ADAM10 Research Articles

MicroRNA-574-3p inhibits the malignant behavior of liver cancer cells by targeting ADAM28.

Liver cancer is one of the most common and aggressive tumors, and usually leads to a poor clinical outcome. Increasing evidence has demonstrated the important functions of microRNAs (miRs) in tumor progression. miR-574-3p has been reported as a tumor suppressor and potential therapeutic target in various types of cancer. However, the underlying mechanism of the effects of miR-574-3p in liver cancer remains unknown. In the present study, reverse transcription-quantitative PCR was performed to detect miR-574-3p expression in liver cancer tissues, and the influence of miR-574-3p on cell growth was evaluated using the Cell Counting Kit-8 assay, and cell migration and flow cytometry analyses. The present study revealed that miR-574-3p expression was downregulated in liver cancer tissues and cell lines. miR-574-3p overexpression, achieved by transfecting miR-574-3p mimics into liver cancer cells, reduced cell proliferation and migration, and promoted cell apoptosis. Mechanistically, ADAM metallopeptidase domain 28 (ADAM28) was identified as a miR-574-3p target via binding to the 3'-untranslated region of the ADAM28 mRNA. Gain-of-function of miR-574-3p downregulated the expression levels of ADAM28 in liver cancer cells. Additionally, overexpression of ADAM28 significantly attenuated the suppressive effect of miR-574-3p on the growth of liver cancer cells. The present results provide novel insights into the function of the miR-574-3p/ADAM28 signaling pathway in liver cancer.

Cooperative binding of the tandem WW domains of PLEKHA7 to PDZD11 promotes conformation-dependent interaction with tetraspanin 33

Pleckstrin homology domain–containing A7 (PLEKHA7) is a cytoplasmic protein at adherens junctions that has been implicated in hypertension, glaucoma, and responses to Staphylococcus aureus α-toxin. Complex formation between PLEKHA7, PDZ domain–containing 11 (PDZD11), tetraspanin 33, and the α-toxin receptor ADAM metallopeptidase domain 10 (ADAM10) promotes junctional clustering of ADAM10 and α-toxin–mediated pore formation. However, how the N-terminal region of PDZD11 interacts with the N-terminal tandem WW domains of PLEKHA7 and how this interaction promotes tetraspanin 33 binding to the WW1 domain is unclear. Here, we used site-directed mutagenesis, glutathione S-transferase pulldown experiments, immunofluorescence, molecular modeling, and docking experiments to characterize the mechanisms driving these interactions. We found that Asp-30 of WW1 and His-75 of WW2 interact through a hydrogen bond and, together with Thr-35 of WW1, form a binding pocket that accommodates a polyproline stretch within the N-terminal PDZD11 region. By strengthening the interactions of the ternary complex, the WW2 domain stabilized the WW1 domain and cooperatively promoted the interaction with PDZD11. Modeling results indicated that, in turn, PDZD11 binding induces a conformational rearrangement, which strengthens the ternary complex, and contributes to enlarging a “hydrophobic hot spot” region on the WW1 domain. The last two lipophilic residues of tetraspanin 33, Trp-283 and Tyr-282, were required for its interaction with PLEKHA7. Docking of the tetraspanin 33 C terminus revealed that it fits into the hydrophobic hot spot region of the accessible surface of WW1. We conclude that communication between the two tandem WW domains of PLEKHA7 and the PLEKHA7–PDZD11 interaction modulate the ligand-binding properties of PLEKHA7.

OxLDL promotes podocyte migration by regulating CXCL16, ADAM10 and ACTN4.

