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Identification of an Endogenous Dominant-Negative Short Isoform of Caspase-9 That Can Regulate Apoptosis¹

Center for Apoptosis Research and Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [S. M. S., M. A., Y. G., Y. Z., T. F-A., E. S. A.]; and Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 [Y. L.]

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Alternatively spliced isoforms of certain apoptosis regulators, such as Bcl-x, Ced-4, and Ich-1, have been shown to play opposing roles in regulating apoptosis. Here, we describe the identification of an endogenous alternatively spliced isoform of caspase-9, named caspase-9b, which lacks the central large subunit caspase domain. Caspase-9b is detectable in many cell lines by PCR and at the mRNA and protein levels. Caspase-9b can interact with the caspase recruitment domain of Apaf-1, and like the active site mutant of caspase-9, it can inhibit multiple forms of apoptosis, including those triggered by oligomerization of death receptors. It can also block activation of caspase-9 and -3 by Apaf-1 in an in vitro cytochrome c-dependent caspase activation assay. These results suggest that caspase-9b functions as an endogenous apoptosis inhibitory molecule by interfering with the formation of a functional Apaf-1-caspase-9 complex.

	Introduction

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The cytochrome c-dependent apoptotic pathway is initiated by release of cytochrome c from the mitochondria in response to an apoptotic stimulus (reviewed in Refs. 1 and 2 ). Cytochrome c, in the presence of dATP or ATP, can form a complex with the human CED-4 homologue Apaf-1, enabling it to recruit the initiator procaspase-9 to the complex (3) . The association of procaspase-9 and Apaf-1 is mediated by the interaction of the NH₂-terminal prodomain of procaspase-9 with the NH₂-terminal CARD⁴ of Apaf-1 (3) . This results in activation of procaspase-9 by Apaf-1-mediated oligomerization and activation of the downstream executioner caspases-3 and -7 by the activated caspase-9 (4) . Activation of caspase-3 and -7 is essential for dismantling important structural and regulatory components of the cell, leading to the final morphological changes of apoptosis, such as chromatin condensation (reviewed in Refs. 5, 6, 7, 8 ).

Activation of the cytochrome c/Apaf-1/caspase-9 pathway is crucial for most forms of apoptosis. As demonstrated with a dominant-negative caspase-9 mutant, caspase-9 activity is necessary for induction of apoptosis by the proapoptotic Bcl-2 family members, UV irradiation, and death receptors and their ligands in transfected MCF-7 cells (3 , 4 , 9 , 10) . Furthermore, in casp-9^-/- knockout mice, the loss of caspase-9 causes perinatal death due to a reduction in apoptosis at an earlier stage of brain development (11 , 12) . This is also associated with resistance of thymocytes, embryonic stem cells, and embryonic fibroblasts to several apoptotic stimuli (11 , 12) .

A potential role for alternative splicing in apoptosis was suggested by the discovery of functionally active alternatively spliced variants of major apoptosis regulators, such as Bcl-x, Ced-4, and Ich-1 (caspase-2; Refs. 13, 14, 15 ). In all these cases, the alternatively spliced isoforms play opposing roles in apoptosis. For example, the long isoforms of Bcl-x (Bcl-xL) and Ced-4 (Ced-4L) protect cells against apoptosis, whereas their short isoforms (Bcl-xS and Ced-4S, respectively) promote cell death (13 , 15) . On the other hand, the long isoform of Ich-1 (Ich-1L) induces apoptosis, whereas the short isoform (Ich-1S) inhibits it (14) . Thus far, there is no known endogenous antiapoptotic molecule that has been clearly shown to disrupt the Apaf-1-procaspase-9 complex. Here, we show that an alternatively spliced isoform of procaspase-9 can interact with Apaf-1 CARD and block apoptosis in vivo and activation of procaspase-9 and -3 in vitro, by a dominant-negative mechanism. We predict that up-regulation of the expression of this isoform in certain instances may contribute to increased resistance of cells to most forms of apoptosis.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Cloning of Caspase-9b.
Poly(A)⁺ RNA from the human Jurkat T-cell line was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Promega) with dT(17) primer. The reverse transcription products were then amplified by PCR using two caspase-9-specific primers, Mch6-ATG (ATGGACGAAGCGGATCGG) and Mch6-TAA (CCCTGGCCTTATGATGTT). The PCR products were subcloned into the pBluescript II SK(+) SmaI-digested vector and sequenced.

