Sesame oil downregulates the expression of ADAMTS-4 in high-fat diet-induced atherosclerosis.

Atherosclerosis is a chronic inflammatory disease forming plaques in medium and large-sized arteries. ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs-4) is an extracellular-matrix remodelling enzyme involved in the degradation of versican in the arterial wall. Recent reports indicated that increased expression of ADAMTS-4 is associated with plaque progression and vulnerability. Bioactive components of dietary oil, like sesame oil, are reported to have anti-inflammatory and antioxidant properties. Here, we studied the effect of sesame oil on regulating ADAMTS-4 in high-fat diet-induced atherosclerosis rat model. Our results indicated that sesame oil supplementation improved the anti-inflammatory and anti-oxidative status of the body. It also reduced atherosclerotic plaque formation in high-fat diet-fed rats. Our results showed that the sesame oil supplementation significantly down-regulated the expression of ADAMTS-4 in serum and aortic samples. The versican, the large proteoglycan substrate of ADAMTS-4 in the aorta, was downregulated to normal control level on sesame oil supplementation. This study, for the first time, reveals that sesame oil could down-regulate the expression of ADAMTS-4 in high-fat diet-induced atherosclerosis, imparting a new therapeutic potential for sesame oil in the management of atherosclerosis.

miR-126a-5p inhibits H1N1-induced inflammation and matrix protease secretion in lung fibroblasts by targeting ADAMTS-4.

Upregulation of ADAMTS-4 has been reported to have an important role in lung injury, and ADAMTS-4 expression is regulated by miR-126a-5p in abdominal aortic aneurysms. The aim of this study was to investigate whether miR-126a-5p/ADAMTS-4 plays a role in influenza-virus-induced lung injury. Lung fibroblasts were infected with H1N1 influenza virus to detect changes in miR-126a-5p and ADAMTS-4 expression, and cell viability was measured by CCK-8 assay. Inflammatory factors and matrix protease levels were examined using ELISA kits, and cell apoptosis was assessed by measuring the levels of apoptosis-related proteins. A dual luciferase assay was used to verify the regulatory relationship between miR-126a-5p and ADAMTS-4. H1N1 influenza virus reduced fibroblast viability, inhibited miR-126a-5p expression, and promoted ADAMTS-4 expression. Overexpression of miR-126a-5p attenuated the cellular inflammatory response, apoptosis, matrix protease secretion, and virus replication. Luciferase reporter assays revealed that miR-126a-5p inhibited ADAMTS-4 expression by targeting ADAMTS-4 mRNA. Further experiments showed that overexpression of ADAMTS-4 significantly reversed the inhibitory effects of miR-126a-5p on fibroblast inflammation, apoptosis, matrix protease secretion, and virus replication. Upregulation of miR-126a-5p inhibits H1N1-induced apoptosis, inflammatory factors, and matrix protease secretion, as well as virus replication in lung fibroblasts.

Chrysin downregulates the expression of ADAMTS-4 in foam cells.

BACKGROUND: Chrysin, a polyphenolic compound, possesses antioxidant and anti-inflammatory properties. In this study, we investigated the effect of chrysin on the expression of A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), a protease enzyme involved in degrading extracellular matrix associated with atherosclerosis. METHODS AND RESULTS: We have studied the cell viability by MTT assay and foam cell formation by oil red O staining. The mRNA and protein expression of ADAMTS-4 was studied using quantitative polymerase chain reaction (qPCR) and Western blotting, respectively. Our study showed that chrysin significantly downregulates the expression of ADAMTS-4 in foam cells. CONCLUSION: Chrysin's ability to downregulate the expression of ADAMTS-4, a protease involved in degrading the extracellular matrix, bestows upon it a new therapeutic potential for managing atherosclerosis.

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.

MiR-126a-5p limits the formation of abdominal aortic aneurysm in mice and decreases ADAMTS-4 expression.

