We have used the unique and potent bacterial toxin, PMT, which perturbs bone remodeling in vivo, to investigate the potential signal transduction pathways involved in osteoblast differentiation and have shown that cells of the osteoblast lineage are bona fide cellular targets for PMT. More importantly, the data show that PMT stimulates the Rho-ROK pathway, resulting in inhibition of osteoblast differentiation and bone formation in vitro and that specific inhibition of Rho and ROK stimulates bone formation.
PMT is a potent inhibitor of osteoprogenitor cell differentiation
Continuous treatment of primary mouse calvarial cells with PMT inhibited the formation of mineralized bone nodules, and this inhibitory effect was manifested early during differentiation, where as little as a 6-h exposure to PMT was sufficient to inhibit nodule formation. These data suggest that the effects of PMT on osteoprogenitors are rapid and that PMT may decrease the proportion of precursors that are capable of forming nodules. This was further supported by the time-lapse studies where early nodule condensations treated with PMT exhibited morphological changes from ∼10 h of treatment and failed to progress further to form 3D bone nodules.
Our data indicate that PMT is a mitogen that inhibits bone formation. Numerous hormones and growth factors that act through diverse receptor signaling pathways are mitogenic for osteoblasts and can inhibit bone formation in vitro.(3, 40) However, it is difficult to establish a simple correlation between proliferation and differentiation because primary calvarial cultures are heterogeneous, and nodule-forming osteoprogenitor cells exist at a very low frequency; thus, changes in the proliferative potential of these cells may not be observed.(41) Nevertheless, at the mass population level, several lines of evidence support the notion that these events may not be sequential in PMT-treated cultures. First, we observed clear inhibitory effects on bone nodule formation at PMT concentrations that failed to stimulate cell proliferation. Second, a short-term pulse of PMT inhibited the number of nodules but did not stimulate proliferation (data not shown). Third, inhibition of Ras, which is known to mediate growth factor-induced proliferation through the Erk/MAPK pathway, blocked PMT-induced BrdU incorporation but not PMT-induced stress fiber formation, which is mediated by Rho.(26, 27) Furthermore, preliminary data showed that the MEK inhibitor, PD98059, failed to block the inhibitory effects of PMT and had no effect on its own on osteoblast differentiation (data not shown), although inhibition of MEK has recently been shown to enhance BMP-2 effects in the MC3T3-E1 cell line.(42) Thus, it is tempting to speculate that the effects of PMT on osteoblastic cell proliferation and differentiation may be mediated by different signaling pathways, although this needs to be further clarified.
It is not yet known whether the inhibition of osteoblast marker gene expression by PMT is direct or indirect, although notably, the reduction in Cbfa1/Runx2 expression, which is essential for osteoblast differentiation,(2) correlated well with the inhibition of differentiation. Thus, one possibility is that the reduction in the expression of Cbfa1/Runx2 may represent one mechanism by which PMT inhibits osteoblast differentiation. Indeed, the downregulation of BMP-2 and −4 expression by PMT would suggest that the inhibition of Cbfa1/Runx2 expression may be indirect, because these BMPs are known to regulate Cbfa1/Runx2 transcription.(43) However, because we only observed ∼50% inhibition of Cbfa1/Runx2 expression, it remains possible that the PMT-dependent signaling events that inhibit osteoblast differentiation lie downstream or are independent of Cbfa1/Runx2.
We have eliminated the possibility that the effects observed are caused by possible contamination of our PMT preparations by bacterial products, such as a lipopolysaccharide (LPS), which can synergize with some bacterial toxins. LPS was the first bacterial component shown to induce osteoclastic bone resorption in vitro,(44) and it has been shown that LPS from Porphyromonas gingivalis inhibits bone nodule formation and gene expression in primary rat calvarial osteoblasts.(45) However, in contrast to LPS, PMT is heat labile,(17) and we have confirmed that heat inactivation of PMT abolished the inhibitory effects on osteoblast differentiation (data not shown). Moreover, removal of any potential LPS contamination using polymixin B did not inhibit the PMT effects, suggesting there is no synergy between LPS and PMT (data not shown). Together with the fact that the inactive mutant C1165S had no effects on nodule formation or proliferation, this shows that the effects of PMT on osteoblast differentiation are caused by the recombinant protein and not a result of other bacterial factors.
