(Received for publication, October 5, 1995; and in revised form, December 27, 1995)
From the
The plasma membrane Ca-ATPase pump (PMCA) is
an integral component of the Ca
signaling system
which participates in signal transduction during agonist stimulated
cell activation. To better understand the physiological function of the
pump, isoform 1a (PMCA1a) was over-expressed in rat aortic endothelial
cells using a stable transfection system under the control of a
cytomegalovirus promoter. The cell lines selected after transfection
with PMCA1a construct, expressed 3-4-fold increased pump protein
which was mostly targeted to the plasma membrane as indicated by
immunoperoxidase staining. Ca
uptake assays in a
membrane preparation indicated a 3-4-fold increase in
Ca
pumping activity in the transfected cells, and the
expressed PMCA1a showed typical dependence on Ca
and
calmodulin for stimulation of activity. Measurement of
[Ca
]
and
[Ca
]
showed that expression
of PMCA1a had a profound effect on different aspects of the
Ca
signal. The peak increase in
[Ca
]
evoked by ATP
and/or thapsigargin was lower but the plateau phase was similar in the
PMCA1a expressing cells. Accordingly, titration with ionomycin of
Ca
content of internal stores, measurement of
Ca
uptake into the thapsigargin- and
oxalate-sensitive pool (endoplasmic reticulum) of isolated microsomes,
Ca
uptake into streptolysin O-permeabilized
cells, and analysis of SERCA mRNA and protein, showed that expression
and activity of the SERCA pump was down-regulated in cells expressing
PMCA1a pump. Expression of PMCA1a also down-regulated expression of the
inositol 1,4,5-trisphosphate (IP
)-activated Ca
channel and the rate of IP
-mediated Ca
release in permeable cells, without affecting the affinity of the
channel for IP
. On the other hand the rate of store
depletion-dependent Ca
and Mn
influx (Ca
entry) into PMCA1a expressing cells
was increased by about 2.6-fold. These changes prevented estimating the
rate of pump-mediated Ca
efflux from changes in
[Ca
]
. Measurement of
[Ca
]
showed that the rate of
Ca
efflux in cells expressing PMCA1a was about
1.45-fold higher than Neo controls, despite the 4-fold increase in the
amount of functional pump protein. The overall study points to the
flexibility, interdependence, and adaptability of the different
components of the Ca
signaling systems to regulate
the expression and activity of each component and maintain a nearly
constant Ca
signal.
The plasma membrane Ca pump (PMCA) (
)is the sole high affinity Ca
extrusion
mechanism in the plasma membrane(1) . In non-muscle cells, the
pump plays a prominent role in Ca
extrusion than
other mechanisms such as the Na
/Ca
exchanger(2, 3, 4, 5, 6, 7) .
PMCA activity is believed to be activated during hormonal stimulation
of cells (2) and the increased Ca
efflux is
concomitant with an enhanced phosphorylation of the Ca
pump protein(8, 9) .
cDNA cloning indicates
the presence of at least four major isoforms for the PMCA, which are
encoded by distinct genes numbered ``1 to 4'' (1, 10, 11, 12) . The primary
transcripts of each gene may be alternatively spliced at their 3` ends
in a different way, further increasing the variety of pumps as denoted
by letters ``a to d''(1) . While these isozymes show
distinct tissue and cellular distribution, isoform 1b (PMCA1b) appears
to be the major isoform that is expressed in all tissues
examined(1, 13) . Recent attempts to over-express the
human gene 4 in COS cells have demonstrated the usefulness of transient
transfection in relating the structure of the pump to its biochemical
activity(14, 15, 16, 17) . However,
a transient expression system does not allow the study of intracellular
Ca signaling in a metabolically active cell type,
such as endothelial cells, due to the low transfection efficiency
(normally 15% or less of the cells were transfected by the transient
transfection system). (
)In this report we focus on the
stable transfection of the PMCA1a isoform of the pump in rat aortic
endothelial cells (RAECs), which endogenously contain a low level of
PMCA1b as the predominant species, while isoform 1a is either absent or
undetectable by reverse transcriptase-polymerase chain
reaction(13) . This over-expression system allowed us to study
the physiological function of PMCA during agonist stimulation. In
addition, since no information is available regarding how
over-expression of one calcium transporter may influence the expression
of other calcium transporters, we have examined the effect of PMCA1a
over-expression on the four major Ca
transport
pathways responsible for cellular Ca
homeostasis,
which includes intracellular pump (SERCA), the IP
-activated
Ca
channel, the plasma membrane calcium entry
pathway, and the PMCA(18, 19) . The overall findings
demonstrate the flexibility and coordinated regulation of all pathways,
possibly on the gene level, to maintain constant Ca
signaling.
