Iron oxide labeling does not affect differentiation potential of human bone marrow mesenchymal stem cells exhibited by their differentiation into cardiac and neuronal cells

Osteoarthritis (OA) is characterized by articular cartilage degradation and joint inflammation. The purpose of the present study is to elucidate the role of the specific function of PRMT1 in chondrocytes and its association with the pathophysiology of OA. We observed that the expression of PRMT1 was apparently upregulated in OA cartilage, as well as in chondrocytes stimulated with IL-1β. Additionally, knockdown of PRMT1 suppressed interleukin 1 beta (IL-1β)-induced extracellular matrix (ECM) metabolic imbalance by regulating the expression of MMP-13, ADAMTS-5, COL2A1, and ACAN. Furthermore, silencing of PRMT1 dramatically declined the production of prostaglandin E2 (PGE2) and nitric oxide as well as the level of pro-inflammatory cytokine IL-6 and TNF-α. Mechanistic analyses further revealed that IL-1β-induced activation of the Hedgehog/Gli-1 signaling is suppressed upon PRMT1 knockdown. However, the effects of inhibition of PRMT1-mediated IL-1β-induced cartilage matrix degradation and inflammatory response in OA chondrocytes were obviously abolished by Hedgehog agonist Purmorphamine (Pur). Our data collectively suggest that silencing of PRMT1 exerts anti-catabolic and anti-inflammatory effects on IL-1β-induced chondrocytes via suppressing the Gli-1 mediated Hedgehog signaling pathway, indicating that PRMT1 plays a critical role in OA development and serves as a promising therapeutic target for OA.     

Matrix metalloproteinase (MMP)-12 regulates MMP-9 expression in interleukin-1beta-treated articular chondrocytes

Limited information is available on the expression and role of matrix metalloproteinase (MMP)-12 in chondrocytes. We characterized the expression mechanism of MMP-12 and possible function in chondrocytes. Interleukin (IL)-1beta induced the expression and activation of MMP-12 in primary culture chondrocytes and cartilage explants via mitogen-activated protein (MAP) kinase signaling pathways. Among MAP kinases, extracellular signal-regulated kinase and p38 kinase are necessary for MMP-12 expression, whereas c-jun N-terminal kinase is required for the activation of MMP-12. The possibility that MMP-12 acts as a modulator of other MMP was examined. MMP-12 alone did not affect other MMP expressions. However, MMP-12 enhanced expression and activation of MMP-9 in the presence of IL-1beta. Our results indicate that IL-1beta in chondrocytes induces the expression and activation of MMP-12, which, in turn, augments MMP-9 expression and activation.     

Glucosamine prevents <Emphasis Type="Italic">in vitro</Emphasis> collagen degradation in chondrocytes by inhibiting advanced lipoxidation reactions and protein oxidation

Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction. Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.     

Proliferation of rabbit chondrocyte and inhibition of IL-1β-induced apoptosis through MEK/ERK signaling by statins

Chondrocyte plays a critical role in endochondral ossification and cartilage repair by maintaining the cartilaginous matrix. Statins have been widely used to lower the cholesterol level in patients with cardiovascular disorders. Previous research has demonstrated potential role of statins in chondrocyte proliferation. This study addresses the proliferation-regulatory effect of lovastatin in rabbit chondrocytes as well as the underlying signaling mechanisms, thereby exploring its potential application in chondrocyte-related disorders, such as cartilage damage and osteoarthritis. Rabbit chondrocytes were treated with lovastatin at multiple concentrations, and the proliferation rate was measured by CCK-8 test. The results showed significant increase in chondrocyte proliferation under lovastatin treatment. Using real-time quantitative PCR, it was observed that the expression levels of COL2A1, SOX-9, Caspase-3, and MMP-3 genes were significantly changed by lovastatin treatment. Western blotting analysis showed that the abundance of COL2A1, SOX-9, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, Caspase-3, and MMP-3 proteins was also significantly influenced by lovastatin treatment. Interleukine-1 beta (IL-1β) is involved in the progression of osteoarthritis (OA) by inducing articular cartilage and chondrocyte aging and senescence. In this study, we observed that lovastatin treatment inhibited IL-1β-induced chondrocyte apoptosis, while the combined treatment of lovastatin and U0126 evidently offset the apoptosis-inhibiting effect of lovastatin in chondrocyte proliferation. The expressional level and protein abundance of COL2A1, SOX-9, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, caspase-3, and MMP-3 genes showed significant alterations under the combined treatment. Together, our results suggested that lovastatin significantly promoted proliferation and inhibited the IL-1β-induced apoptosis in rabbit chondrocytes, which was mediated by the MEK/ERK signaling.     

