Receptor Shedding Research Articles

New insights into methoxetamine mechanisms of action: Focus on serotonergic 5-HT2 receptors in pharmacological and behavioral effects in the rat

Methoxetamine (MXE) is a dissociative substance of the arylcyclohexylamine class that has been present on the designer drug market as a ketamine-substitute since 2010. We have previously shown that MXE (i) possesses ketamine-like discriminative and positive rewarding effects in rats, (ii) affects brain processing involved in cognition and emotional responses, (iii) causes long-lasting behavioral abnormalities and neurotoxicity in rats and (iv) induces neurological, sensorimotor and cardiorespiratory alterations in mice. To shed light on the mechanisms through which MXE exerts its effects, we conducted a multidisciplinary study to evaluate the various neurotransmitter systems presumably involved in its actions on the brain. In vivo microdialysis study first showed that a single administration of MXE (0.25 and 0.5 mg/kg, i.v.) is able to significantly alter serotonin levels in the rat medial prefrontal cortex (mPFC) and nucleus accumbens. Then, we observed that blockade of the serotonin 5-HT2 receptors through two selective antagonists, ketanserin (0.1 mg/kg, i.p.) and MDL 100907 (0.03 mg/kg, i.p.), at doses not affecting animals behavior per se, attenuated the facilitatory motor effect and the inhibition on visual sensory responses induced by MXE (3 mg/kg, i.p.) and ketamine (3 mg/kg, i.p.), and prevented MXE-induced reduction of the prepulse inhibition in rats, pointing to the 5-HT2 receptors as a key target for the recently described MXE-induced sensorimotor effects. Finally, in-vitro electrophysiological studies revealed that the GABAergic and glutamatergic systems are also likely involved in the mechanisms through which MXE exerts its central effects since MXE inhibits, in a concentration-dependent manner, NMDA-mediated field postsynaptic potentials and GABA-mediated spontaneous currents. Conversely, MXE failed to alter both the AMPA component of field potentials and presynaptic glutamate release, and seems not to interfere with the endocannabinoid-mediated effects on mPFC GABAergic synapses. Altogether, our results support the notion of MXE as a NMDA receptor antagonist and shed further lights into the central mechanisms of action of this ketamine-substitute by pointing to serotonin 5-HT2 receptors as crucial players in the expression of its sensorimotor altering effects and to the NMDA and GABA receptors as potential further important targets of action.

Ectodomain shedding of low-density lipoprotein receptor and atherosclerosis

Background and Aims: Plasma levels of low-density lipoprotein cholesterol (LDL-C) are positively correlated with risk of atherosclerosis. LDL receptor (LDLR) is mainly resposible for LDL-C clearance. LDLR can be proteolytically cleaved to release its soluble ectodomain (sLDLR) into extracellular milieu. Plasma sLDLR levels are positively correlated with plasma LDL-C levels. However, the proteinase responsible for LDLR cleavage is unknown. Membrane type 1-matrix metalloproteinase (MT1-MMP) is a Zn2+-dependent endopeptidase that can cleave extracellular matrix and non-matrix substrates.

Membrane-type I matrix metalloproteinase (MT1-MMP), lipid metabolism, and therapeutic implications.

Lipids exert many essential physiological functions, such as serving as a structural component of biological membranes, storing energy, and regulating cell signal transduction. Dysregulation of lipid metabolism can lead to dyslipidemia related to various human diseases, such as obesity, diabetes, and cardiovascular disease. Therefore, lipid metabolism is strictly regulated through multiple mechanisms at different levels, including the extracellular matrix. Membrane-type I matrix metalloproteinase (MT1-MMP), a zinc-dependent endopeptidase, proteolytically cleaves extracellular matrix components, and non-matrix proteins, thereby regulating many physiological and pathophysiological processes. Emerging evidence supports the vital role of MT1-MMP in lipid metabolism. For example, MT1-MMP mediates ectodomain shedding of low-density lipoprotein receptor and increases plasma low-density lipoprotein cholesterol levels and the development of atherosclerosis. It also increases the vulnerability of atherosclerotic plaque by promoting collagen cleavage. Furthermore, it can cleave the extracellular matrix of adipocytes, affecting adipogenesis and the development of obesity. Therefore, the activity of MT1-MMP is strictly regulated by multiple mechanisms, such as autocatalytic cleavage, endocytosis and exocytosis, and post-translational modifications. Here, we summarize the latest advances in MT1-MMP, mainly focusing on its role in lipid metabolism, the molecular mechanisms regulating the function and expression of MT1-MMP, and their pharmacotherapeutic implications.

