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Gerhard K. H. Przemeck,2
Manuela Schneider,1
Stéphanie G. Moreno,3
Nadja Mayr,3
Antonis K. Hatzopoulos,3
Martin Hrabé de Angelis,2
Wolfgang Wurst,4,5
Georg W. Bornkamm,3
Markus Brielmeier,1 and
Marcus Conrad3*
Department of Comparative Medicine,1 Institute of Experimental Genetics,2 Institute of Developmental Genetics, GSF Research Centre for Environment and Health, Neuherberg,4 Max Planck Institute of Psychiatry,5 Institute of Clinical Molecular Biology and Tumor Genetics, GSF, Munich, Germany3
Received 30 July 2004/ Returned for modification 1 October 2004/ Accepted 24 December 2004
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ABSTRACT |
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INTRODUCTION |
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B (34) and AP-1 (20), mediate peroxiredoxin antioxidant properties (35), and are putatively involved in DNA synthesis. The activity of Txn is controlled by thioredoxin reductases (Txnrd) together with NADPH as a cofactor. Three mammalian thioredoxin reductases are known, including a cytosolic (Txnrd1) (10), a mitochondrial (Txnrd2) (9), and a testis-specific (41) isoform. Thioredoxin reductases are homodimeric flavoproteins with two N- and C-terminally located interacting catalytic centers (12, 26). The C-terminal redox center contains a selenocysteine (Sec) residue which is part of a conserved Gly-Cys-Sec-Gly motif that is crucial for Txnrd function. Besides thioredoxins, thioredoxin reductases can also reduce other substrates, such as lipoic acid, NK-lysin, ascorbate, and ubiquinone (1, 31, 45). Several gene targeting approaches with mice have been performed to investigate the participation of the Txn/Txnrd systems in development and adult physiology. The results revealed that cytosolic (Txn1) and mitochondrial (Txn2) thioredoxins are indispensable for embryonic development (24, 30). In Txn1–/– mutants, early embryonic death (by embryonic day 6.5 [E6.5]) is associated with a dramatically reduced proliferation of inner mass cells. Txn2-deficient embryos develop exencephaly, show markedly increased apoptosis, and die during midgestation around E10.5. Using a conditional targeting approach, we have shown recently that Txnrd2 is also essential for embryonic development (5). Txnrd2-null embryos die around E13.0 due to defects in hematopoiesis and heart development. The cardiac-specific deletion of Txnrd2 leads to fatal dilated cardiomyopathy and morphological abnormalities of cardiomyocytes. These results support and extend the observation that the heart-specific overexpression of dominant-negative Txn1 leads to oxidative stress and cardiac hypertrophy (46).
Txnrd1 is expressed in cells with a strong proliferative activity, such as neoplastic tissues and tumor cell lines (13, 23), and it has also been shown to be a c-Myc target in a human B-cell line (39), indicating a role in cell proliferation. The presence of several thioredoxin reductases obscures the contribution of the individual thioredoxin reductases to cellular growth and survival, and it is unknown to what extent Txnrd1 and Txnrd2 can complement each other. To study the impact of Txnrd1 on apoptosis and proliferation in vivo, we used cre/loxP technology to create mice with a floxed Txnrd1 allele. Ubiquitous Cre-mediated deletion resulted in early embryonic death around E10.5 due to severe growth retardation and widespread developmental abnormalities in most tissues, excluding the heart. This notion was further supported by the heart-specific deletion of Txnrd1, which resulted in viable mice with no obvious phenotypes. It thus appears that only Txnrd2 is crucial for heart development and function, whereas Txnrd1 activity is required in other developing tissues.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR analysis. Total RNAs were isolated from embryos at gestational day 9.5 by the use of peqGOLDTriFast (peqLab, Erlangen, Germany). DNase-treated total RNAs (DNase I and RNase I free; Roche, Mannheim, Germany) were reverse transcribed by use of a reverse transcription (RT) system (Promega Corporation, Madison, Wis.). Primer pairs were designed to amplify products from exon 13 to exon 15 (Oligo Txnrd1-E13f, 5'-TTG GCC ATT GGA ATG GAC AGT CC-3'; Oligo Txnrd1-E15r, 5'-AGC ACC TTG AAT TGG CGC CTA GG-3') and from exon 5 to exon 11 (Oligo Txnrd1-59, 5'-CGA AGA CAC AGT GAA GCA TGA CTG GG-3'; Oligo Txnrd1-60, 5'-TCC CCT CCA GGA TGT CAC CGA TGG CG-3'). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control (GAPDH1, 5'-CTT CAT TGA CCT CAA CTA CAT GG-3'; GAPDH2, 5'-GCC TGC TTC ACC ACC TTC TTG-3').
