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J Biol Chem
2004 Feb 27;2799:7629-35. doi: 10.1074/jbc.M306217200.
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Xenopus death receptor-M1 and -M2, new members of the tumor necrosis factor receptor superfamily, trigger apoptotic signaling by differential mechanisms.
Tamura K
,
Noyama T
,
Ishizawa YH
,
Takamatsu N
,
Shiba T
,
Ito M
.
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Signaling through the tumor necrosis factor receptor (TNFR) superfamily can lead to apoptosis or promote cell survival, proliferation, and differentiation. A subset of this family, including TNFR1 and Fas, signals cell death via an intracellular death domain and therefore is termed the death receptor (DR) family. In this study, we identified new members of the DR family, designated xDR-M1 and xDR-M2, in Xenopus laevis. The two proteins, which show high homology (71.7% identity), have characteristics of the DR family, that is, three cysteine-rich domains, a transmembrane domain, and a death domain. To elucidate how members of xDR-M subfamily regulate cell death and survival, we examined the intracellular signaling mediated by these receptors in 293T and A6 cells. Overexpression of xDR-M2 induced apoptosis and activated caspase-8, c-Jun N-terminal kinase, and nuclear factor-kappaB, although its death domain to a greater extent than did that of xDR-M1 in 293T cells. A caspase-8 inhibitor potently blocked this apoptosis induced by xDR-M2. In contrast, xDR-M1 showed a greater ability to induce apoptosis through its death domain than did xDR-M2 in A6 cells. Interestingly, a general serine protease inhibitor, but not the caspase-8 inhibitor, blocked the xDR-M1-induced apoptosis. These results imply that activation of caspase-8 or serine protease(s) may be required for the xDR-M2- or xDR-M1-induced apoptosis, respectively. Although xDR-M1 and xDR-M2 are very similar to each other, the difference in their death domains may result in diverse signaling, suggesting distinct roles of xDR-M1 and xDR-M2 in cell death or survival.
FIG. 1.
Structures of xDR-M1 and xDR-M2.A, predicted amino acid sequences of xDR-M1 and xDR-M2. The N-terminal signal peptides, cysteine-rich repeats, transmembrane regions, and death domains are wavy underlined, double underlined, single underlined, and boxed, respectively. B, sequence comparisons of the ligand-binding regions and death domains among xDR-M1, xDR-M2, chick CAR1, mouse Fas, mouse DR5, and mouse TNFR1. Multiple alignments were performed using the ClustalW program. Identical amino acids of xDR-M1 and xDR-M2, and amino acids identical with them in other death receptors, are shown in white against black.
FIG. 2.
Expression of xDR-M1 and xDR-M2 mRNA by RT-PCR. The cDNA was synthesized with 1 μg of total RNA isolated from various Xenopus tissues (A) or A6 cells (B). xDR-M1 or xDR-M2 cDNA fragment was amplified by PCR using one-fortieth of the cDNA with specific primer (see “Experimental Proceduresâ€). 10 ng of pEF1 xDR-M1-Myc-His or pEF1 xDR-M2-Myc-His plasmid was used as templates to verify specific amplification of xDR-M1 or xDR-M2.
FIG. 3.
Apoptosis induced by overexpression of xDR-M1 and xDR-M2 in 293T and A6 cells.A, 293T cells were cotransfected with 1.0 μg of pEF1 xDR-M1-Myc-His (panels aâc), pEF1 xDR-M2-Myc-His (panels dâf), pEF1 Fas-Myc-His (panels gâi), or pEF1 empty vector (panels jâl), and 0.1 μg of pcDNA3 FLAG-EGFP. After 24 h, they were fixed and treated with the anti-Myc antibody followed by incubation with Alexa-conjugated secondary anti-mouse antibody (panels a, d, g, and j) and Hoechst 33258 (panels b, e, h, and k), which stained nuclei. Expression of EGFP (panels c, f, i, and l) was also observed in the same fields. Arrowheads indicate apoptotic cells with nuclear shrinkage. The data represent the means ± S.E. (n = 3). B and C, 293T and A6 cells were cotransfected with pEF1 and pCI expression plasmid, respectively, of Myc-His (vector), xDR-M1-Myc-His (Full), xDR-M1-LBR-Myc-His (LBR), xDR-M1-DD-Myc-His (DD), xDR-M2-Myc-His (Full), xDR-M2-LBR-Myc-His (LBR), or xDR-M2-DD-Myc-His (DD) under the same condition as described above in A. The percentage of apoptotic cells among the transfected cells that expressed EGFP is shown. The data represent the means ± S.E. (n = 3). Full, full-length; LBR, ligand-binding region; DD, death domain.
