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• W2045307092 abstract "The Drosophila steroid hormone ecdysone mediates cell death during metamorphosis by regulating the transcription of a number of cell death genes. The apical caspase DRONC is known to be transcriptionally regulated by ecdysone during development. Here we demonstrate that ecdysone also regulates the transcription of DRICE, a major effector caspase and a downstream target for DRONC in the fly. Using RNA interference in an ecdysone-responsive Drosophila cell line, we show that drice up-regulation is essential for apoptosis induced by ecdysone. We also show that drice expression is specifically controlled by the ecdysone-regulated transcription factor BR-C. Combined with previous observations, our results indicate that transcriptional regulation of the components of the core apoptotic machinery plays a key role in hormone-regulated programmed cell death during Drosophila development. The Drosophila steroid hormone ecdysone mediates cell death during metamorphosis by regulating the transcription of a number of cell death genes. The apical caspase DRONC is known to be transcriptionally regulated by ecdysone during development. Here we demonstrate that ecdysone also regulates the transcription of DRICE, a major effector caspase and a downstream target for DRONC in the fly. Using RNA interference in an ecdysone-responsive Drosophila cell line, we show that drice up-regulation is essential for apoptosis induced by ecdysone. We also show that drice expression is specifically controlled by the ecdysone-regulated transcription factor BR-C. Combined with previous observations, our results indicate that transcriptional regulation of the components of the core apoptotic machinery plays a key role in hormone-regulated programmed cell death during Drosophila development. Programmed cell death is necessary to delete superfluous cells in metazoans and to maintain homeostasis (reviewed in Refs. 1Lockshin R.A. Zakeri Z. Nat. Rev. Mol. Cell. Biol. 2001; 2: 545-550Crossref PubMed Scopus (273) Google Scholar and 2Zakeri Z. Lockshin R.A. J. Immunol. Methods. 2002; 265: 3-20Crossref PubMed Scopus (82) Google Scholar). The core cell death machinery, consisting of the BH3-only proteins, BCL-2 family, caspase adaptors, and caspases, is highly conserved and is present in all metazoan cells (reviewed in Refs. 3Adams J.M. Genes Dev. 2003; 17: 2481-2495Crossref PubMed Scopus (680) Google Scholar, 4Kumar S. Cakouros D. Trends Biochem. Sci. 2004; 29: 193-199Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 5Baehrecke E.H. Nat. Rev. Mol. Cell. Biol. 2002; 3: 779-787Crossref PubMed Scopus (335) Google Scholar, 6Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4060) Google Scholar). As most components of the cell death machinery are present constitutively within a cell, it is widely believed that execution of apoptosis is primarily regulated post-transcriptionally, that is apoptotic signals somehow feed into and activate preexisting caspase machinery. However, recent data suggest that many components of the core apoptosis machinery, including some caspases, are transcriptionally regulated during cell death and that the levels of the prosurvival and proapoptotic factors in the cell may be crucial to activate the apoptotic program (reviewed in Ref. 4Kumar S. Cakouros D. Trends Biochem. Sci. 2004; 29: 193-199Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Consistent with this, there is evidence that various signals such as cytotoxic insults, hormones, and growth factors regulate the activation of the death program by controlling the balance between prosurvival and proapoptotic proteins of the core cell death machinery (4Kumar S. Cakouros D. Trends Biochem. Sci. 2004; 29: 193-199Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To understand cell death regulation, it is thus essential to understand the transcriptional control of apoptosis execution. Steroid hormones are known to regulate cell survival and cell death in many tissues. Drosophila melanogaster is an ideal model system to study steroid hormone-regulated apoptosis as a single steroid hormone, 20-hydroxyecdysone, regulates cell death during development (reviewed in Refs. 5Baehrecke E.H. Nat. Rev. Mol. Cell. Biol. 2002; 3: 779-787Crossref PubMed Scopus (335) Google Scholar and 7Thummel C.S. Trends Genet. 