Nephrotic syndrome (NS) is one of the most common causes of chronic kidney disease in the pediatric population. Hyperlipidemia is one of the main features of NS. The present study investigated the role of CXC motif chemokine ligand 16 (CXCL16) and ADAM metallopeptidase domain 10 (ADAM10) in oxidized low-density lipoprotein (oxLDL)-stimualted podocytes and the underlying mechanisms. CXCL16 and ADAM10 expression levels in oxLDL-treated podocytes were measured via reverse transcription-quantitative PCR and western blotting. Cell migration assays were conducted to assess the migration of oxLDL-treated podocytes. CXCL16 or ADAM10 overexpression and knockdown assays were conducted. The results indicated that oxLDL stimulation increased ADAM10 and CXCL16 expression levels, and enhanced podocyte migration compared with the control group. Moreover, CXCL16 and ADAM10 overexpression significantly increased podocyte migration and the expression of actinin-α4 (ACTN4) compared with the control groups. By contrast, CXCL16 and ADAM10 knockdown significantly reduced podocyte migration and the expression of ACTN4 compared with the control groups. The results suggested that oxLDL promoted podocyte migration by regulating CXCL16 and ADAM10 expression, as well as by modulating the actin cytoskeleton. Therefore, CXCL16 and ADAM10 may serve as novel therapeutic targets for primary nephrotic syndrome in children.

Differentiation of NSC-34 cells is characterized by expression of NGF receptor p75, glutaminase and NCAM L1, activation of mitochondria, and sensitivity to fatty acid intervention

Motor neuronal damage due to diseases, traumatic insults or de-afferentation of the spinal cord is often incurable because of poor intrinsic regenerative capacity. Hence, medical basic research has to provide a better understanding of development-/regeneration-related cellular processes as only way to develop new and successful therapeutic strategies. Here, we investigated the neuronal differentiation of the NSC-34 hybrid cell line, which is an accepted model for spinal cord motor neurons. Their differentiation was stimulated by switching from normal to differentiation medium and by supplementation with palmitic and oleic acid. To characterize neuro-differentiation of NSC-34 cells, expression of nicotinic acetylcholine receptor alpha 4, NGF p75 receptor, IGF I alpha receptor, glutaminase, NCAM L1, ADAM10 and myelin basic protein as well as activation of mitochondria were analyzed. Both switch from normal to differentiation medium and fatty acid application stimulated NSC-34 differentiation. Differentiation was characterized by diminishing expression of the nicotinic acetylcholine receptor alpha 4 and enhancing expression of the NGF receptor p75, of glutaminase, of NCAM L1 and it’s partially transformation from the cell surface into the cell. Fatty acid intervention stabilized the expression of the nicotinic acetylcholine receptor alpha 4, diminished the expression of the NGF receptor p75, consolidated the expression profile of NCAM L1, and intensified the expression of the relevant for NCAM L1 cleavage ADAM10. However, NCAM L1 cleavage itself was unaffected by fatty acid intervention, as was the differentiation-relevant activation of mitochondria and their transformation into neuronal filopodia.

