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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. [ABSTRACT FROM AUTHOR]

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 ADAM17expression, were coupled with emphysema-prone gp130F/F mice to produce gp130F/F:Adam17ex/ex. Both Adam17ex/ex and wildtype 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary Interleukin (IL)-11 has been shown to be a crucial factor for intestinal tumorigenesis, lung carcinomas, and asthma. IL-11 is thought to exclusively mediate its biological functions through cell-type-specific expression of the membrane-bound IL-11 receptor (IL-11R). Here, we show that the metalloprotease ADAM10, but not ADAM17, can release the IL-11R ectodomain. Chimeric proteins of the IL-11R and the IL-6 receptor (IL-6R) revealed that a small juxtamembrane portion is responsible for this substrate specificity of ADAM17. Furthermore, we show that the serine proteases neutrophil elastase and proteinase 3 can also cleave the IL-11R. The resulting soluble IL-11R (sIL-11R) is biologically active and binds IL-11 to activate cells. This IL-11 trans-signaling pathway can be inhibited specifically by the anti-inflammatory therapeutic compound sgp130Fc. In conclusion, proteolysis of the IL-11R represents a molecular switch that controls the IL-11 trans-signaling pathway and widens the number of cells that can be activated by IL-11. [ABSTRACT FROM AUTHOR]

Abstract: ADAM17 has been molecularly cloned as the enzyme responsible for cleavage of the transmembrane protein TNFα (TNFα converting enzyme, TACE). Later it was realized that ADAM17 was also responsible for the processing of cell adhesion proteins, cytokine and growth factor receptors and many ligands of the EGF receptor. Since TNFα is a target of anti-inflammatory therapies, it was speculated that inhibition of ADAM17 might be a therapeutic strategy in the treatment of inflammation or inflammation associated cancer. Meanwhile it has been recognized that ADAM17 governs many vital functions in the body and loss of ADAM17 leads to severe defects in the skin and to high susceptibility of the intestine to inflammation. Here I summarize data on the physiologic role of ADAM17 and the feasibility of specific blockade of this enzyme. [Copyright &y& Elsevier]

Abstract: A great number of physiological processes are regulated by the release of ectodomains of membrane proteins. A Disintegrin And Metalloprotease 17 (ADAM17) is one of the important enzymes, which mediate this process called shedding. Today, more than 70 substrates of this transmembrane metalloprotease are known. This broad spectrum raises the question how ADAM17 recognizes its substrates specifically. Differently tagged ADAM17 deletion variants were used to demonstrate that exclusively the extracellular domains of ADAM17 are needed for interaction with two of its substrates, the IL-6R and the IL-1RII; whereas the transmembrane- and cytoplasmic-region are dispensable for this process. In the extracellular part solely the membrane-proximal domain of ADAM17 is mandatory for recognition of the two type-I transmembrane proteins, but not for the interaction with the type-II transmembrane molecule TNF-α. Structured summary of protein interactions: IL-6R physically interacts with ADAM17 by anti bait coimmunoprecipitation ( View interaction ) ADAM17 physically interacts with IL-1RII by anti tag coimmunoprecipitation ( View interaction ) [Copyright &y& Elsevier]

