C3 Rho-Inhibitor for Targeted Pharmacological Manipulation of Osteoclast-Like Cells

et al. (2013) C3 Rho-Inhibitor for Targeted Pharmacological Manipulation of Osteoclast-Like Cells. PLoS ONE 8(12): e85695. doi:10.1371/journal.pone.0085695 C3 Rho-Inhibitor for Targeted Pharmacological Manipulation of Osteoclast-Like Cells Andrea Tautzenberger 0 Christina Frtsch 0 Christian Zwerger 0 Lydia Dmochewitz 0 Ludwika Kreja 0 Anita Ignatius 0 Holger Barth 0 Michel R. Popoff, Institute Pasteur, France 0 1 Institute of Orthopaedic Research and Biomechanics, Centre of Musculoskeletal Research, University of Ulm , Ulm, Germany , 2 Institute of Pharmacology and Toxicology, University of Ulm Medical Center , Ulm , Germany The C3 toxins from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) as well as C3-derived fusion proteins are selectively taken up into the cytosol of monocytes/macrophages where the C3-catalyzed ADPribosylation of Rho results in inhibition of Rho-signalling and characteristic morphological changes. Since the fusion toxin C2IN-C3lim was efficiently taken up into and inhibited proliferation of murine macrophage-like RAW 264.7 cells, its effects on RAW 264.7-derived osteoclasts were investigated. C2IN-C3lim was taken up into differentiated osteoclasts and decreased their resorption activity. In undifferentiated RAW 264.7 cells, C2IN-C3lim-treatment significantly decreased their differentiation into osteoclasts as determined by counting the multi-nucleated, TRAPpositive cells. This inhibitory effect was concentration- and time-dependent and most efficient when C2IN-C3lim was applied in the early stage of osteoclast-formation. A single-dose application of C2IN-C3lim at day 0 and its subsequent removal at day 1 reduced the number of osteoclasts in a comparable manner while C2IN-C3limapplication at later time points did not reduce the number of osteoclasts to a comparable degree. Control experiments with an enzymatically inactive C3 protein revealed that the ADP-ribosylation of Rho was essential for the observed effects. In conclusion, the results indicate that Rho-activity is crucial during the early phase of osteoclastdifferentiation. Other bone cell types such as pre-osteoblastic cells were not affected by C2IN-C3lim. Due to their cell-type selective and specific mode of action, C3 proteins and C3-fusions might be valuable tools for targeted pharmacological manipulation of osteoclast formation and activity, which could lead to development of novel therapeutic strategies against osteoclast-associated diseases. - Funding: The work was supported by the Medical Faculty of the University of Ulm and the International Graduate School in Molecular Medicine Ulm (IGradU). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. The C3 toxins (~25 kDa) from Clostridium botulinum (C3bot1) [1] and Clostridium limosum (C3lim) [2] selectively mono-ADP-ribosylate the small guanosine triphosphate (GTP)binding proteins Rho A, -B, and C at Asn-41, which inhibits Rho-signalling in mammalian cells [3]. Among a variety of cellular responses, C3-treatment protects cells from apoptosis and inhibits proliferation [3]. Interestingly, C3 toxins are not efficiently taken up into most eukaryotic cell types including epithelial cells and fibroblasts and it was suggested that uptake of C3 toxin into cells might only occur by non-specific pinocytosis when large amounts of C3 are applied for incubation periods longer than 24 h [4]. We discovered recently that monocytes/macrophages are the target cells for the clostridial C3 toxins [5]. These cells internalize comparatively low concentrations of C3 toxins within approx. 3 h, most likely by a specific uptake mechanism including receptor-mediated endocytosis and subsequent translocation from acidified endosomal vesicles into the host cell cytosol [5]. In these cells, the C3-catalyzed Rho-modification leads to re-organization of the actin cytoskeleton and characteristic morphological changes [5]. Enzymatically inactive C3bot1E174Q [6] is internalized into monocytes/macrophages comparable to wildtype C3 proteins [5] and due to lacking adverse effects on cells, it serves as carrier for selective delivery of foreign proteins into the cytosol of monocytes/macrophages [7,8]. In order to deliver C3 Rho-inhibitor into the cytosol of various cell types, we previously developed the recombinant fusion toxin C2IN-C3lim (~50 kDa), which exploits the binary C2 toxin from C. botulinum for its transport into cells [9]. The C2 toxin consists of the actin ADP-ribosylating enzyme component C2I and the separate transport component C2IIa, which delivers C2I into the cytosol of all tested cell types (for review see [10]). The fusion toxin C2IN-C3lim consists of enzymatically active C3lim and the enzymatically inactive N-terminal domain of C2I (C2IN, ~25 kDa) [9]. When applied together with C2IIa, C2INC3lim is efficiently delivered into the cytosol of all mammalian cell types tested so far because its C2IN domain interacts with C2IIa and this triggers specific internalization via the C2 toxin uptake pathway [9,11]. However, in the absence of C2IIa, C2IN-C3lim is taken up into monocytes/macrophages but not into other cell types [5]. Like the clostridial C3 toxins, C2INC3lim is selectively taken up into macrophage-like cells by the C3-specific uptake mechanism via acidified endosomal vesicles [5,9]. Regarding its Rho-selective ADPribosyltransferase activity and the cellular effects, C2IN-C3lim behaves like C3lim [9]. Since C3 toxins are the only known Rho-inhibitors and selectively target cells from the monocyte/macrophage-line, C3 toxins and C3-derived fusion toxins such as C2IN-C3lim are ideal tools for investigation and targeted pharmacological manipulation of Rho-dependent signal transduction in cells which are related to this cell line such as osteoclasts. In vivo, monocytes/macrophages and osteoclasts are derived from pluripotent hematopoietic stem cells [12] and in vitro, osteoclasts differentiate from macrophage-like RAW 264.7 cells. Osteoclasts form a tight sealing zone on mineralized surfaces which is essential for resorption of matrix, e.g. during re-organization of bone tissue [13]. It was reported earlier that the C3-catalyzed ADP-ribosylation of Rho in osteoclasts resulted in disruption of their sealing zone and their resorption activity [14], indicating that Rho plays a crucial role for osteoclast activity. Here, we demonstrate that C2IN-C3lim is efficiently taken up into the cytosol of RAW 264.7 cells and derived osteoclasts. Treatment with C2IN-C3lim inhibited the resorption activity of already differentiated osteoclasts but also the formation of osteoclasts from RAW 264.7 cells due to the C3-catalyzed ADP-ribosylation of Rho. In contrast, C2IN-C3lim had no effects on other cultured bone cell types such as murine pre-osteoblastic MC3T3 cells, confirming its monocyte/ macro (...truncated)