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Konstantinos Konstantopoulos - One of the best experts on this subject based on the ideXlab platform.

  • Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-D 12,14-PGJ 2
    2013
    Co-Authors: Pu Wang, Fei Zhu, Konstantinos Konstantopoulos
    Abstract:

    Background: Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E2, PGD2 and its dehydration end product 15-deoxy-D 12,14-PGJ2 (15d-PGJ2) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE2 directly stimulates IL-6 production in human articular Chondrocytes. However, the effects of PGD2 and 15d-PGJ2 in the absence or presence of PGE 2 on IL-6 synthesis in human Chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients. Methodology/Principal Findings: Using the T/C-28a2 Chondrocyte Cell Line as a model system, we report that exogenous PGE 2 and PGD 2/15d-PGJ 2 exert antagonistic effects on IL-6 synthesis in human T/C-28a2 Chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE2 stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-kB p65 subunit leading to NF-kB activation. Binding of the activated NF-kB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 Chondrocytes. PGD 2 or 15d-PGJ 2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE2-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-kB-dependent IL-6 synthesis in Chondrocytes

  • the antagonistic actions of endogenous interleukin 1β and 15 deoxy δ12 14 prostaglandin j2 regulate the temporal synthesis of matrix metalloproteinase 9 in sheared Chondrocytes
    Journal of Biological Chemistry, 2012
    Co-Authors: Pu Wang, Konstantinos Konstantopoulos
    Abstract:

    Mechanical overloading of articular cartilage producing hydrostatic stress, tensile strain, and fluid flow results in irreversible cartilage erosion and osteoarthritis (OA). Application of high fluid shear to Chondrocytes recapitulates the earmarks of OA as evidenced by the induction of proinflammatory cytokines and prostaglandins, which are capable of inducing the expression of matrix-degrading enzymes. Matrix metalloproteinase-9 (MMP-9) synthesis is detected at early but not late stages of OA. However, the underlying mechanism(s) of the MMP-9 temporal regulation remains unknown. Using the T/C-28a2 Chondrocyte Cell Line as a model system, we demonstrated that high fluid shear induces a marked increase in MMP-9 expression at short shear exposure times (3–6 h), which falls below basal levels after prolonged shear exposure (12–48 h). High fluid shear stress induced the rapid and sustained synthesis of IL-1β, activating PI3K, ERK1/2, and JNK, which are in turn responsible for MMP-9 expression. Prolonged shear exposure (>12 h) induced 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) synthesis, which exerted an antagonistic effect on IL-1β-mediated PI3K-, ERK1/2-, and JNK-dependent NF-κB activation, thereby suppressing MMP-9 expression in human Chondrocytes. Reconstructing the signaling network that regulates shear-mediated MMP-9 expression in human Chondrocytes may provide insights for developing strategies to treat arthritic disorders.

  • interleukin 6 synthesis in human Chondrocytes is regulated via the antagonistic actions of prostaglandin pg e2 and 15 deoxy δ12 14 pgj2
    PLOS ONE, 2011
    Co-Authors: Pu Wang, Konstantinos Konstantopoulos
    Abstract:

    Background Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E2, PGD2 and its dehydration end product 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE2 directly stimulates IL-6 production in human articular Chondrocytes. However, the effects of PGD2 and 15d-PGJ2 in the absence or presence of PGE2 on IL-6 synthesis in human Chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients. Methodology/Principal Findings Using the T/C-28a2 Chondrocyte Cell Line as a model system, we report that exogenous PGE2 and PGD2/15d-PGJ2 exert antagonistic effects on IL-6 synthesis in human T/C-28a2 Chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE2 stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 Chondrocytes. PGD2 or 15d-PGJ2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE2-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in Chondrocytes. Conclusions/Significance We have deLineated the signaling cascade by which PGE2 and PGD2/15d-PGJ2 exert opposing effects on IL-6 synthesis in human Chondrocytes. Elucidation of the molecular pathway of IL-6 synthesis and secretion by Chondrocytes will provide insights for developing strategies to reduce inflammation and pain in RA patients.

  • interleukin 6 synthesis in human Chondrocytes is regulated via the antagonistic actions of prostaglandin pg e2 and 15 deoxy δ12 14 pgj2
    PLOS ONE, 2011
    Co-Authors: Pu Wang, Fei Zhu, Konstantinos Konstantopoulos
    Abstract:

    Background Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E(2), PGD(2) and its dehydration end product 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE(2) directly stimulates IL-6 production in human articular Chondrocytes. However, the effects of PGD(2) and 15d-PGJ(2) in the absence or presence of PGE(2) on IL-6 synthesis in human Chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients. Methodology/principal findings Using the T/C-28a2 Chondrocyte Cell Line as a model system, we report that exogenous PGE(2) and PGD(2)/15d-PGJ(2) exert antagonistic effects on IL-6 synthesis in human T/C-28a2 Chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE(2) stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 Chondrocytes. PGD(2) or 15d-PGJ(2) concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE(2)-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in Chondrocytes. Conclusions/significance We have deLineated the signaling cascade by which PGE(2) and PGD(2)/15d-PGJ(2) exert opposing effects on IL-6 synthesis in human Chondrocytes. Elucidation of the molecular pathway of IL-6 synthesis and secretion by Chondrocytes will provide insights for developing strategies to reduce inflammation and pain in RA patients.

