Copyright © 2007, European Society of Cardiology
ERK and Smads: Getting together for angiogenic sprouting
Signal Transduction Laboratory, Methodist Research Institute, Indianapolis, IN, USA
Department of Pharmacology Indiana University School of Medicine, Indianapolis, IN, USA
*Signal Transduction Laboratory, Methodist Research Institute, Clarian Health Partners, 1800 N. Capitol Blvd, Noyes Bldg, Room E504, Indianapolis, IN, 46202, USA. Tel.: +1 317 962 6891; fax: +1 317 962 9369. mrizzo{at}clarian.org
See article by Zhou et al. [8] (pages 390–399) in this issue.
Sprouting of new blood vessels from preexisting capillaries is a multi-faceted and highly orchestrated process that is dependent on a finely tuned balance between pro-angiogenic and anti-angiogenic microenvironmental clues [1]. Perturbations in the equilibrium between pro-angiogenic and anti-angiogenic factors can lead to excessive or defective vascularization [1,2]. Inputs from the microenvironment impinge upon receptive endothelial cells to initiate a multi-step response characterized by degradation of the basement membrane, sprout formation, proliferation, migration along the sprouts and organization into a tubular network of vascular-like structures that ultimately, with the recruitment of mural cells, acquire the anatomical and functional characteristics of mature blood vessels [1]. Microenvironmental clues originating from angiogenic protein and lipid growth factors, tissue oxygenation, adjacent cells, or components of the extracellular matrix are processed by the endothelial cells via intracellular and intercellular signaling pathways that are temporally and spatially segregated to coordinate the initiation, progression and completion of each stage of the angiogenic response [1,3,4]. These signaling pathways, rather than following a linear path, are organized via a cascade of signaling molecules, scaffolding proteins and multi-domain adaptor proteins into highly interconnected networks that provide endothelial cells with a myriad of opportunities for signal integration and crosstalk [5–7]. These complex signaling networks serve to confer a high degree of specificity to endothelial cell angiogenic decisions and to ensure that a sophisticated cellular response, such as the formation of new blood vessels, is successfully carried out. Despite considerable progress in our understanding of the players and dynamics of the angiogenic response, little is known on the signaling networks that govern this response during physiological conditions, and more importantly, on whether and how these networks are distorted in disease-associated angiogenesis.
The study by Zhou et al. published in this issue of Cardiovascular Research provides evidence that a crosstalk between the extracellular-signal regulated kinase (ERK) and Smad signaling pathways optimizes endothelial cell sprouting in response to bone morphogenic protein-4 (BMP-4) [8].
ERK and the signal transducer proteins Smads play fundamental roles in signaling networks [9,10]. ERK acts in a coordinated fashion to allow the transfer of signals from the extracellular milieu to the cytosol or the nucleus, leading to activation of specific targets and gene programs, including those involved in angiogenesis [9]. Several studies have emphasized the crucial role of ERK activation in regulation of endothelial cell functions during angiogenesis [11,12]. Smad proteins act as transcription factors and transduce signals generated upon cell activation by members of the transforming growth factor-β (TGF-β) superfamily, including BMP-4 [10]. Upon binding to its receptors, BMP-4 phosphorylates Smad1 and Smad5, which then associate with Smad4 and form a complex that translocates into the nucleus to regulate transcription of target genes, including Smad6 [10]. Smad6 functions as a negative feedback molecule by shutting off BMP-4-dependent signaling [10]. Because of several interactive motifs in their MH1 and MH2 domains, Smad proteins can interact and crosstalk with diverse signaling partners, including ERK [13]. The presence and functional consequences of ERK and Smad interactions in endothelial cell functions are presently unknown.
In their work, Zhou et al. showed that ERK and Smad 1/5 are rapidly and transiently phosphorylated upon challenge of human umbilical vein endothelial cells with BMP-4. Inhibition of MEK markedly decreased ERK activation and capillary sprouting in response to BMP-4, but had no effect on Smad 1/5 phosphorylation. Moreover, siRNA-mediated knockdown of Smad4 did not affect the ability of BMP-4 to stimulate vessel sprouting [8]. These findings differ from a recent report indicating that targeted deletion of endothelial Smad4, obtained using the Cre-Lox system, attenuated endothelial cell tube formation in vitro [14]. One potential explanation for this discrepancy is that Smad4 contribution to angiogenesis varies depending on the stimulus, cell type and context. The findings of Zhou et al. suggest that BMP-4-dependent signals leading to vessel sprouting are transduced by ERK. However, it is tempting to speculate that, in addition to ERK, Smad 1/5 could mediate BMP-4-induced capillary sprouting by forming a complex not with Smad4 but with other partners, similar to the recently proposed model for TGF-β-induced differentiation of hematopoietic cells [15]. Notably, Zhou and colleagues provide the first evidence that Smad6 functions as an endogenous inhibitor of ERK activation. Accordingly, overexpression of Smad6 had no effect on BMP-4-induced Smad 1/5 phosphorylation, but abrogated BMP-4-induced ERK activation in a MEK-independent fashion. The stimulatory effect of BMP-4 on capillary sprouting was also blunted by overexpression of Smad6. Conversely, silencing of Smad6 enhanced capillary sprouting in the absence of BMP-4. Importantly, stimulation of endothelial cells with BMP-4 increased endogenous Smad6 expression [8]. Additional modes of interaction between ERK and Smad pathways were investigated. Surprisingly, while pharmacological inhibition of MEK had no effect on BMP-4-induced Smad 1/5 phosphorylation, overexpression of a constitutively active MEK construct led to robust Smad 1/5 phosphorylation in the absence of BMP-4 stimulation [8]. Aside from the caution that one must exert when interpreting results obtained with overexpression of constitutively active constructs to study ligand-dependent responses, the findings of Zhou et al. suggest the fascinating possibility that endogenous activation of the inhibitory Smad6 by BMP-4 serves to control the magnitude and duration of ERK activation, while MEK-dependent phosphorylation of Smad 1/5 may lead, via activation of Smad4, to stimulation of Smad6 expression, thus contributing to the maintenance of the inhibitory feedback loop.
The work of Zhou et al. raises several intriguing questions. What are the mechanisms of ERK inhibition by Smad6? What are the functional consequences of this inhibitory feedback loop to angiogenesis in vivo? To which degree can the data obtained in human umbilical vein endothelial cells be generalized to endothelial cells of other tissue origins? While answers to these questions await future studies, the findings of Zhou et al. provide a paradigm for the contribution of signaling crosstalk in dictating the angiogenic outcome of BMP-4. Given the side effects and potential resistance associated with the currently known anti-angiogenic agents [2], a better understanding of the wiring of angiogenic signaling circuits should accelerate the development of more selective and less toxic therapies for the treatment of dysregulated angiogenesis.
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Acknowledgement |
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Support from the Showalter Cardiovascular Foundation is gratefully acknowledged. Conflict of Interest: none declared.
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