Prompted by our earlier findings that clostridial C3bot1 and C3lim toxins are selectively taken up by cells of the monocyte/ macrophage line, we have performed a series of experiments to investigate the effects of C3-treatment on osteoclasts which were generated by RANKL-induced differentiation of murine osteoclastic RAW 264.7 cells. Like the clostridial C3 toxins, the recombinant C2IN-C3lim fusion toxin was selectively internalized into PD325901 undifferentiated RAW 264.7 cells and already differentiated osteoclasts by the C3-specific uptake mechanism. Interestingly, C2IN-C3lim exhibited a stronger effect than C3lim alone or C3bot on undifferentiated RAW 264.7 cells. Although the reason for this unexpected effect is not known, one could speculate that the C2IN portion enhances the uptake of the C3 GDC-0879 side effects ADP-ribosyltransferase into the cytosol of the macrophages. In particular, C2IN could enhance the transport of internalized C2IN-C3lim protein across endosomal membranes from the endosomal lumen into the cytosol since C2IN mediates this translocation step of the C2I ADP-ribosyltransferase through C2IIa-pores across endosomal membranes. Moreover, C2IN could serve as a scaffold protein which may facilitate refolding of C3lim in the cytosol if an unfolding of C3lim is required for membrane translocation, which is not clear so far. Therefore, C2IN-C3lim was used to investigate the effects mediated by C3-catalyzed Rho-inhibition in differentiating osteoclasts and in already differentiated osteoclasts. By using this fusion toxin, we confirmed earlier results by another group that C3-catalyzed Rho inhibition in already differentiated osteoclasts decreases the resorption activity of these cells. It was reported by various groups that Rho activity regulates the formation of the actin ring in osteoclasts which is a prerequisite for bone resorption by these cells. Moreover, we discovered that application of C2INC3lim to RAW 264.7 cells inhibited their RANKL-induced differentiation into osteoclasts in a time- and concentrationdependent manner which might be a consequence of the inhibited proliferation of C2IN-C3lim-treated RAW 264.7 cells. A weaker inhibitory effect on osteoclast-differentiation was observed when C3bot1 was used instead of C2IN-C3lim while enzymatically inactive C3bot1E174Q had no effect on the morphology of RAW 264.7 cells. Moreover, these results confirmed that C2IN-C3lim is an attractive tool to investigate the specific C3-mediated effects in such cells. The concentration- and time-dependent inhibition of osteoclastformation by C2IN-C3lim with the strongest effect after a single-dose of C2IN-C3lim at day 0 or C2IN-C3lim-treatment from day 0 on implies an essential role of Rho in the early phase of osteoclast-differentiation. Although the results imply that a time-dependent Rho-inhibition seems to be crucial for osteoclast-formation, the reason for this effect is not known so far. Interestingly, in contrast to RhoU, the expression of RhoA, B and �CC, which are the selective targets of C3 proteins is not upregulated during RANKL-induced osteoclastogenesis. However, it is not clear whether the merely constant expression of RhoA, -B, and -C over time is related to the strong effect that is exerted by C3 in early osteoclast differentiation. Besides its role as a specific inhibitor to investigate the role of Rho-signalling in osteoclastogenesis and osteoclast functions, the finding that C2IN-C3lim is taken up into the cytosol of osteoclasts but not of other bone cell types such as pre-osteoblastic cells might have a pharmacological impact. The observation that C3-derived recombinant fusion toxins such as C2IN-C3lim are taken up into osteoclasts is an essential prerequisite for exploiting enzymatically inactive C3 protein.