Expression Constructs.
cDNAs encoding caspase-9b or caspase-9 prodomain were subcloned in the mammalian expression vectors pcDNA3 or pRSC-lacZ. Constructs encoding procaspase-9, caspase-9-pro, procaspase-3, Apaf-CARD, Bax, Bik, and Blk have been described previously (3 , 4 , 9 , 16) .

Assay of Cytochrome c/dATP-dependent Activation of Procaspase-9 and Procaspase-3.
S100 cellular extracts (2 µg/µl, total protein) were prepared from 293 or MCF-7 cells transfected with empty construct or constructs encoding caspase-9b or caspase-9 prodomain, as described previously (3 , 4) . The extracts (5 µl) were incubated in a 10-µl total reaction volume with in vitro translated ³⁵S-labeled procaspase-9 (0.5 µl) or procaspase-3 (0.5 µl) in the presence of cytochrome c (5 ng/µl) and dATP (1 mM). SDS sample buffer was then added to each sample, and the samples were boiled and subjected to SDS-PAGE analysis, followed by autoradiography.

Transfection and Northern and Western Analyses.
Transfection and Northern and Western analyses were performed as described previously (3 , 16 , 17) .

Apoptosis Assays.
MCF-7-Fas cells were transiently cotransfected with ß-galactosidase reporter and test plasmids as described (3 , 17) . The percentage of round blue apoptotic cells (mean ± SD) was determined by phase contrast microscopy and then represented as a function of total blue cells under each condition (n 3).

	Results and Discussion

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To identify novel isoforms of caspase-9 that could participate in regulation of the cytochrome c/Apaf-1-dependent apoptotic pathway, we performed RT-PCR on Jurkat mRNA with two procaspase-9-specific primers spanning the initiation and stop codons of procaspase-9. In addition to the full-length procaspase-9 (1259 bp), we identified a short amplification product (809 bp) that represents 10% of the full-length product. Sequence analysis revealed that the short PCR product encodes an alternatively spliced 266-residue isoform of procaspase-9 (Fig. 1A) . Following the caspase nomenclature, this isoform was designated caspase-9b. Caspase-9b contains the first 139-residue prodomain fused inframe to the last 127-residue linker and small subunit caspase domain (Fig. 1, B and C) ; that is, it lacks the entire large subunit caspase domain (residues 140–289), which contains the active site pentapeptide QACGG (Fig. 1B) . Its predicted molecular mass is 30.1 kDa.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A, nucleotide and predicted amino acid sequences of human caspase-9b. Indicated are the primer sequences used to PCR amplify this sequence (underlined) and the splice junction (arrow). B, sequence alignment of procaspase-9 and caspase-9b. The large subunit caspase domain in procaspase-9, which contains the active site pentapeptide QACGG, is highlighted. This domain is not present in caspase-9b. C, domain organization of procaspase-9 and caspase-9b. PD, prodomain; LCD, large subunit caspase domain; LD, linker domain; SCD, small subunit caspase domain.

