Reciprocity of Action of Increasing Oct4 and Repressing p53 in Transdifferentiation of Mouse Embryonic Fibroblasts into Cardiac Myocytes

Abstract

p53 is a barrier to somatic cell reprogramming. Deletion or transient suppression of p53 increases the efficiency of reprogramming of somatic cells into induced pluripotent stem cells. Whether p53 represents an obstacle to a similar process transdifferentiation of somatic cells is unknown. However, it is predicted that inhibition of p53 would promote transdifferentiation of fibroblasts into cardiomyocytes. In this study, the effect of p53 on the capacity of cardiogenic transdifferentiation is evaluated using p53 wild-type (p53^+/+), p53 heterozygous mutant (p53^+/-), and p53 homozygous mutant (p53^-/-) mouse embryonic fibroblasts (MEFs). Repression of p53 in MEFs increases the expression level of mesoderm transcription factors Brachyury (T) and MESP1. The cardiac-specific markers, Myh6 (Myosin, Heavy Chain 6), Myh7 (Myosin, Heavy Chain 7), and cTnI (cardiac muscle troponin I), show elevated expression in p53^+/- and p53^-/- MEFs compared with wild-type MEFs, but cardiac muscle troponin T (cTnT) showed a lower expression level when p53 was inhibited. After induction to cardiac differentiation, cTnT expression increased and markers of endoderm and ectoderm decreased in p53^+/- and p53^-/- MEFs. The effect of an important reprogramming factor Oct4 on cardiac transdifferentiation was also evaluated in the allelic series of p53 MEFs. We found that overexpression of Oct4 significantly enhanced Mesp1, Tbx5, and Isl1 expression in p53^+/+ and p53^+/- MEFs. Oct4 also enhanced cTnT expression in all three cell lines, especially in p53^+/- MEFs. Thus, inhibition of p53 expression and viral expression of Oct4 both promote transdifferentiation of MEFs into cardiomyocytes, establishing reciprocity of action in the process.

Keywords: Oct4; cardiomyocyte differentiation; fibroblast; p53; reprogramming; transdifferentiation.

FIG. 1.

Characteristics of p53^+/+, p53^+/−, and p53^−/− MEFs. (A–C) The morphologies of p53^+/+, p53^+/−, and p53^−/− MEFs at early passages (not more than three passages), respectively. (D–F) The morphologies of p53^+/+, p53^+/−, and p53^−/− MEFs in long-term culture (more than 15 days), respectively. (G) The levels of p53 mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. (H) The levels of p53 protein expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by western blot. (I) Growth curves of the in p53^+/+, p53^+/−, and p53^−/− MEFs. (J) The levels of Mesp1 mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. (K) The levels of T mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. (L) The levels of Oct4 mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. *Indicates no statistical significance compared with p53^+/+ MEFS with p ≥ 0.05; **indicates statistical significance compared with p53^+/+ MEFS with p < 0.05. Scale bar = 100 μm. MEF, mouse embryonic fibroblast; qRT-PCR, quantitative real-time polymerase chain reaction.

FIG. 2.

The expression of cardiac lineage marker genes at early passages (not more than three passages). (A) The levels of Myh7 mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. (B) The levels of Myh6 mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR, respectively. (C) The levels of cTnT mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. (D) The levels of cTnI mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. cTnI, cardiac muscle troponin I; cTnT, cardiac muscle troponin T; Myh6, Myosin, Heavy Chain 6; Myh7, Myosin, Heavy Chain 7. **Indicates statistical significance compared with p53^+/+ MEFs with p < 0.05.

FIG. 3.

Expression of marker genes of the three germ layers after 21 days of induction. (A) cTnT-positive cells in p53^+/+ isotype control MEFs was analyzed by FACS. (B) cTnT-positive cells in p53^+/+ MEFs was analyzed by FACS. (C) cTnT-positive cells in p53^+/− isotype control MEFs was analyzed by FACS. (D) cTnT-positive cells in p53^+/− MEFs was analyzed by FACS. (E) cTnT-positive cells in p53^−/− isotype control MEFs were analyzed by FACS. (F) cTnT-positive cells in p53^−/− MEFs were analyzed by FACS. (G) The chart of the percentage for cTnT-positive cells among p53^+/+, p53^+/−, and p53^−/− MEFs. (H) The levels of AFP mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. (I) The levels of Sox17 mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. (J) The levels of Nestin mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. (K) The levels of Gfap mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. AFP, Alpha fetoprotein; FACS, fluorescence-activated cell sorting; Gfap, Glial fibrillary acidic protein. *Indicates no statistical significance compared with p53^+/+ MEFs with p ≥ 0.05; **indicates statistical significance compared with p53^+/+ MEFs with p < 0.05.

FIG. 4.

The effect of Oct4 on the expression of cardiac transcription factors verified by qRT-PCR. (A) T expression. (B) Mesp1 expression. (C) Tbx5 expression. (D) Isl1 expression. (E) cTnT expression. *Indicates no statistical significance with p ≥ 0.05; **indicates statistical significance with p < 0.05.

FIG. 5.

Functional reciprocity between p53 and Oct4 in promoting cardiac transdifferentiation. (A) The levels of endogenous and total Oct4 mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs were verified by qRT-PCR. (B) The levels of p53 mRNA expression in p53^+/+, p53^+/−, and p53^−/− MEFs with Oct4 overexpression and with mock were verified by qRT-PCR. (C, D) FACS results for cTnT-positive cells in p53^+/+, p53^+/−, and p53^−/− MEFs with Oct4 overexpression and with mock. (E) cTnT protein expression in p53^+/+, p53^+/−, and p53^−/− MEFs at day 0 (d0) without Oct4 transfection and at day 21 (d21) with Oct4 transfection and with mock. *Indicates no statistical significance with p ≥ 0.05; **indicates statistical significance with p < 0.05.