It was realized in the 1990s that some membrane proteins such as TNFα, both TNF receptors, ligands of the EGF-R and the Interleukin-6 receptor are proteolytically cleaved and are shed from the cell membrane as soluble proteins. The major responsible protease is a metalloprotease named ADAM17. So far, close to 100 substrates, including cytokines, cytokine receptors, chemokines and adhesion molecules of ADAM17 are known. Therefore, ADAM17 orchestrates many different signaling pathways and is a central signaling hub in inflammation and carcinogenesis. ADAM17 plays an important role in the biology of Interleukin-6 (IL-6) since the generation of the soluble Interleukin-6 receptor (sIL-6R) is needed for trans-signaling, which has been identified as the pro-inflammatory activity of this cytokine. In contrast, Interleukin-6 signaling via the membrane-bound Interleukin-6 receptor is mostly regenerative and protective. Probably due to its broad substrate spectrum, ADAM17 is essential for life and most of the few human individuals identified with ADAM17 gene defects died at young age. Although the potential of ADAM17 as a therapeutic target has been recognized, specific blockade of ADAM17 is not trivial since the metalloprotease domain of ADAM17 shares high structural homology with other proteases, in particular matrix metalloproteases. Here, the critical functions of ADAM17 in IL-6, TNFα and EGF-R pathways and strategies of therapeutic interventions are discussed. • Many transmembrane proteins are shed by limited proteolysis. • Shed soluble proteins constitute a transition from local to systemic responses. • ADAM17 is one of the major shedding proteins. • ADAM17 orchestrates the signaling pathways of IL-6, TNFα and EGF-R. • Therapeutic blockade of ADAM17 could be beneficial in many diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Although extensively investigated, cancer is still one of the most devastating and lethal diseases in the modern world. Among different types, colorectal cancer (CRC) is most prevalent and mortal, making it an important subject of research. The metalloprotease ADAM17 has been implicated in the development of CRC due to its involvement in signaling pathways related to inflammation and cell proliferation. ADAM17 is capable of releasing membrane-bound proteins from the cell surface in a process called shedding. A deficiency of ADAM17 activity has been previously shown to have protective effects against CRC in mice, while an upregulation of ADAM17 activity is suspected to facilitate tumor development. In this study, we characterize ADAM17 variants found in tissue samples of cancer patients in overexpression studies. We here focus on point mutations identified within the catalytic domain of ADAM17 and could show a functional dysregulation of the CRC-associated variants. Since the catalytic domain of ADAM17 is the only region structurally determined by crystallography, we study the effect of each point mutation not only to learn more about the role of ADAM17 in cancer, but also to investigate the structure–function relationships of the metalloprotease. [ABSTRACT FROM AUTHOR]

All ligands of the epidermal growth factor receptor (EGF-R) are transmembrane proteins, which need to be proteolytically cleaved in order to be systemically active. The major protease responsible for this cleavage is the membrane metalloprotease ADAM17, which also has been implicated in cleavage of TNFα and interleukin-6 (IL-6) receptor. It has been recently shown that in the absence of ADAM17, the main protease for EGF-R ligand processing, colon cancer formation is largely abrogated. Intriguingly, colon cancer formation depends on EGF-R activity on myeloid cells rather than on intestinal epithelial cells. A major activity of EGF-R on myeloid cells is the stimulation of IL-6 synthesis. Subsequently, IL-6 together with the ADAM17 shed soluble IL-6 receptor acts on intestinal epithelial cells via IL-6 trans-signaling to induce colon cancer formation, which can be blocked by the inhibitor of IL-6 trans-signaling, sgp130Fc. Blockade of IL-6 trans-signaling therefore offers a new therapeutic window downstream of the EGF-R for the treatment of colon cancer and possibly of other EGF-R related neoplastic diseases. [ABSTRACT FROM AUTHOR]

Lung cancer is the leading cause of cancer-related mortality, which histologically is classified into small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 85% of all lung cancer diagnoses, with the majority of patients presenting with lung adenocarcinoma (LAC). KRAS mutations are a major driver of LAC, and are closely related to cigarette smoking, unlike mutations in the epidermal growth factor receptor (EGFR) which arise in never-smokers. Although the past two decades have seen fundamental progress in the treatment and diagnosis of NSCLC, NSCLC still is predominantly diagnosed at an advanced stage when therapeutic interventions are mostly palliative. A disintegrin and metalloproteinase 17 (ADAM17), also known as tumour necrosis factor-α (TNFα)-converting enzyme (TACE), is responsible for the protease-driven shedding of more than 70 membrane-tethered cytokines, growth factors and cell surface receptors. Among these, the soluble interleukin-6 receptor (sIL-6R), which drives pro-inflammatory and pro-tumourigenic IL-6 trans-signaling, along with several EGFR family ligands, are the best characterised. This large repertoire of substrates processed by ADAM17 places it as a pivotal orchestrator of a myriad of physiological and pathological processes associated with the initiation and/or progression of cancer, such as cell proliferation, survival, regeneration, differentiation and inflammation. In this review, we discuss recent research implicating ADAM17 as a key player in the development of LAC, and highlight the potential of ADAM17 inhibition as a promising therapeutic strategy to tackle this deadly malignancy. [ABSTRACT FROM AUTHOR]

