Involvement of Ca²⁺ in the Apoptotic Process – “Friends or Foes”

Over the past two decades, the ubiquitous position of Ca²⁺ signaling in cellular apoptosis has been carefully investigated. The body of work described here highlights the complex role played by Ca²⁺ in generation, regulation and coordination apoptosis.

Introduction

The word ‘apoptosis’ is derived from a Greek word meaning ‘falling leaves’ and is used to describe a form of cell death occurring in multicellular organisms. It is defined as programmed cell death that involves altruistic suicide of individual cells in favor of the organism as a whole. Apoptosis is a tightly regulated, highly efficient and energyrequiring process which engages multiple cell signaling pathways. The apoptotic network components are genetically encoded and are usually in place in a cell ready to be activated by a death-inducing stimulus^1,3.

Apoptotic activity is desirable during organism development and morphological changes especially at the embryonic stage (for example the formation of digits), as well as during the activation of the immune system⁴. Additionally, this process is essential in the homeostasis of cell number in organs in order to determine the cell numbers of the whole organism⁵. Defects in apoptosis or apoptotic prevention can result in cancer, autoimmune diseases and spreading of viral infections, while neurodegenerative disorders, AIDS and ischemic diseases are caused or enhanced by excessive apoptosis⁵.

In contrast to the necrotic mode of cell death, apoptosis results in a loss of membrane integrity, swelling and disruption of the cell undergoing apoptosis without causing damage to the surrounding cells. Apoptotic cells can be characterized by stereotypical morphological changes which include cell shrinkage, loss of cell surface shape, chromatin condensation, DNA degradation, protein fragmentation, organelle disassembly and degradation of cells into small apoptotic bodies, which are disposed of by the immune system¹.

The apoptotic process can be driven by various stimuli from outside or inside the cell; in some cases, absence of survival factors is enough to propel a cell into apoptosis, but it can also be stimulated by DNA damage, oxidative stress, treatment with cytotoxic drugs or irradiation, interruption in cell cycle signaling and death receptor ligands (TNF and Fas ligand). Cell signaling modulators, such as the universal protein kinase inhibitor Staurosporine (#S-350)⁶ (Fig. 1) and the tyrosine kinase inhibitor Genistein (#G-300)⁷ are usually used to induce apoptosis experimentally. Apoptosis occurs through two types of pathways: the death receptor pathway (extrinsic apoptotic pathways) and the mitochondrial pathway (intrinsic apoptotic pathways). The apoptotic signal involves activation of several powerful enzymes (mainly the Bcl and Caspase families). The Bcl family has a controlling role in maintaining the apoptotic signal, while the Caspase family plays a central role in cell protein degradation^8,9.

Ca²⁺ is a fundamental second messenger in cell signaling. Stimulation of cells by Ca²⁺-linked signaling agents increases Ca²⁺ levels within the cell cytosol and the nucleus. This can modulate numerous Ca²⁺-regulated enzymes which have different subcellular localizations and create a wide range of spatial and temporal signals. Cytosolic Ca²⁺ has been implicated in the activity of Ca²⁺/calmodulin-dependent protein kinase (CaMK), protein kinase A (PKA) and C (PKC), mitogen-activated protein kinase (MAPK), and phosphatidylinositol (PI)-3 kinase^10-14. Ca²⁺ metabolism plays a crucial role during the propagation of apoptosis. However, the role of Ca²⁺ in generation of apoptosis is controversial, in view of the fact that in different systems, Ca²⁺ was observed as both a survival supporter and apoptosis inducer. For instance, high K⁺ depolarization and subsequent Ca²⁺ entry into the cytosol helps to sustain the survival of granule cells^15,18 On the other hand, compounds that chronically elevate intracellular Ca²⁺, such as Ca²⁺ ionophores, have been observed to induce apoptosis in a variety of cell lines^19-21.

Moreover, processing of the apoptotic signal, involves intracellular Ca²⁺ elevation as an obligatory factor for reactive oxygen generation and mitochondrial cytochrome c release, which are both considered as apoptotic triggers²².

The following paper reviews the divisive role of Ca²⁺ as either a positive or negative generator of the apoptotic processes and its involvement in their progression.