  • shear induced interleukin 6 synthesis in Chondrocytes roles of e prostanoid ep 2 and ep3 in camp protein kinase a and pi3 k akt dependent nf κb activation
    Journal of Biological Chemistry, 2010
    Co-Authors: Pu Wang, Fei Zhu, Norman H Lee, Konstantinos Konstantopoulos
    Abstract:

    Mechanical overloading of cartilage producing hydrostatic stress, tensile strain, and fluid flow can adversely affect Chondrocyte function and precipitate osteoarthritis (OA). Application of high fluid shear stress to Chondrocytes recapitulates the earmarks of OA, as evidenced by the release of pro-inflammatory mediators, matrix degradation, and Chondrocyte apoptosis. Elevated levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E2, and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated Chondrocytes in vitro. Although PGE2 positively regulates IL-6 synthesis in Chondrocytes, the underlying signaling pathway of shear-induced IL-6 expression remains unknown. Using the human T/C-28a2 Chondrocyte Cell Line as a model system, we demonstrate that COX-2-derived PGE2 signals via up-regulation of E prostanoid (EP) 2 and down-regulation of EP3 receptors to raise intraCellular cAMP, and activate protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. PKA and PI3-K/Akt transactivate the NF-κB p65 subunit via phosphorylation at Ser-276 and Ser-536, respectively. Binding of p65 to the IL-6 promoter elicits IL-6 synthesis in sheared Chondrocytes. Selective knockdown of EP2 or ectopic expression of EP3 blocks PKA- and PI3-K/Akt-dependent p65 activation and markedly diminishes shear-induced IL-6 expression. Similar inhibitory effects on IL-6 synthesis were observed by inhibiting PKA, PI3-K, or NF-κB using pharmacological and/or genetic interventions. Reconstructing the signaling network regulating shear-induced IL-6 expression in Chondrocytes may provide insights for developing therapeutic strategies for arthritic disorders and for culturing artificial cartilage in bioreactors.

Pu Wang - One of the best experts on this subject based on the ideXlab platform.

  • Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-D 12,14-PGJ 2
    2013
    Co-Authors: Pu Wang, Fei Zhu, Konstantinos Konstantopoulos
    Abstract:

    Background: Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E2, PGD2 and its dehydration end product 15-deoxy-D 12,14-PGJ2 (15d-PGJ2) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE2 directly stimulates IL-6 production in human articular Chondrocytes. However, the effects of PGD2 and 15d-PGJ2 in the absence or presence of PGE 2 on IL-6 synthesis in human Chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients. Methodology/Principal Findings: Using the T/C-28a2 Chondrocyte Cell Line as a model system, we report that exogenous PGE 2 and PGD 2/15d-PGJ 2 exert antagonistic effects on IL-6 synthesis in human T/C-28a2 Chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE2 stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-kB p65 subunit leading to NF-kB activation. Binding of the activated NF-kB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 Chondrocytes. PGD 2 or 15d-PGJ 2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE2-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-kB-dependent IL-6 synthesis in Chondrocytes

  • the antagonistic actions of endogenous interleukin 1β and 15 deoxy δ12 14 prostaglandin j2 regulate the temporal synthesis of matrix metalloproteinase 9 in sheared Chondrocytes
    Journal of Biological Chemistry, 2012
    Co-Authors: Pu Wang, Konstantinos Konstantopoulos
    Abstract:

    Mechanical overloading of articular cartilage producing hydrostatic stress, tensile strain, and fluid flow results in irreversible cartilage erosion and osteoarthritis (OA). Application of high fluid shear to Chondrocytes recapitulates the earmarks of OA as evidenced by the induction of proinflammatory cytokines and prostaglandins, which are capable of inducing the expression of matrix-degrading enzymes. Matrix metalloproteinase-9 (MMP-9) synthesis is detected at early but not late stages of OA. However, the underlying mechanism(s) of the MMP-9 temporal regulation remains unknown. Using the T/C-28a2 Chondrocyte Cell Line as a model system, we demonstrated that high fluid shear induces a marked increase in MMP-9 expression at short shear exposure times (3–6 h), which falls below basal levels after prolonged shear exposure (12–48 h). High fluid shear stress induced the rapid and sustained synthesis of IL-1β, activating PI3K, ERK1/2, and JNK, which are in turn responsible for MMP-9 expression. Prolonged shear exposure (>12 h) induced 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) synthesis, which exerted an antagonistic effect on IL-1β-mediated PI3K-, ERK1/2-, and JNK-dependent NF-κB activation, thereby suppressing MMP-9 expression in human Chondrocytes. Reconstructing the signaling network that regulates shear-mediated MMP-9 expression in human Chondrocytes may provide insights for developing strategies to treat arthritic disorders.

