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

Background: Estrogen receptor alpha (ER alpha) has been suggested to regulate anti-inflammatory signaling in brain microglia, the only resident immune cells in the brain. ER alpha 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 ER alpha to regulate migration. Given the implication of this phosphorylation in immune regulation, ER alpha 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 ER alpha in ER alpha KI brains microglia inflamed, confirming that phosphorylation confers ER alpha with anti-inflammatory capability. ER alpha 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 ER alpha (alpha P-S216) was used to detect phosphorylated ER alpha in double immunofluorescence staining with ER alpha antibodies and a microglia maker Iba-1 antibody. A knock-in (KI) mouse line bearing the phosphorylation-blocked ER alpha S216A mutation (ER alpha KI) was generated to examine inflammation-regulating functions of phosphorylated ER alpha 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 ER alpha 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 ER alpha KI brains, indicating the spontaneous activation of microglia as observed with those of lipopolysaccharide (LPS)-treated ER alpha WT brains. Pro-inflammatory cytokines were up-regulated in the brain of ER alpha 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 ER alpha KI brains. Times of ER alpha KI mice on rod were shortened in Rotarod tests. Conclusions: Blocking of Ser216 phosphorylation aggravated microglia activation and inflammation of mouse brain, thus confirming that phosphorylated ER alpha exerts anti-inflammatory functions. ER alpha KI mice enable us to further investigate the mechanism by which phosphorylated ER alpha regulates brain immunity and inflammation and brain diseases.