteins were transferred electrophoretically to nitrocellulose
membranes (Schleicher & Schuell, Dassel, Germany). The membrane was
incubated with 5% nonfat dry milk for 1 h at room temperature in
Tris-buffered saline (TBS, in mM concentrations: NaCl, 500;
Tris-base, 20, pH 7.5) to block the remaining binding sites. The blots
were incubated with anti-active JNK antibody (1:5000) diluted in TBS
containing 5% nonfat dry milk at 4 °C overnight. The blots were
then incubated with peroxidase-conjugated anti-rabbit antibody (1:10
000) for 2 h at room temperature, and the proteins were detected
using the ECL Western blotting detection kit according to the
manufacturer's instructions.
Measurement of [3H]Thymidine Incorporation in
FRTL-5 Cells--
The cells were plated onto 35-mm dishes and grown in
6H medium for 2-3 days. Then the cells were washed twice with PBS and grown in 0H (Coon's medium without hormones or serum) containing 0.2%
bovine serum albumin for 2 days. The medium was then changed to
0H/bovine serum albumin containing the appropriate concentrations of
the test compounds and [3H]thymidine (0.4 µCi/ml), and
the cells were incubated for 24 h (38). The cells were washed
twice with cold PBS solution and once with cold 5% trichloroacetic
acid. The trichloroacetic acid-insoluble precipitate was dissolved in
0.1 N NaOH, and the radioactivity was measured by
scintillation counting.
Statistics--
The results are expressed as the means ± S.E. Statistical analysis was made using Student's t test
for paired observations. When three or more means were tested, analysis
of variance was used.
 |
RESULTS |
TNF-
, SMase, and Cell-permeable Ceramides Inhibit DNA
Synthesis--
Previous studies have shown that TNF-
and
C2- and C6-ceramides potently inhibit both the
TSH- and the insulin-evoked incorporation of
[3H]thymidine in DNA, i.e. DNA synthesis
(6-8, 20). We confirmed these results and further showed that SMase
also inhibited the incorporation of [3H]thymidine in
response to TSH and insulin (data not shown). The inactive ceramides
dihydro-C2 and dihydro-C6 had only marginal effects (data not shown).
TNF-
Inhibits Calcium Entry--
In FRTL-5 cells, as well as in
other cell types, changes in [Ca2+]i are probably
important events in the initialization of cell proliferation (39, 40).
We thus investigated the effect of TNF-
on
[Ca2+]i and on the entry of calcium in FRTL-5
cells. TNF-
(100 ng/ml, the highest dose tested) did not per
se affect [Ca2+]i in these cells (data not
shown). To avoid any possible effects of TNF-
on receptor-mediated
events, we activated calcium entry by stimulating the cells with the
Ca2+ATPase inhibitor thapsigargin (Tg) (41). Tg activates a
rapid store-operated calcium entry in FRTL-5 cells (42). Pretreatment of the cells with TNF-
for 10-30 min potently attenuated the Tg-evoked calcium entry in both a calcium-containing buffer and in a
calcium-free buffer in a concentration-dependent manner
(Fig. 1). We also observed that TNF-
did not inhibit the Tg-evoked mobilization of sequestered calcium. In
cells pretreated with 100 ng/ml TNF-
for 10 min, the Tg-evoked
release of intracellular calcium was 178 ± 15 nM,
compared with 155 ± 10 nM in control cells. These
experiments were performed in a calcium-free buffer to avoid any
interference of Tg-evoked calcium entry.

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Fig. 1.
Effect of TNF- on
thapsigargin-evoked calcium entry in FRTL-5 cells. The cells were
harvested and loaded with Fura 2 as described under "Experimental
Procedures." A, control cells (trace a) or
cells pretreated with TNF- (100 ng/ml for 10 min; trace
b) were stimulated with 2 µM thapsigargin
(large arrowhead). B, control cells (trace
a) or cells pretreated with TNF- (100 ng/ml for 10 min;
trace b) were stimulated with 2 µM
thapsigargin (small arrowhead) in a calcium-free buffer, and
then Ca2+ (final concentration 1 mM) was added
to the cells (large arrowhead). C, cells
pretreated for 10 min with the indicated concentrations of TNF- were
stimulated with 2 µM thapsigargin in a calcium-free
buffer, and then Ca2+ (final concentration 1 mM) was added to the cells. The calcium-evoked spike ( )
and plateau level ( ) of [Ca2+]i were then
measured. Each point is the mean ± S.E. of 4-7 separate
experiments.