  • interleukin 6 synthesis in human Chondrocytes is regulated via the antagonistic actions of prostaglandin pg e2 and 15 deoxy δ12 14 pgj2
    PLOS ONE, 2011
    Co-Authors: Pu Wang, Konstantinos Konstantopoulos
    Abstract:

    Background Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E2, PGD2 and its dehydration end product 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE2 directly stimulates IL-6 production in human articular Chondrocytes. However, the effects of PGD2 and 15d-PGJ2 in the absence or presence of PGE2 on IL-6 synthesis in human Chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients. Methodology/Principal Findings Using the T/C-28a2 Chondrocyte Cell Line as a model system, we report that exogenous PGE2 and PGD2/15d-PGJ2 exert antagonistic effects on IL-6 synthesis in human T/C-28a2 Chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE2 stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 Chondrocytes. PGD2 or 15d-PGJ2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE2-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in Chondrocytes. Conclusions/Significance We have deLineated the signaling cascade by which PGE2 and PGD2/15d-PGJ2 exert opposing effects on IL-6 synthesis in human Chondrocytes. Elucidation of the molecular pathway of IL-6 synthesis and secretion by Chondrocytes will provide insights for developing strategies to reduce inflammation and pain in RA patients.

  • interleukin 6 synthesis in human Chondrocytes is regulated via the antagonistic actions of prostaglandin pg e2 and 15 deoxy δ12 14 pgj2
    PLOS ONE, 2011
    Co-Authors: Pu Wang, Fei Zhu, Konstantinos Konstantopoulos
    Abstract:

    Background Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E(2), PGD(2) and its dehydration end product 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE(2) directly stimulates IL-6 production in human articular Chondrocytes. However, the effects of PGD(2) and 15d-PGJ(2) in the absence or presence of PGE(2) on IL-6 synthesis in human Chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients. Methodology/principal findings Using the T/C-28a2 Chondrocyte Cell Line as a model system, we report that exogenous PGE(2) and PGD(2)/15d-PGJ(2) exert antagonistic effects on IL-6 synthesis in human T/C-28a2 Chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE(2) stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 Chondrocytes. PGD(2) or 15d-PGJ(2) concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE(2)-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in Chondrocytes. Conclusions/significance We have deLineated the signaling cascade by which PGE(2) and PGD(2)/15d-PGJ(2) exert opposing effects on IL-6 synthesis in human Chondrocytes. Elucidation of the molecular pathway of IL-6 synthesis and secretion by Chondrocytes will provide insights for developing strategies to reduce inflammation and pain in RA patients.

  • shear induced interleukin 6 synthesis in Chondrocytes roles of e prostanoid ep 2 and ep3 in camp protein kinase a and pi3 k akt dependent nf κb activation
    Journal of Biological Chemistry, 2010
    Co-Authors: Pu Wang, Fei Zhu, Norman H Lee, Konstantinos Konstantopoulos
    Abstract:

    Mechanical overloading of cartilage producing hydrostatic stress, tensile strain, and fluid flow can adversely affect Chondrocyte function and precipitate osteoarthritis (OA). Application of high fluid shear stress to Chondrocytes recapitulates the earmarks of OA, as evidenced by the release of pro-inflammatory mediators, matrix degradation, and Chondrocyte apoptosis. Elevated levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E2, and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated Chondrocytes in vitro. Although PGE2 positively regulates IL-6 synthesis in Chondrocytes, the underlying signaling pathway of shear-induced IL-6 expression remains unknown. Using the human T/C-28a2 Chondrocyte Cell Line as a model system, we demonstrate that COX-2-derived PGE2 signals via up-regulation of E prostanoid (EP) 2 and down-regulation of EP3 receptors to raise intraCellular cAMP, and activate protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. PKA and PI3-K/Akt transactivate the NF-κB p65 subunit via phosphorylation at Ser-276 and Ser-536, respectively. Binding of p65 to the IL-6 promoter elicits IL-6 synthesis in sheared Chondrocytes. Selective knockdown of EP2 or ectopic expression of EP3 blocks PKA- and PI3-K/Akt-dependent p65 activation and markedly diminishes shear-induced IL-6 expression. Similar inhibitory effects on IL-6 synthesis were observed by inhibiting PKA, PI3-K, or NF-κB using pharmacological and/or genetic interventions. Reconstructing the signaling network regulating shear-induced IL-6 expression in Chondrocytes may provide insights for developing therapeutic strategies for arthritic disorders and for culturing artificial cartilage in bioreactors.

Mary B Goldring - One of the best experts on this subject based on the ideXlab platform.

  • www.mdpi.com/journal/ijms Article Mechanical Forces Induce Changes in VEGF and VEGFR-1/sFlt-1 Expression in Human Chondrocytes
    2014
    Co-Authors: Rainer Beckmann, Mary B Goldring, Astrid Houben, Mersedeh Tohidnezhad, Nisreen Kweider, Athanassios Fragoulis, Christoph J. Wruck, Benita Hermanns-sachweh, Thomas Pufe
    Abstract:

    Abstract: Expression of the pro-angiogenic vascular endothelial growth factor (VEGF) stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1 % to 16 % for 12 h, respectively, induced expression of VEGF in human Chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human Chondrocyte Cell Line C-28/I2 than in primary articular Chondrocytes. Twelve hours of 8 % stretch induced VEGF expression to 175 % of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA) OPEN ACCESS Int. J. Mol. Sci. 2014, 15 15457 studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducibl

  • Mechanical Forces Induce Changes in VEGF and VEGFR-1/sFlt-1 Expression in Human Chondrocytes
    MDPI AG, 2014
    Co-Authors: Rainer Beckmann, Mary B Goldring, Astrid Houben, Mersedeh Tohidnezhad, Nisreen Kweider, Athanassios Fragoulis, Christoph J. Wruck, Benita Hermanns-sachweh, Lars O. Brandenburg, Thomas Pufe
    Abstract:

