Expression In Articular Cartilage Research Articles

Notch Signaling Regulates MMP-13 Expression via Runx2 in Chondrocytes

Notch signaling is involved in the early onset of osteoarthritis. The aim of this study was to investigate the role of Notch signaling changes during proliferation and differentiation of chondrocyte, and to testify the mechanism of MMP-13 regulation by Notch and Runx2 expression changes during osteoarthritis. In this study, Chondrocytes were isolated from rat knee cartilages. Notch signaling was activated/inhibited by Jagged-1/DAPT. Proliferative capacity of Chondrocytes was analyzed by CCK-8 staining and EdU labeling. ColX, Runx2 and MMP-13 expressions were analyzed as cell differentiation makers. Then, Runx2 gene expression was interfered using lentivirus transfection (RNAi) and was over-expressed by plasmids transfected siRNA in chondrocytes, and MMP-13 expression was analyzed after Jagged-1/DAPT treatment. In vivo, an intra-articular injection of shRunx2 lentivirus followed with Jagged1/DAPT treatments was performed in rats. MMP-13 expression in articular cartilage was detected by immunohistochemistry. Finally, MMP-13 expression changes were analyzed in chondrocytes under IL-1β stimulation. Our findings showed that, CCK-8 staining and EdU labeling revealed suppression of cell proliferation by Notch signaling activation after Jagged-1 treatment in chondrocytes. Promoted differentiation was also observed, characterized by increased expressions of Col X, MMP-13 and Runx2. Meanwhile, Sox9, aggrecan and Col II expressions were down-regulated. The opposite results were observed in Notch signaling inhibited cells by DAPT treatment. In addition, Runx2 RNAi significantly attenuated the ‘regulatory sensitivity’ of Notch signaling on MMP-13 expression both in vitro and in vivo. However, we found there wasn’t significant changes of this ‘regulatory sensitivity’ of Notch signaling after Runx2 over-expression. Under IL-1β circumstance, MMP-13 expression could be reduced by both DAPT treatment and Runx2 RNAi, while Runx2 interference also attenuated the ‘regulatory sensitivity’ of Notch in MMP-13 under IL-1β stimulation. In conclusion, Notch signaling is an important regulator on rat chondrocyte proliferation and differentiation, and this regulatory effect was partially mediated by proper Runx2 expression under both normal and IL-1β circumstances. In the meanwhile, DAPT treatment could effectively suppress expression of MMP-13 stimulated by IL-1 β.

The Effect and Mechanism of Continuous Passive Exercise Combined with Platelet-Rich Plasma on Rabbit Knee Osteoarthritis Model

Osteoarthritis (OA) is a disease in orthopedics. Continuous passive exercise (CPM) and intraarticular injection of platelet-rich plasma (PRP) have certain effects on OA. However, the effect of these two combination on OA remains unclear. The present study aims to evaluate the treatment efficacy of this combination on OA. 50 New Zealand white rabbits were randomly divided into control group, OA group, CPM group, PRP group and CPM + PRP group followed by analysis of the pathological changes of chondrocyte by H&E staining and Mankin's score, level of IL-1 and TNF-α by ELISA, as well as MMP-1 and MMP-13 protein expression by western blot. Compared with control group, the Mankin's score of OA group was significantly increased (P < 0.05), along with increased expressions of MMP-1 and MMP-13 in the joint fluid, levels of IL-1 and TNF-α (P < 0.05). CPM group and PRP group presented significantly lower Mankin's scores, reduced IL-1 and TNF-α levels in synovial fluid as well as decreased MMP-1 and MMP-13 protein expression in articular cartilage compared with OA group (P < 0.05). Compared with CPM group and PRP group, the Mankin's score of CPM + PRP group was significantly decreased (P < 0.05), along with reduced IL-1 and TNF-α levels in joint fluid, and decreased expression of MMP-1 and MMP-13 protein in articular cartilage (P < 0.05). The efficacy of CPM combined with PRP in treating OA is better than that of CPM or PRP alone, which may be related to the decreased expression of IL-1, TNF-α , MMP-1 and MMP-13.

Differential gene expression in articular cartilage between rheumatoid arthritis and endemic Kashin-Beck disease.

