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Notch signaling is known to modulate macrophage polarization. Hu and colleagues show that the Notch–RBP-J axis controls the expression of M1 macrophage–specific genes by promoting translation of the transcription factor IRF8. 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.

Key Points Proteases function as molecular switches in signalling circuits on the cell surface and in the extracellular milieu. In light of the many proteases that are encoded by the genome, and the even larger number of bioactive substrates, it is crucial to identify which substrates individual enzymes cleave and which proteases cleave a particular substrate. Four general approaches are discussed that are commonly used to link proteases and relevant substrates: biochemistry, cell biology, proteomics and animal models (with a focus on mouse models). Biochemical studies use purified enzymes and substrates, and provide valuable information on which peptide and proteins a protease can cleave, the substrate's cleavage sites, and the inhibitor profile for small molecules and naturally occurring protease inhibitors. Cell-biological assays help determine the function of enzymes in the context of an intact cell. Gain- and loss-of-function experiments can link enzymes and substrates and can help build hypotheses about enzyme function. Regulation of enzymes by activators and inhibitors of signal transduction as well as by transcriptional activation can be evaluated. Degradomics studies of proteolysis use liquid chromatography or gel-based approaches for the mass spectrometric analysis of proteolysis. Degradomics enables the identification of hundreds or thousands of proteins in complex proteomes that have been moulded by proteolysis. Through isotope tagging, changes in the abundance levels of multiple peptides of a sample enables the identification of cleaved native substrates in cell-based systems to define the substrate degradome of a protease. Mouse models allow an analysis of a protease's function in the context of an intact organism and help establish its expression pattern and relevance in development, adult homeostasis and disease models. Loss-of-function models help evaluate the contribution of enzymes to development and disease in vivo , whereas gain-of-function models yield insights into the consequences of dysregulated enzyme activity. The combined application of these different approaches provides insights that exceed the sum of the individual approaches, and help resolve questions that arise from individual approaches. For example, they can help resolve the question: which of several candidate enzymes are relevant for processing a substrate in different cells and tissues or under different circumstances during development and in disease? Extracellular proteases regulate cell function through proteolytic switching of signalling circuits and through shedding of membrane proteins. It is crucial to link proteases to their substrates to understand their functions in development and disease, and to validate proteases as drug targets. Proteases function as molecular switches in signalling circuits at the cell surface and in the extracellular milieu. In light of the many proteases that are encoded by the genome, and the even larger number of bioactive substrates, it is crucial to identify which proteases cleave a particular substrate and which substrates individual proteases cleave. Elucidating the substrate degradomes of proteases will help us to understand the function of proteases in development and disease and to validate proteases as drug targets.

The metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) controls EGF receptor (EGFR) signaling by liberating EGFR ligands from their membrane anchor. Consequently, a patient lacking ADAM17 has skin and intestinal barrier defects that are likely caused by lack of EGFR signaling, and Adam17 ⁻/⁻ mice die perinatally with open eyes, like Egfr ⁻/⁻ mice. A hallmark feature of ADAM17-dependent EGFR ligand shedding is that it can be rapidly and posttranslationally activated in a manner that requires its transmembrane domain but not its cytoplasmic domain. This suggests that ADAM17 is regulated by other integral membrane proteins, although much remains to be learned about the underlying mechanism. Recently, inactive Rhomboid 2 (iRhom2), which has seven transmembrane domains, emerged as a molecule that controls the maturation and function of ADAM17 in myeloid cells. However, iRhom2 ⁻/⁻ mice appear normal, raising questions about how ADAM17 is regulated in other tissues. Here we report that iRhom1/2 ⁻/⁻ double knockout mice resemble Adam17 ⁻/⁻ and Egfr ⁻/⁻ mice in that they die perinatally with open eyes, misshapen heart valves, and growth plate defects. Mechanistically, we show lack of mature ADAM17 and strongly reduced EGFR phosphorylation in iRhom1/2 ⁻/⁻ tissues. Finally, we demonstrate that iRhom1 is not essential for mouse development but regulates ADAM17 maturation in the brain, except in microglia, where ADAM17 is controlled by iRhom2. These results provide genetic, cell biological, and biochemical evidence that a principal function of iRhoms1/2 during mouse development is to regulate ADAM17-dependent EGFR signaling, suggesting that iRhoms1/2 could emerge as novel targets for treatment of ADAM17/EGFR-dependent pathologies. Significance The skin and intestinal barrier are controlled by signaling scissors, termed ADAM17 (a disintegrin and metalloprotease 17), that reside in the membrane on the surface of cells. The main purpose of these signaling scissors is to liberate growth factors from their membrane anchor, allowing them to activate their receptors, including the epidermal growth factor receptor (EGFR). The ADAM17/EGFR signaling axis is tightly regulated, yet little is known about the underlying mechanism. Here we use genetic, cell biological, and biochemical approaches to identify two membrane proteins termed iRhoms 1 and 2 (inactive Rhomboid-like proteins) as crucial upstream regulators of ADAM17-dependent EGFR signaling. This uncovers the iRhoms as attractive novel targets to treat ADAM17/EGFR-dependent diseases such as cancer.

