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. 1997 Sep 22;138(6):1219-28.
doi: 10.1083/jcb.138.6.1219.

Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2

H He  1 , M Lam, T S McCormick, C W Distelhorst
Affiliations

Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2

H He et al. J Cell Biol. .

Abstract

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca2+) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca2+ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca2+-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca2+ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca2+ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca2+ pool in untreated cells when extracellular Ca2+ is low. However, low extracellular Ca2+ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca2+ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca2+ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.

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Figures

Figure 1
Figure 1
Bcl-2 mediates ER Ca2+ uptake. Uptake of 45Ca2+ was measured in digitonin-permeabilized cells in the absence (A) or presence of oxalate (B–F), and in the absence (A and B) or presence of 5 nM TG (C), 10 nM TG (D), 20 nM TG (E), or 100 nM TG (F). Open symbols, WEHI7.2 cells; closed symbols, W.Hb12 cells. Symbols represent the mean of duplicate determinations in a single experiment that was repeated at least twice with a similar result.
Figure 2
Figure 2
Bcl-2 does not increase expression of Ca2+-binding proteins. Extracts from untreated cells listed on the horizontal axis were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Lanes 1–4 were subjected to Western blotting using anti-Bcl-2 antibody (lanes 1 and 2) and anticalbindin antibody (lanes 3 and 4) as probes. Lanes 5–8 were incubated with 45Ca2+ and then exposed to x-ray film. Molecular mass standards are in lane 9. An unidentified protein that immunoreacts with anticalbindin antibody is detected at ∼80-kD in lane 4, and an unidentified Ca2+-binding protein was detected in CaBP20 cells (lane 5) midway between the 49.5- and 80-kD markers. The appearance of these proteins was variable among experiments and therefore not considered significant.
Figure 3
Figure 3
Bcl-2 prevents TG-induced delay in cathepsin D processing. Pulse–chase analysis of cathepsin D processing in wild-type S49 cells (−Bcl-2) and Bcl-2 transfectants (+Bcl-2) in the presence or absence of 200 nM TG. [35S]methionine-labeled cathepsin D was immunoprecipitated, resolved by PAGE under reducing and denaturing conditions, and analyzed by autoradiography.
Figure 4
Figure 4
Bcl-2 prevents ER Ca2+ pool depletion. WEHI7.2 cells (A–D) and W.Hb12 cells (E–H) were incubated for 3 h (A, B, E, and F) or 18 h (C, D, G, and H) in medium with a Ca2+ concentration of 1.3 mM (A, C, E, and G) or medium with a Ca2+ concentration of 0.13 mM (B, D, F, and H) and then loaded with Fura2-AM. Shown are continuous fluorometric tracings in which addition of 100 nM TG (right hand arrow) induces an increase in cytosolic Ca2+ concentration. Note that EGTA (left hand arrow) was added immediately before TG to chelate extracellular Ca2+; hence, the increase in cytosolic Ca2+ that follows TG addition represents release of Ca2+ from the ER pool. Large vertical oscillations in fluorescence near the beginning of each tracing correspond to brief light exposure during EGTA and TG additions.
Figure 5
Figure 5
Effect of Bcl-2 on cell growth and viability. WEHI7.2 cells (A, B, E, and F) and W.Hb12 cells (C, D, G, and H) were incubated in medium containing 1.3 mM Ca2+ (open symbols) or 0.13 mM Ca2+ (closed symbols), in either the absence of TG (A–D) or presence of 100 nM TG (E–H), for 32 h. At various time points, the number of viable cells, defined as cells that exclude trypan blue dye, and the percentage of apoptotic cells, based on nuclear apoptotic changes detected on fluorescence microscopy of cells stained with acridine orange, were measured. Symbols represent the mean of duplicate determinations in a single experiment that was repeated three times with a similar result.
Figure 6
Figure 6
Low extracellular Ca2+ inhibits cytosolic Ca2+ elevation in TG-treated cells. WEHI7.2 cells (A) and W.Hb12 cells (B) were incubated in medium containing 1.3 mM Ca2+ or 0.13 mM Ca2+, in the presence or absence of 100 nM TG, for 4 h before loading with Fura-2 AM. Cytosolic Ca2+ concentration was measured according to Fura-2 fluorescence. Symbols represent the mean ± SE of multiple determinations in three separate experiments.
Figure 7
Figure 7
Model: Bcl-2 regulation of Ca2+ homeostasis in the ER. The relationship between Ca2+ pools in the cytoplasm and ER is illustrated. A–D represent cells cultured in medium containing a physiologic concentration of Ca2+ (1.3 mM), and E–H represent cells cultured in medium containing a low concentration of Ca2+ (0.13 mM). A, C, E, and G represent cells that lack Bcl-2, and B, D, F, and H represent cells that express Bcl-2. A, B, E, and F represent untreated cells in which Ca2+ is pumped into the ER by the Ca2+-ATPase, and C, D, G, and H represent cells in which the Ca2+-ATPase pump is inhibited by TG. Bcl-2 is represented by two parallel lines anchored in the ER membrane.
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