Kashin–beck disease (KBD) is endemic chronic osteoarthrosis and its pathogenesis is still unclear. The present study aimed to explore differential gene expression in articular cartilage between patients with rheumatoid arthritis (RA) and KBD. Articular cartilages were collected from KBD and RA patients, and differentially expressed genes (DEGs) were analyzed by RNA-seq. The signaling pathway and biological process (BP) of the DEGs were identified by enrichment analysis. The protein–protein interaction (PPI) network of DEGs and the key genes of KBD were identified by network analysis with STRING and cytoscape software. We identified 167 immune-related DEGs in articular cartilage samples from KBD patients compared with RA. The up-regulation of MAPK signaling pathway and the down-regulation of signaling pathways such as toll-like receptor, janus kinase-signal transducers and activators of transcription, leukocyte migration, T-cell receptor and chemokine, and antigen processing and presentation were involved in KBD. We identified 137 genes nodes related with immune and mapped the PPI network diagram. BP analysis revealed that immune response, calcium ion homeostasis, blood vessel morphogenesis, inflammatory response, lymphocyte proliferation, and MAPK activation were involved in KBD. In conclusion, gene expression profiling can be used to identify the different mechanism of pathogenesis between KBD and RA.

MiR-9 facilitates cartilage regeneration of osteoarthritis in rabbits through regulating Notch signaling pathway.

To explore the effect of micro ribonucleic acid (miR-9) on cartilage regeneration of osteoarthritis in rabbits through the regulation of the Notch signaling pathway. A total of 30 specific pathogen-free Sprague-Dawley rabbits were randomly divided into control group (healthy rabbits, n=10), model group (osteoarthritis model, n=10) and miR-9 group (osteoarthritis model + miR-9 interference, n=10). The degeneration degree of rabbit knee articular cartilage in three groups was assessed through the Mankin's score. The morphology of cartilage tissues was observed under an optical microscope. Expressions of the Notch1, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) proteins, and collagen II (CII) in chondrocytes were detected via the immunohistochemical assay. In rabbits of control group, the articular cartilage had a smooth surface and complete structure, and the cells were arranged orderly with a clear tidal line. A large number of articular chondrocytes died in model group, while it was improved in miR-9 group. The Mankin's score was 3.52±0.79 points in control group, 6.73±0.95 points in model group, and 5.37±0.61 points in miR-9 group, showing significant differences among the three groups (p<0.05). Results of immunohistochemistry showed that the protein expressions of Notch1 and Bax were higher in model group, but lower in control group and miR-9 group (p<0.05). The protein expression of Bcl-2 was lower in model group but was upregulated in control group and miR-9 group (p<0.05). The results of Reverse Transcription-Polymerase Chain Reaction (RT-PCR) revealed that the expressions of Notch1 and Bax in control group were lower than those in model group and miR-9 group (p<0.05), while the expression of Bcl-2 in model group was lower than that in control group and miR-9 group (p<0.05). According to the results of immunohistochemistry, the CII optical density (OD) value was 0.18±0.013, 0.25±0.05 and 0.22±0.009 in control group, model group, and miR-9 group, respectively. It could be seen that the CII OD value was the highest in model group, indicating that the CII expression in articular cartilage in osteoarthritis was negatively correlated with the severity of osteoarthritis. MiR-9, through the down-regulation of the expressions of Notch and Bax, can activate the Bcl-2 to promote the differentiation and regeneration of chondrocytes. It can facilitate the cartilage regeneration of osteoarthritis in rabbits through the mediation of the CII expression.

Long non-coding RNA reprogramming (lncRNA-ROR) regulates cell apoptosis and autophagy in chondrocytes.

