Báo cáo khoa học: Involvement of caspase 1 and its activator Ipaf upstream of mitochondrial events in apoptosis

Involvement of caspase 1 and its activator Ipaf upstream of mitochondrial events in apoptosis Subhash Thalappilly, Subhashini Sadasivam, Vegesna Radha and Ghanshyam Swarup

Centre for Cellular and Molecular Biology, Hyderabad, India

Keywords apoptosis; caspase 1; Ipaf; p53; protein tyrosine phosphatase

Correspondence G. Swarup, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India Fax: +91 40 2716 0591 Tel: +91 40 2716 022 E-mail: gshyam@ccmb.res.in

(Received 2 January 2006, revised 1 March 2006, accepted 25 April 2006)

doi:10.1111/j.1742-4658.2006.05293.x

PTP-S2 ⁄ TC45 is a nuclear protein tyrosine phosphatase that activates p53 and induces caspase 1-dependent apoptosis. We analyzed the role of ICE protease-activating factor (Ipaf), an activator of caspase 1 in p53-depend- ent apoptosis. We also determined the sequence of events that lead to apoptosis upon caspase 1 activation by Ipaf. PTP-S2 expression induced Ipaf mRNA in MCF-7 cells which was dependent on p53. PTP-S2-induced apoptosis was inhibited by a dominant-negative mutant of Ipaf and also by an Ipaf-directed short-hairpin RNA. Doxorubicin-induced apoptosis was potentiated by the expression of caspase 1 (but not by a catalytic mutant of caspase 1) and required endogenous Ipaf. Doxorubicin treatment of MCF-7 cells resulted in activation of exogenous caspase 1, which was partly dependent on endogenous Ipaf. An activated form of Ipaf induced caspase 1-dependent apoptosis that was inhibited by Bcl2 and also by a dominant inhibitor of caspase 9 (caspase 9s). Caspase 1-dependent apopto- sis induced by doxorubicin was also inhibited by Bcl2 and caspase 9s, but caspase 1 activation by activated Ipaf was not inhibited by Bcl2. Mitoch- ondrial membrane permeabilization was induced by caspase 1 and activated Ipaf, which was inhibited by Bcl2, but not by caspase 9s. Expression of caspase 1 with activated Ipaf resulted in the activation of Bax at mitochon- dria. Our results suggest that Ipaf is involved in PTP-S2-induced apoptosis and that caspase 1, when activated by Ipaf, causes release of mitochondrial proteins (cytochrome c and Omi) through Bax activation, thereby function- ing as an initiator caspase.

Abbreviations CARD, caspase-recruitment domain; DAPI, 4¢,6-diamidino-2-phenylindoledihydrochloride; DN-Ipaf, dominant-negative Ipaf; GFP, green fluorescent protein; IL-1b, interleukin-1 beta; Ipaf, ICE protease-activating factor; LRR, leucine-rich repeats; TNF, tumor necrosis factor.

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Apoptosis can be triggered in cells in response to either extrinsic signals (perceived through death receptors) or intrinsic signals, which generally act by causing the release of mitochondrial components [1]. These signals cause activation of initiator caspases through protein interaction domain-mediated molecular interactions. These caspases then cleave and activate the effector or executioner caspases which are responsible for target proteolysis and subsequent morphological changes associated with apoptosis [2,3]. Most of the mammalian caspases have been characterized with respect to their function as either initiator or executioner caspases [4]. Caspase 1 was the first member of the family to be identified [5] and several studies have shown its role in apoptosis in addition to its involvement in inflamma- tion [6,7]. Overexpression of caspase 1 induces apopto- sis in Rat1 cells and this cell death was inhibited by Bcl2 and CrmA [8]. Thymocytes of caspase 1-null mice were resistant to Fas-induced apoptosis. Interleukin-1 beta (IL-1b), IL-1a, tumor necrosis factor (TNF) and IL-6 export was impaired in caspase 1-null mice and these mice showed high resistance to endotoxic shock [9,10]. But the mechanism by which caspase 1 engages is apoptotic pathways in response to various stimuli

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Caspase 1 ⁄ Ipaf acts upstream of mitochondria

inhibitor of caspase 1. These observations implicated caspase 1 as an important mediator of PTP-S2-induced apoptosis [30,31].

In this study we investigated the mechanism by which caspase 1 is activated during p53-dependent apoptosis and how it engages apoptotic pathways. We show that Ipaf is induced and is involved in apoptosis caused by PTP-S2 overexpression. Caspase 1 activated by Ipaf acts upstream of mitochondria to cause release of proteins that are known to mediate apoptosis, sug- gesting its role as an apical caspase.

Results

Induction of Ipaf gene expression by PTP-S2 not understood. Like caspases 2 and 9, which are initi- ator caspases, caspase 1 has a caspase-recruitment domain (CARD) at the N-terminus, which is a protein interaction domain through which it is known to asso- ciate with several regulatory molecules [11]. Whereas some of these interacting molecules like ICE protease- activating factor (Ipaf) [12–14], Rip2 [15], ASC [16], PAK [17] and Nod1 [18] activate caspase 1, others such as COP [19], Iceberg [20] and CARD-8 [21] inhi- bit its activity, suggesting that its activation can be regulated stringently in response to various stimuli [6,11]. Caspase 1 expression is restricted to certain cell types. Although some stimuli are known to cause its direct activation, it is also regulated at the level of gene expression as it is induced in a variety of cell types in response to various stimuli.

cells apoptosis in MCF-7

Different activators may be involved in the activa- tion of caspase 1 in response to various stimuli. Ipaf is a recently characterized member of the NOD family of proteins that interacts with caspase 1 and activates it [12–14]. Ipaf has an N-terminal CARD, a central leucine-rich repeats NBD domain and a C-terminal (LRR) domain. Although the full-length Ipaf molecule can bind to caspase 1 through its CARD, the interac- tion does not result in processing or activation of caspase 1. However, the C-terminal truncated form of Ipaf that lacks the LRR domain can induce processing of caspase 1, a feature reminiscent of WD domain- deleted Apaf-1 molecule [12]. Ipaf knockout mice did show activation of caspase 1 in response to not Salmonella typhimurium infection [22]. Expression of Ipaf in immune cells also indicates a role for it in inflammatory process. It is involved in modulating IL-1b production by macrophages stimulated with lipopolysaccharide, peptidoglycan and invasive bacteria [23]. Ipaf is involved in caspase 8-mediated apoptosis when coexpressed with ASC [24]. TNF-a has been shown to induce Ipaf gene expression [25]. We have previously shown that both caspase 1 and Ipaf are direct transcriptional targets of p53 and that they play a role in p53-induced apoptosis [26,27].

