Cryo-EM reveals that iRhom2 restrains ADAM17 protease activity to control the release of growth factor and inflammatory signals.

A disintegrin and metalloprotease 17 (ADAM17) is a membrane-tethered protease that triggers multiple signaling pathways. It releases active forms of the primary inflammatory cytokine tumor necrosis factor (TNF) and cancer-implicated epidermal growth factor (EGF) family growth factors. iRhom2, a rhomboid-like, membrane-embedded pseudoprotease, is an essential cofactor of ADAM17. Here, we present cryoelectron microscopy (cryo-EM) structures of the human ADAM17/iRhom2 complex in both inactive and active states. These reveal three regulatory mechanisms. First, exploiting the rhomboid-like hallmark of TMD recognition, iRhom2 interacts with the ADAM17 TMD to promote ADAM17 trafficking and enzyme maturation. Second, a unique iRhom2 extracellular domain unexpectedly retains the cleaved ADAM17 inhibitory prodomain, safeguarding against premature activation and dysregulated proteolysis. Finally, loss of the prodomain from the complex mobilizes the ADAM17 protease domain, contributing to its ability to engage substrates. Our results reveal how a rhomboid-like pseudoprotease has been repurposed during evolution to regulate a potent membrane-tethered enzyme, ADAM17, ensuring the fidelity of inflammatory and growth factor signaling.

ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

Ectodomain shedding of PLA2R1 is mediated by the metalloproteases ADAM10 and ADAM17.

Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer and is the major autoantigen in membranous nephropathy, a rare but severe autoimmune kidney disease. A soluble form of PLA2R1 has been detected in mouse and human serum. It is likely produced by proteolytic shedding of membrane-bound PLA2R1 but the mechanism is unknown. Here, we show that human PLA2R1 is cleaved by A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 in HEK293 cells, mouse embryonic fibroblasts, and human podocytes. By combining site-directed mutagenesis and sequencing, we determined the exact cleavage site within the extracellular juxtamembrane stalk of human PLA2R1. Orthologs and paralogs of PLA2R1 are also shed. By using pharmacological inhibitors and genetic approaches with RNA interference and knock-out cellular models, we identified a major role of ADAM10 in the constitutive shedding of PLA2R1 and a dual role of ADAM10 and ADAM17 in the stimulated shedding. We did not observe evidence for cleavage by ß- or γ-secretase, suggesting that PLA2R1 may not be a substrate for regulated intramembrane proteolysis. PLA2R1 shedding occurs constitutively and can be triggered by the calcium ionophore ionomycin, the protein kinase C activator PMA, cytokines, and lipopolysaccharides, in vitro and in vivo. Altogether, our results show that PLA2R1 is a novel substrate for ADAM10 and ADAM17, producing a soluble form that is increased in inflammatory conditions and likely exerts various functions in physiological and pathophysiological conditions including inflammation, cancer, and membranous nephropathy.

Analysis of the function of ADAM17 in iRhom2 curly-bare and tylosis with esophageal cancer mutant mice.

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.

The metalloproteinase ADAM10 sheds angiotensin-converting enzyme (ACE) from the pulmonary endothelium as a soluble, functionally active convertase.

The renin-angiotensin-aldosterone system (RAAS) plays a critical role in the regulation of blood pressure and fluid balance, with angiotensin-converting enzyme (ACE) being a key transmembrane enzyme that converts angiotensin I to angiotensin II. Hence, ACE activity is an important drug target in cardiovascular pathologies such as hypertension. Our study demonstrates that human pulmonary microvascular endothelial cells (HPMECs) are an important source of proteolytically released ACE. The proteolytic release of transmembrane proteins, a process known as ectodomain shedding, is facilitated by membrane proteases called sheddases. By knockout and inhibition studies, we identified ADAM10 (A disintegrin and metalloprotease 10) as a primary sheddase responsible for ACE release in HEK293 cells. The function of ADAM10 as primary, constitutive sheddase of ACE was confirmed in HPMECs. Moreover, we demonstrated the physiological relevance of ADAM10 for ACE shedding in ex vivo precision cut lung slices (PCLS) from human and mouse lungs. Notably, ADAM17 activity is not directly involved in ACE shedding but indirectly by regulating ACE mRNA and protein levels, leading to increased ADAM10-mediated ACE shedding. Importantly, soluble ACE generated by shedding is enzymatically active and can thereby participate in systemic RAAS functions. Taken together, our findings highlight the critical role of ADAM10 (directly) and ADAM17 (indirectly) in ACE shedding and RAAS modulation.

