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Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement of both the ER and mitochondria in ceramide-mediated caspase-independent programmed cell death
© Deerberg et al; licensee BioMed Central Ltd. 2009
Received: 22 August 2009
Accepted: 9 October 2009
Published: 9 October 2009
Programmed cell death (PCD) is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase-independent PCD (ciPCD). Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER). Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist.
We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA), the ER (Bcl-2 cb5), both (Bcl-2 WT) or the cytosol/nucleus (Bcl-2 ΔTM) and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology.
Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD.
Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide.
The survival and homeostasis of multicellular organisms critically depends on programmed cell death (PCD) to correctly remove cells that are potentially harmful or which have fulfilled their function. Although caspase-dependent apoptosis, the most well-studied form of PCD, represents the principal suicide program in most physiological settings , many cells have the ability to commit suicide by caspase-independent modes of PCD (ciPCD) . ciPCD fulfills vital functions in processes such as the negative selection of lymphocytes, the termination of immune responses, embryonic removal of interdigital webs, regulation of bone growth, ovulation, and cellular turnover in the intestine . Furthermore, ciPCD has been implicated the pathology of hyperacute shock , pancreatitis [5, 6], cerebral and myocardial ischemia-reperfusion injury, epilepsy, Alzheimer's disease and other inflammatory injuries, as well as in the destruction of cells by pathogens such as HIV, vaccinia virus, Shigella and Salmonella [3, 7]. In contrast to apoptosis, the molecular mechanisms of ciPCD are just beginning to be unraveled, e. g. by the recent identification of RIP3, enzymes of the energy metabolism, Nox1, CYLD, Bmf, and cathepsin and calpain proteases as candidate mediators of ciPCD [2, 8–10]. However, and despite these advances, a coherent picture of the molecular steps in ciPCD is still lacking.
Mitochondria have been identified not only as essential elicitors of apoptosis, but also of ciPCD, e.g. by release of proteins such as AIF, EndoG and HtrA2/OMI, as well as by production of reactive oxygen species (ROS) [2, 11–13]. It has been proposed that excess formation of ROS triggers ciPCD by activation of the DNA repair enzyme PARP, followed by intracellular depletion of NAD+ and ATP, nuclear translocation of AIF and finally, death . Similar to mitochondria, the endoplasmic reticulum (ER) - as the main site for critical cellular functions such as protein folding, lipid biosynthesis, and calcium storage in the cell - has also been implicated in the induction of both apoptosis  and ciPCD [2, 12, 13]. In fact, the ER may play a key role in certain types of ciPCD, as intracellular calcium influx caused by ER stress induces activation of calpains, a family of calcium-dependent cytosolic proteases that can elicit ciPCD .
Members of the Bcl-2 family of proteins have long been recognized as central regulators of mitochondrial apoptosis, primarily by controlling the permeabilization of the outer mitochondrial membrane . They have also been found localized at the ER, where they regulate apoptosis in response to a range of cellular stresses, and also in the nucleus , where they may fulfill yet unknown functions. Similar to the established protective functions of mitochondrial Bcl-2, expression of Bcl-2 at the ER has been shown to protect cells from apoptosis, e. g. by regulating the release of proapoptotic calcium, reducing calcium-uptake by mitochondria and subsequent calpain-dependent apoptosis . The role of Bcl-2 in mitochondrial vs. ER-mediated apoptosis has been further addressed in a number of studies by the use of Bcl-2 constructs that specifically localize to mitochondria or to the ER [16–23]. In these studies, Bcl-2 localizing to the ER was shown to interfere with apoptosis induction by some (c-myc overexpression, etoposide, staurosporine, tunicamycin, brefeldin A, ceramide, ionizing radiation, thapsigargin, Bax, Bad) but not all stimuli (reviewed in [24, 25]).