SO026HUMAN KIDNEY PROXIMAL TUBULAR 3D SPHEROIDS TO STUDY THE ROLE OF ADAM17 IN DIABETIC NEPHROPATHY

Abstract Background and Aims ADAM17 is a disintegrin and metalloproteinase initially described to cleave the tumor necrosis factor α (TNFα). Currently, it is known that it can also release ectodomains of a diverse variety of molecules such as, transforming growth factor α (TGFα), L-selectin, and angiotensin-converting enzyme 2 (ACE2). It has been shown that ADAM17 protein expression increases in kidney mesangial cells after incubation with high glucose media mimicking what has been observed in diabetic patients and experimental models of diabetic nephropathy. We now studied the effect ADAM17 deletion on human kidney cells (HKC-8) in a 3D spheroids in vitro cell culture incubated with high glucose, low glucose and mannitol medium resembling the in vivo human kidney diabetic environment. Method ADAM17 deletion was performed using the CRISPR/Cas9 technology. HKC8 cells grew inside a RGD-functionalized dextran hydrogel to obtain 3D spheroids. 13 days post-seeding, the spheroids were incubated with 35mM of D-glucose (HG), 5mM of D-glucose (LG) or 35mM of mannitol as osmotic control for 6h, 24h or 72h. The quality of the established 3D cell culture of mature HKC-8 spheroids was assessed by Aquoporin-1 and Glut-1 staining. After incubations quantitative-PCR analyses were performed for fibrotic and inflammatory markers. Immunofluorescence for fibrotic markers was performed on HKC-8 spheroids incubated for 72h. Results High glucose (HG) medium induced CCL5 gene expression on wild-type HKC-8 spheroids after 6h and 24h of incubation in comparison with the control group. Interestingly, in the ADAM17-deleted spheroids, CCL5 gene expression maintained similar to control after 6h of incubation with HG medium and tended to decrease after 24h of incubation in comparison with the wild-type. Collagen IV gene expression was increased in the wild-type spheroids incubated with HG in comparison with the control group. In ADAM17-deleted spheroids, Collagen IV gene expression was significantly decreased in the cells incubated with HG in comparison with the wild-type cells incubated with HG. HG increased the expression of α-SMA, fibronectin and Collagen IV in wild-type spheroids. Adam17 deletion blocked the increase of α-SMA, fibronectin and Collagen IV expression compared with wild-type cells after 72h of incubation. Conclusion ADAM17 blockade protects against fibrosis and inflammation in human kidney tubular spheroids under high glucose.

Macrolets: Outsized Extracellular Vesicles Released from Lipopolysaccharide-Stimulated Macrophages that Trap and Kill Escherichia coli.

SummaryMacrophages release a variety of extracellular vesicles (EVs). Here we describe a previously unreported class of EVs that are released from macrophages in response to Escherichia coli endotoxin, lipopolysaccharide (LPS), that we have named "macrolets" since they are extruded as large "droplets" released from macrophages. Morphologically, macrolets are anuclear, bounded by a single lipid membrane and structurally dependent on an actin cytoskeleton. Macrolets are enriched in tetraspanins and separable on this basis from their parent macrophages. Macrolets are distinguished from classic exosomes by their larger size (10–30 μm), discoid shape, and the presence of organelles. Macrolets are rich in both interleukin 6 (IL-6) and interleukin 6 receptor (IL-6R),and are capable of trapping and killing E. coli in association with production of reactive oxygen species. Our observations offer insights into the mechanisms by which macrophage activities may be amplified in sites of infection, inflammation, and healing.

ADAM10 and ADAM17 proteases mediate proinflammatory cytokine-induced and constitutive cleavage of endomucin from the endothelial surface

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte-EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.

Combination of pembrolizumab and 125I attenuates the aggressiveness of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated mortality. Therapies targeting programmed cell death 1 ligand 1 (PD1L1) have promising effects on NSCLC. However, resistance to targeted therapy has become the main problem and the underling molecular mechanism remains unclear. In the present study, the expression of PD1L1 in NSCLC was determined and the association with clinicopathological characteristics was analyzed. A combination therapy was also constructed, including pembrolizumab (Pem) and iodine-125 (125I), which represented an efficient strategy for the treatment of NSCLC. The expression of PD1L1 was upregulated in NSCLC tissues and positively correlated with the Ki-67 index, pathological subtypes and risk stages. A higher level of PD1L1 expression was associated with poorer survival in patients with NSCLC, which could be used as a prognostic indicator. When NSCLC cells were cultured in the presence of Pem and 125I seeds, the combination treatment significantly abrogated the tumor proliferation and aggressiveness through the inhibition of matrix metalloproteinase-2 and -9 secretion. Flow cytometry analysis revealed pembrolizumab combined with 125I contributed to a higher rate of apoptosis and cell cycle arrest, indicating that the combination treatment improved the resistance to immunotherapy. Furthermore, the associated molecular mechanism was the dysregulation of ADAM metallopeptidase domain 17. The findings from the present study revealed that PD1L1 could be used as a predictive biomarker, and the application of combination treatment of pembrolizumab and 125I showed promising effects on NSCLC.