1996; 12: 306-310Abstract Full Text PDF PubMed Scopus (413) Google Scholar, 8Baehrecke E.H. Cell Death Differ. 2000; 7: 1057-1062Crossref PubMed Scopus (81) Google Scholar, 9Truman J.W. Riddiford L.M. Annu. Rev. Entomol. 2002; 47: 467-500Crossref PubMed Scopus (322) Google Scholar). Ecdysone binds to its heterodimeric EcR/Usp receptor and transcriptionally regulates a number of primary response genes. Waves of ecdysone, produced at various times during fly development, regulate molting, cell proliferation, differentiation, and death in a highly controlled manner (5Baehrecke E.H. Nat. Rev. Mol. Cell. Biol. 2002; 3: 779-787Crossref PubMed Scopus (335) Google Scholar, 7Thummel C.S. Trends Genet. 1996; 12: 306-310Abstract Full Text PDF PubMed Scopus (413) Google Scholar, 8Baehrecke E.H. Cell Death Differ. 2000; 7: 1057-1062Crossref PubMed Scopus (81) Google Scholar, 9Truman J.W. Riddiford L.M. Annu. Rev. Entomol. 2002; 47: 467-500Crossref PubMed Scopus (322) Google Scholar). During the transition of larva into pupa, an ecdysone pulse toward the end of the third larval instar stage signals puparium formation, followed by a second pulse ∼12 h later, which initiates head eversion. During this process obsolete larval tissues, such as salivary glands and midgut, are deleted and replaced by adult tissues (5Baehrecke E.H. Nat. Rev. Mol. Cell. Biol. 2002; 3: 779-787Crossref PubMed Scopus (335) Google Scholar, 7Thummel C.S. Trends Genet. 1996; 12: 306-310Abstract Full Text PDF PubMed Scopus (413) Google Scholar, 8Baehrecke E.H. Cell Death Differ. 2000; 7: 1057-1062Crossref PubMed Scopus (81) Google Scholar, 9Truman J.W. Riddiford L.M. Annu. Rev. Entomol. 2002; 47: 467-500Crossref PubMed Scopus (322) Google Scholar). Cell death in the larval midgut begins in response to the late larval pulse of ecdysone while the salivary glands undergo removal around 15 h later in response to the second hormone pulse (5Baehrecke E.H. Nat. Rev. Mol. Cell. Biol. 2002; 3: 779-787Crossref PubMed Scopus (335) Google Scholar, 7Thummel C.S. Trends Genet. 1996; 12: 306-310Abstract Full Text PDF PubMed Scopus (413) Google Scholar, 8Baehrecke E.H. Cell Death Differ. 2000; 7: 1057-1062Crossref PubMed Scopus (81) Google Scholar, 9Truman J.W. Riddiford L.M. Annu. Rev. Entomol. 2002; 47: 467-500Crossref PubMed Scopus (322) Google Scholar). Recent data suggest that EcR/Usp and ecdysone-induced transcription factors including βFTZ-F1, BR-C, E74, E75, and E93 play a role in ecdysone-mediated cell death in the larval salivary gland and midgut (5Baehrecke E.H. Nat. Rev. Mol. Cell. Biol. 2002; 3: 779-787Crossref PubMed Scopus (335) Google Scholar, 8Baehrecke E.H. Cell Death Differ. 2000; 7: 1057-1062Crossref PubMed Scopus (81) Google Scholar, 10Jiang C. Baehrecke E.H. Thummel C.S. Development. 1997; 124: 4673-4683Crossref PubMed Google Scholar, 11Quinn L.M. Dorstyn L. Mills K. Colussi P.A. Chen P. Coombe M. Abrams J. Kumar S. Richardson H. J. Biol. Chem. 2000; 275: 40416-40424Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 12Chew S.K. Akdemir F. Chen P. Lu W.J. Mills K. Daish T. Kumar S. Rodriguez A. Abrams J.M. Dev. Cell. 2004; 7: 897-907Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 13Daish T.J. Mills K. Kumar S. Dev. Cell. 2004; 7: 909-915Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Studies, mostly with salivary glands, indicate that ecdysone controls the up-regulation of a number of proapoptotic genes such as rpr, hid, dark and dronc, and down-regulates the expression of death inhibitors such as diap1 and diap2 (14Jiang C. Lamblin A.F. Steller H. Thummel C.S. Mol. Cell. 2000; 5: 445-455Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 15Lee C. Cooksey B. Baehrecke E. Dev. Biol. 2002; 250: 101-111Crossref PubMed Scopus (180) Google Scholar, 16Lee C.Y. Baehrecke E.H. Cell Res. 2000; 10: 193-204Crossref PubMed Scopus (20) Google Scholar). Among the seven Drosophila caspases (17Kumar S. Doumanis J. Cell Death Differ. 