Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis

The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy-pertrophy.     

Coenzyme Q10 Ameliorates Pain and Cartilage Degradation in a Rat Model of Osteoarthritis by Regulating Nitric Oxide and Inflammatory Cytokines

Objective

To investigate the effect of CoenzymeQ10 (CoQ10) on pain severity and cartilage degeneration in an experimental model of rat osteoarthritis (OA).

Materials and Methods

OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA) to the knee. Oral administration of CoQ10 was initiated on day 4 after MIA injection. Pain severity was assessed by measuring secondary tactile allodynia using the von Frey assessment test. The degree of cartilage degradation was determined by measuring cartilage thickness and the amount of proteoglycan. The mankin scoring system was also used. Expressions of matrix metalloproteinase-13 (MMP-13), interleukin-1β (IL-1β), IL-6, IL-15, inducible nitric oxide synthase (iNOS), nitrotyrosine and receptor for advanced glycation end products (RAGE) were analyzed using immunohistochemistry.

Results

Treatment with CoQ10 demonstrated an antinociceptive effect in the OA animal model. The reduction in secondary tactile allodynia was shown by an increased pain withdrawal latency and pain withdrawal threshold. CoQ10 also attenuated cartilage degeneration in the osteoarthritic joints. MMP-13, IL-1β, IL-6, IL-15, iNOS, nitrotyrosine and RAGE expressions were upregulated in OA joints and significantly reduced with CoQ10 treatment.

Conclusion

CoQ10 exerts a therapeutic effect on OA via pain suppression and cartilage degeneration by inhibiting inflammatory mediators, which play a vital role in OA pathogenesis.     

Eupatilin Exerts Antinociceptive and Chondroprotective Properties in a Rat Model of Osteoarthritis by Downregulating Oxidative Damage and Catabolic Activity in Chondrocytes

Increases in oxidative stress are thought to be associated with the development of osteoarthritis (OA). Eupatilin, one of the major compounds present in artemisia species, was shown to have both anti-oxidative and anti-inflammatory properties. Here, we investigated the in vivo effects of eupatilin on pain severity and cartilage degradation in an experimental rat model of OA, along with the mechanisms of action underlying these effects. Experimental OA was induced via an intra-articular injection of monosodium iodoacetate (MIA), with oral administration of eupatilin initiated on the day of MIA injection. Pain was assessed by measuring the paw withdrawal latency and threshold. Cartilage destruction was analyzed macroscopically and histomorphologically. The effects of eupatilin on mRNA expression were investigated in interleukin-1β (IL-1β)-stimulated human OA chondrocytes. Eupatilin treatment exhibited clear antinociceptive effects, along with an attenuation of cartilage degradation in OA rats. Additionally, the number of osteoclasts present in the subchondral bone region was significantly decreased following eupatilin treatment. Eupatilin reduced the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), nitrotyrosine and inducible nitric oxide synthase (iNOS) in cartilage. mRNA levels of matrix metalloproteinase-3 (MMP-3), MMP13, and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) were reduced in IL-1β-stimulated human OA chondrocytes, while tissue inhibitor of metalloproteinases-1 (TIMP-1) was induced. Phosphorylated protein levels of the c-jun N-terminal kinase (JNK) was reduced by eupatilin. Taken together, these results suggest that eupatilin suppresses oxidative damage and reciprocally enhances extracellular matrix production in articular chondrocytes, making eupatilin a promising therapeutic option for the treatment of OA.     

JUNB‐FBXO21‐ERK axis promotes cartilage degeneration in osteoarthritis by inhibiting autophagy

Osteoarthritis (OA) is a heterogeneous disease that is extremely hard to cure owing to its complex regulation network of pathogenesis, especially cartilage degeneration. FBXO21 is a subunit of ubiquitin E3 ligases that degrades P‐glycoprotein and EID1 by ubiquitination and activates the JNK and p38 pathways; however, its role in OA remains unknown. Here, the main objective of this study was to evaluate the potential effects and mechanism of FBXO21 in OA degeneration, we revealed that FBXO21 is upregulated in the cartilage of patients with OA, aging, and monosodium iodoacetate‐induced OA rats, and chondrocytes treated with interleukin‐1β, tumor necrosis factor‐α, and lipopolysaccharide. Moreover, the in vivo and in vitro knockdown of FBXO21 suppressed OA‐related cartilage degeneration, as evidenced by activated autophagy, upregulated anabolism, alleviated apoptosis, and downregulated catabolism. In contrast, its overexpression promoted OA‐related cartilage degeneration. In addition, using mass spectrometry and co‐immunoprecipitation assay, we demonstrated that the downstream mechanism of FBXO21 inhibits autophagy by interacting with and phosphorylating ERK. Furthermore, FBXO21 alleviated anabolism and enhanced apoptosis and catabolism by inhibiting autophagy in rat chondrocytes. Interestingly, for its upstream mechanism, JUNB promoted FBXO21 expression by directly targeting the FBXO21 promoter, thus further accelerating cartilage degeneration in SW1353 cells and rat chondrocytes. Overall, our findings reveal that the JUNB‐FBXO21‐ERK axis regulates OA apoptosis and cartilage matrix metabolism by inhibiting autophagy. Therefore, FBXO21 is an attractive target for regulating OA pathogenesis, and its knockdown may provide a novel targeted therapy for OA.     