|
|
To investigate whether the observed effect of TNF-
was due to
activation of a sphingomyelinase, we preincubated the cells with the
phosphatidylcholine-phospholipase C inhibitor D609. Previous investigations have shown that D609 effectively inhibits TNF-
-evoked events (16). However, we observed that D609 was a very potent modulator
of calcium entry in FRTL-5 cells (data not shown). Furthermore, D609
also mobilized sequestered calcium in our cells (data not shown). Thus,
D609 is apparently not a suitable compound for studies using intact
cells, as its effects on calcium fluxes probably will affect a
multitude of cellular events.
Activation of protein kinases, including PKC, is an important part of
the signaling cascade evoked by TNF-
(15). We preincubated the cells
with 200 nM staurosporin for 10 min prior to addition of
100 ng/ml TNF-
. In these cells, we were unable to detect any TNF-
-evoked inhibition of calcium entry in cells stimulated with Tg
(Fig. 2). We next investigated the effect
of the PKC inhibitor calphostin C, and we treated the cells with 100 nM calphostin C for 10 min prior to addition of 100 ng/ml
TNF-
. In these cells, the effect of TNF-
on the Tg-evoked
increase in [Ca2+]i was abolished (Fig. 2). We
have also shown earlier that stimulating FRTL-5 cells with the phorbol
ester PMA attenuates store-operated calcium entry (42). In the present
study, pretreatment with 200 nM PMA significantly decreased
the plateau level of [Ca2+]i after readdition of
calcium to cells stimulated with Tg in a calcium-free buffer (Fig. 2).
Pretreatment of the cells with both PMA and 100 ng/ml TNF-
decreased
both the transient increase in [Ca2+]i as well as
the new plateau level of [Ca2+]i (Fig. 2). In
addition, in these cells the increase in the plateau level of
[Ca2+]i was of lower magnitude than in cells
treated with PMA only, suggesting an additive effect of PMA and TNF-
on calcium entry (Fig. 2).

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Fig. 2.
Protein kinase mediates the effect of
TNF- on thapsigargin-evoked calcium entry in
FRTL-5 cells. The cells were harvested and loaded with Fura 2 as
described under "Experimental Procedures." A, the cells
were preincubated with staurosporin (200 nM for 10 min)
prior to addition of vehicle or TNF- (100 ng/ml). Control cells
(trace a) or cells pretreated with TNF- for 10 min (in
the continuous presence of staurosporin; trace b) were
stimulated with 2 µM thapsigargin (small
arrowhead) in a calcium-free buffer, and then Ca2+
(final concentration 1 mM) was added to the cells
(large arrowhead). B, the cells were preincubated
with calphostin C (100 nM for 10 min) prior to addition of
vehicle or TNF- (100 ng/ml). Control cells (trace a) or
cells pretreated with TNF- for 10 min (in the continuous presence of
calphostin C; trace b). The cells were then stimulated
exactly as described in A. The traces shown in A
and B are representative of 4-7 separate experiments.
C, the cells were pretreated with PMA (200 nM
for 2 min; ) or both PMA and TNF- ( ); thereafter the cells
were stimulated with 2 µM thapsigargin in a calcium-free
buffer. Then Ca2+ (final concentration 1 mM)
was added, and the peak increase and the new plateau level of
[Ca2+]i were measured. Control cells, . Each
bar gives the mean ± S.E. of 6-7 separate
determinations. *, p < 0.05 compared with control
cells; +, p < 0.05 compared with PMA-treated
cells.
|
|
SMase and Cell-permeable Ceramides Inhibit Calcium Entry--
To
investigate further whether the effect of TNF-
on calcium entry was
mediated via activation of SMase, we preincubated the cells with
different concentrations of exogenous SMase for 30 min. As shown in
Fig. 3, SMase inhibited calcium entry in
a concentration-dependent manner very similar to that of
TNF-
. Furthermore, SMase did not affect the amount of sequestered
calcium. In cells treated with SMase (1 units/ml) for 30 min, the
increase in [Ca2+]i evoked by Tg in a
calcium-free buffer was 130 ± 23 nM, compared with
155 ± 10 nM in control cells.

