Interferon-
induces a decrease in the intracellular
calcium pump in a human salivary gland cell line
Sean
Meehan,
Ava J.
Wu,
Elaine C.
Kang,
Takayuki
Sakai, and
Indu
S.
Ambudkar
Secretory Physiology Section, Gene Therapy and Therapeutics Branch,
National Institute of Dental Research, National Institutes of
Health, Bethesda, Maryland 20892
 |
ABSTRACT |
Interferon-
(IFN-
) ± tumor necrosis factor-
(TNF-
) induces antiproliferation and intracellular
Ca2+ store depletion in a human
submandibular ductal cell line (HSG), which can be reversed on cytokine
removal [A. J. Wu, G. C. Chen, B. J. Baum, and I. S. Ambudkar. Am. J. Physiol. 270 (Cell Physiol. 39): C514-C521,
1996]. Here we have examined a possible mechanism for the
IFN-
-induced intracellular Ca2+
store depletion. There was a time-dependent decrease in
thapsigargin-dependent internal
Ca2+ release after exposure of the
cells to the cytokines. The intracellular Ca2+ pump
[sarco(endo)plasmic reticulum
Ca2+-ATPase
(SERCA)] protein in lysates and membranes of cells
treated with IFN-
± TNF-
, but not with TNF-
alone, showed
a similar time-dependent decrease (examined using a SERCA2
antibody). Removal of the cytokines, which resulted in
recovery of cell growth and refill of internal
Ca2+ stores, also increased the
level of SERCA protein. The decrease in SERCA is not a result of
decreased cell proliferation, since thapsigargin,
2,5-di-(t-butyl)-1,4-hydroquinone, or
serum-free growth conditions induced antiproliferative effects on HSG
cells without any corresponding decrease in SERCA. We suggest that the IFN-
-induced decrease in the level of SERCA accounts for the depleted state of internal Ca2+
stores in cytokine-treated HSG cells. These data suggest a novel mechanism for the inhibition of HSG cell growth by IFN-
.
sarco(endo)plasmic reticulum calcium adenosinetriphosphatase
protein; intracellular calcium ion store; salivary cell line; cell
proliferation
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INTRODUCTION |
-INTERFERON (IFN-
) is an immunoregulatory
cytokine that exerts well-documented effects on the growth and
differentiation of cells of the immune system (2, 15). It is now
recognized that IFN-
also decreases the proliferative ability of
several types of nonimmune cells (21, 22). More recently, IFN-
treatment has been reported to induce alterations in the physiological
characteristics of epithelial cells. For example, IFN-
induces
expression of the immunomodulatory cell surface proteins, HLA-DR and
intracellular adhesion molecule-1 (ICAM-1), and loss of epithelial
barrier function in colonic epithelial cells (4, 6). In addition, a
number of ion transport functions are altered in these cells. The exact mechanism(s) involved in these pleiotropic effects of IFN-
on immune
and nonimmune cell types is not yet clearly understood.
The cytokines, IFN-
and tumor necrosis factor-
(TNF-
), have
been suggested to be involved in salivary gland pathogenesis of
Sjogren's syndrome, an autoimmune disease associated with a dramatic
decrease in salivary gland function (i.e., fluid secretion) and chronic
inflammation of major and minor salivary glands. Loss of salivary
acinar tissue, extensive lymphocytic infiltrates, elevated levels of
IFN-
and TNF-
, and expression of HLA-DR and ICAM-1 have been
reported in the diseased glands (7, 20). We have previously reported
that IFN-
± TNF-
treatment of a human salivary gland ductal
cell line (HSG) induces a strong antiproliferative effect and decreases
the progression of cells through the cell cycle (25, 26). Although the
IFN-
-induced effect on HSG cell growth is enhanced in the presence
of TNF-
, TNF-
alone does not alter HSG cell growth.
IFN-
-treated HSG cells express the cell surface proteins HLA-DR and
ICAM-1, indicating an alteration in the physiological status of these
cells, which would make them more "immune responsive" (26).