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Analyses of the expression of caspase-9b. A, Northern blot analysis of the expression of procaspase-9 and caspase-9b mRNA in normal tissues and tumor cell lines. Poly(A)+ RNA blots of selected human tissues or tumor cell lines (Clontech) were hybridized to 32P-labeled riboprobes complementary to caspase-9b (left) or caspase-9 large subunit (453–873 bp of procaspase-9 cDNA; right). Numbers on the left, size markers (kb). B, PCR analysis of the expression of procaspase-9 and caspase-9b in selected tumor cell lines. Total RNA from human tumor cell lines was reverse transcribed and then amplified by PCR with caspase-9-specific primers (shown in Fig. 1A ). The PCR products were then analyzed on an agarose gel and stained with ethidium bromide. Numbers on the left, size markers (kb); right, the two caspase-9 isoforms (procaspase-9, 1259 bp; caspase-9b, 809 bp). The identity of the two isoforms were verified by sequencing. The 1.08-kb band is a nonspecific PCR product. The PCR product in the control lane was generated with procaspase-9 cDNA template. C, Western blot analysis of the expression of procaspase-9 (Pcasp-9) and caspase-9b (Casp-9b) in 293 and MCF-7 cells. Cellular extracts were prepared from 293 (200 µg) and MCF-7 (200 µg) cell lines or from 293 cells transfected with a construct encoding caspase-9b (Casp-9b; 20 µg) and then fractionated by SDS-PAGE and detected by immunoblot analysis with a caspase-9-specific monoclonal antibody directed against the prodomain of caspase-9. Numbers on the left, size markers (kb); right, procaspase-9 (Pcasp-9) and caspase-9b (Casp-9b). A549, lung carcinoma; SW480, colorectal adenocarcinoma; Raji, Burkitt’s lymphoma; K-562, chronic myelogenous leukemia; HeLa, S3 cervical carcinoma; Jurkat, T-lymphoblastic leukemia; 293, embryonic kidney fibroblast; MCF-7, breast adenocarcinoma; CEM-C7, T-lymphoblastic leukemia; 697, pre-B lymphoblastic leukemia.

To demonstrate that caspase-9b can interact with Apaf-1 CARD (residues 1–97), we incubated in vitro translated caspase-9b with Apaf-1 CARD (Fig. 3) . Apaf-1 CARD was able to bind to the same extent both procaspase-9 and caspase-9b but not procaspase-9 without its prodomain, suggesting that both isoforms can be recruited by Apaf-1 (Fig. 3) .

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. In vitro interaction of procaspase-9 and caspase-9b with Apaf-1-CARD. 35S-labeled procaspase-9 (Pcasp-9), caspase-9b (Casp-9b), and procaspase-9 lacking its prodomain (pro) were incubated with a bacterially produced Apaf-1-CARD-His6 or a mock-purified material immobilized on a Ni2+-affinity resin. After extensive washing, the bound proteins were eluted by adding SDS sample buffer to the resin and analyzed by SDS-PAGE and autoradiography. Input, 35S-labeled translation products. Mock, Ni2+-affinity resin-bound lysates from empty vector transformed bacteria. CARD, Ni2+-affinity resin-bound Apaf-1-CARD-His6 from Apaf-1-CARD-His6-expressing bacteria.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Activity of caspase-9b in vitro and in vivo. A, 293T and MCF-7 cells were transiently transfected with an empty vector or constructs encoding caspase-9b (Casp-9b) or caspase-9 prodomain (Casp-9-pro) and then harvested after 36 h. S100 extracts were prepared from these cells and supplemented with 35S-labeled procaspase-9 (top) or procaspase-3 (bottom). The extracts were then incubated with cytochrome c plus dATP for 1 h at 30°C (Lanes 2–7, left to right). Samples were then analyzed by SDS-PAGE and autoradiography. Lane 1, control procaspase-9 or procaspase-3 without extract. B, 293 cells were transiently transfected with a construct encoding caspase-9b and then harvested after 36 h. S100 extracts (2 µg/µl, total protein) were prepared from these cells and serially diluted with S100 extracts (2 µg/µl, total protein) from nontransfected 293 cells, to maintain the concentration of the endogenous procaspase-9 constant and vary the concentration of recombinant caspase-9b. The resulting samples (10 µl/sample) were then fractionated by SDS-PAGE and immunoblotted with anti-caspase-9 antibody and the relative amounts of procaspase-9 and caspase-9b were determined by densitometric scanning of the immunoreactive bands. The same samples (5 µl/reaction) were also incubated with 35S-labeled procaspase-3 (0.5 µl) and cytochrome c plus dATP for 1 h at 30°C and then analyzed by SDS-PAGE, autoradiography, and densitometric scanning. The extent of procaspase-3 activation was determined from the intensity of the cleavage products relative to the total input and expressed as percentage of activation. The ratios of caspase-9b to procaspase-9 were then plotted against the percentages of procaspase-3 activation in all of the samples. C, MCF-7 cells were transiently transfected with an empty vector or a construct expressing caspase-9b in combination with a ß-gal reporter gene at a ratio of 4:1 and then treated 30 h after transfection for 6 h with agonist Fas-antibody (50 ng/ml), human TNF- (TNF; 10 ng/ml), TRAIL (200 ng/ml), or UV (40 mJ) using Stratagene’s UV Stratalinker 1800. MCF-7 cells were also transiently transfected with pRSC-lacZ plasmids encoding Bax, Bik, or Blk in combination with 4-fold excess of caspase-9b or empty vector. Columns, percentage of round blue apoptotic cells (mean), determined by phase contrast microscopy and then represented as a function of total blue cells under each condition (n 3).

