Notch4 intracellular domain binding to Smad3 and inhibition of the TGF-β signaling

The Tumor Growth Factor Section, Mammary Biology and Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD 20892, USA.
Oncogene (Impact Factor: 8.46). 09/2005; 24(34):5365-74. DOI: 10.1038/sj.onc.1208528
Source: PubMed


We present evidence that Notch4ICD attenuates TGF-beta signaling. Cells expressing the activated form of the Notch4 receptor (ICD4) were resistant to the growth-inhibitory effects of TGF-beta. Notch4ICD was found to bind to Smad2, Smad3 and Smad4 but with higher affinity to Smad3. Deletion analysis showed that binding of Smad3 to ICD4 was mediated by its MH2 domain and was not dependent on the presence of the RAM23 region in ICD4. Using two TGF-beta/Activin reporter luciferase assays, RT-PCR and Western blot analysis, we demonstrate that ICD4 and ICD4 deltaRAM23 inhibit Smad-binding element and 3TP luciferase reporter activity and PAI-1 gene expression. MCF-7 human breast cancer cells express Notch4ICD (ICD4) and are resistant to the growth-inhibitory effects of TGF-beta. Blockage of Notch4 processing to ICD4 by gamma-secretase inhibitor renders MCF-7 cells sensitive to growth inhibition by TGF-beta. The interplay between these two signaling pathways may be a significant determinant during mammary tumorigenesis.

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Available from: Morihisa Hirota, Aug 14, 2014
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    • "However, in our studies, we did not observe Notch4 staining in either human (Figure 1c) or murine tumor cells (Figure 1a, ix, xii and 1b, iii), but rather observed Notch4 upregulation in the tumor vasculature. We used a well-characterized Notch4-specific antibody [28] and verified its specificity using Notch4 knockout tissues (Additional file 1: Figure S1). Our study demonstrates Notch4 upregulation in the vasculature of both mouse models of mammary adenocarcinoma and human breast cancer. "
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    ABSTRACT: Background Notch4 is a member of the Notch family of receptors that is primarily expressed in the vascular endothelial cells. Genetic deletion of Notch4 does not result in an overt phenotype in mice, thus the function of Notch4 remains poorly understood. Methods We examined the requirement for Notch4 in the development of breast cancer vasculature. Orthotopic transplantation of mouse mammary tumor cells wild type for Notch4 into Notch4 deficient hosts enabled us to delineate the contribution of host Notch4 independent of its function in the tumor cell compartment. Results Here, we show that Notch4 expression is required for tumor onset and early tumor perfusion in a mouse model of breast cancer. We found that Notch4 expression is upregulated in mouse and human mammary tumor vasculature. Moreover, host Notch4 deficiency delayed the onset of MMTV-PyMT tumors, wild type for Notch4, after transplantation. Vessel perfusion was decreased in tumors established in Notch4-deficient hosts. Unlike in inhibition of Notch1 or Dll4, vessel density and branching in tumors developed in Notch4-deficient mice were unchanged. However, final tumor size was similar between tumors grown in wild type and Notch4 null hosts. Conclusion Our results suggest a novel role for Notch4 in the establishment of tumor colonies and vessel perfusion of transplanted mammary tumors.
    Vascular Cell 04/2013; 5(1):7. DOI:10.1186/2045-824X-5-7
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    • "The effect of crosstalk between Notch and other signalling pathways can vary depending on the Notch paralogue analysed (Foltz et al., 2002; Dahlqvist et al., 2003; Espinosa et al., 2003; Sun et al., 2005; Beres et al., 2011). We therefore investigated whether Dishevelled could inhibit each one of the four Notch receptors. "
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    ABSTRACT: Notch and Wnt are highly conserved signalling pathways that are used repeatedly throughout animal development to generate a diverse array of cell types. However, they often have opposing effects on cell-fate decisions with each pathway promoting an alternate outcome. Commonly, a cell receiving both signals exhibits only Wnt pathway activity. This suggests that Wnt inhibits Notch activity to promote a Wnt-ON/Notch-OFF output; but what might underpin this Notch regulation is not understood. Here, we show that Wnt acts via Dishevelled to inhibit Notch signalling, and that this crosstalk regulates cell-fate specification in vivo during Xenopus development. Mechanistically, Dishevelled binds and directly inhibits CSL transcription factors downstream of Notch receptors, reducing their activity. Furthermore, our data suggest that this crosstalk mechanism is conserved between vertebrate and invertebrate homologues. Thus, we identify a dual function for Dishevelled as an inhibitor of Notch signalling and an activator of the Wnt pathway that sharpens the distinction between opposing Wnt and Notch responses, allowing for robust cell-fate decisions.
    Development 12/2012; 139(23):4405-15. DOI:10.1242/dev.081885 · 6.46 Impact Factor
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    • "Notch4 ICD -mediated TGF-β activity and expression of Notch4 ICD in fibrotic kidneys Defining novel regulators of the pro-fibrotic activity of TGF-β in diabetic kidney disease is critical towards understanding the molecular basis of this disease process. Previous reports indicate a role for Notch4 activity in TGF-β function in epithelial-derived breast tumor, smooth muscle, and endothelial cell lines [14] [15] [16] "
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    ABSTRACT: Notch proteins (Notch 1-4) are a family of trans-membrane cell surface receptors that are converted into transcriptional regulators when activated by interactions with cell surface ligands on adjacent cells. Ligand-binding stimulates proteolytic cleavage of the trans-membrane domain, releasing an active intracellular domain (ICD) that translocates to the nucleus and impacts transcription. In transit, the ICD may interact with regulatory proteins that modulate the expression and transcriptional activity. We have found that Notch4(ICD) expression is enhanced in the tubule cells of fibrotic kidneys from diabetic mice and humans and identified Notch4(ICD) interacting proteins that could be pertinent to normal and pathological functions. Using proteomic techniques, several components of the Elongin C complex were identified as candidate Notch4(ICD) interactors. Elongin C complexes can function as ubiquitin ligases capable of regulating proteasomal degradation of specific protein substrates. Our studies indicate that ectopic Elongin C expression stimulates Notch4(ICD) degradation and inhibits its transcriptional activity in human kidney tubule HK11 cells. Blocking Elongin C mediated degradation by MG132 indicates the potential for ubiquitin-mediated Elongin C regulation of Notch4(ICD). Functional interaction of Notch4(ICD) and Elongin C provides novel insight into regulation of Notch signaling in epithelial cell biology and disease.
    Biochimica et Biophysica Acta 12/2011; 1814(12):1748-57. DOI:10.1016/j.bbapap.2011.09.010 · 4.66 Impact Factor
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