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Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signalling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g., HB-EGF) are known to bind to heparan sulphate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-treated secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e., HB-EGF, CX3CL1) and 14 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting., (© 2024 The Author(s). PROTEOMICS published by Wiley‐VCH GmbH.)

The autoimmune disease lupus erythematosus (lupus) is characterized by photosensitivity, where even ambient ultraviolet radiation (UVR) exposure can lead to development of inflammatory skin lesions. We have previously shown that Langerhans cells (LCs) limit keratinocyte apoptosis and photosensitivity via a disintegrin and metalloprotease 17 (ADAM17)-mediated release of epidermal growth factor receptor (EGFR) ligands and that LC ADAM17 sheddase activity is reduced in lupus. Here, we sought to understand how the lupus skin environment contributes to LC ADAM17 dysfunction and, in the process, differentiate between effects on LC ADAM17 sheddase function, LC ADAM17 expression, and LC numbers. We show through transcriptomic analysis a shared IFN-rich environment in non-lesional skin across human lupus and three murine models: MRL/lpr, B6.Sle1yaa, and imiquimod (IMQ) mice. IFN-I inhibits LC ADAM17 sheddase activity in murine and human LCs, and IFNAR blockade in lupus model mice restores LC ADAM17 sheddase activity, all without consistent effects on LC ADAM17 protein expression or LC numbers. Anti-IFNAR-mediated LC ADAM17 sheddase function restoration is associated with reduced photosensitive responses that are dependent on EGFR signaling and LC ADAM17. Reactive oxygen species (ROS) is a known mediator of ADAM17 activity; we show that UVR-induced LC ROS production is reduced in lupus model mice, restored by anti-IFNAR, and is cytoplasmic in origin. Our findings suggest that IFN-I promotes photosensitivity at least in part by inhibiting UVR-induced LC ADAM17 sheddase function and raise the possibility that anifrolumab ameliorates lupus skin disease in part by restoring this function. This work provides insight into IFN-I-mediated disease mechanisms, LC regulation, and a potential mechanism of action for anifrolumab in lupus., Competing Interests: TL, VZ, ES, MD, YC, KV, DO, PC, YL, WS, ST, KO, MR, IR No competing interests declared, AD, PL is an employee of AMPEL BioSolutions, but has no financial conflicts of interest to report, NS was awarded the Lupus Therapeutics: The Clinical Trial Network Infrastructure Grant, received by The Albert Einstein College of Medicine. The author received payment for lectures at the Congress of Clinical Rheumatology East and the Congress of Clinical Rheumatology West. The author has no other competing interests to declare, JL has received the grants F31 NIH GM136144 and T32 NIH GM008539. The author has received stock or stock options from NASDAQ/NYSE Ticker: FULC, ABCL, AVXL, VOR, MRNA, BNTX, SAVA, OCGN, CTMX, BCEL, GE. The author has no other competing interests to declare, WA received support for travel and attending Lupus 21st century meeting in 2021. The author has no other competing interests to declare, JZ received a grant from NIH NIAMS, and consulting fees from Hoth Therapeutics and Pfizer. The author received payment for participation on a Data Safety Monitoring Board/ Advisory Board for Hoth Therapeutics and acts as President elect for PASPCR. The author holds stock options from Hoth Therapeutics, FoxWayne Inc and YouV labs. The author has no other competing interests to declare, JK has received grant support from AbbVie, Akros, Allergan, Amgen, Avillion, Biogen, Botanix, Boehringer Ingelheim, Bristol-Myers Squibb, Exicure, Innovaderm, Incyte, Janssen, Kyowa Kirin, Lilly, Nimbus Lackshmi, Novan, Novartis, PAREXEL, Pfizer, Regeneron, UCB, Vitae Pharmaceuticals. The author received consulting fees from AbbVie, Aclaris, Allergan, Almirall, Amgen, Artax Biopharma, Arena, Aristea, Asana, Aurigene, Biogen Idec, Boehringer Ingelheim, Bristol-Myers Squibb, Escalier, Galapagos, Janssen, Kyowa Kirin, Lilly, MoonLake Immunotherapeutics, Nimbus, Novartis, Pfizer, Sanofi, Sienna Biopharmaceuticals, Sun Pharma, Target-Derm, UCB, Valeant, Ventyx. The author has no other competing interests to declare, NA has received the following grants: NIAMS AR080436-01, NIAMS R56AR078686-01 and NIH NIAMS 5R01 GRANT AR070234-05. The author received consulting fees from Immunitas, Shennon Bio and Janssen. The author received payment as a lecturer from 23 and me, Cellino and Bristol Meyer Squibb Genomics. They are also a board member of the Society of Investigative Dermatology. The author has no other competing interests to declare, AJ has received grants from the NIH and the VA and consulting fees from Pfizer. The author has no other competing interests to declare, CB The patent number is US10024844B2 and the title of the patent is "Identification of an inhibitor of iRhom1 or an inhibitor of iRhom2", which is also what the patent relates to. Carl Blobel and the Hospital for Special Surgery have identified iRhom2 inhibitors and have co-founded the start-up company SciRhom in Munich to commercialize these inhibitors, TL has received the following grants: NIH R01AI079178, NIH R21 AR081493, Department of Defense W81XWH-21-LRP-IPA, Lupus Research Alliance Lupus Innovation Award grant, Barbara Volcker Center for Women and Rheumatic Diseases grant. She has also received funding support from the St. Giles Foundation and A Lasting Mark Foundation. She has received consulting fees from Pfizer, and has a received payment from Bristol Meyers Squibb for giving a lecture. The author has received payment for attending Lupus 21st Century meeting. The author has no other competing interests to declare, (© 2024, Li, Zyulina, Seltzer et al.)

The cell surface metalloprotease ADAM17 (a disintegrin and metalloprotease 17) and its binding partners iRhom2 and iRhom1 (inactive Rhomboid-like proteins 1 and 2) modulate cell-cell interactions by mediating the release of membrane proteins such as TNFα (Tumor necrosis factor α) and EGFR (Epidermal growth factor receptor) ligands from the cell surface. Most cell types express both iRhoms, though myeloid cells exclusively express iRhom2, and iRhom1 is the main iRhom in the mouse brain. Here, we report that iRhom2 is uniquely expressed in olfactory sensory neurons (OSNs), highly specialized cells expressing one olfactory receptor (OR) from a repertoire of more than a thousand OR genes in mice. iRhom2-/- mice had no evident morphological defects in the olfactory epithelium (OE), yet RNAseq analysis revealed differential expression of a small subset of ORs. Notably, while the majority of ORs remain unaffected in iRhom2-/- OE, OSNs expressing ORs that are enriched in iRhom2-/- OE showed fewer gene expression changes upon odor environmental changes than the majority of OSNs. Moreover, we discovered an inverse correlation between the expression of iRhom2 compared to OSN activity genes and that odor exposure negatively regulates iRhom2 expression. Given that ORs are specialized G-protein coupled receptors (GPCRs) and many GPCRs activate iRhom2/ADAM17, we investigated if ORs could activate iRhom2/ADAM17. Activation of an olfactory receptor that is ectopically expressed in keratinocytes (OR2AT4) by its agonist Sandalore leads to ERK1/2 phosphorylation, likely via an iRhom2/ADAM17-dependent pathway. Taken together, these findings point to a mechanism by which odor stimulation of OSNs activates iRhom2/ADAM17 catalytic activity, resulting in downstream transcriptional changes to the OR repertoire and activity genes, and driving a negative feedback loop to downregulate iRhom2 expression.

