Human Molecular Genetics, 2002, Vol. 11, No. 24 2997-3006
© 2002 Oxford University Press
Identification of the coding sequences responsible for Tsc2-mediated tumor suppression using a transgenic rat system
1Department of Experimental Pathology, Cancer Institute, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, Japan, 2Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo-ku, Kyoto, Japan and 3YS New Technology Institute Inc., 519 Shimo-Ishibashi, Ishibashi-cho, Shimotuga-gun, Tochigi, Japan
Received June 11, 2002; Revised August 11, 2002; Accepted September 17, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hereditary renal carcinomas in the Eker rat are caused by germline retrotransposon insertion in the tuberous sclerosis-2 (Tsc2) gene. We established previously a transgenic Eker rat model into which was introduced a wild-type Tsc2 gene. The embryonic lethality of mutant homozygotes and renal carcinogenesis of heterozygotes were completely suppressed by this transgene (Tg). The function of the Tsc2 product (tuberin) is not fully understood, although several findings have been obtained mainly in vitro. Therefore, to elucidate the functional domains of Tsc2 in vivo, we generated transgenic Eker rats carrying deletion mutants of the Tsc2 gene. A Tg coding for the C-terminal region (amino acids 1425–1755) suppressed renal carcinogenesis in the Eker rat and interestingly the degree of this suppression correlated with the level of expression of the Tg. Notably, the product of this Tg lacks the ability to bind to the Tsc1 product (hamartin). Surprisingly, while a Tg lacking the C-terminus of tuberin (amino acids 1–1755) completely suppressed renal carcinogenesis, it partially rescued homozygous mutants from embryonic lethality. In conclusion, we have determined the minimal region of tuberin necessary for tumor suppression but the suppressive effect was quantitative. Tuberin could function as a tumor suppressor without binding to hamartin. The requirement of the functional domain(s) of tuberin might differ for prevention of embryonic lethality and for suppression of renal carcinogenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tuberous sclerosis (TSC) is an autosomal dominantly inherited disease which affects
1 in 6000 individuals (1). Its clinical features mainly include hamartomas of various organs (angiomyolipomas of kidney and liver, lymphangioleiomyomatosis of lung, etc.) and neurological dysfunction (seizures, mental retardation, etc.). There are two genes associated with TSC: TSC1 on chromosome at 9q34 (2) and TSC2 at 16p13.3 (3). The TSC1 product (hamartin) has a putative transmembrane domain in the N-terminal region and a coiled-coil domain in the C-terminal end. Hamartin was recently reported to be a regulator of the small GTPase Rho and to bind to members of the ERM (ezrin/radixin/moesin) family to regulate cell adhesion (4). In the TSC2 product (tuberin), a homologous region for Rap1 GTPase activating protein (GAP), Rap1GAP, is located at the C-terminal region and possesses weak GAP activity for Rap1 (5). The following findings have also been reported for tuberin: specific GAP activity for Rab5 (6); two transcriptional activating domains (7); and several binding partners—hamartin (8,9), Rabaptin5 (6) and steroid hormone receptors (10). Except for the binding region for hamartin at the N-terminus, the reported domains are all located in the C-terminal region. Recent studies have revealed that hamartin and tuberin might be involved in the insulin signaling pathway in Drosophila and lack of function of either gene results in deregulation of cell growth, cell proliferation and organ size (11–13).
The functional relationship between hamartin and tuberin might be important because of the similarity of symptoms among TSC patients. In addition to forming a complex, hamartin prevented the ubiquitin-mediated degradation of tuberin, when tuberin was highly overexpressed by transfection (14). The possibility of tuberin acting as a chaperone for hamartin has been mentioned (15).
There is a naturally occurring animal model, the Eker rat, which possesses a germline mutation in the middle of Tsc2 (Eker mutation) (16,17). Heterozygous mutant Eker rats (Tsc2Ek/+) develop multiple lesions in various organs such as kidney (renal carcinoma), spleen (hemangiosarcoma) and brain (subependymal and cortical hamartomas) (18–20). Homozygosity of the mutant allele in the Eker embryo (Tsc2Ek/Ek) causes embryonic lethality. The cause of embryonic lethality is not clear, but it has been documented that dysraphia and disrupted neuroepithelial growth are caused by loss of tuberin function though both were strain-dependent (21). We recently reported that, using transgenic technology, introduction of a wildtype Tsc2 (
exon 25 and 31) rescued homozygous Eker mutants from embryonic lethality and suppressed renal carcinogenesis of heterozygous Eker rats (22). Thus, analysis using transgenic Eker rats can be expected to elucidate Tsc2 gene function in vivo.