Figure 6:
SERCA pump activity and mRNA levels. Panel A, ATP-mediated, oxalate-dependent Ca uptake was measured by incubating microsomes in
Ca
uptake medium with and without Tg and in the presence of 7.5 mM oxalate. At the indicated times samples were removed to analyze
Ca
content in microsomes. The figure
shows the fraction of the Tg-sensitive
Ca
uptake. Panel B shows a Northern blot analysis of total
RNA prepared form Neo control and clone 8 cells. SERCA mRNA (4.4
kilobases) was identified by using a specific cDNA probe representing
SERCA 3, the major species in RAEC (T. H. Kuo, unpublished data). The
same blot was hybridized with an actin cDNA probe to show the equal
loading of RNA samples. Similar reductions in SERCA mRNA were obtained
in four additional experiments using clone 8 or 35
cells.
Figure 1: Western blot analysis of PMCA1a over-expressing clones. Lysates from the different RAEC clones stably over-expressing PMCA1a (A) and from BSC-1 cells transfected with a vaccinia virus system that includes the PMCA1a construct (B) were prepared and separated by SDS-polyacrylamide gel electrophoresis as described under ``Materials and Methods.'' The blots were probed with monoclonal antibody 5F10. Lanes a and b in panel A show membranes from control nontransfected and control transfected with vector only, respectively. Lanes 2-7 show membrane from selected clones. Note the higher PMCA1a levels in clones 8, 26, 33, and 34. Panel B illustrates the difference in molecular weight between PMCA1a and PMCA1b. The left lane shows membranes from control BSC-1 cells and the right lane shows membranes from BSC-1 cells transfected with the PMCA1a construct.
The present study represents the first report that a PMCA isoform 1 cDNA was successfully over-expressed in mammalian cells. This is different from an earlier report (14) describing the failed attempt to express a full-length human PMCA isoform 1 cDNA. Presently, the only other successful expression of the PMCA protein is with the human isoform 4b(14, 17) . However, in one report there was a problem in the correct targeting of the protein to the plasma membrane of the COS cells(14) . Most recently isoform PMCA4b was successfully expressed and targeted to the plasma membrane(27) .
Figure 2: Immunolocalization of PMCA1a. Clone 8 cells (panel A) and control cells transfected with the vector (panel B) were fixed and stained with monoclonal antibody 5F10 as described under ``Materials and Methods.'' Note the punctate peripheral staining and absence of cytosolic, ER-like staining in clone 8 cells over-expressing the PMCA1a pump. The amount of pump protein in the control cells was below the detection limit at the concentration of antibody used.
Additional
support for correct targeting of PMCA1a comes from the assay of PMCA
activity using isolated microsomes. It was reported previously (15) that over-expression of human PMCA4 led to an increased
Ca uptake in microsomes prepared from COS-1 cells,
and this activity is stimulated by oxalate. Stimulation by oxalate was
interpreted as an evidence that PMCA4 was retained in the
ER(15) . An assay of Ca
uptake in microsomes
obtained from control and PMCA1a over-expressing RAECs indicated that
over-expression of PMCA1a led to an increase in the Ca
uptake that is not stimulated by oxalate (see below). Thus the
combined evidence from immunoperoxidase staining and the properties of
microsomal Ca
uptake suggest that the expressed
PMCA1a is not retained in the ER but rather localized to the plasma
membrane.
