(Received for publication, August 10, 1995; and in revised form, November 30, 1995)
From the
The widely distributed and highly conserved
Ca-binding protein calreticulin has been suggested to
play a role as a Ca
storage protein of intracellular
Ca
stores. To test this hypothesis, we have generated
a mouse L fibroblast cell line stably transfected with a calreticulin
expression vector. The calreticulin content of the overexpressers was
increased by 1.6 ± 0.2-fold compared with mock-transfected
cells. The total cellular Ca
content of
calreticulin-overexpressing and control cells, as assessed by
equilibrium
Ca
uptake, was 141 ±
8 and 67 ± 6 pmol of Ca
/10
cells,
respectively (i.e. a 2.1 ± 0.2-fold increase in the
Ca
content of calreticulin-overexpressing cells).
Over 80% of the increased Ca
content was found within
thapsigargin-sensitive Ca
stores. The pattern of
calreticulin distribution, revealed by immunofluorescence microscopy,
showed an endoplasmic reticulum-like pattern and was identical in
overexpressers and control cells. In overexpressers, cytosolic free
[Ca
] elevations due to Ca
release were enhanced when either ATP or a combination of
ionomycin and thapsigargin was used as a stimulus. In contrast,
thapsigargin-induced Ca
and Mn
influxes from the extracellular space were markedly diminished in
calreticulin-overexpressing cells, suggesting an active involvement of
calreticulin in the regulation of store-operated Ca
influx.
Ca stores are intracellular compartments that
are characterized by (i) their high intraluminal Ca
content and (ii) their participation in the regulation of the
cytosolic free Ca
concentration
([Ca
]
) (
)through rapid Ca
accumulation and
Ca
release (1, 2, 3) .
Intracellular Ca
stores are faced with the dilemma to
store large amounts of Ca
within a restricted
fraction of the cellular volume. For this reason, it is widely assumed
that intracellular Ca
stores contain intraluminal
Ca
buffers that allow the accumulation of large
amounts of Ca
without an excessive increase in the
free intraluminal Ca
concentration. The protein that
has been suggested to make the physiologically most relevant
contribution to Ca
storage in nonmuscle
Ca
stores is
calreticulin(2, 4, 5, 6, 7) .
Calreticulin is able to bind Ca
with high capacity
and low affinity (8) and was found in many (but not all)
studies to colocalize with other markers of intracellular
Ca
stores. However, while circumstantial evidence
suggested a role for calreticulin in Ca
storage, it
turned out to be extremely difficult to provide scientific proof for
this hypothesis.
Besides playing a crucial role in Ca uptake and Ca
release, intracellular
Ca
stores appear to regulate, in a large variety of
cell types, the divalent cation permeability of the plasma membrane: a
decrease in the store Ca
concentration activates the
so-called store-operated Ca
influx(9, 10) . The intraluminal Ca
sensor that detects the drop in the Ca
concentration within intracellular stores is not known, nor is
the biochemical machinery that mediates the signaling from
Ca
stores to the plasma membrane.
In this study,
we demonstrate that overexpression of calreticulin in L fibroblasts
leads to an increase in the Ca content of
thapsigargin-sensitive Ca
stores and to a decrease in
store-operated Ca
influx.
Mouse L fibroblasts were stably transfected with a calreticulin expression vector or a control vector as described under ``Experimental Procedures.'' To assess the level of calreticulin overexpression, we performed quantitative immunoblotting of calreticulin-transfected and control cells (Fig. 1A). Immunoblots were scanned, and the slope of the correlation between cell number and optical density was determined. The slope of calreticulin-transfected cells divided by the slope of control cells yielded directly the increase in calreticulin content due to expression of calreticulin in transfected cells (see also (16) ). Fig. 1B shows that cells transfected with the calreticulin expression vector had a 1.6 ± 0.2-fold increase in the level of immunoreactive calreticulin.
Figure 1:
Calreticulin levels and total cellular
Ca content of calreticulin overexpressers and of
control cells. Mouse L fibroblasts were stably transfected with a
calreticulin expression vector or a control vector containing only the
Geneticin resistance gene. A, to quantitate calreticulin,
calreticulin overexpressers and control cells were analyzed by Western
blotting with polyclonal anti-calreticulin antibodies. For each
condition, three different amounts of cells (0.06, 0.08, and 0.11
10
cells/well) were analyzed. The immunblots were
developed by a chemiluminescence assay. B, the immunoblots
were scanned by a densitometer. The slope of the cell number versus optical density plot was obtained by a linear fit; it is a direct
measure of the relative cellular calreticulin content. The calreticulin
content of transfected cells is shown as percent of the calreticulin
content of control cells. C, the total cellular Ca
content was determined using equilibrium incubation with
Ca
. The total cellular Ca
content is given as picomoles of Ca
/10
cells. Data shown in B and C are means ±
S.E. of three separate experiments performed in
duplicate.
