By using calcium ions as an intracellular messenger, cells walk a tight rope between life and death. Because critical cellular functions depend on the precise delivery of Ca²⁺ at the right time and place, calcium ions must navigate at all times between intracellular calcium stores and target proteins located in the cytosol, the mitochondria, or the nucleus. Due to the toxicity of high Ca²⁺ concentrations, even slight disruption of the elaborate calcium signaling machinery can have devastating consequences on cell functions: too much or too little calcium at the wrong time and place might lead to rapid cell death by necrosis, or to the induction of the cell death program of apoptosis. ER chaperones, and most notably calreticulin, play a key role in the making and decoding of both normal and pathological calcium signals. Calreticulin is the main Ca²⁺-binding protein residing in the ER, and as such contributes most of the ER Ca²⁺ buffering capacity. Calreticulin also acts as a chaperone for several ER Ca²⁺ transport proteins, and thus indirectly modulates Ca²⁺ fluxes across the ER membrane. Accordingly, over- or underexpression of calreticulin leads to rapid and severe alterations in ER Ca²⁺ homeostasis. Calreticulin expression levels are controlled by the ER Ca²⁺ levels, thus enabling cells to mount an appropriate response during long-term perturbations in ER Ca²⁺ storage. However, calreticulin levels are also increased by a variety of cellular stress conditions, and this upregulation might contribute to the Ca²⁺ signaling defects leading to apoptosis. In this chapter, we will review the role of calreticulin and of other ER chaperones in the control of Ca²⁺-mediated apoptosis.

Introduction

Apoptosis, a process first described in 1972 by Kerr et al¹ has changed radically our perception of cell death. In this elaborated form of cellular suicide, cells sacrifice themselves for the well being of the whole organism by dying in a quiet manner, without undergoing cell lysis. In contrast, during necrosis cell membranes are disrupted and the release of the intracellular contents contributes to the generation of inflammation and tissue damage. Apoptosis is in fact a highly regulated process of cell deletion involved during development, during normal cell turnover and during cell elimination following injury. This programmed cell death is defined morphologically by a typical sequence of events: cytoplasmic shrinkage, loss of intercellular contacts, organelle compaction and chromatine condensation and, finally, cytoplasmic blebbing with generation of apoptotic bodies that are phagocytosed by neighboring cells. A family of cysteine proteases known as caspases are tightly involved in this process. These enzymes appear to be essential components in both the initial signaling events and the downstream proteolytic cleavage that results in the apoptotic phenotype. Ca²⁺ seems to modulate the role of some proteases like caspase 3² and is also implicated in the activation of other players of apoptosis like calpain and Ca²⁺ dependent endonucleases. The first evidence that Ca²⁺ was involved in triggering apoptosis came from the evidence that glucocorticoid-stimulated apoptosis was associated with enhanced Ca²⁺ influx.³ Afterwards, Ca²⁺ ionophores were shown to mimick the cytolytic effects of glucocorticoids on lymphocytes^4,5 and overexpression of the Ca²⁺-binding protein calbindin in thymoma cells was able to prevent ionophore induced apoptosis.⁶ Since these initial observations, numerous studies have shown that conditions that preclude cytosolic Ca²⁺ elevations, such as removing Ca²⁺ from the external medium, buffering the intracellular [Ca²⁺]_cyt, or inhibiting plasma membrane Ca²⁺ channels, protect cells from apoptosis.⁷¹⁰ Conversely, the SERCA inhibitor thapsigargin, which generates long-lasting Ca²⁺ elevations by depleting Ca²⁺ stores and activating capacitative Ca²⁺ entry, triggers all the morphological and biochemical events of apoptosis in numerous cell types.¹¹¹³ All these data illustrate the pivotal role of Ca²⁺ in the apoptotic process. However the precise mechanism leading to the apoptotic response are not yet understood. While excessive [Ca²⁺]_cyt elevations are pro-apoptotic, moderate [Ca²⁺]cyt elevations appear to be anti-apoptotic.¹⁴ The beneficial effects of Ca²⁺ signals on cell survival might involve the activation of Ca²⁺/calmodulin-dependent kinase kinase, protein kinase B and phosphorylation of Bad.¹⁵