Immune control of Mycobacterium tuberculosis is dependent on both soluble TNFRp55 and soluble TNFRp75.

Tuberculosis presents a global health challenge, and tumour necrosis factor (TNF) signalling is required for host immunity against Mycobacterium tuberculosis (Mtb). TNF receptor shedding, however, compromises effective immunity by reducing bioactive TNF through the formation of inactive complexes. In this study, we first compared the effect of total soluble TNF receptors using a transgenic p55ΔNS /p75-/- murine strain on host protection during a low-dose aerosol Mtb H37Rv challenge. We report that the presence of membrane-bound TNFRp55 alone in the absence of TNFRp75 results in superior control of a primary Mtb infection where p55ΔNS /p75-/- hyperactive dendritic cells displayed an increased capacity to induce a hyperactive Mtb-specific CD4+ T-cell response. p55ΔNS /p75-/- dendritic cells expressed a higher frequency of MHCII and increased MFIs for both CD86 and MHCII, while CD4+ T cells had higher expression of CD44 and IFN-γ. Next, the relative contributions of soluble TNFRp55 and soluble TNFRp75 to host protection against either primary Mtb infection or during reactivation of latent tuberculosis were delineated by comparing the experimental outcomes of control C57BL/6 mice to transgenic p55ΔNS /p75-/- , p55ΔNS and p75-/- mouse strains. We found that soluble TNFRp55 is redundant for immune regulation during the chronic stages of a primary Mtb infection. However, TNFRp55 together with soluble TNFRp75 has a crucial role in immune regulation of reactivation of latent tuberculosis.

Extracellular domain shedding of the ALK receptor mediates neuroblastoma cell migration.

SUMMARYAlthough activating mutations of the anaplastic lymphoma kinase (ALK) membrane receptor occur in ~10% of neuroblastoma (NB) tumors, the role of the wild-type (WT) receptor, which is aberrantly expressed in most non-mutated cases, is unclear. Both WT and mutant proteins undergo extracellular domain (ECD) cleavage. Here, we map the cleavage site to Asn654-Leu655 and demonstrate that cleavage inhibition of WT ALK significantly impedes NB cell migration with subsequent prolongation of survival in mouse models. Cleavage inhibition results in the downregulation of an epithelial-to-mesenchymal transition (EMT) gene signature, with decreased nuclear localization and occupancy of β-catenin at EMT gene promoters. We further show that cleavage is mediated by matrix metalloproteinase 9, whose genetic and pharmacologic inactivation inhibits cleavage and decreases NB cell migration. Together, our results indicate a pivotal role for WT ALK ECD cleavage in NB pathogenesis, which may be harnessed for therapeutic benefit.

Airway monocyte modulation relates to tumour necrosis factor dysregulation in neutrophilic asthma.