We next investigated whether calreticulin overexpression leads to a change in the pattern of calreticulin localization. Both control cells (Fig. 2A) and overexpressers (Fig. 2B) were heavily stained by anti-calreticulin antibodies. In both cells types, calreticulin appears to be located in the perinuclear system of the membrane corresponding in localization to the endoplasmic reticulum. More important, neither in control cells nor in overexpressers was nuclear, cytoplasmic, or surface staining observed.
Figure 2: Localization of calreticulin in overexpressers and in control cells by immunofluorescence confocal microscopy. Control cells (A) and overexpressers (B) were stained with goat anti-calreticulin antibodies. In both cell types, calreticulin localized predominantly to an endoplasmic reticulum-like intracellular network. Calreticulin also delineates the nuclear envelope in these cells. We did not observe nuclear, cytoplasmic, or cell surface staining. When the preimmune serum was substituted for the calreticulin antibody, no labeling was observed (data not shown).
To assess whether the Ca content of intracellular
Ca
stores is modified by the overexpression of
calreticulin, we performed equilibrium loading experiments with
Ca
. Calreticulin overexpressers and
control cells were cultured for 54 h in the regular culture medium
containing 10 µCi/ml
Ca
. The time
required to obtain isotopic equilibrium was within 36-48 h and
was not significantly different between overexpressers and control
cells (data not shown). The total cellular Ca
content
was then calculated based on the cell-associated radioactivity and on
the specific activity of Ca
in the culture medium.
Control cells contained 67 ± 6 pmol of
Ca
/10
cells, whereas the overexpressers
contained 141 ± 8 pmol of Ca
/10
cells (Fig. 1C). Thus, the 1.6 ± 0.2-fold
increase in cellular content of calreticulin protein led to a 2.1
± 0.2-fold increase in cellular Ca
content.
We next wanted to know whether the increased cellular Ca content reflected an increased Ca
load of
agonist-sensitive, rapidly exchangeable intracellular Ca
stores. Agonist-sensitive, rapidly exchangeable intracellular
Ca
stores accumulate Ca
through
sarcoplasmic-endoplasmic reticulum Ca
-ATPases. We
therefore used thapsigargin, an inhibitor of sarcoplasmic-endoplasmic
reticulum Ca
-ATPases(17) , to measure the
amount of Ca
associated with rapidly exchangeable
intracellular Ca
stores. To assess the residual
amount of Ca
contained within
thapsigargin-insensitive luminal Ca
stores, we added
the Ca
ionophore ionomycin. Finally, as ionomycin has
been shown to be inactive in releasing Ca
from acidic
intracellular compartments, we added the sodium proton ionophore
monensin. For these experiments, cells were equilibrium-loaded with
Ca
isotope as described above and
resuspended in a nonradioactive Ca
-free buffer.
Unidirectional fluxes to the extracellular medium after addition of the
respective compounds were then measured. For more details concerning
this approach, see (18) . Fig. 3shows that in both
calreticulin overexpressers and control cells,
60% of the total
cellular Ca
could be released by thapsigargin. The
remaining Ca
was almost completely released by
ionomycin. The increase in cellular Ca
content of
calreticulin overexpressers was mostly due to an increase in the size
of thapsigargin-sensitive Ca
stores (Fig. 3B). The monensin-induced Ca
release was very small, suggesting that neither controls nor
calreticulin-transfected cells contained relevant quantities of
Ca
stored within acidic compartments.
Figure 3:
Characterization of Ca pools in calreticulin overexpressers and in control cells. Cells
were cultured for 54 h with
Ca
to reach
isotopic equilibrium, detached from the culture flask, and resuspended
in Ca
-free medium. Cell suspensions were preincubated
for 3 min at 37 °C and sequentially stimulated with thapsigargin
(100 nM), ionomycin (2 µM), and monensin (2
µM). Aliquots of the suspensions (corresponding to
10
cells) were collected at the indicated times and
centrifuged. The radioactivity in the supernatant (i.e. the
amount of Ca
released from the cells) was measured.
Background values (i.e. counts/minute in the supernatant
before addition of thapsigargin) were substracted. A, typical
experiment; B, mean ± S.E. of three separate
experiments performed in duplicate. Ionomycin- and monensin-induced
Ca
release were defined as the additional release
caused by the application of the respective
compound.
