T-2 toxin induces articular cartilage damage by increasing the expression of MMP-13 via the TGF-ß receptor pathway.

T-2 toxin pre-disposes individuals to osteoarthritis, Kashin-Beck disease (KBD). The major pathological change associated with KBD is the degradation of the articular cartilage matrix. Herein, we investigated the key molecules that regulate T-2 toxin-mediated cartilage degradation. Potential KBD treatments were also investigated. Sprague Dawley rats were divided into the T-2 toxin group and the control group. The T-2 toxin group received 100 ng/g BW/day, whereas the control group received a similar dose of PBS. The expression of matrix metalloproteinase-13 (MMP-13) and TGF-ß receptor I/II (TGF-ßRI/II) was analyzed using immunohistochemical staining. C28/I2 chondrocytes were exposed to TGF-ßRI/II binding inhibitor (GW788388) for 24 h before incubation in different T-2 toxin concentrations (0, 6, 12, and 24 ng/mL for 72 h). The expression of mRNA for TGF-ßRI/II, MMP-13 and proteins for MMP-13, and Smad-2 in chondrocytes were analyzed using RT-PCR and western blot, respectively. Safranin O staining revealed that T-2 toxin treatment modulated the expression of articular cartilage matrix. On the other hand, T-2 toxin treatment sharply increased the expression of MMP-13, TGF-ßRI, and TGF-ßRII in the rat cartilages. Interestingly, blocking the TGF-ßRs-smad 2 signaling pathway using GW788388 abrogated the effect of T-2 toxin on upregulating MMP-13 expression. The expression of MMP-13 in chondrocytes induced with T-2 toxin is regulated via the TGF-ßRs signaling pathway. As such, inhibiting the expression of TGF-ßRs is a potential KBD treatment.

The effect of opine on matrix metalloproteinase expression in mice with breast cancer.

Regarding the anti-inflammatory and anti-tumour effects of arginine and its derivatives, this study evaluates matrix metalloproteinase (MMPs) expression in an animal model of breast cancer following administration of octopine. In this study, 40 animals of Balb/C mice were divided into 5 groups: the healthy control, the cancer control, the cancer group receiving 50 mg of octopine, the cancer group receiving 100 mg of octopine and the cancer group receiving 150 mg of octopine for 3 weeks. 4T1 cell line was used to induce cancer. Biopsy specimens were enrolled from mice and MMP-1, MMP-3 and MMP-9 gene expression evaluated using real-time PCR, while these protein amounts were measured using immunohistochemistry and ELISA methods. Data were analysed using one-way ANOVA, Kruskal-Wallis and Mann-Whitney U tests (p < .05). The results showed that 100 mg octopine consumption had significant decreasing effect on MMP-9 expression (p = .02) in the treatment group compared with cancerous non-treated mice. Furthermore, results from immunohistochemistry and ELISA confirmed this effect, the protein amount of MMP-9 was significantly decreased in group treating with 100 mg octopine (.005). The use of octopine has a beneficial effect on reducing MMP-9 in mice breast cancer.

Colchicine protects against cartilage degeneration by inhibiting MMP13 expression via PLC-γ1 phosphorylation.

OBJECTIVE: Low molecular weight compounds that reduce the expression of MMP13 at the mRNA level might serve as disease-modifying osteoarthritis (OA) drugs (DMOADs). The objective of this study was to identify a candidate DMOAD that targets MMP13 expression. DESIGN: High-throughput screening was performed to identify compounds that suppress inflammatory cytokine-induced MMP13 expression. Ingenuity pathway analysis (IPA) using isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analysis was conducted to identify signaling pathways related to cytokines. MMP13 expression in chondrocytes was evaluated through RT-qPCR and western blotting analyses. Additionally, 10-week-old mice were subjected to destabilization of the medial meniscus (DMM) surgery to induce OA and were sacrificed 12 weeks post-surgery for pathological examination. OA was evaluated using the OARSI scoring system. RESULTS: Colchicine was identified as a DMOAD candidate as it inhibited inflammatory cytokine-induced MMP13 expression in vitro, and the colchicine-administered mice with DMM presented significantly lower OARSI scores (adjusted P: 0.0242, mean difference: 1.6, 95% confidence interval (CI) of difference: 0.1651-3.035) and significantly lower synovial membrane inflammation scores (adjusted P: 0.0243, mean difference: 0.6, 95% CI of difference: 0.06158-1.138) than mice with DMM. IPA further revealed that components of the Rho signaling pathways are regulated by cytokines and colchicine. IL-1ß and TNF-α activate RAC1 and SRC signals, respectively, leading to the phosphorylation of PLC-γ1 and synergistic induction of MMP13 expression. Most notably, colchicine abrogates inflammatory cytokine-induced phosphorylation of PLC-γ1, leading to the induction of MMP13 expression. CONCLUSIONS: Colchicine is a potential DMOAD candidate that inhibits MMP13 expression and consequent cartilage degradation by disrupting the SRC/RAC1-phospho-PLCγ1-Ca2+ signaling pathway.

