Inactivation of S6 ribosomal protein gene in T lymphocytes activates a p53-dependent checkpoint response

Abstract

Ribosome biogenesis has been associated with regulation of cell growth and cell division, but the molecular mechanisms that integrate the effect of ribosome biogenesis on these processes in mammalian cells remain unknown. To study the effect of impaired ribosome functions in vivo, we conditionally deleted one or two alleles of the 40S ribosomal protein S6 gene in T cells in the mouse. While complete deletion of S6 abrogated T-cell development, hemizygous expression did not have any effect on T-cell maturation in the thymus, but inhibited the accumulation of T cells in the spleen and lymph nodes, as a result of their decreased survival in the peripheral lymphoid organs. Additionally, TCR-mediated stimulation of S6-heterozygous T cells induced a normal increase in their size, but cell cycle progression was impaired. Genetic inactivation of p53 tumor suppressor rescued development of S6-homozygous null thymocytes and proliferative defect of S6-heterozygous T cells. These results demonstrate the existence of a p53-dependent checkpoint mechanism that senses changes in the fidelity of the translational machinery to prevent aberrant cell division or eliminate defective T cells in vivo. Failure to activate this checkpoint response could potentially lead to a development of pathological processes such as tumors and autoimmune diseases.

Figure 1.

Conditional deletion of the S6 gene in the thymus. (A) Total number of thymocytes in S6^lox/lox/CD4-Cre^- and S6^lox/lox/CD4-Cre⁺ mice (n = 14 and 10, respectively). (B) Southern blot analysis of genomic DNA from pooled thymi of S6^lox/lox/CD4-Cre⁺ and S6^lox/lox/CD4-Cre^- mice using S6 intron-specific probe. The positions of DNA fragments representing S6^del and S6^lox are shown. (C) Southern blot analysis of genomic DNA from S6^wt/lox/CD4-Cre^- and S6^wt/lox/CD4-Cre⁺ thymi using S6 intron-specific probe. The positions of DNA fragments representing S6^del, S6^lox, and S6^wt alleles are indicated. (D) Northern blot analysis of total RNA from S6^wt/lox/CD4-Cre^- and S6^wt/lox/CD4-Cre⁺ thymi using S6 cDNA as a probe. The L11 cDNA probe was used as a control for loading the same quantity of total RNA on the gel. (E) Total number of thymocytes from S6^wt/lox/CD4-Cre^- and S6^wt/lox/CD4-Cre⁺ mice. (F) S6^wt/lox/CD4-Cre^- and S6^wt/lox/CD4-Cre⁺ thymocytes were stained with CD4 and CD8 antibodies (left panel) or anti-CD3 antibodies (right panel), and analyzed by FACS. Cell surface markers are shown as coordinates. The numbers in the quadrant refer to the percentage of a particular T-cell population of live cells. (G) The protein expression of ribosomal proteins S6, L7a, and L11 in thymocytes from S6^wt/lox/CD4-Cre⁺ and S6^wt/lox/CD4-Cre^- mice was analyzed by Western blot. Reprobing with antibodies to actin served as a loading control. (H) Total RNA from 15 × 10⁶ S6^wt/lox/CD4-Cre⁺ and S6^wt/lox/CD4-Cre^- thymocytes was separated in agarose gel containing formaldehyde, transferred to a nitrocellulose membrane, and stained with ethidium bromide. Positions of 28S and 18S rRNAs are shown.

Figure 2.

Decreased number of S6^wt/del T cells in the spleen and lymph nodes is a consequence of their decreased survival. (A) Total cell number in spleens from female (F) and male (M) S6^wt/lox/CD4-Cre^- and S6^wt/lox/CD4-Cre⁺ mice (n = 10, for each group). Error bars represent standard deviation. For females, (^*) p < 0.05 versus S6^wt/lox/CD4-Cre^- splenocytes; and for males, (^**) p > 0.05 versus S6^wt/lox/CD4-Cre^- splenocytes (Mann Whitney U-test). (B,D) FACS analysis of cells from S6^wt/lox/CD4-Cre^- and S6^wt/lox/CD4-Cre⁺ spleens. (C,E) FACS analysis of cells from S6^wt/lox/CD4-Cre^- and S6^wt/lox/CD4-Cre⁺ lymph nodes (n = 35, for each group). (^*) p < 0.001 versus S6^wt/lox/CD4-Cre^- CD3⁺ cells (t-test). Genotypes are as indicated above the panels. Cell surface markers are shown as coordinates. The numbers in quadrants refer to percentage of respective cell population of live cells. (F) Southern blot analysis of genomic DNA from S6^wt/lox/CD4-Cre^- and S6^wt/lox/CD4-Cre⁺ T cells purified from lymph nodes of 8-wk-old mice using the S6 intron-specific probe. (G) Six-week-old S6^wt/_lox/CD4-Cre^- and S6^wt/lox/CD4-Cre⁺ mice were daily injected intraperitoneally with 100 μg/g of body weight 5-BrdU (Sigma) in 200 μL of PBS during the indicated time periods; 5-BrdU incorporation into DNA was determined by flow cytometry. The percentages of 5-BrdU-positive lymph node T cells are shown. The experiment is a representative of four independent experiments. (H) CFSE-labeled S6^wt/lox/CD4-Cre⁺ or S6^wt/lox/CD4-Cre⁺ T cells (10 × 10⁶) were transferred into wild-type recipients by intravenous injection, and the percentage of CFSE-labeled T cells was determined in spleens. The total number of T cells in different recipients was similar. The experiment is a representative of three independent experiments.

Figure 3.

