Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening

In this paper we describe novel fluorescent substrates for the human ADAM family members ADAM17, ADAM10, ADAM8, and ADAM12 that have good specificity constants and are useful for high-throughput screening of inhibitors. The fluorescence resonance energy transfer substrates contain a 4-(4-dimethylaminophenylazo)benzoyl and 5-carboxyfluorescein (Dabcyl/Fam) pair and are based on known cleavage sequences in precursor tumor necrosis factor-alpha (TNF-alpha) and CD23. The precursor TNF-alpha-based substrate, Dabcyl-Leu-Ala-Gln-Ala-Homophe-Arg-Ser-Lys(Fam)-NH2, is a good substrate for all the ADAMs tested, including ADAM12 for which there is no reported fluorescent substrate. The CD23-based substrate, Dabcyl-His-Gly-Asp-Gln-Met-Ala-Gln-Lys-Ser-Lys(Fam)-NH2, is more selective, being hydrolyzed efficiently only by ADAM8 and ADAM10. The substrates were used to obtain inhibition constants for four inhibitors that are commonly used in shedding assays: TMI-1, GM6001, GW9471, and TAPI-2. The Wyeth Aerst compound, TMI-1, is a potent inhibitor against all of the ADAMs tested and is slow binding against ADAM17.     

Identification of ADAM10 as a major TNF sheddase in ADAM17-deficient fibroblasts

ADAM17 (a disintegrin and metalloprotease)-deficient murine fibroblasts stably transfected with proTNF cDNA release significant amounts of biologically active soluble TNF. The enzyme responsible for this activity is a membrane protein that hydrolyzes the peptide bond Ala⁷⁶:Val⁷⁷ within proTNF. Its activity is inhibited by 1,10-phenantroline and GM6001, insusceptible to TIMP-2 (tissue inhibitor of metalloproteinases-2), and stimulated by ionomycin. These characteristics match ADAM10. The moderate silencing of ADAM10 by shRNA resulted in a significant inhibition of TNF shedding. There was no correlation between the level of ADAM10 expression and the presence of active ADAM17. Our results indicate that ADAM10 may function as the TNF sheddase in cells which lack ADAM17 activity.     

Overexpression of CDKN2B is involved in poor gastric cancer prognosis

The objective of this investigation is to elucidate the clinical significance of cyclin-dependent kinase inhibitor 2B (CDKN2B) expression regarding gastric cancer (GC), as well as to detect the involvement of CDKN2B expression in the clinicopathological indexes and prognosis of GC. Immunohistochemical analysis was used for identification of CDKN2B expression in GC specimens. Chi-square (χ²) test was applied to detect the association of CDKN2B expression and clinicopathological parameters of GC. The involvement of CDKN2B expression in the prognosis was analyzed via univariate and multivariate analysis. It was indicated that relative to the corresponding para-carcinoma tissues, CDKN2B expression was notably upregulated in GC specimens. Moreover, the expression of CDKN2B was strongly correlated with the differentiation (r = −0.182; P = .015), invasion (r = −0.157; P = .038), distant metastases (r = −0.196; P = .004), and TNM stage (r = −0.204; P = .005). Nevertheless, no remarkable variance was related to age, tumor loci, or sex. Kaplan-Meier survival curve and univariate analysis showed that CDKN2B overexpression predicted poorer disease-free survival (P = .007) and overall survival (P = .005) in those with GC. In addition, Cox proportional hazards regression model revealed that CDKN2B was an isolated biomarker of disease-free survival and overall survival in patients with GC. Taken together, our data demonstrated that the overexpression of CDKN2B could be an isolated factor for GC prognostic in patients. CDKN2B gene may be a useful target and new treatment for improving the prognosis of GC.     

EGF promotes the shedding of soluble E-cadherin in an ADAM10-dependent manner in prostate epithelial cells

