Neurocan is a New Substrate for the ADAMTS12 Metalloprotease: Potential Implications in Neuropathies.

BACKGROUND/AIMS: The composition of the extracellular matrix (ECM) in the central nervous system (CNS) has several features that make it unique. For instance, it is remarkable for the presence of proteoglycans such as versican, brevican, and neurocan, some of which have been identified as substrates of different members of the ADAMTS family of secreted metalloproteases. Previous studies have associated ADAMTSs with the repair of the CNS, including recovery following degradation of glial scar tissue and the stimulation of axonal growth after brain injury. However, the involvement of ADAMTSs in diseases of the CNS is complex and not understood fully, and a current challenge is unraveling the precise roles of these metalloproteases in the brain. METHODS: ADAMTS12 and neurocan gene expression was examined by quantitative PCR. Western blot analysis was employed to detect ADAMTS12 and neurocan protein expression in cell lines, and immunostaining techniques were used to detect neurocan in mouse brain tissues. Neurocan cleavage using recombinant ADAMTS1, ADAMTS4, ADAMTS5, and ADAMTS12 metalloproteases was evaluated by western blotting. Cell adhesion and migration were assessed using uncoated culture dishes or dishes coated with Matrigel or ECM components. RESULTS: We identified neurocan as a novel component of brain ECM that can be cleaved by ADAMTS12. In addition, we showed that neurocan cleavage by ADAMTS12 altered the adhesive properties of the human neuroglioma H4 cell line. Moreover, immunohistochemical analysis of Adamts12-deficient mice revealed the significant accumulation of neurocan in the brain of neonatal mice. CONCLUSION: Overall, our results suggest that ADAMTS12 could be involved in the repair of the CNS through its ability to degrade neurocan. Moreover, it can be inferred that alterations in neurocan degradation processes could be associated with the pathogenesis of neurological disorders.

Relationship Between ADAMTS8, ADAMTS18, and ADAMTS20 (A Disintegrin and Metalloproteinase with Thrombospondin Motifs) Expressions and Tumor Molecular Classification, Clinical Pathological Parameters, and Prognosis in Breast Invasive Ductal Carcinoma.

BACKGROUND The aim of this study was to investigate the correlations between ADAMTSs expression and breast invasive ductal carcinoma (IDC), and to offer a theoretical basis for novel treatment methods for IDC patients. MATERIAL AND METHODS Non-proliferative catheter of breast fibroadenoma (FA) and IDC were used as the normal control and experimental group, respectively. Immunohistochemical (IHC) staining and Western blot (WB) analysis was used to assess protein expression levels of ADAMTS8, ADAMTS18, and ADAMTS20 in both FA and IDC tissues. The results of IHC, the relationship between the protein expression and the tumor molecular classification, and clinical pathological parameters were all evaluated. RESULTS IHC and WB results showed that the expression of ADAMTS8/18 in IDC samples was higher than in FA samples, while the expression of ADAMTS20 in IDC samples was lower than that in FA samples. According to the results of WB, the level of ADAMTS8 was higher in the HER2+ group than in the HER2- group and FA group. The expression of ADAMTS18 in the HR+ (including ER+ and PR+) group was significantly higher than in the HR- group and FA group. The expression of ADAMTS18 protein was also higher in the Ki67+ group than in the Ki67- group. ADAMTS20 was higher in HER2+ IDC compared with the basal subtype of IDC. CONCLUSIONS ADAMTS8/18/20 levels were not significantly correlated to the molecular subtype of IDC. ADAMTS18/20 was significantly associated with histological grade of IDC. ADAMTS8 may predict poor prognosis results of IDC patients.

Healthy and Osteoarthritic Synovial Fibroblasts Produce a Disintegrin and Metalloproteinase with Thrombospondin Motifs 4, 5, 7, and 12: Induction by IL-1ß and Fibronectin and Contribution to Cartilage Damage.