In contrast to this relative wealth of available data on apoptosis, the role of ER- vs. mitochondrially localized Bcl-2 in ciPCD has not been investigated so far. Here, we utilize ciPCD elicited by ceramide as a model system, a lipid second messenger that has been recognized as important in radiation-induced elimination of tumor cells [26, 27]. We demonstrate for the first time that in contrast to wildtype Bcl-2, restricted expression of Bcl-2 solely at mitochondria-, the ER-, or the cytosol/nucleus is insufficient to prevent ciPCD. Therefore, our data suggest a combined requirement of mitochondria and the ER in ceramide-mediated ciPCD and implicate the existence of a molecular crosstalk between both organelles.
Reagents and constructs
Highly purified human recombinant TNF (hTNF) was supplied by BASF Bioresearch. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) was obtained from Bachem. Cycloheximide (CHX) was purchased from Sigma. Expression constructs cloned in the vector pRc/CMV (Invitrogen) encoding wildtype Bcl-2 (Bcl-2 WT), cytosolic Bcl-2 lacking the transmembrane domain (Bcl-2 ΔTM) and Bcl-2 mutants restricted to the outer mitochondrial membrane (Bcl-2 ActA) or to the endoplasmic reticulum (Bcl-2 cb5) were kindly provided by B. Leber (McMaster University, Hamilton, Canada).
Cell culture and transfections
Wildtype human leukemic Jurkat cells were originally obtained from the American Type Culture Collection. Jurkat cells stably transfected with the plasmid pSFFV-Bcl-2, overexpressing full length human Bcl-2 at levels 10-20-fold over untransfected cells  were kindly provided by S. Korsmeyer (Harvard Medical School, Boston, USA). Cells were maintained in a mixture of Click's/RPMI 1640 (50/50% v/v) supplemented with 10% v/v FCS, 2 mM glutamine and 50 μg/ml each of streptomycin and penicillin in a humidified incubator containing 5% w/v CO2. Transient transfections of wildtype Jurkat cells were performed by Amaxa nucleofection (Lonza Cologne), using solution V and program C-16. Jurkat cells stably expressing the various versions of Bcl-2 in pRc/CMV were obtained by electroporation using a Gene pulser II (Bio-Rad) followed by selection with Geneticin (Invitrogen) and were used as pool transfectants.
Cells were lysed in a buffer containing 10 mM Hepes pH 7.4, 142 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.2% v/v NP40, 1 mM DTT and 2 mM Pefabloc. To generate positive controls for activation, cytosolic extracts of untreated cells were equilibrated for 1 h at 30°C after the addition of 1 mM dATP and 10 μM cytochrome c to permit activation of caspases and subsequent cleavage of substrate proteins. To measure caspase activity, 100 μl of caspase buffer (20 mM Pipes, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% w/v CHAPS, 10% w/v sucrose, pH 7.2) containing 100 μM zDEVD-afc or zIETD-afc (Calbiochem) were added to 5 μl of cytosolic extract (50 μg protein) and incubated at 37°C. The release of afc was measured as emission at 505 nm upon excitation at 405 nm using a Labsystems Fluoroskan II fluorimeter equipped with a thermostated plate reader.
For documentation of cell morphology, images from unfixed cells were obtained using an Axiovert 100 microscope (Zeiss) and a DS-5 M camera (Nikon).
For flow cytometric measurement of cell death, cells were seeded in six-well plates at 5 × 105 cells/well. Following treatment, cells were collected by centrifugation and resuspended in PBS/5 mM EDTA containing 2 μg/ml propidium iodide (PI), and the red fluorescence was measured on a FACSCalibur flow cytometer (BD Biosciences). Since we observed that measurement exclusively of PI-positive cells did not account for a significant fraction of highly disintegrated dead cells that - due to diffusion of the dye - had already become PI-negative again (shown e. g. in Fig. 1C, right panels, lower left quadrants), we alternatively measured the fraction of large, PI negative cells (lower right quadrants) which represents viable, intact cells. For each measurement, a total of 10.000 cells was analyzed.