The combination of mitogenic stimulation and DNA damage induces chondrocyte senescence.

OBJECTIVE: Cellular senescence is a phenotypic state characterized by stable cell-cycle arrest, enhanced lysosomal activity, and the secretion of inflammatory molecules and matrix degrading enzymes. Senescence has been implicated in osteoarthritis (OA) pathophysiology; however, the mechanisms that drive senescence induction in cartilage and other joint tissues are unknown. While numerous physiological signals are capable of initiating senescence, one emerging theme is that damaged cells convert to senescence in response to sustained mitogenic stimulation. The goal of this study was to develop an in vitro articular cartilage explant model to investigate the mechanisms of senescence induction. DESIGN: This study utilized healthy cartilage derived from cadaveric equine stifles and human ankles. Explants were irradiated to initiate DNA damage, and mitogenic stimulation was provided through serum-containing medium and treatment with transforming growth factor ß1 and basic fibroblastic growth factor. Readouts of senescence were a quantitative flow cytometry assay to detect senescence-associated ß galactosidase activity (SA-ß-gal), immunofluorescence for p16 and γH2AX, and qPCR for the expression of inflammatory genes. RESULTS: Human cartilage explants required both irradiation and mitogenic stimulation to induce senescence as compared to baseline control conditions (7.16% vs 2.34% SA-ß-gal high, p = 0.0007). These conditions also resulted in chondrocyte clusters within explants, a persistent DNA damage response, increased p16, and gene expression changes. CONCLUSIONS: Treatment of cartilage explants with mitogenic stimuli in the context of cellular damage reliably induces high levels of SA-ß-gal activity and other senescence markers, which provides a physiologically relevant model system to investigate the mechanisms of senescence induction.

Positive Effects of a Young Systemic Environment and High Growth Differentiation Factor 11 Levels on Chondrocyte Proliferation and Cartilage Matrix Synthesis in Old Mice.

OBJECTIVE: To investigate the effects of a young systemic environment and growth differentiation factor 11 (GDF-11) on aging cartilage. METHODS: A heterochronic parabiosis model (2-month-old mouse and 12-month-old mouse [Y/O]), an isochronic parabiosis model (12-month-old mouse and 12-month-old mouse [O/O]), and 12-month-old mice alone (O) were evaluated. Knee joints and chondrocytes from old mice were examined by radiography, histology, cell proliferation assays, immunohistochemistry, Western blotting, and quantitative reverse transcriptase-polymerase chain reaction 16 weeks after parabiosis surgery. GDF-11 was injected into 12-month-old mouse joints daily for 16 weeks. Cartilage degeneration, cell proliferation, and osteoarthritis-related gene expression were evaluated. RESULTS: Osteoarthritis Research Society International scores in old mice were significantly lower in the Y/O group than in the O/O and O groups (both P < 0.05). The percentage of 5-ethynyl-2'-deoxyuridine-positive chondrocytes in old mice was significantly higher in the Y/O group than in the other groups (P < 0.05). Type II collagen (CII) and SOX9 messenger RNA levels differed in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). RUNX-2, CX, and matrix metalloproteinase 13 levels were significantly lower in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). Similar results were obtained for protein expression levels and after GDF-11 treatment in vitro and in vivo. Phosphorylated Smad2/3 (pSmad2/3) levels were higher in the recombinant GDF-11-treated group than in the control group. CONCLUSION: A young systemic environment promotes chondrocyte proliferation and cartilage matrix synthesis in old mice. GDF-11, a "young factor," contributes to these effects through the up-regulation of pSmad2/3.

