Cytochrome c is sufficient to activate caspases and induce apoptosis in naïve but not in neuronally differentiated PC12 cells
PC12 cells can exist either as mitotic naïve cells, or can be differentiated with NGF treatment into postmitotic, neuronal-like cells (Greene and Tischler, 1976
; Greene, 1978
). Both naïve and neuronally differentiated PC12 cells undergo a Bcl-2 inhibitable, caspase-dependent apoptosis in response to a variety of apoptotic stimuli (Mesner et al., 1992
; Batistatou et al., 1993
; Pittman et al., 1993
; Mills et al., 1995
; Troy et al., 1996
). To determine whether cellular differentiation of mitotic cells into postmitotic neurons induces a change in the postcytochrome c
regulation of apoptosis, we examined whether cytosolic cytochrome c
was sufficient to induce apoptosis in naïve or neuronally differentiated PC12 cells. Naïve PC12 cells were differentiated with NGF for 12 d, during which the cells become postmitotic and extend neurites ( A) (Greene and Tischler, 1976
; Pittman et al., 1993
Figure 1. Addition of exogenous cytochrome c induces caspase activation and apoptosis in naïve but not neuronally differentiated PC12 cells. (A) Naïve and neuronally differentiated PC12 cells were injected with 10–15 mg/ml cytochrome c or (more ...)
Cytosolic microinjection of cytochrome c
induced extensive cell death within 30 min in naïve cells. In contrast, upon neuronal differentiation, these cells became remarkably resistant, with 90% of cells viable 30 min after microinjection of cytochrome c
(). Even 3 h after the injections, >60% of neuronally differentiated cells remained alive ( B), with viability slowly decreasing at later (12–24 h) time points (unpublished data). This resistance to cytochrome c
was acquired gradually, with 50% of maximum resistance seen only after 8 d of differentiation with NGF (Fig. S1, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200406073/DC1
We examined whether the differential ability of cytochrome c
to induce apoptosis in naïve and neuronally differentiated PC12 cells was also seen in cell-free extracts. Although addition of cytochrome c
was sufficient to induce robust caspase activation in cytosolic lysates of naïve cells, no significant caspase activation was detected in cytosolic lysates of neuronally differentiated cells ( C). Control yeast cytochrome c
protein, which is unable to bind Apaf-1 (Ellerby et al., 1997
; Kluck et al., 2000
), did not activate caspases in either naïve or neuronally differentiated cells. We examined whether the resistance to cytochrome c
in neuronally differentiated cells was because of a deficit in procaspase-3. Levels of procaspase-3 were in fact found to be slightly higher in neuronally differentiated cells compared with naïve cells (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200406073/DC1
) (Rong et al., 1999
). Importantly, addition of granzyme B, which can directly activate caspase-3 (Trapani and Sutton, 2003
), induced robust caspase activation in lysate from both naïve and neuronally differentiated PC12 cells ( C). Thus, the inability of cytochrome c
to activate caspases in neuronally differentiated cells was not because of a defect in caspase-3.
Apoptosome-mediated cleavage of caspase-9 generates a 35-kD and a 12-kD fragment (Liu et al., 1996
; Cain et al., 2002
). Activated caspase-9 then cleaves and activates caspase-3, which can also feedback on unprocessed procaspase-9 to cleave it into a 37-kD and a 10-kD fragment (Srinivasula et al., 1998
; Zou et al., 2003
). In naïve cell lysates, the addition of bovine cytochrome c
, but not yeast cytochrome c
, induced significant processing of caspase-9 and caspase-3 ( D). In contrast, in neuronally differentiated PC12 cell lysates, consistent with the inability of cytochrome c
to induce apoptosis, no significant processing of caspase-9 or caspase-3 was detected. Addition of granzyme B induced robust and equivalent processing of caspase-9 and caspase-3 in both naïve and neuronally differentiated PC12 cells ( D). Together, these results show that although cytosolic cytochrome c
was sufficient to activate caspases and induce death in naïve cells, upon postmitotic differentiation these cells became markedly resistant as shown in both intact cells and in cell-free extracts.
