Incubation of osteoclasts with 100M ALN in the presence of 100 ng mL RANKL for 48 hours did not prevent the accumulation of unprenylated Rap1A. RANKL increases the level of Mcl 1 in rabbit osteoclasts Western blot analysis of purified rabbit worldwide distributors osteoclasts showed that treatment with 100 ng mL RANKL, 100M ALN, or ALN RANKL had no effect on the level of Bcl 2 protein. However, RANKL alone consistently caused a threefold increase in the level of Mcl 1 protein in osteoclasts. Treatment with ALN caused a decrease Inhibitors,Modulators,Libraries of approximately 90% in Mcl 1, although co treatment with RANKL almost completely pre vented this effect and maintained the level of Mcl 1 similar to that in control cells.

Loss of Mcl 1 precedes apoptosis during cytokine deprivation of mouse osteoclasts but is prevented Inhibitors,Modulators,Libraries by pro survival factors To further examine the importance of Mcl 1 in osteoclast sur vival, multinucleated osteoclasts were generated from M CSF dependent mouse bone marrow macrophages by culturing the latter Inhibitors,Modulators,Libraries cells for 5 days with M CSF RANKL. When the osteo clasts were starved of these cytokines, morphological changes indicative of apoptosis were apparent after 6 to 8 hours, consistent with the appearance in Western blots of the cleaved form of caspase 3 after 6 hours of cytokine starvation. The appearance of apoptotic osteoclasts and cleaved caspase 3 was preceded by a decrease in the level of Mcl 1. Mcl 1 was almost completely absent after Inhibitors,Modulators,Libraries 12 hours of cytokine starvation, although the levels of Bcl 1 and Bcl xL did not change during this time.

The loss of Mcl 1 that occurred in mouse osteoclasts following cytokine starvation Inhibitors,Modulators,Libraries could be prevented by the addition of M CSF, RANKL, TNF, or LPS. Discussion BPs have become the mainstay of treatment for post meno pausal osteoporosis, Paget disease, and tumour associated osteolysis and have been shown to prevent generalised bone loss in patients with RA treated with corticosteroids. However, apart from a recent clinical study using the highly potent BP zoledronic acid in patients with RA and two studies of zoledronic acid in animal models of RA, the effective ness of BPs at preventing focal bone loss has been less con vincing. It has recently been suggested that the reason for this relative lack of effect on local, inflammatory bone loss is due to factors in the inflamed joint, such as TNF, that antagonise the ability of BPs to inhibit osteoclasts.

Zhang and colleagues, using TNF transgenic mice, showed that Bcl xL levels were markedly higher in osteoclasts, an effect that appeared to be caused by TNF induced expression of Ets 2. Furthermore, overexpression of Ets 2 or Bcl xL protected osteoclasts those from ALN induced apoptosis in vitro. RANKL is also abundant in the rheumatoid microenviron ment and drives the enhanced osteoclastogenesis and hence excessive osteoclast mediated destruction of bone.