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Fig. 3.
Effect of SMase on thapsigargin-evoked
calcium entry in FRTL-5 cells. The cells were harvested and loaded
with Fura 2 as described under "Experimental Procedures."
A, control cells (trace a) or cells pretreated
with SMase (1 unit/ml for 30 min; trace b) were stimulated
with 2 µM thapsigargin (large arrowhead).
B, control cells (trace a) or cells pretreated
with SMase (1 unit/ml for 30 min; trace b) were stimulated
with 2 µM thapsigargin (small arrowhead) in a
calcium-free buffer, and then Ca2+ (final concentration 1 mM) was added to the cells (large arrowhead).
C, cells pretreated for 30 min with the indicated
concentrations of SMase were stimulated with 2 µM
thapsigargin in a calcium-free buffer, and then Ca2+ (final
concentration 1 mM) was added to the cells. The
calcium-evoked spike ( ) and plateau level ( ) of
[Ca2+]i were then measured. Each point is the
mean ± S.E. of 4-7 separate experiments.
|
|
In the next series of experiments, the cells were incubated with 200 nM staurosporin for 10 min prior to addition of SMase (1 units/ml for 30 min). In these experiments, pretreatment with staurosporin totally abolished the effect of SMAse (Fig.
4), in a manner similar to what was
observed in cells treated with both staurosporin and TNF-
. To
investigate whether the effect of SMase was mediated via activation of
PKC, we pretreated the cells with 100 nM calphostin C. In
these experiments calphostin C also abolished the effect of SMase (Fig.
4). However, in cells in which PKC was down-regulated by preincubating
the cells with 2 µM PMA for 24 h, SMase (1 milliunit/ml for 30 min) still attenuated the Tg-evoked calcium entry
(Fig. 4).

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Fig. 4.
Modulation of the effect of SMase on the
thapsigargin-evoked calcium entry in FRTL-5 cells. The cells were
harvested and loaded with Fura 2 as described under "Experimental
Procedures." A, the cells were preincubated with
staurosporin (200 nM for 10 min) prior to addition of
vehicle or SMase (1 unit/ml). Control cells (trace a) or
cells pretreated with SMase (1 unit/ml for 30 min; trace b)
were stimulated with 2 µM thapsigargin (small
arrowhead) in a calcium-free buffer, and then Ca2+
(final concentration 1 mM) was added to the cells
(large arrowhead). B, the cells were preincubated
with calphostin C (100 nM for 10 min) prior to addition of
vehicle or SMase. Control cells (trace a) or cells
pretreated with SMase (1 units/ml for 30 min; trace b) were
stimulated with 2 µM thapsigargin (small
arrowhead) in a calcium-free buffer, and then Ca2+
(final concentration 1 mM) was added to the cells
(large arrowhead). C, the cells were preincubated
with PMA (2 µM for 24 h) prior to addition of
vehicle or SMase (1 unit/ml). Control cells (trace a) or
cells pretreated with SMase (1 unit/ml for 30 min; trace b)
were stimulated with 2 µM thapsigargin (small
arrowhead) in a calcium-free buffer, and then Ca2+
(final concentration 1 mM) was added to the cells
(large arrowhead). The traces shown are
representative of 4-5 separate experiments.
|
|
To investigate whether the observed effect of SMase was due to
inhibition of calcium entry, and not due to an enhanced calcium extrusion (43), we tested the effect of SMase on barium entry. As
barium is not a substrate for Ca2+ATPase, barium cannot be
extruded from the cells (44). In cells pretreated with SMase (1 units/ml for 30 min), and then stimulated with Tg, the entry of barium
was clearly attenuated (Fig. 5). Thus,
our results suggest that pretreatment of the cells with SMase indeed
resulted in a decreased store-operated entry of calcium.

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Fig. 5.
SMase inhibits the thapsigargin-evoked barium
entry in FRTL-5 cells. The cells were harvested and loaded with
Fura 2 as described under "Experimental Procedures." Control cells
(trace a) or cells pretreated with SMase (1 unit/ml for 30 min; trace b) were stimulated with 2 µM
thapsigargin (small arrowhead) in a calcium-free buffer, and
then Ba2+ (final concentration 1 mM) was added
to the cells (large arrowhead). The traces shown
are representative of 3-4 separate experiments.
|
|
We then investigated the effect of two membrane-permeable ceramide
analogs, C2- and C6-ceramide. These compounds
per se were without any significant effects on basal
[Ca2+]i levels. As seen in Fig.