SIRT1 mediates the inflammatory response of macrophages and regulates the TIMP3/ADAM17 pathway in atherosclerosis.

OBJECTIVE: Macrophage polarization and the resulting phenotype have versatile roles in atherosclerosis. The study aims to decipher the role of SIRT1 in regulating macrophage phenotypes and atherosclerosis development. METHODS: Two mouse lines of SIRT1△Mac/ApoE-/- and SIRT1fl/fl/ApoE-/- were fed with high-fat diet to generate atherosclerotic lesion. Mouse peritoneal macrophages were isolated and transfected with SIRT1-overexpressing vector or vector-null. RESULTS: The SIRT1△Mac/ApoE-/- mice exhibited greater atherosclerotic lesions, stronger immunofluorescence staining for M1-like macrophage marker, iNOS, and weaker immunofluorescence staining for M2-like macrophage marker, Arginase-1, than the SIRT1fl/fl/ApoE-/- littermates. The gene expressions of M1 markers (IL-1ß, IL-6, and iNOS) were increased and those of M2 markers (IL-10 and Arg-1) decreased in both aortic roots and peritoneal macrophages from SIRT1△Mac/ApoE-/- mice, whereas SIRT1 overexpression rectified the changes in M1/M2 expression. A declined expression of TIMP3 with an increased expression of ADAM17 was noted in SIRT1△Mac/ApoE-/- macrophages, whereas SIRT1 overexpression rescued TIMP3 expression and inhibited ADAM17 expression. CONCLUSION: Our data suggest that SIRT1 deficiency may promote macrophage M1 polarization and regulate the TIMP3/ADAM17 pathway thus favoring atherosclerosis development, indicating an anti-atherosclerotic role of macrophage SIRT1.

iRhom2 regulates ectodomain shedding and surface expression of the major histocompatibility complex (MHC) class I.

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.

Pathological mutations reveal the key role of the cytosolic iRhom2 N-terminus for phosphorylation-independent 14-3-3 interaction and ADAM17 binding, stability, and activity.

The protease ADAM17 plays an important role in inflammation and cancer and is regulated by iRhom2. Mutations in the cytosolic N-terminus of human iRhom2 cause tylosis with oesophageal cancer (TOC). In mice, partial deletion of the N-terminus results in a curly hair phenotype (cub). These pathological consequences are consistent with our findings that iRhom2 is highly expressed in keratinocytes and in oesophageal cancer. Cub and TOC are associated with hyperactivation of ADAM17-dependent EGFR signalling. However, the underlying molecular mechanisms are not understood. We have identified a non-canonical, phosphorylation-independent 14-3-3 interaction site that encompasses all known TOC mutations. Disruption of this site dysregulates ADAM17 activity. The larger cub deletion also includes the TOC site and thus also dysregulated ADAM17 activity. The cub deletion, but not the TOC mutation, also causes severe reductions in stimulated shedding, binding, and stability of ADAM17, demonstrating the presence of additional regulatory sites in the N-terminus of iRhom2. Overall, this study contrasts the TOC and cub mutations, illustrates their different molecular consequences, and reveals important key functions of the iRhom2 N-terminus in regulating ADAM17.

Blockade of the protease ADAM17 ameliorates experimental pancreatitis.

