Role of Calcium in Glucocorticosteroid-Induced Apoptosis of Thymocytes and Lymphoma Cells: Resurrection of Old Theories by New Findings

Recognition that purinergic receptors are involved in ACS-induced apoptosis evolved out of an effort by Owens et al17 to identify differentially expressed mRNAs associated with ACS-induced apoptosis in thymocytes. One of the differentially expressed mRNAs identified by these investigators, termed RP-2, was induced early in the course of ACS-induced apoptosis. However, the identity of the RP-2 sequence was not elucidated until 5 years later, when it was discovered that RP-2 corresponds to a partial sequence of a gene encoding an ATP-gated cation channel, termed purinergic receptor or P_2X receptor.18,19 This discovery is particularly intriguing, because extracellular ATP has been reported to induce apoptosis in thymocytes by increasing the intracellular concentration of calcium.20,21 

The P_2X receptors constitute a family of at least seven members, distributed throughout central and peripheral neurons, smooth muscles, epithelia, developing skeletal neuromuscular junctions, lymphocytes, platelets, and macrophages. P_2X receptors are nonselective cation channels with significant permeability to calcium. P_2X receptors engage in intercellular communication by detecting the regulated (synaptic) or lytic (cell death) release of intracellular metabolites such as ATP. Unlike other well-known ion channels gated by extracellular ligands (eg, nicotinic, serotonin, 5-HT₃), P_2X receptors are characterized by only two transmembrane domains with intracellular amino- and carboxy-termini.22 RP-2 corresponds to the subfamily member P2X₁ (originally termed P_2XR1), recently cloned from vas deferens and PC12 cells.18,19 Curiously, P_2X receptors are structurally similar to some of theCaenorhabditis elegans gene products implicated in neuronal cell selection and death.

The link between P2X₁ receptor expression and ACS-induced apoptosis in rat thymocytes was recently established by Chvatchko et al.15 These investigators found that P2X₁receptors were upregulated in thymocytes during ACS-induced apoptosis. Furthermore, extracellular ATP enhanced ACS-induced apoptosis and antagonists of ATP substantially reduced ACS-induced apoptosis, suggesting that ACS-induced apoptosis is dependent on P2X₁receptor activation by extracellular ATP.

The other ACS-inducible gene implicated in mediating apoptosis of both thymocytes and lymphoma cells encodes the Type 3 IP₃R.16 The IP₃R has been extensively characterized and plays a central role in calcium signaling.23,24 Typically, IP₃ generated in response to G-protein–coupled receptors and receptor tyrosine kinases binds to the IP₃ receptor that spans the endoplasmic reticulum (ER) membrane.23,24 The binding of IP₃ induces transient opening of the receptor, allowing calcium to flow from the ER lumen into the cytoplasm, thereby producing a transient elevation of cytosolic calcium that in turn activates signal transduction kinases. Although IP₃R are most prominently located on the ER membrane, there is evidence for localization of the IP₃R to the plasma membrane, enabling the IP₃R to mediate entry of extracellular calcium into the cytoplasm16 (and references therein). In cells undergoing apoptosis in response to ACS, immunocytochemical studies localized the IP₃R (specifically subtype 3) to the plasma membrane.16 Upregulation of IP₃R in S49 lymphoma cells was associated with increased cytosolic calcium concentration, suggesting that increased expression of IP₃R on the plasma membrane increased calcium entry.16Significantly, repression of ACS-induced IP₃R expression by transfecting cells with an IP₃R antisense plasmid not only decreased the induction of IP₃R, but also inhibited the induction of apoptosis by ACS.16 

The involvement of calcium in ACS-induced apoptosis was first suggested by Kaiser and Edelman,25 who discovered that extracellular calcium is necessary for induction of apoptosis in thymocytes by ACS. This observation, which has been confirmed by others,12,13suggests that extracellular calcium uptake mediates ACS-induced apoptosis. The concept that calcium signals ACS-induced apoptosis is further supported by evidence that the calmodulin inhibitor, calmidazolium, interferes with ACS-induced thymocyte apoptosis.26,27 However, in contrast to the situation in thymocytes, extracellular calcium is unnecessary for ACS-induced apoptosis of peripheral lymph node lymphocytes and lymphoma cells.28-33 

These earlier findings are fully consistent with the patterns of expression of the P2X₁ receptor. P2X₁expression was detected in thymocytes, but not in peripheral (lymph node) T lymphocytes.15 Furthermore, there is a strong correlation between P2X₁ expression and the susceptibility of individual thymocyte subsets to ACS-induced apoptosis. In the thymus gland, immature CD4⁺CD8⁺ thymocytes residing within the cortex are programmed to undergo apoptosis in response to ACS, whereas the more mature CD4⁺CD8⁻ or CD4⁻CD8⁺ thymocytes located in the medulla and circulating T lymphocytes are less sensitive to ACS-induced apoptosis.34-37 Significantly, P2X₁ expression was detected only in CD4⁺CD8⁺ thymocytes, but not in peripheral (lymph node) T lymphocytes.15 

The earlier findings suggesting a role of extracellular calcium in mediating ACS-induced apoptosis of thymocytes are also consistent with evidence that ACS treatment increases the expression of the Type 3 IP₃R in cortical, but not medullary, thymocytes.16 However, evidence that ACS induce the expression of Type 3 IP₃R on the plasma membrane of S49 cells, a T-cell lymphoma line,16 appears to be less consistent with earlier evidence indicating the extracellular calcium is not required for induction of apoptosis in lymphoma cells by ACS.29-32 Moreover, it is possible that the localization of the IP₃R to plasma membrane was a consequence of apoptotic bleb formation in ACS-treated lymphoma cells. The apoptotic blebs that form at the cell surface during apoptosis are membranous structures composed of ER membrane.38 Thus, further work will be required to determine whether IP₃R induced by ACS treatment are located primarily on the plasma membrane, or are located on the ER membrane and then relocated to the cell surface as apoptotic blebs form.

