A Disintegrin And Metalloproteinase 17 Research Articles

IRhom2 regulates ectodomain shedding and surface expression of the major histocompatibility complex (MHC) class I

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.

Porcine reproductive and respiratory syndrome virus infection activates ADAM17 to induce inflammatory responses

Porcine reproductive and respiratory syndrome (PRRS), which has posed substantial threats to the swine industry worldwide, is primarily characterized by interstitial pneumonia. A disintegrin and metalloproteinase 17 (ADAM17) is a multifunctional sheddase involved in various inflammatory diseases. Herein, our study showed that PRRS virus (PRRSV) infection elevated ADAM17 activity, as demonstrated in primary porcine alveolar macrophages (PAMs), an immortalized PAM cell line (IPAM cells), and the lung tissues of PRRSV-infected piglets. We found that PRRSV infection promoted ADAM17 translocation from the endoplasmic reticulum to the Golgi by enhancing its interaction with inactive rhomboid protein 2 (iRhom2), a newly identified ADAM17 regulator, which in turn elevated ADAM17 activity. By screening for PRRSV-encoded structural proteins, viral envelope (E) and nucleocapsid (N) proteins were identified as the predominant ADAM17 activators. E and N proteins bind with both ADAM17 and iRhom2 to form ternary protein complexes, ultimately strengthening their interactions. Additionally, we demonstrated, using an ADAM17-knockout cell line, that ADAM17 augmented the shedding of soluble TNF-α, a pivotal inflammatory mediator. We also discovered that ADAM17-mediated cleavage of porcine TNF-α occurred between Arg-78 and Ser-79. By constructing a precision mutant cell line with Arg-78-Glu/Ser-79-Glu substitution mutations in TNF-α, we further revealed that the ADAM17-mediated production of soluble TNF-α contributed to the induction of inflammatory responses by PRRSV and its E and N proteins. Taken together, our results elucidate the mechanism by which PRRSV infection activates the iRhom2/ADAM17/TNF-α axis to enhance inflammatory responses, providing valuable insights into the elucidation of PRRSV pathogenesis.

CircCDYL Contributes to Apoptosis, Ferroptosis, and Oxidative Stress of Ang II-Induced Vascular Smooth Muscle Cells in Thoracic Aortic Aneurysm.

Circular RNAs (circRNAs) have important regulation in thoracic aortic aneurysm (TAA). The function and mechanism of circCDYL (circ_0008285) was explored in TAA here. Angiotensin II (Ang II) was used to construct a TAA model. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for the detection of circCDYL, miR-1270, and a disintegrin and metalloproteinase 10 (ADAM10). Cell viability was examined via cell counting kit-8 (CCK-8) assay and proliferation was analyzed using Ethynyl-2'-deoxyuridine (EdU) assay. Apoptosis rate was assessed via flow cytometry. Western blot was used for protein detection. Oxidative stress was evaluated by commercial kits. CircCDYL was upregulated in TAA tissues and Ang II-induced circCDYL upregulation in vascular smooth muscle cells (VSMCs). Knockdown of circCDYL weakened Ang II-aroused inhibition of viability, proliferation, and promotion of apoptosis, ferroptosis, and oxidative stress in VSMCs. CircCDYL served as a miR-1270 sponge. The mitigated regulation of circCDYL knockdown for Ang II-induced injury was restored after miR-1270 downregulation. CircCDYL positively regulated ADAM10 through interacting with miR-1270. Overexpression of miR-1270 abated Ang II-induced injury by downregulating ADAM10. In conclusion, circCDYL was involved in the Ang II-induced VSMC injury in TAA via the miR-1270/ADAM10 axis.

