Estrogens regulate neuroinflammatory genes via estrogen receptors α and β in the frontal cortex of middle-aged female rats

Abstract

Background: Estrogens exert anti-inflammatory and neuroprotective effects in the brain mainly via estrogen receptors α (ERα) and β (ERβ). These receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors. This study was aimed at the elucidation of the effects of ERα and ERβ agonists on the expression of neuroinflammatory genes in the frontal cortex of aging female rats.

Methods: To identify estrogen-responsive immunity/inflammation genes, we treated middle-aged, ovariectomized rats with 17β-estradiol (E2), ERα agonist 16α-lactone-estradiol (16α-LE2) and ERβ agonist diarylpropionitrile (DPN), or vehicle by Alzet minipump delivery for 29 days. Then we compared the transcriptomes of the frontal cortex of estrogen-deprived versus ER agonist-treated animals using Affymetrix Rat230 2.0 expression arrays and TaqMan-based quantitative real-time PCR. Microarray and PCR data were evaluated by using Bioconductor packages and the RealTime StatMiner software, respectively.

Results: Microarray analysis revealed the transcriptional regulation of 21 immunity/inflammation genes by 16α-LE2. The subsequent comparative real-time PCR study analyzed the isotype specific effects of ER agonists on neuroinflammatory genes of primarily glial origin. E2 regulated the expression of sixteen genes, including down-regulation of complement C3 and C4b, Ccl2, Tgfb1, macrophage expressed gene Mpeg1, RT1-Aw2, Cx3cr1, Fcgr2b, Cd11b, Tlr4 and Tlr9, and up-regulation of defensin Np4 and RatNP-3b, IgG-2a, Il6 and ER gene Esr1. Similar to E2, both 16α-LE2 and DPN evoked up-regulation of defensins, IgG-2a and Il6, and down-regulation of C3 and its receptor Cd11b, Ccl2, RT1-Aw2 and Fcgr2b.

Conclusions: These findings provide evidence that E2, 16α-LE2 and DPN modulate the expression of neuroinflammatory genes in the frontal cortex of middle-aged female rats via both ERα and ERβ. We propose that ERβ is a promising target to suppress regulatory functions of glial cells in the E2-deprived female brain and in various neuroinflammatory diseases.

Figure 1

The effects of estrogen replacements on uterus weight (A) and body weight (B). E2 and ERα agonist 16α-lactone-estradiol (LE2) changed the uterus weight significantly (p values for E2 and LE2 were p = 0.0001 in both cases), while DPN had no effect. In a similar way, E2 and LE2 decreased body weight significantly (p values for E2 and LE2 were p = 0.0035 and p = 0.0001, respectively). Change in body weight represents the weight difference between the weight of the animal before and after the chronic treatment. Asterisks mark statistically significant changes compared to vehicle. Error bars correspond to standard deviations.

Figure 2

Microarray analysis revealed 87 ERα agonist-regulated genes which were categorized based on function. ERα agonist-regulated genes included twenty-one immunity/inflammation genes. The cluster contained S100 calcium-binding (S100a9, S100a8) and defense proteins (RatNP-3b, Np4, Defa, Camp), complement C3 and Serping1, Ig chains (Igha, IgG-2a, Igj), mast cell proteases (Mcpt8, Mcpt9) and MHC antigens (RT1-Aw2, RT1-N1).