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8 + T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding., (© 2024. The Author(s).)

Tylosis with oesophageal cancer (TOC) is a rare familial disorder caused by cytoplasmic mutations in inactive rhomboid 2 (iRhom2 or iR2, encoded by Rhbdf2). iR2 and the related iRhom1 (or iR1, encoded by Rhbdf1) are key regulators of the membrane-anchored metalloprotease ADAM17, which is required for activating EGFR ligands and for releasing pro-inflammatory cytokines such as TNFα (or TNF). A cytoplasmic deletion in iR2, including the TOC site, leads to curly coat or bare skin (cub) in mice, whereas a knock-in TOC mutation (toc) causes less severe alopecia and wavy fur. The abnormal skin and hair phenotypes of iR2cub/cub and iR2toc/toc mice depend on amphiregulin (Areg) and Adam17, as loss of one allele of either gene rescues the fur phenotypes. Remarkably, we found that iR1-/- iR2cub/cub mice survived, despite a lack of mature ADAM17, whereas iR2cub/cub Adam17-/- mice died perinatally, suggesting that the iR2cub gain-of-function mutation requires the presence of ADAM17, but not its catalytic activity. The iR2toc mutation did not substantially reduce the levels of mature ADAM17, but instead affected its function in a substrate-selective manner. Our findings provide new insights into the role of the cytoplasmic domain of iR2 in vivo, with implications for the treatment of TOC patients., Competing Interests: Competing interests C.P.B. and G.W. are listed as inventors on patents on inhibitors of iRhom2. C.P.B. and the Hospital for Special Surgery have co-founded the start-up company SciRhom in Munich to commercialize these inhibitors., (© 2023. Published by The Company of Biologists Ltd.)

The metalloprotease ADAM17 is a key regulator of the TNFα, IL-6R and EGFR signaling pathways. The maturation and function of ADAM17 is controlled by the seven-membrane-spanning proteins iRhoms1 and 2. The functional properties of the ADAM17/iRhom1 and ADAM17/iRhom2 complexes differ, in that stimulated shedding of most ADAM17 substrates tested to date can be supported by iRhom2, whereas iRhom1 can only support stimulated shedding of very few ADAM17 substrates, such as TGFα. The first transmembrane domain (TMD1) of iRhom2 and the sole TMD of ADAM17 are important for the stimulated shedding of ADAM17 substrates by iRhom2. However, little is currently known about how the iRhoms interact with different substrates to control their stimulated shedding by ADAM17. To provide new insights into this topic, we tested how various chimeras between iRhom1 and iRhom2 affect the stimulated processing of the EGFR-ligands TGFα (iRhom1- or 2-dependent) and EREG (iRhom2-selective) by ADAM17. This uncovered an important role for the TMD7 of the iRhoms in determining their substrate selectivity. Computational methods utilized to characterize the iRhom1/2/substrate interactions suggest that the substrate selectivity is determined, at least in part, by a distinct accessibility of the substrate cleavage site to stimulated ADAM17. These studies not only provide new insights into why the substrate selectivity of stimulated iRhom2/ADAM17 differs from that of iRhom1/ADAM17, but also suggest new approaches for targeting the release of specific ADAM17 substrates.

The severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19, but host cell factors contributing to COVID-19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS-CoV-2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID-19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS-CoV-2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease-targeted inhibitors severely impair lung cell infection by the SARS-CoV-2 variants of concern alpha, beta, delta, and omicron and also reduce SARS-CoV-2 infection of primary human lung cells in a TMPRSS2 protease-independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Proteolytic ectodomain shedding of membrane proteins is a fundamental mechanism to control the communication between cells and their environment. A key protease for membrane protein shedding is ADAM17, which requires a non-proteolytic subunit, either inactive Rhomboid 1 (iRhom1) or iRhom2 for its activity. While iRhom1 and iRhom2 are co-expressed in most tissues and appear to have largely redundant functions, the brain is an organ with predominant expression of iRhom1. Yet, little is known about the spatio-temporal expression of iRhom1 in mammalian brain and about its function in controlling membrane protein shedding in the nervous system. Here, we demonstrate that iRhom1 is expressed in mouse brain from the prenatal stage to adulthood with a peak in early postnatal development. In the adult mouse brain iRhom1 was widely expressed, including in cortex, hippocampus, olfactory bulb, and cerebellum. Proteomic analysis of the secretome of primary neurons using the hiSPECS method and of cerebrospinal fluid, obtained from iRhom1-deficient and control mice, identified several membrane proteins that require iRhom1 for their shedding in vitro or in vivo. One of these proteins was 'multiple-EGF-like-domains protein 10' (MEGF10), a phagocytic receptor in the brain that is linked to the removal of amyloid β and apoptotic neurons. MEGF10 was further validated as an ADAM17 substrate using ADAM17-deficient mouse embryonic fibroblasts. Taken together, this study discovers a role for iRhom1 in controlling membrane protein shedding in the mouse brain, establishes MEGF10 as an iRhom1-dependent ADAM17 substrate and demonstrates that iRhom1 is widely expressed in murine brain., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Notch signaling is critical for controlling a variety of cell fate decisions during metazoan development and homeostasis. This unique, highly conserved signaling pathway relies on cell-to-cell contact, which triggers the proteolytic release of the cytoplasmic domain of the membrane-anchored transcription factor Notch from the membrane. A disintegrin and metalloproteinase (ADAM) proteins are crucial for Notch activation by processing its S2 site. While ADAM10 cleaves Notch1 under physiological, ligand-dependent conditions, ADAM17 mainly cleaves Notch1 under ligand-independent conditions. However, the mechanism(s) that regulate the distinct contributions of these ADAMs in Notch processing remain unclear. Using cell-based assays in mouse embryonic fibroblasts (mEFs) lacking ADAM10 and/or ADAM17, we aimed to clarify what determines the relative contributions of ADAM10 and ADAM17 to ligand-dependent or ligand-independent Notch processing. We found that EDTA-stimulated ADAM17-dependent Notch1 processing is rapid and requires the ADAM17-regulators iRhom1 and iRhom2, whereas the Delta-like 4-induced ligand-dependent Notch1 processing is slower and requires ADAM10. The selectivity of ADAM17 for EDTA-induced Notch1 processing can most likely be explained by a preference for ADAM17 over ADAM10 for the Notch1 cleavage site and by the stronger inhibition of ADAM10 by EDTA. The physiological ADAM10-dependent processing of Notch1 cannot be compensated for by ADAM17 in Adam10-/- mEFs, or by other ADAMs shown here to be able to cleave the Notch1 cleavage site, such as ADAMs9, 12, and 19. Collectively, these results provide new insights into the mechanisms underlying the substrate selectivity of ADAM10 and ADAM17 towards Notch1.