Furthermore, we had also reported that IFN-
± TNF-
treatment
of HSG cells induces depletion of intracellular
Ca2+ stores, which is reversed
when the cytokines are removed from the growth medium (24). It has been
previously suggested that internal
Ca2+ stores have a role in the
maintenance of cell proliferation and protein synthesis (11, 14). Loss
of cell proliferation has been associated with the depletion of
internal Ca2+ stores in smooth
muscle cells treated with inhibitors of the intracellular
Ca2+ pump
[sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA)],
thapsigargin or
2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) (8, 19, 23). A similar association between the internal Ca2+ store status and cell
proliferation has been seen in several other cell types after treatment
with antimycotic agents, e.g., miconazole, (3). We have also previously
shown that treatment of HSG cells with thapsigargin, which results in a
persistent depletion of internal
Ca2+ stores, exerts a strong
antiproliferative effect (24). On the basis of these previous findings,
we have suggested that the depletion of internal
Ca2+ stores in IFN-
± TNF-
-treated cells may have a role in the cytokine-induced
inhibition of cell growth.
The Ca2+ content of internal
Ca2+ stores is regulated by a
number of Ca2+ transporters that
transport Ca2+ either in or out of
the endoplasmic reticulum (ER) and by proteins that retain
Ca2+ in the ER lumen (1). SERCA
are located in the ER membrane and mediate the active transport of
Ca2+ from the cytosol into the
lumen of the ER. The isoform SERCA2b appears to be ubiquitously present
in nonexcitable cells, whereas SERCA3 is less widely distributed. The
other isoforms, SERCA1 and SERCA2a, are more specifically distributed
in excitable cells (27). The present study was aimed at understanding
the mechanism by which internal
Ca2+ store depletion is induced
after treatment of HSG cells with IFN-
and TNF-
. To this end, we
have examined the status of internal Ca2+ stores and the level of SERCA
protein in cytokine-treated cells. The data demonstrate that
cytokine-induced internal Ca2+
store depletion is associated with a decrease in the SERCA protein level. We suggest that the IFN-
-induced decrease in SERCA protein accounts for the depleted state of the internal
Ca2+ stores in cytokine-treated
cells and contributes to the antiproliferative effect exerted by the
cytokine on HSG cells.
 |
MATERIALS AND METHODS |
Cell culture. The HSG cell line was a
gift of Dr. Mitsunabo Sato, Tokushima University, Shikoku, Japan (18).
HSG cells were cultured in Eagle's minimal essential medium
(Biofluids, Rockville, MD) supplemented with 10% bovine calf serum
(Biofluids),
L-glutamine (2 mM),
100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C with 5%
CO2, as described previously
(24-26). Cells (5 × 105) were plated onto tissue
culture plates (Falcon, NJ). After 24 h, cytokines or other agents, as
specified, and fresh media were added at the following concentrations:
1,000 U/ml IFN-
(Genzyme), 20 U/ml TNF-
(Genzyme), and 200 nM
thapsigargin or 4 µM BHQ (both from Calbiochem). For the serum-free
condition, the cell medium was replaced with serum-free medium
containing 0.1% fatty acid-free bovine serum albumin, and the cells
were grown for the number of days specified. The concentration of
cytokines used in this study was based on our previous studies
(24-26). Media cytokines (or other agents) were changed every 3 days for 7-9 days. For the "reversal" condition, cells were
grown in cytokine-containing media for 6 days as described above.
Thereafter, the media were removed and replaced with media without
cytokines. After 4 days of "recovery" in cytokine-free media,
cells displayed normal proliferative activity and morphology.
Cell proliferation assay. Assays were
performed as described previously (24-26). Briefly,
104 cells were plated onto 12-well
tissue culture plates and treated with cytokines or other conditions,
as described in the text, for 7 days. Cell proliferation was calculated
from optical density measurements at 410 nm with a Gilford
spectrophotometer (Gilford, Oberlin, OH) to detect
bromodeoxyuridine incorporated into cellular DNA (Cell
Proliferation Assay; Amersham Life Science, Arlington Heights, IL).
Cell number and viability were determined using the 0.4% trypan blue
exclusion technique (24-26). All assays were done in triplicate,
and experiments were repeated at least three times.
Cytosolic
Ca2+
concentration measurements.