    Expression of the pro-angiogenic vascular endothelial growth factor (VEGF) stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1% to 16% for 12 h, respectively, induced expression of VEGF in human Chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human Chondrocyte Cell Line C-28/I2 than in primary articular Chondrocytes. Twelve hours of 8% stretch induced VEGF expression to 175% of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA) studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducible than its ligand, VEGF-A, in these Cells. ELISA assays demonstrated, for the first time, a stretch-mediated suppression of sVEGFR-1 secretion 24 h after stretching. Overall, strained Chondrocytes activate their VEGF expression, but in contrast, strain appears to suppress the secretion of the major VEGF decoy receptor (sVEGFR-1/sFlt-1). The latter may deplete a biologically relevant feedback regulation to inhibit destructive angiogenesis in articular cartilage. Our data suggest that mechanical stretch can induce morphological changes in human Chondrocytes in vitro. More importantly, it induces disturbed VEGF signaling, providing a molecular mechanism for a stress-induced increase in angiogenesis in cartilage pathologies

  • adeno associated vector mediated gene transfer of transforming growth factor beta1 to normal and osteoarthritic human Chondrocytes stimulates cartilage anabolism
    European Cells & Materials, 2005
    Co-Authors: Michael Ulrichvinther, Mary B Goldring, Carsten Stengaard, Edward M Schwarz, Kjeld Soballe
    Abstract:

    The objective of the present study was to investigate whether cartilage anabolism in human primary osteoarthritic Chondrocytes could be improved by adeno-associated virus (AAV) vector-mediated gene transduction of transforming growth factor TGF-beta1 (TGF-beta1). A bi-cistronic AAV-TGF-beta1-IRES-eGFP (AAV-TGF-beta1) vector was generated and used for transduction of a normal human articular Chondrocyte Cell Line (tsT/AC62) and primary human osteoarthritic articular Chondrocytes harvested from 8 patients receiving total knee joint arthroplasty. Transduction efficiency was detected by fluorescent microscopy for gene expression of enhanced green fluorescent protein (eGFP). TGF-beta1 synthesis was determined by ELISA. To assess the influence of TGF-beta1 gene therapy on Chondrocyte cartilage metabolism, mRNA expressions of type II collagen, aggrecan, and matrix metalloproteinase 3 (MMP-3) were determined by quantitative real-time PCR. AAV-TGF-beta1 transduction resulted in increased synthesis of TGF-beta1 in both osteoarthritic Chondrocytes and the normal articular Chondrocyte Cell Line. The expression levels of the transduced genes were correlated to "multiplicity of infection" (MOI) and post-infectious time. In both osteoarthritic Chondrocytes and the normal articular Chondrocyte Cell Line, AAV-TGF-beta1 treatment increased mRNA expression of both type II collagen and aggrecan, but decreased MMP-3 mRNA expression. Osteoarthritic Chondrocytes and the normal articular Chondrocyte Cell Line could be transduced with equal efficiencies. In conclusion, it was demonstrated that AAV-TGF-beta1 gene transfer stimulates cartilage anabolism and decreases expression of enzymes responsible for cartilage degradation in human osteoarthritic Chondrocytes. The results indicate that the AAV vector is an efficient mediator of growth factors to human articular Chondrocytes, and that it might be useful in future Chondrocyte gene therapy.

  • divergent stress responses to il 1β nitric oxide and tunicamycin by Chondrocytes
    Journal of Cellular Physiology, 2005
    Co-Authors: Bonnie L Oliver, Mary B Goldring, Chunxia G Cronin, Yahui Zhangbenoit, Marvin L Tanzer
    Abstract:

    As the only Cell in cartilage responsible for matrix synthesis, the Chondrocyte's viability is crucial to healthy tissue. It must tolerate stresses from both mechanical and Cellular sources. This study examines the endoplasmic reticulum (ER) stress response in Chondrocytes after exposure to IL-1beta, nitric oxide, or tunicamycin in order to determine whether this form of stress causes Cell death. Cultures of the immortalized human juvenile costal Chondrocyte Cell Line, C-28/I2, were treated with IL-1beta, S-nitroso-N-acetylpenicillamine (SNAP), and tunicamycin. Increasing intraCellular nitric oxide levels by SNAP treatment or inhibiting protein folding in the ER lumen by tunicamycin induced the ER stress response as evidenced by increased protein and gene expression of GADD153 as well as PERK and eIF2-alpha phosphorylation, and resulted in apoptosis. IL-1beta treatment induced PERK and eIF2-alpha phosphorylation, but not GADD153 expression or apoptosis. The ER stress signaling pathway of IL-1beta involved iNOS because blocking its expression, inhibited ER stress gene expression. Therefore, inducing the ER stress response in Chondrocytes results in divergent responses depending on the agent used. Even though IL-1beta, a common proinflammatory cytokine, induces the ER stress response, it is not proapoptotic to Chondrocytes. On the other hand, exposure to high levels of intraCellular nitric oxide induce Chondrocyte apoptosis as part of the ER stress response.

