Down-regulation of inhibitor of apoptosis levels provides competence for steroid-triggered cell death - PubMed (original) (raw)

Down-regulation of inhibitor of apoptosis levels provides competence for steroid-triggered cell death

Viravuth P Yin et al. J Cell Biol. 2007.

Abstract

A pulse of the steroid hormone ecdysone triggers the destruction of larval salivary glands during Drosophila metamorphosis through a transcriptional cascade that converges on reaper (rpr) and head involution defective (hid) induction, resulting in caspase activation and cell death. We identify the CREB binding protein (CBP) transcriptional cofactor as essential for salivary gland cell death. We show that CBP acts 1 d before the onset of metamorphosis in apparent response to a mid-third instar ecdysone pulse, when CBP is necessary and sufficient for down-regulation of the Drosophila inhibitor of apoptosis 1 (DIAP1). It is only after DIAP1 levels are reduced that salivary glands become competent to die through rpr/hid-mediated cell death. Before this time, high levels of DIAP1 block salivary gland cell death, even in the presence of ectopic rpr expression. This study shows that naturally occurring changes in inhibitor of apoptosis levels can be critical for regulating cell death during development. It also provides a molecular mechanism for the acquisition of competence in steroid signaling pathways.

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Figures

Figure 1.

Figure 1.

Mutations in CBP block ecdysone-triggered programmed cell death of larval salivary glands. (A–D) Living animals assayed 24 h APF with a GFP reporter expressed in larval salivary glands. (A) Salivary glands are no longer detectable in control pupae (w; UAS-GFP; SG-GAL4) but persist in CBP mutants (CBPG0350; UAS-GFP/+; SG-GAL4/+) (B) or salivary glands in which CBP is inactivated by RNAi (w; UAS-CBP-RNAi/ UAS-GFP; SG-GAL4/+) (C) or in which diap1 is overexpressed (w; UAS-diap1/UAS-GFP; SG-GAL4/+) (D). (E and F) Larval salivary glands dissected at 0 or 13.5 h APF from controls or 0-, 13.5-, or 16-h CBPG0350 animals (G–I). (J and K) Control and CBPG0350 mutant (L–N) salivary glands stained for the cleaved active form of caspase-3.

Figure 2.

Figure 2.

CBP regulates diap1 mRNA levels in larval salivary glands. Northern blots from control (left) and CBPG0350 mutant (right) larval salivary glands staged at 10, 12, 14, or 16 h APF, probed for key cell death regulators. Death activators rpr and hid, the caspase gene dronc, and the Apaf-1 gene dark are all induced normally in mutant salivary glands, whereas diap1 mRNA is selectively up-regulated. rp49 is a loading control. The kinetics of rpr and hid induction in control and mutant salivary glands were confirmed by Northern blot analysis of four independent RNA samples.

Figure 3.

Figure 3.

CBP is necessary and sufficient for diap1 down-regulation in response to ecdysone during the mid-third instar transition. (A–F) DIAP1 antibody stains of larval salivary glands dissected from animals that span the mid-L3 transition (larva staged relative to the L2–L3 molt). (A and B) DIAP1 levels drop between 20–24 and 34–38 h in control glands (w) but not in CBPG0350 mutant salivary glands (C and D). (E) Ectopic expression of CBP (w; hs-CBP) in 12-h L3 animals is sufficient to down-regulate DIAP1 expression at 20–24 h, whereas removal of EcR by RNAi blocks DIAP1 down-regulation (F). (G) Northern blots of RNA isolated from wild-type salivary glands from before (20–24 h) or after (34–38 h) the mid-L3 transition, probed to detect CBP, diap1, ng-1, and Sgs-4 mRNA, with rp49 as a control. (H) Organs dissected from L3 were cultured in the presence of 20-hydroxyecdysone (20E), cycloheximide (cyc), or 20E and cycloheximide for 2 or 6 h, and total RNA was extracted for Northern blot hybridization to detect CBP and rp49 mRNA.

Figure 4.

Figure 4.

Down-regulation of diap1 confers competence for _rpr_-mediated cell death. Salivary glands dissected from control (w) (A–D) first (L1), second (L2), early third and late third (L3) instar larvae or animals at the same stages after heat-induced rpr overexpression (w; hs-rpr) (E–H) were stained for DIAP1 protein (A–D) or cleaved active caspase-3 (E–H). DIAP1 levels drop during L3 development. Cell death in response to rpr overexpression only occurs when DIAP1 levels are low in late L3.

Figure 5.

Figure 5.

Competence for _rpr_-mediated cell death occurs after the mid-third instar transition. Salivary glands were dissected from heat-treated third instar larvae (L3) at either 20–24 h after the L2–L3 molt (A, C, and E) or 34–38 h after the molt (B, D, and F) and stained with antibodies directed against cleaved active caspase-3. Heat-treated control (w) L3 (A and B) and hs-rpr transformants at 20–24 h after the molt (C) display no staining for active caspase-3, whereas salivary glands at 34–38 h after the molt are susceptible to _hs-rpr–_mediated cell death (D). This switch in competence to respond to rpr, however, fails to occur in CBP mutant glands (CBPG0350; hs-rpr) (E and F).

Figure 6.

Figure 6.

A model for the temporal regulation of salivary gland cell death by sequential pulses of ecdysone. Ecdysone (20E)–induced CBP expression at the mid-L3 transition directs down-regulation of DIAP1 in larval salivary glands to a critical threshold level, establishing competence for cell death. Later, ecdysone-induced rpr and hid expression at pupation (12 h APF) eliminates the remaining DIAP1, allowing caspase activation and salivary gland destruction.

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