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Endocrinology, doi:10.1210/en.2007-1591
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Endocrinology Vol. 149, No. 7 3379-3389
Copyright © 2008 by The Endocrine Society

Tumor Necrosis Factor-{alpha} Attenuates Thyroid Hormone-Induced Apoptosis in Vascular Endothelial Cell Line XLgoo Established from Xenopus Tadpole Tails

Shuuji Mawaribuchi, Kei Tamura, Saori Okano, Shutaro Takayama, Yoshio Yaoita, Tadayoshi Shiba, Nobuhiko Takamatsu and Michihiko Ito

Department of Biosciences (S.M., K.T.,S.O., S.T., T.S., N.T., M.I.), School of Science, Kitasato University, Sagamihara, Kanagawa 228-8555 Japan; and Division of Embryology and Genetics (Y.Y.), Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Hiroshima 734-8551, Japan

Address all correspondence and requests for reprints to: Michihiko Ito, Department of Biosciences, School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Japan. E-mail: ito{at}jet.sci.kitasato-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Amphibian metamorphosis induced by T3 involves programmed cell death and the differentiation of various types of cells in degenerated and reconstructed tissues. However, the signaling pathway that directs the T3-dependent cell-fate determinations remains unclear. TNF-{alpha} is a pleiotropic cytokine that affects diverse cellular responses. Engagement of TNF-{alpha} with its receptor (TNFR1) causes intracellular apoptotic and/or survival signaling. To investigate TNF signaling functions during anuran metamorphosis, we first identified Xenopus laevis orthologs of TNF (xTNF)-{alpha} and its receptor. We found that xTNF-{alpha} activated nuclear factor-{kappa}B in X. laevis A6 cells through the Fas-associated death domain and receptor-interacting protein 1. Interestingly, xTNF-{alpha} mRNA in blood cells showed prominent expression at prometamorphosis during metamorphosis. Next, to elucidate the apoptotic and/or survival signaling induced by xTNF-{alpha} in an in vitro model of metamorphosis, we established a vascular endothelial cell line, XLgoo, from X. laevis tadpole tail. XLgoo cells formed actin stress fibers and elongated in response to xTNF-{alpha}. T3 induced apoptosis in these cells, but the addition of xTNF-{alpha} blocked the T3-induced apoptosis. In addition, treatment of the cells with T3 for 2 d induced the expression of thyroid hormone receptor-β and caspase-3, and this thyroid hormone receptor-β induction was drastically repressed by xTNF-{alpha}. Furthermore, in organ culture of the tail, xTNF-{alpha} significantly attenuated the tail degeneration induced by T3. These findings suggested that xTNF-{alpha} could protect vascular endothelial cells from apoptotic cell death induced by T3 during metamorphosis and thereby participate in the regulation of cell fate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AMPHIBIAN METAMORPHOSIS is characterized by dynamic morphologic changes as the larval body is remodeled into the adult form; these changes may be closely related to interphylogenic changes among vertebrates during evolution. In anurans, morphological changes, such as tail degeneration, the appearance of extremities, and gill resorption, are induced by increased blood levels of thyroid hormone (1, 2). Generally, thyroid hormone, including T4 and T3, enhance the basic metabolic rate and energy metabolism, thereby contributing to the regulation of growth and development (3). Thyroid hormone receptor (TR), which belongs to the nuclear receptor superfamily, forms a complex with thyroid hormone and regulates gene expression through binding to specific sites on DNA (4). Xenopus is widely used to investigate embryogenesis and metamorphosis, and recent reports on the role of TR in morphogenesis have used a transgenic X. laevis that expresses a dominant-negative Xenopus TR (xTR)-{alpha}; these reports verify that TR plays critical roles in ontogenesis, including metamorphosis (5, 6, 7, 8, 9). One TR-regulated aspect of metamorphosis is programmed cell death; however, little is known about the intracellular pathway that mediates the apoptotic signal from the TR-T3 complex.

Programmed cell death is essential during animal development (10) and is often apoptotic in character, with dying cells exhibiting nuclear condensation or fragmentation. Recently apoptosis induced by signals from death receptor (DR) family members was shown to be involved in organogenesis (11, 12, 13). TNF-{alpha} belongs to the TNF superfamily. The DRs are a subfamily of the TNF receptor (TNFR) superfamily, and the TNFR 1 (TNFR1) is a DR (14). When TNFR1 is engaged by TNF-{alpha}, death domain-containing proteins, such as TNFR-associated death domain, receptor-interacting protein 1 (RIP1), and Fas-associated death domain (FADD), are recruited to form a complex, called the death-inducing signal complex (DISC) (15, 16, 17). The DISC activates survival and/or apoptotic signaling pathways, including those mediated by nuclear factor-{kappa}B (NF-{kappa}B), MAPKs, or caspase-8, whose activation is followed by the activation of other caspases (18, 19).

We previously showed that the Xenopus DR (xDR) family members (xDR-Ms), can activate caspase-8, NF-{kappa}B, and/or c-Jun N-terminal kinase (JNK; which belongs to the MAPK family) (20). We also found that Xenopus FADD and RIP1, which are both members of the DISC, can synergistically induce NF-{kappa}B activation (21). The NF-{kappa}B activation was involved in cell survival signaling, whereas the activation of JNK and caspases led to apoptotic cell death (19, 22). However, contradictory results involving these molecules have been reported (23, 24). TNF-{alpha} has been studied in vascular endothelial cells, in which it elicits the enhanced transcription of several adhesion molecules, after cytoskeletal changes (25, 26). However, little is known about TNF-{alpha} functioning during early development.

