Expression Of Matrix-degrading Enzymes Research Articles

Targeting ROS in osteoclasts within the OA environment: A novel therapeutic strategy for osteoarthritis management

This study investigated the therapeutic potential of a manganese dioxide-polymer dot (MnO2-PD)-incorporated hydrogel, designated as M-PD hydrogel, for modulating reactive oxygen species (ROS) within the osteoarthritis (OA) environment. Our research highlights the ability of the hydrogel to scavenge ROS, thereby influencing the differentiation of osteoclasts and protecting chondrocytes, offering a novel approach to osteoarthritis (OA) management. Our results indicated that the M-PD hydrogel increased electrical resistance and fluorescence recovery in the presence of osteoclasts, correlating with decreased ROS levels and suppressed expression of osteoclast differentiation markers. Coculture experiments revealed the protective effects of the hydrogel on chondrocytes by reducing the expression of matrix-degrading enzymes. In vivo application in burr holes and/or OA-induced mice revealed a significant reduction in osteoclast formation and cartilage destruction, suggesting the dual therapeutic action of the hydrogel in altering the joint microenvironment. These findings highlight the potential of targeting ROS in osteoclasts as a comprehensive therapeutic approach, offering not only symptomatic relief but also targeting the underlying mechanisms of disease progression in OA.

The role of lactoferrin in bone remodeling: evaluation of its potential in targeted delivery and treatment of metabolic bone diseases and orthopedic conditions.

Lactoferrin (Lf) is a multifunctional protein that is synthesized endogenously and has various biological roles including immunological regulation, antibacterial, antiviral, and anticancer properties. Recently, research has uncovered Lf's critical functions in bone remodeling, where it regulates the function of osteoblasts, chondrocytes, osteoclasts, and mesenchymal stem cells. The signaling pathways involved in Lf's signaling in osteoblasts include (low density lipoprotein receptor-related protein - 1 (LRP-1), transforming growth factor β (TGF-β), and insulin-like growth factor - 1 (IGF-1), which activate downstream pathways such as ERK, PI3K/Akt, and NF-κB. These pathways collectively stimulate osteoblast proliferation, differentiation, and mineralization while inhibiting osteoclast differentiation and activity. Additionally, Lf's inhibitory effect on nuclear factor kappa B (NF-κB) suppresses the formation and activity of osteoclasts directly. Lf also promotes chondroprogenitor proliferation and differentiation to chondrocytes by activating the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphoinositide 3-kinase/protein kinase B(PI3K/Akt)signaling pathways while inhibiting the expression of matrix-degrading enzymes through the suppression of the NF-κB pathway. Lf's ability to stimulate osteoblast and chondrocyte activity and inhibit osteoclast function accelerates fracture repair, as demonstrated by its effectiveness in animal models of critical-sized long bone defects. Moreover, studies have indicated that Lf can rescue dysregulated bone remodeling in osteoporotic conditions by stimulating bone formation and suppressing bone resorption. These beneficial effects of Lf on bone health have led to its exploration in nutraceutical and pharmaceutical applications. However, due to the large size of Lf, small bioactive peptides are preferred for pharmaceutical applications. These peptides have been shown to promote bone fracture repair and reverse osteoporosis in animal studies, indicating their potential as therapeutic agents for bone-related diseases. Nonetheless, the active concentration of Lf in serum may not be sufficient at the site requiring bone regeneration, necessitating the development of various delivery strategies to enhance Lf's bioavailability and target its active concentration to the site requiring bone regeneration. This review provides a critical discussion of the issues mentioned above, providing insight into the roles of Lf in bone remodeling and the potential use of Lf as a therapeutic target for bone disorders.