Resistance to cytochrome c–induced apoptosis in neuronally differentiated cells is mediated by endogenous IAPs
In primary sympathetic neurons, the resistance to cytochrome c
–induced apoptosis is mediated by endogenous IAPs (Deshmukh et al., 2002
; Potts et al., 2003
). To determine whether a similar IAP-mediated regulation was promoting resistance to cytochrome c
in neuronally differentiated PC12 cells, we examined whether addition of Smac, an IAP inhibitor, could relieve the inhibition and permit cytochrome c
to activate caspases in these cells. Addition of wild-type mature Smac (AVPI-Smac) and cytochrome c
together resulted in increased caspase activation in cytosolic extracts from neuronally differentiated cells that are otherwise resistant to addition of cytochrome c
or AVPI-Smac alone ( A; unpublished data). The concentration of Smac used in these experiments was maximal, as a 10-fold increase in Smac did not result in any further increase in caspase activation in cell extracts. Importantly, unlike wild-type AVPI-Smac, mutant MVPI-Smac, which is unable to bind to IAPs and inhibit their function (Chai et al., 2000
), did not enhance cytochrome c
–mediated caspase activation in these cell extracts ( A).
Figure 2. Exogenous addition of Smac that can relieve IAP inhibition permits cytochrome c to activate caspases and induce death in neuronally differentiated PC12 cells. Neuronally differentiated (A and B) or naïve (C and D) PC12 cells were treated with (more ...)
We also examined whether excess Smac could allow cytochrome c to induce death in intact neuronally differentiated cells. Coinjection of cytochrome c with wild-type AVPI-Smac induced significantly greater death in neuronally differentiated cells than seen with injection of cytochrome c alone or mutant MVPI-Smac and cytochrome c together ( B). These results show that endogenous IAPs effectively inhibited cytochrome c–mediated caspase activation and death in neuronally differentiated cells. In contrast, endogenous IAPs did not appear to regulate apoptosis in the naïve, undifferentiated cells. Not only was cytochrome c alone sufficient to induce rapid cell death in naïve cells, but inhibiting IAPs with addition of Smac did not enhance the rate of caspase activation or cell death in these cells ().
Apoptosome becomes highly inefficient in neuronally differentiated cells
The selective ability of endogenous IAPs to block caspase activation in neuronally differentiated but not naïve cells was not due to elevated expression of IAPs in the neuronally differentiated cells. Naïve and neuronally differentiated PC12 cells expressed similar levels of multiple IAPs, including XIAP, cIAP-1, cIAP-2, and NAIP ().
Figure 3. Naïve and neuronally differentiated cells express similar levels of IAPs. (A) Protein levels of XIAP, cIAP-1, cIAP-2, NAIP, and lactate dehydrogenase (LDH) were examined in naïve and neuronally differentiated PC12 cells (equal total protein (more ...)
Because inhibition of IAPs with Smac permitted cytochrome c
to activate caspases in neuronally differentiated cells (), we examined the processing of caspases when Smac was added to the naïve and neuronally differentiated extracts. Cytochrome c
–mediated activation of the apoptosome was very efficient in naïve cell extracts as the apoptosome-processed, p35-kD form of processed caspase-9 predominated over the caspase-3 feedback-cleaved, p37-kD form of caspase-9 ( C). Because cleavage of caspase-9 may not necessarily correlate with caspase-9 activity (Stennicke et al., 1999
; Srinivasula et al., 2001
), we also examined the processing of caspase-3 as an indicator of caspase-9 activity. Cytochrome c
induced robust processing of caspase-3 in naïve cells, and relieving IAPs with Smac did not enhance this processing ( C).
