The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity.

A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1) is a protease involved in fertilization, cancer, cardiovascular development, and thoracic aneurysms. Proteoglycans such as versican and aggrecan have been identified as ADAMTS1 substrates, and Adamts1 ablation in mice typically results in versican accumulation; however, previous qualitative studies have suggested that ADAMTS1 proteoglycanase activity is weaker than that of other family members such as ADAMTS4 and ADAMTS5. Here, we investigated the functional determinants of ADAMTS1 proteoglycanase activity. We found that ADAMTS1 versicanase activity is approximately 1000-fold lower than ADAMTS5 and 50-fold lower than ADAMTS4 with a kinetic constant (kcat/Km) of 3.6 × 103 M-1 s-1 against full-length versican. Studies on domain-deletion variants identified the spacer and cysteine-rich domains as major determinants of ADAMTS1 versicanase activity. Additionally, we confirmed that these C-terminal domains are involved in the proteolysis of aggrecan as well as biglycan, a small leucine-rich proteoglycan. Glutamine scanning mutagenesis of exposed positively charged residues on the spacer domain loops and loop substitution with ADAMTS4 identified clusters of substrate-binding residues (exosites) in ß3-ß4 (R756Q/R759Q/R762Q), ß9-ß10 (residues 828-835), and ß6-ß7 (K795Q) loops. This study provides a mechanistic foundation for understanding the interactions between ADAMTS1 and its proteoglycan substrates and paves the way for development of selective exosite modulators of ADAMTS1 proteoglycanase activity.

Elevated glucose promotes MMP13 and ADAMTS5 production by osteoarthritic chondrocytes under oxygenated but not hypoxic conditions.

Type 2 diabetes is linked with increased incidence and severity of osteoarthritis. The purpose of this study was to determine the effect of extracellular glucose within the normal blood glucose and hyperglycemic range on catabolic enzyme production by chondrocytes isolated from osteoarthritic (OA) and macroscopically normal (MN) human cartilage under oxygenated (18.9% oxygen) and hypoxic (1% oxygen) conditions. OA and MN chondrocytes were maintained in 4, 6, 8, or 10 mM glucose for 24 h. Glucose consumption, GLUT1 glucose transporter levels, MMP13 and ADAMTS5 production, and levels of RUNX2, a transcriptional regulator of MMP13, ADAMTS5, and GLUT1, were assessed by enzyme-linked assays, RT-qPCR and/or western blot. Under oxygenated conditions, glucose consumption and GLUT1 protein levels were higher in OA but not MN chondrocytes in 10 mM glucose compared to 4 mM. Both RNA and protein levels of MMP13 and ADAMTS5 were also higher in OA but not MN chondrocytes in 10 mM compared to 4 mM glucose under oxygenated conditions. Expression of RUNX2 was overall lower in MN than OA chondrocytes and there was no consistent effect of extracellular glucose concentration on RUNX2 levels in MN chondrocytes. However, protein (but not RNA) levels of RUNX2 were elevated in OA chondrocytes maintained in 10 mM versus 4 mM glucose under oxygenated conditions. In contrast, neither RUNX2 levels or MMP13 or ADAMTS5 expression were increased in OA chondrocytes maintained in 10 mM compared to 4 mM glucose in hypoxia. Elevated extracellular glucose leads to increased glucose consumption and increased RUNX2 protein levels, promoting production of MMP13 and ADAMTS5 by OA chondrocytes in oxygenated but not hypoxic conditions. These findings suggest that hyperglycaemia may exacerbate chondrocyte-mediated cartilage catabolism in the oxygenated superficial zone of cartilage in vivo in patients with undertreated type 2 diabetes, contributing to increased OA severity.

Modulation of early osteoarthritis by tibiofemoral re-alignment in sheep.

