Abstract
The pathology of joint destruction is associated with elevated production of basic fibroblast growth factor (bFGF) and matrix metalloproteinase-13 (MMP-13). In osteoarthritic joint disease, expression of bFGF and MMP-13 in chondrocytes and their release into the synovial fluid are significantly increased. We have previously found that the capacity for cartilage repair in human adult articular chondrocytes is severely compromised by minimal exposure to bFGF because bFGF reduces responsiveness to bone morphogenetic protein-7 and insulin-like growth factor-1 and induces MMP-13 through protein kinase Cdelta-dependent activation of multiple mitogen-activated protein kinase (MAPK) signaling pathways. Here we show using biochemical and molecular approaches that transcription factor Elk-1, a direct downstream target of MAPK, is a critical transcriptional activator of of MMP-13 by bFGF in human articular chondrocytes. We also provide evidence that Elk-1 is a direct target of NFkappaB and induces MMP-13 expression upon activation of the NFkappaB signaling pathway. Taken together, our results suggest that elevated expression of MMP-13 occurs through Elk-1 activation of both MAPK and NFkappaB signaling pathways, thus revealing a two-pronged biological mechanism by which bFGF controls the production of catabolic enzymes that are associated with excessive degradation of the cartilage matrix in degenerative joint diseases such as osteoarthritis.
Highlights
Physiological significance and potential clinical impact on cartilage homeostasis, comparatively little is known about the role of growth factors, cytokines, and inflammatory mediators in the pathogenesis of the disease
BFGF Stimulates matrix metalloproteinase-13 (MMP-13) via Elk-1 Activation decreased level of promoter activity is similar to the activity observed for the Ϫ186MMP-13 promoter that contains minimal core transcription factor response elements, including motifs for Runx2, ETS, and AP-1. These results suggest that the distal Ϫ736 to Ϫ370 promoter region of the matrix metalloproteinases (MMPs)-13 gene encompasses key elements that regulate MMP-13 promoter activity in human adult articular chondrocytes
Because Basic FGF (bFGF) activates the MAPKAP-1 pathway to stimulate MMP-13 expression in human adult articular chondrocytes [18], and because Elk-1 is an MAPKresponsive factor in other biological contexts (19 –24), we examined the role of Elk-1 in MMP-13 expression
Summary
Cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 containing 10% fetal bovine serum and antibiotics (complete media) for 5 days before the experiments. In samples containing combinations of plasmids (i.e. co-transfections with Elk-1 or pCMV-IB␣M 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 set of experiments to minimize toxic effects observed at higher DNA concentrations. The cells were washed and incubated with green fluorescent-labeled anti-mouse secondary antibody (prepared in 1% bovine serum albumin-PBS) for 1 h at 4 °C in the dark. Histology—Safranin Orange staining was performed using full thickness cartilage slices prepared from OA knee joint tissues and a 4-mm diameter puncher These explants were cultured in 1.0 ml of Dulbecco’s modified Eagle’s medium/F-12 containing 10% fetal bovine serum. The effect of siRNA was evaluated by using anti-Elk-1 or anti-GAPDH antibody
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