A well-controlled innate immune response is characterized by a rapid yet self-limiting inflammatory response. Although much is known about the range of inflammatory stimuli capable of triggering an innate immune response, the mechanisms which govern the degree of inflammation induced by inflammatory insults and the mechanisms in place to reset or maintain homeostasis are poorly understood. Tumor necrosis factor (TNF) is a potent early response pro-inflammatory cytokine produced by immune cells following a broad range of insults spanning autoimmunity and metabolic diseases to pathogenic infections. Previous studies have shown that a disintegrin and metalloproteinase (ADAM) 17 controls the release of soluble TNF and epidermal growth factor receptor signaling. Utilizing a genetic model of ADAM17 deficiency through the deletion of its regulator, the inactive rhomboid 2 (iRhom2), we show that loss of ADAM17 activity in innate immune cells leads to decreased expression of various cytokines in response to low levels of pathogen-associated molecular pattern (PAMP) stimulation but not at high-dose stimulation. In addition, TNF receptor (TNFR ) 1/2 -deficient bone marrow-derived macrophages yielded significantly reduced TNF expression following low levels of PAMP stimulation, suggesting that signaling through the TNFRs in immune cells drives a feed-forward regulatory mechanism wherein low levels of TNF allow sustained enhancement of TNF expression in an iRhom2/ADAM17-dependent manner. Thus, we demonstrate that inflammatory expression of  TNF and IL1β  is differentially regulated following high or low doses of PAMP stimulation, invoking the activation of a previously unknown regulatory mechanism of inflammation., Competing Interests: CB and the Hospital for Special Surgery have identified iRhom2 inhibitors and have co-founded the start-up company SciRhom in Munich to commercialize these inhibitors. CB and TM hold a patent on a method of identifying agents for combination with inhibitors of iRhoms. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Skurski, Dixit, Blobel, Issuree and Maretzky.)

A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17-/- mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function., Competing Interests: Conflict of interest Drs Maretzky and Blobel hold a patent on a method of identifying agents for combination with inhibitors of iRhoms. Dr Blobel and the Hospital for Special Surgery have identified iRhom2 inhibitors and have cofounded the start-up company SciRhom in Munich to commercialize these inhibitors., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Growth of the axial and appendicular skeleton depends on endochondral ossification, which is controlled by tightly regulated cell-cell interactions in the developing growth plates. Previous studies have uncovered an important role of a disintegrin and metalloprotease 17 (ADAM17) in the normal development of the mineralized zone of hypertrophic chondrocytes during endochondral ossification. ADAM17 regulates EGF-receptor signaling by cleaving EGFR-ligands such as TGFα from their membrane-anchored precursor. The activity of ADAM17 is controlled by two regulatory binding partners, the inactive Rhomboids 1 and 2 (iRhom1, 2), raising questions about their role in endochondral ossification. To address this question, we generated mice lacking iRhom2 ( iR2-/- ) with floxed alleles of iRhom1 that were specifically deleted in chondrocytes by Col2a1-Cre ( iR1∆Ch ). The resulting iR2-/-iR1∆Ch mice had retarded bone growth compared to iR2-/- mice, caused by a significantly expanded zone of hypertrophic mineralizing chondrocytes in the growth plate. Primary iR2-/-iR1∆Ch chondrocytes had strongly reduced shedding of TGFα and other ADAM17-dependent EGFR-ligands. The enlarged zone of mineralized hypertrophic chondrocytes in iR2-/-iR1∆Ch mice closely resembled the abnormal growth plate in A17∆Ch mice and was similar to growth plates in Tgfα-/- mice or mice with EGFR mutations. These data support a model in which iRhom1 and 2 regulate bone growth by controlling the ADAM17/TGFα/EGFR signaling axis during endochondral ossification.

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.

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) regulates EGF-receptor and TNFα signaling, thereby not only protecting the skin and intestinal barrier, but also contributing to autoimmunity. ADAM17 can be rapidly activated by many stimuli through its transmembrane domain (TMD), with the seven membrane-spanning inactive Rhomboids (iRhom) 1 and 2 implicated as candidate regulatory partners. However, several alternative models of ADAM17 regulation exist that do not involve the iRhoms, such as regulation through disulfide bond exchange or through interaction with charged phospholipids. Here, we report that a non-activatable mutant of ADAM17 with the TMD of betacellulin (BTC) can be rescued by restoring residues from the ADAM17 TMD, but only in Adam17 -/- cells, which contain iRhoms, not in iRhom1/2 -/- cells. We also provide the first evidence that the extracellular juxtamembrane domains (JMDs) of ADAM17 and iRhom2 regulate the stimulation and substrate selectivity of ADAM17. Interestingly, a point mutation in the ADAM17 JMD identified in a patient with Tetralogy of Fallot, a serious heart valve defect, affects the substrate selectivity of ADAM17 toward Heparin-binding epidermal growth factor like growth factor (HB-EGF), a crucial regulator of heart valve development in mice. These findings provide new insights into the regulation of ADAM17 through an essential interaction with the TMD1 and JMD1 of iRhom2., (© 2020 Federation of American Societies for Experimental Biology.)

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) is a key regulator of tumor necrosis factor α (TNFα), interleukin 6 receptor (IL-6R), and epidermal growth factor receptor (EGFR) signaling. ADAM17 maturation and function depend on the seven-membrane-spanning inactive rhomboid-like proteins 1 and 2 (iRhom1/2 or Rhbdf1/2). Most studies to date have focused on overexpressed iRhom1 and -2, so only little is known about the properties of the endogenous proteins. Here, we show that endogenous iRhom1 and -2 can be cell surface-biotinylated on mouse embryonic fibroblasts (mEFs), revealing that endogenous iRhom1 and -2 proteins are present on the cell surface and that iRhom2 also is present on the surface of lipopolysaccharide-stimulated primary bone marrow-derived macrophages. Interestingly, very little, if any, iRhom2 was detectable in mEFs or bone marrow-derived macrophages lacking ADAM17, suggesting that iRhom2 is stabilized by ADAM17. By contrast, the levels of iRhom1 were slightly increased in the absence of ADAM17 in mEFs, indicating that its stability does not depend on ADAM17. These findings support a model in which iRhom2 and ADAM17 are obligate binding partners and indicate that iRhom2 stability requires the presence of ADAM17, whereas iRhom1 is stable in the absence of ADAM17., (© 2020 Weskamp et al.)

