Abstract
CD4+CD8+ double-positive (DP) thymocytes, which are extremely sensitive to apoptosis, specifically up-regulate Bcl-xL to extend their lifespan. Deletion of the Bcl-xL gene leads to premature apoptosis of the thymocytes. In this study, we show that stabilization of β-catenin, a critical coactivator for T cell factor (TCF), enhances DP thymocyte survival via up-regulating Bcl-xL. Spontaneous or glucocorticoid-induced thymocyte apoptosis was associated with reduced levels of β-catenin and Bcl-xL. Transgenic expression of a stabilized β-catenin protected DP thymocytes from both spontaneous and glucocorticoid-induced apoptosis, resulting in significantly increased thymic cellularity. Compared with the wild-type mice, both protein and transcript levels of Bcl-xL were significantly increased in thymocytes of β-catenin transgenic mice. In addition, TCF-1 as well as β-catenin were able to stimulate transcriptional activity of the reporter driven by a Bcl-xL promoter. β-Catenin/TCF is thus able to act as a signal to up-regulate Bcl-xL levels in DP thymocytes, resulting in their enhanced survival.
Developing T cells are subject to two critical checkpoints that determine whether they can eventually survive and differentiate into mature T cells (1, 2). The earliest T cells are double-negative (DN)3 because they express neither CD4 nor CD8. Survival of the early DN cells depends on IL-7 until a TCRβ chain is successfully rearranged and assembled with pTα (pre-TCRα) to form pre-TCR. DN cells expressing a functional pre-TCR receive survival signal and continue to differentiate into CD4+CD8+ double-positive (DP) stage (pre-TCR checkpoint), and cells with nonproductive TCRβ rearrangement undergo apoptosis. The TCRα gene is rearranged at the DP stage, and results in expression of an αβ TCR. Less than 5% of DP thymocytes with αβ TCRs that recognize foreign Ags presented by self-MHC are selected and mature into CD4 or CD8 single-positive (SP) T cells (TCRαβ checkpoint) (3). The lifespan of DP thymocytes limits the progression of TCRα chain rearrangement and thus controls the opportunity for assembling a functional TCR (4). Bcl-xL, an anti-apoptotic molecule, is specially up-regulated in DP thymocytes to ensure their survival, as thymocytes deficient in Bcl-xL undergo premature apoptosis (5, 6). However, little is known about the signals required for stimulating Bcl-xL expression in DP cells.
β-catenin is a central effector of the Wnt signaling pathway, and a powerful regulator of development and differentiation (7, 8). The Wnt-β-catenin pathway has been shown to regulate multiple developmental processes ranging from regeneration of stem cells to organogenesis of the kidney and reproductive systems (9). β-Catenin is usually regulated at the protein levels. In the absence of Wnt signaling, several serines and threonines located at the N terminus of β-catenin (aa 31–45) are phosphorylated by glycogen synthase-3β (GSK-3β) bound to scaffolding proteins axin and adenomatous polyposis coli. The phosphorylated β-catenin is a target for ubiquitination and degradation by 26S proteosome (10). In addition, there are reports that β-catenin can also be degraded in a phosphorylation-independent manner (11, 12). Activation of Wnt signaling leads to inactivation of GSK-3β and accumulation of nonphosphorylated β-catenin in cytoplasm. Accumulated β-catenin is then available to bind to and activate T cell factor (TCF) and lymphoid enhancer factor (LEF), which translocate to nucleus and regulate target gene expression.
Examination of various components of the Wnt pathway has indicated its critical function in the regulation of DN to DP transition at the pre-TCR checkpoint. By both gain and loss of function approaches, Wnt1 and Wnt4 have been shown to be required for transition from the DN-to-DP stage by regulating the proliferation of these early thymocytes (13, 14). In agreement, disruption of TCF-1 blocks T cell development also at this transition stage (15). Moreover, TCF-1 and LEF-1 double-knockout leads to a complete block of T cell development at the DN-to-DP transition stage (16). Consistent with the critical role of β-catenin pathway at the DN stage, Xu et al. (17) reported that knockout of β-catenin impairs the DN to DP transition. However, using an inducible knockout strategy, Cobas et al. (18) showed that β-catenin is dispensable for T cell development. It remains unknown whether the discrepancy between these two lines of knockout mice is a result of the different strategies used in gene targeting.
