Heparin-binding Fibronectin Fragment Research Articles

AB0107 AKT activation by fibronectin fragment leads to NF-KB up-regulation in rheumatoid arthritic chondrocytes

BackgroundIncreased fibronectin fragments are thought to contribute to joint destruction in rheumatoid arthritis (RA). However, the mechanism whereby fibronectin fragments cause catabolic activities is not totally understood. While COOH-terminal heparin-binding...

Activation of Akt leading to NF-κB up-regulation in chondrocytes stimulated with fibronectin fragment

Increased fibronectin fragments are thought to contribute to joint destruction in osteoarthritis (OA). However, the mechanism whereby fibronectin fragments cause catabolic activities is not totally understood. While COOH-terminal heparin-binding fibronectin fragment (HBFN-f) has been shown to activate nuclear factor (NF)-κB pathway, intracellular upstream events that cause NF-κB up-regulation in response to HBFN-f remain unclear. Thus, this study was aimed to elucidate the involvement of phosphoinositide-3-OH kinase (PI3K)/Akt pathway in NF-κB activation by HBFN-f in OA chondrocytes. In chondrocyte monolayer cultures, HBFN-f stimulated nitric oxide (NO) production in association with phosphorylation of NF-κB and Akt. Inhibition studies using LY294002 revealed the requirement of PI3K/Akt pathway for NO production and NF-κB activation by HBFN-f. Anti-CD44 treatment with anti-CD44 antibody and hyaluronan resulted in significant inhibition of HBFN-f actions on NO, NF-κB, and Akt. Herein, we provided the first evidence that HBFN-f activates PI3K/Akt pathway leading to up-regulation of NF-κB through interaction with CD44.

Hyaluronan inhibits p38 mitogen-activated protein kinase via the receptors in rheumatoid arthritis chondrocytes stimulated with fibronectin fragment

This study was aimed to examine the inhibitory mechanism of high molecular weight hyaluronan (HA) on nitric oxide (NO) production by NH2-terminal heparin-binding fibronectin fragment (FN-f) in rheumatoid arthritis (RA) chondrocytes. When the RA cartilage explants or the isolated RA chondrocytes in monolayer were incubated with FN-f, the fragment stimulated NO production with induction of inducible nitric oxide synthase (iNOS) and activation of p38 mitogen-activated protein kinase. Pretreatment with 2,700 kDa HA resulted in significant suppression of FN-f-stimulated NO production in RA cartilage as well as in chondrocyte monolayer cultures in association with iNOS down-regulation. Inhibition studies with p38 inhibitor indicated the requirement of p38 for FN-f-induced NO production. HA suppressed p38 activation by the FN-f, leading to a decrease in NO production. Immunofluorescence cytochemistry revealed HA association with intercellular adhesion molecule-1 (ICAM-1) and CD44. While the individual antibody to ICAM-1 or CD44 partially reversed HA effect on the FN-f action, both antibodies in combination completely blocked the HA effect. The present study clearly demonstrated that the high molecular weight of HA suppressed the FN-f-activated p38 via ICAM-1 and the CD44 in RA chondrocytes. HA could down-regulate the catabolic action of FN-f in RA joints through the mechanism demonstrated in this study.

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

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.

Inhibition of nuclear factor-κB by hyaluronan in rheumatoid chondrocytes stimulated with COOH-terminal heparin-binding fibronectin fragment

The aim of this study was to examine the inhibitory effect of high molecular weight hyaluronan (HA) on nuclear factor (NF)-kappaB activation by COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in rheumatoid arthritis (RA) chondrocytes. When RA chondrocytes in monolayer or cartilage explants were cultured with HBFN-f, the fragment stimulated the phosphorylation and nuclear translocation of NF-kappaB, leading to nitric oxide (NO) production in association with inducible form of NO synthase (iNOS) up-regulation. Inhibition studies with NF-kappaB inhibitors indicated the requirement of NF-kappaB for HBFN-f-induced NO production. Pretreatment with 2700 kDa HA resulted in significant suppression of NF-kappaB activation by HBFN-f. HA also inhibited HBFN-f-stimulated NO production with down-regulation of iNOS. The present study clearly demonstrated that high molecular weight HA suppressed HBFN-f-activated NF-kappaB in RA chondrocytes. HA could down-regulate the catabolic action of fibronectin fragments like HBFN-f in RA joints as a potent NF-kappaB inhibitor.

