P36 Hydrogen sulfide inhibits RANKL-induced ROS production and osteoclast differentiation

Abstract

Osteoclasts (OC), the bone resorbing cells, are multinucleated cells arising from the fusion of circulating monocytic precursors. Once differentiated, they settle into bone and break down bone mineral matrix, thereby playing a vital role in physiological bone turnover.In post-menopausal osteoporosis or in inflammatory bone disease, OC are activated leading to an excess bone erosion which in not balanced by an equal bone formation by osteoblasts, a process that leads to a loss of bone mass, increased bone fragility and fracture risk. The rate of monocyte differentiation into OC has been shown to be a critical step in pathological bone erosion.Here we sought to investigate whether hydrogen sulfide affect OC differentiation and function. Human osteoclast precursors were isolated from peripheral blood obtained from healthy donors by immunomagnetic positive selection of CD11b+ cells. Mature OC were obtained in vitro by culturing CD11b+ cells for 8–10 days in the presence of M-CSF (10 ng/ml) and RANKL (75 ng/ml). The effect of hydrogen sulfide was evaluated by treating the cells with NaHS in the range of concentrations between 50 and 300 μg/ml.We first assessed whether NaHS affect cell survival and proliferation. LDH activity assay revealed no cytotoxic effect of NaHS at 50, 10, 200 or 300 μg/ml at 24 h after stimulation, while 72 h after stimulation the concentration of 300 μg/ml induced a 25% increase in cell death. Cell proliferation was evaluated by 3 H-timidine uptake: consistent with citotoxicity data, NaHS did not affect the proliferation of CD11b+ cells in response to M-CSF stimulation up to a concentration of 200 μg/ml. Evaluation of apoptosis by Annexin V/PI further confirmed that NaHS did not induced cell death at 50, 100 or 200 μg/ml while induced a 30% increase of apoptosis at the concentration of 300 μg/ml. To assess the effect of NaHS on OC differentiation, TRAP + mature OC were counted at the end of the culture. NaHS dose-dependently inhibited OC formation, leading to a significant decrease in OC number starting a 100 μg/ml. Functional Pit Assay confirmed that NaHS significantly inhibits the ability of OC to break down a bone-like mineral matrix. Attesting to specificity, similar data were obtained in the presence of the H 2 S-donor GYY4137. Therefore, hydrogen sulfide inhibits OC differentiation and function in a range of concentration which does not affect cell survival. As RANKL signaling is critically dependent on ROS generation in OC progenitor cells, we evaluated the effect of NaHS on RANKL-dependent ROS generation by Dichlorofluorescein staining. As expected, RANKL (100 μg/ml, 30 min) induced a significant production of intracellular ROS in OC precursors cells; however, NaHS (200 μg/ml), significantly inhibited RANKL-induced ROS generation.In conclusion, Hydrogen sulfide dose-dependently prevents OC differentiation and function and this effect is associated to a significant inhibition of RANKL-induced ROS production. These findings suggest a possible therapeutical role for hydrogen sulfide donors in the treatment of pathological bone loss.