Heterozygous loss of TSC2 alters p53 signaling and human stem cell reprogramming

Abstract

Tuberous sclerosis complex (TSC) is a pediatric disorder of dysregulated growth and differentiation caused by loss of function mutations in either the TSC1 or TSC2 genes, which regulate mTOR kinase activity. To study aberrations of early development in TSC, we generated induced pluripotent stem cells using dermal fibroblasts obtained from patients with TSC. During validation, we found that stem cells generated from TSC patients had a very high rate of integration of the reprogramming plasmid containing a shRNA against TP53. We also found that loss of one allele of TSC2 in human fibroblasts is sufficient to increase p53 levels and impair stem cell reprogramming. Increased p53 was also observed in TSC2 heterozygous and homozygous mutant human stem cells, suggesting that the interactions between TSC2 and p53 are consistent across cell types and gene dosage. These results support important contributions of TSC2 heterozygous and homozygous mutant cells to the pathogenesis of TSC and the important role of p53 during reprogramming.

Figure 1.

Heterozygous nonsense mutations in TSC2 result in reduced tuberin mRNA levels and protein. (A) Sequencing confirms single nucleotide changes causes premature stop codons in TSC2+/− fibroblasts. DNA sequencing reveals wild type and mutant allele but only wild-type allele was detected in mRNA. (B) TSC1 and TSC2 mRNA quantified by qPCR in control and TSC patient (TSP) fibroblasts (n = 3 average of four experimental replicates; P = 0.039, t-test). (C) Tuberin and hamartin protein quantified by immunoblot (n = 3; P = 0.016, t-test).

Figure 2.

Increased integration of reprogramming plasmids in TSP iPSC lines. (A) PCR for continued presence of the reprogramming plasmids at passage ≥10. Multiple iPSC clones generated from five individual patients and five individual controls were analysed. Plasmid bands were detectable in eight of ten TSC2 heterozygous mutant clones analysed and two of ten control clones from control volunteers (P = 0.023, Fisher‘s exact test). (B) PCR to distinguish between the three reprogramming plasmids (C) Protein levels of p53 were measured by immunoblot in non-integrated or integrated control and TSP iPSC lines (average of 2–3 experimental replicates; P = 0.259, t-test). See also Supplementary Material, Figure S2.

Figure 3.

TSC patient fibroblasts display increased p53 in response to UV light. (A) Three control and two TSC patient fibroblast lines were challenged with UV light and then immunostained for p53. Cells were treated with 0.2 nM rapamycin for 24 h before through 24 h after UV challenge. Mean fluorescent intensity of p53 was quantified in nuclei defined by Hoechst staining. Nuclear p53 was analysed by two-way ANOVA in UV-treated cells (n = 2-3; interaction P = 0.532, genotype P = 0.012, rapamycin treatment P = 0.008; Bonferroni post-test control UV vs TSP UV P < 0.01). (B) Fibroblast lines were exposed to UV light and protein isolated at 0, 8 16, and 24 h post-exposure was analysed by immunoblot. (n = 3, average of 3–4 experimental replicates; two-way ANOVA). See also Supplementary Material, Figure S3.

Figure 4.

Impaired reprogramming and increased apoptosis in TSC patient fibroblasts. (A) Control (CA, CC) and patient (T20, T31, T23) fibroblasts were reprogrammed with the three plasmids including either the Oct4 plasmid containing shRNA to TP53 or the Oct4 plasmid alone. Pluripotent colonies were stained for alkaline phosphatase. Alkaline phosphatase positive colonies were counted using gray scale images in ImageJ (n = 2 controls and 3 patients, average of 1–2 wells per individual; P = 0.015, t-test). (B) Fibroblast lines were challenged with UV light and apoptosis measured by Annexin V staining by flow cytometry. Annexin V versus propidium iodide is plotted for untreated and UV challenged fibroblasts; control (blue) and TSP (green) samples have been combined for display purposes. (n = 3 controls and 2 TSP, average of 2 experimental replicates; interaction P = 0.048, genotype P = 0.189, UV treatment P = 0.007; Bonferroni post-test control UV vs TSP UV P < 0.05). (C) Fibroblasts were treated with 0.2 nM rapamycin or DMSO for a total of 48 h, starting 24 h before UV challenge and ending 24 h after. Apoptosis was measured by Annexin V staining by flow cytometry. (n = 3, average of 2 experimental replicates; interaction P = 0.012, genotype P = 0.678, rapamycin treatment P = 0.032; Bonferroni post-test Control Vehicle vs Rapa P < 0.05).

Figure 5.

Stability, mRNA and translation of p53 in TSC2+/− fibroblasts. (A) mRNA levels of TP53 in fibroblasts were measured by qPCR at baseline using primers directed to the 3’ end of the gene. Samples were normalized to ACTIN and PGK1. (n = 3, 4 technical replicates; P = 0.334, t-test) (B) Fibroblasts were challenged with UV light. Levels of TP53 mRNA were measured 24 h later by qPCR using primers directed to the 3’ end (n = 3, 4 technical replicates; P = 0.023, t-test) (C) Fibroblasts were treated with the proteasome inhibitor MG132 for 6 h with or without UV challenge. p53 protein levels were analysed by immunoblot. (D) Fibroblasts were challenged with UV light and 15 h later treated with the translation inhibitor cycloheximide (100 μM) for 2 h. P53 protein levels were analysed by immunoblot. See also Supplementary Material, Figure S4.

Figure 6.

Increased p53 in heterozygous and homozygous TSC2 mutant stem cells. (A) Non-integrated iPSC lines generated from TSC2 heterozygous patient fibroblast lines treated with the DNA damaging agent neocarzinostatin for 1 h show increased p53 relative to controls. (n = 6, 1–3 experimental repeats per sample; P = 0.04 t-test) (B) Protein levels of p53 and mTORC1/mTORC2-related proteins were measured at baseline in homozygous knockout and control stem cells (n = 14, 2 clones with 7 experimental repeats; P = 0.03 t-test). (C) Homozygous TSC2^−/− and isogenic control stem cells were nutrient starved for 2.5 h in DMEM without glucose or HBSS media. Protein levels of pS6 were elevated in TSC2^−/− iPSCs following nutrient starvation. (n = 4, 2 clones with 2 experimental repeats; P = 0.0002 t-test) See also Supplementary Material, Figure S5.