ER Ca2+ depletion triggers apoptotic signals for endoplasmic reticulum (ER) overload response induced by overexpressed reticulon 3 (RTN3/HAP)

Perturbance of endoplasmic reticulum (ER) function, either by the mutant proteins not folding correctly, or by an excessive accumulation of proteins in the organelle, will lead to the unfolded protein response (UPR) or ER overload response (EOR). The signal-transducing pathways for UPR have been identified, whereas the pathway for EOR remains to be elucidated. Our previous study demonstrated that the overexpression of reticulon 3 (RTN3, also named HAP, homologue of ASY protein) caused apoptosis with the depletion of ER Ca(2+) stores. In present research, we characterized RTN3 as a novel EOR-induced protein, triggering the apoptotic signals through the release of ER Ca(2+) and the elevation of cytosolic Ca(2+). Our studies showed that overexpressed RTN3 induced EOR, eliciting ER-specific apoptosis with activation of caspase-12 and mitochondrial dysfunction through ER Ca(2+) depletion and the sustained elevation of cytosolic Ca(2+). Furthermore, we demonstrated that overexpressed RTN3 and stimuli that activate both EOR and UPR, not UPR only, were able to induce up-regulation of inducible nitric oxide synthase (iNOS) in HeLa cells through ER Ca(2+) release and reactive oxygen intermediates (ROIs), resulting in endogenous calcium-dependent nitric oxide protecting cells against ER specific apoptosis, which suggested that the nitric oxide and iNOS represented a likely protective response to EOR, not the UPR. These results supported that the release of ER Ca(2+) stores triggered the initial signal-transducing pathways for EOR induced by overexpressed RTN3.     

The endoplasmic reticulum (ER)-target protein Bik induces Hep3B cells apoptosis by the depletion of the ER Ca2+ stores

Bik, a BH3-only protein, was identified to induce cells apoptosis. In this study, we reported that Bik exclusively localized to endoplasmic reticulum rather than mitochondria. The apoptosis induced by Bik was inhibited in Hep3B cells, when TM domain of Bik was truncated. The ectopic overexpression of Bik protein caused the rapid and sustained elevation of the intracellular cytosolic Ca²⁺, which originated from the ER Ca²⁺ stores releasing. The Hep3B cells apoptosis induced by Bik was not prevented by establishing the clamped cytosolic Ca²⁺ condition, or by buffering of the extracellular Ca²⁺ with EGTA, suggesting that the depletion of ER Ca²⁺ stores rather than the elevation of cytosolic Ca²⁺ or the extracellular Ca²⁺ entry contributed to Bik-induced Hep3B cells apoptosis. The authors Xiaoping Zhao and Li Wang contributed equally to this work.     

Cytosolic Ca(2+) signal is involved in regulating UV-induced apoptosis in hela cells

Results of recent studies using BAPTA/AM have raised a serious question on whether Ca(2+) signal is truly involved in regulating the progression of apoptosis. To resolve this question, we examined the differential effects of three different Ca(2+) signaling blockers (BAPTA/AM, membrane-impermeant BAPTA, and heparin) on UV-induced apoptosis in HeLa cells. We found that although the membrane-permeable form of BAPTA (i.e., BAPTA/AM) could not inhibit cell death, the membrane-impermeant form of BAPTA, loaded into the cytosol by electroporation, clearly protected cells from entering apoptosis. Furthermore, when we injected heparin to block Ca(2+) release from the endoplasmic reticulum (ER) to cytosol, apoptosis was greatly suppressed. These findings strongly suggest that elevation of cytosolic Ca(2+) is part of the signal that drives the progression of apoptosis. The negative result of BAPTA/AM is probably due to its dual effect on subcellular Ca(2+) distribution; besides suppressing the Ca(2+) elevation in cytosol, BAPTA/AM can also enter into the ER to reduce the free Ca(2+) level there. The depletion of Ca(2+) in ER is believed to stimulate apoptosis and thus would counterbalance the protection effect of BAPTA/AM in suppressing the cytosolic Ca(2+) elevation.     

Characterization of calcium release-activated apoptosis of LNCaP prostate cancer cells

Apoptosis inhibition rather than enhanced cellular proliferation occurs in prostate cancer (CaP), the most commonly diagnosed malignancy in American men. Therefore, it is important to characterize residual apoptotic pathways in CaP cells. When intracellular Ca(2+) stores are released and plasma membrane "store-operated" Ca(2+) entry channels subsequently open, cytosolic [Ca(2+)] increases and is thought to induce apoptosis. However, cells incapable of releasing Ca(2+) stores are resistant to apoptotic stimuli, indicating that Ca(2+) store release is also important. We investigated whether release of intracellular Ca(2+) stores is sufficient to induce apoptosis of the CaP cell line LNCaP. We developed a method to release stored Ca(2+) without elevating cytosolic [Ca(2+)]; this stimulus induced LNCaP cell apoptosis. We compared the apoptotic pathways activated by intracellular Ca(2+) store release with the dual insults of store release and cytosolic [Ca(2+)] elevation. Earlier processing of caspases-3 and -7 occurred when intracellular store release was the sole Ca(2+) perturbation. Apoptosis was attenuated in both conditions in stable transfected cells expressing antiapoptotic proteins Bclx(L) and catalytically inactive caspase-9, and in both scenarios inactive caspase-9 became complexed with caspase-7. Thus, intracellular Ca(2+) store release initiates an apoptotic pathway similar to that elicited by the dual stimuli of cytosolic [Ca(2+)] elevation and intracellular store release.     

Uncoupling protein 3 (UCP3) modulates the activity of Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) by decreasing mitochondrial ATP production

The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca(2+) entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca(2+) uniporter. To clarify the role of UCPs in mitochondrial Ca(2+) handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca(2+) and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca(2+) uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca(2+) elevations evoked by histamine. The reduced Ca(2+) elevations were due to inhibition of store-operated Ca(2+) entry and reduced depletion of endoplasmic reticulum (ER) Ca(2+) stores. UCP3 depletion accelerated the ER Ca(2+) refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca(2+) (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca(2+) responses. Our results indicate that UCP3 is not a mitochondrial Ca(2+) uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca(2+) thus reflect metabolic alterations that impact on cellular Ca(2+) homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca(2+) uptake and release sites.     

Apoaequorin monitors degradation of endoplasmic reticulum (ER) proteins initiated by loss of ER Ca(2+)

Apoaequorin was targeted to the cytosol, nucleus, and endoplasmic reticulum of HeLa cells in order to determine the effect of Ca(2+) release from the ER on protein degradation. In resting cells apoaequorin had a rapid half-life (ca. 20-30 min) in the cytosol or nucleus, but was relatively stable for up to 24 h in the ER (t(1/2) > 24 h). However, release of Ca(2+) from the ER, initiated by the addition of inhibitors of the ER Ca(2+)/Mg(2+) ATPase such as 2 microM thapsigargin or 1 microM ionomycin, initiated rapid loss of apoaequorin in the ER, but had no detectable effect on apoaequorin turnover in the cytosol nor the nucleus. This loss of apoprotein was not the result of secretion into the external fluid, and could not be inhibited by inhibitors of protein degradation by proteosomes. Proteolysis of apoaequorin in cell extracts (t(1/2) < 20 min) was completely inhibited in the presence of 1 mM Ca(2+), and this effect was independent of the ER retention signal KDEL at the C-terminus. Proteolysis was unaffected by the presence of selected serine protease inhibitors, or 10 microM Zn(2+), a known caspase-3 inhibitor. The results show that apoaequorin can monitor proteolysis of ER proteins activated by loss of ER Ca(2+). Several Ca(2+)-binding proteins exist in the ER, acting as the Ca(2+) store and chaperones. Our results have important implications both for the role of ER Ca(2+) in cell activation and stress and when using aequorin for monitoring free ER Ca(2+) over long time periods.     

Synergistic movements of Ca(2+) and Bax in cells undergoing apoptosis.

Apoptosis is a physiological counterbalance to mitosis and plays important roles in tissue development and homeostasis. Cytosolic Ca(2+) has been implicated as a proapoptotic second messenger involved in both triggering apoptosis and regulating cell death-specific enzymes. A critical early event in apoptosis is associated with the redistribution of Bax from cytosol to mitochondria and endoplasmic reticulum (ER) membranes; however, the molecular mechanism of Bax translocation and its relationship to Ca(2+) is largely unknown. Here we provide functional evidence for a synergistic interaction between the movements of intracellular Ca(2+) and cytosolic Bax in the induction of apoptosis. Overexpression of Bax in cultured cells causes a loss of ER Ca(2+) content. Depletion of ER Ca(2+) through activation of the ryanodine receptor enhances the participation of Bax into the mitochondrial membrane. Neither Bax translocation nor Bax-induced apoptosis is affected by buffering of cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, suggesting that depletion of ER Ca(2+) rather than elevation of cytosolic Ca(2+) is the signal for cell apoptosis. This dynamic interplay of Ca(2+) and Bax movements may serve as an amplifying factor in the initial signaling steps of apoptosis.     

