Tie1 deficiency induces endothelial–mesenchymal transition (original) (raw)
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The FASEB Journal, 2007
A critical role for Tie1, an orphan endothelial receptor, in blood vessel morphogenesis has emerged from mutant mouse studies. Moreover, it was recently demonstrated that certain angiopoietin (Ang) family members can activate Tie1. We report here that Ang1 induces Tie1 phosphorylation in endothelial cells. Tie1 phosphorylation was, however, Tie2 dependent because 1) Ang1 failed to induce Tie1 phosphorylation when Tie2 was down-regulated in endothelial cells; 2) Tie1 phosphorylation was induced in the absence of Ang1 by either a constitutively active form of Tie2 or a Tie2 agonistic antibody; 3) in HEK 293 cells Ang1 phosphorylated a form of Tie1 without kinase activity when coexpressed with Tie2, and Ang1 failed to phosphorylate Tie1 when coexpressed with kinase-defective Tie2. Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2 mediated, and Tie1 down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven signaling and endothelial survival. Our new observations help to explain why Tie1 null embryos have increased capillary densities in several organ systems. The experiments also constitute a paradigm for how endothelial integrity is fine-tuned by the interplay between closely related receptors by a single growth factor.-Yuan, H. T., Venkatesha, S., Chan, B., Deutsch, U., Mammoto, T., Sukhatme, V. P., Woolf, A. S., Karumanchi, S. A. Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival. FASEB J. 21, 3171-3183 (2007)
Akt promotes Endocardial-Mesenchyme Transition
Journal of Angiogenesis Research, 2009
Endothelial to mesenchyme transition (EndMT) can be observed during the formation of endocardial cushions from the endocardium, the endothelial lining of the atrioventricular canal (AVC), of the developing heart at embryonic day 9.5 (E9.5). Many regulators of the process have been identified; however, the mechanisms driving the initial commitment decision of endothelial cells to EndMT have been difficult to separate from processes required for mesenchymal proliferation and migration. We have several lines of evidence that suggest a central role for Akt signaling in committing endothelial cells to enter EndMT. Akt1 mRNA was restricted to the endocardium of endocardial cushions while they were forming. The PI3K/Akt signaling pathway is necessary for mesenchyme outgrowth, as sprouting was inhibited in AVC explant cultures treated with the PI3K inhibitor LY294002. Furthermore, endothelial marker, VE-cadherin, was downregulated and mesenchyme markers, N-cadherin and Snail, were induced in response to expression of a constitutively active form of Akt1 (myrAkt1) in endothelial cells. Finally, we isolated the function of Akt1 signaling in the commitment to the transition using a transgenic model where myrAkt1 was pulsed only in endocardial cells and turned off after EndMT initiation. In this way, we determined that increased Akt signaling in the endocardium drives EndMT and discounted its other functions in cushion mesenchymal cells.
Molecular and Cellular Biology, 2010
One essential downstream signaling pathway of receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor receptor (VEGFR) and the Tie2 receptor, is the phosphoinositide-3 kinase (PI3K)phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade that plays a critical role in development and tumorigenesis. However, the role of PDK1 in cardiovascular development remains unknown. Here, we deleted PDK1 specifically in endothelial cells in mice. These mice displayed hemorrhage and hydropericardium and died at approximately embryonic day 11.5 (E11.5). Histological analysis revealed defective vascular remodeling and development and disrupted integrity between the endothelium and trabeculae/myocardium in the heart. The atrioventricular canal (AVC) cushion and valves failed to form, indicating a defect in epithelial-mesenchymal transition (EMT), together with increased endothelial apoptosis. Consistently, ex vivo AVC explant culture showed impeded mesenchymal outgrowth. Snail protein was reduced and was absent from the nucleus in AVC cells. Delivery of the Snail S6A mutant to the AVC explant effectively rescued EMT defects. Furthermore, adenoviral Akt delivery rescued EMT defects in AVC explant culture, and deletion of PTEN delayed embryonic lethality of PDK1 endothelial deletion mice by 1 day and rendered normal development of the AVC cushion in the PDK1-deficient heart. Taken together, these results have revealed an essential role of PDK1 in cardiovascular development through activation of Akt and Snail.
Octamer-dependent in vivo expression of the endothelial cell-specific TIE2 gene
Journal of Biological Chemistry, 1999
The TIE2 gene, also known as TEK, encodes a tyrosine kinase receptor that is required for the normal development of the vascular system during embryogenesis. TIE2 is specifically expressed in endothelial cells; however, the transcriptional mechanisms that regulate this highly restricted pattern of expression remain unknown. Here we demonstrate that a consensus octamer element located in the 5-flanking region of TIE2 is required for normal expression in embryonic endothelial cells. Transgenic embryos carrying a TIE2/LacZ construct spanning 2.1 kilobases of upstream regulatory sequences exhibit expression of the reporter transgene specifically in endothelial cells. Site-directed mutagenesis of a consensus octamer element located in this region results in the loss of enhancer activity and significantly impairs the endothelial expression of the reporter transgene. Consistent with the in vivo data, in vitro DNA-protein binding studies show that the consensus octamer element displays an endothelial cell-specific pattern of binding, suggesting an interaction with a protein complex consisting of Oct1 and an endothelial cell-restricted cofactor. These data identify a novel role for the octamer element as an essential regulator of TIE2 expression, define the first known transcriptional pathway that mediates the expression of a developmental endothelial cell gene, and provide insights into the transcriptional mechanisms that regulate development of the vasculature during embryogenesis.
