Notch-1 signalling is activated in brain arteriovenous malformations in humans.

Zhuge Q, Zhong M, Zheng W, Yang GY, Mao X, Xie L, Chen G, Chen Y, Lawton MT, Young WL, Greenberg DA, Jin K.

Brain Advance Access published online on October 7, 2009, doi:10.1093/brain/awp246

A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that, compared with control brain vascular tissue, Notch-1 signalling was activated in smooth muscle and endothelial cells of the lesional tissue. Western blotting showed an activated form of Notch-1 in brain arteriovenous malformations, irrespective of clinical presentation and with or without preoperative embolization, but not in normal cerebral vessels from controls. In addition, the Notch-1 ligands Jagged-1 and Delta-like-4 and the downstream Notch-1 target Hes-1 were increased in abundance and activated in human brain arteriovenous malformations. Finally, increased angiogenesis was found in adult rats treated with a Notch-1 activator. Our findings suggest that activation of Notch-1 signalling is a phenotypic feature of brain arteriovenous malformations, and that activation of Notch-1 in normal vasculature induces a pro-angiogenic state, which may contribute to the development of vascular malformation

Notch Signal Activates Hypoxia Pathway through HES1-Dependent SRC/Signal Transducers and Activators of Transcription 3 Pathway.

Lee JH, Suk J, Park J, Kim SB, Kwak SS, Kim JW, Lee CH, Byun B, Ahn JK, Joe CO.

Mol Cancer Res. 2009 Oct 6. [Epub ahead of print] doi: 10.1158/1541-7786.MCR-09-0191

We report a Notch signal–induced pathway that leads to transcriptional activation of HIF1-α gene. HeLa/rtTAA/TRE-N1-IC cell line capable of doxycycline-induced expression of human Notch1-IC was established. The induction of Notch signaling activates HIF1-α and its target gene expression in HeLa/rtTAA/TRE-N1-IC cells. Notch signaling enhanced signal transducers and activators of transcription 3 (STAT3) phosphorylation required for HIF1-α expression. SRC kinase was found to be responsible for the enhanced STAT3 phosphorylation in response to Notch signaling. Activation of SRC/STAT3 pathway by Notch signaling was dependent on the expression of Notch effector HES1 transcription factor. The induction of HES1 enhanced STAT3 phosphorylation at Tyr 705 as well as SRC phosphorylation at Tyr 416 in inducible HeLa/rtTAA/TRE-HES1 cells, which express HES1 in response to doxycycline treatment. However, the treatment of Trichostatin A that interferes with HES1 transcriptional regulation did not affect STAT3 phosphorylation, and the expression of dominant negative HES1 failed to interfere with HES1-dependnent SRC/STAT3 pathway. These observations have led us to the conclusion that HES1-dependent activation of SRC/STAT3 pathway is independent of HES1 transcription regulation. This study first reports HES1-dependent SRC/STAT3 pathway that provides a functional link between Notch signaling and hypoxia pathway.

Shear Stress Increases Expression of the Arterial Endothelial Marker EphrinB2 in Murine ES Cells via the VEGF-Notch Signaling Pathways

Tomomi Masumura, Kimiko Yamamoto, Nobutaka Shimizu, Syotaro Obi, Joji Ando

Arterioscler Thromb Vasc Biol, doi: 10.1161/ATVBAHA.109.193185

Objective—Arterial-venous specification in the embryo has been assumed to depend on the influence of fluid mechanical forces, but its cellular and molecular mechanisms are still poorly understood. Our previous in vitro study revealed that fluid shear stress induces endothelial cell (EC) differentiation by murine embryonic stem (ES) cells. In the present study we investigated whether shear stress regulates the arterial-venous specification of ES-cell-derived ECs.

Methods and Results—When murine ES cell– derived VEGFR2⁺ ES cells were exposed to shear stress, expression of the arterial EC marker protein ephrinB2 increased dose-dependently. The ephrinB2 mRNA levels also increased in response to shear stress, whereas the mRNA levels of the venous EC marker EphB4 decreased. Notch cleavage and translocation of the Notch intracellular domain (NICD) into the nucleus occurred as early as 30 minutes after the start of shear stress and increased with time. Gamma-Secretase inhibitors (DAPT and L685 458), and the recombinant extracellular domain of the Notch ligand DLL4 abolished the shear stress–induced NICD translocation, and that, in turn, blocked the shear stress–induced upregulation of ephrinB2 expression. In addition, the VEGF receptor kinase inhibitor SU1498 was found to suppress both the shear-stress-induced Notch cleavage and up-regulation of ephrinB2 expression.