Cells were collected and lysed in TNE buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% v/v NP40, 2 mM EDTA) containing 10 μg/ml pepstatin/aprotinin/leupeptin, 1 mM sodium orthovanadate and 5 mM NaF. After removal of insoluble material by centrifugation at 10.000 × g and 4°C for 15 min, the protein concentration of the supernatants was measured using a BCA assay (Pierce). 30 μg of cell protein per lane were resolved by electrophoresis on 12.5% w/v SDS polyacrylamide gels (SDS-PAGE). After electrophoretic transfer to Protran nitrocellulose (Whatman), reactive proteins were detected using a monoclonal IgG1 antibody specific for human Bcl-2 (sc-7382, Santa Cruz) and the ECL detection kit (GE Healthcare).
Wildtype Bcl-2 protects from ceramide-mediated ciPCD
Transient expression of Bcl-2 constructs with restricted subcellular localization
Wildtype, but not organelle-specific Bcl-2 protects from ceramide-mediated ciPCD in stably transfected Jurkat cells
In this study, we show for the first time that Bcl-2-mediated protection from non-apoptotic, caspase-independent PCD requires the full-length, wildtype protein whereas a limited expression of Bcl-2 at mitochondria, the ER, or the cytosol/nucleus is not sufficient to prevent ciPCD elicited by ceramide. The failure of the organelle-specific constructs to confer protection is not due to an inefficient level of expression (Fig. 3A) or an incorrect localization , and their functionality has been previously demonstrated for apoptotic PCD using the same stable transfectants as analyzed here [21, 22]. Moreover, the protection by wildtype Bcl-2 was independently seen in two distinct stably transfected Jurkat lines, expressed from different vector backbones (pSFFV-neo, pRc/CMV).
The fact that expression of Bcl-2 restricted to mitochondria, the ER (or the cytosol/nucleus, if the results with Bcl-2 ΔTM are additionally taken into account) is not effectively suppressing ceramide-induced ciPCD may suggest that wildtype Bcl-2 interferes with this form of PCD at yet another cellular site. However, this is unlikely, as wildtype Bcl-2 has been detected only at mitochondria, the ER and the nucleus in numerous previous studies (reviewed in [15, 35]). Alternatively, the exclusive protection by wildtype Bcl-2 may be explained by a combined requirement of Bcl-2 at both mitochondria and at the ER, suggesting that both organelles coordinately participate in ciPCD via a molecular crosstalk. This hypothesis appears especially attractive because a similar crosstalk has been shown for apoptotic PCD, where the ER controls mitochondrial apoptosis by releasing proapoptotic calcium . Although calcium likewise appears as an attractive candidate molecule for ER-mitochondrial crosstalk in ciPCD (especially when considering that the calcium-dependent calpain proteases can elicit ciPCD ), the precise nature of this crosstalk will have to be clarified in future studies.
Notably, in autophagy, a distinct form of ciPCD, Bcl-2 has been recognized to act as a negative regulator specifically at the ER by binding to the autophagy-inducing protein Beclin-1 .
Even though the role of Bcl-2 has been most intensively studied in mitochondria and the ER, Bcl-2 also localizes to the nucleus. However, apart from a recent study where it was shown that nuclear Bcl-2 forms an integral part of mitotic chromosomes , information on the functions of nuclear Bcl-2 are scarce. Therefore, nuclear functions of Bcl-2 may likewise be required for suppression of ciPCD in concert with its mitochondrial and ER-specific functions.
In summary, the further exploration of the exact roles of mitochondrial, ER- and nucleus-specific Bcl-2 as well as their potential crosstalk will provide further insight into the molecular mechanisms by which ceramide-mediated ciPCD is executed. At the clinical level, this may directly prove beneficial also for radiation therapy by providing novel options to eliminate tumor cells that have become resistant to apoptotic death signals.
We thank D. Kabelitz for his continuous support and encouragement. This work was supported by grants from the from the Forschungskommission der Medizinischen Fakultät and from the Deutsche Forschungsgemeinschaft to D. A. (SFB 415, A4). The authors have no conflicting financial interests.
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