2000; 7: 1039-1044Crossref PubMed Scopus (130) Google Scholar), ecdysone is well known to regulate dronc expression (18Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (240) Google Scholar, 19Cakouros D. Daish T. Martin D. Baehrecke E.H. Kumar S. J. Cell Biol. 2002; 157: 985-995Crossref PubMed Scopus (85) Google Scholar). DRONC is a CED3/caspase-9-like apical caspase that is essential for several programmed cell death pathways in the fly (17Kumar S. Doumanis J. Cell Death Differ. 2000; 7: 1039-1044Crossref PubMed Scopus (130) Google Scholar, 18Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (240) Google Scholar, 20Richardson H. Kumar S. J. Immunol. Methods. 2002; 265: 21-38Crossref PubMed Scopus (89) Google Scholar). Our previous data with DRONC suggest that ecdysone-mediated up-regulation of dronc is an important regulator of hormone-dependent cell death in Drosophila cells (19Cakouros D. Daish T. Martin D. Baehrecke E.H. Kumar S. J. Cell Biol. 2002; 157: 985-995Crossref PubMed Scopus (85) Google Scholar). One of the downstream targets of DRONC is the effector caspase DRICE (17Kumar S. Doumanis J. Cell Death Differ. 2000; 7: 1039-1044Crossref PubMed Scopus (130) Google Scholar). As DRICE is the major caspase-3-like effector enzyme in Drosophila, we tested whether it is also regulated by ecdysone. We report here that drice up-regulation by ecdysone plays an important role in hormone-dependent cell death. Ecdysone Treatment of Salivary Glands—Animals (W1118) were grown on bromphenol blue-supplemented food and staged by the gut clearance technique as previously described (13Daish T.J. Mills K. Kumar S. Dev. Cell. 2004; 7: 909-915Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 21Daish T.J. Cakouros D. Kumar S. Cell Death Differ. 2003; 10: 1348-1356Crossref PubMed Scopus (31) Google Scholar). Late third instar larvae with empty guts were collected, dissected in Schneiders cell medium (Invitrogen), and incubated with or without 1 mm ecdysone (Sigma) for 1 h at 25 °C. Cell Culture—Drosophila l(2)mbn cells, a kind gift from Dr. A. Dorn (Johannes Gutenburg University, Mainz, Germany) (22Ress C. Holtmann M. Maas U. Sofsky J. Dorn A. Tissue Cell. 2000; 32: 464-477Crossref PubMed Scopus (23) Google Scholar) were maintained as described previously (19Cakouros D. Daish T. Martin D. Baehrecke E.H. Kumar S. J. Cell Biol. 2002; 157: 985-995Crossref PubMed Scopus (85) Google Scholar). Cells, at 1 × 106/well, were seeded in six-well plates in triplicate or quadruplicate and allowed to recover for 3 days. Where necessary, ecdysone (10 μm) (Sigma) was added for the desired time. For RNA interference (RNAi) 1The abbreviations used are: RNAi, RNA interference; dsRNA, double-stranded RNA; DAPI, 4′-6-diamidino-2-phenylindole; amc, amino-methylcoumarin; RT-PCR, reverse transcription PCR. experiments, cells were treated with ecdysone until ∼50% of control cells were apoptotic as the rate of l(2)mbn apoptosis varies between batches. Apoptosis Detection—To assess apoptosis, cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) fluorescent dye. Briefly, cells were fixed and stained with 1 μg/ml DAPI, 20% formaldehyde, and mounted 1:1 with 80% glycerol. Changes in nuclear morphology were observed by fluorescent microscopy and apoptosis (%) was calculated as the proportion of cells with condensed chromatin in a total count of at least 300 cells. Data were derived from three or four experiments. Caspase Assays—Cell lysates were prepared by freeze thawing and clarified by centrifugation at 13,000 rpm for 5 min at 4 °C. Equal amounts of lysate were assayed for caspase activity using 100 μm VDVAD-amc and DEVD-amc substrates as described previously (23Dorstyn L. Mills K. Lazebnik Y. Kumar S. J. Cell Biol. 2004; 167: 405-410Crossref PubMed Scopus (99) Google Scholar), and the release of amc was measured using a fluorometric plate reader (PerkinElmer Life Sciences) (excitation 385 nm, emission 460 nm). RT-PCR Analysis—Total RNA was extracted from l(2)mbn cells using TRIzol reagent (Invitrogen) as per manufacturer's protocol. 