Saturated very long-chain fatty acids regulate macrophage plasticity and invasiveness

Saturated very long-chain fatty acids (VLCFA, ≥ C22), enriched in brain myelin and innate immune cells, accumulate in X-linked adrenoleukodystrophy (X-ALD) due to inherited dysfunction of the peroxisomal VLCFA transporter ABCD1. In its severest form, X-ALD causes cerebral myelin destruction with infiltration of pro-inflammatory skewed monocytes/macrophages. How VLCFA levels relate to macrophage activation is unclear. Here, whole transcriptome sequencing of X-ALD macrophages indicated that VLCFAs prime human macrophage membranes for inflammation and increased expression of factors involved in chemotaxis and invasion. When added externally to mimic lipid release in demyelinating X-ALD lesions, VLCFAs did not activate toll-like receptors in primary macrophages. In contrast, VLCFAs provoked pro-inflammatory responses through scavenger receptor CD36-mediated uptake, cumulating in JNK signalling and expression of matrix-degrading enzymes and chemokine release. Following pro-inflammatory LPS activation, VLCFA levels increased also in healthy macrophages. With the onset of the resolution, VLCFAs were rapidly cleared in control macrophages by increased peroxisomal VLCFA degradation through liver-X-receptor mediated upregulation of ABCD1. ABCD1 deficiency impaired VLCFA homeostasis and prolonged pro-inflammatory gene expression upon LPS treatment. Our study uncovers a pivotal role for ABCD1, a protein linked to neuroinflammation, and associated peroxisomal VLCFA degradation in regulating macrophage plasticity.

Epigenetic modification of miR-217 promotes intervertebral disc degeneration by targeting the FBXO21-ERK signalling pathway

BackgroundNumerous potential therapeutic alternatives for intervertebral disc degeneration (IDD) have been investigated, the most promising of which are based on biological variables such as microRNAs (miRNAs). Therefore, we verified the hypothesis that miRNAs modulate IDD by affecting the FBXO21-ERK signalling pathway.MethodsMicroarray and quantitative real-time polymerase chain reaction (RT–qPCR) tests were used to examine the expression profiles of miRNAs in nucleus pulposus (NP) cells between patients with IDD and controls. Western blotting and luciferase reporter assays were used to identify the miRNA targets.ResultsMicroarray and RT–qPCR assays confirmed that the expression level of miR-217 was significantly decreased in degenerative NP cells. CpG islands were predicted in the miR-217 promoter region. The IDD group had considerably higher methylation than the control group. Gain- and loss-of-function experiments revealed that miR-217 mimics inhibited apoptosis and extracellular matrix (ECM) breakdown in NP cells. Bioinformatic analyses and luciferase assays were used to determine the connection between miR-217 and FBXO21. In vitro tests revealed that miR-217 mimics inhibited the expression of FBXO21, pERK, MMP13, and ADAMTS5 proteins, successfully protecting the ECM from degradation. Additionally, in vivo investigation using the IDD mouse model demonstrated that the miR-217 agonist may sufficiently promote NP cell proliferation, decrease apoptosis, promote ECM synthesis, and suppress the expression of matrix-degrading enzymes in NP cells.ConclusionsOverexpression of miR-217 inhibits IDD via FBXO21/ERK regulation.Trial registrationThis study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and was approved by the human research ethics committee of Wuhan University Renmin Hospital (Approval No. RMHREC-D-2020-391), and written informed consent was obtained from each participant.

Exploration of microRNA-106b-5p as a therapeutic target in intervertebral disc degeneration: a preclinical study.

MicroRNA (miRNA) has emerge as a vital regulator in the pathogenesis of intervertebral disc degeneration (IDD). However, miR-106b-5p expression in the human nucleus pulposus (NP) and potential mechanisms remain to be elucidated. In this study, the aim was to verify the potential therapeutic mechanisms of miR-106b-5p for IDD. Key miRNAs were screened for in degenerative and normal human intervertebral disc samples. qRT-PCR and fluorescence in situ hybridization (FISH) were used to verify the miR-106b-5p differential expression. The targeting link between miR-106b-5p and Sirtuin 2 (SIRT2) was identified using the luciferase reporter assay and bioinformatics. Flow cytometry, EdU method, and cell scratching were all performed to determine the NP cell function and IDD models were constructed for in vivo experiments. SIRT2, MMP13, ADAMTS5, Col II, Aggrecan, Ras, ERK1/2, and p-ERK1/2 protein levels were assayed by western blotting. Overexpression of miR-106b-5p in NP cells decreased cell growth, induced apoptosis, hindered extracellular matrix formation, and increased the expression of matrix-degrading enzymes through the SIRT2/MAPK/ERK signaling pathway. Importantly, intradiscal delivery of antagomiR-106b-5p significantly attenuated IDD development. Our findings demonstrate that targeting miR-106b-5p in intervertebral disc has therapeutic effects on IDD.

