Genetic regulation of mammalian heart size is poorly understood. Hippo signaling represents an organ-size control pathway in Drosophila, where it also inhibits cell proliferation and promotes apoptosis in imaginal discs. To determine whether Hippo signaling controls mammalian heart size, we inactivated Hippo pathway components in the developing mouse heart. Hippo-deficient embryos had overgrown hearts with elevated cardiomyocyte proliferation. Gene expression profiling and chromatin immunoprecipitation revealed that Hippo signaling negatively regulates a subset of Wnt target genes. Genetic interaction studies indicated that β-catenin heterozygosity suppressed the Hippo cardiomyocyte overgrowth phenotype. Furthermore, the Hippo effector Yap interacts with β-catenin on Sox2 and Snai2 genes. These data uncover a nuclear interaction between Hippo and Wnt signaling that restricts cardiomyocyte proliferation and controls heart size.
SUMMARYWe performed an in depth analysis of Bmp4, a critical regulator of development, disease, and evolution, in cranial neural crest (CNC). Conditional Bmp4 overexpression, using a tetracycline-regulated Bmp4 gain-of-function allele, resulted in facial skeletal changes that were most dramatic after an E10.5 Bmp4 induction. Expression profiling uncovered a signature of Bmp4-induced genes (BIG) composed predominantly of transcriptional regulators that control self-renewal, osteoblast differentiation and negative Bmp autoregulation. The complimentary experiment, CNC inactivation of Bmp2, Bmp4 and Bmp7, resulted in complete or partial loss of multiple CNC-derived skeletal elements, revealing a crucial requirement for Bmp signaling in membranous bone and cartilage development. Importantly, the BIG signature was reduced in Bmp loss-of-function mutants, indicating Bmpregulated target genes are modulated by Bmp dose. Chromatin immunoprecipitation (ChIP) revealed a subset of the BIG signature, including Satb2, Smad6, Hand1, Gadd45 and Gata3, that was bound by Smad1/5 in the developing mandible, revealing direct Smad-mediated regulation. These data support the hypothesis that Bmp signaling regulates craniofacial skeletal development by balancing self-renewal and differentiation pathways in CNC progenitors.
Summary MicroRNAs (miRNA) are small, non-coding RNAs that regulate gene expression post-transcriptionally. We investigated the hypothesis that Bone Morphogenetic Protein (Bmp)-signaling regulates miRNAs in cardiac progenitor cells. Bmp2 and Bmp4 regulate OFT myocardial differentiation via regulation of the miRNA 17-92 cluster. In Bmp mutant embryos, myocardial differentiation was delayed and multiple miRNAs encoded by miRNA 17-92 were reduced. We uncovered functional miRNA17-92 seed sequences within the 3′ UTR of cardiac progenitor genes such as Isl1 and Tbx1. In both Bmp and miRNA 17-92 mutant embryos, Isl1and Tbx1 expression failed to be correctly downregulated. Transfection experiments indicated that miRNA 17 and miRNA 20a directly repressed Isl1and Tbx1. Genetic interaction studies uncovered a synergistic interaction between miRNA 17-92 cluster and Bmp4 providing direct in vivo evidence for the Bmp-miRNA 17-92 regulatory pathway. Our findings indicate that Bmp-signaling directly regulates a miRNA-mediated effector mechanism that downregulates cardiac progenitor genes and enhances myocardial differentiation.
Müllerian duct differentiation and development into the female reproductive tract is essential for fertility, but mechanisms regulating these processes are poorly understood. WNT signaling is critical for proper development of the female reproductive tract as evident by the phenotypes of Wnt4, Wnt5a, Wnt7a, and β-catenin (Ctnnb1) mutant mice. Here we extend these findings by determining the effects of constitutive CTNNB1 activation within the mesenchyme of the developing Müllerian duct and its differentiated derivatives. This was accomplished by crossing Amhr2-Cre knock-in mice with Ctnnb1 exon (ex) 3(f/f) mice. Amhr2-Cre(Δ/+); Ctnnb1 ex3(f/+) females did not form an oviduct, had smaller uteri, endometrial gland defects, and were infertile. At the cellular level, stabilization of CTNNB1 in the mesenchyme caused alterations within the epithelium, including less proliferation, delayed uterine gland formation, and induction of an epithelial-mesenchymal transition (EMT) event. This EMT event is observed before birth and is complete within 5 days after birth. Misexpression of estrogen receptor α in the epithelia correlated with the EMT before birth, but not after. These studies indicate that regulated CTNNB1 in mesenchyme is important for epithelial cell differentiation during female reproductive tract development.
