Published online before print July 2, 2009, doi: 10.1161/CIRCRESAHA.109.201186
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2009 American Heart Association, Inc.
Molecular Medicine |
G Protein-Coupled Receptor APJ and Its Ligand Apelin Act Downstream of Cripto to Specify Embryonic Stem Cells Toward the Cardiac Lineage Through Extracellular Signal-Regulated Kinase/p70S6 Kinase Signaling Pathway
From the Stem Cell Fate Laboratory (C.D., E.L., S.I., O.G., G.M.), Institute of Genetics and Biophysics "A. Buzzati-Traverso," Consiglio Nazionale delle Ricerche, Naples, Italy; Institute of Genetics and Biophysics "A. Buzzati-Traverso" (C.D., E.L., S.I., O.G., A.M.L., G.L.L., G.M.), Consiglio Nazionale delle Ricerche, Naples, Italy; Vesalius Research Center (M.A., P.C.), VIB, Leuven, Belgium; and Vesalius Research Center (P.C.), Katholieke Universiteit Leuven, Belgium.
Correspondence to Gabriella Minchiotti, Institute of Genetics and Biophysics "A. Buzzati-Traverso," CNR, Via Pietro Castellino 111, 80131 Naples, Italy. E-mail minchiot{at}igb.cnr.it
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Rationale: Pluripotent stem cells represent a powerful model system to study the early steps of cardiac specification for which the molecular control is largely unknown. The EGF-CFC (epidermal growth factor-Cripto/FRL-1/Cryptic) Cripto protein is essential for cardiac myogenesis in embryonic stem cells (ESCs).
Objective: Here, we study the role of apelin and its G protein-coupled receptor, APJ, as downstream targets of Cripto both in vivo and in ESC differentiation.
Methods and Results: Gain-of-function experiments show that APJ suppresses neuronal differentiation and restores the cardiac program in Cripto–/– ESCs. Loss-of-function experiments point for a central role for APJ/apelin in the gene regulatory cascade promoting cardiac specification and differentiation in ESCs. Remarkably, we show for the first time that apelin promotes mammalian cardiomyogenesis via activation of mitogen-activated protein kinase/p70S6 through coupling to a Go/Gi protein.
Conclusions: Together our data provide evidence for a previously unrecognized function of APJ/apelin in the Cripto signaling pathway governing mesoderm patterning and cardiac specification in mammals.
Key Words: embryonic stem cells • cardiomyogenesis • cripto • apelin • APJ/msr1
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The earliest event in cardiogenesis is commitment of mesodermal cells to a cardiogenic fate and their migration into the anterolateral region of the embryo during gastrulation.1 It is therefore important to understand how mesodermal cells are instructed to assume a cardiac fate to elucidate the molecular mechanisms later in heart development. In mammals, these instructive events are largely unknown. Their identification could provide insights into pathways governing cell lineage specification and differentiation, including transcription factor network and extracellular cues that activate them.2 In addition, understanding early cardiogenesis is of particular interest because cardiomyocyte loss from damage in mammals is largely irreversible and frequently underlies impaired cardiac function in individuals with heart disease. Although there are still multiple barriers to successful regenerative therapies for cardiac disease using embryonic or adult stem cells, cell-based therapeutic approaches remain a valuable goal, particularly when using strategies that do not cross species barriers.3,4 In this light, embryonic stem cells (ESCs), which faithfully recapitulate early stages of cardiac cell commitment and differentiation, provide a powerful model for investigating how best to control the earliest events in mammalian cardiomyogenesis and ultimately enhance differentiation efficiency.
Proteins essential for heart induction have been studied extensively in ESCs, which includes Wnt/β-catenin, transforming growth factor-β family, bone morphogenetic proteins and Cripto.5–7 Cripto is a glycosylphosphatidylinositol-anchored multifunctional protein that is involved in the activation of a complex network of signaling pathways both in development and tumorigenesis.8,9 Cripto stimulates signaling by the transforming growth factor β-family member Nodal or related ligands growth/differentiation factor (GDF)1 and -3,10,11 through activin type IB (activin receptor-like kinase [ALK]-4) and activin type IIB serine/threonine kinase receptors.10,12,13 Besides its well-documented stimulatory effect on the canonical Nodal-GDF1-3/ALK-4/Smad2 pathway, Nodal/ALK-4-independent Cripto activities have also been described.9,14 Notably, recent data highlight a novel role of Cripto as Activin/transforming growth factor-β antagonist.15,16
We have previously showed that Cripto acts through the Nodal/ALK-4/Smad2 pathway to negatively regulate neural differentiation and to permit the entry of ESCs into a cardiac lineage.17 Accordingly, recent data pointed for a key role of Nodal/Cripto-dependent early activation of Smad2, which was indispensable for mesendodermal induction and the subsequent cardiac differentiation of ESCs.18 However, little is yet known about the mechanisms of action and the identity of the factors downstream of Cripto/Smad2 in mammalian cardiomyogenesis.