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PTP-S2 ⁄ TC45 is a nuclear protein tyrosine phospha- tase that, upon overexpression, activates p53 to induce caspase 1 gene expression and apoptosis [30,31]. This apoptosis is strongly inhibited by blocking caspase 1 function, suggesting activation of caspase 1 under these conditions. The level of caspase 1 induced by PTP-S2 expression is very low compared with that achieved by caspase 1 overexpression. Because expres- sion of PTP-S2 induced much more apoptosis than did caspase 1 expression [31], it was likely that an acti- vator of caspase 1 may be induced by PTP-S2. Ipaf is an activator of caspase 1 which is induced by p53 [27]. We analyzed the expression of Ipaf mRNA during by PTP-S2-induced RT-PCR. The identity of the Ipaf PCR product was confirmed by Southern blot analysis with 32P-labeled Ipaf cDNA as the probe. Ipaf and caspase 1 mRNA levels were induced in cells that were transfected with PTP-S2 (Fig. 1A). The levels of PTP-S2 achieved by overexpression were shown by RT-PCR as well as western blotting. The antibody used in this experiment detects only murine proteins and therefore no endog- enous protein is seen (Fig. 1B) [32]. Quantitation of the PCR products showed an approximately 10-fold increase in levels of PTP-S2 upon overexpression. When p53DD (a dominant inhibitor of p53) [33] was coexpressed with PTP-S2 it reduced levels of caspase 1 and Ipaf mRNAs suggesting that Ipaf induction by PTP-S2 was dependent on p53. This was further exam- ined by using MCF-7 mp53 cells, a clone of MCF-7 cells expressing the His273 mutant of p53 which func- tions as dominant-negative mutant of p53 [31]. Induc- tion of Ipaf gene expression by PTP-S2 was abrogated in MCF-7 mp53 cells compared with the induction observed in MCF-7 cells suggesting that in these cells Ipaf induction is dependent on presence of functional p53 (Fig. 1C). PTP-S2 ⁄ TC45 is a ubiquitously expressed tyrosine phosphatase that binds to DNA and localizes to the nucleus in association with chromatin [28]. It plays a role in cell proliferation [29] and induces apoptosis upon overexpression in a p53-dependent manner [30]. We have previously shown that PTP-S2 expression results in p53 protein stabilization and activation of its targets. Forced expression of PTP-S2 caused caspase 1 gene upregulation dependent on functional p53 [31]. Apoptosis induced under these conditions was inhib- ited by Bcl2 as well as by peptide inhibitors of dominant-negative caspase 1 expression and of

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shRNA function in MCF-7

apoptosis was

shRNA-expressing of

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PTP-S2-induced apoptosis. This shRNA has previously been shown to inhibit expression of Ipaf protein upon coexpression with Ipaf construct [27]. PTP-S2 induced expression of cotransfected Ipaf mRNA in cells [34] when compared with with pro-mU6 vector untransfected MCF-7 cells. Cotransfection of shRNA- expressing vector abolished this upregulation, whereas a mutant shRNA construct did not have this effect. Levels of other mRNAs, such as caspase 1 and Apaf-1 which were upregulated by overexpression of PTP-S2, were not influenced by shRNA for Ipaf, showing the specificity of cells (Fig. 2B). The consequence of downregulating Ipaf on PTP-S2-induced examined. When cotransfected with pro-mU6 vector, 27% of PTP-S2- expressing cells were found to be apoptotic. But vector with cotransfection PTP-S2 (in a ratio of 1:1) reduced the apoptosis to (cid:1)12% (55% inhibition; P ¼ 1.97 · 10)5) (Fig. 2C). However, expression of mutant shRNA did not have any effect on PTP-S2-induced apoptosis as approxi- mately 27% of PTP-S2-expressing cells were found to exhibit apoptotic morphology. When cells were trans- fected with PTP-S2 and Ipaf shRNA at a ratio of 1:2, only (cid:1)9% cells were found to be apoptotic (66.6% inhibition, P ¼ 3.8 · 10)7) (Fig. 2C).

Fig. 1. p53-Dependent induction of Ipaf gene expression by PTP- S2. (A) MCF-7 cells were transfected with 500 ng each of Tet-Off and TRE-PTP-S2 plasmids, with 500 ng of pcDNA or p53DD. RNA was isolated from the cells 48 h after transfection and RT-PCRs were performed with respective primers for GAPDH, PTP-S2, Ipaf and caspase 1. The PTP-S2-specific primers amplify the rat and human isoform. To confirm the identity of Ipaf PCR product, South- ern blot analysis was carried out with 32P-labeled Ipaf cDNA as the probe. (B) Transfections were carried out as in (A) and lysates were prepared 48 h later. Western blots were carried out to detect levels of PTP-S2 and cdk2 proteins. (C) MCF-7 and MCF-7 mp53 cells were transfected with PTP-S2 expression plasmids and induced (+) or not induced (–) for PTP-S2 expression. Induction of PTP-S2 expression occurs upon removal of tetracycline. After 48 h of trans- fection, RNA was isolated and RT-PCR analysis was carried out for Ipaf and GAPDH.

Role of caspase 1 and Ipaf in doxorubicin-induced apoptosis

Inhibition of Ipaf function compromises PTP-S2-induced apoptosis

Dominant-negative Ipaf (DN-Ipaf), a truncated mole- cule lacking the central nucleotide-binding domain, can inhibit the function of full-length Ipaf [27]. In order to analyze the role of Ipaf in PTP-S2-induced apoptosis, PTP-S2 was cotransfected with DN-Ipaf or Ipaf. After 48 h of transfection, (cid:1)25% of PTP-S2- expressing cells showed apoptosis (Fig. 2A). Although expression of full-length Ipaf did not enhance PTP-S2- induced apoptosis, transfection of DN-Ipaf reduced to (cid:1)11% (56% inhibition, P ¼ this apoptosis 2.3 · 10)4) (Fig. 2A).

