Angiotensins

Angiotensins are a second class of hormone that induce calcium-dependent apoptosis, specifically Angiotensin II (Cigola et al. 1997; Kajstura et al. 1997; Palomeque et al. 2009; Yamada et al. 1996). Angiotensins are oligomeric peptides released in response to steroid hormones, such as glucocorticoids and estrogen. They are powerful vasoconstrictors, and consequently, angiotensin receptors are targets for antihypertensive medications (Gradman 2009). In a cardiomyocyte, muscle contraction is stimulated by the opening of an L-type calcium channel that enables calcium release via ryanodine receptors (Fig. 2). Angiotensins bind to their receptors (AT1 and AT2) resulting in the generation of IP₃ followed by transient calcium elevations (Mattiazzi 1997). While ryanodine receptors are more abundant than IP₃Rs in cardiomyocytes, both calcium and IP₃ are required for IP₃R channel opening. While still not universally accepted, it is likely that calcium release via neighboring ryanodine receptors facilitates IP₃R-opening, enabling both channels to function cooperatively in response to angiotensin ligands (Kockskamper et al. 2008).

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Figure 2.

Calcium signaling mediated by angiotensin II hormone in a cardiomyocyte. In a cardiomyocyte, calcium signaling is mediated by the influx of calcium through L-type calcium channels (LTCC). The Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA) is responsible for maintaining the appropriate concentration of luminal calcium by actively transporting calcium ion across the SR membrane, while ryanodine receptors and IP₃Rs promote its release into the cytosol. Angiotensin, a peptide hormone, binds to the AT-1 receptor (AT-1R), a G-protein coupled receptor that activates phospholipase C (PLC) following GTP hydrolysis, thereby generating IP₃ and diacylglycerol (DAG). It should be noted that ryanodine receptors are 50- to 100-fold more abundant than IP₃Rs in cardiomyocytes. Therefore, calcium release via ryanodine receptors have shown to be much more robust compared to calcium responses that are mediated by IP₃Rs (Kockskamper et al. 2008). Nevertheless, there is unequivocal evidence for the contribution of IP₃Rs during SR-calcium release, which is likely due to synergy between the two calcium channels in mediating calcium-induced calcium release.

Much like glucocorticoids, treatment with higher concentrations of angiotensin II results in apoptosis that can be blocked by receptor antagonists (Andreka et al. 2004). As depicted in Figure 2, stimulation of cardiomyocytes with angiotensin II causes an acute release of cytosolic calcium, and several reports suggest that calcium elevation contributes to apoptosis (Kajstura et al. 1997). For instance, verapamil, an L-type calcium channel blocker, inhibits angiotensin-induced apoptosis (Goldenberg et al. 2001). Further, ectopic expression of angiotensin receptors (AT1 and AT2) results in apoptosis by a calcium-dependent mechanism (Aranguiz-Urroz et al. 2009).

Testosterone

Testosterone is a steroid hormone that has rapid effects on cardiomyocytes. In one study, it was shown that testosterone increased cytoplasmic calcium concentrations in 1–7 minutes by an IP₃R-dependent fashion (Vicencio et al. 2006). A second study reported that testosterone signals activate the extracellular signal-regulated kinase (ERK), and this activation could be inhibited by 2-aminoethyldiphenyl borate and the phospholipase C inhibitor U-73122, suggesting that testosterone generates IP₃ in cardiomyocytes (Altamirano et al. 2009). Similar rapid non-genomic effects of testosterone have been observed in T cells, where calcium influx was observed within seconds (Benten et al. 1997). However, it remains to be determined whether these calcium signals would eventually lead to apoptosis. Interestingly, in neuronal cells, low concentrations of testosterone result in calcium oscillations, whereas higher concentrations induce apoptosis, also by a mechanism that is IP₃R dependent (Estrada et al. 2006). Thus, steroid hormones can have direct apoptotic effects on multiple cells types, as is the case for glucocorticoids and androgens, and perhaps indirectly via glucocorticoid regulation of angiotensin.

Thapsigargin

Thapsigargin decreases the ER calcium pool by inhibiting SERCA pumps, which results in ER stress and apoptosis (Lam et al. 1993). Apoptosis induced by thapsigargin occurs by a mechanism that is dependent on the activation of caspase-12, a mammalian protease that localizes to the ER and is important for mediating apoptosis in response to ER stress (Szegezdi et al. 2003). This is exemplified by experiments performed in mice in which caspase-12 had been deleted (Nakagawa et al. 2000). In vitro studies have demonstrated that a calpain activates caspase-12 leading to the subsequent activation of caspase-9 (Morishima et al. 2002; Nakagawa and Yuan 2000; Rao et al. 2002). These data suggest the possibility that apoptosis induced by thapsigargin can occur independently of cytochrome c release and is thus directly induced by calcium via calpain activation.

