Roles for ADAM17 in TNF-R1 Mediated Cell Death and Survival in Human U937 and Jurkat Cells.

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer.

ADAM17 Deficiency Protects against Pulmonary Emphysema.

Pulmonary emphysema is the major debilitating component of chronic obstructive pulmonary disease (COPD), which is a leading cause of morbidity and mortality worldwide. The ADAM17 (A disintegrin and metalloproteinase 17) protease mediates inflammation via ectodomain shedding of numerous proinflammatory cytokines, cytokine receptors, and adhesion molecules; however, its role in the pathogenesis of emphysema and COPD is poorly understood. This study aims to define the role of the protease ADAM17 in the pathogenesis of pulmonary emphysema. ADAM17 protein expression and activation was investigated in lung biopsies from patients with emphysema, as well as lungs of the emphysematous gp130F/F mouse model and an acute (4 d) cigarette smoke (CS)-induced lung pathology model. The Adam17ex/ex mice, which display significantly reduced global ADAM17 expression, were coupled with emphysema-prone gp130F/F mice to produce gp130F/F:Adam17ex/ex. Both Adam17ex/ex and wild-type mice were subjected to acute CS exposure. Histological, immunohistochemical, immunofluorescence, and molecular analyses as well as lung function tests were performed to assess pulmonary emphysema, inflammation, and alveolar cell apoptosis. ADAM17 was hyperphosphorylated in the lungs of patients with emphysema and also in emphysematous gp130F/F and CS-exposed mice. ADAM17 deficiency ameliorated the development of pulmonary emphysema in gp130F/F mice by suppressing elevated alveolar cell apoptosis. In addition, genetic blockade of ADAM17 protected mice from CS-induced pulmonary inflammation and alveolar cell apoptosis. Our study places the protease ADAM17 as a central molecular switch implicated in the development of pulmonary emphysema, which paves the way for using ADAM17 inhibitors as potential therapeutic agents to treat COPD and emphysema.

Inactivation of Endothelial ADAM17 Reduces Retinal Ischemia-Reperfusion Induced Neuronal and Vascular Damage.

Retinal ischemia contributes to visual impairment in ischemic retinopathies. A disintegrin and metalloproteinase ADAM17 is implicated in multiple vascular pathologies through its ability to regulate inflammatory signaling via ectodomain shedding. We investigated the role of endothelial ADAM17 in neuronal and vascular degeneration associated with retinal ischemia reperfusion (IR) injury using mice with conditional inactivation of ADAM17 in vascular endothelium. ADAM17Cre-flox and control ADAM17flox mice were subjected to 40 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Albumin extravasation and retinal leukostasis were evaluated 48 h after reperfusion. Retinal morphometric analysis was conducted 7 days after reperfusion. Degenerate capillaries were assessed by elastase digest and visual function was evaluated by optokinetic test 14 and 7 days following ischemia, respectively. Lack of ADAM17 decreased vascular leakage and reduced retinal thinning and ganglion cell loss in ADAM17Cre-flox mice. Further, ADAM17Cre-flox mice exhibited a remarkable reduction in capillary degeneration following IR. Decrease in neurovascular degeneration in ADAM17Cre-flox mice correlated with decreased activation of caspase-3 and was associated with reduction in oxidative stress and retinal leukostasis. In addition, knockdown of ADAM17 resulted in decreased cleavage of p75NTR, the process known to be associated with retinal cell apoptosis. A decline in visual acuity evidenced by decrease in spatial frequency threshold observed in ADAM17flox mice was partially restored in ADAM17-endothelial deficient mice. The obtained results provide evidence that endothelial ADAM17 is an important contributor to IR-induced neurovascular damage in the retina and suggest that interventions directed at regulating ADAM17 activity can be beneficial for alleviating the consequences of retinal ischemia.

Proximal Tubule-Derived Amphiregulin Amplifies and Integrates Profibrotic EGF Receptor Signals in Kidney Fibrosis.