MiR-20a inhibits the progression of human arthritis fibroblast-like synoviocytes and inflammatory factor expression by targeting ADAM10.

MiR-20a has been reported as a key regulator to pro-inflammatory factor release in fibroblast-like synoviocytes (FLS), which caused rheumatoid arthritis (RA). However, the molecular mechanism of miR-20a in RA remains to be further elucidated. This study aimed to investigate the roles of miR-20a in RA pathology. RA (n = 24) and osteoarthritis (OA, n = 20) and normal healthy tissues (n = 16) were collected from operation. TargetScan and dual-luciferase reporter were performed to predict and confirm the potential binding sites of miR-20a on ADAM metallopeptidase domain 10 (ADAM10). Pearson's analysis was adopted to evaluate the correlation between miR-20a and ADAM10 expression. It was found that MiR-20a was downregulated in RA tissues, and overexpressed miR-20a inhibited cell viability, migration and invasion, and the expression of inflammatory factors in RA-FLS MH7A cells. ADAM10 was identified as the target gene of miR-20a, and upregulation of ADAM10 reversed the inhibitory effects of miR-20a. In conclusion, miR-20a inhibits the progression of RA-FLS as well as the inflammatory factor expression by targeting ADAM10.

Effect of miR-26a-5p targeting ADAM17 gene on apoptosis, inflammatory factors and oxidative stress response of myocardial cells in hypoxic model.

The aim of this study was to explore the effect of miR-26a-5p targeting and regulating ADAM17 gene on myocardial cells in hypoxic model. Myocardial cells from 1 day old Sprague-Dawley rats were isolated and cultured for 3 days, and were used for experiment. The hypoxia model of myocardial cells was established after cell grouping transfection. The targeting relationship between miR-26a-5p and ADAM17 was verified by bioinformatics website prediction and double luciferase report experiment. The double luciferase report experiment showed that miR-26a-5p had a targeted relationship with ADAM17, and miR-26a-5p could target and bind ADAM17, down-regulate its expression, and the transfection efficiency of each group was good (P < 0.05). After overexpression of miR-26a-5p, cell activity was increased (P < 0.05), apoptosis was decreased (P < 0.05), and the expression levels of TNF-α, IL-1β and IL-6 were significantly decreased (all P < 0.05). The release of creatine kinase-MB and the expression level of malondialdehyde were significantly decreased (both P < 0.05), and the expression level of superoxide dismutase was significantly increased (all P < 0.05). After overexpression of ADAM17, the results were reversed (all P < 0.05). MiR-26a-5p could target and regulate ADAM17, reduce the apoptosis of myocardial cells and the expression of inflammatory factors in acute myocardial infarction, and reduce the occurrence of oxidative stress.

ADAM12 silencing promotes cellular apoptosis by activating autophagy in choriocarcinoma cells.

ADAM metallopeptidase domain 12 (ADAM12) has been demonstrated to mediate cell proliferation and apoptosis resistance in several types of cancer cells. However, the effect of ADAM12 silencing on the proliferation and apoptosis of choriocarcinoma cells remains unknown. The present study revealed that ADAM12 silencing significantly inhibited cellular activity and proliferation in the human choriocarcinoma JEG3 cell line and increased the rate of apoptosis. In addition, ADAM12 silencing significantly increased the expression levels of the autophagy proteins microtubule-associated protein-light-chain 3 (LC3B) and autophagy related 5 (ATG5) and the fluorescence density of LC3B in JEG-3 cells. However, the suppression of autophagy by 3-methyladenine could block ADAM12 silencing-induced cellular apoptosis. ADAM12 silencing reduced the levels of the inflammatory factors interleukin-1β, interferon-γ and TNF-α, and inactivated nuclear p65-NF-κB and p-mTOR in JEG-3 cells. The downregulation of p-mTOR expression by ADAM12 silencing was rescued in 3-methyladenine-treated JEG-3 cells, indicating that mTOR might participate in the autophagy-mediated pro-apoptotic effect of ADAM12 silencing. In conclusion, ADAM12 silencing promoted cellular apoptosis in human choriocarcinoma JEG3 cells, which might be associated with autophagy and the mTOR response. These findings indicate that ADAM12 silencing might be a potential novel therapeutic target for choriocarcinoma.

