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. 2019 Sep;18(9):1782-1795.
doi: 10.1074/mcp.RA119.001492. Epub 2019 Jun 27.

NOTCH Activation Promotes Valve Formation by Regulating the Endocardial Secretome

Rebeca Torregrosa-Carrión  1 , Luis Luna-Zurita  1 , Fernando García-Marqués  2 , Gaetano D'Amato  3 , Rebeca Piñeiro-Sabarís  1 , Elena Bonzón-Kulichenko  4 , Jesús Vázquez  4 , José Luis de la Pompa  5
Affiliations

NOTCH Activation Promotes Valve Formation by Regulating the Endocardial Secretome

Rebeca Torregrosa-Carrión et al. Mol Cell Proteomics. 2019 Sep.

Abstract

The endocardium is a specialized endothelium that lines the inner surface of the heart. Functional studies in mice and zebrafish have established that the endocardium is a source of instructive signals for the development of cardiac structures, including the heart valves and chambers. Here, we characterized the NOTCH-dependent endocardial secretome by manipulating NOTCH activity in mouse embryonic endocardial cells (MEEC) followed by mass spectrometry-based proteomics. We profiled different sets of soluble factors whose secretion not only responds to NOTCH activation but also shows differential ligand specificity, suggesting that ligand-specific inputs may regulate the expression of secreted proteins involved in different cardiac development processes. NOTCH signaling activation correlates with a transforming growth factor-β2 (TGFβ2)-rich secretome and the delivery of paracrine signals involved in focal adhesion and extracellular matrix (ECM) deposition and remodeling. In contrast, NOTCH inhibition is accompanied by the up-regulation of specific semaphorins that may modulate cell migration. The secretome protein expression data showed a good correlation with gene profiling of RNA expression in embryonic endocardial cells. Additional characterization by in situ hybridization in mouse embryos revealed expression of various NOTCH candidate effector genes (Tgfβ2, Loxl2, Ptx3, Timp3, Fbln2, and Dcn) in heart valve endocardium and/or mesenchyme. Validating these results, mice with conditional Dll4 or Jag1 loss-of-function mutations showed gene expression alterations similar to those observed at the protein level in vitro These results provide the first description of the NOTCH-dependent endocardial secretome and validate MEEC as a tool for assaying the endocardial secretome response to a variety of stimuli and the potential use of this system for drug screening.

Keywords: Cardiac Valve; Cardiovascular Function or Biology; Developmental Biology; EMT; Endocardium; NOTCH; RNA SEQ; Secretome; Signal Transduction.

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Conflict of interest statement