Raloxifene retards cartilage degradation and improves subchondral bone micro-architecture in ovariectomized rats with patella baja-induced - patellofemoral joint osteoarthritis.

OBJECTIVE: Abnormal remodeling of subchondral bone (SB) induced by estrogen deficiency has been shown to be involved in osteoarthritis (OA). Raloxifene (RAL) is commonly used to treat postmenopausal osteoporosis (OP). However, little is known about its effects on OA combined with estrogen deficiency. This study was performed to evaluate the efficacy of RAL on patella baja-induced patellofemoral joint OA (PFJOA) in an ovariectomized rat model. DESIGN: Patellar ligament shortening (PLS) and ovariectomy (OVX) were performed simultaneously in 3-month-old female Sprague-Dawley rats, which were treated with RAL (10 mg/kg/day) or vehicle at 72 h postoperatively for 10 weeks. PFJOA was assessed by immunohistochemistry (IHC), real-time polymerase chain reaction (PCR), tartrate-resistant acid phosphatase (TRAP) staining, enzyme-linked immunosorbent assay (ELISA), micro-computed tomography (µCT), histomorphology and behavioral analyses. RESULTS: X-ray examinations showed that patella baja was successfully established by PLS. Histomorphological analysis revealed that PFJOA was significantly exacerbated by OVX and markedly alleviated by RAL. Moreover, RAL improved cartilage metabolism by decreasing MMP-13, ADAMTS-4, and caspase-3 and increasing Col-II and aggrecan at both the protein and mRNA levels. Furthermore, RAL markedly improved bone mass and SB microarchitecture and reduced osteoclast numbers and the serum osteocalcin and CTX-I levels. Although RAL showed a trend toward reducing pain sensitivity based on mechanical allodynia testing, this result was not statistically significant. CONCLUSION: These findings demonstrate that RAL treatment retards PFJOA progression in an ovariectomized rat model, suggesting that it may be a potential candidate for amelioration of the progression of PFJOA accompanied by postmenopausal OP.

Intra-articular injection of magnesium chloride attenuates osteoarthritis progression in rats.

OBJECTIVE: To explore the effects of Mg2+ on the expression of osteoarthritic markers in human cartilage and synovium tissue explants. To investigate the therapeutic effect of intra-articular injection of Mg2+ in an established rat OA (Osteoarthritis) model of anterior cruciate ligament transection with partial medial meniscectomy (ACLT + PMM). DESIGN: Human cartilage and synovium explants were collected from total knee replacement surgeries and incubated with MgCl2 (20 mmol/L) in vitro. A rat OA model was established by ACLT + PMM surgery in 450-500 g male Sprague Dawley (SD) rats. To select the optimal dose, intra-articular injections of MgCl2 (0.05, 0.5, 5 mol/L) were performed at 4 weeks after the surgery every 3 days for 2 weeks. The effect of optimized MgCl2 was further determined by histology, immunohistochemistry, and quantitative real-time polymerase chain reaction. RESULTS: The expressions of osteoarthritic markers in human cartilage and synovium explants were inhibited by Mg2+in vitro. Immunohistochemical analysis further suggested the inhibitory effects of Mg2+ on the expression of MMP-13 and IL-6 in the human tissue explants. Cartilage degeneration and synovitis in ACLT + PMM rats were significantly improved by intra-articular injections of Mg2+ (0.5 mol/L). Immunohistochemical analysis also showed the regulatory effects of Mg2+ on osteoarthritic markers in both cartilage and synovium in rats, consistent with in vitro results. CONCLUSION: Intra-articular injections of Mg2+ at 0.5 mol/L attenuate the progression of OA in the ACLT + PMM rat model. Such effect was at least in part explained by the promotion of cartilage matrix synthesis and the suppression of synovial inflammation.