Here, we report the identification of the apelin receptor APJ (also known as angiotensin type I-like receptor [AGTRL-1] and msr1) and its ligand apelin as previously unrecognized downstream targets of Cripto and provide evidence that the APJ/apelin pathway redirects the neural fate of ESCs in the absence of Cripto and promotes cardiac differentiation. APJ is a G protein-coupled receptor, and its ligand apelin is a peptide originating from the larger precursor preproapelin molecule19; recently, emerging as an important regulator of cardiovascular homeostasis and angiogenesis.20–23 Remarkably, recent data in Zebrafish provided the first evidence that the APJ/apelin pathway is required, at the onset of gastrulation, to mediate migration of myocardial progenitors to the correct position into the anterior lateral plate mesoderm24,25; nevertheless, their function in mammalian cardiomyogenesis is still unproven. Here, we reveal a previously undescribed functional link between Cripto/Smad2 and APJ/apelin in the signaling pathways that redirect the neural fate of ESCs and promote cardiomyogenesis in mammals.
|
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
ESC Differentiation, Western Blot, and Immunofluorescence Analysis
The ESC lines RI and Cripto–/– DE717 were used throughout the study. Western blot and immunofluorescence analysis were performed as previously described.17 Anti-βIII-tubulin (Sigma), anti-V5 (Invitrogen), anti-GAPDH (AbCam), anti-phospho-extracellular signal-regulated kinase (ERK), anti-ERK, and anti-p70S6 kinase (p70S6K) (Cell Signaling) antibodies were used according to the instructions of the manufacturers. Monoclonal anti-βIII-tubulin (1:400; Sigma-Aldrich) and MF20 (1:50; obtained from the Developmental Studies of Hybridoma Bank, University of Iowa) were used as previously described.17 Apelin (13) peptide was purchased from Sigma-Aldrich.
ESC Transfection and Plasmids
Murine msr1 cDNA was subcloned into the pEF1a vector (Clontech) to generate an msr1-V5 fusion protein and then cloned into the pallino βA vector for expression in ESCs.17
Short hairpin (sh)RNAs vectors (pGIPZ lentiviral, pLKO vectors; OpenBiosystem) were used accordingly to the instructions of the manufacturer. Nucleofector Technology (AMAXA) was used for ESC transfection, according to the instructions of the manufacturer.
RNA Preparation and RT-PCR
Total RNAs were isolated using RNeasy Mini Kit (Qiagen) and reverse-transcribed using SuperScript II reverse transcriptase (Invitrogen Life Technologies Inc) and random hexamers, according to the instructions of the manufacturer. Quantitative real-time PCR was performed using SYBR Green PCR master mix (EuroClone). The primers used are described in Table I in the Online Data Supplement, available at http://circres.ahajournals.org.
Whole-Mount In Situ Hybridization
Embryos were dissected in PBS and fixed in 4% paraformaldehyde in PBS at 4°C for 2 to 16 hours; whole-mount in situ hybridization was performed as previously described.26
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Complementary Expression of APJ, Apelin, and Cripto in ESC Cardiac Differentiation and Gastrulating Embryos
In searching for genes that might act in concert with Cripto to promote cardiomyogenesis, we were interested in the G protein-coupled receptor APJ (also known as angiotensin type I-like receptor [AGTRL-1]) and its ligand apelin. APJ and apelin have been recently described as important regulators of cardiovascular function both in Xenopus21 and in Zebrafish,24,25 yet a role for these proteins in mammalian cardiogenesis is still unproven. We therefore determined whether they were involved in cardiogenesis in concert with Cripto. Notably, both Apj and apelin mRNAs were upregulated in Cripto–/– embryoid bodies (EBs) exposed to recombinant Cripto for 24 hours in a microarray analysis (data not shown). We thus compared the expression profile of both Apj and apelin in wild-type and Cripto–/– ESCs that were allowed to differentiate toward cardiomyocytes. Real-time PCR showed that Apj and apelin expression was reduced in Cripto–/– ESCs, whereas both genes were markedly upregulated in wild-type ESCs (Figure 1A).