Manipulation of the β-catenin pathway affects the early development of thymocytes at the pre-TCR checkpoint, thus interfering in the analysis of the function of this pathway in regulating later stages of T cell development. By an elegant gene targeting strategy, Gounari et al. (19) expressed a stabilized β-catenin starting at the DN stage from an endogenous gene locus, and showed that the forced stabilization of β-catenin allows development of thymocytes lacking productive TCRβ rearrangement, suggesting that β-catenin mediates the critical signals essential for pre-TCR checkpoint. However, it is difficult to determine from such studies the function of β-catenin in DP cells due to disturbed earlier T cell development. In this study, we show that thymocyte apoptosis is coupled with down-regulation of β-catenin. Furthermore, transgenic mice expressing a stabilized β-catenin under the control of a CD4 promoter were established. Because CD4 promoter targets transgene primarily to DP cells, the development of DN cells was not affected in transgenic mice, allowing us to analyze the function of β-catenin in DP cells. Stabilized β-catenin extended the lifespan of DP thymocytes by transcriptionally up-regulating Bcl-xL. In addition, β-catenin was able to directly activate Bcl-xL promoter activity. Thus, we provide direct evidence that β-catenin is able to enhance thymocyte survival by transcriptionally regulating Bcl-xL levels.
Materials and Methods
Plasmids and reagents
The following Abs for FACS analyses were purchased from BD Pharmingen: PE-anti-TCRβ (catalog no. 553172), biotin-anti-CD4 (catalog no. 553649), streptavidin-PE-Cy5 (catalog no. 554062), annexin V-PE (catalog no. 556421), PE-anti-CD8 (catalog no. RM2204), biotin-anti-CD25 (catalog no. 553070), PE-anti-CD44 (catalog no. 553134), and anti-Bcl-xL. Anti-β-catenin Ab (catalog no. 610153) was from Upstate Cell Signaling, and anti-actin Ab (catalog no. SC-8422) was from Santa Cruz Biotechnology. The TCF knockout mice and expression plasmids for TCF-1 were obtained from the laboratory of Dr. H. Clevers (Hubrecht Laboratory, Center for Biomedical Genetics, Utrecht, The Netherlands). Topflash (TOP) and Fopflash (FOP) reporters were gifts from Dr. P. Howe (Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH). The DNA fragments containing Bcl-xL promoter and Bcl-xL cDNA originally were obtained from Dr. S. Korsmeyer (Dana-Farber Cancer Institute, Harvard, Boston, MA). Drs. F. McCormick and O. Tetsu (University of California, San Francisco, CA) provided β-catenin expression plasmids.
Generation of transgenic mice
The transgenic construct was generated as shown in Fig. 2 a, encoding a stabilized β-catenin driven by a CD4 promoter. The NotI DNA fragment was microinjected into fertilized eggs by Transgenic Production Service at University of Illinois. The founder mice were screened by Southern blot and FACS analyses of GFP expression. All the mice used in this study were housed in the specific pathogen-free facility at the University of Illinois.
Apoptosis assays
Thymocytes isolated from each genotype of mice were cultured in RPMI 1640 medium supplemented with 10% FBS at 37°C for different time periods. The apoptotic cells were then detected by annexin V-PE and propidium iodide (PI) staining for 20 min as described previously (6).
Western blot analysis
Thymi were removed and passed through a 70-μm cell strainer (BD Biosciences), and thymocytes were collected by centrifugation. A total of 107 thymocytes were cultured in RPMI 1640 medium supplemented with 10% FBS and treated with 25 mM lithium or 10−7 M dexamethasone (Dex) for various time periods. Thymocytes were then lysed in 200 μl of lysis buffer (150 mM NaCl, 50 mM Tris, 4 mM KCl, 1 mM MgCl2, 1 mM Na3VO4, 10% glycerol, 1% Nonidet P-40, and protease inhibitors). The cell lysates were then analyzed by immunoblot with anti-Bcl-xL, anti-β-catenin, or anti-actin Abs.
Northern blot analysis
For Northern blot, thymocytes were collected from wild-type and β-catenin transgenic mice as described in Western blot analysis. Total RNA was purified by TRIzol (Invitrogen Life Technologies). Ten to 20 μg of total RNA were separated by electrophoresis on a 1.0% agarose-formaldehyde gel, transferred onto nylon filters, and cross-linked in an UV chamber. Radioactive DNA probes for Bcl-xL and hypoxanthine phosphoribosyltransferase were prepared by random-primed labeling of the mouse cDNAs with [α-32P]dCTP, respectively. Hybridization and washing were performed under high-stringency conditions. The filters were exposed to an imaging plate for Model 810-UNV (Molecular Dynamics) to calculate the radioactivity.