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shRNA targeted to Ipaf was also used to down- in expression and study its Ipaf role regulate Levels of caspase 1 and Ipaf are extremely low in MCF-7 cells and treatment with doxorubicin induces expression of caspase 1 and Ipaf. Although we were not able to detect changes in endogenous protein lev- els, we have previously shown the involvement of both in doxorubicin-induced apoptosis these molecules [26,27,31]. To determine whether expression of caspase 1 and Ipaf sensitizes cells to doxorubicin- induced apoptosis, MCF-7 cells were transfected with caspase 1 in the presence or absence of Ipaf, and after 24 h were treated with doxorubicin for a further 24 h. Cells were fixed and stained for caspase 1 and DNA, and apoptosis quantitated in expressing and non- expressing cells. Caspase 1 expression alone induced apoptosis in 10.4% of the cells; this was increased to 25.6% upon doxorubicin treatment (Fig. 3A). Expres- sion of Ipaf with caspase 1 further increased this apop- tosis to 33.5%. Under these conditions, doxorubicin alone induced apoptosis in (cid:1)15% cells. A catalytic mutant of procaspase 1 (Ala285) did not increase doxorubicin-induced apoptosis suggesting that the cat- alytic activity of caspase 1 was required to increase doxorubicin-induced apoptosis (Fig. 3A). Because Ipaf

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Fig. 2. Inhibition of Ipaf gene expression by shRNA and its effect on PTP-S2-induced apoptosis. (A) MCF-7 cells were transfected with 125 ng each of Tet-Off and TRE-PTP-S2 plasmids with 250 ng of Ipaf or DN-Ipaf expression vectors. Cells were fixed 48 h after transfection and immunostained for PTP-S2. DNA was stained with DAPI. The percentage of PTP-S2-expressing cells undergoing apop- tosis, averaged from three experiments, is represented. In order to determine the effect of Ipaf and DN-Ipaf expression alone on apop- tosis, these plasmids were transfected into MCF-7 cells with 10 ng GFP expression plasmid. Percentage of apoptotic cells among GFP-expressing cells were determined. (B) MCF-7 cells were trans- fected with 500 ng each of Tet-Off and TRE-PTP-S2 with 500 ng of pro mU6 vector or shRNA for Ipaf or mutant shRNA and expres- sion of various genes analyzed by RT-PCR as described in the legend to Fig. 1. 32P-labeled Ipaf cDNA was used as probe for Ipaf Southern blot analysis. (C) MCF-7 cells were transfected with TRE-PTP-S2 and Tet-Off with pro-mU6 vector or shRNA or mutant shRNA as required. For a 1:1 ratio, 125 ng of each plasmid was used, whereas for a 1:2 ratio, 100 ng each of Tet-Off and TRE-PTP-S2 plasmids and 200 ng of the other plasmids were used as required. Cells were fixed 48 h after transfection and immuno- stained for PTP-S2 and stained for DNA with DAPI. The data repre- sent percentage of PTP-S2-expressing cells undergoing apoptosis (n ¼ 3).

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gene expression is induced by doxorubicin, we ana- lyzed the role of Ipaf in caspase 1-dependent apoptosis induced by doxorubicin. Expression of Ipaf-directed shRNA resulted in a reduction in caspase 1-dependent apoptosis induced by doxorubicin from 16 to 7% (58% inhibition) (Fig. 3B). Mutant shRNA was used as a control for shRNA.

Activation of exogenous caspase 1 by doxorubicin

Activated Ipaf and caspase 1 induce apoptosis, which is inhibited by Bcl2 and caspase 9s

Ipaf, but not

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Because exogenous caspase 1 was able to increase doxorubicin-induced apoptosis, we determined whether caspase 1 was activated in this process. MCF-7 cells were transfected with caspase 1 and IL-1b. The pre- cursor of IL-1b is a known substrate for caspase 1 and processing of proIL-1b was used to determine caspase 1 activation [35]. After 24 h transfection, cells were treated with doxorubicin. Cell lysates were pre- pared 24 h later and western blot analysis was carried out to determine IL-1b levels. The level of mature IL-1b increased upon doxorubicin treatment in cells, suggesting that caspase 1 was activated by doxorubicin (Fig. 4A). Caspase 1-dependent formation of mature IL-1b was reduced upon coexpression of Ipaf-directed shRNA (Fig. 4B). These results suggest that activation of exogenous caspase 1 by doxorubicin treatment of cells was dependent, at least in part, on endogenous Ipaf. A truncated form of Ipaf (amino acids 1–656) lacking the C-terminal LRR domain has been shown to acti- vate caspase 1 by proteolytic autoprocessing [12]. This activated form of full-length Ipaf, increased caspase 1-induced apoptosis in MCF-7 cells (Fig. 5A). Even at a low plasmid concentration (10 ng), activated Ipaf and caspase 1 induced apoptosis (Fig. 5B). Expression of activated Ipaf alone (Fig. 5A) or with catalytically inactive mutant of caspase 1 (data not shown) did not induce any significant level of apoptosis in MCF-7 cells. The ability of activated Ipaf to cause caspase 1 processing and activation was

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Fig. 3. Effect of caspase 1 and Ipaf on doxorubicin-induced apoptosis. (A) Caspase 1 expression plasmid (10 ng) alone, or with 10 ng Ipaf expression plasmid, was transfected into MCF-7 cells. Transfected cells were left untreated or treated with 500 ngÆmL)1 doxorubicin 24 h after transfection. Cells were fixed 24 h later, stained for caspase 1 and DNA. Apoptosis was determined in caspase 1-expressing and -non- expressing cells (n ¼ 3). (B) MCF-7 cells were transfected with 10 ng caspase 1 expression plasmid with 500 ng shRNA for Ipaf or mutant shRNA. Cells were treated with doxorubicin as in (A) and apoptosis was quantitated. Bars represent the percentage of caspase 1-expressing cells undergoing apoptosis after subtraction of background apoptosis induced by doxorubicin in nonexpressing cells.

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determined by its ability to process IL-1b. Processing of caspase 1 was induced by activated Ipaf but not by full-length Ipaf as evidenced by the reduced levels of procaspase 1 (Fig. 5C). Enhanced formation of mature IL-1b showed that caspase 1 activity was increased by the activated form of Ipaf but not by full-length Ipaf (Fig. 5C).

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Fig. 4. Activation of exogenous caspase 1 by doxorubicin (A) MCF- 7 cells were transfected with 1 lg IL-1b plasmid alone or with 100 ng caspase 1 expression plasmid. After 24 h of transfection cells were either left untreated or treated with doxorubicin for 24 h. Cell lysates were subjected to western blotting to detect IL-1b and caspase 1 protein levels. (B) MCF-7 cells were cotransfected with 1 lg IL-1b and 100 ng caspase 1 expression plasmids with 900 ng of shRNA targeted to Ipaf or mutant shRNA. Cells were treated with doxorubicin for 24 h after transfection. Cell lysates were pre- pared after 24 h of treatment and western blotting was carried out to detect IL-1b and caspase 1 protein levels.