1-5 μg of total RNA was used as a template for cDNA synthesis, in a 20-μl reaction with 500 ng of oligo(dT)18, using a Superscript II RNase H- Reverse Transcriptase kit (Invitrogen), according to manufacturer's protocol. Using 1.5 μl of 1:3 diluted cDNA template, PCR amplification was performed using appropriate primers in a 50-μl reaction employing 27-33 cycles. Drosophila rp49 was used as a control. Aliquots of PCR products (20 μl) were electrophoresed on 1.5-2% agarose gel for analysis. Immunoblotting—Cell lysates were separated by 10% SDS-PAGE, transferred onto polyvinylidene difluoride membrane (Schleicher & Schuell) and blocked for 1 h in 5% skim milk (pH 7.5). Affinity-purified anti-DRONC (24Dorstyn L. Read S. Cakouros D. Huh J.R. Hay B.A. Kumar S. J. Cell Biol. 2002; 156: 1089-1098Crossref PubMed Scopus (162) Google Scholar) was used at a 1:300 dilution, and DRICE antibody (24Dorstyn L. Read S. Cakouros D. Huh J.R. Hay B.A. Kumar S. J. Cell Biol. 2002; 156: 1089-1098Crossref PubMed Scopus (162) Google Scholar) was used at a 1:500 dilution. Secondary alkaline phosphatase-conjugated anti-rabbit antibody (Amersham Biosciences) was used at 1:2000 dilution. Signals were detected using ECF system (Amersham Biosciences). A cytochrome c antibody was purchased from Pharmingen and used at a 1:2000 dilution as described (23Dorstyn L. Mills K. Lazebnik Y. Kumar S. J. Cell Biol. 2004; 167: 405-410Crossref PubMed Scopus (99) Google Scholar, 24Dorstyn L. Read S. Cakouros D. Huh J.R. Hay B.A. Kumar S. J. Cell Biol. 2002; 156: 1089-1098Crossref PubMed Scopus (162) Google Scholar). RNAi—Regions of cDNA for drice (nucleotides 451-968), dcp-1 (nucleotides 921-1303), dronc (nucleotides 781-1047), E74A (nucleotides 2929-3248), E74B (nucleotides 1367-1724), E75A (nucleotides 706-1060), E75B (nucleotides 893-1240), E93 (nucleotides 397-862), βFTZ-F1 (nucleotides 39-530), and BR-C (nucleotides 392-1060) were PCR-amplified and cloned into pGEM-T Easy (Promega). Plasmids were linearized, and RNA was synthesized using T7 and SP6 Megascript kits (Ambion). Sense and antisense strands were annealed to generate dsRNA, and the quality of RNA was analyzed on a 2.2 m formaldehyde gel. dsRNA (∼40 nm) was added to cells in 1 ml of serum-free medium and mixed vigorously as described previously (23Dorstyn L. Mills K. Lazebnik Y. Kumar S. J. Cell Biol. 2004; 167: 405-410Crossref PubMed Scopus (99) Google Scholar, 24Dorstyn L. Read S. Cakouros D. Huh J.R. Hay B.A. Kumar S. J. Cell Biol. 2002; 156: 1089-1098Crossref PubMed Scopus (162) Google Scholar, 25Cakouros D. Daish T.J. Kumar S. J. Cell Biol. 2004; 165: 631-640Crossref PubMed Scopus (81) Google Scholar). Cells were incubated for 1 h followed by the addition of 2 ml of medium supplemented with 15% fetal bovine serum. Cells were incubated at 27 °C for 3 days and then harvested for 0-h time points or treated with ecdysone for the required length of time. drice Is Up-regulated in Salivary Glands Treated with Ecdysone—It has previously been shown that ecdysone treatment of late second instar larvae salivary glands results in the up-regulation of dronc mRNA (18Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (240) Google Scholar). Because the effector caspases are necessary for salivary gland death to occur (5Baehrecke E.H. Nat. Rev. Mol. Cell. Biol. 2002; 3: 779-787Crossref PubMed Scopus (335) Google Scholar, 8Baehrecke E.H. Cell Death Differ. 2000; 7: 1057-1062Crossref PubMed Scopus (81) Google Scholar) we investigated whether the major downstream effector caspase DRICE was also regulated by ecdysone. To determine whether drice mRNA expression is induced by ecdysone, dissected salivary glands from staged late third instar larvae were treated with ecdysone. After a 1-h ecdysone treatment a dramatic increase in drice mRNA levels was observed, demonstrating that ecdysone can induce drice expression in larval salivary glands (Fig. 1A). Caspase assays performed with lysates from ecdysone-treated salivary glands showed elevated caspase activity (Fig. 1B). In particular