Cells were detached from the plates with Versene (Biofluids) and then
were washed twice with Hanks' balanced salt solution (HBSS) buffered
with
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) containing 0.01% bovine serum albumin. Cells were incubated in HBSS at 30°C for 15 min. Fura 2-acetoxymethyl ester (2 µM; Molecular Probes, Eugene, OR) was then added to the medium, and
the cells were further incubated for 45 min at 30°C (10, 24). Cells
were then washed two times with HBSS and maintained at 30°C until
use. Immediately before use, aliquots of cells were removed, spun
gently (500 g for 30 s), washed, and
resuspended in the required incubation medium at 37°C. Fluorescence
was measured using an SLM 8000-DMX 100 spectrofluorometer. Cells were
gently stirred in a cuvette maintained at 37°C. The emission
wavelength was set at 510 nm, and excitation wavelengths were 340 and
380 nm. Cytosolic Ca2+
concentration
([Ca2+]i)
was calculated from the 340/380 nm fluorescence ratio. Each experiment
was repeated at least three times.
Crude membrane preparation. Cells,
treated as specified in the legends to Figs. 1-5,
were detached from the plates by incubation with Versene as described
above. Cells were washed with ice-cold buffer containing 50 mM
tris(hydroxymethyl)aminomethane (Tris)-HEPES, pH 7.4, and 0.1 mM
phenylmethylsulfonyl fluoride. Cells were homogenized in the same
medium using a Polytron homogenizer and then were centrifuged at low
speed [3,000 revolutions/min (rpm)]. The pellet was
rehomogenized as described above and recentrifuged at 3,000 rpm. The
supernatants were combined and centrifuged at 40,000 rpm. The resulting
pellet was washed once with buffer, suspended in a minimal volume of
buffer, divided into aliquots, and stored at
80°C after
quick-freezing. As needed, aliquots were thawed on ice and
then used. Protein was estimated using the Bio-Rad protein assay kit.
Gel electrophoresis and Western blot
procedures. Control HSG cells or cells treated with the
agents, as described, were lysed with buffer containing 300 mM NaCl, 50 mM Tris-HCl (pH 7.6), 0.5% Triton X-100, 0.1% sodium dodecyl sulfate
(SDS), 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride. The lysates were centrifuged to remove
cellular debris, and the supernatants were stored at
80°C
(it should be noted that all reactivity to the antibody was found in
the supernatant, which is referred to as cell lysate below). Cell
lysates (50 µg protein) or crude membranes (3-5 µg protein)
were treated with SDS-containing sample buffer for 30 min at 37°C
and electrophoresed on 8% acrylamide gels (12). To allow comparison
between the various samples, equal amounts of protein were loaded on
gels. The protein was transferred electrophoretically to polyvinylidene
difluoride filters and immunoblotted with a monoclonal antibody
(1:1,000 dilution) to the SERCA pump (obtained from Affinity
Bioreagents, Golden, CO), which has been reported to react with the
SERCA2 isoform of the protein. The proteins reacting with the antibody
were detected by using the enhanced chemiluminescence (ECL) assay
(Amersham). Autoradiographs were scanned on a Pharmacia Biotech imaging
system, and the band volume corresponding to SERCA was calculated using
an Image Master program.
 |
RESULTS |
Time-dependent effect of IFN-
and
TNF-
on the thapsigargin-sensitive internal
Ca2+ store in
HSG cells.
We had previously reported that internal
Ca2+ stores are depleted in HSG
cells treated for 7 days with IFN-
± TNF-
, at which time maximal effects on cell number are also observed (24, 26). To
examine when internal Ca2+ store
depletion occurs during exposure of cells to the cytokines, we have
examined thapsigargin-stimulated internal
Ca2+ release in control cells and
cells treated with the cytokines for a various numbers of days. In
these experiments, cells were allowed to attach to the plate for 24 h
before the cytokines were added to the growth medium. Control cells
were treated with vehicle (dimethyl sulfoxide) and grown in the absence
of cytokines for the same time period. The status of the internal
Ca2+ stores was assessed by
measuring the elevation of
[Ca2+]i
stimulated by thapsigargin in cells suspended in medium containing 5 mM
ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) to prevent Ca2+
influx. Under these conditions, addition of thapsigargin (2 µM) to
HSG cells induces a sharp rise in
[Ca2+]i,
which is transient and returns to resting
[Ca2+]i
within 100-150 s (data not shown here; see Refs. 10 and 24). This
increase in
[Ca2+]i
is due to Ca2+ release from
internal stores, and the magnitude of the increase is directly related
to the Ca2+ content of the store.
