Program for DAP Activation
Cell Death and Differentiation volume 15, pages — Download Citation Edited by DC Rubinsztein Subjects Abstract Damage to endoplasmic reticulum ER homeostasis that cannot be corrected by the unfolded protein response activates cell death. Here, we identified death-associated protein kinase DAPk as an important component in the ER stress-induced cell death pathway. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells. Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion.
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Cell Death and Differentiation volume 15, pages — Download Citation Edited by DC Rubinsztein Subjects Abstract Damage to endoplasmic reticulum ER homeostasis that cannot be corrected by the unfolded protein response activates cell death.
Here, we identified death-associated protein kinase DAPk as an important component in the ER stress-induced cell death pathway. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells.
Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion.
We further show that ER stress promotes the catalytic activity of DAPk by causing dephosphorylation of an inhibitory autophosphorylation on Ser by a PP2A-like phosphatase. Thus, DAPk constitutes a critical integration point in ER stress signaling, transmitting these signals into two distinct directions, caspase activation and autophagy, leading to cell death.
Main Disturbances of endoplasmic reticulum ER homeostasis that lead to extensive and irreparable damage activate ER-specific cell death mechanisms. Additionally, cytosolic calcium concentrations are increased during ER stress, contributing to cell death activation. Knockout of DAPk provides significant protection from ER stress-induced cell death, and both apoptosis and autophagy activation are attenuated.
Both processes, shown here to be activated concurrently within the same cells, lead to cellular demise in response to ER stress, as simultaneous disruption of apoptosis and autophagy is required to obtain maximal cell viability.
Thus, these studies implicate DAPk as a common upstream integrator of the two types of programmed cell death pathways that are induced by ER stress.
Results ER stress leads to both apoptosis and autophagy in the same cell A cell culture model of ER stress was used to thoroughly assess the role of autophagy in this phenomenon. This involved treatment of primary mouse embryonic fibroblasts MEFs with drugs that induce ER stress through accumulation of misfolded proteins, including thapsigargin, an ER calcium channel blocker, and tunicamycin, an N-linked glycosylation inhibitor.
An assay measuring the autophagy-mediated degradation of radiolabelled long-lived proteins showed a 3. The accumulation of LC3-II was significantly augmented by addition of the lysosomal inhibitors E64d and pepstatin A, indicating that the shift observed resulted from an increase in autophagic activity and not from a block in lysosomal-mediated degradation of autophagosomes.
Figure 1B , right panel. The presence of autophagosomes was confirmed by transmission electron microscopic TEM analysis Figure 1C.
Typical double or multimembrane autophagic vesicles engulfing cytoplasmic components and organelles accumulated in cells treated with ER stress inducers Figure 1Cd—f , arrowheads.
Figure 1 ER stress induces autophagy and caspase activation. A Increased rate of autophagic degradation of long-lived proteins after tunicamycin treatment. MEFs were metabolically labeled with radiolabeled valine, treated with DMSO or tunicamycin for 16 h and protein degradation was measured 4 h later after 20 h of tunicamycin treatment. The results are presented as percent 3-MA inhibited degradation of total-labeled protein.
Left panel, kinetics of LC3-II and active caspase-3 accumulation following tunicamycin treatment. Right panel, the effect of lysosomal protease inhibitors on the tunicamycin-induced LC3-II conversion. Scale bars, a—c, nm; d—f, nm; m, mitochondrion; n, nucleus; black arrow, condensed chromatin; white arrowheads, double membrane autophagic vesicles; black arrowheads, multimembrane autophagic vesicles. D Simultaneous activation of autophagy and caspase-3 by ER stress.
A minimum of cells were scored at each time point, and only cells with more than five puncta were considered positive. Western blotting indicated proteolytic activation of caspase-3 starting at 16 h after addition of tunicamycin Figure 1B , left panel. The fact that apoptosis appeared later than the earliest indications of autophagy suggests that the autophagy observed here is not a reaction to, or a compensatory mechanism for, the apoptotic response.
To determine whether the two phenomena occurred within the same cell populations, MEFs from a transgenic mouse line expressing the autophagy marker protein GFP-LC3 19 were used. The treatment of cells with either thapsigargin or tunicamycin caused a punctate staining of GFP-LC3, reflecting the recruitment of LC3 to autophagic vesicles Figure 1D , similar to the pattern observed in cells starved of amino acids and serum data not shown.
Thus a significant portion of the population exhibited signs of increased autophagic activity following ER stress. Notably, immunostaining of GFP-LC3 fibroblasts with an antibody recognizing only the cleaved, activated form of caspase-3 showed that ER stress caused simultaneous activation of autophagy and caspase-3 cleavage in the same cell Figure 1D.
