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(Circulation. 2002;105:402.)
© 2002 American Heart Association, Inc.

Editorials

Apoptosis Signal-Regulating Kinase/Nuclear Factor-B

A Novel Signaling Pathway Regulates Cardiomyocyte Hypertrophy

Thomas Force, MD; Syed Haq, MD; Heiko Kilter, MD; Ashour Michael, PhD

From the Molecular Cardiology Research Institute, New England Medical Center and the Department of Medicine, Tufts University School of Medicine, Boston, Mass.

Correspondence to Thomas Force, MD, Molecular Cardiology Research Institute, New England Medical Center, 750 Washington St, Box 8486, Boston, MA 02111. E-mail tforce{at}lifespan.org

Key Words: Editorials • angiotensin • heart failure • signal transduction • oxidant stress

Since 1993, when Sadoshima et al¹ published their observations on the role of Angiotensin II (Ang II) in the stretch-induced hypertrophic response of cardiomyocytes, we have known that peptide hormones were important in the development of hypertrophy stimulated by cell stretch, the initiating stimulus in pressure overload-induced hypertrophy and in the hypertrophy that occurs in the noninfarcted myocardium following a myocardial infarction. Ang II, as well as endothelin-1 (ET-1) and -adrenergic agents, activate intracellular pathways by binding to 7 transmembrane-spanning receptors coupled to heterotrimeric G proteins of the Gq class. The critical role of this class of receptors and G proteins in pressure overload-induced hypertrophy was elegantly demonstrated by Ahkter et al² who showed that cardiac specific expression in mice of a peptide that blocked signal transmission by Gq markedly blunted the hypertrophic response to aortic banding.

See p 509

The question that has dominated the field of cardiomyocyte biology is how do these hormones, acting via their cognate receptors and Gq, trigger the hypertrophic response? One clear conclusion has evolved thus far: the hypertrophic response of cardiomyocytes is regulated by an enormously complex network of interacting cytosolic signaling pathways.³ Gq activation ultimately results in the production of intermediates that increase cytosolic free [Ca²⁺] and activate members of the protein kinase C family, and leads to the recruitment of several protein kinases, including the mitogen-activated protein kinases (MAPKs), calcium calmodulin-dependent protein kinases, Akt/PKB, and the Janus kinases. These kinases phosphorylate a number of transcription factors, increasing their transcriptional activating activity and, in some cases, DNA binding activity. Elevated [Ca²⁺] can also activate the protein phosphatase, calcineurin,⁴ which dephosphorylates members of the nuclear factor-activated T cell (NF-AT) family of transcription factors, causing their nuclear translocation. Activated transcription factors bind to specific DNA sequences within the promoter regions of genes and recruit cofactors and the basal transcription machinery, leading to the induction of gene expression. This reprogramming of gene expression drives the hypertrophic response.

Given the intense interest in the study of hypertrophic signaling, it is always surprising when researchers identify an entirely new pathway that plays a role in this response. In this issue of Circulation, Hirotani et al⁵ do just that. The authors explore the role of reactive oxygen species (ROS), the apoptosis signal-regulating kinase, ASK1, which is activated by ROS, and the transcription factor NF-B in the hypertrophic response to Ang II, ET-1, and phenylephrine. Earlier work had shown that Ang II acts as a relatively potent inducer of ROS (Nakamura et al⁶ and references therein), and ROS play a critical role in Ang II-induced hypertrophic responses.⁶ Additionally, Ang II has been implicated in the activation of NF-B, both in vascular smooth muscle cells and cardiac myocytes (reviewed in Braiser et al⁷). Hirotani et al now connect these observations by showing that the vasoactive peptides induce hypertrophy, at least in part, via ROS-dependent activation of NF-B. This is, in itself, an important finding that significantly extends earlier work demonstrating that NF-B regulates mechanical strain-induced expression of BNP in cardiomyoyctes⁸ and corroborates findings published while Hirotani et al was in press.⁹ In addition to defining a role for NF-B in hypertrophy, however, Hirotani et al also implicate an entirely new pathway, ROS-induced activation of ASK1, a kinase best known as a mediator of TNF-induced apoptosis, as the mechanism by which vasoactive peptides both activate NF-B and induce hypertrophy. Utilizing adenovirus-mediated gene transfer of dominant inhibitory or constitutively active mutants of ASK1, they report that ASK-1 is both necessary and sufficient for NF-B activation and the hypertrophic response to Ang II, ET-1, and phenylephrine. These are potentially important observations for understanding not only mechanisms regulating hypertrophy and vascular remodeling, but also basic mechanisms of signal transduction.