|
3). B and B', Whole-mount in situ hybridization of Apj, apelin, and Cripto in wild-type embryos at 6.7 days postconception (B) and Apj and apelin in Cripto–/– embryos at 7 days postconception (B'). C and D, Smad2 inhibition on Apj, apelin, and Cripto expression. Two-day-old wild-type EBs treated (24 hours) with increasing amounts of SB-431542 (SB) or DMSO (vehicle); expression of Apj, apelin, Cripto (C), and NeuroD (D) by QRT-PCR. mRNA was normalized to GAPDH and presented as fold change in gene expression relative to the control (DMSO); data are means±SE (nTo extend our analysis in vivo, we compared the expression profile of Cripto with that of Apj and apelin in mouse embryos. To our knowledge, no previous studies have described apelin/Apj expression in early embryogenesis; ie, before the late head-fold stages (embryonic day 8).27 Whole-mount in situ hybridization on gastrulating wild-type and Cripto–/– embryos revealed that the expression of both Apj and apelin correlates with that of Cripto (Figure 1B). Apelin mRNA clearly identifies the developing primitive streak; whereas, Apj expression domain is detectable both in the primitive streak and adjacent mesoderm, rather similar to Cripto. Of note, both Apj and apelin mRNA were also detected in the extraembryonic mesoderm (Figure 1B). In line with ESCs results, Apj and apelin expression was almost undetectable in Cripto–/– embryos, specifically in the posterior mesoderm. Cripto is essential for both primitive streak and anterior-posterior axis formation; however, Cripto–/– embryos express posterior markers such as Brachyury and form anterior neural structures and extraembryonic mesoderm.26,28 Remarkably, Apj and apelin expression persisted in the extraembryonic tissue of Cripto–/– embryos, thus indicating that their expression is Cripto-dependent in the embryonic mesoderm and Cripto-independent in the extraembryonic tissue (Figure 1B').
Early Activation of Smad2 Is Critical for the Expression of Cripto, Apj, and Apelin in Cardiomyogenesis of ESCs
Recent data indicated that early activation of Smad2 was indispensable for mesendodermal induction and subsequent cardiac differentiation of ESCs.18 We also previously showed that Cripto acts via the Nodal/ALK-4/Smad2 pathway to induce ESC cardiomyogenesis.17 We thus asked whether early activation of Smad2 induced Apj and/or apelin expression. Two-day-old wild-type EBs were therefore treated with SB-431542, a specific inhibitor of ALK-4–, -5-, -7-dependent Smad2 activation29 or left untreated as control. SB-431542 reduced both Apj and apelin expression (Figure 1C). Notably, recent data showed that human Cripto is a direct target of Smad430; accordingly, SB-431542 affected Cripto expression as well. By contrast, as previously described, expression of neuroectodermal marker Neurod1 was upregulated (Figure 1D).18 Comparable results were obtained with Smad2-silenced ESCs (Online Results and Online Figures I and II).
Together, our data indicate that that early activation of Smad2 is critical for induction of Cripto, Apj, and apelin expression and the consequent inhibition of the neuronal fate.
APJ Overexpression Redirects the Neural Fate of Cripto–/– ESCs
Previous data revealed an essential role of Cripto in redirecting the neuronal fate and promoting cardiomyogenesis in ESCs.17 Therefore, we evaluated whether forced overexpression of Apj might redirect the neural fate of ESCs and thus compensate for the lack of Cripto in cardiomyogenesis. A recombinant vector encoding an APJ/V5-tagged protein was transfected into Cripto–/– ESCs and APJ/V5 overexpression, and its membrane localization was verified in 3 independent clones (Figure 2A and 2B). Following characterization, both control and APJ-overexpressing Cripto–/– ESCs were allowed to differentiate toward cardiomyocytes.17 As expected, when Cripto–/– EBs were plated onto an adhesive substrate, a population of cells with a neuron-like morphology was observed, which produce a dense neural network. This characteristic morphology was never observed either in wild-type or in Cripto–/– APJ/V5 EBs (data not shown). Notably, overexpression of Apj in Cripto–/– ESCs reached levels comparable to that of WT ESCs, as shown by quantitative RT-PCR (QRT-PCR) (Figure 1A and Online Figure III). These data suggested that Apj overexpression likely prevented neuronal differentiation of Cripto–/– ESCs.
|
To address this issue directly, immunofluorescence analysis was performed using anti-βIII-tubulin antibodies, which recognize the neuron-specific form of class III β-tubulin. To semiquantify the activity of APJ, we arbitrarily defined 4 grades of neuronal differentiation ranging from the absence of neurons (grade 0) to full neuronal differentiation (grade 3), ie, presence of a dense network of βIII-tubulin-positive cells. The presence of either few isolated neurons or areas of βIII-tubulin-positive cells defined intermediate phenotypes, named grade 1 and grade 2, respectively (Figure 3A). Results clearly showed a negative effect of APJ on neuronal differentiation of Cripto–/– ESCs. Indeed, most, if not all, APJ/V5 Cripto–/– EBs scored showed poor neuronal differentiation (Figure 3B). Accordingly, βIII-tubulin expression was reduced, as shown by Western blot analysis (Figure 3C). Notably, in 1 of 3 clones, βIII-tubulin levels were comparable to control cells, which was likely attributable to an atypically high percentage of grade 1 EBs. Finally, we found downregulated expression of transcripts encoding neuronal transcription factors NeuroD and neurofilament M (Figure 3D and data not shown).
