| First Author | Shindo S | Year | 2020 |
| Journal | Cell Commun Signal | Volume | 18 |
| Issue | 1 | Pages | 117 |
| PubMed ID | 32727504 | Mgi Jnum | J:300412 |
| Mgi Id | MGI:6502165 | Doi | 10.1186/s12964-020-00578-x |
| Citation | Shindo S, et al. (2020) Estrogen receptor alpha phosphorylated at Ser216 confers inflammatory function to mouse microglia. Cell Commun Signal 18(1):117 |
| abstractText | BACKGROUND: Estrogen receptor alpha (ERalpha) has been suggested to regulate anti-inflammatory signaling in brain microglia, the only resident immune cells in the brain. ERalpha 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 ERalpha to regulate migration. Given the implication of this phosphorylation in immune regulation, ERalpha 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 ERalpha in ERalpha KI brains microglia inflamed, confirming that phosphorylation confers ERalpha with anti-inflammatory capability. ERalpha 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 ERalpha (alphaP-S216) was used to detect phosphorylated ERalpha in double immunofluorescence staining with ERalpha antibodies and a microglia maker Iba-1 antibody. A knock-in (KI) mouse line bearing the phosphorylation-blocked ERalpha S216A mutation (ERalpha KI) was generated to examine inflammation-regulating functions of phosphorylated ERalpha 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 ERalpha 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 ERalpha KI brains, indicating the spontaneous activation of microglia as observed with those of lipopolysaccharide (LPS)-treated ERalpha WT brains. Pro-inflammatory cytokines were up-regulated in the brain of ERalpha 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 ERalpha KI brains. Times of ERalpha 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 ERalpha exerts anti-inflammatory functions. ERalpha KI mice enable us to further investigate the mechanism by which phosphorylated ERalpha regulates brain immunity and inflammation and brain diseases. Video abstract. |