BACKGROUND: Sustained activation of EGF receptor (EGFR) in proximal tubule cells is a hallmark of progressive kidney fibrosis after AKI and in CKD. However, the molecular mechanisms and particular EGFR ligands involved are unknown. METHODS: We studied EGFR activation in proximal tubule cells and primary tubular cells isolated from injured kidneys in vitro. To determine in vivo the role of amphiregulin, a low-affinity EGFR ligand that is highly upregulated with injury, we used ischemia-reperfusion injury or unilateral ureteral obstruction in mice with proximal tubule cell-specific knockout of amphiregulin. We also injected soluble amphiregulin into knockout mice with proximal tubule cell-specific deletion of amphiregulin's releasing enzyme, the transmembrane cell-surface metalloprotease, a disintegrin and metalloprotease-17 (ADAM17), and into ADAM17 hypomorphic mice. RESULTS: Yes-associated protein 1 (YAP1)-dependent upregulation of amphiregulin transcript and protein amplifies amphiregulin signaling in a positive feedback loop. YAP1 also integrates signals of other moderately injury-upregulated, low-affinity EGFR ligands (epiregulin, epigen, TGFα), which also require soluble amphiregulin and YAP1 to induce sustained EGFR activation in proximal tubule cells in vitro. In vivo, soluble amphiregulin injection sufficed to reverse protection from fibrosis after ischemia-reperfusion injury in ADAM17 hypomorphic mice; injected soluble amphiregulin also reversed the corresponding protective proximal tubule cell phenotype in injured proximal tubule cell-specific ADAM17 knockout mice. Moreover, the finding that proximal tubule cell-specific amphiregulin knockout mice were protected from fibrosis after ischemia-reperfusion injury or unilateral ureteral obstruction demonstrates that amphiregulin was necessary for the development of fibrosis. CONCLUSIONS: Our results identify amphiregulin as a key player in injury-induced kidney fibrosis and suggest therapeutic or diagnostic applications of soluble amphiregulin in kidney disease.

Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

TNF-α-converting enzyme, a member of the ADAM (A disintegrin and metalloproteinase) protease family and also known as ADAM17, regulates inflammation and regeneration in health and disease. ADAM17 targets are involved in pain development and hypersensitivity in animal models of inflammatory and neuropathic pain. However, the role of ADAM17 in the pain pathway is largely unknown. Therefore, we used the hypomorphic ADAM17 (ADAM17ex/ex) mouse model to investigate the importance of ADAM17 in nociceptive behavior, morphology, and function of primary afferent nociceptors. ADAM17ex/ex mice were hyposensitive to noxious stimulation, showing elevated mechanical thresholds as well as impaired heat and cold sensitivity. Despite these differences, skin thickness and innervation were comparable to controls. Although dorsal root ganglia of ADAM17ex/ex mice exhibited normal morphology of peptidergic and nonpeptidergic neurons, a small but significant reduction in the number of isolectin ß-4-positive neurons was observed. Functional electrical properties of unmyelinated nociceptors showed differences in resting membrane potential, afterhyperpolarization, and firing patterns in specific subpopulations of sensory neurons in ADAM17ex/ex mice. However, spinal cord morphology and microglia activity in ADAM17ex/ex mice were not altered. Our data suggest that ADAM17 contributes to the processing of painful stimuli, with a complex mode of action orchestrating the function of neurons along the pain pathway.-Quarta, S., Mitric, M., Kalpachidou, T., Mair, N., Schiefermeier-Mach, N., Andratsch, M., Qi, Y., Langeslag, M., Malsch, P., Rose-John, S., Kress, M. Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation.

Macrophages are prominent cells in acute and chronic inflammatory diseases. Recent studies highlight a role for macrophage proliferation post-monocyte recruitment under inflammatory conditions. Using an acute peritonitis model, we identify a significant defect in macrophage proliferation in mice lacking the leukocyte transmembrane protease ADAM17. The defect is associated with decreased levels of macrophage colony-stimulating factor 1 (CSF-1) in the peritoneum and is rescued by intraperitoneal injection of CSF-1. Cell surface CSF-1 (csCSF-1) is one of the substrates of ADAM17. We demonstrate that both infiltrated neutrophils and macrophages are major sources of csCSF-1. Furthermore, acute shedding of csCSF-1 following neutrophil extravasation is associated with elevated expression of iRhom2, a member of the rhomboid-like superfamily, which promotes ADAM17 maturation and trafficking to the neutrophil surface. Accordingly, deletion of hematopoietic iRhom2 is sufficient to prevent csCSF-1 release from neutrophils and macrophages and to prevent macrophage proliferation. In acute inflammation, csCSF-1 release and macrophage proliferation are self-limiting due to transient leukocyte recruitment and temporally restricted csCSF-1 expression. In chronic inflammation, such as atherosclerosis, the ADAM17-mediated lesional macrophage proliferative response is prolonged. Our results demonstrate a novel mechanism whereby ADAM17 promotes macrophage proliferation in states of acute and chronic inflammation.

