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

Shindo, Sawako; Sakuma, Tsutomu; Negishi, Masahiko; Squires, James

doi:10.1016/j.steroids.2012.01.001

Cited by 18 publications

(10 citation statements)

References 12 publications

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“…In contrast, phosphorylation of ERα Ser 212 ( Fig. 1A) did not compromise ERα activity in gene reporter assays, and a gene array study suggested that a distinct set of genes are activated by the phosphomimic S212D ERα mutant, compared to S212A ERα 27 . A phospho-specific antibody developed to specifically detect this Ser phosphorylation of ERα found positive staining in peripheral blood neutrophils, and detected migration and infiltration of these cells to the mouse uterus 5,6 .…”

Section: Discussionmentioning

confidence: 86%

A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice

Sueyoshi

Sakuma

Shindo

et al. 2019

Laboratory Investigation

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Retinoid X receptor α (RXRα) has a conserved phosphorylation motif at threonine 162 (humans) and threonine 167 (mice) within the DNA binding domain. Here we have generated RXRα knockin mice (Rxrα T167A) bearing a single mutation of Thr167 to alanine and examined the roles of Thr167 in the regulation of energy metabolism within adipose, muscle and liver tissues. Rxrα T167A mice exhibited down-regulation of metabolic pathways converting glucose to fatty acids, such as acetyl-CoA carboxylase in white adipose tissue (WAT) and ATP citrate lyase in muscle. They also reduced gene expression for genes related to fatty acid catabolism and triglyceride synthesis in WAT and controlled heat factors such as adrenergic receptor β1 in muscles. In contrast, hepatic gluconeogenic pathways and synthetic pathways related to fatty acids remained unaffected by this mutation. Expression of multiple genes that were affected by the Thr 167 mutation in adipose tissue exhibited clear response to LG100268, a synthetic RXR agonist. Thus, the altered gene expression in mutant mice adipose appeared to be a direct effect of RXRα Thr 167 mutation and by some secondary effect of the mutation. Blood glucose levels remained normal in Rxrα T167A during feeding, as observed with RXRα WT mice. However, Rxrα T167A mice exhibited an attenuated decrease of blood glucose levels that occurred after fasting. This attenuation was correlated with a concomitant down-regulation of lipid metabolism in WAT and was associated with RXRα phosphorylation at Thr167. Thus, Thr167 enabled RXRα to coordinate these three organs for regulation of energy metabolism and maintaining glucose homeostasis.

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Section: Discussionmentioning

confidence: 86%

A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice

Sueyoshi

Sakuma

Shindo

et al. 2019

Laboratory Investigation

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“…If chemicals that specifically bind and activate or inactivate phosphorylated ERα are identified, they may be effective in microglia but not the other cells in which ERα is not phosphorylated. Our previous study showed that human ERα S212D mutant activated ERE-reporter gene as observed with ERα WT and ERα S212A in Huh-7 cells [ 9 ], suggesting that cell-based reporter assays can be utilized to develop a high-throughput assay for identifying ligands specific to phosphorylated ERα, with cells such as Huh-7 and/or mouse microglia-derived BV-2 cells. In addition to the brain, other tissues such as skin (Langerhans cells in) and liver (Kupffer cells) house resident macrophages.…”

Section: Discussionmentioning

confidence: 99%

“…Post-translational modifications are known to be important for protein activities. We previously showed that human ERα S212 mutants regulated the different group of genes form those regulated when they were overexpressed in Huh-7 cells [9]. Moreover, it was revealed that serine 216 of ERα was phosphorylated in vivo in neutrophils infiltrating the mouse uterus using a specific phosphorylated ERα recognition antibody [10].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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“…If chemicals that specifically bind and activate or inactivate phosphorylated ERα are identified, they may be effective in microglia but not the other cells in which ERα is not phosphorylated. Our previous study showed that human ERα S212D mutant activated ERE-reporter gene as observed with ERα WT and ERα S212A in Huh-7 cells [26], suggesting that cell-based reporter assays can be established for highthroughput assays to identify ligands specific to phosphorylated ERα, using cells such as Huh-7 and/or mouse microglia-derived BV-2 cells. In addition to the brain, other tissues such as skin (Langerhans cells in) and liver (Kupffer cells) house resident macrophages.…”

Section: Microglia Are Critically Involved In the Development Of Varimentioning

confidence: 88%

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

Shindo

Chen

Gotoh³

et al. 2019

Preprint

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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.

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

Cited by 18 publications

References 12 publications

A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice

A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice

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

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

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