REST, RCOR1 and RCOR2 expression is reduced in osteoarthritic chondrocytes and contributes to increasing MMP13 and ADAMTS5 expression through upregulating HES1.

Expression of key transcriptional regulators is altered in chondrocytes in osteoarthritis (OA). This contributes to an increase in production of cartilage-catabolizing enzymes such as MMP13 and ADAMTS5. RCOR1 and RCOR2, binding partners for the transcriptional repressor REST, have previously been found to be downregulated in OA chondrocytes although their function in chondrocytes is unclear. HES1 is a known REST/RCOR1 target gene and HES1 has been shown to promote MMP13 and ADAMTS5 expression in murine OA chondrocytes. The purpose of this study was to determine whether reduced REST/RCOR levels leads to increased HES1 expression in human OA chondrocytes and whether HES1 also promotes ADAMTS5 and MMP13 expression in these cells. Chondrocytes were isolated from osteoarthritic and adjacent macroscopically normal cartilage obtained from patients undergoing total knee arthroplasty. RNA and protein levels of REST, RCOR1 and RCOR2 were lower, but levels of HES1 higher, in chondrocytes isolated from osteoarthritic compared to macroscopically normal cartilage. Over-expression of either REST, RCOR1 or RCOR2 resulted in reduced HES1 levels in OA chondrocytes whereas knockdown of REST, RCOR1 or RCOR2 led to increased HES1 expression in chondrocytes from macroscopically normal cartilage. In OA chondrocytes, ADAMTS5 and MMP13 expression were reduced following HES1 knockdown, but further enhanced following HES1 over-expression. Levels of phosphorylated CaMKII were higher in chondrocytes from OA cartilage consistent with previous findings that HES1 only promotes gene transcription in the presence of active CaMKII. These findings identify the REST/RCOR/HES1 pathway as a contributing factor leading to increased ADAMTS5 and MMP13 expression in OA chondrocytes.

Synergetic enrichment of aggrecan in nucleus pulposus cells by scAAV6-shRNA-mediated knockdown of aggrecanase-1 and aggrecanase-2.

Degenerative disk disease (DDD) that aggravates structural deterioration of intervertebral disks (IVDs) can be accompanied by painful inflammation and immunopathological progressions. Current surgical or pharmacological therapies cannot repair the structure and function of IVDs. Nucleus pulposus (NP) cells are crucial for the preservation or restoration of IVDs by balancing the anabolic and catabolic factors affecting the extracellular matrix. Imbalanced anabolic and catabolic factors cause increased degradation of aggrecan. Aggrecanases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)4 and ADAMTS5 are the main degrading enzymes of aggrecan. Previously, we characterized adeno-associated virus (AAV6) as the most suitable serotype with marked NP cellular tropism and demonstrated that ADAMTS4 could be silenced by self-complementary adeno-associated virus grade 6 small helix ribonucleic acid (scAAV6-shRNA) in NP cells of degeneration grade III, which resulted in enrichment of aggrecan. Nonetheless, neither scAAV6-shRNA-mediated inhibition of ADAMTS5 nor joint inhibitions of ADAMTS4 and ADAMTS5 have been investigated, although both enzymes are regulated by analogous proinflammatory cytokines and have the same cleavage sites in aggrecan. Therefore, we attempted scAAV6-shRNA-mediated inhibitions of both enzymes in NP cells of degeneration grade IV to increase efficacies in treatments of DDD. The degeneration grade of IVDs in patients was determined by magnetic resonance imaging (MRI) before surgical operations. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting ADAMTS4 or ADAMTS5. Transduced cells were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence microscopy, flow cytometry-assisted cell sorting (FACS), MTT assay (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay), immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Joint transduction of NP cells exhibited high transduction efficacies (98.1%), high transduction units (TU) (1381 TU/Cell), and no effect on cell viability or proliferation. Above all joint treatments resulted in effective knockdown of ADAMTS4 (92.8%) and ADAMTS5 (93.4%) along with additive enrichment of aggrecan (113.9%). Treatment effects were significant for more than 56 days after transduction (P < 0.001). In conclusion, scAAV6-shRNA-mediated combined molecular therapy could be very valuable for more effective, durable, and less immunogenic treatment approaches in DDD.