Colorectal cancer is one of the most commonly diagnosed malignancies in the Western world and is associated with elevated expression and activity of epidermal growth factor receptors (EGF-R). The metalloproteinase ADAM17 is involved in EGF-R activation by processing EGF-R ligands from membrane-bound pro-ligands. Underlining the link between colon cancer and ADAM17, genetic intestinal cancer models in ADAM17-deficient mice show a reduced tumor burden. In this study, we characterize point mutations within the ADAM17 gene found in the tissue of colon cancer patients. In order to shed light on the role of ADAM17 in cancer development, as well as into the mechanisms that regulate maturation and cellular trafficking of ADAM17, we here perform overexpression studies of four ADAM17 variants located in the pro-, membrane-proximal- and cytoplasmic-domain of the ADAM17 protein in ADAM10/17-deficient HEK cells. Interestingly, we found a cancer-associated point mutation within the pro-domain of ADAM17 (R177C) to be most impaired in its proteolytic activity and trafficking to the cell membrane. By comparing this variant to an ADAM17 construct lacking the entire pro-domain, we discovered similar functional limitations and propose a crucial role of the pro-domain for ADAM17 maturation, cellular trafficking and thus proteolytic activity. [ABSTRACT FROM AUTHOR]

Deregulated gp130-dependent STAT3 signalling by the pleiotropic cytokine interleukin (IL)-11 has been implicated in the pathogenesis of gastric cancer (GC), the third most common cancer worldwide. While the IL-11-gp130-STAT3 signalling axis has traditionally been thought to exclusively use the membrane-bound IL-11 receptor (mIL-11R), recent evidence suggests that mIL-11R can be proteolytically cleaved to generate a soluble form (sIL-11R) which can elicit trans-signalling. Since the role of IL-11 trans-signalling in disease pathogenesis is unknown, here we have employed the IL-11-driven gp130 F/F spontaneous model of GC to determine whether IL-11 trans-signalling promotes gastric tumourigenesis. sIL-11R protein was detectable in gastric tissue from GC patients, and sIL-11R levels were elevated in tumours of gp130 F/F mice compared to matched non-tumours. Among candidate proteases associated with the generation of sIL-11R, ADAM10 and the related metalloprotease ADAM17 were significantly upregulated in tumours of both gp130 F/F mice and GC patients compared to matched non-tumour tissues. The genetic blockade of IL-11 trans-signalling in gp130 F/F mice upon the transgenic over-expression of the trans-signalling antagonist, sgp130Fc, failed to suppress gastric inflammation and associated tumour growth, and also had no effect on reducing hyper-activated STAT3 levels. Furthermore, a non-essential role for ADAM17 in IL-11-driven gastric tumourigenesis was supported by the observation that the tumour burden was unaffected in gp130 F/F : Adam17 ex/ex mice in which ADAM17 expression levels have been substantially reduced. Collectively, these findings suggest that classic signalling rather than trans-signalling is the mode by which IL-11 promotes gastric tumourigenesis. [ABSTRACT FROM AUTHOR]

Highlights: [•] The IL-6 receptor can be shed from the cellular membrane by the protease ADAM17. [•] The complex of IL-6 and IL-6 receptor induces signaling on cells only expressing gp130, a process has been named trans-signaling. [•] IL-6 trans-signaling can be specifically blocked by soluble gp130. [•] IL-6 trans-signaling is pro-inflammatory whereas IL-6 signaling via the membrane bound IL-6 receptor is regenerative and protects from bacterial infections. [•] A soluble gp130Fc protein has been developed as an anti-inflammatory drug, which is currently tested as in phase I clinical trials. [Copyright &y& Elsevier]