To investigate whether TNF-{alpha} signaling could be involved in the T3-induced apoptosis that characterizes metamorphosis, we first identified X. laevis homologs of TNF-{alpha} and TNFR1 (xTNF-{alpha} and xTNFR1, respectively) and established a vascular endothelial cell line, XLgoo, from premetamorphic X. laevis tadpole tails. Exposure to T3 could cause apoptotic cell death in the XLgoo cultures. Interestingly, xTNF-{alpha} suppressed this cell death. Moreover, in tail organ culture, the T3-induced degeneration of the tails was attenuated by xTNF-{alpha}. Taken together, these data suggest a role for TNF-{alpha} as a regulator of cell fate during metamorphosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Antimouse IgG and antirabbit IgG horseradish-peroxide-conjugated antibodies were purchased from Sigma (St. Louis, MO). Mouse anti-c-Myc monoclonal antibody (9E10) was purchased from Roche Diagnostics (Stockholm, Sweden).

Animal care and use
The Institutional Animal Care and Use Committee of Kitasato University approved all experimental procedures involving X. laevis.

Isolation of xTNF-{alpha} and xTNFR1 cDNAs
A Xenopus database (Axeldb) was screened for sequence similarity with human TNF-{alpha} (hTNF-{alpha}) and human TNFR1 (hTNFR1) cDNA using the blastn algorithm. We designed primers from the sequence data and amplified the full-length xTNF-{alpha} and xTNFR1 by PCR from cDNAs of unfertilized X. laevis eggs (xTNF-{alpha}) or tadpole tails (xTNFR1). The products were subcloned into the pBluescript vector (Stratagene, Tokyo, Japan). The nucleotide sequences were confirmed by DNA sequencing.

Plasmid constructs
The coding sequence for xTNF-{alpha} was amplified by PCR from the xTNF-{alpha} cDNA in pBluescript using the specific primers 5'-GTACCAGGATCCCAAGCAATGAAAG-3' and 5'-CCACCCTCGAGGTTGTCTCA-3'. The resulting xTNF-{alpha} product contained a BamHI site at the 5' end and an XhoI site at the 3' end of the sense strand. It was digested with BamHI and XhoI. The resultant cDNA fragment was subcloned into a BamHI/XhoI-digested pcDNA3-FLAG vector to generate pcDNA3-FLAG-xTNF-{alpha}. The plasmid pGEX-GST-FLAG-xTNF-{alpha} was constructed from the pGEX-4T vector (GE Healthcare, Piscataway, NJ) using pcDNA3-FLAG-xTNF-{alpha}. The open reading frame of xTNFR1 was amplified by PCR from tadpole tail cDNA using the specific primers 5'-ACCATGATTGGCCACCTGATGC-3' and 5'-GGCAACTCTAGACAGTCCTG-3'. The resultant cDNA fragment was subcloned into the pEF1-Myc-His vector to generate pEF1-Myc-His-xTNFR1. pEF1-Myc-His-xTNFR1 was digested with EcoRV to delete its death domain and then self-ligated to generate pEF1-Myc-His-xTNFR{Delta}DD.

Establishment of cell lines
Cell lines from X. laevis tadpole tails were established basically as described by Yaoita and Nakajima (27). Tail tips from X. laevis tadpoles at stage 55 were treated with 0.25% trypsin and 0.5% collagenase in 0.7 x PBS for 30 min at 20 C. After trituration with a pipette, the cells were spun, resuspended in 0.7 x L-15 medium supplemented with 20% fetal calf serum that had been treated with the resin AG1-X8 (Bio-Rad, Hercules, CA) to deplete the thyroid hormone, and cultured at 20 C on a PRIMARIA culture dish (Becton Dickinson, Lincoln Park, NJ). After 2 wk, the surviving cells were transferred to a new dish. Once the cells became confluent, they were passaged once a week. Several cell lines proliferated and were maintained for more than 3 yr. One of the cell lines was recloned and named XLgoo; we used this line for the current study.

Cell culture and transfection
X. laevis A6 kidney epithelial cells and human embryonic kidney 293T cells were cultured as described previously (20). XLgoo cells were maintained as described above. They were transfected using TransIT (Mirus, Madison, WI) according to the manufacturer’s instructions.

