The role of chromosomal translocation-derived chimeric transcription factor PAX3-FKHR in ARMS development is well characterized.2,3
However, studies have indicated that the mechanism of PAX3-FKHR in ARMS oncogenesis works, at least in part, by preventing myogenic cells from completing the terminal differentiation program.19,22,23
A key event in the myogenic differentiation process is the activation of the AKT signaling pathway, which acts as a promyogenic signals for myogenic gene activation.25–28
Despite activated AKT pathway in ARMS cells,38
they are defective in terminal myogenic differentiation.17,40,41
In ARMS, it is well recognized that PAX3-FKHR works by the gain of transcriptional power.2,50,51
The present study was focused on deciphering the molecular mechanism underlying activated AKT signaling in association with PAX3-FKHR transactivation in the suppression of the myogenic differentiation program in ARMS. To investigate the above mechanism, we have exploited conditional mouse models of ARMS cells.18,21
Here, we show that mouse ARMS cells reflect the defective terminal myogenic phenotype as revealed by no sign of MyHC expression similar to their human counterpart under differentiation conditions (DM).17,40,41
In this context, studies have implicated that impaired MyoD transcriptional activity-mediated myogenic gene expression, but not the absence of MyoD, is associated with the failure of ARMS cells to differentiate terminally.31,42,52
However, the data here indicated that impaired MyoD transactivation function is coupled with decreased levels of MyoD when these ARMS cells are under DM. Because MyoD is a transcriptional target of PAX3-FKHR,13,15
we evaluated the status of PAX3-FKHR transcriptional activity in these ARMS cells under DM. Interestingly, PAX3-FKHR transcriptional activity is downregulated in ARMS cells grown under DM, which coincided with decreased expression MyoD at both the mRNA and protein levels. Although, a numerous studies have delineated the transactivation function of PAX3-FKHR in attenuating myogenic differentiation,15,19,20,22,23,39,52,53
to our knowledge, downregulation of PAX3-FKHR transactivation function under DM has not been previously reported. This discovery led us to investigate the mechanism by which PAX3-FKHR transcriptional activity is downregulated in ARMS cells grown in DM. Interestingly, the data here demonstrate that decreased PAX3-FKHR transcriptional activity is not due to decreased levels of mRNA or protein nor to altered nuclear localization. However, we did perceive, during the assessment of PAX3-FKHR transactivation potency in ARMS cells, that PAX3-FKHR appears as a doublet of a faster and slower migrating form. Interestingly, the data showed the shift of PAX3-FKHR to a slower migrating form in ARMS cells grown in DM. This finding provided us a hint that the transcriptional activity of PAX3-FKHR may be modulated through its post-translational modification, which includes phosphorylation.54,55
Since induced PAX3-FKHR phosphorylation occurred when cells were grown in DM, we hypothesized that phosphorylation could be responsible for the decreased PAX3-FKHR transcriptional activity. Several studies have reported phosphorylation in the PAX3 domain of PAX3-FKHR in association with its transcriptional activity.1,54–58
However, none of the above studies reported phosphorylation-induced downregulation of transcriptional activity under differentiation conditions.
We investigated putative kinase signaling-pathway(s) induced under conditions suitable to induce terminal muscle differentiation. Our prime suspect in this context was the PI3/AKT pathway, since this pathway is activated in normal myoblasts when cultured in DM.28,29,59
In addition, PAX3-FKHR retains two consensus AKT phosphorylation sites in the FKHR domain.47
Although activated AKT is present in ARMS,38
we found hyper-activated AKT in ARMS cells grown in DM. Most significantly, the data generated using a pharmacological PI3/AKT inhibitor (LY294002) and a constitutive or dominant-negative version of AKT in ARMS cells showed that (1) the transactivation potency of PAX3-FKHR is preserved by basal activated AKT levels in cells under growth conditions, and (2) hyperactivated AKT as acts as a regulatory switch for PAX3-FKHR, changing it from a transcriptionally active to an inactive state under conditions of differentiation. In contrast, a previous study performed on NIH3T3 cells claimed that AKT activity fails to regulate the transcriptional activity of PAX3-FKHR.47
However, the previous finding does not negate our current findings, since PAX3-FHR activity and significance is cell type-specific,2,53
with a significant role almost exclusively in ARMS.2,3
Taken together, the data presented here uncover a molecular link between AKT activity and PAX3-FKHR transcriptional response, which can be modulated by manipulating the level of AKT activity.