Long Non-Coding RNA Reprogramming (lncRNA-ROR) plays an important role in regulating various biologic processes, whereas the effect of lncRNA-ROR in osteoarthritis (OA) is little studied. This study aimed to explore lncRNA-ROR expression in articular cartilage and identify the functional mechanism of lncRNA-ROR in OA. OA cartilage tissues were obtained from 15 OA patients, and 6 normal cartilage tissues were set as controls. Chondrocytes were isolated from the collected cartilage tissues. lncRNA-ROR was knockdown in normal cells and overexpressed in OA cells. Cell viability was determined with Cell Counting Kit-8 assay, and apoptosis was measured using flow cytometric analysis. Moreover, proteins and mRNAs involved in this study were also measured using Western blotting and quantitative real-time PCR (qPCR). Level of lncRNA-ROR was decreased in OA compared with normal chondrocytes, and overexpression of lncRNA-ROR dramatically promoted cell viability of OA chondrocytes. In addition, knockdown lncRNA-ROR inhibited apoptosis and promoted autophagy of normal chondrocytes. Moreover, lncRNA-ROR inhibited the expression of p53 in both mRNA and protein levels. Furthermore, we revealed that lncRNA-ROR regulated apoptosis and autophagy of chondrocytes via HIF1α and p53. The results indicated that lncRNA-ROR played a critical role in the pathogenesis of OA, suggesting that lncRNA-ROR could serve as a new potential therapeutic target for OA.

Indian hedgehog signaling is in response to the blunt trauma in articuar cartilage

Purpose: Posttraumatic osteoarthritis (PTOA) represents end-stage organ level failure of an injured joint, especially in an intra-articular fracture. The severity of the PTOA is associated with the degree of the fracture. The initial impact injury to the cartilage, in the combination with secondary pathomechanical and pathobiologic response of the cartilage, is prone to result in the final PTOA. Inhibition of Indian Hedgehog (IHH) signaling, one of a main signaling regulating the chondrocytes hypertrophy, attenuates the severity of OA in OA cartilage explants in vitro, implying that the OA development might be involved in the expression of IHH signaling. How to prevent the development of PTOA after the occurrence of a fracture in early time seems to be essential. However, none of the previous studies had focused on the response of IHH signaling expression in articular cartilage in early stage after initial severe impact. Recently, bovine cartilage plugs were proved to express most common catabolic markers of osteoarthritic degeneration after impacted by our newly designed spring-loaded impact device. Therefore, with the help of this PTOA model, it is more meaningful and clinical relevant to test the IHH signaling response in the articular cartilage to the initial impact loading. Methods: Articular cartilage explants were isolated from the distal femoral condylar of the hind-leg stifle of 2- to 3-year-old bovine within 24 hours of slaughter. The punch protocol were followed our previous study. The blunt impact to the cartilage surface was applied using a custom-designed impact device to induce trauma to cartilage plugs as described in our recently work. Briefly, the spring-loaded impact device fitted with a hemispherical tip was used to deliver traumatic, injurious impacts of calibrated magnitude and rate into the cartilage piece, resulting in increased catabolic activities and tissue degeneration. The traumatized cartilage developed lesions similar to cartilage lesions seen in PTOA. Samples were randomly distributed into 3 different impact load magnitude groups those are 0, 17 and 36 MPa. (n=3) The samples were collected in Day 1, 4 and 7. Both gene and protein of IHH signaling were extracted and tested. The immunohistology was also performed to test the IHH signaling expression. Results: HE staining showed that the 36Mpa impact injury is a very severe trauma to the articular cartilage and an apparent reduction of matrix sulfated GAG in areas below the superficial zone of the articular cartilage by Safranin-O staining. From the PCR result, comparing with the control group of 0 Mpa, gene of IHH/HHIP/GLi-1/PTCH were increased on day 4, especially after 36 Mpa impact. The differences were significant, P<0.05. On the other hand, the IHH/Gli-1 protein was tested by western blot, which validate the PCR results. The differences were significant, P<0.05. The immunohistology results showed that on day4, the IHH protein expression was apparently increased after 34Mpa impact injury. Conclusions: We firstly proved that IHH signaling is sensitively in response to the mechanical blunt trauma on the articular cartilage. Early after the blunt trauma of 36Mpa, compared with 0 Mpa, the IHH signaling significantly increased on day 4. Our results can be utilized for the future study on the mechanism of PTOA development after the intra-articular fractures.