Cell cycle analysis
Thymocytes were fixed in 70% ethanol, and kept in darkness for at least 2 h. They were then washed once with ice-cold PBS and resuspended in PI staining solution (20 μg/ml RNase, 2 μg/ml PI, 0.1% Triton X-100). Thymocytes were subjected to FACS analysis to determine DNA content 30 min after PI staining.
Cell culture, transient transfection, and reporter assay
293T cells were cultured in DMEM medium supplemented with 10% FBS, penicillin and streptomycin, and glutamine. A total of 24 × 105 293T cells were plated in a 12-well dish. After 20 h, the cells were transfected with 5 ng of reporter, 200 ng of expression plasmids for TCF or ΔTCF, or β-catenin by calcium phosphate method. After 16 h, the DNA precipitates were removed by washing with ice-cold PBS and replenished with fresh medium. Cells were collected after 24 h and lysed in 200 μl of lysis buffer (137 mM NaCl, 50 mM Tris, 0.5% Nonidet P-40). A luciferase assay was then performed. Renilla luciferase assay was used as transfection internal control.
Results
Thymocyte apoptosis is associated with reduced levels of β-catenin
When separated from thymic stromal cells that express various Wnt family members (20), thymocytes undergo accelerated spontaneous apoptosis. To determine whether β-catenin plays a role in thymocyte survival, we first monitored changes in β-catenin activity during spontaneous apoptosis. Activation of the Wnt pathway leads to accumulation of β-catenin, which is then available to bind to TCF and stimulate its transcriptional activity, whereas inhibition of the Wnt signals results in ubiquitination and degradation of β-catenin (10). Therefore, the protein levels of β-catenin can be considered an indicator for activity of the Wnt pathway. Isolated thymocytes were cultured in medium for different times, and the protein levels of β-catenin and Bcl-xL were determined by Western blot analysis (Fig. 1,a). The intracellular levels of anti-apoptotic molecule Bcl-xL were gradually reduced (Fig. 1,a, middle panel) with increased spontaneous apoptosis (Fig. 1,b). The changes in β-catenin levels (Fig. 1 a, top panel) mirrored those of Bcl-xL, suggesting a positive correlation between the two.
Bcl-xL has also been shown to regulate glucocorticoid-induced apoptosis (21). Therefore, we examined β-catenin levels in thymocytes treated with Dex (Fig. 1,c). Dex induced rapid reduction of Bcl-xL (Fig. 1,c, middle panel) and correspondingly increased apoptosis (Fig. 1,d). Dex treatment also resulted in a decrease in β-catenin levels (Fig. 1 c, top panel) in a time course similar to that of Bcl-xL. These results suggest that down-regulation of β-catenin is coupled with Bcl-xL-regulated thymocyte apoptosis.
Activation of β-catenin pathway by lithium inhibits apoptosis
It is possible that the decreased β-catenin is a result of apoptosis. To determine whether reduced β-catenin plays a role in the apoptosis, we examined the effects of lithium chloride (LiCl) on thymocyte survival. LiCl is an inhibitor of GSK-3β (22), and thus prevents phosphorylation and degradation of β-catenin. Thymocytes were cultured in medium containing different concentrations of LiCl for 6 h, and levels of Bcl-xL and β-catenin were then determined by Western blot analysis (Fig. 1,e). Consistent with Fig. 1,a, β-catenin levels were reduced in the absence of LiCl (Fig. 1,e, comparing first two lanes of top panel). As expected, LiCl treatment lead to accumulation of β-catenin in a dose-dependent manner (Fig. 1,e, top panel). In control cells not treated with LiCl, Bcl-xL was reduced similar to β-catenin after 6 h in culture (Fig. 1,e, first two lanes of middle panel), which was consistent with the results presented in Fig. 1,a. LiCl treatment resulted in an increase in Bcl-xL levels in a dose-dependent manner similar to that of β-catenin (Fig. 1,e, middle panel). Correspondingly, thymocyte apoptosis was also greatly inhibited by LiCl treatment (Fig. 1 f). These results suggest that up-regulation of β-catenin inhibits apoptosis likely by increasing the levels of Bcl-xL.