SUMMARYCongenital heart disease (CHD) is a devastating anomaly that affects ~1% of live births. Defects of the outflow tract (OFT) make up a large percentage of human CHD. We investigated Bmp signaling in mouse OFT development by conditionally deleting both Bmp4 and Bmp7 in the second heart field (SHF). SHF Bmp4/7 deficiency resulted in defective epithelial to mesenchymal transition (EMT) and reduced cardiac neural crest ingress, with resultant persistent truncus arteriosus. Using a candidate gene approach, we found that Vegfa was upregulated in the Bmp4/7 mutant hearts. To determine if Vegfa is a downstream Bmp effector during EMT, we examined whether Vegfa is transcriptionally regulated by the Bmp receptor-regulated Smad. Our findings indicate that Smad directly binds to Vegfa chromatin and represses Vegfa transcriptional activity. We also found that Vegfa is a direct target for the miR-17-92 cluster, which is also regulated by Bmp signaling in the SHF. Deletion of miR-17-92 reveals similar phenotypes to Bmp4/7 SHF deletion. To directly address the function of Vegfa repression in Bmp-mediated EMT, we performed ex vivo explant cultures from Bmp4/7 and miR-17-92 mutant hearts. EMT was defective in explants from the Bmp4/7 double conditional knockout (dCKO; Mef2c-Cre;Bmp4/7 f/f ) and miR-17-92 null. By antagonizing Vegfa activity in explants, EMT was rescued in Bmp4/7 dCKO and miR-17-92 null culture. Moreover, overexpression of miR-17-92 partially suppressed the EMT defect in Bmp4/7 mutant embryos. Our study reveals that Vegfa levels in the OFT are tightly controlled by Smad-and microRNA-dependent pathways to modulate OFT development.
E2F3 is a transcription factor that may initiate tumorigenesis if overexpressed. Previously, we demonstrated that E2F3 mRNA is overexpressed in breast cancer and that E2F3 overexpression results in centrosome amplification and unregulated mitosis, which can promote aneuploidy and chromosome instability to initiate and sustain tumors. Further, we demonstrated that E2F3 leads to overexpression of the mitotic regulator Shugoshin-1, which until recently had unknown roles in cancer. This study aims to evaluate the roles of E2F3 and Shugoshin-1 in breast cancer metastatic potential. Here we demonstrated that E2F3 and Shugoshin-1 silencing leads to reduced cell invasion and migration in two mesenchymal triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and Hs578t). Moreover, E2F3 and Shugoshin-1 modulate the expression of epithelial-to-mesenchymal transition-associated genes such as Snail, E-Cadherin, and multiple matrix metalloproteinases. Furthermore, E2F3 depletion leads to reductions in tumor growth and metastasis in NOD- scid Gamma mice. Results from this study suggest a key role for E2F3 and a novel role for Shugoshin-1 in metastatic progression. These results can further help in the improvement of TNBC targeted therapies by interfering with pathways that intersect with the E2F3 and Shugoshin-1 signaling pathways.
Multiple studies suggest that chronic stress accelerates the growth of existing tumors by activating the sympathetic nervous system. Data suggest that sustained adrenergic signaling can induce tumor growth, secretion of pro-inflammatory cytokines, and macrophage infiltration. Our goal was to study the role of adrenergic-stimulated macrophages in ovarian cancer biology. Cytokine arrays were used to assess the effect of adrenergic stimulation in pro-tumoral cytokine networks. An orthotopic model of ovarian cancer was used to assess the in vivo effect of daily restraint stress on tumor growth and adrenergic-induced macrophages. Cytokine analyses showed that adrenergic stimulation modulated pro-inflammatory cytokine secretion in a SKOV3ip1 ovarian cancer cell/U937 macrophage co-culture system. Among these, platelet-derived growth factor AA (PDGF-AA), epithelial cell-derived neutrophil-activating peptide (ENA-78), Angiogenin, vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-5 (IL-5), Lipocalin-2, macrophage migration inhibitory factor (MIF), and transferrin receptor (TfR) were upregulated. Enriched biological processes included cytokine-mediated signaling pathways and positive regulation of cell proliferation. In addition, daily restraint stress increased ovarian cancer growth, infiltration of CD68+ macrophages, and expression of PDGF-AA in orthotopic models of ovarian cancer (SKOV3ip1 and HeyT30), while zoledronic acid, a macrophage-depleting agent, abrogated this effect. Furthermore, in ovarian cancer patients, high PDGFA expression correlated with worse outcomes. Here, it is shown that the adrenergic regulation of macrophages and PDGFA might play a role in ovarian cancer progression.
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