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TOPABSTRACTINTRODUCTIONRESULTDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of transgenic rats and detection of each transgene-derived product
We constructed four Tgs to examine functional domains (Fig. 1A). In Tsc2–DCT55–flag, the last 55 amino acids, which corresponds to exon 41, were deleted. The region (1425–1755 amino acids) including the Rap1GAP homologous domain was deleted in Tsc2–DRG and Tsc2–DCT385–flag and the latter Tg was also deleted for the last 55 residues. Conversely, 1425–1755 amino acids were retained with the first 73 residues in Tsc2–RGH. We obtained the following number of transgenic rats; four transgenic founders of Tsc2–DCT55–flag, three of Tsc2–DCT385–flag, six of Tsc2–DRG and three of Tsc2–RGH (Fig. 1B). We selected two independent lines of each type of transgenic rat for further analysis. There was strain mixing, a Wister background and a Eker background, as a result of the transgenesis procedure. In each type, the Tg-specific RT–PCR product was amplified both in embryo and in kidney (data not shown). Sequence analysis confirmed that the products were derived from each Tg (data not shown). We next detected Tg-derived proteins by western blot analysis in both embryos and adults (Fig. 2). In each line, specific bands with expected molecular weights of
195 kD, 160 kD and 45 kDa for Tgs of Tsc2–DCT55–flag, Tsc2–DRG and Tsc2–RGH products, respectively, were detected. The product from Tsc2–DCT385–flag could not be detected by western blot analysis though the mRNA was expressed. The reason for this was unclear. Thus, further analysis was performed using Tsc2–DCT55–flag, Tsc2–DRG and Tsc2–RGH. There was no general effect of the transgene expression on growth and development in these rats at several points in time (data not shown).
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/HindIII) are shown on the side.
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Suppression of renal carcinogenesis by Tsc2–RGH correlating with expression level of Tg
Exposure of rat embryos to N-ethyl-N-nitrosourea (ENU) via the dam placenta accelerates renal carcinogenesis (23). Therefore, we used this system to examine which Tg suppresses renal carcinomas (RCs). We mated Eker rats with wildtype rats in the presence of Tg in either of the parents. Dams were given a single i.p. administration of ENU on the 15th day of gestation. Offspring (non-transgenic Eker rats and transgenic Eker rats) were then macroscopically and histologically examined at 8 weeks of age (Fig. 3A). An one-way analysis of variance (ANOVA) was applied to the numbers of small (<2 mm) and large (>2 mm) renal tumors with respect to the genotypes from Table 1. No significant main effect of sex or interaction between sex and genotype was found by two-way ANOVA. Applying the one-way ANOVA to the same combinations of sexes and genotypes, no significant differences were observed by multiple comparison. Applying the two-way ANOVA to total tumors and size (Table 2), no significant main effect of sex or interaction between sex and genotype was found. Hence, the one-way ANOVA was applied to these quantities with respect to genotype and multiple comparisons. Tsc2–DCT55–flag, which lacks only exon 41, completely suppressed ENU-induced RCs of the Eker rat (n=8). In contrast, Tsc2–DRG did not suppress RCs (n=12) and there was no significant difference in non-transgenic Eker rats and Tsc2–DRG–carrying Eker rats. Also there was no statistical significance in the mean of size and total number of tumors. On microscopic analysis, we found no phenotypically altered tubules in kidneys from Eker rats carrying Tsc2–DCT55–flag (data not shown). There was no apparent difference in either the number or the size of tumors between two independent lines in Tsc2–DCT55–flag- and Tsc2–DRG-carrying Eker rats. Surprisingly, Tsc2–RGH, which possesses the Rap1GAP homologous domain but lacks the N-terminal region, suppressed RCs, although not completely. In addition, Eker rats carrying Tsc2–RGH exhibited varying extent of RCs. In one line (no. 26), transgenic Eker rats manifested macroscopic RCs clearly smaller than non-transgenic Eker rats (n=8). In another line (no. 15), there were no macroscopic RCs, although we found some focal adenomas microscopically (n=8). A multiple comparison by Tukey's method of the number of both sizes of renal tumors identified macroscopically yields significant differences between Tsc2Ek/+ (control) and Tsc2Ek/+ rats in line no. 15, but not line no. 26 (Table 1; the level of significance was 0.05). Histologically, there was statistical significance both in the mean of size and in the total number of tumors for both lines (Table 2; the levels of significance were 0.05). There was an apparent difference in suppressive activity between the two lines. Line no. 15 had a stronger expression of Tg than line no. 26 (Fig. 3B). Thus, the level of expression of the Tsc2–RGH-derived product was correlated with the extent of tumor suppression.