Development of matrix metalloproteinase-13 inhibitors - A structure-activity/structure-property relationship study.

A structure-activity/structure-property relationship study based on the physicochemical as well as in vitro pharmacokinetic properties of a first generation matrix metalloproteinase (MMP)-13 inhibitor (2) was undertaken. After systematic variation of inhibitor 2, compound 31 was identified which exhibited microsomal half-life higher than 20 min, kinetic solubility higher than 20 µM, and a permeability coefficient greater than 20 × 10-6 cm/s. Compound 31 also showed excellent in vivo PK properties after IV dosing (Cmax = 56.8 µM, T1/2 (plasma) = 3.0 h, Cl = 0.23 mL/min/kg) and thus is a suitable candidate for in vivo efficacy studies in an OA animal model.

Proprotein convertase furin inhibits matrix metalloproteinase 13 in a TGFß-dependent manner and limits osteoarthritis in mice.

Cartilage loss in osteoarthritis (OA) results from altered local production of growth factors and metalloproteases (MMPs). Furin, an enzyme involved in the protein maturation of MMPs, might regulate chondrocyte function. Here, we tested the effect of furin on chondrocyte catabolism and the development of OA. In primary chondrocytes, furin reduced the expression of MMP-13, which was reversed by treatment with the furin inhibitor α1-PDX. Furin also promoted the activation of Smad3 signaling, whereas activin receptor-like kinase 5 (ALK5) knockdown mitigated the effects of furin on MMP-13 expression. Mice underwent destabilization of the medial meniscus (DMM) to induce OA, then received furin (1 U/mice), α1-PDX (14 µg/mice) or vehicle. In mice with DMM, the OA score was lower with furin than vehicle treatment (6.42 ± 0.75 vs 9.16 ± 0.6, p < 0.01), and the number of MMP-13(+) chondrocytes was lower (4.96 ± 0.60% vs 20.96 ± 8.49%, p < 0.05). Moreover, furin prevented the increase in ALK1/ALK5 ratio in cartilage induced by OA. Conversely, α1-PDX had no effect on OA cartilage structure. These results support a protective role for furin in OA by maintaining ALK5 receptor levels and reducing MMP-13 expression. Therefore, furin might be a potential target mediating the development of OA.

Chondroprotective activity of N-acetyl phenylalanine glucosamine derivative on knee joint structure and inflammation in a murine model of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by joint structure changes and inflammation, both mediated by the IκB kinase (IKK) signalosome complex. The ability of N-acetyl phenylalanine derivative (NAPA) to increase cartilage matrix components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity, has been observed in vitro. The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM). DESIGN: 26 mice were divided into three groups: (1) DMM surgery without treatment; (2) DMM surgery treated after 2 weeks with one intra-articular injection of NAPA (2.5 mM) and (3) no DMM surgery. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and microhardness of subchondral bone (SB) tests. RESULTS: The injection of NAPA significantly improved cartilage thickness (CT) and reduced Chambers and Mankin modified scores and fibrillation index (FI), with weaker MMP13, ADAMTS5, MMP10 and IKKα staining. The microhardness measurements did not shown statistically significant differences between the different groups. CONCLUSIONS: NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity in vivo.

IL37 dampens the IL1ß-induced catabolic status of human OA chondrocytes.