Ca²⁺ Signaling: Suppressor and Promoter

Ca²⁺/Calmodulin-Dependent Protein Kinase in Apoptosis

Ca²⁺ signaling is the center of multifunctional CaMK family regulation and activation. This family, which phosphorylates a large variety of substrates, has activation properties that allow it to discriminate between Ca²⁺ signals that differ in spike frequency, amplitude and duration^23-25. The CaMKI, II and IV subfamilies have been detected within the cell nucleus and suggested as mediators of nuclear Ca²⁺ signals. These kinases were implicated in control of gene transcription since they phosphorylate several transcription factors²⁶. Several reports have indicated that both kinases negatively modulate apoptosis^27,28. For instance, CaMKII up-regulation increased the resistance of glioma cells to apoptosis and KN-93, a CaMKII inhibitor, annulled this effect. In cerebellar granule cells, Ca²⁺ entry through L-type voltage gated Ca²⁺ channels supported neuronal survival in a CaMKIV-dependent manner^29,30. During apoptosis, CaMKIV was reported to be negatively controlled by specific cleavage caused by caspase-3-like activity³⁰. Moreover the serine-threonine protein kinase B, which is positively activated by the CaMKs-dependent protein kinase pathway, plays an important role in neuronal survival⁹. Similarly, in neuronal NG108 cells, expression of a peptide located in the noncatalytic N-terminal domain of CaMK-like kinase facilitated apoptosis (Figure 2)^31,32.

Nuclear Transcription Factor-κB in Apoptosis

Intracellular Ca²⁺ is a coordinating factor that positively regulates the activity of nuclear transcription factor-κB (NF-κB). This transcription factor is considered an anti apoptotic agent and plays a key role in cell survival by up-regulating expression of several apoptosis inhibitor genes and negative regulating the activity of caspase-3^33-35. Several reports illuminate the function of this intracellular pathway in prevention of apoptosis, for instance: In hippocampal pyramidal neurons, activation of glutamate receptors leads to membrane depolarization and Ca²⁺ influx followed by activation of NF-κB³⁶. In pancreatic β- cells, depolarization and L-type Ca²⁺ influx induce MEK/ERK dependent NF-κB related survival³⁷.

Protein Phosphatase – Calcineurin in Apoptosis

Cytosolic Ca²⁺ increase has a pivotal role in activation of the serine threonine Ca²⁺ -calmodulin-regulated phosphatase – calcineurin (also called protein phosphatase 2B). This phosphatase is a critical transducer of Ca²⁺ signals in most cell types particularly in the immune system and in the heart, due to its specific responsiveness to sustained low frequency Ca²⁺ signals. Calcineurin was suggested to be both a promoter and a supportive agent during apoptosis.

Sustained increase in cytosolic Ca²⁺ has been reported to induce apoptosis in some susceptible cell types through activation of calcineurin^38-40. High level forced activity of this phosphatase induced cytochrome c/caspase-3-dependent apoptosis in neurons4 and treatment with calcineurin inhibitors Cyclosporin A (#C-900) and FK-506 (#F-900) reduced susceptibility to apoptosis⁴¹. Additionally, the apoptosis-associated tyrosine kinase (AATYK) is highly phosphorylated during the normal cell cycle. Activation of calcineurin in response to high extracellular K⁺ mediated L-type Ca²⁺ influx, dephosphorylates AATYK and leads to apoptotic cell death⁴².

In hippocampal neurons, L-glutamate induced Ca²⁺ influx activates calcineurin which triggered transcription-independent apoptosis by mitochondrial targeting of BAD- a pro-apoptotic protein member of the Bcl-2 family. The Ca²⁺-induced dephosphorylation of BAD was correlated with its dissociation from the cytosol, translocation to mitochondria and enhancement of BAD Bcl-x_L heterodimerization promoting apoptosis³⁹.

Some reports suggest that the Ca²⁺-calcineurin pathway is critical in the progression of heart failure by regulating cardiomyocyte apoptosis. Moreover, induced ischemia caused more apoptosis in the hearts of transgenic mice expressing high levels of calcineurin than in the hearts of wild-type mice. Pharmacologic inhibitors of calcineurin activity block hypertrophy in vivo and in vitro and some of them (Cyclosporine A, FK-506 and Rapamycin (#R-900)) (Figure 3) were even suggested as candidates for treatment of heart disease^43-44.

