Expression Of ADAMTS9 Research Articles

Anti-osteoarthritis effects of injectable hyaluronic acid hydrogel loaded with platelet lysate and cerium oxide nanoparticle by regulating TNF-α: An in vitro model

The most prevalent type of joint illness is osteoarthritis, also known as OA. The inflammation of the joint is strongly linked to both the beginning and the progression of osteoarthritis. The use of hydrogels as an OA treatment has the potential to go beyond the administration of anti-inflammatory components to have an inherent immunomodulatory role. Platelet lysate (PL) contains a cocktail of growth factors that actively participates in cartilage repair. Moreover, cerium oxide-nanoparticles (CeO2-NPs) have been broadly studied owing to their powerful antioxidant properties and potential preventive and therapeutic effects against chronic diseases. The current study was designed to determine the effect and mechanism of injectable hyaluronic acid (HA) hydrogel loaded with PL and CeO2-NPs on OA. The results showed that the obtained PL and synthesized CeO2-NPs were effectively incorporated into HA hydrogel and the resultant hydrogel was injectable and have a porous micro structure with interconnected pores. The biological evaluation by qPCR revealed that the fabricated hydrogel exhibited a significantly increased expression of Col2 and Aggrecan (TNF-α-suppressed anabolic molecules) and decreased expression of Col10, Mmp13, Adamts5, and Adamts9 (TNF-α-activated catabolic molecules). Also, ELISA results confirmed this potent antioxidant activity of hydrogel and also increased the growth of chondrocytes over the culturing period.

Dynamic and broad expression of adamts9 in developing and adult zebrafish.

Previous studies showed that Adamts9 is involved in multiple functions including ovulation, spine formation, primordial germ cell migration, and development of primary ovarian follicles in animals. However, systemic examination and high-resolution analyses of adamts9 expression are missing due to lack of a sensitive reporter assay. In the present study, we created a new transgenic zebrafish reporter line Tg(adamts9:EGFP) and assayed its expression in various tissues and cells during development and in adults at high-resolution using confocal imaging. Reporter expression was validated with real-time quantitative PCR, whole mount in situ hybridization, and immunohistochemistry for endogenous adamts9. Strong expression of the adamts9:EGFP transgene was found in a wide range of adult and embryonic zebrafish tissues/cells including ovaries, testes, brains, eyes, pectoral fins, intestine, skin, gill, muscle, and heart; while lower expression was observed in the liver and growing ovarian follicles (stages II and III). Our results of a broad and dynamic expression pattern for this evolutionary conserved metalloprotease suggest involvement of adamts9 in the development and physiological functions of various tissues in animals.

Differential gene expression of ADAMTS-1, ADAMTS-9 and TIMP-3 in periodontitis

ABSTRACT A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) are metalloproteinases that bind to components of the extracellular matrix (ECM) to regulate tissue remodeling and homeostasis. ADAMTS can be inhibited by tissue inhibitors of metalloproteinases (TIMPs). Expression of ADAMTS increases under inflammatory conditions. We investigated the mRNA expression of ADAMTS-1, ADAMTS-9 and TIMP-3 genes in both healthy gingival tissues and periodontitis. Clinical periodontal measurements were conducted and gingival biopsies were obtained from stage IIIgrade C generalized periodontitis and healthy (control) groups. mRNA expression was evaluated using real-time quantitative polymerase chain reaction (RTqPCR). All clinical periodontal parameters were significantly higher in the periodontitis group than for the control group. ADAMTS-1 levels were significantly higher in the periodontitis group and were significantly correlated with clinical attachment level and probing pocket depth. Differences in ADAMTS-9 and TIMP-3 mRNA in the periodontitis group compared to the control group were not statistically significant. Increased ADAMTS-1 mRNA expression in periodontitis indicates that members of the ADAMTS family of metalloproteinases are associated with pathogenesis and progression of periodontal disease. Maintaining balance between ADAMTS and TIMP is important for limiting ECM catabolism and preventing tissue damage.