Abdominal aortic aneurysm (AAA) is a serious vascular disease featured by inflammatory infiltration in aortic wall, aortic dilatation and extracellular matrix (ECM) degradation. Dysregulation of microRNAs (miRNAs) is implicated in AAA progress. By profiling miRNA expression in mouse AAA tissues and control aortas, we noted that miR-126a-5p was down-regulated by 18-fold in AAA samples, which was further validated with real-time qPCR. This study was performed to investigate miR-126a-5p's role in AAA formation. In vivo, a 28-d infusion of 1 µg/kg/min Angiotensin (Ang) II was used to induce AAA formation in Apoe-/- mice. MiR-126a-5p (20 mg/kg; MIMAT0000137) or negative control (NC) agomirs were intravenously injected to mice on days 0, 7, 14 and 21 post-Ang II infusion. Our data showed that miR-126a-5p overexpression significantly improved the survival and reduced aortic dilatation in Ang II-infused mice. Elastic fragment and ECM degradation induced by Ang II were also ameliorated by miR-126a-5p. A strong up-regulation of ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS-4), a secreted proteinase that regulates matrix degradation, was observed in smooth muscle cells (SMCs) of aortic tunica media, which was inhibited by miR-126a-5p. Dual-luciferase results demonstrated ADAMTS-4 as a new and valid target for miR-126a-5p. In vitro, human aortic SMCs (hASMCs) were stimulated by Ang II. Gain- and loss-of-function experiments further confirmed that miR-126-5p prevented Ang II-induced ECM degradation, and reduced ADAMTS-4 expression in hASMCs. In summary, our work demonstrates that miR-126a-5p limits experimental AAA formation and reduces ADAMTS-4 expression in abdominal aortas.

Raloxifene retards cartilage degradation and improves subchondral bone micro-architecture in ovariectomized rats with patella baja-induced - patellofemoral joint osteoarthritis.

OBJECTIVE: Abnormal remodeling of subchondral bone (SB) induced by estrogen deficiency has been shown to be involved in osteoarthritis (OA). Raloxifene (RAL) is commonly used to treat postmenopausal osteoporosis (OP). However, little is known about its effects on OA combined with estrogen deficiency. This study was performed to evaluate the efficacy of RAL on patella baja-induced patellofemoral joint OA (PFJOA) in an ovariectomized rat model. DESIGN: Patellar ligament shortening (PLS) and ovariectomy (OVX) were performed simultaneously in 3-month-old female Sprague-Dawley rats, which were treated with RAL (10 mg/kg/day) or vehicle at 72 h postoperatively for 10 weeks. PFJOA was assessed by immunohistochemistry (IHC), real-time polymerase chain reaction (PCR), tartrate-resistant acid phosphatase (TRAP) staining, enzyme-linked immunosorbent assay (ELISA), micro-computed tomography (µCT), histomorphology and behavioral analyses. RESULTS: X-ray examinations showed that patella baja was successfully established by PLS. Histomorphological analysis revealed that PFJOA was significantly exacerbated by OVX and markedly alleviated by RAL. Moreover, RAL improved cartilage metabolism by decreasing MMP-13, ADAMTS-4, and caspase-3 and increasing Col-II and aggrecan at both the protein and mRNA levels. Furthermore, RAL markedly improved bone mass and SB microarchitecture and reduced osteoclast numbers and the serum osteocalcin and CTX-I levels. Although RAL showed a trend toward reducing pain sensitivity based on mechanical allodynia testing, this result was not statistically significant. CONCLUSION: These findings demonstrate that RAL treatment retards PFJOA progression in an ovariectomized rat model, suggesting that it may be a potential candidate for amelioration of the progression of PFJOA accompanied by postmenopausal OP.

Sox4 is involved in osteoarthritic cartilage deterioration through induction of ADAMTS4 and ADAMTS5.

Osteoarthritis is a common disease in joint cartilages. Because the molecular pathogenesis of osteoarthritis remains elusive, early diagnostic markers and effective therapeutic agents have not been developed. To understand the molecular mechanisms, we attempted to identify transcription factors involved in the onset of osteoarthritis. Microarray analysis of mouse articular cartilage cells indicated that retinoic acid, a destructive stimulus in articular cartilage, up-regulated expression of sex-determining region Y-box (Sox)4, a SoxC family transcription factor, together with increases in Adamts4 and Adamts5, both of which are aggrecanases of articular cartilages. Overexpression of Sox4 induced a disintegrin-like and metallopeptidase with thrombospondin type 4 and 5 motif (ADAMTS4 and ADAMTS5, respectively) expression in chondrogenic cell lines C3H10T1/2 and SW1353. In addition, luciferase reporter and chromatin immunoprecipitation assays showed that Sox4 up-regulated ADAMTS4 and Adamts5 gene promoter activities by binding to their gene promoters. Another SoxC family member, Sox11, evoked similar effects. To evaluate the roles of Sox4 and Sox11 in articular cartilage destruction, we performed organ culture experiments using mouse femoral head cartilages. Sox4 and Sox11 adenovirus infections caused destruction of articular cartilage associated with increased Adamts5 expression. Finally, SOX4 and SOX11 mRNA expression was increased in cartilage of patients with osteoarthritis compared with nonosteoarthritic subjects. Thus, Sox4, and presumably Sox11, are involved in osteoarthritis onset by up-regulating ADAMTS4 and ADAMTS5.-Takahata, Y., Nakamura, E., Hata, K., Wakabayashi, M., Murakami, T., Wakamori, K., Yoshikawa, H., Matsuda, A., Fukui, N., Nishimura, R. Sox4 is involved in osteoarthritic cartilage deterioration through induction of ADAMTS4 and ADAMTS5.

