iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFR signaling

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.ADAM17 (a disintegrin and metalloprotease 17) is a membrane-anchored metalloproteinase that controls two major signaling pathways with important roles in development and disease, the EGF receptor (EGFR) pathway and the proinflammatory tumor necrosis factor α (TNF-α) pathway (1–5). Mice lacking ADAM17 resemble mice with defects in EGFR signaling in that they have open eyes at birth, enlarged semilunar heart valves, and enlarged hypertrophic zones in long bone growth plates, most likely caused by a lack of ADAM17-dependent release of the EGFR ligands transforming growth factor α (TGF-α) and heparin-binding epidermal growth factor (HB-EGF) (3, 6–14). In humans, defects in skin and intestinal barrier protection have been reported in a patient lacking ADAM17 (15) and in patients treated with EGFR inhibitors (16, 17), and similar skin defects were recently identified in a patient with defective EGFR signaling (18). Mouse models of ADAM17/EGFR signaling appear to recapitulate these mechanisms, because defects in skin barrier protection can be observed by inactivating either ADAM17 or the EGFR in keratinocytes (19), as well as in mice expressing very low levels of ADAM17, which also have increased susceptibility to intestinal inflammation (20). A hallmark feature of ADAM17 is its rapid response to various activators of cellular signaling pathways (21–23), which is presumably important to allow a rapid response to injury and to maintain the skin and intestinal barrier. The rapid activation of ADAM17 is controlled by its transmembrane domain whereas the cytoplasmic domain is dispensable in this context (22), suggesting that ADAM17 is regulated by one or more other membrane proteins, yet the underlying mechanism has remained enigmatic.Recent studies have shown that the maturation and function of ADAM17 in myeloid cells depend on inactive Rhomboid 2 (iRhom2), a catalytically inactive member of the Rhomboid family of seven membrane-spanning intramembrane serine proteinases (24–28). Myeloid cells lacking iRhom2 release very little TNF-α in response to activation of Toll-like receptor 4 by lipopolysaccharide (LPS) (24, 26, 28). Therefore, mice lacking iRhom2 are protected from the detrimental effects of TNF-α in mouse models for septic shock and inflammatory arthritis, similar to conditional knockout mice lacking ADAM17 in myeloid cells (11, 26, 29). However, iRhom2^−/− (iR2^−/−) mice are viable with no evident spontaneous pathological phenotypes (26, 29), whereas Adam17^−/− (A17^−/−) mice die shortly after birth (3). A major unresolved question has therefore been whether iRhom2 and the related iRhom1 are the long-sought-after regulators of the function of ADAM17-dependent EGFR signaling in vivo. Here we generate iRhom1^−/− (iR1^−/−) mice, which are viable and healthy, and report that iR1/2^−/− double knockout mice closely resemble mice lacking ADAM17 or the EGFR, providing the first genetic evidence, to our knowledge, that the principal function of iRhoms1/2 during mouse development is to control ADAM17/EGFR signaling.