Knockdown of NEK7 alleviates anterior cruciate ligament transection osteoarthritis (ACLT)-induced knee osteoarthritis in mice via inhibiting NLRP3 activation

Osteoarthritis is thought to be a NLRP3-related disease. NEK7 is an essential mediator for NLRP3 inflammasome activation. This study aimed to demonstrate whether NEK7 has regulatory roles in the pathogenesis of osteoarthritis. C57BL/6 mice were subjected to anterior cruciate ligament transection osteoarthritis (ACLT) for constructing animal models of osteoarthritis. Injection of adeno-associated virus (AAV) expressing NEK7-specific shRNA into the knee joints of mice, following of which immunohistochemistry, qRT-PCR, western blotting, Safranin-O Fast Green staining, ELISA, and co-immunoprecipitation were performed to determine the effects of NEK7. NEK7 was highly expressed in the joint tissues of ACLT mice. As compared with shScr, AAV delivery of NEK7 shRNA significantly inhibited cartilage degeneration, OARSI score, and serum CTX-II and COMP levels. AAV delivery of NEK7 shRNA downregulated the expression of matrix-degrading enzymes (ADAMTS-4, MMP3, and MMP13) and upregulated the expression of ECM-related molecules (SOX9, collagen II, and aggrecan). In addition, AAV delivery of NEK7 shRNA alleviated ACLT-induced synovial inflammation, as was evidenced by the decreased levels of TNF-α, IL-6, IL-1β, and IL-18 and increased levels of IL-10. In the joint tissues of ACLT mice, NEK7 interacted with NLRP3 proteins. AAV delivery of NEK7 shRNA inhibited the protein interaction, and thereby inhibited the activation of the NLRP3 inflammasome. AAV delivery of NEK7 shRNA has no significant effects on cartilage degeneration and synovial inflammation in Nlrp3−/− mice. In conclusion, knockdown of NEK7 exerted anti-osteoarthritic effects, possibly via inhibiting the activation of the NLRP3 inflammasome. This study provided a novel mechanism of NEK7-NLRP3 interaction affecting osteoarthritis.

Matrix-Degrading Enzyme Expression and Aortic Fibrosis During Continuous-Flow Left Ventricular Mechanical Support

BackgroundThe effects of nonphysiological flow generated by continuous-flow (CF) left ventricular assist devices (LVADs) on the aorta remain poorly understood. ObjectivesThe authors sought to quantify indexes of fibrosis and determine the molecular signature of post–CF-LVAD vascular remodeling. MethodsPaired aortic tissue was collected at CF-LVAD implant and subsequently at transplant from 22 patients. Aortic wall morphometry and fibrillar collagen content (a measure of fibrosis) was quantified. In addition, whole-transcriptome profiling by RNA sequencing and follow-up immunohistochemistry were performed to evaluate CF-LVAD–mediated changes in aortic mRNA and protein expression. ResultsThe mean age was 52 ± 12 years, with a mean duration of CF-LVAD of 224 ± 193 days (range 45-798 days). There was a significant increase in the thickness of the collagen-rich adventitial layer from 218 ± 110 μm pre-LVAD to 410 ± 209 μm post-LVAD (P < 0.01). Furthermore, there was an increase in intimal and medial mean fibrillar collagen intensity from 22 ± 11 a.u. pre-LVAD to 41 ± 24 a.u. post-LVAD (P < 0.0001). The magnitude of this increase in fibrosis was greater among patients with longer durations of CF-LVAD support. CF-LVAD led to profound down-regulation in expression of extracellular matrix–degrading enzymes, such as matrix metalloproteinase-19 and ADAMTS4, whereas no evidence of fibroblast activation was noted. ConclusionsThere is aortic remodeling and fibrosis after CF-LVAD that correlates with the duration of support. This fibrosis is due, at least in part, to suppression of extracellular matrix–degrading enzyme expression. Further research is needed to examine the contribution of nonphysiological flow patterns on vascular function and whether modulation of pulsatility may improve vascular remodeling and long-term outcomes.