  • up regulation of microsomal prostaglandin e synthase 1 in osteoarthritic human cartilage critical roles of the erk 1 2 and p38 signaling pathways
    Arthritis & Rheumatism, 2004
    Co-Authors: Kayo Masukohongo, Mary B Goldring, Francis Berenbaum, Lydie Humbert, Colette Salvat, Sylvie Thirion
    Abstract:

    Objective Microsomal prostaglandin E synthase 1 (mPGES-1) is the final enzyme of the cascade that produces prostaglandin E2 (PGE2), a key actor in arthritis. To study mPGES-1 synthesis in human cartilage and its regulation by interleukin-1β (IL-1β), we used human cartilage and an immortalized human Chondrocyte Cell Line. Furthermore, we investigated the signaling pathways involved in mPGES-1 expression. Methods We used real-time quantitative reverse transcription–polymerase chain reaction, Northern blotting, and Western blotting to measure mPGES-1 messenger RNA (mRNA) and protein expression in human Chondrocytes. PGE2 production was measured by enzyme-linked immunosorbent assay. Results Cartilage specimens from osteoarthritis (OA) patients contained far greater amounts of mPGES-1 and cyclooxygenase 2 (COX-2) mRNA than did normal cartilage. Incubation with IL-1β markedly increased mPGES-1 mRNA and protein in a dose-dependent and time-dependent manner, in parallel with an increase in PGE2 levels. Both PD98059, an ERK pathway inhibitor, and SB203580, a p38α/β MAPK inhibitor, abolished the increases in mPGES-1 mRNA and protein in response to IL-1β. The specific p38α MAPK inhibitor SC906 suppressed IL-1β–induced COX-2 expression but not IL-1β–induced mPGES-1 expression, suggesting preferential involvement of p38β MAPK in IL-1β–induced mPGES-1 expression. Conclusion This study is the first to show that mPGES-1 is stimulated in human Chondrocytes by the proinflammatory cytokine IL-1β via activation of both ERK-1/2 and p38 MAPK in an isoform-specific manner. We postulate that mPGES-1 may be a novel target for OA therapy.

Oreste Gualillo - One of the best experts on this subject based on the ideXlab platform.

  • E74-Like Factor (ELF3) and Leptin, a Novel Loop Between Obesity and Inflammation Perpetuating a Pro-Catabolic State in Cartilage
    Karger Publishers, 2018
    Co-Authors: Javier Conde, Morena Scotece, Veronica Lopez, Jesus Pino, Rodolfo Gomez, Miguel Otero, Vanessa Abella, Antonio Mera, Mary b. Goldring, Oreste Gualillo
    Abstract:

    Background/Aims: The E74-like factor 3 (ELF3) is an inflammatory mediator that participates in cartilage destruction in osteoarthritis. Leptin and other adipokines negatively impact articular cartilage, triggering catabolic and inflammatory responses in Chondrocytes. Here, we investigated whether leptin induces ELF3 expression in Chondrocytes and the signaling pathway involved in this process. Methods: We determined mRNA and protein levels of ELF3 by RT-qPCR and Western blotting using cultured human primary Chondrocytes and the human T/C-28a2 Chondrocyte Cell Line. Further, we measured luciferase activities of different reporter constructs, and we assessed the contribution of leptin to the induction of ELF3 mRNA by knocking down hLEPR gene expression using siRNA technology. Results: Leptin synergizes with IL-1β in inducing ELF3 expression in Chondrocytes. We also found that PI3K, p38, and JAK2 signaling pathways are at play in the leptin-driven induction of ELF3. Moreover, we confirm the participation of NFΚB in the leptin/IL-1β synergistic induction of ELF3. Conclusion: Here we show, for the first time, the regulation of ELF3 expression by leptin, suggesting that this transcription factor likely mediates the inflammatory responses triggered by leptin in articular Chondrocytes

  • Adiponectin and Leptin Induce VCAM-1 Expression in
    2016
    Co-Authors: Murine Chondrocytes, Morena Scotece, Javier Conde, Francisca Lago, Oreste Gualillo
    Abstract:

    Background: Osteoarthritis (OA) and rheumatoid arthritis (RA), the most common rheumatic diseases, are characterized by irreversible degeneration of the joint tissues. There are several factors involved in the pathogenesis of these diseases including pro-inflammatory cytokines, adipokines and adhesion molecules. Objective: Up to now, the relationship between adipokines and adhesion molecules at cartilage level was not explored. Thus, the aim of this article was to study the effect of leptin and adiponectin on the expression of VCAM-1 in human and murine Chondrocytes. For completeness, intraCellular signal transduction pathway was also explored. Methods: VCAM-1 expression was assessed by quantitative RT-PCR and western blot analysis upon treatment with leptin, adiponectin and other pertinent reagents in cultured human primary Chondrocytes. Signal transduction pathways have been explored by using specific pharmacological inhibitors in the adipokine-stimulated human primary Chondrocytes and ATDC5 murine Chondrocyte Cell Line. Results: Herein, we demonstrate, for the first time, that leptin and adiponectin increase VCAM-1 expression in human and murine Chondrocytes. In addition, both adipokines have additive effect with IL-1b. Finally, we demonstrate that several kinases, including JAK2, PI3K and AMPK are at a play in the intraCellular signalling of VCAM-1 induction. Conclusions: Taken together, our results suggest that leptin and adiponectin could perpetuate cartilage-degradin

