RANKL protects osteoclasts from the anti resorptive effects of bisphosphonates The effect of RANKL on the morphology of BP treated rabbit osteoclasts was also studied by scanning EM. After 24 www.selleckchem.com/products/Perifosine.html hours of culture on ivory discs, 71% of osteoclasts in control cultures were spread on the mineral surface, often located in or adja cent to extensive and deep resorption cavities. However, after treatment with 50M CLO for 24 hours, few, shallow resorption pits were present and 50% of the osteo clasts were rounded and lacked areas of spreading. Many of these rounded Inhibitors,Modulators,Libraries osteoclasts lacked membrane ruffles or micro villi but contained numerous blebs, indicative of cells undergo ing apoptosis. The morphology of other cell types, such as stromal cells, in the culture did not appear Inhibitors,Modulators,Libraries to be affected by CLO.

The appearance of apoptotic Inhibitors,Modulators,Libraries osteoclasts was prevented by the presence of RANKL since 30% of oste oclasts Inhibitors,Modulators,Libraries were rounded when cultured with CLO RANKL, few of these had membrane blebbing, and 70% of the osteoclasts appeared similar to those in control cultures, associated with numerous resorption pits. Treatment with 50M ALN for 24 hours caused the appear ance of osteoclasts that appeared retracted, with long cell processes, and were associated with only minor resorp tion pits. These osteoclasts often lacked microvilli but exhibited ridges on the basolateral membrane, and few apoptotic Inhibitors,Modulators,Libraries osteoclasts were observed with the obvious mem brane blebbing observed in CLO treated cultures. After cul ture with ALN RANKL, osteoclasts were mostly well spread with membrane ruffles and surface microvilli, and resorption cavities were more evident.

Some osteoclasts also retained the presence of membrane ridges on the basolateral surface, but few had the retracted morphology of ALN treated cells or the blebbed morphology of CLO treated cells. As expected, when the area of bone inhibitor Romidepsin resorption was quantified, 100M ALN or CLO significantly inhibited the resorptive activity of rabbit osteoclasts cultured on ivory discs in vitro. However, the inhibitory effect of ALN or CLO on bone resorp tion was significantly overcome by the presence of 100 ng mL RANKL. RANKL reduces caspase 9 activity in osteoclasts A fluorescent, cell permeable caspase 9 inhibitor was used to identify single cells with caspase 9 activity. In con trol cultures of purified rabbit osteoclasts, approximately 10% of the cells had detectable caspase 9 activity. After treatment with 100M ALN for 48 hours, the pro portion of caspase 9 positive osteoclasts increased signifi cantly. This was significantly reduced, almost to the proportion in control cultures, in the presence of 100 ng mL RANKL.