Upregulated endonuclease Regnase-1 suppresses osteoarthritis by forming a negative feedback loop of catabolic signaling in chondrocytes

BackgroundRibonucleases (RNases) play central roles in the post-transcriptional regulation of mRNA stability. Our preliminary results revealed that the endonuclease Regnase-1 is specifically upregulated in osteoarthritic chondrocytes. We herein explored the possible functions and regulatory mechanisms of Regnase-1 in a mouse model of osteoarthritis (OA).MethodsThe expression and target genes of Regnase-1 were identified by microarray analysis in primary-culture mouse articular chondrocytes. Experimental OA in mice was induced by destabilization of the medial meniscus (DMM). The function of Regnase-1 in DMM-induced post-traumatic OA mice was examined by adenovirus-mediated overexpression or knockdown in knee joint tissues, and also by using Regnase-1 heterozygous knockout mice (Zc3h12a+/−).ResultsAmong the RNases, Regnase-1 was exclusively upregulated in chondrocytes stimulated with OA-associated catabolic factors. Adenovirus-mediated overexpression or knockdown of Regnase-1 alone in joint tissues did not cause OA-like changes. However, overexpression of Regnase-1 in joint tissues significantly ameliorated DMM-induced post-traumatic OA cartilage destruction, whereas knockdown or genetic ablation of Regnase-1 exacerbated DMM-induced cartilage destruction. Mechanistic studies suggested that Regnase-1 suppresses cartilage destruction by modulating the expression of matrix-degrading enzymes in chondrocytes.ConclusionOur results collectively suggest that upregulated Regnase-1 in OA chondrocytes may function as a chondro-protective effector molecule during OA pathogenesis by forming a negative feedback loop of catabolic signals, such as matrix-degrading enzyme expression, in OA chondrocytes.

Compressive mechanical stress enhances susceptibility to interleukin-1 by increasing interleukin-1 receptor expression in 3D-cultured ATDC5 cells

BackgroundMechanical overload applied on the articular cartilage may play an important role in the pathogenesis of osteoarthritis. However, the mechanism of chondrocyte mechanotransduction is not fully understood. The purpose of this study was to assess the effects of compressive mechanical stress on interleukin-1 receptor (IL-1R) and matrix-degrading enzyme expression by three-dimensional (3D) cultured ATDC5 cells. In addition, the implications of transient receptor potential vanilloid 4 (TRPV4) channel regulation in promoting effects of compressive mechanical loading were elucidated.MethodsATDC5 cells were cultured in alginate beads with the growth medium containing insulin-transferrin-selenium and BMP-2 for 6 days. The cultured cell pellet was seeded in collagen scaffolds to produce 3D-cultured constructs. Cyclic compressive loading was applied on the 3D-cultured constructs at 0.5 Hz for 3 h. The mRNA expressions of a disintegrin and metalloproteinases with thrombospondin motifs 4 (ADAMTS4) and IL-1R were determined with or without compressive loading, and effects of TRPV4 agonist/antagonist on mRNA expressions were examined. Immunoreactivities of reactive oxygen species (ROS), TRPV4 and IL-1R were assessed in 3D-cultured ATDC5 cells.ResultsIn 3D-cultured ATDC5 cells, ROS was induced by cyclic compressive loading stress. The mRNA expression levels of ADAMTS4 and IL-1R were increased by cyclic compressive loading, which was mostly prevented by pyrollidine dithiocarbamate. Small amounts of IL-1β upregulated ADAMTS4 and IL-1R mRNA expressions only when combined with compressive loading. TRPV4 agonist suppressed ADAMTS4 and IL-1R mRNA levels induced by the compressive loading, whereas TRPV4 antagonist enhanced these levels. Immunoreactivities to TRPV4 and IL-1R significantly increased in constructs with cyclic compressive loading.ConclusionCyclic compressive loading induced mRNA expressions of ADAMTS4 and IL-1R through reactive oxygen species. TRPV4 regulated these mRNA expressions, but excessive compressive loading may impair TRPV4 regulation. These findings suggested that TRPV4 regulates the expression level of IL-1R and subsequent IL-1 signaling induced by cyclic compressive loading and participates in cartilage homeostasis.

