ADAM10 Expression Research Articles

Alternatively Spliced Isoforms of MUC4 and ADAM12 as Biomarkers for Colorectal Cancer Metastasis.

There is a pertinent need to develop prognostic biomarkers for practicing predictive, preventive and personalized medicine (PPPM) in colorectal cancer metastasis. The analysis of isoform expression data governed by alternative splicing provides a high-resolution picture of mRNAs in a defined condition. This information would not be available by studying gene expression changes alone. Hence, we utilized our prior data from an exon microarray and found ADAM12 and MUC4 to be strong biomarker candidates based on their alternative splicing scores and pattern. In this study, we characterized their isoform expression in a cell line model of metastatic colorectal cancer (SW480 & SW620). These two genes were found to be good prognostic indicators in two cohorts from The Cancer Genome Atlas database. We studied their exon structure using sequence information in the NCBI and ENSEMBL genome databases to amplify and validate six isoforms each for the ADAM12 and MUC4 genes. The differential expression of these isoforms was observed between normal, primary and metastatic colorectal cancer cell lines. RNA-Seq analysis further proved the differential expression of the gene isoforms. The isoforms of MUC4 and ADAM12 were found to change expression levels in response to 5-Fluorouracil (5-FU) treatment in a dose-, time- and cell line-dependent manner. Furthermore, we successfully detected the protein isoforms of ADAM12 and MUC4 in cell lysates, reflecting the differential expression at the protein level. The change in the mRNA and protein expression of MUC4 and ADAM12 in primary and metastatic cells and in response to 5-FU qualifies them to be studied as potential biomarkers. This comprehensive study underscores the importance of studying alternatively spliced isoforms and their potential use as prognostic and/or predictive biomarkers in the PPPM approach towards cancer.

Neuroprotective effect of quercetin through targeting key genes involved in aluminum chloride induced Alzheimer’s disease in rats

ABSTRACT Alzheimer’s disease (AD) is a neurodegenerative disorder that is clinically characterized by deteriorating cognitive function. Quercetin (Q), a bioflavonoid, has been reported to slow down AD progression. Q at a dose of 50 mg/kg-1 shows an important therapeutic effect in aluminum chloride (AlCl3)-induced Alzheimer’s disease, which has been previously published by us. Here, this study aimed to highlight the neuroprotective effect of quercetin on hallmark genes in AlCl3-induced Alzheimer’s disease in rats. Wistar male rats were subjected to a vehicle group, AlCl3 group, and co-administration with AlCl3 + Q50 for 60 sequential days. Behavioral tests and qPCR were performed to assess the efficacy of Q. The co-administration of quercetin (50 mg kg-1) has a significant effect on memory deficits. Furthermore, AlCl3 + Q50 group resulted in significantly decreased amyloid precursor protein levels (APP), β-amyloid converting enzyme 1 (BACE1), and presenilin I (PSEN1) and increased the expression of ADAM17 in the hippocampus tissue compared to AlCl3 group (p < 0.05). The current study showed that the quercetin’s neuroprotective properties may involve its ability to target the most significant Alzheimer’s disease-related genes and slow the progression of cognitive impairment.

A Bioengineering Strategy to Control ADAM10 Activity in Living Cells.

A Disintegrin and Metalloprotease 10, also known as ADAM10, is a cell surface protease ubiquitously expressed in mammalian cells where it cuts several membrane proteins implicated in multiple physiological processes. The dysregulation of ADAM10 expression and function has been implicated in pathological conditions, including Alzheimer's disease (AD). Although it has been suggested that ADAM10 is expressed as a zymogen and the removal of the prodomain results in its activation, other potential mechanisms for the ADAM10 proteolytic function and activation remain unclear. Another suggested mechanism is post-translational modification of the cytoplasmic domain, which regulates ADAM10-dependent protein ectodomain shedding. Therefore, the precise and temporal activation of ADAM10 is highly desirable to reveal the fine details of ADAM10-mediated cleavage mechanisms and protease-dependent therapeutic applications. Here, we present a strategy to control prodomain and cytosolic tail cleavage to regulate ADAM10 shedding activity without the intervention of small endogenous molecule signaling pathways. We generated a series of engineered ADAM10 analogs containing Tobacco Etch Virus protease (TEV) cleavage site (TEVcs), rendering ADAM10 cleavable by TEV. This strategy revealed that, in the absence of other stimuli, the TEV-mediated removal of the prodomain could not activate ADAM10. However, the TEV-mediated cleavage of the cytosolic domain significantly increased ADAM10 activity. Then, we generated ADAM10 with a minimal constitutively catalytic activity that increased significantly in the presence of TEV or after activating a chemically activatable TEV. Our results revealed a bioengineering strategy for controlling the ADAM10 activity in living cells, paving the way to obtain spatiotemporal control of ADAM10. Finally, we proved that our approach of controlling ADAM10 promoted α-secretase activity and the non-amyloidogenic cleavage of amyloid-β precursor protein (APP), thereby increasing the production of the neuroprotective soluble ectodomain (sAPPα). Our bioengineering strategy has the potential to be exploited as a next-generation gene therapy for AD.