|
60 (n
APJ and Apelin Drive ESCs Toward the Cardiac Lineage
Given the ability of APJ to redirect the neural fate of ESCs in the absence of Cripto, we examined whether APJ overexpression might rescue the genetic program of cardiac differentiation in Cripto–/– EBs. To this end, both control and APJ-overexpressing Cripto–/– ESCs were allowed to differentiate toward cardiomyocytes and the expression profile of selected markers was evaluated (Figure 4). Notably, APJ overexpression cells resulted in the induction of the pan-mesodermal marker T/Bra, as well as the earliest cardiac marker Mesp1. Mesp1 functions subsequent to Bra as the earliest molecular marker of cardiac precursors that migrate through the primitive streak and is essential for cardiac myogenesis in committed mesodermal cells.31,32 Consistent with a restored cardiogenic program in APJ-overexpressing Cripto–/– ESCs, the transcription factor Mef2C, Nkx2.5, Tbx5, and Gata4, as well as the cardiac structural gene Mlc2v were upregulated, and their expression profile correlates with that of wild-type EBs (Figure 4).2 Furthermore, upregulation of Afp (extraembryonic endoderm marker) also suggested that APJ overexpression induced the mesendodermal program in the absence of Cripto (Figure 4). By contrast, beating EBs did not form, even after long-term culture, and 
-myosin heavy chain (MHC)-positive cells were almost absent (data not shown).
|
Of note, apelin expression increased on APJ overexpression and persisted throughout ESC differentiation (Online Figure III, A). Nevertheless, the transient nature of apelin accumulation in wild-type EBs suggested that its activity might be required at a defined step in cardiomyocyte differentiation (Figure 1A). We thus determined whether addition of apelin might redirect the neural fate of Cripto–/– ESCs and/or improve the effect of APJ overexpression on cardiomyogenesis. To this end, apelin was added to the cells every 24 hours during the 2- to 4-day interval of differentiation, which reflected the peak of endogenous apelin expression. Interestingly, expression of the cardiac structural gene Mlc2v significantly increased (Online Figure III, B); however, it did not induce beating EBs. Notably, apelin treatment of Cripto–/– EBs reduced the expression of NeuroD (Online Figure III, B), suggesting that it is antagonizing neuronal differentiation. Apelin activity in Cripto–/– EBs was likely attributable to residual Apj expression; however, we cannot completely rule out the possibility of an APJ-independent apelin activity. Remarkably, addition of apelin to wild-type EBs did not further enhance cardiac differentiation, as revealed by expression Mlc2v and MHC, which was comparable in control and apelin-treated EBs (Online Figure III, C). Most likely, APJ/apelin signaling becomes saturated in wild-type EBs, and thus addition of apelin cannot further activate the pathway.
To assess the role of APJ/apelin signaling on cardiomyogenesis, silencing experiments were performed, using 2 shRNAs, which targeted nonoverlapping apelin mRNA sequences. To minimize effects of clone-to-clone variation, independent ESC clones for each shRNA were isolated, with a silencing efficacy in the range of 80% to 95% (Figure 5A). These clones showed suppression of cardiac differentiation (Figure 5B), with few and isolated, if any, MHC immunoreactive cells (Figure 5C). Morphological observation was supported by molecular analysis, showing downregulation of MHC expression (data not shown and Figure 6B).
|
|
The transient nature of apelin expression suggested that its activity might be required at a defined step in cardiomyogenesis. To address this issue, apelin signaling was reconstituted by adding apelin peptide to the EBs (Figure 5B'). This treatment restored cardiac differentiation of apelin-silenced ESCs (Figure 5B and 5C), thus providing the first direct evidence that apelin is required in an early window for priming differentiation of ESCs to a cardiac fate. We then went on to determine whether apelin silencing might be able to promote neuronal differentiation, as in Cripto–/– ESCs. As expected, βIII-tubulin accumulated in Cripto–/– EBs, whereas it was undetectable in EBs derived from ESCs expressing either nontargeting shRNA or apelin shRNAs (Figure 6A), thus indicating that apelin was not able to redirect the neural fate of ESCs.
Next, we examined at which step of cardiac myogenesis apelin functions (Figure 6B). Remarkably, apelin shRNA did not significantly affect expression of Brachyury/T. By contrast, the earliest cardiac marker Mesp1 and the cardiac genes Nkx2.5 and MHC were downregulated in apelin silenced compared to control ESCs. Together, these data indicated that APJ/apelin signaling was pivotal in promoting ESC cardiac specification and differentiation. Accordingly, Apj silencing impaired ESC cardiac differentiation (Online Figure IV).