The data in Fig. 1 show that thapsigargin induces a 100 nM increase in
[Ca2+]i
in control cells (above the resting level of ~80 nM). A
similar increase in
[Ca2+]i
is seen in cells treated with cytokines for 1 or 2 days. On day 4 after treatment, the
thapsigargin-stimulated
[Ca2+]i
increase in cytokine-treated cells is decreased by ~50% compared with that in control cells, whereas resting
[Ca2+]i
in both sets of cells are similar and not altered. A similar 50%
reduction in the thapsigargin-stimulated
[Ca2+]i
elevation is also observed on day 7 of
treatment. The thapsigargin-stimulated internal
Ca2+ release in control cells on
days 1 through
7 are similar even though the cells
are at different densities (i.e., on day
1 they are <25% as confluent as on
day 7). Thus it appears that the
difference in thapsigargin-stimulated
Ca2+ release observed between
control and cytokine-treated cells is not due to a difference in cell
densities of two sets of cells. These results are consistent with our
previous report (24) and suggest that the thapsigargin-sensitive
Ca2+ stores are depleted (~50%)
when HSG cells are exposed to IFN-
± TNF-
. Furthermore, the
results demonstrate that store depletion is achieved between
days 2 and
4 of treatment. It should be noted that some other effects of IFN-
were observed earlier. For example, activation of JAK2 and STAT-1 is seen within 10 min (unpublished data),
whereas expression of HLA-DR and ICAM-1 is seen by 3 days (24, 26). Thus there does not appear to be a
temporal correlation between these different effects induced by IFN-
on HSG cells.

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Fig. 1.
Time course of internal Ca2+ store
depletion in cytokine-treated human submandibular ductal cell line
(HSG) cells. Cells were grown for number of days indicated in presence
of interferon- (IFN- ) and tumor necrosis factor- (TNF- ), as
described in MATERIALS AND METHODS.
For cytosolic Ca2+ concentration
([Ca2+]i)
measurements, cells were detached from the plates and loaded with fura
2. Two-milliliter aliquots of cells were washed in Hanks' balanced
salt solution medium and maintained in a cuvette at 37°C, with
gentle stirring. EGTA (5 mM) was added to the medium followed 30 s
later by 2 µM thapsigargin. Thapsigargin induced the typical
transient
[Ca2+]i
elevation as previously described (34). Changes in
[Ca2+]i
values (peak minus resting) determined from 4-5 experiments are
shown as means ± SE for control cells (open bars) and
cytokine-treated cells (solid bars). Resting
[Ca2+]i
in control and cytokine cells were not significantly different from
each other at any time point. Thapsigargin-stimulated
[Ca2+]i
increases on days 4 and
7 are significantly different
(P < 0.01, Student's
t-test) from all other values. All
other values are not significantly different from each other.
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SERCA levels in cytokine-treated HSG cell lysates and
isolated membranes. To determine a possible mechanism
to account for the depletion of internal
Ca2+ stores, we examined the
levels of the SERCA protein in control and cytokine-treated cells by
using a monoclonal antibody against the SERCA2 isoform (28). Western
blots of total cell lysates run on 8% polyacrylamide gels are shown in
Fig. 2A. A
protein of molecular mass of ~100 kDa reacts with the antibody in the samples from control cells, which is consistent with the reported molecular mass of SERCA2 (28). Because SERCA2a is not likely to be
present in HSG cells, we suggest that the protein detected by the
antibody is probably SERCA2b. However, a contribution of SERCA3 to this
reactivity cannot be presently ruled out, since the antibody we have
used has been recently reported to also recognize the SERCA3 subtype of
the Ca2+ pump (13). Thus further
studies using a SERCA3-specific antibody are needed to investigate the
presence of SERCA3 in HSG cells. In the results described below, we
have referred to the protein(s) that reacts with this antibody as
SERCA. The level of SERCA does not appear to be altered in lysates of
HSG cells grown for various days in a normal growth medium (i.e., in
control cells). However, the level is significantly decreased to 85 ± 4% of control (P < 0.01, by
paired Student's t-test,
n = 6) determined by densitometric scanning of Western blots (6 gels, 3 different experiments) of lysates
of HSG cells treated with IFN-
± TNF-
for 4 days and further
decreased to 49 ± 3.5% of controls
(P < 0.001, paired Student's
t-test,
n = 6) in cells treated for 7 days.