Current description of osteoarthritis includes the involvement of synovial inflammation. Studies contributing to understanding the mechanisms of cross-talk and feedback among the joint tissues could be relevant to the development of therapies that block disease progression. During osteoarthritis, synovial fibroblasts exposed to anomalous mechanical forces and an inflammatory microenvironment release factors such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) metalloproteinases that mediate tissue damage and perpetuate inflammation. We therefore studied the production of ADAMTS by synovial fibroblasts and their contribution to cartilage degradation. Moreover, we analyzed the implication of two mediators present in the osteoarthritis joint, IL-1ß as proinflammatory cytokine, and 45-kDa fibronectin fragments as products of matrix degradation. We reported that synovial fibroblasts constitutively express and release ADAMTS 4, 5, 7, and 12. Despite the contribution of both mediators to the stimulation of Runx2 and Wnt/ß-catenin signaling pathways, as well as to ADAMTS expression, promoting the degradation of aggrecan and cartilage oligomeric matrix protein from cartilage, fibronectin fragments rather than IL-1ß played the major pathological role in osteoarthritis, contributing to the maintenance of the disease. Moreover, higher levels of ADAMTS 4 and 7 and a specific regulation of ADAMTS-12 were observed in osteoarthritis, suggesting them as new potential therapeutic targets. Therefore, synovial fibroblasts provide the biochemical tools to the chronicity and destruction of the osteoarthritic joints.

Progesterone receptor and prostaglandins mediate luteinizing hormone-induced changes in messenger RNAs for ADAMTS proteases in theca cells of bovine periovulatory follicles.

Little is known about the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of extracellular proteases in ovarian follicles of non-rodent species, particularly in theca cells. In the present study, temporal changes in the abundance of mRNA encoding four ADAMTS subtypes and hormonal regulation of mRNA encoding two subtypes were investigated in theca interna cells during the periovulatory period in cattle. Gonadotropin-releasing hormone (GnRH) was injected into animals to induce a luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge, and follicles were obtained at 0 hr post-GnRH (preovulatory) or at 6, 12, 18, or 24 hr (periovulatory). ADAMTS1, -2, -7, and -9 transcript abundance was then determined in the isolated theca interna. ADAMTS1 and -9 mRNA levels were up-regulated at 24 hr post-GnRH, whereas ADAMTS2 mRNA was higher at 12-24 hr post-GnRH and ADAMTS7 mRNA increased transiently at 12 hr post-GnRH compared to other time points. Subsequent in vitro experiments using preovulatory theca interna (0 hr post-GnRH) showed that application of LH in vitro can mimic the effects of the gonadotropin surge on mRNAs encoding ADAMTS1 and -9 and that progesterone/progesterone receptor and/or prostaglandins may regulate the levels of mRNA encoding ADAMTS1 and -9 in theca interna, downstream of the LH surge. Time- and subtype-specific changes in ADAMTS mRNA abundance in vivo, and their regulation in vitro by hormones, indicate that ADAMTS family members produced by theca cells may play important roles in follicle rupture and the accompanying tissue remodeling in cattle. Mol. Reprod. Dev. 84: 55-66, 2017. © 2016 Wiley Periodicals, Inc.

Adamts 1, 4, 5, 8, and 9 in Early Pregnancies.

INTRODUCTION: The aim of this study was to immunohistochemically investigate the presence and localization of ADAMTS 1, 4, 5, 8 and 9 in decidual and chorionic tissues in first trimester pregnancy losses. MATERIALS AND METHODS: This study was conducted with early pregnancy failure decidual and chorionic tissue samples from 36 pregnant women in the first trimester of pregnancy (ongoing pregnancies, missed miscarriages, anembryonic pregnancies) Results: It was observed that the decidual and chorionic tissue levels of ADAMTS 1, 4, 5, and 8 in ongoing pregnancies were more intensely expressed when compared with miscarriages. ADAMTS 1 expression was not observed in the anembryonic pregnancies, ADAMTS 4, 5, and 8 were less intensely expressed. ADAMTS 9 showed no staining in any group. CONCLUSION: ADAMTS 1 may be necessary during the decidualization and implantation stages of early normal pregnancy.

Inflammasome components and ADAMTS4 in premature rupture of membranes.