Apelin Promotes Cardiomyogenesis via Mitogen-Activated Protein Kinase/p70S6 Through Coupling to a Pertussis Toxin-Sensitive GTP-Binding Protein
Previous data showed that apelin induced activation of ERKs and AKT and that this resulted in the activation of p70S6K.33 To gain insight into the molecular basis of APJ/apelin signaling in cardiomyogenesis, we first evaluated whether addition of apelin induced mitogen-activated protein kinase (MAPK) and AKT signaling. Interestingly, phosphorylation ERK but not AKT (data not shown) was transiently induced in 4-day-old EBs treated with apelin (Figure 7A); accordingly, p70 S6K was rapidly phosphorylated at residues T421/S424 but not at T389, which is selectively phosphorylated on AKT activation (Figure 7A).33 We thus performed the rescue assay described above (Figure 5B and 5B') in the presence of U0126, a specific MAPK inhibitor (Figure 7B). As expected, apelin rescued cardiac differentiation. Most remarkably, this effect was fully abolished in the presence of U0126 (Figure 7C), thus providing evidence that apelin promotes cardiomyogenesis through the activation of MAPK. Finally, because the apelin receptor is a G protein-coupled receptor, we decided to characterize the G protein that transduced its activation in cardiomyogenesis. Pretreatment of EBs with pertussis toxin (PTX) fully abrogated the ability of apelin to promote cardiomyogenesis (Figure 7D), thus extending previous findings on APJ overexpressing cells, on the PTX-sensitive activation of ERKs by apelin.34
|
In conclusion, our data provide the first direct evidence that the MAPK is activated by apelin through coupling to a Go/Gi protein, which contributes to mammalian cardiomyogenesis.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Dissecting the extracellular signals and their intracellular effectors controlling the early steps of mammalian cardiomyogenesis provide important insight into the mechanisms underlying cardiac fate specification.
Our findings identify APJ and apelin as novel extracellular signals that act downstream of Cripto in the early phase of mesoderm patterning and cardiac specification in mammals. Expression analysis both in ESCs and gastrulating embryos showed that apj and apelin expression correlated with that of Cripto. More interestingly, using the chemical inhibitor of ALK receptors SB-431542 and an shRNA-based approach, we provide evidence that expression of both genes is Cripto/Smad2-dependent. In line with our results, it has been recently shown that early activation of Smad2 is required for mesendodermal induction and patterning in ESCs and that once this pathway is inhibited, cardiomyogenesis is reduced and neuroectodermal induction is augmented.18 Our data identify Apj and apelin as effectors of Cripto/Smad2 pathway in these circuits. Accordingly, gain-of-function experiments show that APJ redirects the neural fate of Cripto–/– ESCs and restores mesendodermal patterning and the cardiogenic program. Expression of Mesp1, which is the earliest cardiac transcription factor required for cardiac morphogenesis,31,32 is induced by APJ overexpression. Notably, Mesp1 acts as a key molecular switch in the specification of multipotent cardiovascular progenitors from ESCs, residing at the top of the hierarchy of the cardiovascular transcriptional network.31,35,36 In line with these findings, APJ overexpression is able to restore the correct timing of cardiac gene expression; however, it fails to induce beating EBs in the absence of Cripto. The incomplete rescue of cardiomyogenesis in APJ-overexpressing Cripto–/– ESCs could have different reasons. First of all, the APJ ligand apelin might represent the major limiting factor. However, apelin is induced in APJ-overexpressing Cripto–/– EBs, suggesting that APJ and apelin interact through a positive regulatory loop. Moreover, further addition of apelin peptide to APJ-overexpressing EBs does not induce the beating phenotype. Remarkably, overexpression experiments do not allow the modulation of receptor signaling either in terms of timing or signal strength; thus, we can only measure the effect of constitutive but not transient activation of APJ signaling. In line with this hypothesis, we show that whereas apelin is transiently expressed in wild-type ESC differentiation, it accumulates throughout differentiation in APJ-overexpressing Cripto–/– ESCs. Interestingly, the introduction of excess of apelin in zebrafish embryos was found to impair gastrulation and block myocardial differentiation. These findings thus suggest that loss of a localized source of apelin did not allow cells fated to form the myocardium to reach their localization.25,37
In line with a functional role of APJ/apelin pathway in mammalian cardiomyogenesis, we provide evidence that apelin is crucial for cardiac differentiation in ESCs; however, unlike Cripto, apelin is not able to redirect the neural fate of ESCs. Remarkably, expression of Mesp1 but not Brachyury/T was reduced in apelin-silenced EBs, thus indicating that the action of apelin is preferential for mesoderm patterning more than mesoderm formation. Similarly, both the HMG-box transcription factor Sox17 and Notch signaling have been described as key players in the molecular events, which lead to Mesp1 induction in ESCs and in the embryo, respectively.2,38
In line with our findings, apelin-null mice do not show any early embryonic patterning defects,37,39 nor have Apj and apelin-null mice been reported to have congenital cardiac defects.39,40 Possibly, either cardiac differentiation was insufficiently examined or there might be genetic redundancy. It has been recently hypothesized that in the embryo, cardiac myogenesis might rely on a more complex ensemble of signals and mediators and be easier to compensate than in ESC differentiation; thus, embryonic development might be less vulnerable than EBs to loss of genes of early cardiomyogenesis.2
Remarkably, although recent experiments in zebrafish showed a critical role for APJ/apelin pathway in myocardial cell specification and heart development,24,25 the molecular mechanism involved was unproven. Our study demonstrates that apelin induces p70S6K phosphorylation through the activation of the ERK cascade in mammalian cardiac differentiation. Most remarkably, we have found that the blockade of ERKs, by the specific inhibitor U0126, not only abrogates the phosphorylation of p70S6K but also prevents apelin-induced cardiomyogenesis. Moreover, we directly prove that there is a PTX-sensitive GTP-binding protein in the transduction cascade leading to apelin-induced cardiomyogenesis.
