Oestrogen prevents cardiomyocyte apoptosis by suppressing p38α-mediated activation of p53 and by down-regulating p53 inhibition on p38β

Abstract

Aims: we have previously shown that 17-β-estradiol (E2) protects cardiomyocytes exposed to simulated ischaemia-reperfusion (I/R) by differentially regulating pro-apoptotic p38α mitogen-activated protein kinase (p38α MAPK) and pro-survival p38β. However, little is known about how E2 modulation of these kinases alters apoptotic signalling. An attractive downstream target is p53, a well-known mediator of apoptosis and a substrate of p38α MAPK. The aim of this study was to determine whether the cytoprotective actions of oestrogen involve regulation of p53 via cardiac p38 MAPKs.

Methods and results: cultured rat cardiomyocytes underwent hypoxia followed by reoxygenation (H/R) to simulate I/R. We found that inhibiting p53 significantly reduced apoptosis. Phosphorylation of p53 at serine 15 [p-p53(S15)] increased after H/R in a p38α MAPK- and reactive oxygen species (ROS)-dependent manner. E2 at 10 nM effectively inhibited p-p53(S15) and mitochondrial translocation of p53. Blocking p53 led to augmented p38β activity and attenuated ROS, suggesting suppression of this antioxidant kinase by p53. The use of a specific agonist for each oestrogen receptor (ER) isoform, ERα and ERβ, demonstrated that both isoforms participate in preventing cell death by inhibiting p53 in the mitochondria-centred apoptotic processes.

Conclusion: our results demonstrate that during H/R stress, cardiomyocytes undergo p53-dependent apoptosis following phosphorylation of p53 by p38α MAPK, leading to p38β suppression. E2 protects cardiomyocytes by inhibiting p38α-p53 signalling in apoptosis.

Figure 1

(A) Apoptosis assay. After H/R with or without PFT (1 µM) or siRNA p53, apoptotic cardiomyocytes were identified by annexin V-FITC. The apoptotic cells appeared green, and the nuclei co-stained with Hoechst 33342 blue. N, normoxia. The white scale bar represents 25 µm. Mean% apoptotic cells ± standard error (n = 3) are shown in a bar graph. *P < 0.05 vs. normoxia and ^##P < 0.05 vs. H/R. (B) Cyt c release into cytosol. After indicated treatments, immunoblotting was performed on the mitochondrial (Mito) and cytosolic (Cyto) fraction for Cyt c and a marker protein (CoxIV for mitochondria and actin for cytosol). Representative immunoblots are shown with quantitative analysis (n = 3). *P < 0.05 vs. normoxia and ^##P < 0.05 vs. H/R. (C) p53 inhibition with siRNA p53. To confirm inhibition of p53 expression by siRNA p53, immunoblotting for p53 was done 2 days following siRNA transfection, with actin as a loading control. Representative blots are shown with quantitative analysis (n = 3). *P < 0.05 vs. normoxia and ^##P < 0.05 vs. H/R. (D) Time course of E2 exposure on cell survival. Cardiomyocytes were treated with 10 nM E2 for 30 min prior to hypoxia (before H), at the time of starting hypoxia (at H), or at the start of reoxygenation (at R). Apoptotic cells were identified in a manner similar to (A). The white scale bar represents 25 µm. Mean% apoptotic cells ± standard error (n = 3) are shown in a bar graph. *P < 0.05 vs. normoxia, ^##P < 0.01 vs. H/R, and ⁺P < 0.05 vs. H/R. (E) The effect of E2 on p-p53(S15). Immunoblotting was performed for p-p53(S15), p53, and actin. The concentration of E2 and ICI was 10 and 100 nM, respectively. Representative immunoblots are shown, with quantitative analysis (n = 3). *P < 0.05 vs. normoxia, ^##P < 0.05 vs. H/R, and ⁺P < 0.05 vs. H/R + E2.

Figure 2

The effect of p38α MAPK on p-p53(S15). Western blotting of p-p53(S15), p53, and actin was done after treatment with p38αDN or SB (1 µM). Representative immunoblots are shown with quantitative analysis (n = 3) of p-p53(S15). *P < 0.05 vs. normoxia and ^##P < 0.05 vs. H/R.