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.

Loss of MEN1 leads to renal fibrosis and decreases HGF-Adamts5 pathway activity via an epigenetic mechanism.

BACKGROUND: Renal fibrosis is a serious condition that results in the development of chronic kidney diseases. The MEN1 gene is an epigenetic regulator that encodes the menin protein and its role in kidney tissue remains unclear. METHODS: Kidney histology was examined on paraffin sections stained with hematoxylin-eosin staining. Masson's trichrome staining and Sirius red staining were used to analyze renal fibrosis. Gene and protein expression were determined by quantitative real-time PCR (qPCR) and Western blot, respectively. Immunohistochemistry staining in the kidney tissues from mice or patients was used to evaluate protein levels. Flow cytometry was used to analyze the cell cycle distributions and apoptosis. RNA-sequencing was performed for differential expression genes in the kidney tissues of the Men1f/f and Men1∆/∆ mice. Chromatin immunoprecipitation sequencing (ChIP-seq) was carried out for identification of menin- and H3K4me3-enriched regions within the whole genome in the mouse kidney tissue. ChIP-qPCR assays were performed for occupancy of menin and H3K4me3 at the gene promoter regions. Luciferase reporter assay was used to detect the promoter activity. The exacerbated unilateral ureteral obstruction (UUO) models in the Men1f/f and Men1∆/∆ mice were used to assess the pharmacological effects of rh-HGF on renal fibrosis. RESULTS: The expression of MEN1 is reduce in kidney tissues of fibrotic mouse and human diabetic patients and treatment with fibrotic factor results in the downregulation of MEN1 expression in renal tubular epithelial cells (RTECs). Disruption of MEN1 in RTECs leads to high expression of α-SMA and Collagen 1, whereas MEN1 overexpression restrains epithelial-to-mesenchymal transition (EMT) induced by TGF-ß treatment. Conditional knockout of MEN1 resulted in chronic renal fibrosis and UUO-induced tubulointerstitial fibrosis (TIF), which is associated with an increased induction of EMT, G2/M arrest and JNK signaling. Mechanistically, menin recruits and increases H3K4me3 at the promoter regions of hepatocyte growth factor (HGF) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (Adamts5) genes and enhances their transcriptional activation. In the UUO mice model, exogenous HGF restored the expression of Adamts5 and ameliorated renal fibrosis induced by Men1 deficiency. CONCLUSIONS: These findings demonstrate that MEN1 is an essential antifibrotic factor in renal fibrogenesis and could be a potential target for antifibrotic therapy.

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.

Selection and characterization of DNA aptamers inhibiting a druggable target of osteoarthritis, ADAMTS-5.

There is a critical need for the development of more potent inhibitors for osteoarthritis (OA) therapy given the poor life quality of arthritis patients. Aggrecanase ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) is an established drug target identified for osteoarthritis. In this study, we evolved and characterized two new DNA aptamer inhibitors of ADAMTS-5, namely apt21 and apt25. The aptamers exhibited nanomolar binding affinity and high specificity against ADAMTS-5. KD values of apt21 and apt25 were determined by the Enzyme-linked Oligonucleotide Assay (ELONA) at 1.54 ± 0.16 nM and 1.79 ± 0.08 nM, respectively. Circular Dichroism (CD) analysis demonstrated that both aptamers formed monovalent cation dependent G-quadruplex structures. Calcium ions did not affect the binding of the aptamers to ADAMTS-5. The inhibitory effects of apt21 and apt25 on ADAMTS-5 were evaluated by the Förster Resonance Energy Transfer (FRET) assay, in which IC50 values of apt21 and apt25 were estimated at 52.76 ± 6.70 µM and 61.14 ± 9.67 µM, respectively. These two aptamers are the first DNA G-quadruplex aptamers demonstrated to inhibit ADAMTS-5 and could have value for OA therapy.