ASK1: A Mediator of Cytokine and ROS Signaling

ASK1 was first identified as a kinase upstream of the stress response MAP kinases, p38-MAPK and JNKs, and it is believed to be specific to these MAPK pathways.¹⁰ When overexpressed, ASK1 induces apoptosis via the mitochondrial-dependent caspase pathway and, based on studies in mice deleted for ASK1, it is essential for TNF- and oxidant stress-induced, but not Fas-induced, apoptosis.¹¹ Because TNF can cause apoptosis via mitochondrial-independent mechanisms, ASK1 must act via additional mechanisms to induce apoptosis in response to TNF. In support of this, ASK1 has been reported to trigger apoptosis via association with Daxx, a putative mediator of Fas-induced apoptosis, via phosphorylation and inactivation of antiapoptotic Bcl-2, and by phosphorylation and stabilization of c-Myc (mediated by JNK). In addition, ASK1, in contrast to other kinases, induces sustained activation of the JNKs and p38-MAPKs and this appears to be critical for TNF- and ROS-induced apoptosis.¹¹ Oddly, the catalytic activity of ASK1 does not appear to be essential for a caspase-independent form of cell death mediated by ASK1 and Daxx, which is characterized by "crumpled" as opposed to the fragmented nuclei of caspase-dependent death.¹²

An early clue to the mechanisms of regulation of ASK1 was the observation that when overexpressed, ASK1 was constitutively active, suggesting that overexpression was titrating out an endogenous repressor. Indeed, the redox sensitive molecule, thioredoxin, was found to bind to ASK1, holding it in an inactive state.¹³ With ROS production, thioredoxin dissociates from ASK1. Thioredoxin binds to the amino-terminal region of ASK1, and deletion of this region produces the constitutively active mutant used by Hirotani et al. A host of other molecules, including 14-3-3 proteins, c-Raf-1, and HIV-1 Nef protein, have been found to inhibit ASK1 by similar mechanisms, although the binding sites may differ and not all are redox sensitive. In all cases, cells are protected from apoptosis. Of note in Hirotani et al,⁵ vasoactive peptide-induced generation of ROS appears to be of sufficient degree to lead to the dissociation of thioredoxin from ASK1 and activation of the kinase. This adds ASK1 to the list of mediators of Ang II signaling, a finding with significant implications for cardiovascular biology beyond the study of hypertrophy.

ASK1, NF-B, and Hypertrophy

The NF-B family comprises 5 family members that can homo- or heterodimerize with one another (see Baldwin¹⁴ and subsequent Perspective Series). NF-B is regulated by subcellular localization. It is retained in the cytosol by being bound to inhibitors of B (IBs), which mask the nuclear localization sequence of NF-B, and is released to translocate to the nucleus when IB is phosphorylated by the IB kinase (IK) complex, targeting IB for ubiquitination and degradation by the 26S proteasome.

Using an inhibitory mutant of IB that cannot be degraded by the proteasome and persistently binds to NF-B, blocking its translocation to the nucleus and thus its activity, Hirotani et al show that NF-B is necessary for the hypertrophic response to the vasoactive peptides. Although the authors did not determine whether NF-B is sufficient to induce hypertrophy, leaving open the possibility that persistent inhibition of NF-B signaling may be toxic to cardiomyocytes, Purcell et al⁹ did demonstrate that overexpression of NF-B alone was sufficient to induce modest ANF expression and cardiomyocyte enlargement, suggesting sufficiency.

