Regulation of osteoblast differentiation by Pasteurella multocida toxin (PMT): a role for Rho GTPase in bone formation.
The role of the Rho-Rho kinase signaling pathway on osteoblast differentiation was investigated using primary mouse calvarial cells. The bacterial toxin PMT inhibited, whereas Rho-ROK inhibitors stimulated, osteoblast differentiation and bone nodule formation. These effects correlated with altered BMP-2 and -4 expression. These data show the importance of Rho-ROK signaling in osteoblast differentiation and bone formation.


BACKGROUND
The signal transduction pathways controlling osteoblast differentiation are not well understood. In this study, we used Pasteurella multocida toxin (PMT), a unique bacterial toxin that activates the small GTPase Rho, and specific Rho inhibitors to investigate the role of Rho in osteoblast differentiation and bone formation in vitro.


METHODS
Primary mouse calvarial osteoblast cultures were used to investigate the effects of recombinant PMT and Rho-Rho kinase (ROK) inhibitors on osteoblast differentiation and bone nodule formation. Osteoblast gene expression was analyzed using Northern blot and RT-PCR, and actin rearrangements were visualized after phalloidin staining and confocal microscopy.


RESULTS
PMT stimulated the proliferation of primary mouse calvarial cells and markedly inhibited the differentiation of osteoblast precursors to bone nodules with a concomitant inhibition of osteoblastic marker gene expression. There was no apparent causal relationship between the stimulation of proliferation and inhibition of differentiation. PMT caused cytoskeletal rearrangements because of activation of Rho, and the inhibition of bone nodules was completely reversed by the Rho inhibitor C3 transferase and partly reversed by inhibitors of the Rho effector, ROK. Interestingly, Rho and ROK inhibitors alone potently stimulated osteoblast differentiation, gene expression, and bone nodule formation. Finally, PMT inhibited, whereas ROK inhibitors stimulated, bone morphogenetic protein (BMP)-2 and -4 mRNA expression, providing a possible mechanism for their effects on bone nodule formation.


CONCLUSIONS
These results show that PMT inhibits osteoblast differentiation through a mechanism involving the Rho-ROK pathway and that this pathway is an important negative regulator of osteoblast differentiation. Conversely, ROK inhibitors stimulate osteoblast differentiation and may be potentially useful as anabolic agents for bone.
Harmey
Dympna
D
Departments of Craniofacial Development and Orthodontics, King's College London, Guy's Hospital, London, United Kingdom.
Stenbeck
Gudrun
G
Nobes
Catherine D
CD
Lax
Alistair J
AJ
Grigoriadis
Agamemnon E
AE
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D036002:ADP Ribose Transferases / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis; D001905:Botulinum Toxins / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology*; D010012:Osteogenesis / Q000187:drug effects* / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D012886:Skull / Q000166:cytology / Q000502:physiology; D016212:Transforming Growth Factor beta; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054460:rho-Associated Kinases
D000577:Amides; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; C062258:Pasteurella multocida toxin; D011725:Pyridines; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D001905:Botulinum Toxins; D020741:rho GTP-Binding Proteins; C049347:fasudil