The pleiotropic cytokine interleukin-6 (IL-6) is involved in numerous physiological and pathophysiological functions that include development, immune cell differentiation, inflammation and cancer. IL-6 can signal via the membrane-bound IL-6 receptor (IL-6R, classic signaling) or via soluble forms of the IL-6R (sIL-6R, trans-signaling). Both modes of signaling induce the formation of a homodimer of the signal transducing β-receptor glycoprotein 130 (gp130) and the activation of several intracellular signaling cascades, e.g. the Jak/STAT pathway. Intriguingly, only IL-6 trans-signaling is required for the pro-inflammatory properties of IL-6, while regenerative and anti-inflammatory functions are mediated via classic signaling. The sIL-6R is generated by different molecular mechanisms, including alternative mRNA splicing, proteolysis of the membrane-bound IL-6R and the release of extracellular vesicles. In this review, we give an in-depth overview on these molecular mechanisms with a special emphasize on IL-6R cleavage by the metalloprotease ADAM17 and other proteases. We discuss the biological functions of the sIL-6R and highlight attempts to selectively block IL-6 trans-signaling in pre-clinical animal models as well as in clinical studies in patients with inflammatory bowel disease. • Interleukin-6 can signal via membrane-bound and soluble forms of the Interleukin-6 receptor. • ADAM17 is the most important protease for IL-6R cleavage. • Olamkicept (sgp130Fc) blocks IL-6 trans-signaling, but not classic signaling. • Olamkicept is currently in clinical development. [ABSTRACT FROM AUTHOR]

• Systemic ADAM17-deficiency promotes functional recovery after spinal cord injury. • ADAM17-deficiency does not change the macrophage/microglia activation phenotype. • ADAM17-deficiency on macrophages or endothelium has no effect on SCI recovery. • Microglial ADAM17-deficiency promotes phagocytosis after SCI. • Only microglial ADAM17-deficiency enhances functional outcome after SCI. A disintegrin and metalloproteinase 17 (ADAM17) is the major sheddase involved in the cleavage of a plethora of cytokines, cytokine receptors and growth factors, thereby playing a substantial role in inflammatory and regenerative processes after central nervous system trauma. By making use of a hypomorphic ADAM17 knockin mouse model as well as pharmacological ADAM10/ADAM17 inhibitors, we showed that ADAM17-deficiency or inhibition significantly increases clearance of apoptotic cells, promotes axon growth and improves functional recovery after spinal cord injury (SCI) in mice. Microglia-specific ADAM17-knockout (ADAM17flox+/+-Cx3Cr1 Cre+/−) mice also showed improved functional recovery similar to hypomorphic ADAM17 mice. In contrast, endothelial-specific (ADAM17flox+/+-Cdh5Pacs Cre+/−) and macrophage-specific (ADAM17flox+/+-LysM Cre+/−) ADAM17-knockout mice or bone marrow chimera with transplanted ADAM17-deficient macrophages, displayed no functional improvement compared to wild type mice. These data indicate that ADAM17 expression on microglia cells (and not on macrophages or endothelial cells) plays a detrimental role in inflammation and functional recovery after SCI. [ABSTRACT FROM AUTHOR]

The cytokine interleukin-6 (IL-6) has considerable pro-inflammatory properties and is a driver of many physiological and pathophysiological processes. Cellular responses to IL-6 are mediated by membrane-bound or soluble forms of the IL-6 receptor (IL-6R) complexed with the signal-transducing subunit gp130. While expression of the membrane-bound IL-6R is restricted to selected cell types, soluble IL-6R (sIL-6R) enables gp130 engagement on all cells, a process termed IL-6 trans-signalling and considered to be pro-inflammatory. sIL-6R is predominantly generated through proteolytic processing by the metalloproteinase ADAM17. ADAM17 also liberates ligands of the epidermal growth factor receptor (EGFR), which is a prerequisite for EGFR activation and results in stimulation of proliferative signals. Hyperactivation of EGFR mostly due to activating mutations drives cancer development. Here, we reveal an important link between overshooting EGFR signalling and the IL-6 trans-signalling pathway. In epithelial cells, EGFR activity induces not only IL-6 expression but also the proteolytic release of sIL-6R from the cell membrane by increasing ADAM17 surface activity. We find that this derives from the transcriptional upregulation of iRhom2, a crucial regulator of ADAM17 trafficking and activation, upon EGFR engagement, which results in increased surface localization of ADAM17. Also, phosphorylation of the EGFR-downstream mediator ERK mediates ADAM17 activity via interaction with iRhom2. In sum, our study reveals an unforeseen interplay between EGFR activation and IL-6 trans-signalling, which has been shown to be fundamental in inflammation and cancer. • EGFR activation induces IL-6 expression and sIL-6R release. • Activity of ADAM17 is regulated by EGFR signalling and ERK phosphorylation. • iRhom2 is an EGFR target gene. • EGFR signalling stimulates inflammatory gene expression. [ABSTRACT FROM AUTHOR]