In contrast, the apoptosome appeared highly inefficient in neuronally differentiated cells as very little caspase-9 processing, neither apoptosome mediated nor feedback cleaved by caspase-3, was detected when cytochrome c was added to these extracts ( C). Relieving IAP inhibition with wild-type AVPI-Smac in neuronally differentiated extracts permitted cytochrome c to activate a limited amount of caspase-3, as some caspase-3 feedback-cleaved form of caspase-9 (p37) was detected under these conditions ( C). Importantly however, the apoptosome remained highly inefficient even under these conditions, as no significant increase in the apoptosome-mediated processing of caspase-9 (p35) was observed even when Smac was added to neuronally differentiated extracts. Furthermore, the amount of processed caspase-3 that was observed in neuronally differentiated cells with Smac addition, although adequate to induce cell death, was significantly lower than seen in naïve cells ( C). Thus, although Smac addition resulted in an increase in caspase-3 activation sufficient for cell death in neuronally differentiated cells, a marked decrease in the efficiency of apoptosome activity occurred when naïve cells became neuronally differentiated.
Efficiency of apoptosome sets the threshold for IAPs to inhibit apoptosis in cells
If the differential ability of endogenous IAPs to regulate cytochrome c–mediated caspase activation in naïve and neuronally differentiated cells is linked to the differential efficiencies of the apoptosome in these two cell types, two simple predictions result from this model. First, reducing the efficiency of the apoptosome in naïve cells should allow endogenous IAPs to now tightly regulate cytochrome c–mediated caspase activation. Second, increasing the efficiency of the apoptosome in neuronally differentiated cells should eliminate the stringent IAP-mediated regulation of caspase activation seen normally in these cells.
To reduce apoptosome function in naïve cells, we simply decreased the amount of exogenous cytochrome c, thereby limiting the amount of apoptosome formation that could occur in these cells. Serial dilutions of cytochrome c were added to cytosolic lysate from naïve PC12 cells, causing a dose-dependent decrease in caspase activation ( A). To determine whether the reduced caspase activation was because of an increased ability of endogenous IAPs to inhibit caspase activation in these cells, we examined whether relieving IAP inhibition with Smac addition enhanced caspase activation seen with suboptimum cytochrome c. Indeed, addition of wild-type AVPI-Smac to lysates with suboptimum cytochrome c (100 nM) effectively increased the amount of caspase activation as compared with lysates with suboptimum cytochrome c alone ( B).
Figure 4. Reducing the apoptosome efficiency with addition of suboptimum cytochrome c permits endogenous IAPs to effectively block caspase activation and apoptosis in naïve cells. (A) Cytosolic lysates from naïve cells were incubated with serial (more ...)
Additionally, serial dilutions of cytochrome c were injected into intact naïve cells to identify a concentration of cytochrome c that was ineffective in inducing cell death ( C). At the suboptimal dose of 100 μg/ml cytochrome c, ~65% of naïve cells were still viable at 3 h. Importantly, coinjection of 100 μg/ml cytochrome c with wild-type AVPI-Smac significantly enhanced the amount of cell death, with <25% of cells remaining viable at 3 h ( D). Together, these results indicate that reducing the activity of the apoptosome alone in naïve cells was sufficient to allow endogenous IAPs to now effectively regulate caspase activation and apoptosis.
Neuronally differentiated cells have decreased apoptosome activity because of markedly reduced Apaf-1 levels
Next, we examined whether the reduced apoptosome activity in neuronally differentiated cells was due to limiting levels of Apaf-1 or procaspase-9. We found that levels of Apaf-1, but not caspase-9, were reduced by a striking 50% when naïve cells were neuronally differentiated for 12 d ( A).
Figure 5. A marked reduction in Apaf-1 levels causes the reduced apoptosome activity in neuronally differentiated cells. (A) Western blots comparing the levels of Apaf-1, caspase-9, and lactate dehydrogenase (LDH) in naïve and neuronally differentiated (more ...)
