leupeptins and Osteoarthritis

In this study, the role of ubiquitin conjugating enzyme E2 M (UBE2M) and molecular mechanisms associated with osteoarthritis (OA) were explored. Cartilage tissues and corresponding healthy tissues from OA patients were isolated. Our data suggested that the expression level of UBE2M in OA patients was significantly higher compared to that in healthy individuals (P < 0.01). The apoptosis of human OA chondrocytes was inhibited when silencing UBE2M and increased when overexpressing UBE2M. XAV939, as a tankyrase 1 inhibitor, could block the signaling pathway of Wnt/β-catenin, which significantly reversed the change introduced by UBE2M. The expression level of cytoplasmic β-catenin in siUBE2M cells dramatically increased, and the expression levels of nuclear β-catenin, cleaved caspase-3 (C-caspase-3), and MMP13 remarkably downregulated. Moreover, the ubiquitination of Axin was enhanced by the overexpression of UBE2M. The expression level of Axin significantly decreased in OA chondrocytes with UBE2M overexpression and increased after MG132 treatment. Moreover, UBE2M enhanced the apoptosis of OA chondrocytes by activating the Axin-dependent Wnt/β-catenin pathway. In this process, UBE2M downregulated Axin in an ubiquitination-dependent degradation pathway and subsequently activated Wnt/β-catenin signaling.

Topics: Apoptosis; Axin Protein; beta Catenin; Cartilage, Articular; Case-Control Studies; Caspase 3; Chondrocytes; Femur; Gene Expression Regulation; Heterocyclic Compounds, 3-Ring; Humans; Leupeptins; Matrix Metalloproteinase 13; Osteoarthritis; Primary Cell Culture; RNA, Small Interfering; Signal Transduction; Tankyrases; Ubiquitin-Conjugating Enzymes; Wnt Proteins

Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood. In our current study, we found that palmitate inhibits autophagy by modulating the protein levels of autophagy-related genes-5 (ATG5) that is associated with decreased lipidation of LC3 and increased activation of cleaved caspase 3. Pretreatment of meniscus cells with 4-phenyl butyric acid, a small molecule chemical chaperone that alleviates ER stress, or with MG-132, a proteasome inhibitor, restored normal levels of ATG5 and autophagosome formation, and decreased expression of cleaved caspase 3. Thus, our data suggest that palmitate downregulates autophagy in meniscus cells by degrading ATG5 protein via ER-associated protein degradation, and thus promotes apoptosis. This is the first study to demonstrate that palmitate-induced endoplasmic reticulum stress negatively regulates autophagy.

Topics: Animals; Autophagy; Autophagy-Related Protein 5; Cells, Cultured; Down-Regulation; Endoplasmic Reticulum Stress; Humans; Leupeptins; Meniscus; Osteoarthritis; Palmitic Acid; Phenylbutyrates; Proteasome Inhibitors; Proteolysis; Swine

Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases.

Topics: Adenomatous Polyposis Coli Protein; Animals; Axin Protein; beta Catenin; Casein Kinase I; Cell Line; Chondrocytes; Gases; Glycogen Synthase Kinase 3; HCT116 Cells; HeLa Cells; HT29 Cells; Humans; Hydrogen; Leupeptins; Lithium Chloride; Male; Microscopy, Fluorescence; Osteoarthritis; Phosphorylation; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; SOX9 Transcription Factor; Water; Wnt Signaling Pathway; Wnt3A Protein

To determine whether the process of ubiquitination and/or activity of the 26S proteasome are involved in the induction of osteoarthritis (OA).. Bovine cartilage resorption assays, chondrocyte cell-line SW1353 and primary human articular chondrocytes were used with the general proteasome inhibitor MG132 or vehicle to identify a role of the ubiquitin-proteasome system (UPS) in cartilage destruction and matrix metalloproteinase-13 (MMP13) expression. In vivo, MG132 or vehicle, were delivered subcutaneously to mice following destabilisation of the medial meniscus (DMM)-induced OA. Subsequently, DMM was induced in Lys-to-Arg (K48R and K63R) mutant ubiquitin (Ub) transgenic mice. Cytokine signalling in SW1353s was monitored by immunoblotting and novel ubiquitinated substrates identified using Tandem Ubiquitin Binding Entities purification followed by mass spectrometry. The ubiquitination of TRAFD1 was assessed via immunoprecipitation and immunoblotting and its role in cytokine signal-transduction determined using RNA interference and real-time RT-PCR for MMP13 and interleukin-6 (IL6).. Supplementation with the proteasome inhibitor MG132 protected cartilage from cytokine-mediated resorption and degradation in vivo in mice following DMM-induced OA. Using transgenic animals only K48R-mutated Ub partially protected against OA compared to wild-type or wild-type Ub transgenic mice, and this was only evident on the medial femoral condyle. After confirming ubiquitination was vital for NF-κB signalling and MMP13 expression, a screen for novel ubiquitinated substrates involved in cytokine-signalling identified TRAFD1; the depletion of which reduced inflammatory mediator-induced MMP13 and IL6 expression.. Our data for the first time identifies a role for ubiquitination and the proteasome in the induction of OA via regulation of inflammatory mediator-induced MMP13 expression. These data open avenues of research to determine whether the proteasome, or K48-linked ubiquitination, are potential therapeutic targets in OA.