The coronary vasculature is crucial for normal heart function, yet much remains to be learned about its development, especially the maturation of coronary arterial endothelium. Here, we show that endothelial inactivation of ADAM10, a key regulator of Notch signaling, leads to defects in coronary arterial differentiation, as evidenced by dysregulated genes related to Notch signaling and arterial identity. Moreover, transcriptome analysis indicated reduced EGFR signaling in A10ΔEC coronary endothelium. Further analysis revealed that A10ΔEC mice have enlarged dysfunctional hearts with abnormal myocardial compaction, and increased expression of venous and immature endothelium markers. These findings provide the first evidence for a potential role for endothelial ADAM10 in cardioprotective homeostatic EGFR signaling and implicate ADAM10/Notch signaling in coronary arterial cell specification, which is vital for normal heart development and function. The ADAM10/Notch signaling pathway thus emerges as a potential therapeutic target for improving the regenerative capacity and maturation of the coronary vasculature.

The vasculature is a remarkably interesting, complex, and interconnected organ. It provides a conduit for oxygen and nutrients, filtration of waste products, and rapid communication between organs. Much remains to be learned about the specialized vascular beds that fulfill these diverse, yet vital functions. This review was prompted by the discovery that Notch signaling in mouse endothelial cells is crucial for the development of specialized vascular beds found in the heart, kidneys, liver, intestines, and bone. We will address the intriguing questions raised by the role of Notch signaling and that of its regulator, the metalloprotease ADAM10, in the development of specialized vascular beds. We will cover fundamentals of ADAM10/Notch signaling, the concept of Notch-dependent cell fate decisions, and how these might govern the development of organ-specific vascular beds through angiogenesis or vasculogenesis. We will also consider common features of the affected vessels, including the presence of fenestra or sinusoids and their occurrence in portal systems with two consecutive capillary beds. We hope to stimulate further discussion and study of the role of ADAM10/Notch signaling in the development of specialized vascular structures, which might help uncover new targets for the repair of vascular beds damaged in conditions like coronary artery disease and glomerulonephritis.

Hemophilic arthropathy (HA) is a debilitating degenerative joint disease that is a major manifestation of the bleeding disorder hemophilia A. HA typically begins with hemophilic synovitis that resembles inflammatory arthritides, such as rheumatoid arthritis, and frequently results in bone loss in patients. A major cause of rheumatoid arthritis is inappropriate release of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) by the TNF-α convertase (TACE; also referred to as ADAM17) and its regulator, iRhom2. Therefore, we hypothesized that iRhom2/ADAM17-dependent shedding of TNF-α also has a pivotal role in mediating HA. Here, we show that addition of blood or its components to macrophages activates iRhom2/ADAM17-dependent TNF-α shedding, providing the premise to study the activation of this pathway by blood in the joint in vivo. For this, we turned to hemophilic FVIII- deficient mice ( F8 -/- mice), which develop a hemarthrosis following needle puncture injury with synovial inflammation and significant osteopenia adjacent to the affected joint. We found that needle puncture-induced bleeding leads to increased TNF-α levels in the affected joint of F8 -/- mice. Moreover, inactivation of TNF-α or iRhom2 in F8 -/- mice reduced the osteopenia and synovial inflammation that develops in this mouse model for HA. Taken together, our results suggest that blood entering the joint activates the iRhom2/ADAM17/TNF-α pathway, thereby contributing to osteopenia and synovitis in mice. Therefore, this proinflammatory signaling pathway could emerge as an attractive new target to prevent osteoporosis and joint damage in HA patients., (© 2018 by The American Society of Hematology.)

Photosensitivity, or skin sensitivity to ultraviolet radiation (UVR), is a feature of lupus erythematosus and other autoimmune and dermatologic conditions, but the mechanistic underpinnings are poorly understood. We identify a Langerhans cell (LC)-keratinocyte axis that limits UVR-induced keratinocyte apoptosis and skin injury via keratinocyte epidermal growth factor receptor (EGFR) stimulation. We show that the absence of LCs in Langerin-diphtheria toxin subunit A (DTA) mice leads to photosensitivity and that, in vitro, mouse and human LCs can directly protect keratinocytes from UVR-induced apoptosis. LCs express EGFR ligands and a disintegrin and metalloprotease 17 (ADAM17), the metalloprotease that activates EGFR ligands. Deletion of ADAM17 from LCs leads to photosensitivity, and UVR induces LC ADAM17 activation and generation of soluble active EGFR ligands, suggesting that LCs protect by providing activated EGFR ligands to keratinocytes. Photosensitive systemic lupus erythematosus (SLE) models and human SLE skin show reduced epidermal EGFR phosphorylation and LC defects, and a topical EGFR ligand reduces photosensitivity. Together, our data establish a direct tissue-protective function for LCs, reveal a mechanistic basis for photosensitivity, and suggest EGFR stimulation as a treatment for photosensitivity in lupus erythematosus and potentially other autoimmune and dermatologic conditions., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The transmembrane protein, ADAM10 (a disintegrin and metalloprotease 10), has key physiologic functions-for example, during embryonic development and in the brain. During transit through the secretory pathway, immature ADAM10 (proADAM10) is converted into its proteolytically active, mature form (mADAM10). Increasing or decreasing the abundance and/or activity of mADAM10 is considered to be a therapeutic approach for the treatment of such diseases as Alzheimer's disease and cancer. Yet biochemical detection and characterization of mADAM10 has been difficult. In contrast, proADAM10 is readily detected-for example, in immunoblots-which suggests that mADAM10 is only a fraction of total cellular ADAM10. Here, we demonstrate that mADAM10, but not proADAM10, unexpectedly undergoes rapid, time-dependent degradation upon biochemical cell lysis in different cell lines and in primary neurons, which prevents the detection of the majority of mADAM10 in immunoblots. This degradation required the catalytic activity of ADAM10, was efficiently prevented by adding active site inhibitors to the lysis buffer, and did not affect proADAM10, which suggests that ADAM10 degradation occurred in an intramolecular and autoproteolytic manner. Inhibition of postlysis autoproteolysis demonstrated efficient cellular ADAM10 maturation with higher levels of mADAM10 than proADAM10. Moreover, a cycloheximide chase experiment revealed that mADAM10 is a long-lived protein with a half-life of approximately 12 h. In summary, our study demonstrates that mADAM10 autoproteolysis must be blocked to allow for the proper detection of mADAM10, which is essential for the correct interpretation of biochemical and cellular studies of ADAM10.-Brummer, T., Pigoni, M., Rossello, A., Wang, H., Noy, P. J., Tomlinson, M. G., Blobel, C. P., Lichtenthaler, S. F. The metalloprotease ADAM10 (a disintegrin and metalloprotease 10) undergoes rapid, postlysis autocatalytic degradation.