Differential expression of "A Disintegrin and Metalloproteinase 10" in hepatocellular carcinoma and the noncancerous hepatic tissues: Contribution to HCV-promoted hepatocarcinogenesis.

A disintegrin and metalloproteinases (ADAMs) have emerged as therapeutic targets in many cancers. ADAM10 was particularly studied in hepatocellular carcinoma (HCC) for its potential role in hepatocarcinogenesis and HCC progression. To investigate the immunohistochemical (IHC) expression of ADAM10 in HCCs and the adjacent noncancerous tissues from 70 HCC patients, attempting to elucidate any association between ADAM10 and HCC development and/or progression. IHC staining for anti-ADAM10 was performed using horseradish peroxidase technique. An extent and intensity-dependent scoring was applied dividing samples into high- and low-expression groups. HCCs were statistically compared in relation with gender, age, cirrhosis, hepatitis C virus (HCV) status, alpha-fetoprotein (AFP) serum level, tumor size, multiplicity, encapsulation/invasion, grade, histological pattern and variant, mitosis, necrosis, vascular emboli, portal thrombosis, stage, recurrence, and mortality. Kaplan-Meier's method was used to analyze disease-free and overall survival (DFS and OS). ADAM10 was expressed in 77.1% of HCCs compared with 42.9% of noncancerous tissues. Differential expression showed significant statistical difference (P = 0.02), as 38.6% of HCCs showed high expression, whereas 92.8% of noncancerous samples showed low expression. No significant differences were observed when high- and low-ADAM10 expression HCCs were compared with respect to all tested prognostic parameters except the HCV status. Patients whose tumors showed high-ADAM10 expression had relatively longer DFS and OS times, but with insignificant log-rank differences. ADAM10 is frequently expressed in HCCs compared with noncancerous hepatic tissues suggesting its role in hepatocarcinogenesis, especially in association with HCV. It has no association with HCC progression or survival. Further studies should be sought to investigate its validity as a therapeutic target.

S-39-3: ENDOTHELIAL INFLAMMATION AND DYSFUNCTION INDUCED BY SARS-COV-2 SPIKE PROTEIN 1 INVOLVE ADAM17 AND INTERFERON ACTIVATION PATHWAYS

Objective: COVID19 is associated with vascular inflammation. IFN-alpha (IFNα) and IFN-lambda3 (IFNλ3) are potent cytokines produced in viral infections. Their effects involve interferon-stimulated genes (ISGs) and may influence expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. We hypothesized that S1P-induced immune/inflammatory responses in endothelial cells (EC) are mediated via IFN-activated pathways Design and methods: Human ECs were stimulated with S1P (1 μg/mL), IFNα (100ng/mL) or IFNλ3 (100IU/mL). Because ACE2, ADAM17 and TMPRSS2 are important for SARS-CoV-2 infection, we used inhibitors of ADAM17 (marimastat, 3.8 nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50 μM). Gene and protein expression was investigated by real-time PCR and immunoblotting, respectively. Vascular function was assessed in mesenteric arteries from wild-type (WT) normotensive and hypertensive (LinA3) mice and in ISG15-deficient (ISG15KO) mice. Results: S1P increased expression of IFNα (3-fold), IFNλ3 (4-fold) and ISGs (2-fold) in EC (p &lt; 0.05). EC responses to IFNα (ISG15: 16-fold) were greater than to IFNλ3 (ISG15: 1.7-fold) (p &lt; 0.05). S1P increased gene expression of IL-6 (1.3-fold), TNFα (6.2-fold) and IL-1β (3.3-fold), effects that were amplified by IFNs. Only the ADAM17 inhibitor marimastat inhibited S1P effects. IFNα and IFNλ3 increase protein expression of ADAM17 (27%) and TMPRSS2 (38%). No changes were observed on ACE2 expression. This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). EC production of IL-6 was increased by IFNα (1,230pg/mL) and IFNλ3 (1,124pg/mL) vs control (591pg/mL). Nitric oxide generation and eNOS phosphorylation (Ser1177) were reduced by IFNα (40%) and IFNλ3 (40%). Vascular functional responses demonstrated that endothelium-dependent vasorelaxation (% Emax) in vessels from WT-mice stimulated with IFNα (67%) and IFNλ3 (71%) were reduced vs control (82%) (p &lt; 0.05). Responses were not altered in vessels from ISG15KO mice. Increased contraction was observed only in vessels from hypertensive mice treated with IFNα (9.1 ± 0.5mN vs control: 7.3 ± 0.3mN) (p &lt; 0.05). Conclusions: In ECs, S1P, IFNα and IFNλ3 increased ISG15 and IL-6 by mechanisms dependent on ADAM17. IFNs amplifies endothelial cell inflammatory responses and induced vascular dysfunction through ISG15-dependent mechanisms, with augmented effects in hypertension. Our findings demonstrate that S1P induces immune/inflammatory responses that may be important in endotheliitis associated with COVID-19. This may be especially important in the presence of cardiovascular risk factors, including hypertension.