Long noncoding RNA MALAT-1 inhibits apoptosis and matrix metabolism disorder in interleukin-1β-induced inflammation in articular chondrocytes via the JNK signaling pathway

Proinflammatory cytokine such as interleukin (IL)-1β causes inflammation of articular cartilage. In this current study, we explored the chondroprotective effects of long noncoding RNA (lncRNA) MALAT-1 on cell proliferation, apoptosis, and matrix metabolism in IL-1β-induced inflammation in articular chondrocytes. Articular chondrocytes from knee joints of normal rats were isolated and cultured, followed by identification through observation of toluidine blue and COL II immunocytochemical stainings. The proliferation of chondrocytes at passage 2 was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The inflammatory chondrocytes induced by 10 ng/mL IL-1β were observed and identified by toluidine blue and COL II immunocytochemical stainings. pcDNA 3.1 and pcDNA-MALAT-1 were transfected in the chondrocytes. Ultrastructure of chondrocytes was observed by using a transmission electron microscope. The MTT assay was carried out to evaluate chondrocyte viability. Hoechst 33258 staining and flow cytometry were adopted to assess chondrocyte apoptosis. The chondrocytes at passage 2 with the biological characteristics of chondrocytes were used for subsequent experiments. In IL-1β-treated chondrocytes, the growth rate of chondrocytes slowed down, the cells became narrow and long, the vacuoles were seen in the cells, and the morphology of the chondrocytes was irregular. The toluidine blue staining and the immunohistochemical staining of COL II became weaker. In response to IL-1β induction, articular chondrocytes showed reduced MALAT-1 expression; moreover, obvious cartilage injury was observed with decreased chondrocyte viability and Col II expression and elevated chondrocyte apoptosis, MMP-13 expression, and p-JNK expression. With the treatment of pcDNA-MALAT-1, the cartilage injury was alleviated with increased chondrocyte viability and type II collagen (Col II) expression and reduced chondrocyte apoptosis, MMP-13 expression and p-JNK expression. Taken together these results, lncRNA MALAT-1 blocked the activation of the JNK signaling pathway; thereby, IL-1β-induced inflammation in articular chondrocytes was reduced with enhanced chondrocyte proliferation and suppressed chondrocyte apoptosis and extracellular matrix degradation.     

Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1

Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1β is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1β-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1β increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1β-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.     

Interferonregulatoryfactor-8(IRF-8) regulates the expression of matrix metalloproteinase-13 (MMP-13) in chondrocytes

Low levels of inflammation-induced expression of matrix metalloproteinase (MMP) play a crucial role in articular cartilage matrix destruction in osteoarthritis (OA) patients. Interferon regulatory factor-8 (IRF-8), an important member in the IRF family, plays a key role in regulating the inflammation-related signaling pathway. The aim of this study is to investigate the physiological roles of IRF-8 in the pathological progression of OA. We found that IRF-8 was expressed in human primary chondrocytes. Interestingly, the expression of IRF-8 was upregulated in OA chondrocytes. In addition, IRF-8 was increased in response to interleukin-1β (IL-1β) treatment, mediated by the Janus kinase 2 (JAK2) pathway. Overexpression of IRF-8 in human chondrocytes by transduction with lentiviral-IRF-8 exacerbated IL-1β-induced expression of matrix metalloproteinase-13 (MMP-13) in human chondrocytes. In contrast, knockdown of IRF-8 inhibited IL-1β-induced expression of MMP-13. Importantly, IRF-8 could bind to the promoter of MMP-13 and stimulate its activity. Additionally, overexpression of IRF-8 exacerbated IL-1β-induced degradation of type II collagen. However, silencing IRF-8 abrogated the degradation of type II collagen. Taken together, our findings identified a novel function of IRF-8 in regulating articular cartilage matrix destruction by promoting the expression of MMP-13.     

Development of selective tolerance to interleukin-1beta by human chondrocytes in vitro

Interleukin-1 induces release of NO and PGE(2) and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1beta. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1beta (0.05-10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1beta declined over the pretreatment period. In response to IL-1beta (0.1 ng/ml), NO and PGE(2) release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1beta on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1beta-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1beta increased glucose consumption and lactate production throughout the treatment. IL-1beta stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE(2) and MMP-13 in response to IL-1beta than OA cultures. Like the OA, IL-1beta-induced NO and PGE(2) release decreased over time. In conclusion, with prolonged exposure to IL-1beta, human chondrocytes develop selective tolerance involving NO and PGE(2) release but not MMP-13 production, metabolic activity, or matrix metabolism.     