Generation of transgenic mice expressing a stabilized β-catenin in T cell compartments
To determine whether activation of β-catenin regulates thymocyte survival in vivo, we generated transgenic mice expressing a stabilized form of β-catenin. Deletion of the N terminus of β-catenin generates a nondegradable and thus stabilized β-catenin, because the deleted portion contains four critical phosphorylation sites (Ser45, Thr41, Ser37, and Ser31) that are targets for a ubiquitination-mediated degradation pathway (23). To minimize the disturbance on β-catenin structure, we chose to use a version of β-catenin that only has an internal deletion of 20 aa (29–48) instead of deleting the entire N terminus. A CD4 promoter was used to target transgene to T cell compartments including CD4+CD8+ thymocytes (24). The cDNA encoding the stabilized β-catenin was cloned downstream of a CD4 promoter along with an internal ribosome entry site (IRES) and an enhanced GFP coding region (Fig. 2,a). The IRES permits both transgene and GFP to be expressed from a single bicistronic mRNA. Thus, we were able to analyze GFP expression in thymocytes. Two founder mice were identified based on GFP expression and Southern blot analyses. Progeny from two lines (β-catTg6101 and β-catTg6102) of transgenic mice (β-catTg) exhibited a similar phenotype, and in this study, results from one line (β-catTg6102) are reported. More than 95% of thymocytes were GFP positive in β-catTg (Fig. 2,b), suggesting that transgene was successfully expressed in thymocytes. A CD4 promoter without the silencer was used in generating transgenic mice; therefore, the transgene is expected to be primarily targeted to DP and SP cells. Indeed, obvious GFP expression is detected in the DP and SP, but not in DN cells of transgenic mice when GFP expression in different subset of thymocytes was compared between transgenic and wild-type mice (Fig. 2,c). β-Catenin levels in thymus were then examined (Fig. 2 d). As a result of deletion of 20 aa, m.w. of the β-catenin expressed from transgene was lower than that of the wild type. The levels of transgenic β-catenin (β-catTg) were relatively high as expected most likely because of its intrinsic resistance to ubiquitination-mediated degradation. Interestingly, the levels of wild-type β-catenin are also higher in transgenic mice, suggesting that expression of a stabilized β-catenin may competitively prevent degradation of the endogenous β-catenin.
Increased DP thymocytes in β-catTg mice
To determine whether β-catTg affects T cell development, thymocytes were analyzed. We first noticed considerably increased thymic cellularity in β-catTg mice. The total thymocyte number of β-catTg mice was two to three times more than that of the wild-type mice (Fig. 3,a). Further analysis of each subset of thymocytes indicated that the number of DP cells significantly increased in β-catTg mice, whereas the DN and SP cell number did not have such obvious changes (Fig. 3,a), suggesting that the increased thymic cellularity of β-catTg mice resulted mostly from the increased DP, but not the DN and SP population. In accordance, flow cytometric analysis indicated increased percentage of DP cells (92%) compared with the wild type (82%) (Fig. 3,b). An increase in the levels of surface CD4 on thymocytes were observed in β-catTg mice (Fig. 3,b), presumably due to enhanced TCF activity by stabilized β-catenin. A TCF/LEF binding site was previously identified within the CD4 proximal enhancer, and was found to activate CD4 gene by stimulating the enhancer activity (25). Consistent with increased CD4 levels, CD4 to CD8 SP cells ratio was increased in β-catTg mice, suggesting that lineage differentiation skewed to CD4 SP cells. Flow cytometric analysis of surface TCR did not reveal differences in surface TCR levels in TCR high population (Fig. 3,c). However, compared with the wild type, β-catTg mice displayed slightly lower levels of TCR in TCR intermediate and low population, which are mostly DN and DP thymocytes. Because the transgene was primarily targeted to DP cells by a CD4 promoter, it was not expected to have significant effects on DN cells. DN cells are divided into four different stages (DN1–DN4) according to surface expression of CD44 and CD25. Indeed, analysis of surface CD44 and CD25 on DN population did not reveal obvious differences between wild-type and β-catTg mice (Fig. 3 d). Therefore, T cell development was affected in β-catTg mice at the DP stage. The most obvious effect observed was a greatly increased number of DP cells.