RT-PCR
Total RNA was isolated from several X. laevis cell lines, embryos at various stages, and tadpoles, using the RNeasy mini kit (QIAGEN, Valencia, CA). RNA (1 µg) was reverse transcribed using PowerScript (CLONTECH, Palo Alto, CA), according to the manufacturer’s instructions. Using a part of the first-strand cDNA as the template, PCR was carried out for 30 cycles to amplify cDNA fragments with specific primer pairs as follows: xTNF-{alpha}, 5'-TGAAGACGTGAACCAAGTGG-3' and 5'-CCTGTAAAATGAGCTGCCAG-3'; xTNFR1, 5'-TGCAACAGTCCAACCTGTCG-3' and 5'-CTGATATCGAATTCACCGGC-3'; xVE-cadherin or XL-fli as a vascular endothelial marker, 5'-GAGGAAGGCGGTGGAGAAATG-3' and 5'-ATGTGAAGGGTGTCATATGG-3' or 5'-AGGAGGGTTATTGTACCTGC-3' and 5'-ATATGGATGTGCTCCGCAGG-3', respectively; xTR{alpha} or xTRβ, 5'-AAATCAGTGCCAGCTCTGCC-3' and 5'-AGACCTCCGTTCGTTCTTAAGCTG-3' or 5'-ATGCCAAGCAGTATGTCAG-3' and 5'-CCACCTTCAGGCGCATTAAC-3', respectively; xCaspase3, 5'-TCAGGCATTGAAACAGACAG-3' and 5'-AGCTACCATATGATTTACACAGG-3'; 18S rRNA cDNA as a control, 5'-GGCCCTGTAATTGGAATGAG-3' and 5'-CCCAAGATCCAACTACGAGC-3'. The amplified PCR products were subjected to electrophoresis on 2% agarose gels.

Protein purification
Expression vectors for GST-FLAG or GST-FLAG-xTNF-{alpha} fusion protein were expressed in Escherichia coli and purified using glutathione agarose beads (GE Healthcare). Protein immobilized on the glutathione agarose beads was used for ligand-receptor interaction experiments. GST-FLAG-xTNF-{alpha} was treated with biotinylated thrombin (Merck Biosciences, Darmstadt, Germany) and then incubated with streptavidin-agarose (Invitrogen, Carlsbad, CA) and polymyxin B agarose (Sigma) to remove biotinyl thrombin and endotoxin, respectively. Purified FLAG-xTNF-{alpha} was used in experiments on cultured cells and tail organ culture.

Analysis of ligand-receptor interactions in vitro
c-Myc-tagged DR was expressed in human 293T cells. The cell extracts were mixed with GST-FLAG or GST-FLAG-xTNF-{alpha} fusion protein immobilized on beads in buffer B [20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Triton X-100], rotated overnight at 4 C, spun, and washed three times with buffer B. The precipitates were separated by SDS-PAGE and analyzed by immunoblotting with an anti-c-Myc monoclonal antibody 9E10.

NF-{kappa}B luciferase reporter assay
Human 293T and X. laevis A6 cells were seeded into 12-well dishes at 1 x 105 cells/well. They were then transiently cotransfected with reporter constructs and various amounts of the testing plasmids. The pNF-{kappa}B-Luc reporter plasmid (Stratagene) was used to measure the NF-{kappa}B activity. The pRL-TK plasmid (Promega, Madison, WI), which expresses Renilla luciferase, was used for normalization of the transfection efficiency. Empty control plasmid was added to keep the amount of total DNA in each transfection constant. After 24 h, the cells were harvested, and their luciferase activities were measured using the dual-luciferase reporter assay system (Promega). The data represent the means ± SEM, and each transfection was performed in triplicate.

Fluorescence
XLgoo cells were cultured in the presence or absence of 100 ng/µl xTNF-{alpha}, fixed in 4% paraformaldehyde in 0.7x PBS for 30 min, and then exposed to 0.2% Triton X-100/5% skim milk in 0.7x PBS for 30 min. They were then treated with Alexa 488-phalloidin and 5 µg/ml Hoechst 33258 in 0.7x PBS for 30 min. After being washed with 0.7x PBS three times, they were examined with fluorescence microscopy.

Analysis of apoptosis
XLgoo cells were grown in 35-mm dishes and then treated with 10 nM thyroid hormone (T3) or left untreated. After the adherent cells were fixed and stained with 5 µg/ml Hoechst 33258, apoptosis was determined by evaluating the morphological features of nuclear condensation or fragmentation. Floating cells were counted as apoptotic cells because the nucleus of these cells showed condensation or fragmentation.

Organ culture of tadpole tails
X. laevis tadpoles at stage 51 were gently stirred in 0.1% sodium sulfatiazole. After 30 min, the tadpoles were washed with water for 2 min. The tails were severed with a scissors, rinsed four times in 0.7x PBS, and transferred to culture dishes containing 0.7x L-15 medium supplemented with 100 U/ml penicillin G, 100 µg/ml streptomycin, and 50 µg/ml gentamicin. The cultures were incubated at 25 C in the presence or absence of 100 nM T3 for 2 wk. The medium was changed every 2 d. The left sides of tails before and after the culture were photographed. Then the surface areas were measured by counting dots at a resolution of 150 dpi. The percentage of the surface area was calculated as the proportion of a treated tail to that of the same individual before the culture.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of xTNF-{alpha} and xTNFR1 cDNAs
To clarify the roles of TNF-{alpha} signaling during development in X. laevis, we first needed to isolate xTNF-{alpha} cDNA. We searched the Axeldb database for sequences similar to hTNF-{alpha}. We designed specific primers corresponding to the most similar sequence obtained and used them to amplify by PCR the full-length Xenopus cDNA for TNF-{alpha}, designated xTNF-{alpha}, from unfertilized egg cDNA. The xTNF-{alpha} cDNA encoded a protein consisting of 225 amino acid residues that contained a C-terminal TNF domain (Fig. 1Go, A and B). A comparison of xTNF-{alpha} with hTNF-{alpha} showed an identity of 33% for the whole protein and 34% for the TNF domain.