Objective: A crucial feature of OA is cartilage degradation. This process is mediated by pro-inflammatory cytokines, among other factors, via induction of matrix-degrading enzymes. Interleukin 37 (IL37) is an anti-inflammatory cytokine and is efficient in blocking the production of pro-inflammatory cytokines during innate immune responses. We hypothesize that IL37 is therapeutic in treating the inflammatory cytokine cascade in human OA chondrocytes and can act as a counter-regulatory cytokine to reduce cartilage degradation in OA. Methods: Human OA cartilage was obtained from patients undergoing total knee or hip arthroplasty. Immunohistochemistry was applied to study IL37 protein expression in cartilage biopsies from OA patients. Induction of IL37 expression by IL1ß, OA synovium-conditioned medium and TNFα was investigated in human OA chondrocytes. Adenoviral overexpression of IL37 followed by IL1ß stimulation was performed to investigate the anti-inflammatory potential of IL37. Results: IL37 expression was detected in cartilage biopsies of OA patients and induced by IL1ß. After IL1ß stimulation, increased IL1ß, IL6 and IL8 expression was observed in OA chondrocytes. Elevated IL37 levels diminished the IL1ß-induced IL1ß , IL6 and IL8 gene levels and IL1ß and IL8 protein levels. In addition to the reduction in pro-inflammatory cytokine expression, IL37 reduced MMP1 , MMP3 , MMP13 and disintegrin and metalloproteinase with thrombospondin motifs 5 gene levels and MMP3 and MMP13 protein levels. Conclusion: IL37 is induced by IL1ß, and IL37 itself reduced IL1ß, IL6 and IL8 production, indicating that IL37 is able to induce a counter-regulatory anti-inflammatory feedback loop in chondrocytes. In addition, IL37 dampens catabolic enzyme expression. This supports IL37 as a potential therapeutic target in OA.

Hydroxytyrosol modulates the levels of microRNA-9 and its target sirtuin-1 thereby counteracting oxidative stress-induced chondrocyte death.

OBJECTIVE: Nutraceutical compounds, such as hydroxytyrosol (HT), have been found to exert protective effects in osteoarthritis (OA) by affecting a variety of key molecular and cellular processes in chondrocytes. However, to our knowledge, no relationship has been reported between nutraceuticals and microRNA (miR) network in OA models. Here, we identified a miR that is implicated in HT-mediated chondroprotection following oxidative stress condition by targeting sirtuin-1 (SIRT-1). METHODS: Human primary and C-28/I2 chondrocytes were pre-treated with 100 µM HT 30 min before 100 µM H2O2 addition. In silico analyses were exploited to select putative candidate miRs able to target SIRT-1 mRNA. Luciferase-based gene reporter assay was employed to demonstrate the direct link between miR-9 and its putative mRNA target. Transient transfection approach was performed to examine the effects of miR-9 levels on caspase activity, cell viability and expression of OA-related genes. RESULTS: MiR-9 was identified and confirmed as a post-transcriptional regulator of SIRT-1. MiR-9 and SIRT-1 levels showed opposite changes in chondrocytes following H2O2 and HT treatment. Moreover mir-9 silencing inhibited cell death induced by H2O2 partly through down-regulation of SIRT-1, whereas miR-9 overexpression markedly reduced the protective effect of HT. The manipulation of miR-9 levels also resulted in the modulation of OA-related gene expression, including MMP-13, VEGF and RUNX-2. CONCLUSIONS: These results show that miR-9 is a critical mediator of the deleterious and OA-related effects of oxidative stress in chondrocytes and that modulation of miR expression may be a crucial mechanism underlying the protective action of HT.

Role of the C-C chemokine receptor-2 in a murine model of injury-induced osteoarthritis.

OBJECTIVE: We previously found in our embryonic studies that proper regulation of the chemokine CCL12 through its sole receptor CCR2, is critical for joint and growth plate development. In the present study, we examined the role of CCR2 in injury-induced-osteoarthritis (OA). METHOD: We used a murine model of injury-induced-OA (destabilization of medial meniscus, DMM), and systemically blocked CCR2 using a specific antagonist (RS504393) at different times during disease progression. We examined joint degeneration by assessing cartilage (cartilage loss, chondrocyte hypertrophy, MMP-13 expression) and bone lesions (bone sclerosis, osteophytes formation) with or without the CCR2 antagonist. We also performed pain behavioral studies by assessing the weight distribution between the normal and arthritic hind paws using the IITS incapacitance meter. RESULTS: Testing early vs delayed administration of the CCR2 antagonist demonstrated differential effects on joint damage. We found that OA changes in articular cartilage and bone were ameliorated by pharmacological CCR2 blockade, if given early in OA development: specifically, pharmacological targeting of CCR2 during the first 4 weeks (wks) following injury, reduced OA cartilage and bone damage, with less effectiveness with later treatments. Importantly, our pain-related behavioral studies showed that blockade of CCR2 signaling during early, 1-4 wks post-surgery or moderate, 4-8 wks post-surgery, OA was sufficient to decrease pain measures, with sustained improvement at later stages, after treatment was stopped. CONCLUSIONS: Our data highlight the potential efficacy of antagonizing CCR2 at early stages to slow the progression of post-injury OA and, in addition, improve pain symptoms.