BackgroundDysregulation of tumour necrosis factor-α (TNF-α) signalling is implicated in neutrophilic asthma. TNF-α signalling involves membrane-bound and soluble ligand (TNF-α) and receptors (TNFRs); however, little is known about how these proteins are altered in asthma. We hypothesised that intercompartment-, immune cell- and/or asthma inflammatory phenotype-dependent regulation could relate to TNF dysregulation in neutrophilic asthma.MethodsMeasurements were made in 45 adults with asthma (36 non-neutrophilic, 9 neutrophilic) and 8 non-asthma controls. Soluble TNF-α, TNF receptor 1 (TNFR1) and TNFR2 were quantified in plasma and sputum supernatant by ELISA, and membrane-bound TNF-α/TNFR1/TNFR2 measured on eosinophils, neutrophils, monocytes, and macrophages in blood and sputum by flow cytometry. Marker expression was compared between inflammatory phenotypes and compartments, and relationship of membrane-bound and soluble TNF markers and immune cell numbers tested by correlation.ResultsSoluble sputum TNFR1 and TNFR2 were increased in neutrophilic versus non-neutrophilic asthma (p=0.010 and p=0.029). Membrane-bound TNF-α expression was upregulated on sputum versus blood monocytes, while TNFR1 and TNFR2 levels were reduced on airway versus blood monocytes and neutrophils. Soluble TNFR1 and TNFR2 in sputum significantly correlated with the number of airway monocytes (p=0.016, r=0.358 and p=0.029, r=0.327).ConclusionOur results imply that increased sputum soluble TNF receptor levels observed in neutrophilic asthma relate to the increased recruitment of monocytes and neutrophils into the airways and their subsequent receptor shedding. Monocytes also increase TNF-α ligand expression in the airways. These results suggest an important contribution of airway monocytes to the altered inflammatory milieu in neutrophilic asthma.

Novel TREM2 splicing isoform that lacks the V-set immunoglobulin domain is abundant in the human brain.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immunoglobulin-like receptor expressed by certain myeloid cells, such as macrophages, dendritic cells, osteoclasts, and microglia. In the brain, TREM2 plays an important role in the immune function of microglia, and its dysfunction is linked to various neurodegenerative conditions in humans. Ablation of TREM2 or its adaptor protein TYROBP causes polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (also known as Nasu-Hakola disorder) with early onset of dementia, whereas some missense variants in TREM2 are associated with an increased risk of late-onset Alzheimer's disease. The human TREM2 gene is subject to alternative splicing, and its major, full-length canonic transcript encompasses 5 exons. Herein, we report a novel alternatively spliced TREM2 isoform without exon 2 (Δe2), which constitutes a sizable fraction of TREM2 transcripts and has highly variable inter-individual expression in the human brain (average frequency 10%; range 3.7-35%). The protein encoded by Δe2 lacks a V-set immunoglobulin domain from its extracellular part but retains its transmembrane and cytoplasmic domains. We demonstrated Δe2 protein expression in TREM2-positive THP-1 cells, in which the expression of full-length transcript was precluded by CRISPR/Cas9 disruption of the exon 2 coding frame. Similar to the full-length TREM2, Δe2 is sorted to the plasma membrane and is subject to receptor shedding. In "add-back" experiments, Δe2 TREM2 had diminished capacity to restore phagocytosis of amyloid beta peptide and promote IFN-I response as compared to full-length TREM2. Our findings suggest that changes in the balance of two mutually exclusive TREM2 isoforms may modify the dosage of full-length transcript potentially weakening some TREM2 receptor functions in the human brain.

Platelet α‐granule cargo packaging and release are affected by the luminal proteoglycan, serglycin