MiR-4299 inhibits tumor progression in pancreatic cancer through targeting ADAM17.

Pancreatic cancer (PC) is one of the most aggressive malignant tumors in human beings. Tumor capacity of evading immune-mediated lysis is a critical step in PC malignant progression. We aimed to evaluate the underlying regulatory mechanism of miR-4299 in the proliferation, metastasis, apoptosis, and immune escape in PC. miR-4299 and ADAM17 expressions in PC tissues and cell lines were detected using qRT-PCR. MTT assay and flow cytometry were used to detect cell viability and apoptosis, respectively. A luciferase reporter gene assay was conducted to confirm the targeted relationship between miR-4299 and ADAM17. Xenograft tumors in nude mice were used to detect tumorigenesis in vivo. PC cells were co-cultured with NK cells for determining the immune escape ability. NKG2D-positive rate of NK cells was detected using flow cytometry; NK cell-killing ability was detected using MTT assay. miR-4299 was downregulated in PC tissues and cell lines. miR-4299 inhibited PC cell proliferation and invasion, promoted cell apoptosis, and reduced PC tumor growth in vivo. ADAM17 3'UTR directly bound to miR-4299. ADAM17 overexpression could reverse miR-4299 effects on PC cell viability, invasion, apoptosis, and immune escape. miR-4299 exerted suppressive effects on PC cell proliferation, invasion, and immune escape via targeting ADAM17 expression. This study revealed a novel miR-4299/ADAM17 axis-modulating PC progression and proposed to concern the immune regulatory mechanism of miRNAs in PC development.

Association of Inherited Copy Number Variation in ADAM3A and ADAM5 Pseudogenes with Oropharynx Cancer Risk and Outcome.

Inherited copy number variations (CNVs) can provide valuable information for cancer susceptibility and prognosis. However, their association with oropharynx squamous cell carcinoma (OPSCC) is still poorly studied. Using microarrays analysis, we identified three inherited CNVs associated with OPSCC risk, of which one was validated in 152 OPSCC patients and 155 controls and related to pseudogene-microRNA-mRNA interaction. Individuals with three or more copies of ADAM3A and ADAM5 pseudogenes (8p11.22 chromosome region) were under 6.49-fold increased risk of OPSCC. ADAM5 shared a highly homologous sequence with the ADAM9 3'-UTR, predicted to be a binding site for miR-122b-5p. Individuals carrying more than three copies of ADAM3A and ADAM5 presented higher ADAM9 expression levels. Moreover, patients with total deletion or one copy of pseudogenes and with higher expression of miR-122b-5p presented worse prognoses. Our data suggest, for the first time, that ADAM3A and ADAM5 pseudogene-inherited CNV could modulate OPSCC occurrence and prognosis, possibly through the interaction of ADAM5 pseudogene transcript, miR-122b-5p, and ADAM9.

Antiviral Potential of Small Molecules Cordycepin, Thymoquinone, and N6, N6-Dimethyladenosine Targeting SARS-CoV-2 Entry Protein ADAM17.