GTS-21 attenuates ACE/ACE2 ratio and glycocalyx shedding in lipopolysaccharide-induced acute lung injury by targeting macrophage polarization derived ADAM-17

Acute lung injury (ALI) has received considerable attention in intensive care owing to its high mortality rate. It has been demonstrated that the selective alpha7 nicotinic acetylcholine receptor agonist Gainesville Tokushima scientists (GTS)-21 is promising for treating ALI caused by lipopolysaccharides (LPS). However, the precise underlying mechanism remains unknown. This study aimed to investigate the potential efficacy of GTS-21 in the treatment of ALI. We developed mouse models of ALI and alveolar epithelial type II cells (AT2s) injury following treatment with LPS and different polarized macrophage supernatants, respectively. Pathological changes, pulmonary edema, and lung compliance were assessed. Inflammatory cells count, protein content, and pro-inflammatory cytokine levels were analysed in the bronchoalveolar lavage fluid. The expression of angiotensin-converting enzyme (ACE), ACE2, syndecan-1 (SDC-1), heparan sulphate (HS), heparanase (HPA), exostosin (EXT)-1, and NF-κB were tested in lung tissues and cells. GTS-21-induced changes in macrophage polarization were verified in vivo and in vitro. Polarized macrophage supernatants with or without recombination a disintegrin and metalloproteinase-17 (ADAM-17) and small interfering (si)RNA ADAM-17 were used to verify the role of ADAM-17 in AT2 injury. By reducing pathological alterations, lung permeability, inflammatory response, ACE/ACE2 ratio, and glycocalyx shedding, as well as by downregulating the HPA and NF-κB pathways and upregulating EXT1 expression in vivo, GTS-21 significantly diminished LPS-induced ALI compared to that of the LPS group. GTS-21 significantly attenuated macrophage M1 polarization and augmented M2 polarization in vitro and in vivo. The destructive effects of M1 polarization supernatant can be inhibited by GTS-21 and siRNA ADAM-17. GTS-21 exerted a protective effect against LPS-induced ALI, which was reversed by recombinant ADAM-17. Collectively, GTS-21 alleviates LPS-induced ALI by attenuating AT2s ACE/ACE2 ratio and glycocalyx shedding through the inhibition of macrophage M1 polarization derived ADAM-17.

Exploring crucial structural attributes of quinolinyl methoxyphenyl sulphonyl-based hydroxamate derivatives as ADAM17 inhibitors through classification-dependent molecular modelling approaches

ABSTRACT A Disintegrin and Metalloproteinase 17 (ADAM17), a Zn2+-dependent metalloenzyme of the adamalysin family of the metzincin superfamily, is associated with various pathophysiological conditions including rheumatoid arthritis and cancer. However, no specific inhibitors have been marketed yet for ADAM17-related disorders. In this study, 94 quinolinyl methoxyphenyl sulphonyl-based hydroxamates as ADAM17 inhibitors were subjected to classification-based molecular modelling and binding pattern analysis to identify the significant structural attributes contributing to ADAM17 inhibition. The statistically validated classification-based models identified the importance of the P1' substituents such as the quinolinyl methoxyphenyl sulphonyl group of these compounds for occupying the S1' - S3' pocket of the enzyme. The quinolinyl function of these compounds was found to explore stable binding of the P1' substituents at the S1' – S3' pocket whereas the importance of the sulphonyl and the orientation of the P1' moiety also revealed stable binding. Based on the outcomes of the current study, four novel compounds of different classes were designed as promising ADAM17 inhibitors. These findings regarding the crucial structural aspects and binding patterns of ADAM17 inhibitors will aid the design and discovery of novel and effective ADAM17 inhibitors for therapeutic advancements of related diseases.

Curcumin Modulates the Membrane Raft Integrity via Phase Separation and Induces CD44 Shedding in Tumor Cells.

CD44 is a transmembrane cell adhesion molecule that is cleaved by the membrane proteinase, a disintegrin and metalloproteinase 10 (ADAM10), on the cell surface via ectodomain shedding after cholesterol depletion. Lipid raft-mediated CD44 shedding is essential for cancer cell invasion. As cell-cell and cell-matrix adhesions are critical for cancer progression, lipid raft-targeting agents may be effective for cancer therapy. Here, we found that curcumin and its derivatives induced the ADAM10-mediated shedding of CD44 in tumor cells. We also found that curcumin and the derivatives are membrane-active compounds whose effect depends on its planar backbone and the spatial arrangement of methoxy groups substituted on the two aromatic rings using giant unilamellar and plasma membrane vesicles. Curcumin and its derivatives with rigid backbones and hydroxy groups exerted membrane-domain-modulating activity, which may account for their pleiotropic effects via multiple signaling pathways involving membrane receptors. This study provides a basis for the use of membrane-active compounds, such as curcuminoids, to elucidate the roles of lipid rafts in cellular signaling, regulation of membrane-bound ADAM metalloproteinases, and the development of novel membrane lipid-based therapies.

