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The GTPase rho controls a p53-dependent survival checkpoint during thymopoiesis.

Costello PS, Cleverley SC, Galandrini R, Henning SW, Cantrell DA - J. Exp. Med. (2000)

Bottom Line: Previous work has shown that the guanine nucleotide binding protein Rho controls cell survival in T cell progenitors.Here we define the survival pathways controlled by Rho in pre-T cells and show that this GTPase is a pivotal regulator of the p53-mediated checkpoint operating at the time of beta selection: loss of Rho function results in apoptosis in pre-T cells, but this cell death is prevented by loss of p53.The prevention of cell death by loss of p53 restored numbers of early T cell progenitors but did not fully restore thymic cellularity.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.

ABSTRACT
During the early stages of thymopoiesis, cell survival is controlled by cytokines that regulate the expression of antiapoptotic proteins such as Bcl-2. At the pre-T cell stage, a critical checkpoint for beta chain selection is monitored by the tumor suppressor p53: pre-T cells can survive and differentiate when p53 is removed genetically or when its proapoptotic function is inactivated physiologically as a consequence of signaling through the pre-T cell receptor complex. Previous work has shown that the guanine nucleotide binding protein Rho controls cell survival in T cell progenitors. Here we define the survival pathways controlled by Rho in pre-T cells and show that this GTPase is a pivotal regulator of the p53-mediated checkpoint operating at the time of beta selection: loss of Rho function results in apoptosis in pre-T cells, but this cell death is prevented by loss of p53. The prevention of cell death by loss of p53 restored numbers of early T cell progenitors but did not fully restore thymic cellularity. Further analysis revealed that loss of Rho function caused survival defects in CD4/8 double-positive thymocytes that is independent of p53 but can be prevented by ectopic expression of Bcl-2. These studies highlight that the GTPase Rho is a crucial component of survival signaling pathways in at least two different thymocyte subpopulations: Rho controls the p53 survival checkpoint in pre-T cells and is also crucial for a p53 independent survival signaling pathway in CD4/8 double positives.

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DP thymocytes are rescued from apoptosis in Bcl-2/lck-C3 double-transgenic mice, and the normal SP/DP ratio is restored. (A) Percent of annexin V–positive ex vivo thymocytes staining simultaneously with CD4–PE/CD8–FITC and Th1.2–allophycocyanin. (B) Thy1.2-gated CD4/8 profiles of lck-C3/Bcl-2 double transgenics.
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Figure 5: DP thymocytes are rescued from apoptosis in Bcl-2/lck-C3 double-transgenic mice, and the normal SP/DP ratio is restored. (A) Percent of annexin V–positive ex vivo thymocytes staining simultaneously with CD4–PE/CD8–FITC and Th1.2–allophycocyanin. (B) Thy1.2-gated CD4/8 profiles of lck-C3/Bcl-2 double transgenics.