Role of ER Calcium in Apoptosis

Ca²⁺ is a ubiquitous intracellular messenger involved in many cellular processes ranging from muscle contraction to hormone secretion, synaptic transmission, and gene transcription. The bewildering array of functions controlled by this simple ion stems from the complexity and versatility of intracellular calcium signals, which can be encoded in time, space, frequency, and amplitude. This plasticity allows cells to generate subtle and diverse patterns of Ca²⁺ signals, both on a local (sparks and puffs) or global scale (transients and waves). To generate such complex Ca²⁺ signals, cells rely on the rapid release of the Ca²⁺ stored within the endo/sarcoplasmic reticulum (ER/SR) as well as on the controlled influx of Ca²⁺ from the extracellular medium. Opening of Ca²⁺ release or Ca²⁺ influx channels transiently increases the averaged cytosolic Ca²⁺ concentration, from ˜100 nM to ˜1 μM, but much higher values can be achieved close to the mouth of the channels. Because of the toxicity of such high Ca²⁺ concentrations, high Ca²⁺ levels are reached only transiently and Ca²⁺ is rapidly removed from the cytosol by Ca²⁺ pumps and exchangers. Both the plasma membrane Ca²⁺-ATPase (PMCA) and the Na/Ca²⁺ exchanger drive Ca²⁺ out to the external medium, whereas the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) recycle Ca²⁺ to the ER/SR in order to replenish the stores. This ensures that a high Ca²⁺ concentration is maintained at all times within the lumen of the ER, a condition that is crucial not only for the generation of further Ca²⁺ signals but also for the proper function of ER-resident proteins. The release of Ca²⁺ from the ER stores occurs by the opening of Ca²⁺ release channels belonging to two families: the inositol 1, 4, 5 trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Activation of these intracellular channels generate local Ca²⁺ signals (puffs and sparks, respectively) which, by a positive feed-back mechanism of Ca²⁺-induced Ca²⁺-release, further activate the Ca²⁺-release channels to produce regenerative Ca²⁺ oscillations and waves (for review see ref. 16).

Recently, mitochondria have also emerged as bona-fide Ca²⁺ signaling organelles, able to encode and decode Ca²⁺ signals. Mitochondria are often located close to the ER, and therefore exposed to the Ca²⁺ released by the IP₃R¹⁷ and the RyR.¹⁸ The high Ca²⁺ levels achieved at these contact sites favors Ca²⁺ uptake into mitochondria, via a Ca²⁺ uniporter driven by the very negative mitochondrial membrane potential (-150 mV). Indeed, Ca²⁺-clamp experiments in permeabilized hepatocytes have revealed that Ca²⁺ influx into mitochondria occurs at cytosolic Ca²⁺ concentrations exceeding 300 μM. Mitochondrial Ca²⁺ transients, nicknamed Ca²⁺ “marks”, can be observed during elementary Ca²⁺ release events,¹⁹ indicating that such high Ca²⁺ concentration are indeed achieved around individual mitochondria during physiological Ca²⁺ signals. The Ca²⁺ taken up by mitochondria is subsequently released to the cytosol where it can diffuse locally or return back to the ER, allowing mitochondria to shape cytosolic Ca²⁺ signals and to prevent the depletion of the ER Ca²⁺ stores.^20,21 Thus, mitochondria located close to Ca²⁺ release or influx channels handle a large part of the Ca²⁺ used for signaling. Mitochondria also play a critical role in apoptosis, by providing the energy required for the ordered execution of cells and by delivering apoptogenic proteins. One crucial step in the apoptotic process is the irreversible opening of the mitochondrial permeability transition pore (in its high conductance state), and the collapse of the mitochondria membrane potential, Δψ_m. This phenomenon, controlled by members of the Bcl-2 family,²² is associated with an increased permeability of the outer mitochondrial membrane and a swelling of the mitochondria inner membrane. As a result, soluble proteins are released from the intermembrane space such as the proapoptotic cytochrome c,^23,24 procaspase 2, 3 and 9,^25,26 the apoptosis inducing factor (AIF)²⁷ and Smac/Diablo,^28,29 initiating the caspase cascade leading to the cleavage of a large quantity of proteins and eventually to the ordered disassembly of the cell.