CircGLCE alleviates intervertebral disc degeneration by regulating apoptosis and matrix degradation through the targeting of miR-587/STAP1.

The purpose of this study was to identify a specific circular RNA and to investigate its regulatory mechanism in intervertebral disc degeneration (IDD). CircGLCE was selected after microarray analyses and was further analysed by RT-qPCR and FISH. CircGLCE was found to stably exist in the cytoplasm of nucleus pulposus (NP) cells. It was downregulated in IDD. After silencing CircGLCE, its function was assessed with RT-qPCR, immunofluorescence analysis and flow cytometry. Knockdown of CircGLCE promoted apoptosis and induced the expression of matrix-degrading enzymes in NP cells. CircGLCE served as a miR-587 sponge in NP cells. Inhibiting miR-587 counteracted the IDD-enhancing effect caused by silencing CircGLCE. STAP1 served as the miRNA target that mediated the functions of miR-587. In an IDD mouse model, the in vivo effects of overexpressing CircGLCE on IDD were confirmed by imaging techniques, TUNEL staining, FISH, western blotting, H&E staining and immunohistochemistry. Thus, CircGLCE attenuates IDD by inhibiting the apoptosis of NP cells and ECM degradation through the targeting of miR-587/STAP1. CircGLCE may be a potential therapeutic target for IDD treatments.

Lentinan Inhibits AGE-Induced Inflammation and the Expression of Matrix-Degrading Enzymes in Human Chondrocytes.

BackgroundChondrocyte-mediated inflammation is an important pathological component of osteoarthritis (OA) development. There are currently no therapies that completely reverse the development of OA. Lentinan, a type of polysaccharide derived from Lentinus edodes, has been demonstrated to possess significant anti-viral, anti-cancer, and anti-inflammatory effects, and has been recently used in the treatment of several inflammatory diseases. However, little research has focused on the pharmacological effect of lentinan in human OA.Materials and MethodsWe evaluated the anti-inflammatory and anti-ROS effects of lentinan in SW1353 chondrocytes treated with AGEs using real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and the nitro oxide-specific stain DAF-FM DA. The regulatory effects of lentinan on NF-κB and MAPK p38 signaling were investigated via promoter assay and Western blot analysis.ResultsWe found that lentinan inhibits the production of pro-inflammatory cytokines, including IL-1β, TNF-α, IL-8 and the secretion of PGE2 and NO, by reducing the expression of COX-2 and iNOS in AGE-challenged chondrocytes. Lentinan also reduces AGE-induced increased expression of matrix metalloproteinases-1, −3, and −13 (MMP-1, MMP-3, MMP-13). Furthermore, lentinan has a similar effect on a disintegrin and metalloproteinase with thrombospondin motifs-4 and −5 (ADAMTS-4, ADAMTS-5). Mechanistically, lentinan reduces the activation of NF-κB.ConclusionOur findings indicate that lentinan shows a protective effect against AGE-induced inflammatory response in chondrocytes. These findings suggest that lentinan is a promising agent for the treatment of OA that could be used as a dietary supplement for patients with OA.

MiR-296-5p inhibits IL-1β-induced apoptosis and cartilage degradation in human chondrocytes by directly targeting TGF-β1/CTGF/p38MAPK pathway