The principal function of glomeruli is to filter blood through a highly specialized filtration barrier consisting of a fenestrated endothelium, the glomerular basement membrane and podocyte foot processes. Previous studies have uncovered a crucial role of endothelial a disintegrin and metalloprotease 10 (ADAM10) and Notch signaling in the development of glomeruli, yet the resulting defects have not been further characterized nor understood in the context of kidney development. Here, we used several different experimental approaches to analyze the kidneys and glomeruli from mice lacking ADAM10 in endothelial cells (A10ΔEC mice). Scanning electron microscopy of glomerular casts demonstrated enlarged vascular diameter and increased intussusceptive events in A10ΔEC glomeruli compared to controls. Consistent with these findings, genes known to regulate vessel caliber (Apln, AplnR and Vegfr3) are significantly upregulated in A10ΔEC glomeruli. Moreover, transmission electron microscopy revealed the persistence of diaphragms in the fenestrae of A10ΔEC glomerular endothelial cells, which was corroborated by the elevated expression of the protein PLVAP/PV-1, an integral component of fenestral diaphragms. Analysis of gross renal vasculature by light sheet microscopy showed no major alteration of the branching pattern, indicating a localized importance of ADAM10 in the glomerular endothelium. Since intussusceptions and fenestrae with diaphragms are normally found in developing, but not mature glomeruli, our results provide the first evidence for a crucial role of endothelial ADAM10, a key regulator of Notch signaling, in promoting the development and maturation of the glomerular vasculature.

Lupus nephritis (LN) often results in progressive renal dysfunction. The inactive rhomboid 2 (iRhom2) is a newly identified key regulator of A disintegrin and metalloprotease 17 (ADAM17), whose substrates, such as TNF-α and heparin-binding EGF (HB-EGF), have been implicated in the pathogenesis of chronic kidney diseases. Here, we demonstrate that deficiency of iRhom2 protects the lupus-prone Fcgr2b-/- mice from developing severe kidney damage without altering anti-double-stranded DNA (anti-dsDNA) Ab production by simultaneously blocking HB-EGF/EGFR and TNF-α signaling in the kidney tissues. Unbiased transcriptome profiling of kidneys and kidney macrophages revealed that TNF-α and HB-EGF/EGFR signaling pathways are highly upregulated in Fcgr2b-/- mice, alterations that were diminished in the absence of iRhom2. Pharmacological blockade of either TNF-α or EGFR signaling protected Fcgr2b-/- mice from severe renal damage. Finally, kidneys from LN patients showed increased iRhom2 and HB-EGF expression, with interstitial HB-EGF expression significantly associated with chronicity indices. Our data suggest that activation of iRhom2/ADAM17-dependent TNF-α and EGFR signaling plays a crucial role in mediating irreversible kidney damage in LN, thereby uncovering a target for selective and simultaneous dual inhibition of 2 major pathological pathways in the effector arm of the disease.

The xenoestrogens bisphenol-A (BPA) and nonylphenol (NP) are endocrine disruptors used in the plastic polymer industry to manufacture different products for human use. Previous studies have suggested a role of these compounds in the shedding of signaling molecules, such as tumor necrosis factor α (TNF-α). The aim of this work was to evaluate the effect of BPA and NP on the sheddase ADAM17 and its newly discovered regulators iRhom1 and iRhom2 in the release of EGFR-ligands. We report that BPA and NP can stimulate the release of the ADAM17-substrates HB-EGF and TGF-α. In cells lacking ADAM17 (Adam17 -/- mEFs) BPA-stimulated release of HB-EGF, but not TGF-α, was strongly reduced, whereas NP-stimulated shedding of HB-EGF and TGF-α was completely abolished. Inactivation of both ADAM17 and the related ADAM10 (Adam10/17 -/- mEFs) completely prevented the release of these substrates. In the absence of iRhom1, BPA- or NP-stimulated release of HB-EGF or TGF-α was comparable to wild-type control mEFs, conversely the BPA-induced release of HB-EGF was abolished in iRhom2 -/- mEFs. The defect in shedding of HB-EGF in iRhom2 -/- mEF cells could be rescued by overexpressing iRhom2. Interestingly, the NP-stimulated release of HB-EGF was not affected by the absence of iRhom2, suggesting that NP could potentially activate both ADAM10 and ADAM17. We tested this hypothesis using betacellulin (BTC), an EGFR-ligand that is a substrate for ADAM10. We found that NP, but not BPA stimulated the release of BTC in Adam17 -/- , iRhom2 -/- , or iRhom1/2 -/- , but not in Adam10/17 -/- cells. Taken together, our results suggest that BPA and NP stimulate the release of EGFR-ligands by differentially activating ADAM17 or ADAM10. The identification of specific effects of these endocrine disruptors on ADAM10 and ADAM17 will help to provide a better understanding of their roles in cell signaling and proinflammatory processes, and provide new potential targets for treatment of reproductive or inflammatory diseases such as asthma or breast cancer that are promoted by xenoestrogens., (© 2017 Wiley Periodicals, Inc.)

Theta-defensins (θ-defensins) are macrocyclic peptides expressed exclusively in granulocytes and selected epithelia of Old World monkeys. They contribute to anti-pathogen host defense responses by directly killing a diverse range of microbes. Of note, θ-defensins also modulate microbe-induced inflammation by affecting the production of soluble tumor necrosis factor (sTNF) and other proinflammatory cytokines. Here, we report that natural rhesus macaque θ-defensin (RTD) isoforms regulate sTNF cellular release by inhibiting TNF-α-converting enzyme (TACE; also known as ad isintegrin a nd m etalloprotease 17; ADAM17), the primary pro-TNF sheddase. Dose-dependent inhibition of cellular TACE activity by RTDs occurred when leukocytes were stimulated with live Escherichia coli cells as well as numerous Toll-like receptor agonists. Moreover, the relative inhibitory potencies of the RTD isoforms strongly correlated with their suppression of TNF release by stimulated blood leukocytes and THP-1 monocytes. RTD isoforms also inhibited ADAM10, a sheddase closely related to TACE. TACE inhibition was abrogated by introducing a single opening in the RTD-1 backbone, demonstrating that the intact macrocycle is required for enzyme inhibition. Enzymologic analyses showed that RTD-1 is a fast binding, reversible, non-competitive inhibitor of TACE. We conclude that θ-defensin-mediated inhibition of pro-TNF proteolysis by TACE represents a rapid mechanism for the regulation of sTNF and TNF-dependent inflammatory pathways. Molecules with structural and functional features mimicking those of θ-defensins may have clinical utility as TACE inhibitors for managing TNF-driven diseases., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10 nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20 nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

A disintegrin and metalloproteinase 17 (ADAM17) controls the release of the pro-inflammatory cytokine tumor necrosis factor α (TNFα, also known as TNF) and is crucial for protecting the skin and intestinal barrier by proteolytic activation of epidermal growth factor receptor (EGFR) ligands. The seven-membrane-spanning protein called inactive rhomboid 2 (Rhbdf2; also known as iRhom2) is required for ADAM17-dependent TNFα shedding and crosstalk with the EGFR, and a point mutation (known as sinecure, sin) in the first transmembrane domain (TMD) of Rhbdf2 (Rhbdf2 sin ) blocks TNFα shedding, yet little is known about the underlying mechanism. Here, we used a structure-function analysis informed by structural modeling to evaluate the interaction between the TMD of ADAM17 and the first TMD of Rhbdf2, and the role of this interaction in Rhbdf2-ADAM17-dependent shedding. Moreover, we show that double mutant mice that are homozygous for Rhbdf2 sin/sin and lack Rhbdf1 closely resemble Rhbdf1/2 -/- double knockout mice, highlighting the severe functional impact of the Rhbdf2 sin/sin mutation on ADAM17 during mouse development. Taken together, these findings provide new mechanistic and conceptual insights into the critical role of the TMDs of ADAM17 and Rhbdf2 in the regulation of the ADAM17 and EGFR, and ADAM17 and TNFα signaling pathways., (© 2017. Published by The Company of Biologists Ltd.)