Degranulation of rough endoplasmic reticulum in M phase and adenosine-triphosphate-treated interphase cells is reversible.

In the preferential harvesting of rounded mitotic (M phase) cells of human Chang liver monolayer cultures by mechanical agitation in Ca(2+)-free phosphate-buffered saline, degranulation of endoplasmic reticulum (ER) was observed. Mitotic cells are known to have a series of Ca2+ transients and, without being subjected to Ca(2+)-free washings, did not have degranulated ER. Quiescent cells incubated with 0.7 mM adenosine 5'-triphosphate (ATP) in Ca(2+)-free HEPES-buffered saline produced very similar ER degranulations. Confocal argon laser imaging of fluo-3-loaded cells showed a Ca2+ transient peaking at 2 min after ATP treatment. In the absence of extracellular Ca2+, transients of Ca2+ elevation in the cytosol would exit the cell in a down-gradient, draining the ER Ca2+ stores. Substituting ATP with 1 microM brominated A23187 calcium ionophore in the incubation that contained 1-100 mM CaCl2, respectively, did not produce ER degranulation, thereby excluding raised cytosolic Ca2+ per se as the cause of ER degranulation. In fact, incubation with 0.7 mM ATP in the presence of 1-5 mM CaCl2 failed to produce ER degranulation. ER degranulated cells, from treatment with ATP without extracellular Ca2+ as well as from Ca(2+)-free washings at M phase, could be rescued by subsequent incubation in growth medium that contains Ca2+ whereupon the rounded cells re-flatten (a round-to-flat change) and have well-defined rough ER. It therefore seems possible for Ca2+ depletion, or at least a reduction, to be causally related to ER degranulation. If that were the case, ER granularity would appear to be a facultative rather than a constitutive state.     

Endoplasmic reticulum stress-induced apoptosis in Leishmania through Ca2+-dependent and caspase-independent mechanism

Numerous reports have shown that mitochondrial dysfunctions play a major role in apoptosis of Leishmania parasites, but the endoplasmic reticulum (ER) stress-induced apoptosis in Leishmania remains largely unknown. In this study, we investigate ER stress-induced apoptotic pathways in Leishmania major using tunicamycin as an ER stress inducer. ER stress activates the expression of ER-localized chaperone protein BIP/GRP78 (binding protein/identical to the 78-kDa glucose-regulated protein) with concomitant generation of intracellular reactive oxygen species. Upon exposure to ER stress, the elevation of cytosolic Ca(2+) level is observed due to release of Ca(2+) from internal stores. Increase in cytosolic Ca(2+) causes mitochondrial membrane potential depolarization and ATP loss as ablation of Ca(2+) by blocking voltage-gated cation channels with verapamil preserves mitochondrial membrane potential and cellular ATP content. Furthermore, ER stress-induced reactive oxygen species (ROS)-dependent release of cytochrome c and endonuclease G from mitochondria to cytosol and subsequent translocation of endonuclease G to nucleus are observed. Inhibition of caspase-like proteases with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or metacaspase inhibitor antipain does not prevent nuclear DNA fragmentation and phosphatidylserine exposure. Conversely, significant protection in tunicamycin-induced DNA degradation and phosphatidylserine exposure was achieved by either pretreatment of antioxidants (N-acetyl-L-cysteine, GSH, and L-cysteine), chemical chaperone (4-phenylbutyric acid), or addition of Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester). Taken together, these data strongly demonstrate that ER stress-induced apoptosis in L. major is dependent on ROS and Ca(2+)-induced mitochondrial toxicity but independent of caspase-like proteases.     