BackgroundSerglycin (SRGN) is an intragranular, sulfated proteoglycan in hematopoietic cells that affects granule composition and function. ObjectiveTo understand how SRGN affects platelet granule packaging, cargo release, and extra‐platelet microenvironments. MethodsPlatelets and megakaryocytes from SRGN−/− mice were assayed for secretion kinetics, cargo levels, granule morphology upon activation, and receptor shedding. ResultsMetabolic, 35SO4 labeling identified SRGN as a major sulfated macromolecule in megakaryocytes. SRGN colocalized with α‐granule markers (platelet factor 4 [PF4], von Willebrand factor [VWF], and P‐selectin), but its deletion did not affect α‐granule morphology or number. Platelet α‐granule composition was altered, with a reduction in basic proteins (pI ≥8; e.g., PF4, SDF‐1, angiogenin) and constitutive release of PF4 from SRGN−/− megakaryocytes. P‐Selectin, VWF, and fibrinogen were unaffected. Serotonin (5‐HT) uptake and β‐hexosaminidase (HEXB) were slightly elevated. Thrombin‐induced exocytosis of PF4 from platelets was defective; however, release of RANTES/CCL5 was normal and osteopontin secretion was more rapid. Release of 5‐HT and HEXB (from dense granules and lysosomes, respectively) were unaffected. Ultrastructural studies showed distinct morphologies in activated platelets. The α‐granule lumen of SRGN−/− platelet had a grainy staining pattern, whereas that of wild‐type granules had only fibrous material remaining. α‐Granule swelling and decondensation were reduced in SRGN−/− platelets. Upon stimulation of platelets, a SRGN/PF4 complex was released in a time‐ and agonist‐dependent manner. Shedding of GPVI from SRGN−/− platelets was modestly enhanced. Shedding of GP1b was unaffected. ConclusionThe polyanionic proteoglycan SRGN influences α‐granule packaging, cargo release, and shedding of platelet membrane proteins.

Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis

Plasma low-density lipoprotein (LDL) is primarily cleared by LDL receptor (LDLR). LDLR can be proteolytically cleaved to release its soluble ectodomain (sLDLR) into extracellular milieu. However, the proteinase responsible for LDLR cleavage is unknown. Here we report that membrane type 1-matrix metalloproteinase (MT1-MMP) co-immunoprecipitates and co-localizes with LDLR and promotes LDLR cleavage. Plasma sLDLR and cholesterol levels are reduced while hepatic LDLR is increased in mice lacking hepatic MT1-MMP. Opposite effects are observed when MT1-MMP is overexpressed. MT1-MMP overexpression significantly increases atherosclerotic lesions, while MT1-MMP knockdown significantly reduces cholesteryl ester accumulation in the aortas of apolipoprotein E (apoE) knockout mice. Furthermore, sLDLR is associated with apoB and apoE-containing lipoproteins in mouse and human plasma. Plasma levels of sLDLR are significantly increased in subjects with high plasma LDL cholesterol levels. Thus, we demonstrate that MT1-MMP promotes ectodomain shedding of hepatic LDLR, thereby regulating plasma cholesterol levels and the development of atherosclerosis.

Constitutive and Regulated Shedding of Soluble FGF Receptors Releases Biologically Active Inhibitors of FGF-2.

The identification of soluble fibroblast growth factor (FGF) receptors in blood and the extracellular matrix has led to the prediction that these proteins modulate the diverse biological activities of the FGF family of ligands in vivo. A recent structural characterization of the soluble FGF receptors revealed that they are primarily generated by proteolytic cleavage of the FGFR-1 ectodomain. Efforts to examine their biological properties are now focused on understanding the functional consequences of FGFR-1 ectodomain shedding and how the shedding event is regulated. We have purified an FGFR-1 ectodomain that is constitutively cleaved from the full-length FGFR-1(IIIc) receptor and released into conditioned media. This shed receptor binds FGF-2; inhibits FGF-2-induced cellular proliferation; and competes with high affinity, cell surface FGF receptors for ligand binding. FGFR-1 ectodomain shedding downregulates the number of high affinity receptors from the cell surface. The shedding mechanism is regulated by ligand binding and by activators of PKC, and the two signaling pathways appear to be independent of each other. Deletions and substitutions at the proposed cleavage site of FGFR-1 do not prevent ectodomain shedding. Broad spectrum inhibitors of matrix metalloproteases decrease FGFR-1 ectodomain shedding, suggesting that the enzyme responsible for constitutive, ligand-activated, and protein kinase C-activated shedding is a matrix metalloprotease. In summary, shedding of the FGFR-1 ectodomain is a highly regulated event, sharing many features with a common system that governs the release of diverse membrane proteins from the cell surface. Most importantly, the FGFR ectodomains are biologically active after shedding and are capable of functioning as inhibitors of FGF-2.