Delay in primordial germ cell migration in adamts9 knockout zebrafish

Adamts9 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 9) is one of a few metalloproteinases structurally conserved from C. elegans to humans and is indispensable in germ cell migration in invertebrates. However, adamts9′s roles in germ cell migration in vertebrates has not been examined. In the present study, we found zygotic expression of adamts9 started around the germ ring stage and reached peak levels at 3 days post fertilization (dpf) in zebrafish. The migration of primordial germ cells (PGC) was completed within 24 hours (h) in wildtype siblings, while a delay in PGC migration was found at 15 and 24-h post-fertilization (hpf) in the Adamts9 knockout (KO). However, the delayed PGC migration in Adamts9 KO disappeared at 48 hpf. Our study suggests a conserved function of Adamts9 in germ cell migration among invertebrates and vertebrates. In addition, our results also suggest that Adamts9 is not essential for germ cell migration as reported in C. elegans, possibly due to expansion of Adamts family members and compensatory roles from other metalloproteinases in vertebrates. Further studies are required in order to elucidate the functions and mechanisms of metalloproteinases in germ cell migration and gonad formation in vertebrates.

Global transcriptomic analysis reveals Lnc-ADAMTS9 exerting an essential role in myogenesis through modulating the ERK signaling pathway

BackgroundLong non-coding RNAs (lncRNAs) are emerging key regulators involved in a variety of biological processes such as cell differentiation and development. The balance between myogenesis and adipogenesis is crucial for skeletal muscle homeostasis in humans and meat quality in farm animals. The present study aimed to reveal the global transcriptomic profiles of adipogenic (Adi-) and myogenic (Myo-) precursors derived from porcine skeletal muscle and identify lncRNAs involved in the modulation of myogenesis homeostasis in porcine skeletal muscle.ResultsIn this study, a total of 655 novel individual lncRNAs including differentially expressed 24 lncRNAs, and 755 differentially expressed mRNAs were identified (fold change ≥2 or ≤ 0.5 and adjusted P < 0.05). Integrated results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis accompanied by the variation of intracellular Ca2+ concentration highlighted Lnc-ADAMTS9 involved in the modulation of myogenesis homeostasis in porcine skeletal muscle. Although Lnc-ADAMTS9 knock-down did not alter the mRNA expression of ADAMTS9, we demonstrated that Lnc-ADAMTS9 can promote myogenic proliferation and myogenic differentiation of myogenic precursors through inhibiting the ERK/MAPK signaling pathway.ConclusionWe deciphered a comprehensive catalog of mRNAs and lncRNAs that might be involved in the regulation of myogenesis and adipogenesis homeostasis in the skeletal muscle of pigs. The Lnc-ADAMTS9 exerts an essential role in myogenesis through the ERK signaling pathway.

Mining TCGA database for tumor mutation burden and their clinical significance in bladder cancer.

Background: Bladder cancer is the ninth most-common cancer worldwide and it is associated with high morbidity and mortality. Tumor mutational burden (TMB) is an emerging biomarker in cancer characterized by microsatellite instability. TMB has been described as a powerful predictor of tumor behavior and response to immunotherapy.Methods: A total of 443 bladder cancer samples obtained from The Cancer Genome Atlas (TCGA) were analyzed for mutation types, TMB values, and prognostic value of TMB. Differentially expressed genes (DEGs) were identified from the TMB groupings. Functional analysis was performed to assess the prognostic value of the first 30 core genes. CIBERSORT algorithm was used to determine the correlation between the immune cells and TMB subtypes.Results: Single nucleotide polymorphism (SNP) and C>T were reported as the most common missense mutations and we also identified a high rate of mutations in TP53, TTN, KMT2D. Bladder cancer patients with high TMB showed a better prognosis. Enrichment analysis of the DEGs revealed that they were involved in the regulation of the P13K-Akt signaling pathway, cytokine–cytokine receptor interaction, and Ras signaling pathway. The high expression of hub genes ADRA2A, CXCL12, S1PR1, ADAMTS9, F13A1, and SPON1 was correlated with poor overall survival. Besides, significant differences in the composition of the immune cells of T cells CD8, T cells CD4 memory activated, NK cells resting and Mast cells resting were observed.Conclusions: The present study provides a comprehensive and systematic analysis of the prediction of TMB in bladder cancer and its clinical significance. Also, the study provides additional prognostic information and opportunities for immunotherapy in bladder cancer.