Mentions: This study shows that the cell death initiated by loss of Rho function in pre-T cells can be prevented by the removal of p53. However, despite this rescue within the pre-T cell compartment, examination of the gross morphology of the thymus and of total thymocyte numbers revealed that the absence of p53 did not restore cellularity in lck-C3 transgenics (Table ). This result is different from the results seen when ectopic Bcl-2 is used to prevent cell death in lck-C3–transgenic mice. These results were published previously and revealed that overexpression of Bcl-2 in lck-C3–transgenic mice restored the pre-T cell population to normal levels but also partially rescued total thymic cellularity 24. One explanation for this discrepancy is that Rho is required for survival of thymocytes at later stages of development and that these later pathways for thymocyte survival are p53 independent but can be modulated by Bcl-2. In this context, not only are the lck-C3 thymi extremely small but they also have a very abnormal ratio of CD4/8 DPs and SPs. Typically, DPs should represent 80% of total thymocytes, but in lck-C3 thymi, they approximated 50% of the population. SPs in a normal mouse usually represent 15% of normal thymocytes, whereas in lck-C3 thymi, they were frequently 30–40% of the total population. It should be remembered that total numbers of thymocytes in lck-C3 thymi are severely reduced, so the skewing of DP to SP percentages does not mean that there are more SPs in the lck-C3 thymi. However, the skewing of the ratios of DPs/SPs indicated that there might be selective depletion of DPs in the lck-C3–transgenic mice. We have never before looked at the effects of Rho inhibition on the survival characteristics of DPs. The data in Fig. 4A and Fig. B, address this point. Cells were analyzed by flow cytometry, with apoptotic cells being identified by 7AAD staining 30 and with annexin V 29. The results revealed an increase in apoptotic cells in the CD4/8 DPs isolated from lck-C3–transgenic mice compared with wild-type controls. The data in Fig. 4 D show that the loss of p53 did not prevent apoptosis in CD4/8 DPs from lck-C3–transgenic mice, which was in marked contrast to the protective effect of p53 removal on the cell death caused by loss of Rho function in DN cells (Fig. 4 C). Strikingly, the increased rate of cell death in DPs is reduced to wild-type levels in the lck-C3/Bcl-2 double transgenics (Fig. 5 A). We had hypothesized that increased cell death was responsible for selective loss of DPs in lck-C3–transgenic mice and hence responsible for the skewed ratio of DP/SP seen in lck-C3 mice (Fig. 5 B). Consistent with this hypothesis, cell death in DPs was prevented by ectopic expression of Bcl-2, and the ratio of DP/SP in the lck-C3 mice was restored to normal (Fig. 5 B). Expression of Bcl-2 also resulted in an increase in the total numbers of DP thymocytes: normally, there are only 4–6 million DPs in lck-C3 mice, compared with 100–150 million DPs in normal littermate controls. Expression of Bcl-2 in the lck-C3 mice restored DPs to 26–34 million. Total numbers of SPs were not changed by Bcl-2 expression.


The GTPase rho controls a p53-dependent survival checkpoint during thymopoiesis.

Costello PS, Cleverley SC, Galandrini R, Henning SW, Cantrell DA - J. Exp. Med. (2000)

DP thymocytes are rescued from apoptosis in Bcl-2/lck-C3 double-transgenic mice, and the normal SP/DP ratio is restored. (A) Percent of annexin V–positive ex vivo thymocytes staining simultaneously with CD4–PE/CD8–FITC and Th1.2–allophycocyanin. (B) Thy1.2-gated CD4/8 profiles of lck-C3/Bcl-2 double transgenics.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887705&req=5