Figure 1
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  		FIG. 1. Structural comparisons of TNF-{alpha} or xTNFR1 between X. laevis and mammals. A, Schematic representation of X. laevis (x) and human (h) TNF-{alpha} (upper panel) and TNFR1 (lower panel). The amino acid identity of the TNF domain between xTNF-{alpha} and hTNF-{alpha} and the cysteine-rich domains and death domains between xTNFR1 and hTNFR1 are shown. B, Amino acid sequence comparisons between xTNF-{alpha} and hTNF-{alpha} (upper panel) or between xTNFR1 and hTNFR1 (lower panel). Multiple sequence alignments were performed using the CLUSTALW program. Identical amino acids between xTNF-{alpha} and hTNF-{alpha} or xTNFR1 and hTNFR1 are shown in white lettering on black. Similar amino acids are shown in black lettering on gray. C, A phylogenetic tree of TNF or TNFR family members. Xl, Xenopus laevis; Xt, X. tropicalis; Hs, Homo sapiens; Mm, Mus musculus.

 
We also isolated the full-length xTNFR1 cDNA, from tadpole tail cDNA, by PCR using primers based on an hTNFR1-like sequence from the database. xTNFR1 consisted of 412 amino acid residues with an identity to hTNFR1 of approximately 35%. The N-terminal cysteine-rich domain and C-terminal death domain of xTNFR1 had relatively high similarities (42 and 49% identity, respectively) to those of hTNFR1 (Fig. 1Go, A and B). Phylogenetic trees of the TNF and the TNFR superfamilies showed that xTNF-{alpha} or xTNFR1 could be X. laevis orthologs of TNF-{alpha} or TNFR1, respectively (Fig. 1CGo). The nucleotide sequences for xTNF-{alpha} and xTNFR1 have been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases under the accession numbers AB298595 and AB298596, respectively.

xTNF-{alpha} is a ligand for xTNFR1
To determine whether xTNF-{alpha} could bind xTNFR1, myc-tagged xTNFR1, or a different myc-tagged death receptor, xDR-M2 (20), was expressed in human 293T cells. Each cell extract was then mixed with glutathione-S-transferase (GST)-FLAG or a fusion protein consisting of GST, FLAG, and the extracellular region of xTNF-{alpha} (GST-FLAG-xTNF-{alpha}). After a GST pull-down with glutathione Sepharose 4B, we performed a Western blot analysis with an anti-Myc antibody and detected the band only in the combination of xTNFR1-Myc and GST-xTNF-{alpha} (Fig. 2AGo), indicating that xTNF-{alpha} could directly bind to xTNFR1 but not xDR-M2.


Figure 2
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  		FIG. 2. NF-{kappa}B activation by xTNF-{alpha} is specifically mediated through xTNFR1. A, Interactions between xTNF-{alpha} and xTNFR1 in vitro. Left panel, Coomassie brilliant blue (CBB) staining of the GST-FLAG or GST-FLAG-xTNF-{alpha} fusion protein. Right panel, Immunoblot of cell lysates and precipitates from a GST pull-down (see Materials and Methods) probed with the anti-c-Myc monoclonal antibody 9E10. WB, Western blot. B, xTNF-{alpha} induced NF-{kappa}B activation via xTNFR1 in human 293T cells. Human 293T cells were transfected with an NF-{kappa}B firefly luciferase reporter plasmid (300 ng of pNF-{kappa}B-Luc), Renilla luciferase vector (50 ng of pRL-TK), and an expression vector for xTNFR1 or xDR-M2 as a negative control. At 24 h after transfection, the cells were treated with 10 ng/ml xTNF-{alpha} and cultured another 24 h. The luciferase activities were measured by the dual-luciferase reporter system. The data represent the means ± SEM, and each transfection was performed in triplicate. C, Repression of xTNF-{alpha}-induced NF-{kappa}B activation by dominant-negative xTNFR1 in Xenopus A6 cells. Xenopus A6 cells were transfected with the NF-{kappa}B firefly luciferase reporter plasmid (250 ng of pNF-{kappa}B-Luc), Renilla luciferase vector (50 ng of pRL-TK), and an expression vector for the xTNFR1-{Delta}DD and treated with xTNF-{alpha} as in B. The data represent the means ± SEM, and each transfection was performed in triplicate.

 
Because xTNF-{alpha} specifically bound to xTNFR1 in vitro, we next examined whether xTNF-{alpha} could specifically bind xTNFR1 in intact cells, resulting in NF-{kappa}B activation. Human 293T cells were transfected with an NF-{kappa}B luciferase reporter plasmid and an expression vector for xTNFR1 or for xDR-M2 as the negative control, and the transfected cells were then treated with xTNF-{alpha} for 24 h. The luciferase assay indicated that xTNF-{alpha} induced the activation of NF-{kappa}B in cells expressing exogenous xTNFR1 but not in those expressing xDR-M2 (Fig. 2BGo). Next, we examined whether xTNF-{alpha} could induce NF-{kappa}B activation in X. laevis A6 cells, which produce endogenous xTNFR1. After transfection with the NF-{kappa}B luciferase reporter plasmid, the A6 cells were exposed to xTNF-{alpha} for 24 h. The xTNF-{alpha} treatment increased the luciferase activity. To confirm that this NF-{kappa}B activation was mediated through endogenous xTNFR1, a dominant-negative xTNFR1 mutant that lacked its death domain (xTNFR1{Delta}DD), was expressed in the A6 cells. The xTNF-{alpha}-induced NF-{kappa}B activation was significantly inhibited by the expression of xTNFR1{Delta}DD (Fig. 2CGo). These results indicated that xTNF-{alpha} is a ligand for xTNFR1.