Cytosolic [Ca2+] transients in dictyostelium discoideum depend on the filling state of internal stores and on an active sarco/endoplasmic reticulum calcium ATPase (SERCA) Ca2+ pump

Stimulation of Dictyostelium discoideum with cAMP evokes a change of the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). We analyzed the role of the filling state of Ca(2+) stores for the [Ca(2+)] transient. Parameters tested were the height of the [Ca(2+)](i) elevation and the percentage of responding amoebae. After loading stores with Ca(2+), cAMP induced a [Ca(2+)](i) transient in many cells. Without prior loading, cAMP evoked a [Ca(2+)](i) change in a few cells only. This indicates that the [Ca(2+)](i) elevation is not mediated exclusively by Ca(2+) influx but also by Ca(2+) release from stores. Reducing the Ca(2+) content of the stores by EGTA preincubation led to a cAMP-activated [Ca(2+)](i) increase even at low extracellular [Ca(2+)]. Moreover, the addition of Ca(2+) itself elicited a capacitative [Ca(2+)](i) elevation. This effect was not observed when stores were emptied by the standard technique of inhibiting internal Ca(2+) pumps with 2,5-di-(t-butyl)-1,4-hydroquinone. Therefore, in Dictyostelium, an active internal Ca(2+)-ATPase is absolutely required to allow for Ca(2+) entry. No influence of the filling state of stores on Ca(2+) influx characteristics was found by the Mn(2+)-quenching technique, which monitors the rate of Ca(2+) entry. Both basal and cAMP-activated Mn(2+) influx rates were similar in control cells and cells with empty stores. By contrast, determination of extracellular free Ca(2+) concentration ([Ca(2+)](e)) changes, which represent the sum of Ca(2+) influx and efflux, revealed a higher rate of [Ca(2+)](e) decrease in EGTA-treated than in control amoebae. We conclude that emptying of Ca(2+) stores does not change the rate of Ca(2+) entry but results in inhibition of the plasma membrane Ca(2+)-ATPase. Furthermore, the activities of the Ca(2+) transport ATPases of the stores are of crucial importance for the regulation of [Ca(2+)](i) changes.     

BiP-mediated closing of the Sec61 channel limits Ca(2+) leakage from the ER

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic protein-conducting channel, the Sec61 complex. Previous work has characterized the Sec61 channel as a potential ER Ca(2+) leak channel and identified calmodulin as limiting Ca(2+) leakage in a Ca(2+)-dependent manner by binding to an IQ motif in the cytosolic aminoterminus of Sec61α. Here, we manipulated the concentration of the ER lumenal chaperone BiP in cells in different ways and used live cell Ca(2+) imaging to monitor the effects of reduced levels of BiP on ER Ca(2+) leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca(2+) leakage via the Sec61 complex. When we replaced wild-type Sec61α with mutant Sec61αY344H in the same model cell, however, Ca(2+) leakage from the ER increased and was no longer affected by manipulation of the BiP concentration. Thus, BiP limits ER Ca(2+) leakage through the Sec61 complex by binding to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344.     

HAP的凋亡诱发功能对其内质网定位的依赖性

hap是从人胚肺细胞系 (WI 38)cDNA文库中克隆的ASY相互作用蛋白基因 ,它为已知基因RTN3的同源体 .hap过表达能引起多种细胞凋亡 .激光共聚焦显微观察显示N端带有EGFP标签的HAP蛋白定位于内质网上 ,C端带有EGFP标签的HAP蛋白则游离分布于整个细胞中 .用Hoechst33342染色观察、DNAladder分析以及流式细胞仪检测均表明 ,定位在内质网上的HAP蛋白高表达的HeLa细胞呈现明显的凋亡特征 ,而游离的HAP高表达的HeLa细胞则没有明显的凋亡现象 .结果表明 ,HAP蛋白的亚细胞定位决定其是否具有诱导细胞凋亡的功能 .推测HAP蛋白可能是内质网上的钙通道的重要组分     

Neurotransmitter release from bovine adrenal chromaffin cells is modulated by capacitative Ca(2+)entry driven by depleted internal Ca(2+)stores