Activation of Rho-ROK mediates the PMT effects on osteoblasts and inhibits bone formation
The marked cytoskeletal rearrangements induced by PMT, particularly in cuboidal cells lining bone nodules, clearly indicated that PMT stimulates the Rho pathway in osteoblastic cells, because actin rearrangements and stress fiber formation are mediated by Rho GTPases. Thus, we have demonstrated a novel link between osteoblast differentiation, PMT, and Rho-induced actin rearrangements. However, it is not yet clear whether the altered cytoskeleton is causally involved in regulating osteoblast differentiation, because Rho GTPases can also activate other signaling pathways and transcription factors.(46) Indeed, a recent report has demonstrated that Rho-ROK can regulate the lineage commitment of mesenchymal cells and that this may be independent of the cytoskeleton.(47) Nevertheless, the activation of Rho played a functional role in osteoblast differentiation because the Rho inhibitor, C3 transferase, abrogated the PMT inhibition of bone nodule formation. It is not yet known how PMT induces Rho-dependent signaling in osteogenic cells, although it is well accepted that Rho is not a direct target of PMT. Rather, it is likely that heterotrimeric G proteins are involved, in particular the Gq/11 family, which are known to be activated by PMT and which have been shown recently to activate Rho, or alternatively, the G12/13 family, which is known to activate Rho through RhoGEFs.(20-22, 48, 49) We also cannot rule out that other signaling pathways stimulated by PMT downstream of Gq/11 or G12/13 may contribute to its inhibitory effects, although our data would suggest that it is unlikely that other Rho GTPases are involved (e.g., Rac, cdc42) because the inhibitory effects of CNF were reversed by the specific ROK inhibitor Y-27632. Last, previous reports have suggested that the increase in osteoblast activity after mechanical stimulation is linked to cytoskeletal rearrangements and induction of cyclo-oxygenase-2 (Cox-2) and may be blocked by inhibiting Rho activity.(50-52) We have recently demonstrated that CNF, which activates Rho, Rac, and cdc42, also stimulates Cox-2 expression in a manner that is dependent on Rho activation but independent of ROK, indicating that other Rho effectors mediate the transcriptional regulation of Cox-2.(53) Our preliminary evidence has indicated that inhibition of Cox-2 and prostaglandin synthesis by indomethacin does not block the inhibitory effects of PMT on osteoblasts (data not shown), suggesting that PMT inhibits bone nodule formation through a prostaglandin-independent pathway.
While Rho is important for mediating the PMT effects, we have shown additionally that the Rho effector, ROK, is important. Because nodule number was not fully restored by the ROK inhibitors, it remains possible that there exist different subpopulations within these cultures that respond differently to PMT and HA-1077/Y-27632 or that another Rho effector may play an important role.(46) However, the importance of ROK as a critical regulator of bone formation was highlighted by the fact that the ROK inhibitors, HA-1077 and Y-27632 alone, markedly stimulated osteoblast marker expression and caused precocious formation of bone nodules that mineralized earlier than control cultures. Moreover, BMPs may play a key role downstream of the Rho-ROK pathway because Y-27632 stimulated, whereas PMT inhibited, both BMP-2 and −4 expression, consistent with their stimulatory and inhibitory roles on nodule formation, respectively. These results are interesting in view of the observed stimulation of bone formation by statins,(54) which inhibit HMG-CoA reductase, eventually leading to inhibition of small GTPases by preventing their prenylation, although it is not established whether the observed anabolic effects by statins are directly caused by inhibition of Rho activity. Indeed, the possible mechanisms may differ as statins have been reported to stimulate BMP-2, but not BMP-4, expression.(54) Finally, a recent study has indicated that inhibition of ROK stimulates osteoblast gene expression, although bone formation was not assessed in that study.(55) Our data show for the first time that direct inhibition of Rho and ROK stimulates bone nodule formation and that this may be through enhanced BMP expression; we have also implicated specific heterotrimeric G proteins in the regulation of bone formation by PMT.
In conclusion, we have established that the bacterial toxin, PMT, targets osteogenic cells. There are a large number of bacterial products, some of which are protein toxins, that affect bone cell function, but the majority of them lead to bone destruction either by targeting osteoclastic bone resorption, inhibiting osteoblast proliferation, and/or stimulating apoptosis.(44) The main in vivo effect of PMT is bone loss, suggesting the involvement of osteoclasts, and indeed, our recent data has demonstrated that PMT has direct independent effects on the osteoclast lineage (unpublished data). However, the data presented here would suggest that a major contributing factor in the pathogenesis of atrophic rhinitis may be the inhibition of osteoblast differentiation and bone formation. The use of PMT as a tool to study intracellular signal transduction pathways has led to the demonstration that activation of the Rho-ROK pathway potently inhibits osteoblast differentiation. Moreover, inhibition of Rho-ROK signaling results in marked stimulatory effects on bone nodule formation, thus providing potential therapeutic targets for metabolic diseases characterized by bone loss.