Staurosporine

Staurosporine is a natural apoptosis-inducing alkaloid originally isolated from Streptomyces staurosporeus. It directly provokes calcium leak from the ER by activating caspase-3 mediated cleavage of IP₃R1 (Hirota et al. 1999). Additionally, it was shown that cleavage of IP₃R1 contributed, in part, to the induction of apoptosis by accelerating calcium leak (Assefa et al. 2004; Verbert et al. 2008). In these experiments, transfection of a mutant IP₃R resistant to caspase-mediated cleavage partially inhibited apoptosis induction by staurosporine in B cells lacking wild type IP₃Rs. A recent study by Mikoshiba and colleagues further identified IP₃Rs as being important mediators of apoptosis induction by staurosporine. They determined that G protein-coupled receptor kinase interacting proteins (GITs) bind to IP₃Rs to inhibit their function and suppress apoptosis in the presence of staurosporine (Zhang et al. 2009b). Finally, staurosporine also promotes the activation of a mitochondrial calpain that positively regulates apoptosis (Norberg et al. 2008), thus illustrating similarities with ER stress-driven pathways.

The Bcl-2-IP₃ Receptor Interaction

Our laboratory was the first to show that Bcl-2 directly interacts with IP₃Rs to inhibit IP₃-dependent calcium flux (Fig. 3) (Chen et al. 2004). This interaction, as well as an interaction of the Bcl-2 homologue Bcl-xL with the IP₃R, has subsequently been detected by a number of laboratories (Rong and Distelhorst 2007). Bcl-2 directly inhibits IP₃R channel opening in vitro in lipid bilayer experiments and also inhibits IP₃-induced calcium release in T cells (Chen et al. 2004; Zhong et al. 2006). We have now further elucidated the mechanism of the Bcl-2-IP₃R interaction by demonstrating that the BH4 domain of Bcl-2 associates with the regulatory and coupling domain of IP₃R1, specifically an 80 amino acid sequence within domain 3 (Rong et al. 2008). Further analysis indicated that the BH4 domain was both necessary and sufficient to inhibit IP₃-mediated calcium signals and subsequently apoptosis in T cells (Rong et al. 2009). This is particularly interesting given that the BH4 domain of Bcl-2 and Bcl-xL seem to be involved in preventing apoptosis. Although our work has emphasized the interaction of Bcl-2 with the regulatory and coupling domain of the IP₃R, there is also evidence that Bcl-2 and/or Bcl-xL may interact with the C-terminus of the IP₃R, and through this interaction, enhance IP₃-mediated calcium oscillations (White et al. 2005). In light of these discoveries, there is now increased interest in using small molecules to inhibit Bcl-2 and enhance proapoptotic calcium transients. One example of this was the use of a Bcl-2 inhibitor HA14-1 that induced cytochrome c release and apoptosis by a calcium-dependent mechanism in myeloid leukemia (An et al. 2004).

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Figure 3.

The Bcl-2-IP₃R interaction inhibits ER-calcium release. Bcl-2 localizes to the ER where it binds IP₃Rs to inhibit calcium transients. In T cells, calcium transients are activated in response to strong T-cell receptor ligation, which results in apoptosis that can be inhibited by Bcl-2. In contrast, calcium oscillations that are associated with cell survival are promoted by Bcl-2 and Bcl-xL. In addition, Bcl-2 regulates the level of ER luminal calcium by increasing membrane permeability or by interacting with the Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA). Bcl-2 also interacts with calcineurin, thereby forming a complex with both calcineurin and IP₃Rs on the ER membrane.

Bcl-2 and IP₃R Phosphorylation

Other proteins within the Bcl-2-IP₃R complex may regulate ER calcium release by altering phosphorylation of IP₃Rs. As shown in Figure 3, Bcl-2 binds the phosphatase calcineurin (Erin et al. 2003; Shibasaki et al. 1997), and calcineurin also interacts with IP₃Rs (Cameron et al. 1995). Because IP₃R channel activity is positively regulated by phosphorylation (DeSouza et al. 2002), it is reasonable to speculate that Bcl-2 may facilitate the dephosphorylation of IP₃R by interacting with calcineurin. Although such a mechanism has not been definitively established, we have observed that IP₃R phosphorylation is decreased in Bcl-2 overexpressing T cells (Chen et al. 2004). Furthermore, Bcl-2 has been reported to regulate IP₃R phosphorylation in Bax/Bak double knockout cells (Oakes et al. 2005). Moreover, Xu et al. have implicated protein phosphatase 1 in the regulation of IP₃R phosphorylation by Bcl-2 (Xu et al. 2007). Thus, much remains to be determined regarding the specific kinases and phosphatases that regulate IP₃R channel opening, how these are regulated by Bcl-2-IP₃R interaction, and how they contribute overall to the regulation of calcium signals by Bcl-2 and its relatives.