Whole-mount in situ hybridization. C57BL/6j embryos were dissected at E8.5, E9.5, and E10.5. Sense and antisense RNA probes were generated by the use of DIG RNA labeling mix (Roche Molecular Biochemicals) according to the manufacturer's instructions. Whole-mount in situ hybridization was performed as previously described (40). Embryos were stained with BM Purple AP substrate (Roche Molecular Biochemicals) and postfixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline. A Txnrd1-specific probe corresponding to exons 5 to 11 was generated from mouse testis cDNA by use of the primer pair OligoTxnrd1-59 and OligoTxnrd1-60. A probe specific for T (brachyury) was used as described previously (15).
In situ end-labeling method (ISEL). The detection of apoptotic cells was performed with an ApopTag kit (S7100; Serologicals Corporation, Norcross, Ga.) on 4% PFA-fixed paraffin-embedded sections according to the manufacturer's instructions.
Immunohistochemistry. An antibody against phospho-histone H3 (Ser10) (New England Biolabs GmbH, Frankfurt am Main, Germany) was used to detect mitotic chromosome condensations at a dilution of 1:50 on 4% PFA-fixed paraffin-embedded sections according to the manufacturer's instructions.
Immunoblotting. After dissection, the cytosolic fraction of cardiac tissue was prepared as described previously (27). Extracts were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-Txnrd1 (LF-PA0023; LabFrontier, Seoul, Korea). The protein concentration was quantified by the Bradford assay (Bio-Rad, Hercules, Calif.).
Isolation and culture of embryonic cells. Embryos were isolated at E9.5 or E10.5. The head and the primitive heart were removed from the body trunk. The heart and the remaining body trunk were minced separately, trypsinized, treated with DNase, and cultivated under standard conditions in Dulbecco's modified Eagle's medium (Invitrogen GmbH, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany), 2 mM glutamine, 100 U of penicillin-streptomycin/ml, and 0.1 mM ß-mercaptoethanol.
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RESULTS |
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Targeted disruption of Txnrd1 results in early embryonic lethality. The intercross of hemizygous Txnrd1+/– mice, obtained by the mating of floxed Txnrd1 mice with ubiquitously expressing Cre deletion mice, never resulted in viable homozygous Txnrd1 KO mice. Of 79 pups, 32 (40.5%) were WT and 47 (59.5%) were hemizygous for Txnrd1. The genotyping of embryos dissected from hemizygous intercrosses on different days of gestation revealed that the expected Mendelian ratio was maintained up to E10.5, although the resorption of Txnrd1 KO embryos was frequently observed between gestational days 9.5 and 10.5 (Table 1).
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Proliferation defects in Txnrd1 KO embryos. To investigate whether increased apoptosis or reduced proliferation was responsible for the obvious growth retardation of Txnrd1–/– embryos, we performed ISEL immunostaining of serial sections of paraffin-embedded embryos. In contrast to the case for WT sections at E10.5, only a few ISEL-positive cells could be detected in sections of Txnrd1-deficient embryos of the same age (data not shown). This was in line with the findings for WT sections at E8.5, indicating that apoptosis levels were comparable in WT and KO embryos. In contrast, cell proliferation in Txnrd1 knockout embryos was markedly reduced compared to that in their wild-type siblings. Using phospho-histone H3 (PH3) as a marker (6, 43), we observed clear differences in the numbers and densities of PH3-positive cells between Txnrd1 KO and wild-type siblings at E9.5 (Fig. 4N and O) as well as E10.5 (data not shown). The number of proliferating cells in KO embryos was clearly reduced and comparable to the number of PH3-positive cells in WT embryos at earlier stages.
To further investigate the effect of Txnrd1 deficiency on cell proliferation, we isolated mouse embryonic fibroblast (MEFs) from WT and KO cells. Whereas the cultivation of MEFs isolated from hemizygous Txnrd1 embryos at E8.5 and E9.5 was always successful, the establishment of proliferating MEFs from homozygous knockout embryos invariably failed (data not shown). Although some cells attached to the culture dish after seeding, they were not capable of proliferating. Notably, the isolation and cultivation of spontaneously contracting cardiomyocytes from E10.5 Txnrd1 KO embryos were possible (data not shown).