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Deletion of C-terminal 55 residues of tuberinaffected embryonic development
Since the Tg of wildtype Tsc2 rescues homozygous Tsc2 mutants from embryonic lethality (22), we next examined whether the Tgs constructed in this study could rescue the homozygous mutants. We mated transgenic Eker rats with non-transgenic animals and obtained at least 5 litters in each independent line and thus at least 10 litters for each type of Tg. Although we confirmed that some of each Tg-carrying Tsc2Ek/Ek mice were alive and expressing the Tg product at E12.5 (Fig. 2B), we could not obtain Tsc2Ek/Ek live born mice carrying Tsc2–DRG or Tsc2–RGH (Table 3). In the case of Tsc2–DCT55–flag, we obtained four Tsc2Ek/Ek offspring. On comparison of two independent lines carrying Tsc2–DCT55–flag (
no. 21 and no. 51), we obtained four Tg-carrying Tsc2Ek/Ek mice in line no. 21 but not in line no. 51. These four were of normal appearance until six months of age. By Mendel's law, the probability of Tsc2Ek/Ek offspring is less than or equal to 0.125. The observed number of cases was 4/67. In the most conservative case, i.e. a probability of 0.125, a binomial test with the null hypothesis P=0.125 versus the alternative hypothesis P<0.125 was give a P-value of 0.067. The null hypothesis was rejected at a level of significance of 0.1 but not 0.05. Interestingly, there were four stillborn offspring in one litter in line no. 21, three of which proved to be Tg-carrying Tsc2Ek/Ek. The genotype of the remaining pups could not be determined. We examined Tg expression in these lines to explore any differences. The expression level was higher in line no. 21 than no. 51 (Fig. 4). Thus, the level of Tg expression of Tsc2–DCT55–flag was correlated with the rate of birth of Tsc2Ek/Ek offspring carrying Tsc2–ins.flag. Therefore, the presence of the 55 C-terminal residues of tuberin might influence the development of embryos.
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Association between hamartin and each Tg-derived product
We examined the association between hamartin and endogenous tuberin for each Tg-derived product in vivo. First, we examined the ability of the Tg-derived product to bind with hamartin. Endogenous tuberin and products of both Tsc2–DCT55–flag and Tsc2–DRG bound to hamartin in vivo (Fig. 5A and B). Though the level of expression of Tsc2–DRG product was much higher than that of endogenous tuberin (Fig. 2B), the amount of Tsc2–DRG product binding to hamartin was smaller than that of endogenous tuberin (Fig. 5B). It is likely that the ability to bind hamartin or the stability of the complex depends on the integrity of the C-terminal region of tuberin. As expected from the reported binding of tuberin to hamartin (9), the Tsc2–RGH product did not bind to hamartin (Fig. 5C).
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The effect of each deletion mutant on the level of expression of hamartin was also examined. Among the Tsc2+/+ embryos and each transgenic and non-transgenic Tsc2Ek/Ek embryo, the protein expression of hamartin gradually decreased with deletion of the C-terminal region of tuberin (Fig. 6A). Furthermore, the level of expression of hamartin in the Tsc2Ek/Ek embryos carrying Tsc2–RGH was equal to that in the Tsc2Ek/Ek embryos (Fig. 6B). However, the levels of Tsc1 mRNA expression in these embryos did not significantly differ (Fig. 6C), suggesting that the decrease in amount of protein was due to a translational or post-translational mechanism.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Although the relationship between the structure and activity of tuberin has been explored in previous studies, the functional domains important for its tumor suppression in vivo remain to be elucidated. In this study, we applied a transgenic system to determine the domains of tuberin which function in vivo, by analysing effects of deletion mutants on the phenotype of Eker rats. In addition, we examined the association between hamartin and tuberin mutants in rat tissues in relation to the phenotypic consequences of Tg expression.