Bile and urine peptide marker profiles: access keys to molecular pathways and biological processes in cholangiocarcinoma.

BACKGROUND: Detection of cholangiocarcinoma (CCA) remains a diagnostic challenge. We established diagnostic peptide biomarkers in bile and urine based on capillary electrophoresis coupled to mass spectrometry (CE-MS) to detect both local and systemic changes during CCA progression. In a prospective cohort study we recently demonstrated that combined bile and urine proteome analysis could further improve diagnostic accuracy of CCA diagnosis in patients with unknown biliary strictures. As a continuation of these investigations, the aim of the present study was to investigate the pathophysiological mechanisms behind the molecular determinants reflected by bile and urine peptide biomarkers. METHODS: Protease mapping and gene ontology cluster analysis were performed for the previously defined CE-MS based biomarkers in bile and urine. For that purpose, bile and urine peptide profiles (from samples both collected at the date of endoscopy) were investigated from a representative cohort of patients with benign (n = 76) or CCA-associated (n = 52) biliary strictures (verified during clinical follow-up). This was supplemented with a literature search for the association of the individual biomarkers included in the proteomic patterns with CCA or cancer progression. RESULTS: For most of the peptide markers, association to CCA has been described in literature. Protease mapping revealed ADAMTS4 activity in cleavage of both bile and urine CCA peptide biomarkers. Furthermore, increased chymase activity in bile points to mast cell activation at the tumor site. Gene ontology cluster analysis indicates cellular response to chemical stimuli and stress response as local and extracellular matrix reorganization by tissue destruction and repair as systemic events. The analysis further supports that the mapped proteases are drivers of local and systemic events. CONCLUSIONS: The study supports connection of the CCA-associated peptide biomarkers to the molecular pathophysiology and indicates an involvement in epithelial-to-mesenchymal transition, generation of cancer-associated fibroblasts and activation of residual immune cells. Proteases, extracellular matrix components, inflammatory cytokines, proangiogenic, growth and vasoactive factors released from the tumor microenvironment are drivers of systemic early events during CCA progression.

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)-1ß 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.

Velvet antler polypeptide partially rescue facet joint osteoarthritis-like phenotype in adult ß-catenin conditional activation mice.

BACKGROUND: Wnt/ß-catenin signaling pathway is closely related to osteoarthritis. In our preliminary study, ß-catenin conditional activation (cAct) mice that specifically over-express ß-catenin gene in cartilage chondrocyte exhibits osteoarthritis-like phenotype in the lumbar disc and knee joint. Therefore, we used the mice to model FJ-OA and test the potential curative effect of Velvet Antler Polypeptide (VAP) on this mice model. METHODS: We tested the effect of VAP on ß-catenin conditional activation mice, and used Cre negative littermates as controls. Micro-CT, histology and histomorphometry analysis were performed to evaluate the curative effect of VAP on mice facet joint-like phenotype. Expression of ß-catenin and collagen II was detected by immunohistochemistry (IHC) and western-blot., MMP13, ADAMTS4 and ADAMTS5 was detected by immunofluorescence (IF). RT-PCR analysis was preformed to detect mRNA expression of cartilage degrading enzymes, such as MMP13, ADAMTS4 and ADAMTS5. RESULTS: Results of micro-CT (µCT) analysis showed that VAP could partially reverse lumbar disc osteophyte formation observed in ß-catenin(ex3)Col2ER mice. Histology data revealed VAP partially improved facet joint cartilage tissue invades. Histomorphometry analysis showed an increase in total cartilage area after VAP treatment. IHC show that VAP reduced ß-catenin protein levels and moderately up-regulated collagen II protein levels. RT-PCR and IF data showed that VAP down-regulated the expression of extracellular matrix synthesis (ECM) degradation enzymes MMP13, ADAMTS4 and ADAMTS5. CONCLUSION: Taken together, VAP may modulate ECM by inhibits MMP13, ADAMTS4 and ADAMTS5 via Wnt /ß-catenin signaling pathway. Velvet Antler Polypeptide may be a potential medicine for FJ-OA.

