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.
- MeSH
- cytokinový receptor gp130 genetika MeSH
- epitelové buňky metabolismus MeSH
- erbB receptory genetika metabolismus MeSH
- interleukin-6 * genetika metabolismus MeSH
- lidé MeSH
- protein ADAM17 * MeSH
- signální transdukce * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
Membrane-tethered signalling proteins such as TNFα and many EGF receptor ligands undergo shedding by the metalloproteinase ADAM17 to get released. The pseudoproteases iRhom1 and iRhom2 are important for the transport, maturation and activity of ADAM17. Yet, the structural and functional requirements to promote the transport of the iRhom-ADAM17 complex have not yet been thoroughly investigated. Utilising in silico and in vitro methods, we here map the conserved iRhom homology domain (IRHD) and provide first insights into its structure and function. By focusing on iRhom2, we identified different structural and functional factors within the IRHD. We found that the structural integrity of the IRHD is a key factor for ADAM17 binding. In addition, we identified a highly conserved motif within an unstructured region of the IRHD, that, when mutated, restricts the transport of the iRhom-ADAM17 complex through the secretory pathway in in vitro, ex vivo and in vivo systems and also increases the half-life of iRhom2 and ADAM17. Furthermore, the disruption of this IRHD motif was also reflected by changes in the yet undescribed interaction profile of iRhom2 with proteins involved in intracellular vesicle transport. Overall, we provide the first insights into the forward trafficking of iRhoms which is critical for TNFα and EGF receptor signalling.
- MeSH
- aminokyselinové motivy MeSH
- buněčné linie MeSH
- epidermální růstové faktory metabolismus MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- membránové proteiny genetika metabolismus MeSH
- mutageneze MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- poločas MeSH
- protein ADAM17 chemie metabolismus MeSH
- proteinové domény MeSH
- RNA interference MeSH
- signální transdukce MeSH
- TNF-alfa metabolismus MeSH
- transport proteinů MeSH
- transportní proteiny antagonisté a inhibitory genetika metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Uptake of bacteria by phagocytes is a crucial step in innate immune defence. Members of the disintegrin and metalloproteinase (ADAM) family critically control the immune response by limited proteolysis of surface expressed mediator molecules. Here, we investigated the significance of ADAM17 and its regulatory adapter molecule iRhom2 for bacterial uptake by phagocytes. Inhibition of metalloproteinase activity led to increased phagocytosis of pHrodo labelled Gram-negative and -positive bacteria (E. coli and S. aureus, respectively) by human and murine monocytic cell lines or primary phagocytes. Bone marrow-derived macrophages showed enhanced uptake of heat-inactivated and living E. coli when they lacked either ADAM17 or iRhom2 but not upon ADAM10-deficiency. In monocytic THP-1 cells, corresponding short hairpin RNA (shRNA)-mediated knockdown confirmed that ADAM17, but not ADAM10, promoted phagocytosis of E. coli. The augmented bacterial uptake occurred in a cell autonomous manner and was accompanied by increased release of the chemokine CXCL8, less TNFα release and only minimal changes in the surface expression of the receptors TNFR1, TLR6 and CD36. Inhibition experiments indicated that the enhanced bacterial phagocytosis after ADAM17 knockdown was partially dependent on TNFα-activity but not on CXCL8. This novel role of ADAM17 in bacterial uptake needs to be considered in the development of ADAM17 inhibitors as therapeutics.
- MeSH
- antigeny CD36 genetika metabolismus MeSH
- Escherichia coli patogenita MeSH
- fagocytóza MeSH
- fagocyty metabolismus mikrobiologie MeSH
- interleukin-8 metabolismus MeSH
- intracelulární signální peptidy a proteiny genetika metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- myši MeSH
- protein ADAM17 genetika metabolismus MeSH
- RAW 264.7 buňky MeSH
- receptory TNF - typ I genetika metabolismus MeSH
- Staphylococcus aureus patogenita MeSH
- THP-1 buňky MeSH
- toll-like receptor 6 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
