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Phosphorylation of serine 212 confers novel activity to human estrogen receptor ?.

ABSTRACT: Estrogen receptor ? (ER?) can be phosphorylated at various residues, one of which is serine 212 in the DNA binding domain. The majority of human nuclear receptors conserves, as a motif, this serine residue within their DNA binding domain. Among these nuclear receptors, phosphorylation of the corresponding threonine 38 in the nuclear receptor CAR is essential for determining its activity [9]. Here, we have investigated the role of phosphorylated serine 212 in the regulation of ER? activity by comparing it with serine 236, another potential phosphorylation site within the DNA binding domain, and demonstrated that phosphorylation of serine 212 confers upon ER? a distinct activity regulating gene expression in Huh-7 cells. In Western blot analysis, wild type ER? and mutants ER? S212A, ER? S212D, ER? S236A and ER? S236D were equally expressed in the nucleus, thus indicating that phosphorylation does not determine nuclear localization of ER?. ER? S212D, but not ER? S236D, retained its capability of activating an ERE-reporter gene in luciferase assays. Similar results were also obtained for human ER?; the ER? S176D mutant retained its trans-activation activity, but the ER? S200D mutant did not. cDNA microarray and Ingenuity Pathway Analysis, employed on Huh-7 cells ectopically expressing either ER? S212A or ER? S212D, revealed that phosphorylation of serine 212 enabled ER? to regulate a unique set of genes and cellular functions.

SUBMITTER: Shindo S

PROVIDER: S-EPMC4033595 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Phosphorylation of serine 212 confers novel activity to human estrogen receptor α.

Shindo Sawako S Sakuma Tsutomu T Negishi Masahiko M Squires James J

Steroids 20120115 5

Estrogen receptor α (ERα) can be phosphorylated at various residues, one of which is serine 212 in the DNA binding domain. The majority of human nuclear receptors conserves, as a motif, this serine residue within their DNA binding domain. Among these nuclear receptors, phosphorylation of the corresponding threonine 38 in the nuclear receptor CAR is essential for determining its activity [9]. Here, we have investigated the role of phosphorylated serine 212 in the regulation of ERα activity by com ...[more]

PMID: 22266331

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Project description:BackgroundEstrogen 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.MethodsMixed 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.ResultsDouble 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.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 and brain diseases. Video abstract.

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Phosphorylation of serine 212 confers novel activity to human estrogen receptor ?.

Publications

Phosphorylation of serine 212 confers novel activity to human estrogen receptor α.

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