The level of SERCA in cytokine-treated cells on various days has been
expressed relative to the respective control (i.e., untreated cells on
same day of culture, as shown in Figs. 1 and
2A). In Fig.
2A, the blots were subjected to the
ECL reaction for a longer period of time (5-10 min, until the band
on day 7 was clearly detected) to
allow comparison of the SERCA level in the different samples.
Shortening the time of the ECL reaction allowed for better resolution
of the decrease in SERCA on day 4;
e.g., with 2-5 min of exposure, a 40-50% decrease was noted.
However, in this case, the band on day
7 was not clearly detected. Thus there appears to be a
greater decrease in SERCA on day 7 compared with day 4. Although it is not possible from the present data to directly quantitate the SERCA
levels, the temporal pattern of the decrease in SERCA is similar to the
cytokine-induced decrease in the internal
Ca2+ store(s) content in HSG
cells. Furthermore, when the cytokines are removed from the medium and
cells are allowed to recover for 7 days, the SERCA level is increased
to levels comparable with that in control cells. The recovery of SERCA
on cytokine removal is greater in Fig.
2A than in
2B, since the cells were grown for a
longer time in regular medium (7 days vs. 4 days). Thus the cytokine-induced decrease in the SERCA protein level appears to be a
reversible process. These results are consistent with our previous
report (24), showing recovery of internal
Ca2+ stores under the same
conditions. Furthermore, in cells treated with TNF-
alone, there is
no decrease in SERCA, whereas, in those treated with IFN-
, the level
of SERCA is similar to that in IFN-
± TNF-
-treated cells
(Fig. 2B). These results are similar
to the effects of these cytokines on HSG cell growth; TNF-
alone does not alter the proliferative activity of HSG cells, whereas IFN-
alone reduces proliferation (25, 26).

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Fig. 2.
IFN- -induced decrease in sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) protein levels
of HSG cells. Cells were treated with IFN- and TNF- for various
days, as described for Fig. 1. On days noted (A), cells
were exposed to lysis buffer. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and Western blotting were performed as
described in MATERIALS AND METHODS.
Control cell lysates exhibit a protein with a molecular mass of ~98
kDa that reacts with SERCA2 antibody (control blots not treated with
primary antibody showed no reactivity and are not shown). Levels of
this protein in control cells (c) and IFN- + TNF- -treated cells
(t) on various days (1, 2, 4, and 7) after treatment are shown. Last
lane shows reactivity in samples from "reversed cells" (r) (i.e.,
grown with cytokines for 7 days and then in normal medium for 7 days).
B: Western blot of cell lysates (50 µg protein/sample) from control cells (C) 7 days after plating, cells
treated with IFN- + TNF- (I+T) for 7 days, cells treated with
IFN- (I) for 7 days, cells treated with TNF- (T) for 7 days, and
cells treated with both cytokines for 7 days and then grown in control
medium for 4 days (R). Results are representative of 3 similar
experiments. C: levels of SERCA in
membranes isolated from cytokine-treated cells. Cells were treated for
7 days with IFN- ± TNF- , as described previously. A crude
membrane (40,000 g pellet, see
MATERIALS AND METHODS) was prepared
from control cells (C) and cells treated with IFN- (I) or IFN-
and TNF- (I+T) for 7 days. SDS-PAGE and Western blotting with
anti-SERCA antibody were performed as described above with 3 µg
protein/lane. Results are representative of 3 similar experiments.
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|
To further examine this decrease in SERCA levels in cytokine-treated
HSG cells, we prepared a crude membrane fraction (40,000 g pellet) from control,
IFN-
-treated, and IFN-
± TNF-
-treated (for 7 days) cells.
Figure 2C shows the reactivity of an
~100 kDa protein in these membranes to the SERCA antibody. The level of this protein (SERCA) is markedly lower in membranes prepared from
IFN-
± TNF-
-treated cells compared with that in membranes from untreated cells. Membranes from cells treated with IFN-
alone
show a similar decrease in SERCA. Silver staining of comparable gels
further demonstrates that the general protein profile of membranes from
cytokine-treated and control cells is similar (data not shown).
Internal Ca2+
store content and SERCA levels in HSG cells grown in the presence of
thapsigargin, BHQ, or serum-free conditions.