Polydatin suppresses nucleus pulposus cell senescence, promotes matrix homeostasis and attenuates intervertebral disc degeneration in rats.

Intervertebral disc degeneration (IVDD) is one of the major causes of low back pain. Polydatin (PD) has been shown to exert multiple pharmacological effects on different diseases; here, we test the therapeutic potential of PD for IVDD. In in-vitro experiments, we confirmed PD is nontoxic to nucleus pulposus cells (NPCs) under the concentration of 400 µmol/L. Furthermore, PD was able to decrease the level of senescence in TNF-α-treated NPCs, as indicated by ß-gal staining as well as senescence markers p53 and p16 expression. In the aspect of extracellular matrix (ECM), PD not only reduced metalloproteinase 3 (MMP-3), metalloproteinase 13 (MMP-13) and a disintegrin-like and metalloproteinase thrombospondin type 1 motif 4 (ADAMTS-4) expression, but also increased aggrecan and collagen II levels. Mitochondrion is closely related to cellular senescence and ECM homeostasis; mechanistically, we found PD may rescue TNF-α-induced mitochondrial dysfunction, and it may also promote Nrf2 expression and activity. Silencing Nrf2 partly abolished the protective effects of PD on mitochondrial homeostasis, senescence and ECM homeostasis in TNF-α-treated NPCs. Correspondingly, PD ameliorated IVDD in rat model by promoting Nrf2 activity, preserving ECM and inhibiting senescence in nucleus pulposus cells. To sum up, our study suggests that PD exerts protective effects in NPCs against IVDD and reveals the underlying mechanism of PD on Nrf2 activation in NPCs.

Leptin induces MMP1/13 and ADAMTS 4 expressions through bone morphogenetic protein-2 autocrine effect in human chondrocytes.

The induction of bone morphogenetic protein (BMP)2 in injured and arthritis articular cartilage has been proposed, but the precise mechanism has not been clearly clarified. Our previous study has found that leptin could stimulate the BMP2 autocrine effect to increase the anabolic collagen II expression when it initiates the catabolic response in human chondrocytes. It has been suggested that this BMP2 autocrine effect contributes to a reparative role in leptin-stimulated human chondrocytes. In this study, we further determined whether this BMP2 autocrine effect also affect the expressions of catabolic matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS). Human primary and SW1353 chondrocytes were used in this study. It was shown that leptin could induce the expressions of MMP1, 3, and 13 and ADAMTS4 and 5 in both human primary and SW1353 chondrocytes. Leptin-increased MMP1/13 (not MMP3) and ADAMTS4 (not ADAMTS5) expressions were affected by the leptin-upregulated BMP2 and its specific downstream Smad1/5 signaling. Moreover, both HDAC3 and 4 are involved in regulating leptin-induced BMP2 upregulation and then affect MMP1 and 13 and ADAMTS4 expression. Both HDAC3 and 4 also affect leptin-increased MMP3 mRNA expression but not through BMP2 autocrine effect of leptin induction. Our results further elucidated the role of BMP2 autocrine effect in matrix-degrading enzymes expressions under leptin stimulation. The findings in this study provide new insights into the possible mechanism of BMP2 induction in leptin-stimulated chondrocytes and in leptin-induced OA development.

IL-1ß-induced miR-34a up-regulation inhibits Cyr61 to modulate osteoarthritis chondrocyte proliferation through ADAMTS-4.

