Matrix Metalloproteinase Production by COOH-Terminal Heparin-Binding Fibronectin Fragment in Rheumatoid Synovial Cells

2010

Biomed. Res.

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High molecular weight hyaluronan (HA) is widely used in the treatment of osteoarthritis (OA) and rheumatoid arthritis (RA) by intra-articular injection. However, comparative studies of HA actions on catalytically activated cartilages in different pathologic conditions have rarely been investigated. This study was aimed to compare the inhibitory effects of HA on nitric oxide (NO) production by COOH-terminal heparin-binding fibronectin fragment (HBFN-f) between normal and diseased cartilages. When articular cartilage explants from normal, OA, or RA joints were incubated with HBFN-f, the RA and OA cartilages produced higher levels of NO compared with normal cartilage. Pretreatment with 2700 kDa HA resulted in significant suppression of HBFN-f-stimulated NO production in OA and RA cartilages. While CD44 was up-regulated in OA and RA cartilages, anti-CD44 antibody reversed HA inhibition of HBFN-f action in those cartilages. The present results clearly demonstrated that HA blocked HBFN-f actions in OA and RA cartilages through interaction with CD44. HA, which targets CD44 highly expressed on OA and RA chondrocytes, could suppress catabolic actions by fibronectin fragments like HBFN-f in diseased cartilage.Fibronectin (FN) is a component of normal cartilage matrix. FN consists predominantly of three types of homologous repeating segments (designated I, II, and III). While FN is encoded by a single gene, significant protein heterogeneity results from alternative RNA splicing at three sites, termed Extra type III Domain A (ED-A), Extra type III Domain B (ED-B), and the variable (V) region. The V region is also referred to as the connecting segment between III14-15 (IIICS). There are 10 principal isoforms predicted by RNA splicing patterns, each of which contains amino (NH 2 )-terminal heparin-, gelatin-, cell-and carboxyl (COOH)-terminal heparinbinding domains. Elevated levels of FN are found in osteoarthritic cartilage (12, 21) and in both synovial fluid and plasma of osteoarthritis (OA) and rheumatoid arthritis (RA) (29,34). FN is readily degraded into fragments by proteinases. In OA and RA, activation of extracellular proteolysis may lead to the fragmentation of FN. Indeed, increased levels of 30-200 kDa fibronectin fragments (FN-fs) are found in cartilage and synovial fluid from patients with OA and RA (9, 34). Native FN has no catabolic effect on cartilage. Once FN is fragmented, those proteolytic fragments acquire catalytic activities whereby increased FN-fs are thought to cause cartilage destruction in OA and RA (35). Of FN-fs, the fragments with the central cell-, NH 2 -terminal heparin-and NH 2 -terminal gelatin-binding domains have been extensively investigated. Those FN-fs can stimulate proteoglycan breakdown in cultured articular cartilage explants (8). We have recently found that another FN-f, 40 kDa COOH-terminal heparin-binding fragment containing both the III12-14 and IIICS domains (HBFN-f), enhances type II collagen cleavage by collagenase in association with enhanced production

Section: Correlation Of Cd44 Levels With Hbfn-f-stimulated No Productmentioning

confidence: 79%

Comparison of hyaluronan effects among normal, osteoarthritis, and rheumatoid arthritis cartilages stimulated with fibronectin fragment

2010

Biomed. Res.

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“…DNA content in proteinase K digests was determined as described previously (25,26). We found no significant difference of DNA content in U937 cells between any treatment groups (data not shown).…”

Section: Dna Assaymentioning

confidence: 97%

“…Immunoblot analysis was performed as described before (16,18,25). The cell lysates prepared as described above were heated with SDS-PAGE sample buffer [0.125 M Tris-HCl (pH 6.8), 10% 2-mercaptoethanol, 4% SDS, 10% sucrose, and 0.004% bromophenol blue] at 80°C for 20 min and subjected to SDS-PAGE under reducing conditions, separated by SDS-PAGE after standardization, and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA).…”