Silencing of circ_0000205 mitigates interleukin-1ß-induced apoptosis and extracellular matrix degradation in chondrocytes via targeting miR-766-3p/ADAMTS5 axis.

The aim of this study was to explore the role of hsa_circRNA_0000205 (circ_0000205) in chondrocyte injury in osteoarthritis (OA) and the underlying mechanism. Expression of circ_0000205, microRNA (miR)-766-3p and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 was detected by quantitative real time (qRT)-polymerase chain reaction (PCR) and Western blot assays. Cell proliferation, apoptosis, and extracellular matrix (ECM) synthesis were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 5-ethynyl-2-deoxyuridine assays, flow cytometry, and qRT-PCR and Western blot assays. The target relationship between miR-766-3p and circ_0000205 or ADAMTS5 was confirmed by luciferase reporter assay and RNA immunoprecipitation. IL-1ß treatment could attenuate cell viability of primary chondrocytes and proliferating cell nuclear antigen (PCNA) and collagen II type alpha-1 (COL2A1) levels, and elevate apoptosis rate and cleaved caspase-3, ADAMTS5 and matrix metalloproteinase-13 (MMP13) levels, suggesting that IL-1ß induced chondrocyte apoptosis and ECM degradation. Expression of circ_0000205 was up-regulated in OA tissues and IL-1ß-induced primary chondrocytes, accompanied with miR-766-3p down-regulation and ADAMTS5 up-regulation. Knockdown of circ_0000205 could mitigate IL-1ß-induced above effects and improve cell proliferation. Moreover, both depleting miR-766-3p and promoting ADAMTS5 could partially counteract circ_0000205 knockdown roles in IL-1ß-cultured primary chondrocytes. Notably, circ_0000205 was verified as a sponge for miR-766-3p via targeting, and ADAMTS5 was a direct target for miR-766-3p. Silencing circ_0000205 could protect chondrocytes from IL-1ß-induced proliferation reduction, apoptosis, and ECM degradation by targeting miR-766-3p/ADAMTS5 axis.

Differential DNA methylation analysis of SUMF2, ADAMTS5, and PXDN provides novel insights into colorectal cancer prognosis prediction in Taiwan.

BACKGROUND: Patients with colorectal cancer (CRC) undergo surgery, as well as perioperative chemoradiation or adjuvant chemotherapy primarily based on the tumor-node- metastasis (TNM) cancer staging system. However, treatment responses and prognostic outcomes of patients within the same stage vary markedly. The potential use of novel biomarkers can improve prognostication and shared decision making before implementation into certain therapies. AIM: To investigate whether SUMF2, ADAMTS5, and PXDN methylation status could be associated with CRC prognosis. METHODS: We conducted a Taiwanese cohort study involving 208 patients with CRC recruited from Tri-Service General Hospital and applied the candidate gene approach to identify three genes involved in oncogenesis pathways. A methylation-specific polymerase chain reaction (MS-PCR) and EpiTYPER DNA methylation analysis were employed to detect methylation status and to quantify the methylation level of candidate genes in tumor tissue and adjacent normal tissue from participants. We evaluated SUMF2, ADAMTS5, and PXDN methylation as predictors of prognosis, including recurrence-free survival (RFS), progression-free survival (PFS), and overall survival (OS), using a Cox regression model and Kaplan-Meier analysis. RESULTS: We revealed various outcomes related to methylation and prognosis. Significantly shorter PFS and OS were associated with the CpG_3+CpG_7 hypermethylation of SUMF2 from tumor tissue compared with CpG_3+CpG_7 hypomethylation [hazard ratio (HR) = 2.24, 95% confidence interval (CI) = 1.03-4.85 for PFS, HR = 2.56 and 95%CI = 1.08-6.04 for OS]. By contrast, a significantly longer RFS was associated with CpG_2 and CpG_13 hypermethylation of ADAMTS5 from normal tissue compared with CpG_2 and CpG_13 hypomethylation [HR (95%CI) = 0.15 (0.03-0.71) for CpG_2 and 0.20 (0.04-0.97) for CpG_13]. The relationship between the methylation status of PXDN and the prognosis of CRC did not reach statistical significance. CONCLUSION: Our study found that CpG_3+CpG_7 hypermethylation of SUMF2 from tumor tissue was associated with significantly shorter PFS and OS compared with CpG_3+CpG_7 hypomethylation. CpG_2 and CpG_13 hypermethylation of ADAMTS5 from normal tissue was associated with a significantly longer RFS compared with CpG_2 and CpG_13 hypomethylation. These methylation-related biomarkers which have implications for CRC prognosis prediction may aid physicians in clinical decision-making.