CSF1R (colony stimulating factor 1 receptor) is the main receptor for CSF1 and has crucial roles in regulating myelopoeisis. CSF1R can be proteolytically released from the cell surface by ADAM17 (A disintegrin and metalloprotease 17). Here, we identified CSF1R as a major substrate of ADAM17 in an unbiased degradomics screen. We explored the impact of CSF1R shedding by ADAM17 and its upstream regulator, inactive rhomboid protein 2 (iRhom2, gene name Rhbdf2), on homeostatic development of mouse myeloid cells. In iRhom2-/- mice, we found constitutive accumulation of membrane-bound CSF1R on myeloid cells at steady state, although cell numbers of these populations were not altered. However, in the context of mixed bone marrow (BM) chimera, under competitive pressure, iRhom2-/- BM progenitor-derived monocytes, tissue macrophages and lung DCs showed a repopulation advantage over those derived from wild-type (WT) BM progenitors, suggesting enhanced CSF1R signaling in the absence of iRhom2. In vitro experiments indicate that iRhom2-/- Lin - SCA-1 + c-Kit + (LSKs) cells, but not granulocyte-macrophage progenitors (GMPs), had faster growth rates than WT cells in response to CSF1. Our results shed light on an important role of iRhom2/ADAM17 pathway in regulation of CSF1R shedding and repopulation of monocytes, macrophages and DCs., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rationale: Endothelial Notch signaling is critical for early vascular development and survival. Yet, previously described mice lacking endothelial a disintegrin and metalloproteinase 10 (ADAM10), a key regulator of Notch signaling, survived into adulthood with organ-specific vascular defects. These findings raised questions about whether these vascular defects were related to Notch signaling or other functions of ADAM10., Objective: The aims of the study are to determine whether compensatory or redundant functions of ADAM17 in Notch signaling can explain the survival of Adam10ΔEC mice, explore the contribution of different Tie2-Cre transgenes to the differences in survival, and establish whether the Adam10ΔEC vascular phenotypes can be recapitulated by inactivation of Notch receptors in endothelial cells., Methods and Results: Mice lacking ADAM10 and ADAM17 in endothelial cells (Adam10/Adam17ΔEC), which survived postnatally with organ-specific vascular defects, resembled Adam10ΔEC mice. In contrast, Adam10ΔEC mice generated with the Tie2Cre transgene previously used to inactivate endothelial Notch (Adam10ΔEC(Flv)) died by E10.5. Quantitative polymerase chain reaction analysis demonstrated that Cre-mediated recombination occurs earlier in Adam10ΔEC(Flv) mice than in the previously described Adam10ΔEC mice. Finally, mice lacking endothelial Notch1 (Notch1ΔEC) share some organ-specific vascular defects with Adam10ΔEC mice, whereas Notch4(-/-) mice lacking endothelial Notch1 (Notch1ΔEC/Notch4(-/-)) had defects in all vascular beds affected in Adam10ΔEC mice., Conclusions: Our results argue against a major role for ADAM17 in endothelial Notch signaling and clarify the difference in phenotypes of previously described mice lacking ADAM10 or Notch in endothelial cells. Most notably, these findings uncover new roles for Notch signaling in the development of organ-specific vascular beds., (© 2016 American Heart Association, Inc.)

The protease ADAM17 (a disintegrin and metalloproteinase 17) catalyzes the shedding of various transmembrane proteins from the surface of cells, including tumor necrosis factor (TNF) and its receptors. Liberation of TNF receptors (TNFRs) from cell surfaces can dampen the cellular response to TNF, a cytokine that is critical in the innate immune response and promotes programmed cell death but can also promote sepsis. Catalytically inactive members of the rhomboid family of proteases, iRhom1 and iRhom2, mediate the intracellular transport and maturation of ADAM17. Using a genetic screen, we found that the presence of either iRhom1 or iRhom2 lacking part of their extended amino-terminal cytoplasmic domain (herein referred to as ΔN) increases ADAM17 activity, TNFR shedding, and resistance to TNF-induced cell death in fibrosarcoma cells. Inhibitors of ADAM17, but not of other ADAM family members, prevented the effects of iRhom-ΔN expression. iRhom1 and iRhom2 were functionally redundant, suggesting a conserved role for the iRhom amino termini. Cells from patients with a dominantly inherited cancer susceptibility syndrome called tylosis with esophageal cancer (TOC) have amino-terminal mutations in iRhom2. Keratinocytes from TOC patients exhibited increased TNFR1 shedding compared with cells from healthy donors. Our results explain how loss of the amino terminus in iRhom1 and iRhom2 impairs TNF signaling, despite enhancing ADAM17 activity, and may explain how mutations in the amino-terminal region contribute to the cancer predisposition syndrome TOC., (Copyright © 2015, American Association for the Advancement of Science.)

Hemophilia A is an X-linked bleeding disorder that can be largely controlled by treatment with recombinant factor VIII. However, this treatment is only partially effective in preventing hemophilic arthropathy (HA), a debilitating degenerative joint disease that is caused by intra-articular bleeding events. The disease progression of HA has several distinct steps, beginning with hemophilic synovitis (HS), a hyperplasia of the synovial lining coupled with a neovascular response, followed by joint erosion with cartilage destruction and erosion of the underlying bone. The early stages of HA have certain features in common with arthritides such as rheumatoid arthritis (RA), whereas the later degenerative stages of HA have some similarities with osteoarthritis (OA). The main purpose of this review is to explore the similarities between HA with RA and OA and discuss how this information could potentially help understand the pathogenesis of HA and uncover new treatment opportunities., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Introduction: Engagement of the homotypic cell-to-cell adhesion molecule cadherin-11 on rheumatoid arthritis (RA) synovial fibroblasts with a chimeric molecule containing the cadherin-11 extracellular binding domain stimulated cytokine, chemokine, and matrix metalloproteinases (MMP) release, implicating cadherin-11 signaling in RA pathogenesis. The objective of this study was to determine if cadherin-11 extracellular domain fragments are found inside the joint and if a physiologic synovial fibroblast cleavage pathway releases those fragments., Methods: Cadherin-11 cleavage fragments were detected by western blot in cell media or lysates. Cleavage was interrupted using chemical inhibitors or short-interfering RNA (siRNA) gene silencing. The amount of cadherin-11 fragments in synovial fluid was measured by western blot and ELISA., Results: Soluble cadherin-11 extracellular fragments were detected in human synovial fluid at significantly higher levels in RA samples compared to osteoarthritis (OA) samples. A cadherin-11 N-terminal extracellular binding domain fragment was shed from synovial fibroblasts after ionomycin stimulation, followed by presenilin 1 (PSN1)-dependent regulated intramembrane proteolysis of the retained membrane-bound C-terminal fragments. In addition to ionomycin-induced calcium flux, tumor necrosis factor (TNF)-α also stimulated cleavage in both two- and three-dimensional fibroblast cultures. Although cadherin-11 extracellular domains were shed by a disintegrin and metalloproteinase (ADAM) 10 in several cell types, a novel ADAM- and metalloproteinase-independent activity mediated shedding in primary human fibroblasts., Conclusions: Cadherin-11 undergoes ectodomain shedding followed by regulated intramembrane proteolysis in synovial fibroblasts, triggered by a novel sheddase that generates extracelluar cadherin-11 fragments. Cadherin-11 fragments were enriched in RA synovial fluid, suggesting they may be a marker of synovial burden and may function to modify cadherin-11 interactions between synovial fibroblasts.

