SummaryProtein ectodomain shedding is crucial for cell-cell interactions because it controls the bioavailability of soluble tumor necrosis factora (TNFa) and ligands of the epidermal growth factor (EGF) receptor, and the release of many other membrane proteins. Various stimuli can rapidly trigger ectodomain shedding, yet much remains to be learned about the identity of the enzymes that respond to these stimuli and the mechanisms underlying their activation. Here, we demonstrate that the membrane-anchored metalloproteinase ADAM17, but not ADAM10, is the sheddase that rapidly responds to the physiological signaling pathways stimulated by thrombin, EGF, lysophosphatidic acid and TNFa. Stimulation of ADAM17 is swift and quickly reversible, and does not depend on removal of its inhibitory pro-domain by pro-protein convertases, or on dissociation of an endogenous inhibitor, TIMP3. Moreover, activation of ADAM17 by physiological stimuli requires its transmembrane domain, but not its cytoplasmic domain, arguing against inside-out signaling via cytoplasmic phosphorylation as the underlying mechanism. Finally, experiments with the tight binding hydroxamate inhibitor DPC333, used here to probe the accessibility of the active site of ADAM17, demonstrate that this inhibitor can quickly bind to ADAM17 in stimulated, but not quiescent cells. These findings support the concept that activation of ADAM17 involves a rapid and reversible exposure of its catalytic site.

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.

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/EGFRdependent pathologies.inactive Rhomboid proteins | a disintegrin and metalloprotease 17 | epidermal growth factor receptor | heparin-binding epidermal growth factor | transforming growth factor alpha

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.

During development, progenitor cells with binary potential give rise to daughter cells that have distinct functions. Heritable epigenetic mechanisms then lock in gene expression programs that define lineage identity. Cd4 regulation in helper and cytotoxic T cells exemplifies this process, with enhancer- and silencer-regulated establishment of epigenetic memories for stable gene expression and repression, respectively. Using a genetic screen, we identified the DNA methylation machinery as essential for maintaining Cd4 silencing in the cytotoxic lineage. Further, we found a requirement for the proximal enhancer in mediating removal of Cd4 DNA methylation marks, allowing for stable expression in T helper cells. These findings suggest that stage-specific methylation and demethylation events in Cd4 regulate its heritable expression in response to the distinct signals that dictate lineage choice during T cell development.

The inheritance of gene expression patterns is dependent on epigenetic regulation, but the establishment and maintenance of epigenetic landscapes during T cell differentiation are incompletely understood. Here we show that two stage-specific Cd4 cis-elements, the previously characterized enhancer E4p and a novel enhancer E4m, coordinately promote Cd4 transcription in mature thymic MHC-II-specific T cells, in part through the canonical Wnt pathway. Specifically, E4p licenses E4m to orchestrate DNA demethylation by TET1 and TET3, which in turn poises the Cd4 locus for transcription in peripheral T cells. Cd4 locus demethylation is important for subsequent Cd4 transcription in activated peripheral T cells wherein these cis-elements become dispensable. By contrast, in developing thymocytes the loss of TET1/3 does not affect Cd4 transcription, highlighting an uncoupled event between transcription and epigenetic modifications. Together our findings reveal an important function for thymic cis-elements in governing gene expression in the periphery via a heritable epigenetic mechanism.

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The anti-inflammatory activity of the phytoalexin resveratrol (RSV) was evaluated in C5 anaphylatoxin (C5a)-stimulated primary neutrophils and in a mouse model of acute peritonitis. Pretreatment of human and mouse neutrophils with RSV significantly blocked oxidative burst, leukocyte migration, degranulation, and inflammatory cytokine production. The anti-inflammatory activity of RSV was a function of inhibition of sphingosine kinase (SphK) activity (IC(50) approximately 20 microM) within 5 min of exposure, its membrane localization, and SphK1-mediated Ca(2+) release. As an experimental control, the SphK1 pharmacological inhibitor N,N-dimethyl sphingosine (DMS) was used to compare the inhibitory effect of RSV. We also provide evidence that the SphK inhibitory effect of RSV was mediated via its ability to block phospholipase D (PLD) activity and membrane recruitment. Furthermore, RSV blocked ERK1/2 phosphorylation, which functioned independently of SphK1 in this study. To provide in vivo relevance to these data, C5a-induced model of acute peritonitis was established, and the effects of prior injection of RSV were investigated. Indeed, prior injection of RSV virtually completely attenuated the effects of C5a on vascular permeability, neutrophil migration, release of interleukin 1beta, tumor necrosis factor alpha, interleukin 6, and the chemokine MIP-1alpha. Taken together, these data demonstrate strong anti-inflammatory activity of RSV in vitro and in vivo and highlight SphK1 as a potential target of this remarkable phytoalexin. These data could have tremendous implications for the clinical use of RSV in inflammatory pathologies.