ABSTRACT
Calcium (Ca(2+)) is a secondary messenger in cells and Ca(2+) flux initiated from endoplasmic reticulum (ER) stores via inositol 1,4,5-triphosphate (IP3) binding to the IP3 receptor (IP3R) is particularly important for the activation and function of immune cells. Previous studies demonstrated that genetic deletion of selenoprotein K (Selk) led to decreased Ca(2+) flux in a variety of immune cells and impaired immunity, but the mechanism was unclear. Here we show that Selk deficiency does not affect receptor-induced IP3 production, but Selk deficiency through genetic deletion or low selenium in culture media leads to low expression of the IP3R due to a defect in IP3R palmitoylation. Bioinformatic analysis of the DHHC (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) family of enzymes that catalyze protein palmitoylation revealed that one member, DHHC6, contains a predicted Src-homology 3 (SH3) domain and DHHC6 is localized to the ER membrane. Because Selk is also an ER membrane protein and contains an SH3 binding domain, immunofluorescence and coimmunoprecipitation experiments were conducted and revealed DHHC6/Selk interactions in the ER membrane that depended on SH3/SH3 binding domain interactions. DHHC6 knockdown using shRNA in stably transfected cell lines led to decreased expression of the IP3R and impaired IP3R-dependent Ca(2+) flux. Mass spectrophotometric and bioinformatic analyses of the IP3R protein identified two palmitoylated cysteine residues and another potentially palmitoylated cysteine, and mutation of these three cysteines to alanines resulted in decreased IP3R palmitoylation and function. These findings reveal IP3R palmitoylation as a critical regulator of Ca(2+) flux in immune cells and define a previously unidentified DHHC/Selk complex responsible for this process.
Subject(s)
Acyltransferases/physiology , Inositol 1,4,5-Trisphosphate Receptors/physiology , Protein Processing, Post-Translational , Selenoproteins/physiology , T-Lymphocyte Subsets/metabolism , Acyltransferases/antagonists & inhibitors , Acyltransferases/chemistry , Animals , Bone Marrow Cells/metabolism , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cysteine/chemistry , Endoplasmic Reticulum/enzymology , HEK293 Cells , Humans , Inositol 1,4,5-Trisphosphate Receptors/chemistry , Inositol 1,4,5-Trisphosphate Receptors/genetics , Jurkat Cells , Lipoylation , Mice , Mice, Knockout , Multiprotein Complexes , Mutagenesis, Site-Directed , Protein Interaction Mapping , RNA, Small Interfering/pharmacology , Rats , Recombinant Fusion Proteins/metabolism , Selenium/physiology , Selenoproteins/chemistry , Selenoproteins/deficiency , Thapsigargin/pharmacology , Transfection , src Homology DomainsABSTRACT
Cancer-associated fibroblasts (CAFs) drive tumour progression, but the emergence of this cell state is poorly understood. A broad spectrum of metalloproteinases, controlled by the Timp gene family, influence the tumour microenvironment in human cancers. Here, we generate quadruple TIMP knockout (TIMPless) fibroblasts to unleash metalloproteinase activity within the tumour-stromal compartment and show that complete Timp loss is sufficient for the acquisition of hallmark CAF functions. Exosomes produced by TIMPless fibroblasts induce cancer cell motility and cancer stem cell markers. The proteome of these exosomes is enriched in extracellular matrix proteins and the metalloproteinase ADAM10. Exosomal ADAM10 increases aldehyde dehydrogenase expression in breast cancer cells through Notch receptor activation and enhances motility through the GTPase RhoA. Moreover, ADAM10 knockdown in TIMPless fibroblasts abrogates their CAF function. Importantly, human CAFs secrete ADAM10-rich exosomes that promote cell motility and activate RhoA and Notch signalling in cancer cells. Thus, Timps suppress cancer stroma where activated-fibroblast-secreted exosomes impact tumour progression.