Because of their tight coupling to ER Ca²⁺ stores, mitochondria are highly susceptible to abnormalities in Ca²⁺ signaling. Recent evidences suggest that the amount of Ca²⁺ going through mitochondria is crucial in triggering Ca²⁺-dependent apoptotic responses. The amount of Ca²⁺ sensed by mitochondria depends on several factors, notably:

1. the activity of Ca²⁺-release channels in the ER membrane (IP₃R or RyR), which controls the flux across the ER membrane,
2. the ER Ca²⁺ load, which determines the total amount of Ca²⁺ that can be released from the ER,
3. the free ER Ca²⁺ concentration, [Ca²⁺]_ER, which determine the driving force for Ca²⁺ release, and
4. the proximity between mitochondria and the ER, which determines the magnitude of the Ca²⁺ microdomain sensed by mitochondria.^17,30

Increase in any of these parameters will increase the Ca²⁺ flowing through mitochondria, and induce a switch from the cell survival to the cell death program. Increased expression of thetype 3 IP₃R has been reported in lymphocytes undergoing cell death,³¹ and was also observed during developmental apoptosis in several post natal tissues.³² The apoptosis associated with IP₃-dependent IP3-dependent Ca²⁺ signals in lymphocytes appears to be mediated by calcineurin, a Ca²⁺-regulated phosphatase that can dephosphorylate and activate the pro-apoptotic factor Bad.^33,34 Numerous procedures that reduce the ER Ca²⁺ load, such as lowering extracellular Ca²⁺, depleting the stores with low doses of the SERCA inhibitor tBuBHQ, or overexpressing the plasma membrane Ca²⁺ pump, protect HeLa cells from ceramide-induced apoptosis.³⁵ Similarly, deletion of the calreticulin gene, by removing the major Ca²⁺-bufferring protein from the ER lumen, reduces the total amount of Ca²⁺ stored in the ER and increases cell survival.³⁶ Importantly, the free ER Ca²⁺ concentration, [Ca²⁺]_ER, was not altered in the calreticulin knock-out cells, but the ability of these cells to generate Ca²⁺ transients upon stimulation with agonists was markedly reduced. Conversely, overexpression of calreticulin or of SERCA ATPases increased both the total ER Ca²⁺ load as well as [Ca²⁺]_ER, and enhanced the sensitivity of cells to ceramide-induced apoptosis.^35,37

In the presence of ceramide, a classical apoptotic stimulus, even physiological IP₃-dependent Ca²⁺ signals are able to trigger the apoptotic process, probably by the opening of a sensitized state of the mPTP. The switch from the life to the death program might involve coincident detection of pro-apoptotic stimuli and calcium signals,³⁸ reviewed in.³⁹ As mentioned above, the opening of the mPTP promotes the release of apoptotic factors, most notably cytochrome c, which forms a complex with pro-caspase 9, Apaf-1 and dATP.^40,41 This results in the activation of caspase 9, which dissociates from the complex and activates other executioner caspases such as caspase 3. By integrating Ca²⁺ and apoptotic stimuli, mitochondria thus function as a central checkpoint in determining cell survival or cell death. The release of cytochrome c from mitochondria is prevented by the anti-apoptotic factor Bcl-2, one of the most widely studied proto-oncogene, whose mechanism of action is still debated. Several Bcl-2 family members have been identified: anti-apoptotic factors such as Bcl-2, Bcl-X_L and Mcl-1 pro-apoptotic factors such as Bax, Bad and Bid. Bcl-2 can prevent the opening of the mPTP and protect cell from apoptosis, whereas Bax has the opposite effect. Most Bcl-2 family members are anchored by a hydrophobic stretch of amino-acids in the outer mitochondrial membrane,⁴² but Bcl-2 has also been detected in the membrane of the ER, suggesting that this organelle is also implicated in the apoptosis program. Recently, the expression of recombinant Bcl-2 was shown to reduce the ER Ca²⁺ concentration by increasing the passive leak across the ER membrane.^43,44 This effect of Bcl-2 is consistent with the three-dimensional structure of Bcl-X_L proteins, which is reminiscent of pore forming bacterial toxins,^45,46 and with the observation that Bcl-2 can function as an ion channel in artificial lipid bilayers.^47,48 In this model, Bcl-2 insertion in the ER membrane increases the passive ER Ca²⁺ permeability, thus reducing both the total amount of stored Ca²⁺ and the free ER Ca²⁺ concentration, [Ca²⁺]_ER. The ensuing Ca²⁺ depletion of the ER is an integral part of the mechanism of action of Bcl-2.

