The studies of novel regulators of bone cell function

Abstract

The current study investigates novel factors in bone cell biology by using both in vitro and in vivo models. The results show that murine receptor activator of nuclear factor-KB ligand (muRANKL) and rat RANKL (rRANKL) stimulated, while human osteoprotegerin (hOPG) inhibited, 45Ca release and [3H]-thymidine incorporation in rat calvarial organ cultures, osteoclastogenesis from mouse bone marrow cultures and bone resorption in rat isolated mature osteoclast assays. Interestingly, truncated rRANKL(246-318) became an antagonist for non-truncated rRANKL in isolated osteoclast assays. Fibroblast growth factor (FGF)-8a at 25 ng/mL or greater potently stimulated osteoblast proliferation and mildly inhibited osteoblast differentiation. An autocrine mechanism may be involved in the regulation of osteoblasts based on detection of the expression of FGF-8 in these cells. Osteoclastogenesis was significantly reduced by FGF-8a at 0.05— 25 ng/mL, while the ratio of RANKL/OPG was elevated by this agent. α-melanocyte stimulating hormone (α-MSH) markedly stimulated both osteoblast proliferation (at 10-9 M or greater) and osteoclastogenesis (at 10-8 M or greater) in vitro. The stimulatory effect on osteoclasts was associated with the increased ratio of RANKL/OPG. Systemic administration of α-MSH resulted in a decrease in trabecular volume in tibiae. α-MSH also stimulated canine chondrocyte proliferation, but had no direct interaction with mature osteoclasts. Milk basic fraction (MBF)-1, MBF-2, milk acidic fraction (MAF)-1 and MAF-2 were found to be mitogenic to osteoblasts in vitro. While MAF-1 inhibited and MAF-2 stimulated bone resorption in calvarial cultures, the peptic hydrolysates of the both fractions showed inhibitory effects in this model. Among the individual components identified from the milk fractions, lactoferrin was found to be an interesting novel anabolic factor, potently stimulating osteoblast proliferation at 1-10 µg/mL or greater in both isolated osteoblast and calvarial organ cultures, as well as potently inhibiting osteoclast formation from bone marrow cells via a RANKL/OPG-independent mechanism. In summary, this study has revealed the possibility of the development of a RANKL antagonist through truncation of the molecule. In addition, the osteoblastic and osteoclastic effects of FGF-8a, α-MSH and lactoferrin have been demonstrated for the first time. However, further study is still needed to better understand the mechanisms of their functioning and to evaluate their potential for clinical application.