ADAM17 (a disintegrin and metalloproteinase) is a membrane-anchored metalloproteinase that regulates the release of EGFR-ligands, TNFα and other membrane proteins from cells. ADAM17 can be rapidly activated by a variety of signaling pathways, yet little is known about the underlying mechanism. Several studies have demonstrated that the cytoplasmic domain of ADAM17 is not required for its rapid activation by a variety of stimuli, including phorbol esters, tyrosine kinases and some G-protein coupled receptors. However, phosphorylation of cytoplasmic residue T735 was recently reported as a crucial step for activation of ADAM17 by IL-1β and by the p38 MAP-kinase pathway. One possible mechanism to reconcile these results would be that T735 has an inhibitory role and that it must be phosphorylated as a pre-requisite for the activation of ADAM17, which would then proceed via a mechanism that is independent of its cytoplasmic domain. To test this hypothesis, we performed rescue experiments of Adam17-/- cells with wild type and mutant forms of ADAM17. However, these experiments showed that an inactivating mutation (T735A) or an activating mutation (T735D) of cytoplasmic residue T735 or the removal of the cytoplasmic domain of ADAM17 did not significantly affect the stimulation of ADAM17 by IL-1β or by activation of MAP-kinase with anisomycin. Moreover, we found that the MAP-kinase inhibitor SB203580 blocked activation of cytoplasmic tail-deficient ADAM17 and of the T735A mutant by IL-1β or by anisomycin, providing further support for a model in which the activation mechanism of ADAM17 does not rely on its cytoplasmic domain or phosphorylation of T735.

Protein ectodomain shedding by ADAM17 (a disintegrin and metalloprotease 17), a principal regulator of EGF-receptor signaling and TNFα release, is rapidly and posttranslationally activated by a variety of signaling pathways, and yet little is known about the underlying mechanism. Here, we report that inactive rhomboid protein 2 (iRhom2), recently identified as essential for the maturation of ADAM17 in hematopoietic cells, is crucial for the rapid activation of the shedding of some, but not all substrates of ADAM17. Mature ADAM17 is present in mouse embryonic fibroblasts (mEFs) lacking iRhom2, and yet ADAM17 is unable to support stimulated shedding of several of its substrates, including heparin-binding EGF and Kit ligand 2 in this context. Stimulated shedding of other ADAM17 substrates, such as TGFα, is not affected in iRhom 2 ⁻/⁻ mEFs but can be strongly reduced by treating iRhom2 ⁻/⁻ mEFs with siRNA against iRhom1. Activation of heparin-binding EGF or Kit ligand 2 shedding by ADAM17 in iRhom2 ⁻/⁻ mEFs can be rescued by wild-type iRhom2 but not by iRhom2 lacking its N-terminal cytoplasmic domain. The requirement for the cytoplasmic domain of iRhom2 for stimulated shedding by ADAM17 may help explain why the cytoplasmic domain of ADAM17 is not required for stimulated shedding. The functional relevance of iRhom2 in regulating shedding of EGF receptor (EGFR) ligands is established by a lack of lysophasphatidic acid/ADAM17/EGFR-dependent crosstalk with ERK1/2 in iRhom2 ⁻/⁻ mEFs, and a significant reduction of FGF7/ADAM17/EGFR-stimulated migration of iRhom2 ⁻/⁻ keratinocytes. Taken together, these findings uncover functions for iRhom2 in the regulation of EGFR signaling and in controlling the activation and substrate selectivity of ADAM17-dependent shedding events.

iRHOM2, encoded by the gene Rhbdf2, regulates the maturation of the TNF-α convertase (TACE), which controls shedding of TNF-α and its biological activity in vivo. TACE is a potential target to treat TNF-α-dependent diseases, such as rheumatoid arthritis, but there are concerns about potential side effects, because TACE also protects the skin and intestinal barrier by activating EGFR signaling. Here we report that inactivation of Rhbdf2 allows tissue-specific regulation of TACE by selectively preventing its maturation in immune cells, without affecting its homeostatic functions in other tissues. The related iRHOM1, which is widely expressed, except in hematopoietic cells, supported TACE maturation and shedding of the EGFR ligand TGF-α in Rhbdf2-deficient cells. Remarkably, mice lacking Rhbdf2 were protected from K/BxN inflammatory arthritis to the same extent as mice lacking TACE in myeloid cells or Tnfa-deficient mice. In probing the underlying mechanism, we found that two main drivers of K/BxN arthritis, complement C5a and immune complexes, stimulated iRHOM2/TACE-dependent shedding of TNF-α in mouse and human cells. These data demonstrate that iRHOM2 and myeloid-expressed TACE play a critical role in inflammatory arthritis and indicate that iRHOM2 is a potential therapeutic target for selective inactivation of TACE in myeloid cells.