Knockdown of circ-PRKCH alleviates IL-1ß-treated chondrocyte cell phenotypic changes through modulating miR-502-5p/ADAMTS5 axis.

BACKGROUND: Osteoarthritis (OA) is a common joint disease characterized by progressive cartilage degradation. Circular RNAs (circRNAs) are involved in the initiation and development of OA. This study aimed to explore the potential role and mechanism of circRNA protein kinase C eta (circ-PRKCH) in OA. METHODS: A total of 30 cartilage specimens were collected from OA patients or normal subjects. Human chondrocytes (CHON-001) were stimulated with interleukin-1ß (IL-1ß) to establish an in vitro OA model. The expression levels of circ-PRKCH, microRNA-502-5p (miR-502-5p) and circ-PRKCH or A disintegrin and metalloproteases metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) in cartilage specimens and IL-1ß-treated chondrocytes were detected by quantitative real-time PCR or Western blot, and their correlation in OA cartilage specimens was analysed by Spearman's correlation coefficient. The targeted relationship between miR-502-5p and circ-PRKCH or ADAMTS5 was verified by dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EDU), flow cytometry, wound healing and enzyme-linked immunosorbent assay (ELISA) assays were applied to evaluate cell proliferation, apoptosis, migration and inflammatory response in IL-1ß-treated chondrocytes. Exosomes were identified by transmission electron microscope (TEM) and Western blot. RESULTS: Circ-PRKCH and ADAMTS5 expression levels were up-regulated, while miR-502-5p expression was down-regulated in OA cartilage tissues and IL-1ß-treated chondrocytes. Depletion of circ-PRKCH relieved IL-1ß-treated chondrocyte cell phenotypic changes by promoting cell proliferation and migration, as well as inhibiting apoptosis and inflammatory response. Mechanically, circ-PRKCH acted as a sponge for miR-502-5p to regulate ADAMTS5 expression, thereby contributing to IL-1ß-treated chondrocyte cell phenotypic changes. Moreover, exosomes derived from IL-1ß-treated chondrocytes could transfer circ-PRKCH across cells. CONCLUSION: Circ-PRKCH contributed to IL-1ß-treated cell phenotypic changes in chondrocytes via modulating miR-502-5p/ADAMTS5 pathway, which might provide a promising biomarker for OA treatment.

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.

Elevation of MMP1 and ADAMTS5 mRNA expression in glenohumeral synovia of patients with hypercholesterolemia.

BACKGROUND: Epidemiological studies have reported a positive association between hypercholesterolemia and shoulder disease. Previous studies have focused on the effect of hypercholesterolemia on tendinopathy. Moreover, hypercholesterolemia has also been linked to joint pathology in the knee and hand. However, the effect of hyperlipidemia on glenohumeral joint remain unclear. A hypercholesterolemic condition has been reported to alter levels of A Disintegrin and Metalloprotease with Thrombospondin Motifs (ADAMTSs) and matrix metalloproteases (MMPs) in synovium of the knee joint. Here, we evaluated the mRNA expression of ADAMTSs and MMPs in the glenohumeral synovium of patients with and without hypercholesterolemia. METHODS: Study participants were 73 patients who underwent arthroscopic rotator cuff repair for degenerative rotator cuff tears. They were divided into two groups according to total cholesterol (TC) and triglyceride levels. Synovial membrane samples were harvested at the rotator interval during surgery, and mRNA expression levels of the aggrecanases ADAM-TS4 and ADAM-TS5 and MMPs (MMP-1, 3, 9, and 13) were analyzed quantitatively. RESULTS: ADAM-TS5 and MMP1 mRNA levels were significantly higher in the high TC group than in the low TC group (P = 0.023 and P = 0.025, respectively). In contrast, no significant differences were observed in ADAMTS4 or MMPs 3, 9, and 13 (ADAMTS4, P = 0.547; MMP3, P = 0.55; MMP9, P = 0.521; and MMP13, P = 0.785). CONCLUSION: Hypercholesterolemia may alter MMP1 and ADAMTS5 expression in the synovium of the glenohumeral joint.