All these results are consistent with the hypothesis that a moderate reduction in ER Ca²⁺ protects cells from apoptotic stimuli, by decreasing the amount of toxic Ca²⁺ ions sensed by the cytosol and mitochondria. In contrast, an ER Ca²⁺ overload sensitizes cells to apoptosis by the opposite mechanism. This scheme is in apparent contradiction with the well-known pro-apoptotic effects of agents such as the SERCA inhibitor thapsigargin or the Ca²⁺ ionophore A23187, which induce a massive Ca²⁺ store depletion. These conflicting observations can be reconciled by postulating that a severe ER Ca²⁺ depletion, in itself, is sensed as a stress signal by the cell and causes apoptosis.⁴⁹ Alternatively, the massive and long-lasting increase in cytosolic Ca²⁺ caused by these agents might bypass the protective effect of the ER Ca²⁺ depletion and trigger apoptosis. In the former scenario, transduction of the ER -induced apoptosis signal might be mediated by caspase 12, an ER-associated caspase. This particular ER caspase is activated by a variety of ER stress conditions, including not only the disruption of ER Ca²⁺ homeostasis but also the accumulation of excess protein in the ER.⁵⁰

Role of ER Chaperones in Apoptosis

Besides its role as the most prominent intracellular Ca²⁺ store, the ER compartment plays a crucial role in protein maturation, folding, transport, and storage. These two roles are closely intricated, and alterations in ER functions that perturb either the protein folding process or change the Ca²⁺ level in the ER lead to a situation called ER stress response. Depending on the severity of such ER stress, this process can path either to an adaptive response or to apoptosis. In general, the ER stress is due to an accumulation of misfolded proteins in the lumen of the ER that, in turn, yields to a phenomenon referred as “unfolded protein response” (UPR). The UPR is characterized by a general decrease of protein synthesis whereas the expression of specific sets of proteins, mainly the ER resident chaperones, is increased. Both events constitute responsive processes that will eventually result in the normalization of the folding process machinery, or, if unsuccessful, trigger apoptosis (for reviewed see ref. 51,52).

Chaperones are found in every cell compartment and aid to protein maturation in two ways: chaperones bind to unfolded proteins in order to prevent their further aggregation and degradation and, in addition, actively promote protein folding.⁵¹ Many ER-resident proteins function as molecular chaperones that belong to the glucose-regulated proteins (GRP) family. The major ER chaperones are GRP 78/BiP GRP94, the protein disulfide isomerase (PDI) and its homologue ERp57, as well as the two lectin-like proteins calreticulin and calnexin that bind newly synthesized glycosylated proteins. Several of the ER chaperones, including calreticulin, calnexin, GRP78/BiP, and GRP94 are major ER Ca²⁺ binding proteins, able to bind large quantities of Ca²⁺ ions. The GRPs are constitutively expressed but their transcription can be enhanced by various stimuli that disrupt ER homeostasis, such as an exhaustive ER Ca²⁺ depletion by Ca²⁺ ionophores or SERCA inhibitors, inhibition of N-glycosylation by tunacamycin, and the prevention of ER-Golgi protein trafficking by brefeldin A. Malign/prolonged Ca²⁺ depletion promotes the accumulation of misfolded proteins, as the function of several chaperones is controlled by the concentration of luminal Ca²⁺. It was shown that calreticulin and calnexin,⁵³ as well as GRP78/BiP activities are decreased by low luminal ER Ca²⁺ concentrations.⁵⁴ Furthermore, Ca²⁺ also modulate the interactions between different chaperones. For instance, the association between CRT and PDI is promoted at low luminal Ca²⁺ concentration, ⁵⁵ thereby reducing the activity of PDI. The chaperoning function of PDI is thus inhibited under conditions of ER Ca²⁺ depletion. Conversely, CRT dissociates from PDI at higher Ca²⁺ concentrations and thus the activity of PDI is promoted when Ca²⁺ stores are full. Therefore, it is tempting to speculate that alterations of the ER Ca²⁺ concentration result in the accumulation of misfolded protein, and that this condition will trigger the UPR.

