Estrogen regulates estrogen receptors and antioxidant gene expression in mouse skeletal muscle

Abstract

Background: Estrogens are associated with the loss of skeletal muscle strength in women with age. Ovarian hormone removal by ovariectomy in mice leads to a loss of muscle strength, which is reversed with 17beta-estradiol replacement. Aging is also associated with an increase in antioxidant stress, and estrogens can improve antioxidant status via their interaction with estrogen receptors (ER) to regulate antioxidant gene expression. The purpose of this study was to determine if ER and antioxidant gene expression in skeletal muscle are responsive to changes in circulating estradiol, and if ERs regulate antioxidant gene expression in this tissue.

Methodology/principal findings: Adult C57BL/6 mice underwent ovariectomies or sham surgeries to remove circulating estrogens. These mice were implanted with placebo or 17beta-estradiol pellets acutely or chronically. A separate experiment examined mice that received weekly injections of Faslodex to chronically block ERs. Skeletal muscles were analyzed for expression of ER genes and proteins and antioxidant genes. ERalpha was the most abundant, followed by Gper and ERbeta in both soleus and EDL muscles. The loss of estrogens through ovariectomy induced ERalpha gene and protein expression in the soleus, EDL, and TA muscles at both the acute and chronic time points. Gpx3 mRNA was also induced both acutely and chronically in all 3 muscles in mice receiving 17beta-estradiol. When ERs were blocked using Faslodex, Gpx3 mRNA was downregulated in the soleus muscle, but not the EDL and TA muscles.

Conclusions/significance: These data suggest that Gpx3 and ERalpha gene expression are sensitive to circulating estrogens in skeletal muscle. ERs may regulate Gpx3 gene expression in the soleus muscle, but skeletal muscle regulation of Gpx3 via ERs is dependent upon muscle type. Further work is needed to determine the indirect effects of estrogen and ERalpha on Gpx3 expression in skeletal muscle, and their importance in the aging process.

Figure 1. ER gene expression in skeletal muscles of 4-mo-old female wild-type mice.

Data normalized to ERβ in the soleus. Values are means ± SEM. *Signifies different from ERα within a muscle type. ^#Signifies different from ERβ within a muscle type. ^$Signifies different from soleus muscle.

Figure 2. ER gene expression in skeletal muscle following ovariectomy and 48 hours of 17β-estradiol replacement.

A. ERα gene expression. B. ERβ gene expression. C. Gper gene expression. Data are normalized to sham mice within each muscle. Values are means ± SEM. *Signifies different from sham. ^#Signifies different from OVX + Placebo.

Figure 3. ERα protein expression in the TA muscle following ovariectomy and 48 hours of 17β-estradiol replacement.

A. Preliminary work examining ERα expression in uterine tissue and skeletal muscle. Lane 1 = 10 µg of uterine homogenate. Lanes 2-4 = 10, 20, and 40 µg of skeletal muscle homogenate. B. ERα protein expression in muscle from sham, OVX + Placebo, and OVX + E₂ mice. Data are normalized to sham mice. Values are means ± SEM. *Signifies different from sham. ^#Signifies different from OVX + Placebo.

Figure 4. Chronic ovariectomy and 17β-estradiol replacement on ER and antioxidant gene expression in skeletal muscle.

A. ERα, ERβ, and Gper gene expression. B. Gpx1, Gpx3, Nox4, and Txnip gene expression. ER and antioxidant gene expression were measured in the soleus, EDL, and TA muscles after 3 weeks of ovariectomy (OVX + Placebo) or in ovariectomized mice immediately replaced with 17β-estradiol (OVX + E₂). Data are normalized to OVX + Placebo mice within each muscle. Values are means ± SEM. ND = not detected. *Signifies different from OVX + Placebo.

Figure 5. Effects of chronic ER inhibition on uterus and skeletal muscle.

A. Body mass. B. Voluntary wheel running activity. C. Uterine wet mass. D. Uterine gene expression. E. Skeletal muscle mass. F. Skeletal muscle ER gene expression. G. Skeletal muscle Gpx3 expression. ERs were blocked by administering Faslodex for 1 month to female mice. Data are normalized to oil-injected mice. Values are means ± SEM. *Signifies different from Oil. $Main effect of time.

Figure 6. MyoD and Glut-4 gene expression in ovariectomized mice with and without 17β-estradiol supplementation.

A. MyoD and Glut-4 after 48 hours of estrogen replacement. B. MyoD and Glut-4 mRNA expression after 3 weeks of estrogen replacement. Values are means ± SEM. *Signifies different from OVX + Placebo.