Two known inhibitors of the mitochondrial pathway of apoptosis, Bcl2 [1] and caspase 9s (an inhibitor of caspase 9) [36] were used to determine the mechanism by which activated Ipaf and caspase 1 induced apopto- sis. Caspase 1 and activated Ipaf cotransfected cells showed (cid:1)41% apoptotic cells 24 h after transfection (Fig. 6A). However, Bcl2 cotransfection reduced apop- tosis in these cells to (cid:1)13% (Fig. 6A,C). Thus Bcl2 is a potent inhibitor of apoptosis induced by activated Ipaf and caspase 1. Caspase 9s coexpression also reduced apoptosis by 50% (Fig. 6A). Similarly, both Bcl2 and caspase 9s inhibited apoptosis induced by caspase 1 and Ipaf upon treatment with doxorubicin (Fig. 6B). Because Bcl2 and caspase 9s

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inhibited activated Ipaf- and caspase 1-induced apoptosis, one possibility was that they inhibited the processing and activation of caspase 1 even in the presence of activated Ipaf. To examine this possibility, activated Ipaf, caspase 1 and IL-1b were transfected in MCF-7 cells and the effect of Bcl2 or caspase 9s was determined on processing of IL-1b. The level of mature IL-1b was not reduced by Bcl2 or caspase 9s (Fig. 6D). These results showed that Bcl2 or caspase 9s did not inhibit caspase 1 activation by activated Ipaf suggesting that caspase 1 activation takes place upstream of the sites of action of Bcl2 and caspase 9.

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Activated Ipaf and caspase 1 coexpression induces permeabilization of mitochondrial outer membrane

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Inhibition of activated Ipaf- and caspase 1-induced apoptosis by Bcl2 suggested the involvement of mitochondrial proteins [37,38]. In order to detect any mitochondrial permeability change during activated Ipaf–caspase 1-induced apoptosis, we monitored the release of cytochrome c and Omi. In activated Ipaf- and caspase 1-transfected cells, cytochrome c and Omi were present in cytoplasm at higher levels than in con- trol plasmid-transfected cells (Fig. 7B). The release of cytochrome c and Omi ⁄ HtrA2 from mitochondria dur- ing activated Ipaf–caspase 1-induced apoptosis was inhibited by the coexpression of Bcl2 but not by caspase 9s (Fig. 7B). These results showed that the release of mitochondrial proteins by expression of acti- vated Ipaf–caspase 1 does not require caspase 9 activ- ity. Cytochrome c release was also observed upon PTP-S2 overexpression (Fig. 7C). Coexpression of mutant caspase 1-inhibited cytochrome c release sug- gesting that activated caspase 1 mediates this event.

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non-GFP-expressing cells mostly

The effect of caspase 1 and activated Ipaf on mitochondrial permeability changes was also checked using the localization of cytochrome c and Omi by immunofluorescence. Cells transfected with caspase 1 and activated Ipaf with 10 ng green fluorescent protein (GFP) expression plasmid were fixed 24 h after trans- fection. They were then stained for cytochrome c and Omi. Several GFP-expressing cells showed diffused cytoplasmic staining of Omi as well as cytochrome c. The exhibited speckle-like staining of these proteins indicating their mitochondrial localization (Fig. 7A).

Fig. 5. Induction of caspase 1 activation and apoptosis by activated Ipaf. (A) MCF-7 cells were transfected with 250 ng of the indicated plasmids and extent of apoptosis determined in caspase 1-expres- sing cells. Apoptosis induced by Ipaf or activated Ipaf alone was determined by coexpressing these proteins with 10 ng of GFP and quantitating apoptosis in GFP-expressing cells. (B) Apoptosis induced by expression of 10 ng each of caspase 1 and activated Ipaf. (C) MCF-7 cells were transfected with indicated plasmids (IL-1b, 1 lg; caspase 1, Ipaf, activated Ipaf, 200 ng each). Lysates were prepared 24 h after transfection. Western blotting was carried out to determine levels of procaspase 1 and IL-1b. Tubulin levels were used as loading control.

The Bid protein, upon cleavage and activation by caspases, functions to initiate mitochondrial permeabil- ity changes [39]. Bid activation has been found to occur downstream of caspase 1 activation by Rip2 [40]. Therefore, we examined the possibility of induc- tion of Bid cleavage by caspase 1 activated by Ipaf. We observed a small fraction of processed Bid when cells were transfected with caspase 1 and activated Ipaf (Fig. 8A). Expression of caspase 1 alone resulted in lesser cleavage of Bid than observed in presence of caspase 1 and activated Ipaf. Co-expression of Bcl2 or caspase 9s did not affect Bid processing (Fig. 8B).

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Mitochondrial permeabilization generally requires the function of either Bax or Bak [39]. Activation of Bax involves a conformational change involving expo- sure of the N-terminal region [41]. In order to look at the activation of Bax downstream of caspase 1 activa- tion, we transfected MCF-7 cells with 200 ng activated

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Fig. 6. Bcl2 and caspase 9s inhibit caspase 1-mediated apoptosis. (A) Caspase 1 and activated Ipaf expression plasmids (125 ng each) were transfected in MCF-7 cells with 250 ng of pcDNA or Bcl2 or caspase 9s expression plasmids. Apoptosis among caspase 1-expressing cells was determined 24 h after transfection. (B) Caspase 1 and Ipaf plasmids (10 ng each) were transfected with 100 ng of pcDNA or Bcl2 or caspase 9s expression plasmids in MCF-7 cells. Cells were treated with doxorubicin for 24 h beginning the day after transfection. Cells were subsequently fixed and stained for caspase 1 by immunofluorescence using Cy3-conjugated secondary antibody and DNA was stained using DAPI. The data represent the percentage of caspase 1-expressing cells undergoing apoptosis after subtraction of background apoptosis induced by doxorubicin in nonexpressing cells. (C) Representative field of transfected cells showing morphology of caspase 1-expressing cells in the presence or absence of Bcl2. The experiment was performed as in (A). Arrows indicate apoptotic cells. (D) Caspase 1, activated Ipaf (250 ng each), IL-1b (1 lg) and GFP (100 ng) expression plasmids were transfected with 500 ng of pcDNA (lane 3) or Bcl2 (lane 4) or caspase 9s (lane 5) expression plasmids. In one set of cells, caspase 1, IL-1b and GFP plasmid were transfected with pcDNA (lane 2) and another set was transfected with GFP and IL-1b plasmids alone (lane 1). Lysates were made after 18 h and western blotting was carried out to detect levels of procaspase 1 and tubulin.

p53 is not activated. The cytoplasmic speckles formed by Bax were found to colocalize with mitochondria that were stained with Mitotracker Red (Fig. 9D).