Identification of prolargin expression in articular cartilage and its significance in rheumatoid arthritis pathology

Qualitative 2D gel-electrophoresis (2DE) protein profiling for osteoarthritis (OA) and rheumatoid arthritis (RA) is challenging because of selective protein loss due to discrepancies in protein precipitation methodologies. Thus, we aimed at developing qualitative protein representation from OA/RA articular cartilage without protein precipitation towards identification of clinically relevant proteins. Chondroitinase digested human articular cartilages from RA patients were subjected to protein extraction using guanidinium hydrochloride (GuHCl) or 8 M urea with 10 or 2% ASB-14-4 or 0.45 M urea with 2% ASB-14-4 with cetylpyridinium chloride (CPC). The GuHCl extract is further protein precipitated with acetone or ammonium acetate-methanol or centricon-fractionated using 100 kDa cut filters and protein precipitated using ethanol. Processed extracts were subjected to 2DE to identify protein profiles. Poor proteins representations were observed in 2D gels with protein precipitated samples compared to qualitative protein representations seen in 2D gels of 0.45 M urea and 2%ASB-14-4 extraction procedure reproducibly. The strategy circumventing protein precipitation generated qualitative 2D gels. RA vs OA gel comparison showed elevated prolargin levels in RA with biglycan levels remaining unaltered. Up regulation of prolargin in RA suggests the likelihood of an adaptive mechanism to control the increased osteoclastogenesis in RA and may have therapeutic value in controlling the disease.

Interleukin-1β and tumor necrosis factor-α augment acidosis-induced rat articular chondrocyte apoptosis via nuclear factor-kappaB-dependent upregulation of ASIC1a channel

The acute-phase proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) demonstrate high-level expression and pleiotropic biological effects, and contribute to the progression and persistence of rheumatoid arthritis (RA). Acid hydrarthrosis is also an important pathological characteristic of RA, and the acid-sensing ion channel 1a (ASIC1a) plays a critical role in acidosis-induced chondrocyte cytotoxicity. However, the roles of IL-1β and TNF-α in acid-induced apoptosis of chondrocytes remain unclear. Rat adjuvant arthritis and primary articular chondrocytes were used as in vivo and in vitro model systems, respectively. ASIC1a expression in articular cartilage was increased and highly colocalized with nuclear factor (NF)-κB expression in vivo. IL-1β and TNF-α could upregulate ASIC1a expression. These cytokines activated mitogen-activated protein kinase and NF-κB pathways in chondrocytes, while the respective inhibitors of these signaling pathways could partially reverse the ASIC1a upregulation induced by IL-1β and TNF-α. Dual luciferase and gel-shift assays and chromatin immunoprecipitation-polymerase chain reaction demonstrated that IL-1β and TNF-α enhanced ASIC1a promoter activity in chondrocytes by increasing NF-κB DNA-binding activities, which was in turn prevented by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate. IL-1β and TNF-α also decreased cell viability but enhanced LDH release, intracellular Ca2+ concentration elevation, loss of mitochondrial membrane potential, cleaved PARP and cleaved caspase-3/9 expression, and apoptosis in acid-stimulated chondrocytes, which effects could be abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1), ASIC1a-short hairpin RNA or calcium chelating agent BAPTA-AM. These results indicate that IL-1β and TNF-α can augment acidosis-induced cytotoxicity through NF-κB-dependent up-regulation of ASIC1a channel expression in primary articular chondrocytes.

Chondromodulin‑I expression and correlation with angiogenesis in human osteoarthritic cartilage.