Matrix Metalloproteases and Tissue Inhibitors of Metalloproteinases in Medial Plica and Pannus-like Tissue Contribute to Knee Osteoarthritis Progression

Osteoarthritis (OA) is characterized by degradation of the cartilage matrix, leading to pathologic changes in the joints. However, the pathogenic effects of synovial tissue inflammation on OA knees are not clear. To investigate whether the inflammation caused by the medial plica is involved in the pathogenesis of osteoarthritis, we examined the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the medial plica and pannus-like tissue in the knees of patients with medial compartment OA who underwent either arthroscopic medial release (stage II; 15 knee joints from 15 patients) or total knee replacement (stage IV; 18 knee joints from 18 patients). MMP-2, MMP-3, MMP-9, IL-1β, and TNF-α mRNA and protein levels measured, respectively, by quantitative real-time PCR and Quantibody human MMP arrays, were highly expressed in extracts of medial plica and pannus-like tissue from stage IV knee joints. Immunohistochemical staining also demonstrated high expression of MMP-2, MMP-3, and MMP-9 in plica and pannus-like tissue of stage IV OA knees and not in normal cartilage. Some TIMP/MMP ratios decreased significantly in both medial plica and pannus-like tissue as disease progressed from stage II to stage IV. Furthermore, the migration of cells from the pannus-like tissue was enhanced by IL-1β, while plica cell migration was enhanced by TNF-α. The results suggest that medial plica and pannus-like tissue may be involved in the process of cartilage degradation in medial compartment OA of the knee.     

Resistance to Inhibitors of Cholinesterase 3 (Ric-3) Expression Promotes Selective Protein Associations with the Human α7-Nicotinic Acetylcholine Receptor Interactome

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel widely expressed in vertebrates and is associated with numerous physiological functions. As transmembrane ion channels, α7-nAChRs need to be expressed on the surface of the plasma membrane to function. The receptor has been reported to associate with proteins involved with receptor biogenesis, modulation of receptor properties, as well as intracellular signaling cascades and some of these associated proteins may affect surface expression of α7-nAChRs. The putative chaperone resistance to inhibitors of cholinesterase 3 (Ric-3) has been reported to interact with, and enhance the surface expression of, α7-nAChRs. In this study, we identified proteins that associate with α7-nAChRs when Ric-3 is expressed. Using α-bungarotoxin (α-bgtx), we isolated and compared α7-nAChR-associated proteins from two stably transfected, human tumor-derived cell lines: SH-EP1-hα7 expressing human α7-nAChRs and the same cell line further transfected to express Ric-3, SH-EP1-hα7-Ric-3. Mass spectrometric analysis of peptides identified thirty-nine proteins that are associated with α7-nAChRs only when Ric-3 was expressed. Significantly, and consistent with reports of Ric-3 function in the literature, several of the identified proteins are involved in biological processes that may affect nAChR surface expression such as post-translational processing of proteins, protein trafficking, and protein transport. Additionally, proteins affecting the cell cycle, the cytoskeleton, stress responses, as well as cyclic AMP- and inositol triphosphate-dependent signaling cascades were identified. These results illuminate how α-bgtx may be used to isolate and identify α7-nAChRs as well as how the expression of chaperones such as Ric-3 can influence proteins associating with α7-nAChRs. These associating proteins may alter activities of α7-nAChRs to expand their functionally-relevant repertoire as well as to affect biogenesis and membrane trafficking of α7-nAChRs.     

MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis of osteoarthritis

Background

Even though osteoarthritis (OA) is the most common musculoskeletal dysfunction, there are no effective pharmacological treatments to treat OA due to lack of understanding in OA pathology. To better understand the mechanism in OA pathogenesis and investigate its effective target, we analyzed miRNA profiles during OA pathogenesis and verify the role and its functional targets of miR-488.

Results

Human articular chondrocytes were obtained from cartilage of OA patients undergoing knee replacement surgery and biopsy samples of normal cartilage and the expression profile of miRNA was analyzed. From expression profile, most potent miR was selected and its target and functional role in OA pathogenesis were investigated using target validation system and OA animal model system. Among miRNAs tested, miR-488 was significantly decreased in OA chondrocytes Furthermore, we found that exposure of IL-1β was also suppressed whereas exposure of TGF-β3 induced the induction of miR-488 in human articular chondrocytes isolated from biopsy samples of normal cartilages. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation.

Conclusions

miR-488 acts as a positive role for chondrocyte differentiation/cartilage development by inhibiting MMP-13 activity through targeting ZIP-8.     

Single Particle Tracking of α7 Nicotinic AChR in Hippocampal Neurons Reveals Regulated Confinement at Glutamatergic and GABAergic Perisynaptic Sites

α7 neuronal nicotinic acetylcholine receptors (α7-nAChR) form Ca²⁺-permeable homopentameric channels modulating cortical network activity and cognitive processing. They are located pre- and postsynaptically and are highly abundant in hippocampal GABAergic interneurons. It is unclear how α7-nAChRs are positioned in specific membrane microdomains, particularly in cultured neurons which are devoid of cholinergic synapses. To address this issue, we monitored by single particle tracking the lateral mobility of individual α7-nAChRs labeled with α-bungarotoxin linked to quantum dots in live rat cultured hippocampal interneurons. Quantitative analysis revealed different modes of lateral diffusion of α7-nAChR dependent on their subcellular localization. Confined receptors were found in the immediate vicinity of glutamatergic and GABAergic postsynaptic densities, as well as in extrasynaptic clusters of α-bungarotoxin labeling on dendrites. α7-nAChRs avoided entering postsynaptic densities, but exhibited reduced mobility and long dwell times at perisynaptic locations, indicative of regulated confinement. Their diffusion coefficient was lower, on average, at glutamatergic than at GABAergic perisynaptic sites, suggesting differential, synapse-specific tethering mechanisms. Disruption of the cytoskeleton affected α7-nAChR mobility and cell surface expression, but not their ability to form clusters. Finally, using tetrodotoxin to silence network activity, as well as exposure to a selective α7-nAChR agonist or antagonist, we observed that α7-nAChRs cell surface dynamics is modulated by chronic changes in neuronal activity. Altogether, given their high Ca²⁺-permeability, our results suggest a possible role of α7-nAChR on interneurons for activating Ca²⁺-dependent signaling in the vicinity of GABAergic and glutamatergic synapses.     

Zinc protects chondrocytes from monosodium iodoacetate-induced damage by enhancing ATP and mitophagy

Osteoarthritis (OA) is characterized with articular cartilage degradation, and monosodium iodoacetate (MIA)-treated chondrocyte is the most commonly used model for mimicking OA progression. Zinc protects chondrocytes from MIA-induced damage. Here, we explored the protective effects of 25 μM zinc on 5 μM MIA-treated SW1353 cells (human chondrosarcoma cell line) through the analysis of energy metabolism- and autophagy-related parameters. We found that the exposure of SW1353 cells to MIA decreased ATP levels, expression of glycolysis-related proteins, including glucose transporter 1, hexokinase 2, and pyruvate dehydrogenase E1 component subunit alpha, and the levels of mitochondrial complex I, II, IV, and V subunits of the oxidative phosphorylation pathway. MIA treatment also decreased the expression of autophagy-related proteins, including autophagic elongation protein 5 (ATG5), ATG7, and microtubule-associated protein 1A/1B light chain 3B (LC3-II) and mitophagy-related proteins, including phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), ubiquitin, and p62. These results indicate that MIA interferes with energy metabolism and the autophagic clearance of dysfunctional mitochondria (so called mitophagy). Interestingly, zinc exposure could almost completely reverse the effects of MIA, suggesting its potential protective role against OA progression.     

Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC₅₀ ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,—non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC₅₀ ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH₄C_l cells, but does not compete with ¹²⁵I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to ‘metabotropic’ signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.     

Human Osteoarthritic Cartilage Shows Reduced In Vivo Expression of IL-4, a Chondroprotective Cytokine that Differentially Modulates IL-1β-Stimulated Production of Chemokines and Matrix-Degrading Enzymes In Vitro

Background

In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β.