Discussion

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Ipaf and 20 ng GFP with 200 ng of caspase 1 or transfection, we mutant caspase 1. After 18 h of observed a punctate staining pattern of Bax in several cells transfected with caspase 1 and activated Ipaf upon immunostaining (Fig. 9A,B). This staining pat- tern was observed in very few cells transfected with activated Ipaf and mutant caspase 1 or with GFP alone. Observation of Bax staining in activated Ipaf- and caspase 1-expressing cells was not due to an increase in the levels of Bax protein, as shown in Fig. 9C. Under these conditions we also did not observe stabilization of p53 protein suggesting that Induction of apoptosis by tumor suppressor p53 involves transcriptional activation of various target genes. Caspase 1 and Ipaf gene transcription is induced by p53 through binding sites in the minimal promoter [26,27]. A nuclear protein tyrosine phosphatase, PTP- S2 ⁄ TC45, which stabilizes and activates p53, induces

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Fig. 7. Coexpression of caspase 1 and activated Ipaf induces mitochondrial outer membrane permeability in MCF-7 cells. (A) Caspase 1 and activated Ipaf (100 ng each) were transfected in MCF-7 cells with 10 ng GFP expression plasmid. Cells were fixed 24 h after transfection and stained for cytochrome c or Omi by immunofluorescence using Cy3-conjugated secondary antibody to visualize the pattern of staining in GFP-expressing cells. DNA was stained with DAPI. Phase-contrast images of the corresponding fields are shown. Arrows indicate expres- sing cells that show release of cytochrome c or Omi. (B) Caspase 1 and activated Ipaf expression plasmids (500 ng each) were transfected in MCF-7 cells with 1 lg of pcDNA or Bcl2 expression plasmids. The cells were permeabilized 24 h after transfection with digitonin-contain- ing lysis buffer. The supernatants were resolved by SDS ⁄ PAGE and western blots were carried out for cytochrome c, Omi and mitochondrial protein Hsp60. A pellet of untransfected cells was used as a positive control for Hsp60. (C) Cells were transfected with 500 ng each of Tet- Off and pTRE-PTP-S2 plasmids with 1 lg of pcDNA or mutant caspase 1 expression plasmid. 42 h after transfection cells were fractionated as in (B) and western blots were carried out for cytochrome c and mitochondrial protein Hsp60. Tubulin was used as loading control.

is likely that it

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caspase 1 gene expression [31]. PTP-S2-induced apop- tosis is strongly inhibited by mutant caspase 1 [31]. Because expression of caspase 1 alone induced much less apoptosis, we looked for induction of an activator of caspase 1. We have shown that Ipaf gene expression was induced by PTP-S2 expression in a p53-dependent manner. Only a marginal increase in levels of ASC, another activator of caspase 1 was observed (data not shown). In addition, Ipaf gene expression contributes to PTP-S2-induced apoptosis as shown by the use of a dominant-negative mutant and an Ipaf-directed shRNA. The relative contribution of specific p53 tar- gets to apoptosis is cell type-, as well as inducer-, dependent because only a subset of the targets may be induced. We have previously shown that several other genes which are known p53 targets such as caspase 6, caspase 8, caspase 10, Bax and Puma do not show sig- nificant increase upon PTP-S2 overexpression [31]. Therefore, significant effects on apoptosis seen upon expression of Ipaf-directed shRNA reflect the contribu- tion of an Ipaf-mediated pathway. This pathway invol- ving caspase 1 and Ipaf may be a major player in PTP-S2-induced apoptosis in MCF-7. The Ipaf-direc- ted shRNA is quite specific because it did not inhibit the induction of caspase 1 gene expression by PTP-S2. Because both caspase 1 and Ipaf were induced by PTP-S2, and both are required for PTP-S2-induced apoptosis, these two proteins act together to induce apoptosis. However full-length Ipaf increase PTP-S2-induced apoptosis. These did not results suggest that the molecule(s) that activates Ipaf (or Ipaf–caspase 1 complex) [6] is induced by PTP-S2

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meabilization. The ability of PTP-S2 to trigger cyto- chrome c release dependent on caspase 1 function is indicative of a similar sequence of events being initi- ated by an inducer of caspase 1 and Ipaf.

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Fig. 8. Bid is processed in cells expressing caspase 1 and activated Ipaf. (A) MCF-7 cells were transfected with 500 ng caspase 1 and 1.5 lg pcDNA or 500 ng caspase 1 with 500 ng activated Ipaf and lysates were prepared, resolved by 1 lg pcDNA. After 18 h, cell SDS ⁄ PAGE and western blotting was carried out to detect proc- essed form of Bid. (B) Bid processing is not inhibited by coexpres- sion of either Bcl2 or caspase 9s with caspase 1 and activated Ipaf. Activated Ipaf and caspase 1 were transfected with 1 lg each of indicated plasmids and cell lysates were prepared 18 h later. Level of processed Bid was determined by western blotting.

Some other studies have also shown that caspase 1 can play a role upstream of mitochondria during apop- tosis. Studies on fumarylacetoacetate-induced apopto- sis in V79 cells have identified a role for caspase 1 upstream of mitochondria [43]. Apoptosis induced by oxygen–glucose deprivation of mouse cortical neurons depends on caspase 1 activation and Rip2 [40]. Bid activation was found to occur downstream of caspase 1 in these cells. Inhibition of caspase 1 has ear- lier been shown to prevent caspase 9 and caspase 3 activation in a transgenic mouse model [44]. In this system, they found that the time course of Bid clea- vage is more consistent with that of caspase 1 activa- tion rather than that of caspase 8. Because caspase 1 activated by Rip2 is known to function upstream of Bid activation to initiate mitochondrial permeability changes [40], a similar mechanism may be envisaged in the Ipaf ⁄ caspase 1-mediated pathway of apoptosis. Because only a minor component of Bid is cleaved, other mechanisms leading to mitochondrial changes may also be engaged.

and that Ipaf induced by PTP-S2 is not a limiting fac- tor for apoptosis. Under these conditions, exogenous Ipaf is unable to enhance the extent of apoptosis because either an activator of Ipaf or caspase 1 may be limiting. It has also been suggested that various components of the multiprotein complex must be pre- sent in precise stoichiometric concentrations for opti- mal assembly and activation of caspase 1 [42].