On introduction of Tsc2–RGH, the ENU-induced renal tumorigenesis in the Eker rat was significantly, though not completely, inhibited. Although suppression of the proliferation of established tumor cell lines by introducing the C-terminal region of tuberin was reported (24), its significance for in vivo tumorigenesis has remained elusive. The present study clearly showed that the C-terminal region of tuberin including the Rap1GAP homology domain plays a pivotal role in tumor suppression in vivo and that its level of expression correlated with the degree of tumor suppression. In contrast to the effect on renal tumorigenesis, however, Tsc2–RGH alone was not sufficient to rescue homozygous Tsc2 mutants from embryonic lethality. The product of Tsc2–RGH lost hamartin-binding activity in rat tissues, as expected from a previous study (9). Recently, stabilization of hamartin by tuberin upon the formation of their complex was suggested by transfection experiments (14,15). In the present study, we reproducibly observed a significant decrease in the amount of hamartin in homozygous Tsc2 mutant embryos compared with wildtype controls. With the functional loss of tuberin, the amount of hamartin may be reduced by translational or post-translational mechanisms such as the facilitation of processes of degradation in vivo and Tsc2–RGH may not be able to reverse this reduction. The reduction would be tightly linked to the functional association between tuberin and hamartin and related to the cause of embryonic mortality. These observations suggested that the mechanism underlying the suppression of renal tumorigenesis by Tsc2–RGH did not include the formation of a stable complex between hamartin and tuberin or the presence of normal amounts of hamartin. On immunohistochemical analysis, a limited co-localization of tuberin and hamartin was demonstrated in rat and human tissues (25,26). Possibly, some of the functional activity of wild-type tuberin may not depend on binding with hamartin.
Because the suppression of renal tumor development by Tsc2–RGH was not complete, the N-terminal two-thirds of tuberin including the hamartin-binding domain appeared to be necessary for the complete tumor suppressor function in vivo. Why the competitive kinetics of tumor suppression observed in Tsc2–RGH between two independent lines (no. 15 and no. 26) occurred was currently unknown. However, one possibility may be the following. From an observation that the increased expression amount of Tsc2–RGH reflects stronger tumor suppression, it was suggested that high concentration of Tsc2–RGH resulted in increased accessibility of tuberin to the target molecule by diffusion. In other words, binding of N-terminal region of tuberin to hamartin might bring tuberin in appropriate localization efficiently. In contrast to Tsc2–RGH, Tsc2–DRG lacking the C-terminal region of tuberin, even at high level, had no apparent effect on either ENU-induced renal tumorigenesis or embryonic lethality. Double transgenic Eker rats carrying both Tsc2–DRG and Tsc2–RGH are being investigated. Primarily we have observed a complete suppression of ENU-induced renal carcinogenesis microscopically (our unpublished data). Together, these findings suggest that a complete suppression of renal tumorigenesis, as well as rescue from embryonic lethality, requires functions of both the N-terminal and C-terminal regions, probably in a combinational fashion. Thus, the N-terminal half may have a supportive function. In the Eker rat, there is a transposon insertion in intron 30 of the Tsc2 gene. Hence aberrant mRNAs which lack from exon 31 onwards are produced. This might cause aberrant truncation of tuberin. However, though we tried to identify product derived from the Eker mutant allele, we could not detect it by western blotting (data not shown). Therefore, we believe that there would be no interaction between Eker mutant protein and Tg-derived product. Perhaps, the progression or growth of tumor cells is inhibited in the presence of the C-terminal region of tuberin but not the initiation of tumorigenesis. Searches for TSC2 mutations among human tuberous sclerosis patients revealed that missense mutations and in-frame deletions were distributed throughout the coding region (27,28). However, those mutations were found more frequently in the C-terminal region of tuberin, especially in the region corresponding to Tsc2–RGH (28). Although, the apparent phenotype–genotype correlation has not been described in tuberous sclerosis, the possibility that C-terminal mutations in tuberin lead to more severe phenotypes than those caused by N-terminal mutations should be carefully re-considered.
