Because cardiac hypertrophy eventually plateaus even in the setting of continued prohypertrophic stimulation and is reversible when these stimuli abate, it is evident that the regulation of cardiomyocyte size depends on the balance between factors that either promote or antagonize cellular enlargement. Our previous studies identified a role for atrogin-1 in limiting pathological cardiomyocyte hypertrophic responses in vivo and in vitro by targeting calcineurin for proteasome-dependent degradation (8
), which provides a regulatory mechanism to counterbalance pro- and antigrowth mechanisms in cardiac hypertrophy (12
). The results of our present studies indicate that this counterregulatory role for atrogin-1 exists for calcineurin-independent cardiac growth as well. Akt is a proximal component of the physiologic, calcineurin-independent hypertrophic pathway (1
). We found using a combination of knockdown and overexpression approaches that atrogin-1 has no effect on Akt activation in response to IGF-1 or insulin challenge in cardiomyocytes, but nevertheless represses Akt-dependent hypertrophy by activating the Forkhead transcription factors. The transcriptional coactivation of Foxo1 and Foxo3a by atrogin-1 and upregulation of Forkhead-dependent transcriptional targets allows the heart to uncouple the inhibition of Akt-dependent hypertrophic signaling from other essential effects of Akt on cell survival and metabolism.
Activated Forkhead transcription factors participate in repression of both Akt-dependent and calcineurin-dependent hypertrophy (12
). Thus, whereas prohypertrophic signaling is segregated exquisitely in physiologic and pathologic circumstances (1
), the pathways that antagonize cardiac hypertrophic responses through transcriptional (Forkhead proteins) and protein stability (atrogin-1) mechanisms are common to both types of cardiac hypertrophy. This shared role is consistent with studies implicating both protein synthesis and protein degradation in antigrowth mechanisms in the heart in vivo (20
). Importantly, because Forkhead proteins regulate atrogin-1 expression in skeletal and cardiac muscle (11
), our results indicate the presence of a feed-forward mechanism in which atrogin-1 is activated by, and in turn coactivates, Foxo3a and Foxo1. The presence of such a mechanism suggests that postnatal cardiac growth is carefully regulated at several levels through inhibitory processes, with atrogin-1 acting as both a transcriptional coactivator and ubiquitin ligase to coordinate multiple steps in this process. These studies also suggest the existence of additional layers of regulation that suppress this feed-forward pathway, and the elucidation of these mechanisms remains a fruitful topic for further inquiry. One potential mechanism to regulate this feedforward loop is through Foxo-dependent activation of Akt itself, which has recently been described (13
Because canonical ubiquitylation of proteins via lysine 48–linked chains targets proteins for degradation via the proteasome, the observation that atrogin-1 binds and activates the Forkhead transcription factors raised the possibility of non–proteasome-dependent activities for atrogin-1. The Forkhead transcription factors are regulated through several well-defined posttranslational mechanisms. When phosphorylated by Akt, these proteins are retained in the cytoplasm, where they are transcriptionally inactive and susceptible to proteasome-dependent degradation that is triggered by a Skp2-containing ubiquitin ligase (22
). In contrast, the dephosphorylated forms of Forkhead proteins are nuclear localized and transcriptionally active, and p300-dependent acetylation additionally enhances their activity (14
). Atrogin-1 blocks Akt-dependent Foxo1 and Foxo3a phosphorylation and enforces their localization in the nucleus, which may account in part for the transcriptional effects of atrogin-1 on these proteins. However, atrogin-1 also enhances the transcriptional activity of phosphorylation-defective constitutively active mutants of Foxo1 and Foxo3a that spontaneously localize to the nucleus, indicating that atrogin-1 modifies the activity of Forkhead proteins within the nucleus directly and not simply as a consequence of altered subcellular trafficking.
The coactivation of transcriptionally active proteins by ubiquitin ligases may seem paradoxical, but there is precedent for ubiquitin modifications regulating transcription. Monoubiquitylation of histones, transcription factors, and components of the core RNA polymerase machinery is a well-described mechanism for regulation of transcription (24
). Indeed, the Forkhead protein Foxo4 was recently shown to be monoubiquitylated in response to hydrogen peroxide stimulation (28
). However, the effects of atrogin-1 on Foxo1 and Foxo3a we observed here are distinctive in that atrogin-1 regulation is dependent on assembly of lysine 63–linked ubiquitin chains. Lysine 63–linked ubiquitin chains have typically been associated with intracellular signaling events by serving as a signal for recruitment of accessory proteins (15
), but a clear role for this noncanonical ubiquitylation event as a regulatory mechanism for mammalian transcription factors has not been established. The promiscuity of atrogin-1 with respect to chain linkage topology is not unprecedented for a ubiquitin ligase (29
) and provides an elegant means for a ubiquitin ligase to mediate activities that are both dependent on and independent of proteasomes. In the case of atrogin-1, the ability to assemble ubiquitin chains of different linkages permits the inactivation of calcineurin through targeting to the proteasome at the same time that transcriptional activation of Forkhead transcription factors is favored. The assembly of noncanonical ubiquitin chains by atrogin-1 provides what we believe to be a new muscle-specific regulatory modification that regulates nuclear functions to suppress hypertrophic signaling within the heart.