Previous data in primary endothelial cells showed that stimulation of p70S6K by apelin proceeds through activation of two signaling cascades, implicating AKT and ERK pathways, which are linked to a different pattern of p70S6K phosphorylation.33 Our data suggest that apelin activates ERK-dependent but not AKT-dependent p70S6K cascade in cardiomyogenesis. p70S6K is implicated in the regulation of cell cycle progression, and several studies have documented its central role in endothelial cell proliferation33,41; it is thus tempting to speculate that this mitogenic effect might be extended to cardiac precursors.
Besides its crucial role in embryo development and ESC differentiation,8,13 Cripto overexpression has been associated with tumor initiation and progression, including tumor angiogenesis9,42; however, the molecular mechanism is still not clear. Intriguingly, apelin has recently attracted considerable interest as potent inducer of tumor angiogenesis.43–45 It would thus be important in the future to determine whether Cripto activity in tumor angiogenesis correlates with the activation of the APJ/apelin system.
Taken together, our data provide new insights into understanding the molecular mechanisms underlying early cardiogenesis in mammals. This might be helpful in improving understanding of the etiology of cardiac disease and lead to ways of enhancing conversion of ESCs to cardiomyocytes, still an important bottleneck to their use in drug discovery and regenerative medicine. Further analysis of the regulatory networks that involve Apj/apelin will be required both in mouse and human ESCs, to reprogram ESCs toward a cardiovascular fate for cell therapy and cardiovascular tissue engineering.
|
Acknowledgments |
|---|
We thank Y. Audigier for the kind gift of APJ plasmid.
Sources of Funding
This work was supported by grants from the Associazione Italiana Ricerca sul Cancro (to G.M.).
Disclosures
None.
|
Footnotes |
|---|
Original received September 16, 2008; resubmission received May 21, 2009; revised resubmission received June 22, 2009; accepted June 23, 2009.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Garry DJ, Olson EN. A common progenitor at the heart of development. Cell. 2006; 127: 1101–1104.[CrossRef][Medline] [Order article via Infotrieve]
2. Liu Y, Asakura M, Inoue H, Nakamura T, Sano M, Niu Z, Chen M, Schwartz RJ, Schneider MD. Sox17 is essential for the specification of cardiac mesoderm in embryonic stem cells. Proc Natl Acad Sci U S A. 2007; 104: 3859–3864.
3. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac repair. J Clin Invest. 2005; 115: 572–583.[CrossRef][Medline] [Order article via Infotrieve]
4. Passier R, van Laake LW, Mummery CL. Stem-cell-based therapy and lessons from the heart. Nature. 2008; 453: 322–329.[CrossRef][Medline] [Order article via Infotrieve]
5. Olson EN, Schneider MD. Sizing up the heart: development redux in disease. Genes Dev. 2003; 17: 1937–1956.
6. Foley A, Mercola M. Heart induction: embryology to cardiomyocyte regeneration. Trends Cardiovasc Med. 2004; 1: 121–125.
7. Martin-Puig S, Wang Z, Chien KR. Lives of a heart cell: tracing the origins of cardiac progenitors. Cell Stem Cell. 2008; 2: 320–331.[CrossRef][Medline] [Order article via Infotrieve]
8. Minchiotti G. Nodal-dependant Cripto signaling in ES cells: from stem cells to tumor biology. Oncogene. 2005; 24: 5668–5675.[CrossRef][Medline] [Order article via Infotrieve]
9. Strizzi L, Bianco C, Normanno N, Salomon D. Cripto-1: a multifunctional modulator during embryogenesis and oncogenesis. Oncogene. 2005; 24: 5731–5741.[CrossRef][Medline] [Order article via Infotrieve]
10. Cheng SK, Olale F, Bennett JT, Brivanlou AH, Schier AF. EGF-CFC proteins are essential coreceptors for the TGF-beta signals Vg1 and GDF1. Genes Dev. 2003; 17: 31–36.
11. Chen C, Ware SM, Sato A, Houston-Hawkins DE, Habas R, Matzuk MM, Shen MM, Brown CW. The Vg1-related protein Gdf3 acts in a Nodal signaling pathway in the pre-gastrulation mouse embryo. Development. 2006; 133: 319–329.
12. Reissmann E, Jornvall H, Blokzijl A, Andersson O, Chang C, Minchiotti G, Persico MG, Ibanez CF, Brivanlou AH. The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development. Genes Dev. 2001; 15: 2010–2022.