Contribution of ADAM17 and related ADAMs in cardiovascular diseases.

A disintegrin and metalloproteases (ADAMs) are key mediators of cell signaling by ectodomain shedding of various growth factors, cytokines, receptors and adhesion molecules at the cellular membrane. ADAMs regulate cell proliferation, cell growth, inflammation, and other regular cellular processes. ADAM17, the most extensively studied ADAM family member, is also known as tumor necrosis factor (TNF)-α converting enzyme (TACE). ADAMs-mediated shedding of cytokines such as TNF-α orchestrates immune system or inflammatory cascades and ADAMs-mediated shedding of growth factors causes cell growth or proliferation by transactivation of the growth factor receptors including epidermal growth factor receptor. Therefore, increased ADAMs-mediated shedding can induce inflammation, tissue remodeling and dysfunction associated with various cardiovascular diseases such as hypertension and atherosclerosis, and ADAMs can be a potential therapeutic target in these diseases. In this review, we focus on the role of ADAMs in cardiovascular pathophysiology and cardiovascular diseases. The main aim of this review is to stimulate new interest in this area by highlighting remarkable evidence.

Label-free multimodal quantitative imaging flow assay for intrathrombus formation in vitro

Microfluidics in vitro assays recapitulate a blood vessel microenvironment using surface-immobilized agonists under biofluidic flows. However, these assays do not quantify intrathrombus mass and activities of adhesive platelets at the agonist margin and use fluorescence labeling, therefore limiting clinical translation potential. Here, we describe a label-free multimodal quantitative imaging flow assay that combines rotating optical coherent scattering microscopy and quantitative phase microscopy. The combined imaging platform enables real-time evaluation of membrane fluctuations of adhesive-only platelets and total intrathrombus mass under physiological flow rates in vitro. We call this multimodal quantitative imaging flow assay coherent optical scattering and phase interferometry (COSI). COSI records intrathrombus mass to picogram accuracy and shape changes to a platelet membrane with high spatial-temporal resolution (0.4 μm/s) under physiological and pathophysiological fluid shear stress (1800 and 7500 s−1). With COSI, we generate an axial slice of 4 μm from the coverslip surface, approximately equivalent to the thickness of a single platelet, which permits nanoscale quantification of membrane fluctuation (activity) of adhesive platelets during initial adhesion, spreading, and recruitment into a developing thrombus (mass). Under fluid shear, pretreatment with a broad range metalloproteinase inhibitor (250 μM GM6001) blocked shedding of platelet adhesion receptors that shown elevated adhesive platelet activity at average of 42.1 μm/s and minimal change in intrathrombus mass.

NK/T cell ratios associate with interleukin-2 receptor alpha chain expression and shedding in multiple sclerosis

NK/T-cell ratios predict disease activity in relapsing remitting multiple sclerosis (RRMS). We investigated in 50 RRMS patients whether interleukin-2 receptor alpha-chain (IL-2Rα) expression and shedding associates with NK/T-cell balance, as suggested by daclizumab-trials in RRMS. A subsample (N = 31) was genotyped for IL2RA-associated MS risk SNPs. CD56bright NK-cell/IL-17A+CD4+ T-cell ratios correlated negatively with plasma and PBMC-culture supernatant sIL-2Rα-levels [R = -0.209; p = 0.038 and R = -0.254; p = 0.012, resp.], and with CD4+ T-cell CD25 MFI [R = -0.341; p = 0.001]. Carriers of the rs3118470 risk-allele showed higher sIL-2Rα-levels (P = 0.031) and a lower CD56bright NK-cell/IL-17A+CD4+ T-cell ratio (P = 0.038). Therefore, IL-2Rα may be involved in the interplay between NK-cells and T-cells.