M6A‑modified HOXC10 promotes HNSCC progression via co‑activation of ADAM17/EGFR and Wnt/β‑catenin signaling.

The homeobox (HOX) gene family plays a fundamental role in carcinogenesis. However, the oncogenic mechanism of HOXC10 in head and neck squamous cell carcinoma (HNSCC) remains unclear. In the present study, it was revealed that HOXC10 expression was significantly higher in HNSCC tissues than in adjacent tissues, and a high level of HOXC10 was closely associated with worse clinical outcomes. HOXC10 overexpression promoted HNSCC cell proliferation, migration, and invasion, both invitro and invivo. Mechanistically, chromatin immunoprecipitation sequencing revealed that HOXC10 drove the transcriptional activation of a disintegrin and metalloproteinase17 (ADAM17), and the ADAM17/epidermal growth factor receptor (EGFR)/ERK1/2 signaling pathway facilitating the proliferation of HNSCC. Furthermore, mass spectrometric analysis indicated that HOXC10 interacted with ribosomal proteinS15A (RPS15A) and enhanced RPS15A protein expression, activating the Wnt/β‑catenin pathway and contributing to invasion and metastasis of HNSCC. Additionally, the methylated RNA immune precipitation and RNA antisense purification assays showed that N6‑methyladenosine (m6A) writer, methyltransferase‑like 3, catalyzed m6A modification of the HOXC10 transcript, m6A reader insulin like growth factor2 mRNA binding protein (IGF2BP)1 and IGF2BP3 involved in recognizing and stabilizing m6A‑tagged HOXC10 mRNA. In summary, the present study identified HOXC10 as a promising candidate oncogene in HNSCC. The m6A modification‑mediated HOXC10 promoted proliferation, migration, and invasion of HNSCC through co‑activation of ADAM17/EGFR and Wnt/β‑catenin signaling, providing a novel diagnostic and prognostic biomarker and a potential therapeutic target for HNSCC.

Using siRNA Silencing to Analyze ADAM17 in Macrophages.

Silencing expression with short interfering RNA (siRNA) is a rapid and cost-effective way to analyze the involvement of target genes in a range of biological processes. Here we describe isolation of primary human monocytes from peripheral blood and their in vitro differentiation to macrophages, followed by electroporation with siRNA to silence expression of a disintegrin and metalloproteinase 17 (ADAM17). This enables evaluation of ADAM17's role in cleaving transmembrane proteins, such as its prototypic substrate tumor necrosis factor (TNF), by enzyme-linked immunosorbent assay (ELISA), flow cytometry, or immunoblotting.

Neuron-specific gene NSG1 binds to and positively regulates sortilin ectodomain shedding via a metalloproteinase-dependent mechanism

Increasing evidence suggests that aberrant regulation of sortilin ectodomain shedding can contribute to amyloid-β pathology and frontotemporal dementia, although the mechanism by which this occurs has not been elucidated. Here we probed for novel binding partners of sortilin using multiple and complementary approaches and identified two proteins of the neuron-specific gene (NSG) family, NSG1 and NSG2, that physically interact and colocalize with sortilin. We show both NSG1 and NSG2 induce subcellular redistribution of sortilin to NSG1- and NSG2-enriched compartments. However, using cell surface biotinylation we found only NSG1 reduced sortilin cell surface expression, which caused significant reductions in uptake of progranulin (PGRN), a molecular determinant for frontotemporal dementia. In contrast, we demonstrate NSG2 has no effect on sortilin cell surface abundance or PGRN uptake, suggesting specificity for NSG1 in the regulation of sortilin cell surface expression. Using metalloproteinase inhibitors and A Disintegrin And Metalloproteinase 10 (ADAM10) knockout cells, we further show that NSG1-dependent reduction of cell surface sortilin occurred via proteolytic processing by ADAM10 with a concomitant increase in shedding of sortilin ectodomain to the extracellular space. This represents a novel regulatory mechanism for sortilin ectodomain shedding that is regulated in a neuron-specific manner. Furthermore, this finding has implications for the development of strategies for brain-specific regulation of sortilin and possibly sortilin-driven pathologies.