COVID-19 is an acute respiratory disease caused by SARS-CoV-2 that has spawned a worldwide pandemic. ADAM17 is a sheddase associated with the modulation of the receptor ACE2 of SARS-CoV-2. Studies have revealed that malignant phenotypes of several cancer types are closely relevant to highly expressed ADAM17. However, ADAM17 regulation in SARS-CoV-2 invasion and its role on small molecules are unclear. Here, we evaluated the ADAM17 inhibitory effects of cordycepin (CD), thymoquinone (TQ), and N6, N6-dimethyladenosine (m62A), on cancer cells and predicted the anti-COVID-19 potential of the three compounds and their underlying signaling pathways by network pharmacology. It was found that CD, TQ, and m62A repressed the ADAM17 expression upon different cancer cells remarkably. Moreover, CD inhibited GFP-positive syncytia formation significantly, suggesting its potential against SARS-CoV-2. Pharmacological analysis by constructing CD-, TQ-, and m62A-based drug-target COVID-19 networks further indicated that ADAM17 is a potential target for anti-COVID-19 therapy with these compounds, and the mechanism might be relevant to viral infection and transmembrane receptors-mediated signal transduction. These findings imply that ADAM17 is of potentially medical significance for cancer patients infected with SARS-CoV-2, which provides potential new targets and insights for developing innovative drugs against COVID-19.

ADAM12 abrogation alters immune cell infiltration and improves response to checkpoint blockade therapy in the T11 murine model of triple-negative breast cancer

ABSTRACT Immunosuppressive tumor microenvironment (TME) impedes anti-tumor immune responses and contributes to immunotherapy resistance in triple-negative breast cancer (TNBC). ADAM12, a member of cell surface metalloproteases, is selectively upregulated in mesenchymal/claudin-low TNBCs, where its expression is largely restricted to tumor cells. The role of cancer cell-expressed ADAM12 in modulating the immune TME is not known. We show that Adam12 knockout in the T11 mouse syngeneic transplantation model of claudin-low TNBC leads to decreased numbers of tumor-infiltrating neutrophils (TINs)/polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and increased numbers of tumor-infiltrating B cells and T cells. ADAM12 loss in cancer cells increases chemotaxis of B cells in vitro and this effect is eliminated by inhibition of CXCR4, a receptor for CXCL12, or anti-CXCL12 blocking antibody. Importantly, ADAM12 loss in T11 cancer cells sensitizes tumors to anti-PD1/anti-CTLA4 combination therapy, although the initial responsiveness is followed by acquired therapy resistance. Depletion of B cells in mice eliminates the improved response to immune checkpoint blockade of Adam12 knockout T11 tumors. Analysis of gene expression data for claudin-low TNBCs from the METABRIC patient cohort shows significant inverse correlations between ADAM12 and gene expression signatures of several anti-tumor immune cell populations, as well as a significant positive correlation between ADAM12 and gene expression signature of TINs/PMN-MDSCs. Collectively, these results implicate ADAM12 in immunosuppression within the TME in TNBC.

Characterization of pulmonary vascular remodeling and MicroRNA-126-targets in COPD-pulmonary hypertension

BackgroundDespite causing increased morbidity and mortality, pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) patients (COPD-PH) lacks treatment, due to incomplete understanding of its pathogenesis. Hypertrophy of pulmonary arterial walls and pruning of the microvasculature with loss of capillary beds are known features of pulmonary vascular remodeling in COPD. The remodeling features of pulmonary medium- and smaller vessels in COPD-PH lungs are less well described and may be linked to maladaptation of endothelial cells to chronic cigarette smoking (CS). MicroRNA-126 (miR126), a master regulator of endothelial cell fate, has divergent functions that are vessel-size specific, supporting the survival of large vessel endothelial cells and inhibiting the proliferation of microvascular endothelial cells. Since CS decreases miR126 in microvascular lung endothelial cells, we set out to characterize the remodeling by pulmonary vascular size in COPD-PH and its relationship with miR126 in COPD and COPD-PH lungs.MethodsDeidentified lung tissue was obtained from individuals with COPD with and without PH and from non-diseased non-smokers and smokers. Pulmonary artery remodeling was assessed by ⍺-smooth muscle actin (SMA) abundance via immunohistochemistry and analyzed by pulmonary artery size. miR126 and miR126-target abundance were quantified by qPCR. The expression levels of ceramide, ADAM9, and endothelial cell marker CD31 were assessed by immunofluorescence.ResultsPulmonary arteries from COPD and COPD-PH lungs had significantly increased SMA abundance compared to non-COPD lungs, especially in small pulmonary arteries and the lung microvasculature. This was accompanied by significantly fewer endothelial cell markers and increased pro-apoptotic ceramide abundance. miR126 expression was significantly decreased in lungs of COPD individuals. Of the targets tested (SPRED1, VEGF, LAT1, ADAM9), lung miR126 most significantly inversely correlated with ADAM9 expression. Compared to controls, ADAM9 was significantly increased in COPD and COPD-PH lungs, predominantly in small pulmonary arteries and lung microvasculature.ConclusionBoth COPD and COPD-PH lungs exhibited significant remodeling of the pulmonary vascular bed of small and microvascular size, suggesting these changes may occur before or independent of the clinical development of PH. Decreased miR126 expression with reciprocal increase in ADAM9 may regulate endothelial cell survival and vascular remodeling in small pulmonary arteries and lung microvasculature in COPD and COPD-PH.