NF-{kappa}B activation by xTNF-{alpha} may be mediated through X. laevis FADD (xFADD) as well as X. laevis RIP1 (xRIP1)
In mammals, TNF-{alpha} induces the activation of JNK, p38 MAPK, and/or NF-{kappa}B via its ligation with TNFR1, which, when activated, recruits death domain proteins, such as TNFR-associated death domain, RIP1, and FADD (15, 16, 17). In X. laevis, we previously showed that xRIP1 and xFADD could interact to activate NF-{kappa}B synergistically (21). Here we examined whether NF-{kappa}B activation by xTNF-{alpha} was mediated through xRIP1 and xFADD in X. laevis A6 cells. The cells were transfected with an expression vector for the xRIP1 mutant containing only the death domain (xRIP1-DD) or an xFADD mutant containing only the death domain (xFADD-DD) along with the NF-{kappa}B luciferase reporter plasmid. The assay revealed that xTNF-{alpha} induced NF-{kappa}B activation dose-dependently, and this induction was repressed by xFADD-DD or xRIP1-DD (Fig. 3Go). Interestingly, the overexpression of xRIP1-DD almost completely suppressed the reporter activation, but xFADD-DD blocked it only partially, even when the amounts of expression vector were increased. These results indicated that both xRIP1 and xFADD could participate in xTNF-{alpha}-induced NF-{kappa}B signaling in X. laevis A6 cells, although there could be an xFADD-independent pathway that was an important contributor to NF-{kappa}B activation.


Figure 3
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  		FIG. 3. xTNF-{alpha} induces NF-{kappa}B activation via xFADD and xRIP1. Xenopus A6 cells were transfected with an expression vector for an xRIP1 mutant containing only the DD (xRIP1-DD) or an xFADD mutant containing only the DD (xFADD-DD) along with the NF-{kappa}B firefly luciferase reporter plasmid (250 ng of pNF-{kappa}B-Luc) and the Renilla luciferase vector (50 ng of pRL-TK). The cells were treated with xTNF-{alpha} as in Fig. 2Go. The data represent the means ± SEM, and each transfection was performed independently in triplicate.

 
xTNF-{alpha} is highly expressed at prometamorphosis
To examine the expression patterns of xTNF-{alpha} and xTNFR1 before and during metamorphosis, RT-PCR was performed with RNAs from whole tadpoles at stages 51 to 61 (Fig. 4AGo). The amplified cDNA fragments for both xTNF-{alpha} and xTNFR1 were easily detected at stage 51 before premetamorphosis. However, the amplified fragments for the two genes were faintly detected at stage 55 (i.e. premetamorphosis). Interestingly, the both expressions increased at stage 57 (i.e. prometamorphosis) and then decreased at stage 61, a climax stage of metamorphosis. We next examined the expression of xTNF-{alpha} in whole blood cells including macrophages and leukocytes, which might produce xTNF-{alpha} because TNF-{alpha} is known to be secreted from these cells in mammals. The xTNF-{alpha} mRNA was scarcely and faintly expressed in the blood cells at stages 51 and 55, respectively. Then the prominent expression of xTNF-{alpha} was observed at stage 57 and decreased at stage 61 (Fig. 4BGo). The result indicated that xTNF-{alpha} expression in blood cells was connected to prometamorphosis.


Figure 4
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  		FIG. 4. RT-PCR analyses for xTNF-{alpha} and xTNFR1 mRNA during X. laevis metamorphosis. An xTNF-{alpha}, xTNFR1, or 18S rRNA cDNA fragment was amplified by PCR using a specific primer pair (see Materials and Methods) using reverse-transcribed cDNA from whole tadpoles (A) and blood cells (B).

 
XLgoo cells are derived from tadpole-tail vascular endothelial cells
XLgoo cells were established from stage 55 (i.e. premetamorphosis). X. laevis tadpole tails. Because XLgoo cells did not appear morphologically to be muscle, neural, or epidermal cells, we examined whether they were from vascular endothelial cells. RT-PCR was carried out using primers for vascular endothelial markers, xVE-cadherin and XL-fli, with RNAs from XLgoo cells and another type of X. laevis cell line, kidney epithelial A6 cells (Fig. 5AGo). The two marker genes were expressed only in the XLgoo cells, and not in the A6 cells. Interestingly, xTNFR1 was more abundant in the XLgoo cells than in the A6 cells, which is similar to the finding that mammalian vascular endothelial cells respond to TNF-{alpha} (25, 26, 28). In contrast, xTNF{alpha} expression could not be detected in the XLgoo cells (Fig. 5AGo). From these results, we concluded that XLgoo cells were derived from vascular endothelial cells of the premetamorphic tadpole tail.