Upregulation of adamts9 by gonadotropin in preovulatory follicles of zebrafish

Previously we had identified adamts9 as a downstream target of Pgr, which is essential for ovulation in zebrafish. The primary goal of this study is to determine whether human chorionic gonadotropin (hCG, LH analog) also regulate adamts9 expression prior to ovulation. The expression of adamts9 was induced by hCG in a dose and time dependent manner in zebrafish preovulatory follicles in vitro. Interestingly, the stimulatory effect of hCG on adamts9 expression was not blocked in pgr−/− follicles but blocked in lhcgr−/−. This effect of hCG was via Lhcgr and its associated cAMP and PKC signaling pathways. Reduced fecundity and reduced expression of adamts9 were also found in lhcgr−/− females in vivo. Therefore, we have provided the first evidence of gonadotropin (hCG) regulated adamts9 in zebrafish, which could be important for ovulation.

Mechanical strain attenuates cytokine-induced ADAMTS9 expression via transient receptor potential vanilloid type 1

The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1β and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.

Adamts9 is necessary for ovarian development in zebrafish

Expression of adamts9 (A disintegrin and metalloprotease with thrombospondin type-1 motif, member 9) increases dramatically in the somatic cells surrounding oocytes during ovulation in vertebrates from zebrafish to human. However, the function of Adamts9 during ovulation has not been determined due to the embryonic lethality of knockouts in mice and Drosophila. To identify the role of Adamts9 during ovulation we generated knockout (adamts9−/−) zebrafish using CRISPR/Cas9 and characterized the effects of the mutation. From 1047 fish generated by crossing adamts9+/− pairs, we found significantly fewer adult adamts9−/− fish (4%) than predicted by Mendelian ratios (25%). Of the mutants found, there was a significant male bias (82%). Only 3 female mutants were identified (7%), and they had small ovaries with few stage III and IV oocytes compared to wildtype (wt) counterparts of comparable size and age. Astoundingly, the remaining mutants (11%) did not appear to have normal testis or ovaries. Instead there was a pair of transparent, ovarian-like membranous shells that filled the abdominal cavity. Histological examination confirmed that shells were largely empty with no internal structure. Surprisingly, seminiferous tubules and various spermatocytes including mature spermatozoa were observed on the periphery of these transparent shells. No female or female like knockouts were observed to release eggs, and no ovulated oocytes were observed in histological sections. To our knowledge, this is the first report of an adamts9 global knockout model in any adult vertebrates and the first description of how gonadal sex and structure are affected- highlighting the importance of Adamts9 during gonadal development and the value of zebrafish as a model organism.

MicroRNA‐190b regulates lipid metabolism and insulin sensitivity by targeting IGF‐1 and ADAMTS9 in non‐alcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is characterized by ectopic lipid accumulation and insulin resistance, yet the underlying molecular mechanisms are poorly understood. MiR-190b is thought to play a role in hepatocellular carcinoma by modulating insulin resistance; however, its role in NAFLD remains unknown. Here, we found that miR-190b expression was significantly increased in the liver tissues of patients with NAFLD, compared to normal tissues. Moreover, miR-190b was upregulated in a high-fat diet NAFLD mouse model and a free fatty acid-induced NAFLD cellular model. Knockdown of miR-190b decreased aspartate transaminase (AST), alanine transaminase (ALT), triglyceride (TG), and total cholesterol (TC). It also reduced expression of the lipogenic genes fatty acid synthase (FAS) and 3-hydroxy-3-methylglutarylCoA reductase (HMGCR), alleviated hepatic steatosis, improved glucose tolerance, elevated insulin sensitivity, and activated insulin receptor substrate (IRS)2/Akt signaling in vivo and/or in vitro. Furthermore, we confirmed that miR-190b directly targeted IGF-1 and ADAMTS9. MiR-190b overexpression suppressed expression of IGF-1 and ADAMTS9, which were increased by miR-190b inhibition. Expression of IGF-1 and ADAMTS9 was inversely correlated with miR-190b in liver tissues of patients with NAFLD, respectively. We also found that IGF-1 or ADAMTS9 inhibition partially reversed the effects of miR-190b on lipid metabolism and insulin signaling in vitro. Taken together, the data reveal that miR-190b inhibition suppressed lipid accumulation and improved insulin sensitivity by targeting IGF-1 and ADAMTS9, suggesting that miR-190b inhibition may be a therapeutic strategy against NAFLD.