The present study aimed to evaluate the expression and localization of chondromodulin‑I (ChM‑I) in human osteoarthritic cartilage and its correlation with vascular invasion during osteoarthritis (OA) progression. Osteochondral specimens were collected from patients with OA, as well as from young and aged donors without joint diseases. The grade and the number of vascular channels terminating in non‑calcified cartilage of these collected specimens were assessed by Safranin‑O/Fast green staining. ChM‑I expression in articular cartilage was examined by immunohistochemistry, western blotting and reverse transcription‑quantitative polymerase chain reaction analyses. ChM‑I protein and mRNA levels in articular cartilage appeared to be consistent between the normal young and aged groups (P>0.05). In mildly degene-rated cartilage, ChM‑I expression decreased in the extracellular matrix (ECM) of the superficial zone and in the cytoplasm of the superficial and middle zone compared with normal cartilage (P<0.05). In moderately degenerated cartilage, ChM‑I protein expression was reduced in the ECM of all zones of articular cartilage, but the immunostaining intensity in the cytoplasm was increased. In severely degenerated cartilage, ChM‑I expression was detected primarily in the cytoplasm of the cluster‑forming chondrocytes. The density of vascular channels was correlated with the ChM‑I expression levels in cartilage ECM. ChM‑I expression was reduced in OA cartilage matrix, compared with normal cartilage (both young and aged), and correlated with angiogenesis, indicating that loss of ChM‑I may promote angiogenesis in OA cartilage. Expression of ChM‑I protein in the cytoplasm was decreased in mildly degenerated cartilage, whereas ChM‑I expression increased in moderately degenerated cartilage accompanied by chondrocyte proliferation. These findings suggested that attenuation of ChM‑I in the cartilage ECM may be due to decreased expression of ChM‑I in cytoplasm of early stage OA and increased depletion of ChM‑I in the ECM of advanced stage OA.

Hedgehog signal expression in articular cartilage of rat temporomandibular joint and association with adjuvant‐induced osteoarthritis

The aim of this study was to investigate the changes in hedgehog (Hh) expression and its possible effects on cartilage degeneration in adjuvant-induced temporomandibular joint osteoarthritis (TMJOA) of rats. Forty-eight male Sprague-Dawley rats were randomly divided into experimental osteoarthritis (OA) and sham control groups. The bilateral TMJs of six rats from each group were harvested at three, seven, 14, and 28 days. Histological changes in condylar cartilage were assessed by hematoxylin and eosin, toluidine blue, and safranin O staining. The expression of Hh signal-related proteins including Indian hedgehog (Ihh), patched-1 (Ptch1), smoothened (Smo), glioma-associated oncogene homologue1 (Gli1) in cartilage was assessed by immunohistochemistry and Western blot. The protein expression of matrix metalloproteinase-13 (MMP-13), type X collagen, and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) in cartilage was evaluated by Western blot. The histological analysis showed marked cartilage degeneration in adjuvant-induced OA groups, including reduced cartilage cellular density, thinner and degraded cartilage, and decreased proteoglycan content in the extracellular matrix. Compared with matched control groups, the expression of Ihh, Ptch1, Smo, and Gli1 in the OA groups was higher in a time-dependent manner. The protein levels of MMP-13, type X collagen, and ADAMTS-5 were substantially increased in OA cartilage compared with those in matched control rats. These results indicate that the activation of Ihh signaling may be correlated with pathological changes of condylar cartilage in adjuvant-induced TMJOA.

Circular RNA Related to the Chondrocyte ECM Regulates MMP13 Expression by Functioning as a MiR-136 'Sponge' in Human Cartilage Degradation.

Circular RNAs (circRNAs) are involved in the development of various diseases, but there is little knowledge of circRNAs in osteoarthritis (OA). The aim of study was to identify circRNA expression in articular cartilage and to explore the function of chondrocyte extracellular matrix (ECM)-related circRNAs (circRNA-CER) in cartilage. To identify circRNAs that are specifically expressed in cartilage, we compared the expression of circRNAs in OA cartilage with that in normal cartilage. Bioinformatics was employed to predict the interaction of circRNAs and mRNAs in cartilage. Loss-of-function and rescue experiments for circRNA-CER were performed in vitro. A total of 71 circRNAs were differentially expressed in OA and normal cartilage. CircRNA-CER expression increased with interleukin-1 and tumor necrosis factor levels in chondrocytes. Silencing of circRNA-CER using small interfering RNA suppressed MMP13 expression and increased ECM formation. CircRNA-CER could compete for miR-136 with MMP13. Our results demonstrated that circRNA-CER regulated MMP13 expression by functioning as a competing endogenous RNA (ceRNA) and participated in the process of chondrocyte ECM degradation. We propose that circRNA-CER could be used as a potential target in OA therapy.

AAV8-mediated expression of N-acetylglucosamine-1-phosphate transferase attenuates bone loss in a mouse model of mucolipidosis II.