Section: Immunoblot Analysismentioning

confidence: 99%

Hyaluronan Inhibits Akt, Leading to Nuclear Factor-κB Down-Regulation in Lipopolysaccharide-Stimulated U937 Macrophages

2011

J Pharmacol Sci

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Abstract. Hyaluronan (HA) of high molecular weight is used in the treatment of osteoarthritis and rheumatoid arthritis by intra-articular injection. While HA has been shown to suppress nuclear factor (NF)-κB activation by proinflammatory cytokines and lipopolysaccharide (LPS), intracellular upstream events that cause NF-κB down-regulation in response to HA remain unclear. Thus, this study was performed to investigate the involvement of phosphoinositide-3-OH kinase (PI3K)/Akt in the inhibition of the LPS-activated NF-κB pathway by HA in U937 macrophages. In adherent U937 macrophage cultures, pretreatment with HA of 2700 kDa (1 mg/ml, 1 h) significantly inhibited interleukin-6 (IL-6) production by LPS (200 ng/ml, 24 h)-stimulated U937 cells. LPS (200 ng/ ml) activated Akt and NF-κB, whereas HA (1 mg/ml) down-regulated LPS-stimulated phosphorylation of Akt and NF-κB. Inhibition studies using LY294002 (20 μM) revealed the requirement of the PI3K/Akt pathway for LPS-stimulated IL-6 production and NF-κB activation. Pretreatment with anti-intercellular adhesion molecule-1 (ICAM-1) antibody (20 μg/ml) reversed the inhibitory effects of HA on LPS-induced production of IL-6 and activation of Akt and NF-κB. Herein, we provided the first evidence that HA suppresses the LPS-activated PI3K/Akt pathway, leading to down-regulation of NF-κB with diminished IL-6 production through interaction with ICAM-1.

“…However, little is known about the effect of HA on the actions of type II collagen fragment through interaction with the HA receptor. Because activation of the p38 pathway leads to the expression of a variety of inflammatory genes including MMP-13 (13,14,26,27), p38 activation by type II collagen fragment and its inhibition by HA via the receptor are important issues to be addressed. In the present study, therefore, I investigated the role of p38 in MMP-13 induction by the CB12-II peptide and its inhibition by HA through ICAM-1 in articular chondrocytes.…”

Section: Introductionmentioning

confidence: 99%

Activation of p38 Mitogen-Activated Protein Kinase Is Inhibited by Hyaluronan via Intercellular Adhesion Molecule-1 in Articular Chondrocytes Stimulated With Type II Collagen Peptide

2012

J Pharmacol Sci

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Abstract. This study examined the activation of p38 mitogen-activated protein kinase with matrix metalloproteinase-13 (MMP-13) production by a synthetic peptide derived from type II collagen (CB12-II) and its inhibition by high molecular weight hyaluronan (HA) in chondrocytes. When cartilage explants or isolated chondrocytes in monolayer were incubated with CB12-II, the peptide (50 μM, 72 h) activated p38 in association with enhanced MMP-13 production. Inhibition studies with SB203580 (0.1 -1 μM) indicated the requirement of p38 for CB12-II-induced MMP-13 production. Pretreatment with 2700 kDa HA (1 mg/ml, 1 h) resulted in significant suppression of CB12-II-stimulated MMP-13 production in cartilage as well as in chondrocyte monolayer cultures. HA (1 mg/ml) suppressed p38 activation by CB12-II, leading to a decrease in MMP-13 production. The antibody (20 μg/ml) to intercellular adhesion molecule-1 (ICAM-1), which has been recognized as a receptor of HA on chondrocytes, reversed the HA effect on CB12-II action. Thus, the present study clearly demonstrated that high molecular weight HA suppressed CB12-IIactivated p38 via ICAM-1 in articular chondrocytes. HA could down-regulate the catabolic action of type II collagen fragments in osteoarthritic joints through the mechanism demonstrated in this study.