During the progression of prostate cancer, the epithelial adhesion molecule E-cadherin is cleaved from the cell surface by ADAM15 proteolytic processing, generating an extracellular 80 kDa fragment referred to as soluble E-cadherin (sE-cad). Contrary to observations in cancer, the generation of sE-cad appears to correlate with ADAM10 activity in benign prostatic epithelium. The ADAM10-specific inhibitor INCB8765 and the ADAM10 prodomain inhibit the generation of sE-cad, as well as downstream signaling and cell proliferation. Addition of EGF or amphiregulin (AREG) to these untransformed cell lines increases the amount of sE-cad shed into the conditioned media, as well as sE-cad bound to EGFR. EGF-associated shedding appears to be mediated by ADAM10 as shRNA knockdown of ADAM10 results in reduced shedding of sE-cad. To examine the physiologic role of sE-cad on benign prostatic epithelium, we treated BPH-1 and large T immortalized prostate epithelial cells (PrEC) with an sE-cad chimera comprised of the human Fc domain of IgG₁, fused to the extracellular domains of E-cadherin (Fc-Ecad). The treatment of untransformed prostate epithelial cells with Fc-Ecad resulted in phosphorylation of EGFR and downstream signaling through ERK and increased cell proliferation. Pre-treating BPH-1 and PrEC cells with cetuximab, a therapeutic monoclonal antibody against EGFR, decreased the ability of Fc-Ecad to induce EGFR phosphorylation, downstream signaling, and proliferation. These data suggest that ADAM10-generated sE-cad may have a role in EGFR signaling independent of traditional EGFR ligands.     

ADAM10 cleavage of N-cadherin and regulation of cell-cell adhesion and beta-catenin nuclear signalling

Cadherins are critically involved in tissue development and tissue homeostasis. We demonstrate here that neuronal cadherin (N-cadherin) is cleaved specifically by the disintegrin and metalloproteinase ADAM10 in its ectodomain. ADAM10 is not only responsible for the constitutive, but also for the regulated, shedding of this adhesion molecule in fibroblasts and neuronal cells directly regulating the overall levels of N-cadherin expression at the cell surface. The ADAM10-induced N-cadherin cleavage resulted in changes in the adhesive behaviour of cells and also in a dramatic redistribution of beta-catenin from the cell surface to the cytoplasmic pool, thereby influencing the expression of beta-catenin target genes. Our data therefore demonstrate a crucial role of ADAM10 in the regulation of cell-cell adhesion and on beta-catenin signalling, leading to the conclusion that this protease constitutes a central switch in the signalling pathway from N-cadherin at the cell surface to beta-catenin/LEF-1-regulated gene expression in the nucleus.     

ADAM8 expression is associated with increased invasiveness and reduced patient survival in pancreatic cancer

ADAM8 belongs to a family of transmembrane proteins implicated in cell-cell interactions, proteolysis of membrane proteins, and various aspects of carcinogenesis. In the present study, we aimed to evaluate the expression and function of ADAM8 in pancreatic cancer. ADAM8 mRNA levels were analysed by quantitative RT-PCR and correlated to patient survival. Immunohistochemistry was performed to localize ADAM8 in pancreatic tis-sues. Silencing of ADAM8 expression was carried out by transfection with specific siRNA oligonucleotides. Cell growth and invasion assays were used to assess the functional consequences of ADAM8 silencing. SELDI-TOF-MS was performed to detect the proteolytic activity of ADAM8 in pancreatic cancer cells. ADAM8 mRNA was significantly overexpressed in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissues (5.3-fold increase; P= 0.0008), and high ADAM8 mRNA and protein expression levels correlated with reduced survival time of PDAC patients (P= 0.048 and P= 0.065, respectively). Silencing of ADAM8 expression did not significantly influence pancreatic cancer cell growth but suppressed invasiveness. In addition, decreased proteolytic activity was measured in cell culture supernatants following silencing of ADAM8. In conclusion, ADAM8 is overexpressed in PDAC, influences cancer cell invasiveness and correlates with reduced survival, suggesting that ADAM8 might be a potential target in pancreatic cancer therapy.     

Overexpression of sigma-1 receptor inhibits ADAM10 and ADAM17 mediated shedding in vitro

The sigma-1 receptor is a molecular chaperone protein highly enriched in the brain. Recent studies linked it to many diseases, such as drug addition, Alzheimer’s disease, stroke, depression, and even cancer. Sigma-1 receptor is enriched in lipid rafts, which are membrane microdomains essential in signaling processes. One of those signaling processes is ADAM17- and ADAM10-dependent ectodomain shedding. By using an alkaline phosphatase tagged substrate reporter system, we have shown that ADAM10-dependent BTC shedding was very sensitive to both membrane lipid component change and sigma-1 receptor agonist DHEAS treatment while ADAM17-dependent HB-EGF shedding was not; and overexpression of sigma-1 receptor diminished ADAM17- and ADAM10-dependent shedding. Our results indicate that sigma-1 receptor plays an important role in modifying the function of transmembrane proteases.     