On the other hand, Ca²⁺-calcineurin activation by 2-deoxyglucose and Staurosporine prevents apoptosis of cardiac myocytes⁴⁵. Calcineurin, which dephosphorylates the transcription factor NF-AT3, enables it to translocate into the nucleus, leading to prevention of apoptosis both in vitro and in vivo. Furthermore, calcineurin and protein kinase C suppressed apoptosis induced by tumor necrosis factor, through activation of NF-κB in fibroblasts and lymphoma cells⁴⁶. Finally, Ca²⁺-mobilizing compounds such as the Ca²⁺ ionophore A23187 (#A-600) or the ER Ca²⁺ ATPase inhibitor Thapsigargin (#T-650), which both trigger calcineurin activity, can either suppress or induce apoptosis in the same cells⁴⁷. The reason for this dichotomy is unknown at present, but from the data summarized here we conclude that it depended on pathways downstream from calcineurin. This idea is supported by the fact that suppression or induction of apoptosis by opposing pathways such p38 and p44/42 mitogen-activated protein kinases are located downstream to Ca²⁺-activated calcineurin⁴⁸.

C-Jun N-terminal Kinase in Apoptosis

A distinctive feature of apoptosis is the requirement for de novo RNA synthesis. A key transcription factor for apoptosis is c-Jun, an immediate-early gene⁴⁹. Ca²⁺ influx has been reported to be involved in c-jun N-terminal kinase (JNK) signaling pathway mediated IL-1β-induced apoptosis. Pharmacological blockers of L- and T-type Ca²⁺ channels, such as dihydropyridine, suppressed IL-1β-induced c-jun phosphorylation. Treatment with Ca²⁺ mobilizing compounds such as A23187 and Ionomycin (#I-700) caused an amplification of IL-1β-induced JNK activation and lead to apoptosis⁵⁰.

Regulation of Intracellular Ca²⁺ Levels and Apoptosis

Control of cytosolic Ca²⁺ levels involves coordination of diverse intracellular and extracellular Ca²⁺ currents and it plays a vital role in the initiation of the apoptotic process. Manipulation of cytosolic Ca²⁺ by different experimental methods may interfere with cellular process to induce apoptosis. For example, sustained increase of Ca²⁺ by Ca²⁺-mobilizing compounds such as the Ca²⁺ ionophore A23187 (Figure 4) and Ionomycin were reported to act as apoptosis inducers in some cell lines^47,51,8.

Ligand Gated Ca²⁺ Permeability

Some receptors that function as ligand gated Ca²⁺ permeable channels play a key role in Ca²⁺ homeostasis and have been formally implicated in mediating apoptosis. This role is reported to be taken by the purinergic receptor P2X (P2X R)⁵², inositol 1,4,5-triphosphate receptor (IP₃R)⁵³. and ryanodine receptor (RyR).54 Whereas the P2XRs are cell surface membrane Ca²⁺ channel, the IP₃R and RyR function as Ca²⁺ release channels in the ER membrane.

The P2XRs are non selective cation channels with significant permeability to Ca²⁺. They induce signaling by allowing Ca²⁺ entry relative to the extracellular concentration of ATP. The first clue to the involvement of P2XRs was the finding that high concentrations of ATP induced apoptosis by increasing the intracellular Ca²⁺level^55,56. P2X1 channel proteins were up-regulated in thymocytes during dexamethasone-induced apoptotic cell death, while pharmacological inhibition of the P2XR1 protect thymocytes from apoptosis^52,55. P2X channels were reported to mediate the apoptotic ATP signal by Ca²⁺ overload in blood, brain and skin cells⁵⁷.

Intracellular organelles such as the ER and mitochondria have higher Ca²⁺ concentrations than the cytosol and they are considered cellular Ca²⁺ stores. Regulation of ER Ca²⁺ release controls many cellular functions. Enzymatic cascades which are dependent on Ca²⁺ concentration in the ER lumen integrate rapid Ca²⁺signaling with longlasting adaptive responses through modifications of protein synthesis and processing. Several studies have suggested a central role for participation of ER and intracellular Ca²⁺ levels in the regulation of mitochondrial cytochrome c release that leads to induction of apoptosis⁵⁸.

IP₃Rs are intracellular ion channels that mediate the release of Ca²⁺ from the ER and play a central role in control of intracellular Ca²⁺ homeostasis. Cytosolic IP₃ is produced in response to GPCR stimulated phospholipase C. Binding of IP₃ to the receptor induces transient opening of the receptor channel, facilitating Ca²⁺ flow from the ER lumen into the cytoplasm, thereby producing a transient elevation of cytosolic Ca²⁺ that in turn activates signal transduction kinases. Although IP₃Rs are mostly located on the ER membrane, evidence suggests that they are also located on the plasma membrane, enabling the IP₃R to mediate entry of extracellular Ca²⁺ into the cytoplasm. The IP₃R has been suggested to be involved in adrenal corticosteroid related apoptosis^58,59 through its downstream target, calcineurin⁶⁰. Cytosolic Ca²⁺ levels are tightly controlled by IP₃R channel activity. Intermediate Ca²⁺ levels facilitate the channel closure, and prevent Ca²⁺overload^54,62.