The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant. [ABSTRACT FROM AUTHOR]

Abstract: Proteolysis of transmembrane molecules is an irreversible post-translational modification enabling autocrine, paracrine and endocrine signaling of many cytokines. The pro-inflammatory activities of membrane bound TNFα (pro-TNFα) strongly depend on ectodomain shedding mediated by the A Disintegrin And Metalloprotease family member ADAM17. Despite the well-documented role of ADAM17 in pro-TNFα cleavage during inflammation, little is known about its regulation. Mitogen-activated protein kinase-induced phosphorylation of the ADAM17 cytoplasmic tail has been described to be required for proper activation. To address, if pro-TNFα shedding depends on cytosolic phosphorylation we analyzed ADAM17 mutants lacking the cytoplasmic domain. ADAM17 mediated shedding of pro-TNFα was induced by PMA, Anisomycin and the phosphatase inhibitors Cantharidin and Calyculin A. Deletion of the entire cytoplasmic portion of ADAM17 abolished furin-dependent proteolytic maturation and pro-TNFα cleavage. Interestingly, we could exclude that resistance to proconvertase processing is the reason for the enzymatic inactivity of ADAM17 lacking the cytoplasmic portion as furin-resistant ADAM17 mutants rescued genetic ADAM17 deficiency after mitogen-activated protein kinase activation. Adding only 6 cytoplasmic amino acids completely restored ADAM17 maturation and shedding of pro-TNFα as well as of both TNF-receptors Finally, we showed that a pro-TNFα mutant lacking the cytoplasmic portion was also shed from the cell surface. We conclude that pro-TNFα cleavage by its major sheddase ADAM17 does not depend on cytosolic phosphorylation and/or interaction. These results have general implications on understanding the activation mechanism controlling the activity of ADAM17. [Copyright &y& Elsevier]

Abstract: Interleukin-6 (IL6) signals are mediated by classic and trans-signaling. In classic signaling, IL6 first binds to the membrane bound Interleukin-6 Receptor (IL6R) whereas in trans-signaling, IL6 acts via a soluble form of the IL6R. Trans-signaling via the soluble IL6R (sIL6R) was linked to chronic inflammation and cancer. The release of the IL6R is mediated by the disintegrin and metalloproteinases ADAM10 and ADAM17. To analyze the fate of the C-terminal cleavage fragment after ectodomain shedding we fused the IL6R C-terminally to two Z-domains of Protein-A (2Z-tag) or to GFP. A specific C-terminal fragment of the IL6R protein could be detected after ADAM17-induced shedding. Using γ-secretase inhibitors and gene-deficient cells, we demonstrate that after ADAM17 mediated cleavage, the IL6R C-terminal fragment was cleaved by the γ-secretase at the plasma membrane. We were, however, not able to detect an IL6R intracellular domain. After γ-secretase cleavage IL6R cell surface expression was lost and γ-secretase cleavage product(s) of the IL6R were endocytosed. No GFP-fluorescence of a γ-secretase-cleaved IL6R-GFP fusion protein was observed in the nucleus. We therefore hypothesize that a potential IL6R intracellular domain fragment is not involved in nuclear signaling but rapidly degraded. [Copyright &y& Elsevier]