Circular RNA circ_0020014 contributes to osteoarthritis progression via miR-613/ADAMTS5 axis.

Circular RNAs (circRNAs) have been shown to be significant regulators in osteoarthritis (OA), whereas the functional effect of circ_0020014 in OA remains unclear. Our goal was to try and understand the underlying regulatory mechanism of circ_0020014 in OA. The cartilage tissue was obtained from OA patients and trauma patients. Interleukin-1ß (IL-1ß)-treated chondrocytes (CHON-001) were used as the in vitro cellular model for OA. The expression levels of circ_0020014, microRNA-613 (miR-613), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were examined by real-time quantitative polymerase chain reaction (RT-qPCR). The protein level was detected using the western blot assay. Cell viability and apoptosis were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) and flow cytometry assays, respectively. The secretion of inflammatory cytokine was determined by enzyme-linked immunosorbent assay (ELISA). Circ_0020014 was upregulated in OA cartilage tissues and IL-1ß-treated CHON-001 cells, compared with that in healthy cartilage tissues and untreated cells. IL-1ß treatment induced cell injury by promoting inflammation and apoptosis, and inhibiting cell viability and extracellular matrix (ECM) accumulation in chondrocytes. Circ_0020014 knockdown significantly protected CHON-001 cells from IL-1ß-induced cell dysfunction. MiR-613 was targeted by circ_0020014 and negatively regulated ADAMTS5 expression. In addition, miR-613 downregulation or ADAMTS5 overexpression partly lessened the protective effect of circ_0020014 knockdown on IL-1ß-treated CHON-001 cells. Collectively, circ_0020014 acted as a miR-613 sponge to regulate ADAMTS5 expression, thereby protecting chondrocytes from IL-1ß-induced inflammatory damage, which might be a novel diagnostic marker for OA.

IL-33 induces ADAMTS5 expression and cell migration in glioblastoma multiforme.

Glioblastoma multiforme (GBM), characterized by a high rate of proliferation and migration capacity, is an incurable brain tumor in adults. Interleukin-33 (IL-33), a member of the IL-1 cytokine superfamily, and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), a family of zinc dependent metalloproteinases, are known to have essential roles in GBM migration and invasion. Previous studies have separately revealed elevated expressions of IL-33 and ADAMTS5 in GBM; however, the interaction between IL-33 and ADAMTS5 in GBM remains unclear. Here, using publically available GlioVis and GEPIA programs, we showed that mRNA expressions of IL-33 and ADAMTS5 are significantly high in GBM cells, and a positive correlation between IL-33 and ADAMTS5 was also determined in these cells. In parallel with the mRNA data of IL-33 and ADAMTS5, by Western blot analysis, protein levels were found to be elevated in GBM tissues and increased gradually with the disease progression. Primary GBM cells and low-grade glioma cells were then treated with IL-33 to examine its stimulating effect on ADAMTS5 expression. Exposure to IL-33 raised ADAMTS5 protein levels in a dose-dependent manner. Finally, the wound-healing method was performed to confirm the impact of IL-33 on migration in primary GBM cells. IL-33 promoted migration of primary GBM cells three times higher than untreated GBM cells. Thus, the current study suggests for the first time that IL-33 might have a role in playing a part in GBM progression through induction of ADAMTS5 expression and promotion of migration in GBM cells.

Fermentation supernatant of Staphylococcus aureus drives catabolism in chondrocytes via NF-κB signaling mediated increase of cholesterol metabolism.