ABSTRACT Osteoarthritis (OA) is characterized by apoptosis of chondrocytes and an imbalance of extracellular matrix (ECM) synthesis and catabolism. Emerging evidence has demonstrated that miRNAs are involved in OA pathologies, but the role of miR-296-5p in OA remains unclear. The present study proposes to reveal the functions and mechanisms of miR-296-5p in a cell model of OA. In this study, human chondrocytes were treated with 5 ml interleukin-1 beta (IL-1β) to induce apoptosis and cartilage degradation. Our results showed that miR-296-5p was downregulated in chondrocytes stimulated with IL-1β. Overexpressed miR-296-5p enhanced cell proliferation and inhibited apoptosis and matrix degrading enzyme expression in response to IL-1β stimulation, and knockdown of miR-296-5p showed the opposite effect. Further, we found that miR-296-5p directly targeted the 3′-untranslated region (3′-UTR) of TGF-β1 mRNA, and miR-296-5p inactivated the TGF-β1/CTGF/p38MAPK signaling pathway. Overexpression of TGF-β1 alleviated the inhibition of miR-296-5p on chondrocyte apoptosis and cartilage degradation. In conclusion, miR-296-5p inhibited the progression of OA through the CTGF/p38MAPK pathway by directly targeting TGF-β1.

Expression and Circulating Levels of Perlecan in Breast Cancer - Implications for Oestrogen Dependent Stromal Remodeling

Localised breast cancer can be cured by surgery and adjuvant treatments, but mortality remains high as some tumours metastasize early. Perlecan is a basement membrane (BM) protein involved in tumour development and progression. Here, mRNA and protein expression of perlecan, and mRNA expression of matrix degrading enzymes were studied in normal breast and invasive breast cancer, and correlated to prognostic risk factors, in particular oestrogen status. Moreover, plasma levels of perlecan were measured in patients with breast cancer and compared with controls. mRNA data was extracted from the Cancer Genome Atlas database. Perlecan protein expression was visualized using immunofluorescence and plasma levels measured by ELISA assay. Perlecan mRNA levels were twice as high in normal breast compared with breast cancer tissue. A strong correlation was found between mRNA expression of perlecan and several matrix-degrading enzymes in oestrogen receptor positive (ER+) tumours. Perlecan protein was localized to both epithelial and vascular BMs, but absent in the stroma in normal breast. In breast cancer, the expression of perlecan in epithelial BM was fragmented or completely lost, with a marked upregulation of perlecan expression in the stroma. Significantly higher levels of perlecan were found in plasma of ER+ patients when compared with ER- patients. This study shows that perlecan expression and degradation in breast cancer may be linked to the ER status of the tumour.

The pro-inflammatory effect of NR4A3 in osteoarthritis.

NR4A3 is a member of nuclear receptor subfamily 4, which is an important regulator of cellular function and inflammation. In this study, high expression of NR4A3 in human osteoarthritis (OA) cartilage was firstly observed. To explore the relationship between NR4A3 and OA, we used a lentivirus overexpression system to simulate its high expression and study its role in OA. Additionally, siRNA‐mediated knockdown of NR4A3 was used to confirm the findings of overexpression experiments. The results showed the stimulatory effect of IL‐1β on cartilage matrix‐degrading enzyme expression such as MMP‐3, 9, INOS and COX‐2 was enhanced in NR4A3‐overexpressed chondrocytes and decreased in NR4A3‐knockdown chondrocytes at both mRNA and protein levels, while IL‐1β‐induced chondrocyte‐specific gene (collagen 2 and SOX‐9) degradation was only regulated by NR4A3 at protein level. Furthermore, overexpression of NR4A3 would also enhance EBSS‐induced chondrocytes apoptosis, while knockdown of NR4A3 decreased apoptotic level after EBSS treatment. A pathway study indicated that IL‐1β‐induced NF‐κB activation was enhanced by NR4A3 overexpression and reduced by NR4A3 knockdown. We suggest that NR4A3 plays a pro‐inflammatory role in the development of OA, and we also speculate that NR4A3 mainly regulates cartilage matrix‐degrading gene expression under inflammatory conditions via the NF‐κB pathway.