Acute and chronic pancreatitis, the latter associated with fibrosis, are multifactorial inflammatory disorders and leading causes of gastrointestinal disease-related hospitalization. Despite the global health burden of pancreatitis, currently, there are no effective therapeutic agents. In this regard, the protease A Disintegrin And Metalloproteinase 17 (ADAM17) mediates inflammatory responses through shedding of bioactive inflammatory cytokines and mediators, including tumor necrosis factor α (TNFα) and the soluble interleukin (IL)-6 receptor (sIL-6R), the latter of which drives proinflammatory IL-6 trans-signaling. However, the role of ADAM17 in pancreatitis is unclear. To address this, Adam17ex/ex mice-which are homozygous for the hypomorphic Adam17ex allele resulting in marked reduction in ADAM17 expression-and their wild-type (WT) littermates were exposed to the cerulein-induced acute pancreatitis model, and acute (1-wk) and chronic (20-wk) pancreatitis models induced by the cigarette smoke carcinogen nicotine-derived nitrosamine ketone (NNK). Our data reveal that ADAM17 expression was up-regulated in pancreatic tissues of animal models of pancreatitis. Moreover, the genetic (Adam17ex/ex mice) and therapeutic (ADAM17 prodomain inhibitor [A17pro]) targeting of ADAM17 ameliorated experimental pancreatitis, which was associated with a reduction in the IL-6 trans-signaling/STAT3 axis. This led to reduced inflammatory cell infiltration, including T cells and neutrophils, as well as necrosis and fibrosis in the pancreas. Furthermore, up-regulation of the ADAM17/IL-6 trans-signaling/STAT3 axis was a feature of pancreatitis patients. Collectively, our findings indicate that the ADAM17 protease plays a pivotal role in the pathogenesis of pancreatitis, which could pave the way for devising novel therapeutic options to be deployed against this disease.

Development of a Proteomic Workflow for the Identification of Heparan Sulphate Proteoglycan-Binding Substrates of ADAM17.

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.

Proinflammatory microglial activation impairs in vitro cortical tissue repair via zinc-dependent ADAM17 cleavage of the CSF-1 receptor.

Infection and subsequent inflammatory processes negatively impact prognosis in individuals with traumatic brain injury (TBI). Tissue repair following TBI is tightly regulated by microglia, promoting or, importantly, preventing astrocyte-mediated repair processes, depending on the activation state of the neuroimmune cells. This study investigated the poorly understood mechanism linking proinflammatory microglia activation and astrocyte-mediated tissue repair using an in vitro mechanical injury model in mixed cortical cultures of rat neurons and glia. We hypothesized that proinflammatory activation disrupts the microglial response to colony-stimulating factor 1 (CSF-1), which stimulates microglia migration and proliferation, both essential for astrocyte-mediated tissue repair. Following mechanical damage, cultures were treated with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) to induce a proinflammatory state. Immunocytochemical and biochemical analyses were used to evaluate glial repair. Proinflammatory activation dramatically impeded wound closure, reducing microglial levels via upregulation of the zinc-dependent disintegrin and metalloprotease 17 (ADAM17), leading to the cleavage of the CSF-1 receptor (CSF-1R). Indeed, pharmacological inhibition of ADAM17 effectively promoted wound closure during inflammation. Moreover, zinc chelation prevented ADAM17-mediated cleavage of CSF-1R and induced the release of trophic factors, dramatically improving tissue recovery. Our findings strongly identify ADAM17 as a primary regulator of CSF-1R-mediated signaling and establish a mechanism defining the association between pro-inflammatory microglial activation and tissue repair following injury.

Photoreceptors inhibit pathological retinal angiogenesis through transcriptional regulation of Adam17 via c-Fos.

Pathological retinal angiogenesis profoundly impacts visual function in vascular eye diseases, such as retinopathy of prematurity (ROP) in preterm infants and age-related macular degeneration in the elderly. While the involvement of photoreceptors in these diseases is recognized, the underlying mechanisms remain unclear. This study delved into the pivotal role of photoreceptors in regulating abnormal retinal blood vessel growth using an oxygen-induced retinopathy (OIR) mouse model through the c-Fos/A disintegrin and metalloprotease 17 (Adam17) axis. Our findings revealed a significant induction of c-Fos expression in rod photoreceptors, and c-Fos depletion in these cells inhibited pathological neovascularization and reduced blood vessel leakage in the OIR mouse model. Mechanistically, c-Fos directly regulated the transcription of Adam17 a shedding protease responsible for the production of bioactive molecules involved in inflammation, angiogenesis, and cell adhesion and migration. Furthermore, we demonstrated the therapeutic potential by using an adeno-associated virus carrying a rod photoreceptor-specific short hairpin RNA against c-fos which effectively mitigated abnormal retinal blood vessel overgrowth, restored retinal thickness, and improved electroretinographic (ERG) responses. In conclusion, this study highlights the significance of photoreceptor c-Fos in ROP pathology, offering a novel perspective for the treatment of this disease.

ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration.

Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. Here, we reveal an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age-related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age-related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. We show that the glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but that in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, we demonstrate the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, we have discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina.

Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits.

PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits. Only a limited number of PP2A-regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site-specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A-B56 and PP2A-B55 holoenzymes. Strikingly, we find that B-subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A-B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A-B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.

iRhom2 regulates ERBB signalling to promote KRAS-driven tumour growth of lung cancer cells.

Dysregulation of the ERBB/EGFR signalling pathway causes multiple types of cancer. Accordingly, ADAM17, the primary shedding enzyme that releases and activates ERBB ligands, is tightly regulated. It has recently become clear that iRhom proteins, inactive members of the rhomboid-like superfamily, are regulatory cofactors for ADAM17. Here, we show that oncogenic KRAS mutants target the cytoplasmic domain of iRhom2 (also known as RHBDF2) to induce ADAM17-dependent shedding and the release of ERBB ligands. Activation of ERK1/2 by oncogenic KRAS induces the phosphorylation of iRhom2, recruitment of the phospho-binding 14-3-3 proteins, and consequent ADAM17-dependent shedding of ERBB ligands. In addition, cancer-associated mutations in iRhom2 act as sensitisers in this pathway by further increasing KRAS-induced shedding of ERBB ligands. This mechanism is conserved in lung cancer cells, where iRhom activity is required for tumour xenograft growth. In this context, the activity of oncogenic KRAS is modulated by the iRhom2-dependent release of ERBB ligands, thus placing the cytoplasmic domain of iRhom2 as a central component of a positive feedback loop in lung cancer cells. This article has an associated First Person interview with the first authors of the paper.

An ADAM17 selective inhibitor promotes glucose uptake by activating AMPK.

AMPK activation promotes glucose and lipid metabolism. Here, we found that our previously reported ADAM17 inhibitor SN-4 activates AMPK and promotes membrane translocation and sugar uptake of GLUT4. AMPK inhibitor dorsomorphin reversed this effect of SN-4, confirming that the effect is mediated by AMPK activation. In addition, SN-4 inhibited lipid accumulation in HepG2 under high glucose conditions by promoting lipid metabolism and inhibiting lipid synthesis. Although lactic acidosis is a serious side effect of biguanides such as metformin, SN-4 did not affect lactate production. Furthermore, SN-4 was confirmed to inhibit the release of TNF-α, a causative agent of insulin resistance, from adipocytes. In diabetes treatment, it is important to not only regulate blood sugar levels but also prevent complications. Our findings reveal the therapeutic potential of SN-4 as a new antidiabetic drug that can also help prevent future complications.

GRWD1 Over-Expression Promotes Gastric Cancer Progression by Activating Notch Signaling Pathway via Up-Regulation of ADAM17.

BACKGROUND: Glutamate-rich WD repeat containing 1 (GRWD1) is over-expressed in a variety of malignant tumors and is considered to be a potential oncogene. However, its mechanism of action in gastric cancer (GC) is still unclear. METHODS: Data analysis, Immunohistochemistry, and Western Blot (WB) were performed to verify the expression of GRWD1 in GC and para-cancerous tissues. The association between GRWD1 expression and tumor size, tissue differentiation, lymph node metastasis, TNM stage, and prognosis was analyzed according to the high and low expression levels of GRWD1. The relationship between GRWD1 and Notch pathway was verified by data analysis and WB. The effects of GRWD1 on the proliferation, migration, and invasion of GC cells were verified by cell proliferation, migration, and invasion assays. We confirmed that the high expression of GRWD1 promoted the proliferation of GC cells in vivo through the tumor formation assay in nude mice. RESULTS: The expression of GRWD1 was higher in GC tissues than in para-cancerous tissues, and its expression was positively correlated with tumor size, lymph node metastasis, and TNM stage, but negatively correlated with differentiation grade and prognosis. GRWD1 over-expression increased ADAM metallopeptidase domain 17 (ADAM17) expression and promoted Notch1 intracellular domain (NICD) release to promote GC cell proliferation, migration, and invasion in vitro. Results from animal studies have shown that high GRWD1 expression could promote GC cell proliferation in vivo by activating the Notch signaling pathway. CONCLUSION: GRWD1 promotes GC progression through ADAM17-dependent Notch signaling, and GRWD1 may be a novel tumor marker and therapeutic target.

A structural model of the iRhom-ADAM17 sheddase complex reveals functional insights into its trafficking and activity.