Our analysis also showed that the C-terminal 55 residues of tuberin encoded by exon 41 were not required for the suppression of ENU-induced renal tumorigenesis. These C-terminal 55 residues contain a binding region for steroid hormone receptor superfamily members (10) as well as a potent transcriptional activating domain (7). These activities, if they are expressed in vivo, are not required for tumor suppression. Interestingly, loss of the C-terminal 55 residues affected normal embryonic development, although homozygous Tsc2 mutants carrying Tsc2–DCT55–flag were occasionally born. Thus, the requirement of the C-terminal 55 residues may differ for renal tumor suppression and for prevention of embryonic lethality. Among the 55 residues encoded by exon 41, the first 18 from the C-terminal are highly conserved in vertebrates, suggesting functional importance (3,29). However, they are not conserved in the fly homologue (30). This limited degree of conservation may reflect some modifying and not essential role for the C-terminal end in the overall function of tuberin in vertebrates. Since some homozygous Tsc2 mutants carrying Tsc2–DCT55–flag were born and grew with a normal appearance, the function of these 55 residues is not indispensable for normal embryonic development. The reason for the viability of these rescued homozygous Tsc2 mutants is currently unknown. However, a difference in the level of expression of Tg between two independent lines was observed. In addition, extension of the C-terminal end of tuberin with no alteration of the wild-type sequence, caused by a tuberous sclerosis-associated mutation, diminished hamartin-binding activity (31). Thus, the amount of expression and C-terminal structure of tuberin might affect the development of embryos. Strizheva and co-workers investigated TSC2 mutations in lymphangioleiomyomatosis (LAM) patients and suggested an association of mutations in exons 40 and 41 with the pathogenesis of LAM (32). Therefore, the functional importance of exon 41 in the TSC2 gene would be conserved among these species and further studies will be needed to elucidate the function of this domain. We are thus currently investigating phenotypes of homozygous Tsc2 mutant rats rescued by Tsc2–DCT55–flag to determine whether they develop characteristic lesions. Though all of the non-transgenic homozygous mutants died by E13.5, the state of homozygous mutants carrying Tsc2–DCT55–flag as well as Tsc2–DRG and Tsc2–RGH is under investigation to explore the cause of embryonic lethality and observe whether there is any retardation of the timing of their death.
Products of Tsc2–DCT55–flag and Tsc2–DRG, both of which contain the N-terminal region, were capable of binding to hamartin, as expected from a previous study (9). The N-terminal two-thirds of tuberin has been thought to be responsible for the stabilization of hamartin as revealed by transfection experiments (14). However, in our analysis, despite expression of this N-terminal two-thirds at a high level, hamartin expression was not fully restored by Tsc2–DRG in homozygous Tsc2 mutants. In those carrying Tsc2–DCT55–flag, a reduced amount of hamartin was also observed. These results suggest that not only the N-terminal but also the C-terminal region of tuberin is required to maintain normal hamartin levels in vivo. Recently, it was reported that phosphorylation of the C-terminal region of tuberin regulates the formation of a complex with hamartin (33,34). Our observations, together with these reports, suggest that the secondary structure and modification of the C-terminal region determine the hamartin-binding activity in wildtype tuberin and are important for the maintenance of a stable hamartin–tuberin complex.
The involvement of tuberin and hamartin in a common pathway was expected, given the similar symptoms of TSC1 and TSC2 patients. In this study, using a transgenic rat system, we observed the association of tuberin and hamartin in vivo and determined the functional domains of tuberin summarized in Figure 7. Many aspects of the pathogenesis of tuberous sclerosis, such as phenotypic variety among patients with the same mutation, remain to be examined. Our transgenic system provides new insights, most notably, that the amount of gene expression might affect the variety of symptoms in patients who carry the same mutation of the TSC2 gene. Further study with this system should facilitate our understanding of the pathogenic mechanisms associated with mutations in TSC2 genes through the analysis of phenotype–genotype relationships.