The 3p14.2 tumour suppressor ADAMTS9 is inactivated by promoter CpG methylation and inhibits tumour cell growth in breast cancer.

Chromosome region 3p12‐14 is an important tumour suppressor gene (TSG) locus for multiple cancers. ADAMTS9, a member of the metalloprotease large family, has been identified as a candidate 3p14.2 TSG inactivated by aberrant promoter CpG methylation in several carcinomas, but little known about its expression and function in breast cancer. In this report, ADAMTS9 expression and methylation was analysed in breast cancer cell lines and tissue samples. ADAMTS9 RNA was significantly down‐regulated in breast cancer cell lines (6/8). After treating the cells with demethylation agent Aza and TSA,ADAMTS9 expression was dramatically increased. Bisulphite genomic sequencing and methylation‐specific PCR detected promoter methylation, which was associated with decreased ADAMTS9 expression. Hypermethylation was also detected in 130/219 (59.4%) of primary tumours but only in 4.5% (2/44) of paired surgical margin tissues. Ectopic expression of ADAMTS9 in tumor cells induced significant growth suppression, cell cycle arrest at the G0/G1 phase, enhanced apoptosis and reduced cell migration and invasion. Conditioned culture medium from ADAMTS9‐transfected BT549 cells markedly disrupted tube formation ability of human umbilical vein endothelial cell (HUVEC) in Matrigel. Furthermore, ADAMTS9 inhibited AKT signaling and its downstream targets (MDM2, p53, p21, p27, E‐cadherin, VIM, SNAIL, VEGFA, NFκB‐p65 and MMP2). In addition, we demonstrated, for the first time, that ADAMTS9 inhibits AKT signaling, through suppressing its upstream activators EGFR and TGFβ1/TβR(I/II) in breast cancer cells. Our results suggest that ADAMTS9 is a TSG epigenetically inactivated in breast cancer, which functions through blocking EGFR‐ and TGFβ1/TβR(I/II)‐activated AKT signaling.

Hemoglobin stimulates the expression of ADAMTS-5 and ADAMTS-9 by synovial cells: a possible cause of articular cartilage damage after intra-articular hemorrhage

BackgroundADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) proteins play an important pathological role in matrix degeneration. Aggrecan degradation is a significant and critical event in early-stage osteoarthritis. To determine the effect of hemoglobin (Hb) on the ability of synovial tissues to produce ADAMTS family members, we examined the influence of Hb by synovial cells in an in vitro experimental system.MethodsSynovial tissues were obtained from five young patients with meniscal injury under arthroscopic surgery. Primary cultures of human knee synovial cells were treated with different doses of human Hb (0, 25, 50, 100 μg/ml). The culture media were collected 24 h after Hb-treatment. In the time-course studies, cells were treated with and without 100 μg/ml Hb, and culture media were taken at 6, 12, and 24 h. To identify the proteins responsible for aggrecanase activity, Western blot analysis using antibodies against human ADAMTS-5, −8, −9, and −10; enzyme-linked immunosorbent assay (ELISA); and gene expression for ADAMTS-5 and -9 were examined. Statistical comparisons between each group were performed using paired t-tests.ResultsWestern blot analysis revealed that Hb-treatment resulted in the expression of ADAMTS-5 and -9. Neither control group nor Hb-treated medium showed immunoreactivity against ADAMTS-8 or −10. In a dose-dependency study, the Hb-treated group showed significantly higher levels of ADAMTS-5 and -9 compared with the control (p < 0.05). There was no significant difference between 25, 50, and 100 μg/ml Hb-treated groups. In a time-course study, the ADAMTS-5 and -9 levels in the conditioned medium had significantly increased expression at 6, 12, and 24 h in the Hb-treated group (p < 0.05). Hb evoked significant expression of ADAMTS-9 mRNA at 12 and 24 h (p < 0.05).ConclusionsThese findings indicate that Hb induces the expression of ADAMTS-5 and -9 by synovial cells at low doses, even at an acute phase, and suggests a possible role for Hb in cartilage damage after intra-articular hemorrhage. The results also suggest a new potential therapeutic target by inhibiting the activities of ADAMTS-5 and -9 to prevent cartilage damage after intra-articular hemorrhage.

