Detection and Functional Analysis of Estrogen Receptor α Phosphorylated at Serine 216 in Mouse Neutrophils

Part of the Methods in Molecular Biology book series (MIMB, volume 1366)


Serine 216 constitutes a protein kinase C phosphorylation motif located within the DNA binding domain of estrogen receptor α (ERα). In this chapter we present experimental procedures confirming that mouse ERα is phosphorylated at serine 216 in peripheral blood neutrophils and in neutrophils that infiltrate the uterus, as well as the role of phosphoserine 216 in neutrophil migration. A phospho-peptide antibody (αP-S216) was utilized in Western blot, immunohistochemistry, and double immunofluorescence staining to detect this phosphorylation of an endogenous ERα. Both immunohistochemistry (with αP-S216 or neutrophil marker Ly6G antibody) and double immunofluorescence staining of mouse uterine sections prepared from C3H/HeNCrIBR females revealed that phosphorylated ERα was expressed in all infiltrating neutrophils during hormonal cycles but not in any other of the other uterine cells. Neutrophils infiltrate the uterus from the blood stream. White blood cells (WBC) were prepared from peripheral blood of C3H/HeNCrIBR females or males and double immunostained. Blood neutrophils also expressed phosphorylated ERα but in only about 20 % of cells in both sexes. Only the neutrophils expressing phosphorylated ERα spontaneously migrated in in vitro Transwell migration assays and infiltrated the uterus in mice.

Key words

Estrogenreceptorα (ERα) Phosphorylation Neutrophils Migration Infiltration Mouse uterus Immunohistochemistry Immunofluorescencestaining 



This work was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences: Z01ES1005-01. We would like to acknowledge Dr. Kenneth Korach’s lab members and also Drs. Gordon Flake and Hideki Nakano for various experimental protocols.


  1. 1.
    Murphy LC, Seekallu SV, Watson PH (2011) Clinical significance of estrogen receptor phosphorylation. Endocr Relat Cancer 18:R1–R14CrossRefPubMedGoogle Scholar
  2. 2.
    Atsriku C, Britton DJ, Held JM et al (2009) Systematic mapping of posttranslational modifications in human estrogen receptor-alpha with emphasis on novel phosphorylation sites. Mol Cell Proteomics 8:467–480CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Shindo S, Sakuma T, Negishi M, Squires J (2012) Phosphorylation of serine 212 confers novel activity to human estrogen receptor alpha. Steroids 77:448–453CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Shindo S, Moore R, Flake G, Negishi M (2013) Serine 216 phosphorylation of estrogen receptor alpha in neutrophils: migration and infiltration into the mouse uterus. PLoS One 8, e84462CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Tibbetts TA, Conneely OM, O'Malley BW (1999) Progesterone via its receptor antagonizes the pro-inflammatory activity of estrogen in the mouse uterus. Biol Reprod 60:1158–1165CrossRefPubMedGoogle Scholar
  6. 6.
    Daimon E, Wada Y (2005) Role of neutrophils in matrix metalloproteinase activity in the preimplantation mouse uterus. Biol Reprod 73:163–171CrossRefPubMedGoogle Scholar
  7. 7.
    Wood GA, Fata JE, Watson KL, Khokha R (2007) Circulating hormones and estrous stage predict cellular and stromal remodeling in murine uterus. Reproduction 133:1035–1044CrossRefPubMedGoogle Scholar
  8. 8.
    Cunningham M, Gilkeson G (2011) Estrogen receptors in immunity and autoimmunity. Clin Rev Allergy Immunol 40:66–73CrossRefPubMedGoogle Scholar
  9. 9.
    Baumgarten SC, Frasor J (2012) Minireview: Inflammation: an instigator of more aggressive estrogen receptor (ER) positive breast cancers. Mol Endocrinol 26:360–371CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Lang TJ (2004) Estrogen as an immunomodulator. Clin Immunol 113:224–230CrossRefPubMedGoogle Scholar
  11. 11.
    Mutoh S, Osabe M, Inoue K et al (2009) Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3). J Biol Chem 284:34785–34792CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Mutoh S, Sobhany M, Moore R et al (2013) Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by Inhibition of epidermal growth factor receptor signaling. Sci Signal 6(274):ra31CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Reproductive and Developmental Biology LaboratoryNational Institute of Environmental Health Sciences, National Institutes of HealthResearch Triangle ParkUSA

Personalised recommendations