ADAM17 is required for EGF-R-induced intestinal tumors via IL-6 trans-signaling.

Colorectal cancer is treated with antibodies blocking epidermal growth factor receptor (EGF-R), but therapeutic success is limited. EGF-R is stimulated by soluble ligands, which are derived from transmembrane precursors by ADAM17-mediated proteolytic cleavage. In mouse intestinal cancer models in the absence of ADAM17, tumorigenesis was almost completely inhibited, and the few remaining tumors were of low-grade dysplasia. RNA sequencing analysis demonstrated down-regulation of STAT3 and Wnt pathway components. Because EGF-R on myeloid cells, but not on intestinal epithelial cells, is required for intestinal cancer and because IL-6 is induced via EGF-R stimulation, we analyzed the role of IL-6 signaling. Tumor formation was equally impaired in IL-6-/- mice and sgp130Fc transgenic mice, in which only trans-signaling via soluble IL-6R is abrogated. ADAM17 is needed for EGF-R-mediated induction of IL-6 synthesis, which via IL-6 trans-signaling induces ß-catenin-dependent tumorigenesis. Our data reveal the possibility of a novel strategy for treatment of colorectal cancer that could circumvent intrinsic and acquired resistance to EGF-R blockade.

Hepatocyte specific TIMP3 expression prevents diet dependent fatty liver disease and hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of conditions, ranging from non-progressive bland steatosis to hepatocarcinoma. Tissue inhibitor of metalloproteinase 3 (Timp3) has a role in the pathogenesis of fatty liver disease associated with obesity and is silenced during metabolic disorders and liver cancer. We generated an hepatocyte-specific TIMP3 'gain-of-function' mouse model under the control of the Albumin promoter (AlbT3) and investigated its effects during high-fat diet (HFD). After 16 weeks of HFD, TIMP3 overexpression significantly improved glucose metabolism, hepatic fatty acid oxidation and cholesterol homeostasis. In AlbT3 mice CYP7A1, MDR3 and MRP2 gene expressions were observed, consistent with higher bile acid synthesis and export. Next, to evaluate the role of A Disintegrin and Metalloproteinase 17 (ADAM17), a crucial target of TIMP3, in these processes, we created mice deficient in Adam17 specifically in hepatocyte (A17LKO) or in myeloid lineage (A17MKO), founding that only A17LKO showed improvement in liver steatosis induced by HFD. Moreover, both, AlbT3 and A17LKO significantly reduced diethylnitrosamine-initiated, HFD-promoted hepatic tumorigenesis assessed by tumor multiplicity and total tumor area. Taken together, these data indicate that hepatic TIMP3 can slow progression of NAFLD, and tumorigenesis, at least in part, through the regulation of ADAM17 activity.

The shedding protease ADAM17: Physiology and pathophysiology.

The disintegrin metalloprotease ADAM17 has been a matter of intense studies aiming to unravel structure, function and regulation of protease expression, maturation and activity. In this review, we summarize data on the physiological role of ADAM17 in health and disease. Here we provide an overview of ADAM17 substrates, mouse models of ADAM17-deficiencies and discuss recent findings of ADAM17 function in the immune system and central nervous system as well as in cancer. Whereas ADAM17 function in EGF-R-, in Interleukin-6 (IL-6)- and in TNFα-biology has been shown to play a decisive role in regulation of the immune system as well as cancer development, the role of ADAM17 in the central nervous system and neurodegeneration still remains elusive. We show ADAM17 expression in human dopaminergic neurons derived from induced pluripotent stem cells and we discuss how this state-of-the-art technology can be further exploited to study the function of this important protease in the brain and other tissues. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Inactivation of Rab11a GTPase in Macrophages Facilitates Phagocytosis of Apoptotic Neutrophils.

