Leta K. Nutt

Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis

Previous studies have demonstrated that Ca2+ is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca2+ pool in human PC-3 prostate cancer cells. This mobilization of ER Ca2+ was associated with a concomitant increase in mitochondrial Ca2+ levels, and neither ER Ca2+ mobilization nor mitochondrial Ca2+ uptake occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145 Bax expression via adenoviral gene transfer restored ER Ca2+ release and mitochondrial Ca2+ uptake and dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax expression in this model system. In addition, an inhibitor of the mitochondrial Ca2+ uniporter (RU-360) attenuated mitochondrial Ca2+ uptake, cytochrome crelease, and DNA fragmentation, directly implicating the mitochondrial Ca2+ changes in cell death. Together, our data demonstrate that Bax-mediated alterations in ER and mitochondrial Ca2+ levels serve as important upstream signals for cytochrome c release in some examples of apoptosis.

Cis-urocanic acid, a sunlight-induced immunosuppressive factor, activates immune suppression via the 5-HT2A receptor.

Exposure to UV radiation induces skin cancer and suppresses the immune response. To induce immune suppression, the electromagnetic energy of UV radiation must be absorbed by an epidermal photoreceptor and converted into a biologically recognizable signal. Two photoreceptors have been recognized: DNA and trans-urocanic acid (UCA). Trans-UCA is normally found in the outermost layer of skin and isomerizes to the cis isomer upon exposure to UV radiation. Although UCA was identified as a UV photoreceptor years ago, and many have documented its ability to induce immune suppression, its exact mode of action remains elusive. Particularly vexing has been the identity of the molecular pathway by which cis-UCA mediates immune suppression. Here we provide evidence that cis-UCA binds to the serotonin [5-hydroxytryptamine (5-HT)] receptor with relatively high affinity (Kd = 4.6 nM). Anti-cis-UCA antibody precipitates radiolabeled 5-HT, and the binding is inhibited by excess 5-HT and/or excess cis-UCA. Similarly, anti-5-HT antibody precipitates radiolabeled cis-UCA, and the binding is inhibited by excess 5-HT or excess cis-UCA. Calcium mobilization was activated when a mouse fibroblast line, stably transfected with the human 5-HT2A receptor, was treated with cis-UCA. Cis-UCA-induced calcium mobilization was blocked with a selective 5-HT2A receptor antagonist. UV- and cis-UCA-induced immune suppression was blocked by antiserotonin antibodies or by treating the mice with 5-HT2A receptor antagonists. Our findings identify cis-UCA as a serotonin receptor ligand and indicate that the immunosuppressive effects of cis-UCA and UV radiation are mediated by activation of the 5-HT2A receptor.

Bax and Bak are required for cytochrome c release during arsenic trioxide-induced apoptosis

Arsenic trioxide is a potent chemotherapeutic agent that selectively triggers apoptosis in tumor cells. Previous studies have demonstrated that arsenicals cause direct damage to mitochondria, but it is not clear that these effects initiate apoptosis. Here we used Bak-/- mouse liver mitochondria and virally immortalized Bax-/-Bak-/- mouse embryonic fibroblasts (MEFs) to investigate whether or not multidomain proapoptotic Bcl-2 family proteins were required for arsenic-induced mitochondrial damage and cell death. Near clinically achievable concentrations, arsenic stimulated cytochrome c release and apoptosis via a Bax/Bak- dependent mechanism. At higher concentrations (125 mM- 1 mM), cells died via a Bax/Bak-independent mechanism mediated by oxidative stress that resulted in necrosis. Consistent with previous reports, arsenic directly inhibited complex I of the mitochondrial electron transport chain, which resulted in mitochondrial permeability transition (MPT), the generation of reactive oxygen species (ROS), and thiol oxidation. However, these effects only occurred at concentrations of arsenic trioxide of 50 mM and higher, and the oxidative stress associated with them blocked caspase activation. Our data demonstrate for the first time that the cytochrome c release which initiates apoptosis in cells exposed to this classic mitochondrial poison occurs indirectly via the activation of Bax/Bak rather than via direct mitochondrial damage. Furthermore, the results implicate reactive oxygen species in a concentration-dependent mechanistic switch between apoptosis and necrosis.