The authors declare that they have no conflict of interest

Figures

None
Graphical abstract
Fig. 1.
Fig. 1.
Profiling the endocardial-derived secretome through in vitro modulation of the NOTCH pathway in MEEC. (A) Experimental design of MEEC proteome analysis. MEEC were stimulated with the recombinant NOTCH ligands DLL4-His10 (green) or JAG1-hIgG1Fc, in combination with vehicle (DMSO) or the γ-secretase inhibitor RO4929097 (RO). At the end of the treatment period, cells were collected for qRT-PCR analysis and protein content in conditioned media was analyzed by LC-MS/MS. (B, C) qRT-PCR analysis of canonical Notch target genes Hey1, Hey2, HeyL, and Nrarp in DLL4-stimulated MEEC (B) and JAG1-stimulated MEEC (C). Data are means of triplicate measures of each sample and are presented as mean ± s.d. (Student's t test; *p < 0.05; **p < 0.01, ***p < 0.005). (D) Workflow of sequential bioinformatics analysis with the DAVID, TMHMM, SignalP, and SecretomeP servers to identify secreted proteins. Circles show the number of proteins in each group; orange indicates secreted proteins.
Fig. 2.
Fig. 2.
Global NOTCH-associated secretory profile. (A) Hierarchical clustering of the 129 secreted factors displaying significant abundance changes (t test, p < 0.05) in comparisons of NOTCH activation versus inhibition or of NOTCH activation versus the combination of control plus inhibition. Dashed lines mark the delineation of four clusters; yellow clusters (1, 3) show increased abundance upon NOTCH activation; purple clusters (2, 4) show decreased abundance upon NOTCH activation. (B, C) Charts showing enrichment analysis for GO terms and KEGG pathways for proteins hypersecreted (B) or hyposecreted (C) in response to NOTCH activation. Colored boxes show names of secreted factors in each cluster pair.
Fig. 3.
Fig. 3.
Interactome of NOTCH-associated secretory profile. (A) NOTCH-dependent interactome, showing 78 nodes (differentially secreted proteins in at least one comparison: NOTCH activation versus inhibition, or NOTCH activation versus the combination of control plus inhibition. The nodes are connected by 229 edges (physical or functional interactions). The purple-yellow gradient of the edges reflects their degree of interaction (from high to low). Dashed lines delineate those nodes forming the most densely connected clusters. Red and blue indicate higher and lower secretion upon NOTCH activation, respectively. Bold names represent ECM-remodeling proteins.
Fig. 4.
Fig. 4.
DLL4 and JAG1 elicit distinct secretory profiles. (A) Venn diagram showing overlap of differentially secreted proteins (t test, p < 0.05) in at least one comparative analysis (NOTCH activation versus inhibition or NOTCH activation versus the combination of control plus inhibition), with JAG1-specific and DLL4-specific signaling considered separately. (B–D) Heatmaps showing z-score-normalized fold changes for proteins differentially secreted exclusively in response to signaling by JAG1 (B), DLL4 (C), or by both ligands (D), in at least one comparison. In D, protein names are given alongside each row, and the dashed line distinguishes between three major clusters based on their secretory behavior. (E) Heatmap showing the GO-Elite server-generated list of enriched GO terms and KEGG pathways for hypersecreted (yellow dashed line in B and C) and hyposecreted proteins (purple dashed line) in response to stimulation with JAG1 (orange) and DLL4 (green) stimulation. Color scale represents z-enrichment scores. Colored boxes show names of secreted factors in each cluster.
Fig. 5.
Fig. 5.
mRNA-level validation of candidate NOTCH-regulated secreted factors. (A) MEEC endogenous expression of candidate NOTCH-responsive effectors selected for validation by qRT-PCR: Fn1, Tgfβ2, Timp1, Timp3, Loxl2, Ctgf, Col4a1, Mmp2, Sema3e, Sdc4, Ptx3, and Sema3a. Data are means of triplicate measures for each sample and are presented as mean ± s.d. (Student's t test *p < 0.05; **p < 0.01, ***p < 0.005). (B) Heatmap showing mean z-score-normalized fold changes for differentially secreted proteins selected for gene expression analysis. The dashed line distinguishes between two major clusters based on their secretory behavior. (C) Cartoon depicting MEEC-derived proteins whose transcript levels (measured by RNA-Seq analysis) reflect the same protein abundance variations found in the secretome, with JAG1- and DLL4-specific signaling considered separately. UP refers to “up-regulated” and DOWN to “downregulated” during NOTCH activation.
Fig. 6.
Fig. 6.
In vivo validation of Tgfβ2, Loxl2, Fbln2, and Ptx3 as NOTCH-target genes in NOTCH pathway mouse mutants. (A–F) Whole-mount ISH analysis in E9.5 WT and Dll4flox;Tie2-Cre embryos, showing a general view of the heart. Tgfβ2 expression in AVC myocardium (A) is lost in mutants (B, asterisk). Loxl2 expression in AVC endocardium (C, arrowheads) is decreased in mutants (D, asterisk). Fbln2 expression in AVC endocardial and mesenchymal cells (E, arrowheads) is markedly downregulated in mutants (F, asterisk). (G–L″) ISH analysis in heart sections from E14.5 WT and Jag1flox;Nkx2.5-Cre embryos. Expression of Loxl2 (G–H″) and Fbln2 (I–J″) is lower in endocardial and mesenchymal cells of mutant atrioventricular valves, whereas Ptx3 is up-regulated in mesenchymal cells (K–L″). Boxed areas are magnified in the panels on the right (′ tricuspid valve; ″ mitral valve). Scale bars: 100 μm. a (atria), avc (atrioventricular canal), ivs (interventricular septum), lv (left ventricle), mv (mitral valve), tv (tricuspid valve).
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References

    1. Ma L., Lu M. F., Schwartz R. J., and Martin J. F. (2005) Bmp2 is essential for cardiac cushion epithelial-mesenchymal transition and myocardial patterning. Development 132, 5601–5611 - PubMed
    1. Rivera-Feliciano J., and Tabin C. J. (2006) Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field. Dev. Biol. 295, 580–588 - PMC - PubMed
    1. Del Monte G., Grego-Bessa J., Gonzalez-Rajal A., Bolos V., and De La Pompa J. L. (2007) Monitoring Notch1 activity in development: Evidence for a feedback regulatory loop. Dev. Dyn. 236, 2594–2614 - PubMed
    1. Luna-Zurita L., Prados B., Grego-Bessa J., Luxán G., del Monte G., Benguría A., Adams R. H., Pérez-Pomares J. M., and de la Pompa J. L. (2010) Integration of a Notch-dependent mesenchymal gene program and Bmp2-driven cell invasiveness regulates murine cardiac valve formation. J. Clin. Invest. 120, 3493–3507 - PMC - PubMed
    1. Papoutsi T., Luna-Zurita L., Prados B., Zaffran S., and de la Pompa J. L. (2018) Bmp2 and Notch cooperate to pattern the embryonic endocardium. Development 145, pii - PubMed

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