Figure 3

(A) The effect of ROS on p-p53(S15). Western blotting of p-p53(S15) and p53 after treatment with rotenone (2.5 µM) or Mito-Q^® (10 µM) was done, with representative immunoblots and quantitative analysis (n = 3) shown. *P < 0.05 vs. normoxia and ^##P < 0.05 vs. H/R. (B) The effect of p53 on ROS. Intracellular ROS was detected by ROS-dependent deacetylation and oxidation of 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (D₂CFDA) to green fluorescein. Representative images of cells are shown with quantitative analysis (n = 3). The bars represent mean ± standard error. *P < 0.05 vs. normoxia and ^##P < 0.05 vs. H/R. N, normoxia. The white scale bar represents 25 µm. The ROS-positive cells appeared green fluorescent, whereas nuclei stained blue with Hoechst 33342. The concentration of PFT and rotenone was 1 and 2.5 µM, respectively.

Figure 4

(A) The effect of p53 on p38β activity. Following treatment with PFT (1 µM) or siRNAp53, the p38β kinase activity was assessed in an in vitro kinase assay, with purified ATF2 as a substrate and p38β immunoprecipitated from the cell lysate. A representative image of radiolabelled ATF2 is shown with quantitative analysis (n = 3). *P < 0.05 vs. H/R. Western blotting of immunoprecipitated p38β used in the kinase assay is shown. (B) The effect of p38α MAPK on p38β activity. The p38β kinase activity was assessed in a similar manner as in (A) after blocking p38α MAPK with p38αDN. A representative radiograph of phosphorylated ATF2 is shown with quantitative analysis (n = 3). *P < 0.05 vs. H/R. Western blotting of immunoprecipitated p38β protein used in the kinase assay is shown.

Figure 5

(A) The effect of ER isoforms on p-p53(S15). Immunoblotting was performed for p-p53(S15) and total p53 after treatment with E2, PPT or DPN, each at 10 nM. Representative blots are shown, with quantitative analysis (n = 3) of p-p53(S15). *P < 0.01 vs. normoxia and ^##P < 0.05 vs. H/R. (B) The effect of ER isoforms on Bax translocation. After treating with E2, PPT, or DPN (each at 10 nM), the mitochondrial (Mito) and cytosolic (Cyto) fractions were separated, and immunoblotted for Bax and a marker protein (CoxIV for mitochondria and actin for cytosol). Representative blots are shown, with quantitative analysis (n = 3) of the mitochondrial Bax-to-cytosolic Bax ratio. *P < 0.01 vs. normoxia, ^##P < 0.01 vs. H/R, and ⁺P < 0.05 vs. H/R. (C) The effect of ER isoforms on Cyt c release. The mitochondrial (Mito) and cytosolic (Cyto) fractions were separated and immunoblotted for Cyt c and a marker protein (CoxIV for mitochondria and actin for cytosol) in a manner similar to (B). Representative immunoblots are shown with quantitative analysis (n = 3) of Cyt c released into cytosol. *P < 0.05 vs. normoxia and ^##P < 0.05 vs. H/R. (D) The effect of ER isoforms on caspase-9 activation. Immunoblotting was performed for procaspase-9 or cleaved caspase-9 after treatment with 10 nM E2, PPT, or DPN. Representative blots are shown, with quantitative analysis (n = 3) of the cleaved caspase-9 to procaspase-9 ratio. *P < 0.05 vs. normoxia and ^##P < 0.05 vs. H/R. (E) The effect of ER isoforms on cardiomyocyte survival. Apoptotic cells were detected in a similar manner as in Figure 1A. The final concentration of E2, PPT, and DPN was 10 nM. Representative images of apoptotic cells are shown, with the white scale bar representing 25 µm. Mean% (±standard error) of apoptotic cells is presented with quantitative analysis (n = 3). *P < 0.01 vs. normoxia and ^##P < 0.05 vs. H/R. (F) E2 alters subcellular translocation of p53. After H/R with or without E2 (10 nM), cardiomyocytes were labelled with MitoTracker, fixed, permeabilized and stained with anti-p53 primary antibody, followed by FITC-conjugated secondary antibody, and visualized under a fluorescence microscope. The photographs of the same cells labelled with anti-p53 antibody (green) or MitoTracker (red) were taken and merged to demonstrate co-localization (yellow) of p53 and mitochondria. A white scale bar represents 25 µm.

Figure 6

A working model of E2-dependent protection of a cardiomyocyte under H/R stress. Following H/R or simulated I/R, there is a burst of mitochondrial ROS. The oxygen radicals stimulate p38α MAPK, which phosphorylates and activates p53. p53 in turn represses p38β. E2 binds and activates ERs (possibly in heterodimers), and the E2/ER complex activates p38β via PI3K/Akt.