Purification of ADAM 10 from bovine spleen as a TNFα convertase

We have purified a protease with characteristics of TNFα convertase from bovine spleen membranes. Peptide sequencing of the purified protein identified it as ADAM 10 (Genbank accession no. Z21961). This metalloprotease cleaves a recombinant proTNFα substrate to mature TNFα, and can cleave a synthetic peptide substrate to yield the mature TNFα amino terminus in vitro. The enzyme is sensitive to a hydroxamate inhibitor of MMPs, but insensitive to phosphoramidon. In addition, cloned ADAM 10 mediates proTNFα processing in a processing-incompetent cell line.     

The Effect of ADAM8 on the Proliferation and Apoptosis of Hepatocytes and Hepatoma Carcinoma Cells

This study was undertaken to evaluate the effect of ADAM8 on the proliferation and apoptosis of hepatocytes and hepatoma carcinoma cells during hepatocellular carcinoma (HCC) progression. The expression of ADAM8 was significantly increased with good correlation of PCNA expression increasing and cells apoptosis decreasing during the progression of HCC in the liver of mice. Proliferation experiment in vitro showed that recombinant ADAM8 could induce the expression of PCNA in L02 cells, but not in HepG2 cells. Apoptosis experiment in vitro showed that recombinant ADAM8 did not induce or inhibit the expression of apoptosis‐related factors Bcl2, Bax, and Caspase3 in L02 cells, but significantly induced the expression of Bcl2, inhibited the expression of Bax and Caspase3 in HepG2 cells. In conclusion, our study suggested that ADAM8 could promote the proliferation of normal hepatocytes and render hepatoma carcinoma cells more resistant to apoptosis to play important roles during the progression of HCC. ADAM8; Proliferation; Apoptosis     

ADAM10 correlates with uveal melanoma metastasis and promotes in vitro invasion

Uveal melanoma (UM) is a rare ocular tumor that may lead to deadly metastases in 50% of patients. A disintegrin and metalloproteinase (ADAM)10, ADAM17, and the HGF‐receptor c‐Met support invasiveness in different tumors. Here, we report that high ADAM10, MET, and, to a lesser extent, ADAM17 gene expression correlates with poor progression‐free survival in UM patients (hazard ratio 2.7, 2.6, and 1.9, respectively). About 60% of primary UM expresses c‐Met and/or ADAM10 proteins. Four UM cell lines display high levels of ADAM10 and ADAM17, which constitutively cleave c‐Met, inducing the release of soluble c‐Met. ADAM10/17 pharmacological inhibition or gene silencing reduces c‐Met shedding, but has limited impact on surface c‐Met, which is overexpressed. Importantly, ADAM10 silencing inhibits UM cell invasion driven by FCS or HGF, while ADAM17 silencing has a limited effect. Altogether our data indicate that ADAM10 has a pro‐invasive role and may contribute to UM progression.     

ADAMl0在胰腺癌患者血清中表达及其临床意义

目的：探讨ADAM10在胰腺癌患者外周血中的表达及其临床意义。方法：应用酶联免疫吸附试验法（ELISA）检测40例胰腺癌患者和20例健康体检者的外周血ADAM10的表达水平,分析其与临床病理特征的关系。结果：胰腺癌患者血清ADAM10水平显著高于正常对照组（P〈0.01）;胰腺癌患者血清中ADAM10的表达水平与胰腺癌淋巴结转移、远处转移及TNM分期密切相关（P〈0.05）,且行根治性手术切除的胰腺癌患者ADAM10表达水平低于姑息性手术切除的患者（P〈0.05）;ADAM10对胰腺癌诊断的敏感度、特异性分别为51.7%、76.9%,联合检测CA19-9有助于提高胰腺癌诊断敏感度,但特异性有所下降;根治性切除后胰腺癌患者血清中ADAM10水平明显下降。结论：胰腺癌癌患者血清中ADAM10水平明显增高,检测血清ADAM10有助于胰腺癌的诊断和治疗。     

The role of CXCL16 and its processing metalloproteinases ADAM10 and ADAM17 in the proliferation and migration of human mesangial cells

In this study, we analyzed the regulation and functional role of CXCL16 in human mesangial cells (hMCs). We can show, that CXCL16 is constitutively expressed in hMCs and is further up-regulated by cytokine mix (IFNγ, TNFα, and IL1β). The constitutive release of CXCL16 from hMCs was rapidly induced by the stimulation with cytokines. We identified ADAM10 and ADAM17 as being responsible for the cytokine-induced shedding of CXCL16. Notably, targeting ADAM10 and ADAM17 in hMCs decreased the chemotaxis of T-Jurkat cells, whereas the inhibition of CXCL16 had no significant influence. This suggests that both proteases are important players in the recruitment of immune cells into the glomerulus, but other substrates than CXCL16 are involved in this process. Finally, we could show that the inhibition of CXCL16, ADAM10, and ADAM17 led to a strong reduction of cell proliferation and migration of hMCs. This finding could be important to develop novel diagnostic and therapeutic strategies to treat mesangial proliferative kidney diseases.     