Unfavorable immunotherapy plus tyrosine kinase inhibition outcome of metastatic renal cell carcinoma after radical nephrectomy with increased ADAM9 expression.

Immunotherapy plus tyrosine kinase inhibitor (IO-TKI) has become the standard first-line therapy for advanced renal cell carcinoma (RCC). However, the modest response rate of IO-TKI therapy and the absence of biomarkers limited the selection of treatment strategies for RCC patients. There were three cohorts enrolled: two from our facility (ZS-MRCC and ZS-HRRCC) and one from a clinical study (JAVELIN-101). By RNA sequencing, the expression of ADAM9in each sample was measured. By flow cytometry and immunohistochemistry, immune infiltration and T cell function were examined. Primary outcomes were established as treatmentresponse and progression-free survival (PFS). Patients with low-ADAM9 expression had a higher objective response rate (56.5% vs 13.6%, P = 0.01) and longer PFS in both cohorts. In the ZS-HRRCC cohort, the expression of ADAM9 was associated with increased tumor-infiltrating T cells, which was proved by immunohistochemistry (P < 0.05) and flow cytometry (Spearman's ρ = 0.42, P < 0.001). In the high-ADAM9 group, CD8+ and CD4+ T cells revealed an exhausted phenotype with decreased GZMB (Spearman's ρ = - 0.31, P = 0.05, and Spearman's ρ = - 0.49, P < 0.001, respectively), and fewer Macrophages were identified. A predictive RFscore was further constructed by random forest approach, involving ADAM9 and immunologic genes. Only in the subgroup with the lower RFscore did IO-TKI outperform TKI monotherapy. High-ADAM9 expression was associated with immunosuppression and IO-TKI resistance. Expression of ADAM9 was also associated with the exhaustion and dysfunction of T cells. ADAM9-based RFscore has the potential to be used as a biomarker to distinguish the optimal patient treatment methods between IO-TKI and TKI monotherapy.

MicroRNA-149 Regulates Proliferation, Migration, and Invasion of Pituitary Adenoma Cells by Targeting ADAM12 and MMP14

ObjectivePituitary adenomas (PAs) can adapt an aggressive phenotype by invading adjacent brain structures with rapid cellular proliferation. Previous studies demonstrated that excessive expression of metalloproteases ADAM12 and MMP-14 is instrumental for the active proliferation and invasiveness of PA cells in vitro and of tumors in vivo. However, the mechanisms regulating ADAM12 and MMP-14 expression in PAs remain unclear.MethodsTarget gene prediction and transcriptomic profiling of invasive vs. noninvasive human PA samples were performed to identify miRNA species potentially involved in the regulation of ADAM12 and MMP14. For cellular analyses of miRNA functions, two mouse PA cell lines (AtT20 and TtT/GF) were transfected with miR-149-3p and miR-149-5p, respectively. The effects of miR-149 (3p and 5p) on expression levels of ADAM12 and MMP14 were determined by Western blotting followed by an analysis of proliferation and colony formation assays, scratch migration assays, and invasion assays.ResultsA significant downregulation of miRNA-149 was observed in invasive vs. noninvasive PA (0.32 vs. 0.09, P<0.0001). In AtT-20 and TtT/GF mouse PAs cells, transfection of mimic miRNA-149 (3p and 5p) caused a significantly reduced cell proliferation and matrigel invasion, whilst the effect on cell migration was less pronounced. Both strands of miRNA-149 (3p and 5p) markedly reduced protein levels of ADAM12 and MMP-14 by at least 40% in both cell lines.ConclusionThis study proved that the invasiveness of PA cells is, at least partly, regulated by miRNA-149-dependent expression of ADAM12 and MMP-14.