To determine whether the expressed protein was functional,
the properties of ATP-dependent Ca uptake were
measured. A 70-fold excess concentration of thapsigargin (Tg, 200
nM) was included in the uptake reaction to ensure complete
inhibition of SERCA pump activity, without affecting the
calmodulin-dependent Ca
uptake mediated by the
PMCA(15) . As shown in Fig. 3A, the rate of
ATP-dependent Ca
uptake in the membrane preparation
from the PMCA-transfected cells (clone 8) was about 3-fold higher than
the vector transfected controls (0.47 versus 0.16
nmol/mg/min). Similar results were obtained with microsomes prepared
from clone 35 (not shown). Thus the activity assay shows that the
over-expressed PMCA1a is functional in Ca
transport.
Figure 3:
Ca uptake in isolated
microsomes. Isolated microsomes were incubated in uptake medium
containing
Ca and 200 nM Tg. Uptake was initiated
by addition of ATP. Panel A shows the time course of
Ca
uptake in microsomes prepared from Neo control
(
) or clone 8 (
) cells. Panels B and C show the dependence of Ca
uptake on
[Ca
] in the presence (
) or absence
(
) of 0.24 µM calmodulin in microsomes prepared
from clone 8 (B) or Neo control (C) cells. Pump
activity was evaluated from 10 min uptake and calculated as
nanomoles/mg of protein/10 min.
A critical parameter for the correct functioning of the PMCA is its
affinity for Ca and response to calmodulin.
Therefore, Ca
transport activities of microsomes
isolated from the transfected cells were tested as a function of free
Ca
concentration and in the presence or absence of
calmodulin. As shown in Fig. 3B, in membranes prepared
from the over-expressing cells (clone 8), the activity of PMCA1a was
dependent on Ca
concentration. Addition of calmodulin
greatly enhanced the activity. Thus, the expressed isoform 1a shows a
characteristic dependence on Ca
and calmodulin for
activation. Comparison of the PMCA over-expressing cells (Fig. 3B) with the vector-transfected cells (Fig. 3C) indicated a 3-4-fold increase in PMCA
activity at low and saturating Ca
concentrations.
Figure 4:
Measurement of
[Ca]
in single cells.
Neo control (traces a-d) or clone 8 (traces e-h)
cells grown on glass coverslips were loaded with Indo 1 (a, c, e, and g) or Fura 2 like (b, d, f, and h)
for measurements of
[Ca
]
. The cells were
continuously perfused with warm solution A. Where indicated 100
µM ATP or 2 µM Tg were included in the
perfusion medium. Similar results were obtained by imaging at least 10
cells in each coverslip in five separate experiments with Fura 2-loaded
cells.
To verify
this finding and obtain an averaged behavior of the cells we measured
[Ca]
of a suspension of about
10
cells. Resting [Ca
]
was similar in control and clone 8 cells and averaged 77.5
± 7.5 (n = 51) and 75.8 ± 6.4 nM (n = 56), respectively. Hence, despite a 4-fold
over-expression of active PMCA1a in the plasma membrane, the cells were
able to maintain the same resting
[Ca
]
. Fig. 5shows that
when control cells were exposed to a mixture of ATP and Tg to discharge
most of the agonist-mobilizable Ca
pool,
[Ca
]
increased to about 358
± 21 (n = 46) nM. ATP and Tg increased
[Ca
]
of clone 8 cells only to
266 ± 22 (n = 37) nM. The difference in
peak [Ca
]
was highly
significant (p < 0.01, Student's t test). To
exclude the possibility that modification of signal transduction was
responsible for the reduced response to agonist we estimated the size
of the intracellular Ca
pool with the Ca
ionophore ionomycin (Fig. 5, b and d).