13. Schier AF. Nodal signaling in vertebrate development. Annu Rev Cell Dev Biol. 2003; 19: 589–621.[CrossRef][Medline] [Order article via Infotrieve]
14. D'Andrea D, Liguori GL, Le Good JA, Lonardo E, Andersson O, Constam DB, Persico MG, Minchiotti G. Cripto promotes A-P axis specification independently of its stimulatory effect on Nodal autoinduction. J Cell Biol. 2008; 180: 597–605.
15. Shani G, Fischer WH, Justice NJ, Kelber JA, Vale W, Gray PC. GRP78 and Cripto form a complex at the cell surface and collaborate to inhibit transforming growth factor beta signaling and enhance cell growth. Mol Cell Biol. 2008; 28: 666–677.
16. Gray PC, Shani G, Aung K, Kelber J, Vale W. Cripto binds transforming growth factor beta (TGF-beta) and inhibits TGF-beta signaling. Mol Cell Biol. 2006; 26: 9268–9278.
17. Parisi S, D'Andrea D, Lago CT, Adamson ED, Persico MG, Minchiotti G. Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells. J Cell Biol. 2003; 163: 303–314.
18. Kitamura R, Takahashi T, Nakajima N, Isodono K, Asada S, Ueno H, Ueyama T, Yoshikawa T, Matsubara H, Oh H. Stage-specific role of endogenous Smad2 activation in cardiomyogenesis of embryonic stem cells. Circ Res. 2007; 101: 78–87.
19. Choe H. Chemokine receptors in HIV-1 and SIV infection. Arch Pharm Res. 1998; 21: 634–639.[Medline] [Order article via Infotrieve]
20. Chen MM, Ashley EA, Deng DX, Tsalenko A, Deng A, Tabibiazar R, Ben-Dor A, Fenster B, Yang E, King JY, Fowler M, Robbins R, Johnson FL, Bruhn L, McDonagh T, Dargie H, Yakhini Z, Tsao PS, Quertermous T. Novel role for the potent endogenous inotrope apelin in human cardiac dysfunction. Circulation. 2003; 108: 1432–1439.
21. Inui M, Fukui A, Ito Y, Asashima M. Xapelin and Xmsr are required for cardiovascular development in Xenopus laevis. Dev Biol. 2006; 298: 188–200.[CrossRef][Medline] [Order article via Infotrieve]
22. Sorli SC, van den Berghe L, Masri B, Knibiehler B, Audigier Y. Therapeutic potential of interfering with apelin signalling. Drug Discov Today. 2006; 11: 1100–1106.[CrossRef][Medline] [Order article via Infotrieve]
23. Japp AG, Newby DE. The apelin-APJ system in heart failure: pathophysiologic relevance and therapeutic potential. Biochem Pharmacol. 2008; 75: 1882–1892.[CrossRef][Medline] [Order article via Infotrieve]
24. Scott IC, Masri B, D'Amico LA, Jin SW, Jungblut B, Wehman AM, Baier H, Audigier Y, Stainier DY. The G protein-coupled receptor agtrl1b regulates early development of myocardial progenitors. Dev Cell. 2007; 12: 403–413.[CrossRef][Medline] [Order article via Infotrieve]
25. Zeng XX, Wilm TP, Sepich DS, Solnica-Krezel L. Apelin and its receptor control heart field formation during zebrafish gastrulation. Dev Cell. 2007; 12: 391–402.[CrossRef][Medline] [Order article via Infotrieve]
26. Liguori GL, Echevarria D, Improta R, Signore M, Adamson E, Martinez S, Persico MG. Anterior neural plate regionalization in cripto null mutant mouse embryos in the absence of node and primitive streak. Dev Biol. 2003; 264: 537–549.[CrossRef][Medline] [Order article via Infotrieve]
27. Devic E, Rizzoti K, Bodin S, Knibiehler B, Audigier Y. Amino acid sequence and embryonic expression of msr/apj, the mouse homolog of Xenopus X-msr and human APJ. Mech Dev. 1999; 84: 199–203.[CrossRef][Medline] [Order article via Infotrieve]
28. Ding J, Yang L, Yan YT, Chen A, Desai N, Wynshaw-Boris A, Shen MM. Cripto is required for correct orientation of the anterior-posterior axis in the mouse embryo. Nature. 1998; 395: 702–707.[CrossRef][Medline] [Order article via Infotrieve]
29. Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2002; 62: 65–74.
30. Mancino M, Strizzi L, Wechselberger C, Watanabe K, Gonzales M, Hamada S, Normanno N, Salomon DS, Bianco C. Regulation of human Cripto-1 gene expression by TGF-beta1 and BMP-4 in embryonal and colon cancer cells. J Cell Physiol. 2008; 215: 192–203.[CrossRef][Medline] [Order article via Infotrieve]
31. Kitajima S, Takagi A, Inoue T, Saga Y. MesP1 and MesP2 are essential for the development of cardiac mesoderm. Development. 2000; 127: 3215–3226.[Abstract]
32. David R, Brenner C, Stieber J, Schwarz F, Brunner S, Vollmer M, Mentele E, Muller-Hocker J, Kitajima S, Lickert H, Rupp R, Franz WM. MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling. Nat Cell Biol. 2008; 10: 338–345.[CrossRef][Medline] [Order article via Infotrieve]
33. Masri B, Morin N, Cornu M, Knibiehler B, Audigier Y. Apelin (65-77) activates p70 S6 kinase and is mitogenic for umbilical endothelial cells. FASEB J. 2004; 18: 1909–1911.
