Fine tuning endothelial Notch: SIRT-ainly an unexpected mechanism

Abstract

Guarini et al. (2011), reporting in Nature, identify a mechanism for fine-tuning endothelial Notch signaling by SIRT1-mediated deacetylation. This regulation is critical for vascular sprouting and raises intriguing questions about therapeutic targeting of SIRT1 in angiogenesis and the potential mechanisms that link vascular growth and energy homeostasis.

What makes the Notch pathway tick? The unique mechanism of Notch activation by proteolytic cleavage and the ability of Notch to regulate transcription is well appreciated (Kopan and Ilagan, 2009). Unlike a clock that must function with steadfast regularity, you cannot simply wind the spring that drives Notch and watch it go. Notch signaling must change constantly in response to local environmental cues. Notch researchers know that having the right level of Notch signaling at the right time is all-important, but the ways in which Notch is controlled in such an exquisitely precise fashion is still a subject of wonder, and mystification. Recently, we learned that each and every time an endothelial cell breaks off from an existing blood vessel to follow its VEGF-driven calling in angiogenesis, it must decide what the status of its integral Notch signal is (Jakobsson et al., 2010). This status may change at a moment's notice, necessitating equally rapid changes in Notch activity.

In a recent issue of Nature, Guarani et al. (2011) report a mechanism for fine-tuning Notch signaling in endothelial cells. The authors identified reversible acetylation of the intracellular domain of Notch1 as a “molecular mechanism to adapt the dynamics of Notch signaling”. This mechanism acts through SIRT1, a protein deacetylase that has been linked to diverse biological processes from metabolism to aging to cancer (Brooks and Gu, 2009). A member of the sirtuin family of proteins, SIRT1 is distinguished by a unique dependence on NAD+ for catalysis. SIRT1 is induced by caloric restriction, and in turn, the deacetylase activity of SIRT1 targets diverse proteins involved in metabolism and energy homeostasis. SIRT1-dependent physiological changes under caloric restriction have been linked to increased longevity (Brooks and Gu, 2009).

Using a very comprehensive and definitive series of biochemical experiments, the authors show that acetylation of the Notch1 intracellular domain (N1ICD) occurs on conserved lysines, and that SIRT1 binds to N1ICD and promotes deacetylation. Notch protein stability is controlled by ubiquitin-mediated degradation (Wu et al., 2001) and acetylation of proteins can impair ubiquitination (Brooks and Gu, 2009). Thus, the authors demonstrate that the state of N1ICD acetylation controls the level of protein and thus Notch activity. The implication for cellular behavior in response to Notch signaling is clear: SIRT1 activity may control the amplitude and duration of Notch responses. To explore this concept, the authors turn to the study of angiogenesis, choosing to conduct experiments with cultured endothelial cells or in zebrafish and mice. Guarini et al. (2011) demonstrate that the development of angiogeneic sprouts depends on SIRT1 activity to complete the process of building new vasculature. Endothelial cells lacking SIRT1 activity display stronger responses to Notch signal activation; these cells have stalk cell phenotypes, indicative of high Notch signaling. In loss of function studies in vivo, reduced Sirt1 in zebrafish and mice caused reduced vascular branching and density as a consequence of enhanced Notch signaling.

Can the fact that SIRT1 fine-tunes the Notch pathway in endothelial cells be harnessed for therapeutic effect? Notch is an intensely explored area in tumor angiogenesis, with the Notch ligand DLL4 representing a key target in tumor endothelium. DLL4 blockade causes excessive angiogenic sprouting, leading to poorly developed vessels that lack perfusion and thus poorly nourished tumors (Thurston and Kitajewski, 2008). In considering the targeting of tumor vasculature, one may propose to increase SIRT1 activity with agonists to reduce Notch signaling. This in turn would promote the type of abnormal sprouting elicited by Dll4 blockade or tumor phenotypes associated with combined blockade of DLL4 and JAGGED1 (Funahashi et al., 2008). Guarini et al. (2011) focus their analysis on endothelial cells and vasculature growth; however, they do not establish if SIRT1 is a general regulator of N1ICD protein levels in multiple tissue or cell types. The relationship between SIRT1 and tumor biology is complex. SIRT1 has a repressive action against p53. Thus, activators of SIRT1, such as resveratrol, may act to reduce mutated p53 at the protein level, possibly restoring the balance of tumor suppressor activity (Brooks and Gu, 2009). However, the relevance of such effects in vivo may not be so simple, and one must consider that the therapeutic potential of SIRT1 may depend on the genetic state of the tumor cell (Brooks and Gu, 2009). Would SIRT1 agonists be appropriate in situations where Notch activation drives oncogenic growth, such as in T-cell acute lymphoblastic leukemia (T-ALL) (Paganin and Ferrando, 2011)? To complicate matters, SIRT1 can have activities that promote Notch signaling, in one case through the regulation of the metalloprotease ADAM10 in neuronal cells (Donmez et al., 2010). However, although there are potential caveats, as agonists of SIRT1 could both promote development of non-functional tumor vasculature and the reduction of mutant p53, SIRT1 activation does represents the most promising approach to pursue for therapeutics against tumorigenesis.

Certainly one of the most interesting considerations regarding SIRT1-mediated regulation of endothelial Notch is the prospect that the authors have discovered a link between vascular growth and energy homeostasis. SIRT1 catalytic activity is dependent on NAD+ and thus its activity is responsive to changes in the metabolic and redox state of the cell. Little is known regarding cellular sensors responsible for coupling energy and oxygen homeostasis to angiogenesis. Could SIRT1 fine-tune Notch-dependent control of sprouting angioenesis in response to caloric restriction? Could this occur through the generation of metabolites by endothelial cells that affect NAD+ levels in response to angiogenic factors, such as VEGF? These questions will likely soon be addressed by new studies, and the answers will no doubt have consequences for both our understanding of vascular growth and of complex human diseases with vascular components, such as diabetes and obesity.