Inhibition of nuclear factor-κB by hyaluronan in rheumatoid chondrocytes stimulated with COOH-terminal heparin-binding fibronectin fragment

The aim of this study was to examine the inhibitory effect of high molecular weight hyaluronan (HA) on nuclear factor (NF)-κB activation by COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in rheumatoid arthritis (RA) chondrocytes. When RA chondrocytes in monolayer or cartilage explants were cultured with HBFN-f, the fragment stimulated the phosphorylation and nuclear translocation of NF-κB, leading to nitric oxide (NO) production in association with inducible form of NO synthase (iNOS) up-regulation. Inhibition studies with NF-κB inhibitors indicated the requirement of NF-κB for HBFN-f-induced NO production. Pretreatment with 2700 kDa HA resulted in significant suppression of NF-κB activation by HBFN-f. HA also inhibited HBFN-f-stimulated NO production with down-regulation of iNOS. The present study clearly demonstrated that high molecular weight HA suppressed HBFN-f-activated NF-κB in RA chondrocytes. HA could down-regulate the catabolic action of fibronectin fragments like HBFN-f in RA joints as a potent NF-κB inhibitor.

Cyclophilin C-associated Protein Is a Mediator for Fibronectin Fragment-induced Matrix Metalloproteinase-13 Expression

The function of cyclophilin C-associated protein (CyC-AP) on expression of extracellular matrix and matrix metalloproteinases (MMPs) was studied in CyC-AP-null mice. Fibronectin showed increased expression of the 53- and 29-kDa fragments in skin and wounds from CyC-AP-null mice. Type I collagen had an initial degraded pattern in the skin of CyC-AP-null mice, which did not occur in wild-type mice. MMP-3, MMP-13, MMP-14, and tumor necrosis factor-alpha (TNFalpha) had a higher expression in CyC-AP-null skin. During wound healing, MMP-13 and TNFalpha were stimulated to an even higher level, suggesting they are regulated by multiple factors. To understand the regulatory mechanisms of the up-regulated MMPs, the direct effects of TNFalpha, IL-1beta, 45-kDa fibronectin fragment (FN-45), and the 70-kDa fibronectin fragments (FN-70) on the expression of MMPs were studied. MMP-13 expression increased significantly in both CyC-AP-null and wild-type dermal fibroblasts after treatment with IL-1beta or with TNFalpha. However, MMP-13 expression did not increase in CyC-AP-null fibroblasts but did increase only in wild-type fibroblasts after FN-45 and FN-70 treatment. MMP-3 activation was induced by FN-45 and did not show a difference between CyC-AP-null and wild-type fibroblasts, suggesting different regulatory pathways for FN-45 on MMP-13 and MMP-3 expression. Our data are the first to demonstrate that deletion of CyC-AP can abolish fibronectin fragment-induced MMP-13 expression through an unknown mechanism. CyC-AP is an important factor for the regulation of MMP-13 expression.

COOH-terminal heparin-binding fibronectin fragment induces nitric oxide production in rheumatoid cartilage through CD44.

To examine the mechanism of nitric oxide (NO) production by a COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in rheumatoid arthritis (RA) cartilage. Articular cartilage slices from RA knee joints and normal hip joints were cultured with HBFN-f. Secreted NO levels in conditioned media were determined. Cultures were pretreated with anti-CD44 antibody or HBFN-f-derived synthetic peptide (peptide V; WQPPRARI) to evaluate the role of CD44 in HBFN-f action. Immunofluorescence histochemistry was performed using fluorescein isothiocyanate-conjugated anti-CD44 antibody. HBFN-f stimulated NO production in a dose-dependent manner. Whereas CD44 expression was up-regulated in RA cartilage, anti-CD44 antibody blocked HBFN-f-stimulated NO production. Peptide V with heparin-binding ability significantly reduced NO levels elevated by HBFN-f. Compared with normal cartilage, cartilage response to HBFN-f and the blocking effects of anti-CD44 antibody on HBFN-f action were stronger in RA cartilage. The present study clearly demonstrated that HBFN-f stimulated NO production through CD44 in RA cartilage. Increased expression of CD44 in RA cartilage may play a pathological role in joint destruction through enhanced NO production by binding to fibronectin fragments such as HBFN-f.