FIG. 4.
Apoptosis induced by xDR-M2 is dependent on activation of caspase-8. 293T cells were cotransfected with 0.25 μg of pcDNA3 FLAG-xCaspase-8 and 1.0 μg of pEF1 Myc-His (vector), pEF1 xDR-M1-Myc-His (Full), pEF1 xDR-M1-LBR-Myc-His (LBR), pEF1 xDR-M1-DD-Myc-His (DD), pEF1 xDR-M2-Myc-His (Full), pEF1 xDR-M2-LBR-Myc-His (LBR), or pEF1 xDR-M2-DD-Myc-His (DD). After 24 h, the cell lysates were prepared and procaspase-8 (white arrowhead) or processed one (black arrowhead) was detected by Western blotting with anti-FLAG antibody. Full, full-length; LBR, ligand-binding region; DD, death domain.
FIG. 5.
Effects of caspase-8 and serine protease inhibitors on apoptosis induced by xDR-M1 and xDR-M2.A and C, 293T cells were transfected with pEF1 expression plasmid of xDR-M1, xDR-M2, or Fas under the same condition with the experiments of Fig. 3A. 3 h later, they were treated with or without a caspase-8-specific inhibitor, Z-IETD-FMK (20 μM) or a serine protease inhibitor, AEBSF (0.3 mM), respectively. 24 h post-transfection, the cells were fixed. The data represent the means of the percentage of apoptosis ± S.E. (n = 3). B and D, A6 cells were transfected with pCI xDR-M1-DD, pCI xDR-M2-DD, or pCI empty vector under the same condition with the experiments of Fig. 3C. They were analyzed with the same way as described above in A or C, except that a final concentration of AEBSF was 0.2 mM.
FIG. 6.
Activation of JNK by xDR-M1 and xDR-M2. 293T cells were cotransfected with 0.5 μg of pcDNA3 His-S-JNK2 and 1.0 μg of pEF1 xDR-M1-Myc-His, pEF1 xDR-M2-Myc-His, pEF1 Fas-Myc-His, or pEF1 Myc-His empty vector. After 40 h, 9/10 of the cell lysates were precipitated with S-protein-linked agarose (S-PA) and analyzed by immunoblotting with phospho-specific-JNK antibody (upper panel) and anti-His antibody (middle panel). Expression of the death receptors was examined by immunoblotting 1/10 of the cell lysates with anti-Myc antibody (lower panel).
FIG. 7.
Differential activation of NF-κB by xDR-M1 and xDR-M2 in 293T cells or A6 cells. 293T (A) and A6 (B) cells were transfected with pEF1 and pCI expression plasmids, respectively, of xDR-M1-Myc-His (Full), xDR-M1-LBR-Myc-His (LBR), xDR-M1-DD-Myc-His (DD), xDR-M2-Myc-His (Full), xDR-M2-LBR-Myc-His (LBR), or xDR-M2-DD-Myc-His (DD) (open bars, 50 ng; hatched bars, 100 ng; filled bars, 200 ng), and pNF-κB-luc reporter plasmid (0.3 μg) together with Renilla luciferase vector, pRL-TK (50 ng) as a control for transfection efficiency. Total DNA was kept at 0.55 μg per transfection with pEF1 Myc-His empty vector. After 24 h, luciferase activity were measured by dual luciferase reporter system. Each firefly luciferase activity was normalized to Renilla luciferase activity. Each relative luciferase activity is shown as the fold increase compared with the value obtained with 0.2 μg of pEF1 Myc-His empty vector. The data represent the mean ± S.E. from three separate experiments.