As a response to a pathological stimulus, Ca²⁺ together with cytochrome c participates in self amplifying feedback to promote apoptosis⁶¹. Low levels of cytochrome c prevent the closing of the IP₃R channel during Ca²⁺ overload, leading to a burst of Ca²⁺ flow from the ER lumen into the cytosol. This in turn potentiates release of all the mitochondrial cytochrome c, which is sufficient to operate the caspase cascade to promote apoptosis⁶¹.

RyRs operate as Ca²⁺ release channels particularly in skeletal and cardiac ER, they are also located in the cell membrane of a variety of cells and are highly expressed in T- and B-lymphocytes. These channels provide a pathway for fast Ca²⁺ release from intracellular stores into the cell cytosol. In some cases, RyR channels transmit Ca²⁺ signals directly to closely associated mitochondria⁵⁴. In pancreatic β-cells RyR2 was found to be involved in buffering of Ca²⁺ influx during stimulation with glucose or high K⁺. Blocking of Ca²⁺ flux through the type 2 RyR in these cells markedly increased apoptosis in calpain-dependent death pathways⁶². On the other hand, depletion of intracellular Ca²⁺ stores and cytosolic Ca²⁺ overload via caffeine or Ryanodine (#R-500) activated RyR channels in CHO cells, observed to enhance translocation of the proapoptotic factor Bax into the mitochondrial membrane and induction of apoptosis^3,63,64.

The maintenance of ER Ca²⁺ stores is result of equilibrium between Ca²⁺ release and Ca²⁺ refill which involves opening of cation channels as well as activity of Ca²⁺ pumps. The Sarco Endoplasmic Reticulum Ca²⁺ATPase (SERCA) is a critical molecule which pumps Ca²⁺ from the cytosol into the ER lumen in order to maintain low intracellular Ca²⁺⁶⁵. Dysfunction of this pump by inhibition with Thapsigargin caused ER Ca²⁺depletion and cytosolic Ca²⁺ overload, followed by extensive DNA fragmentation and apoptosis in a variety of cells (Figure 5)⁶⁶. Increase of the cholesterol phospholipid ratio which occurs under pathological conditions changes the fluidity of the ER membrane and inhibits SERCA. This effect can be avoided by pharmacologic manipulations that block cholesterol trafficking to the ER⁶⁷.

Ca²⁺ Channels

Ca²⁺ influx in nonexcitable cells regulates such diverse processes as exocytosis, enzyme activity, gene regulation and cell proliferation. Overload of extracelluar Ca²⁺ can damage tissues, this effect may attribute to apoptotic effect caused by Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels and by Transient Receptor Potential (TRP) non selective ion channels.

L-type Ca²⁺ channels were exclusively upregulated as a response to chronic cell membrane high K⁺ depolarization, leading to cell apoptosis in some cell lines⁶⁸. Such an observation might be explained by the fact that a steep rise in intracellular Ca²⁺ is buffered to some degree by mitochondrial Ca²⁺ uptake. However, a continuous increase in cytosolic Ca²⁺, such as in the case of channel upregulation, revokes the buffering capacity and causes dysfunction of these organelles^69-71. On the other hand, high K⁺ induced transmembrane Ca²⁺– influx was observed to be essential in the maintaining of nerve and renal cells since blockage of Ca²⁺influx might prevent cell survival. Moreover, the L-type Ca²⁺ channel agonist – (±)-Bay K8644 (#B-350) is a potent agent that prevents nerve cell loss during low K⁺ enhanced apoptosis (Figs. 6-7)^72-74.

The TRPM2 channels which belong to the TRP family were shown to contribute to apoptotic cell death. This Ca²⁺ permeable channel was reported to be gated by adenosine diphosphate-ribose (ADP-ribose) and H2O2 which are both evoked during oxidative stress, resulting in a disruption of intracellular free Ca²⁺homeostasis^75,76.