OBJECTIVE: To investigate whether tibiofemoral alignment influences early knee osteoarthritis (OA). We hypothesized that varus overload exacerbates early degenerative osteochondral changes, and that valgus underload diminishes early OA. METHOD: Normal, over- and underload were induced by altering alignment via high tibial osteotomy in adult sheep (n = 8 each). Simultaneously, OA was induced by partial medial anterior meniscectomy. At 6 weeks postoperatively, OA was examined in five individual subregions of the medial tibial plateau using Kellgren-Lawrence grading, quantification of macroscopic OA, semiquantitative histopathological OA and immunohistochemical type-II collagen, ADAMTS-5, and MMP-13 scoring, biochemical determination of DNA and proteoglycan contents, and micro-computed tomographic evaluation of the subchondral bone. RESULTS: Multivariate analyses revealed that OA cartilaginous changes had a temporal priority over subchondral bone changes. Underload inhibited early cartilage degeneration in a characteristic topographic pattern (P ≥ 0.0983 vs. normal), in particular below the meniscal damage, avoided alterations of the subarticular spongiosa (P ≥ 0.162 vs. normal), and prevented the disturbance of otherwise normal osteochondral correlations. Overload induced early alterations of the subchondral bone plate microstructure towards osteopenia, including significantly decreased percent bone volume and increased bone surface-to-volume ratio (all P ≤ 0.0359 vs. normal). CONCLUSION: The data provide high-resolution evidence that tibiofemoral alignment modulates early OA induced by a medial meniscus injury in adult sheep. Since underload inhibits early OA, these data also support the clinical value of strategies to reduce the load in an affected knee compartment to possibly decelerate structural OA progression.

Inhibition of SMAD3 effectively reduces ADAMTS-5 expression in the early stages of osteoarthritis.

OBJECTIVE: As one of the most important protein-degrading enzymes, ADAMTS-5 plays an important role in the regulation of cartilage homeostasis, while miRNA-140 is specifically expressed in cartilage, which can inhibit the expression of ADAMTS-5 and delay the progression of OA (osteoarthritis). SMAD3 is a key protein in the TGF-ß signaling pathway, inhibiting the expression of miRNA-140 at the transcriptional and post-transcriptional levels, and studies have confirmed the high expression of SMAD3 in knee cartilage degeneration, but whether SMAD3 can mediate the expression of miRNA-140 to regulate ADAMTS-5 remains unknown. METHODS: Sprague-Dawley (SD) rat chondrocytes were extracted in vitro and treated with a SMAD3 inhibitor (SIS3) and miRNA-140 mimics after IL-1 induction. The expression of ADAMTS-5 was detected at the protein and gene levels at 24 h, 48 h, and 72 h after treatment. The OA model of SD rats was created using the traditional Hulth method in vivo, with SIS3 and lentivirus packaged miRNA-140 mimics injected intra-articularly at 2 weeks, 6 weeks and 12 weeks after surgery. The expression of miRNA-140 and ADAMTS-5 in the knee cartilage tissue was observed at the protein and gene levels. Concurrently, knee joint specimens were fixed, decalcified, and embedded in paraffin prior to immunohistochemical, Safranin O/Fast Green staining, and HE staining analyses for ADAMTS-5 and SMAD3. RESULTS: In vitro, the expression of ADAMTS-5 protein and mRNA in the SIS3 group decreased to different degrees at each time point. Meanwhile, the expression of miRNA-140 in the SIS3 group was significantly increased, and the expression of ADAMTS-5 in the miRNA-140 mimics group was also significantly downregulated (P < 0.05). In vivo, it was found that ADAMTS-5 protein and gene were downregulated to varying degrees in the SIS3 and miRNA-140 mimic groups at three time points, with the most significant decrease at the early stage (2 weeks) (P < 0.05), and the expression of miRNA-140 in the SIS3 group was significantly upregulated, similar to the changes detected in vitro. Immunohistochemical results showed that the expression of ADAMTS-5 protein in the SIS3 and miRNA-140 groups was significantly downregulated compared to that in the blank group. The results of hematoxylin and eosin staining showed that in the early stage, there was no obvious change in cartilage structure in the SIS3 and miRNA-140 mock groups. The same was observed in the results of Safranin O/Fast Green staining; the number of chondrocytes was not significantly reduced, and the tide line was complete. CONCLUSION: The results of in vitro and in vivo experiments preliminarily showed that the inhibition of SMAD3 significantly reduced the expression of ADAMTS-5 in early OA cartilage, and this regulation might be accomplished indirectly through miRNA-140.