Bcl-2 and Mitochondrial Cross Talk

Another important function of Bcl-2 and Bcl-xL is to inhibit calcium-mediated cross talk between ER and mitochondria (Kruman and Mattson 1999; Pinton et al. 2008). Because both organelles are in close proximity, calcium is rapidly taken up by mitochondria via the calcium uniporter on the outer mitochondrial membrane (Hajnoczky et al. 2006; Hanson et al. 2004; Rizzuto et al. 2003; Szalai et al. 1999; Szlufcik et al. 2006). Bcl-2 and Bcl-xL inhibit calcium redistribution to the mitochondria, thereby limiting calcium uptake (Hanson et al. 2008; Pinton et al. 2008) (Fig. 3). In the context of apoptosis, it was shown that Bcl-2 inhibited mitochondrial calcium uptake following IL-3 and serum withdrawal in hematopoietic cells and fibroblasts, respectively (Baffy et al. 1993; Magnelli et al. 1994). Interestingly, Bcl-2 inhibited apoptosis in cardiomyocytes exposed to ceramide and staurosporine, which caused depolarization of the mitochondrial membrane and cytochrome c release (Pacher and Hajnoczky 2001). A more recent study suggests that Bcl-2 inhibits calcium release through L-type channels, thereby preventing mitochondrial calcium uptake (Diaz-Prieto et al. 2008). Finally, Mcl-1, an anti-apoptotic protein overexpressed in myeloid and lymphoid leukemia, also blocks calcium redistribution following exposure to staurosporine (Minagawa et al. 2005).

Bcl-2 Regulation of Luminal Calcium

In contrast to experiments reporting that Bcl-2 inhibits IP₃R opening, there is also substantial evidence that Bcl-2 and Bcl-xL directly regulate the concentration of luminal calcium (Fig. 3). Initial studies showed that Bcl-2 increased membrane permeability, thereby resulting in calcium leak and decreased signaling in response to ATP (Foyouzi-Youssefi et al. 2000; Pinton et al. 2000). Another study documented that knocking down Bcl-2 prevented the loss of ER luminal calcium (Oakes et al. 2005). While these studies also cite the significance of the Bcl-2-IP₃R interaction in regulating the calcium pool, others have found that Bcl-2 depletes luminal calcium by interacting with SERCA (Dremina et al. 2004; Dremina et al. 2006; Vanden Abeele et al. 2002). In spite of these differences in experimental findings, it cannot be refuted that Bcl-2 localizes to the ER to inhibit calcium signaling. Thus, mechanistic differences may be attributed to cell type in which the relative expression and localization of Bcl-2 family members are considerably distinct. Accordingly, the effect of luminal calcium is dependent upon the predominant IP₃R isoform expressed, yet not necessary for Bcl-2 or Bcl-xL mediated effects on calcium signaling (Li et al. 2007).

Calcium Regulation by Bax and Bak

Like Bcl-2 and Bcl-xL, Bax and Bak also localize to the ER where they regulate calcium homeostasis. In Bax/Bak knockout cells, ER luminal calcium is decreased compared to wild type cells, which compromises ER calcium release as well as mitochondrial uptake (Oakes et al. 2005; Scorrano et al. 2003). Consistent with these observations, the Bax Inhibitor-1 protein facilitates ER calcium leak, depleting the available calcium pool (Chae et al. 2004; Kim et al. 2008). Although the mechanism by which Bax and Bak decrease luminal calcium has not been determined, it is generally inferred that Bax/Bak ordinarily prevents Bcl-2-mediated ER calcium leak, and thus their deficiency promotes depletion of ER luminal calcium by Bcl-2. Moreover, Bax alone is required for calcium elevation in response to cytotoxic agents, such as staurosporine (Nutt et al. 2002a). For example, reconstitution of Bax in a prostate cancer cell line augmented cytosolic calcium elevation and restored mitochondrial uptake. The same group also reported that Bax/Bak overexpression induced calcium elevation followed by cytochrome c release and apoptosis (Nutt et al. 2002b). Interestingly, when Bak is targeted to the ER, it facilitates cytosolic calcium elevation and activates caspase-12; yet this does not occur when Bak is specifically targeted to mitochondria (Zong et al. 2003). In addition, ER-targeted Bak requires calcium in conjunction with ER stress for apoptosis to occur (Klee et al. 2009). This suggests that the localization of Bax and/or Bak may determine which effector pathway is induced. It is possible that Bax/Bak localization to the ER is favored when cells undergo apoptosis induced by ER stress.

