THE SMALL GTP-BINDING PROTEIN, RHO-P21, IS INVOLVED IN BONE-RESORPTION BY REGULATING CYTOSKELETAL ORGANIZATION IN OSTEOCLASTS

Rho protein (rho p21), a p21(ras)-related small guanine nucleotide bin ding protein, regulates cytoskeletal organization in a number of diffe rent types of cells. Evidence has indicated that Clostridium botulinum -derived ADP-ribosyltransferase (C3 exoenzyme) specifically ADP-ribosy lates rho p21 at Asn41 and renders it functionally inactive. In this s tudy, we examined the involvement of rho p21 in osteoclastic bone reso rption using the C3 exoenzyme. When osteoclast-like multinucleated cel ls obtained from cocultures of mouse osteoblastic cells and bone marro w cells were placed on dentine slices, they formed ringed structures o f podosomes containing F-actin (corresponding to the clear zone) withi n 8 hours. Many resorption pits were formed on dentine slices after cu lture for 24 hours. The C3 exoenzyme at 0.15-10 mu g/ml added to the c ulture medium disrupted the ringed structure of podosomes in osteoclas tlike cells in a dose-dependent manner. Correspondingly, pit formation by osteoclast-like cells on dentine slices was dose-dependently inhib ited also by adding the C3 exoenzyme. Microinjection of the C3 exoenzy me into osteoclast-like cells placed on culture dishes completely disr upted the ringed podosome structure within 20 minutes. The amount of t he rho p21 which was ADP-ribosylated by the C3 exoenzyme in vitro was much greater in purified osteoclastlike cells than in osteoblastic cel ls. Prior exposure of the purified osteoclast-like cell preparation to the C3 exoenzyme in vivo markedly decreased the amount of unribosylat ed rho p21. This indicated that the C3 exoenzyme incorporated into ost eoclast-like cells effectively ADP-ribosylates rho p21 in vivo, Isoele ctric focusing and immunoprecipitation studies revealed that the prefe rentially expressed rho p21 in osteoclast-like cells was rhoA. These r esults suggested that rho p21, probably rhoA, plays an important role in bone resorption by regulating cytoskeletal organization in osteocla sts.