Our results show that caspase 1, activated by Ipaf, can cause Bid processing which might be partially responsible for triggering mitochondrial permeability changes. The processed form of Bid has been shown to induce cytochrome c release from mitochondria by causing Bax or Bak oligomerization [38,40]. Localiza- tion of N-terminus-exposed Bax to mitochondria in cells expressing caspase 1 and activated Ipaf implies its activation and possible function in release of cyto- chrome c [41,45]. Catalytic activity of caspase 1 was required for Bax activation because expression of an active site mutant of caspase 1 with activated Ipaf did not result in Bax activation. As activation of proapop- totic Bcl2 family proteins precedes mitochondrial per- meabilization during apoptosis, Bid cleavage and Bax activation provides additional evidence for role of Ipaf and caspase 1 upstream of mitochondria during apop- tosis.

caspase 9 was proteins, but

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An activated form of Ipaf lacking the C-terminal LRR domain was able to induce processing and acti- vation of caspase 1. This form of Ipaf could also increase caspase 1-dependent apoptosis at a fairly low level of expression. Because the mechanism by which Ipaf is activated in response to various stimuli is not understood, we studied the consequence of caspase 1 activation using forced expression of activated Ipaf. The apoptosis induced by activated Ipaf and caspase 1 involves the release of mitochondrial proteins and is also dependent on caspase 9. Bcl2 inhibited release of mitochondrial not required. Neither Bcl2 nor caspase 9s could protect against the processing of caspase 1 induced by activa- ted Ipaf. Our results suggest that caspase 1 activated by Ipaf acts upstream of mitochondrial membrane per- Earlier work from our laboratory has shown the involvement of caspase 1 and Ipaf in doxorubicin- induced apoptosis [26,27,31]. Ectopic expression of caspase 1, alone or with Ipaf, enhances the sensitivity of MCF-7 cells to doxorubicin-induced apoptosis. This suggests that these molecules are also activated upon doxorubicin treatment and can aid p53-dependent apoptosis. Using shRNA to reduce Ipaf expression, we recently demonstrated the role of endogenous Ipaf in apoptosis induced by doxorubicin treatment or p53

S. Thalappilly et al.

Caspase 1 ⁄ Ipaf acts upstream of mitochondria

B

A

C

D

Fig. 9. Bax activation is induced by expression of caspase 1 and activated Ipaf. (A) Cells were transfected with 200 ng caspase 1 or mutant caspase 1 and 200 ng activated Ipaf with 20 ng GFP or with GFP alone. The cells were fixed 18 h later and immunostained for Bax using Cy3-conjugated secondary antibody. DNA was stained using DAPI. Arrows indicate cells exhibiting punctate cytoplasmic staining. (B) Magni- fied image of cells with punctate Bax staining corresponding to the boxed region in (A). (C) Cells were transfected with 500 ng of activated Ipaf expression plasmid with 500 ng of caspase 1 or mutant caspase 1 expression plasmid. Cell lysates were prepared 18 h later and west- ern blots were carried out to detect the levels of Bax, p53 and cdk2 proteins. pcDNA3-transfected cells and untransfected cells were inclu- (D) Cells were ded as controls. Doxorubicin treated MCF-7 cells were used as positive control to show stabilization of p53 protein. transfected with 200 ng each of caspase 1 and activated Ipaf and stained for mitochondria using Mitotracker Red. Cells were subsequently fixed and immunostained for Bax using Alexa Fluor 488 conjugated secondary antibody and analyzed for Bax localization relative to mitochon- dria using a confocal microscope.

transfection for a further 24 h. The MCF-7-derived cell line MCF-7 mp53 has been described previously [26].

Expression vectors

overexpression in U2OS and A549 cells [27]. Earlier studies have shown the involvement of Ipaf in bacterial pathogen-induced caspase 1 activation and cell death [23]. In this study, we show for the first time its role in synergizing with caspase 1 and acting upstream of mitochondria to induce apoptosis.

Experimental procedures

Full-length Ipaf cDNA cloned in pcDNA3 with T7 tag at the N-terminus was a kind gift from E. S. Alnemri (Thomas Jefferson University, Philadelphia, PA). DN-Ipaf, which lacks the central nucleotide-binding domain, has been des- cribed previously [27]. Activated Ipaf lacking the C-terminal LRR domain was made by introducing a stop codon after 656 amino acids using PCR-based site-directed mutagenesis. Plasmids for expressing catalytically inactive mutant of human caspase 1, wild-type caspase 1 and PTP-S2 have been described previously [31]. PTP-S2 overexpression was achieved by transfection of pTet-off and TRE-PTP-S2 plas- mids, which result in PTP-S2 expression in the absence of tetracycline [31]. The shRNA expression vector, which spe- cifically downregulates Ipaf mRNA, has been described

line was maintained at 37 (cid:1)C in a CO2 The MCF-7 cell incubator (5% CO2) in Dulbecco’s modified Eagle’s med- ium with 10% fetal bovine serum (Invitrogen, San Diego, CA) supplemented with penicillin, streptomycin and kana- mycin (Sigma, St Louis, MO). Transfections were carried out with Lipofectamine 2000 reagent (Invitrogen) as per the manufacturer’s instructions. For doxorubicin treatment, 500 ngÆmL)1 doxorubicin (Sigma) was added 24 h after

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Cell culture and transfections

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Caspase 1 ⁄ Ipaf acts upstream of mitochondria

previously [27]. This is a U6 promoter-based vector that targets Ipaf sequence from nucleotides 1294 to 1312 (Acces- sion no. AY035391). A nonfunctional mutant of this shRNA, made by replacing two nucleotides, has also been described [27].

carried out

to detect

trypsinized after 24 h and suspended in permeabilization buffer (NaCl ⁄ Pi with 250 mm sucrose, 40 mm KCl, 10 mm Hepes and 100 lgÆmL)1 digitonin; Sigma). Under these conditions, > 95% cells were found to be permeabilized when stained with 0.2% Trypan Blue solution. After incu- bation for 1 min on ice, the cells were centrifuged at 12 000 g for 5 min at 4 (cid:1)C. Supernatant was collected as cytosol, separated on 13% SDS polyacrylamide gel and blotted to Immobilon-P (Millipore Corp., Bedford, MA). Western blotting was relevant proteins.

Immunofluorescence and apoptosis assays

(DAPI)

Western blotting analyzes to detect proteins were carried out as described previously [30]. Briefly, cells were lysed in Laemli’s SDS sample buffer. The proteins were resolved by SDS ⁄ PAGE and transferred on to nitrocellulose membrane or Immobilon-P by semidry electroblotting. The blots were blocked with 5% Blotto (Santa Cruz Biotechnologies) and incubated with required antibodies and developed using chemiluminescence (NEN, Boston, MA). Caspase 1, IL-1b, and cytochrome c antibodies were from Santa Cruz. Tubu- lin antibodies were from Amersham Pharmacia Biotech (Piscataway, NJ). Bid antibody that recognizes both full- length Bid and t-Bid was obtained from R&D Systems the (Minneapolis, MN). Bax antibody raised against N-terminal epitope was obtained from Upstate Biotechnol- ogy (Lake Placid, NY). The overexpressed murine PTP-S2 protein was detected on western blot using the G11 mouse monoclonal antibody [32] which does not recognize the endogenous human protein.

