The small GTP-binding protein, rho p21, is involved in bone resorption by regulating cytoskeletal organization in osteoclasts.
Rho protein (rho p21), a p21ras-related small guanine nucleotide binding protein, regulates cytoskeletal organization in a number of different types of cells. Evidence has indicated that Clostridium botulinum-derived ADP-ribosyltransferase (C3 exoenzyme) specifically ADP-ribosylates rho p21 at Asn41 and renders it functionally inactive. In this study, we examined the involvement of rho p21 in osteoclastic bone resorption using the C3 exoenzyme. When osteoclast-like multinucleated cells obtained from cocultures of mouse osteoblastic cells and bone marrow cells were placed on dentine slices, they formed ringed structures of podosomes containing F-actin (corresponding to the clear zone) within 8 hours. Many resorption pits were formed on dentine slices after culture for 24 hours. The C3 exoenzyme at 0.15-10 micrograms/ml added to the culture medium disrupted the ringed structure of podosomes in osteoclast-like cells in a dose-dependent manner. Correspondingly, pit formation by osteoclast-like cells on dentine slices was dose-dependently inhibited also by adding the C3 exoenzyme. Microinjection of the C3 exoenzyme into osteoclast-like cells placed on culture dishes completely disrupted the ringed podosome structure within 20 minutes. The amount of the rho p21 which was ADP-ribosylated by the C3 exoenzyme in vitro was much greater in purified osteoclast-like cells than in osteoblastic cells. Prior exposure of the purified osteoclast-like cell preparation to the C3 exoenzyme in vivo markedly decreased the amount of unribosylated rho p21. This indicated that the C3 exoenzyme incorporated into osteoclast-like cells effectively ADP-ribosylates rho p21 in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhang
D
D
Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan.
Udagawa
N
N
Nakamura
I
I
Murakami
H
H
Saito
S
S
Yamasaki
K
K
Shibasaki
Y
Y
Morii
N
N
Narumiya
S
S
Takahashi
N
N
D000199:Actins / Q000502:physiology; D000818:Animals; D001853:Bone Marrow / Q000502:physiology; D001854:Bone Marrow Cells; D001862:Bone Resorption; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D015726:Giant Cells / Q000502:physiology; D008297:Male; D051379:Mice; D010010:Osteoclasts / Q000502:physiology*; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology; D020741:rho GTP-Binding Proteins
D000199:Actins; D011065:Poly(ADP-ribose) Polymerases; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins