Abstracts of 2nd Scientific Meeting on Bone Disease in Multiple Myeloma
Bone Resorption is Regulated Locally in Bone by the Relative Expression of RANK Ligand and Osteoprotegerin
Colin R Dunstan, Amgen Inc. Thousand Oaks, California
In 1981, Rodan and Martin proposed that osteoblasts mediate the regulation of bone resorption by calciotropic hormones and other pro-resorptive factors. However, determination of the nature of this mediating signal proved to be elusive. The key that lead to identification of this factor was unexpected. In 1997, two groups[1,2] independently identified a novel protein, which was named osteoprotegerin (OPG) that inhibited the differentiation of osteoclasts. OPG was found to be a large (120kD) dimeric protein with an N-terminal region with homology to the ligand binding domains of the tumor necrosis factor receptor (TNFR) family. However the protein had no transmembrane domain or apparent signal transduction sequences. Instead OPG was a secreted protein with a large C-terminal domain which served to covalently dimerize the molecule and provide heparin binding capability. It was found that only the ligand-binding domain was required for inhibition of osteoclast differentiation.
These groups again working independently, were able to use OPG as a probe to identify a TNF family member. This molecule was found to be identical to a previously described molecule known as RANKL[3,4]. RANKL had been previously identified as a molecule with potential activity in the immune system regulating interactions of T-cells and dendritic cells. RANKL can, with mCSF, replace the requirement for stromal cells for osteoclast generation in vitro. In addition RANKL, when injected into mice, produced profound hypercalcemia and osteoclast mediated bone destruction. Thus RANKL appears to be a potent osteoclast differentiation, activation and survival factor, which when administered systemically is potent stimulator of bone resorption. RANK has been definitively demonstrated to be the relevant signaling receptor for RANKL effects on osteoclasts.
OPG and RANKL have been found to be expressed by marrow stroma derived cells, osteoblast-like cell lines and by primary cultures of osteoblasts derived from mouse calvaria. Calciotropic hormones and cytokines interact with their respective receptors in osteoblasts, or their precursors within the bone marrow, to alter the expression of RANKL and OPG such that there is an increase in the level of RANKL relative to OPG. RANKL is then available to interact with its receptor RANK on osteoclast precursors to promote differentiation, and on mature osteoclasts to activate bone resorption. Factors, such as estrogen, increase expression of OPG relative to RANKL and downregulate bone resorption.
Targeted disruption of the OPG gene in mice produces animals which develop a severe osteoporotic phenotype[5]. These animals are characterized by skeletal deformities, pathological fractures, high bone turnover and increased cortical porosity and thus share many features with human osteoporosis. Another interesting characteristic of the homozygous gene knockout phenotype was a high incidence of aortic calcification. Mice lacking a RANKL[6] or RANK gene have profoundly severe osteopetrosis with characteristic dense bones, failed tooth eruption and club shaped long bones. There is a complete absence of osteoclasts. Mice deficient in RANKL also have no lymph nodes indicating a role for RANKL in lymph-node organogenesis.
OPG is able to oppose the hypercalcemic effects of administered PTH, PTHrp, I,25(OH)2D3, IL-1b, and TNFa, and to oppose the normal homeostatic mechanisms for bone calcium mobilization in animal models. In each case this was associated with inhibition of the increase in osteoclast number produced by these treatments. OPG has also been shown to be effective in reversing the hypercalcemia in a rodent model of humoral hypercalcemia of malignancy, and to block the lytic effects of solid tumor cells growing in bone. OPG is also able to inhibit bone resorption following ovariectomy indicating that the loss of estrogen does not affect bone resorption by a mechanism independent of OPG ligand.
Osteoclast precursors in the human peripheral circulation are responsive to RANKL and can be induced to form functional osteoclasts. In addition these factors are expressed in human tissues and detectable levels of OPG are present in the circulation of adult humans. The human congenital condition Familial Expansile Osteolysis has been found to result from an activating mutation in the RANK gene. Subcutaneous injection of a single dose of OPG in healthy post-menopausal women, dose dependently suppressed the surrogate marker of bone resorption, urinary N-telopeptide of collagen (N-Tx). Suppression of N-Tx was rapid with significant effects seen 12 hours after dosing, with maximal suppression of approximately 80% occurring three to four days after treatment[7], indicating a central role for RANKL signaling in human bone metabolism.
It is likely that in human bone disease, dysregulation of calciotropic hormones, inflammatory cytokines or other factors also increase the expression of RANKL relative to that of OPG producing increased bone resorption. Some forms of human osteopetrosis may be found to be due to a defect in RANKL signaling and this is an attractive avenue of research. Pagetic bone with its woven matrix, fibrotic changes and high bone turnover is superficially similar to that seen both in mice with OPG deficiency, or in mice administered recombinant RANKL. This raises the intriguing possibility that Pagets disease results from some local constitutive activation of the RANKL signaling pathway.
While OPG and RANK are widely expressed, the expression of RANKL is restricted to areas of bone resorption and to organs of the immune system. Indeed RANKL has been described as a protein produced by T cell cells. T cell expressed RANKL could be involved in osteoclast recuitment[8], but may also be involved in the interaction of T cells and dendritic cells. RANKL deficient mice do lack lymph nodes, indicating an essential role for RANKL in the embryonic development of the immune system. OPG has been reported to also bind and inactivate TRAIL (with much lower affinity than with RANKL), another TNF family member with potential cytotoxic activity. However in adult animals, the role of RANKL and OPG in the immune system remains unclear. In OPG transgenic animals with high circulating levels of OPG throughout life, organs of the immune system were normal and mice had a normal life expectancy, implying a functional immune system.
The discoveries of OPG, RANKL and RANK have demonstrated the existence of a central pathway in the regulation of osteoclast differentiation and activation. These discoveries are likely to lead to targeted anti-resorptive therapies that are more rationally directed at the dysregulation of bone resorption that occurs in disease.
References
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