6, C6-ceramide attenuated Tg-evoked calcium entry in FRTL-5 cells in a
concentration-dependent manner. The inactive analogue
dihydro-C6 was without an effect. In addition, neither
C6-ceramide nor dihydro-C6 had any effects on
the Tg-evoked release of sequestered calcium from these cells. The
Tg-evoked increase in [Ca2+]i in a calcium-free
buffer was 145 ± 12 nM in control cells, 142 ± 18 nM in cells treated with 30 µM
C6-ceramide, and 149 ± 15 nM in cells
treated with 30 µM dihydro-C6. Furthermore, in PKC down-regulated cells, C6-ceramide attenuated the
Tg-evoked calcium entry (data not shown). We also tested
C2-ceramide and obtained a decreased calcium entry in
Tg-stimulated cells. However, the effect of C2 was smaller
than that observed with C6-ceramide (data not shown).
Dihydro-C2 had no effect on calcium entry (data not
shown).

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Fig. 6.
Effect of C6-ceramide on
thapsigargin-evoked calcium entry in FRTL-5 cells. The cells were
harvested and loaded with Fura 2 as described under "Experimental
Procedures." A, C6-ceramide (final
concentration 30 µM; trace c) or
DHC6-ceramide (final concentration 30 µM;
trace b) was added to the cells (white
arrowhead), and then the cells were stimulated with 2 µM thapsigargin (large arrowhead). Control
cells, trace a. B, C6-ceramide (final
concentration 30 µM; trace c) or
DHC6-ceramide (final concentration 30 µM;
trace b) was added to cells (white arrowhead)
incubated in a calcium-free buffer. After 1 min, the cells were
stimulated with 2 µM thapsigargin (small
arrowhead), and then Ca2+ (final concentration 1 mM) was added to the cells (large arrowhead).
Control cells, trace a. C, cells pretreated for 1 min with the indicated concentrations of C6-ceramide were
stimulated with 2 µM thapsigargin in a calcium-free
buffer, and then Ca2+ (final concentration 1 mM) was added to the cells. The calcium-evoked spike ( )
and plateau level ( ) of [Ca2+]i were then
measured. Each point is the mean ± S.E. of 4-7 separate
experiments.
|
|
TNF-
, C6-ceramide, and DHC6-ceramide and
the Activation of PKC--
In addition to activating the
sphingomyelinase pathway, TNF-
may activate PKC (15). Recent studies
have shown that FRTL-5 cells express the
,
,
, and
isoforms of PKC (45), and our initial experiments confirmed these
findings (data not shown). However, we were unable to show an
activation of PKC in cells stimulated with neither TNF-
,
C6-ceramide, nor DHC6-ceramide (Table
I). In control experiments PMA
significantly activated PKC (Table I).
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Table I
Lack of an effect of TNF- , C6-ceramide, and
DHC6-ceramide on PKC activity in FRTL-5 cells
The cells were incubated in 0H medium containing either
C6-ceramide (final concentration 30 µM),
DHC6-ceramide (final concentration 30 µM), or PMA
(final concentration 1 µM) for 1 min or with TNF-
(final concentration 100 ng/ml) for 10 min, and the activation of PKC
was determined as described under "Experimental Procedures." The
results are given as mean ± S.E. of 3-8 separate determinations.
|
|
Importance of c-Jun N-terminal Kinase and p38 Mitogen-activated
Protein Kinase--
TNF-
may activate JNK (15) and p38
mitogen-activated protein kinase (46) in several cell types.