Supermaximal concentration of ionomycin (10 µM) increased
[Ca
]
of Neo controls by 668
± 43 (n = 4) nM and that of clone 8
cells by 372 ± 19 (n = 4) nM.
Figure 5:
Measurement of
[Ca]
in suspension of
cells. Fura 2-loaded Neo controls (traces a and b) or
clone 8 (traces c and d) cells were suspended in
solution A containing 1.5 mM CaCl
(a and c) or Ca
-free solution A containing 0.1
mM EGTA (b and d). Where indicated the cells
were exposed to a mixture of 100 µM ATP and 2 µM Tg and then 1.8 mM EGTA (a and c).
Cells in Ca
-free medium were treated with 10
µM ionomycin to determine maximal Ca
content of internal stores (b and d).
An
additional finding illustrated in Fig. 5, a and c, is that the second phase increase in
[Ca]
appears to be somewhat
more prominent in clone 8 cells, suggesting a possible difference in
Ca
influx rate. It is difficult to estimate the
difference in plasma membrane Ca
pumping from the
rate of reduction in [Ca
]
after
ATP and Tg treatment, since the agonists increased
[Ca
]
to different levels in the
two cell types and Ca
influx appears to be modified
by over-expression of the pump. Nonetheless, removal of external
Ca
with EGTA caused faster reduction in
[Ca
]
in clone 8 relative to
control. Although not conclusive, this provides an indication that the
rate of Ca
pumping across the plasma membrane of
clone 8 cells was higher than in controls.
Due to the apparent
multiple effects of PMCA1a over-expression on Ca signaling we proceeded to examine the expression and activity of
the different Ca
transporting pathways involved in
regulating
[Ca
]
(18, 19) .
Figure 8:
Ca uptake and
IP
-mediated Ca
release in
SLO-permeabilized cells. Neo control (A and
) or clone 8
(B and
) cells were washed and incubated in the SLO-containing
permeabilization medium. After stabilization of medium
[Ca
] the cells were exposed to increasing
concentrations of IP
as indicated and finally to 5
µM ionomycin. The resulting
[Ca
] was plotted as a function of
[IP
] (C) or the released Ca
(Ca
increase above resting) was calculated as a
% of the ionomycin-mobilizable pool (D). Two experiments with
each subpassage and four experiments from different subpassages showed
the same apparent affinity for IP
in control and clone 8
cells.
Figure 7:
Western blot analysis of SERCA and
IPR proteins. Lysates prepared from Neo control and clone 8
cells were separated by SDS-polyacrylamide gel electrophoresis and
Western blotted. SERCA protein (100 kDa) and IP
R (250 kDa)
were identified on separate blots by using specific antibody followed
by a chemiluminescence methods. SERCA antibody (monoclonal anti-SERCA2,
clone 2A7-A1 which cross-reacts with SERCA3) was purchased from
Affinity BioReagents and IP
R (type 1) antibody was a gift
from Dr. Richard J. H. Wojcikiewicz, State University of New York at
Syracuse. The blots were hybridized with an actin antibody from Bio-Rad
to control for protein loading (not shown). Similar results were
obtained in one additional experiment.
To evaluate
the impact of reduced levels of the IP-activated channel on
Ca
release we used streptolysin O (SLO)-permeabilized
cells and measured Ca
uptake and release in the
presence of mitochondrial inhibitors. Under these conditions
Ca
uptake was completely dependent on the presence of
ATP in the permeabilization medium and was inhibited better than 96% by
0.1 µM Tg (not shown). Fig. 8, A and B, shows that the rate of Ca
uptake by clone
8 cells was slower by about 38 ± 1.2% (n = 9)
compared to that measured in control cells. In addition, clone 8 cells
reduced medium [Ca
] to 307 ± 26
nM (n = 9) as compared to control cells, which
reduced medium [Ca
] to 203 ± 15
nM (n = 9). These results further support the
reduced SERCA pump activity in clone 8 cells.