To determine whether the reduction in Apaf-1 levels was specifically responsible for the decreased apoptosome activity in neuronally differentiated cells, we examined the ability of wild-type, Apaf-1−/−, and caspase-9−/− cell lysates to complement the deficit in neuronally differentiated cell lysates. Cytosolic lysates from either wild-type, Apaf-1−/−, or caspase-9−/− fibroblast cells were incubated with neuronally differentiated cell lysates and activated in the presence of cytochrome c and dATP. Addition of wild-type or caspase-9−/− cell lysate to neuronally differentiated lysate allowed robust caspase activation with cytochrome c ( B), indicating that one or more components from these lysates were sufficient to complement the deficiency and activate caspases in neuronally differentiated cell lysate, without the need to overcome any IAP inhibition. Lysates from naïve cells also complemented the defect in neuronally differentiated cells (Fig. S2 B). In contrast, addition of Apaf-1−/− cell lysates failed to complement the apoptosome deficiency of neuronally differentiated cells, as no activation of caspases with cytochrome c were seen under these conditions ( B). The inability of Apaf-1−/− lysates to complement in this experiment was not because of a nonspecific defect in the preparation of these lysates, as addition of Apaf-1−/− lysates to caspase-9−/− lysates fully complemented each other and activated caspases with cytochrome c (Fig. S2 C). These complementation studies indicate that the inability of cytochrome c alone to activate caspases in neuronally differentiated cells was because of a specific reduction in Apaf-1 function in these cells.
Restoration of Apaf-1 levels in neuronally differentiated cells eliminates regulation by endogenous IAPs and sensitizes cells to cytochrome c
If the reduced apoptosome activity was crucial in enabling endogenous IAPs to effectively regulate apoptosis postcytochrome c
in neuronally differentiated cells, then increasing Apaf-1 levels should be sufficient to overcome the deficit in the apoptosome and permit cytochrome c
alone to activate caspases in neuronally differentiated cells. Although cytochrome c
alone did not induce caspase activation, addition of Apaf-1 protein was remarkably effective in permitting cytochrome c
to induce robust caspase activation in the neuronally differentiated cell lysates, without the need to overcome IAP inhibition ( C). The amount of Apaf-1 protein added (10 nM) was in the physiological range for restoring Apaf-1 levels in the neuronally differentiated lysates, as it was equivalent to the amount required for 50% of maximal activation in Apaf-1 knock-out fibroblast lysates (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200406073/DC1
). Also, the processing of caspase-9 seen in neuronally differentiated extracts to which Apaf-1 was added was indicative of its restored apoptosome efficiency with the p35 form (apoptosome processed) predominating just as was seen in naïve cell extracts.
We also examined whether expression of Apaf-1 permitted cytochrome c to induce apoptosis in intact neuronally differentiated cells. Neuronally differentiated cells were injected with plasmids expressing GFP and either Apaf-1 or vector alone. After allowing 24 h for expression, the GFP-positive cells were injected with cytochrome c to examine the ability of cytosolic cytochrome c to induce apoptosis in these cells. As anticipated, the neuronally differentiated cells expressing GFP and vector alone were resistant to cytosolic injection of cytochrome c. In contrast, increasing the levels of Apaf-1 permitted cytochrome c to induce apoptosis, as <20% of the Apaf-1–expressing cells remained viable 3 h after cytochrome c injections ( D). Injection of rhodamine dextran dye alone did not induce cell death in Apaf-1–overexpressing cells. To determine whether this effect was specific to Apaf-1, we examined whether overexpression of procaspase-9 also made the neuronally differentiated cells more vulnerable to cytosolic cytochrome c. No appreciable increase in cell death was observed when cytochrome c was injected in procaspase-9–overexpressing cells ( D). Thus, restoring Apaf-1 levels eliminated the stringent postcytochrome c regulation of apoptosis and allowed cytochrome c to induce apoptosis rapidly and without the need to overcome any endogenous IAP inhibition in neuronally differentiated cells.