Given the complexity of the hypertrophic response, how is it possible that this single transcription factor could be both necessary and sufficient for the hypertrophic response? NF-B is extremely promiscuous, interacting with a large number of transcription factors, including AP1 and Ets family members.¹⁵ These interactions are often necessary for full induction of target genes. In addition, cooperative interactions between NF-B and NF-ATs amplify gene expression. Thus NF-B, by itself, induces expression of a wide array of genes, and acting in concert with other transcription factors, amplifies expression of a second large set of genes. When the necessity and sufficiency of NF-B for the hypertrophic response is viewed in the context of the variety of transcription factors with which it interacts, including some believed to play a role in hypertrophy (eg, AP1 and NF-ATs),^4,16 the incredible number and variety of genes activated by NF-B, including some known to regulate cellular growth in metazoans (eg, cyclin D1 and c-Myc), and the number of disease processes for which dysregulation of NF-B is believed to play a role,¹⁴ the findings do not seem quite so surprising. In addition, NF-B is a mediator of induction of angiotensinogen and regulates the expression of the prohypertrophic cytokines TNF and IL-6.¹⁴ This creates a positive feedback loop that can be expected to perpetuate the hypertrophic response.⁷

As is usually the case with reports of novel findings, there are a number of questions that will need to be addressed in the future. One of the more surprising findings in the paper is that ASK1 positively regulates NF-B. Several kinases, including NF-B-inducing kinase (NIK), MEKK-1, -2, and -3, and TGFß-activated kinase (TAK1) can activate IKs leading to degradation of IB and activation of NF-B. However, ASK1 does not activate IKs or lead to IB degradation and has been reported to be either a negative regulator or to have no effect on NF-B activity.¹⁷ How then could ASK1 be activating NF-B? NF-B can also be activated by novel IB-independent mechanisms involving phosphorylation events that modulate both DNA binding and transcriptional activating activity of NF-B.¹⁸ These phosphorylations might, under certain conditions, be catalyzed directly by ASK1 or by a downstream target of ASK1 such as p38-MAPK.⁸ The mechanism by which ASK1 activates NF-B in cardiomyocytes will be critically important to define. In addition, identifying the mechanism will allow a determination of whether ASK1 could be a general route to the activation of NF-B by many agents that induce ROS production or specific to cardiomyocytes and hypertrophic agonists.

Second, it is not clear that NF-B is the major target of ASK1 because Hirotani et al did not demonstrate that dominant negative IB blocked ASK1-induced hypertrophy. It is likely that ASK1 regulates hypertrophy via mechanisms in addition to NF-B. ASK1 is a potent activator of the JNK and p38-MAPK pathways. These pathways have been reported to be both sufficient and necessary for hypertrophic responses of cardiomyocytes in culture,¹⁹ and in the case of the JNKs, necessary for the hypertrophic response to pressure overload in vivo.²⁰ Given the size of the regulatory domain of ASK1 and the multiple proteins with which it interacts, multiple additional pathways could be involved. Similarly, the necessity of ASK1 in hypertrophy needs to be confirmed in the ASK1 knockout mouse because overexpression of dominant inhibitory mutants of this protein could nonspecifically block signaling down pathways that are ordinarily not regulated by ASK1.

In summary, Hirotani et al⁵ have added ASK1 to the list of mediators of vasoactive peptide signaling and have identified ASK1 as a positive regulator of NF-B. Finally, they have identified ASK1 and NF-B as a novel pathway regulating cardiomyocyte hypertrophy. It will, of course, be a major challenge to determine the role of the pathway in vivo in the various stages of progression from hypertrophy to heart failure. Given the involvement of one potent activator, Ang II, at various stages of the progression, and the role of others, including TNF and possibly Fas, in the late progression of disease, this pathway could serve as a target in future attempts to alter the progression of heart failure. With the aggressive pursuit of inhibitors of this pathway by the pharmaceutical industry, the hypothesis should soon be ready for testing.

Acknowledgments

We thank Drs John Kyriakis and Jeffery Molkentin for their helpful comments.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

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