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The TNF-family molecule, Receptor Activator of Nuclear factor κ B Ligand (RANKL) is known as a key regulator for bone remodeling, and is essential for the development and activation of osteoclasts. In this study, we examined the regulation of RANKL in primary human bone marrow adipocytes and the relationship between bone marrow adipocytes and bone metabolism. RANKL expression and the RANKL/osteoprotegerin (OPG) mRNA ratio in marrow adipocytes increased following dexamethasone treatment. In co-cultures of human osteoclast precursors and bone marrow adipocytes with dexamethasone, osteoclast precursors differentiated to TRAP-positive multinuclear cells. Moreover, the ability of bone resorption was confirmed in co-culture in flasks coated with calcium phosphate film. Osteoclast precursor differentiation and bone resorption were blocked by RANKL antibody pretreatment. TRAP-positive multinuclear cells did not form in coculture without cell-to-cell contact conditions. We conclude that primary human bone marrow adipocytes have the ability to promote osteoclast differentiation and activities, similar to osteoblasts and other RANKL-expressing cells.There are various hypotheses about the function of bone marrow adipocytes, which are present in large quantities in the choke marrow space. Some hold that bone marrow adipocytes function as 1) a spacer between hematopoietic cells and bone tissue, 2) a fat metabolism organ, 3) an energy storehouse, or 4) support cells of hematopoiesis and bone metabolism (14). In contrast, it is clear that subcutaneous and visceral fats are not only simple spacers, but also energy storehouses. Subcutaneous and visceral fats also serve as metabolic organs secreting physiologically active substances named adipokines, which are associated with hypertension and metabolic syndrome (23). The adipokines, such as leptin, adiponectin, and TNF-α, act on osteoclasts and osteoblasts that are associated with bone metabolism (1,8,10,17,22). However, their function has not yet been fully elucidated.It is known that with aging, the composition of the bone marrow shifts to favor the presence of adipocytes, and osteoclast activity increases while osteoblast function declines, resulting in osteoporosis (14). Several reports have described the relationship between bone marrow adipocytes and bone metabolism. The BMS2 adipocyte cell line from murine bone marrow stromal cells promotes the differentiation of osteoclast-like cells in the presence of vitamin D (7). C3 protein, which is secreted during the initial stage of adipogenesis in murine bone marrow stromal cells, enhances osteoclast differentiation (15). Receptor Activator of Nuclear factor κ B Ligand (RANKL) is an essential molecule in osteoclast differentiation, as is the decoy receptor molecule os-

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BackgroundOsteonecrosis of the femoral head is a common complication of high-dose glucocorticoid treatment. Intravascular thrombosis is thought to be associated with the ischemic state of the femoral head. Plasminogen activator inhibitor-1 (PAI-1) is an adipokine, which are physiologically active substances secreted from visceral and subcutaneous adipocytes. PAI-1 suppresses fibrinolysis by binding tissue-type plasminogen activator. Several reports have described the relationship between PAI-1 and steroid-induced osteonecrosis of the femoral head, and the preventive effects of lipid-lowering agents (statins) against steroid-induced osteonecrosis of the femoral head. We previously reported that adipokines and dexamethasone induced PAI-1 secretion from bone marrow adipocytes. The purpose of the present study is to examine the effects of simvastatin on PAI-1 secretion from human bone marrow adipocytes in vitro.MethodsPrimary bone marrow adipocytes were extracted from collagenase-treated bone marrow fluid obtained from the femoral necks of 40 patients (6 men, 34 women; age range, 52-81 years) undergoing hip joint replacement surgery. After suspended culture with or without dexamethasone or simvastatin, PAI-1 mRNA expression was assessed by real-time RT-PCR. Total PAI-1 protein secretion in culture medium was assessed by enzyme-linked immunosorbent assay.ResultsPAI-1 mRNA expression was up-regulated by 388% (P = 0.002) with dexamethasone, and down-regulated by 45% (P = 0.002) with simvastatin, as compared to control levels. Dexamethasone increased total PAI-1 secretion by 166% (P = 0.001) and simvastatin decreased total PAI-1 secretion by 64% (P = 0.002). No significant changes were observed in adiponectin mRNA expression and secretion by dexamethasone and simvastatin, while pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion by 89%, as compared to control levels.ConclusionThe present study confirmed the suppressive effects of simvastatin on PAI-1 expression and secretion from bone marrow adipocytes. Furthermore, pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion. Simvastatin may thus exhibit preventive effects against steroid-induced osteonecrosis of the femoral head by suppressing PAI-1 secretion.

Several studies have demonstrated the association of plasminogen activator inhibitor-1 (PAI-1) with osteonecrosis, but the underlying mechanism of osteonecrosis and its relationship with local PAI-1 is not clear. The objective of this study was to evaluate PAI-1 production by primary human bone marrow adipocytes and the effects of glucocorticoid administration. Bone marrow was obtained from 25 individuals during prosthetic insertion. Mature adipocytes were cultured for 24 h with or without dexamethasone. PAI-1, adiponectin, tumor necrosing factor-α (TNFα) expression were measured by latex photometric immunoassay or RT-PCR. Adiponectin, TNFα and PAI-1 were detected in all culture media. PAI-1 expression was significantly increased by treatment with 10 −6 mol/L dexamethasone up to 24 h in protein and mRNA levels, while the levels of other adipokines did not change by dexamethasone. These results suggest that bone marrow adipocytes may play important roles for the development of glucocorticoid-induced osteonecrotic diseases by enhancing PAI-1 expression.