Neuron-secreted NLGN3 ameliorates ischemic brain injury via activating Gαi1/3-Akt signaling

We here tested the potential activity and the underlying mechanisms of neuroligin-3 (NLGN3) against ischemia-reperfusion-induced neuronal cell injury. In SH-SY5Y neuronal cells and primary murine cortical neurons, NLGN3 activated Akt-mTOR and Erk signalings, and inhibited oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R)-induced cytotoxicity. Akt activation was required for NLGN3-induced neuroprotection. Gαi1/3 mediated NLGN3-induced downstream signaling activation. NLGN3-induced Akt-S6K1 activation was largely inhibited by Gαi1/3 silencing or knockout. Significantly, NLGN3-induced neuroprotection against OGD/R was almost abolished by Gαi1/3 silencing or knockout. In vivo, the middle cerebral artery occlusion (MCAO) procedure induced NLGN3 cleavage and secretion, and increased its expression and Akt activation in mouse brain tissues. ADAM10 (A Disintegrin and Metalloproteinase 10) inhibition blocked MCAO-induced NLGN3 cleavage and secretion, exacerbating ischemic brain injury in mice. Neuronal silencing of NLGN3 or Gαi1/3 in mice also inhibited Akt activation and intensified MCAO-induced ischemic brain injury. Conversely, neuronal overexpression of NLGN3 increased Akt activation and alleviated MCAO-induced ischemic brain injury. Together, NLGN3 activates Gαi1/3-Akt signaling to protect neuronal cells from ischemia-reperfusion injury.

Optimization of fluorescent substrates for ADAM17 and their utility in the detection of diabetes

ADAM17 (a disintegrin and metalloproteinase 17) is a sheddase that releases various types of membrane-associated proteins, including adhesive molecules, cytokines and their receptors, and inflammatory mediators. Evidence suggests that the enzyme is involved in the proteolytic cleavage of antiaging transmembrane protein Klotho (KL). What is more, reduced serum and urinary KL levels are observed in the early stages of chronic kidney disease. This study aimed to optimise the ADAM17 specific and selective fluorescent substrates. Then, the obtained substrate was used to detect the enzyme in urine samples of patients diagnosed with diabetes. It turned out that in all cases we were able to detect proteolytic activity, which was the opposite of the healthy samples.

Biomolecular interactions between the antibacterial ceftolozane and the human inflammatory disease target ADAM17: a drug repurposing study

Inhibition of a disintegrin and metalloproteinase-17 (ADAM17), a metzincin, is proposed as a novel therapeutic strategy to suppress overproduction of the proinflammatory cytokine TNF-α in rheumatoid arthritis and inflammatory bowel disease. Existing ADAM17 inhibitors generate toxic metabolites in-vivo or haven’t progressed in clinical trials. Previous studies suggest that ligands which bind to ADAM17 active site by interacting with the Zn ion and L-shaped hydrophobic S1’- and S3’-pockets and forming favorable hydrogen bonds could act as potential ADAM17 inhibitors. Here, we investigated whether the FDA-approved anti-bacterial drug ceftolozane, a cephalosporin containing aromatic groups and carboxyl groups as probable zinc binding groups (ZBGs), forms non-covalent interactions resulting in its binding in the active site of ADAM17. In this study, the density functional theory (DFT), molecular docking and molecular dynamics calculations with the catalytic chain of ADAM17 show that carboxyl group of ceftolozane acts as moderate ZBG, and its extended geometry forms hydrogen bonds and hydrophobic interactions resulting in a binding affinity comparable to the co-crystallized known ADAM17 inhibitor. The favorable binding interactions identified here suggest the potential of ceftolozane to modulate ADAM17 activity in inflammatory diseases. ADAM17 cleaves and releases epidermal growth factor (EGF) ligands from the cell surface. The shed EGF ligands then bind to the EGF receptors to drive embryonic development. Therefore, our findings also suggest that use of ceftolozane during pregnancy may inhibit ADAM17-mediated shedding of EGF and thus increase the risk of birth defects in humans. Communicated by Ramaswamy H. Sarma

The protease ADAM17 at the crossroads of disease: revisiting its significance in inflammation, cancer, and beyond.