Radiolabeled Selective Matrix Metalloproteinase 13 (MMP-13) Inhibitors: (Radio)Syntheses and in Vitro and First in Vivo Evaluation.

The noninvasive imaging of MMP activity in vivo could have a high impact in basic research as well as in clinical applications. This approach can be established using radiolabeled MMP inhibitors (MMPIs) as tracers for the detection of activated MMPs by means of PET. However, the complexity of diseases associated with dysregulated MMP expression necessitates the imaging of distinct MMPs or MMP subgroups to distinguish their individual role in specific diseases. To this end, selective and potent MMP-13 inhibitors based on a N,N'-bis(benzyl)pyrimidine-4,6-dicarboxamide core have been synthesized and successfully radiolabeled with carbon-11, fluorine-18, and gallium-68. Selected radiolabeled candidates were evaluated in vitro and in vivo regarding their pharmacokinetic properties and metabolic stability.

Salubrinal Suppresses IL-17-Induced Upregulation of MMP-13 and Extracellular Matrix Degradation Through the NF-kB Pathway in Human Nucleus Pulposus Cells.

Matrix metalloproteinase 13 (MMP-13) plays an important role in the process of pro-inflammatory cytokine-induced intervertebral disc degeneration (IDD). This study examined the effect of IL-17 on the regulation of MMP-13 and the extracellular matrix (ECM) in the intervertebral disc (IVD). We then examined whether salubrinal, a known inhibitor of eIF2α dephosphorylation, inhibited the IL-17-induced changes mentioned above. Furthermore, we demonstrated a potential therapeutic role for salubrinal in alleviating the chronic inflammatory-dependent degenerative state commonly observed in IDD. After inflammatory distress with IL-17, RT-PCR and western blot were employed to investigate the expression of MMP-13, collagen type II (COL2A1), collagen type I (COL1A1), and aggrecan (ACAN) in nucleus pulpous (NP) tissue. Activation of the NF-kB pathway was measured by western blot and immunocytochemistry following IL-17 treatment. We also examine the level of eIF2α phosphorylation after IL-17 treatment with or without salubrinal. Then, we investigated interactions of the NF-kB pathway to eIF2α phosphorylation. Moreover, we employed salubrinal and a specific inhibitor of NF-kB (BAY11-7082) to evaluate their effects on IL-17-driven regulation of MMP-13 and the ECM, as well as on the activation of NF-kB. The results showed that IL-17 increased the production of MMP-13 and decreased expression of COL2A1 and ACAN via the NF-kB pathway. Either IL-17 or salubrinal increased the level of eIF2α phosphorylation, but the effects of BAY11-7082 on the level of p-eIF2α were not detectable. BAY11-7082 and salubrinal significantly suppressed IL-17-driven intervertebral disc degeneration. Furthermore, salubrinal produced stronger effects than BAY11-7082. These results imply the potential involvement of IL-17 in IDD through activation of NF-kB signaling, which successively upregulated the expression of MMP-13 and led to the degradation of the ECM. Furthermore, salubrinal can inhibit this process through inhibition of NF-kB activation that is not directly linked to eIF2α phosphorylation, suggesting a potential therapeutic role in IDD.

Histone Deacetylase Inhibitor Vorinostat (SAHA) Suppresses IL-1ß-Induced Matrix Metallopeptidase-13 Expression by Inhibiting IL-6 in Osteoarthritis Chondrocyte.

