The number of apoptotic rabbit osteo clasts was significantly lower in cultures treated for 48 hours with ALN or CLO in the presence of RANKL selleck chemical Vandetanib than in cultures treated with the BPs alone. Consistent with this, RANKL pre served the total number of osteoclasts Inhibitors,Modulators,Libraries in cultures treated with the BPs and also significantly overcame the anti resorptive effect of the BPs when osteoclasts were cultured on dentine discs. This ability of RANKL to rescue osteoclasts from the effects of BPs was also observed morphologically by using scanning EM. Treatment of osteoclasts with CLO for 24 hours caused morphological changes associated with apoptotic cell death, consistent with the ability of CLO to induce osteoclast apoptosis via the formation and rapid accumulation of a cyto toxic metabolite.

The morphological changes asso ciated with ALN treatment for 24 hours are consistent with the inhibition of FPP synthase. This leads to the inhibition of protein prenylation and the slow accumulation of unpre nylated small GTPases such as Rap1A. The morpho logical appearance of osteoclasts after 24 hours of treatment with ALN therefore Inhibitors,Modulators,Libraries probably indicates cells with altered cytoskeletal arrangement and vesicular trafficking, as well as cells in the very early stages of apoptosis, as a result of abnor mal small GTPase signalling. In the presence of RANKL, these effects of ALN and CLO on osteoclast morphol ogy were largely overcome, at least over a 24 hour culture period. In the case of ALN, this was not due to any ability of RANKL to prevent inhibition of FPP synthase, since RANKL treatment did not prevent the accumulation of unprenylated Rap1A in osteoclasts.

We have previously shown that BP induced osteoclast apop tosis involves loss of mitochondrial membrane potential, lead ing to the cleavage and activation of procaspase 3. To identify a potential mechanism by which RANKL prevents Inhibitors,Modulators,Libraries BP induced apoptosis, we examined in more detail the events involved in ALN induced apoptosis. Co treatment with RANKL prevented the increase in the proportion of osteoclasts with active caspase 9 seen after ALN treatment, suggesting that RANKL either prevents the mitochondrial changes that lead to activation of procaspase 9 or prevents procaspase 9 activation subsequent to the release of cytochrome C.

Although expression of XIAP and cIAP1 2 can be stimulated via nuclear factor Inhibitors,Modulators,Libraries kappa B, which is a major signalling Inhibitors,Modulators,Libraries pathway acti vated by RANKL, we did not observe any effect of RANKL selleck compound on the level of XIAP, cIAP1, or cIAP2 in rabbit osteoclasts by Western blotting, consistent with earlier studies on murine osteoclasts. This suggests that RANKL probably prevents apoptosis by preventing mitochondrial changes prior to caspase activation, perhaps via members of the Bcl 2 family of proteins that regulate the mitochondrial pathway of apoptosis.