Consequences of Estrogen Receptor Beta Phosphorylation in the Aged Female Brain and Heart

Female life expectancy in the United States has steadily increased and now averages 81 years. Meanwhile, the age at which women reach menopause remains constant at 51 years. Both neurological and cardiovascular disorders increase following menopause, affecting women’s quality of life and leading to large health care costs. Estrogens are neuroprotective and cardioprotective for women, however when administered long after menopause, they fail to improve their health. The effects of estrogen are mediated by Estrogen Receptor α (ERα) and Estrogen Receptor β (ERβ). Evidence supports that ERβ plays an important role in neuroprotection and cardioprotection, but aspects of ERβ molecular signaling are still not fully understood. One possibility is that aging and estrogen deprivation alter ERβ post translational modifications, such as phosphorylation. Indeed the mitogen activated protein kinases (MAPKs) responsible for phosphorylation of ERβ are modulated by aging and steroid hormones. Therefore, I hypothesized that MAPKs are sensitive to menopause leading to a differential phosphorylation of ERβ which results in altered transcriptional regulation. First, phosphorylated ERβ was detected in the heart and brain of aged female rats prompting further investigation on functional consequences of this modification. Phosphorylation of ERβ was found to alter both its ligand dependent and ligand independent regulation of transcription. Therefore, ERβ phosphorylation would lead to differential gene regulation both in absence of estrogen or following estrogen treatment. Using an animal model of menopause in aged female rats, we found that the expression and activation of the MAPKs that phosphorylate ERβ are sensitive to estrogen deprivation and treatment in the brain and heart. Altered kinase activity could have important physiological consequences as they control many cell signaling required for cell function. Furthermore altered kinase activation could result in differential ERβ phosphorylation and downstream effects on gene regulation. To detect and measure phosphorylated ERβ in vivo I designed a targeted mass spectrometry method that has been successful for the detection of lowly expressed proteins. Taken together, the data presented in my dissertation demonstrate that alternative regulation of MAPKs signaling in the brain and heart could provide a novel mechanism explaining the variable effects of estrogen treatment following menopause

Differential Effects of E2 on MAPK Activity in the Brain and Heart of Aged Female Rats

<div><p>Aging and the coincident loss of circulating estrogens at menopause lead to increased risks for neurological and cardiovascular pathologies. Clinical studies show that estrogen therapy (ET) can be beneficial in mitigating these negative effects, in both the brain and heart, when it is initiated shortly after the perimenopausal transition. However, this same therapy is detrimental when initiated >10 years postmenopause. Importantly, the molecular mechanisms underlying this age-related switch in ET efficacy are unknown. Estrogen receptors (ERs) mediate the neuroprotective and cardioprotective functions of estrogens by modulating gene transcription or, non-genomically, by activating second messenger signaling pathways, such as mitogen activated protein kinases (MAPK). These kinases are critical regulators of cell signaling pathways and have widespread downstream effects. Our hypothesis is that age and estrogen deprivation following menopause alters the expression and activation of the MAPK family members p38 and ERK in the brain and heart. To test this hypothesis, we used a surgically induced model of menopause in 18 month old rats through bilateral ovariectomy (OVX) followed by an acute dose of 17β-estradiol (E2) administered at varying time points post-OVX (1 week, 4 weeks, 8 weeks, or 12 weeks). Age and E2 treatment differentially regulated kinase activity in both the brain and heart, and the effects were also brain region specific. MAPK signaling plays an integral role in aging, and the aberrant regulation of those signaling pathways might be involved in age-related disorders. Clinical studies show benefits of ET during early menopause but detrimental effects later, which might be reflective of changes in kinase expression and activation status.</p></div

Statistical analysis.

<p>F and p-values from two-factor ANOVA in hypothalamus, dorsal hippocampus, ventral hippocampus, and heart left ventricle. Light gray shading indicates analysis of ERK and white background indicates analysis of p38.</p

Effects of age and E2 treatment on p38 mRNA expression.

<p>P38 mRNA was measured using RT-qPCR. in the hypothalamus (A), dorsal hippocampus (B), ventral hippocampus (C), and left ventricle (D). Data are expressed as mean fold change ± SEM compared to vehicle-treated animals at one week post-OVX. An * indicates statistically significant difference from 1-week time point; # indicates significant difference within the same time point.</p

Effects of age and E2 treatment on ERK mRNA expression.

<p>ERK mRNA was measured using RT-qPCR. in the hypothalamus (A), dorsal hippocampus (B), ventral hippocampus (C), and left ventricle (D). Data are expressed as mean fold change ± SEM compared to vehicle-treated animals at one week post-OVX. An * indicates statistically significant difference from 1-week time point; # indicates significant difference within the same time point.</p

Effects of age and E2 treatment on ERK and p38 activation in the dorsal hippocampus.

<p>Calculated ratio of phospho:total-ERK or p38 (A, C), and percent change from vehicle following E2 treatment (B, D). Data are expressed as mean fold change ± SEM compared to vehicle-treated animals at one week post-OVX (A, C). An * indicates statistically significant difference from 1-week time point; # indicates significant difference within the same time point.</p

Diagram of the animal paradigm.

<p>Female Fisher 344 rats were ovariectomized at day 0 (= 18 months old) and subjected to increasingly longer periods of hormone deprivation (1, 4, 8, and 12 weeks). Following the assigned length of deprivation, animals were treated with either vehicle (safflower oil) or 17β-estradiol (E2; 2.5 μg/kg) by subcutaneous injection once daily for 3 consecutive days (n = 10/treatment group/deprivation time). Animals were euthanized 24 hours following the last treatment.</p

Effects of age and E2 treatment on ERK and p38 activation in the hypothalamus.

<p>Calculated ratio of phospho:total-ERK or p38(A, C), and percent change from vehicle following E2 treatment (B, D). Data are expressed as mean fold change ± SEM compared to vehicle-treated animals at one week post-OVX (A, C). An * indicates statistically significant difference from 1-week time point; # indicates significant difference within the same time point.</p

Effects of age and E2 treatment on ERK and p38 activation in the left ventricle.

<p>Calculated ratio of phospho:total-ERK or p38 (A, C), and percent change from vehicle following E2 treatment (B, D). Data are expressed as mean fold change ± SEM compared to vehicle-treated animals at one week post-OVX (A, C). An * indicates statistically significant difference from 1-week time point; # indicates significant difference within the same time point.</p