ADAM17 Genetic Variants and the Response of TNF-α Inhibitor in Rheumatoid Arthritis Patients.

PurposeTNF-α is a transmembrane protein which requires cleavage by ADAM17 in order to act systemically. The activation of ADAM17 to generate soluble TNF‑α results in an increased inflammatory activity. We hypothesized that variants spanning the ADAM17 gene contribute towards the observed variation in patient response defined by the number of changes in TNF‑α inhibitors.Patients and MethodsSeven single-nucleotide polymorphisms (SNPs) of ADAM17 in 63 patients with rheumatoid arthritis who received TNF-α inhibitors were analyzed: rs57467365, rs62117540, rs117645314, rs6432013, rs532704607, rs117179141, and rs12692386. Univariate and multivariate regression analysis were employed to investigate the independent predictable factors for changes in TNF-α inhibitors.ResultsADAM17 rs117645314 and rs117179141 showed significant association with the number of changes in TNF-α inhibitors. Patients with GA in rs117645314 and AT in rs117179141 had significantly higher chance of two or more changes of TNF-α inhibitors than those with wild homozygous alleles. Multivariate analysis showed that rs117179141 explained 5.7% of the 23.8% variability in TNF-α inhibitor response.ConclusionThis study showed that the number of changes in TNF-α inhibitor is associated with ADAM17 SNPs.

Chronic Mild Stress Modified Epigenetic Mechanisms Leading to Accelerated Senescence and Impaired Cognitive Performance in Mice.

Cognitive and behavioural disturbances are a growing public healthcare issue for the modern society, as stressful lifestyle is becoming more and more common. Besides, several pieces of evidence state that environment is crucial in the development of several diseases as well as compromising healthy aging. Therefore, it is important to study the effects of stress on cognition and its relationship with aging. To address these queries, Chronic Mild Stress (CMS) paradigm was used in the senescence-accelerated mouse prone 8 (SAMP8) and resistant 1 (SAMR1). On one hand, we determined the changes produced in the three main epigenetic marks after 4 weeks of CMS treatment, such as a reduction in histone posttranslational modifications and DNA methylation, and up-regulation or down-regulation of several miRNA involved in different cellular processes in mice. In addition, CMS treatment induced reactive oxygen species (ROS) damage accumulation and loss of antioxidant defence mechanisms, as well as inflammatory signalling activation through NF-κB pathway and astrogliosis markers, like Gfap. Remarkably, CMS altered mTORC1 signalling in both strains, decreasing autophagy only in SAMR1 mice. We found a decrease in glycogen synthase kinase 3 β (GSK-3β) inactivation, hyperphosphorylation of Tau and an increase in sAPPβ protein levels in mice under CMS. Moreover, reduction in the non-amyloidogenic secretase ADAM10 protein levels was found in SAMR1 CMS group. Consequently, detrimental effects on behaviour and cognitive performance were detected in CMS treated mice, affecting mainly SAMR1 mice, promoting a turning to SAMP8 phenotype. In conclusion, CMS is a feasible intervention to understand the influence of stress on epigenetic mechanisms underlying cognition and accelerating senescence.

Protein secondary structure determines the temporal relationship between folding and disulfide formation

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, β2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

Development of flow cytometry assays for measuring cell-membrane enzyme activity on individual cells

Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable.Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes.Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells.Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.