Western blotting

Acknowledgements

Analysis of apoptosis was carried out as described previ- ously [30,46]. Cells grown on cover slips were transfected with the required plasmids, and fixed in 3.7% formaldehyde (Qualigens, Mumbai, India) for the specified time. For im- munostaining of proteins, the cells were permeabilized with 0.5% Triton X-100 (Sigma) and 0.05% Tween 20 (Sigma) in NaCl ⁄ Pi. Blocking was carried out with 2% bovine serum albumin (Sigma) in NaCl ⁄ Pi for 1 h. Cells were incu- bated with the appropriate primary and secondary anti- bodies as required and mounted in 90% glycerol with 1 mgÆmL)1 paraphenylenediamine (Sigma) and 0.5 lgÆmL)1 4¢,6-diamidino-2-phenylindoledihydrochloride for visualizing DNA. Primary antibodies used were against cas- pase 1 (Santa Cruz Biotechnologies, Santa Cruz, CA), cyto- chrome c (Santa Cruz Biotechnologies) and Omi. Detection of these proteins was carried out using FITC- (Bangalore Genie, Bangalore, India) or Cy3- (Amersham Pharmacia Biotech, Piscataway, NJ) conjugated secondary antibodies. In some experiments GFP coexpression (10 ng) was used to detect expressing cells. Apoptotic cells were detected by shrinkage, lower refractility and condensed chromatin. At least 200 expressing cells were counted from each cover slip. Mean and standard deviation of percentage of apoptotic cells from at least three independent experiments performed in duplicate were used in analysis. Untransfected apoptotic cells were also counted from each cover slip and this was used to calculate background apoptosis. For mitotracker staining, transfected cells, after the required time were incu- bated with 500 nm Mitotracker Red (Molecular Probes, Eugene, OR) for 25 min and subsequently fixed and proc- essed for immunofluorescence.

This work was supported by a grant from Department of Biotechnology, Government of India to GS and VR. ST and SS gratefully acknowledge research fellow- ship from Council for Scientific and Industrial Research, India. RT-PCR

References

1 Danial NN & Korsmeyer SJ (2004) Cell death: critical

control points. Cell 116, 205–219.

Total RNA was isolated from appropriately treated or transfected cells grown in 35 mm dishes using TRIzol rea- gent (Invitrogen) Semiquantitative PCR was performed as described previously [26]. Primers used for amplifying dif- ferent genes have been described previously [27]. GAPDH amplicons were used as controls for input RNA.

2 Boatright KM & Salvesen GS (2003) Mechanisms of caspase activation. Curr Opin Cell Biol 15, 725–731. 3 Boatright KM, Renatus M, Scott FL, Sperandio S, Shin H, Pedersen IM, Ricci JE, Edris WA, Sutherlin DP, Green DR et al. (2003) A unified model for apical caspase activation. Mol Cell 11, 529–541.

4 Degterev A, Boyce M & Yuan J (2003) A decade of cas-

pases. Oncogene 22, 8543–8567.

A digitonin-based permeabilization technique was used to release cytosol from cells [47]. Transfected cells were

FEBS Journal 273 (2006) 2766–2778 ª 2006 The Authors Journal compilation ª 2006 FEBS

2776

Preparation of cytosolic extracts for western blot analysis of cytochrome c

S. Thalappilly et al.

Caspase 1 ⁄ Ipaf acts upstream of mitochondria

with pro-caspase-1. Biochem Biophys Res Commun 299, 652–658.

19 Lee SH, Stehlik C & Reed JC (2001) Cop, a caspase

5 Yuan J, Shaham S, Ledoux S, Ellis HM & Horvitz HR (1993) The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1b-converting enzyme. Cell 75, 641–652.

recruitment domain-containing protein and inhibitor of caspase-1 activation processing. J Biol Chem 276, 34495–34500.

6 Martinon F & Tschopp J (2004) Inflammatory caspases: linking an intracellular innate immune system to autoin- flammatory diseases. Cell 117, 561–574.

20 Humke EW, Shriver SK, Starovasnik MA, Fairbrother WJ & Dixit VM (2000) ICEBERG: a novel inhibitor of interleukin-1beta generation. Cell 103, 99–111.

21 Razmara M, Srinivasula SM, Wang L, Poyet JL, Ged- des BJ, DiStefano PS, Bertin J & Alnemri ES (2002) CARD-8 protein, a new CARD family member that regulates caspase-1 activation and apoptosis. J Biol Chem 277, 13952–13958.

22 Mariathasan S, Newton K, Monack DM, Vucic D,

7 Kondo S, Barna BP, Morimura T, Takeuchi J, Yuan J, Akbasak A & Barnett GH (1995) Interleukin-1 beta- converting enzyme mediates cisplatin-induced apoptosis in malignant glioma cells. Cancer Res 55, 6166–6171. 8 Miura M, Zhu H, Rotello R, Hartwieg EA & Yuan J (1993) Induction of apoptosis in fibroblasts by IL-1b- converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75, 653–660.

9 Kuida K, Lippke JA, Ku G, Harding MW, Livingston

DJ, Su MS & Flavell RA (1995) Altered cytokine export and apoptosis in mice deficient in interleukin-1b converting enzyme. Science 267, 2000–2003.

French DM, Lee WP, Roose-Girma M, Erickson S & Dixit VM (2004) Differential activation of the inflam- masome by caspase-1 adaptors ASC and Ipaf. Nature 430, 213–218.

10 Li P, Allen H, Banerjee S & Seshadri T (1997) Charac- terization of mice deficient in interleukin-1b converting enzyme. J Cell Biochem 64, 27–32.

23 Damiano JS, Newman RM & Reed JC (2004) Multiple roles of CLAN (caspase-associated recruitment domain, leucine-rich repeat, and NAIP CIIA HET-E, and TP1- containing protein) in the mammalian innate immune response. J Immunol 173, 6338–6345.

11 Reed JC, Doctor KS & Godzik A (2004) The domains of apoptosis: a genomics perspective. Sci STKE 239, doi: 10.1126/stke.2392004re9.

12 Poyet JL, Srinivasula SM, Tnani M, Razmara M, Fern- andes-Alnemri T & Alnemri ES (2001) Identification of Ipaf, a human caspase-1-activating protein related to Apaf-1. J Biol Chem 276, 28309–28313.

24 Masumoto J, Dowds TA, Schaner P, Chen FF, Ogura Y, Li M, Zhu L, Katsuyama T, Sagara J, Taniguchi S et al. (2003) ASC is an activating adaptor for NF-kappa B and caspase-8-dependent apoptosis. Biochem Biophys Res Commun 303, 69–73.