How do cells sense the accumulation of misfolded proteins in the lumen of the ER and transmit this information to the cytosol and nucleus? In yeast, the signal transduction pathway involves an ER transmembrane ser/thr kinase (Ire1p) that senses unfolded proteins in the ER and stimulates a downstream transcription factor. Upon ER stress, Ire1p oligomerizes and trans-phosphorylates, which activates its endonuclease activity. The target of the endonuclease is the mRNA of HAC. Once cleaved by Ire1p, HAC becomes active as a transcription factor and specifically bind to promoter regions containing an unfolded protein response element (UPRE). In mammals, even if many similarities were noticed, the pathways appear to be more complex and are so far not fully understood. Similar to yeast, the sensor is a transmembrane protein, Ire1α or Ire1β, that is able to trans-phosphorylate and possesses an endonuclease activity. In resting conditions, the chaperone GRP78/BiP binds to Ire1a, preventing its oligomerization. In case of unfolded protein accumulation, BiP binds misfolded proteins and releases Ire1α, which then becomes activated.⁵⁶ The target of Ire1α is almost certainly a mRNA, but no definitive target has been found so far. A likely candidate is ATF6, a basic leucine zipper (bZIP) transcription factor that belongs to the same family of ATF/CREB protein as HAC in yeast. No direct interaction between Ire1α and ATF6 has been described so far, but ATF6 is regulated by ER stress.⁵⁷ ATF6 recognizes specific sequence on promoter regions called ERSE (ER stress response element). Several chaperones, including GRP78/BiP, GRP94, and calreticulin contain this region within their promoter,⁵⁷ and the induction of these chaperones by tunacamycin or ER Ca²⁺ depletion required the presence of ERSE.

This mechanism might account for the increased expression of ER chaperones during cellular stress such as oxygen deprivation, glucose starvation, and treatments that inhibit protein glycosylation (tunicamycin) or induce a ER Ca²⁺ depletion (thapsigargin)^58,59 (reviewed in ref. 60). In addition, an ER stress caused by Ca²⁺ depletion or misfolded protein accumulation induces the expression of other proteins such as SERCA2b, the ubiquitous Ca²⁺-ATPase of intracellular Ca²⁺ stores.⁶¹ SERCA2b contains in its promoter region an ERSE element, suggesting that this Ca²⁺ transporter protein responds to an ER stress in a similar manner as ER chaperones. Interestingly, Ca²⁺ depletion does not seem to be the only initiator of the response, as tunicamycin, which does not change the ER Ca²⁺ levels, is also able to increase SERCA2b expression. This suggests that a generic response is induced regardless of the type of ER stress, which leads to the upregulation of several ER proteins. However, all the chaperones are not induced by an ER stress, and some chaperones respond only to specific stress stimuli. In WEHI7.2 mouse lymphoma cells, GRP78/GRP94 are not induced by treatment with thapsigargin, but increase in response to tunicamycin. Thus, an ER stress caused by ER Ca²⁺ depletion or unglycosylated proteins accumulation does not necessarily trigger the same signaling pathway.⁶²

The increased expression of chaperones and of Ca²⁺-regulatory proteins is clearly beneficial for the cell, and has been shown to protect cells against further stress-induced apoptosis. The protection conferred by specific chaperones, however, might be restricted to a specific stress condition. While GRP78 expression is enhanced by several ER stress stimuli, it seems to specifically protect cells against ER Ca²⁺ depletion-induced apoptosis, but not against tunicamycin-induced apoptosis.⁶³ A broader protection might be conferred by the general attenuation of protein synthesis, which occurs during an ER stress together with the specific increase in ER chaperones expression. In this case, the response occurs at the translational level. Another ER transmembrane protein, PERK, was shown to phosphorylate the eukaryotic translation initiation factor 2 (eIF2α), resulting in a reduction of the translation initiation.^64,65 The reduced protein synthesis diminishes the load of putative misfolded protein during ER stress, and thus serves as a protection against cell death. Accordingly, Harding⁶⁶ showed that Perk-/- cells are more susceptible to cell death during an ER stress. These cells have much higher levels of activated caspase-12 during treatment with thaspigargin or tunicamycin, which might explain their higher susceptibility to apoptotic stimuli (see below).