Topics: Animals; Cysteine Proteinase Inhibitors; Disease Models, Animal; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Leupeptins; Lysine; Matrix Metalloproteinase 13; Mice, Inbred C57BL; Mice, Transgenic; Osteoarthritis; Proteasome Endopeptidase Complex; Signal Transduction; Ubiquitination; Zinc Fingers

To evaluate the effects of the proteasome inhibitor MG-132 on the expression of nuclear factor (NF)-κB p65, inhibitor (I)-κB, tumour necrosis factor (TNF)-α, and interleukin (IL)-1β in the cartilage and synovial tissues of rats with osteoarthritis (OA), and to investigate the role that the ubiquitin/proteasome system (UPS) plays in the OA process.. A total of 144 adult male Sprague Dawley rats were randomly assigned to four groups: anterior cruciate ligament transaction (ACLT) + MG-132 (ACLT/M), ACLT + dimethylsulfoxide (ACLT/D), sham surgery (Sham), and naïve + MG-132 (naïve/M). Pathological morphology was undertaken. mRNA expression levels of NF-κB p65, I-κB, TNF-α, and IL-1β were determined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The activities of the 20S proteasome chymotrypsin-like and peptidylglutamyl-peptide hydrolase-like enzymes were measured using fluorospectrophotometry.. The Mankin scores at all time points in ACLT/M rats were significantly lower than those in ACLT/D rats (p < 0.05). Despite the NF-κB p65 in the synovial tissue at 2 weeks after surgery and IL-1β in the cartilage tissue at 12 weeks after surgery, mRNA expression levels of NF-κB p65, IL-1β, and TNF-α at other time points in ACLT/M were significantly lower than those in ACLT/D (p < 0.05). mRNA levels of I-κB in the cartilage tissue in ACLT/M were significantly higher than those in ACLT/D at 2 weeks after surgery (p < 0.05). mRNA levels of I-κB in the synovial tissue in ACLT/M were higher than those in ACLT/D at all time points, and the difference was significant at 4 weeks after surgery (p < 0.05). MG-132 decreased the activities of the 20S proteasome chymotrypsin-like and peptidylglutamyl-peptide hydrolase-like enzymes in the cartilage and synovial tissues of rats.. The proteasome inhibitor MG-132 delays the progress of OA by alleviating synovial inflammation and protecting the articular cartilage tissue.

Topics: Animals; Anterior Cruciate Ligament; Cartilage, Articular; Chymotrypsin; Cysteine Proteinase Inhibitors; Disease Models, Animal; Endopeptidases; I-kappa B Proteins; Interleukin-1beta; Leupeptins; Male; Osteoarthritis; Physical Conditioning, Animal; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Signal Transduction; Synovial Membrane; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation. The ideal anti-OA drug should have immunomodulatory effects while at the same time having limited or no toxicity. We examined the anti-inflammatory effects of Ginkgo biloba extract (EGb) in interleukin-1 (IL-1)-stimulated human chondrocytes. Chondrocytes were prepared from cartilage specimens taken from patients with osteoarthritis who had received total hip or total knee replacement. The concentrations of chemokines and the degree of cell migration were determined by ELISA and chemotaxis assays, respectively. The activation of inducible nitric oxide synthase (iNOS), mitogen-activated protein kinases (MAPKs), activator protein-1 (AP-1), and nuclear factor-kappaB (NF-κB) was determined by immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay. We found that EGb inhibited IL-1-induced production of chemokines, which in turn resulted in attenuation of THP-1 cell migration toward EGb-treated cell culture medium. EGb also suppressed IL-1-stimulated iNOS expression and release of nitric oxide (NO). The EGb-mediated suppression of the iNOS-NO pathway correlated with the attenuation of activator protein-1 (AP-1) but not nuclear factor-kappaB (NF-κB) DNA-binding activity. Of the mitogen-activated protein kinases (MAPKs), EGb inhibited only c-Jun N-terminal kinase (JNK). Unexpectedly, EGb selectively caused degradation of c-Jun protein. Further investigation revealed that EGb-mediated c-Jun degradation was preceded by ubiquitination of c-Jun and could be prevented by the proteosome inhibitor MG-132. The results imply that EGb protects against chondrocyte degeneration by inhibiting JNK activation and inducing ubiquitination-dependent c-Jun degradation. Although additional research is needed, our results suggest that EGb is a potential therapeutic agent for the treatment of OA.