Figure 5
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  		FIG. 5. Characterization of XLgoo cells established from X. laevis tadpole tail. A, RT-PCR analysis of the XLgoo cell line. An xVE-cadherin, XL-fli, xTNF{alpha}, xTNFR1, or 18S rRNA cDNA fragment was amplified by PCR using a specific primer pair with reverse-transcribed cDNA from the XLgoo or A6 cell lines (see Materials and Methods). The xVE-cadherin and XL-fli cDNAs were amplified as specific markers for vascular endothelial cells. XLgoo cells, vascular endothelial cell-derived line (this study); A6, X. laevis kidney epithelial cell-derived line. B, Responses of XLgoo cells to xTNF-{alpha}. XLgoo cells were treated with 100 ng/ml xTNF-{alpha} for 1 h or 5 d, fixed, and stained with Alexa 488-phalloidin and 5 µg/ml Hoechst 33258. Phase-contrast (upper panels), fluorescence (middle panels), and confocal fluorescence microscope (lower panels) images are shown. Scale bar, 25 µm.

 
XLgoo cells are elongated after actin stress fiber formation in response to xTNF-{alpha}
Because XLgoo cells strongly expressed xTNFR1 mRNA (Fig. 5AGo), we asked whether they would respond to xTNF-{alpha}. When cultured in the presence of xTNF-{alpha} for 5 d, most XLgoo cells displayed an elongated cell morphology, compared with untreated control cultures (Fig. 5BGo). Next, we investigated whether actin stress fibers would form in XLgoo cells in response to a short-term exposure to xTNF-{alpha}. Fluorescence and confocal microscopy of Alexa 488-phaloidin-stained cells indicated that actin stress fibers formed completely within 1 h (Fig. 5BGo) of treatment with TNF-{alpha}. This response to TNF-{alpha} is the same as reported for mammalian vascular endothelial cells (28) and indicated that xTNF-{alpha} caused the actin stress fiber formation in the XLgoo cells.

T3 induces apoptosis in the vascular endothelial XLgoo cells
Amphibian metamorphosis is induced by high levels of T3 in the blood (2). Because the XLgoo cells were derived from the X. laevis premetamorphic tadpole tail, we first verified that T3 induced apoptotic cell death in these cells (Fig. 6Go, A and B). The percentage of floating cells in the total number of the T3-treated cells or untreated cells was 24 or 14%, respectively. Fluorescence microscopy showed nuclear condensation and/or fragmentation in 32% of the T3-treated cells; the same phenotypes were seen in 16% of the control, untreated cells (Fig. 6Go, A and B). Furthermore, the pan-caspase inhibitor Z-VAD-FMK substantially suppressed the T3-induced apoptosis (Fig. 6BGo).


Figure 6
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  		FIG. 6. Thyroid hormone (T3) induced apoptosis in endothelial XLgoo cells. A, XLgoo cells treated with 10 nM T3 for 1 wk and stained with Hoechst 33258. Scale bar, 50 µm. Arrows indicate apoptotic nuclei. B, Apoptosis induced in XLgoo cells treated with T3, in the presence or absence of pan-caspase inhibitor Z-VAD-FMK (50 µM), or with or without T3 in the presence of 0.25% dimethyl sulfoxide, the Z-VAD-FMK vehicle. The floating cells were collected and counted as apoptotic cells, and the apoptotic status of the adherent cells was determined by examining the Hoechst 33258-stained nuclei for condensation or fragmentation. The percentage of floating cells (left panel) or apoptosis (right panel) was calculated as the proportion of floating cells or floating cells plus adherent apoptotic cells, respectively, in the total number of cells counted. Each analysis was independently performed in triplicate. Two additional sets of experiments using the same method yielded similar results. C, PCR results showing cDNA fragments of xTR{alpha}, xTRβ, xCaspase3, xTNF-{alpha}, xTNFR1, or 18S rRNA amplified from XLgoo cells that were treated with 10 nM T3 for 24 or 48 h.

 
We next examined the expression of TR genes, xCaspase3, xTNF-{alpha}, and xTNFR1 in response to T3 by RT-PCR analysis. xTR{alpha} mRNA as well as xTNFR1 mRNA was constitutively expressed in the presence and absence of T3, but xTRβ mRNA was dramatically increased in response to T3. The level of xCaspase3 transcripts gradually increased over time with exposure to T3 (Fig. 6CGo). These results indicated that T3 induced the expression of apoptosis-related genes such as xTRβ and xCaspase3, which could participate in caspase-dependent apoptosis. xTNF{alpha} expression could not be detected in the presence and absence of T3.

xTNF-{alpha} suppresses T3-induced apoptosis in XLgoo cells
Because xTNF-{alpha} activated the NF-{kappa}B survival signal in A6 and XLgoo cells (Fig. 3Go and data not shown) and induced the formation of actin stress fibers and the elongation of the XLgoo cells (Fig. 5BGo), we hypothesized that xTNF-{alpha} might regulate the T3-induced apoptosis of XLgoo cells. Therefore, we investigated whether xTNF-{alpha} suppressed the T3-induced apoptosis of these cells. XLgoo cells were pretreated with xTNF-{alpha} and then treated with T3. These cells showed significantly less T3-induced apoptosis than cells that were not pretreated with xTNF-{alpha} (Fig. 7Go, A and B).


