The beneficial influence of estradiol in the maintenance of healthy bone is well recognized. However, the way in which the actions of this hormone are mediated is less clearly understood. We have demonstrated that the well characterised 66kDa ER was only one of the ER? isoforms present in bone. We describe that a 46kDa isoform of ER, expressed at a level similar to the 66kDa isoform, is also present in human primary osteoblasts. This shorter isoform is generated by alternative splicing of an ER gene product, that results in exon 1 being skipped with a start codon in exon 2 used to initiate translation of the protein. Consequently, the transactivation domain AF-1 of this ER isoform is absent. Functional analysis revealed that hER 46 is able to heterodimerize with the full-length ER and also with Er(beta). We have shown that hER 46 are a strong inhibitor of hER 66 when they are co-expressed in the human osteosarcoma cell line SaOs. As a functional consequence, proliferation of the transfected cells is inhibited when increasing amounts of hER 46 are cotransfected with hER 66. In addition to human bone, the expression of the alternatively spliced ER mRNA variant is also detectable in bone of ER knockout (ERKO) mice. These data suggest that, in osteoblasts, estradiol can act in part through an ER isoform that is markedly different to the 66kDa receptor. The expression of two ER protein isoforms may account in part for the differential action that estrogens and estrogen analogues have in different tissues. In particular, the current models of the action of estrogens should be re-evaluated to take account of the presence of at least two ER protein isoforms in bone and perhaps in other tissues.
The long-term objective of this proposal has been to provide an integrated understanding of the genetic mechanisms that underlay the pathophysiology of osteoporosis. In the framework of this consortium, we wanted also to transfer directly this new knowledge to diagnostic, therapeutic and preventive applications that might be beneficial for the ageing population in Europe and in the world. The GENOSPORA's outcome include the following results: - Specific anti-cathepsin K monoclonal antibodies that inhibit enzyme activity: Specific anti-cathepsin K monoclonals have been produced, which partially inhibit cathepsin K activity in vitro. Cathepsin K plays an important role in bone resorption and several pharmaceutical groups are actively seeking inhibitors of this enzyme as potential therapies for osteoporosis, a condition associated with loss of bone density as a result of an imbalance of bone resorption and bone formation. Inhibitory antibodies could provide an alternative approach to therapy. - Protocol for density screening in mice: We developed protocols for a standardised protocol for the screening of mice with abnormalities in bone metabolism. The protocol is divided in four parts: - Anatomical inspection; - Physiology (calcium and phosphate level, alkaline phosphatase); - X-ray analysis; - Bone densitometry. The adaptation of a micro-CT for the use in life mice is in process. Baseline experiments for the bone density of C3HeB/FeJ and C57BL6/J mice were carried out at different age steps. The protocols have been used in the current project to screen for mice with phenotypes in bone development and homeostasis. As the protocols are standardised operating procedures, they are applicable for any other research institution. The comparability of the results is ensured. - New mouse models with altered bone density: Four mutant mouse lines have been established with altered bon density, developmental bone abnormalities and/or abnormal bone metabolism. Further characterisation and evaluation for the use as model organisms of the mutants is needed for a potential licensing by the pharmaceutical industries. For the further characterisation involving other specialised research institutes might be of benefit. - Regulation of estrogen receptor expression during differentiation of osteoclasts: The steroid hormone estrogen is a key component in the maintenance of healthy bone. Estrogens act to decrease bone resorption, thereby contributing to the prevention of osteoporosis. Bone resorption is mediated by the action of osteoblasts and osteoclasts, however, the detailed cellular and molecular mechanisms underlying the effects of estrogens on this cell type are poorly defined. Our studies aimed to evaluate the effects of estrogens on differentiation of osteoclasts from monocytes and of ER mRNA variant expression during this differentiation process. We have shown that the expression levels of ER correlate with the expression of TGF and IL-6, known estrogen responsive target genes. We investigated the role of estrogens on osteoclast differentiation and function to obtain a better understanding of the process of bone turnover. - Serum cathepsin K level not suitable bone marker candidate as it has no discriminatory power: Using specific monoclonal antibodies raised against recombinant cathepsin K, both a competitive enzymeimmunoassay and an immunoblot assay showed very little difference in the concentration of cathepsin K in a panel of human serum samples taken from a broad age group. Hence, serum cathepsin K levels were non-discriminatory and not a suitable bone marker candidate. - Anti-mouse osteocalcin peptide monoclonal: We have developed monoclonal antibodies against osteocalcin. A high affinity monoclonal antibody against a mouse osteocalcin peptide is currently being evaluated for use in a competitive enzyme immunoassay for mouse osteocalcin. This antibody might find applications in diagnostic and research.