MiR-126 acts as a tumor suppressor in pancreatic cancer cells via the regulation of ADAM9

The epithelial-mesenchymal transition (EMT) is a critical step for pancreatic cancer cells as an entry of metastatic disease. Wide variety of cytokines and signaling pathways are involved in this complex process while the entire picture is still cryptic. Recently, miRNA was found to regulate cellular function including EMT by targeting multiple mRNAs. We conducted comprehensive analysis of miRNA expression profiles in invasive ductal adenocarcinoma (IDA), intraductal papillary mucinous adenoma, intraductal papillary mucinous carcinoma, and human pancreatic cancer cell line to elucidate essential miRNAs which regulate invasive growth of pancreatic cancer cells. Along with higher expression of miR-21 which has been shown to be highly expressed in IDA, reduced expression of miR-126 in IDA and pancreatic cancer cell line was detected. The miR-126 was found to target ADAM9 (disintegrin and metalloproteinase domain-containing protein 9) which is highly expressed in pancreatic cancer. The direct interaction between miR-126 and ADAM9 mRNA was confirmed by 3' untranslated region assay. Reexpression of miR-126 and siRNA-based knockdown of ADAM9 in pancreatic cancer cells resulted in reduced cellular migration, invasion, and induction of epithelial marker E-cadherin. We showed for the first time that the miR-126/ADAM9 axis plays essential role in the inhibition of invasive growth of pancreatic cancer cells.     

CXCL16 Promotes Gastric Cancer Tumorigenesis via ADAM10-Dependent CXCL16/CXCR6 Axis and Activates Akt and MAPK Signaling Pathways

Abnormal expression of CXC motif chemokine ligand 16 (CXCL16) has been demonstrated to be associated with tumor progression and metastasis, served as a prognostic factor in many cancers, with higher relative expression behaving as a marker of tumor progression. However, its role and mechanisms underlying progression and metastasis of gastric cancer (GC) are yet to be elucidated. In our investigation, public datasets and human GC tissue samples were used to determine the CXCL16 expression levels. Our results revealed that CXCL16 was upregulated in GC. The high expression CXCL16 in GC was significantly associated with histologic poor differentiation and pTNM staging. And high CXCL16 was positively correlated with the poor survival of GC patients. Gain-and loss-of-function experiments were employed to investigate the biological role of CXCL16 in proliferation and migration both in vitro and in vivo. Mechanically, Gene set enrichment analysis (GSEA) revealed that the epithelial‑mesenchymal transition (EMT), Akt and MAPK signal pathway related genes were significantly enriched in the high CXCL16 group, which was confirmed by western blot. Moreover, overexpression CXCL16 promoted the disintegrin and metalloproteases (ADAM10) and the CXC motif chemokine receptor 6 (CXCR6) expression, which mediated the CXCL16/CXCR6 positive feedback loop in GC, with activating Akt and MAPK signaling pathways. Knocking down ADAM10 would interrupted the CXCL16/CXCR6 axis in the carcinogenesis and progression of GC. In conclusion, our findings offered insights into that CXCL16 promoted GC tumorigenesis by enhancing ADAM10-dependent CXCL16/CXCR6 axis activation.     

TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions: EVIDENCE FOR DISTINCT BINDING MECHANISMS FOR DIFFERENT TspanC8 PROTEINS*

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting.     

Retracted: Vinculin promotes gastric cancer proliferation and migration and predicts poor prognosis in patients with gastric cancer