MicroRNA-200c-5p Regulates Migration and Differentiation of Myoblasts via Targeting Adamts5 in Skeletal Muscle Regeneration and Myogenesis.

Skeletal muscle, as a regenerative organization, plays a vital role in physiological characteristics and homeostasis. However, the regulation mechanism of skeletal muscle regeneration is not entirely clear. miRNAs, as one of the regulatory factors, exert profound effects on regulating skeletal muscle regeneration and myogenesis. This study aimed to discover the regulatory function of important miRNA miR-200c-5p in skeletal muscle regeneration. In our study, miR-200c-5p increased at the early stage and peaked at first day during mouse skeletal muscle regeneration, which was also highly expressed in skeletal muscle of mouse tissue profile. Further, overexpression of miR-200c-5p promoted migration and inhibited differentiation of C2C12 myoblast, whereas inhibition of miR-200c-5p had the opposite effect. Bioinformatic analysis predicted that Adamts5 has potential binding sites for miR-200c-5p at 3'UTR region. Dual-luciferase and RIP assays further proved that Adamts5 is a target gene of miR-200c-5p. The expression patterns of miR-200c-5p and Adamts5 were opposite during the skeletal muscle regeneration. Moreover, miR-200c-5p can rescue the effects of Adamts5 in the C2C12 myoblast. In conclusion, miR-200c-5p might play a considerable function during skeletal muscle regeneration and myogenesis. These findings will provide a promising gene for promoting muscle health and candidate therapeutic target for skeletal muscle repair.

Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling.

BACKGROUND: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. METHODS: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. RESULTS: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin ß1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of ß-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that ß1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with ß-blockers had a distinct cardiac ECM profile. CONCLUSIONS: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that ß-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

lncRNA PSMB8-AS1 promotes colorectal cancer progression through sponging miR-1299 to upregulate ADAMTS5.

Long non-coding RNAs (lncRNAs) have been reported to be vital participants in tumor progression. Recently, lncRNA PSMB8-AS1 has been uncovered to facilitate pancreatic cancer progression by regulating miR-382-3p/STAT1/PD-L1 network. Nonetheless, the role of PSMB8-AS1 and its underlying mechanism have not been well-explored in colorectal cancer (CRC). The expression of RNAs or proteins was detected via qRT-PCR or western blot assays. Functional assays were involved in evaluating the effects of PSMB8-AS1/miR-1299/ADAMTS5 on the malignant behaviors of CRC cells. The molecular mechanism of PSMB8-AS1 was explored via mechanism analyses in CRC cells. Based on experimental results, PSMB8-AS1 expression was notably higher in CRC cell lines than in normal cells. The downregulation of PSMB8-AS1 repressed cell viability, proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC while promoting cell apoptosis. It was also revealed that PSMB8-AS1 could sponge miR-1299 to upregulate ADAMTS5 in CRC cells. In rescue assays, we further discovered that miR-1299 inhibition or ADAMTS5 overexpression abrogated the suppressive influence of PSMB8-AS1 deficiency on CRC cell growth. In addition, PSMB8-AS1 was validated to induce M2 polarization. In conclusion, PSMB8-AS1 sponges miR-1299 to increase PSMB8-AS1 expression, thus promoting CRC cell growth.

MicroRNA-148a targets ADAMTS5 to inhibit proliferation of endometriosis cells.