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MATERIALS AND METHODS |
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Construction of Tgs
Deletion mutants of rat Tsc2 Tgs were generated from the wildtype Tsc2 Tg (
exon 25 and 31) as previously described (22). The wildtype Tsc2 Tg consists of a genomic DNA fragment covering the 710 bp 5' upstream region to exon 2, rat Tsc2 cDNA (lacking exons 25 and 31 by alternative splicing) and an additional SV40 poly(A) signal sequence (nucleotide numbers of Tsc2 cDNA follow those for GeneBank accession no. D50413). For construction of Tsc2–DCT55–flag ( nt 5300–5573), the wildtype Tsc2 Tg was partially digested by BamHI, and a linker (5'-GACTACAAGGATGACGATGACAAGGACTACAAGGATGACGATGACAAGTGAGAATTC-3' sense strand) was introduced after nt 5299 of the BamHI site. For construction of Tsc2–DCT385–flag ( nt 4303–5573), the wildtype Tsc2 Tg was also partially digested by BamHI and the same linker was introduced after nt 4303 of the BamHI site. For construction of Tsc2–RGH ( nt 239–4302 and nt 5300–5573), Tsc2–DCT55–flag was partially digested with BamHI and BglII. The linker described above was designed to encode two tandem FLAG tags and an in-frame stop codon. It also contains an EcoRI site used for detection of transgenes. For construction of Tsc2–DRG ( nt 4303–5299), the wildtype Tsc2 Tg was partially digested by BamHI and self-ligated to remove a 1 kb BamHI fragment. All constructions were confirmed by sequence analysis.
Generation of transgenic founders
All of the Tgs were completely digested with NotI for linearization and then partially digested with KpnI. Before injection, full-length objective Tgs were purified using GENECLEAN II KIT (BIO 101) after separation by SeaKem agarose (FMC Bioproducts) gel electrophoresis. The methods for injection into fertilized eggs obtained by crossing Eker male and Wister females were described previously in detail (22).
Southern blot analysis
DNA was isolated from rat tail or Reichert's membrane of embryo for genotyping. 10 µg of each DNA was digested with BamHI or EcoRI and BglII, or EcoRI for Tsc2–DCT55–flag and Tsc2–DRG, Tsc2–DCT385–flag and Tsc2–RGH, respectively. Each digested DNA was separated on a 1% agarose gel and transfered onto a nylon membrane, as previously described (17). The probes used were as follows: a 1.1 kb EcoRI–BamHI fragment covering exons 26–33 for Tsc2–DCT55–flag and Tsc2–DRG, a 280 bp fragment covering exons 32 and 33 for Tsc2–DCT55–flag and a 610 bp fragment covering exons 32–34 for Tsc2–RGH. The 280 bp fragment was amplified by PCR using primers RTSC34 (5'-GAGGACTTTGAGGCAGCATT-3') and RTSC04 (5'-CGTCCAATGTCAGTCTTGT-3'). The 610 bp fragment was also amplified using primers RTSC34 and RTSC69 (5'-CAAAGCTAGGGTTGATGCCT-3'). Conditions for PCR were described previously (17).
RNA analysis
Total RNA was isolated from brain, kidney and embryos using TRIZOL Reagent (Life Technologies, Inc.,) and was treated with 2.5 units of DNase I (TAKARA Biochemicals). 2 µg of total RNAs were subjected to a reverse transcription (RT) reaction in 20 µl. 1 µl of this reaction mixture was used for PCR analysis. The PCR conditions were as described previously (17). The primer sets used were as follows. For Tsc2–DCT55–flag, RTSC33 (5'-CCGCAACCTGTCCTTTGTG-3') and RTSC11 (5'-CATCCACAGAGGAAATGAGG-3') were used to amplify exon 40 and exon 41. For Tsc2–DCT385–flag and Tsc2–DRG, RTSC05 (5'-GACAAGACTGACATTGGACG-3') and RTSC11 were used to amplify the sequence from exon 33 to exon 41 deleting the fragment between BamHI (nt 4303) and BamHI (nt 5299). For Tsc2–RGH, RTSC30 (5'-CAAAGCTAGGGTTGATGCCT-3') and RTSC61 (5'-AGAACTGATGTGTGAATGC-3') were used to detect the fragment with exon 2 and exon 33. For northern blot analysis, 10 µg of total RNA was electrophoresed through a denaturing 0.8% agarose gel containing 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0, and 6% (v/v) formaldehyde. The gel was blotted onto a nylon membrane by capillary action and the membrane was hybridized as described for Southern blotting. The Tsc1 cDNA probe was a 1.7 kb EcoRI fragment covering exons 8–18.