ADAM10: Possible functions in enamel development.

ADAM10 is A Disintegrin And Metalloproteinase (ADAM) family member that is membrane bound with its catalytic domain present on the cell surface. It is a sheddase that cleaves anchored cell surface proteins to shed them from the cell surface. ADAM10 can cleave at least a hundred different proteins and is expressed in most tissues of the body. ADAM10 is best characterized for its role in Notch signaling. Interestingly, ADAM10 is transported to specific sites on the cell surface by six different tetraspanins. Although the mechanism is not clear, tetraspanins can regulate ADAM10 substrate specificity, which likely contributes to the diversity of ADAM10 substrates. In developing mouse teeth, ADAM10 is expressed in the stem cell niche and subsequently in pre-ameloblasts and then secretory stage ameloblasts. However, once ameloblasts begin transitioning into the maturation stage, ADAM10 expression abruptly ceases. This is exactly when ameloblasts stop their movement that extends enamel crystallites and when the enamel layer reaches its full thickness. ADAM10 may play an important role in enamel development. ADAM10 can cleave cadherins and other cell-cell junctions at specific sites where the tetraspanins have transported it and this may promote cell movement. ADAM10 can also cleave the transmembrane proteins COL17A1 and RELT. When either COL17A1 or RELT are mutated, malformed enamel may occur in humans and mice. So, ADAM10 may also regulate these proteins that are necessary for proper enamel development. This mini review will highlight ADAM10 function, how that function is regulated by tetraspanins, and how ADAM10 may promote enamel formation.

Effects of Subtoxic Concentrations of Atrazine, Cypermethrin, and Vinclozolin on microRNA-Mediated PI3K/Akt/mTOR Signaling in SH-SY5Y Cells.

Endocrine-disrupting chemicals (EDCs) are different natural and synthetic chemicals that may interfere with several mechanisms of the endocrine system producing adverse developmental, metabolic, reproductive, and neurological effects in both human beings and wildlife. Among pesticides, numerous chemicals have been identified as EDCs. MicroRNAs (miRNAs) can regulate gene expression, making fine adjustments in mRNA abundance and regulating proteostasis. We hypothesized that exposure to low doses of atrazine, cypermethrin, and vinclozolin may lead to effects on miRNA expression in SH-SY5Y cells. In particular, the exposure of SH-SY5Y cells to subtoxic concentrations of vinclozolin is able to downregulate miR-29b-3p expression leading to the increase in the related gene expression of ADAM12 and CDK6, which may promote a pro-oncogenic response through the activation of the PI3K/Akt/mTOR pathway and counteracting p53 activity. A better understanding of the molecular mechanisms of EDCs could provide important insight into their role in human disease.

Does Vitamin D Work Synergistically with Anti-Asthmatic Drugs in Airway Remodeling?

Vitamin D is commonly known for its properties of airway remodeling inhibition. Due to this, we decided to analyze the action of calcitriol with anti-asthmatic drugs in airway remodeling. The HFL1 cell line was treated with calcitriol, beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β in different combinations. Real-time PCR was used to analyzed the expression of ACTA2, CDH-1, Vimentin, ADAM33, MMP-9 and CysLTR1 on the mRNA level, whereas Western blot was used to analyze gene expression on the protein level. One-way analysis variants, the Kruskal-Wallis test, Student's t-test or Welch's t-test were used for statistical analysis. Concerning the results, pre-treatment with calcitriol increased the inhibitory effect of beclomethasone 17-propionate and montelukast sodium on the expression of ACTA2 (p = 0.0072), Vimentin (p = 0.0002) and CysLTR1 (p = 0.0204), and 1,25(OH)2D3 had an influence on the effects of beclomethasone 17-propionate and montelukast sodium and of CDH1 expression (p = 0.0076). On the protein level, pre-treatment with calcitriol with beclomethasone 17-propionate and montelukast sodium treatment decreased ACTA2 expression in comparison to the LT (LTD4 and TGF-β) control group (p = 0.0191). Hence, our study not only confirms that vitamin D may inhibit airway remodeling, but also shows that vitamin D has a synergistic effect with anti-asthmatic drugs.