Osteoarthritis (OA) is the most prevalent degenerative joint disease with multifactorial etiology caused by risk factors. The degradation of aggrecan by upregulated ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) is the key event in the development of OA. ADAMTS-4 contributes to aggrecan degradation in human OA. Cysteine-rich angiogenic inducer 61 (Cyr61), which is associated with diseases related to chronic inflammation, is found in articular cartilage from patients with osteoarthritis and appears to suppress ADAMTS-4 activity, possibly leading to chondrocyte cloning. Herein, we first revealed that Cyr61 and ADAMTS-4 protein levels were remarkably increased in OA cartilage tissues and OA chondrocytes, and verified Cyr61 regulation of ADAMTS-4 in normal and OA chondrocyte. Further, we revealed that Cyr61 could promote OA chondrocyte proliferation through inhibiting ADAMTS-4. Overproduction of inflammatory cytokines plays a vital role in the pathological development of OA; herein, we demonstrated that IL-1ß inhibited Cyr61, while promoted ADAMTS-4 expression. By using online tools and luciferase assays, we confirmed that miR-34a, a regulatory miRNA of chondrocyte proliferation, could directly bind to the 3'-UTR of Cyr61 to inhibit its expression; further, IL-1ß regulated Cyr61 and ADAMTS-4 expression through miR-34a. In OA cartilage tissues, miR-34a, and IL-1ß mRNA expression was up-regulated and positively correlated; miR-34a and Cyr61 mRNA was positively correlated, further indicating that suppressing miR-34a expression might rescue IL-1ß-induced Cyr61 suppression, and promote OA chondrocyte proliferation. Taken together, we provided novel experimental basis for rescuing OA chondrocyte proliferation through miR-34a/Cyr61 axis.

Promotion of Tumor Growth by ADAMTS4 in Colorectal Cancer: Focused on Macrophages.

BACKGROUND/AIMS: ADAMTSs (A disintegrin and metalloprotease domains with thrombospondins motifs) are a family of extracellular proteases that have been related to both oncogenic and tumor-suppressive functions. The aim of the present study was to investigate: 1) the mutation, copy-number alterations, and expression profile of ADAMTSs in colorectal cancer and 2) whether ADAMTSs participate in colorectal cancer (CRC) progression and invasion. METHODS: The mutation, copy-number alterations, and expression profile of ADAMTSs in CRC were analyzed in the TCGA cohort using cBioportal. ADAMTS4 expression in tumor tissues and cell lines were determined by immunostaining and real-time quantitative PCR. The role of ADAMTS-4 in CRC progression and the underlying mechanisms were studied by using short hairpin RNA-mediated knockdown of ADAMTS4. The effects of ADAMTS4 in cell proliferation and invasion were determined by clone formation assay and transwell migration assay, respectively. Macrophages were depleted by liposomal clodronate in immune-competent BALB/c mice and tumor growth was analyzed. RESULTS: ADAMTS4 was differentially expressed in CRC and predicted a poor prognosis. Elevated ADAMTS4 expression was closely associated with larger tumor size, enhanced TNM stage, and a poor clinical outcome in patients with CRC. ADAMTS4 knockdown had no inhibitory implications on cell proliferation and invasion in vitro, but significantly attenuated tumor growth in vivo. Mechanistically, we revealed that ADAMTS4 was associated macrophages infiltration and polarization in the tumor microenvironment of CRC. Macrophage depletion largely abolished the promotive effect of ADAMTS4 on tumor growth in the immune competent BALB/c mice. CONCLUSION: ADAMTS4 seemed to be a promising prognostic indicator in CRC. The novel link between ADAMTS4 and macrophages mirrors the potential regulatory roles of ADAMTSs in the inflammatory microenvironment of cancers.

Lycorine Suppresses Endplate-Chondrocyte Degeneration and Prevents Intervertebral Disc Degeneration by Inhibiting NF-κB Signalling Pathway.

BACKGROUND/AIMS: Cartilaginous endplate (CEP) degeneration is an important cause for intervertebral disc (IVD) degeneration that leads to low-back pain. The identification of compounds that may prevent CEP degeneration is of interest for the prevention of IVD degeneration. METHODS: Catabolic protease expression in the CEP of disc degeneration patients was first assessed. The toxicity, function and underlying mechanism of lycorine (LY) on CEP-derived chondrocytes degeneration were assessed in vitro by flow cytometry analysis and western blotting. The concentration and function of LY in rat-tail disc-degeneration models were also assessed by HPLC (High Performance Liquid Chromatography) quantification and histological analysis. RESULTS: In CEP cells, Interleukin (IL)-1ß upregulated the expression of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 that is critical for the degradation of cartilage extracellular matrix. Interestingly, LY suppressed the expression of these enzymes via the inhibition of nuclear factor-κB (NFκB) signalling and thus prevented IL-1ß-induced endplate cell degeneration in vitro. More importantly, LY also reduced the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in CEP and exerted a protective effect on both CEP and nucleus pulposus (NP) degeneration. In addition to its inhibitory effect on matrix-degrading protease expression, LY treatment also reduced positive regulators of proinflammatory cytokines, such as MIF, which can be secreted by CEP cells and subsequently target NP cells. CONCLUSION: LY could serve as a potential drug for treating IVD disease.

LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes.

Reduced SIRT1 activity and levels during osteoarthritis (OA) promote gradual loss of cartilage. Loss of cartilage matrix is accompanied by an increase in matrix metalloproteinase (MMP) 13, partially because of enhanced LEF1 transcriptional activity. In this study, we assessed the role of SIRT1 in LEF1-mediated MMP13 gene expression in human OA chondrocytes. Results showed that MMP13 protein levels and enzymatic activity decreased significantly during SIRT1 overexpression or activation by resveratrol. Conversely, MMP13 gene expression was reduced in chondrocytes transfected with SIRT1 siRNA or treated with nicotinamide (NAM), a sirtuin inhibitor. Chondrocytes challenged with IL-1ß, a cytokine involved in OA pathogenesis, enhanced LEF1 protein levels and gene expression, resulting in increased MMP13 gene expression; however, overexpression of SIRT1 during IL-1ß challenge impeded LEF1 levels and MMP13 gene expression. Previous reports showed that LEF1 binds to the MMP13 promoter and transactivates its expression, but we observed that SIRT1 repressed LEF1 protein and mRNA expression, ultimately reducing LEF1 transcriptional activity, as judged by luciferase assay. Finally, mouse articular cartilage from Sirt1-/- presented increased LEF1 and MMP13 protein levels, similar to human OA cartilage. Thus, demonstrating for the first time that SIRT1 represses MMP13 in human OA chondrocytes, which appears to be mediated, at least in part, through repression of the transcription factor LEF1, a known modulator of MMP13 gene expression.-Elayyan, J., Lee, E.-J., Gabay, O., Smith, C. A., Qiq, O., Reich, E., Mobasheri, A., Henrotin, Y., Kimber, S. J., Dvir-Ginzberg, M. LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes.

ADAMTS-4 in oligodendrocytes contributes to myelination with an impact on motor function.

Myelination is a late developmental process regulated by a set of inhibitory and stimulatory factors, including extracellular matrix components. Accordingly, chondroitin sulfate proteoglycans (CSPGs) act as negative regulators of myelination processes. A disintegrin and metalloproteinase with thrombospondin motifs type 4 (ADAMTS-4) is an extracellular protease capable of degrading CSPGs. Although exogenous ADAMTS-4 has been proven to be beneficial in several models of central nervous system (CNS) injuries, the physiological functions of endogenous ADAMTS-4 remain poorly understood. We first used Adamts4/LacZ reporter mice to reveal that ADAMTS-4 is strongly expressed in the CNS, especially in the white matter, with a cellular profile restricted to mature oligodendrocytes. Interestingly, we evidenced an abnormal myelination in Adamts4-/- mice, characterized by a higher diameter of myelinated axons with a shifting g-ratio. Accordingly, lack of ADAMTS-4 is accompanied by motor deficits and disturbed nervous electrical activity. In conclusion, we demonstrate that ADAMTS-4 is a new marker of mature oligodendrocytes contributing to the myelination processes and thus to the control of motor capacities.

Crosstalk Between T Lymphocytes and Lung Fibroblasts: Generation of a Hyaluronan-Enriched Extracellular Matrix Adhesive for Monocytes.