Taken together, the results show that Txnrd1 knockout embryos display general growth retardation with the exception of cardiomyocytes, most likely due to a global proliferation defect, and reach a developmental stage comparable to E8.5 wild-type littermates when dissected at gestational day E10.5.
No phenotype after heart-specific elimination of Txnrd1. Thioredoxin has recently attracted considerable interest in cardiac research (47). The heart-specific overexpression of dominant-negative Txn1 was shown to be associated with increased oxidative stress and cardiac hypertrophy in mice, whereas the overexpression of wild-type Txn1 was beneficial (46). Yet the expression studies of Txnrd1 by in situ hybridization described above and analyses of the Txnrd1 null phenotype did not provide evidence for the suspected role of Txnrd1 in the heart. There are two possibilities to explain this discrepancy. Firstly, Txnrd1 might contribute to proper heart functioning at later stages of development or eventually only after birth, when an entirely self-sustaining circulatory system must be maintained by the heart. The second possibility is that Txnrd2, and not Txnrd1, plays a decisive role in proper heart development and function. To discriminate between these possibilities, we specifically deleted Txnrd1 from the heart. To this end, we first crossed Txnrd1+/– mice with MLC2a-Cre-expressing mice (44). The resulting Txnrd1+/– MLC2a-Cre mice were subsequently crossed with Txnrd1loxP/loxP mice to obtain Txnrd1loxP/– MLC2a-Cre mice. The efficiency of the heart-specific deletion of Txnrd1 was monitored by RT-PCR analysis and Western blotting (Fig. 5A) and appeared to be similarly effective as the MLC2a-Cre-mediated elimination of Txnrd2. Heart-specific Txnrd1 KO mice were viable and fully fertile. The preparation of cardiac tissue did not show any signs of cardiac insufficiency (data not shown). Similarly, a histological examination did not reveal any differences in heart shape or in the cellular morphology of cardiomyocytes between WT and heart-specific Txnrd1 KO mice (Fig. 5B to E). These results indicate that Txnrd1 is dispensable for embryonic heart development. We conclude that Txnrd2 rather than Txnrd1 plays the decisive role in heart development.
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DISCUSSION |
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Only the last of 15 exons of the Txnrd1 gene was deleted in our gene targeting approach. This exon includes the selenocysteine-containing active site and the SECIS element required for selenocysteine incorporation. The strategy of deleting only a small part of the gene bears the risk that a truncated protein will be synthesized from the targeted allele and that the truncated protein may impose a gain-of-function rather than a loss-of-function phenotype. Given the facts that small amounts of truncated transcripts were indeed detected and that selenium-compromised Txnrd1 was shown to induce apoptosis (2), it was particularly important to test whether a truncated protein that might be responsible for the phenotype observed was made from the targeted allele. A Western blot analysis of heart tissue obtained from Txnrd1loxP/– MLC2a-Cre mice (and Txnrd1loxP/– mice as controls) revealed that the deletion of the Txnrd1 gene in the heart was indeed very effective and apparently did not lead to the formation of a truncated protein (Fig. 5A). The notion that our targeting strategy led to a loss- and not to a gain-of-function phenotype is corroborated by the fact that hemizygous Txnrd1+/– and Txnrd1loxP/– mice were normal and did not show any evidence of increased cell death in vivo.
The Txnrd1–/– phenotype described here clearly differs from that of Txnrd2–/– embryos (5). Txnrd2–/– embryos were smaller and pale compared to their wild-type siblings and died of severe anemia and improper heart development at E13.5. The importance of Txnrd2 for heart development and function was emphasized by the heart-specific deletion of Txnrd2 leading to congestive dilated cardiomyopathy and perinatal death, whereas mice with a heart-specific elimination of Txnrd1 developed normally and appeared healthy. The importance of thioredoxin for proper heart function has recently been noticed by Yamamoto et al. (46). They showed that the heart-specific overexpression of a dominant-negative mutant of Txn1 in mice results in increased oxidative stress and cardiac hypertrophy. Since dominant-negative Txn1 most likely also interferes with Txn2 function, the transgenic approach did not determine which of the thioredoxin/thioredoxin reductase systems is responsible for the observed phenotype. Thus, our data clearly indicate a pivotal role for Txnrd2 in proper heart function but do not exclude a potential contribution of Txn1. If Txn1, in addition to Txn2, is also of critical importance in the heart, then Txn1 should be a substrate of Txnrd2.