To study the
effect of the increased Ca content of intracellular
Ca
stores on the regulation of
[Ca
]
, we next performed
experiments with the Ca
-sensitive fluorescent dye
fura-2. Cells were loaded with fura-2/AM under conditions preventing
sequestration of the dye into subcellular organelles (e.g. endoplasmic reticulum, lysosomes). (i) Indicator loading was
performed at room temperature; (ii) pluronic acid was added during
incubation with fura-2/AM; (iii) sulfinpyrazone, an inhibitor of
organic anion transport, was included in the buffer for dye loading and
[Ca
]
measurements(19) .
To document the efficacy of these precautions, we measured the amount
of fura-2 that was released by digitonin (20 µM) or Triton
X-100 (0.1%); >90% of the fura-2 from both control and
calreticulin-overexpressing cells was released by digitonin,
demonstrating the cytosolic localization of the dye (Fig. 4, A and B). Note also that the maximal fluorescence in
control cells and overexpressers was comparable (Fig. 4A), suggesting that both cell lines contained
the same amount of cytosolic fura-2.
Figure 4:
Fura-2 loading of calreticulin
overexpressers and control cells. Cells were loaded with the
fluorescent Ca indicator fura-2, taking precautions
to avoid dye sequestration (see ``Experimental Procedures''
for details). A, fura-2 fluorescence (
= 340 nm) was measured in unstimulated cells in
Ca
-free medium after addition of (i) 1 mM EGTA and 20 µM digitonin (EGTA+Dig.),
(ii) 0.1% Triton X-100 and 5 mM Tris (min), and (iii)
10 mM HCl and 3 mM CaCl
(max).
Data are means ± S.E. of four determinations of three
independent experiments. B, cells were centrifuged after 5 min
of incubation with 20 µM digitonin in
Ca
-free medium at 37 °C. The supernatant was
transferred to a cuvette, and 1 mM Ca
was
added. The pellet was resuspended in an identical volume of
Ca
medium, and cells were lysed with 0.1% Triton
X-100. Ca
-dependent fura-2 fluorescence (
= 340 nm) was measured in the supernatant and in the
resuspended pellet. Results are shown as percent of total fluorescence.
Data are means ± S.D. of two independent
experiments.
We next examined the effect of
calreticulin overexpression on [Ca]
elevations in response to Ca
release from
intracellular stores. We first investigated the effect of the receptor
agonist ATP, which in mouse L fibroblasts has been shown to activate
P2y purinergic receptors linked to phospholipase C(19) . ATP
stimulation of cells suspended in Ca
-free medium
caused a rapid and transient [Ca
]
increase that was totally abolished by thapsigargin pretreatment
of the cells (data not shown). The amplitude of the
[Ca
]
elevation elicited by ATP
was increased by a factor 1.5 in calreticulin overexpressers as
compared with control cells (Fig. 5, A and E).
Similarly, increased [Ca
]
elevations due to Ca
release were observed when
calreticulin overexpressers were concomitantly stimulated by
thapsigargin and ionomycin (Fig. 5, D and E).
In contrast, when cells were stimulated separately either by the
sarcoendoplasmic reticulum Ca
-ATPase inhibitor or by
the ionophore, the peak and the duration of the
[Ca
]
elevations due to
Ca
release were comparable in overexpressers and in
control cells (Fig. 5, B, C, and E). Fig. 5F shows the amount of Ca
that
was released under the identical conditions in
Ca
experiments. Note that the increased
amount of Ca
that is mobilized by either thapsigargin
or ionomycin alone is not reflected by increased
[Ca
]
elevations (Fig. 5,
compare E and F; see also ``Discussion'').
No significant
Ca
release was detected
after ATP stimulation of either control cells or overexpressers,
suggesting that most of the released Ca
was rapidly
reaccumulated by intracellular Ca
stores.
Figure 5:
[Ca]
elevations in response to Ca
release from
intracellular stores. Calreticulin overexpressers and control cells
were loaded with the fluorescent Ca
indicator fura-2. A-D, typical traces showing the stimulation of cells in
Ca
-free medium by the receptor agonist ATP (100
µM) followed by the Ca
-ATPase inhibitor
thapsigargin (100 nM) (A), by 100 nM thapsigargin only (B), by 2 µM ionomycin (C), or by a combination of 2 µM ionomycin and 50
nM thapsigargin (D). E,
[Ca
]
([Ca
]
)
elevations in response to stimulation by ATP, thapsigargin, ionomycin,
and a combination of ionomycin and thapsigargin in calreticulin
overexpressers, expressed as percent of values seen in control cells.