Antibodies
For immunoblotting and immunoprecipitaion, the following antibodies were used. For hamartin, C-Tsc1, which recognizes 16 amino acid residues at the C-terminal of rat Tsc1, was used (25). Anti-tuberin antibody (C20) and anti-actin antibody (N19) were obtained from Santa Cruz Biotechnology. For the FLAG tag, anti-FLAG M2 monoclonal antibody and M2 affinity gel were obtained from Sigma-Aldrich, Inc.
Immunoblotting
Whole tissue of brain, kidney and whole embryo were lysed in a buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 30 mM NaPPi, 50 mM NaF and 2% NP40, 1 mM Na3VO4, 2.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µM pepstatin and 0.23 units/ml aprotinin (Buffer A). The debris was removed by centrifugation at 12 000 g for 30 min at 4°C. The protein concentration was determined with a DC Protein Assay kit (BioRad) and 30 µg/lane of sample was separated by SDS–polyacrylamide gel electrophoresis. Then proteins were electrophoretically transferred from gels onto polyvinylidene difluoride membranes (Millipore), for incubation with antibodies. Biotinylated anti-mouse and anti-goat IgGs (Amersham Pharmacia Biotech) were used as secondary antibodies and streptavidin-conjugated horseradish peroxidase (Amersham Pharmacia Biotech) and ECL (Amersham Pharmacia Biotech) were used for visualization. In the case of rabbit primary antibodies, the polymer immunocomplex method was applied to the secondary antibody complex (25).
Co-immunoprecipitation assay
Whole embryo at E12.5 was lysed in Buffer A as used for immunoblotting. 1 mg of each lysate was precleared with Protein G PLUS/Protein A-Agarose (Oncogene Research Products) for 1 h. Each appropriate antibody was added to the precleared lysate and incubated for 1 h and then immunoprecipitation was performed overnight by adding Protein G PLUS/Protein A-Agarose. Immune complexes were analysed by immunoblotting with each appropriate primary antibody.
ENU treatment via the placenta and histological examination
ENU (N-ethyl-N-nitrosourea, Nacalai Tesque) was dissolved in saline and dams were treated with 80 mg/kg body weight of ENU at the 15th day of gestation (23). Eight weeks after birth, Tg-carrying Tsc2Ek/+ offspring were sacrificed to observe the kidneys bilaterally. In addition, Tsc2+/+and Tsc2Ek/+ non-Tg-carrying offspring were sacrificed as controls. Tissues were fixed in 10% buffered formalin and embedded in paraffin. Midsagittal sections (5 µm) of kidneys were stained with hematoxylin and eosin and were examined microscopically.
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ACKNOWLEDGEMENTS |
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We thank Y. Hirayama, E. Kobayashi, S. Honda, T. Fukuda, and M. Yamazaki for technical assistance, S. Miyata, M. Ushijima and T. Umeda for assistance with statistical analysis and H. Sugano, T. Kitagawa and A.G. Knudson for encouragement throughout this work. The work was supported in part by grants from the Ministry of Education, Science, Culture, Sports, Science and Technology and the Ministry of Health, Labor and Welfare of Japan and the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research.
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FOOTNOTES |
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* To whom correspondence should be addressed: Tel: +81 339180111 x 4331; Fax: +81 353943815; Email: ohino{at}ims.u-tokyo.ac.jp
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REFERENCES |
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2
van Slegtenhorst, M., de Hoogt, R., Hermans, C., Nellist, M., Janssen, B., Verhoef, S., Lindhout, D., van den Ouweland, A., Halley, D., Young, J. et al. (1997) Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science, 277, 805–808.
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5
Wienecke, R., Konig, A. and DeClue, J.E. (1995) Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity. J. Biol. Chem., 270, 16409–16414.
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Xiao, G.H., Shoarinejad, F., Jin, F., Golemis, E.A. and Yeung, R.S. (1997) The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis. J. Biol. Chem., 272, 6097–6100.
7
Tsuchiya, H., Orimoto, K., Kobayashi, K. and Hino, O. (1996) Presence of potent transcriptional activation domains in the predisposing tuberous sclerosis (Tsc2) gene product of the Eker rat model. Cancer Res., 56, 429–433.
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