To determine whether the decrease in SERCA in cytokine-treated HSG
cells is due to more general effects of antiproliferation, e.g.,
decreased protein synthesis, we examined the status of internal Ca2+ stores and SERCA levels of
cells grown 1) in the presence of thapsigargin or BHQ, two strong SERCA inhibitors, or
2) in a serum-free medium. Figure
3 shows that all three conditions induce
strong antiproliferative effects on HSG cells. Compared with control cells, the cell number after 7 days is 10, 15, and 20%, respectively, in the +thapsigargin, +BHQ, and serum-free growth conditions. These
data are consistent with an earlier report (24) and further demonstrate
that BHQ, a SERCA inhibitor like thapsigargin, also induces
antiproliferative effects on HSG cells. Both thapsigargin and BHQ also
induce total depletion of the thapsigargin-sensitive internal
Ca2+ stores (data not shown).
Importantly, lysates of cells grown for 7 days in medium containing BHQ
or thapsigargin do not show any decrease in SERCA protein levels (data
not shown).

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Fig. 3.
Effect of SERCA inhibitors, cytokines, and serum-free conditions on HSG
cell growth. Cell number was determined by trypan blue exclusion.
Bromodeoxyuridine incorporation also gave similar results (not shown).
All conditions for cell culture were as described in
MATERIALS AND METHODS. Cells were
grown for 7 days in 1) control
medium (Con), 2) medium with 4 µM
2,5-di-(t-butyl)-1,4-hydroquinone
(BHQ), 3) medium with 200 nM
thapsigargin (Tg), 4) medium without
fetal bovine serum ( serum), and
5) medium with IFN- + TNF-
(I+T). Data are means ± SE from 3-5 experiments. All values
are significantly different from control condition
(P < 0.005, by Student's
t-test).
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Fetal bovine serum exerts a strong mitogenic effect on HSG cells, and,
in the absence of serum in the culture medium, HSG cell growth is
greatly reduced (Fig. 3). However, the thapsigargin-induced Ca2+ release in these cells is
similar to those in control cells that are maintained in a
serum-containing medium (Fig. 4),
demonstrating the intactness of the internal
Ca2+ store(s). Consistent with
this observation, we have observed that the SERCA level in these cells
is not decreased. These data demonstrate that the decrease in SERCA in
IFN-
-treated cells is not a result of a decrease in cell
proliferation, protein synthesis, or decrease in intracellular
Ca2+ stores.

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Fig. 4.
Effect of SERCA inhibitors and serum-free growth conditions on SERCA
protein and intracellular Ca2+
stores in HSG cells. Thapsigargin-stimulated
[Ca2+]i
elevation was measured as described for Fig. 1, in control cells (open
bar) and in cells grown in a serum-free condition (solid bar). Values
were not statistically different from each other.
[Ca2+]i
increase was undetectable in cells grown in presence of thapsigargin or
BHQ and is not shown.
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Effect of BHQ on the recovery of HSG cell growth
induced by removal of the cytokines from the culture
medium. Figure 5 shows the
effect of TNF-
and IFN-
on HSG cell growth. As reported by us
previously (24-26), treatment of HSG cells with these cytokines induces a persistent decrease (>95%) of cell growth (compare
curves 1 and
2). When the cytokines are removed
from the growth medium on day 6, the
cells resume normal growth after day
10 (curve 3). Notably, at this time, the SERCA level shows a recovery (see Fig. 2B), and the internal
Ca2+ store(s) is also partially
refilled (24). To examine whether functional SERCA is required for the
reversal of cell growth in cytokine-treated cells, we treated the cells
with 4 µM BHQ when the cytokine was removed on day
6. Under these conditions, the recovery of cell growth
is completely inhibited and cell number remains >95% lower than that
of controls (curve 4 ). However, when BHQ is removed from
the medium on day 12, increased cell growth is seen after day 17 (curve 5 ). These data are consistent with the effects of
SERCA inhibitors, e.g., BHQ and thapsigargin, on cell growth reported
by us and others (8, 19, 23, 24). The data suggest that functional
SERCA is required 1) for normal growth of HSG cells and 2) for
recovery of growth of HSG cells after IFN-
treatment.

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Fig. 5.