Osteoarthritis (OA) is the most common whole-joint disease and is characterized by progressive loss of the cartilage matrix. Matrix metallopeptidase-13 (MMP-13) is a highly active and an abundantly expressed protease in OA cartilage and chondrocytes and degrades type II collagen and proteoglycans. We investigated the mechanism of MMP-13 suppression by histone deacetylase inhibitor vorinostat (SAHA). OA chondrocytes were obtained from knee cartilage after enzymatic digestion and treated with IL-1ß in the absence or presence of various histone deacetylase inhibitors. Gene expression was quantified using quantitative RT-PCR. Protein expression and chromatin modifications were determined by Western immunoblotting using specific antibodies. The effect of IL-6 on the expression of MMP-13 was determined by treating chondrocytes with recombinant IL-6 or by IL6 knockdown using IL6-specific siRNA. We found that SAHA is a potent suppressor of IL-1ß-induced MMP-13, tumor necrosis factor-α, and other catabolic marker expression in OA chondrocytes. Interestingly, SAHA rescued the COL2A1 and ACAN expression in OA chondrocytes that was down-regulated by IL-1ß. Of importance is our finding that IL-6-stimulated MMP-13 expression was independent of IL-1ß stimulation and was blocked by SAHA, suggesting that SAHA inhibits IL-6 signaling in OA chondrocytes. Taken together, our results suggest that SAHA could be used as a therapeutic agent for the management of OA.

Mineralization Effect of Hyaluronan on Dental Pulp Cells via CD44.

INTRODUCTION: CD44 is a cell-surface glycoprotein involved in various cellular functions. Recent studies have suggested that CD44 is involved in early mineralization of odontoblasts. Hyaluronic acid (HA) is the principal ligand for receptor CD44. Whether and how HA regulated the mineralization process of dental pulp cells were investigated. METHODS: The effects of high-molecular-weight HA on differentiation and mineral deposition of dental pulp cells were tested by using alkaline phosphatase (ALP) activity assay and alizarin red S staining. Osteogenesis real-time polymerase chain reaction array, quantitative polymerase chain reaction, and Western blotting were performed to identify downstream molecules involved in the mineralization induction of HA. CD44 was knocked down and examined to confirm whether the mineralization effect of HA was mediated by receptor CD44. Immunohistochemistry was used to understand the localization patterns of CD44 and the identified downstream proteins in vivo. RESULTS: Pulse treatment of HA enhanced ALP activity and mineral deposition in dental pulp cells. Tissue-nonspecific ALP, bone morphogenetic protein 7 (BMP7), and type XV collagen (Col15A1) were upregulated via the HA-CD44 pathway in vitro. Immunohistochemistry of tooth sections showed that the staining pattern of BMP7 was very similar to that of CD44. CONCLUSIONS: Results of this study indicated that high-molecular-weight HA enhanced early mineralization of dental pulp cells mediated via CD44. The process involved important mineralization-associated molecules including tissue-nonspecific ALP, BMP7, and Col15A1. The findings may help develop new strategies in regenerative endodontics.

Restriction of spontaneous and prednisolone-induced leptin production to dedifferentiated state in human hip OA chondrocytes: role of Smad1 and ß-catenin activation.

OBJECTIVE: The aetiology of OA is not fully understood although several adipokines such as leptin are known mediators of disease progression. Since leptin levels were increased in synovial fluid compared to serum in OA patients, it was suggested that joint cells themselves could produce leptin. However, exact mechanisms underlying leptin production by chondrocytes are poorly understood. Nevertheless, prednisolone, although displaying powerful anti-inflammatory properties has been recently reported to be potent stimulator of leptin and its receptor in OA synovial fibroblasts. Therefore, we investigated, in vitro, spontaneous and prednisolone-induced leptin production in OA chondrocytes, focusing on transforming growth factor-ß (TGFß) and Wnt/ß-catenin pathways. DESIGN: We used an in vitro dedifferentiation model, comparing human freshly isolated hip OA chondrocytes cultivated in monolayer during 1 day (type II, COL2A1 +; type X, COL10A1 + and type I collagen, COL1A1 -) or 14 days (COL2A1 -; COL10A1 - and COL1A1+). RESULTS: Leptin expression was not detected in day1 OA chondrocytes whereas day14 OA chondrocytes produced leptin, significantly increased with prednisolone. Activin receptor-like kinase 1 (ALK1)/ALK5 ratio was shifted during dedifferentiation, from high ALK5 and phospho (p)-Smad2 expression at day1 to high ALK1, endoglin and p-Smad1/5 expression at day14. Moreover, inactive glycogen synthase kinase 3 (GSK3) and active ß-catenin were only found in dedifferentiated OA chondrocytes. Smad1 and ß-catenin but not endoglin stable lentiviral silencing led to a significant decrease in leptin production by dedifferentiated OA chondrocytes. CONCLUSIONS: Only dedifferentiated OA chondrocytes produced leptin. Prednisolone markedly enhanced leptin production, which involved Smad1 and ß-catenin activation.

Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses.

OBJECTIVE: Norepinephrine (NE) was measured in synovial fluid of trauma patients and sympathetic nerve fibers were detected in healthy and osteoarthritic (OA) joint tissues indicating that cartilage pathophysiology might be influenced by sympathetic neurotransmitters. The aim of this study was to elucidate the mostly unknown role of NE in OA chondrocyte metabolism and inflammatory responses. METHODS: Articular cartilage was received after total knee replacement surgery from OA patients. Expression of adrenergic receptors (AR) and tyrosine hydroxylase (TH) was tested with end point polymerase chain reaction (PCR) and immunohistochemistry. 3-dimensional (3D) cell cultures were employed to analyze effects of NE on chondrocyte cell metabolism and the expression of interleukins (ILs), matrix metalloproteases (MMPs), tissue inhibitor of metalloproteases (TIMPs), glycosaminoglycan (GAG) and collagen II under non- and inflammatory conditions. Chondrocyte monolayer cultures were used to specify AR subtypes, to analyze cell cycle distribution and to determine catecholamines in cell culture supernatants. RESULTS: AR subtypes and TH were detected in chondrocytes, whereas NE was not released in measurable amounts. 10(-6) M NE reversed IL-1ß induced changes in IL-8, MMP-13, GAG and collagen II expression/production indicating for ß-AR signaling. Additionally, NE caused cell cycle slow down and decreased proliferation via ß-AR signaling. 10(-8) M NE increased the number of proliferating cells and induced apoptosis via α1-AR signaling. CONCLUSIONS: NE affects chondrocytes from OA cartilage regarding inflammatory response and its cell metabolism in a dose dependent manner. The sympathetic nervous system (SNS) may have a dual function in OA pathology with preserving a stable chondrocyte phenotype via ß-AR signaling and OA pathogenesis accelerating effects via α-AR signaling.

Cysteine-Mediated Redox Regulation of Cell Signaling in Chondrocytes Stimulated With Fibronectin Fragments.

OBJECTIVE: Oxidative posttranslational modifications of intracellular proteins can potentially regulate signaling pathways relevant to cartilage destruction in arthritis. In this study, oxidation of cysteine residues to form sulfenic acid (S-sulfenylation) was examined in osteoarthritic (OA) chondrocytes and investigated in normal chondrocytes as a mechanism by which fragments of fibronectin (FN-f) stimulate chondrocyte catabolic signaling. METHODS: Chondrocytes isolated from OA and normal human articular cartilage were analyzed using analogs of dimedone that specifically and irreversibly react with protein S-sulfenylated cysteines. Global S-sulfenylation was measured in cell lysates with and without FN-f stimulation by immunoblotting and in fixed cells by confocal microscopy. S-sulfenylation in specific proteins was identified by mass spectroscopy and confirmed by immunoblotting. Src activity was measured in live cells using a fluorescence resonance energy transfer biosensor. RESULTS: Proteins in chondrocytes isolated from OA cartilage were found to have elevated basal levels of S-sulfenylation relative to those of chondrocytes from normal cartilage. Treatment of normal chondrocytes with FN-f induced increased levels of S-sulfenylation in multiple proteins, including the tyrosine kinase Src. FN-f treatment also increased the levels of Src activity. Pretreatment with dimedone to alter S-sulfenylation function or with Src kinase inhibitors inhibited FN-f-induced production of matrix metalloproteinase 13. CONCLUSION: These results demonstrate for the first time the presence of oxidative posttranslational modification of proteins in human articular chondrocytes by S-sulfenylation. Due to the ability to regulate the activity of a number of cell signaling pathways, including catabolic mediators induced by fibronectin fragments, S-sulfenylation may contribute to cartilage destruction in OA and warrants further investigation.