Vinculin is a highly conserved protein involved in cell proliferation, migration, and adhesion. However, the effects of vinculin on gastric cancer (GC) remain unclear. Therefore, we aimed to explore the functional role of vinculin in GC, as well as its underlying mechanism. Expression of vinculin in patients with GC was analyzed by real-time polymerase chain reaction, Western blot analysis, and immunohistochemistry. Overall survival was evaluated by the Kaplan-Meier method with the log-rank test. The relationship between vinculin and clinicopathological characteristics of patients with GC was further identified. In addition, we assessed the expression of vinculin in GC cell lines. Besides, vinculin was suppressed or overexpressed by transfection with small interfering (si-vinculin) or pcDNA-vinculin and then cell viability, cell apoptosis, and/or migration was respectively examined by the 3-(4, 5-dimethylthiazole-2-yl)-2, 5-biphenyl tetrazolium bromide assay, flow cytometer, and scratch assay, respectively. Moreover, the cell cycle- and apoptosis-related proteins were detected by Western blot analysis. The expression of vinculin was significantly increased in the GC tissues and cells compared with the nontumor tissues or cells. Vinculin protein positive staining was mainly located in the cell membrane and cytoplasm. Moreover, vinculin was significantly associated with Tumor Node Metastasis (TNM) and poor differentiation. Patients with high vinculin levels had significantly worse overall survival than those with low levels. Suppression of vinculin significantly decreased cell viability and migration and promoted cell apoptosis. However, overexpression of vinculin statistically increased cell viability but had no effects on cell apoptosis. Vinculin promotes GC proliferation and migration and predicts poor prognosis in patients with GC.     

成年大脑神经细胞特异性ADAM10基因敲除小鼠模型的建立与鉴定

去整合素和金属蛋白酶10(ADAM10)是一种能够水解30余种跨膜蛋白质的“脱落酶”(sheddase), 参与诸多生理过程和致病机制, 如胚胎发育、细胞粘附、信号转导、免疫反应、癌症和阿尔茨海默病。迄今, 已报道的ADAM10完全基因敲除小鼠和大脑神经前体细胞特异性ADAM10基因敲除小鼠分别于胚胎期或围产期死亡, 致使无法研究成年小鼠大脑神经细胞ADAM10基因的功能。文章利用本研究小组建立的CaMKIIα-Cre转基因小鼠与ADAM10^loxP/loxP转基因小鼠杂交, 获得了CaMKIIα-Cre/ADAM10^loxP/loxP小鼠, 并对其进行鉴定。利用PCR方法检测成年ADAM10 cKO小鼠大脑基因组DNA表明, ADAM10基因缺失主要发生在前脑皮层和海马中。荧光定量PCR检测结果显示, ADAM10 mRNA的表达水平在前脑皮层和海马中分别降低55.7%和60.8% ; 使用Western blotting方法研究发现, ADAM10成熟蛋白质的含量在前脑皮层和海马中分别减少63%和84.8% 。采用免疫组织化学方法检测表明, 成年ADAM10 cKO小鼠与野生型小鼠相比, 其大脑皮层和海马神经细胞的ADAM10免疫染色明显减弱, 而其它细胞如胶质细胞的免疫染色基本一致。总之, 文章成功制备了首个存活至成年的大脑神经细胞特异性ADAM10基因敲除(cKO)小鼠, 克服了小鼠因ADAM10缺失在胚胎期或围产期死亡的弊端, 为研究成年小鼠大脑神经细胞ADAM10基因的功能奠定了坚实的基础。     

KIF14 promotes tumor progression and metastasis and is an independent predictor of poor prognosis in human gastric cancer

The kinesin family member 14 (KIF14) is a potential oncogene and is involved in the metastasis of various cancers. Nevertheless, its function in gastric cancer (GC) remains poorly defined. The expression of KIF14 was examined in GC cell lines and a clinical cohort of GC specimens by qPCR, western blotting and immunohistochemistry (IHC) staining. The relationship between KIF14 expression and the clinicopathological features was analyzed. The effect of KIF14 on cell proliferation, colony formation, invasion and migration were investigated in vitro and in vivo. The expression of KIF14 was significantly increased in the GC tissues and cell lines. High KIF14 expression was associated with tumor stage, tumor-node-metastasis (TNM) stage and metastasis. KIF14 was an independent prognostic factor for the overall survival of GC, and a higher expression of KIF14 predicted a poorer survival. KIF14 silencing resulted in attenuated proliferation, invasion and migration in human gastric cancer cells, whereas KIF14 ectopic expression facilitated these biological abilities. Notably, the depressed expression of KIF14 inhibited Akt phosphorylation, while overexpressed KIF14 augmented Akt phosphorylation. Additionally, there was a significant correlation between the expression of KIF14 and p?Akt in GC tissues. Importantly, the proliferation, invasion and migration of the GC cells, which was promoted by KIF14 overexpression, was abolished by the Akt inhibitor MK-2206, while Akt overexpression greatly rescued the effects induced by KIF14 knockdown. Our findings are the first to demonstrate that KIF14 is overexpressed in GC, is correlated with poor prognosis and plays a crucial role in the progression and metastasis of GC.