Phosphatidylserine exposure in Fas type I cells is mitochondria-dependent

Previous studies have demonstrated that Fas‐triggered activation of effector caspases and subsequent nuclear apoptosis either is mitochondria‐independent (type I cells) or relies on mitochondrial amplification of the initial stimulus (type II cells). We show herein that Bcl‐2 overexpression in a prototypic type I cell line (SKW6.4) promotes mitochondrial generation of ATP and blocks Fas‐triggered plasma membrane externalization of phosphatidylserine (PS). Moreover, overexpression of Bcl‐2 attenuates macrophage engulfment of Fas‐triggered cells. Fas‐mediated DNA fragmentation, on the other hand, remains unaffected in SKW6.4‐bcl‐2 cells. These studies thus demonstrate that PS externalization and clearance of cell corpses are mitochondria‐dependent events, and show that these events can be dissociated from other features of the apoptotic program, in Fas type I cells.

Metabolic Activation of CaMKII by Coenzyme A

Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis by directly activating CaMKII. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca(2+) to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes but also in Murine oocytes. These findings uncover a direct mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.

Calcium flux measurements in apoptosis.

Early studies in apoptosis implicated an increase in cytosolic Ca2+ as a direct mediator of DNA fragmentation. However, efforts to delineate targets for this increase in Ca2+ have been slow in evolving. Several previous studies have implicated ER Ca2+ pool depletion in the initiation of apoptosis. Our own preliminary studies confirm that many (but not all) apoptotic stimuli empty the ER store via a mechanism that is blocked by BCL-2 expression. Furthermore, ER pool depletion is not affected by broad spectrum caspase inhibitors, indicating that it occurs via a caspase-independent mechanism. Finally, our data demonstrate that ER pool depletion occurs prior to release of cytochrome c from mitochondria. Given previous work demonstrating close coordination of ER and mitochondrial Ca2+ levels, we speculate that ER-dependent changes in mitochondrial Ca2+ serve as important signals for cytochrome c release. Alternative mechanisms include activation of caspase-12 and/or the JNK pathway, both of which can be directly stimulated by depletion of the ER Ca2+ pool. Although substantial improvements in intracellular Ca2+ imaging have emerged, compelling answers to many of the present questions related to the role of Ca2+ in apoptosis await future technical improvements. The development of organelle-specific, recombinant Ca2+ probes (targeted aequorins and cameleons) certainly should facilitate some of this work, although the target cell of interest must be amenable to molecular manipulation (transfection), which precludes straightforward analysis of primary cells. Pharmacological tools (i.e., thapsigargin and DBHQ) can provide conclusive data on ER pool status without requiring an overly sophisticated image analysis system. However, confocal microscopy allows for the effective analysis of Ca2+ pools as long as dye localization is homogeneous and properly controlled. However, current techniques should be considered semiquantitative at best and will remain so until specific organelle-targeted fluorescent dyes are developed and widely available.

Metabolic regulation of CaMKII protein and caspases in Xenopus laevis egg extracts

Background: In the basal state, oocytes produce lactate from G6P even in the presence of oxygen. Results: Addition of G6P to egg extracts inhibits PP1, preventing dephosphorylation/inactivation of CaMKII and initiation of apoptotic pathways. Conclusion: Normal oocyte metabolism suppresses apoptosis by inhibiting PP1 and activating CaMKII. Significance: These mechanistic insights suggest potential targets for modulating cell death. The metabolism of the Xenopus laevis egg provides a cell survival signal. We found previously that increased carbon flux from glucose-6-phosphate (G6P) through the pentose phosphate pathway in egg extracts maintains NADPH levels and calcium/calmodulin regulated protein kinase II (CaMKII) activity to phosphorylate caspase 2 and suppress cell death pathways. Here we show that the addition of G6P to oocyte extracts inhibits the dephosphorylation/inactivation of CaMKII bound to caspase 2 by protein phosphatase 1. Thus, G6P sustains the phosphorylation of caspase 2 by CaMKII at Ser-135, preventing the induction of caspase 2-mediated apoptotic pathways. These findings expand our understanding of oocyte biology and clarify mechanisms underlying the metabolic regulation of CaMKII and apoptosis. Furthermore, these findings suggest novel approaches to disrupt the suppressive effects of the abnormal metabolism on cell death pathways.