In immunity and inflammation, T cells are often associated with stromal mesenchymal cells such as fibroblasts. Hyaluronan and proteins that associate with hyaluronan such as versican and tumor necrosis factor-inducible gene-6 (TSG-6) are extracellular matrix (ECM) components that promote leukocyte adhesion, accumulation, and activation. However, the factors responsible for producing this specialized ECM and its impact on inflammatory events are not well understood. In this study, we explored the role of T cells in stimulating lung fibroblasts to produce an ECM that impacts monocyte adhesion. We found that CD3/CD28-activated human CD4+ T cells when co-cultured with human lung fibroblasts stimulated the expression of mRNA for hyaluronan synthase 2 (HAS2) and decreased the expression of hyaluronidase 2 (HYAL2). This led to an increase in the deposition of hyaluronan that formed cable-like structures within the ECM. Co-culturing activated T cells with fibroblasts also led to increased expression and accumulation of TSG-6. Surprisingly, addition of activated CD4+ T cells to the fibroblasts reduced the expression of mRNA for versican, and increased the expression of enzymes that degrade versican, such as ADAMTS4 and ADAMTS9 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif) leading to a decrease in versican in the ECM of the co-cultures. Furthermore, addition of human monocytes to these co-cultures resulted in elevated monocyte adhesion to the cable-like structures in the ECM when compared to controls. These results illustrate the importance of crosstalk between T cells and fibroblasts in promoting the generation of a matrix that is adhesive for monocytes. J. Cell. Biochem. 118: 2118-2130, 2017. © 2016 Wiley Periodicals, Inc.

Licochalcone A Inhibits MMPs and ADAMTSs via the NF-κB and Wnt/ß-Catenin Signaling Pathways in Rat Chondrocytes.

BACKGROUND: Osteoarthritis (OA) is a joint disease in which cartilage degradation is the central pathological change. In this study, we investigated the anti-osteoarthritic effects of licochalcone A (Lico A) in rat chondrocytes. METHODS: Polymerase chain reaction and Western blotting were performed to evaluate the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5, ADAMTS-4, collagen II, matrix metalloproteinase (MMP)-13 and MMP-1 at both the gene and protein levels, respectively. In addition, the wnt/ß-catenin and nuclear factor kappa B (NF-κB) signaling pathways were also analyzed by Western blotting. RESULTS: Lico A downregulated ADAMTS-5, ADAMTS-4,MMP-13 and MMP-1 expression, and diminished the IL-1ß-induced inhibition of collagen II. In addition, the activation of ß-catenin and phosphorylation of p65 and IKKα/ß were suppressed by Lico A. CONCLUSIONS: Our results suggest that Lico A inhibits MMPs and ADAMTS partly via the NF-κB and wnt/ß-catenin signaling pathways in rat chondrocytes. Thus, Lico A may have therapeutic effects in OA.

MicroRNA-92a-3p Regulates Aggrecanase-1 and Aggrecanase-2 Expression in Chondrogenesis and IL-1ß-Induced Catabolism in Human Articular Chondrocytes.

BACKGROUND/AIMS: Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) are secreted enzymes belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family that play significant roles in the progression of osteoarthritis (OA). Here, we aimed to determine whether the expression of ADAMTS-4/5 in chondrogenesis and inflammation is regulated by microRNA-92a-3p (miR-92a-3p). METHODS: MiR-92a-3p and ADAMTS-4/5 expressions were determined by quantitative polymerase chain reaction (qPCR). To investigate the repressive effect of miR-92a-3p on ADAMTS-4/5 expression, chondrogenic human mesenchymal stem cells (hMSCs) and human chondrocytes were transfected with mature miR-92a-3p or an antisense inhibitor (anti-miR-92a-3p), respectively. ADAMTS-4/5 protein production was quantified by enzyme-linked immunosorbent assay (ELISA), and miR-92a-3p involvement in IL-1ß-mediated catabolic effects was examined by immunoblotting. The roles of activated MAP kinases (MAPK) and nuclear factor (NF)-κB were evaluated by using specific inhibitors. Interaction between miR-92a-3p and its putative binding site in the 3'-untranslated region (3'-UTR) of ADAMTS-4/5 mRNA was confirmed by luciferase reporter assay. RESULTS: miR-92a-3p expression was elevated in chondrogenic hMSCs, with significantly lower expression in OA cartilage than in normal cartilage. Stimulation with IL-1ß significantly reduced miR-92a-3p expression in primary human chondrocytes (PHCs). Transfection of chondrocytes with miR-92a-3p downregulated IL-1ß-induced ADAMTS-4/5 expression, and the activity of a reporter construct containing the 3'-UTR of human ADAMTS-4/5 mRNA. MiR-92a-3p expression was suppressed upon IL-1ß-induced activation of MAPK and NF-κB in chondrocytes. CONCLUSION: MiR-92a-3p is an important regulator of ADAMTS-4/5 in human chondrocytes and may contribute to the development of OA.