Intrauterine ischemic reperfusion switches the fetal transcriptional pattern from HIF-1α- to P53-dependent regulation in the murine brain

Abstract

Ischemic reperfusion (IR) during the perinatal period is a known causative factor of fetal brain damage. So far, both morphologic and histologic evidence has shown that fetal brain damage can be observed only several hours to days after an IR insult has occurred. Therefore, to prevent fetal brain damage under these circumstances, a more detailed understanding of the underlying molecular mechanisms involved during an acute response to IR is necessary. In the present work, pregnant mice were exposed to IR on day 18 of gestation by clipping one side of the maternal uterine horn. Simultaneous fetal electrocardiography was performed during the procedure to verify that conditions resulting in fetal brain damage were met. Fetal brain sampling within 30 minutes after IR insult revealed molecular evidence that a fetal response was indeed triggered in the form of inhibition of the Akt-mTOR-S6 synthesis pathway. Interestingly, significant changes in mRNA levels for both HIF-1α and p53 were apparent and gene regulation patterns were observed to switch from a HIF-1α-dependent to a p53-dependent process. Moreover, pre-treatment with pifithrin-α, a p53 inhibitor, inhibited protein synthesis almost completely, revealing the possibility of preventing fetal brain damage by prophylactic pifithrin-α treatment.

Figure 1. An ischemic reperfusion in pregnant mice.

(A) Experimental design (B) No IR treatment pregnant mice and Ischemic reperfusion (IR) mice underwent surgery on day 18 of gestation. (C). Fetal electrocardiography (FECG) was used to monitor the conditions of both the mother and the fetuses in either clipped or non-clipped uterine horns (n = 5 fetuses from at least 5 individual pregnant mice). (C) Measurement of oxygen concentration in the amniotic fluid before and after IR (p<0.01 indicated as two stars, n = 5 fetuses from at least 3 individual pregnant mice).

Figure 2. IR changed signaling pathways in the fetal brain.

(A) The phosphorylation of JNK1/2 was activated in the fetal brain without (−) or with IR (+). The phosphorylation of both Akt and ERK1/2 were suppressed in the fetal brain by IR. (B, C, D) Measurement and Graph data p<0.01 indicated as two stars (n  = 5 fetuses from at least 3 individual pregnant mice). White means fetal brain and black means fetal heart.

Figure 3. IR inhibited protein synthesis in the fetal brain.

(A) Phosphorylation of Akt, (B) Phosphorylation of mTOR, (C) Phosphorylation of S6, and (D) Cleaved-Parp of either fetal brain or heart was measured. (n = 4 fetuses from at least 3 individual pregnant mice). White means fetal brain and black means fetal heart.

Figure 4. P53 mRNA was increased response to IR in the fetal brain.

Within 30 minutes post-IR, the mRNA levels of Mapk associated proteins, except Jnk2 and Trp53 (p53), were noted to have increased, while Hif-1α, Mdm2 and Ccm3/Pdcd10 were not increased as compared to the housekeeping gene Hprt1. (n = 12 from at least 3 individual pregnant mice). p<0.05 indicated as single star and p<0.01 indicated as two stars.

Figure 5. P53 was increased response to IR in the fetal brain.

Even though no significant change in protein level, total p53 and phosphorylation of P53 at ser15 were increased compared with HIF-1α in fetal brains response to IR. Numbers in the pictures show the average of three independent experiments (n = 3 from 3 individual pregnant mice).

Figure 6. Transcriptional pattern was changed in the fetal brain.

Binding to gene promoters by two transcriptional factors, HIF-1α (A) and P53 (B), was measured by ChIP assay. Fold change from 0.5 to 2 was considered as not significant. Gene No. 1.P53; 2.Mdm2; 3.Akt1; 4.Pdcd10; 5.Pfkfb4; 6.S100A10; 7.Cox4i1; 8.Sirt1. Binding Folds =  (After IR)/(Before IR) (n = 6∼8 from at least 3 individual pregnant mice, each dot means one sample). (C) HIF-1α release and P53 binding on the promoter of indicated genes. From left to right according the number of x-axis, they are trp53 (purple), Akt1 (sky blue), mdm2 (blue), Sirt1 (White), cox4i1 (green), s100A10 (yellow), pdcd10 (red) and pfkfb4 (black).

Figure 7. In vivo, PFT-α inhibition of protein synthesis after IR.

(A) PFT-α inhibited phosphorylation of S6 in fetal brain. (B) Measurement phosphorylated S6 (Vs. beta actin, n = 4∼6 from at least 3 individual pregnant mice). White means fetal brain and black means fetal heart.