Absolute values of [Ca
]
elevations in control cells were 123 ± 45 nM (ATP), 193 ± 16 nM (thapsigargin), 226 ± 36
nM (ionomycin), and 269 ± 33 nM (ionomycin
+ thapsigargin). Data are means ± S.E. of 8-12
independent experiments. F, Ca
release from
Ca
-loaded control and
calreticulin-overexpressing cells in response to stimulation by
thapsigargin, ionomycin, and a combination of thapsigargin and
ionomycin.
Ca
experiments were performed
as described in the legend of Fig. 3. Values shown are
counts/minute recovered in the supernatant 5 min after stimulation
subtracted from the counts present in the medium before addition of
thapsigargin (100 nM), ionomycin (2 µM), or a
combination of thapsigargin (50 nM) and ionomycin (2
µM). Unstimulated
Ca
release was negligible in the period investigated. Data are means
± S.E. of three independent
experiments.
In many
cell types, the depletion of intracellular Ca stores
leads to the activation of a Ca
influx across the
plasma membrane, a phenomenon referred to as store-operated
Ca
entry. We therefore investigated the effect of
calreticulin overexpression on the activation of Ca
influx in response to store depletion.
[Ca
]
elevations after addition
of Ca
to cells stimulated by thapsigargin in
Ca
-free medium were markedly decreased in
calreticulin overexpressers (Fig. 6). These results raise the
possibility that calreticulin overexpression diminishes store-operated
Ca
influx. However,
[Ca
]
elevations in response to
Ca
readdition experiments are the complex result of
the activity of a variety of Ca
transport pathways
and may, at least partially, be explained by an inhibition of the
plasma membrane Ca
-ATPase through extracellular
Ca
. To measure more specifically the activation of
the Ca
influx pathway, we studied
thapsigargin-induced Mn
influx. Mn
is able to permeate through store-operated Ca
channels, but is not transported by Ca
transport ATPases. Mn
influx is detected as a
quenching of the fluorescence of cytosolic fura-2. In both control
cells and calreticulin overexpressers, there was a relatively important
Mn
influx without thapsigargin stimulation (Fig. 7). This most likely reflects the previously observed
presence of mechanisms that allow Mn
entry
independent of store-operated Ca
influx(20) .
However, in control cells, thapsigargin clearly increased the amplitude
of the fura-2 quenching after addition of Mn
,
demonstrating the presence of store-operated Ca
influx in control mouse L fibroblasts. In contrast, no
thapsigargin activation of Mn
influx was observed in
the overexpressers (Fig. 7). The suppression of
thapsigargin-induced Mn
influx was even observed when
Mn
was added 10 min after stimulation (Fig. 7D), i.e. at times when there was an
almost complete emptying of thapsigargin-sensitive Ca
stores (see Fig. 3A).
Figure 6:
[Ca]
changes in response to Ca
influx across
the plasma membrane. Fura-2-loaded cells were suspended in
Ca
-free medium at 37 °C and exposed to either 100
nM thapsigargin or dimethyl sulfoxide. Five minutes after the
stimulation, 3 mM Ca
was added. A and B, typical traces showing
[Ca
]
([Ca
]
)
increases in both control cells (solid lines) and calreticulin
overexpressers (dotted lines) in response to the
Ca
readdition. C and D,
[Ca
]
values
immediately before and at different times after the Ca
readdition in control and calreticulin-overexpressing cells. Data
are means ± S.E. of five independent
experiments.
Figure 7:
Thapsigargin-induced Mn
influx. Cells were loaded with the fluorescent Ca
indicator fura-2. Recordings were performed at the
Ca
-independent excitation wavelength of fura-2 (360
nm). A and B, typical traces showing spontaneous and
thapsigargin-induced fura-2 quenching after addition of Mn
to the extracellular medium in control cells (A) and in
overexpressers (B). C and D,
thapsigargin-induced Mn
influx
(Mn
-induced fura-2 quenching after thapsigargin
stimulation minus Mn
-induced fura-2
quenching without thapsigargin stimulation) in calreticulin
overexpressers and in control cells (mean ± S.E.).
Mn
was added either 5 min (C; n = 6) or 10 min (D; n = 3) after
thapsigargin stimulation. The small amplitude of the fluorescence
increase after DTPA addition witnesses the low amount of extracellular
fura-2. Unstimulated fura-2 quenching (as percent fluorescence
decrease/80 s) was 15.3 ± 0.8 (5 min after dimethyl sulfoxide (DMSO)) and 18.7 ± 1.7 (10 min after dimethyl
sulfoxide) in control cells and 15.8 ± 0.4 (5 min after dimethyl
sulfoxide) and 18.8 ± 1.1 (10 min after dimethyl sulfoxide) in
overexpressers.