Although there is considerable evidence that extracellular calcium is not required for apoptosis induction by ACS in lymphoma cells, a role for calcium in mediating ACS-induced apoptosis of lymphoma cells has been supported by several findings. First, calmodulin gene expression is increased in T-cell lymphoma cells after treatment with ACS.27 Second, calmidazolium interferes with apoptosis in ACS-treated lymphoma cells.27 Third, stable expression of a cDNA encoding the high-affinity calcium-binding protein, calbindin, inhibited ACS-induced apoptosis in lymphoma cells.39 Where, then, might the calcium come from that mediates ACS-induced apoptosis in these cells? We and others have detected a diminution of the ER calcium pool in ACS-treated lymphoma cells.33,40 Thus, one theory is that calcium release from the ER, perhaps via IP₃R located on the ER membrane, may be involved in signaling apoptosis in ACS-treated lymphoma cells.

Although the novel findings of the past year have provided molecular evidence of a role for calcium in ACS-induced apoptosis, the specific role that calcium plays in death induction and the signal transduction pathway initiated by cytosolic calcium elevation and how it leads to apoptosis are unknown. In the case of T-cell receptor (TCR)-mediated apoptosis, calcium in combination with calmodulin activates calcineurin, a cytosolic protein phosphatase that dephosphorylates and thereby activates the transcription factor NF-AT_C, leading to increased transcription of calcium-regulated, immediate-early genes, including Nur77.41 Calcineurin function, and hence TCR-mediated apoptosis, is inhibited by the potent immunosuppressants cyclosporin A and FK506.42 However, these agents do not inhibit ACS-induced apoptosis.42 Furthermore, ACS- and TCR-mediated apoptotic pathways are mutually antagonistic42and calcineurin activation protects T cells from ACS-induced apoptosis.43 Also, Nur77 is not significantly induced by ACS.41 One possible lead is a recently identified calcium-binding protein, ALG-2, that has been implicated as necessary for ACS-induced apoptosis, but its precise role in the apoptotic process has not been defined.44 

Another concept deserving further consideration is that while calcium mediates apoptosis of ACS-treated cells, it might not be necessary for ACS-induced cell death. This concept is based on a recent report by Iseki et al,45 who found that high concentrations of the intracellular calcium chelator Quin-2/AM inhibited DNA fragmentation in ACS-treated thymocytes, but did not inhibit cell death. In their hands, calmodulin inhibitors blocked DNA fragmentation, but markedly enhanced cytolysis. One interpretation of these findings is that calcium might be required for DNA cleavage during apoptosis, but not for cell death. Thus, calcium might be involved in endonuclease activation, as part of the degradation phase of apoptosis, but might not be necessary for cell death.

However, the role of calcium in endonuclease activation varies among different types of lymphocytes. Whereas the endonuclease responsible for apoptotic DNA fragmentation in ACS-treated thymocytes requires calcium for maximal activity,5,13,31,46-49 DNA is cleaved by a calcium-independent endonuclease in the CEM human lymphoblast cell line.50 These observations correlate with the evidence that extracellular calcium uptake is required for ACS-induced thymocyte apoptosis, but not for ACS-induced apoptosis of lymphoma cell.12,13,25,28 

Another potential role for calcium in ACS-induced apoptosis might be in protease activation. The calcium-dependent neutral protease, calpain, is activated in the course of apoptosis induction in ACS-treated thymocytes, and a calpain inhibitor appears to inhibit ACS-induced thymocyte cell death.51 Moreover, destruction of the nuclear structural protein lamin, an event that appears to precede endonucleolytic DNA cleavage during ACS-induced apoptosis, is inhibited by a calpain inhibitor.52,53 Currently, there is little mechanistic insight into how ACS-induced cytosolic calcium elevation might lead to activation of interleukin-1β converting enzyme–like proteases, which clearly play a prominent role in ACS-induced apoptosis, as well as other forms of apoptosis.54 

Since the initial observations by Kaiser and Edelman,25,28interest in the role of calcium in ACS-induced apoptosis has wavered, in part because of the fact that extracellular calcium is only necessary for induction of apoptosis in thymocytes, but not in peripheral lymphocytes or lymphoma cells. Now, as a result of molecular evidence implicating two separate ligand-gated calcium channels in ACS-induced apoptosis, interest in the role of calcium is sure to be renewed. The major challenge lies in determining the signal transduction pathway through which ACS-induced calcium fluxes mediate apoptosis.

Recent results (Jayaraman T, Marks AR: Mol Cell Biol17:3005, 1997) indicate that T cells deficient in IP₃RI are resistant to ACS-induced apoptosis, providing additional evidence for a role of calcium in signaling ACS-induced apoptosis.