Inflammation may be responsible for the development of premature rupture of membranes (PROM) including preterm PROM (PPROM) and mature PROM (MPROM). A total of four classic receptor proteins have been confirmed to assemble inflammasomes: NLR family pyrin domain containing (NLRP)1, NLRP3 and NLR family CARD­domain containing 4 (NLRC4) and absent in melanoma 2 (AIM2). The activation and expression of these receptor­modulated inflammasomes in placenta and fetal membrane of PROM pregnancies requires investigation. In addition, a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a risk factor for PROM, but whether its expression is associated with inflammasome activation remains to be elucidated. In the present study, the placenta and fetal membrane tissues of patients who had suffered PPROM and MPROM and healthy pregnancies were investigated. Reverse transcription­quantitative PCR was used to determine the mRNA expression of inflammasomes and ADAMTS4. Western blotting, immunohistochemistry and ELISA were used to investigate the protein expression levels of inflammasomes and ADAMTS4. The results demonstrated that all four inflammasomes were elevated in placenta and fetal membrane of PPROMs as were mRNA and protein expression levels of IL­18 and IL­1ß (compared with controls). A further increase of inflammasomes and interleukins was observed in MPROMs compared with controls. Similar results were also observed in ADAMTS4 expression in PPROM and MPROM groups. However, immunohistochemistry results revealed no significant difference of inflammasome receptor expression in PPROMs compared with controls. Finally, a general positive correlation between ADAMTS4 and all four inflammasome receptors in placenta and fetal membrane of PPROMs and MPROMs was observed. The present study revealed that NLRP1, NLRP3, AIM2 and NLRC4 inflammasome activation in PROM was increased. Promoted ADAMTS4 level was further observed in PROM group and was significantly correlated with inflammasome expression. Inhibition of inflammasome activation may provide a therapeutic target for clinical PROM treatment.

DNA methylation, microRNA expression profiles and their relationships with transcriptome in grass-fed and grain-fed Angus cattle rumen tissue.

Rumen is an organ for supplying nutrients for the growth and production of bovine, which might function differently under grass-fed and grain-fed regimens considering the association of gene expression, DNA methylation, and microRNA expression. The objective of this study was to explore the potential mechanism influencing rumen function of grass-fed and grain-fed animals. Methylated DNA binding domain sequencing (MBD-Seq) and microRNA-Seq were respectively utilized to detect the DNA methylation and microRNA expression in rumen tissue of grass-fed and grain-fed Angus cattle. Combined analysis revealed that the expression of the differentially expressed genes ADAMTS3 and ENPP3 was correlated with the methylation abundance of the corresponding differentially methylated regions (DMRs) inside these two genes, and these two genes were reported to be respectively involved in biosynthesis and regulation of glycosyltransferase activity; the differentially expressed microRNA bta-mir-122 was predicted to possibly target the differentially expressed genes OCLN and RBM47, potentially affecting the rumen function; the microRNA bta-mir-655 was exclusively detected in grain-fed group; its targets were significantly enriched in insulin and TGF-beta signaling pathways, which might worked together to regulate the function of rumen, resulting in different characteristics between grass-fed and grain-fed cattle. Collectively, our results provided insights into understanding the mechanisms determining rumen function and unraveled the biological basis underlying the economic traits to improve the productivity of animals.

Overexpression of ADAMTS-2 in tumor cells and stroma is predictive of poor clinical prognosis in gastric cancer.

ADAMTS-2 is a member of the ADAMTS family and is a procollagen N-proteinase. The objective of our research is to explore the prognostic significance of ADAMTS-2 in gastric carcinoma. A total of 655 samples with full clinicopathological data were investigated in this study. Tissue microarray immunohistochemistry analysis was used to analyze the relationship between clinicopathological characteristics and ADAMTS-2 expression. Oncomine and Kaplan-Meier plotters were performed for the relationship analysis between prognosis and ADAMTS-2 expression in patients with gastric cancer. Compared with that of normal tissues, the ADAMTS-2 protein expression was remarkably higher in gastric cancer cells and fibroblast cells. The results of univariate analysis indicated that the expression of ADAMTS-2 in tumor cells and fibroblast cells, Laurén classification, TNM grade, and carcinoembryonic antigen level in gastric cancer were all correlated with overall survival. The results of multivariate analysis indicated that the high expression of ADAMTS-2 in gastric cancer cells and fibroblast cells both were independent prognostic factors. Therefore, ADAMTS-2 may be a potential biomarker for assessing the prognosis of gastric carcinoma.