  • nucb2 nesfatin 1 a new adipokine expressed in human and murine Chondrocytes with pro inflammatory properties an in vitro study
    Journal of Orthopaedic Research, 2014
    Co-Authors: Morena Scotece, Javier Conde, Veronica Lopez, Jesus Pino, Juan J Gomezreino, Francisca Lago, Vanessa Abella, Oreste Gualillo
    Abstract:

    Nesfatin-1 is a recently discovered satiety-inducing adipokine identified in hypothalamic regions that regulates energy balance. So far, no data exist on NUCB2/nesfatin-1 localization in human and murine Chondrocytes. Here, we therefore investigated NUCB2/nesfatin-1 gene and protein expression in human and murine Chondrocytes and the effect of nesfatin-1 on pro-inflammatory cytokines expression. Peptide localization was performed by laser confocal microscopy, NUCB2 mRNA expression was studied by RT-PCR and protein secretion was measured by XMap technology and Western blot analysis. First, we demonstrated cytoplasmic localization of NUCB2/nesfatin-1 peptide in both human and murine Chondrocytes. We present evidence that both mRNA and protein expression of NUCB2 were increased during the differentiation of ATDC5 murine Chondrocyte Cell Line. Furthermore, we demonstrated that nesfatin-1 induces IL-6 and MIP-1α mRNA expression and protein secretion in ATDC-5 Cells challenged with IL-1, and also increases COX-2 mRNA expression in these Cells. Finally, nesfatin-1 provoked a clear induction of pro-inflammatory agents, such as COX-2, IL-8, IL-6, and MIP-1α in human primary Chondrocytes from OA patients. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:653–660, 2014.

  • fri0029 nucb2 nesfatin 1 a new adipokine expressed in human and murine Chondrocytes with pro inflammatory properties
    Annals of the Rheumatic Diseases, 2013
    Co-Authors: Morena Scotece, Javier Conde, Veronica Lopez, Jesus Pino, Juan J Gomezreino, Oreste Gualillo
    Abstract:

    Background Nesfatin-1 is a recently identified satiety-inducing adipokine determined in hypothalamic regions that regulates energy balance [1]. It is an integral regulator of energy homeostasis and a putative glucose-dependent insulin coadjuvant [2]. Nesfatin-1 has strong similarities in terms of metabolic actions with other members of adipokine superfamily, such as leptin, adiponectin and lipocalin-2. These factors have been recently identified as active players in the regulation of physiological and pathological processes, such as inflammation and immune response in whole joint tissues and in rheumatic diseases, such as osteoarthritis (OA) and/or rheumatoid arthritis (RA). Objectives So far, no data exist on nesfatin expression, regulation, and secretion in human and murine Chondrocytes. We therefore investigated NUCB2/nesfatin-1 gene and protein expression in human and murine Chondrocytes. For completeness, the effect of NUCB-2 on pro-inflammatory cytokines expression was also studied Methods NUCB-2 mRNA expression was studied by qRT-PCR. Protein secretion was measured by BioPlex assay and Western blot analysis. Peptide localization was performed by laser confocal microscopy. Results First, we demonstrated cytoplasmic localization of NUCB-2 peptide in both human and murine Chondrocytes. We present evidence that both mRNA and protein expression of NUCB-2 were increased during the differentiation of ATDC5 murine Chondrocyte Cell Line Furthermore, we demonstrated that nesfatin-1 induces IL-6 and MIP-1α mRNA expression and protein secretion in ATDC5 Cells challenged with IL-1. Finally, nesfatin-1 provoked a clear and a direct induction of pro-inflammatory agents, such as COX-2 and IL-8 in human Chondrocytes. Conclusions NUCB-2/Nesfatin-1 is synthesized and produced by human and murine Chondrocytes, in which it exerts a clear pro-inflammatory activity. References Oh-I S, Shimizu H, Satoh T, Okada S, 2006 Identification of nesfatin-1 as a satiety molecule in the hypothalamus. Nature 443:709-71 Ramanjaneya M, Chen J, Brown JE, Tripathi G, 2010 Identification of nesfatin-1 in human and murine adipose tissue: a novel depot-specific adipokine with increased levels in obesity. Endocrinology 151(7):3169-80 Disclosure of Interest None Declared

  • expanding the adipokine network in cartilage identification and regulation of novel factors in human and murine Chondrocytes
    Annals of the Rheumatic Diseases, 2011
    Co-Authors: Javier Conde, Morena Scotece, Juan J Gomezreino, Rodolfo Gomez, Giuseppe Bianco, Pamela V Lear, Carlos Dieguez, Francisca Lago, Oreste Gualillo
    Abstract:

    Background Obesity is a major risk factor for a plethora of diseases including joint disorders associated with cartilage destruction. Recently, it has been demonstrated that adipose tissue might contribute to degenerative joint diseases via the secretion of potent bioactive molecules termed adipokines. Objective To study expression of the novel adipokines chemerin, lipocalin 2 (LCN2) and serum amyloid A3 (SAA3) in murine and human Chondrocytes, under basal conditions, in response to a range of biological and pharmacological treatments, and during Chondrocyte differentiation. Methods Chemerin, LCN2 and SAA3 mRNA and protein expression were evaluated by quantitative real-time reverse transcription PCR and western blot analysis, respectively, in the ATDC-5 murine Chondrocyte Cell Line, a human immortalised Chondrocyte Cell Line (T/C-28a2) and primary cultured human Chondrocytes. Results Human and murine Chondrocytes expressed chemerin, LCN2 and SAA3 mRNA; interleukin (IL)-1β was a potent inducer of these novel adipokines. Moreover, dexamethasone, lipopolysaccharides (LPS) and other relevant adipokines such as leptin and adiponectin were able to modulate chemerin, LCN2 and SAA3 mRNA expression alone and when coadministered. IntraCellular signal transducers involved in the IL-1β-mediated upregulation of LCN2 and SAA3 included Janus kinase (JAK) 2, phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein (MAP) kinases. Finally, expression of chemerin, LCN2 and SAA3 mRNA expression were modulated throughout Chondrocyte differentiation. Conclusion Chemerin, LCN2 and SAA3 are implicated in Chondrocyte pathophysiology, and regulated by other relevant factors that drive inflammatory process such as IL-1β, LPS and adipokines including leptin and adiponectin. It seems likely that JAK2, PI3K and MAP kinases are involved in mediating these responses.

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  • E74-Like Factor (ELF3) and Leptin, a Novel Loop Between Obesity and Inflammation Perpetuating a Pro-Catabolic State in Cartilage
    Karger Publishers, 2018
    Co-Authors: Javier Conde, Morena Scotece, Veronica Lopez, Jesus Pino, Rodolfo Gomez, Miguel Otero, Vanessa Abella, Antonio Mera, Mary b. Goldring, Oreste Gualillo
    Abstract:

    Background/Aims: The E74-like factor 3 (ELF3) is an inflammatory mediator that participates in cartilage destruction in osteoarthritis. Leptin and other adipokines negatively impact articular cartilage, triggering catabolic and inflammatory responses in Chondrocytes. Here, we investigated whether leptin induces ELF3 expression in Chondrocytes and the signaling pathway involved in this process. Methods: We determined mRNA and protein levels of ELF3 by RT-qPCR and Western blotting using cultured human primary Chondrocytes and the human T/C-28a2 Chondrocyte Cell Line. Further, we measured luciferase activities of different reporter constructs, and we assessed the contribution of leptin to the induction of ELF3 mRNA by knocking down hLEPR gene expression using siRNA technology. Results: Leptin synergizes with IL-1β in inducing ELF3 expression in Chondrocytes. We also found that PI3K, p38, and JAK2 signaling pathways are at play in the leptin-driven induction of ELF3. Moreover, we confirm the participation of NFΚB in the leptin/IL-1β synergistic induction of ELF3. Conclusion: Here we show, for the first time, the regulation of ELF3 expression by leptin, suggesting that this transcription factor likely mediates the inflammatory responses triggered by leptin in articular Chondrocytes

  • Adiponectin and Leptin Induce VCAM-1 Expression in
    2016
    Co-Authors: Murine Chondrocytes, Morena Scotece, Javier Conde, Francisca Lago, Oreste Gualillo
    Abstract:

    Background: Osteoarthritis (OA) and rheumatoid arthritis (RA), the most common rheumatic diseases, are characterized by irreversible degeneration of the joint tissues. There are several factors involved in the pathogenesis of these diseases including pro-inflammatory cytokines, adipokines and adhesion molecules. Objective: Up to now, the relationship between adipokines and adhesion molecules at cartilage level was not explored. Thus, the aim of this article was to study the effect of leptin and adiponectin on the expression of VCAM-1 in human and murine Chondrocytes. For completeness, intraCellular signal transduction pathway was also explored. Methods: VCAM-1 expression was assessed by quantitative RT-PCR and western blot analysis upon treatment with leptin, adiponectin and other pertinent reagents in cultured human primary Chondrocytes. Signal transduction pathways have been explored by using specific pharmacological inhibitors in the adipokine-stimulated human primary Chondrocytes and ATDC5 murine Chondrocyte Cell Line. Results: Herein, we demonstrate, for the first time, that leptin and adiponectin increase VCAM-1 expression in human and murine Chondrocytes. In addition, both adipokines have additive effect with IL-1b. Finally, we demonstrate that several kinases, including JAK2, PI3K and AMPK are at a play in the intraCellular signalling of VCAM-1 induction. Conclusions: Taken together, our results suggest that leptin and adiponectin could perpetuate cartilage-degradin

  • nucb2 nesfatin 1 a new adipokine expressed in human and murine Chondrocytes with pro inflammatory properties an in vitro study
    Journal of Orthopaedic Research, 2014
    Co-Authors: Morena Scotece, Javier Conde, Veronica Lopez, Jesus Pino, Juan J Gomezreino, Francisca Lago, Vanessa Abella, Oreste Gualillo
    Abstract:

    Nesfatin-1 is a recently discovered satiety-inducing adipokine identified in hypothalamic regions that regulates energy balance. So far, no data exist on NUCB2/nesfatin-1 localization in human and murine Chondrocytes. Here, we therefore investigated NUCB2/nesfatin-1 gene and protein expression in human and murine Chondrocytes and the effect of nesfatin-1 on pro-inflammatory cytokines expression. Peptide localization was performed by laser confocal microscopy, NUCB2 mRNA expression was studied by RT-PCR and protein secretion was measured by XMap technology and Western blot analysis. First, we demonstrated cytoplasmic localization of NUCB2/nesfatin-1 peptide in both human and murine Chondrocytes. We present evidence that both mRNA and protein expression of NUCB2 were increased during the differentiation of ATDC5 murine Chondrocyte Cell Line. Furthermore, we demonstrated that nesfatin-1 induces IL-6 and MIP-1α mRNA expression and protein secretion in ATDC-5 Cells challenged with IL-1, and also increases COX-2 mRNA expression in these Cells. Finally, nesfatin-1 provoked a clear induction of pro-inflammatory agents, such as COX-2, IL-8, IL-6, and MIP-1α in human primary Chondrocytes from OA patients. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:653–660, 2014.