The timely and efficient clearance of apoptotic neutrophils by macrophages (efferocytosis) is required for the resolution of inflammation and tissue repair, but the regulatory mechanisms remain unclear. In this study, we investigated the role of the small GTPase Ras-related protein in brain (Rab)11a in regulating efferocytosis, and on this basis the resolution of inflammatory lung injury. We observed that apoptotic neutrophil feeding induced a rapid loss of Rab11a activity in bone marrow-derived macrophages and found that depletion of Rab11a in macrophages by small interfering RNA dramatically increased the phagocytosis of apoptotic neutrophils compared with control cells. Additionally, overexpression of wild-type Rab11a inhibited macrophage efferocytosis, whereas overexpression of dominant-negative Rab11a (Rab11a S25N) increased the clearance of apoptotic neutrophils. Rab11a knockdown also increased the surface level of CD36 in macrophages, but it reduced cell surface expression of a disintegrin and metalloproteinase (ADAM) 17. Depletion of ADAM17 rescued the decreased surface CD36 expression found in macrophages overexpressing wild-type Rab11a. Also, blockade of CD36 abolished the augmented efferocytosis seen in Rab11a-depleted macrophages. In mice challenged with endotoxin, intratracheal instillation of Rab11a-depleted macrophages reduced neutrophil count in bronchoalveolar lavage fluid, increased the number of macrophages containing apoptotic neutrophils, and prevented inflammatory lung injury. Thus, Rab11a inactivation in macrophages as a result of apoptotic cell binding initiates phagocytosis of apoptotic neutrophils via the modulation of ADAM17-mediated CD36 cell surface expression. Our results raise the possibility that inhibition of Rab11a activity in macrophages is a promising strategy for activating the resolution of inflammatory lung injury.

Adam17 Deficiency Promotes Atherosclerosis by Enhanced TNFR2 Signaling in Mice.

OBJECTIVE: ADAM17 (a disintegrin and metalloproteinase 17) is a sheddase releasing different types of membrane-bound proteins, including adhesion molecules, cytokines, and their receptors as well as inflammatory mediators. Because these substrates modulate important mechanisms of atherosclerosis, we hypothesized that ADAM17 might be involved in the pathogenesis of this frequent disease. APPROACH AND RESULTS: Because Adam17-knockout mice are not viable, we studied the effect of Adam17 deficiency on atherosclerosis in Adam17 hypomorphic mice (Adam17ex/ex), which have low residual Adam17 expression. To induce atherosclerosis, mice were crossed onto the low-density lipoprotein receptor (Ldlr)-deficient background. We found that Adam17ex/ex.Ldlr-/- mice developed ≈1.5-fold larger atherosclerotic lesions, which contained more macrophages and vascular smooth muscle cells than wild-type littermate controls (Adam17wt/wt.Ldlr-/-). Reduced Adam17-mediated shedding led to significantly increased protein levels of membrane-resident TNFα (tumor necrosis factor) and TNFR2 (tumor necrosis factor receptor 2), resulting in a constitutive activation of TNFR2 signaling. At the same time, Adam17 deficiency promoted proatherosclerotic cellular functions, such as increased proliferation and reduced apoptosis in cultured macrophages and vascular smooth muscle cells and increased adhesion of macrophages to vascular endothelial cells. Because siRNA (small interfering RNA)-mediated knockdown of Tnfr2 rescued from aberrant proliferation and from misregulation of apoptosis in Adam17-depleted cells, our data indicate that TNFR2 is an important effector of ADAM17 in our mouse model. CONCLUSIONS: Our results provide evidence for an atheroprotective role of ADAM17, which might be mediated by cleaving membrane-bound TNFα and TNFR2, thereby preventing overactivation of endogenous TNFR2 signaling in cells of the vasculature.

Serum Starvation Induces BACE1 Processing and Secretion.

BACKGROUND: ß-secretase (BACE1) is a type 1 transmembrane protein implicated in Alzheimer's Disease (AD) pathogenesis. Cleavage of Amyloid Precursor Protein (APP), initiated by BACE1 and followed by γ-secretase, leads to the formation of toxic Aß peptides. Increased levels of BACE1 have been detected in the CSF of AD patients compared to age-matched healthy controls indicating that neurodegenerative conditions induce shedding of BACE1. OBJECTIVE: To mimic such conditions, we examined whether serum deprivation stimulates proteolysis-dependent secretion of BACE1. METHOD: Detection of BACE1 secretion in BACE1 overexpressing cells or ADAM10/ADAM17 knockout fibroblasts cultured under serum deprivation conditions, using western blot analysis. RESULTS: We found that serum deprivation of U251 neuroblastoma or HEK293T cells overexpressing BACE1 stimulated secretion of BACE1. Using ADAM10/ADAM17 knockout fibroblasts and inhibitors of both ADAM10 and ADAM17, we obtained data indicating that these proteases are involved in serum-starvation induced shedding of BACE1. This is unexpected since BACE1 is localized mainly in lipid rafts while ADAM10 is localized mainly in nonlipid raft domains. We hypothesized that serum deprivation results in alterations in the lipid composition of the membrane which can alter the localization of ADAM10 and BACE1. In support, we obtained results indicating that extraction of membrane cholesterol following incubation with methyl ß cyclodextrin potentiated the effect of serum deprivation. Secreted BACE1 was also found to be enzymatically active towards immunoprecipiated full length APP. CONCLUSION: Serum starvation induces ADAM10-mediated BACE1 secretion.