On the role of bacterial metalloproteases in COVID-19 associated cytokine storm

The cytokine release syndrome or cytokine storm, which is the hyper-induction of inflammatory responses has a central role in the mortality rate of COVID-19 and some other viral infections. Interleukin-6 (IL-6) is a key player in the development of cytokine storms. Shedding of interleukin-6 receptor (IL-6Rα) results in the accumulation of soluble interleukin-6 receptors (sIL-6R). Only relatively few cells express membrane-bound IL-6Rα. However, sIL-6R can act on potentially all cells and organs through the ubiquitously expressed gp130, the coreceptor of IL-6Rα. Through this, so-called trans-signaling, IL-6–sIL-6R is a powerful factor in the development of cytokine storms and multiorgan involvement. Some bacteria (e.g., Serratia marcescens, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes), commonly considered to cause co-infections during viral pneumonia, can directly induce the shedding of membrane receptors, including IL-6Rα, or enhance endogenous shedding mechanisms causing the increase of sIL-6R level. Here we hypothesise that bacteria promoting shedding and increase the sIL-6R level can be an important contributing factor for the development of cytokine storms. Therefore, inhibition of IL-6Rα shedding by drastically reducing the number of relevant bacteria may be a critical element in reducing the chance of a cytokine storm. Validation of this hypothesis can support the consideration of the prophylactic use of antibiotics more widely and at an earlier stage of infection to decrease the mortality rate of COVID-19.B1YsZKRExp1rTeKvgh-6-gVideo abstract

Functional impact of intramolecular cleavage and dissociation of adhesion G protein–coupled receptor GPR133 (ADGRD1) on canonical signaling

GPR133 (ADGRD1), an adhesion G protein–coupled receptor (GPCR) whose canonical signaling activates GαS-mediated generation of cytosolic cAMP, has been shown to be necessary for the growth of glioblastoma (GBM), a brain malignancy. The extracellular N terminus of GPR133 is thought to be autoproteolytically cleaved into N-terminal and C- terminal fragments (NTF and CTF, respectively). However, the role of this cleavage in receptor activation remains unclear. Here, we used subcellular fractionation and immunoprecipitation approaches to show that the WT GPR133 receptor is cleaved shortly after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant GPR133 (H543R) in patient-derived GBM cultures and HEK293T cells. After cleavage, the resulting NTF and CTF remain noncovalently bound to each other until the receptor is trafficked to the plasma membrane, where we demonstrated NTF–CTF dissociation occurs. Using a fusion of the CTF of GPR133 and the N terminus of thrombin-activated human protease-activated receptor 1 as a controllable proxy system to test the effect of intramolecular cleavage and dissociation, we also showed that thrombin-induced cleavage and shedding of the human protease-activated receptor 1 NTF increased intracellular cAMP levels. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. These findings add depth to our understanding of the molecular life cycle and mechanism of action of GPR133 and provide critical insights that will inform therapeutic targeting of GPR133 in GBM.

Device-Induced Hemostatic Disorders in Mechanically Assisted Circulation

Mechanically assisted circulation (MAC) sustains the blood circulation in the body of a patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) or on ventricular assistance with a ventricular assist device (VAD) or on extracorporeal membrane oxygenation (ECMO) with a pump-oxygenator system. While MAC provides short-term (days to weeks) support and long-term (months to years) for the heart and/or lungs, the blood is inevitably exposed to non-physiological shear stress (NPSS) due to mechanical pumping action and in contact with artificial surfaces. NPSS is well known to cause blood damage and functional alterations of blood cells. In this review, we discussed shear-induced platelet adhesion, platelet aggregation, platelet receptor shedding, and platelet apoptosis, shear-induced acquired von Willebrand syndrome (AVWS), shear-induced hemolysis and microparticle formation during MAC. These alterations are associated with perioperative bleeding and thrombotic events, morbidity and mortality, and quality of life in MCS patients. Understanding the mechanism of shear-induce hemostatic disorders will help us develop low-shear-stress devices and select more effective treatments for better clinical outcomes.

Neutrophil injury and function alterations induced by high mechanical shear stress with short exposure time.