Hemoglobin Stimulates the Expression of Adamts-5 and -9 by Synovial Cells; A Possible Cause of Cartilage Damage after Intra-articular Bleeding

Objectives:A disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-5 and -9 play an important pathologic role in matrix degeneration. Aggrecanase-mediated aggrecan degradation is a significant and critical event in early-stage osteoarthritis. To determine the effect of hemoglobin (Hb) on the ability of synovial tissues to produce ADAMTS family that play important roles in the degradation of articular cartilage, examining the influence of Hb by synovial cells in vitro experimental system.Methods:Synovial tissues were obtained from five young patients with meniscus injury. The mean age of patients was 14 years (range: 10-16). The experimental design was reviewed and approved (Accession No. 405) by the Committee for Ethics at the Faculty of Medicine, Miyazaki University. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. We obtained written, informed consent from all patients before entering the study. Primary cultures of human knee synovial cells were treated with different doses of human Hb (0, 25, 50, 100 μg/ml) for the preliminary examination. The culture media were collected 24 h after Hb-treatment. In the time-course studies, cells were treated with 100 μg/ml Hb and culture media were taken at 6, 12, and 24 h. To identify the proteins those are responsible for aggrecanase activity, Western blot analysis using antibodies against human ADAMTS-5, -8, -9, and -10, Enzyme-Linked ImmunoSorbent Assay (ELISA) and gene expression for ADAMTS-5, and -9 were examined.Results:Western blot analysis revealed that Hb-treatment resulted in the detection of ADAMTS-5 and -9 proteins in a dose-, and time-dependent manner. Neither control nor Hb-treated medium showed immunoreactivity against anti-ADAMTS-8, and -10 IgG. In a dose-dependency study which were measured by ELISA, the peak immunoreactivity was confirmed at Hb concentration of 100μg/ml. Even if 25μg/ml treated with Hb at 24h, there are significantly increasing expression compared with the control group both in the results of ADAMTS-5, and -9 (p<0.05). In a time-course study analyzed, the levels of ADAMTS-5 and -9 antigen level in the conditioned medium which were measured by ELISA reveals significantly increasing expression at 6, 12, and 24h in the Hb-treated group compared with the results of control group (p<0.05). Hb evoked significantly expression of ADAMTS-9 mRNA at 12 and 24 hours (p<0.05). Hb induced highly expression of ADAMTS-9 at 6 hours without significant difference. The mRNA level of ADAMTS-5 was not enhanced by Hb, significantly.Conclusion:Our results suggest a possible role of Hb for joint damage after intra-articular bleeding. Hb induces the expression of ADAMTS family proteinase such as ADAMTS-5 and -9 by synovial cells at low doses even at an acute phase. These findings suggest that single instance of intra-articular bleeding during trauma or sports injury could affect on articular cartilage metabolism deleteriously during an acute phase. Immediate treatment for intra-articular bleeding, such as washing out and removing the blood from the joint cavity, may be required regardless of whether it occurs once or repeatedly.

Vascular signal transducer and activator of transcription-3 promotes angiogenesis and neuroplasticity long-term after stroke.

Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3(floxed/KO) and Tie2-Cre(ERT2) mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration.

Expression of ADAMTS-9 in the central nervous system in multiple sclerosis and its relationship to cleavage of aggrecan and versican

A Disintegrin And Metalloproteinase with Thrombospondin motifs (ADAMTSs) are proteases which are able to cleave aggregating chondroitin sulphate proteoglycans (CSPGs), including aggrecan and versican. Extracellular matrix (ECM) alterations have been reported in the central nervous system (CNS) in multiple sclerosis (MS) in post mortem white matter tissue, due to both the release and activation of matrix metalloproteinases (MMPs) and ADAMTSs as well as increased synthesis of ECM components. To elucidate the potential pathophysiological role of ADAMTS-9 in ECM changes in MS, we investigated expression of aggrecan and versican by dual label immunohistochemistry (IHC) and western blotting (WB) using antibodies which specifically recognise cleavage-derived aggrecan and versican neoepitopes or intact proteins.