Autophagy Induction by ER Stress Pathways

The link between calcium and autophagy was initially discovered by several groups reporting that autophagy could be provoked by ER stress (Bernales et al. 2006; Ogata et al. 2006; Yorimitsu et al. 2006). For example, both thapsigargin and tunicamycin stimulate autophagy (Ogata et al. 2006). ER stress affects Autophagy-Related Genes (atg), which are evolutionarily conserved and indispensable for autophagy in many cell systems. In yeast, the transcription factor Hac-1 (an ortholog of the ER-stress mammalian XBP-1) transactivates atg8 during the unfolded protein response (Bernales et al. 2006). Other studies have shown that mutations in ER stress-related proteins such as PERK or EIF2α can inhibit autophagy (Kouroku et al. 2007). Further, knockout of several atg genes prevents autophagy-mediated survival in the presence of tunicamycin (Ogata et al. 2006). While these data indisputably demonstrate that ER stress induces autophagy, a direct role for calcium had not been implicated at this point in time.

Autophagy Regulation by Calcium Signaling

Direct evidence that calcium signaling stimulates autophagy was first reported by Jaattela and colleagues (Hoyer-Hansen et al. 2007). They demonstrated that ER calcium mobilization induces autophagy when stimulated by agents such as vitamin D, ionomycin, and thapsigargin. Moreover, GFP-LC3 aggregates were inhibited with BAPTA-AM, suggesting that autophagosome formation was calcium dependent. They provided further evidence that autophagy occurred by the calcium-dependent activation of AMP activated protein kinase, which required upstream activation of the calcium/calmodulin kinase kinase β. AMPK is activated during nutrient deprivation to inhibit activity of the target of rapamycin (mTOR), a negative regulator of autophagy (Hoyer-Hansen and Jaattela 2007). Further evidence supporting a direct role for calcium in the induction of autophagy was the finding that calcium phosphate precipitates could induce autophagy when transfected into HEK293 cells (Gao et al. 2008). Importantly, autophagy mediated by calcium phosphate was also Beclin-dependent. Beclin is a newly discovered BH3-only protein that mediates autophagy by forming a complex between the class III PI3 kinase Vps34 and p150, which facilitates assembly of the autophagosome (Sinha and Levine 2008).

Autophagy in the Context of T-cell Activation

As previously described, significant contributions in the calcium field have been made by investigating signal transduction pathways in activated lymphocytes. Accordingly, autophagy may also be important for regulating lymphocyte activation. For example, T cells from atg5 knockout mice do not proliferate following ligation of the T-cell receptor, nor do they survive in the periphery (Pua et al. 2007), suggesting that autophagy is required for T-cell activation. Interestingly, it was shown that T-cell activation increases autophagy by NFkB-dependent transcription of Beclin-1 (Copetti et al. 2009a; Copetti et al. 2009b). In this study, the authors provide evidence that NFκB directly binds to the Beclin-1 promoter following activation of Jurkat T cells. Although a direct link has not been observed, it is possible that calcium-dependent activation of calcineurin stimulates this process, thus implicating a role for calcium in Beclin-1 transcription and autophagy.

In addition to producing IP₃, T-cell activation also generates reactive oxygen species (Devadas et al. 2002; Hildeman et al. 2003). Thiol groups are found on IP₃Rs and ryanodine receptors, and oxidation of both calcium channels favors their opening (Bootman et al. 1992; Bultynck et al. 2004; Joseph et al. 2006; Sun et al. 2001; Xia et al. 2000). Further, cyclic ADP ribose and NAADP govern redox reactions and are also endogenous ligands for ryanodine receptors and natural 2nd messengers produced by T-cell activation (Fliegert et al. 2007; Guse 2009). These studies have implicated a role for reactive oxygen species in regulating calcium signals. Not surprisingly, reactive oxygen species also contribute to the induction of autophagy. For example, hydrogen peroxide directly facilitates formation of the autophagosome by oxidizing Atg4 (Scherz-Shouval et al. 2007). Another study demonstrated that neurons undergo autophagy when mitochondrial fission is induced by nitric oxide (Barsoum et al. 2006). This observation is attractive in light of the fact that nitric oxide protects cardiomyocytes from apoptosis. Together, these data suggest the possibility that oxidative metabolites function as signaling molecules by activating calcium and triggering autophagy in lymphocytes, although more definitive data is necessary to support this important conclusion.

We thank Karen McColl for editing and proofreading this manuscript as well as Hisashi Fujioka of the electron microscopy core facility at Case Western Reserve University.