title: C3 rho-inhibitor for targeted pharmacological manipulation of osteoclast-like cells.
abstract: 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 ADP-ribosylation 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, TRAP-positive 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-C3lim-application 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 osteoclast-differentiation. 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.
authors.affiliation: Institute of Orthopaedic Research and Biomechanics, Centre of Musculoskeletal Research, University of Ulm, Ulm, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Tautzenberger
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Förtsch
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Zwerger
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.
authors.forename: Lydia
authors.initials: L
authors.lastname: Dmochewitz
authors.affiliation: Institute of Orthopaedic Research and Biomechanics, Centre of Musculoskeletal Research, University of Ulm, Ulm, Germany.
authors.forename: Ludwika
authors.initials: L
authors.lastname: Kreja
authors.affiliation: Institute of Orthopaedic Research and Biomechanics, Centre of Musculoskeletal Research, University of Ulm, Ulm, Germany.
authors.forename: Anita
authors.initials: A
authors.lastname: Ignatius
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Barth
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D003014:Clostridium botulinum / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D051379:Mice; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D013997:Time Factors; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000246:Adenosine Diphosphate Ribose; D001905:Botulinum Toxins; D020741:rho GTP-Binding Proteins