Figure 2.
Figure 2. Ablation of Trp53 in TECs limits the expression and responsiveness of RANK. (A) The expression of Tnfrsf11a, Tnfrsf5, Tnfrsf3, and Tnfrsf11b was assessed by qRT-PCR in FACS-sorted mTECs (UEA+Ly51−) from 2-week-old Ctr and p53cKO mice. Values were normalized to 18s ribosomal RNA. Graphs represent data from 4 independent experiments (top). Fold difference in the relative mRNA levels between Ctr (dashed line) and p53cKO mTECs (bottom). (B) E15.5 dGuo-treated FTOCs from Ctr and p53cKO mice were cultured for 4 days with anti-RANK (αRANK). Expression of CD80 and Aire was analyzed in mTECs (UEA+Ly51−) by flow cytometry. Numbers indicate the mean percentage of gated cells. Graphs show the cellularity of mTEC subsets per thymic lobe. Number of asterisks compare mature mTECs from Ctr with p53cKO mice (top). Results are presented as mean ± SEM of 10 to 12 thymic lobes per group from 5 independent experiments. (C) The expression of Tnfrsf11a was analyzed by qRT-PCR in FACS-sorted TECs from Ctr and p53cKO E15.5 dGuo-treated FTOC stimulated over 24 hours with αRANK or αRANK plus Pifithrin-α. Values were normalized as in panel A, and those in TECs from nonstimulated dGuo-treated FTOC were set to 1. Graphs represent data from 3 to 6 independent experiments (mean ± SEM). (D) The region upstream of the Tnfrsf11a (RANK) TSS contains putative p53 response elements (REs) (triangles), identified on the basis of the p53 RE matrix logo (RRRC-A/T-A/T-GYYY motifs, in which R is a purine and Y is a pyrimidine) (MatInspector, rVista, or both, software tolls). DNA fragments (A-D) from the Tnfrsf11a (RANK) and Cdkn1a (p21) loci were cloned into the pGL3-Promoter reporter plasmid. p53 KO MEFs were transiently transfected with the indicated luciferase plasmids along with a p53 overexpressing construct (p53) or an empty construct (Empty). Luciferase reporter activity was normalized to the relative pGL3-promoter signal. Represented is the average of 3 independent experiments (±SEM). (E) The expression of Trp53 was analyzed by qRT-PCR in FACS-sorted TECs from Ctr and p53cKO E15.5 dGuo-treated FTOC stimulated over 24 hours with anti-RANK, recombinant CD40L, and anti-LTβR at different combinations. Values were normalized as in panel A, and those in TECs from nonstimulated dGuo-treated FTOC were set to 1. Data are representative of 4 independent experiments (mean ± SEM). A.U., arbitrary units; MHC, major histocompatibility complex. *P < .05; **P < .01.

Ablation of Trp53 in TECs limits the expression and responsiveness of RANK. (A) The expression of Tnfrsf11a, Tnfrsf5, Tnfrsf3, and Tnfrsf11b was assessed by qRT-PCR in FACS-sorted mTECs (UEA+Ly51−) from 2-week-old Ctr and p53cKO mice. Values were normalized to 18s ribosomal RNA. Graphs represent data from 4 independent experiments (top). Fold difference in the relative mRNA levels between Ctr (dashed line) and p53cKO mTECs (bottom). (B) E15.5 dGuo-treated FTOCs from Ctr and p53cKO mice were cultured for 4 days with anti-RANK (αRANK). Expression of CD80 and Aire was analyzed in mTECs (UEA+Ly51−) by flow cytometry. Numbers indicate the mean percentage of gated cells. Graphs show the cellularity of mTEC subsets per thymic lobe. Number of asterisks compare mature mTECs from Ctr with p53cKO mice (top). Results are presented as mean ± SEM of 10 to 12 thymic lobes per group from 5 independent experiments. (C) The expression of Tnfrsf11a was analyzed by qRT-PCR in FACS-sorted TECs from Ctr and p53cKO E15.5 dGuo-treated FTOC stimulated over 24 hours with αRANK or αRANK plus Pifithrin-α. Values were normalized as in panel A, and those in TECs from nonstimulated dGuo-treated FTOC were set to 1. Graphs represent data from 3 to 6 independent experiments (mean ± SEM). (D) The region upstream of the Tnfrsf11a (RANK) TSS contains putative p53 response elements (REs) (triangles), identified on the basis of the p53 RE matrix logo (RRRC-A/T-A/T-GYYY motifs, in which R is a purine and Y is a pyrimidine) (MatInspector, rVista, or both, software tolls). DNA fragments (A-D) from the Tnfrsf11a (RANK) and Cdkn1a (p21) loci were cloned into the pGL3-Promoter reporter plasmid. p53 KO MEFs were transiently transfected with the indicated luciferase plasmids along with a p53 overexpressing construct (p53) or an empty construct (Empty). Luciferase reporter activity was normalized to the relative pGL3-promoter signal. Represented is the average of 3 independent experiments (±SEM). (E) The expression of Trp53 was analyzed by qRT-PCR in FACS-sorted TECs from Ctr and p53cKO E15.5 dGuo-treated FTOC stimulated over 24 hours with anti-RANK, recombinant CD40L, and anti-LTβR at different combinations. Values were normalized as in panel A, and those in TECs from nonstimulated dGuo-treated FTOC were set to 1. Data are representative of 4 independent experiments (mean ± SEM). A.U., arbitrary units; MHC, major histocompatibility complex. *P < .05; **P < .01.

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