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

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

Up-regulated HIF-2α contributes to the Osteoarthritis development through mediating the primary cilia loss

BackgroundsUp-regulated HIF-2α (hypoxia induced factor 2) had been demonstrated to contribute to Osteoarthritis (OA) development via inducing the expression of matrix-degrading enzymes. However, the HIF-2α also could promote primary cilia loss through HIF-2α/AURKA (Aurora kinase A)/NEDD9 pathway. And the primary cilia dysfunction is another characteristic of the OA. Thus, we investigated here whether the HIF-2α also contributes the OA development through mediating the primary cilia loss. MethodsThe primary chondrocytes were isolated from the experimental OA mice induced by destabilization of the medial meniscus (DMM). Chondrocytes were cultured under normoxia (21% O2) or hypoxia (2% O2) conditions. The HIF-1α and HIF-2α expressions were assessed by western blot. The cilia formation was counted by immuno-staining the acetylated tubulin. The contribution of HIF-1α or HIF-2α to the primary cilia loss was assessed by knocking-down the HIF-1α or HIF-2α individually. The HIF-2α/AURKA/NEDD9 pathway was validated through over-expressing or knocking-down specific components of the pathway and then counting the primary cilia number. Finally, the pathway was further confirmed in the OA mice. ResultsHypoxia could induce the expression of both HIF-1α and HIF-2α, and also reduce the number of primary cilia on the chondrocytes isolated from the experimental OA mice. Knocking-down or over-expressing HIF-1α or HIF-2α individually showed that the HIF-2α could induce the primary cilia reduction rather than the HIF-1α. Manipulating the HIF-2α expression could positively affect the AURKA and NEDD9 expression. Manipulating the AURKA and NEDD9 expressions could reverse the function of HIF-2α on primary cilia. In the mice, knocking-down both AURKA and NEDD9 could alleviate the OA development significantly. ConclusionUp-regulated HIF-2α contributes to the Osteoarthritis development through mediating the primary cilia loss, which might be developed as therapeutic targets for OA treatment.

Interleukin 1 is a key driver of inflammatory bowel disease-demonstration in a murine IL-1Ra knockout model.

Interleukin 1 (IL-1) is an important mediator of inflammation and tissue damage in inflammatory bowel disease (IBD). The balance between IL-1 and IL-1Ra as a natural inhibitor plays a vital role in a variety of diseases. Here, we investigated whether changes seen during IBD are induced spontaneously in mice lacking a functional IL-1rn gene. Histological staining was performed on the jejunum and ileum of BALB/c IL-1rn+/+ and IL-1rn-/- mice to characterize crypt-villus height, villus width, and number of goblet cells per villus. Pro-inflammatory cytokines, immune cell infiltration and matrix-degrading enzymes, together with the production of intestinal enzymes and the integrity of tight and adherent junction proteins were determined using immunohistochemistry. In the small intestine of BALB/c IL-1rn-/- mice the villus heights were significantly reduced; and in the ileum this was accompanied by a decrease in villi width. There was also an increase in goblet cell number and mucin production compared to wild-type mice. IL-1α and IL-1β immunopositivity were increased, whilst IL-1R1 expression was decreased in IL-1rn-/- mice. IL-15 and TNFα were also increased in older IL-1rn-/- mice. Increased polymorphonuclear and macrophage infiltration were seen in IL-1rn-/- mice, whilst expression of matrix-degrading enzymes and digestive enzymes were unchanged, except for dipeptidyl peptidase IV which was increased in younger IL-1rn-/- mice compared to wild type mice. The expression of tight and adhesion junctions were also dramatically decreased in IL-1rn-/- mice. In conclusion, IL-1rn-/- mice developed spontaneous abnormalities which displayed features associated with IBD, demonstrating a clear role for IL-1 in IBD.

A crucial role of arginase-2 in osteoarthritis pathogenesis

Purpose: Osteoarthritis (OA) appears to be associated with various metabolic disorders. However, a role of amino acids metabolism in OA pathogenesis has not been clearly elucidated to date. Here, we explored whether alterations in amino acid metabolism in chondrocytes regulates OA pathogenesis. Methods: Expression profiles of genes regulating amino acid metabolism in primary-culture mouse chondrocytes was examined by microarray analysis. Expression pattern of the identified genes were further characterized in mouse OA chondrocytes and OA cartilage of human and mouse models. Experimental OA in mice was induced by destabilization of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing catabolic regulators. The function of Arg2 was examined by analyzing Arg2-/- mice and those subjected to IA injection of an adenovirus encoding Arg2(Ad-Arg2). Results: Microarray analysis revealed that arginase-2 (Arg2), an arginine metabolizing enzyme, was specifically upregulated in chondrocytes under various pathological conditions and in OA cartilage from human OA patients and various mouse models. Adenovirus-mediated overexpression of Arg2 in mouse joint tissues caused OA pathogenesis, whereas genetic ablation of Arg2 in mice (Arg2-/-) abolished all manifestations of DMM-induced OA. Mechanistically, Arg2 appears to cause OA cartilage destruction at least partly by upregulating the expression of matrix-degrading enzymes (MMP3 and MMP13) in chondrocytes via the NF-κB pathway. Conclusions: Our results collectively indicate that Arg2 is a crucial catabolic regulator of OA pathogenesis, supporting the notion that Arg2 could be a therapeutic target for the treatment of OA pathogenesis.

