Sakamaki K et al. (2004), The adaptor molecule FADD from Xenopus laevis d...

XB-ART-2672

Genes Cells 2004 Dec 01;912:1249-64. doi: 10.1111/j.1365-2443.2004.00802.x.

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The adaptor molecule FADD from Xenopus laevis demonstrates evolutionary conservation of its pro-apoptotic activity.

Sakamaki K , Takagi C , Kominami K , Sakata S , Yaoita Y , Kubota HY , Nozaki M , Yonehara S , Ueno N .

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FADD is an adaptor protein that transmits apoptotic signals from death receptors such as Fas to downstream initiator caspases in mammals. We have identified and characterized the Xenopus orthologue of mammalian FADD (xFADD). xFADD contains both a death effector domain (DED) and a death domain (DD) that are structurally homologous to those of mammalian FADD. We observed xFADD binding to Xenopus caspase-8 and caspase-10 as well as to human caspase-8 and Fas through interactions with their homophilic DED and DD domains. When over-expressed, xFADD was also able to induce apoptosis in wild-type mouse embryonic fibroblasts (MEF), but not in caspase-8-deficient MEF cells. In contrast, DED-deficient xFADD (xFADDdn) acted as a dominant-negative mutant and prevented Fas-mediated apoptosis in mammalian cell lines. These results indicate that xFADD transmits apoptotic signals from Fas to caspase-8. Furthermore, we found that transgenic animals expressing xFADD in the developing heart or eye under the control of tissue-specific promoters show abnormal phenotypes. Taken together, these results suggest that xFADD can substitute functionally for its mammalian homologue in death receptor-mediated apoptosis, and we suggest that xFADD functions as a pro-apoptotic adaptor molecule in frogs. Thus, the structural and functional similarities between xFADD and mammalian FADD provide evidence that the apoptotic pathways are evolutionally conserved across vertebrate species.

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Species referenced: Xenopus laevis
Genes referenced: casp10 casp8 cdca5 fadd fas gchfr hamp myc myl2 pnhd tff3.7 Upk3b