Subject(s)
Fibroblasts/metabolism , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/pathology , Tissue Inhibitor of Metalloproteinases/genetics , ADAM Proteins/metabolism , ADAM10 Protein , Amyloid Precursor Protein Secretases/metabolism , Animals , Cell Line, Tumor , Cell Movement , Exosomes/physiology , Female , Fibroblasts/pathology , Humans , Lung Neoplasms/enzymology , Mammary Neoplasms, Experimental/enzymology , Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Neoplasm Transplantation , Phenotype , Signal Transduction , Tissue Inhibitor of Metalloproteinases/deficiency , Tumor BurdenABSTRACT
Acute lung injury (ALI) is associated with increased vascular permeability, leukocyte recruitment, and pro-inflammatory mediator release. We investigated the role of the metalloproteinase ADAM17 in endotoxin-induced ALI with focus on endothelial ADAM17. In vitro, endotoxin-mediated induction of endothelial permeability and IL-8-induced transmigration of neutrophils through human microvascular endothelial cells required ADAM17 as shown by inhibition with GW280264X or shRNA-mediated knockdown. In vivo, ALI was induced by intranasal endotoxin-challenge combined with GW280264X treatment or endothelial adam17-knockout. Endotoxin-triggered upregulation of ADAM17 mRNA in the lung was abrogated in knockout mice and associated with reduced ectodomain shedding of the junctional adhesion molecule JAM-A and the transmembrane chemokine CX3CL1. Induced vascular permeability, oedema formation, release of TNF-α and IL-6 and pulmonary leukocyte recruitment were all markedly reduced by GW280264X or endothelial adam17-knockout. Intranasal application of TNF-α could not restore leukocyte recruitment and oedema formation in endothelial adam17-knockout animals. Thus, activation of endothelial ADAM17 promotes acute pulmonary inflammation in response to endotoxin by multiple endothelial shedding events most likely independently of endothelial TNF-α release leading to enhanced vascular permeability and leukocyte recruitment.
Subject(s)
ADAM Proteins/metabolism , Endothelial Cells/enzymology , Inflammation/pathology , Lipopolysaccharides/immunology , Lung/drug effects , Pneumonia/chemically induced , ADAM17 Protein , Animals , Cell Adhesion Molecules/metabolism , Cells, Cultured , Chemokine CX3CL1/metabolism , Humans , Interleukin-6/metabolism , Leukocytes/immunology , Lipopolysaccharides/toxicity , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Cell Surface/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.
Subject(s)
Chemokine CX3CL1/metabolism , Leukocytes/physiology , Transendothelial and Transepithelial Migration/physiology , ADAM Proteins/genetics , ADAM Proteins/metabolism , ADAM10 Protein , ADAM17 Protein , Amino Acid Sequence , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Animals , CX3C Chemokine Receptor 1 , Calcium Signaling/physiology , Cell Line, Tumor , Cells, Cultured , Chemokine CX3CL1/genetics , Chemotaxis/physiology , Endothelial Cells/cytology , Endothelial Cells/physiology , Epithelial Cells/cytology , Epithelial Cells/physiology , Humans , Leukocytes/cytology , Ligands , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Protein Structure, Secondary , Receptors, Chemokine/chemistry , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolismABSTRACT
Syndecans are cell surface proteoglycans that bind and modulate various proinflammatory mediators and can be proteolytically shed from the cell surface. Within the lung, syndecan-1 and -4 are expressed as transmembrane proteins on epithelial cells and released in the bronchoalveolar fluid during inflammation. We here characterize the mechanism leading to the generation of soluble syndecan-1 and -4 in cultured epithelial cells and murine lung tissue. We show that the bladder carcinoma epithelial cell line ECV304, the lung epithelial cell line A459 and primary alveolar epithelial cells express and constitutively release syndecan-1 and -4. This release involves the activity of the disintegrin-like metalloproteinase ADAM17 as demonstrated by use of specific inhibitors and lentivirally transduced shRNA. Stimulation of epithelial cells with PMA, thrombin, or proinflammatory cytokines (TNFalpha/IFNgamma) led to the down-regulation of surface-expressed syndecan-1 and -4, which was associated with a significant increase of soluble syndecans and cell-associated cleavage fragments. The enhanced syndecan release was not related to gene induction of syndecans or ADAM17, but rather due to increased ADAM17 activity. Soluble syndecan-1 and -4 were also released into the bronchoalveolar fluid of mice. Treatment with TNFalpha/IFNgamma increased ADAM17 activity and syndecan release in murine lungs. Both constitutive and induced syndecan shedding was prevented by the ADAM17 inhibitor. ADAM17 may therefore be an important regulator of syndecan functions on inflamed lung epithelium.
