Localization of estrogen receptor beta protein expression in adult human bone

Evidence suggests that the newly described estrogen receptor beta (ER-beta) may be important for estrogen (17beta-estradiol) action on the skeleton, but its cellular localization in adult human bone requires clarification. We addressed this by using indirect immunoperoxidase with a novel affinity purified polyclonal antibody to human ER-beta, raised to hinge domain (D) sequences from the human receptor. Bone was demineralized in 20% EDTA and all biopsy specimens were formalin-fixed and wax-embedded. Vigorous retrieval was essential for ER-beta detection. In sections (5 microm) of benign prostate hyperplasia, used as positive control, clear nuclear immunoreactivity was seen in glandular epithelial cells, with a 1:500 dilution of ER-beta40. For bone sections, optimal antibody dilutions were 1:100-1:250. We found that in normal bone (from graft operations), in fracture callus from both men and women (>25 years old), pagetic bone, osteophytes, and secondary hyperparathyroid bone, all from older patients, ER-beta was expressed clearly in osteoclast nuclei, with little cytoplasmic immunoreactivity. Nuclear immunoreactivity was still prominent in osteoclasts, with antibody diluted 1:500, although it faded in other cells. Osteoblasts, in areas of active bone formation or bone remodeling, also expressed ER-beta, as did some osteocytes. However, hypertrophic chondrocytes were negative, unlike mesenchymal cells, adjacent to the osteogenesis. Megakaryocytes and some capillary blood vessels cells were receptor positive. All ER-beta expression was blocked totally by preincubation of antibody with antigen. We conclude that ER-beta is expressed in cells of osteoblast lineage and in osteoclasts. The latter appear relatively abundant in this receptor and this might provide a means for direct action of estrogen on osteoclasts.