Conclusion—Exposure to shear stress induces an increase in expression of ephrinB2 in murine ES cells via VEGF-Notch signaling pathways. (Arterioscler Thromb Vasc Biol. 2009;29:00-00.)

The metalloprotease ADAM10 is required for notch1 S2 cleavage.

van Tetering G, van Diest P, Verlaan I, van der Wall E, Kopan R, Vooijs M.

JBC Papers in Press published on September 2, 2009 as doi:10.1074/jbc.M109.006775

Notch signaling is controlled by ligand binding which unfolds a negative control region to induce proteolytic cleavage of the receptor. First, a membrane proximal cleavage is executed by a metalloprotease, removing the extracellular domain. This allows gamma-secretase to execute a second cleavage within the Notch transmembrane domain which releases the intracellular domain to enter the nucleus. Here we show that the ADAM10 metalloprotease Kuzbanian, but not ADAM17/TACE, plays an essential role in executing ligand induced extracellular cleavage at site 2 (S2) in cells and localize this step to the plasma membrane. Importantly, genetic or pharmacological inhibition of metalloproteases still allowed extracellular cleavage of Notch, indicating the presence of unknown proteases with the ability to cleave at S2. Gain of function mutations identified in human cancers and in model organisms that map to the negative control region alleviate the requirement for ligand binding for extracellular cleavage to occur. Since cancer-causing Notch1 mutations also depend on (rate-limiting) S2 proteolysis, the identity of these alternative proteases have important implications for understanding Notch activation in normal and cancer cells.

Notch signalling and haematopoietic stem cell formation during embryogenesis.

Gering M, Patient R.

Journal of Cellular Physiology, Published Online: 1 Sep 2009, doi: 10.1002/jcp.21905

The Notch signalling pathway is repeatedly employed during embryonic development and adult homeostasis of a variety of tissues. In particular, its frequent involvement in the regulation of stem and progenitor cell maintenance and proliferation, as well as its role in binary fate decisions in cells that are destined to differentiate, is remarkable. Here, we review its role in the development of haematopoietic stem cells during vertebrate embryogenesis and put it into the context of Notch’s functions in arterial specification, angiogenic vessel sprouting and vessel maintenance. We further discuss interactions with other signalling cascades, and pinpoint open questions and some of the challenges that lie ahead.

Notch pathway regulation of chondrocyte differentiation and proliferation during appendicular and axial skeleton development

Timothy J. Mead and Katherine E. Yutzey

PNAS 2009 106:14420-14425, doi:10.1073/pnas.0902306106

The role of Notch signaling in cartilage differentiation and maturation in vivo was examined. Conditional Notch pathway gain and loss of function was achieved using a Cre/loxP approach to manipulate Notch signaling in cartilage precursors and chondrocytes of the developing mouse embryo. Conditional overexpression of activated Notch intracellular domain (NICD) in the chondrocyte lineage results in skeletal malformations with decreased cartilage precursor proliferation and inhibited hypertrophic chondrocyte differentiation. Likewise, expression of NICD in cartilage precursors inhibits sclerotome differentiation, resulting in severe axial skeleton abnormalities. Furthermore, conditional loss of Notch signaling via RBP-J gene deletion in the chondrocyte lineage results in increased chondrocyte proliferation and skeletal malformations consistent with the observed increase in hypertrophic chondrocytes. In addition, the Notch pathway inhibits expression of Sox9 and its target genes required for normal chondrogenic cell proliferation and differentiation. Together, our results demonstrate that appropriate Notch pathway signaling is essential for proper chondrocyte progenitor proliferation and for the normal progression of hypertrophic chondrocyte differentiation into bone in the developing appendicular and axial skeletal elements.

DLL4 blockade inhibits tumor growth and reduces tumor-initiating cell frequency.

Hoey T, Yen WC, Axelrod F, Basi J, Donigian L, Dylla S, Fitch-Bruhns M, Lazetic S, Park IK, Sato A, Satyal S, Wang X, Clarke MF, Lewicki J, Gurney A.

Cell Stem Cell. 2009 Aug 7;5(2):168-77

And Comment:

Molecular targeting of cancer stem cells.
Gupta R, Vyas P, Enver T.

Cancer stem cells may be important targets for new anticancer drugs. In two recent articles in Cell Stem Cell, Jin et al. (2009) and Hoey et al. (2009) provide proof of principle for this idea in experimental models of solid tumors and leukemias, respectively.