Casticin protects against IL-1ß-induced inflammation in human osteoarthritis chondrocytes.

Casticin, an active compound isolated from Vitex rotundifolia L., was reported to possess anti-inflammatory activity. However, the effect of casticin on inflammatory response in human osteoarthritis (OA) chondrocytes remains unclear. In the current study, we examined the anti-inflammatory effects of casticin on chondrocytes exposed to interleukin-1ß (IL-1ß). Our results demonstrated that casticin treatment significantly improved cell viability in chondrocytes exposed to IL-1ß. Casticin significantly inhibited IL-1ß-induced NO and PGE2 production, and iNOS and COX-2 expression in human OA chondrocytes. It also suppressed the levels of TNF-α and IL-6, as well as decreased production of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in IL-1ß-stimulated chondrocytes. Furthermore, casticin prevented IL-1ß-induced NF-κB activation in chondrocytes. Taken together, these findings showed that casticin attenuates inflammatory responses in chondrocytes stimulated with IL-1ß, possibly through the NF-κB signaling pathway. Thus, casticin may serve as a potential anti-inflammatory agent in the treatment of OA.

Downregulation of ADAMTS18 May Serve as a Poor Prognostic Biomarker for Cervical Cancer Patients.

ADAMTS18 is a member of ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family proteins which have been found to play important roles in cancer progression and metastasis in different cancer types. The present study aims at evaluating the prognostic significance of ADAMTS18 in cervical cancer. The expression levels of ADAMTS18 was evaluated by real-time PCR in normal (N=28) and cervical cancer tissues from patients at different stages as well as in tumor tissues from 35 cervical cancer patients with primary cancer and 29 patients with metastasis. The correlation between serum ADAMTS18 expression levels and clinicopathologic features or overall survival of cervical cancer patients was analyzed. ADAMTS18 expression levels were significantly decreased in cervical cancer tissues compared with normal tissues and it decreased gradually from early stage to late stage. Low expression of ADAMTS18 was positively associated with high tumor stage (P=0.0239), positive lymph node metastasis (P=0.0388), and distant metastasis (P=0.0004). Tissue levels of ADAMTS18 in patients with primary cancer were significantly lower compared with those with metastasis. Moreover, patients with low ADAMTS18 expression levels had shorter overall survival (P=0.0119) or disease-free survival (P=0.0033). Multivariate analysis demonstrated that ADAMTS18 was an independent prognostic factor for cervical cancer. Our study suggests that ADAMTS18 is downregulated in cervical cancer and ADAMTS18 may serve as a potential prognostic biomarker for cervical cancer.

Local intra­articular injection of vascular endothelial growth factor accelerates articular cartilage degeneration in rat osteoarthritis model.

In the pathophysiology of osteoarthritis (OA), articular cartilage degeneration exhibits a significant role. Vascular endothelial growth factor (VEGF) is considered to be an effective angiogenic factor and a crucial regulator of articular cartilage degeneration in the development of OA. Therefore, the present study aimed to investigate the underlying influences of exogenous VEGF on articular cartilage degeneration in OA model rat. A total of 24 male Sprague­Dawley rats were randomly allocated into 3 groups. In the normal saline (NS) and VEGF groups, animals received bilateral anterior cruciate ligament (ACL) transection to establish the OA model; at 4 weeks post­surgery, the rats received local intra­articular injections of 100 µl NS or VEGF solution, respectively, every week for 4 weeks. The Control group received neither surgery nor injections. All animals were sacrificed at 12 weeks following surgery. Prominent cartilage degeneration was observed in rats in the NS­ and VEGF­injected groups. The extent and the grade of cartilage damage in the VEGF­injected group were notably more severe compared with those in the NS­treated group. Western blotting results demonstrated that the expression levels of aggrecan and type II collagen were significantly reduced in OA model rats that were treated with VEGF. In addition, the expression levels of matrix metalloproteinase (MMP)­3, MMP­9, MMP­13, a disintegrin and metalloproteinase with thrombospondin motifs (a disintegrin and metalloproteinase; ADAMTS)­4, ­5 and ­12, type III collagen and transforming growth factor­ß1 were significantly increased following VEGF administration. Results from the present study indicated that VEGF may exhibit a promoting role in the development of OA by destroying articular cartilage matrix.