Phosphatidylserine exposure is required for ADAM17 sheddase function.

ADAM17, a prominent member of the 'Disintegrin and Metalloproteinase' (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Here we present evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. PS exposure is tightly coupled to substrate shedding provoked by diverse ADAM17 activators. PS dependency is demonstrated in the following: (a) in Raji cells undergoing apoptosis; (b) in mutant PSA-3 cells with manipulatable PS content; and (c) in Scott syndrome lymphocytes genetically defunct in their capacity to externalize PS in response to intracellular Ca(2+) elevation. Soluble phosphorylserine but not phosphorylcholine inhibits substrate cleavage. The isolated membrane proximal domain (MPD) of ADAM17 binds to PS but not to phosphatidylcholine liposomes. A cationic PS-binding motif is identified in this domain, replacement of which abrogates liposome-binding and renders the protease incapable of cleaving its substrates in cells. We speculate that surface-exposed PS directs the protease to its targets where it then executes its shedding function.

Targeting ADAM17 in leukocytes increases neutrophil recruitment and reduces bacterial spread during polymicrobial sepsis.

A rapid and robust recruitment of circulating neutrophils at sites of infection is critical for preventing bacterial spread. The efficiency of this process, however, is greatly diminished during sepsis, a severe systemic inflammatory response to infection. The proteolytic activity of a disintegrin and metalloprotease-17 is induced in the cell membrane of leukocytes upon their activation, resulting in the conversion of membrane to soluble TNF-α and the release of assorted receptors from the surface of neutrophils important for their effector functions. We show that conditional knockout mice lacking a disintegrin and metalloprotease-17 in all leukocytes had a survival advantage when subjected to polymicrobial sepsis. Bacteremia and the levels of circulating proinflammatory cytokines, key determinants of sepsis severity, were significantly reduced in conditional a disintegrin and metalloprotease-17 knockout mice during sepsis. Although cecal bacterial microbiota and load were similar in unmanipulated conditional a disintegrin and metalloprotease-17 knockout and control mice, peritoneal spread of bacteria was significantly reduced in conditional a disintegrin and metalloprotease-17 knockout mice following sepsis induction, which was associated with an amplified recruitment of neutrophils. Taken together, our findings suggest that extensive a disintegrin and metalloprotease-17 induction during sepsis may tip the balance between efficient and impaired neutrophil recruitment.

Skin Barrier Defects Caused by Keratinocyte-Specific Deletion of ADAM17 or EGFR Are Based on Highly Similar Proteome and Degradome Alterations.

Keratinocyte-specific deletion of ADAM17 in mice impairs terminal differentiation of keratinocytes leading to severe epidermal barrier defects. Mice deficient for ADAM17 in keratinocytes phenocopy mice with a keratinocyte-specific deletion of epidermal growth factor receptor (EGFR), which highlights the role of ADAM17 as a "ligand sheddase" of EGFR ligands. In this study, we aim for the first proteomic/degradomic approach to characterize the disruption of the ADAM17-EGFR signaling axis and its consequences for epidermal barrier formation. Proteomic profiling of the epidermal proteome of mice deficient for either ADAM17 or EGFR in keratinocytes at postnatal days 3 and 10 revealed highly similar protein alterations for ADAM17 and EGFR deficiency. These include massive proteome alterations of structural and regulatory components important for barrier formation such as transglutaminases, involucrin, filaggrin, and filaggrin-2. Cleavage site analysis using terminal amine isotopic labeling of substrates revealed increased proteolytic processing of S100 fused-type proteins including filaggrin-2. Alterations in proteolytic processing are supported by altered abundance of numerous proteases upon keratinocyte-specific Adam17 or Egfr deletion, among them kallikreins, cathepsins, and their inhibitors. This study highlights the essential role of proteolytic processing for maintenance of a functional epidermal barrier. Furthermore, it suggests that most defects in formation of the postnatal epidermal barrier upon keratinocyte-specific ADAM17 deletion are mediated via EGFR.