  • fri0029 nucb2 nesfatin 1 a new adipokine expressed in human and murine Chondrocytes with pro inflammatory properties
    Annals of the Rheumatic Diseases, 2013
    Co-Authors: Morena Scotece, Javier Conde, Veronica Lopez, Jesus Pino, Juan J Gomezreino, Oreste Gualillo
    Abstract:

    Background Nesfatin-1 is a recently identified satiety-inducing adipokine determined in hypothalamic regions that regulates energy balance [1]. It is an integral regulator of energy homeostasis and a putative glucose-dependent insulin coadjuvant [2]. Nesfatin-1 has strong similarities in terms of metabolic actions with other members of adipokine superfamily, such as leptin, adiponectin and lipocalin-2. These factors have been recently identified as active players in the regulation of physiological and pathological processes, such as inflammation and immune response in whole joint tissues and in rheumatic diseases, such as osteoarthritis (OA) and/or rheumatoid arthritis (RA). Objectives So far, no data exist on nesfatin expression, regulation, and secretion in human and murine Chondrocytes. We therefore investigated NUCB2/nesfatin-1 gene and protein expression in human and murine Chondrocytes. For completeness, the effect of NUCB-2 on pro-inflammatory cytokines expression was also studied Methods NUCB-2 mRNA expression was studied by qRT-PCR. Protein secretion was measured by BioPlex assay and Western blot analysis. Peptide localization was performed by laser confocal microscopy. Results First, we demonstrated cytoplasmic localization of NUCB-2 peptide in both human and murine Chondrocytes. We present evidence that both mRNA and protein expression of NUCB-2 were increased during the differentiation of ATDC5 murine Chondrocyte Cell Line Furthermore, we demonstrated that nesfatin-1 induces IL-6 and MIP-1α mRNA expression and protein secretion in ATDC5 Cells challenged with IL-1. Finally, nesfatin-1 provoked a clear and a direct induction of pro-inflammatory agents, such as COX-2 and IL-8 in human Chondrocytes. Conclusions NUCB-2/Nesfatin-1 is synthesized and produced by human and murine Chondrocytes, in which it exerts a clear pro-inflammatory activity. References Oh-I S, Shimizu H, Satoh T, Okada S, 2006 Identification of nesfatin-1 as a satiety molecule in the hypothalamus. Nature 443:709-71 Ramanjaneya M, Chen J, Brown JE, Tripathi G, 2010 Identification of nesfatin-1 in human and murine adipose tissue: a novel depot-specific adipokine with increased levels in obesity. Endocrinology 151(7):3169-80 Disclosure of Interest None Declared

  • expanding the adipokine network in cartilage identification and regulation of novel factors in human and murine Chondrocytes
    Annals of the Rheumatic Diseases, 2011
    Co-Authors: Javier Conde, Morena Scotece, Juan J Gomezreino, Rodolfo Gomez, Giuseppe Bianco, Pamela V Lear, Carlos Dieguez, Francisca Lago, Oreste Gualillo
    Abstract:

    Background Obesity is a major risk factor for a plethora of diseases including joint disorders associated with cartilage destruction. Recently, it has been demonstrated that adipose tissue might contribute to degenerative joint diseases via the secretion of potent bioactive molecules termed adipokines. Objective To study expression of the novel adipokines chemerin, lipocalin 2 (LCN2) and serum amyloid A3 (SAA3) in murine and human Chondrocytes, under basal conditions, in response to a range of biological and pharmacological treatments, and during Chondrocyte differentiation. Methods Chemerin, LCN2 and SAA3 mRNA and protein expression were evaluated by quantitative real-time reverse transcription PCR and western blot analysis, respectively, in the ATDC-5 murine Chondrocyte Cell Line, a human immortalised Chondrocyte Cell Line (T/C-28a2) and primary cultured human Chondrocytes. Results Human and murine Chondrocytes expressed chemerin, LCN2 and SAA3 mRNA; interleukin (IL)-1β was a potent inducer of these novel adipokines. Moreover, dexamethasone, lipopolysaccharides (LPS) and other relevant adipokines such as leptin and adiponectin were able to modulate chemerin, LCN2 and SAA3 mRNA expression alone and when coadministered. IntraCellular signal transducers involved in the IL-1β-mediated upregulation of LCN2 and SAA3 included Janus kinase (JAK) 2, phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein (MAP) kinases. Finally, expression of chemerin, LCN2 and SAA3 mRNA expression were modulated throughout Chondrocyte differentiation. Conclusion Chemerin, LCN2 and SAA3 are implicated in Chondrocyte pathophysiology, and regulated by other relevant factors that drive inflammatory process such as IL-1β, LPS and adipokines including leptin and adiponectin. It seems likely that JAK2, PI3K and MAP kinases are involved in mediating these responses.