β-catTg inhibited thymocyte apoptosis by regulating Bcl-xL levels
To determine whether the increased DP thymocyte number in β-catTg mice results from decreased apoptosis or increased proliferation, we performed apoptosis and cell cycle analyses. Apoptosis was examined by annexin V and PI staining. Spontaneous apoptosis was greatly inhibited in β-catTg mice compared with the wild type (Fig. 4,a). Two days after cultured in medium, >60% of the β-catTg thymocytes were alive, whereas only <20% of the wild-type thymocytes were alive. Next, Dex-induced apoptosis was examined (Fig. 4,b), and it was found that there were only 30% of live cells in wild-type thymocytes 6 h after Dex treatment relative to ∼80% in the β-catTg mice. Examination of the spontaneous apoptosis of DN cells did reveal significant differences between wild-type and β-catTg mice (Fig. 4 c). Stabilization of β-catenin thus specifically enhanced DP thymocyte survival.
We next determined Bcl-xL levels that are critical for DP thymocyte survival. Compared with the wild-type mice, β-catTg thymocytes had significantly higher levels of Bcl-xL in either freshly isolated thymocytes or at various times after cultured in medium (Fig. 4,d, left panel). Twenty-four hours after cultured in medium, thymocytes obtained from wild-type mice had almost undetectable levels of Bcl-xL, whereas a significant amount of Bcl-xL was detected in thymocytes from β-catTg mice. The levels of Bcl-2, which has a minimal role in DP thymocyte survival (26), was lower if any in β-catTg mice (Fig. 4,d, right panel), presumably from high levels of Bcl-xL. In agreement, it has been observed that forced expression of Bcl-xL down-regulated endogenous Bcl-2 in thymus (27). In addition, we did not detect significant differences in expression of Bcl-xL or Bcl-2 in SP cells between wild-type and β-catTg mice (Fig. 4 e). Thus, transgenic expression of a stabilized β-catenin results in significantly increased levels of Bcl-xL known to regulate DP thymocyte survival.
To determine whether β-catTg affects cell cycle progression, thymocytes from both wild-type and β-catTg mice were fixed and stained with PI, which allows for detection of DNA content. Flow cytometric analysis indicated that in wild-type mice, 7.5% of thymocytes had >2 N DNA, representing the cells in S/G2 phase of the cell cycle (Fig. 4 f). β-catTg mice had slightly more thymocytes in S/G2 phase (11.6%), suggesting that β-catTg may have minor effects on cell cycle progression.
β-catenin transcriptionally stimulates the expression of Bcl-xL
One of the functions for β-catenin is to bind and activate the transcription factor, TCF-1, which then regulates target gene expression (8). To determine whether β-catenin transcriptionally regulates Bcl-xL expression, we first compared Bcl-xL mRNA levels between wild-type and β-catTg mice (Fig. 5 a). Northern blot analysis of the RNA purified from thymocytes was performed using Bcl-xL cDNA as a probe. Levels of the Bcl-xL mRNA were much higher in β-catTg mice compared with the wild type, indicating that stabilization of β-catenin is sufficient to stimulate the expression of Bcl-xL at the transcriptional level.
To determine whether β-catenin activates Bcl-xL expression by directly stimulating its promoter activity, we cloned a 1-kb DNA fragment upstream of Bcl-xL start codon to a luciferase reporter. A TOP reporter containing three TCF-binding sites was used as a positive control, whereas a FOP reporter containing three mutant TCF-binding sites was used as a negative control (Fig. 5 b). As expected, both TCF-1 and stabilized β-catenin, same as the one used in generating transgenic mice, greatly stimulated TOP but not FOP activity. However, ΔTCF, which has β-catenin-binding domain deleted, was not able to stimulate TOP activity, confirming the critical role of β-catenin in TCF-1-mediated transcriptional activation. We next examined Bcl-xL promoter activity under the same conditions used to stimulate TOP. Stabilized β-catenin or TCF-1, but not ΔTCF, stimulated the activity of the reporters containing a 1-kb Bcl-xL promoter, suggesting that the β-catenin/TCF pathway can directly stimulate Bcl-xL promoter activity.
Discussion
β-Catenin is a highly conserved and important signaling molecule that regulates multiple developmental processes (9). Various gain and loss of function studies revealed a role for β-catenin and the components of β-catenin pathway in the regulation of early T cell development at the DN to DP transition (13, 15, 16, 17, 19, 28). Disturbed earlier T cell development prevented analysis of β-catenin function at later stages of T cell development. We chose to express a stabilized β-catenin specifically in DP cells to avoid interfering with the development of DN cells, and we clearly demonstrated that stabilized β-catenin enhances the survival of DP thymocytes by up-regulating Bcl-xL.