Two potential mechanisms by which the intracellular Ca(2 stores might modulate catecholamine release from bovine adrenal chromaffin cells were investigated: (i) that the cytosolic Ca(2+)transient caused by Ca(2+)release from the intracellular stores recruits additional chromaffin granules to a readily releasable pool that results in augmented catecholamine release when this is subsequently evoked, and (ii) that the Ca(2+)influx that follows depletion of intracellular stores (i.e. store-operated Ca(2+)entry) triggers release per se thereby augmenting evoked catecholamine release. When histamine or caffeine were applied in Ca(2+)-free perfusion media, a transient elevation of intracellular free Ca(2+)occurred owing to mobilization of Ca(2+)from the stores. When Ca(2+)was later readmitted to the perfusing fluid there followed a prompt and maintained rise in intracellular Ca(2+)concentrations of magnitude related to the degree of store mobilization. In parallel experiments, increased catecholamine secretion was measured under the conditions when Ca(2+)influx following store-mobilization occurred. Furthermore, the size of the catecholamine release increment correlated with the degree of Ca(2+)influx. Store-operated Ca(2+)entry evoked by mobilization with histamine and/or caffeine did not augment nicotine-evoked secretion per se; that is, it augmented evoked catecholamine release only to the extent that it increased basal catecholamine release. The nicotine-evoked catecholamine release was sensitive to cytosolic BAPTA, which, at the concentration used (50 microM BAPTA-AM), reduced release by approximately 25%. However, the increment in basal catecholamine release which followed Ca(2+)influx triggered by Ca(2+)store mobilization was not reduced by intracellular BAPTA. This finding is inconsistent with the hypothesis that the elevated cytosolic Ca(2+)from store mobilization recruits additional vesicles of catecholamine to the sub-plasmalemmal release sites to augment subsequently evoked secretion. This position is supported by the observation that histamine (10 microM) in Ca(2+)-free medium caused a pronounced elevation of cytosolic free Ca(2+), but this caused no greater catecholamine release when Ca(2+)was re-introduced than did prior exposure to Ca(2+)-free medium alone, which caused no elevation of cytosolic free Ca(2+). It is concluded that intracellular Ca(2+)stores can modulate secretion of catecholamines from bovine chromaffin cells by permitting Ca(2+)influx through a store-operated entry pathway. The results do not support the notion that the Ca(2+)released from intracellular stores plays a significant role in the recruitment of vesicles into the ready-release pool under the experimental conditions reported here.     

Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor

Recent studies have suggested a central role for Ca(2+) in the signaling pathway of apoptosis and certain anti-apoptotic effects of Bcl-2 family of proteins have been attributed to changes in intracellular Ca(2+) homeostasis. Here we report that depletion of Ca(2+) from endoplasmic reticulum (ER) leads to apoptosis in Chinese hamster ovary cells. Stable expression of ryanodine receptor (RyR) in these cells enables rapid and reversible changes of both cytosolic Ca(2+) and ER Ca(2+) content via activation of the RyR/Ca(2+) release channel by caffeine and ryanodine. Sustained depletion of the ER Ca(2+) store leads to apoptosis in Chinese hamster ovary cells, whereas co-expression of Bcl-xL and RyR in these cells prevents apoptotic cell death but not necrotic cell death. The anti-apoptotic effect of Bcl-xL does not correlate with changes in either the Ca(2+) release process from the ER or the capacitative Ca(2+) entry through the plasma membrane. The data suggest that Bcl-xL likely prevents apoptosis of cells at a stage downstream of ER Ca(2+) release and capacitative Ca(2+) entry.     

Intracellular Ca(2+) release triggers translocation of membrane marker FM1-43 from the extracellular leaflet of plasma membrane into endoplasmic reticulum in T lymphocytes

Stimulation of T cell receptor in lymphocytes enhances Ca(2+) signaling and accelerates membrane trafficking. The relationships between these processes are not well understood. We employed membrane-impermeable lipid marker FM1-43 to explore membrane trafficking upon mobilization of intracellular Ca(2+) in Jurkat T cells. We established that liberation of intracellular Ca(2+) with T cell receptor agonist phytohemagglutinin P or with Ca(2+)-mobilizing agents ionomycin or thapsigargin induced accumulation of FM1-43 within the lumen of the endoplasmic reticulum (ER), nuclear envelope (NE), and Golgi. FM1-43 loading into ER-NE and Golgi was not mediated via the cytosol because other organelles such as mitochondria and multivesicular bodies located in close proximity to the FM1-43-containing ER were free of dye. Intralumenal FM1-43 accumulation was observed even when Ca(2+) signaling in the cytosol was abolished by the removal of extracellular Ca(2+). Our findings strongly suggest that release of intracellular Ca(2+) may create continuity between the extracellular leaflet of the plasma membrane and the lumenal membrane leaflet of the ER by a mechanism that does not require global cytosolic Ca(2+) elevation.     

