Abstract
Excessive release of basic fibroblast growth factor (bFGF) during loading and/or injury of the cartilage matrix may contribute to the onset or progression of osteoarthritis. This pathological role may be related to the ability of bFGF to decrease proteoglycan synthesis and to antagonize the activity of anabolic growth factors in cartilage such as insulin-like growth factor-1 and bone morphogenetic protein 7 (BMP7 or OP-1). Matrix metalloproteinase-13 (MMP-13), a catabolic cartilage-degrading enzyme, is dramatically up-regulated by inflammatory cytokines or by fibronectin fragments in articular chondrocytes. In this study, we investigated MMP-13 production by bFGF using human articular chondrocytes. Endogenous concentration of bFGF in synovial fluids collected from arthritis patients and asymptomatic subjects showed a good linear correlation with the endogenous levels of MMP-13. bFGF stimulation of MMP-13 was mediated at the transcriptional level and, at least in part, by stimulation of interleukin-1 production. Also, our findings suggest that bFGF stimulation of MMP-13 required the activation of multiple MAPKs (ERK, p38, and JNK) by bFGF, and more importantly, bFGF activation of protein kinase C (PKC) delta played a key role in the MMP-13 stimulation. Indeed, PKCdelta is the only isoform associated with MMP-13 stimulation among the PKC isoforms tested. PKCdelta controls the bFGF response by regulating multiple MAPK pathways. Our results suggest that PKCdelta activation is a principal rate-limiting event in the bFGF-dependent stimulation of MMP-13 in human adult articular chondrocytes. We propose that deregulation of cross-talk between MAPK and PKCdelta signaling may contribute to the etiology of osteoarthritis in human patients.
Highlights
Osteoarthritis (OA)2 involves the progressive destruction of the cartilage extracellular matrix (ECM) by a pathological imbalance in the normal metabolic functions of articular chondrocytes
Endogenous Levels of bFGF and matrix metalloproteinases (MMPs)-13 Are Increased in Arthritic Patients—Evidence suggests that bFGF is pathologically associated with joint destruction and MMP-13 stimulation (10 –12)
Accumulated evidence has indicated that in arthritic cartilage the overproduction of collagenases, in particular MMP-13, by chondrocytes plays a central role in cartilage degeneration
Summary
Adenovirus Infection—For adenovirus infection, primary human chondrocytes were plated at a density of 2 ϫ 106 cells per well in 12-well plates and incubated for Ͼ16 h in serum-free media by modifying our previous methodology (26 –28) Briefly, cells were infected with a replication-defective adenovirus encoding PKC-␦ wild type, PKC-␦ DN, or dominant negative forms of other PKC isoforms (PKC␣ and PKC⑀) at a multiplicity of infection of 200. In samples containing combinations of plasmids (i.e. co-transfections with MEKK cDNA construct with MMP-13 promoter/luciferase reporter construct), we adjusted the total amount of DNA concentration to Ͻ5 g per 100 l of cell-nucleofector solution complex for the entire sets of experiments to minimize the toxic effect observed at higher DNA concentrations.
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