Question

Moderate increases in cytoplasmic Ca2+ concentration cause apoptosis, extreme increases in cytoplasmic Ca2+ concentration cause necrosis. Describe the two mechanisms involved.

Answer 1

NECROSIS

Up until 1971, the term “necrosis” was used for all types of cell death. A common definition of necrosis is that of a catastrophic derangement of cell integrity following exposure to different types of cell injury and leading to the activation of Ca2+-activated hydrolyzing enzymes. The concept that necrosis is un-programmed was reinforced by the fact that necrotic cell death can be caused by exposures of cells to supraphysiological conditions such as mechanical force, heat, or cold. Thus, necrosis has long been described as a consequence of physio-chemical stress, accidental and uncontrolled. Recently, it is becoming clear that necrotic cell death is as well controlled and programmed as caspase-dependent apoptosis, and that it may be an important cell death mode that is both pathologically and physiologically relevant. Necrotic cell death is not the result of a single well-described signaling cascade but is the consequence of extensive crosstalk between several biochemical and molecular events at different cellular levels. Necrosis is characterized by cytoplasmic swelling, irreversible plasma membrane damage, and organelle breakdown. In necrosis, the cellular content leaks into the extracellular environment, where it may act as a “danger signal”. Consequently, necrosis is usually associated with inflammation. Intracellular Ca2+ is an important signaling molecule also in necrosis. Indeed, in certain pathological conditions, extracellular ligands can induce Ca2+-dependent necrosis.

Calcium-mediated programmed necrosis. Intracellular calcium increases in response to the activation of ionotropic glutamate receptors or through other calcium channels on the plasma membrane or the ER membrane. An intracellular calcium spike induces the activation of Ca2+-dependent proteases and stimulates mitochondrial TCA cycle activity and ROS production. If sustained, the resulting ROS leads to mPT that is dependent on CypD. mPT then leads to the loss of ATP production and necrosis.

Apoptosis

The word ‘apoptosis’ is derived from a Greek word meaning ‘the gentle falling of leaves’ and is used to describe a form of cell death occurring in multicellular organisms. It is defined as a form of cell death that involves altruistic suicide of individual cells in favour of the organism as a whole. Apoptosis is a tightly regulated, highly efficient and energy requiring 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. Apoptotic activity is desirable during organism development and morphological changes especially at the embryonic stage, as well as during the activation of the immune system. Additionally, this process is essential for organ homeostasis by keeping under control cell number and tissue tropism. Defects in apoptosis can result in cancer, autoimmune diseases, neurodegenerative disorders, AIDS and ischemic diseases. Apoptosis results in an orchestrated collapse of a cell, staging cell shrinkage, chromatin condensation, DNA and protein cleavage, fragmentation in the apoptotic-bodies followed by phagocytosis by neighboring cells. 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 drive 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).

Apoptosis occurs through two types of pathways: the death receptor pathway (extrinsic apoptotic pathways) and the mitochondrial pathway (intrinsic apoptotic pathways), and requires the activity of dedicated enzymes (caspases) and regulatory proteins (such as the Bcl2-related family). As to Ca2+, experimental works of the past decade has highlighted its importance in the regulation of apoptosis. However, the role of Ca2+ of apoptosis is very complex, given that in different systems Ca2+-linked stimuli were shown to represent both survival signals and apoptosis inducers. This is not surprising, given the vast array of Ca2+ transducers present in the various compartments of the cell. Ca2+ signaling is the focus of the regulation and activation of the multifunctional Ca2+/calmodulindependent protein kinase (CaMK) family. 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. 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 the control of gene transcription since they phosphorylate several transcription factors. Several reports have indicated that these kinases negatively modulate apoptosis. Intracellular Ca2+ is a coordinating factor that positively regulates the activity of the nuclear transcription factor-kB (NF-kB). This transcription factor is considered an anti-apoptotic agent and plays a key role in cell survival by upregulating expression of several apoptosis inhibitor genes and negatively regulating the activity of caspase-3. Cytosolic Ca2+ increase has a pivotal role in activating 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. Some reports suggest that the Ca2+-calcineurin pathway is critical in the progression of heart failure by regulating cardiomyocyte apoptosis. On the other hand, Ca2+- calcineurin activation by 2-deoxyglucose and staurosporine prevents apoptosis of cardiac myocytes. 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. Another distinctive feature of apoptosis is the requirement for de novo RNA synthesis. A key transcription factor for apoptosis is c-Jun, an immediate-early gene. Ca2+ influx has been reported to be involved in c-jun N-terminal kinase (JNK) signaling pathway mediated IL-1ß-induced apoptosis. Ca2+ mediated regulation, however, is not restricted to the cytosolic compartment. The switch into a death signal often involves the coincidental detection of Ca2+ and proapoptotic stimuli and depends on the amplitude of the mitochondrial Ca2+ signal. Several studies indicate that the Ca2+ content of the Endoplasmic Reticulum (ER) determines the cell’s sensitivity to apoptotic stress and perturbation of ER Ca2+ homeostasis appears to be a key component in the development of several pathological situations. The ER is the main intracellular agonist-sensitive Ca2+ store capable of rapid Ca2+ exchange. The potential of the ER to function as a rapidly exchanging Ca2+ store is due to the presence of three main components: i) ATP dependent pumps for Ca2+ uptake (called SERCAs: Sarco/Endoplasmic Reticulum Ca2+ ATPases), ii) channels for Ca2+ release such as the ubiquitous inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR), and iii) Ca2+ binding proteins for Ca2+ storage, the best characterized being calreticulin and calsequestrin [6]. Procedures that decrease the Ca2+ loading of the ER, such as genetic ablation of the ER Ca2+-buffering protein calreticulin or overexpression of plasma membrane Ca2+ ATPases, protect cells from apoptosis. Conversely, procedures that increase the ER Ca2+ load, such as overexpression of SERCA or calreticulin, sensitize cells to apoptotic stress. Sensitivity to apoptosis correlates with the total ER Ca2+ load, rather than with the free ER Ca2+ concentration, and depends on the ability of cells to transfer Ca2+ from the ER to the mitochondria. Accordingly, procedures that enhance the transfer of Ca2+ from the ER to mitochondria augment ceramide-induced cell death.