Requirement of mitogen-activated protein kinase for collagenase production by the fibronectin fragment in human articular chondrocytes in culture

Fibronectin fragments have been shown to up-regulate matrix metalloproteinase production in chondrocytes. We investigated the roles of mitogen-activated protein kinase (MAPK) pathways activated by the COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in collagenase production by human chondrocytes in culture. In articular cartilage explant culture, HBFN-f stimulated type II collagen cleavage by collagenase in association with increased secretion of MMP-1 and MMP-13. In human articular chondrocytes, HBFN-f induced the collagenases with activation of the extracellular signal-regulated kinase (ERK), p38, and the c-Jun NH2-terminal kinase (JNK). PD98059 that inhibits the ERK pathway blocked HBFN-f-stimulated production of MMP-1 and MMP-13 in explant culture. SB203580 at 1 µM, the concentration that inhibits p38 only, partially suppressed HBFN-f-induced collagenase production, whereas at 10 µM, the inhibitor that blocks both p38 and JNK almost completely inhibited collagenase induction. PD98059 and SB203580 individually blocked HBFN-f-increased cleavage of type II collagen in the explant culture, although 10 µM SB203580 strongly inhibited the collagen cleavage compared with 1 µM of the inhibitor. These results indicate that collagenase production leading to type II collagen cleavage in cartilage explants requires ERK, p38, and JNK.

Requirement of mitogen-activated protein kinase for collagenase production by the fibronectin fragment in human articular chondrocytes in culture

Fibronectin fragments have been shown to up-regulate matrix metalloproteinase production in chondrocytes. We investigated the roles of mitogen-activated protein kinase (MAPK) pathways activated by the COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in collagenase production by human chondrocytes in culture. In articular cartilage explant culture, HBFN-f stimulated type II collagen cleavage by collagenase in association with increased secretion of MMP-1 and MMP-13. In human articular chondrocytes, HBFN-f induced the collagenases with activation of the extracellular signal-regulated kinase (ERK), p38, and the c-Jun NH(2)-terminal kinase (JNK). PD98059 that inhibits the ERK pathway blocked HBFN-f-stimulated production of MMP-1 and MMP-13 in explant culture. SB203580 at 1 microM, the concentration that inhibits p38 only, partially suppressed HBFN-f-induced collagenase production, whereas at 10 microM, the inhibitor that blocks both p38 and JNK almost completely inhibited collagenase induction. PD98059 and SB203580 individually blocked HBFN-f-increased cleavage of type II collagen in the explant culture, although 10 microM SB203580 strongly inhibited the collagen cleavage compared with 1 microM of the inhibitor. These results indicate that collagenase production leading to type II collagen cleavage in cartilage explants requires ERK, p38, and JNK.

Involvement of CD44 in induction of matrix metalloproteinases by a COOH-terminal heparin-binding fragment of fibronectin in human articular cartilage in culture.