Although the cellular responses described above might allow cells to overcome an ER stress, an ER stress can also, in mammalian cells, lead to programmed cell death. Caspase 12 might be an important mediator in this process, as this caspase was recently found to be involved in ERmediated apoptosis.⁵⁰ mediated apoptosis.⁵⁰ Caspase-12 is an ER membrane bound protease that is located at the cytosolic side of the ER.⁶⁷ Treatment with thapsigargin, A23187, tunamycin, or brefeldin A cleaves procaspase-12, while other apoptotic stimuli that do not involve an ER stress (i.e., staurosporine) are ineffective.⁶⁷ In line with this finding, caspase-12 knock out mice are resistant to ER stress-induced apoptosis. m-calpain, a low-affinity Ca²⁺-dependent protease distinct from the caspase family, was recently found to be involved in the activation of caspase-12. The cleavage of caspase-12 required millimolar Ca²⁺ concentrations, consistent with its activation by m-calpain. In addition, m-calpain also cleaves Bcl-xL, likely transforming this protein from an anti-apoptotic to an apoptotic agent.^68,69 Interestingly, in cells treated with etoposide (a topoisomerase II inhibitor), GRP 94 appeared to be cleaved by calpain and to generate a fragment of 80 KDa. This cleavage was selective for GRP94, as other chaperones did not get cleaved.⁷⁰ More recently, it was shown that tumor necrosis factor receptor-associated factor 2 (TRAF-2) is involved in the activation of caspase-12.⁷¹ TRAF2 acts downstream from Ire1α and was shown to stimulate components of the c-Jun N-terminal kinase (JNK) pathway. In resting conditions, TRAF2 is associated with procaspase-12, but the complex dissociates during an ER stress, thus favoring the cleavage of procaspase-12 by proteins such as m-calpain. However, the precise Ca²⁺-dependency of this process is not well established in vivo. Another pathway leading to apoptosis involves the transcription factor Gadd 153/CHOP, by an as yet unknown mechanism. Transcription of CHOP is induced by the UPR and follows a similar kinetic as GRP78/BiP (reviewed in ref.51). In cells overexpressing CHOP the level of the anti-apoptotic protein Bcl-2 is dramatically reduced, possibly accounting for the susceptibility of these cells to apoptosis.⁷² Similarly, in microglial MG5 cells, NO induced apoptosis, which is linked to an ER Ca²⁺ decrease, is also mediated by a stimulation of CHOP.⁷³

In summary, the ER is emerging as a central player in apoptosis, being able to detect, transduce, and respond to a variety of stress signals. The ER stress response might enable yeast to survive under stress conditions, and, in mammalian cells, ensures that damaged cells are safely and efficiently removed by apoptosis. The ER stress response invariably interferes with the role of the ER as a protein factory and Ca²⁺ storage organelle, and is often caused by alterations in these two central ER functions. Changes in the Ca²⁺ concentration within the ER lumen, in particular, can both induce and execute the ER stress response, and have a direct impact on cellular function. Excessively high ER Ca²⁺ levels lead to apoptosis by activating Ca²⁺-dependent targets located in the cytosol or in neighboring mitochondria. In contrast, low ER Ca²⁺ levels induce the ER stress response by promoting the accumulation of ER chaperones and of ER Ca²⁺ transport proteins. Calreticulin appears to play a critical role in sensing and correcting alterations in ER Ca²⁺ signals, and changes in calreticulin expression levels alters both the total and the free Ca²⁺ concentration within the ER lumen. The dual role of this Ca²⁺-binding chaperone allows calreticulin to integrate variations in ER Ca²⁺ homeostasis and in protein folding, thereby linking Ca²⁺ signaling to apoptosis.

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