we observed a 4.5-fold increase in activity on DEVD-amc, which is a substrate for DRICE. Consistent with this observation, DRICE protein levels were elevated in extracts from ecdysone-treated salivary glands (Fig. 1C). These findings suggest the involvement of DRICE in ecdysone-regulated salivary gland death and more importantly demonstrate that drice mRNA is up-regulated by ecdysone. drice Is Induced by Ecdysone in l(2)mbn Cells—To further investigate the ecdysone-induction of drice transcription and dissect out its transcriptional regulation by various transcription factors we used the ecdysone-responsive Drosophila cell line l(2)mbn. We first analyzed ecdysone-mediated apoptosis by DAPI staining. As shown in Fig. 2A, apoptotic cells were identified by nuclear fragmentation. In the representative experiment shown in Fig. 2B, low levels of apoptosis were observed at 12- and 24-h post ecdysone treatment. From 36 h the number of cells undergoing apoptosis increased significantly, and by 48 h 70% of cells display condensed nuclei. Northern blot analysis indicated that l(2)mbn cells have low levels of drice transcript. Up-regulation of the drice transcript was evident 6 h after treatment and continued to increase with treatment time, being greatest at 48 h (Fig. 2C). Caspase activity on DEVD-amc and VDVAD-amc substrates was observed from 24 and 12 h, respectively, and subsequently increased with time (Fig. 2D). In addition, increased DRICE processing is observed around 36 h, and the accumulation of processed DRONC is evident at 12 h (Fig. 2E). The results with l(2)mbn cells confirm that drice is transcriptionally regulated by ecdysone treatment. In addition, our observations suggest that following ecdysone treatment of l(2)mbn cells, the increase in drice transcript levels correlates with increased DRICE and its processed form, DEVD-specific caspase activity, and apoptosis. DRICE Is Essential for Execution of Apoptosis in l(2)mbn Cells following Ecdysone Treatment—We next wanted to determine the importance of DRICE in ecdysone-induced apoptosis of l(2)mbn cells. To do this we used RNAi to ablate gene function. Because DCP-1 is also an important effector caspase in Drosophila it was of interest to test whether there was compensation between the two key effector caspases, DRICE and DCP-1. We also tested whether knockdown of both drice and dcp-1 could prevent apoptosis of l(2)mbn cells following ecdysone treatment. As a comparison we silenced the expression of the initiator caspase DRONC, shown previously to be important in ecdysone-induced l(2)mbn death (19Cakouros D. Daish T. Martin D. Baehrecke E.H. Kumar S. J. Cell Biol. 2002; 157: 985-995Crossref PubMed Scopus (85) Google Scholar). The knockdown of drice, dcp-1, both drice and dcp-1, or dronc by RNAi was confirmed by RT-PCR analysis using total RNA prepared from ecdysone-treated cells (Fig. 3A). At a time when ∼50% of control cells were undergoing apoptosis we were unable to detect significant levels of apoptosis in drice dsRNA-treated cells (Fig. 3B). The rate of ecdysone-induced apoptosis in dcp-1 knockdown cells was reduced to ∼50% of controls (Fig. 3B) and was reduced to ∼30% of controls in dronc knockdown cells (Fig. 3B). These results show that DRICE, DCP-1, and DRONC are all required for ecdysone to efficiently induce l(2)mbn apoptosis; however, ablation of drice had the most profound effect on ecdysone-mediated programmed cell death. DRICE Is Necessary for Maximal Ecdysone-induced Caspase Activity—Because DRICE function is essential for the efficient execution of apoptosis in ecdysone-treated l(2)mbn cells we wanted to establish its contribution to caspase activity. Caspase assays were performed with lysates using DEVD-amc and VDVAD-amc substrates (Fig. 4, A and B, respectively). In the absence of DRICE, lysates displayed basal levels of DEVDase activity. Ablation of dcp-1 resulted in ∼25% activity of controls. When both drice and dcp-1 were silenced DEVDase activity was virtually abolished (Fig. 4A). Previous studies have established that DCP-1 is a substrate for DRICE (26Fraser A.G. McCarthy