After stabilization of
medium [Ca] the permeabilized cells were
used to measure the properties of IP
-mediated
Ca
release. Due to differences in SERCA pump activity
between the cells, the ability of IP
to release
Ca
was expressed as total Ca
release (Fig. 8C) and as a percentage of
Ca
mobilized by ionomycin (Fig. 8D).
It can be seen that in both cell types IP
released about
50% of the ionomycin-mobilizable Ca
pool with similar
apparent affinity. This would suggest that the IP
R
remaining in clone 8 cells are not modified and that they are at
sufficiently high density to allow maximal Ca
release.
The lower density of IPR in clone 8 cells
prompted testing the rate of IP
-mediated Ca
release. Fig. 9shows that Ca
release in
clone 8 cells was slower than that of control cells. In four
determinations from two experiments the rate in clone 8 was reduced by
an average of 34 ± 4%.
Figure 9:
Rate
of IP-mediated Ca
release. Experimental
protocol was as in Fig. 8except that after stabilization of
[Ca
]
the cells were exposed
to 2 µM IP
. The integrated fluorescence signal
was recorded at a resolution of 3/s. Interruption in recording was
required for addition of IP
.
Figure 10:
Measurement of Ca and
Mn
influx. Neo control (a and b) or
clone 8 (c and d) cells were suspended in
Ca
-free solution A. Where indicated the cells were
stimulated with a mixture of 100 µM ATP and 2 µM Tg (b and d) and exposed to 1.5 mM CaCl
and then 0.5 mM MnCl
. To
determine maximal quench, at the end of each experiment the cells were
layered with 50 µM digitonin. The calcium signal is the
360/380 ratio and the Mn
signal is the 360 nm portion
of the fluorescence signal.
Figure 11:
Unidirectional Ca efflux in control and clone 8 cells. Neo control (a and c) or clone 8 (b and d) cells were suspended
in Chelex-treated efflux medium and external
[Ca
] was reduced to 115 nM with
0.4 µM EGTA. Within 1 min of EGTA titration the cells were
either stimulated with a mixture of 100 µM ATP and 2
µM Tg or exposed to 5 µM ionomycin.
In previous studies we showed that
agonist stimulation activates the plasma membrane Ca pump(2) . Thus, it was important to show that the faster
Ca
pumping measured in clone 8 cells is independent
of cell stimulation. Fig. 11, c and d, shows
that when internal Ca
was mobilized by ionomycin, the
initial rate of Ca
extrusion by clone 8 cells was
about 1.35 ± 0.03 (n = 3) fold higher than that
of Neo controls. This was despite the lower increase in
[Ca
]
caused by ionomycin in
clone 8 cells (Fig. 5). This experiment also confirmed the
reduced size of the internal stores Ca
pool in clone
8 cells.
Although the biochemical properties of the PMCA have been
extensively studied(1) , its functional role in resting and
stimulated cells is not well understood. In recent years it became
clear that the pump plays a pivotal role in the down-stroke phase of
the [Ca]
signal evoked by
agonist stimulation (2, 18, 19) and during
[Ca
]
oscillations(7) .
Direct (2, 5) and indirect (4, 6) evidence suggests that the PMCA is activated by
agonists, and the activation is accompanied by phosphorylation of the
pump protein(8, 9) .
In an effort to better
understand the role and regulation of the PMCA in Ca signaling, we over-expressed the PMCA1a isoform and isolated
stable transfectant. A major difficulty in using expression systems to
study the role of PMCA in Ca
transport in intact
cells was the correct targeting of the pump to the plasma
membrane(14) . The cell line, vector, transfection, and
selection procedures used in the present studies overcame this problem.
Two types of evidence suggest proper targeting of most of the PMCA1a to
the plasma membrane. Immunocytochemistry showed a typical punctate
staining in the plasma membrane of over-expressing cell lines with
minimal cytosolic staining. The high levels of PMCA1a colocalized with
markers of caveolae, (
)similar to the localization of native
pump protein reported before(28, 29, 30) .