The time of embryonic death and the appearance of Txnrd1 KO embryos closely reflected the expression pattern. At E8.5, Txnrd1 was highly expressed in the developing neural tissues, i.e., the neural tube, rhombomeres, and forebrain, and at E9.5 and E10.5, expression was confined to branchial arches, somites, and the apicoectodermal ridge of the limb buds, but the primitive heart was largely excluded. Notably, Jurado and colleagues showed that Txnrd1 expression peaks around E10.5 (19). The discrepancy in the expression patterns of Txnrd1 and Txnrd2 in the heart, the different subcellular localization of Txnrd1 and Txnrd2, and potential differences in the enzymes themselves suggest the nonredundancy of both thioredoxin reductases (28).
Our analysis of the phenotypes of Txnrd1–/– and Txnrd2–/– embryos has clearly revealed the nonredundant functions of Txnrd1 and Txnrd2, but we could not assess to what extent both thioredoxin/thioredoxin reductase systems might functionally overlap. Additional information was provided by comparing the phenotypes of Txn1–/– to Txnrd1–/– and of Txn2–/– to Txnrd2–/– embryos. Mice deficient in Txn1 show a severely perturbed proliferation of the inner cell mass and embryonic death at around E6.5, which is much earlier than that for Txnrd1–/– embryos (E10.5). Likewise, mice lacking the Txn2 gene die at E10.5 from exencephaly and massive apoptosis, which is significantly earlier than the death of Txnrd2–/– embryos (E13). The fact that Txn1–/– and Txn2–/– mice die much earlier than mice lacking the respective thioredoxin reductases may suggest a partial functional overlap of Txnrd1 and Txnrd2. Alternatively, other pivotal functions of thioredoxins that do not require thioredoxin reductase activity might exist. A C-terminally truncated form of Txn (referred to as Txn80) exerts a potent mitogenic activity on resting human peripheral mononuclear cells (32). This truncated form lacks reductase activity and tends to form homodimers, unlike full-length Txn. It may be possible to discriminate between these possibilities by generating mice with a Txn1 gene that is mutated in the catalytic cysteine residues Cys 32 and Cys 35.
It is remarkable, but not surprising, that the phenotypes of both Txn1–/– and Txnrd1–/– embryos are dominated by a defect in cell proliferation. This supports the notion that Txn1 is the main substrate of Txnrd1 and strongly argues that Txnrd1–/– embryos die from compromised Txn1 activity. An important role of the Txn1/Txnrd1 system in cell proliferation is also suggested by the facts that the Txn/Txnrd system is abundantly expressed in tumors and tumor cell lines (23, 33, 36), that Txnrd1 is a direct target gene of the proto-oncogene c-myc (39), and that it was impossible to establish primary mouse embryonic fibroblast cultures of Txnrd1–/– embryos. It is of crucial importance now to identify the molecular targets of Txn1 in cell proliferation. Due to the pleiotropic activity of Txn1 and its interaction with many different target molecules, it will remain a very difficult task to define the actions of Txn1 and Txnrd1 in diverse biological processes at a biochemical level. Several reports indicate that the Txn/Txnrd1 system—either directly or through substrates such as Ref-1 and peroxiredoxins—participates in the regulation of transcription, replication, and DNA repair (3, 16). Whether the mammalian Txn/Txnrd system provides reducing equivalents for the synthesis of deoxyribonucleotides by ribonucleotide reductase, as shown for Escherichia coli (21), Saccharomyces cerevisiae (25), and Xenopus laevis (14), is still being debated.
Its association with proliferation makes Txnrd1 an interesting drug target for cancer therapy in the future. Our data stress the importance of inhibiting Txnrd1 without affecting Txnrd2 activity. We predict that drugs lacking specificity for Txnrd1 would most likely also impair the activity of Txnrd2, eventually leading to serious cardiac side effects. Since the catalytic centers of Txnrd1 and Txnrd2 are virtually identical, it will be extremely difficult to design chemical drugs with specificity for Txnrd1. Txnrd1 may therefore be an ideal target for inhibition by RNA interference.
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ACKNOWLEDGMENTS |
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We are most grateful to K. R. Chien for providing the MLC2a-Cre mice. We thank S. Lippl and members of the blastocyst injection unit for their excellent technical assistance and animal keepers for their support. We are grateful to A. Banjac, A. Seiler, W. Schmahl, and U. Heinzmann for fruitful discussions and to V. Mostert and R. Chapman for critically reading the manuscript.
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FOOTNOTES |
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Present address: Institut für Rekonstruktive Neurobiologie, Rheinische Friedrich-Wilhelms-Universität, D-53105 Bonn, Germany.
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