Furthermore, at least in human thyroid cells, JNK may be activated by a
PKC-mediated mechanism (47). When our cells were stimulated with
TNF-
(100 ng/ml), a transient activation of JNK was observed after 1 and 3 min of stimulation (data not shown). This effect was absent in
cells pretreated with PMA (1 µM for 24 h, data not
shown). As the effect of TNF-
on Tg-evoked calcium entry still
occurred in cells pretreated with PMA, it is not likely that JNK is
involved in the attenuating effect of TNF-
on store-operated calcium
entry. Furthermore, in cells pretreated for 30 min with the p38 kinase inhibitor SB203580 (final concentration 10 µM), TNF-
still abrogated the Tg-evoked increase in calcium. In cells pretreated
with SB203580 and then with TNF-
(100 ng/ml) for 10 min, the
readdition of calcium to cells stimulated with 2 µM Tg
increased [Ca2+]i transiently by 459 ± 40 nM and stabilized at a plateau level 201 ± 12 nM above the prestimulatory [Ca2+]i
level. In cells treated with SB203580, but not with TNF-
, the values
were 587 ± 35 and 269 ± 18 nM, respectively (p < 0.05 for both values). SB203580 per se
did not affect the Tg-evoked increase in [Ca2+]i,
compared with control cells (data not shown).
Production of Ceramide and Sphingosine in FRTL-5 Cells in Response
to TNF-
--
Stimulating the cells with TNF-
(final
concentration 100 ng/ml) for up to 30 min did not result in a
significant increase in ceramide production, although SMase (100 milliunits/ml) potently increased ceramide content of the cells (Table
II). Furthermore, TNF-
did not
decrease the amount of sphingomyelin in the cells, although this was
clearly obtained with SMase (Table II). In addition, we could not see
an increase in cellular sphingosine content in response to a 30-min
incubation with TNF-
. Thus, the lack of an effect of TNF-
on
ceramide production was not the result of a rapid conversion of
ceramide to sphingosine (data not shown). A similar lack of an increase
in sphingosine content was obtained when the cells were stimulated with
SMase (data not shown).
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Table II
Effects of TNF- and SMase on the ceramide and sphingomyelin content
of FRTL-5 cells
The cells were preincubated with [3H]serine (5 µCi/ml) for
48 h and then pretreated as described under "Experimental
Procedures." The cells were then stimulated with the indicated
concentrations of TNF- or SMase for 30 min. The amounts of lipids
were then determined as described under "Experimental Procedures."
|
|
Ceramides Inhibit ATP-evoked Calcium Entry--
Previous studies
have shown that ATP evokes calcium entry in FRTL-5 cells (48). It was
of interest to investigate whether the tested ceramides also could
attenuate ATP-evoked calcium entry. Neither C2-ceramide nor
dihydro-C2 affected the transient increase in
[Ca2+]i in response to 100 µM ATP
(Fig. 7). The ATP-evoked increase in
[Ca2+]i in control cells was 880 ± 71 nM, in cells treated with 30 µM
C2-ceramide 796 ± 85 nM, and in cells
treated with 30 µm dihydro-C2 733 ± 69 nM. However, C2 -ceramide clearly attenuated the plateau phase of the ATP-evoked change in
[Ca2+]i, i.e. calcium entry.
Dihydro-C2 was without an effect (Fig. 7). A similar lack
of an effect of TNF-
and C2-ceramide on the GTP-evoked
transient increase in [Ca2+]i has also been
reported (12). Furthermore, in cells stimulated with ATP in a
calcium-free buffer, the ATP-evoked increase in
[Ca2+]i was 53 ± 11 nM in
control cells, in cells treated with 30 µM
C2-ceramide 55 ± 10 nM, and in cells
treated with 30 µM dihydro-C2 the response to
ATP was 65 ± 10 nM. Similar results were obtained
using C6 and dihydro-C6 (data not shown). It is interesting to note that the ATP-evoked receptor-mediated, transient calcium entry (48) was not affected by the tested compounds.

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Fig. 7.
Effect of C2-ceramide on the
ATP-evoked calcium entry in FRTL-5 cells. The cells were harvested
and loaded with Fura 2 as described under "Experimental
Procedures." C2-ceramide (final concentration 30 µM; trace c) or DHC2-ceramide
(final concentration 30 µM; trace b) was added
to the cells (white arrowhead), and then the cells were
stimulated with 100 µM ATP (black arrowhead).