Effect of BHQ on recovery of HSG cell growth induced by cytokine
removal. HSG cell growth was determined as described in
MATERIALS AND METHODS for a period of
23 days after plating. Growth conditions were as follows:
1) control growth medium was used
for entire experiment; 2) growth
medium containing IFN- and TNF- was used during entire
experiment; 3) cytokine-containing
medium was replaced by control medium on day
6; 4)
cytokine-containing medium was replaced by 4 µM BHQ-containing medium
on day 6;
5) cytokine-containing medium was
replaced with BHQ-containing medium on day
6 and then BHQ-containing medium was replaced with
control medium on day 12. Values are
means ± SE from 3 experiments, each done in duplicate.
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 |
DISCUSSION |
We have demonstrated here that treatment of HSG cells with IFN-
and
TNF-
induces a time-dependent, persistent depletion of internal
Ca2+ stores. Furthermore, there is
a concurrent time-dependent decrease in the level of the intracellular
Ca2+ pump protein (SERCA).
IFN-
, which by itself induces a substantial reduction in HSG cell
growth and internal Ca2+ pool
depletion, reduces the level of SERCA to the same extent as that in
cells treated with IFN-
± TNF-
. TNF-
, which by itself does
not decrease HSG cell proliferation (24-26), does not alter SERCA
levels. Thus the ability of the cytokines to decrease SERCA appears to
be related to their ability to reduce cell proliferation. This decrease
in SERCA does not appear to be a result of a general decrease in
protein synthesis, reduced cell proliferation, or depletion of
intracellular Ca2+ stores. We have
shown that HSG cells in a growth-arrested state, due to removal of
serum from the growth medium or due to inclusion of
Ca2+ pump inhibitors (thapsigargin
or BHQ), in the growth medium do not exhibit decreases in SERCA levels.
Finally, the levels of SERCA recover to control levels on removal of
IFN-
. Therefore, we suggest that the decrease in SERCA is
specifically induced in response to the treatment of HSG cells with
IFN-
.
The activity of SERCA is involved in the accumulation of
Ca2+ into the ER and the refill of
internal Ca2+ stores, and it is
well documented that inhibition of SERCA activity induces depletion of
internal Ca2+ stores (1, 8, 10).
Thus we suggest that the IFN-
-induced decrease in SERCA protein
accounts for the decreased Ca2+
content of the internal Ca2+
stores detected in IFN-
-treated cells. Another possible mechanism that can be proposed to account for the internal
Ca2+ store depletion is
IFN-
-stimulated internal Ca2+
release, mediated via an increase in inositol trisphosphate generation, as has been previously reported in renal carcinoma cells (9).
However, we can rule out this possibility since we have previously
demonstrated that IFN-
does not induce any acute (within 10 min)
release of Ca2+ from internal
Ca2+ store(s) or increase in
inositol trisphosphate in HSG cells (24). Thus our data are more
consistent with our first suggestion. Although this is the first report
describing an effect of IFN-
on the SERCA protein level, it has been
previously reported (4, 6) that IFN-
reduces the levels of the
Na+-Cl
-K+
cotransporter and the cystic fibrosis transmembrane conductance regulator in the colon-derived epithelial cell lines (T84 and HT-29,
respectively). The mechanism(s) involved in the modulation of gene
expression by IFN-
is presently not completely understood. Both
upregulation and downregulation of various genes have been reported
(17). Possible effects at the promoter level of the genes as well as on
regulation of transcription factors have been described. In addition,
IFN-
has also been suggested to induce posttranscriptional
modifications, such as alterations in mRNA stability (4).
Previous studies with smooth muscle and neuroblastoma cells have
demonstrated that loss of cell proliferation can be correlated with
depletion of internal Ca2+ stores,
whereas synthesis of new Ca2+ pump
protein in the store membrane precedes reentry of cells into the cell
cycle (19, 23). Our present and previous data show that depletion of
internal Ca2+ store(s), decrease
of SERCA protein, loss of cell proliferation, and arrest of cell cycle
are temporally correlated in IFN-
-treated HSG cells (24, 26). We
have also shown here that addition of BHQ to cells before removal of
the cytokines prevents recovery of cell proliferation. Thus the
functional integrity of SERCA appears to be critical for the normal
growth and cycling of HSG cells. Furthermore, the SERCA activity also
appears to be critical for the recovery of HSG cell growth after
removal of the cytokines. Our studies suggest that SERCA inhibitors and
IFN-
essentially induce antiproliferative effects by a similar
mechanism. In both cases, the function of the SERCA protein is
impaired, and the net result is a decrease in
Ca2+ uptake into internal
Ca2+ stores and internal
Ca2+ store depletion. It should be
noted that the cytokine treatment induces >95% inhibition of cell
growth but a maximum depletion of ~50% of the thapsigargin-sensitive
Ca2+ store. This suggests that
total Ca2+ store depletion is not
required for the inhibition of cell growth and that only part of the
thapsigargin-sensitive Ca2+ store
is important for the normal proliferative activity of HSG cells.