N.J. Evan G.I. EMBO J. 1997; 16: 6192-6199Crossref PubMed Scopus (125) Google Scholar) and that DEVD-amc is a substrate for both DRICE and DCP-1. Because drice knockdown completely abolished DEVDase activity, whereas dcp-1 RNAi lysates still display DEVDase activity, these findings imply a necessity for DRICE in DCP-1 activation. Ablating dronc results in DEVDase activity that is ∼60% of controls (Fig. 4A). This is consistent with DRONC being an upstream activator of the effector caspases. When caspase assays were performed using a VDVAD-amc substrate, caspase activity following ecdysone treatment of drice or dcp-1 knockdown cells was only ∼20% of controls (Fig. 4B). Silencing both drice and dcp-1 resulted in basal VDVADase activity (Fig. 4B). In the absence of dronc expression ∼60% of control VDVADase activity was observed (Fig. 4B). Because VDVADase activity in part represents DRONC activity, these data suggest that DRICE and DCP-1 may affect DRONC activity. In addition, because activity on both DEVD-amc and VDVAD-amc substrates is still present in dronc knockdown cells, these data present evidence that DRICE activation may occur by means other than DRONC-mediated activation in l(2)mbn cells. Evidence for a Caspase Activation Loop in l(2)mbn Cells—To further characterize the effect of ablating drice gene function we analyzed protein expression and processing of DRICE and DRONC. In ecdysone-treated control cells we observe up-regulation of DRICE precursor and accumulation of its processed form (Fig. 4C, DRICE proc.). The ablation of either dcp-1 or dronc did not affect DRICE precursor levels; however, we see a reduced processing of DRICE. dronc RNAi had a more profound affect on DRICE processing than dcp-1 RNAi, thus both DRONC and DCP-1 may be capable of activating DRICE. As discussed previously, ecdysone treatment of l(2)mbn cells leads to an accumulation of processed DRONC (Fig. 4C, control lanes). We observe substantially reduced processed DRONC in the absence of DRICE (Fig. 4C). dcp-1 RNAi did not have any significant affect on processed DRONC levels. Because processed DRONC levels are significantly lower in drice-ablated ecdysone-treated cells, we propose that DRICE is required for amplification of DRONC processing following the initial activation of the upstream caspase. Ecdysone-induced Factors E74A, E74B, E75A, E75B, E93, or βFTZ-F1 Are Not Required for drice Up-regulation—Because we established drice up-regulation and the importance of DRICE in ecdysone-mediated apoptosis in l(2)mbn cells, we could now use this system to investigate the role of known ecdysone-induced transcription factors in drice transcription. We therefore carried out the RNAi ablation of several ecdysone-responsive transcription factors in l(2)mbn cells. The transcription factors E74A, E74B, E75A, E75B, E93, βFTZ-F1, and BR-C have been identified as important regulators of ecdysone-mediated salivary gland death (14Jiang C. Lamblin A.F. Steller H. Thummel C.S. Mol. Cell. 2000; 5: 445-455Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 15Lee C. Cooksey B. Baehrecke E. Dev. Biol. 2002; 250: 101-111Crossref PubMed Scopus (180) Google Scholar, 27Lee C.Y. Baehrecke E.H. Development. 2001; 128: 1443-1455PubMed Google Scholar, 28Martin D.N. Baehrecke E.H. Development. 2004; 131: 275-284Crossref PubMed Scopus (222) Google Scholar). In Fig. 5A we show the effect of ablating E74A, E74B, E75A, or E75B on ecdysone-induced l(2)mbn apoptosis. Compared with controls, induction of apoptosis by ecdysone treatment was not significantly affected by silencing any of these transcription factors. When we silence E93, ecdysone-induced apoptosis was reduced by 35% compared with controls (Fig. 5B). βFTZ-F1 RNAi did not affect apoptosis induction (Fig. 5B). Thus, of the transcription factors studied, only E93 was identified as being important for regulation of ecdysone-mediated apoptosis in l(2)mbn cells. We then analyzed drice transcription by RT-PCR. The ability of ecdysone to induce drice up-regulation was not affected by silencing E74A, E74B, E75A, E75B, E93, or βFTZ-F1 (Fig. 5, C and