Selective inhibition of mesenchymal and hematopoietic RANKL

Receptor activator of NF-B ligand (RANKL) is not only an essential cytokine for osteoclast differentiation, activation, and survival, but may also be involved in the pathogenesis of acute myocardial infarction (MI) and heart failure. On the basis of the fundamental role of the RANKL/osteoprotegerin (OPG) system in bone resorption, human monoclonal antibodies against human RANKL (hRANKL) such as denosumab and AMG161, the IgG1 version of denosumab, have been developed. Furthermore, hRANKL knock-in (KI) mice were recently generated which carry the human instead of the murine exon 5 in their RANKL gene. This chimeric RANKL protein is capable of inducing bone resorption in mice, while being fully inhibited by denosumab or AMG161. Furthermore, still unpublished studies in my lab provided compelling evidence that only hematopoietic and endothelial, but not stromal precursors, engraft in host bone marrow after transplantation with unfractionated bone marrow. The current proposal is based on the idea that, together with an anti-hRANKL antibody such as AMG161, hRANKL-KI mice could be used as a powerful tool to separate between the pathophysiological role of RANKL derived from mesenchymal and hematopoietic/endothelial sources. To reach this goal, we propose to lethally irradiate wild-type and hRANKL-KI mice, and to transplant them with bone marrow from hRANKL-KI and wild-type donors, respectively. In irradiated wild-type mice reconstituted with bone marrow from homozygous hRANKL-KI donors, all hematopoietic and some endothelial cells produce chimeric, humanized RANKL, while all mesenchymal cells produce murine RANKL. Thus, because AMG161 blocks human but not murine RANKL, AMG161 will block only chimeric RANKL derived from hematopoietic/endothelial sources in this model. Vice versa, when homozygous hRANKL-KI mice are reconstituted with bone marrow from wild-type mice, only humanized RANKL derived from mesenchymal sources will be blocked. Together with hRANKL-KI mice as a control for the effects of total RANKL inhibition, such a system could provide unprecedented insight into the pathophysiology of diseases in which RANKL signaling plays a role. Although this model could be exploited in a very broad fashion, we plan to focus on osteoimmunology and myocardial infarction in the current proposal. The current proposal aims to answer the following questions: 1) What is the relative contribution of RANKL derived from mesenchymal or immune/endothelial cells in the pathophysiology of sex steroid deficiency-induced bone loss? 2) Does total RANKL inhibition or selective inhibition of hematopoietic/endothelial and mesenchymal cell-derived RANKL have a beneficial effect on the healing of myocardial lesions? If successful, this proposal will significantly advance our knowledge about the pathophysiological role of hematopoietic/endothelial and mesenchymal cell-derived RANKL in osteoporosis and myocardial infarction. Osteoporosis and myocardial infarction are leading causes of disability and death in modern societies with high percentages of elderly. Thus, the proposed work may have important implications for our health care systems.

In this project, we have discovered a novel role of bone lining cells in the regulation of bone turnover. Bone lining cells differentiate from bone-building osteoblasts und cover all quiescent bone surfaces. It is well established that sex hormones, estrogens and androgens, regulate bone turnover through the expression of receptor activator of NF-B ligand (RANKL). RANKL is an essential factor for the differentiation and activation of bone- resorbing osteoclasts. Augmented expression of RANKL leads to increased bone loss and osteoporosis. So far, it has been controversial which target cells in bone increase their expression of RANKL to induce sex hormone deficiency-induced bone loss. By a combination of different analysis methods in mouse models, we were able to show that enhanced expression of RANKL in the cell membrane of bone lining cells causes the estrogen deficiency-induced increase in bone turnover. To narrow down the cellular compartment responsible for increased RANKL expression in sex hormone deficiency-induced osteoporosis, we used a genetically engineered mouse model in combination with an anti-human RANKL antibody by which we were able to selectively block RANKL produced by hematopoietic or mesenchymal cells. There are two large cellular compartments in bone, the hematopoietic compartment comprising all red and white blood cells as well as osteoclasts, and the mesenchymal compartment comprising osteoblasts, osteocytes embedded within the bone matrix, cartilage-building chondrocytes, and bone lining cells. In our experiments in estrogen and androgen deficient mice, only inhibition of RANKL produced by mesenchymal cells influenced bone turnover, inhibition of RANKL produced by hematopoietic cells was without influence on bone. To further refine our findings, we established a novel technology in which specific cells can be excised from frozen bone sections with the help of a laser beam. It turned out that estrogen deficiency selectively upregulated RANKL expression in bone lining cells, not in osteoblasts or osteocytes. It was previously thought that osteocytes and hematopoietic cells orchestrate bone turnover. Thus, the current project adds to our understanding of the molecular and cellular mechanisms involved in the regulation of bone turnover, and improves our understanding of the mechanisms underlying bone diseases such as osteoporosis.