Effects of chondroitin and glucosamine sulfate in a dietary bar formulation on inflammation, interleukin-1beta, matrix metalloprotease-9, and cartilage damage in arthritis

This study examined the effects of chondroitin sulfate (CS) alone and CS plus glucosamine sulfate (GS) in a dietary bar formulation on inflammatory parameters of adjuvant-induced arthritis and on the synthesis of interleukin-1beta (IL-1beta) and matrix metalloprotease-9 (MMP-9). Following 25 days pretreatment with dietary bars containing either CS alone, CS plus GS, or neither CS nor GS, rats were either sham injected or injected with Freund's complete adjuvant into the tail vein. Rats were fed their respective bars for another 17 days after inoculation. Parameters of disease examined included clinical score (combination of joint temperature, edema, hyperalgesia, and standing and walking limb function), incidence of disease, levels of IL-1beta in the serum and paw joints, levels of MMP-9 in the paw joints, paw joint histology, and joint cartilage thickness. Treatment with CS plus GS, but not CS alone, significantly reduced clinical scores, incidences of disease, joint temperatures, and joint and serum IL-1beta levels. Treatment with CS alone and CS plus GS inhibited the production of edema and prevented raised levels of joint MMP-9 associated with arthritis. Similarly, CS alone and CS plus GS treatment also prevented the development of cartilage damage associated with arthritis. Combination CS plus GS treatment in a dietary bar formulation ameliorates clinical, inflammatory, and histologic parameters of adjuvant-induced arthritis. The benefits of CS and GS in combination are more pronounced than those of CS alone. The reduction of arthritic disease by CS plus GS is associated with a reduction of IL-1beta and MMP-9 synthesis.     

Triptolide suppresses proinflammatory cytokine-induced matrix metalloproteinase and aggrecanase-1 gene expression in chondrocytes

A hallmark of rheumatoid- and osteoarthritis (OA) is proinflammatory cytokine-induced degeneration of cartilage collagen and aggrecan by matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS). Effects of the Chinese herb, Tripterygium wilfordii Hook F (TWHF), on cartilage and its anti-arthritic mechanisms are poorly understood. This study investigated the impact of a purified derivative of TWHF, PG490 (triptolide), on cytokine-stimulated expression of the major cartilage damaging proteases, MMP-3, MMP-13, and ADAMTS4. PG490 inhibited cytokine-induced MMP-3, MMP-13 gene expression in primary human OA chondrocytes, bovine chondrocytes, SW1353 cells, and human synovial fibroblasts. Triptolide was effective at low doses and blocked the induction of MMP-13 by IL-1 in human and bovine cartilage explants. TWHF extract and PG490 also suppressed IL-1-, IL-17-, and TNF-alpha-induced expression of ADAMTS-4 in bovine chondrocytes. Thus, PG490 could protect cartilage from MMP- and aggrecanase-driven breakdown. The immunosuppressive, cartilage protective, and anti-inflammatory properties could make PG490 potentially a new therapeutic agent for arthritis.     

Co-localization of integrins and matrix metalloproteinases in the extracellular matrix of chondrocyte cultures

Beta1-integrins were found in the cartilage matrix, suggesting their implication in the assembly of its architectural scaffold, but the mechanism for this event is not yet clear. Matrix metalloproteinases (MMPs) may be involved in an integrin-shedding mechanism and matrix beta1-integrins may act to alter MMP activity. To begin to address this question, this study was designed to determine whether beta1-integrins and MMPs are colocalized in the chondrocytes or in the extracellular matrix of cartilage. We investigated high-density cultures of limb buds of 12-day-old mouse embryos by double immunofluorescence, immunoelectron microscopy and by coimmunoprecipitation assays in order to examine the localization of beta1-integrins and matrix metalloproteinases (MMP-1, MMP-3 and MMP-9) in cartilage. It was found, that all investigated MMPs and beta1-integrins were specifically co-localized in high-density cartilage cultures. Immunogold and immunofluorescence labelling of both beta1-integrins and MMPs were observed not only at the surface of chondrocytes but mainly also in the pericellular space and distributed between collagen fibrils in the extracellular matrix (ECM) as well. Results of immunoprecipitation experiments suggest a functional association of MMPs and beta1-integrins in chondrocytes as already described for other cell types. Further investigations are needed to elucidate the functional association between beta1-integrins and MMPs in chondrocytes.