Previous studies have shown that blocking DLL4 signaling reduced tumor growth by disrupting productive angiogenesis. We developed selective anti-human and anti-mouse DLL4 antibodies to dissect the mechanisms involved by analyzing the contributions of selectively targeting DLL4 in the tumor or in the host vasculature and stroma in xenograft models derived from primary human tumors. We found that each antibody inhibited tumor growth and that the combination of the two antibodies was more effective than either alone. Treatment with anti-human DLL4 inhibited the expression of Notch target genes and reduced proliferation of tumor cells. Furthermore, we found that specifically inhibiting human DLL4 in the tumor, either alone or in combination with the chemotherapeutic agent irinotecan, reduced cancer stem cell frequency, as shown by flow cytometric and in vivo tumorigenicity studies.

Embryonic arrest at midgestation and disruption of Notch signaling produced by the absence of both epsin 1 and epsin 2 in mice

Hong Chen, Genevieve Ko, Alessandra Zatti, Giuseppina Di Giacomo, Lijuan Liu, Elisabetta Raiteri, Ezio Perucco, Chiara Collesi, Wang Min, Caroline Zeiss, Pietro De Camilli, and Ottavio Cremona

PNAS published online before print August 5, 2009, doi:10.1073/pnas.0907008106

Epsins are endocytic adaptors with putative functions in general aspects of clathrin-mediated endocytosis as well as in the internalization of specific membrane proteins. We have now tested the role of the ubiquitously expressed epsin genes, Epn1 and Epn2, by a genetic approach in mice. While either gene is dispensable for life, their combined inactivation results in embryonic lethality at E9.5–E10, i.e., at the beginning of organogenesis. Consistent with studies in Drosophila, where epsin endocytic function was linked to Notch activation, developmental defects observed in epsin 1/2 double knockout (DKO) embryos recapitulated those produced by a global impairment of Notch signaling. Accordingly, expression of Notch primary target genes was severely reduced in DKO embryos. However, housekeeping forms of clathrin-mediated endocytosis were not impaired in cells derived from these embryos. These findings support a role of epsin as a specialized endocytic adaptor, with a critical role in the activation of Notch signaling in mammals.

Pleiotrophin regulates lung epithelial cell proliferation and differentiation during fetal lung development via ß-catenin and Dlk1.

Weng T, Gao L, Bhaskaran M, Guo Y, Gou D, Narayanaperumal J, Chintagari NR, Zhang K, Liu L.

JBC Papers in Press published on August 6, 2009 as doi:10.1074/jbc.M109.052530

The role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, protein tyrosine phosphatase receptor beta/zeta, were highly expressed in mesenchymal and epithelial cells of the fetal lungs, respectively. Using isolated fetal alveolar epithelial type II cells, we demonstrated that pleiotrophin promoted fetal type II cell proliferation and arrested type II cell trans-differentiation into alveolar epithelial type I cells. Pleiotrophin also increased wound healing of injured type II cell monolayer. Knock-down of pleiotrophin influenced lung branching morphogenesis in a fetal lung organ culture model. Pleiotrophin increased the tyrosine phosphorylation of beta-catenin, promoted beta-catenin translocation into the nucleus and activated TCF/LEF transcription factors. Dlk1, a membrane ligand that initiates the Notch signaling pathway, was identified as a down-stream target of the pleiotrophin/beta-catenin pathway by endogenous dlk1 expression, promoter assay and chromatin immunoprecipitation. These results provide evidence that pleiotrophin regulates fetal type II cell proliferation and differentiation via integration of multiple signaling pathways including pleiotrophin, beta-catenin and Notch pathways.

The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis.

Benedito R, Roca C, Sörensen I, Adams S, Gossler A, Fruttiger M, Adams RH.

Cell. 2009 Jun 12;137(6):1124-35. doi:10.1016/j.cell.2009.03.025

The Notch pathway is a highly conserved signaling system that controls a diversity of growth, differentiation, and patterning processes. In growing blood vessels, sprouting of endothelial tip cells is inhibited by Notch signaling, which is activated by binding of the Notch receptor to its ligand Delta-like 4 (Dll4). Here, we show that the Notch ligand Jagged1 is a potent proangiogenic regulator in mice that antagonizes Dll4-Notch signaling in cells expressing Fringe family glycosyltransferases. Upon glycosylation of Notch, Dll4-Notch signaling is enhanced, whereas Jagged1 has weak signaling capacity and competes with Dll4. Our findings establish that the equilibrium between two Notch ligands with distinct spatial expression patterns and opposing functional roles regulates angiogenesis, a mechanism that might also apply to other Notch-controlled biological processes.