Synergistic upregulation of ADAMTS4 (aggrecanase-1) by cytokines and its suppression in knee osteoarthritic synovial fibroblasts.

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family includes nine members with aggrecan-degrading activity, i.e., ADAMTS1, 4, 5, 8, 9, 15, 16, 18, and 20. However, their systematic expression profile in knee osteoarthritis (OA) synovium and effects of cytokines and growth factors on the expression in OA synovial fibroblasts remain elusive. In this study, expression of all nine aggrecanolytic ADAMTS species was assessed by quantitative real-time PCR in OA and control normal synovial tissues. OA synovial fibroblasts were treated with interleukin-1α (IL-1α), IL-1ß, tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), vascular endothelial growth factor165, and heparin-binding epidermal growth factor, and analyzed for the expression of the ADAMTS species. The signaling pathways and inhibition of ADAMTS4 expression by high-molecular-weight hyaluronan, adalimumab, tocilizumab, and signaling molecule inhibitors were studied. ADAMTS1, 4, 5, 9, and 16 were expressed in OA synovium, but only ADAMTS4 expression was significantly higher in OA as compared to normal synovium. IL-1α, TNF-α, and TGF-ß markedly increased ADAMTS4 expression, while their effects were minimal for the other ADAMTS species. ADAMTS4 was synergistically upregulated by treatment with IL-1α and TNF-α, IL-1α and TGF-ß, or IL-1α, TNF-α and TGF-ß. The signaling molecules' inhibitors demonstrated that IL-1α-induced ADAMTS4 expression is predominantly through TGF-ß-associated kinase 1 (TAK1), and the TNF-α-stimulated expression is via TAK1 and nuclear factor-κB (NF-κB). The TGF-ß-promoted expression was through the activin receptor-like kinase 5 (ALK5)/Smad2/3, TAK1, and non-TAK1 pathways. Adalimumab blocked TNF-α-stimulated expression. ADAMTS4 expression co-stimulated with IL-1α, TNF-α and TGF-ß was abolished by treatment with adalimumab, TAK1 inhibitor, and ALK5/Smad2/3 inhibitor. These data demonstrate marked and synergistic upregulation of ADAMTS4 by IL-1α, TNF-α and TGF-ß in OA synovial fibroblasts, and suggest that concurrent therapy with an anti-TNF-α drug and inhibitor(s) may be useful for prevention against aggrecan degradation in OA.

Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization Is Up-Regulated and Involved in Hyaluronan Degradation in Human Osteoarthritic Cartilage.

Hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), also called cell migration-inducing protein (CEMIP; alias KIAA1199), plays a key role in the degradation of HA in skin and arthritic synovial fibroblasts, but its functions in osteoarthritic (OA) cartilage remain elusive. Here, we investigated the expression and roles of HYBID in human OA cartilage. HYBID was highly expressed by chondrocytes in the HA-depleted area of OA cartilage, and HYBID immunoreactivity was correlated with Mankin score, the histopathologic severity of OA lesions of cartilage. Real-time quantitative PCR indicated that HYBID expression was significantly higher in OA cartilage than in control cartilage. In addition, OA chondrocytes exhibited HA-degrading activity, which was abolished by knock-down of HYBID by siRNAs. Although OA chondrocytes also expressed certain levels of hyaluronidases 1 and 2 and CD44, knock-down of these molecules exhibited negligible effects on HA degradation. Double immunostaining of HYBID and clathrin heavy chain revealed that HYBID was localized in the clathrin-coated vesicles, and HA was endocytosed within the vesicles of OA chondrocytes. Among eight factors including cytokines and growth factors examined, only tumor necrosis factor α stimulated OA chondrocytes to overexpress HYBID. These data are the first to demonstrate that HYBID is up-regulated in OA cartilage, and suggest that tumor necrosis factor α-stimulated HYBID plays a role in HA degradation in OA cartilage.

ADAM23 is downregulated in side population and suppresses lung metastasis of lung carcinoma cells.

Cancer cells contain a small population of cancer stem cells or cancer initiating cells, which can be enriched in the side population (SP) after fluorescence activated cell sorting. To examine the members of the ADAM, ADAMTS and MMP gene families related to phenotypes of the SP and the main population (MP), we screened the expression of all the members in the propagated SP and MP of A549 lung adenocarcinoma cells, and found that the relative expression ratio of ADAM23 in the MP to the SP is most highly increased, but none of them are increased in the SP. A similar result on the ADAM23 expression was obtained with another cell line, Calu-3 cells. Overexpression of ADAM23 inhibited colony formation, cell adhesion and migration, and knockdown of ADAM23 by shRNA showed the reverse effects. ADAM23-mediated suppression of colony formation, cell adhesion and migration was greatly reduced by treatment with neutralizing anti-ADAM23 antibody, anti-αvß3 integrin antibody and/or ADAM23 disintegrin peptide. Expression of cancer stem cell-related genes, including AKRC1/2, TM4SF1 and NR0B1, was increased by knockdown of ADAM23. In addition, lung metastasis of A549 transfectants with different levels of ADAM23 expression was negatively regulated by the ADAM23 expression levels. Our data provide evidence that ADAM23 plays a role in suppression of cancer cell progression through interaction with αvß3 integrin, and suggest that downregulation of ADAM23 in SP cells may contribute toward providing a cancer stem cell phenotype by facilitating the activity of integrin αvß3.