Impaired Autophagy in the Fibroblasts by Titanium Particles Increased the Release of CX3CL1 and Promoted the Chemotactic Migration of Monocytes.

Aseptic loosening (AL) is the most frequent cause of failure of total hip arthroplasties (THA). Prosthetic wear particle-induced monocyte recruitment to the periprosthetic tissue and subsequent inflammatory response are thought to be the major contribution to AL. Fibroblast is a dominant cell type in interfacial membrane (IFM) which is the main pathological feature of periprosthetic osteolysis in failed THAs. Considering the role of fibroblasts, as sentinel cells, in the synthesis of chemokines and regulation of inflammation, we hypothesize that fibroblasts might be involved in the monocyte recruitment in the pathogenesis of periprosthetic osteolysis associated with particle debris. This study explored the induction of fibroblasts on the monocyte recruitment. The results showed that titanium (Ti) particle-stimulated fibroblasts isolated from IFMs of loosened THAs significantly promoted the chemotactic migration of THP-1 cells by increasing the release of CX3CL1 (C-X3-C motif chemokine ligand 1). Further investigation demonstrated that Ti particle stimulation increased the expression of ADAM10 (ADAM metallopeptidase domain 10) by impairing autophagy in the fibroblasts and in turn increased the cleavage and shedding of CX3CL1. Thus, we propose a new insight to the pathogenesis of aseptic loosening which implies that autophagy-ADAM10-CX3CL1 signaling pathway in fibroblasts can be leveraged to alleviating inflammation caused by monocyte recruitment in aseptic loosening and improving performance of articulation of the joint device.

Macrophage MerTK Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis

Nonalcoholic steatohepatitis (NASH) is emerging as a leading cause of chronic liver disease. However, therapeutic options are limited by incomplete understanding of the mechanisms of NASH fibrosis, which is mediated by activation of hepatic stellate cells (HSCs). In humans, human genetic studies have shown that hypomorphic variations in MERTK, encoding the macrophage c-mer tyrosine kinase (MerTK) receptor, provide protection against liver fibrosis, but the mechanisms remain unknown. We now show that holo- or myeloid-specific Mertk targeting in NASH mice decreases liver fibrosis, congruent with the human genetic data. Furthermore, ADAM metallopeptidase domain 17 (ADAM17)-mediated MerTK cleavage in liver macrophages decreases during steatosis to NASH transition, and mice with a cleavage-resistant MerTK mutant have increased NASH fibrosis. Macrophage MerTK promotes an ERK-TGFβ1 pathway that activates HSCs and induces liver fibrosis. These data provide insights into the role of liver macrophages in NASH fibrosis and provide a plausible mechanism underlying MERTK as a genetic risk factor for NASH fibrosis.

Methylomic profiling in trisomy 21 identifies cognition- and Alzheimer\u2019s disease-related dysregulation

BackgroundTrisomy 21 (T21) is associated with intellectual disability that ranges from mild to profound with an average intellectual quotient of around 50. Furthermore, T21 patients have a high risk of developing Alzheimer’s disease (AD) early in life, characterized by the presence of senile plaques of amyloid protein and neurofibrillary tangles, leading to neuronal loss and cognitive decline. We postulate that epigenetic factors contribute to the observed variability in intellectual disability, as well as at the level of neurodegeneration seen in T21 individuals.Materials and MethodsA genome-wide DNA methylation study was performed using Illumina Infinium® MethylationEPIC BeadChips on whole blood DNA of 3 male T21 patients with low IQ, 8 T21 patients with high IQ (4 males and 4 females), and 21 age- and sex-matched control samples (12 males and 9 females) in order to determine whether DNA methylation alterations could help explain variation in cognitive impairment between individuals with T21. In view of the increased risk of developing AD in T21 individuals, we additionally investigated the T21-associated sites in published blood DNA methylation data from the AgeCoDe cohort (German study on Ageing, Cognition, and Dementia). AgeCoDe represents a prospective longitudinal study including non-demented individuals at baseline of which a part develops AD dementia at follow-up.ResultsTwo thousand seven hundred sixteen differentially methylated sites and regions discriminating T21 and healthy individuals were identified. In the T21 high and low IQ comparison, a single CpG located in the promoter of PELI1 was differentially methylated after multiple testing adjustment. For the same contrast, 69 differentially methylated regions were identified. Performing a targeted association analysis for the significant T21-associated CpG sites in the AgeCoDe cohort, we found that 9 showed significant methylation differences related to AD dementia, including one in the ADAM10 gene. This gene has previously been shown to play a role in the prevention of amyloid plaque formation in the brain.ConclusionThe differentially methylated regions may help understand the interaction between methylation alterations and cognitive function. In addition, ADAM10 might be a valuable blood-based biomarker for at least the early detection of AD.