D). Because E93 RNAi did not affect drice transcription, the rate of apoptosis in E93 knockdown cells is likely to be reduced as a result of reduced dronc expression, which has been previously reported (15Lee C. Cooksey B. Baehrecke E. Dev. Biol. 2002; 250: 101-111Crossref PubMed Scopus (180) Google Scholar). DEVDase Activity Is Not Directly Affected by the Ecdysone-responsive Transcription Factors E74A, E74B, E75A, E75B, E93, or βFTZ-F1—Although drice transcription was not regulated by the ecdysone-responsive transcription factors E74A, E74B, E75A, E75B, E93, or βFTZ-F1, it was of interest to establish whether there was any effect on caspase activity. Analysis of DEVDase activity shows that E74A, E74B, E75A, or E75B RNAi-treated cells achieve similar levels of DEVDase activity as controls (Fig. 6A). E93 RNAi resulted in DEVDase activity that was ∼50% of control activity (Fig. 6B). Silencing βFTZ-F1 had no significant effect on DEVDase activity (Fig. 6B). E74A, E74B, E75A, E75B, or βFTZ-F1 ablation had no significant effect on VDVADase activity in l(2)mbn cells, whereas E93 RNAi showed significantly reduced levels of activity (Fig. 6, C and D). Thus ablation of E93, but not E74A, E74B, E75A, E75B, or βFTZ-F1, leads to reduced caspase activity in ecdysone-treated l(2)mbn cells consistent with the effects we see on apoptosis induction. In the absence of E74A, E74B, E75A, E75B, or βFTZ-F1, DRICE processing following ecdysone-induction of DRICE was relatively unchanged compared with controls (Fig. 6E). Although we see less processed DRICE in E93 RNAi lysates, induction of DRICE precursor is not significantly affected (Fig. 6E). Thus none of the transcription factors E74A, E74B, E75A, E75B, E93, or βFTZ-F1 regulate ecdysone-induced drice transcription or expression of this effector caspase. BR-C Regulates Ecdysone-induced drice Transcription—The ecdysone-induced transcription factor BR-C plays a key role in salivary gland death and is a regulator of dronc transcription (14Jiang C. Lamblin A.F. Steller H. Thummel C.S. Mol. Cell. 2000; 5: 445-455Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 19Cakouros D. Daish T. Martin D. Baehrecke E.H. Kumar S. J. Cell Biol. 2002; 157: 985-995Crossref PubMed Scopus (85) Google Scholar). There are four isoforms of BR-C protein, Z1-Z4 (29DiBello P.R. Withers D.A. Bayer C.A. Fristrom J.W. Guild G.M. Genetics. 1991; 129: 385-397Crossref PubMed Google Scholar). The Z2 isoform is constitutively present in l(2)mbn cells, and following ecdysone treatment the Z1, Z3, and Z4 isoforms are expressed (19Cakouros D. Daish T. Martin D. Baehrecke E.H. Kumar S. J. Cell Biol. 2002; 157: 985-995Crossref PubMed Scopus (85) Google Scholar). To test whether BR-C regulates ecdysone-induced drice transcription we carried out RNAi ablation of BR-C in l(2)mbn cells. BR-C RNAi significantly inhibited the rate of ecdysone-mediated apoptosis indicating that BR-C is an important regulator of ecdysone-induced apoptosis in l(2)mbn cells (Fig. 7A). Analysis of transcription by RT-PCR using total RNA shows that BR-C RNAi reduces ecdysone-induced transcription of drice (Fig. 7B). Activity on both DEVD-amc and VDVAD-amc substrates was reduced to ∼30% of control activity in l(2)mbn cells treated with BR-C dsRNA (Fig. 7, C and D) correlating with the effects seen on apoptosis induction. Immunoblot analysis indicated that processing of both DRICE and DRONC was markedly reduced upon knockdown of BR-C (Fig. 7E). Additionally, ecdysone-induction of full-length DRICE and DRONC was significantly reduced (Fig. 7E). Our results therefore demonstrate that BR-C is required for ecdysone-mediated regulation of drice transcription. The data presented here demonstrate that drice expression is up-regulated in response to ecdysone in both larval salivary glands and Drosophila l(2)mbn cells. The observations are consistent with a large scale gene expression analysis during salivary gland cell death, which shows that drice, among many other genes, is up-regulated in dying salivary glands (30Lee C.Y. Clough E.A. Yellon P. Teslovich T.M. Stephan D.A. Baehrecke E.H. Curr. Biol. 