Control cells, trace a. The traces shown are
representative of 5 separate experiments.
|
|
 |
DISCUSSION |
In the present investigation we show that TNF-
inhibits
store-operated calcium entry in FRTL-5 thyroid cells. The same effect was observed when the cells were treated with SMase and
membrane-permeable ceramide derivatives. In a recent observation,
Barger et al. (19) showed that TNF-
inhibits calcium
entry in hippocampal neurons in response to glutamate. However, in that
study the importance of SMase or ceramides was not evaluated. Although
we were unable to observe an increase in ceramide production after
incubating the cells with TNF-
for periods relevant for the
inhibition of calcium entry, our observation is the first to suggest
that TNF-
, SMase, and ceramides acutely inhibit calcium entry.
Another mechanism of action has been shown in osteoblasts. In these
cells, several cytokines, including TNF-
, inhibited a parathyroid
hormone-evoked increase in [Ca2+]i by abrogating
the parathyroid hormone-induced formation of inositol
1,4,5-trisphosphate (49). However, this effect required at least 8 h of incubation with TNF-
.
In thyroid cells, TNF-
inhibits an array of different functions.
Some of these, like the inhibition of proliferation (20), the
inhibition of type I 5'-deiodinase (11), and the inhibition of
TSH-evoked production of hydrogen peroxide (12), have clearly been
shown to be mediated via the production of ceramides. Of these events,
at least the activation of proliferation is crucially dependent on
intracellular calcium, and especially on calcium entry (39, 40). Based
on our findings in the present study it is thus tempting to speculate
that the TNF-
-evoked inhibition of calcium entry is one important
mechanism in inhibition of proliferation. Clearly it cannot solely
explain the effects of TNF-
on FRTL-5 cells, especially as TNF-
has cytotoxic effects that probably are mediated via
calcium-independent signaling pathways. Furthermore, TNF-
inhibits
the activation of thyroid peroxidase and the production of thyroid
hormones (4, 6-8, 10, 11). Both the activation of thyroid peroxidase
and the production of thyroid hormones involve calcium-dependent events in FRTL-5 cells (50-54). Thus, it
is possible that the inhibitory effect of TNF-
on these processes
also is, at least in part, mediated via inhibition of calcium entry. It is also interesting to note that TNF-
may be produced by thyroid epithelial cells (55), suggesting an autocrine function for TNF-
.
Our results suggest that the mechanism by which TNF-
attenuated
calcium entry could involve activation of SMase and the production of
ceramides. This signaling pathway is usually connected to the binding
of TNF-
to the p55 TNF-
receptor (13, 14). Although binding of
TNF-
to FRTL-5 cells has been shown (3), there presently exists no
information on the type of TNF-
receptors present in these cells.
Our results show that TNF-
did not induce a measurable increase in
ceramide for up to 30 min of incubation. In a recent study in FRTL-5
cells an effect of TNF-
on ceramide production was observed, but the
first measurements were made 2.5 h after stimulation (12). Thus,
we cannot exclude the possibility that TNF-
induced a small or
localized increase in ceramide production which we were unable to detect.
We do not yet know how the attenuation of calcium entry occurs in
response to stimulation with TNF-
, SMase, or ceramide. In the recent
report by Barger et al. (19), it was suggested that NF
B
transcription factor may be involved in the TNF-
-evoked attenuation
of calcium entry. However, in their experiments the cells were treated
with TNF-
for 24 h prior to testing for an inhibition of
calcium entry. Our experiments show that TNF-
is effective within 10 min of application to the cells and the C2- and
C6-ceramides within a few minutes. Such a rapid activation of NF
B has been reported in HL-60 cells (17, 56). As TNF-
has
been reported to activate NF
B in human thyroid cells (18), we cannot
exclude the possibility that NF
B mediates the TNF-
-evoked inhibition of calcium entry in FRTL-5 cells. In another recent report,
a short (1-2 min) preincubation with C2-ceramide was also shown to attenuate calcium influx evoked with
N-formyl-methionyl-leucyl-phenylalanine (57). The
mechanism by which this inhibition was obtained was not established.
