In vitro effects of arachidonic acid and docosahexaenoic acid on osteoclastogenesis and bone resorption in human CD14+ monocytes

Abstract

Bone remodelling is a continuous physiological process in the body mediated by bone resorbing osteoclasts and bone forming osteoblasts. Osteoclasts are multinucleated cells formed by the fusion of haematopoietic cells of monocytic lineage in the presence of receptor activator of nuclear factor-kB ligand (RANKL) and macrophage colonystimulating factor (M-CSF), which are produced by osteoblasts. Osteoclast over-activity results in increased resorption of bone in some pathological conditions such as osteoporosis. Clinical and animal studies have shown that dietary fatty acids play a role in bone regulation. However there are no reported studies on the effects of long chain polyunsaturated fatty acids (LCPUFAs) on a human osteoclast cell line. The aim of this study was to determine the effects of arachidonic acid (AA), an w-6 LCPUFA, and docosahexaenoic acid (DHA), an w-3 LCPUFA, on osteoclastogenesis and bone resorption in human CD14+ monocytes, in vitro. CD14+ monocytes, isolated from human peripheral blood, were seeded on either glass cover-slips or dentine discs in cell-culture plates in the presence of differentiation factors (RANKL and M-CSF). To test the effects of the LCPUFAs on differentiating osteoclasts, the cells were exposed to the LCPUFAs from day 3. For experiments on mature osteoclasts, the cells were exposed to the LCPUFAs from the onset of resorption (day 11-14). All experiments were terminated 7 days after the onset of resorption. Tartrate-resistant acid phosphatase (TRAP) is an enzyme highly expressed and secreted by mature osteoclasts. The activity of TRAP in culture media and the number of multinucleated TRAP-stained cells were determined. The degree of resorption by mature osteoclasts on dentine was also determined. Cell morphology and actin ring (required for the structural integrity of osteoclasts) formation were also analysed. The expression of prominent osteoclast receptors, vitronectin receptor (VNR) and calcitonin receptor (CTR) were analysed by immunofluorescence. The presence of resorptive enzymes (TRAP, MMP-9, cathepsin K) was determined by western blot and the regulation of genes involved in osteoclast formation and activity were assessed by PCR. Both LCPUFAs decreased osteoclast formation in differentiating osteoclasts resulting in fewer osteoclasts compared to the control. In differentiating osteoclasts, VNR and CTR expression was affected by AA while DHA only affected VNR expression. Both LCPUFAs decreased the expression of all proteins and genes tested in differentiating osteoclasts. AA and DHA were shown to decrease resorption without affecting osteoclast numbers in mature osteoclasts. The integrity of the actin rings formed was unaffected. This may imply that the LCPUFAs have no harmful effect on mature osteoclasts. Both LCPUFAs were shown to affect VNR and CTR expression in mature osteoclasts. AA decreased the expression of all genes and proteins tested in mature osteoclasts, while DHA had no effect on two proteins (MMP-9, TRAP) and two genes (TRAP, CA2). The results suggest that LCPUFAs can affect osteoclastogenesis and resorption through modulation of osteoclast specific genes. This novel study also demonstrates that the LCPUFAs tested can modulate osteoclast formation and function and may potentially strengthen bones and prevent or delay the onset of osteoporosis.