34. Masri B, Lahlou H, Mazarguil H, Knibiehler B, Audigier Y. Apelin (65-77) activates extracellular signal-regulated kinases via a PTX-sensitive G protein. Biochem Biophys Res Commun. 2002; 290: 539–545.[CrossRef][Medline] [Order article via Infotrieve]
35. Bondue A, Lapouge G, Paulissen C, Semeraro C, Iacovino M, Kyba M, Blanpain C. Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification. Cell Stem Cell. 2008; 3: 69–84.[CrossRef][Medline] [Order article via Infotrieve]
36. Lindsley RC, Gill JG, Murphy TL, Langer EM, Cai M, Mashayekhi M, Wang W, Niwa N, Nerbonne JM, Kyba M, Murphy KM. Mesp1 coordinately regulates cardiovascular fate restriction and epithelial-mesenchymal transition in differentiating ESCs. Cell Stem Cell. 2008; 3: 55–68.[CrossRef][Medline] [Order article via Infotrieve]
37. Quertermous T. Apelin and its G protein-coupled receptor regulate cardiac development as well as cardiac function. Dev Cell. 2007; 12: 319–320.[CrossRef][Medline] [Order article via Infotrieve]
38. Takahashi Y, Yasuhiko Y, Kitajima S, Kanno J, Saga Y. Appropriate suppression of Notch signaling by Mesp factors is essential for stripe pattern formation leading to segment boundary formation. Dev Biol. 2007; 304: 593–603.[CrossRef][Medline] [Order article via Infotrieve]
39. Kuba K, Zhang L, Imai Y, Arab S, Chen M, Maekawa Y, Leschnik M, Leibbrandt A, Markovic M, Schwaighofer J, Beetz N, Musialek R, Neely GG, Komnenovic V, Kolm U, Metzler B, Ricci R, Hara H, Meixner A, Nghiem M, Chen X, Dawood F, Wong KM, Sarao R, Cukerman E, Kimura A, Hein L, Thalhammer J, Liu PP, Penninger JM. Impaired heart contractility in apelin gene-deficient mice associated with aging and pressure overload. Circ Res. 2007; 101: e32–e42.
40. Ishida J, Hashimoto T, Hashimoto Y, Nishiwaki S, Iguchi T, Harada S, Sugaya T, Matsuzaki H, Yamamoto R, Shiota N, Okunishi H, Kihara M, Umemura S, Sugiyama F, Yagami K, Kasuya Y, Mochizuki N, Fukamizu A. Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo. J Biol Chem. 2004; 279: 26274–26279.
41. Eyries M, Siegfried G, Ciumas M, Montagne K, Agrapart M, Lebrin F, Soubrier F. Hypoxia-induced apelin expression regulates endothelial cell proliferation and regenerative angiogenesis. Circ Res. 2008; 103: 432–440.
42. Bianco C, Strizzi L, Ebert A, Chang C, Rehman A, Normanno N, Guedez L, Salloum R, Ginsburg E, Sun Y, Khan N, Hirota M, Wallace-Jones B, Wechselberger C, Vonderhaar BK, Tosato G, Stetler-Stevenson WG, Sanicola M, Salomon DS. Role of human cripto-1 in tumor angiogenesis. J Natl Cancer Inst. 2005; 97: 132–141.
43. Cox CM, D'Agostino SL, Miller MK, Heimark RL, Krieg PA. Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo. Dev Biol. 2006; 296: 177–189.[CrossRef][Medline] [Order article via Infotrieve]
44. Sorli SC, Le Gonidec S, Knibiehler B, Audigier Y. Apelin is a potent activator of tumour neoangiogenesis. Oncogene. 2007; 26: 7692–7699.[CrossRef][Medline] [Order article via Infotrieve]
45. Kidoya H, Ueno M, Yamada Y, Mochizuki N, Nakata M, Yano T, Fujii R, Takakura N. Spatial and temporal role of the apelin/APJ system in the caliber size regulation of blood vessels during angiogenesis. EMBO J. 2008; 27: 522–534.[CrossRef][Medline] [Order article via Infotrieve]
Related Article:
Circ. Res. 2009 105: 211-213.
This article has been cited by other articles:
 |
|
 |
|
  M. L. Kirby Why Don't They Beat?: Cripto, Apelin/APJ, and Myocardial Differentiation Circ. Res., July 31, 2009; 105(3): 211 - 213. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||