Figure 7
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  		FIG. 7. Inhibition of T3-induced apoptosis by xTNF-{alpha}. A, XLgoo cells stained with Hoechst 33258. The cells were pretreated with 100 ng/ml xTNF-{alpha} or left untreated. After 24 h, they were treated with 10 nM T3 in the presence or absence of xTNF-{alpha} for 1 wk. Arrows indicate apoptotic nuclei. Scale bar, 50 µm. B, XLgoo cells treated as in A. The percentage of floating cells (left panel) or apoptosis (right panel) was calculated as in Fig. 6BGo. Each analysis was independently performed in triplicate. Two additional sets of experiments using the same method yielded similar results. C, PCR results showing xTR{alpha}, xTRβ, xCaspase3, xTNF-{alpha}, xTNFR1, or 18S rRNA cDNA fragments amplified from XLgoo cells treated with 100 ng/ml xTNF-{alpha} for 24 or 48 h or with 10 nM T3 in the presence or absence of 100 ng/ml TNF-{alpha} for 48 h.

 
We next examined the effect of xTNF-{alpha} on the T3-induced expression of xTRβ and xCaspase3 mRNA in XLgoo cells (Fig. 7CGo). In the absence of T3, xTNF-{alpha} treatment gradually decreased the expression of xCaspase3 transcripts in a time-dependent manner. In contrast, xTR{alpha} and xTNFR1 expression did not appear to be affected by treatment with xTNF-{alpha}. xTNF-{alpha} and xTRβ transcripts could be hardly detected in the presence or absent of xTNF-{alpha}. Interestingly, xTNF-{alpha} treatment led to drastic and slight decreases of the T3-induced xTRβ and xCaspase3 expression, respectively.

xTNF-{alpha} attenuates T3-induced degeneration in tail organ culture
X. laevis tadpole tails in organ culture degenerate in response to T3 treatment (29). We examined whether xTNF-{alpha} could suppress this T3-induced degeneration. Tails from stage 51 tadpoles were treated with T3 in the presence or absence of xTNF-{alpha}. In the presence of xTNF-{alpha}, the T3-induced tail degeneration was partially blocked (Fig. 8Go). Therefore, xTNF-{alpha} may function as a suppressor in the T3-induced degeneration of tadpole tails during metamorphosis.


Figure 8
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  		FIG. 8. Effect of xTNF-{alpha} on the T3-induced degeneration of tadpole tails in organ culture. Organ culture of stage 51 X. laevis tadpole tails incubated with or without 100 ng/ml xTNF-{alpha} for 1 wk and then treated with 100 nM T3 in the presence or absence of 100 ng/ml xTNF-{alpha} for more 2 wk. Representative pictures are shown (A). Scale bar, 2.5 mm. The surface areas of the degenerating tails (n = 15 in each treatment) were measured by counting dots at a resolution of 150 dpi. The percentage of the surface area was calculated as the proportion of a treated tail to that of the same individual before the culture (B). Two additional sets of experiments using the same method yielded similar results.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, xTNF-{alpha} and xTNFR1 were identified and characterized. Structural comparisons between orthologs in mammals and X. laevis showed some evolutionary conservation of sequence, but it was not strongly conserved (Fig. 1AGo). However, phylogenetic trees of the TNF and TNFR superfamilies supported the classification of xTNF-{alpha} and xTNFR1 as members of the TNF-{alpha} and TNFR1 subfamilies, respectively (Fig. 1CGo). It will be interesting to study the molecular evolution of these gene families and their protein functions, particularly in view of the coevolution of interacting proteins and especially of ligand-receptor pairs.

In mammals, TNF-{alpha} triggers intracellular signaling through binding to its receptor TNFR1, which leads to the formation of the DISC and activation of NF-{kappa}B, JNK, and/or caspase-8 (19, 22). Previously, we reported that xRIP1 and xFADD could act cooperatively to activate NF-{kappa}B and JNK (21). The current study suggested that NF-{kappa}B activation by xTNF-{alpha} could be mediated through xRIP1 and xFADD in a Xenopus cell line (Fig. 3Go). In mammals, it is widely accepted that TNF-{alpha}-induced NF-{kappa}B activation is mediated through RIP1, but not FADD, although FADD is implicated in the activation induced by another DR ligand, Fas ligand (30). It will be interesting to clarify the function of xFADD in xTNF-{alpha} signaling during the development of X. laevis.

In amphibians, including X. laevis, thyroid hormone promotes the metamorphosis from the larval stage to the adult. To understand the intracellular signaling involved in a specific cell type with metamorphosis-mimicking capability, we established the XLgoo cell line from the tail of stage 55 X. laevis tadpoles, which are premetamorphic. The XLgoo cells expressed vascular endothelial markers, xVE-cadherin and XL-fli. This is the first report of a vascular endothelial cell line in X. laevis. The XLgoo cells, which were established from stage 55, expressed high levels of xTNFR1 (Fig. 5AGo), although xTNFR1 showed higher expression in the whole tadpoles at stage 57 than at stage 55 (Fig. 4AGo). Other types of cells than vascular endothelial cells in the tadpoles at stage 57 may contribute the high level expression of xTNFR1. Furthermore, and as expected, this cell line had characteristics of metamorphic cells, i.e. T3 induced apoptosis in them (Fig. 6Go, A and B), as it does in myoblast XLT15 cells derived from tadpole tails (27). Further study of the molecular mechanisms of T3-induced apoptosis in XLgoo cells and XLT-15 cells could provide useful information about the intracellular signaling that drives metamorphosis in muscle and endothelial cells.