2003; 13: 350-357Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). When drice expression was ablated by RNAi, we observed low levels of caspase activity and a dramatic reduction in the rate of apoptosis following ecdysone treatment. In addition, our data suggest that DRICE is required for DRONC processing in ecdysone-treated l(2)mbn cells. Recent studies have shown that effector caspase activation in some tissues may occur in the absence of DRONC (13Daish T.J. Mills K. Kumar S. Dev. Cell. 2004; 7: 909-915Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 18Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (240) Google Scholar). Data in this paper complement these findings and supports the observation that some DRICE activation may occur in the absence of DRONC. Expression of the cell death genes rpr, hid, crq, dark, and dronc in salivary glands and midgut has been shown to be regulated by ecdysone-responsive transcription factors BR-C and E93 (14Jiang C. Lamblin A.F. Steller H. Thummel C.S. Mol. Cell. 2000; 5: 445-455Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 15Lee C. Cooksey B. Baehrecke E. Dev. Biol. 2002; 250: 101-111Crossref PubMed Scopus (180) Google Scholar, 16Lee C.Y. Baehrecke E.H. Cell Res. 2000; 10: 193-204Crossref PubMed Scopus (20) Google Scholar). The knockdown of BR-C in l(2)mbn cells reduced ecdysone-induced drice transcription and resulted in reduced DRICE expression, thus demonstrating that BR-C regulates drice transcription in l(2)mbn cells. E93 knockdown did not affect drice regulation following ecdysone treatment of l(2)mbn cells. As E93 regulates dronc expression (15Lee C. Cooksey B. Baehrecke E. Dev. Biol. 2002; 250: 101-111Crossref PubMed Scopus (180) Google Scholar, 21Daish T.J. Cakouros D. Kumar S. Cell Death Differ. 2003; 10: 1348-1356Crossref PubMed Scopus (31) Google Scholar, 31Lee C.Y. Wendel D.P. Reid P. Lam G. Thummel C.S. Baehrecke E.H. Mol. Cell. 2000; 6: 433-443Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the decrease in apoptosis levels that we observed is likely to be a result of reduced dronc transcription and protein expression. Although BR-C and E93 are required in both salivary gland and midgut apoptosis, their functions differ between the tissues. For example, dronc expression is regulated in salivary glands by both BR-C and E93 but only by E93 in the midgut (15Lee C. Cooksey B. Baehrecke E. Dev. Biol. 2002; 250: 101-111Crossref PubMed Scopus (180) Google Scholar, 33Broadus J. McCabe J.R. Endrizzi B. Thummel C.S. Woodard C.T. Mol. Cell. 1999; 3: 143-149Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). It would be interesting to test whether like dronc, BR-C regulation of drice is tissue-specific. The competence factor βFTZ-F1 is needed for the precise developmental re-expression of transcription factors including BR-C and E93 (32Lee C.Y. Simon C.R. Woodard C.T. Baehrecke E.H. Dev. Biol. 2002; 252: 138-148Crossref PubMed Scopus (98) Google Scholar). We investigated its involvement in ecdysone-induced l(2)mbn apoptosis. Our findings demonstrate no clear role for βFTZ-F1 in regulating the apoptosis of l(2)mbn cells. We speculate that in l(2)mbn cells, βFTZ-F1 is not required, because l(2)mbn cells do not undergo developmental transitions. Studies in this paper suggest that ecdysone-regulated DRICE is an essential effector of apoptosis execution in l(2)mbn cells. Based on the data presented here, we propose a model for DRICE-mediated amplification of the caspase cascade in ecdysone-treated cells (Fig. 8). Given that drice mutants are currently unavailable, specific ablation of this caspase in salivary glands and midgut may be required to further investigate the role of DRICE in ecdysone-induced programmed cell death in vivo. We thank Tasman Daish for technical help and Augustus Dorn for l(2)mbn cells." @default.
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• W2045307092 title "Ecdysone-mediated Up-regulation of the Effector Caspase DRICE Is Required for Hormone-dependent Apoptosis in Drosophila Cells" @default.
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