Here, we performed RNAi for Cdc42 and Ect2 and found that depletion of either protein induces prometaphase delay, which is essentially similar to that found in cells subjected to toxin B treatment, overexpression of Cdc42 mutants, and RNAi for mDia3 (Yasuda et al, 2004). Furthermore, we found that expression of MgcRacGAPR386A also interfered with the transition to metaphase in a similar way. This, although apparently paradoxical, is consistent with our finding that both dominant-active and dominant-negative Cdc42 mutant induced the delay in prometaphase, and can be explained by misorientation of the spindle MTs by ectopic expression of an excess amount of active Cdc42. Although dominant-active and dominant-negative Cdc42 mutants induce the opposing actin phenotypes, they cause the same phenotype with respect to MT targeting in interphase cells (Etienne-Manneville and Hall, 2001). These results not only corroborated the role of Cdc42 in the MT attachment to kinetochores but also that Ect2 and MgcRacGAP regulate this Cdc42 function. In contrast, previous studies have shown that interfering with Ect2 and MgcRaGAP causes cytokinesis failure, which led the authors to suggest that Ect2 and MgcRacGAP are linked mainly with RhoA (Tatsumoto et al, 1999; Jantsch-Plunger et al, 2000; Kimura et al, 2000; Hirose et al, 2001). However, though not explicitly described in the previous papers, the tetraploid nucleus in these cells was abnormal in shape. Furthermore, Van de Putte et al. (2001) reported that homozygous disruption of the gene for MgcRacGAP resulted in death of E3/5–4 embryos due to the failure of chromosomal segregation. In addition, expression of active forms of Pak, an effector of Cdc42, resulted in the production of cells with multiple spindles (Vadlamudi et al, 2000), and an endogenous Pak isoform, Pak 1, was shown to localize at the kinetochore in mitotic cells (Li et al, 2002). Recently, Tatsumoto et al. (2003) used Xenopus laevis mitotic extracts and examined in vitro the effect on mitosis of anti-Ect2 antibody as well as dominant-negative forms of Ect2 and Rho GTPases.
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Alendronate is one of the nitrogen-containing bisphosphonates (NBPs) used as anti-bone resorptive drugs. However, NBPs have inflammatory side effects including osteomyelitis and osteonecrosis of the jaw. In the present study, we examined the effects of alendronate on chemokine production by the macrophage-like cell line, J774/1, when incubated with Pam(3)CSK(4) (a Toll-like receptor (TLR) 2 agonist) and Lipid A (a TLR4 agonist). Pretreatment of J774/1 cells with alendronate decreased the production of TLR ligand-induced monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) but did not influence nuclear factor-kappaB (NF-kappaB) activation. While this agent induced caspase-8 activation, a caspase-8 inhibitor did not affect the decrease in MCP-1 production by alendronate and TLR ligands. Thus, the alendronate-mediated decrease in chemokine production was independent of NF-kappaB and caspase-8 activation. Although transforming growth factor-beta1 (TGF-beta1) is known to inhibit chemokine production by various cell types via Smad3 activation, pretreatment with alendronate did not increase TGF-beta1 production by J774/1 cells incubated in the presence or absence of TLR ligands. However, alendronate directly activated Smad3. These results suggest that by down-regulating MCP-1 and MIP-1alpha production via Smad3, long-term use of alendronate might inhibit normal activation and migration of osteoclasts and cause osteonecrosis.