To investigate the mechanism of induction of matrix metalloproteinases (MMPs) by a 40-kd COOH-terminal heparin-binding fibronectin fragment (HBFN-f) containing III12-14 and IIICS domains in human articular cartilage in culture. Human articular cartilage was removed from macroscopically normal femoral heads and cultured with HBFN-f. MMP secretion into conditioned media was analyzed by immunoblotting (MMPs 1 and 13) and by gelatin zymography (MMPs 2 and 9). Type II collagen cleavage by collagenase was monitored in culture by immunoassay. Involvement of specific peptide-binding domains in HBFN-f and the involvement of CD44 were assessed with synthetic peptides and an anti-CD44 antibody. Immunofluorescence histochemistry was performed using fluorescein isothiocyanate-conjugated anti-CD44 antibody. HBFN-f stimulated production of MMPs 1, 2, 9, and 13 in association with type II collagen cleavage by collagenase in human articular cartilage. Peptide V (WQPPRARI) of HBFN-f, which can bind cell surface heparan sulfate proteoglycan (HSPG), blocked MMP induction by HBFN-f, while the scrambled peptide V (RPQIPWAR) had no effect. Peptide CS-1 of 25 amino acids in IIICS of HBFN-f caused no significant effect. Treatment of cartilage with anti-CD44 antibody or HSPG resulted in significant inhibition of HBFN-f-stimulated MMP production. Preincubation with peptide V blocked binding of the anti-CD44 antibody to chondrocytes in cartilage. Interaction of the peptide V sequence in HBFN-f with glycosaminoglycans, such as those in CD44, plays an important role in HBFN-f-stimulated MMP production in articular cartilage. Because CD44 is up-regulated in osteoarthritic and rheumatoid arthritic cartilage, the role of the interaction between CD44 and HBFN-f in these pathologies should be of relevance and should be studied further.

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

Fibronectin with IIICS region is present in rheumatoid synovium, and fibronectin fragments are increased in rheumatoid joints. We investigated the ability of COOH-terminal heparin-binding fibronectin fragment (COOH-HBFN-f) containing IIICS to induce matrix metalloproteinase (MMP) production and the role of mitogen-activated protein kinase (MAPK) pathway and CS-1 sequence that can bind α4β1 integrin in MMP induction by COOH-HBFN-f in rheumatoid synovial fibroblasts (RSF). When RSF in monolayer culture were incubated with COOH-HBFN-f, COOH-HBFN-f stimulated the production of MMP-1, MMP-3, and MMP-13 by RSF in association with activation of extracellular signal-regulated kinase, p38 MAPK, and c-Jun NH2-terminal kinase. Immunoprecipitation of cell lysates demonstrated the presence of α4 integrin in cultured RSF. Similar to COOH-HBFN-f, treatment with CS-1 synthetic peptide derived from IIICS resulted in increased MMP production and activation of the kinases, although the MMP levels were low. Preincubation of RSF with anti-α4 integrin antibody resulted in partial suppression of the COOH-HBFN-f–stimulated MMP production. Inhibition studies using protein kinase inhibitors (PD98059 and SB203580) showed that those MAPK pathways contributed to MMP up-regulation by COOH-HBFN-f and CS-1. Thus, the present results have clearly shown that COOH-HBFN-f and CS-1 stimulate MMP production in association with activation of MAPK pathways in RSF. Integrin α4β1 may be partially involved in the MMP induction by COOH-HBFN-f.

Involvement of chondroitin sulfates on brain-derived tenascin-R in carbohydrate-dependent interactions with fibronectin and tenascin-C

Tenascin-R (TN-R), a matrix glycoprotein of the central nervous system (CNS), has been implicated in a variety of cell–matrix interactions involved in the control of axon growth, myelination and cell adhesion to fibronectin during development and regeneration. While most of the functional analyses have concentrated exclusively on the role of the core protein, the contribution of TN-R glycoconjugates present on many potential sites for N- and O-glycosylation is presently unknown. Here we provide evidence that TN-R derived from adult mouse brain expresses chondroitin sulfate (CS) glycosaminoglycans (GAGs), i.e. C-6S and C-4S, that are recognized by the CS/dermatan sulfate-specific monoclonal antibodies 473 HD and CS-56. Using ligand-binding, cell adhesion and neurite outgrowth assays, we show that TN-R-linked CS GAGs (i) are involved in the interaction with the heparin-binding sites of fibronectin and are responsible for TN-R-mediated inhibition of cell adhesion to a 33/66-kD heparin-binding fibronectin fragment or to FN-C/H I and FN-C/H II peptides, known to participate in fibronectin binding to cell surface proteoglycans; and (ii) partially contribute to the interaction between TN-R and TN-C which, however, does not lead to an interference with TN-R- and TN-C-mediated inhibition of neurite outgrowth when the two molecules are offered as a mixed substrate in culture. Our findings suggest the functional implication of TN-R-linked CS GAGs in matrix interactions with fibronectin and TN-C that are likely to contribute to a modulation of cellular behavior and the macromolecular organization of matrix components in the developing or injured adult CNS.