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	Figure 1 Primary structure of Xenopus FADD. (A) Nucleotide and predicted amino acid sequences of Xenopus FADD cDNA clone db61b04 are shown. Numbers on the left side of the sequence indicate nucleotide positions, and numbers on the right side indicate amino acid positions. The normal and bold lines under the amino acid sequence indicate the regions of the DED and DD domains, respectively. (B) Amino acid sequences of Xenopus and human FADDs are compared. Identical and similar amino acids between Xenopus and human are indicated by black and shaded boxes, respectively.
	Figure 2 Induction of apoptosis in human HeLa-K cells by over-expression of Xenopus FADD (xFADD), caspase-8 (xCaspase-8) and caspase-10 (xCaspase-10). (Aâ G) Plasmids carrying xFADD (B, C), xCaspase-8 (D, E) or xCaspase-10 (F, G) cDNA, or vector DNA alone (A), were co-transfected with the EGFP-expression vector, pCX-EGFP, into cells. After 24 h culture, transfectants were washed, fixed and photographed using phase contrast (left panels) and fluorescence (right panels) microscopy in the same field. Half of these transfectants were cultured in the presence of 100 Î¼m z-VAD-fmk in medium (C, E, G). (H) The DNA content of transfectants was assessed by flow cytometry. Transfectants carrying control vector DNA (a), xFADD cDNA (b), both xFADD cDNA and the p35 gene (c), xCaspase-8 cDNA (d), or xCaspase-10 cDNA (e) were analysed by flow cytometry for the detection of the DNA content stained with propidium iodide (PI). Percentage indicates the cellular population detected in the sub- G1 fraction of the DNA content.
	Figure 3 Induction of apoptosis by xFADD in wild-type MEF cells, but not caspase- 8-deficient MEF cells. (A) A schematic diagram is shown of the full length and truncated mutant xFADD constructs fused to IRES (Internal Ribosomal Entry Site)- EGFP. (B) xFADD-EGFP was transfected into either wild-type MEF cells (b, c) or caspase-8-deficient MEF cells (f ). The truncation mutant xFADDdn-EGFP was introduced into wild-type MEF cells (d), and the control IRES-EGFP gene was transfected into both types of MEF cells (a, e). In caspase-8-deficient MEF cells, mouse caspase-8 cDNA was co-transfected with xFADD-EGFP (g, h) or IRES-EGFP (i). Both wild-type and caspase-8-deficient MEF transfectants were also incubated with 100 Î¼m z-VAD-fmk (c, h). After 24 h culture, cells were washed, fixed and photographed using phase contrast (left panels) and fluorescence (right panels) microscopy in the same field. (C) A summery of the data in (B) is presented. Cell viability was estimated by counting the number of EGFPpositive cells in each experiment. Data present the means and standard deviations from four experiments.
	Figure 4 Transfection of xFADD lacking the DED motif, a dominant-negative inhibitor of Fas-mediated apoptosis. (AâE) HeLa-K cells (AâD) and SK-Hep1 cells (E) were transfected with the control vector pIRESEGFP (A, B and in E) or the vector carrying mutant xFADD (xFADDdn) cDNA (C, D and in E) fused to the IRES-EGFP gene as shown in Fig. 3A. Transfectants were treated with (B, D) or without (A, C) 500 ng/mL of anti-human Fas antibody CH-11 and 10 Î¼g/mL of CHX for 6 h (AâD) or treated with various concentration of CH11 and 10 Î¼g/mL of CHX for 8 h (E). Cell viability was examined under a microscope (AâD) or calculated by measuring fluorescence in cell lysates (E). Data present the means and standard deviations from four experiments (E).
	Figure 5 xFADD interaction with Xenopus and mammalian initiator caspases. (A) 293T cells were transfected with p35 in conjunction with empty vector ( lane 1) or plasmids encoding Flag-xFADD (lanes 2â4), FlagxFADDdn (lanes 5, 6), Flag-mouse FADD (lanes 7, 8), HA-xCaspase-8 (lanes 3, 5 and 7), or Myc-xCaspase-10 (lanes 4, 6 and 8). Twenty-four hours after transfection, cells were lysed and immunoprecipitated with an anti-Flag antibody. Immunoprecipitates and whole cell lysates were analysed by immunoblotting with anti-Flag, anti-HA and anti-Myc antibodies for the detection of xFADD, mouse FADD, xCaspase-8 and xCaspase-10, respectively. Asterisk indicates the presence of a nonspecific anti-Flag immuno-reactive molecule in the cell lysates. (B) 293T cells were transfected with p35 in conjunction with empty vector ( lane 1) or plasmids encoding Flag-xFADD (lanes 2 and 4), Flag-human FADD (lanes 3 and 5), or Myc-human caspase-8 (lanes 4 and 5). Fourteen hours after transfection, cells were lysed and immunoprecipitated with an anti-Flag antibody. Immunoprecipitates and whole cell lysates were analysed by immunoblotting with anti-Myc antibody for the detection of human caspase-8. Baculovirus p35 was introduced into all cells to prevent cell death. Abbreviation, Ig: immunoglobulin.