Bcl-2 and Bax exert opposing effects on Ca2+ signaling, which do not depend on their putative pore-forming region

Recent work has shown that Bcl-2 and other anti-apoptotic proteins partially deplete the endoplasmic reticulum (ER) Ca(2+) store and that this alteration of Ca(2+) signaling reduces cellular sensitivity to apoptotic stimuli. We expressed in HeLa cells Bcl-2, Bax, and Bcl-2/Bax chimeras in which the putative pore-forming domains of the two proteins (alpha 5-alpha 6) were mutually swapped, comparing the effects on Ca(2+) signaling of the two proteins and relating them to defined molecular domains. The results showed that only Bcl-2 reduces ER Ca(2+) levels and that this effect does not depend on the alpha 5-alpha 6 helices of this oncoprotein. Soon after its expression, Bax increased ER Ca(2+) loading, with ensuing potentiation of mitochondrial Ca(2+) responses. Then the cells progressed into an apoptotic phenotype (which included drastic reductions of cytosolic and mitochondrial Ca(2+) responses and alterations of organelle morphology). These results provide a coherent scenario that high-lights a primary role of Ca(2+) signals in deciphering apoptotic stimuli.     

Alteration of ceramide synthase 6/C16-ceramide induces activating transcription factor 6-mediated endoplasmic reticulum (ER) stress and apoptosis via perturbation of cellular Ca2+ and ER/Golgi membrane network

Mechanisms that regulate endoplasmic reticulum (ER) stress-induced apoptosis in cancer cells remain enigmatic. Recent data suggest that ceramide synthase1-6 (CerS1-6)-generated ceramides, containing different fatty acid chain lengths, might exhibit distinct and opposing functions, such as apoptosis versus survival in a context-dependent manner. Here, we investigated the mechanisms involved in the activation of one of the major ER stress response proteins, ATF-6, and subsequent apoptosis by alterations of CerS6/C(16)-ceramide. Induction of wild type (WT), but not the catalytically inactive mutant CerS6, increased tumor growth in SCID mice, whereas siRNA-mediated knockdown of CerS6 induced ATF-6 activation and apoptosis in multiple human cancer cells. Down-regulation of CerS6/C(16)-ceramide, and not its further metabolism to glucosylceramide or sphingomyelin, activated ATF-6 upon treatment with ER stress inducers tunicamycin or SAHA (suberoylanilide hydroxamic acid). Induction of WT-CerS6 expression, but not its mutant, or ectopic expression of the dominant-negative mutant form of ATF-6 protected cells from apoptosis in response to CerS6 knockdown and tunicamycin or SAHA treatment. Mechanistically, ATF-6 activation was regulated by a concerted two-step process involving the release of Ca(2+) from the ER stores ([Ca(2+)](ER)), which resulted in the fragmentation of Golgi membranes in response to CerS6/C(16)-ceramide alteration. This resulted in the accumulation of pro-ATF-6 in the disrupted ER/Golgi membrane network, where pro-ATF6 is activated. Accordingly, ectopic expression of a Ca(2+) chelator calbindin prevented the Golgi fragmentation, ATF-6 activation, and apoptosis in response to CerS6/C(16)-ceramide down-regulation. Overall, these data suggest a novel mechanism of how CerS6/C(16)-ceramide alteration activates ATF6 and induces ER-stress-mediated apoptosis in squamous cell carcinomas.     

Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis

Phosphorylation of BCL-2 within an unstructured loop inhibits its antiapoptotic effect. We found that phosphorylated BCL-2 predominantly localized to the endoplasmic reticulum (ER) and tested whether phosphorylation would control its activity at this organelle, where Ca(2+) dynamics serve as a critical control point for apoptosis. Phosphorylation greatly inhibits the ability of BCL-2 to lower [Ca(2+)](er) and protect against Ca(2+)-dependent death stimuli. Cells expressing nonphosphorylatable BCL-2(AAA) exhibited increased leak of Ca(2+) from the ER and further diminished steady-state [Ca(2+)](er) stores when compared to cells expressing BCL-2(wt). Consequently, when BCL-2 is phosphorylated, Ca(2+) discharge from the ER is increased, with a secondary increase in mitochondrial Ca(2+) uptake. We also demonstrate that phosphorylation of BCL-2 inhibits its binding to proapoptotic family members. This inhibitory mechanism manifested at the ER, where phosphorylated BCL-2 was unable to bind proapoptotic members. [Ca(2+)](er) proved coordinate with the capacity of BCL-2 to bind proapoptotic BH3-only members, further integrating the apoptotic pathway and Ca(2+) modulation. Unexpectedly, the regulation of ER Ca(2+) dynamics is a principal avenue whereby BCL-2 phosphorylation alters susceptibility to apoptosis.