Measurement of changes in intracellular calcium during apoptosis.

The role of Ca2+ changes in the commitment to apoptosis has been appreciated for more than two decades. However, early work focused on increases in cytosolic Ca2+ levels that may not be associated with most examples of programmed cell death. Rather, recent studies indicate that release of Ca2+ from the endoplasmic reticulum (ER) and subsequent mitochondrial Ca2+ uptake plays a more important role by regulating release of cytochrome c from mitochondria. These apoptosis-associated Ca2+ fluxes are regulated by members of the BCL-2 family of proteins and may therefore be critical targets of their evolutionarily conserved actions. Therefore, the availability of reliable techniques for measuring organelle-associated Ca2+ fluxes is critical to ongoing research in the field, yet these techniques present unique challenges not associated with the more routine measurements of cytosolic Ca2+ levels. In this chapter, we provide detailed methods for measuring cytosolic, ER, and mitochondrial Ca2+ levels in whole using commercially available fluorescent dyes, identifying key potential pitfalls and alternative strategies.

Organelle-derived acetyl-CoA promotes prostate cancer cell survival, migration, and metastasis via activation of calmodulin kinase II

Although emerging evidence suggests a potential role of calcium/calmodulin-dependent kinase II (CaMKII) in prostate cancer, its role in prostate cancer tumorigenesis is largely unknown. Here, we examine whether the acetyl CoA-CaMKII pathway, first described in frog oocytes, promotes prostate cancer tumorigenesis. In human prostate cancer specimens, metastatic prostate cancer expressed higher levels of active CaMKII compared with localized prostate cancer. Correspondingly, basal CaMKII activity was significantly higher in the more tumorigenic PC3 and PC3-mm2 cells relative to the less tumorigenic LNCaP and C4-2B4 cells. Deletion of CaMKII by CRISPR/Cas9 in PC3-mm2 cells abrogated cell survival under low-serum conditions, anchorage-independent growth and cell migration; overexpression of constitutively active CaMKII in C4-2B4 cells promoted these phenotypes. In an animal model of prostate cancer metastasis, genetic ablation of CaMKII reduced PC3-mm2 cell metastasis from the prostate to the lymph nodes. Knockdown of the acetyl-CoA transporter carnitine acetyltransferase abolished CaMKII activation, providing evidence that acetyl-CoA generated from organelles is a major activator of CaMKII. Genetic deletion of the β-oxidation rate-limiting enzyme ACOX family proteins decreased CaMKII activation, whereas overexpression of ACOXI increased CaMKII activation. Overall, our studies identify active CaMKII as a novel connection between organelle β-oxidation and acetyl-CoA transport with cell survival, migration, and prostate cancer metastasis.Significance: This study identifies a cell metabolic pathway that promotes prostate cancer metastasis and suggests prostate cancer may be susceptible to β-oxidation inhibitors. Cancer Res; 78(10); 2490-502. ©2018 AACR.

Metabolic regulation of apoptosis via Ca+/Calmodulin Kinase II (CaMKII)

Background The metabolic state of a cell is an important factor in whether or not it engages its apoptotic machinery. Furthermore, reprogramming energy metabolism is now recognized as a hallmark of cancer; so understanding how metabolism regulates apoptosis is crucial. Recently it has been demonstrated that CaMKII serves as a link between metabolic state and apoptosis [1]. Using the Xenopus laevis oocyte extract, it was shown that addition of glucose-6-phosphate (G6P) induced an inhibitory phosphorylation of caspase 2, mediated via CaMKII. However, the mechanisms of how G6P and metabolism regulate CaMKII still remain unclear. The aim of this study is to investigate the underlying mechanisms of how metabolism regulates CaMKII activity.