The ability of caspase-9b to interfere with Apaf-1-mediated activation of procaspase-9 in vitro (Fig. 4A) suggests that it could inhibit apoptosis in vivo. To test this possibility, we transiently transfected MCF-7 cells with a construct encoding caspase-9b. As shown in Fig. 4C , caspase-9b transfected cells were substantially less sensitive than control vector-transfected cells to multiple apoptosis-inducing agents, such as the proapoptotic bcl-2 family members Bax, Bik, and Blk, and the death receptors ligands TRAIL, tumor necrosis factor, and Fas antibody. These cells were also less sensitive to UV-induced apoptosis. These results indicate that caspase-9b can, in fact, negatively regulate apoptosis induced by a wide variety of stimuli and support our earlier observation that the Apaf-1-caspase-9 pathway is a central apoptotic pathway on which most apoptotic signals converge (4) . Inhibition of death receptor-induced apoptosis in MCF-7 cells by caspase-9b and Bcl-xL (Fig. 4C ; Ref. 4 ) suggests that activation of the cytochrome c/Apaf-1/caspase-9 pathway is necessary for induction of apoptosis by death receptor in these cells.

In conclusion, we have identified caspase-9b as an endogenous molecule that negatively regulates the cytochrome c-dependent apoptotic pathway by inhibiting Apaf-1-mediated activation of procaspase-9. Because of the importance of this pathway in many forms of apoptosis, the identification of this molecule may provide clues to the regulation of apoptosis in several cell types. Perhaps, caspase-9b may provide an inhibitory function similar to that of the antiapoptotic Bcl-2 family members in certain cell types that express high levels of caspase-9b. Furthermore, expression of caspase-9b in many cells may establish a threshold that regulates activation of caspase-9 by preventing spontaneous activation of the Apaf-1-caspase-9 complex. Because caspase-9b is generated by an alternative splicing mechanism, signaling pathways that regulate this mechanism may play a key role in apoptosis.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by NIH Research Grants AG13487 and AG14357. The nucleotide sequence reported here has been submitted to the GenBank/EBI Data Bank (accession no. AF093130).

² These authors contributed equally to this work.

³ To whom requests for reprints should be addressed, at Department of Microbiology and Immunology, Thomas Jefferson University, Bluemle Life Sciences Building, Room 904, 233 South 10th Street, Philadelphia, PA 19107. Phone: (215) 503-4632; Fax: (215) 923-1098; E-mail: E_Alnemri{at}lac.jci.tju.edu

⁴ The abbreviations used are: CARD, caspase recruitment domain; RT-PCR, reverse transcription-PCR.

Received 12/ 2/98. Accepted 1/18/99.

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