Methodology/Principal Findings

The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13.

Conclusions/Significance

Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.     

Local ASIC3 modulates pain and disease progression in a rat model of osteoarthritis

Background

Recent data have suggested a relationship between acute arthritic pain and acid sensing ion channel 3 (ASIC3) on primary afferent fibers innervating joints. The purpose of this study was to clarify the role of ASIC3 in a rat model of osteoarthritis (OA) which is considered a degenerative rather than an inflammatory disease.

Methods

We induced OA via intra-articular mono-iodoacetate (MIA) injection, and evaluated pain-related behaviors including weight bearing measured with an incapacitance tester and paw withdrawal threshold in a von Frey hair test, histology of affected knee joint, and immunohistochemistry of knee joint afferents. We also assessed the effect of ASIC3 selective peptide blocker (APETx2) on pain behavior, disease progression, and ASIC3 expression in knee joint afferents.

Results

OA rats showed not only weight-bearing pain but also mechanical hyperalgesia outside the knee joint (secondary hyperalgesia). ASIC3 expression in knee joint afferents was significantly upregulated approximately twofold at Day 14. Continuous intra-articular injections of APETx2 inhibited weight distribution asymmetry and secondary hyperalgesia by attenuating ASIC3 upregulation in knee joint afferents. Histology of ipsilateral knee joint showed APETx2 worked chondroprotectively if administered in the early, but not late phase.

Conclusions

Local ASIC3 immunoreactive nerve is strongly associated with weight-bearing pain and secondary hyperalgesia in MIA-induced OA model. APETx2 inhibited ASIC3 upregulation in knee joint afferents regardless of the time-point of administration. Furthermore, early administration of APETx2 prevented cartilage damage. APETx2 is a novel, promising drug for OA by relieving pain and inhibiting disease progression.     

Three-Dimensional Quantitative Morphometric Analysis (QMA) for In Situ Joint and Tissue Assessment of Osteoarthritis in a Preclinical Rabbit Disease Model

This work utilises advances in multi-tissue imaging, and incorporates new metrics which define in situ joint changes and individual tissue changes in osteoarthritis (OA). The aims are to (1) demonstrate a protocol for processing intact animal joints for microCT to visualise relevant joint, bone and cartilage structures for understanding OA in a preclinical rabbit model, and (2) introduce a comprehensive three-dimensional (3D) quantitative morphometric analysis (QMA), including an assessment of reproducibility. Sixteen rabbit joints with and without transection of the anterior cruciate ligament were scanned with microCT and contrast agents, and processed for histology. Semi-quantitative evaluation was performed on matching two-dimensional (2D) histology and microCT images. Subsequently, 3D QMA was performed; including measures of cartilage, subchondral cortical and epiphyseal bone, and novel tibio-femoral joint metrics. Reproducibility of the QMA was tested on seven additional joints. A significant correlation was observed in cartilage thickness from matching histology-microCT pairs. The lateral compartment of operated joints had larger joint space width, thicker femoral cartilage and reduced bone volume, while osteophytes could be detected quantitatively. Measures between the in situ tibia and femur indicated an altered loading scenario. High measurement reproducibility was observed for all new parameters; with ICC ranging from 0.754 to 0.998. In conclusion, this study provides a novel 3D QMA to quantify macro and micro tissue measures in the joint of a rabbit OA model. New metrics were established consisting of: an angle to quantitatively measure osteophytes (σ), an angle to indicate erosion between the lateral and medial femoral condyles (ρ), a vector defining altered angulation (λ, α, β, γ) and a twist angle (τ) measuring instability and tissue degeneration between the femur and tibia, a length measure of joint space width (JSW), and a slope and intercept (m, Χ) of joint contact to demonstrate altered loading with disease progression, as well as traditional bone and cartilage and histo-morphometry measures. We demonstrate correlation of microCT and histology, sensitive discrimination of OA change and robust reproducibility.     

Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic β-cells: protective role of p62-positive cytoplasmic inclusions

In type II diabetes (T2DM), there is a deficit in β-cells, increased β-cell apoptosis and formation of intracellular membrane-permeant oligomers of islet amyloid polypeptide (IAPP). Human-IAPP (h-IAPP) is an amyloidogenic protein co-expressed with insulin by β-cells. IAPP expression is increased with obesity, the major risk factor for T2DM. In this study we report that increased expression of human-IAPP led to impaired autophagy, due at least in part to the disruption of lysosome-dependant degradation. This action of IAPP to alter lysosomal clearance in vivo depends on its propensity to form toxic oligomers and is independent of the confounding effect of hyperglycemia. We report that the scaffold protein p62 that delivers polyubiquitinated proteins to autophagy may have a protective role against human-IAPP-induced apoptosis, apparently by sequestrating protein targets for degradation. Finally, we found that inhibition of lysosomal degradation increases vulnerability of β-cells to h-IAPP-induced toxicity and, conversely, stimulation of autophagy protects β-cells from h-IAPP-induced apoptosis. Collectively, these data imply an important role for the p62/autophagy/lysosomal degradation system in protection against toxic oligomer-induced apoptosis.     

Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta

Introduction

Although IL-1β is believed to be crucial in the pathogenesis of osteoarthritis (OA), the IL-1β blockade brings no therapeutic benefit in human OA and results in OA aggravation in several animal models. We explored the role of a cytokine signaling 1 (SOCS1) suppressor as a regulatory modulator of IL-1β signaling in chondrocytes.

Methods

Cartilage samples were obtained from patients with knee OA and those without OA who underwent surgery for femur-neck fracture. SOCS1 expression in cartilage was assessed with immunohistochemistry. IL-1β-induced SOCS1 expression in chondrocytes was analyzed with quantitative polymerase chain reaction and immunoblot. The effect of SOCS1 on IL-1β signaling pathways and the synthesis of matrix metalloproteinases (MMPs) and aggrecanase-1 was investigated in SOCS1-overexpressing or -knockdown chondrocytes.

Results

SOCS1 expression was significantly increased in OA cartilage, especially in areas of severe damage (P < 0.01). IL-1β stimulated SOCS1 mRNA expression in a dose-dependent pattern (P < 0.01). The IL-1β-induced production of MMP-1, MMP-3, MMP-13, and ADAMTS-4 (aggrecanase-1, a disintegrin and metalloproteinase with thrombospondin motifs 4) was affected by SOCS1 overexpression or knockdown in both SW1353 cells and primary human articular chondrocytes (all P values < 0.05). The inhibitory effects of SOCS1 were mediated by blocking p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) activation, and by downregulating transforming growth factor-β-activated kinase 1 (TAK1) expression.

Conclusions

Our results show that SOCS1 is induced by IL1-β in OA chondrocytes and suppresses the IL-1β-induced synthesis of matrix-degrading enzymes by inhibiting IL-1β signaling at multiple levels. It suggests that the IL-1β-inducible SOCS1 acts as a negative regulator of the IL-1β response in OA cartilage.     

Exosomes from IL-1β stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes

Introduction

Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells.

Methods

Human synovial fibroblasts (SFB) and articular chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal articular chondrocytes were treated with exosomes from SFB, and Interleukin-1β (IL-1β) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis.

Results

Exosomes from IL-1β stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in articular chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1β stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1β, TNFα MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1β stimulated SFB compared to non-stimulated SFB.

Conclusions

Exosomes from IL-1β stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints.     

Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

Introduction

AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and performed translational studies of AMPK pharmacologic activation.

Methods

We assessed activity (phosphorylation) of LKB1 and AMPKα in mouse knee OA cartilage, in aging mouse cartilage (6 to 24 months), and in chondrocytes after mechanical injury by dynamic compression, via immunohistochemistry or western blot. We knocked down LKB1 by siRNA transfection. Nitric oxide, matrix metalloproteinase (MMP)-3, and MMP-13 release were measured by Griess reaction and ELISA, respectively.

Results

Knockdown of LKB1 attenuated chondrocyte AMPK activity, and increased nitric oxide, MMP-3 and MMP-13 release (P <0.05) in response to IL-1β and TNFα. Both LKB1 and AMPK activity were decreased in mouse knee OA and aged knee cartilage, and in bovine chondrocytes after biomechanical injury. Pretreatment of bovine chondrocytes with AMPK activators AICAR and A-769662 inhibited both AMPKα dephosphorylation and catabolic responses after biomechanical injury.

Conclusion

LKB1 is required for chondrocyte AMPK activity, thereby inhibiting matrix catabolic responses to inflammatory cytokines. Concurrent loss of LKB1 and AMPK activity in articular chondrocytes is associated with OA, aging and biomechanical injury. Conversely, pharmacologic AMPK activation attenuates catabolic responses to biomechanical injury, suggesting a potentially novel approach to inhibit OA development and progression.