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.

Proenzyme maturation is a general mechanism to control the activation of enzymes. Catalytically active members of the A Disintegrin And Metalloprotease (ADAM) family of membrane-anchored metalloproteases are synthesized as proenzymes, in which the latency is maintained by their autoinhibitory pro-domains. A proteolytic processing then transforms the proenzyme into a catalytically active form. The removal of the pro-domain of ADAMs is currently thought to depend on processing at a canonical consensus site for the proprotein convertase Furin (RXXR) between the pro- and the catalytic domain. Here, we demonstrate that this previously described canonical site is a secondary cleavage site to a prerequisite cleavage in a newly characterized upstream PC site embedded within the pro-domain sequence. The novel upstream regulatory site is important for the maturation of several ADAM proenzymes. Mutations in the upstream regulatory site of ADAM17, ADAM10, and ADAM9 do not prevent pro-domain processing between the pro- and metalloprotease domain, but nevertheless, cause significantly reduced catalytic activity. Thus, our results have uncovered a novel functionally relevant PC processing site in the N-terminal part of the pro-domain that is important for the activation of these ADAMs. These results suggest that the novel PC site is part of a general mechanism underlying proenzyme maturation of ADAMs that is independent of processing at the previously identified canonical Furin cleavage site., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The membrane-anchored metalloproteinase a disintegrin and metalloprotease 10 (ADAM10) is required for shedding of membrane proteins such as EGF, betacellulin, the amyloid precursor protein, and CD23 from cells. ADAM10 is constitutively active and can be rapidly and post-translationally enhanced by several stimuli, yet little is known about the underlying mechanism. Here, we use ADAM10-deficient cells transfected with wild type or mutant ADAM10 to address the role of its cytoplasmic and transmembrane domain in regulating ADAM10-dependent protein ectodomain shedding. We report that the cytoplasmic domain of ADAM10 negatively regulates its constitutive activity through an ER retention motif but is dispensable for its stimulated activity. However, chimeras with the extracellular domain of ADAM10 and the transmembrane domain of ADAM17 with or without the cytoplasmic domain of ADAM17 show reduced stimulated shedding of the ADAM10 substrate betacellulin, whereas the ionomycin-stimulated shedding of the ADAM17 substrates CD62-L and TGFα is not affected. Moreover, we show that influx of extracellular calcium activates ADAM10 but is not essential for its activation by APMA and BzATP. Finally, the rapid stimulation of ADAM10 is not significantly affected by incubation with proprotein convertase inhibitors for up to 8 h, arguing against a major role of increased prodomain removal in the rapid stimulation of ADAM10. Thus, the cytoplasmic domain of ADAM10 negatively influences constitutive shedding through an ER retention motif, whereas the cytoplasmic domain and prodomain processing are not required for the rapid activation of ADAM10-dependent shedding events., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Purpose: Retinal neovascularization is found in diseases such as macular degeneration, diabetic retinopathy, or retinopathy of prematurity and is usually caused by alterations in oxygen supply. We have previously described that mice lacking the membrane-anchored metalloproteinase ADAM15 (a Disintegrin and Metalloprotease 15) have decreased pathological neovascularization of the retina in the oxygen-induced retinopathy (OIR) model. The main purpose of the present study was to determine the contribution of the catalytic activity of ADAM15 to OIR., Methods: To address this question, we generated knock-in mice carrying an inactivating Glutamate to Alanine (E>A) point mutation in the catalytic site of ADAM15 (Adam15E>A mice) and subjected these animals to the OIR model and a heterotopic tumor model. Moreover, we used cell-based assays to determine whether ADAM15 can process cell surface receptors involved in angiogenesis., Results: We found that pathological neovascularization in the OIR model in Adam15E>A mice was comparable to that observed in wild type mice, but tumor implantation by heterotopically injected melanoma cells was reduced. In cell-based assays, overexpressed ADAM15 could process the FGFR2iiib, but was unable to process several receptors with roles in angiogenesis., Conclusions: Collectively, these results suggest that the catalytic activity of ADAM15 is not crucial for its function in promoting pathological neovascularization in the mouse OIR model, most likely because of the very limited substrate repertoire of ADAM15. Instead, other noncatalytic functions of ADAM15 must be important for its role in the OIR model., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

One of the main complications of haemophilia A is haemophilic arthropathy (HA), a debilitating disease with a significant negative impact on motility and quality of life. Despite major advances in the treatment of haemophilia A, many patients still suffer from HA. We wish to develop new treatments for HA, but must first better understand its causes. Our laboratory studies molecular scissors that release the pro-inflammatory cytokine tumour necrosis factor alpha (TNFα) from cells. TNFα is considered the 'fire alarm' of the body - it helps to fight infections, but can also cause diseases such as inflammatory arthritis. We know that the molecular scissors, called TNFα convertase (TACE), and its newly discovered regulator termed iRhom2 can be rapidly activated by small amounts of cytokines, growth factors, and pro-inflammatory mediators present in the blood. We hypothesize that the rapid activation of TACE could help explain one of the unsolved mysteries regarding the development of HA, which is how even small amounts of blood can provoke a persistent inflammatory response. We propose that once blood enters the joint, iRhom2 and TACE are activated to release TNFα and that this could promote the development of HA in a similar manner to that in which it promotes rheumatoid arthritis (RA). We are currently using immune cells stimulated with blood degradation products, and mouse models of HA, to test this hypothesis. If successful, our study could provide the rationale for testing anti-TNF antibodies, which are already used to treat RA, for the treatment of HA. In addition, they might uncover iRhom2 and TACE as attractive new candidate targets for the treatment of HA., (© 2014 John Wiley & Sons Ltd.)

