17β-Estradiol alters oxidative stress response protein expression and oxidative damage in the uterus

Abstract

The steroid hormone 17β-estradiol (E2) has profound effects on the uterus. However, with the E2-induced increase in uterine cell proliferation and metabolism comes increased production of reactive oxygen species (ROS). We examined the expression of an interactive network of oxidative stress response proteins including thioredoxin (Trx), Cu/Zn superoxide dismutase (SOD1), apurinic endonuclease (Ape1), and protein disulfide isomerase (PDI). We demonstrated that treatment of ovariectomized C57BL/6J female mice with E2 increased the mRNA and protein levels of Trx, but decreased SOD1 and Ape1 mRNA and protein expression. In contrast, E2 treatment increased PDI protein levels but had no effect on PDI transcript levels. Interestingly, E2 treatment also increased two markers of cellular damage, lipid peroxidation and protein carbonylation. Our studies suggest that the decreased expression of SOD1 and Ape1 caused by E2 treatment may in the long term result in disruption of ROS regulation and play a role in endometrial carcinogenesis.

Keywords: Apurinic endonuclease; Cu/Zn superoxide dismutase; Estrogen receptor ɑ; Oxidative stress; Protein disulfide isomerase; Thioredoxin.

Fig. 1. Oxidative stress response proteins help maintain protein structure and function

SOD1 dismutates superoxide to form H₂O₂ and other cellular enzymes including catalase (Cat) and Trx, which activates peroxiredoxins (Prx), help eliminate H₂O₂. Cat and Prx convert H₂O₂ to H₂O and O₂. Together, Trx, Ape1, and PDI reduce cellular proteins to maintain their native protein structure and function. Adapted from Ref.(Webster et al 2001).

Fig. 2. E2 regulates PR expression

Ovariectomized female mice were treated with oil or E2 for 7 days. (A) ERα and (B) PR expression was examined in uterine sections using immunofluorescent microscopy. (C) Quantification of PR staining of oil- and E2-treated mice demonstrated that PR expression was significantly increased in the uteri of E2-treated animals (*p<0.05). DAPI counter staining was included to identify cell nuclei. Scale bars indicate 25 µm. Six mice were included in the oil- and E2-treated groups. Representative images are shown.

Fig. 3. E2 treatment differentially regulates Trx, SOD1, Ape1, and PDI mRNA levels

RNA was isolated from the uteri of ovariectomized female mice that had been treated with oil or E2. cDNA was synthesized and (A) Trx, (B) SOD1, (C) Ape1, and (D) PDI mRNA levels were determined using quantitative real time PCR. Each sample was normalized to the amount of hypoxanthine phosphorybosyl transferase (HPRT) mRNA present. E2 induced significant differences in the mRNA expression of Trx (*, p<0.05), SOD1 (**, p<0.01), and Ape1 (**, p<0.01), but not PDI (p=0.63). Seven mice were included in the oil- and E2-treated groups as indicated at the base of each treatment group bar.

Fig. 4. E2 Influences Trx, SOD1, Ape1, and PDI protein expression in uterine epithelial and stromal cells

Ovariectomized female mice were treated with oil or E2 for 7 days and (A) Trx, (B) SOD1, (C) Ape1, and (D) PDI expression was examined in uterine sections using immunofluorescent microscopy. DAPI counter staining was included to identify cell nuclei. Scale bars indicate 25 µm. Six mice were included in the oil- and E2-treated groups. Representative images are shown.

Fig. 5. E2 treatment differentially alters SOD1, Ape1, and PDI protein levels

Whole cell extracts were prepared from the uteri of ovariectomized female mice that had been treated with oil or E2 and subjected to quantitative Western blot analysis. The levels of (A) SOD1 (*, p<0.05), (B) Ape1 (**, p<0.01), and (C) PDI (*, p<0.05) protein were significantly different in oil- and E2-treated mice. Expression of each protein was normalized to the amount of GAPDH present. Six mice were included in the oil- and E2-treated groups as indicated at the base of each treatment group bar.

Fig. 6. E2 treatment increases lipid peroxidation and protein carbonylation in the uterus

Ovariectomized female mice were treated with oil or E2. (A) Lipid peroxidation and (B) protein carbonylation were assessed as described in Materials and Methods. Significant increases in the levels of lipid peroxidation (**, p<0.01) and protein carbonylation (*, p<0.05) were observed in the E2-treated mice. Seven mice were included in the oil- and E2-treated groups as indicated at the base of each treatment group bar. (C) The spectrum of carbonylated proteins in whole cell uterine extracts from oil- and E2-treated mice was examined by Western blot analysis. GAPDH served as a loading control.