Extracellular Ca²⁺ Sensing Prevents Apoptosis

Ca²⁺ sensing plays a key role in regulation of the calcitrophic hormone PTH in tissues that maintain systemic Ca²⁺ homeostasis. The G protein-coupled extracellular Ca²⁺-sensing receptor (CaR) has been elucidated as responsible for recognizing and responding to small changes in extracellular Ca²⁺ level. It is becoming clear that the CaR also participates in a variety of non-Ca²⁺ homeostatic functions, such as control of ion channels, hormone secretion, and regulation of cell-cycle events^77,78. The role of CaR in apoptosis has been demonstrated when it was shown that a rise in extracelluar Ca²⁺ protected AT-3 prostate carcinoma cells and CaR-transfected HEK293 cells from apoptotic cell death. Furthermore, anti apoptotic effect of extracellular Ca²⁺was also observed in c-myc overexpressing/serum deprived cells (all the cells were shown to express CaR)⁷⁹. PI3K and p38 MAPK were suggested to be the downstream pathways that mediate the mitogenic response to extracellular Ca²⁺ activated CaR⁸⁰. However, the downstream mechanism by which CaR prevents apoptosis has not been proved yet.

Ca²⁺ and Mitochondrial Apoptosis

Ca²⁺ plays a central role in mitochondrial function and mitochondrial regulation and it acts on several levels within the organelle to stimulate ATP synthesis. Although the luminal level of Ca²⁺ in mitochondria is high, most of the mitochondrial effects of Ca²⁺ require its entry across the double membrane into the matrix. The mitochondrial outer membrane was thought to be permeable to Ca²⁺, even though recent evidence suggests that the outer membrane Voltage-Dependent Anion Channel (VDAC) is a ruthenium red-sensitive Ca²⁺channel. This Mitochondrial Permeability Transition (MPT) pore was also proposed to conduct non specific low molecular insults movement^81,82. However, dysregulation of mitochondrial Ca²⁺ homeostasis is now recognized to play an active role in apoptosis by switching the mitochondrial Ca²⁺ signaling to facilitate Ca²⁺-induced opening of the MPT pore^81-83. For example, mitochondrial matrix Ca²⁺ overload enhances generation of Reactive Oxygen Species (ROS) which trigger the opening of the MPT pore and release of apoptogenic factors such as cytochrome c⁵⁹. Because mitochondrial cytochrome c release during apoptosis is an “all-or-nothing” event occurring within a rapid time frame³, it has been suggested that the Ca²⁺ spike coordinates cytochrome c release by regulation of the VDAC opening⁸⁴. This paradigm is supported by the observation that increased expression of this channel augments the mitochondrial sensitivity to ER-stress caused by thapsigargin-induced apoptosis^85-87,22.

The primary ROS manufactured by the mitochondria is superoxide (O2^–), which is converted to H₂O₂ either by spontaneous dismutation or by the enzyme superoxide dismutase²². Apoptosis is reported to be accompanied by a burst of ROS which damages cellular components such as proteins, lipids, and DNA⁸⁸. At the heart of understanding how Ca²⁺ can be both a physiological and a pathological effector of mitochondrial function is the fact that Ca²⁺ modulates mitochondrial ROS generation. The normal production of ROS is limited only to the amount required for microdomain cell signaling⁸⁹ whereas overload of mitochondrial Ca²⁺ during pathological conditions upregulates the production of QH – a ROS precursor, thereby causing an increase in ROS production. Alternatively, Ca²⁺ can stimulate nitric oxide generation⁵, another generator of ROS, to generate apoptosis⁹⁰.

Conclusion

Apoptotic processes are an integral part of the life cycle: development, differentiation, proliferation, homoeostasis, regulation and function of the body systems and in the removal of defective and therefore harmful cells. Thus, dysfunction or dysregulation of the apoptotic program is implicated in a variety of pathological conditions. Due to its importance in such varied biological processes, programmed cell death is a widespread phenomenon investigated in many areas of biology.

Pathological elevation in cytosolic Ca²⁺ concentrations (above 10 μM) is deleterious and can modulate apoptotic cell death through multiple mechanisms in a specific cells. Understanding the dual role played by Ca²⁺ signaling in initiation of apoptosis is relevant in many clinical situations such inherited muscle diseases and autoimmune diseases^91,5.

In view of data summarized here we conclude that different Ca²⁺ stimuli have distinct effects on stimulation of apoptotic process, dependent on cell type, cell environment, Ca²⁺ source and Ca²⁺ levels. Furthermore, cell apoptosis is strongly dependent on the biochemical situation or pathways that are already activated at the time of the apoptotic stimulus. Moreover, we speculate that Ca²⁺ can serve as either positive or negative trigger, but not as an exclusive factor, for driving apoptosis. The final result will be dictated by the cell situation, which is an integration of the levels, availability and location of certain cell components as well as equilibrium among different signaling pathways that take place during apoptosis modulation.

On the other hand, during progression of the apoptosis process, Ca²⁺ is an active factor that promotes the conversion of proapoptotic components into apoptotic conformation.

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