Our experiments performed in the presence of staurosporin suggest that
a kinase apparently is of importance in mediating the effect of
TNF-
. Some effects of TNF-
and sphingomyelinase have been shown
to be mediated via activation of PKC (13, 58). In FRTL-5 cells, the
,
,
, and
isoforms of PKC have been detected (45). Of
these isoforms,
,
, and
can be down-regulated by PMA, whereas
the
isoform is insensitive to PMA (45). Previous studies have
suggested that the PKC isoform activated by TNF-
is the
isoform
(13, 58). This finding could explain why TNF-
and SMase were
effective in PKC down-regulated cells but ineffective in cells treated
with calphostin C. This could also explain why the abrogating effect of
PMA and TNF-
on calcium entry was additive. In other cell types,
specific isozymes of PKC regulate calcium entry (59, 60). There is,
however, a discrepancy between these observations and the fact that we
could not measure an activation of PKC with either TNF-
or
C6-ceramide (although PMA did so in control experiments).
The PKC experiments might be hampered by the fact that about 60% of
the PKC in our cells was already associated with the particulate
fraction prior to stimulation, making a small effect of either TNF-
or C6-ceramide difficult to detect.
We also observed that TNF-
evoked a transient activation of JNK, and
this effect was absent in cells pretreated with PMA. However, as the
effect of TNF-
on thapsigargin-evoked calcium entry still occurred
in cells pretreated with PMA, it is not likely that JNK is involved in
the TNF-
-evoked attenuation of store-operated calcium entry.
Furthermore, as the p38 kinase inhibitor SB203580 did not inhibit the
effect of TNF-
, we think it is unlikely that p38 kinase is mediating
the effect of TNF-
on store-operated calcium entry.
The effect of ceramide was not due to conversion of ceramide to
sphingosine, as we were unable to detect an increase in sphingosine content after stimulating the cells with either TNF-
or SMase. However, we have recently shown that the PMA-evoked activation of PKC
depolarizes the membrane potential, resulting in decreased calcium
entry due to a decreased electrochemical driving force for calcium
(42). As the effect of TNF-
on store-operated calcium entry was
abolished by inhibitors of PKC activity, an effect of TNF-
(and
ceramide) on membrane potential cannot be excluded. Indeed, preliminary
results suggest that TNF-
and ceramide evoke a depolarization of the
membrane potential and that this effect is attenuated by calphostin
C.2 These observations are
consistent with a recent report showing that ceramide depolarizes the
membrane potential in oligodendrocytes by inhibiting inwardly
rectifying K+ channels (61). Furthermore, we cannot exclude
the possibility that TNF-
also could modulate store-operated calcium
channels, especially as a protein kinase has been shown to inhibit the
calcium release-activated calcium current
(ICRAC) (62). This possibility appears unlikely
as a recent report shows that ceramide does not modulate
ICRAC (26). The identification of the steps
involved in the TNF-
/ceramide-evoked signaling pathway will be of
crucial importance in understanding the mechanism(s) by which TNF-
inhibits calcium entry. Furthermore, this information may help in
understanding the mechanisms regulating calcium entry in cells.
In conclusion, we have defined a novel mechanism of action for TNF-
,
i.e. an inhibition of calcium entry. This observation will
probably help in understanding the effects of this cytokine (and
probably also of SMase and of ceramide) in thyroid cells and in other
cell systems.
 |
ACKNOWLEDGEMENT |
We thank Dr. William Wetsel for the generous
gift of PKC antibodies.
 |
FOOTNOTES |
*
This study was supported by the Sigrid Juselius Foundation,
the Liv och Hälsa Foundation, The Ella and Georg Ehrnrooth
Foundation, the Novo Nordisk Foundation, and the Signal Transduction
Program (Åbo Akademi University).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. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Biology,
Åbo Akademi University, BioCity, Artillerigatan 6, 20520 Åbo, Finland. Fax: 358-2-215 4748; E-mail: kid.tornqvist{at}abo.fi.
2
K. Törnquist and M. Pasternack,
unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
TNF-
, tumor
necrosis factor
;
PKC, protein kinase C;
PMA, phorbol 12-myristate
13-acetate;
SMase, sphingomyelinase;
SP, sphingosine;
SPP, sphingosine
1-phosphate;
DHC2 and DHC6, dihydroceramide
C2 resp C6;
[Ca2+]i, intracellular free calcium concentration;
Tg, thapsigargin;
TSH, thyrotropin;
JNK-1, Jun kinase 1;
DAG, 1,2-sn-dioctanoyl-acylglycerol;
PS, phosphatidylserine;
PBS, phosphate-buffered saline;
NF
B, nuclear factor
B.
 |
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