Likewise, close examination of the electron micrographs of cells containing autophagosomes revealed the presence of several hallmarks of apoptosis, such as chromatin condensation Figure 1Cb, c , black arrows , mitochondrial condensation Figure 1Cb, c and membrane blebbing data not shown.
Thus autophagy and apoptosis are not mutually exclusive events, but rather occur within the same cell following induction of ER stress.
Importantly, evidence of autophagy and apoptosis was observed in a mouse model of ER stress-induced death as well. Systemic injection of tunicamycin into mice causes ER stress-mediated death of kidney tubular cells.
Furthermore, analysis of kidney sections prepared from GFP-LC3 transgenic mice injected with tunicamycin revealed strong punctate and ring-shaped fluorescent staining reflecting the presence of a high level of autophagic activity in the tubular cells Figure 2A. EM sections of these kidneys clearly exhibited the presence of double or multilayer membrane vesicles engulfing a variety of intracellular components, indicative of the presence of autophagosomes Figure 2Bc—f.
The damaged tubular cells also contained many empty vacuoles e. Figure 2Bc , some of which were engulfed together with the surrounding cytosol by the autophagosomes Figure 2Be, f.
They also contained many condensed mitochondria Figure 2Bc, d. Notably, advanced chromatin condensation characteristic of apoptosis could be detected in damaged tubular cells, which also display autophagic vesicle accumulation suggesting that autophagy and apoptosis develop in the same cells Supplementary Figure 1. Taken together these data demonstrate that ER stress activates both apoptosis and autophagy at a single-cell level in cultured primary fibroblasts and in a mouse model system of kidney damage.
Arrows indicate vacuolated cells f, h. Each point represents the mean of multiple high power fields from an individual mouse Full size image Figure 2 ER stress induces hallmarks of apoptosis and autophagy in vivo in the intact kidney. Note the collapse of the tubule structure following tunicamycin treatment, with the disintegration of the tubular cells d , loss of nuclei n and distortion of the lumen L. Arrows indicate autophagic vesicles within individual tubular cells. Arrowheads indicate brush border of tubular cells.
Shown are images of kidneys from DMSO a, b or tunicamycin-injected mice c—f. Arrows, double d, f and multimembraned e autophagic vesicles. Notably, administration of 3-MA did not accelerate cell death. Instead, it significantly augmented cell viability, but only when added in combination with zVAD, a pancaspase inhibitor. By itself, zVAD had no discernible protective effect on cell viability, suggesting that the combined action of caspase activation and autophagy induction contribute to cellular killing Figure 3a.
These observations were confirmed by utilizing double genetic disruptions, which target both the autophagic and apoptotic pathways.
Atg5 and Beclin1 are essential components of the basic autophagy machinery, and Atg5 knockout cells or Beclin1-depleted cells are unable to activate autophagy in response to classical stimuli such as amino acid starvation. Knocking out Atg5 or knocking down Beclin-1 had no significant effect on the death response to ER stress Figure 3b and c.
The fact that inhibition of autophagy by itself did not accelerate cell death, ruled out the possibility that autophagy is death protective in this cellular setting. In contrast, however, in combination with caspase-3 perturbation by either knockout or knockdown the perturbation of Atg5 or of Beclin-1 was clearly cell death protective Figure 3b and c.
Notably, the single perturbation of caspase-3 by itself either protected to some extent from cell death or had no effect pending on whether the gene was knocked out or down, respectively Figure 3b and c. This difference may emerge from residual levels of caspase-3 in the knocked-down cells. Yet, in spite of these differences, in both cases the second perturbation of an autophagic gene further protected from ER-induced cell death.
These findings imply that in primary fibroblasts, autophagy is a death-promoting mechanism, the contribution of which becomes apparent when the caspase-dependent pathway is blocked. Figure 3 Autophagy is necessary for ER stress-induced cell death. In b and c , nonspecific NS band recognized by the antibodies was used as loading controls Full size image DAPk knockout fibroblasts are resistant to ER stress-induced cell death Several signaling molecules, including DAPk, have been shown to mediate apoptotic or autophagic death pathways, depending on the death stimulus and cell type.
Presumably, such dual-nature proteins are likely to have a significant function in ER stress-induced death, which has both apoptotic and autophagic components. Previous studies have indicated that knock-out of DAPk confers protection from several external stresses, including glutamate toxicity to retinal ganglion cells in the intact animal, 23 ceramide treatment of cultured hippocampal neurons 24 and oncogene activation in isolated MEFs.