Several membrane-anchored signal mediators such as cytokines (e.g. TNFα) and growth factors are proteolytically shed from the cell surface by the metalloproteinase ADAM17, which, thus, has an essential role in inflammatory and developmental processes. The membrane proteins iRhom1 and iRhom2 are instrumental for the transport of ADAM17 to the cell surface and its regulation. However, the structure-function determinants of the iRhom-ADAM17 complex are poorly understood. We used AI-based modelling to gain insights into the structure-function relationship of this complex. We identified different regions in the iRhom homology domain (IRHD) that are differentially responsible for iRhom functions. We have supported the validity of the predicted structure-function determinants with several in vitro, ex vivo and in vivo approaches and demonstrated the regulatory role of the IRHD for iRhom-ADAM17 complex cohesion and forward trafficking. Overall, we provide mechanistic insights into the iRhom-ADAM17-mediated shedding event, which is at the centre of several important cytokine and growth factor pathways.

Estradiol mitigates stress-induced cardiac injury and inflammation by downregulating ADAM17 via the GPER-1/PI3K signaling pathway.

Stress-induced cardiovascular diseases characterized by inflammation are among the leading causes of morbidity and mortality in postmenopausal women worldwide. Estradiol (E2) is known to be cardioprotective via the modulation of inflammatory mediators during stress. But the mechanism is unclear. TNFα, a key player in inflammation, is primarily converted to its active form by 'A Disintegrin and Metalloprotease 17' (ADAM17). We investigated if E2 can regulate ADAM17 during stress. Experiments were performed using female FVB wild-type (WT), C57BL/6 WT, and G protein-coupled estrogen receptor 1 knockout (GPER-1 KO) mice and H9c2 cells. The study revealed a significant increase in cardiac injury and inflammation during isoproterenol (ISO)-induced stress in ovariectomized (OVX) mice. Additionally, ADAM17's membrane content (mADAM17) was remarkably increased in OVX and GPER-1 KO mice during stress. However, in vivo supplementation of E2 significantly reduced cardiac injury, mADAM17, and inflammation. Also, administering G1 (GPER-1 agonist) in mice under stress reduced mADAM17. Further experiments demonstrated that E2, via GPER-1/PI3K pathway, localized ADAM17 at the perinuclear region by normalizing ß1AR-Gαs, mediating the switch from ß2AR-Gαi to Gαs, and reducing phosphorylated kinases, including p38 MAPKs and ERKs. Thus, using G15 and LY294002 to inhibit GPER-1 and its down signaling molecule, PI3K, respectively, in the presence of E2 during stress resulted in the disappearance of E2's modulatory effect on mADAM17. In vitro knockdown of ADAM17 during stress significantly reduced cardiac injury and inflammation, confirming its significant inflammatory role. These interesting findings provide novel evidence that E2 and G1 are potential therapeutic agents for ADAM17-induced inflammatory diseases associated with postmenopausal females.

Inhibition of ADAM17 attenuates high glucose-induced angiogenesis and inflammation in endothelial cells partly through down-regulation of GRO-α/CXCR2 expression: implications in peritoneal dialysis.

BACKGROUND: Angiogenesis and inflammation are key events leading to peritoneal morphologic alteration and ultrafiltration failure in patients undergoing peritoneal dialysis (PD). The current study aims to explore the role of ADAM17 in the angiogenetic and inflammatory responses of endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured and treated with a high glucose-containing medium. In parallel experiments, the expression of ADAM17 in HUVECs was inhibited by SiRNA interference. The mRNA and protein expression of ADAM17, GRO-α and CXCR2 were assessed by qPCR and Western blotting, respectively. The concentrations of GRO-α, VEGF, IL-6 and TNF-α in the cellular supernatants were determined by ELISA. Tube formation and migration of HUVECs were evaluated by Matrigel and transwell migration apparatus. RESULTS: High glucose increased the expression of ADAM17, CXCR2 and GRO-α in cultured HUVECs. RNA silencing of ADAM17 abolished high glucose-mediated increase of GRO-α and CXCR2, which were accompanied by reduced secretion of VEGF, IL-6, TNF-α, as well as tube formation and cell migration in HUVECs. CONCLUSIONS: Inhibition of ADAM17 ameliorates high glucose-induced angiogenic and inflammatory responses in endothelial cells partly through down-regulation of GRO-α/CXCR2 expression.