Dysregulated Cell-Cell Communication Characterizes Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of older adults characterized by fibrotic replacement of functional gas exchange units in the lung. The strongest risk factor for IPF is a genetic variantin the promoter region of the gel-forming mucin, MUC5B. To better understand how the MUC5B variant influences development of fibrosis, we used the NicheNet R package and leveraged publicly available single-cell RNA sequencing data to identify and evaluate how epithelia participating in gas exchange are influenced by ligands expressed in control, MUC5B variant, and fibrotic environments. We observed that loss of type-I alveolar epithelia (AECI) characterizes the single-cell RNA transcriptome in fibrotic lung and validated the pattern of AECI loss using single nuclear RNA sequencing. Examining AECI transcriptomes, we found enrichment of transcriptional signatures for IL6 and AREG, which we have previously shown to mediate aberrant epithelial fluidization in IPF and murine bleomycin models. Moreover, we found that the protease ADAM17, which is upstream of IL6 trans-signaling, was enriched in control MUC5B variant donors. We used immunofluorescence to validate a role for enhanced expression of ADAM17 among MUC5B variants, suggesting involvement in IPF pathogenesis and maintenance.

ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

A disintegrin and metalloproteinase (ADAM) family proteins, acting as sheddases, are important factors in a number of pathologies, including cancer, and have been suggested as promising therapeutic targets. The study presented focuses on the involvement of ADAM10 and ADAM17 in retinoblastoma (RB), the most common malignant intraocular childhood tumor. A significant correlation between ADAM17 expression levels and RB laterality and RB staging was observed. Levels of ADAM10 or ADAM17 regulating miRNAs miR-145, -152, and -365 were significantly downregulated in RB cell lines, and reduced miR levels with simultaneously upregulated ADAM10 and ADAM17 expression were found in RB patients. The involvement of both ADAMs analyzed in ectodomain shedding of the neuronal cell adhesion molecule L1 (L1CAM), shown to induce pro-tumorigenic effects in RB, was confirmed. Lentiviral ADAM10 and ADAM17 single or ADAM10/17 double knockdown (KD) induced caspase-dependent apoptosis and reduced cell viability, proliferation, growth, and colony formation capacity of RB cells. Moreover, differential phosphorylation of the serine/threonine kinase AKT was observed following ADAM17 KD in RB cells. Chicken chorioallantoic membrane (CAM) assays revealed that ADAM17 and ADAM10/17 depletion decreases the tumorigenic and migration potential of RB cells in vivo. Thus, ADAMs are potential novel targets for future therapeutic RB approaches.

The GBM Tumor Microenvironment as a Modulator of Therapy Response: ADAM8 Causes Tumor Infiltration of Tams through HB-EGF/EGFR-Mediated CCL2 Expression and Overcomes TMZ Chemosensitization in Glioblastoma.

Simple SummaryResistance to standard therapies impose a huge challenge on the treatment for glioblastoma multiforme (GBM), which is often considered as a cell intrinsic property of either GBM or, more significantly, of GBM stem-like cells. Tumor-associated macrophages and microglia (TAMs) take up the majority of the immune population in the tumor microenvironment of GBM and potentially participating in modulating therapy responses. However, little is known about the mechanisms underlying the effect of TAMs on temozolomide (TMZ) induced chemoresistance. Members of the metzincin superfamily such as Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloprotease (ADAM) proteases are important participants in the process of intercellular communications in the tumor microenvironment. Herein, we revealed a novel concept of an intra-tumoral ADAM8 mediated malignant positive feedback loop constituted by the intimate interaction of tumor associated macrophages (TAMs) and GBM cells under TMZ treatment. These findings provide a convincing example and further support the notion that the tumor microenvironment, in addition to GBM cells and GBM stem-like cells, should be considered as an essential modulator of therapy in GBM. In conclusion, our study provides a rational basis for TAM sparing ADAM8-targeting in GBM to optimize standard chemotherapy.Standard chemotherapy of Glioblastoma multiforme (GBM) using temozolomide (TMZ) frequently fails due to acquired chemoresistance. Tumor-associated macrophages and microglia (TAMs) as major immune cell population in the tumor microenvironment are potential modulators of TMZ response. However; little is known about how TAMs participate in TMZ induced chemoresistance. Members of the metzincin superfamily such as Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloprotease (ADAM) proteases are important mediators of cellular communication in the tumor microenvironment. A qPCR screening was performed to identify potential targets within the ADAM and MMP family members in GBM cells. In co-culture with macrophages ADAM8 was the only signature gene up-regulated in GBM cells induced by macrophages under TMZ treatment. The relationship between ADAM8 expression and TAM infiltration in GBM was determined in a patient cohort by qPCR; IF; and IHC staining and TCGA data analysis. Moreover; RNA-seq was carried out to identify the potential targets regulated by ADAM8. CCL2 expression levels were determined by qPCR; Western blot; IF; and ELISA. Utilizing qPCR; IF; and IHC staining; we observed a positive relationship between ADAM8 expression and TAMs infiltration level in GBM patient tissues. Furthermore; ADAM8 induced TAMs recruitment in vitro and in vivo. Mechanistically; we revealed that ADAM8 activated HB-EGF/EGFR signaling and subsequently up-regulated production of CCL2 in GBM cells in the presence of TMZ treatment; promoting TAMs recruitment; which further induced ADAM8 expression in GBM cells to mediate TMZ chemoresistance. Thus; we revealed an ADAM8 dependent positive feedback loop between TAMs and GBM cells under TMZ treatment which involves CCL2 and EGFR signaling to cause TMZ resistance in GBM.