To examine miR-148a expression in the serum of patients with endometriosis (EMS), and to further explore the target of miR-148a in HS832.Tc cells and the effect of miR-148a on the proliferation of Hs832.Tc cells. The serum of non-EMS patients and EMS patients were collected and real-time quantitative PCR (qRT-PCR) was used to detect miR-148a in serum. The EMS cell line Hs832.Tc was cultured and transfected with miR-148aminic, miR-148a inhibitor to construct over expressing and interfering cell lines. Cell viability and apoptosis were detected. The dual luciferase assay identified a target relationship between miR-148a and ADAMTS5 and whether miR-148a regulates proliferation of endometriosis cells via ADAMTS5 was further validated. Compared with normal subjects, miR-148a was significantly reduced in serum of EMS patients (p<0.05). The area under the receiver operating characteristic curve for miR-148a for diagnosis of EMS was 0.91, which was statistically significant (p<0.01). The proliferation of Hs832.Tc cells was significantly inhibited and the cell apoptosis was increased after miR-148a over expression. The proliferation of Hs832.Tc cells was promoted and apoptosis was reduced by miR-148a down regulation. The dual luciferase report demonstrates that ADAMTS5 is a target gene of miR-148a. The addition of ADAMTS5 can directly promote apoptosis of Hs832.Tc cells. The expression of miR-148a in serum of EMS patients was decreased, and miR-148a targets ADAMTS5 to promote apoptosis in EMS cells.

ADAMTSL5 is an epigenetically activated gene underlying tumorigenesis and drug resistance in hepatocellular carcinoma.

BACKGROUND & AIMS: The tumour microenvironment shapes tumour growth through cellular communications that include both direct interactions and secreted factors. The aim of this study was to characterize the impact of the secreted glycoprotein ADAMTSL5, whose role in cancer has not been previously investigated, on hepatocellular carcinoma (HCC). METHODS: ADAMTSL5 methylation status was evaluated through bisulfite sequencing, and publicly available data analysis. ADAMTSL5 RNA and protein expression were assessed in mouse models and HCC patient samples and compared to data from published datasets. Functional studies, including association of ADAMTSL5 depletion with responsiveness to clinically relevant drugs, were performed in cellular and in vivo models. Molecular alterations associated with ADAMTSL5 targeting were determined using proteomics, biochemistry, and reverse-transcription quantitative PCR. RESULTS: Methylome analysis revealed hypermethylated gene body CpG islands at the ADAMTSL5 locus in both mouse and human HCC, correlating with higher ADAMTSL5 expression. ADAMTSL5 targeting interfered with tumorigenic properties of HCC cells in vitro and in vivo, whereas ADAMTSL5 overexpression conferred tumorigenicity to pre-tumoural hepatocytes sensitized to transformation by a modest level of MET receptor expression. Mechanistically, ADAMTSL5 abrogation led to a reduction of several oncogenic inputs relevant to HCC, including reduced expression and/or phosphorylation levels of receptor tyrosine kinases MET, EGFR, PDGFRß, IGF1Rß, or FGFR4. This phenotype was associated with significantly increased sensitivity of HCC cells to clinically relevant drugs, namely sorafenib, lenvatinib, and regorafenib. Moreover, ADAMTSL5 depletion drastically increased expression of AXL, accompanied by a sensitization to bemcentinib. CONCLUSIONS: Our results point to a role for ADAMTSL5 in maintaining the function of key oncogenic signalling pathways, suggesting that it may act as a master regulator of tumorigenicity and drug resistance in HCC. LAY SUMMARY: The environment of cancer cells has profound effects on establishment, progression, and response of a tumour to treatment. Herein, we show that ADAMTSL5, a protein secreted by liver cancer cells and overlooked in cancer so far, is increased in this tumour type, is necessary for tumour formation and supports drug resistance. Adamtsl5 removal conferred sensitivity of liver cancer cells to drugs used in current treatment. This suggests ADAMTSL5 as a potential marker in liver cancer as well as a possible drug target.