Epigenetic inactivation of ADAMTS9 via promoter methylation in multiple myeloma

A disintegrin‑like and metalloprotease with thrombospondin typeⅠ motifs(ADAMTS) are a family of 19secreted mammalian metalloproteases. ADAMTS9 was reported to be a novel tumor suppressor gene and is downregulated in various types of human cancer due to hypermethylation of promoter CpG islands. In the present study, the silencing mechanism of the ADAMTS9 gene was analyzed in the multiple myeloma (MM) cell lines, KM3 and RPMI‑8226. Control and MM samples were obtained by conventional bone marrow (BM) biopsy of normal and MM adult BM, respectively. RT‑PCR revealed a high expression of the ADAMTS9 gene in normal samples and RPMI‑8226 cells while marked gene silencing of ADAMTS9 was observed in MM patients and KM3 cells. Promoter methylation of ADAMTS9 was detected in the KM3 cell line and 66% (37/56) MM patients by methylation‑specific PCR. In addition, the DNA demethylating agent, 5‑aza‑2'‑deoxycytidine and trichostatinA restored ADAMTS9 expression by suppressing promoter methylation in KM3 cells. Ectopic expression of ADAMTS9 in ADAMTS9‑silenced MM cells was found to significantly suppress cell colony formation and proliferation. In the present study, DNA methylation was found to play a key role in ADAMTS9 gene silencing and the biological behavior of myeloma cells. The results demonstrate that ADAMTS9 silencing by methylation may be a novel tumor marker for MM and the applicability of demethylating agents in the treatment of MM.

Cytokine‐induced increases in ADAMTS‐4 messenger RNA expression do not lead to increased aggrecanase activity in ADAMTS‐5–deficient mice

To compare the regulation of aggrecanase messenger RNA (mRNA) and enzyme activity by proinflammatory cytokines in primary mouse chondrocytes. Primary chondrocytes were isolated from knee epiphyses of 6-8-day-old mice and cultured as monolayers. The cells were incubated with tumor necrosis factor α (TNFα), oncostatin M (OSM), or interleukin-6 (IL-6)/soluble IL-6 receptor, and mRNA levels were measured by quantitative polymerase chain reaction at various time points. To measure aggrecanase activity, the cells were incubated with cytokine in the presence of exogenous aggrecan, and substrate cleavage was measured using antibodies to neoepitopes. Expression of both ADAMTS-4 and ADAMTS-5 mRNA was up-regulated by TNFα and OSM. ADAMTS-5 mRNA expression was also up-regulated by IL-6. Treatment of wild-type mouse chondrocytes with each of the 3 cytokines increased cleavage of aggrecan at Glu(373)↓(374) Ala and Glu(1670)↓(1671) Gly; in chondrocytes lacking ADAMTS-5 activity, there was negligible cleavage at either site despite increased expression of ADAMTS-4 mRNA in the presence of TNFα or OSM. None of the cytokines substantially altered mRNA expression of ADAMTS-1 or ADAMTS-9. Despite substantial increases in the expression of ADAMTS-4 mRNA induced by TNFα and OSM, these cytokines induced little if any increase in aggrecanolysis in ADAMTS-5-deficient mouse chondrocytes. Our data show a poor correlation between the level of cytokine-induced ADAMTS-4 mRNA expression and the level of aggrecan-degrading activity in cultured chondrocytes.

ADAMTS-9 expression is up-regulated following transient middle cerebral artery occlusion (tMCAo) in the rat

The ADAMTS enzymes ( a disintegrin and metalloproteinase with thrombo spondin type 1-like motifs) have important roles in central nervous system (CNS) physiology and pathology. This current study aimed to analyse the expression of ADAMTS-9 following transient middle cerebral artery occlusion (tMCAo) in the rat, a model of focal cerebral ischaemia. Using real-time RT-PCR, ADAMTS-9 mRNA was demonstrated to be significantly up-regulated in tMCAo brain tissue compared to sham-operated at 24 h post-ischaemia. The mature form of the ADAMTS-9 protein was only detected by Western blotting in brains subjected to tMCAo at 24 h. In situ hybridisation demonstrated that ADAMTS-9 mRNA was expressed by neurones in tMCAo tissue. This study indicates that ADAMTS-9 expression is modulated in response to cerebral ischaemia in vivo and further research will resolve whether it plays a role in the subsequent degenerative or repair processes.

Expression of ADAMTS-9 in mouse growth plate cartilage

Expression of ADAMTS-9 in mouse growth plate cartilage

Cell-surface Processing of Pro-ADAMTS9 by Furin

Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.