Septic arthritis induced by Staphylococcus aureus (S. aureus) causes irreversible cartilage degradation and subsequent permanent joint dysfunction. Recently, cartilage degradation in osteoarthritis is recognized to be associated with metabolic disorders. However, whether cholesterol metabolism is linked to septic arthritis pathology remains largely unknown. Here, we found that exposure to fermentation supernatant (FS) of S. aureus in chondrocytes resulted in a significant increase in expression of key modulators involved in cholesterol metabolism, including lectin-type oxidized low density lipoprotein receptor 1 (LOX1), cholesterol 25-hydroxylase (CH25H), 25- hydroxycholesterol 7α-hydroxylase (CYP7B1) as well as retinoic acid-related orphan receptor alpha (RORα), a binding receptor for cholesterol metabolites. We further demonstrated that enhancement of CH25H/CYP7B1/RORα axis resulted from FS exposure was mediated by activation of NF-κB signaling, along with upregulation in catabolic factors including matrix metallopeptidases (MMP3 and MMP13), aggrecanase-2 (ADAMTS5), and nitric oxide synthase-2 (NOS2) in chondrocytes. Exogenous cholesterol acts synergistically with FS in activating NF-κB pathway and increases cholesterol metabolism. While, the addition of tauroursodeoxycholic acid (TUDCA) which promotes cholesterol efflux, resulted in remarkable reduction of intracellular cholesterol level and restoration of balance between anabolism and catabolism in FS treated chondrocytes. Collectively, our data indicated that, in response to FS of S. aureus, NF-κB signaling activation coupled with increased cholesterol metabolism to stimulate catabolic factors in chondrocytes, highlighting cholesterol metabolism as a potential therapeutic target for treating septic arthritis.

Association of polymorphisms in ADAMTS-5 gene with the susceptibility to knee osteoarthritis: A protocol for systematic review and meta-analysis.

BACKGROUND: To systematically review literature evidence to discover the association of ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin-like motifs 5) gene polymorphisms and the risk of developing KOA (knee osteoarthritis). METHODS: We systematically searched the related randomized controlled trials in 4 databases from inception to August 2021, including the Embase, Web of Science, PubMed, and CNKI (Chinese National Knowledge Infrastructure) databases. No language and publication status restrictions. Two reviewers will independently screen all included studies, and the meta-analysis will be conducted using the Review Manager (RevMan 5.3, Cochrane Collaboration, Nordic Cochrane Center, Copenhagen, Denmark). RESULTS: The gathered evidence suggests that there may be a close relationship between the SNP in the ADAMTS5 gene and KOA development. This study will provide a high-quality and convincing evaluation of the treatment of KOA from the consideration of ADAMTS5 gene and will be published in a peer-reviewed journal. CONCLUSION: ADAMTS5 polymorphism is likely an important risk factor for the development of KOA. Our study will provide a more accurate treatment method for the treatment of KOA. TRIAL REGISTRATION NUMBER: CRD42021276317.

The Decisive Case-Control Study Elaborates the Null Association between ADAMTS5 rs226794 and Osteoarthritis in Asians: A Case-Control Study and Meta-Analysis.