Long non-coding RNA XIST regulates chondrogenic differentiation of synovium-derived mesenchymal stem cells from temporomandibular joint via miR-27b-3p/ADAMTS-5 axis.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJOA) is a common degenerative disease in jaw joint, accompanied by articular cartilage destruction. Differentiation of stem cells to cartilage has important therapeutic implications in TMJ cartilage repair. Previous studies revealed that lncRNA XIST participated in various biological processes. However, the effect of XIST on chondrogenic differentiation of synovium-derived mesenchymal stem cells (SMSCs) remains unclear. Our study aimed to investigate the function of XIST in chondrogenic differentiation of human SMSCs from TMJ. METHODS: Alcian blue staining was performed to determine proteoglycan in SMSCs. qPCR, western blotting and immunofluorescence assays were allowed to assess sex determining region Y-box 9 (SOX9), Collagen type II alpha 1 chain (COL2A1) and Aggrecan (ACAN) expression. The direct interaction between miR-27b-3p and XIST or ADAMTS-5 was confirmed by dual luciferase reporter assay or RNA immunoprecipitation (RIP) assay. RESULTS: XIST was remarkably down-regulated in chondrogenic differentiation of SMSCs. Functional analysis demonstrated that XIST silencing promoted chondrogenic differentiation of SMSCs. Dual luciferase reporter and RIP assays identified that XIST acted as a sponge for miR-27b-3p. Moreover, XIST regulated ADAMTS-5 expression by directly binding miR-27b-3p. More importantly, miR-27b-3p/ADAMTS-5 rescued the effects of XIST on chondrogenic differentiation of SMSCs. CONCLUSION: The results suggest that XIST modulates SMSCs chondrogenic differentiation via the miR-27b-3p/ADAMTS-5 axis, which provides new targets for TMJOA treatment.

SNHG5 protects chondrocytes in interleukin-1ß-stimulated osteoarthritis via regulating miR-181a-5p/TGFBR3 axis.

Long noncoding RNAs (lncRNAs) have been considered as important modulators in the development of osteoarthritis. The present study investigates whether there is a link between lncRNA small nucleolar RNA host gene 5 (SNHG5) and osteoarthritis pathogenesis, and the underlying molecular mechanism. To establish an in vitro model of osteoarthritis, interleukin 1ß (IL-1ß) was used to treat chondrocytes (C20/A4 cells) for mimicking the inflammatory condition in osteoarthritis pathogenesis. SNHG5 and miR-181a-5p expression levels were then detected in cartilage tissues of osteoarthritis patients and C20/A4 cells by quantitative polymerase chain reaction (qPCR). Cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays were applied for detecting the viability of chondrocytes, and the apoptosis of chondrocytes was examined through caspase-3 activity assay and flow cytometry analysis. Western blot and qPCR were employed for determining the expression levels of TGFBR3, ADAMTS5, and MMP-13. The regulatory relationships among SNHG5, miR-181a-5p, and TGFBR3 were verified by RNA immunoprecipitation and dual-luciferase reporter assays. The expression levels of SNHG5 and TGFBR3 were markedly decreased, and miR-181a-5p expression was enhanced in osteoarthritis tissues and chondrocytes treated with IL-1ß. SNHG5 knockdown inhibited the viability of chondrocytes, induced apoptosis, and promoted the expression levels of ADAMTS5 and MMP-13. Conversely, SNHG5 overexpression could counteract the effects of IL-1ß, increase the viability of chondrocytes and suppress apoptosis. Mechanically, SNHG5 positively regulated TGFBR3 expression via sponging miR-181a-5p. Moreover, miR-181a-5p overexpression and TGFBR3 knockdown counteracted the effects of SNHG5 on chondrocytes. SNHG5 can probably protect chondrocytes from the inflammatory response and reduce the degradation of the extracellular matrix via modulating the miR-181a-5p/TGFBR3 axis.

IGF-1 Upregulates Biglycan and Decorin by Increasing Translation and Reducing ADAMTS5 Expression.