During metamorphosis of X. laevis, the T3 and TR complex binds specific DNA elements and promotes the expression of various genes (31, 32, 33). xTRβ and xCaspase3 mRNAs are induced during metamorphosis (27, 34, 35). Here RT-PCR analysis showed that T3 induced xTRβ transcription (Fig. 6CGo). Because it is unclear what roles xTRβ plays in metamorphosis, it is necessary to clarify the relationship between xTRβ and the T3-induced apoptosis of XLgoo cells. The T3 treatment also up-regulated xCaspase3 expression in the XLgoo cells, and T3-induced apoptosis of XLgoo cells could be mediated through caspase activation (Fig. 6Go); this finding agrees with the previous results in XLT15 cells (27). It will be interesting to examine how the caspase cascade is activated and how the caspase3 gene is transcriptionally up-regulated during metamorphosis-associated apoptosis.

From the tail of stage 55 X. laevis tadpoles, we established not only the XLgoo cell line but also a myoblast cell line (Takayama, S., T. Ishii, K. Tamura, S. Hawaribuchi, T. Shiba, N. Takamatsu, and M. Ito, unpublished data). One finding that particularly intrigued us was that xTNF-{alpha} repressed the T3-induced apoptosis in XLgoo cells but not the myoblast cells, suggesting that the suppression of T3-induced apoptosis by xTNF-{alpha} may occur specifically in vascular endothelial cells during metamorphosis. The repressive effect of xTNF{alpha} on the T3-induced TR-β induction in XLgoo cells was impressive (Fig. 7CGo). This contrasted the significant but much more limited effects of xTNF{alpha} in preventing the T3-induced apoptosis in XLgoo cells (Fig. 7BGo). This difference might lead to the idea that xTR-β partially participates in the T3-induced apoptosis in XLgoo cells. In the next paragraph, we also discuss the limited effects of xTNF{alpha} on the T3-induced degeneration of premetamorphic tails. In this study, it remains unknown how xTNF{alpha} could repress T3-induced apoptosis in the XLgoo cells. One possibility is that activated NF-{kappa}B by xTNF{alpha} could block T3-induced apoptosis. In this case, activated NF-{kappa}B as a survival factor might enhance transcription of some genes encoding proteins, which inhibit activation of caspase cascades and transcription of xTRβ. It is also possible that xTNF-{alpha} controls the expression and activity of regulates the expression and activity of type II or III iodothyronine deiodinase, which control levels of the thyroid hormones.

xTNF-{alpha} mRNA showed strong expression at prometamorphosis during metamorphosis (Fig. 4BGo). It is possible that the target cells of xTNF-{alpha} secreted by some blood cells such as macrophages and/or leukocytes include vascular endothelial cells at prometamorphosis. Because blood vessels supply oxygen, remove waste, and deliver nutrients and hormones, such as T3, vascular endothelial cells are sure to play a special role in metamorphosis. It is conceivable that xTNF-{alpha} could protect vascular endothelial cells from T3 during prometamorphosis but support apoptosis in other types of cells. In organ culture, xTNF-{alpha} partially blocked T3-dependent tail degeneration (Fig. 8Go). This result strongly suggests xTNF-{alpha} is involved as a regulator during metamorphosis. However, the repression of the tail degeneration by xTNF-{alpha} was not drastic. This may be because tadpole tails consist mostly of muscle cells.

In mammals, TNF-{alpha} is implicated in leukocyte transendothelial migration (36, 37). Because we found that xTNF-{alpha} and xTNFR1 were highly expressed in endothelial XLgoo cells and blood cells, respectively, it is interesting to investigate whether transendothelial migration induced by xTNF-{alpha} could participate in metamorphosis. In general, TNF-{alpha} signal is involved in cell proliferation, survival, differentiation, and apoptosis as well as immunity and inflammation. TNF-{alpha} is expressed in not only blood cells including macrophages but also several types of cells including adipocytes. TNFRs are also expressed in various types of cells, including vascular endothelial cells, muscle cells, keratinocytes, and chondrocytes. It is possible that xTNF-{alpha} regulates cell survival and/or apoptosis of xTNFR1-expressing cells during metamorphosis. To test the possibility, it may be necessary to clarify distribution patterns of xTNFR1 and xTNF-{alpha} during metamorphosis and produce transgenic tadpoles carrying cell type-specific and stage-specific knockdown vector for xTNFR1. In the future, it will be interesting to discuss functional conservation of TNF{alpha} signal during ontogenesis in vertebrate evolution.


   Footnotes
 
Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online April 10, 2008

Abbreviations: DISC, Death-inducing signal complex; DR, death receptor; FADD, Fas-associated death domain; GST, glutathione-S-transferase; hTNF-{alpha}, human TNF-{alpha}; hTNFR1, human TNFR1; JNK, c-Jun N-terminal kinase; NF-{kappa}B, nuclear factor-{kappa}B; RIP1, receptor-interacting protein 1; TNFR, TNF receptor; TNFR1, TNFR 1; TR, thyroid hormone receptor; xDR, Xenopus DR; xDR-M, xDR family member; xFADD, X. laevis FADD; xFADD-DD, xFADD mutant containing the death domain; xRIP1, X. laevis RIP1; xRIP1-DD, xRIP1 mutant containing the death domain; xTNF-{alpha}, X. laevis homolog of TNF-{alpha}; xTNFR1, X. laevis homolog of TNFR1; xTR, Xenopus TR.

Received November 20, 2007.

Accepted for publication April 2, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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