Evidence suggests that the newly described estrogen receptor ␤ (ER-␤) may be important for estrogen (17␤-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-␤, 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-␤ detection. In sections (5 m) of benign prostate hyperplasia, used as positive control, clear nuclear immunoreactivity was seen in glandular epithelial cells, with a 1:500 dilution of ER-␤40. 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-␤ 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-␤, 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-␤ expression was blocked totally by preincubation of antibody with antigen. We conclude that ER-␤ 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. (J Bone Miner Res 2001;16:214 -220)

Oestrogen is recognized as important for maintaining bone mass in men and women. Oestrogen receptor (ER) alpha and the recently described ER-beta are both expressed in bone cells, but have different affinities for oestrogen agonists and plant oestrogens, which could be important in developing treatments for bone loss in both men and women. It is unclear, however, which isoform predominates in bone; cell type and age may influence their relative expression. The present study has compared ER-alpha and ER-beta expression in serial sections of human fracture callus from males (n = 19, age range 5-72 years) and females (n = 15, age range 3-86 years) by indirect immunoperoxidase. Fracture callus was used as it can be readily obtained from individuals over a wide age range and contains a variety of bone cells. Antibody specificity was confirmed by western blotting and comparison of immunoreactivity in sections of breast tumour and benign prostate hyperplasia. No gender difference in ER expression was found in bone from individuals less than 40 years old. Proliferative chondrocytes were positive for both isoforms, but few larger hypertrophic cells were immunoreactive. ER-alpha and ER-beta were co-expressed in osteoclasts, suggesting that oestrogen may act directly on these cells. Osteoblasts, osteocytes, and mesenchymal cells also expressed both isoforms. In women over 40 years of age, however, relatively fewer biopsies contained osteocytes positive for ER-alpha and ER-beta. Likewise, the proportions of osteoblasts and mesenchymal cells expressing ER-beta were reduced but ER-alpha remained unaffected. In contrast, in men over 40 years, only the proportion of biopsies containing ER-beta-positive mesenchymal cells was lower. In these older men and women, ER-alpha and ER-beta expression was retained by the small proliferative chondrocytes. These results demonstrate that gender, age, and cell type are important determinants of ER isoform expression in skeletal cells.

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