High mechanical shear stresses (HMSS) can cause damage to blood, which manifests as morphologic changes, shortened life span, biochemical alterations, and complete rupture of blood cells and proteins, leading to the alterations of normal blood function. The aim of this study is to determine the state of neutrophil activation and function alterations caused by HMSS with short exposure time relevant to ventricular assist devices. Blood from healthy donors was exposed to three levels of HMSS (75Pa, 125Pa, and 175Pa) for a short exposure time (0.5s) using our Couette-type blood-shearing device. Neutrophil activation (Mac-1, platelet-neutrophil aggregates) and surface expression levels of two key functional receptors (CD62L and CD162) on neutrophils were evaluated by flow cytometry. Neutrophil phagocytosis and transmigration were also examined with functional assays. Results showed that the expression of Mac-1 on neutrophils and platelet-neutrophil aggregates increased significantly while the level of CD62L expression on neutrophils decreased significantly after the exposure to HMSS. The Mac-1 expression progressively increased while the CD62L expression progressively decreased with the increased level of HMSS. The level of CD162 expression on neutrophils slightly increased after the exposure to HMSS, but the increase was not significant. The phagocytosis assay data revealed that the ability of neutrophils to phagocytose latex beads coated with fluorescently labeled rabbit IgG increased significantly with the increased level of HMSS. The transmigration ability of neutrophils slightly increased after the exposure to HMSS, but did not reach a significant level. In summary, HMSS with a short exposure time of 0.5seconds could induce neutrophil activation, platelet-neutrophil aggregation, shedding of CD62L receptor, and increased phagocytic ability. However, the exposure to the three levels of HMSS did not cause a significant change in neutrophil transmigration capacity and shedding of CD162 receptor on neutrophils.

Loss of Furin in β-Cells Induces an mTORC1-ATF4 Anabolic Pathway That Leads to β-Cell Dysfunction.

FURIN is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, but its specific role in pancreatic β-cells is largely unknown. The aim of this study was to determine the role of FURIN in glucose homeostasis. We show that FURIN is highly expressed in human islets, whereas PCs that potentially could provide redundancy are expressed at considerably lower levels. β-cell-specific Furin knockout (βFurKO) mice are glucose intolerant as a result of smaller islets with lower insulin content and abnormal dense-core secretory granule morphology. mRNA expression analysis and differential proteomics on βFurKO islets revealed activation of activating transcription factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). βFurKO cells show impaired cleavage or shedding of vacuolar-type ATPase (V-ATPase) subunits Ac45 and prorenin receptor, respectively, and impaired lysosomal acidification. Blocking V-ATPase pharmacologically in β-cells increased mTORC1 activity, suggesting involvement of the V-ATPase proton pump in the phenotype. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation and impaired lysosomal acidification in β-cells lacking Furin, causing β-cell dysfunction.

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.

TNFRp75-dependent immune regulation of alveolar macrophages and neutrophils during early Mycobacterium tuberculosis and Mycobacterium bovis BCG infection.

TNF signalling through TNFRp55 and TNFRp75, and receptor shedding is important for immune activation and regulation. TNFRp75 deficiency leads to improved control of Mycobacterium tuberculosis (M. tuberculosis) infection, but the effects of early innate immune events in this process are unclear. We investigated the role of TNFRp75 on cell activation and apoptosis of alveolar macrophages and neutrophils during M. tuberculosis and M. bovis BCG infection. We found increased microbicidal activity against M. tuberculosis occurred independently of IFNy and NO generation, and displayed an inverse correlation with alveolar macrophages (AMs) apoptosis. Both M. tuberculosis and M. bovis BCG induced higher expression of MHC-II in TNFRp75-/- AMs; however, M bovis BCG infection did not alter AM apoptosis in the absence of TNFRp75. Pulmonary concentrations of CCL2, CCL3 and IL-1β were increased in TNFRp75-/- mice during M, bovis BCG infection, but had no effect on neutrophil responses. Thus, TNFRp75-dependent regulation of mycobacterial replication is virulence dependent and occurs independently of early alveolar macrophage apoptosis and neutrophil responses.