Duration of viral shedding in asymptomatic or mild cases of novel coronavirus disease 2019 (COVID-19) from a cruise ship: A single-hospital experience in Tokyo, Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the cause of novel coronavirus disease 2019 (COVID-19), was first reported in Wuhan, China, and now has spread across the world as a global pandemic. The propagation from asymptomatic polymerase chain reaction (PCR)-positive individuals represents a complicating factor in the efforts to control the COVID-19 pandemic. We examined the course of PCR assays and the duration of viral shedding in 23 asymptomatic or mild COVID-19 patients from the cruise ship who were admitted to our hospital. Among these 23 cases, the median duration of viral shedding was 19 days (range, 6-37 days) from initial viral detection. Eight cases (35%) had another positive PCR result after testing negative once. Although the duration of viral shedding was approximately three weeks, the infectivity and transmissibility period from asymptomatic and mild COVID-19 cases is unclear. Further studies are needed to determine how long such asymptomatic and mild COVID-19 cases have infectivity.

Selective Inhibition of ADAM28 Suppresses Lung Carcinoma Cell Growth and Metastasis.

ADAM28 (a disintegrin and metalloproteinase 28) is overexpressed by carcinoma cells in non-small cell lung carcinomas (NSCLC) and plays an important role in cancer cell proliferation and metastasis by reactivation of insulin-like growth factor-1 (IGF-1) and escaping from von Willebrand factor (VWF)-induced apoptosis through digestion of IGF-binding protein-3 and VWF, respectively. To aim for new target therapy of NSCLC patients, we developed human neutralizing antibodies 211-12 and 211-14 against ADAM28, which showed IC50 values of 62.4 and 37.5 nmol/L, respectively. Antibody 211-14 recognized the junctional region between cysteine-rich domain and secreted-specific domain and showed a KD value of 94.7 pmol/L for the epitope-containing peptide. This antibody detected monkey and human secreted-form ADAM28s, although it was not reactive with mouse membrane-anchored ADAM28m. Antibody 211-14 effectively inhibited IGF-1-stimulated cell proliferation of lung adenocarcinoma cell lines with ADAM28 expression, including PC-9 cells, and promoted VWF-induced cell death in these cell lines. In lung metastasis models, antibody 211-14 significantly reduced tumor growth and metastases of PC-9 cells and prolonged survivals in the antibody-treated mice compared with the control IgG-treated ones. Combination therapy of the antibody and docetaxel was more effective than that of bevacizumab and docetaxel and showed further elongation of survival time compared with monotherapy. No adverse effects were observed even after administration of 10-fold more than effective dose of anti-ADAM28 antibody to normal mice. Our data demonstrate that antibody 211-14 is a neutralizing antibody specific to ADAM28s and suggest that this antibody may be a useful treatment remedy for NSCLC patients. Mol Cancer Ther; 17(11); 2427-38. ©2018 AACR.

ADAM28 is expressed by epithelial cells in human normal tissues and protects from C1q-induced cell death.

ADAM28 (disintegrin and metalloproteinase 28), which was originally reported to be lymphocyte-specific, is over-expressed by carcinoma cells and plays a key role in cell proliferation and progression in human lung and breast carcinomas. We studied ADAM28 expression in human normal tissues and examined its biological function. By using antibodies specific to ADAM28, ADAM28 was immunolocalized mainly to epithelial cells in several tissues, including epididymis, bronchus and stomach, whereas lymphocytes in lymph nodes and spleen were negligibly immunostained. RT-PCR, immunoblotting and ELISA analyses confirmed the expression in these tissues, and low or negligible expression by lymphocytes was found in the lymph node and spleen. C1q was identified as a candidate ADAM28-binding protein from a human lung cDNA library by yeast two-hybrid system, and specific binding was demonstrated by binding assays, immunoprecipitation and surface plasmon resonance. C1q treatment of normal bronchial epithelial BEAS-2B and NHBE cells, both of which showed low-level expression of ADAM28, caused apoptosis through activation of p38 and caspase-3, and cell death with autophagy through accumulation of LC3-II and autophagosomes, respectively. C1q-induced cell death was attenuated by treatment of the cells with antibodies against the C1q receptor gC1qR/p33 or cC1qR/calreticulin. Treatment of C1q with recombinant ADAM28 prior to addition to culture media reduced C1q-induced cell death, and knockdown of ADAM28 using siRNAs increased cell death. These data demonstrate that ADAM28 is expressed by epithelial cells of several normal organs, and suggest that ADAM28 plays a role in cell survival by suppression of C1q-induced cytotoxicity in bronchial epithelial cells.