NSUN2-mediated mRNA m5C Modification Regulates the Progression of Hepatocellular Carcinoma

RNA modifications affect many biological processes and physiological diseases. The 5-methylcytosine (m5C) modification regulates the progression of multiple tumors. However, its characteristics and functions in hepatocellular carcinoma (HCC) remain largely unknown. Here, we found that HCC tissues had a higher m5C methylation level than the adjacent normal tissues. Transcriptome analysis revealed that the hypermethylated genes mainly participated in the phosphokinase signaling pathways, such as the Ras and PI3K-Akt pathways. The m5C methyltransferase NSUN2 was highly expressed in HCC tissues. Interestingly, the expression of many genes was positively correlated with the expression of NSUN2, including GRB2, RNF115, AATF, ADAM15, RTN3, and HDGF. Real-time PCR assays further revealed that the expression of the mRNAs of GRB2, RNF115, and AATF decreased significantly with the down-regulation of NSUN2 expression in HCC cells. Furthermore, NSUN2 could regulate the cellular sensitivity of HCC cells to sorafenib via modulating the Ras signaling pathway. Moreover, knocking down NSUN2 caused cell cycle arrest. Taken together, our study demonstrates the vital role of NSUN2 in the progression of HCC.

Pulmonary Delivery of Levamisole Nanoparticles as an Immunomodulator Affecting Th and a Potential ADAM10 Inhibitor to Ameliorate Severe Allergic Asthma.

Asthma is a common chronic lung disease without absolute treatment, and hypersensitivity reactions and type 2 immune responses are responsible for asthma pathophysiology. ADAM10 as a metalloproteinase transmembrane protein is critical for development of Th2 responses, and levamisole as an anthelmintic drug has immunomodulatory effects, which not only regulates ADAM10 activity but also can suppress the bone marrow and neutrophil production. Therefore, in the present study, nanoparticles were used as a levamisole delivery system to reduce bone marrow suppression, and the immunomodulatory and ADAM10 inhibitory effects of levamisole were studied in allergic asthma. Asthmatic mice were treated with PLGA-levamisole nanoparticles. Then, AHR, BALF, and blood cell counts, levels of the IgG1 subclass, total and OVA-specific IgE, IL2, IL-4, IL-5, IL-10, IL-13, IL-17, IL-25, IL-33, INF-γ, and TNF-α, gene expression of FoxP3, T-bet, RORγt, PU.1, GATA3, FcεRII, CysLT1R, eotaxin, and ADAM10, and lung histopathology were evaluated. PLGA-LMHCl with considered characteristics could control airway hyper-responsiveness, eosinophils in the BALF, levels of immunoglobulins, Th2-, Th9-, and Th17-derived cytokines and pivotal genes, eosinophilic inflammation, hyperplasia of the goblet cell, and hyperproduction of mucus and could increase Th1- and Treg-derived cytokines and also pivotal genes. It could also modulate the ADAM10 activity and had no effect on the number of neutrophils in the bloodstream. The novel safe nanodrug had no side effect on the bone marrow to produce neutrophils and could control the allegro-immuno-inflammatory response of asthma.