Mucolipidoses II and III (ML II and ML III) are lysosomal disorders in which the mannose 6-phosphate recognition marker is absent from lysosomal hydrolases and other glycoproteins due to mutations in GNPTAB, which encodes two of three subunits of the heterohexameric enzyme, N-acetylglucosamine-1-phosphotransferase. Both disorders are caused by the same gene, but ML II represents the more severe phenotype. Bone manifestations of ML II include hip dysplasia, scoliosis, rickets and osteogenesis imperfecta. In this study, we sought to determine whether a recombinant adeno-associated viral vector (AAV2/8-GNPTAB) could confer high and prolonged gene expression of GNPTAB and thereby influence the pathology in the cartilage and bone tissue of a GNPTAB knock out (KO) mouse model. The results demonstrated significant increases in bone mineral density and content in AAV2/8-GNPTAB-treated as compared to non-treated KO mice. We also showed that IL-6 (interleukin-6) expression in articular cartilage was reduced in AAV2/8-GNPTAB treated ML II mice. Together, these data suggest that AAV-mediated expression of GNPTAB in ML II mice can attenuate bone loss via inhibition of IL-6 production. This study emphasizes the value of the MLII KO mouse to recapitulate the clinical manifestations of the disease and highlights its amenability to therapy.

ASSOCIATION OF BLOOD GENE EXPRESSIONS IN RHEUMATOID ARTHRITIS PATIENTS WITH CLINICAL AND LABORATORY PARAMETERS BEFORE AND AFTER METHOTREXATE THERAPY

The genes, the high basic expression of which indicates the efficiency of methotrexate (MTX) therapy in relieving joint inflammation and destruction in patients with rheumatoid arthritis (RA), have been defined. Objective: to find an association between the initial expression of the genes: mTOR (mammalian target of rapamycin), a major regulator of cell growth and proliferation; ULK1 (an autophagy marker 1); p21 (a cyclin-dependent kinase inhibitor); kaspase-3 (an apoptosis activity indicator); MMP-9 (matrix metalloproteinase 9), and cathepsin K, which are involved in joint destruction, and the cytokines: TNF-α (tumor necrosis factor-α), TGFβ1 (transforming growth factor β1) and Runx2 (Runt-related transcription factor 2) in the blood of RA patients with disease activity and joint destruction before and after MTX therapy during 24 months. Subjects and methods. Forty patients (mean age, 47.5 years) with RA lasting < 2 years) and 26 healthy donors (mean age, 45.1 years) were examined. All the patients took MTX for 2 years. A clinical response was assessed with disease activity score (DAS28); erythrocyte sedimentation rate and the serum levels of anti-cyclic citrullinated peptide antibodies (ACCPA), C-reactive protein (CRP), and rheumatoid factor (RF) were also estimated. Joint destructive changes were assessed by radiography. Furthermore, blood and knee articular cartilage samples from 21 patients (mean age, 50.4 years) with late-stage RA and cartilage samples from 25 healthy individuals were investigated. Gene expression in the cells of peripheral blood and cartilage was determined by real-time reverse transcriptase polymerase chain reaction. Results and discussion. MTX therapy considerably reduced disease activity assessed by DAS28, CRP levels, stiffness, tender and swollen joint counts (TJC and SJC); however, joint space (JS) narrowing (JSN) substantially increased compared with the baseline values. The expression of the ULK1, p21, MMP-9, cathepsin K genes, and TGFβ1 was increased both at the beginning of the investigation and 24 months later whereas the initially higher expression of mTOR, TNF-α, caspase 3, and Runx2 was decreased by the end of therapy to the level of the healthy individuals. The initial expression of the TGFβ1, Runx2, caspase 3, and р21 genes correlated negatively with the RF level measured both at the beginning of the investigation and 24 months later. There was a positive correlation of the initial expression of ULK1 and MMP-9 with CRP levels prior to therapy and that of the initial expression of MMP-9 with baseline DAS28 scores and NSJ. Moreover, the initial expression of the TNF-α gene positively correlated with JSN at the end of therapy and that of the p21, caspase 3, and Runx2 genes with a change in DAS28. There was also a negative correlation of the initial expression of the mTOR gene with the SJC and the number of erosions; the p21 and TNF-α genes correlated negatively with SJC and TJC; and the TNF-α, TGFβ1, Runx2, and cathepsin K genes also negatively correlated to the duration of stiffness at the end of therapy. A positive correlation was found between the expression of the cathepsin K and TGFβ1 genes in the blood and articular cartilage of patients with late-stage RA. Conclusion. The expression of the MMP-9 and ULK1 genes is associated with disease activity. The high initial blood expression of the other examined genes is associated with the more effective action of MTX on stiffness (TNF-α, Runx2, cathepsin K, and TGFβ1), TJC and SJC (mTOR, p21, and TNF-α), and the progression of joint destruction (mTOR) and may play a protective role. The positive correlation of the blood and articular cartilage expression of the TGFβ1 and cathepsin K genes may point to their co-regulation in these tissues in RA.

Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition, but Causes a Transient Reduction of Articular Cartilage Thickness.

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.

Elevated expression of microRNA-30b in osteoarthritis and its role in ERG regulation of chondrocyte.

ERG (ETS-related gene) belongs to the ETS family of transcription factors, and has been recently reported to contribute to homeostatic balance in skeleton cell plasticity. MicroRNA-30 (miR-30) family is also demonstrated to play a role in controlling chondrocyte differentiation. The current study investigated the miR-30b and ERG expression in articular cartilage of osteoarthritis (OA) patients. A total of 20 subjects, with 10 OA patients and 10 healthy participants, were included in this study. Human chondrosarcoma cell line SW1353 was used to explore the relationship of miR-30b and ERG in vitro. In OA patients, a significant increase of miR-30b and a decrease of ERG were observed in articular cartilage compared with Normal ones. MiR-30b mimic down-regulated the ERG mRNA and protein expression levels, while miR-30b inhibitor up-regulated ERG expression. In addition, miR-30b mimic also decreased the mRNA expression of COL2a and aggrecan, while miR-30b inhibitor had the opposite effect. Luciferase reporter assay confirmed that miR-30b targeted ERG. In conclusion, miR-30b was involved in the process of OA, and it probably functioned through its target gene ERG.

Mice Deficient in CIZ/NMP4 Develop an Attenuated Form of K/BxN-Serum Induced Arthritis.

CIZ/NMP4 (Cas interacting zinc finger protein, Nmp4, Zfp384) is a transcription factor that is known to regulate matrix related-proteins. To explore the possible pathophysiological role of CIZ/NMP4 in arthritis, we examined CIZ/NMP4 expression in articular cartilage in arthritis model. CIZ/NMP4 was expressed in the articular chondrocytes of mice at low levels while its expression was enhanced when arthritis was induced. Arthritis induction increased clinical score in wild type mice. In contrast, CIZ/NMP4 deficiency suppressed such rise in the levels of arthritis score and swelling of soft tissue. CIZ/NMP4 deficiency also reduced invasion of inflammatory cells in joint tissue. Quantitative PCR analyses of mRNA from joints revealed that arthritis-induced increase in expressions of IL-1β was suppressed by CIZ/NMP4 deficiency. CIZ/NMP4 bound to IL-1β promoter and activated its transcription. The increase in CIZ/NMP4 in arthritis was also associated with enhancement in bone resorption and cartilage matrix degradation. In fact, RANKL, a signaling molecule prerequisite for osteoclastogenesis and, MMP-3, a clinical marker for arthritis were increased in joints upon arthritis induction. In contrast, CIZ/NMP4 deficiency suppressed the arthritis-induced increase in bone resorption, expression of RANKL and MMP-3 mRNA. Thus, CIZ/NMP4 plays a role in the development of arthritis at least in part through regulation of key molecules related to the arthritis.