	Figure 6 Recruitment of xFADD into the DISC complex after Fas-ligation. 293Fas cells were transfected with p35 (all lanes) together with empty vector ( lanes 1 and 2) or plasmids encoding Flag-xFADD (lanes 3 and 4). Twenty-four hours after transfection, cells were stimulated with ( lanes 2 and 4) or without ( lanes 1 and 3) 500 ng/mL of anti-Fas antibody for 40 min, lysed and immunoprecipitated with an anti-Fas antibody. Immunoprecipitates and whole cell lysates were analysed by immunoblotting with anti- Flag and anti-human Fas antibodies for the detection of xFADD and Fas, respectively.
	Figure 7 Tissue distribution of xFADD and xCaspases-8/10 transcripts in adult frogs. Total RNAs isolated from the brain, heart, kidney, liver, lung, muscle and spleen of adult frogs were subjected to RT-PCR analysis for the detection of xFADD, xCaspase-8 and xCaspase-10 transcripts, and the resulting PCR products were resolved by a 2.5% agarose-gel electrophoresis ( lanes 1â7). As an internal control, EF1Î± transcripts were also examined. Negative control (no first-strand cDNA added) and molecular-weight markers (M.W.M.) are shown in lanes 8 and 9, respectively.
	Figure 8 Effects of xFADD in Xenopus larvae. (A) A schematic structure of the constructed transgenes is shown. The transgene, CÎ±AGFP3, is composed of the fluorescence reporter gene GFP3 and the SV40 polyA signal sequence under the control of the chicken crystallin Î±A (CÎ±A) promoter. A second transgene, CÎ±A-xFADD/EGFP, is composed of the CÎ±A promoter, the xFADD-EGFP construct as shown in Fig. 3A, and the SV40 polyA sequence. A third transgene, XMLC2-xFADD, is composed of xFADD cDNA and SV40 polyA sequence under the control of the Xenopus myosin light chain 2 (XMLC2) promoter. Primers (P1-P5) specific to each of the transgenes were used to genotype animals by PCR. (B) Transgenic tadpoles were generated that expressed GFP3 in the eye. A transgenic tadpole carrying the CÎ±A-GFP3 transgene at stage 45 was imaged using both bright-field (left panel) and fluorescence (right panel) dissecting microscope. (C, D) Morphological and genomic analyses of transgenic tadpoles reveal xFADD expression in the eye. Xenopus embryos were injected with the CÎ±A-xFADD/EGFP transgene and allowed to develop to stage 45. Animals displaying irregular phenotypes of the developing eye were collected and observed under the microscope (C). Two tadpoles (lower and middle) exhibit pinhead-shaped and small left eyes. Transgenic animals were genotyped by PCR-amplifying genomic DNA using two sets of primers-P1 and P2, for the detection of the transgene and P6 and P7, for the detection of the genomic Î³-crystallin gene- followed by 1.5% agarosegel electrophoresis (D). Plasmid DNA carrying CÎ±A-xFADD/EGFP was used as a control. Molecular weight markers (M.W.M.) were applied in lane 1. (Eâ€“G) Genomic and histological analyses were performed on transgenic animals expressing xFADD in the heart. Xenopus embryos were co-injected with the transgenes XMLC2-xFADD and CÎ±A-GFP3. At stage 46/47, six animals expressing GFP3 in the eye were collected as Tg#1-Tg#6, and their anterior and posterior halves were used for histological analysis and for preparation of genomic DNA, respectively. Animals were genotyped by PCR amplification of genomic DNA using two sets of primers-P2 and P3, for detection of the XMLC2-xFADD transgene and P4 and P5, for the detection of GFP3 cDNA- followed by 1.5% agarose-gel electrophoresis (E). Plasmid DNA carrying XMLC2-xFADD or CÎ±A-GFP3 was used as control (lane 8) and molecular weight markers (M.W.M.) were applied in lane 1. Transverse sections containing the heart were prepared and triply stained with azocarmin B, aniline blue and orange G (F). All upper panels show photographs of animals carrying the XMLC2-xFADD transgene, and the lower left panel shows a photograph of an animal not carrying the XMLC2-xFADD transgene, and the lower three right panels are normal tadpoles. Scale bar indicates 100 Î¼m. Abbreviation, v: ventricle. The ventricular volume in each animal was calculated by measuring its area in the serial sections (G). Each bar indicates the ratio of the ventricular volume of each sample to the mean of the ventricular volume of control animals.