and the effect on Ca 2؉ spikes. We conclude that paclitaxel exerts rapid effects on the cytosolic Ca 2؉ signal via the opening of the mitochondrial permeability transition pore. This work indicates that some of the more rapidly developing side effects of chemotherapy might be due to an action of antimitotic drugs on mitochondrial function and an interference with the Ca 2؉ signal cascade.Antimitotic drugs are used extensively for the treatment of cancer. For example, paclitaxel (Taxol) is used in the treatment of breast and ovarian cancers and for AIDS 1 -related Kaposi's sarcoma, and vinblastine is used in the treatment of Hodgkin's disease (1). The mechanism of action of antimitotic drugs, that leads to cancer cell death, is not clear. It is known that paclitaxel stabilizes microtubule dynamics thereby preventing the proper formation of the mitotic spindle apparatus and arresting cancer cells at the G 2 -M phase of the cell cycle (2, 3). While it is thought that this action of paclitaxel on the cell cycle machinery precedes an apoptotic response of cells (4, 5), some recent work has suggested that paclitaxel-induced apoptosis results from more direct effects of the drug on the mitochondria. In this context paclitaxel has been shown to bind to Bcl-2 (6) and this binding may regulate Bcl-2 effects on the mitochondrial permeability transition pore (PTP) (7,8). Furthermore, deletion of the loop region of Bcl-2 (which prevents Bcl-2 phosphorylation) blocks the apoptotic action of paclitaxel on cancer cells (9). Other proteins may also be involved in the paclitaxel effects on mitochondria, such as APAF-1 (10). In isolated mitochondria paclitaxel acts to release cytochrome c (11). This effect is blocked by cyclosporin A providing further evidence that paclitaxel directly targets mitochondria, independent of actions on microtubules.The effect of antimitotic drugs on microtubule dynamics would confer drug specificity on actively dividing cancer cells. However, the actions of these drugs on Bcl-2 and on the mitochondria might be expected to be non-selective and affect all cells. Indeed paclitaxel treatment is associated with serious side effects, including neuropathy (12) and low white blood cell counts (13). These side effects occur rapidly, appear to be due to drug action on terminally differentiated cells, and are slowly reversible. Thus it is unlikely that these side effects are mediated by either mitotic block or apoptosis. Given that the clinical use of antimitotic drugs is limited by these side effects, understanding the mechanisms by which these drugs act is an important step toward optimizing the therapeutic benefits.In our studies, on terminally differentiated epithelial cells, we now show rapid actions of paclitaxel on the cytosolic Ca 2ϩ signal that can be accounted for by effects of paclitaxel on the PTP of the mitochondria. Given the universality of Ca 2ϩ signaling, it is likely that this action of paclitaxel accounts for some of the side effects of antimitotic drugs. EXPERIMENTAL PROCEDURESCell Preparatio...

RANK ligand (RANKL) induces activation of NFB] i elevation using the intracellular Ca 2؉ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) abolished the ability of RANKL to enhance osteoclast survival. Using immunofluorescence, NFB was found predominantly in the cytosol of untreated osteoclasts. RANKL induced transient translocation of NFB to the nuclei, which was maximal at 15 min. U73122 or BAPTA delayed nuclear translocation of NFB. Delays were also observed upon inhibition of calcineurin or protein kinase C. We conclude that RANKL acts through phospholipase C to release Ca 2؉ from intracellular stores, accelerating nuclear translocation of NFB and promoting osteoclast survival. Such cross-talk between NFB and Ca 2؉ signaling provides a novel mechanism for the temporal regulation of gene expression in osteoclasts and other cell types.

Transforming growth factor-␤ (TGF-␤

No abstract

The alpha(v)beta(3) integrin is abundantly expressed in osteoclasts and has been implicated in the regulation of osteoclast function, especially in cell attachment. However, in vivo studies have shown that echistatin, an RGD-containing disintegrin which binds to alpha(v)beta(3), inhibits bone resorption without changing the number of osteoclasts on the bone surface, suggesting inhibition of osteoclast activity. The objective of this study was to examine how occupancy of alpha(v)beta(3) integrins inhibits osteoclast function, using primary rat osteoclasts and murine pre-fusion osteoclast-like cells formed in a co-culture system. We show that: (1) echistatin inhibits bone resorption in vitro at lower concentrations (IC(50)= 0.1 nM) than those required to detach osteoclasts from bone (IC(50) approximately 1 microM); (2) echistatin (IC(50)= 0.1 nM) inhibits M-CSF-induced migration and cell spreading of osteoclasts; (3) alpha(v)beta(3) integrins are localized in podosomes at the leading edge of migrating osteoclasts, whereas, with echistatin treatment (0.1 nM), alpha(v)beta(3) disperses randomly throughout the adhesion surface; and (4) when bone resorption is fully inhibited with echistatin, there is visible disruption of the sealing zone (IC(50)= 13 nM), and alpha(v)beta(3) visualized with confocal microscopy re-distributes from the basolateral membranes to intracellular vesicular structures. Taken together, these findings suggest that alpha(v)beta(3) integrin plays a role in the regulation of two processes required for effective osteoclastic bone resorption: cell migration (IC(50)= 0.1 nM) and maintenance of the sealing zone (IC(50) approximately 10 nM).