13 Damiano JS, Stehlik C, Pio F, Godzik A & Reed JC

(2001) CLAN, a novel human CED-4-like gene. Geno- mics 75, 77–83.

25 Gutierrez O, Pipaon C & Fernandez-Luna JL (2004) Ipaf is upregulated by tumor necrosis factor-alpha in human leukemia cells. FEBS Lett 568, 79–82.

26 Gupta S, Radha V, Furukawa Y & Swarup G (2001) Direct transcriptional activation of human caspase-1 by tumor suppressor p53. J Biol Chem 276, 10585– 10588.

14 Geddes BJ, Wang L, Huang WJ, Lavellee M, Manji GA, Brown M, Jurman M, Cao J, Morgenstern J, Merriam S et al. (2001) Human CARD12 is a novel CED4 ⁄ Apaf-1 family member that induces apoptosis. Biochem Biophys Res Commun 284, 77–82.

27 Sadasivam S, Gupta S, Radha V, Batta K, Kundu TK & Swarup G (2005) Caspase-1 activator Ipaf is a p53- inducible gene involved in apoptosis. Oncogene 24, 627– 636.

28 Kamathar S, Radha V, Nambirajan S, Reddy RS &

15 Thome M, Hofmann K, Burns K, Martinon F, Bodmer JL, Mattmann C & Tschopp J (1998) Identification of CARDIAK, a RIP-like kinase that associates with cas- pase-1. Curr Biol 8, 885–888.

16 Srinivasula SM, Poyet JL, Razmara M, Datta P, Zhang Z & Alnemri ES (2002) The PYRIN-CARD protein ASC is an activating adaptor for caspase-1. J Biol Chem 277, 21119–21122.

Swarup G (1996) Two splice variants of a tyrosine phos- phatase differ in substrate specificity, DNA binding and subcellular location. J Biol Chem 271, 26755–26761. 29 Radha V, Nambirajan S & Swarup G (1997) Overex- pression of a nuclear protein tyrosine phosphatase increases cell proliferation. FEBS Lett 409, 33–36.

30 Radha V, Sudhakar C & Swarup G (1999) Induction of p53 dependent apoptosis upon overexpression of a nuclear protein tyrosine phosphatase. FEBS Lett 453, 308–312.

17 Basak C, Pathak SK, Bhattacharyya A, Mandal D, Pathak S & Kundu M (2005) NF-jB- and C ⁄ EBPb- driven interleukin-1b gene expression and PAK1- mediated caspase-1 activation play essential roles in interleukin-1b release from Helicobacter pylori lipopoly- saccharide-stimulated macrophages. J Biol Chem 280, 4279–4288.

18 Yoo NJ, Park WS, Kim SY, Reed JC, Son SG, Lee JY & Lee SH (2002) Nod1, a CARD protein, enhances pro-interleukin-1beta processing through the interaction

31 Gupta S, Radha V, Sudhakar Ch & Swarup G (2002) A nuclear protein tyrosine phosphatase activates p53 and induces caspase-1-dependent apoptosis. FEBS Lett 532, 61–66.

FEBS Journal 273 (2006) 2766–2778 ª 2006 The Authors Journal compilation ª 2006 FEBS

2777

S. Thalappilly et al.

Caspase 1 ⁄ Ipaf acts upstream of mitochondria

32 Radha V, Nambirajan S & Swarup G (1994) Subcellular

ischemia-induced neuronal cell death. Proc Natl Acad Sci USA 100, 16012–16017.

41 Schinzel A, Kaufmann T, Schuler M, Martinalbo J,

localization of a protein tyrosine phosphatase: evidence for association with chromatin. Biochem J 299, 41–47.

33 Shaulian E, Zauberman A, Ginsberg D & Oren M

Grubb D & Borner C (2004) Conformational control of Bax localization and apoptotic activity by Pro168. J Cell Biol 164, 1021–1032.

(1992) Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding. Mol Cell Biol 12, 5581– 5592.

42 Bruey JM, Bruey-Sedano N, Newman R, Chandler S, Stehlik C & Reed JC (2004) PAN1 ⁄ NALP2 ⁄ PYPAF2, an inducible inflammatory mediator that regulates NF-kappaB and caspase-1 activation in macrophages. J Biol Chem 279, 51897–51907.

34 Yu JY, DeRuiter SL & Turner DL (2002) RNA inter- ference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 99, 6047–6052.

43 Jorquera R & Tanguay RM (1999) Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis. FASEB J 13, 2284–2298.

35 Thornberry NA, Bull HG, Calaycay JR, Chapman KT, Howard AD, Kostura MJ, Miller DK, Molineaux SM, Weidner JR, Aunins J et al. (1992) A novel heterodi- meric cysteine protease is required for interleukin-1b processing in monocytes. Nature 356, 768–774.

36 Seol DW & Billiar TR (1999) A caspase-9 variant miss- ing the catalytic site is an endogenous inhibitor of apop- tosis. J Biol Chem 274, 2072–2076.

37 Breckenridge DG & Xue D (2004) Regulation of

mitochondrial membrane permeabilization by BCL-2 family proteins and caspases. Curr Opin Cell Biol 16, 647–652.

44 Guegan C, Vila M, Teismann P, Chen C, Onteniente B, Li M, Friedlander RM & Przedborski S (2002) Instru- mental activation of bid by caspase-1 in a transgenic mouse model of ALS. Mol Cell Neurosci 20, 553–562. 45 Desagher S, Osen-Sand A, Nichols A, Eskes R, Montes- suit S, Lauper S, Maundrell K, Antonsson B & Marti- nou JC (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 144, 891–901. 46 Shivakrupa R, Radha V, Sudhakar Ch & Swarup G

(2003) Physical and functional interaction between Hck tyrosine kinase and guanine nucleotide exchange factor C3G results in apoptosis, which is independent of C3G catalytic domain. J Biol Chem 278, 52188–52194.

38 Green DR & Kroemer G (2004) The pathophysiology of mitochondrial cell death. Science 305, 626–629. 39 Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ & Schlesinger PH (2000) Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ 7, 1166–1173.

47 Kim HS, Ingermann AR, Tsubaki J, Twigg SM, Walker GE & Oh Y (2004) Insulin-like growth factor-binding protein 3 induces caspase-dependent apoptosis through a death receptor-mediated pathway in MCF-7 human breast cancer cells. Cancer Res 64, 2229–2237.

40 Zhang WH, Wang X, Narayanan M, Zhang Y, Huo C, Reed JC & Friedlander RM (2003) Fundamental role of the Rip2 ⁄ caspase-1 pathway in hypoxia and

FEBS Journal 273 (2006) 2766–2778 ª 2006 The Authors Journal compilation ª 2006 FEBS

2778