Previous studies, although indicating a role of β-catenin in DN cells, failed to demonstrate its function in DP cells. For example, Gounari et al. (19) showed that forced stabilization of β-catenin resulted in increased thymocyte apoptosis, thereby implying the proapoptotic role of β-catenin in thymocytes. However, such a result is likely due to the abnormal development of DN cells that allows generation of thymocytes lacking surface expression of TCR. In addition, there are conflicting reports about the role of β-catenin in thymocyte development. By gene targeting, Xu et al. (17) reported that deletion of β-catenin impairs the DN to DP transition. This result is consistent with the notion that β-catenin regulates the development of DN cells. In contrast to the other reports, Cobas et al. (18) showed that β-catenin is dispensable for lymphopoiesis by knockout β-catenin gene using an IFN-inducible Mx Cre recombinase. It remains to be determined whether such an inducible strategy results in complete deletion of the β-catenin gene. We used a CD4 promoter to target transgene primarily to DP cells so as to avoid interfering with earlier DN stages. Indeed, DN cells were not affected by expression of the transgene (Fig. 3 d). Thus, we excluded the possibility that the observed effects on DP cells in β-catTg mice resulted from disturbed development of earlier T cells.
Mulroy et al. (29) did not observe the effects on DP thymocyte with transgenic expression of a stabilized β-catenin using a Lck proximal promoter, presumably because of insufficient expression of their transgene. Expression of the stabilized β-catenin was detected in DN cells of their transgenic mice (29), but it did not affect the development of DN cells as other studies have demonstrated (17, 19). It suggested that the stabilized β-catenin in their transgenic mice may not be sufficient to compete with endogenous β-catenin. Indeed, the levels of transgenic β-catenin in their transgenic mice were similar to that of the endogenous β-catenin (29). Stabilized β-catenin in our transgenic mice overcame the endogenous β-catenin and enhanced thymocyte survival.
Our observations support the role of β-catenin in the regulation of thymocyte survival. First, spontaneous and glucocorticoid-induced apoptosis was accompanied by reduced levels of β-catenin; whereas, preventing the degradation of β-catenin by lithium treatment inhibited such apoptosis (Fig. 1). Second, transgenic expression of a stabilized β-catenin rendered DP thymocytes resistant to spontaneous and glucocorticoid-induced apoptosis, resulting in significantly increased DP thymocyte number. Last, stabilization of β-catenin led to a significant increase in the anti-apoptotic molecule, Bcl-xL. Stabilized β-catenin has minimal effects on cell cycle progression (Fig. 4 d). Similarly, deletion of TCF-1 gene resulted in apoptosis, but it had no obvious effects on DP cell cycle progression (30). Therefore, the major function of the β-catenin/TCF pathway in DP thymocytes is to regulate survival, but not cell cycle progression. In agreement with our results, Ioannidis et al. (30) showed that a truncated version of TCF-1 lacking N-terminal 116 aa required for binding to β-catenin failed to support DP thymocyte survival in TCF-1-deficient mice, indirectly suggesting the anti-apoptotic role of β-catenin in thymocytes. However, the deleted 116-aa domain may also mediate the interactions with other proteins in addition to β-catenin. Thus, our results directly demonstrate that β-catenin is able to enhance the survival of DP thymocytes.
Where do developing T cells receive Wnt signals? A recent study by Pongracz et al. (20) showed that thymic stromal cells preferentially express Wnt, whereas thymocytes express frizzled receptors, suggesting that thymic stroma provides Wnt signals to thymocytes. In agreement with this result, thymocytes down-regulate β-catenin after they are separated from thymic stroma (Fig. 1). We further linked down-regulation of β-catenin to Bcl-xL-regulated thymocyte apoptosis. All together, Wnt signals provided by thymic stroma are most likely responsible for enhancing DP thymocyte survival by up-regulation of Bcl-xL.