The protease A Disintegrin And Metalloproteinase 17 (ADAM17) plays a central role in the pathophysiology of several diseases. ADAM17 is involved in the cleavage and shedding of at least 80 known membrane-tethered proteins, which subsequently modulate several intracellular signaling pathways, and therefore alter cell behavior. Dysregulated expression and/or activation of ADAM17 has been linked to a wide range of autoimmune and inflammatory diseases, cancer, and cardiovascular disease. In this review, we provide an overview of the current state of knowledge from preclinical models and clinical data on the diverse pathophysiological roles of ADAM17, and discuss the mechanisms underlying ADAM17-mediated protein shedding and the potential therapeutic implications of targeting ADAM17 in these diseases.

ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

Defective efferocytosis by aged macrophages promotes STING signaling mediated inflammatory liver injury

Aged livers have shown aggravated liver ischemia and reperfusion (IR) injury. Timely efferocytosis of apoptotic cells is a key mechanism for avoiding excessive inflammation and tissue injury. Here, we investigated the alteration of efferocytosis by aged macrophages and its role in regulating macrophage STING (stimulator of interferon genes) signaling and liver IR injury. Aged and young mice were subjected to liver partial IR model. Liver injury and inflammation were measured. Efferocytosis by aged macrophages and the underlying regulatory mechanism were analyzed as well. Aged macrophages exhibited impaired efferocytosis with decreased MerTK (c-mer proto-oncogene tyrosine kinase) activation, which was reversed by treatment of the MerTK CRISPR activation plasmid. Increased MerTK cleavage by ADAM17 (a disintegrin and metalloproteinase 17) due to enhanced ROS (reactive oxygen species) levels contributed to defective efferocytosis by aged macrophages. MerTK activation by suppressing ADAM17 or ROS improved aged macrophage efferocytosis, leading to reduced inflammatory liver injury. Moreover, increased apoptotic hepatocytes, DNA accumulation, and macrophage STING activation were observed in aged ischemic livers. Improvement in efferocytosis by aged macrophages via MerTK activation suppressed STING activation and inflammatory liver injury. Our study demonstrates that aging suppresses MerTK- mediated macrophage efferocytosis to promote macrophage STING activation and inflammatory liver IR injury, suggesting a new mechanism and potential therapy to promote inflammation resolution and efferocytosis in aged livers.

Novel structured ADAM17 small-molecule inhibitor represses ADAM17/Notch pathway activation and the NSCLC cells' resistance to anti-tumour drugs.

Background and aims: The outcomes of current treatment for non-small cell lung cancer (NSCLC) are unsatisfactory and development of new and more efficacious therapeutic strategies are required. The Notch pathway, which is necessary for cell survival to avert apoptosis, induces the resistance of cancer cells to antitumour drugs. Notch pathway activation is controlled by the cleavage of Notch proteins/receptors mediated by A disintegrin and metalloproteinase 17 (ADAM17); therefore, ADAM17 is a reliable intervention target for anti-tumour therapy to overcome the drug resistance of cancer cells. This work aims to develop and elucidate the activation of Compound 2b, a novel-structured small-molecule inhibitor of ADAM17, which was designed and developed and its therapeutic efficacy in NSCLC was assessed via multi-assays. Methods and results: A lead compound for a potential inhibitor of ADAM17 was explored via pharmacophore modelling, molecular docking, and biochemical screening. It was augmented by substituting two important chemical groups [R1 and R2 of the quinoxaline-2,3-diamine (its chemical skeleton)]; subsequently, serial homologs of the lead compound were used to obtain anoptimized compound (2b) with high inhibitory activity compared with leading compound against ADAM17 to inhibit the cleavage of Notch proteins and the accumulation of the Notch intracellular domain in the nuclei of NSCLC cells. The inhibitory activity of compound 2b was demonstrated by quantitative polymerase chain reaction and Western blotting. The specificity of compound 2b on ADAM17 was confirmed via point-mutation. Compound 2b enhanced the activation of antitumor drugs on NSCLC cells, in cell lines and nude mice models, by targeting the ADAM17/Notch pathway. Conclusion: Compound 2b may be a promising strategy for NSCLC treatment.

Evaluation of Cisplatin-Induced Acute Renal Failure Amelioration Using Fondaparinux and Alteplase.

