Notch-RBP-J signaling regulates the transcription factor IRF8 to promote inflammatory macrophage polarization.

Emerging concepts suggest that the functional phenotype of macrophages is regulated by transcription factors that define alternative activation states. We found that RBP-J, the main nuclear transducer of signaling via Notch receptors, augmented Toll-like receptor 4 (TLR4)-induced expression of key mediators of classically activated M1 macrophages and thus of innate immune responses to Listeria monocytogenes. Notch-RBP-J signaling controlled expression of the transcription factor IRF8 that induced downstream M1 macrophage-associated genes. RBP-J promoted the synthesis of IRF8 protein by selectively augmenting kinase IRAK2-dependent signaling via TLR4 to the kinase MNK1 and downstream translation-initiation control through eIF4E. Our results define a signaling network in which signaling via Notch-RBP-J and TLRs is integrated at the level of synthesis of IRF8 protein and identify a mechanism by which heterologous signaling pathways can regulate the TLR-induced inflammatory polarization of macrophages.

Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice.

Notch is a critical regulator of angiogenesis, vascular differentiation, and vascular integrity. We investigated whether Notch signaling affects macrophage function during retinal angiogenesis in mice. Retinal macrophage recruitment and localization in mice with myeloid-specific loss of Notch1 was altered, as these macrophages failed to localize at the leading edge of the vascular plexus and at vascular branchpoints. Furthermore, these retinas were characterized by elongated endothelial cell sprouts that failed to anastomose with neighboring sprouts. Using Notch reporter mice, we demonstrate that retinal macrophages localize between Dll4-positive tip cells and at vascular branchpoints, and that these macrophages had activated Notch signaling. Taken together, these data demonstrate that Notch signaling in macrophages is important for their localization and interaction with endothelial cells during sprouting angiogenesis.

Notch1 deficiency results in decreased inflammation during wound healing and regulates vascular endothelial growth factor receptor-1 and inflammatory cytokine expression in macrophages.

We investigated whether Notch signaling plays a role in regulating macrophage responses to inflammation. In a wound healing assay, macrophage recruitment was decreased in Notch1(+/-) mice, and the wounds were characterized by decreased TNF-α expression. As wound healing progressed, Notch1(+/-) wounds had increased vascularization and collagen deposition compared with wild-type wounds. In mice with myeloid-specific Notch1 deletion, wounds had decreased macrophage recruitment as well as decreased TNF-α expression, indicating the specific role of Notch1 in the inflammatory response in these cells. In vitro, we found that vascular endothelial growth factor receptor-1 (VEGFR-1) was upregulated in macrophages in response to LPS/IFN-γ and that this upregulation depended on Notch signaling. Furthermore, macrophages from Notch1(+/-) mice had decreased expression of VEGFR-1 compared with macrophages from wild-type mice, whereas VEGFR-1 expression in Notch4(-/-) macrophages was normal. Inhibition of Notch signaling decreased induction of the inflammatory cytokines IL-6, IL-12, CXCL10, MCP-1, monokine induced by IFN-γ, and TNF-α in macrophages in response to LPS/IFN-γ. Additionally, macrophages from Notch1(+/-) mice demonstrated decreased induction of IL-6, IL-12, and TNF-α in response to stimulation compared with wild-type mice. Thus, both pharmacological inhibition of Notch and genetic analysis demonstrate that Notch1 regulates VEGFR-1 and cytokine expression in macrophages. We have also established that Notch1 is important for the inflammatory response during wound healing in mice.

Autoamplification of Notch signaling in macrophages by TLR-induced and RBP-J-dependent induction of Jagged1.

Several signaling pathways, including the Notch pathway, can modulate TLR activation to achieve responses most appropriate for the environment. One mechanism of TLR-Notch cross-talk is TLR-induced expression of Notch ligands Jagged and Delta that feed back to engage Notch receptors on TLR-activated cells. In this study, we investigated mechanisms by which TLRs induce Notch ligand expression in primary macrophages. TLRs induced Jagged1 expression rapidly and independently of new protein synthesis. Jagged1 induction was augmented by IFN-γ, was partially dependent on canonical TLR-activated NF-κB and MAPK signaling pathways, and elevated Jagged1 expression augmented TLR-induced IL-6 production. Strikingly, TLR-induced Jagged1 expression was strongly dependent on the Notch master transcriptional regulator RBP-J and also on upstream components of the Notch pathway γ-secretase and Notch1 and Notch2 receptors. Thus, Jagged1 is an RBP-J target gene that is activated in a binary manner by TLR and Notch pathways. Early and direct cooperation between TLR and Notch pathways leads to Jagged1-RBP-J-mediated autoamplification of Notch signaling that can modulate later phases of the TLR response.

Physical interaction of two cancer-testis antigens, MAGE-C1 (CT7) and NY-ESO-1 (CT6).

Cancer/testis (CT) antigens are the protein products of germ line-associated genes that are activated in a wide variety of tumors and can elicit autologous cellular and humoral immune responses. CT antigens can be divided between those that are encoded on the X chromosome (CT-X antigens) and those that are not (non-X CT antigens). Among the CT-X antigens, the melanoma antigen gene (MAGE) family, defined by a shared MAGE homology domain (MHD), is the largest. CT-X genes are frequently expressed in a coordinate manner in cancer cells, and their expression appears to be modulated by epigenetic mechanisms. The expression of CT-X genes is associated with advanced disease and poor outcome in different tumor types. We used the yeast two-hybrid system to identify putative MHD-interacting proteins. The MHD of MAGE-C1 (CT7) was used as bait to screen a human testis cDNA library. This study identified NY-ESO-1 (CT6) as a MAGE-C1 binding partner. Immunoprecipitation and immunofluorescence staining confirmed MAGE-C1 interaction with NY-ESO-1, and cytoplasmic co-localization of both proteins in melanoma cells. Co-expression of these two genes was found to occur in cancer cell lines from different origins, as well as in primary tumors (multiple myeloma and non-small cell lung cancer samples). This is the first report of direct interaction between two CT antigens and may be pertinent in the light of the frequently coordinated expression of these proteins.

Notch regulates the angiogenic response via induction of VEGFR-1.

Notch is a critical regulator of angiogenesis and arterial specification. We show that ectopic expression of activated Notch1 induces endothelial morphogenesis in human umbilical vein endothelial cells (HUVEC) in a VEGFR-1-dependent manner. Notch1-mediated upregulation of VEGFR-1 in HUVEC increased their responsiveness to the VEGFR-1 specific ligand, Placental Growth Factor (PlGF). In mice and human endothelial cells, inhibition of Notch signaling resulted in decreased VEGFR-1 expression during VEGF-A-induced neovascularization. In summary, we show that Notch1 plays a role in endothelial cells by regulating VEGFR-1, a function that may be important for physiological and pathological angiogenesis.