T-cell receptor-stimulated ribosome biogenesis is decreased in S6^wt/del T cells, but their growth is normal. (A) FSC analysis of S6^wt/lox and S6^wt/del lymph node T cells stimulated with soluble 1 μg/mL anti-CD3 and 0.1 μg/mL anti-CD28 in vitro was used to determine their size. (B) The protein expression of ribosomal proteins S6 and L7a in lysates from unstimulated (0 h) and stimulated (24 and 36 h) lymph node S6^wt/del and S6^wt/lox T cells was analyzed by Western blot. Reprobing with antibodies to actin served as a loading control. (C) To gain insight into rRNA processing, S6^wt/del and S6^wt/lox T cells were stimulated for 20 h with TCR and CD28 antibodies, pulse-labeled with L-[methyl-³H]methionine for 30 min, and chased in nonradioactive medium for the indicated time. Total RNA normalized to equal cell number was resolved on a formaldehyde-agarose gel, transferred to a nylon membrane, and visualized by autoradiography. The positions of major precursors and 28S and 18S rRNA species are indicated on the left. The experiment is a representative of five independent experiments. (D) Total cellular protein content per 1 × 10⁶ S6^wt/lox or S6^wt/del lymph node T cells before stimulation and after 24 h of stimulation with antibodies against TCR and CD28. The experiment is a representative of six independent experiments.

Figure 4.

Stimulation of S6^wt/del T cells does not increase their cell number. (A) Relative number of S6^wt/lox and S6^wt/del T cells stimulated with either 0.1 μg (open symbols) or 1 μg/mL (closed symbols) of anti-CD3 and fixed concentration of anti-CD28 antibodies (0.1 μg/mL) during 72 h in vitro. The experiment is a representative of 15 independent experiments. (B) FACS analysis of expression levels of CD25, intracellular IL-2, and IFN-γ in S6^wt/lox and S6^wt/del lymph node T cells stimulated for the indicated period of time in vitro. (C) Relative number of S6^wt/lox and S6^wt/del lymph node T cells stimulated with soluble anti-CD3 antibodies and saturating concentration of IL-2 containing MLA-144 supernatant (1:2 dilution). The experiment is a representative of three independent experiments. (D) FACS analysis of lymph node T cells that were stimulated with different concentrations of anti-CD3 (1 μg/mL, upper panel; 0.1 μg/mL, lower panel) and anti-CD28 antibodies (0.1 μg/mL) and labeled with CFSE. The filled area represents CFSE labeling of T cells before they divided for the first time. The number of cell divisions is indicated above the peaks. (E) Southern blot analysis of genomic DNA from S6^wt/lox and S6^wt/del lymph node T-cell cultures at 72 h following stimulation.

Figure 5.

Defect in proliferation of S6^wt/del T cells is due to p21-induced G1/S block. (A) BrdU incorporation into lymph node T cells stimulated with anti-CD3- and IL-2-containing MLA144 supernatant in vitro. The experiment is a representative of four independent experiments. (B) The protein expression of p21 cell cycle inhibitor in lysates from resting and stimulated lymph node S6^wt/del and S6^wt/lox T cells was analyzed by Western blot. Reprobing with antibodies to actin served as a loading control. (C) T cells of indicated genotypes were stimulated for the indicated period of time, labeled with propidium iodide, and analyzed by FACS. The percentage of apoptotic cells was determined by analyzing sub-G1 DNA content. The experiment is a representative of four independent experiments. (D) Relative number of S6^wt/lox, S6^wt/del, S6^wt/lox/Bcl-2⁺, and S6^wt/del/Bcl-2⁺ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 24, 48, and 72 h. The experiment is a representative of three independent experiments.

Figure 6.

A p53-dependent checkpoint is activated following inactivation of one or two S6 alleles in T cells. The percentage (A) and total number (B) of T cells in spleens from S6^wt/lox/CD4-Cre^- (line 1), S6^wt/lox/CD4-Cre⁺ (line 2), S6^wt/wt/p53^lox/lox/CD4-Cre⁺ (line 3), and S6^wt/lox/p53^lox/lox/CD4-Cre⁺ (line 4) 7-wk-old mice (n = 12 mice per each genotype). (C) Relative number of S6^wt/lox/CD4-Cre⁺, S6^wtwt/p53^lox/lox/CD4-Cre⁺, and S6^wt/lox/p53^lox/lox/CD4-Cre⁺ T cells at 24, 48, and 72 h after stimulation with either 0.3 μg/mL (open symbols) or 1 μg/mL (closed symbols) of anti-CD3 and fixed concentration of anti-CD28 antibodies (0.1 μg/mL). The experiment is a representative of six independent experiments. (D) Total number of cells in thymi from S6^lox/lox/CD4-Cre^- (line 1), S6^lox/lox/CD4-Cre⁺ (line 2), S6^wt/wt/p53^lox/lox/CD4-Cre⁺ (line 3), S6^lox/lox/p53^lox/lox/CD4-Cre⁺ (line 4), S6^lox/lox/Bcl-2⁺/CD4-Cre^- (line 5), and S6^lox/lox/Bcl-2⁺/CD4-Cre⁺ (line 6) mice (n = 10 mice per each genotype). (E) S6^wt/wt/p53^lox/lox/CD4-Cre⁺, S6^lox/lox/p53^lox/lox/CD4-Cre⁺, S6^lox/lox/Bcl-2⁺/CD4-Cre^-, and S6^lox/lox/Bcl-2⁺/CD4-Cre⁺ thymocytes were stained with anti-CD4 and anti-CD8 antibodies, and analyzed by FACS. Cell surface markers are shown as coordinates. The numbers in the quadrant refer to the percentage of a particular T-cell population of live cells.