Calreticulin is thought to act as a high capacity
Ca-binding protein within intracellular
Ca
stores(2, 5) . So far, however,
this notion is based on circumstantial
evidence(6, 7, 21, 22) . Here we
demonstrate for the first time that the Ca
content of
cells that stably overexpress calreticulin is augmented. (
)This elevation in stored Ca
is mostly
due to an increase in the size of thapsigargin-sensitive Ca
stores. Thus, our results add novel arguments in favor of a role
for calreticulin as a Ca
storage protein of
agonist-sensitive, rapidly exchangeable Ca
stores.
The precise mechanism through which calreticulin increases the
Ca
content of intracellular Ca
stores is not known. Our results would be compatible with the
concept that the increased intraluminal Ca
buffering
in overexpressers is compensated by an increased total Ca
store content, resulting in an unchanged intraluminal free
Ca
concentration. However, alternatively, one might
consider the possibility that the effect of calreticulin on cellular
Ca
storage could include a regulatory role for this
protein, rather than being a simple function of its Ca
buffering properties. The latter possibility is supported by a
recent study that demonstrates that in Xenopus laevis oocytes,
calreticulin overexpression inhibits repetitive intracellular
Ca
waves and that this inhibition is independent of
the Ca
storage domain of calreticulin (23) .
The amount of calreticulin overexpression found in this study is
relatively modest (i.e. an 1.6 ± 0.2-fold increase over
control), but we consider this relatively low overexpression as an
advantage. Indeed, rather than studying a massive overexpression with
its inherent uncertainties (correct protein targeting, nonspecific
effects due to very high protein concentrations), we have studied an
overexpression that is presumably still within the limits of the
physiological range of calreticulin expression(24) . Our
observation that the increased Ca storage occurred
almost exclusively in thapsigargin-sensitive Ca
stores (Fig. 3) adds weight to this argument.
The
relationship between the increased Ca content of
intracellular stores in calreticulin-transfected cells (as assessed by
Ca
measurements) and the
[Ca
]
elevations in response to
Ca
release (as assessed by fura-2 measurements) is
complex and depends on the applied stimulus. Increased
[Ca
]
elevations in calreticulin
overexpressers are observed with ATP or with a combination of
thapsigargin and ionomycin, but not when thapsigargin or ionomycin is
separately used as a stimulus. We think that, in the case of
thapsigargin, the slowness of Ca
release allows
negative feedback mechanisms (in particular, Ca
extrusion through the plasma membrane
Ca
-ATPase) to obscure the differences in
Ca
release in relation to the level of
[Ca
]
measurements. In the case
of ionomycin, a residual Ca
accumulation through
Ca
stores might dampen
[Ca
]
elevations. The latter
explanation is supported by the observation that, in overexpressers,
increased [Ca
]
elevations in
response to Ca
release are observed when cells are
concomitantly stimulated with ionomycin and thapsigargin. Finally, the
massive and rapid Ca
release that occurs mainly
through the inositol 1,4,5-trisphosphate receptor in cells stimulated
with ATP appears to generate, at least for a short period (
30 s;
see Fig. 5A), [Ca
]
transients whose amplitude is not governed by negative feedback
mechanisms, but rather by the amplitude of the Ca
release itself.
The inhibition of the store-operated
Ca influx pathway by calreticulin overexpression
provides new and independent experimental evidences for the intimate
connection between the Ca
content of rapidly
exchangeable intracellular Ca
stores and the plasma
membrane permeability for divalent cations. The calreticulin inhibition
of Ca
influx might be due to the increased
intraluminal Ca
buffering of intracellular
Ca
stores. However, we have measured
thapsigargin-induced Mn
influx even 10 min after
thapsigargin addition. At this point, thapsigargin-sensitive
Ca
stores were almost completely depleted, as shown
by
Ca
experiments (Fig. 3).
Nevertheless, virtually no store-operated Ca
influx
was observed. Thus, our results suggest that high calreticulin
concentrations do not simply slow down the appearance of store-operated
Ca
influx because of a delayed depletion of
Ca
stores through the increased intraluminal
Ca
buffering. High intraluminal calreticulin
concentrations block store-operated Ca
influx even
after a complete depletion of Ca
stores. This might
be a first indication that calreticulin plays a direct regulatory role
in the mechanism of store-operated Ca
influx.