Topics: Arthroplasty, Replacement, Hip; Arthroplasty, Replacement, Knee; Cell Movement; Chondrocytes; Gene Expression Regulation; Ginkgo biloba; Humans; Interleukin-1; JNK Mitogen-Activated Protein Kinases; Leupeptins; MAP Kinase Kinase 4; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Osteoarthritis; Plant Extracts; Primary Cell Culture; Proteolysis; Signal Transduction; Transcription Factor AP-1; Ubiquitination

Osteoarthritis is a degenerative joint disease with pain and loss of joint function as major pathological features. Recent studies show that proteasome inhibitors reduce pain in various pathological conditions. We evaluated the effects of MG132, a reversible proteasome inhibitor on pain and joint destruction in a rat model of osteoarthritis. Osteoarthritis was induced by intraarticular injection of monosodium iodoacetate into the rat knee. Knee joint stiffness was scored and nociception was evaluated by mechanical pressure applied to the respective hind paw. Knee joint destruction was assessed by radiological and histological analyses. Expression of matrix metalloproteinase-3 (MMP-3) was analyzed by quantitative reverse transcription polymerase chain reaction in the knee articular cartilage. Expression of substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in the dorsal root ganglia (L4-L6) by quantitative reverse transcription polymerase chain reaction and in the knee joints by immunohistochemistry. Our results indicate that daily treatment of osteoarthritic rats with MG132 significantly increases their mobility while the swelling, pain thresholds, and pathological features of the affected joints were reduced. Furthermore, the upregulated expression of MMP-3, SP, and CGRP in the arthritic rats was normalized by MG132 administration. We conclude that the proteasome inhibitor MG132 reduces pain and joint destruction, probably by involving the peripheral nervous system, and that changes in SP and CGRP expression correlate with alterations in behavioural responses. Our findings suggest that nontoxic proteasome inhibitors may represent a novel pharmacotherapy for osteoarthritis.

Topics: Animals; Arthritis, Experimental; Calcitonin Gene-Related Peptide; Cartilage, Articular; Cysteine Proteinase Inhibitors; Female; Ganglia, Spinal; Knee Joint; Leupeptins; Matrix Metalloproteinase 3; Osteoarthritis; Pain; Pain Measurement; Rats; Rats, Inbred Lew; Substance P

Aberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA.. Chondrocytes were stimulated with interleukin-1beta (IL-1beta) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR)-based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription-PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-kappaB was evaluated using specific inhibitors.. In IL-1beta-stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3'-untranslated region (3'-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3'-UTR of human MMP-13 mRNA and inhibited the IL-1beta-induced expression of MMP-13 protein in chondrocytes. NF-kappaB and MAPK activation down-regulated the expression of miR-27b.. Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1beta-induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes.

Topics: 3' Untranslated Regions; Aged; Argonaute Proteins; Chondrocytes; Cysteine Proteinase Inhibitors; DEAD-box RNA Helicases; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factors; Gene Expression Regulation, Enzymologic; Genes, Reporter; Humans; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Leupeptins; Luciferases; Matrix Metalloproteinase 13; MicroRNAs; Middle Aged; NF-kappa B; Osteoarthritis; p38 Mitogen-Activated Protein Kinases; Ribonuclease III; Signal Transduction

Hypoxia and hypoxia-related genes are important factors in articular chondrocytes during cartilage homeostasis and osteoarthritis. We have investigated the various apoptotic factors that show significance in synovial fluid obtained from normal and experimental osteoarthritic animal models and have evaluated the effect of hypoxia on articular chondrocyte apoptosis induced by these apoptotic factors. Mature beagle dogs underwent surgical transections of ligaments and medial meniscectomies to explore the underlying mechanisms of osteoarthritis. Cartilage and synovial fluid obtained from normal animals and those with osteoarthritis were evaluated via proteasome inhibition, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) protein expression, mitochondrial transmembrane potential and levels of reactive oxygen species. Canine chondrocytes were exposed to the proteasome inhibitor N-acetyl-Leu-Leu-Norleu-al and treated with recombinant TRAIL protein under normoxic and hypoxic conditions, measuring chondrocyte cell viability, proteasome activity and levels of apoptotic factors. TRAIL protein expression and ubiquitinated proteins were increased significantly, but the proteasome activity in the synovial fluid of osteoarthritic joints relative to that in normal joints was not. Primary cultured articular chondrocytes cotreated with the proteasome inhibitor and TRAIL progressed to severe apoptosis under normoxic conditions, but the sensitization caused by the combined treatment was suppressed by exposure to hypoxia. Caspase-8 activation, c-Jun N-terminal kinase phosphorylation, the mitochondrial transmembrane potential and the generation of reactive oxygen species involved in cell death regulation were significantly inhibited under hypoxic conditions. These findings suggest that proteasome inhibition and TRAIL may be possible mechanisms in cartilage degradation and joint-related diseases. Furthermore, the maintenance of hypoxic conditions or therapy with hypoxia-related genes in the joint may be successful for the treatment of joint-related diseases, including osteoarthritis.

Topics: Animals; Apoptosis; Cartilage, Articular; Caspase 8; Cells, Cultured; Chondrocytes; Dogs; Female; Homeostasis; Hypoxia; JNK Mitogen-Activated Protein Kinases; Leupeptins; Membrane Potential, Mitochondrial; Osteoarthritis; Proteasome Inhibitors; Reactive Oxygen Species; Synovial Fluid; TNF-Related Apoptosis-Inducing Ligand; Ubiquitinated Proteins