| Background:Osteoporosis is an emerging medical and socioeconomic threat characterised by a systemic impairment of bone mass, strength, and microarchitecture, which increases the propensity of fragility fractures.Osteoporosis has a considerable public health impact in the world, as it affects over200million people, mainly postmenopausal women, and is associated with large expenditures for the health system. According to statistics from the International Osteoporosis Foundation (IOF)34%of postmenopausal women and20%of the male population are affected by this disease.Mortality from fractures produced by bone fragility is significant and may affect up to20%of individuals one year after a hip fracture.The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by an increase in marrow adipose tissue. Indeed an increase in marrow adipocytes is observed in all conditions that lead to bone loss, such as ovariectomy immobilization, or treatment with glucocorticoids. Today the adipose tissue is not considered to be a storage tissue and space filler but is characterized as an endocrine organ with an important role in the secretion adipokines and hormones, involved in the process of bone metabolism。Bone marrow adipocyte tissure has been considered a new target for diagnosis and therapy of osteoporosis.The osteoclast is a specialized macrophage polykaryon, and its differentiation is principally regulated by macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor B ligand (RANKL) and osteoprotegerin (OPG), the latter two cytokines are secreted predominantly by osteoblasts. The RANKL, upon binding to receptor activator of nuclear factor-KB (RANK) on the cell surface of osteoclasts or its precursors, functions to induce differentiation, activation and survival of osteoclasts, whereas OPG, a decoy receptor for RANKL, functions to inhibit osteoclastogenesis. RANKL/RANK/OPG axis plays an important role in bone metabolism.RIS is a nitrogen-containing bisphosphonate and effective inhibitor of bone resorption. It has been commonly used for anti-osteoporosis treatment due to its beneficial clinical outcomes and safety. The function of osteoclast and osteoblast medicated by anti-osteoporosis drugs has frequently been the main concentration of previous studies, but the effect of anti-osteoporosis drugs on bone marrow adipocytes which occupy the most bone marrow cavity has not been fully understood.Objective:The purpose of this study is to investigate the effects of bisphosphonates on bone marrow adipogenesis and the pro-osteoclastic factors produced by adipocytes in bone marrow micro-environment.Materials and Methods:Human mesenchymal stem cells (hMSCs) were obtained and purified from6volunteer donors. Each sample of cells was treated by increasing concentrations of risedronate (RIS) with or without adipogenic induction for14days, and then droplets of the differentiated adipocytes were analyzed. The level of receptor activator of nuclear factor-KB ligand (RANKL) and osteoprotegerin(OPG), as well as pro-osteoclastic inflammatory factors interleukin-1(IL-1), interleukin-6(IL-6) and tumor necrosis factor a(TNF-a) produced by adipocytes were evaluated by Western Blot and ELISA assay. Moreover, the effect of RIS on the activity of mammalian target of rapamycin complex1(mTORCl), a key Ser/Thr kinase for initiation of adipocyte differentiation, was investigated.Results:1. THE ADIPOGENIC AND OSTEOGENIC DIFFERENTIATION POTENTIAL OF PRIMARY HMSCsWe firstly identified the potential of adipogenic and osteogenic differentiation in primary hMSCs. It was found that a variety of droplets has been accumulated in the cellular plasma after2weeks AIM induction. The differentiation of adipocytes from hMSCs was further confirmed by Oil Red O staining and the expression of peroxisome proliferator-activated receptor γ(PPAR-γ), a key transcription factor for adipogenesis. Theosteoblastic differentiation of hMSCs was identified by Alizarin Red staining after3week of OIM induction. These data indicate that primary hMSCs used in our study has multi-potential to differentiate into both adipocytes and osteoblasts by AIM and OIM.2. RISEDRONATE DOSE-DEPENDENTLY INHIBIT ADIPOGENIC DIFFERENTIATION OF HMSCsThe cells in AIM were treated with different concentrations of risedronate for2weeks and stained with the Oil Red O solution. It was found that the number of adipocytes per well decreased significantly upon RIS treatment. The statistical analysis of adipocyte in per visual field revealed the amount of adipocyte decreased following the increasing concentration of RIS. Our finding demonstrated that RIS could inhibit hMSCs adipogenesis in a dose-dependent manner.3. THE RANKL EXPRESSION IS UP-REGULATED DURING ADIPOGENIC DIFFERENTIATION OF HMSCsTo investigate the RANKL and OPG expression during the hMSCs adipogenic differentiation, we assessed the RANKL and OPG expression after2weeks adipogenic induction by western blot assay. The expression of RANKL but not OPG was significantly increased after adipogenic induction for2weeks. The ratio of RANKL/OPG enhanced drastically in AIM induced cells compared with control. It is suggested that excess adipogenesis in bone marrow maypromote osteoclast differentiation through RANKL/OPG system.4. RISEDRONATE SUPPRESSES RANKL EXPRESSION IN ADIPOCYTES DIFFERENTIATED FROM HMSCsTo examine the effect of RIS on RANKL and OPG expression in adipogenic differentiated hMSCs, the adipogenic induced cells were treated at increasing concentrations of RIS for96h and the level of RANKL and OPG were detected by immunoblot analysis. It was revealed that expression of RANKL but not OPG was decreased by RIS treatment in adipocytes differentiated from hMSCs.These data suggested RIS suppressed the RANKL expression during hMSCs adipogenic differentiation in a dose-dependent manner.5. RISEDRONATE COULD NOT MEDIATE RANKL AND OPG EXPRESSION DERIVED FROM HMSCs WITHOUT ADIPOGENIC INDUCTIONTo determine the effect of RIS on RANKL and OPG expression derived from hMSCs without adipogenic induction. HMSCs treated by increasing concentration of RIS, were cultured with maintain medium for96h until western blot assay. It was demonstrated that RIS with concentrations in differ could not influence RANKL and OPG expression significantly6. RISEDRONATE INHIBITS PRO-OSTEOCLASTIC INFLAMMATORY FACTORS SECRETION IN ADIPOCYTE DIFFERENTIATED FROM HMSCsTo detect the effect of RIS on secretion of these inflammatory factors in bone marrow adipocytes, the adipogenic induced hMSCs were treated with10μM RIS for36h in DMEM and IL-1, IL-6,TNF-a in the supernatant were analyzed by commercial ELISA kits. The results revealed that RIS inhibited production of IL-1, TNF-α but not IL-6by with adipogenic induced hMSCs as compared to control.7. RISEDRONATE INHIBITS MTORC1SIGNALING IN ADIGENIC DIFFERENTIATED HMSCsWe further examined the effect of RIS on mTORC1signaling in adipogenic differentiated hMSCs. It was found that RIS were able to inhibit the phosphorylation of S6on S235/236, a direct downstream effector of mTORC1in adipogenic differentiated hMSCs. It is suggested that RIS inhibits mTORC1signaling during adipogenic differentiation of hMSCs.Conclusion:Our findings that RIS influences the crosstalk between bone marrow adipocyte-osteoclast represent a novel mechanism for the anti-osteoporotic effects of risedronate. |
PDF Full Text Request