Proteoglycan (PG) is a glycosaminoglycan (GAG)-conjugated protein essential for maintaining tissue strength and elasticity. The most abundant skin PGs, biglycan and decorin, have been reported to decrease as skin ages. Insulin-like growth factor-1 (IGF-1) is important in various physiological functions such as cell survival, growth, and apoptosis. It is well known that the serum level of IGF-1 decreases with age. Therefore, we investigated whether and how IGF-1 affects biglycan and decorin. When primary cultured normal human dermal fibroblasts (NHDFs) were treated with IGF-1, protein levels of biglycan and decorin increased, despite no difference in mRNA expression. This increase was not inhibited by transcription blockade using actinomycin D, suggesting that it is mediated by IGF-1-induced enhanced translation. Additionally, both mRNA and protein expression of ADAMTS5, a PG-degrading enzyme, were decreased in IGF-1-treated NHDFs. Knockdown of ADAMTS5 via RNA interference increased protein expression of biglycan and decorin. Moreover, mRNA and protein expression of ADAMTS5 increased in aged human skin tissues compared to young tissue. Overall, IGF-1 increases biglycan and decorin, which is achieved by improving protein translation to increase synthesis and preventing ADAMTS5-mediated degradation. This suggests a new role of IGF-1 as a regulator for biglycan and decorin in skin aging process.

Matrix-degrading protease ADAMTS-5 cleaves inter-α-inhibitor and releases active heavy chain 2 in synovial fluids from arthritic patients.

Destruction of the cartilage matrix in joints is an important feature of arthritis. Proteolytic degradation of cartilage glycoproteins can contribute to the loss of matrix integrity. Human inter-α-inhibitor (IαI), which stabilizes the extracellular matrix, is composed of the light-chain serine proteinase inhibitor bikunin and two homologous heavy chains (HC1 and HC2) covalently linked through chondroitin 4-sulfate. Inflammation promotes the transfer of HCs from chondroitin 4-sulfate to hyaluronan by tumor necrosis factor-stimulated gene-6 protein (TSG-6). This reaction generates a covalent complex between the heavy chains and hyaluronan that can promote leukocyte invasion. This study demonstrates that both IαI and the HC-hyaluronan complex are substrates for the extracellular matrix proteases ADAMTS-5 and matrix metalloprotease (MMP) -3, -7, and -13. The major cleavage sites for all four proteases are found in the C terminus of HC2. ADAMTS-5 and MMP-7 displayed the highest activity toward HC2. ADAMTS-5 degradation products were identified in mass spectrometric analysis of 29 of 33 arthropathic patients, indicating that ADAMTS-5 cleavage occurs in synovial fluid in arthritis. After cleavage, free HC2, together with TSG-6, is able to catalyze the transfer of heavy chains to hyaluronan. The release of extracellular matrix bound HC2 is likely to increase the mobility of the HC2/TSG-6 catalytic unit and consequently increase the rate of the HC transfer reaction. Ultimately, ADAMTS-5 cleavage of HC2 could alter the physiological and mechanical properties of the extracellular matrix and contribute to the progression of arthritis.

ADAMTS-5: A difficult teenager turning 20.

A Disintegrin And Metalloproteinase with ThromboSpondin motif (ADAMTS)-5 was identified in 1999 as one of the enzymes responsible for cleaving aggrecan, the major proteoglycan in articular cartilage. Studies in vitro, ex vivo and in vivo have validated ADAMTS-5 as a target in osteoarthritis (OA), a disease characterized by extensive degradation of aggrecan. For this reason, it attracted the interest of many research groups aiming to develop a therapeutic treatment for OA patients. However, ADAMTS-5 proteoglycanase activity is not only involved in the dysregulated aggrecan proteolysis, which occurs in OA, but also in the physiological turnover of other related proteoglycans. In particular, versican, a major ADAMTS-5 substrate, plays an important structural role in heart and blood vessels and its proteolytic processing by ADAMTS-5 must be tightly regulated. On the occasion of the 20th anniversary of the discovery of ADAMTS-5, this review looks at the evidence for its detrimental role in OA, as well as its physiological turnover of cardiovascular proteoglycans. Moreover, the other potential functions of this enzyme are highlighted. Finally, challenges and emerging trends in ADAMTS-5 research are discussed.