The reduction in cell death in response to tunicamycin was similar to that observed in the double knockout of Bax and Bak Figure 4b , previously shown to be necessary for ER stress-induced cell death. Altogether, these results indicate that DAPk has a significant function in cell death activated by ER stress. Tubulin is used as a loading control. Thapsigargin treatment of DAPk knockout cells also resulted in comparable levels of JNK phosphorylation data not shown.
DAPk knockout mice are resistant to kidney toxicity caused by systemic tunicamycin injection As DAPk is highly expressed in the kidney, and especially in renal tubular cells, 27 we assessed whether DAPk is necessary for ER stress-induced tubular cell death in vivo upon injection of tunicamycin.
Many tubular cells were filled with vacuoles occupying almost the entire cytoplasm, pushing the nucleus to the side Figure 5Af , arrows. TUNEL staining was used to quantify cell death. ER stress activates DAPk by causing Ser dephosphorylation In growing cells, DAPk activity is under strict negative control involving auto-inhibition of the catalytic cleft by interaction with its CaM regulatory-binding segment, and an inhibitory autophosphorylation on Ser within this domain. These anti-phosphoSer antibodies were used to immunoprecipitate endogenous DAPk from HEKT cells treated with tunicamycin to determine the abundance of the phosphorylated inactive form relative to total DAPk.
A pronounced, reproducible decrease in the levels of phosphorylated DAPk was observed at 4—8 h Figure 6a and was detectable as early as 1 h after tunicamycin treatment Figure 6b.
Notably, the dephosphorylation persisted for at least 24 h data not shown. This was further supported by measuring kinase activity in vitro using myosin II regulatory light chain MLC as a substrate. In these assays, endogenous DAPk protein was immunoprecipitated with antibodies to the C-terminus of the protein, which recognize both phosphorylated and non-phosphorylated DAPk.
DAPk immunoprecipitated from tunicamycin-treated cells showed significantly higher kinase activity in the presence of 0. TCL, total cell lysate. Tubulin and a non-relevant protein are used as loading controls.
Ionomycin, which causes a fast and robust increase in cytosolic calcium concentrations, has been shown to induce autophagy in MCF7 cells, similar to the calcium mobilizer thapsigargin. Ionomycin treatment led to a rapid dephosphorylation of DAPk Ser after 30 min Figure 6d , lanes 1, 2. In this setting, the addition of the PP2B inhibitor cyclosporine A had no effect on Ser dephosphorylation Figure 6d , lane 4 , whereas protein phosphatase 1 PP1 and PP2A inhibitors calyculin A and okadaic acid blocked it Figure 6d , lanes 3, 7.
It was found that even low concentrations of okadaic acid in the nanomolar range were able to potently inhibit DAPk Ser dephosphorylation, suggesting that a PP2A-like phosphatase is responsible for DAPk activation Figure 6d , lanes 5—7. It strongly reduced the steady state levels of DAPk protein in cells, irrespective of whether they were treated or not by tunicamycin, thus preventing an accurate assessment of the dephosphorylation event Supplementary Figure 7.
Finally, to confirm that PP2A directly dephosphorylates DAPk, an in vitro dephosphorylation assay using purified proteins was performed. DAPk promotes ER stress-induced caspase-activation and autophagy A quantitative analysis of the effects of DAPk deletion on ER stress-induced autophagy and caspase activation was then undertaken. In contrast, the deletion of DAPk did not block caspase activation upon treatment of MEFs with the classical apoptosis inducer staurosporine Supplementary Figure 8A consistent with the lack of protection from staurosporine-induced cell death observed in Figure 4a.
Two different knockout fibroblast preparations gave similar results data not shown. These data demonstrate that activation of DAPk by ER stress signals mediates caspase activation and autophagic induction in primary fibroblasts.
Figure 7 Attenuation of both caspase activity and autophagy in cells lacking DAPk. To gain insight into the molecular mechanism, which integrates these two pathways, we used cells and mice that are deficient of DAPk.
DAPk was previously reported to induce cell death with apoptotic or autophagic characteristics in independent cellular and experimental contexts. We proved that ER stress led to the activation of DAPk catalytic activity by dephosphorylation of a key residue in its calmodulin-regulatory domain. Quantitative measurements of caspase activity and accumulation of autophagic vesicles demonstrated that the hallmarks of both apoptotic and autophagic pathways activated during ER stress were attenuated in DAPk-deficient cells.
This was shown in vitro using isolated fibroblasts in culture and in vivo in a kidney toxicity model.
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