In the context of AKT activity, one would expect that its hyperactivation would lead to terminal myogenic differentiation. This assumption was based on previous studies demonstrating that differentiation-responsive AKT activation is essential for normal myoblasts to differentiate terminally.25,29,31,33
Studies further delineated that AKT signaling promotes differentiation by activating MyoD-regulated myogenic gene transcription.29,30
Concurrently, one would also expect that loss of PAX3-FKHR transactivation function would lead to ARMS cell differentiation. This was based on a recent study that claimed PAX3-FKHR prevents myogenic differentiation of ARMS cells through repression of myogenin expression.39
Their results showed that siRNA-mediated PAX3-FKHR silencing induces myogenin, a downstream MyoD target, to cooperate with it in late gene expression during terminal differentiation16
and late muscle MyHC expression in ARMS cells. However, the expression of MyoD, a direct target of PAX3-FKHR,13,15
was not significantly downregulated by PAX3-FKHR siRNA in their study in ARMS cells. In contrast, a previous study showed that PAX3-FKHR can transactivate myogenin independent of MyoD.60
Moreover, a recent study demonstrated that PAX3-FKHR activates MyoD expression, but at the same time attenuates the MyoD-mediated terminal myogenic program.15
Our data demonstrates that the restoration of PAX3-FKHR activity mediated induction of MyoD by dnAKT is incompetent in gene activation function in ARMS grown in DM, supporting that PAX3-FKHR activity mediated simultaneous induction and inhibition of MyoD regulated the myogenic program. Hence, PAX3-FKHR silencing by its siRNA might induce myogenin expression that cooperates with residual MyoD, leading to expression of late gene in MyoD-mediated myogenic differentiation in ARMS. Our data, however, indicate that hyperactivation of AKT or its activity led to loss of PAX3-FKHR transactivation potency and was not associated with induced expression of either myogenin or MyHC in ARMS cells grown in DM, but expression of MyoD is lost. In this scenario, the absence of MyoD along with myogenin, the latter mediated directly by the loss of PAX3-FKHR transcriptional activity and/or coupled with MyoD eradication, eventually led to the inhibition of MyHC expression. Importantly, restoration of MyoD expression in ARMS cells that possess transactivation-incompetent PAX3-FKHR repairs the defective terminal myogenic program under differentiation conditions. The data further underlines the role of MyoD in the activation of genes, such as growth arrest p21cip1
and muscle early myogenin followed by late MyHC.
In summary, our findings lead to the hypothesis that AKT tailors the PAX3-FKHR mediated block in muscle differentiation through both inhibitions of MyoD expression and function in ARMS cells (). Central to this perspective is our finding that AKT-dependent phosphorylation manipulates the transcriptional activity of PAX3-FKHR in ARMS cells. While activated AKT sustains PAX3-FKHR transcriptional activity-mediated block of MyoD activity and arrests differentiation of ARMS cells, hyperactivated AKT-mediated switching to transactivation-incompetent PAX3-FKHR fails to rescue the differentiation arrest through loss of MyoD expression in these cells. Thus, the activated AKT status could be critical to the control of PAX3-FKHR-positive ARMS cells to maintain undifferentiated state by preventing terminal differentiation.
Model depicting AKT-directed dual strategy in the suppression of MyoD-driven myogenic differentiation through PAX3-FKHR in ARMS cells.