Mice lacking ADAM10 in endothelial cells (Adam10ΔEC mice) have shorter femurs, tibiae, and humeri than controls, raising questions about how endothelial cells could control long bone growth. We performed a histopathological evaluation of the femur and tibia growth plates at different postnatal stages, and assessed the distribution of TRAP-positive osteoclasts and endothelial cells at the growth plate. The growth plates in Adam10ΔEC mice appeared normal at P7 and P14, but a thickened zone of hypertrophic chondrocytes and increased trabecular bone density were apparent by P21 and later. The number of TRAP+ cells at the COJ was normal at P7 and P14, but was strongly reduced at P21 and later. Moreover, the density of endomucin-stained endothelial cells at the COJ was increased starting at P7. The defects in long bone growth in Adam10ΔEC mice could be caused by a lack of osteoclastogenesis at the COJ. Moreover, ADAM10 appears to regulate endothelial cell organization in the developing bone vasculature, perhaps in a similar manner as in the developing retinal vascular tree, where ADAM10 is thought to control Notch-dependent endothelial cell fate decisions. This study provides evidence for the regulation of osteoclast function by endothelial cells in vivo., (© 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Purpose: To characterize the features and possible mechanism of plus disease in the mouse oxygen-induced retinopathy (OIR) model for retinopathy of prematurity., Methods: Wild-type and Adam (A Disintegrin And Metalloproteinase) knockout mice were exposed to 75% oxygen from postnatal day 7 to 12 (P7 to P12) (hyperoxia), then returned to normal air (relative hypoxia). Live fundus imaging and fluorescein angiography at P17 were compared to immunofluorescence analysis of flat-mounted retinas. Two hallmarks of plus disease, arterial tortuosity and venous dilation, were analyzed on fixed retinas (P12-P17). The length of tortuous vessels was compared to a straight line between two points; the diameter of retinal vessels was determined using ImageJ software, and bromo-deoxyuridine (BrdU) labeling was used to visualize proliferation of retinal vascular cells., Results: Mice developed retinal arterial tortuosity and venous dilation after exposure to OIR, which was visible in live fundus images and fixed whole-mounted retinas. Vein dilation, arterial tortuosity, and BrdU incorporation gradually increased over time. Moreover, Adam8(-/-) and Adam9(-/-) mice and mice lacking Adam10 in endothelial cells were partially protected from plus disease compared to controls., Conclusions: The mouse OIR model can be used to study the pathogenesis of plus disease and identify potential therapeutic targets. The severity of plus disease increases over time following OIR and correlates with increased proliferation of endothelial cells, suggesting that proliferation of vascular cells may be a mechanism underlying the development of plus disease. Moreover, our findings suggest that ADAMs 8, 9, and 10 could be targets for treatment of plus disease.

Endochondral ossification is a highly regulated process that relies on properly orchestrated cell-cell interactions in the developing growth plate. This study is focused on understanding the role of a crucial regulator of cell-cell interactions, the membrane-anchored metalloproteinase ADAM17, in endochondral ossification. ADAM17 releases growth factors, cytokines, and other membrane proteins from cells and is essential for epidermal growth factor receptor (EGFR) signaling and for processing tumor necrosis factor alpha. Here, we report that mice lacking ADAM17 in chondrocytes (A17ΔCh) have a significantly expanded zone of hypertrophic chondrocytes in the growth plate and retarded growth of long bones. This abnormality is caused by an accumulation of the most terminally differentiated type of chondrocytes that produces a calcified matrix. Inactivation of ADAM17 in osteoclasts or endothelial cells does not affect the zone of hypertrophic chondrocytes, suggesting that the main role of ADAM17 in the growth plate is in chondrocytes. This notion is further supported by in vitro experiments showing enhanced hypertrophic differentiation of primary chondrocytes lacking Adam17. The enlarged zone of hypertrophic chondrocytes in A17ΔCh mice resembles that described in mice with mutant EGFR signaling or lack of its ligand transforming growth factor α (TGFα), suggesting that ADAM17 regulates terminal differentiation of chondrocytes during endochondral ossification by activating the TGFα/EGFR signaling axis.

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 iRhom2(-/-) 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.

Purpose: Pathological neovascularization is a crucial component of proliferative retinopathies. Previous studies showed that inactivation of A disintegrin and metalloproteinase 17 (ADAM17), a membrane-anchored metalloproteinase that regulates epidermal growth factor receptor (EGFR) signaling, reduces pathological retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Here, we tested how genetic inactivation of a physiological ADAM17 inhibitor, the tissue inhibitor of matrix metalloproteinases-3 (TIMP3), or intravitreal injection of TIMP3 or of the EGFR inhibitor erlotinib influenced the outcome of OIR., Methods: Wild-type mice were subjected to OIR in a chamber with 75% oxygen for 5 days beginning at postnatal day 7 (P7). Upon removal from the oxygen chamber at P12, they received a single intravitreal injection of TIMP3, erlotinib, or control. The central avascular area and neovascular tufts were measured after 5 days in room air (21% oxygen) at P17. Moreover, OIR experiments were performed with Timp3-/- mice and littermate controls., Results: Timp3-/- mice showed greater revascularization of the central avascular area and developed equal or fewer neovascular tufts compared to littermate controls, depending on the genetic background. Wild-type mice injected with TIMP3 or erlotinib developed fewer neovascular tufts when compared to untreated littermates. Moreover, vessel regrowth into the avascular area was reduced in TIMP3-injected mice, but not in erlotinib-injected mice., Conclusions: These studies demonstrate that TIMP3 and erlotinib inhibit pathological neovascularization in the mouse retina, most likely due to inactivation of ADAM17 and the EGFR, respectively. Thus, TIMP3 and erlotinib emerge as attractive candidate antiangiogenic compounds for prevention and treatment of proliferative retinopathies.

ADAM17 (a disintegrin and metalloproteinase 17) is ubiquitously expressed and cleaves membrane proteins, such as epidermal growth factor receptor (EGFR) ligands, l-selectin, and TNF, from the cell surface, thus regulating responses to tissue injury and inflammation. However, little is currently known about its role in skin homeostasis. We show that mice lacking ADAM17 in keratinocytes (A17(ΔKC)) have a normal epidermal barrier and skin architecture at birth but develop pronounced defects in epidermal barrier integrity soon after birth and develop chronic dermatitis as adults. The dysregulated expression of epidermal differentiation proteins becomes evident 2 d after birth, followed by reduced transglutaminase (TGM) activity, transepidermal water loss, up-regulation of the proinflammatory cytokine IL-36α, and inflammatory immune cell infiltration. Activation of the EGFR was strongly reduced in A17(ΔKC) skin, and topical treatment of A17(ΔKC) mice with recombinant TGF-α significantly improved TGM activity and decreased skin inflammation. Finally, we show that mice lacking the EGFR in keratinocytes (Egfr(ΔKC)) closely resembled A17(ΔKC) mice. Collectively, these results identify a previously unappreciated critical role of the ADAM17-EGFR signaling axis in maintaining the homeostasis of the postnatal epidermal barrier and suggest that this pathway could represent a good target for treatment of epidermal barrier defects.