Apaf-1 levels set the threshold for stringent postcytochrome c regulation of apoptosis by IAPs in primary mammalian cells
To test whether our model of coupling between Apaf-1 levels and IAP function can extend beyond the PC12 cell paradigm, we examined this in two different primary cell types. Our model predicts that reducing Apaf-1 levels in a primary cell would engage a strict regulation of cytochrome c–mediated apoptosis by endogenous IAPs where such a regulation is otherwise not detected. We examined this in cytosolic extracts of primary dermal fibroblasts isolated from wild-type and Apaf-1 heterozygous mice. Exogenous cytochrome c induced robust caspase activation in wild-type fibroblast lysates and addition of Smac did not enhance caspase activation, indicating that endogenous IAPs were ineffective in regulating caspase activation in wild-type fibroblasts ( A). In contrast, caspase activation with cytochrome c was significantly reduced in Apaf-1 heterozygous fibroblast lysates. Importantly, this reduction in cytochrome c–mediated caspase activation was not simply because of reduced Apaf-1 levels alone, but rather as a consequence of the resulting increased regulation by IAPs, as addition of Smac allowed greater activation of caspases in Apaf-1 heterozygous extracts ( B).
Figure 6. Reduction of Apaf-1 in primary fibroblasts allows strict IAP-mediated regulation of cytochrome c–mediated caspase activation. Cytosolic lysates were prepared from primary dermal fibroblasts from either wild-type (A) or Apaf-1 heterozygous (B) (more ...)
We also examined whether the ability of endogenous IAPs to strictly regulate apoptosis in primary sympathetic neurons (at P5) (Deshmukh et al., 2002
; Potts et al., 2003
) was a result of decreased Apaf-1 levels with neuronal differentiation. First, we examined sympathetic neurons isolated from embryonic day 16 (E16) mice, a point at which a majority of these neurons have begun postmitotic differentiation and are responsive to NGF (Wyatt and Davies, 1995
). After 2 d in culture, these cells were microinjected with cytochrome c
and were found to undergo rapid apoptosis with cytochrome c
alone ( A). In contrast, P3 sympathetic neurons maintained in culture for 2 d (P5 equivalent) were remarkably resistant to microinjection of cytochrome c
because of strict regulation by IAPs, as described previously (Deshmukh and Johnson, 1998
; Deshmukh et al., 2002
). E16 sympathetic neurons maintained in culture for 8 d (P5 equivalent) also became markedly resistant to cytochrome c
Figure 7. Modulation of Apaf-1 levels determines the strict IAP regulation of caspases in developing primary sympathetic neurons. (A) Sympathetic neurons from E16 and P3 mice were maintained in culture for 2 d and compared for the ability of cytochrome c to induce (more ...)
To determine whether the differential regulation of apoptosis by IAPs was linked to changes in Apaf-1 levels, we examined the levels of Apaf-1 protein in E16 and P3 sympathetic neurons after 2 d in culture. Levels of Apaf-1 were dramatically reduced in P3 as compared with E16 neurons ( B). In contrast, no differences in the levels of caspase-9, caspase-3, or IAPs, as well as Smac and HtrA2, were observed in E16 versus P3 sympathetic neurons ( B; Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200406073/DC1
). We examined whether increasing Apaf-1 levels specifically had the ability to overcome the strict IAP-mediated regulation of apoptosis seen in P5 sympathetic neurons. Primary P5 sympathetic neurons were injected with plasmids expressing GFP and either Apaf-1 or vector alone, and were subsequently reinjected with cytochrome c
to examine the ability of cytosolic cytochrome c
to induce apoptosis in these neurons. Increasing Apaf-1 levels was remarkably effective in permitting cytochrome c
to induce apoptosis, without the need to overcome endogenous IAPs in primary sympathetic neurons. Less than 20% of Apaf-1–injected neurons were viable 6 h after cytochrome c
injections (). Importantly, overexpression of caspase-9 was ineffective in changing the resistance of these neurons to cytochrome c
. These results show that primary sympathetic neurons develop IAP-mediated resistance with neuronal maturation by specifically down-regulating Apaf-1 levels.
Together, the results in primary fibroblasts and neurons are consistent with the data in the PC12 cells and are indicative of a novel common mechanism by which the effectiveness of endogenous IAPs in regulating apoptosis is coupled to Apaf-1 levels in mammalian cells.