Furthermore, likely compartmentalization of intracellular Ca2+ stores and a relatively
heterogeneous distribution of SERCA in these store membranes could
account for the discrepancy between the extent of decrease in SERCA and
content of thapsigargin-sensitive internal
Ca2+ store(s). However, further
studies are required to clarify why the SERCA level continues to
decrease until day 7 while there is no
further detectable depletion of the thapsigargin-sensitive Ca2+ store after
day 4.
We have observed in the present study that
Ca2+ store depletion is induced
between days 2 and
4 and that a significant decrease in
SERCA is also induced within this same time period. In our previous
report (25), we have shown that there is an increase in cell number up
to day 3 of cytokine treatment, after
which the cell number remains unchanged until day
6. This is associated with cell cycle arrest at
G0/G1.
We have recently reported that HSG cells undergo cell death when
treated with IFN-
± TNF-
, but not TNF-
alone, for >4
days (25). It has been suggested that
Ca2+ may be a key
factor in the regulation of cell proliferation (14) and
other functions such as protein synthesis (5), expression of
transcription factors (14, 29), and PKC-dependent activation of
mitogen-activated protein kinase (28). Several recent studies have
suggested that the depletion of internal
Ca2+ stores(s) is also involved in
mediating the process of apoptosis and cell death in several different
cell types (14). Thus IFN-
-induced internal
Ca2+ store depletion could also
have a role in the cell death observed in HSG cells exposed to IFN-
and TNF-
.
The depleted state of the internal
Ca2+ store(s) in salivary cells
exposed to IFN-
and TNF-
can also provide a possible explanation for the two major effects of Sjogren's syndrome on salivary gland cells, i.e., loss of acinar tissue and reduced fluid secretion. Depletion of the internal Ca2+
store(s) could induce a reduction in cell growth or an enhancement in
cell death/apoptosis, both of which would lead to reduced acinar tissue. A reduction in fluid secretion would then result, due to the
loss of acinar tissue, which is the sole site of water movement in
salivary glands. Decreased internal
Ca2+ store(s) could also have more
direct effects on the level of fluid secretion, since intracellular
Ca2+ mobilization events are
required for initiating and maintaining fluid secretion in salivary
glands (1, 16). Further studies are required to determine whether there
are alterations in the status of intracellular
Ca2+ store(s) in salivary glands
during the pathogenesis of Sjogren's syndrome or other inflammatory
conditions and whether these alterations are significant in the
functional damage of the glands induced by the disease.
In summary, we have demonstrated that IFN-
-induced depletion of the
internal Ca2+ store(s) in HSG
cells is temporally associated with the cytokine-induced decrease in
the level of the SERCA protein. Although further studies are required
to determine the mechanism by which IFN-
decreases the level of
SERCA in salivary gland epithelial cells, on the basis of the
correlation observed in the status of internal
Ca2+ store(s), the level of SERCA,
and the proliferation of HSG cells after cytokine treatment, we suggest
that the IFN-
-induced decrease in functional SERCA results in the
depletion of internal Ca2+ stores.
The depletion of the internal Ca2+
store may have a role in the antiproliferative effects and/or apoptosis induced by this cytokine. Thus our present findings suggest a
novel mechanism by which IFN-
induces antiproliferative effects on
salivary gland cells, i.e., by depletion of intracellular Ca2+ stores mediated via a
decrease in the level of SERCA. It will be interesting to see whether
this cytokine induces similar effects on the SERCA and intracellular
Ca2+ stores in other cell types.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Bruce J. Baum for support during the course of this
work. We also thank all our collegues for their help and cooperation.
 |
FOOTNOTES |
Address for reprint requests: I. S. Ambudkar, Rm. 1N-113, Bldg. 10, National Institutes of Health, Bethesda, MD 20892.
Received 14 March 1997; accepted in final form 21 August 1997.
 |
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