Functional Significance of Tie2 Signaling in the Adult Vasculature
2010
Abundant data now demonstrate that the growth of new blood vessels, termed angiogenesis, plays both pathological and beneficial roles in human disease. Based on these data, a tremendous effort has been undertaken to understand the molecular mechanisms that drive blood vessel growth in adult tissues. Tie2 recently was identified as a receptor tyrosine kinase expressed principally on vascular endothelium. Disrupting Tie2 function in mice resulted in embryonic lethality with defects in embryonic vasculature, suggesting a role in blood vessel maturation and maintenance. Based on these studies, we undertook a series of studies to probe the function of Tie2 in adult vasculature that will form the focus of this chapter. Consistent with a role in blood vessel growth in adult vasculature, Tie2 was upregulated and activated in the endothelium of rat ovary and in healing rat skin wounds, both areas of active angiogenesis. Moreover, Tie2 was upregulated in the endothelium of vascular "hot spots" in human breast cancer specimens. Surprisingly, Tie2 also was expressed and activated in the endothelium of all normal rat tissues examined, suggesting a role in maintenance of adult vasculature. To determine the functional role of Tie2 in tumor vasculature, a soluble Tie2 extracellular domain (ExTek) was designed that blocked the activation of Tie2 by its activating ligand, angiopoietin 1 (Ang1). Administration of recombinant ExTek protein or an ExTek adenovirus inhibited tumor growth and metastasis in rodent tumor models, demonstrating a functional role for Tie2 in pathological angiogenesis in adult tissues. To begin to understand the endothelial signaling pathways and cellular responses that mediate Tie2 function, we identified signaling molecules that are recruited to the activated, autophosphorylated Tie2 kinase domain. Two of these molecules, SHP2 and GRB2, are part of the pathway upstream of mitogen-activated protein kinase (MAPK) activation, a pathway that may be responsible for morphogenetic effects of Tie2 on endothelial cells. Another signaling molecule, p85, is responsible for recruitment of phosphatidylinositol 3 kinase (PI3-K) and activation of the Akt/PI3-K pathway. Akt/PI3-K has emerged as a critical pathway downstream of Tie2 that is necessary for cell survival effects as well as for chemotaxis, activation of endothelial nitric oxide synthase, and perhaps for anti-inflammatory effects of Tie2 activation. together, 51 Downloaded from SA, Pasyk KA, Speer MC, Peters KG, Marchuk DA 1999 Allelic and locus heterogeneity in inherited venous malformations. Hum Mol Genet 8:1279 -1289 Chae JK, Kim I, Lim ST, Chung MJ, Kim WH, Kim HG, Ko JK, Koh GY 2000 Coadministration of angiopoietin-1 and vascular endothelial growth factor enhances collateral vascularization. Arterioscler Thromb Vasc Biol 20:2573-2578 Conway EM, Collen D, Carmeliet P 2001 Molecular mechanisms of blood vessel growth. Cardiovasc Res 49:507-521 Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, Jain V, Ryan TE, Bruno J, Radziejewski C, Maisonpierre PC, Yancopoulos GD 1996 Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell
Journal of Clinical Investigation, 1997
Tie2 is a novel receptor tyrosine kinase that is expressed almost exclusively by vascular endothelium. Disruption of Tie2 function in transgenic mice resulted in embryonic lethality secondary to characteristic vascular defects; similar defects occurred after disruption of the Tie2 ligand. These findings indicate that the Tie2/Tie2 ligand pathway plays important roles during development of the embryonic vasculature. To determine whether the Tie2 pathway was involved in pathologic angiogenesis in adult tissues, a soluble form of the extracellular domain of murine Tie2 (ExTek.6His) was developed and used as a Tie2 inhibitor. After a single application of the ExTek.6His protein into a rat cutaneous window chamber, growth of a mammary tumor inside the chamber was reduced by Ͼ 75% ( P Ͻ 0.005), and tumor vascular length density was reduced by 40% when compared with control-treated tumors ( P Ͻ 0.01). In the rat cornea, Ex-Tek.6His blocked angiogenesis stimulated by tumor cell conditioned media. ExTek.6His protein did not affect the viability of cultured tumor cells, indicating that the antitumor effect of ExTek.6His was due to the inhibition of tumor angiogenesis. These data demonstrate a role for the Tie2 pathway in pathologic angiogenesis, suggesting that targeting this pathway may yield effective antiangiogenic agents for treatment of cancer and other angiogenic diseases. ( J. Clin. Invest. 1997. 100:2072-2078.) Key words: endothelium • receptor tyrosine kinase • angiogenesis • Tie2 • cancer
Journal of Clinical Investigation, 2018
Cancer Research Center, Heidelberg, Germany) for valuable suggestions and discussions. This work was supported by the SFB-TR23 Vascular Differentiation and Remodeling project (project A3, to HGA); the SFB873 Maintenance and Differentiation of Stem Cells in Development and Disease project (project B6, to HGA); and the Leducq Transatlantic Network of Excellence-Lymph Vessels in Obesity and Cardiovascular Disease (to HGA).