The increased Ca
pumping activity in microsomes of
PMCA1a over-expressing cells was into an oxalate- and Tg-independent,
but calmodulin-sensitive compartment. These criteria firmly established
that in our cells a large fraction of the PMCA1a pump was correctly
targeted to the plasma membrane. While this work was in progress,
over-expression and successful targeting of the PMCA4b isoform to the
plasma membrane of CHO cells was reported(27) . Also in this
case the vector, transfection, and culture conditions were important in
the successful targeting of the pump(27) . The pattern of
immunostaining of PMCA4b over-expressing cells was similar to that
shown in Fig. 2A.
The successful over-expression of
high levels of PMCA1a in the plasma membrane revealed that pump
activity can be regulated on several levels. The first finding of note
was that despite the correct targeting and over-expression of the
PMCA1a, resting [Ca]
was
identical in control cells and cells of the different clones
over-expressing the PMCA1a. Since resting
[Ca
]
is determined exclusively
by pump/leak turnover across the plasma membrane(18) , it was
clear that either the activity of the over-expressed pumps was
down-regulated in intact cells, Ca
influx rate was
much higher in cells over-expressing the pump or a combination of both.
Measurement of Ca
and Mn
influx
into resting cells (such as those in Fig. 10, a and b) did not reveal major differences in Ca
influx rate among the different cell types. Therefore, it was
likely that the over-expressed pumps were not active at resting
[Ca
]
.
Following
[Ca]
and
[Ca
]
to estimate the rate of
unidirectional Ca
efflux revealed that the activity
of the over-expressed PMCA pumps was also regulated during agonist
stimulation and/or when [Ca
]
was high. Thus, Western blot analysis showed about 4-fold
increase in pump protein in the over-expressing clones used for the
present studies (clone 8 and 35). The over-expressed pumps were fully
functional as they increased Ca
uptake in isolated
microsomes, had the expected apparent affinity for
Ca
, and were regulated by calmodulin. Yet, the rate
of Ca
pumping in intact clone 8 and clone 35 cells
was about 1.4-fold over that measured in control cells. Our
measurements of Ca
pumping activity are not
influenced by the activity of other Ca
transporting
pathways since we measured net Ca
efflux by following
[Ca
]
. The lower than expected
increase in Ca
pumping in intact cells is also not
because the lower increase in [Ca
]
evoked by agonist in these cells. When dCa
/dt
for the two cell types is calculated at the same
[Ca
]
from experiments similar
to those in Fig. 10, b and d, and 11, the
increased rate of pumping due to PMCA1a over-expression is at best
1.67-fold over control. Our findings are further supported by the study
of Guerini et al.(27) . These authors reported a
20-fold increase in Ca
pumping activity in microsomes
prepared from CHO cells over-expressing PMCA4b. Recalculation of the
data in their Fig. 5shows that such over-expression of active
pump protein in the plasma membrane caused only about a 1.7-fold
increase in
Ca
efflux rate(27) .
Considering the fact that agonist-induced
Ca efflux is
influenced by reuptake of Ca
into the ER and reflects
the sum of Ca
extrusion and
Ca
/Ca
exchange through the CRAC
channel(18) , the 1.7-fold increased rate of
Ca
efflux is an overestimation of the increase in Ca
pump rate(27) .
The finding discussed above of the two
independent studies, using different cell type and different pump
isoforms, suggests that cells are capable of controlling Ca pumping rate independent of the number of the Ca
pumps in the plasma membrane. Hence, a 4- (present studies) or a
20- ((27) ) fold increase in the number of Ca
pumps increased Ca
pumping rate in intact cells
to similar extent (1.4-1.7-fold). This suggests a set point for
the maximal possible Ca
pumping rate which is
compatible with cell survival, since over-expression of PMCA strongly
affects the timing of the cell cycle(27) . How the cells
achieve such a set point is not clear at present. Two obvious
possibilities are down-regulation of pumping activity and/or shielding
the pumps from the increase in [Ca
]
by compartmentalizing the Ca
pool and the areas
of Ca
release (31, 32, 33, 34, 35, 36, 37) .
