Original Contribution
Endogenous production of reactive oxygen species is required for stimulation of human articular chondrocyte matrix metalloproteinase production by fibronectin fragments

https://doi.org/10.1016/j.freeradbiomed.2007.01.035Get rights and content

Abstract

The objective of the present study was to determine if reactive oxygen species (ROS) are required as secondary messengers for fibronectin fragment-stimulated matrix metalloproteinase (MMP) production in human articular chondrocytes. Cultured cells were stimulated with 25 μg/ml of the α5β1 integrin-binding 110-kDa fibronectin fragment (FN-f) in the presence and absence of various antioxidants including Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP). FN-f stimulation significantly increased intracellular levels of ROS in articular chondrocytes. Pretreatment of cells with 250 μM MnTBAP or 40 mM N-acetyl-L-cysteine, but not inhibitors of nitric oxide synthase, completely prevented FN-f-stimulated MMP–3, –10, and –13 production. MnTBAP also blocked FN-f-induced phosphorylation of the MAP kinases and NF-κB-associated proteins and blocked activation of an NF-κB promoter-reporter construct. Overexpression of catalase, superoxide dismutase, or glutathione peroxidase also inhibited FN-f-stimulated MMP-13 production. Preincubation of chondrocytes with rotenone, an inhibitor of the mitochondrial electron transport chain, or nordihydroguaiaretic acid (NDGA), a selective 5-lipoxygenase inhibitor, partially prevented FN-f-stimulated MMP-13 production and decreased MAP kinase and NF-κB phosphorylation. These results show that increased production of ROS but not nitric oxide as obligatory secondary messengers in the chondrocyte FN-f signaling pathway leads to the increased production of MMPs, including MMP-13.

Introduction

Osteoarthritis (OA) is a multifactorial disease characterized by a progressive loss of matrix proteins in human articular cartilage and subsequent chondrocyte cell death, with age as the strongest risk factor [1]. The free radical theory of aging suggests that an accumulation of reactive oxygen species (ROS) causes irreparable damage to cells and tissues over time; however, the exact mechanism remains poorly understood [2]. Previous studies implicate ROS as playing an important role in cartilage destruction in arthritis [3]. Excessive ROS production can contribute to chondrocyte death [4], [5], [6], [7]. However, controversy exists as to the exact role of cell death in the development of OA with the vast majority of cell death likely occurring in later stages of the disease [8]. We hypothesized that ROS have additional effects on articular cartilage well before cell death takes place.

ROS have physiologic roles as secondary mediators in multiple cell signaling pathways including those initiated by growth factors, cytokines, and extracellular matrix proteins [2], [9], [10]. Activation of c-Jun NH2-terminal kinase (JNK) by IL-1 and TNF-α in chondrocytes has been shown to require ROS as a signaling intermediate [11]. In synovial fibroblasts, ROS have also been shown to be required for signaling initiated through the α5β1 integrin that results in increased production of matrix metalloproteinase (MMP)-1 [12]. Stimulation of the α5β1 integrin on articular chondrocytes, with either integrin-activating antibodies or fibronectin fragments, also resulted in increased MMP production [13], [14] but the potential role of ROS in this signaling event in chondrocytes has not been determined. Importantly, the role of ROS in the activation of specific downstream signaling proteins in the α5β1 pathway that mediates MMP expression has also not been determined.

Defining the role of ROS in integrin signaling which regulates MMP production is important because excessive MMP production is a key mechanism by which cartilage matrix destruction occurs during the development of arthritis. In chondrocytes, the integrin signaling pathway which mediates increased MMP-13 production includes activation of the three major families of MAP kinases (ERK, JNK, and p38) and increased activity of the NFκB and AP-1 transcription factors [13], [14], [15]. In these studies the chondrocyte α5β1 integrin was stimulated using either an integrin-activating antibody or the 110-kDa fibronectin fragment (FN-f) which contains the RGD-binding site for α5β1. Stimulation of chondrocytes with fibronectin fragments is relevant to cartilage biology because similar fragments have been found in both RA and OA articular cartilage and synovial fluid [16]. In addition, a previous study provided evidence that antioxidants could inhibit the ability of a smaller (29 kDa) FN-f to degrade articular cartilage explants [17].

The aim of the present study was to determine whether FN-f-stimulated MMP production requires ROS as secondary messengers. We chose the 110-kDa FN-f because it contains the α5β1 integrin cell-binding region [18], [19] and because previous studies in our lab using this fragment had shown similar results to chondrocytes stimulated with the α5β1-activating antibody JBS5 [13]. Here we found that ROS are obligatory components of the signal transduction cascade responsible for increased MMP production by cells treated with FN-f. Both antioxidants and overexpession of catalase (CAT) or glutathione peroxidase (GPx) completely block this pathway. These results suggest that ROS can have deleterious effects on human tissues through the increased production of physiologically relevant MMPs.

Section snippets

Reagents

Dulbecco's modified eagle medium (DMEM), Ham's F-12, phosphate-buffered saline (PBS), gentamicin, penicillin G sodium-streptomycin sulfate-amphotericin B, and fetal bovine serum (FBS) were purchased from GibcoBRL (Gaithersburg, MD). Pronase, NG-monomethyl-L-arginine (L-NMMA), L-N6-(1-iminoethyl)lysine (L-NIL), MK-886, 5,8,11,14-eicosatetraynoic acid (ETYA), and manganese (III)-tetrakis (4-benzoic acid) porphyrin (MnTBAP) were obtained from Calbiochem (La Jolla, CA). Collagenase-P was purchased

FN-f-stimulated chondrocytes display increased levels of ROS

The redox-sensitive dye DCFDA was used to detect FN-f-stimulated ROS production in chondrocytes cultured in monolayer. FN-f displayed a 4.5-fold increase in DCFDA intensity at 30 s whereas PBS alone displayed a 2.2-fold increase in DCFDA intensity at the same time point (Fig. 1). The signal intensity began to decrease after the 30 s time point in both control and FN-f treated cells possibly due to photobleaching (data not shown).

Effect of antioxidants on FN-f-stimulated MMP production

The contribution of ROS to FN-f-mediated MMP production was

Discussion

There is accumulating evidence that ROS serve as signaling intermediates in a number of intracellular pathways initiated through a variety of receptors including growth factor, cytokine, and integrin receptors [2], [9], [10], [11]. In the present study, we focused on the chondrocyte α5β1 integrin pathway which is stimulated in response to FN-f and results in increased MMP production. We found that ROS levels in the cell were rapidly increased after exposure to FN-f and that inhibition of ROS

Acknowledgments

This work was supported by Grant AR49003 from the National Institutes of Health. We gratefully acknowledge the Gift of Hope Organ and Tissue Donor Network, the National Disease Research Interchange (NDRI), and the donor families for providing tissue and the assistance of Dr. Arkady Margulis for collecting donor tissues. We also thank Dr. Larry Oberly for providing plasmids and Dr. Kenneth Ingram for fibronectin fragments.

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