Figure 5: DP thymocytes are rescued from apoptosis in Bcl-2/lck-C3 double-transgenic mice, and the normal SP/DP ratio is restored. (A) Percent of annexin V–positive ex vivo thymocytes staining simultaneously with CD4–PE/CD8–FITC and Th1.2–allophycocyanin. (B) Thy1.2-gated CD4/8 profiles of lck-C3/Bcl-2 double transgenics.
Mentions: This study shows that the cell death initiated by loss of Rho function in pre-T cells can be prevented by the removal of p53. However, despite this rescue within the pre-T cell compartment, examination of the gross morphology of the thymus and of total thymocyte numbers revealed that the absence of p53 did not restore cellularity in lck-C3 transgenics (Table ). This result is different from the results seen when ectopic Bcl-2 is used to prevent cell death in lck-C3–transgenic mice. These results were published previously and revealed that overexpression of Bcl-2 in lck-C3–transgenic mice restored the pre-T cell population to normal levels but also partially rescued total thymic cellularity 24. One explanation for this discrepancy is that Rho is required for survival of thymocytes at later stages of development and that these later pathways for thymocyte survival are p53 independent but can be modulated by Bcl-2. In this context, not only are the lck-C3 thymi extremely small but they also have a very abnormal ratio of CD4/8 DPs and SPs. Typically, DPs should represent 80% of total thymocytes, but in lck-C3 thymi, they approximated 50% of the population. SPs in a normal mouse usually represent 15% of normal thymocytes, whereas in lck-C3 thymi, they were frequently 30–40% of the total population. It should be remembered that total numbers of thymocytes in lck-C3 thymi are severely reduced, so the skewing of DP to SP percentages does not mean that there are more SPs in the lck-C3 thymi. However, the skewing of the ratios of DPs/SPs indicated that there might be selective depletion of DPs in the lck-C3–transgenic mice. We have never before looked at the effects of Rho inhibition on the survival characteristics of DPs. The data in Fig. 4A and Fig. B, address this point. Cells were analyzed by flow cytometry, with apoptotic cells being identified by 7AAD staining 30 and with annexin V 29. The results revealed an increase in apoptotic cells in the CD4/8 DPs isolated from lck-C3–transgenic mice compared with wild-type controls. The data in Fig. 4 D show that the loss of p53 did not prevent apoptosis in CD4/8 DPs from lck-C3–transgenic mice, which was in marked contrast to the protective effect of p53 removal on the cell death caused by loss of Rho function in DN cells (Fig. 4 C). Strikingly, the increased rate of cell death in DPs is reduced to wild-type levels in the lck-C3/Bcl-2 double transgenics (Fig. 5 A). We had hypothesized that increased cell death was responsible for selective loss of DPs in lck-C3–transgenic mice and hence responsible for the skewed ratio of DP/SP seen in lck-C3 mice (Fig. 5 B). Consistent with this hypothesis, cell death in DPs was prevented by ectopic expression of Bcl-2, and the ratio of DP/SP in the lck-C3 mice was restored to normal (Fig. 5 B). Expression of Bcl-2 also resulted in an increase in the total numbers of DP thymocytes: normally, there are only 4–6 million DPs in lck-C3 mice, compared with 100–150 million DPs in normal littermate controls. Expression of Bcl-2 in the lck-C3 mice restored DPs to 26–34 million. Total numbers of SPs were not changed by Bcl-2 expression.

Bottom Line: Previous work has shown that the guanine nucleotide binding protein Rho controls cell survival in T cell progenitors.Here we define the survival pathways controlled by Rho in pre-T cells and show that this GTPase is a pivotal regulator of the p53-mediated checkpoint operating at the time of beta selection: loss of Rho function results in apoptosis in pre-T cells, but this cell death is prevented by loss of p53.The prevention of cell death by loss of p53 restored numbers of early T cell progenitors but did not fully restore thymic cellularity.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.

ABSTRACT
During the early stages of thymopoiesis, cell survival is controlled by cytokines that regulate the expression of antiapoptotic proteins such as Bcl-2. At the pre-T cell stage, a critical checkpoint for beta chain selection is monitored by the tumor suppressor p53: pre-T cells can survive and differentiate when p53 is removed genetically or when its proapoptotic function is inactivated physiologically as a consequence of signaling through the pre-T cell receptor complex. Previous work has shown that the guanine nucleotide binding protein Rho controls cell survival in T cell progenitors. Here we define the survival pathways controlled by Rho in pre-T cells and show that this GTPase is a pivotal regulator of the p53-mediated checkpoint operating at the time of beta selection: loss of Rho function results in apoptosis in pre-T cells, but this cell death is prevented by loss of p53. The prevention of cell death by loss of p53 restored numbers of early T cell progenitors but did not fully restore thymic cellularity. Further analysis revealed that loss of Rho function caused survival defects in CD4/8 double-positive thymocytes that is independent of p53 but can be prevented by ectopic expression of Bcl-2. These studies highlight that the GTPase Rho is a crucial component of survival signaling pathways in at least two different thymocyte subpopulations: Rho controls the p53 survival checkpoint in pre-T cells and is also crucial for a p53 independent survival signaling pathway in CD4/8 double positives.

Show MeSH
Related in: MedlinePlus