참당귀 추출분말의 골관절염 흰쥐의 염증성 사이토카인류의 억제활성

The protective effects of extract powder of Angelica gigas on the degeneration of the articular cartilage in rats was investigated with monosodium iodoacetate (MIA)-induced osteoarthritis, The treatment of high concentration (50 µg/mL) of Angelica gigas effectively inhibited nitric oxide (NO) production induced by interleukin-1α (IL-1α) without any cytotoxicity. Specifically, mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were dose dependently reduced by extract powder of Angelica gigas. Importantly, mRNA expression in articular cartilage of inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were clearly reduced. The inflammatory cytokines in blood were also reduced as well. These results suggested that the protective effects on the degeneration of the articular cartilage was derived from the inhibitory effects of mRNA and protein expression of tested inflammatory cytokines which is linked to prevent the degradation of proteoglycan (PG), the main matrix content in articular cartilage. Meanwhile, the 2 hrs incubation of decursin, a major compound of extract powder in rat whole blood rapidely converted decursin into decursinol which shows string anti-inflammatory activity. The coverted decursinol was detected after 8 hrs in whole blood by LC-MS/MS. Conclusively, the inhibitory effects of inflammatory cytokines production in osteoarthritis may be derived from the production of decursinol, which performs against inflammatroy cytokines like TNF-α, IL-1β‚ and IL-6. 요약 : 골관절염에 대한 참당귀 추출분말의 치료효과를 검토하고자 흰쥐의 monosodium iodoacetate (MIA)로 유발된 골관절염 부위에서 시료를 채취하여 염증관련 효소 및 염증성 cytokines의 발현에 대하 여 참당귀 추출분말의 억제효능을 검토하였다. 고농도의 참당귀 추출분말 (50 µg/mL) 투여에서도 독성

Osteopontin induces vascular endothelial growth factor expression in articular cartilage through PI3K/AKT and ERK1/2 signaling.

The expression of osteopontin (OPN) and vascular endothelial growth factor (VEGF) are associated with the severity of cartilage destruction in osteoarthritis. However, the biological connection between OPN and VEGF in osteoarthritis remains to be elucidated. The present study was performed to investigate the effect of OPN on VEGF expression in articular cartilage. Rat articular chondrocytes were isolated and cultured in monolayer conditions, and they were treated with OPN for different time periods (0, 2, 8, 12 or 24 h) and dosages (0, 0.1, 0.25 or 0.5 µM). VEGF expression was assessed by reverse transcription‑quantitative polymerase chain reaction and western blotting. The activation of the phosphoinositide 3‑kinase (PI3K)/AKT and extracellular signal‑regulated kinase (ERK)1/2 pathway was analyzed by detecting the expression of pPI3K, pAKT and pERK1/2. To inhibit the PI3K/AKT and ERK1/2 pathway, LY294002 and PD98059 were used, respectively or in combination. It was identified that OPN increased the expression of VEGF in a dose‑ and time‑dependent manner. The PI3K/AKT and ERK1/2 pathways were activated following OPN stimulation and the effect was concomitant with the upregulation of VEGF. Finally, the regulation of VEGF was inhibited by LY294002 and PD98059, and their combination exhibited a synergistic effect. In conclusion, these findings suggest that OPN may directly upregulate VEGF expression through the PI3K/AKT and ERK1/2 pathway. Further studies are required to reveal the mechanism of action of OPN on cartilage angiogenesis and cartilage destruction.

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.

The role of cyclooxygenase-2, interleukin-1β and fibroblast growth factor-2 in the activation of matrix metalloproteinase-1 in sheared-chondrocytes and articular cartilage.

MMP-1 expression is detected in fluid shear stress (20 dyn/cm2)-activated and osteoarthritic human chondrocytes, however, the precise mechanisms underlying shear-induced MMP-1 synthesis remain unknown. Using primary chondrocytes and T/C-28a2 chondrocytic cells as model systems, we report that prolonged application of high fluid shear to human chondrocytes induced the synthesis of cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β) and fibroblast growth factor-2 (FGF-2), which led to a marked increase in MMP-1 expression. IL-1β, COX-2-dependent PGE2 activated the PI3-K/AKT and p38 signaling pathways, which were in turn responsible for MMP-1 synthesis via NF-κB- and c-Jun-transactivating pathways. Prolonged shear stress exposure (>12 h) induced 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) synthesis. Although 15d-PGJ2 suppressed PI3-K/AKT and p38 signaling pathways, it stimulated MMP-1 expression via activating heme oxygenase 1 (HO-1). The critical role of COX-2 in regulating MMP-1 expression in articular cartilage in vivo was demonstrated using COX-2+/− transgenic mice in the absence or presence of rofecoxib oral administration. These findings provide novel insights for developing therapeutic strategies to combat OA.