Our data suggest that β-catenin is able to transcriptionally stimulate the expression of Bcl-xL. During T cell development, Bcl-xL levels, which are low in both DN and SP cells, are specifically up-regulated in DP cells (5). Mice deficient in Bcl-xL displayed massive DP thymocyte apoptosis (31), whereas overexpression of Bcl-xL extends thymocyte survival (1, 6, 32). Bcl-xL is thus a critical molecule to maintain DP thymocyte survival. However, the signaling pathways required to up-regulate Bcl-xL in DP thymocytes remains elusive. In this study, we show that expression of a stabilized β-catenin is sufficient to increase messenger levels of the Bcl-xL (Fig. 5,a). However, in the absence of TCF-1, the levels of Bcl-xL are significantly reduced (30). β-Catenin/TCF-1 pathway thus acts as a signal to maintain high levels of Bcl-xL messenger in DP thymocytes. Moreover, our results demonstrated that Bcl-xL promoter activity is enhanced by both TCF-1 and β-catenin (Fig. 5 b). Our results do not exclude the possibility that β-catenin regulates Bcl-xL levels by other mechanisms such as stabilizing mRNA or protein of Bcl-xL. However, our data indicate that β-catenin regulates thymocyte survival at least in part by transcriptional regulation of Bcl-xL expression. As NF-κB and Stat5 pathway have previously been shown to regulate Bcl-xL levels (33), it is possible that β-catenin enhances Bcl-xL expression via activating these two pathways. However, comparing the activation status of these two pathways between wild-type and β-catTg mice did not detect significant differences (data not shown). Thus, we excluded such a possibility. More experiments are needed to elucidate how β-catenin/TCF pathway regulates Bcl-xL expression in thymocytes.
The increased thymic cellularity resulting from enhanced thymocyte survival was observed in both β-catTg and Bcl-xL transgenic mice (27), further supporting that β-catenin functions via regulating Bcl-xL. However, there are differences between these two mice. Bcl-xL transgenic mice have increased DP and SP cells. In contrast, β-catTg mice have only increased DP but not SP cells. It may be in part because β-catTg enhances DP but not SP cell survival. In addition, DP thymocytes of β-catTg mice display slightly lower levels of surface TCR, which may result in impaired positive selection. It appears that β-catenin affects other functions in addition to enhancing thymocyte survival.
Besides β-catenin, other molecules are also involved in the regulation of thymocyte survival. Mice lacking NFAT4, which is preferentially expressed in DP thymocytes, have significantly increased DP thymocyte apoptosis (34). Interestingly, GSK-3β, which induces the degradation of β-catenin by phosphorylation, is also a critical regulator for NFAT nuclear localization (35). Therefore, GSK-3β is able to regulate these two pathways essential for thymocyte survival. Similar to β-catenin, RoRγt extends DP thymocyte survival by up-regulating Bcl-xL levels. We, and subsequently Kurebayashi et al. (36), have shown that mice deficient in RoRγt display massive DP thymocyte apoptosis and significantly reduced Bcl-xL levels (6, 36). Forced expression of Bcl-xL prevented apoptosis observed in RoRγt−/− mice (6). However, RoRγt has an additional function in the regulation of cell cycle progression of DP thymocytes, whereas the β-catenin pathway only has a minor effect on cell cycle (6, 30). Egr3 has been shown to negatively regulate thymocyte survival presumably via inhibiting RoRγt (37). It remains to be determined how these molecules coordinate each other in regulating thymocyte survival. β-Catenin, RoRγt, and Egr3 appear to regulate thymocyte survival at least in part by controlling the expression of Bcl-xL. Although β-catenin by itself was able to stimulate Bcl-xL expression, it is very likely that other signaling molecules are also required to cooperate with β-catenin to achieve optimal survival essential for T cell development. Elucidation of the relationship between β-catenin and other signaling molecules in the regulation of thymocyte survival will facilitate the understanding of T cell development.
Acknowledgments
We thank Dr. Phil Howe for providing TOP and FOP reporters, Drs. Frank McCormick and Osamu Tetsu for β-catenin expression plasmids, Drs. Hans Clevers and Marian Waterman for expression plasmids of wild-type TCF and dominant-negative TCF, and Dr. Stanley Korsmeyer for Bcl-xL cDNA and DNA fragment containing Bcl-xL promoter region. Transgenic mice were generated in the University of Illinois transgenic core facility. We also thank Dr. Prasad Kanteti for critically reading the manuscript and providing helpful discussion.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Abbreviations used in this paper: DN, double negative; DP, double positive; TOP, Topflash; FOP, Fopflash; TCF, T cell factor; LEF, lymphoid enhancer factor; PI, propidium iodide; SP, single positive; GSK-3β, glycogen synthase-3β; IRES, internal ribosome entry site; β-catTg, transgenic β-catenin; Dex, dexamethasone; LiCl, lithium chloride.