Heparan sulfate deficiency leads to hypertrophic chondrocytes by increasing bone morphogenetic protein signaling.

OBJECTIVE: Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext-/- mouse model. METHOD: We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice. RESULTS: Hypertrophic chondrocytes were significantly more abundant (P = 0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P = 0.036 for femur; and P < 0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P = 0.007). CONCLUSIONS: HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.

Inflammation and joint destruction may be linked to the generation of cartilage metabolites of ADAMTS-5 through activation of toll-like receptors.

OBJECTIVE: Links between pain and joint degradation are poorly understood. We investigated the role of activation of Toll-like receptors (TLR) by cartilage metabolites in initiating and maintaining the inflammatory loop in OA causing joint destruction. METHODS: Synovial membrane explants (SMEs) were prepared from OA patients' synovial biopsies. SMEs were cultured for 10 days under following conditions: culture medium alone, OSM + TNFα, TLR2 agonist - Pam2CSK4, Pam3CSK4 or synthetic aggrecan 32-mer, TLR4 agonist - Lipid A. Release of pro-inflammatory and degradation biomarkers (acMMP3 and C3M) were measured by ELISA in conditioned media along with IL-6. Additionally, human cartilage was digested with ADAMTS-5, with or without the ADAMTS-5 inhibiting nanobody - M6495. Digested cartilage solution (DCS) and synthetic 32-mer were tested for TLR activation in SEAP based TLR reporter assay. RESULTS: Western blotting confirmed TLR2 and TLR4 in untreated OA synovial biopsies. TLR agonists showed an increase in release of biomarkers - acMMP3 and C3M in SME. Synthetic 32-mer showed no activation in the TLR reporter assay. ADAMTS-5 degraded cartilage fragments activated TLR2 in vitro. Adding M6495 - an anti-ADAMTS-5 inhibiting nanobody®, blocked ADAMTS-5-mediated DCS TLR2 activation. CONCLUSION: TLR2 is expressed in synovium of OA patients and their activation by synthetic ligands causes increased tissue turnover. ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments which activates the TLR2 receptor in vitro. M6495 suppressed cartilage degradation by ADAMTS-5, limiting the activation of TLR2. In conclusion, pain and joint destruction may be linked to generation of ADAMTS-5 cartilage metabolites.

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.

NF-Ä¸ß upregulates ADAMTS5 expression by direct binding after TNF-α treatment in OUMS-27 chondrosarcoma cell line.

Inflammation caused-aggrecan degradation is a critical event in the pathogenesis of osteoarthritis (OA). The aggrecanases like a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) are assumed to be key players in the aggrecan destruction. To develop the comprehensive therapy method for OA, it is essential to elucidate the activation mechanism of ADAMTS5 gene after stimulation of inflammatory cytokines like tumor necrosis factor-α (TNF-α). The cell lines of human chondrosarcoma (OUMS-27) and embryonic kidney (HEK293T) were incubated with tumor necrosis factor-α (TNF-α) for certain time periods, and the expression level of ADAMTS5 was measured in both mRNA and protein levels. Tissue-specific ADAMTS5 activation was founded to be induced after TNF-α treatment. Then, the constructs for the promoter region of ADAMTS5 were prepared and luciferase assay was conducted to understand the involvement mechanism of nuclear factor-kappa beta (NF-ĸß) in ADAMTS5 activation. It was demonstrated that NF-Ä¸ß induces the ADAMTS5 expression level by directly binding the promoter region of ADAMTS5. Although the TNF-α blocker is used for OA treatment, the development of a more comprehensive treatment strategy is an urgent need. Our experimental data contributes in terms of selecting NF-Ä¸ß as a target molecule. Up to date, NF-Ä¸ß has been proven to involve in the ADAMTS5 up-regulation after several pro-inflammatory cytokines stimulation. In conclusion, our findings make important contributions to the knowledge about the roles of NF-Ä¸ß in ADAMTS5 activation under inflammatory conditions. So, NF-Ä¸ß could be considered to be a potential target for OA treatment.

