Oxidative stress contributes to hypermethylation of Histone H3 lysine 9 in placental trophoblasts from preeclamptic pregnancies

Abstract

Background and objective: Aberrant epigenetic regulation and increased oxidative stress in the placenta play a significant role in placental pathophysiology and fetal programming in preeclampsia, a hypertensive disorder in human pregnancy. The purpose of the study is to investigate if hypermethylation of histone H3K9 occurs in placental trophoblasts from preeclampsia.

Methods: Trophoblasts were isolated and cultured from 14 placentas, 7 from normotensive pregnant women and 7 from preeclamptic pregnancies. Methylated H3K9 expression and antioxidant superoxide dismutase expression were determined by Western blot. We also examined consequences of oxidative stress and the downstream effects of histone methyltransferase inhibition on H3K9 expression associated with antioxidant CuZn-SOD and Mn-SOD expression in placental trophoblasts.

Results: We found that expression of mono-, di-, and tri-methylation of histone H3 lysine 9 (H3K9me1, H3K9me2 and H3K9me3) was significantly increased, p<0.01, which correlated with downregulation of antioxidant superoxide dismutase CuZn-SOD and Mn-SOD expression, in trophoblasts from preeclamptic placentas compared to those from uncomplicated control placentas. We further demonstrated hypoxia could promote histone H3K9 methylation in placental trophoblasts, and hypoxia-induced upregulation of H3K9me1, H3K9me2 and H3K9me3 expression was reversible when hypoxic condition was removed. In addition, we also uncovered that inhibition of methyltransferase not only prevented hypoxia-induced upregulation of H3K9me1, H3K9me2 and H3K9me3 expression, but also abolished hypoxia-induced downregulation of CuZn-SOD and Mn-SOD expression in placental trophoblasts.

Conclusions: These findings are noteworthy and provide further evidence that increased oxidative stress in the intrauterine environment is likely a mechanism to induce aberrant histone modification in placental trophoblasts in preeclampsia. Moreover, CuZn-SOD and Mn-SOD expression/activity are possibly H3K9 methylation-dependent in placental trophoblasts, which further suggest that oxidative stress and aberrant histone modification have significant impact on placental trophoblasts/fetal programming in preeclampsia.

Keywords: H3K9 methylation; hypoxia; oxidative stress; placental trophoblast; preeclampsia; superoxide dismutase.

Figure 1

Expression of H3K9m1, H3K9m2, H3K9m2, CuZn-SOD, and Mn-SOD in placental trophoblasts from normal and preeclamptic pregnancies. (A) Expression of H3K9me1, H3K9me2, H3K9me3 in trophoblasts (TCs) from 7 normal (Nor) and 7 preeclamptic (PE) placentas. The bar graphs show relative expression for H3K9me1, H3K9me2, and H3K9me3 after normalized by β-actin expression in each sample. (B) Expression of CuZn-SOD and Mn-SOD in trophoblasts from 7 normal and 7 preeclamptic placentas. The bar graphs show relative expression for CuZn-SOD and Mn-SOD after normalized by β-actin expression in each sample. These data revealed that downregulation of antioxidant CuZn-SOD and Mn-SOD expression are associated with upregulation of methylated H3K9 expression in trophoblasts from preeclamptic vs. normal placentas. **p<0.01: PE-TCs vs. Nor-TCs.

Figure 2

Hypoxia upregulates H3K9 methylation in placental trophoblasts. To determine if H3K9 methylation status is regulated by oxygen environment, H3K9me1, H3K9me2, H3K9me3 expression were examined in trophoblasts cultured under hypoxia and reoxygenation conditions. Hypoxic condition: HTR-8/SVneo cells were cultured 2%O2 for 24 hours; Hypoxic + Re-oxygen condition: HTR-8/SVneo cells were cultured under 2%O2 for 24 hours and then incubated under 21%O2 for 6 hours. Cells cultured under 21%O2 served as control. (A) Expression of H3K9me1, H3K9me2, and H3K9me3 were significantly upregulated in cells cultured under 2%O2 but returned to the control levels after cells were reoxygenated at 21%O2 condition. (B) The bar graphs show relative expression for H3K9me1, H3K9me2, H3K9me3 after normalized by β-actin expression in each sample. Bar graphs shows mean ± SE from 3 independent experiments. *p<0.05 and **p<0.01: Cells cultured under 2% vs. 21%O2; #p<0.05 and ##p<0.01: 2%O2+ re-oxygen vs. 2%O2 alone. These results indicate oxygen environment could regulate methylation status of H3K9 in placental trophoblasts.

Figure 3

Effects of histone methyltransferase on H3K9me1, H3K9me2, H3K9me3, CuZn-SOD, and Mn-SOD expression in placental trophoblasts. To determine if H3K9 methylation regulates CuZn-SOD and Mn-SOD expression in placental trophoblasts under oxidative stress condition, HTR-8/SVneo cells were cultured under 2%O2 treated with or without BIX01294 (5µM) in culture for 24 hours. BIX01294 is an inhibitor of histone methyltransferase. Expression of H3K9me1, H3K9me2, H3K9me3, CuZn-SOD and Mn-SOD were then evaluated. (A) Expression of H3K9me1, H3K9me2, and H3K9me3 were significantly upregulated in cells cultured under 2%O2 alone but no change (H3K9me1 and H3K9me3) or reduced (H3K9me2) in cells treated with BIX01294. In contrast, expression of CuZn-SOD and Mn-SOD were downregulated in cells cultured under 2%O2 alone but increased in cells treated with BIX01294. (B) The bar graphs show relative expression for H3K9me1, H3K9me2, H3K9me3, CuZn-SOD, and Mn-SOD after normalized by β-actin expression in each sample, mean ± SE from 5 independent experiments. *p<0.05 and **p<0.01: Cells cultured under 2% vs. 21%O2; #p<0.05 and ##p<0.01: BIX +2%O2 vs. 2%O2. These results suggest that antioxidant CuZn-SOD and Mn-SOD expression are regulated by methylation status of H3K9 in placental trophoblasts.