Estrogen receptor α phosphorylated at Ser216 confers inflammatory function to mouse microglia

Abstract

Background: Estrogen receptor α (ERα) has been suggested to regulate anti-inflammatory signaling in brain microglia, the only resident immune cells in the brain. ERα conserves the phosphorylation motif at Ser216 within the DNA binding domain. Previously, Ser216 was found to be phosphorylated in neutrophils infiltrating into the mouse uterus and to enable ERα to regulate migration. Given the implication of this phosphorylation in immune regulation, ERα was examined in mouse microglia to determine if Ser216 is phosphorylated and regulates microglia's inflammation. It was found that Ser216 was constitutively phosphorylated in microglia and demonstrated that in the absence of phosphorylated ERα in ERα KI brains microglia inflamed, confirming that phosphorylation confers ERα with anti-inflammatory capability. ERα KI mice were obese and weakened motor ability. Methods: Mixed glia cells were prepared from brains of 2-days-old neonates and cultured to mature and isolate microglia. An antibody against an anti-phospho-S216 peptide of ERα (αP-S216) was used to detect phosphorylated ERα in double immunofluorescence staining with ERα antibodies and a microglia maker Iba-1 antibody. A knock-in (KI) mouse line bearing the phosphorylation-blocked ERα S216A mutation (ERα KI) was generated to examine inflammation-regulating functions of phosphorylated ERα in microglia. RT-PCR, antibody array, ELISA and FACS assays were employed to measure expressions of pro-or anti-inflammatory cytokines at their mRNA and protein levels. Rotarod tests were performed to examine motor connection ability. Results: Double immune staining of mixed glia cells showed that ERα is phosphorylated at Ser216 in microglia, but not astrocytes. Immunohistochemistry with an anti-Iba-1 antibody showed that microglia cells were swollen and shortened branches in the substantial nigra (SN) of ERα KI brains, indicating the spontaneous activation of microglia as observed with those of lipopolysaccharide (LPS)-treated ERα WT brains. Pro-inflammatory cytokines were up-regulated in the brain of ERα KI brains as well as cultured microglia, whereas anti-inflammatory cytokines were down-regulated. FACS analysis showed that the number of IL-6 producing and apoptotic microglia increased in those prepared from ERα KI brains. Times of ERα KI mice on rod were shortened in Rotarod tests.

From Menopause to Neurodegeneration—Molecular Basis and Potential Therapy

“…Stimulation of mouse brain or microglia with lipopolysaccharide induces inflammatory cytokines, and these pro-inflammatory cytokines are upregulated in the brain and in microglia bearing a phosphorylation-blocked ERα S216A mutation. These data validate that estrogen conducts anti-inflammatory action via ERα in microglia [150]. Moreover, patients with higher ERα promoter methylation rates showed decreased ERα mRNA expression and impaired cognition [151], which implies the importance of ERα in AD.…”

Estrogen Receptor β as a Candidate Regulator of Sex Differences in the Maternal Immune Activation Model of ASD

“…Several reports have indicated that 17β-estradiol, a ligand for both ERα and ERβ, can regulate inflammation in myeloid-lineage cells. However, this regulation is not always clear in that some reports have suggested that ER-mediated transcription represses inflammation ( Vegeto et al, 2003 ; Ribas et al, 2011 ), while others have suggested that it does not ( Calippe et al, 2010 ; Shindo et al, 2020 ). While the amino acid sequences of the DNA-binding domains of these two ER isoforms are highly conserved, their ligand-binding domains (LBDs) are much less so (47% in human).…”

A Phosphotyrosine Switch in Estrogen Receptor β Is Required for Mouse Ovarian Function

“…Female mice carrying a mutant S122 ERα had a tissue-preferential impact on adipose tissue mass ( Ohlsson et al, 2020 ). In a separate report, blocking of ERα Ser216 phosphorylation aggravated microglia activation and brain inflammation ( Shindo et al, 2020 ). In a third study, dramatic developmental defects in the reproductive organs, mammary glands, and bones were observed in a Cre-inducible mouse model carrying an ERα Y541 mutation (ERα Y537S in humans; Simond et al, 2020 ).…”