Acute renal failure (ARF) is a deleterious condition with increased mortality or healthcare costs or dialysis-dependent end-stage renal disease. The study aims to compare prophylaxis with fondaparinux (Fund) vs. treatment with alteplase (Alt) in ameliorating cisplatin (Cis)-induced ARF. Sixty male mice were equally divided randomly into six groups of control, Cis, Alt, and Cis + Alt groups receiving normal saline for 10 days. All four groups except for the control received Cis (30 mg/kg, i.p.) on day 7, and 6 h later, both the Alt groups received Alt (0.9 mg/kg, i.v.). The animal groups Fund and Fund + Cis received Fund (5 mg/kg, i.p.) for 10 days, and the Fund + Cis group on day 7 received Cis. All the animal groups were euthanized 72 h after the Cis dose. The Fund + Cis group showed significantly increased expression levels of platelet count, retinoid X receptor alpha (RXR-α) and phosphorylated Akt (p-Akt) in addition to decreased levels of urea, blood urea nitrogen (BUN), uric acid, white blood cells (WBCs), red blood cells (RBCs), relative kidney body weight, kidney injury score, glucose, prothrombin (PT), A Disintegrin And Metalloproteinases-10 (ADAM10), extracellular matrix deposition, protease-activated receptor 2 (PAR-2), and fibrinogen expression when compared to the Cis-only group. Meanwhile, the Cis + Alt group showed increased caspase-3 expression in addition to decreased levels of urea, BUN, uric acid, WBCs, RBCs, glucose, platelet count and PT expression with a marked decrease in PAR-2 protein expression compared to the Cis group. The creatinine levels for both the Fund + Cis and Cis + Alt groups were found to be comparable to those of the Cis-only group. The results demonstrate that the coagulation system's activation through the stimulation of PAR-2 and fibrinogen due to Cis-induced ADAM10 protein expression mediated the apoptotic pathway, as indicated by caspase-3 expression through the p-Akt pathway. This is normally accompanied by the loss of RXR-α distal and proximal tubules as lipid droplets. When the animals were pre-treated with the anticoagulant, Fund, the previous deleterious effect was halted while the fibrinolytic agent, Alt, most of the time failed to treat Cis-induced toxicity.

ADAM17 aggravates the inflammatory response by modulating microglia polarization through the TGF-β1/Smad pathway following experimental traumatic brain injury.

Microglia-mediated neuroinflammatory responses play important roles in secondary neurological injury after traumatic brain injury (TBI). The TGF-β pathway participates in the regulation of M1/M2 phenotype transformation of microglia. TGF-β can activate the Smad pathway by binding to TGF-βRs, which is regulated by the cleavage function of A disintegrin and metalloproteinase 17 (ADAM17). However, the role of ADAM17 and the associated signaling pathways in the pathological process after TBI remain unclear. Herein, we assessed the transformation of microglia M1/M2 phenotype polarization and the neuroinflammatory response after the inhibition of ADAM17. The formation of TGF-βRs and TGF-β1/TGF-βRII complexes on microglia were detected to evaluate the effect of ADAM17 inhibition on the TGF-β1/Smad pathway. ADAM17 was highly expressed after TBI and mainly located in the microglia. the inhibition of ADAM17 improved neurological function after TBI. The neuroprotective effect of ADAM17 inhibition was related to a shift from the M1 microglial phenotype to the M2 microglial phenotype, thus reducing TBI-induced neuroinflammation. ADAM17 inhibition increased expression of TGF-βRs on the microglia membrane, promoted formation of TGF-β1/TGF-βRII complexes, and induced intranuclear translocation of Smads, which activated the TGF-β/Smad pathway. In conclusion, our study suggested that ADAM17 inhibition regulated microglia M1/M2 phenotype polarization through the TGF-β1/Smad pathway and influenced the neuroinflammatory response after TBI.