Caveolin-1 regulates human trabecular meshwork cell adhesion, endocytosis, and autophagy.

Impaired trabecular meshwork (TM) outflow is implicated in the pathogenesis of primary open-angle glaucoma (POAG). We previously identified the association of a caveolin-1 (CAV1) variant with POAG by genome-wide association study. Here we report a study of CAV1 knockout (KO) effect on human TM cell properties. We generated human CAV1-KO TM cells by CRISPR/Cas9 technology, and we found that the CAV1-KO TM cells less adhered to the surface coating than the wildtype TM cells by 69.34% ( P < 0.05), but showed no difference in apoptosis. Higher endocytosis ability of dextran and transferrin was also observed in the CAV1-KO TM cells (4.37 and 1.89-fold respectively, P < 0.001), compared to the wildtype TM cells. Moreover, the CAV1-KO TM cells had higher expression of extracellular matrix-degrading enzyme genes ( ADMTS13 and MMP14) as well as autophagy-related genes ( ATG7 and BECN1) and protein (LC3B-II) than the wildtype TM cells. In summary, results from this study showed that the CAV1-KO TM cells have reduced adhesion with higher extracellular matrix-degrading enzyme expression, but increased endocytosis and autophagy activities, indicating that CAV1 could be involved in the regulation of adhesion, endocytosis, and autophagy in human TM cells.

Recruitment of Brd3 and Brd4 to acetylated chromatin is essential for proinflammatory cytokine-induced matrix-degrading enzyme expression

BackgroundProinflammatory cytokines, which can upregulate the expression of matrix-degrading enzymes in chondrocytes, play important roles in the development of osteoarthritis. BET family proteins, acting as the “readers” of acetylated modifications on histones, have been linked to transcriptional regulation. And a BET protein inhibitor, I-BET151, has been shown to inhibit the induction of matrix-degrading enzymes by proinflammatory cytokines in chondrocytes. Our objective is to clarify the role and mechanism of BET proteins on matrix-degrading enzyme gene expression by using a human chondrosarcoma cell line (SW1353).MethodsWe pretreated SW1353 cells with I-BET151 prior to treatment with IL-1β or TNF-α and then checked the expression of four matrix-degrading enzyme genes (MMP1, MMP3, MMP13, and ADAMTS4). We performed knockdown of BET protein family members (BRD2, BRD3, and BRD4) with corresponding siRNAs in SW1353 cells prior to treatment with IL-1β or TNF-α and checked the expression of the matrix-degrading enzyme genes. We evaluated Brd-mediated transcriptional regulation on the matrix-degrading enzyme genes by ChIP assay.ResultsWe confirmed that I-BET151 could suppress the IL-1β- or TNF-α-induced expression of MMP1, MMP3, MMP13, and ADAMTS4 in SW1353 cells. Brd3 and Brd4 were required for the IL-1β- or TNF-α-induced expression of matrix-degrading enzyme genes in SW1353 cells. We revealed that inducible acetylation of H4k5/8/12 and the recruitment of Brd3, Brd4, and p-TEFb to chromatin were involved in IL-1β- or TNF-α-induced transcription.ConclusionsOur findings suggested that Brd3 and Brd4 were essential for the IL-1β- or TNF-α-induced transcription of matrix-degrading enzyme genes, and recruitment of Brd3 and Brd4 to chromatin of these genes played the main role in this process.