Introduction Since estrogen receptor beta (ESR2) was cloned and characterized in 1990, it has come to the forefront of cancer, cardiovascular, skeletal, autoimmune, hematopoietic, and central nervous system research. It is widely hypothesized that T lymphocytes mediate estrogen signaling and its beneficial effects on the skeleton. Polymorphisms within the ESR2 locus are significantly associated with bone mineral density in men and women and with various other end points of health and disease. We wondered if peripheral blood leukocytes express enough of this receptor to enable its measurement, ligand-binding and transactivation activity and hence provide a relatively non-invasive source of ESR2 protein to further our investigations of ESR2 polymorphisms. Although ESR2 has been detected in human thymus, ovary, testis, all bone cells and several cell lines derived from adenocarcinomas of breast and colon, its expression in circulating leukocytes has not been characterized. The goal of this study was to determine if peripheral blood lymphocytes and monocytes express detectable levels of ESR2 protein.
Methods Peripheral blood mononuclear cells (PBMNCs) were harvested from whole blood obtained from healthy male and female subjects age 20-50 years. PBMNCs were separated by centrifugation, washed free of platelets in PBS, and processed for immoblotting and cytometric analysis. For immunoblotting, cytoplasmic and nuclear fractions were prepared in the presence of a proteinase inhibitor cocktail. Twenty to 50 μg of protein was loaded on a 10% polyacrylamide gel, electrophoresed, transferred to a nitrocellulose membrane, and probed with either anti-human ESR2 monoclonal, monoclonal anti-human estrogen receptor alpha (ESR-1), or polyclonal anti-human ESR2 antisera. Development of the protein bands was performed using peroxidase-conjugated secondary antisera and chemoluminescent substrates. For cytometry, PBMNCs were incubated with ESR2 monoclonal 14C8, washed with PBS containing 1% Tween 20 and 1% BSA, incubated with FITC-conjugated anti-mouse secondary, washed and injected into a Facscalibur flow cytometer.
Results Full-length estrogen receptors alpha and the two principal isoforms of ESR2 were detected in nuclear but not cytoplasmic fractions of peripheral blood mononuclear cells in all subjects tested. A strong ESR2 signal was also detected in the population of permeabilized lymphocytes by cytometyric analysis.
Conclusions The results indicate that both ESR1 and ESR2 are expressed in peripheral blood mononuclear cells harvested from healthy volunteers. Thus, further investigation of protein expression and function can be undertaken using blood samples, avoiding biopsy of human tissue. Our future investigations will seek to quantify ESR2 expression in leukocyte and lymphocyte subsets.
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