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Comparative Study
. 2013 Aug 8;4(8):e758.
doi: 10.1038/cddis.2013.251.

Differential regulation of cell death programs in males and females by Poly (ADP-Ribose) Polymerase-1 and 17β estradiol

N R Jog  1 , R Caricchio
Affiliations
Comparative Study

Differential regulation of cell death programs in males and females by Poly (ADP-Ribose) Polymerase-1 and 17β estradiol

N R Jog et al. Cell Death Dis. .

Abstract

Cell death can be divided into the anti-inflammatory process of apoptosis and the pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP) 1/3 are major mediators. We previously showed that absence or inhibition of PARP-1 protects mice from nephritis, however only the male mice. We therefore hypothesized that there is an inherent difference in the cell death program between the sexes. We show here that in an immune-mediated nephritis model, female mice show increased apoptosis compared to male mice. Treatment of the male mice with estrogens induced apoptosis to levels similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male and female mice are prone to different types of cell death. Our data also suggest that estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore propose that targeting cell death based on sex will lead to tailored and better treatments for each gender.

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Figures

Figure 1
Figure 1
PARP-1 is activated in both male and female mice during nephritis. (a) Kidney sections from (NZWxBXSB)F1 mice were stained with DAPI to identify nuclei and with anti-PARs to identify PARP-1 enzymatic activity; bottom panels shows secondary antibody only. Female mice were injected with AdvIFNα at 8 weeks and were 22 weeks old at the time of the staining. The male mice were 18 weeks old. The figures are representative of six mice. (b) Fluorescence intensities of red channel were measured using ImageJ software. Fluorescence intensities of the secondary controls were subtracted from the stained sections. An average of 5–7 fields was calculated for each mouse. Data are mean±S.E.M. *P<0.05, **P<0.005. (c) NTN was induced in PARP-1+/+ or PARP-1−/− mice by injecting NTS. Kidneys were collected 30 h and 3 days after induction of NTN and stained with anti-PAR, Fluoroscein-conjugated anti-Complement C3 and DAPI. PARs were visualized by Rhodamine-conjugated secondary antibody. The figure shows that although C3 deposition in PARP-1−/− kidneys was same as PARP-1+/+ kidneys, PAR accumulation was absent in PARP-1−/− kidneys. These data show that PARP-1 is responsible for the majority of poly (ADP-ribosyl)-ation in the kidney during nephritis and therefore PARs can be used as a measure of PARP-1 activity in the kidney
Figure 2
Figure 2
Male and female cells show similar susceptibility to H2O2-induced cell death. BMDM from male and female 129s mice were stimulated with various concentrations of H2O2 for 2 h. (a) The cells were harvested and stained for FITC conjugated Annexin V and 7AAD, and acquired on FACS Canto. One representative of two independent cultures is shown. (b) BMDMs were stimulated, harvested, fixed, permeabilized and stained with PE-conjugated active caspase-3 antibodies. Cells were acquired on a FACS Canto and analyzed using FlowJo. One representative of four independent cultures is shown. (c) BMDMs were stimulated with increasing concentrations of H2O2 as in (a). Cells were lysed after 2 h stimulation. Western blot analysis shows that higher concentration of H2O2-induced necrosis as seen by absence of active caspase-3 in cell lysates (upper panel). Lower panel shows the ratios of band density of active caspase-3 to β actin band density, which was used as a loading control. (d) Active caspase-3 was not detected in concentrated culture supernatants from BMDMs treated with H2O2. Upper panel: concentrated supernatants from male BMDMs, lower panel: concentrated supernatants from female BMDMs. ‘−' and ‘+' represent lysates of untreated Jurkat cells (−) or Jurkat cells treated with camptothecin (+). BMDMs from male and female were labeled with Hoechst 33342 and stimulated with H2O2 for 2 h. Cells were fixed and imaged. Apoptotic and necrotic cells were counted. (e) Representative examples of live, apoptotic and necrotic cells are shown. Cells were identified as apoptotic or necrotic according to Figure 2e. Stimulation with 500 μM H2O2-induced necrosis in both male (f) and female BMDMs (g). Caspase-3 inhibition by Z-DEVD-FMK did not inhibit necrosis induced by 500 μM H2O2 in either male (h) or female (i) BMDMs. The data show that both male and female cells show similar susceptibility to H2O2-induced cell death and higher concentrations of H2O2 induce necrosis in both male and female cells
Figure 3
Figure 3
H2O2-induced necrosis is PARP-1 dependent in male BMDM. BMDM from male and female 129s mice or 129 PARP-1−/− mice (a–d); B6 or RIP-3−/− mice (e–h) were stimulated with H2O2. The cells were collected and stained for FITC conjugated Annexin V and Propidium Iodide/7AAD, and acquired on FACS Canto. Data are mean±S.E.M., representative of two independent cultures is shown. The data show that H2O2-induced cell death in male cells is dependent on PARP-1 activation while necrosis in both males and females is independent of RIP-3 activation. *P<0.05, ***P<0.0005
Figure 4
Figure 4
17β Estradiol influences PARP-1 activation and cell death in male and female cells. (a–f) BMDM from male and female 129s mice were pre-treated with various concentrations of E2 for 3 h followed by 2 hours stimulation with H2O2. The cells were harvested and stained for FITC-conjugated Annexin V and Propidium Iodide/7AAD. Results were acquired on FACS Canto. The data show that E2 rescues female cells from death, whereas it shifts cell death program to apoptosis in male cells. Data are mean±S.E.M.; representative of four independent experiments is shown. (g and h) BMDMs were seeded in 96-well plates and pre-treated with E2 for 3 h. Following stimulation with H2O2, PARP-1 activity in the cells was determined by a colorimetric assay as described in Materials and Methods. The data show that E2 inhibits PARP-1 activity in male (g) but not in female cells (h). Data are mean±S.E.M. of triplicates. One representative of three experiments is shown. (i and j) ERα mRNA levels were determined in BMDMs treated with E2 and stimulated with H2O2 as in (a–f) by quantitative real-time PCR. Relative mRNA level calculated from a standard graph was normalized to mRNA levels in untreated cells. The data show that E2 treatment does not alter ERα expression in male (i) or female (j) cells. Data are mean±S.E.M., representative of six mice. *P<0.05, **P<0.005
Figure 5
Figure 5
E2 treatment induces caspase-3 activation following NTN in male but not in female mice. To determine the role of E2 in the induction of cells death, we treated male or female mice with estrogens by implanting E2 pellets s.c. NTS (6 ml/kg) was injected 5 days following pellet implantation, and the kidneys were collected after 30 h. Paraffin-embedded kidney sections were stained for active caspase-3 as a measure of apoptosis (a) or PAR as a measure of PARP-1 activation (b). Fluorescence intensities of red channel were measured using ImageJ software (lower panels). Fluorescence intensities of the secondary controls were subtracted from the stained sections. An average of 7–10 fields was calculated for each mouse. Data are mean±S.E.M., representative of six mice. The data show that E2 treatment of male mice results in increased apoptotic cell death and inhibition of necrosis following induction of nephritis. ERα mRNA expression in the renal tissue from male (c) or female (d) mice was determined by quantitative PCR and normalized to mRNA levels in an untreated mouse of same sex. The results show that E2 treatment of males resulted in the upregulation of ERα in the kidney. The data are representative of six mice. *P<0.05, **P<0.005, ***P<0.0005
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