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.

Osteoblastic differentiation and mRNA analysis of STRO-1-positive human bone marrow stromal cells using primary in vitro culture and poly (A) PCR.

Investigation of osteoblast dysfunction in osteoporosis has been hampered by a poor understanding of normal early osteoblast differentiation, due to a relative lack of markers for the earliest cells in the lineage. Attempts to identify such markers have used cultures of animal or immortalized human cells, of uncertain relevance to human biology, or heterogeneous cultures in which genetic variability precludes the isolation of stage-specific genotypic markers. Primary in vitro generation of clonal populations of human bone marrow stromal cells was used in order to overcome these problems. Fibroblast-like stromal cells were isolated from human sternal bone marrow. They showed differentiation to an osteoblastic phenotype when stimulated with dexamethasone (10(-7) M) and fluorescence activated cell analysis demonstrated immunopositivity for STRO-1 (an antibody that recognizes osteoprogenitor stem cells of the colony-forming unit-fibroblastic) in from 8 to 40 per cent of the cells, dependent on time post-harvest. Cells positive for STRO-1 were immunoselected using magnetic activated cell sorting and seeded at low density (10 cells/cm2) to produce clones. Each clone was subpassaged, osteoblastic differentiation stimulated with dexamethasone, and mRMA-extracted at time points post-stimulation (0 h and 1-14 days). A novel poly (A) reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify cDNA representative of all transcripts expressed at each time point. Differential gene expression within the amplified cDNA was assessed using 3' end cDNA probes to osteocalcin, osteopontin, and collagen type I (positive), demonstrating the acquisition of an osteoblastic phenotype. Time-specific gene products for early osteoblast differentiation have been generated from primary human cultures, utilizing very low density seeding and poly (A) RT-PCR. These products overcome the problems associated with animal, immortalized or heterogeneous culture and can be used to study normal and altered early osteoblast differentiation, indicating the possibility of using the same system to study other disease states.