Neuraminidase causes endothelial phosphatidylserine externalization and ADAM17 activation: implications for shedding of insulin receptor and endothelial insulin resistance

Endothelial insulin resistance represents a causal factor in the pathogenesis of type 2 diabetes (T2D) and vascular disease, thus underscoring the need to identify molecular mechanisms underlying defects in endothelial insulin signaling. We previously showed that a disintegrin and metalloproteinase-17 (ADAM17) is increased while insulin receptor α-subunit (IRα) is decreased in the vasculature of patients with T2D, leading to impaired insulin-induced vasodilation. We also showed that ADAM17 is a sheddase that targets IRα. Externalization of phosphatidylserine (PS) from the inner to the outer leaflet of cell membranes has been proposed as a pivotal step in the activation of ADAM17. However, whether PS externalization increases ADAM17 sheddase activity and, consequently, shedding of endothelial IRα in T2D is not known. Herein, we hypothesized that neuraminidase (Neu), an enzyme we showed is upregulated in diabetes, leads to PS externalization via activation of Ca2+ influx and subsequent induction of ADAM17 sheddase activity. All reported differences are significant at P&lt;0.05. In support of this hypothesis, we report that Neu and ADAM17 activities are increased in plasma of individuals with T2D, relative to non-T2D subjects. In experiments using cultured endothelial cells, we show that exposure to Neu increases Ca2+ influx and ADAM17 activity. We also find that augmenting endothelial Ca 2+ influx using a calcium ionophore (A23187) increases adhesion of lactadherin (a peptide that binds externalized PS) to the surface of endothelial cells, as does treatment with the ADAM17 activator, PMA, which also reduces IRα on the cells. Importantly, overexpression of ADAM17 in endothelial cells reduces IRα in cell lysates and this coincides with a tendency towards increased IRα in the cell culture supernatant, indicative of protein shedding. Collectively, these findings suggest that Neu causes Ca 2+ influx in endothelial cells, favoring PS externalization and, consequently, increased ADAM17 activity and shedding of IRα. Hence, inhibition of Neu and ADAM17 sheddase activities should be considered as novel potential strategies to improve endothelial insulin signaling in T2D. R01HL151384 (to LAML and JP), R01HL153264 (to LAML and JP), and R01HL137769 (to JP). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phosphatidylserine feeding reduces the presence of glycocalyx components glypican-1 and CD44 in the aortic wall of diabetic mice

The vascular glycocalyx, in particular, that which protrudes from the endothelium into the vascular lumen, is an important player in multiple functions of blood vessels. The glycocalyx is a mesh of glycoproteins and proteoglycans present on the outer layer of the cellular plasmalemma. In endothelial cells, the glycocalyx participates in the process of mechanotransduction that converts shear stress forces generated by the flow of blood into intracellular signals that produce functional molecules such as the potent vasodilator, nitric oxide. The glycocalyx is in a constant remodeling process of degradation and synthesis. A major participant in its degradation is a disintegrin and metalloproteinase-17 (ADAM17), a proteolytic cell-anchored enzyme that sheds diverse components of the glycocalyx from the cell surface. ADAM17 is also a proinflammatory enzyme that is upregulated in multiple disease conditions including obesity and diabetes. As the endothelial glycocalyx has been shown to be reduced in such conditions, we hypothesized that ADAM17 inhibition would increase the presence of vascular glycocalyx structures in diabetes. To test this hypothesis while improving the translatability of our study, we fed the dietary supplement and competitive inhibitor of ADAM17, phosphatidylserine, to 9-week-old diabetic db/db mice (n=10) for 4 weeks. Peanut butter vehicle control was fed to 10 db/db mice and an additional 10 wild-type untreated mice served as reference controls. All differences reported here are significant at P&lt;0.05. Contrary to our expectations, neither plasma ADAM17 activity nor its presence in the aortic wall was different between db/db mice vs wild-types or between phosphatidylserine-treated vs. untreated db/db mice. Immunohistochemistry analyses of aortic samples further indicated that compared to wild-types, db/db mice had increased presence of the glycocalyx components glypican-1 and CD44 and that these glycocalyx components were lesser in aortas from db/db mice treated with phosphatidylserine than in those from db/db mice fed vehicle. As for the presence of the major extracellular matrices collagen and elastin, treatment with phosphatidylserine reduced collagen-to-elastin ratios in the aortas of db/db mice. All these results suggest that dietary supplementation with phosphatidylserine decreases the expression of arterial glycocalyx components in diabetic mice. The implications of these specific vascular structural changes on cardiovascular pathophysiology remain to be fully elucidated. National Institutes of Health grant: R01HL153264 to LM-L and JP This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.