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. 2000 Mar 6;148(5):857-62.
doi: 10.1083/jcb.148.5.857.

Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells

P Pinton  1 , D Ferrari, P Magalhães, K Schulze-Osthoff, F Di Virgilio, T Pozzan, R Rizzuto
Affiliations

Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells

P Pinton et al. J Cell Biol. .

Abstract

The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca(2+)-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca(2+) homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca(2+) leak (while leaving Ca(2+) accumulation unaffected), hence reducing the steady-state [Ca(2+)] levels. As a direct consequence, the [Ca(2+)] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca(2+) influx activated by Ca(2+) store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca(2+)-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca(2+) homeostasis may contribute to the protective role of this oncogene against programmed cell death.

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Figures

Figure 1
Figure 1
Cytoplasmic (A) and mitochondrial (B) Ca2+ homeostasis in control and Bcl-2–overexpressing HeLa cells. Parallel batches of HeLa cells were either cotransfected with the appropriate AEQ chimera (cytAEQ and mtAEQ for monitoring the cytoplasm and the mitochondria, respectively) and Bcl-2 (Bcl-2), or transfected with the AEQ chimera alone (control). 36 h after transfection, the measurement of AEQ luminescence was carried out and calibrated into [Ca2+] values, as described in Materials and Methods. Where indicated, the cells, perfused with KRB, were challenged with 100 μM ATP added to the same buffer. These and the following traces are representative of ten experiments that gave similar results.
Figure 2
Figure 2
Ca2+ homeostasis in the lumen of the ER (A) and of the Golgi apparatus (B) in control and Bcl-2–overexpressing HeLa cells. Parallel batches of HeLa cells were either cotransfected with the appropriate AEQ chimera (erAEQ and GoAEQ for monitoring the lumen of the ER and of the Golgi apparatus, respectively) and Bcl-2 (Bcl-2), or transfected with the AEQ chimera alone (control). 36 h after transfection, the organelles were depleted of Ca2+ to optimize AEQ reconstitution. After reconstitution, the cells were transferred to the luminometer chamber and AEQ luminescence was collected and calibrated into [Ca2+] values. Where indicated, the cells were stimulated with 100 μM ATP. All other conditions are as in Fig. 1.
Figure 3
Figure 3
Kinetics of ER Ca2+ uptake and release in control and Bcl-2–transfected cells. A, ER Ca2+ refilling in permeabilized cells. Parallel batches of HeLa cells were either cotransfected with erAEQmut and Bcl-2 (Bcl-2), or transfected with erAEQmut alone (control). 36 h after transfection, depletion of Ca2+ stores and AEQ were carried out as in Fig. 2. The coverslip with the cells was then transferred to the luminometer chamber. The perfusion medium was changed to IB/EGTA, and permeabilization was carried out by perfusing 100 μM digitonin (added to the same medium) for 1 min. After a 2-min wash with IB/EGTA, Ca2+ accumulation in the ER was initiated by switching the medium to IB containing a buffered Ca2+ concentration of 0.2 μM (see text for details). AEQ calibration was carried out as described in Materials and Methods. B, Dependence on [Ca2+]er of the Ca2+ leak rate from the ER. Transfection, depletion of Ca2+ stores, and AEQ reconstitution were carried out as in A, then the coverslip with the cells was transferred to the luminometer and perfused with KRB/Ca2+ until the steady-state [Ca2+]er was reached. Ca2+ release was initiated by treating the cells with 50 μM tBuBHQ. Based on the experimental trace, the maximal rates of Ca2+ release (measured from the first derivative) at different values of [Ca2+]er were calculated and plotted for Bcl-2–transfected and control cells. The plot contains data obtained from ten independent experiments. In each experiment, duplicate samples of control and Bcl-2–overexpressing cells were measured. Due to the mixing time in the luminometer chamber, the kinetics of [Ca2+]er decrease are sigmoidal and the maximal rate is obtained 2–3 s after addition of tBuBHQ. Accordingly, we considered the maximal rates the best approximation for the initial rate of [Ca2+]er decrease. The fitting of the curve shown in B was performed using Microsoft Excel software.
Figure 4
Figure 4
Capacitative Ca2+ influx in control, Bcl-2–transfected, and Ca2+-deprived cells. A, Representitive trace. B, Average [Ca2+] peak. Parallel batches of HeLa cells were either cotransfected with cytAEQ and Bcl-2 (Bcl-2), or transfected with cytAEQ alone (control and Ca2+-deprived). In the Ca2+-deprived cells, the cells were maintained in the 18 h preceding the experiment in KRB containing a lower (0.1 mM) CaCl2 concentration (KRB/lowCa2+). After transferring the coverslip with the cells to the luminometer chamber, the cells, perfused with KRB/EGTA, were challenged with 10 μM tBuBHQ (a specific blocker of the Ca2+ ATPase present in the ER and Golgi apparatus) added to the same medium. After 2 min, capacitative Ca2+ entry was initiated by changing the medium to KRB/Ca2+ (CaCl2). All other conditions as in Fig. 1.
Figure 4
Figure 4
Capacitative Ca2+ influx in control, Bcl-2–transfected, and Ca2+-deprived cells. A, Representitive trace. B, Average [Ca2+] peak. Parallel batches of HeLa cells were either cotransfected with cytAEQ and Bcl-2 (Bcl-2), or transfected with cytAEQ alone (control and Ca2+-deprived). In the Ca2+-deprived cells, the cells were maintained in the 18 h preceding the experiment in KRB containing a lower (0.1 mM) CaCl2 concentration (KRB/lowCa2+). After transferring the coverslip with the cells to the luminometer chamber, the cells, perfused with KRB/EGTA, were challenged with 10 μM tBuBHQ (a specific blocker of the Ca2+ ATPase present in the ER and Golgi apparatus) added to the same medium. After 2 min, capacitative Ca2+ entry was initiated by changing the medium to KRB/Ca2+ (CaCl2). All other conditions as in Fig. 1.
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