Effects of ADAM17 siRNA and doxorubicin on proliferation and apoptosis of prostate cancer PC-3 cells

Objective To investigate the effects of ADAM17 siRNA combined with adriamycin (ADM) on proliferation and apoptosis of human prostate cancer cell line PC-3. Methods PC-3 cells stably transfected with ADAM17 siRNA expression plasmid were treated with ADM. The proliferation rate was observed by MTT colorimetry, the apoptosis rate was detected by acridine orange staining and flow cytometry (FCM), and the activity of apoptotic protein caspase-3 was analyzed. Results Both ADAM17 siRNA and ADM alone and in combination could decrease the proliferation rate and increase the apoptosis rate of PC-3 cells in varying degrees. FCM data showed that the apoptosis rates of ADAM17 siRNA and ADM alone were(6.2±1.4)% and (17.2±1.9)% respectively, which were significantly different from (36.4±3.7)% of ADAM17 siRNA and ADM alone (P<0.01). Both of them also had a good synergistic effect on inducing caspase-3 activity. Conclusions ADAM17 siRNA and ADM have different effects on the proliferation and apoptosis of human prostate cancer PC-3, but the combination of ADAM17 siRNA and ADM is more effective. It is suggested that gene therapy combined with chemotherapy may be an important method to improve the curative effect and reduce the risk of recurrence and metastasis of prostate cancer. Key words: Prostatic Neoplasms; Cells, Cultured; RNA, Small Interfering; Doxorubicin; Cell Proliferation; Apoptosis

Involvement of ADAM10 in acrolein-induced astrocytic inflammation

Acrolein is a neurotoxin produced through lipid peroxidation in the brain affected by ischemic stroke, which results in neuronal cell injury and inflammation. However the mechanism underlying acrolein-induced brain inflammation remains unclear. Therefore we examined how acrolein leads to astrocytic inflammation. It was found that acrolein increased the levels of NLRP3 and cleaved caspase-1, which led to the maturation of interleukin-1β (IL-1β). ELISA assay results, which showed that acrolein increased the secreted IL-1β, further supported acrolein-induced astrocytic inflammation. Acrolein increased ADAM10 protein levels and the cleavage of N-cadherin. The ADAM10 inhibitor, GI 254023X blocked N-cadherin cleavage by acrolein, suggesting that ADAM10 is an upstream of N-cadherin. Furthermore, we found that acrolein activated p38 MAPK and NF-κB p65, while pretreatment with p38 MAPK inhibitor, SB203580 and GI 254023X inhibited NF-κB p65 activation and NLRP3 inflammasome. This suggests that p38 MAPK mediates the activation of NF-κB p65, which is associated with NLRP3 expression. Finally, we showed that acrolein induced cell toxicity and decrease of EAAT1 expression, suggesting that acrolein may induce a loss of glutamate uptake function. In conclusion, we demonstrate that acrolein induces astrocytic inflammation through NLRP3 inflammasome, which is regulated by ADAM10 and attributed to p38 MAPK-activated NF-κB p65 activity.