Fibronectin and fibronectin fragments modulate the expression of proteinases and proteinase inhibitors in human periodontal ligament cells.

Fragments of the matrix molecule fibronectin (FN) have been shown to modulate tissue remodeling activity by inducing matrix metalloproteinases (MMPs) in synovial fibroblasts. These molecules could contribute to the tissue degradation that occurs during periodontal disease if they also modulate the expression of proteinases in cells of the periodontal ligament (PDL). We tested the hypothesis that FN and specific FN fragments induce the expression of specific proteinases in PDL cells. Using substrate zymograms, reverse zymograms and Western immunoblots, we found that PDL cells constitutively express 72 kDa gelatinase, urokinase-type plasminogen activator (uPA) and at least three inhibitors whose molecular masses correspond to those of the tissue inhibitors of metalloproteinases (TIMPs). A fourth, previously uncharacterized, proteinase inhibitor of approximately 22 kDa was also observed in some cell isolates. PDL cells, when exposed to a 120 kDa proteolytic FN fragment containing the cell-binding domain, were induced to express collagenase and stromelysin and also demonstrated an increased secretion of the serine proteinase uPA. Expression of collagenase increased with increasing concentrations (0.001 microM-1 microM) of the 120 kDa FN fragment. This fragment also induced the expression of a 20 kDa inhibitor, but not of the higher-molecular-mass inhibitors, in PDL cells. The observed alterations in proteinases were associated specifically with the 120 kDa FN fragment, since similar responses were not seen when PDL cells were exposed to either a 60 kDa heparin-binding FN fragment or a 45 kDa collagen/gelatin-binding FN fragment. PDL cells exposed to intact FN did not express the proteinases induced by the 120 kDa fragment but did express 92 kDa gelatinase and the 20 kDa proteinase inhibitor. These data suggest that FN and specific FN fragments can differentially induce the expression of proteinases in PDL cells. Thus, functional regions of FN may modulate many of the functions of PDL cells that contribute to periodontal disease, wound healing and maintenance of extracellular matrix in periodontal tissues.

Differential outgrowth of retinal neurites on purified extracellular matrix molecules

Organotypic cultures of the embryonic retina were used to study the influence of extracellular matrix molecules on neurite elongation during development of the central nervous system. Microexplants from the chick retina (embryonic day 6) were grown in medium containing appropriate trophic support on purified matrix molecules adsorbed to plastic at various concentrations. The maximum neurite length obtained on each type of substratum was measured on day 4 of culture. No fiber outgrowth occurred on substrata of vitronectin or a hyaluronate-binding chondroitin sulfate proteoglycan. In contrast, neurite elongation was strongly promoted on laminin in a dose-dependent manner. Fibronectin elicited a neurite outgrowth corresponding to about one-third the length of the outgrowth on laminin. A 31,000-dalton fibronectin fragment representing the heparin-binding domain elicited neurite elongation comparable to that promoted by the intact fibronectin molecule. Other isolated domains of fibronectin, including the 105,000-dalton "cell-binding" domain, did not allow neurite outgrowth. Furthermore, preincubation of fibronectin substratum with antibodies to the heparin-binding fibronectin fragment entirely prevented outgrowth. Fiber outgrowth was also evoked on substrata of platelet factor 4, a protein binding heparan sulfate. Adding increasing concentrations of heparin progressively inhibited the neurite extension on laminin, whereas similar addition of soluble chondroitin sulfate proteoglycan had no effect. The results indicate that growing retinal neurites show strong preference for laminin versus fibronectin. Moreover, the outgrowth-promoting activity of both cell adhesion proteins seems to be localized to their heparin-binding regions. It is suggested that during development of the visual system, elongating retinal neurites can actively discriminate between different extracellular molecules by a mechanism that may involve participation of cell surface heparan sulfate proteoglycans.

Adhesion of glycosaminoglycan-deficient chinese hamster ovary cell mutants to fibronectin substrata.

We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan-deficient cells did not attach to substrate composed of a heparin-binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.