BACKGROUND: Osteoarthritis is an important health issue for the elderly. Many studies indicate that genetics is an important risk factor for osteoarthritis, and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) is one gene that is most frequently implicated. Many recent studies have examined the relationship between a polymorphism in the ADAMTS5 gene (rs226794) and the risk for developing osteoarthritis without definitive results. OBJECTIVE: In this case-control study, we examined the correlation between the ADAMTS5 gene polymorphism, rs226794, and knee osteoarthritis. We used a meta-analysis and trial sequential analysis to determine whether ADAMTS5 rs226794 expression increases susceptibility to osteoarthritis. METHODS: This study consisted of two parts: a case-control study and a meta-analysis. The case-control study included subjects who underwent knee radiography at the Health Examination Center of the Tri Service General Hospital from 2015 to 2019. The Kellgren-Lawrence (KL) grading system was used as diagnostic criteria. Patients with unsuccessful gene sequencing were excluded. There were 606 subjects in the knee osteoarthritis group (KL ≥ 2) and 564 in the control group (KL < 2). Gene sequencing was performed using iPLEX Gold to determine the association between the gene polymorphism of ADAMTS5 rs226794 and knee osteoarthritis. For the meta-analysis, databases such as PubMed, Embase, and Cochrane were queried to identify studies that examined the relationship between ADAMTS5 rs226794 and osteoarthritis. Next, the findings of the meta-analysis were incorporated with the results of the case-control study and samples from the published studies to estimate the association between the genetic polymorphism and osteoarthritis using an odds ratio and a 95% confidence interval. RESULTS: We found a non-significant association between the G allele and knee OA (crude-OR: 0.93 (95% CI: 0.79-1.10) and adjusted-OR: 1.02 (95% CI: 0.76-1.36) in the allele model) in the present study, and the analysis of other genetic models revealed a similar trend. After including five published studies and our case-control study, the results with 2866 Asians indicated a conclusively null association between ADAMTS5 rs226794 and knee OA) OR: 1.09 (95% CI: 0.93-1.26). The results for Caucasians also revealed a null association (OR: 1.21 (95% CI: 0.81-1.82)). CONCLUSIONS: This study indicates that the gene polymorphism, ADAMTS5 rs226794, is not significantly associated with knee osteoarthritis. Additionally, assuming that the cumulative sample size in the allele model is sufficient, we confirmed that the G allele is not a risk factor for osteoarthritis. This study integrated all available evidence to arrive at this conclusion, and it suggests that no additional studies are necessary.

LncRNA MIR22HG promotes osteoarthritis progression via regulating miR-9-3p/ADAMTS5 pathway.

Dysregulation of long non-coding RNAs (lncRNAs) plays a fundamental role in the development and progression of osteoarthritis (OA), but the potential functions of lncRNAs in OA were not fully clarified. In the present work, we want to clarify the underlying functions and mechanisms of MIR22HG in OA. qRT-PCR was employed to detect the mRNA expression of MIR22HG, miR-9-3p, and ADAMTS5, while the protein expressions were measured using Western blot. The cell proliferation was examined through CCK8, while apoptosis was used in flow cytometry. Luciferase reporter assay and RNA immunoprecipitation (RIP) assays were undertaken to investigate the binding relationship among MIR22HG, ADAMTS5, and miR-9-3p. MIR22HG was significantly overexpressed in OA cartilages, OA chondrocytes and IL-1ß-induced chondrocytes. Functionally, MIR22HG knockdown promoted cell proliferation, suppressed apoptosis, and contributed to downregulation of MMP13 and ADAMTS5 and upregulation of COL2A1 and ACAN in IL-1ß-stimulated chondrocytes. Mechanistically, bioinformatic analysis indicated that MIR22HG may serve as a sponge for miR-9-3p and ADAMTS5 may be a potential targeted gene for miR-9-3p, which were subsequently verified through a dual-luciferase reporter assay. Moreover, rescue experiments showed that MIR22HG participated in the regulation of chondrocytes proliferation, apoptosis, and degradation of extracellular matrix via miR-9-3p/ADAMTS5 pathway. In conclusion, our findings illuminated that inhibition of MIR22HG ameliorated IL-1ß-induced apoptosis and ECM degradation of human chondrocytes through miR-9-3p/ADAMTS5 pathway, which may provide a potentially promising target for OA treatment.

Circ-SPG11 knockdown hampers IL-1ß-induced osteoarthritis progression via targeting miR-337-3p/ADAMTS5.

