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

Abstract

Background Estrogen has been suggested to regulate anti-inflammatory signaling in brain microglia through estrogen receptor α (ERα), the only resident immune cells of the brain. The mechanism of how ERα regulates is not well understood. Previously, ERα is phosphorylated at Ser216 in mouse neutrophils, regulating their infiltration into the uterus. Therefore, ERα has now been examined as to its phosphorylation in microglia to regulate their inflammatory functions.MethodsAn antibody against an anti-phospho-S216 peptide of ERα (αP-S216) was used for double immunofluorescence staining to detect to ERα in cultured microglia. A knock-in (KI) mouse line bearing the phosphorylation-blocked ERα mutation S216A (ERα KI) was generated to examine whether this phosphorylation regulate immune functions of microglia.ResultsPhosphorylated ERα at Ser216 was present in microglia but not astrocytes. Staining with an anti-Iba-1 antibody showed that microglia activation was augmented in substantial nigra of ERα KI brains. Lipopolysaccharide (LPS) treatments aggravated microglia activation in ERα KI brains, pro-inflammatory cytokines were increased while anti-inflammatory cytokines were decreased at mRNA and protein levels in whole brain extracts. These increases and decreases of cytokine proteins were also observed in LPS-treated microglia cultured from brains of ERα KI neonates. FACS analysis revealed that ERα KI mutation increased number of IL-6 producing microglia and apoptosis. ERα KI mice decreased motor connection ability in Rotarod tests.ConclusionsBlocking of Ser216 phosphorylation aggravated microglia activation and inflammation of mouse brain, thus confirming that phosphorylated ERα exerts anti-inflammatory functions. ERα KI mice enable us to further investigate the mechanism by which phosphorylated ERα regulates brain immunity and inflammation.

Estrogen receptor 1 appears essential for fetal viability in a murine model of premature birth

“…27 Evidence in adult murine models of cytotoxic inflammation suggest that neuroprotection is mediated by Esr1 signaling. 28,29 Results from CD-1 embryonic brains support alterations of principle estrogen receptors following in utero neuroinflammatory insult. The observed changes in estrogen receptors corresponds with the upregulation of immune genes, in particular mediators of neuroinflammatory injury Il1b and Tnf.…”

“…Protective effects of estrogen signaling are described for neuroinflammatory diseases in adult females and these mechanisms may also apply during development 27 . Evidence in adult murine models of cytotoxic inflammation suggest that neuroprotection is mediated by Esr1 signaling 28,29 . Results from CD‐1 embryonic brains support alterations of principle estrogen receptors following in utero neuroinflammatory insult.…”

Phosphorylation of S122 in ERα is important for the skeletal response to estrogen treatment in male mice

“…In addition to our model investigating the role of a phosphorylation-site in ERα in vivo, two other mouse models have been described 40,41 . Manipulation of ERα signaling is known to affect the body weight of both males and females 38,42 , and in one of the phosphorylation-site models, where a change in serine 216 to alanine results in loss of phosphorylation, body weight is increased in both male and female S216A mice compared to control mice 40 . In the other model, where a Y541S mutation in ERα leads to constitutive activation of the receptor, both male and female mice are growth-retarded and have decreased body weights compared to their controls 41 .…”