The authors report that TACE, the tumor necrosis factor-α–converting enzyme, regulates PNS myelination by affecting neuregulin-1 type III activity. Mice lacking TACE in motor neurons show hypermyelination. Tumor necrosis factor-α–converting enzyme (TACE; also known as ADAM17) is a proteolytic sheddase that is responsible for the cleavage of several membrane-bound molecules. We report that TACE cleaves neuregulin-1 (NRG1) type III in the epidermal growth factor domain, probably inactivating it (as assessed by deficient activation of the phosphatidylinositol-3-OH kinase pathway), and thereby negatively regulating peripheral nervous system (PNS) myelination. Lentivirus-mediated knockdown of TACE in vitro in dorsal root ganglia neurons accelerates the onset of myelination and results in hypermyelination. In agreement, motor neurons of conditional knockout mice lacking TACE specifically in these cells are significantly hypermyelinated, and small-caliber fibers are aberrantly myelinated. Further, reduced TACE activity rescues hypomyelination in NRG1 type III haploinsufficient mice in vivo . We also show that the inhibitory effect of TACE is neuron-autonomous, as Schwann cells lacking TACE elaborate myelin of normal thickness. Thus, TACE is a modulator of NRG1 type III activity and is a negative regulator of myelination in the PNS.

Toll-like receptors (TLRs) trigger intestinal inflammation when the epithelial barrier is breached by physical trauma or pathogenic microbes. Although it has been shown that TLR-mediated signals are ultimately protective in models of acute intestinal inflammation [such as dextran sulfate sodium (DSS)-induced colitis], it is less clear which cells mediate protection. Here we demonstrate that TLR signaling in the nonhematopoietic compartment confers protection in acute DSS-induced colitis. Epithelial cells of MyD88/Trif-deficient mice express diminished levels of the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG), and systemic lipopolysaccharide administration induces their expression in the colon. N-ethyl-N-nitrosourea (ENU)-induced mutations in Adam17 (which is required for AREG and EREG processing) and in Egfr both produce a strong DSS colitis phenotype, and the Adam17 mutation exerts its deleterious effect in the nonhematopoietic compartment. The effect of abrogation of TLR signaling is mitigated by systemic administration of AREG. A TLR→MyD88→AREG/EREG→EGFR signaling pathway is represented in nonhematopoietic cells of the intestinal tract, responds to microbial stimuli once barriers are breached, and mediates protection against DSS-induced colitis.

The fibroblast growth factor receptor 2-IIIb (FGFR2b) and the vascular endothelial growth factor receptor 2 (VEGFR2) are tyrosine kinases that can promote cell migration and proliferation and have important roles in embryonic development and cancer. Here we show that FGF7/FGFR2b-dependent activation of epidermal growth factor receptor (EGFR)/ERK1/2 signalling and cell migration in epithelial cells require stimulation of the membrane-anchored metalloproteinase ADAM17 and release of heparin-binding epidermal growth factor (HB-EGF). Moreover, VEGF-A/VEGFR2-induced migration of human umbilical vein endothelial cells also depends on EGFR/ERK1/2 signalling and shedding of the ADAM17 substrate HB-EGF. The pathway used by the FGF7/FGFR2b signalling axis to stimulate shedding of substrates of ADAM17, including ligands of the EGFR, involves Src, p38 mitogen-activated protein-kinase and PI3K, but does not require the cytoplasmic domain of ADAM17. Based on these findings, ADAM17 emerges as a central component in a triple membrane-spanning pathway between FGFR2b or VEGFR2 and EGFR/ERK1/2 that is required for cell migration in keratinocytes and presumably also in endothelial cells. Activation of the receptor tyrosine kinases FGFR2 and VEGFR2 results in ERK1/2 phosphorylation and cell migration. Here, the authors demonstrate that shedding of HB-EGF—a substrate of the metalloproteinase ADAM17—and activation of EGFR is required for FGFR2 and VEGFR2 mediated cell migration.

During the initiation of pregnancy, the vasculature of the implantation site expands rapidly, yet little is known about this process or its role in fertility. Here, we report that endothelial-specific deletion of a disintegrin and metalloprotease 10 (ADAM10), an essential regulator of Notch signaling, results in severe subfertility in mice. We found that implantation sites develop until 5.5 days post conception (dpc) but are resorbed by 6.5 dpc in A10ΔEC mice. Analysis of the mutant implantation sites showed impaired decidualization and abnormal vascular patterning compared to controls. Moreover, RNA-seq analysis revealed changes in endothelial cell marker expression consistent with defective ADAM10/Notch signaling in samples from A10ΔEC mice, suggesting that this signaling pathways is essential for the physiological function of endometrial endothelial cells during early pregnancy. Our findings raise the possibility that impaired endothelial cell function could be a cause for repeated pregnancy loss (RPL) and infertility in humans.