Discriminating between these and other possibilities requires further
studies. Using the PMCA over-expressing cells it should be now possible
to address such questions.
Another major and significant finding of
the present study was the adaptive regulation of all the major
Ca transporting pathways in response to
over-expression of the PMCA1a. The activity of the CRAC pathway in the
plasma membrane was up-regulated by as much as 2.6-fold, significantly
more than the net increase in Ca
pumping activity.
The large increase in CRAC activity is likely to compensate for the
increase in net pumping rate and the reduced capacity of the
Ca
stores (see below). It is interesting that
activation of CRAC in the PMCA1a over-expressing cells still required
Ca
release from internal stores, indicating
maintained regulation of the CRAC pathway by Ca
content of the ER(18, 38) . Since nothing is
known about the nature of the CRAC pathway (38) we could not
determine on what level CRAC activity was regulated in PMCA1a
over-expressing cells.
Over-expression of PMCA1a down-regulated the
contribution of the internal Ca pool to the
Ca
signal by reducing the level and activity of both
the SERCA pumps and the IP
R. Previous studies similarly
showed that over-expression of PMCA4b in the plasma membrane reduced
the phosphorylated intermediate formed during the turnover cycle of the
SERCA pump in CHO cells(27) . Here we show that the reduced
activity was largely due to a reduction in the number of the pumps.
Also in the case of IP
-mediated Ca
release, the reduced rate of release could be attributed to
reduced number of IP
R, with normal apparent affinity for
IP
. The reduction in the number of SERCA pumps resulted in
reduced rate of Ca
pumping and Ca
content in the ER. This was the case whether Ca
content in the ER was measured in permeable or intact cells and
whether it was released by IP
or ionomycin. The reduced
Ca
content in the ER was reflected in a reduced
initial agonist-evoked [Ca
]
increase.
The regulation of the different Ca transporting pathways by over-expression of PMCA1a appears to be
on the gene level. In the case of the SERCA pump we were able to show
that over-expression of PMCA1a reduced SERCA gene expression by more
than 50%, which is likely to account for the reduced amount of SERCA
pump protein in these cells. The mechanism by which PMCA1a expression
regulates the SERCA gene(s) is not clear at present. Up-regulation of
SERCA2 gene by the hormone T
and by the platelet-derived
growth factor have been reported in cardiac (39) and smooth
muscle cells(40) , respectively. Similarly, we have
demonstrated the stimulation of SERCA3 gene expression (the major
isoform) in RAEC by epidermal growth factor and angiotensin
II(41) . SERCA3 gene is also up-regulated in platelets from
hypertensive rats(42) . In fibroblasts, the expression of a
distinct Tg-resistant Ca
pump is induced by
continuing exposure to Tg(43, 44) . These findings
together with the present studies, suggest that cells can regulate the
level of SERCA in response to external stimuli or a change in the level
or activity of the other Ca
transport pathways.
In
summary, the present studies demonstrate that functional
over-expression of PMCA1a led to a down-regulation of SERCA gene
expression, a reduction in the number of IPR, and an
increase in the activity of the CRAC pathway. It is not clear at
present whether the regulation of all pathways was on the gene level.
However, if this is the case, it is possible that the genes of all the
major Ca
transporting pathways are regulatorally
linked to concomitantly modulate the activities of all pathways. The
link can be provided by the [Ca
]
levels in resting cells. Considering the information encoded in
the [Ca
]
signal and the
involvement of Ca
in numerous vital cell
functions(32, 38) , it is not surprising that its
concentration is regulated on several levels. Regulation on the level
of gene expression is likely to provide a constant
[Ca
]
signal, which is essential
for cell survival. This is emphasized in several recent studies showing
the modulation of the cell cycle (27, 43, 44) by over-expression of PMCA pumps
and/or down-regulation of SERCA pumps by Tg treatment.