Hydrostatic Pressure Regulates Oxidative Stress through microRNA in Human Osteoarthritic Chondrocytes.

Hydrostatic pressure (HP) modulates chondrocytes metabolism, however, its ability to regulate oxidative stress and microRNAs (miRNA) has not been clarified. The aim of this study was to investigate the role of miR-34a, miR-146a, and miR-181a as possible mediators of HP effects on oxidative stress in human osteoarthritis (OA) chondrocytes. Chondrocytes were exposed to cyclic low HP (1-5 MPa) and continuous static HP (10 MPa) for 3 hrs. Metalloproteinases (MMPs), disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5, type II collagen (Col2a1), miR-34a, miR-146a, miR-181a, antioxidant enzymes, and B-cell lymphoma 2 (BCL2) were evaluated by quantitative real-time polymerase chain reaction qRT-PCR, apoptosis and reactive oxygen species ROS production by cytometry, and ß-catenin by immunofluorescence. The relationship among HP, the studied miRNA, and oxidative stress was assessed by transfection with miRNA specific inhibitors. Low cyclical HP significantly reduced apoptosis, the gene expression of MMP-13, ADAMTS5, miRNA, the production of superoxide anion, and mRNA levels of antioxidant enzymes. Conversely, an increased Col2a1 and BCL2 genes was observed. ß-catenin protein expression was reduced in cells exposed to HP 1-5 MPa. Opposite results were obtained following continuous static HP application. Finally, miRNA silencing enhanced low HP and suppressed continuous HP-induced effects. Our data suggest miRNA as one of the mechanisms by which HP regulates chondrocyte metabolism and oxidative stress, via Wnt/ß-catenin pathway.

The Anti-ADAMTS-5 Nanobody® M6495 Protects Cartilage Degradation Ex Vivo.

Osteoarthritis (OA) is associated with cartilage breakdown, brought about by ADAMTS-5 mediated aggrecan degradation followed by MMP-derived aggrecan and type II collagen degradation. We investigated a novel anti-ADAMTS-5 inhibiting Nanobody® (M6495) on cartilage turnover ex vivo. Bovine cartilage (BEX, n = 4), human osteoarthritic - (HEX, n = 8) and healthy-cartilage (hHEX, n = 1) explants and bovine synovium and cartilage were cultured up to 21 days in medium alone (w/o), with pro-inflammatory cytokines (oncostatin M (10 ng/mL) + TNFα (20 ng/mL) (O + T), IL-1α (10 ng/mL) or oncostatin M (50 ng/mL) + IL-1ß (10 ng/mL)) with or without M6495 (1000-0.46 nM). Cartilage turnover was assessed in conditioned medium by GAG (glycosaminoglycan) and biomarkers of ADAMTS-5 driven aggrecan degradation (huARGS and exAGNxI) and type II collagen degradation (C2M) and formation (PRO-C2). HuARGS, exAGNxI and GAG peaked within the first culture week in pro-inflammatory stimulated explants. C2M peaked from day 14 by O + T and day 21 in co-culture experiments. M6495 dose dependently decreased huARGS, exAGNxI and GAG after pro-inflammatory stimulation. In HEX C2M was dose-dependently reduced by M6495. M6495 showed no effect on PRO-C2. M6495 showed cartilage protective effects by dose-dependently inhibiting ADAMTS-5 mediated cartilage degradation and inhibiting overall cartilage deterioration in ex vivo cartilage cultures.