BACKGROUND: Osteoarthritis (OA) is responsible for the impotent disability in old people. Circular RNA (circRNA) has been reported to be related to the development of diseases. The lack of research on the role of circRNA spastic paraplegia 11 (circ-SPG11) results in conducting this study. METHODS: The expression of circ-SPG11, microRNA-337-3p (miR-337-3p), and aggrecanases like a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was used to measure the protein expression of extracellular matrix (ECM) degradation-related markers and ADAMTS5. Ribonuclease R (RNase R) was applied to test the stability of circ-SPG11 in CHON-001 cells. The viability, apoptosis, TNF-α and IL-6 production were determined by cell counting kit-8 (CCK-8) assay, flow cytometry assay, and enzyme-linked immunosorbent assay (ELISA), respectively. Meanwhile, the interaction between miR-337-3p and circ-SPG11 or ADAMTS5 was respectively predicted by Circinteractome or Starbase2.0, which was further verified by dual-luciferase reporter system and RNA binding protein immunoprecipitation (RIP) assay. RESULTS: Circ-SPG11 and ADAMTS5 were upregulated and miR-337-3p was downregulated in OA tissues and OA model cells. Circ-SPG11 knockdown allayed interleukin 1ß (IL-1ß)-induced restraint in viability and promotion in apoptosis, TNF-α, and IL-6 generation and ECM degradation in CHON-001 cells. Anti-miR-337-3p or ADAMTS5 overexpression correspondingly reversed si-circ-SPG11 or miR-337-3p overexpression-mediated facilitation in viability, and inhibition in apoptosis, TNF-α and IL-6 generation and ECM degradation in OA model cells. Moreover, anti-miR-337-3p ameliorated si-circ-SPG11-mediated inhibition in ADAMTS5 mRNA and protein expression in OA model cells. CONCLUSION: Circ-SPG11 facilitated OA development via regulating miR-337-3p/ADAMTS5 axis. This finding might contribute to the improvement of OA therapy.

Internal modulation of proteolysis in vascular extracellular matrix remodeling: role of ADAM metallopeptidase with thrombospondin type 1 motif 5 in the development of intracranial aneurysm rupture.

Intracranial aneurysms (IAs) are common cerebrovascular diseases that carry a high mortality rate, and the mechanisms that contribute to IA formation and rupture have not been elucidated. ADAMTS-5 (ADAM Metallopeptidase with Thrombospondin Type 1 Motif 5) is a secreted proteinase involved in matrix degradation and ECM (extracellular matrix) remodeling processes, and we hypothesized that the dysregulation of ADAMTS-5 could play a role in the pathophysiology of IA. Immunofluorescence revealed that the ADAMTS-5 levels were decreased in human and murine IA samples. The administration of recombinant protein ADAMTS-5 significantly reduced the incidence of aneurysm rupture in the experimental model of IA. IA artery tissue was collected and utilized for histology, immunostaining, and specific gene expression analysis. Additionally, the IA arteries in ADAMTS-5-administered mice showed reduced elastic fiber destruction, proteoglycan accumulation, macrophage infiltration, inflammatory response, and apoptosis. To further verify the role of ADAMTS-5 in cerebral vessels, a specific ADAMTS-5 inhibitor was used on another model animal, zebrafish, and intracranial hemorrhage was observed in zebrafish embryos. In conclusion, our findings indicate that ADAMTS-5 is downregulated in human IA, and compensatory ADAMTS-5 administration inhibits IA development and rupture with potentially important implications for treating this cerebrovascular disease.

BMP5 silencing inhibits chondrocyte senescence and apoptosis as well as osteoarthritis progression in mice.

In this study, we using the in vivo destabilization of the medial meniscus (DMM) mouse model to investigate the role of bone morphogenetic protein 5 (BMP5) in osteoarthritis (OA) progression mediated via chondrocyte senescence and apoptosis. BMP5 expression was significantly higher in knee articular cartilage tissues of OA patients and DMM model mice than the corresponding controls. The Osteoarthritis Research Society International scores based on histological staining of knee articular cartilage sections were lower in DMM mice where BMP5 was knocked down in chondrocytes than the corresponding controls 4 weeks after DMM surgery. DMM mice with BMP5-deficient chondrocytes showed reduced levels of matrix-degrading enzymes such as MMP13 and ADAMTS5 as well as reduced cartilage destruction. BMP5 knockdown also decreased chondrocyte apoptosis and senescence by suppressing the activation of p38 and ERK MAP kinases. These findings demonstrate that BMP5 silencing inhibits chondrocyte senescence and apoptosis as well as OA progression by downregulating activity in the p38/ERK signaling pathway.